Indian Pharmacopoeia Vol-3

INDIAN PHARMACOPOEIA 2007
Volume 3
THE INDIAN PHARMACOPOEIA COMMISSION GHAZIABAD
Volume 3
CONTENTS
Monographs on Drug Substances, Dosage Forms and Pharmaceutical Aids Monographs to Z Monographs on Vaccines and Immunosera for Human Use Monographs on Herbs and Herbal Products Monographs on Blood and Blood-related Products Monographs on Biotechnology Products Monographs on Veterinary Products Index
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GENERAL NOTICES
GENERAL NOTICES
General Statements Name Official and Official Articles Official Standards Added Substances Alternative Methods Meanings of Terms Provisions Applicable to Monographs and Test Methods Expression of Contents Expression of Concentrations Abbreviated Statements Weights and Measures Monographs General Monographs Production Manufacture of Drug Products Excipients Individual Monographs Titles Chemical Formulae Atomic and Molecular Weights Definitions Statement of Contents Descriptions Identification Tests and Assay Tests Other tests Limits Quantities
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Apparatus Reagents and Solutions Indicators Reference Substances Tests Animals Calculation of Results Storage Storage Containers Labelling
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General Notices
General Statements
The General Notices provide the basic guidelines for the interpretation and application of the standards, tests, assays, and other specifications of the Indian Pharmacopoeia (IP), as well as to the statements made in the monographs and other texts of the Pharmacopoeia. A monograph is to be constructed in accordance with any general monograph or notice or any appendix, note or other explanatory material that is contained in this Pharmacopoeia and that is applicable to that monograph. All statements contained in the monograph, except where a specific general notice indicates otherwise and with the exceptions given hereafter, constitute standards for the official articles. An article is not of pharmacopoeial quality unless it complies with all of the requirements stated. Exceptions to the General Notices do exist, and where they do, the wording in the individual monograph or an appendix takes precedence and specifically indicates directions or the intent. Thus, the specific wording of standards, tests, assays and other specifications is binding wherever deviations from the General Notices exist. Likewise, where there is no specific mention to the contrary, the General Notices apply. Name. The full name or title of this book, including addenda thereto, is Indian Pharmacopoeia 2007, abbreviated to IP 2007. In the texts, the term Pharmacopoeia or IP without qualification means the Indian Pharmacopoeia 2007 and any addenda thereto. Official and Official Articles. The word official wherever used in this Pharmacopoeia or with reference thereto, is synonymous with pharmacopoeial, with IP and with compendial. The designation IP in conjunction with the official title on the label of an article is an indication that the article purports to comply with IP standards. The following terms are used where the articles for which monographs are provided are to be distinguished. An official substance is a single drug or a drug entity or a pharmaceutical aid for which the monograph title includes no indication of the nature of a dosage form. An official preparation is a drug product (dosage form) and is the finished or partially finished preparation or product of one or more official substances formulated for use on the patient. An article is an item for which a monograph is provided, whether an official substance or an official preparation. Official Standards. The requirements stated in the monographs apply to articles that are intended for medicinal
use but not necessarily to articles that may be sold under the same name for other purposes. The active pharmaceutical ingredients (drug substances), excipients (pharmaceutical aids), pharmaceutical preparations (dosage forms) and other articles described in the monographs are intended for human and veterinary use (unless explicitly restricted to one of these uses). The requirements given in the monographs are not framed to provide against all possible impurities, contaminants or adulterants; they provide appropriate limitation of potential impurities only. A preparation must comply throughout the shelf-life assigned to it by the manufacturer; for opened or broached containers the maximum period of validity for use may sometimes be stated in the individual monograph. Nevertheless, the responsibility for assigning the period of validity shall be with the manufacturer. Added Substances. An official substance, as distinguished from an official preparation, contains no added substances except when specifically permitted in the individual monograph. Unless otherwise specified in the individual monograph, or elsewhere in the General Notices, suitable substances may be added to an official preparation to enhance its stability, usefulness or elegance, or to facilitate its preparation. Such auxiliary substances shall be harmless in the amounts used, shall not exceed the minimum quantity required to provide their intended effect, shall not impair the therapeutic efficacy or the bioavailability or safety of the preparation and shall not interfere with the tests and assays prescribed for determining compliance with the official standards. Particular care should be taken to ensure that such substances are free from harmful organisms. The freedom to the manufacturers to add auxiliary substances imposes on them the responsibility of satisfying the licensing authorities on the purpose of the addition and the innocuity of such substances. Alternative Methods. The tests and assays described are the official methods upon which the standards of the Pharmacopoeia are based. Alternative methods of analysis may be used for control purposes, provided that the methods used are shown to give results of equivalent accuracy and enable an unequivocal decision to be made as to whether compliance with the standards of the monographs would be achieved if the official methods were used. Automated procedures utilising the same basic chemistry as the test procedures given in the monograph may also be used to determine compliance. Such alternative or automated procedures must be validated. In the event of doubt or dispute, the methods of analysis of the Pharmacopoeia are alone authoritative and only the result obtained by the procedure given in this Pharmacopoeia is conclusive.
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Meanings of Terms Alcohol. The term alcohol without qualification means ethanol (95 per cent). Other dilutions of ethanol are indicated by the term alcohol or alcohol followed by a statement of the percentage by volume of ethanol (C2H6O) required. Desiccator. A tightly-closed container of suitable size and design that maintains an atmosphere of low moisture content by means of silica gel or phosphorus pentoxide or other suitable desiccant. Drying and ignition to constant weight. Two consecutive weighings after the drying or igniting operations do not differ by more than 0.5 mg, the second weighing following an additional period of drying or of ignition respectively appropriate to the nature and quantity of the residue. Ethanol. The term ethanol without qualification means anhydrous ethanol or absolute alcohol. Filtration. Unless otherwise stated, filtration is the passing of a liquid through a suitable filter paper or equivalent device until the filtrate is clear. Freshly prepared. Made not more than 24 hours before it is issued for use. Label. Any printed packing material, including package inserts that provide information on the article. Negligible. A quantity not exceeding 0.50 mg. Solution. Where the name of the solvent is not stated, solution implies a solution in water. The water used complies with the requirements of the monograph on Purified Water. The term distilled water indicates Purified Water prepared by distillation. Temperature. The symbol used without qualification indicates the use of the Celsius thermometric scale. Water. If the term is used without qualification it means Purified Water of the Pharmacopoeia. The term distilled water indicates Purified Water prepared by distillation. Water-bath. A bath of boiling water unless water at another temperature is indicated. Other methods of heating may be used provided the required temperature is approximately maintained but not exceeded. Provisions Applicable To Monographs and Test Methods Expression of Content. Where the content of a substance is defined, the expression per cent is used according to circumstances with one of two meanings: per cent w/w (percentage, weight in weight) expressing the number of grams of substance in 100 grams of final product,
per cent v/v (percentage, volume in volume) expressing the number of millilitres of substance in 100 millilitres of final product. The expression parts per million refers to the weight in weight, unless otherwise stated. Where the content of a substance is expressed in terms of the chemical formula for that substance an upper limit exceeding 100 per cent may be stated. Such an upper limit applies to the result of the assay calculated in terms of the equivalent content of the specified chemical formula. For example, the statement contains not less than 99.0 per cent and not more than 101.0 per cent of C7H6O2 implies that the result of the assay is not less than 99.0 per cent and not more than 101.0 per cent, calculated in terms of the equivalent content of C7H6O2. Where the result of an assay or test is required to be calculated with reference to the dried, anhydrous, ignited substance, or the substance free from solvent, the determination of loss on drying, water content, loss on ignition, content of the specified solvent, respectively is carried out by the method prescribed in the relevant test in the monograph. Expression of Concentrations. The following expressions in addition to the ones given under Expression of Content are also used: per cent w/v (percentage, weight in volume) expressing the number of grams of substance in 100 millilitres of product per cent v/w (percentage, volume in weight) expressing the number of millilitres of substance in 100 grams of product. Usually, the strength of solutions of solids in liquids is expressed as percentage weight in volume, of liquids in liquids as percentage volume in volume, of solids in semi-solid bases (e.g. creams) and of gases in liquids as percentage weight in weight. When the concentration of a solution is expressed as parts of dissolved substance in parts of solution, it means parts by weight (g) of a solid in parts by volume (ml) of the final solution; as parts by weight (g) of a gas in parts by weight (g) of the final solution. When the concentration of a solution is expressed in molarity designated by the symbol M preceded by a number, it denotes the number of moles of the stated solute contained in sufficient Purified Water (unless otherwise stated) to produce 1 litre of solution. Abbreviated Statements. Incomplete sentences are employed in parts of the monographs for directness and brevity (for example, Iodine Value. Not more than ; Relative Density. .to..) Where the tests are abbreviated, it is to be understood that the test method referred to in brackets
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provides the method to be followed and that the values specified are the applicable limits. Weights and Measures. The metric system of weights and measures is employed in the Pharmacopoeia. All measures are required to be graduated at 25 and all measurements in tests and assays, unless otherwise stated, are to be made at that temperature. Graduated glass apparatus used in analytical operations shall comply with the requirements stated in Chapter 2.1.6
Excipients. Any substance added in preparing an official preparation shall be innocuous, shall have no adverse influence in the therapeutic efficacy of the active ingredients and shall not interfere with the tests and assays of the Pharmacopoeia. Care should be taken to ensure that such substances are free from harmful organisms. Individual Monographs Drug products that are the subject of an individual monograph are also required to comply with the tests given in the general monographs. Titles. The main title for a drug substance is the International Non-proprietary Name (INN) approved by the World Health Organization. Subsidiary names and synonyms have also been given in some cases; where included, they have the same significance as the main title. The main titles of drug products are the ones commonly recognised in practice. Synonyms drawn from the full nonproprietary name of the active ingredient or ingredients have also been given. Where, however, a product contains one or the other of different salts of an active molecule, the main title is based on the full name of the active ingredient. For example, Chloroquine Phosphate Tablets and Chloroquine SulphateTablets. Chemical Formulae. When the chemical structure of an official substance is known or generally accepted, the graphic and molecular formulae are normally given at the beginning of the monograph for information. This information refers to the chemically pure substance and is not to be regarded as an indication of the purity of the official material. Elsewhere, in statement of purity and strength and in descriptions of processes of assay, it will be evident from the context that the formulae denote the chemically pure substances. Where the absolute stereochemical configuration is specified, the International Union of Pure and Applied Chemistry (IUPAC) R/S and E/Z systems of designation have been used. If the substance is an enantiomer of unknown absolute stereochemistry, the sign of the optical rotation, as determined in the solvent and under the conditions specified in the monograph, has been attached to the systematic name. An indication of sign of rotation has also been given where this is incorporated in a trivial name that appears on an IUPAC preferred list. Atomic and Molecular Weights. The atomic weight or molecular weight is shown , as and when appropriate at the top right hand corner of the monograph. The atomic and molecular weights and graphic formulae do not constitute analytical standards for the substances described. Definition. The opening statement of a monograph is one that constitutes an official definition of the substance,
Monographs
General Monographs General monographs on dosage forms include requirements of general application and apply to all preparations within the scope of the Introduction section of the general monograph, except where a preamble limits the application. The requirements are not necessarily comprehensive for a given specific preparation; additional requirements may sometimes be given in the individual monograph for it. Production. Statements given under the heading Production relate to particular aspects of the manufacturing process and are not necessarily comprehensive. However, they are mandatory instructions to manufacturers. They may relate, for example, to source materials, to the manufacturing process and its validation and control, to any in-process testing that is to be carried out by the manufacturer on the final product either on selected batches or on each batch prior to release. All this cannot be verified on a sample of the final product by an independent analyst. It is for the licensing authority to verify that the instructions have been followed. The absence of a section on Production does not imply that attention to features such as those given above is not required. An article described in a monograph of the Pharmacopoeia is to be manufactured in accordance with the principles of good manufacturing practice and in accordance with the requirements of the Drugs and Cosmetics Rules, 1945. The general principles applicable to the manufacture and quality assurance of drugs and preparations meant for human use apply equally to veterinary products as well. Manufacture of Drug Products. The opening definitive statement in certain monographs for drug products is given in terms of the active ingredient(s) only. Any ingredient(s) other than those included in the statement, must comply with the general notice on Excipients and the product must conform to the Pharmacopoeial requirements. Official preparations are prepared only from ingredients that comply with the requirements of the pharmacopoeial monographs for those individual ingredients for which monographs are provided.
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preparation or other article that is the subject of the monograph. In certain monographs for pharmaceutical preparations the statement is given in terms of the principal ingredient(s). In monographs on vegetable drugs, the definition indicates whether the subject of the monograph is, for example, the whole drug or the drug in powdered form. Certain pharmaceutical substances and other articles are defined by reference to a particular method of manufacture. A statement that a substance or article is prepared or obtained by a certain method constitutes part of the official definition and implies that other methods are not permitted. A statement that a substance may be prepared or obtained by a certain method, however, indicates that this is one possible method and does not imply that other methods are not permissible. Statement of content. The limits of content stated are those determined by the method described under Assay. Description. The statements under the heading Description are not to be interpreted in a strict sense and are not to be regarded as official requirements. Solubility. Statements on solubility are given in Chapter 2.4.26 and are intended as information on the approximate solubility at a temperature between 15 and 30, unless otherwise stated, and are not to be considered as official requirements. However, a test for solubility stated in a monograph constitutes part of the standards for the substance that is the subject of that monograph. Test Methods References to general methods of testing are indicated by test method numbers in brackets immediately after the heading of the test or at the end of the text. Identification. The tests given under the heading Identification are not necessarily sufficient to establish absolute proof of identity. They provide a means of verifying that the identity of the material under examination is in accordance with the label on the container. In certain monographs alternative series of identification tests are given; compliance with either one or the other set of tests is adequate to verify the identity of the article. When tests for infrared absorption are applied to material extracted from formulated preparations, strict concordance with the specified reference spectrum may not always be possible, but nevertheless a close resemblance between the spectrum of the extracted material and the specified reference spectrum should be achieved. Tests and Assays The tests and assays are the official methods upon which the standards of the Pharmacopoeia depend. The requirements
are not framed to take into account all possible impurities. It is not to be presumed, for example, that an impurity that is not detectable by means of the prescribed tests is tolerated. Material found to contain such an impurity is not of pharmacopoeial quality if the nature or amount of the impurity found is incompatible with good pharmaceutical practice. Pharmacopoeial methods and limits should be used merely as compliance requirements and not as requirements to guarantee total quality assurance. Tests and assays are prescribed for the minimum sample available on which the attributes of the article should be measured. Assurance of quality must be ensured by the manufacturer by the use of statistically valid sampling and testing programmes. Tests. Unless otherwise stated, the assays and tests are carried out at a temperature between 20 and 30. Where it is directed that an analytical operation is to be carried out in subdued light, precautions should be taken to avoid exposure to direct sunlight or other strong light. Where a procedure is directed to be performed protected from light precautions should be taken to exclude actinic light by the use of low-actinic glassware, working in a dark room or similar procedures. For preparations other than those of fixed strength, the quantity to be taken for a test or an assay is usually expressed in terms of the active ingredient. This means that the quantity of the active ingredient expected to be present and the quantity of the preparation to be taken are calculated from the strength stated on the label. Other Tests. In the monographs on dosage forms and certain preparations, under the sub-heading Other tests it is stated that the article complies with the tests stated under the general monograph of the relevant dosage form or preparation. Details of such tests are provided in the general monographs. Limits. The limits given are based on data obtained in normal analytical practice. They take into account normal analytical errors, of acceptable variations in manufacture and of deterioration to an extent that is acceptable. No further tolerances are to be applied to the limits for determining whether or not the article under examination complies with the requirements of the monograph. Quantities. Unless otherwise stated, the quantities to be taken for assays, limit tests and other tests are of the substance under examination. In tests with numerical limits and assays, the quantity stated to be taken for testing is approximate. The amount actually used, which may deviate by not more than 10 per cent from that stated, is accurately weighed or measured and the result of analysis is calculated from this exact quantity. In tests where the limit is not numerical but usually depends upon comparison with the behaviour of a reference in the same
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GENERAL NOTICES
conditions, the stated quantity is taken for testing. Reagents are used in the prescribed amounts. Quantities are weighed or measured with an accuracy commensurate with the indicated degree of precision. For weighings, the precision is plus or minus 5 units after the last figure stated. For example, 0.25 g is to be interpreted as 0.245 g to 0.255 g. For the measurement of volumes, if the figure after the decimal point is a zero or ends in a zero, e.g. 10.0 ml 0r 0.50 ml, the volume is measured using a pipette, a volumetric flask or a burette, as appropriate; in other cases, a graduated measuring cylinder or a graduated pipette may be used. Volumes stated in microlitres are measured using a micropipette or microsyringe. The term transfer is used generally to indicate a quantitative operation. Apparatus. Measuring and weighing devices and other apparatus are described in the chapter entitled Apparatus for Tests and Assays. A specification for a definite size or type of container or apparatus in a test or assay is given merely as a recommendation. Unless otherwise stated, comparative tests are carried out using identical tubes of colourless, transparent, neutral glass with a flat base, commonly known as Nessler cylinders. Reagents and Solutions. The reagents required for the tests and assays of the Pharmacopoeia are defined in the various chapters showing their nature, degree of purity and the strengths of the solutions to be made from them. The requirements set out are not intended to imply that the materials are suitable for use in medicine; regents not covered by monographs in the pharmacopoeia shall not be claimed to be of IP quality. The term analytical reagent grade of commerce implies that the chemical is of a high degree of purity wherein the limits of various impurities are known. Where it is directed to use a general laboratory reagent grade of commerce it is intended that a chemically pure grade material, not necessarily required to be tested for limiting or absence of certain impurities, is to be used. Indicators. Where the use of an indicator solution is mentioned in an assay or test, approximately 0.1 ml of the solution shall be added, unless otherwise directed. Reference Substances. Certain monographs require the use of a chemical reference substance or a biological reference preparation or a reference spectrum These are authentic specimens chosen and verified on the basis of their suitability for intended use as prescribed in the Pharmacopoeia and are not necessarily suitable in other circumstances. IP Reference Substances, abbreviated to IPRS (and referred to as RS in the individual monographs) are issued by the
Indian Pharmacopoeia Commission (IPC). They are the official standards to be used in cases of arbitration. Secondary Standards (Working Standards) may be used for routine analysis, provided they are standardized at regular intervals against the Reference Substances Biological Reference Substances, also abbreviated to IPRS and Standard Preparations of antibiotics are issued by agencies authorised by the IPC. They are standardized against the International Standards and Reference Preparations established by the World Health Organization (WHO). The potency of these preparations is expressed in International Units. Reference spectra are published by the IPC and they are accompanied by information concerning the conditions used for sample preparation and recording of the spectra. Test animals. Unless otherwise directed, animals used in a test or an assay shall be healthy and are drawn from a uniform stock, and have not previously been treated with any material that will interfere with the test or the assay. Calculation of results. In determining compliance with a numerical limit in assay or test, the result should be calculated to one decimal place more than the significant figures stated and then rounded up or down as follows: if the last figure calculated is 5 to 9, the preceding figure is increased by 1; if it is 4 or less, the preceding figure is left unchanged. Storage. Statements under the side-heading Storage constitute non-mandatory advice. The articles of the Pharmacopoeia are to be stored under conditions that prevent contamination and, as far as possible, deterioration. Precautions that should be taken in relation to the effects of the atmosphere, moisture, heat and light are indicated, where appropriate, in the individual monograph. Specific directions are given in some monographs with respect to the temperatures at which Pharmacopoeial articles should be stored, where it is considered that usage at a lower or higher temperature may produce undesirable results. The storage conditions are defined by the following terms: Store in a dry, well-ventilated place at a temperature not exceeding 30 Store in a refrigerator (2 to 8). Do not freeze Store in a freezer (-2 to -18) Store in a deep freezer (Below -18) Storage conditions not related to temperature are indicated in the following terms: Store protected from light Store protected from light and moisture Where no specific storage directions or limitations are given in the monograph or by the manufacturer, it is to be understood
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that the storage conditions include protection from moisture, freezing and excessive heat (any temperature above 40). Storage Containers. The requirements, guidance and information on containers for pharmaceutical use are given in the chapter entitled Containers (6.1) In general, an article should be packed in a well-closed container i.e. one that protects the contents from contamination by extraneous solids, liquids or vapours and from loss of the article under normal conditions of handling and storage. Where, additionally, loss or deterioration of the article from effervescence, deliquescence or evaporation under normal conditions of storage is likely, the container must be capable
of being tightly closed, and re-closed after use. In certain cases, special requirements of pack have been indicated in some monographs under Storage, using expressions that have been defined in chapter 6.1. Labelling. The labelling of drugs and pharmaceuticals is governed by the Drugs and Cosmetics Rules, 1945. The statements that are given in the monographs under the sideheading Labelling are not comprehensive. Only those that are necessary to demonstrate compliance or otherwise with the monograph have been given and they are mandatory. For example, in the monograph on Betamethasone Sodium Tablets the labelling statement is The label states the strength in terms of the equivalent amount of betamethasone. Any other statements are included as recommendations.
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MONOGRAPHS
DRUG SUBSTANCES, DOSAGE FORMS AND PHARMACEUTICAL AIDS

N to Z ..................................................................................................................
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MONOGRAPHS
N
Nalidixic Acid Nalidixic Acid Tablets Nalorphine Hydrochloride Nalorphine Injection Nandrolone Decanoate Nandrolone Decanoate Injection Nandrolone Phenylpropionate Nandrolone Phenylpropionate Injection Naphazoline Nitrate Nelfinavir Mesylate Nelfinavir Mesylate Oral Powder Nelfinavir Tablets Neomycin Sulphate Neomycin Eye Drops Neomycin Eye Ointment Neostigmine Bromide Neostigmine Tablets Neostigmine Methylsulphate Neostigmine Injection Nevirapine Nevirapine Oral Suspension Nevirapine Tablets Niclosamide Niclosamide Tablets Nicotinamide Nicotinamide Tablets Nicotinic Acid Nicotinic Tablets Nicoumalone Nicoumalone Tablets
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MONOGRAPHS
INDIAN PHARMACOPOEIA 2007 2007
Nifedipine Nifedipine Capsules Nifedipine Sustained release-Tablets Nifedipine Tablets Nikethamide Nikethamide Injection Nitrazepam Nitrazepam Tablets Nitrofurantoin Nitrofurantoin Tablets Nitrofurazone Nitrous Oxide Noradrenaline Bitartrate Noradrenaline Bitartrate Injection Norethisterone Norethisterone Tablets Norfloxacin Norfloxacin Eye Drops Norfloxacin Tablets Norgestrel Norgestrel And Ethinyloestradiol Tablets Nortriptyline Hydrochloride Nortriptyline Tablets Noscapine Noscapine Linctus Novobiocin Sodium Nystatin Nystatin Ointment Nystatin Pessaries Nystatin Tablets
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NALIDIXIC ACID TABLETS
Nalidixic Acid
CH3 H3C N N COOH O
C12H12N2O3 Mol. Wt. 232.2
Reference solution (b). A 0.0008 per cent w/v solution of the substance under examination in dichloromethane. Reference solution (c). A 0.1 per cent w/v solution of nalidixic acid RS in dichloromethane. Apply to the plate 10 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with test solution (a) is not more intense than the spot in the chromatogram obtained with reference solution (a) and not more than one such spot is more intense than the spot in the chromatogram obtained with reference solution (b). Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Sulphated ash (2.3.18) Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.15 g, dissolve in 10 ml of dichloromethane, add 30 ml of 2-propanol and 10 ml of carbon dioxide-free water and titrate with 0.1 M ethanolic sodium hydroxide, determining the end-point potentiometrically (2.4.25) and using a glass electrode as the indicator electrode and a silver-silver chloride reference electrode with a sleeve diaphragm or a capillary tip filled with a saturated solution of lithium chloride in ethanol. Throughout the titration keep the temperature of the solution at 15 to 20 and pass a current of nitrogen through the solution. 1 ml of 0.1 M ethanolic sodium hydroxide is equivalent to 0.02322 g of C12H12N2O3. Storage. Store protected from light and moisture.
Nalidixic Acid is 1-ethyl-1,4-dihydro-7-methyl-4-oxo-1,8naphthyridine-3-carboxylic acid. Nalidixic Acid contains not less than 99.0 per cent and not more than 101.0 per cent of C12H12N2O3, calculated on the dried basis. Description. A white to slightly yellow, crystalline powder.
Identification
Test A may be omitted if tests B, C and D are carried out. Tests B, C and D may be omitted if test A is carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with nalidixic acid RS or with the reference spectrum of nalidixic acid. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.0005 per cent w/v solution in 0.1 M sodium hydroxide shows absorption maxima at about 258 nm and 334 nm; ratio of the absorbance at about 258 nm to that at about 334 nm, 2.2 to 2.4. C. In the test for Related substances, the principal spot in the chromatogram obtained with test solution (b) corresponds to that in the chromatogram obtained with reference solution (c). D. Dissolve 0.1 g in 2 ml of hydrochloric acid and add 0.5 ml of a 10 per cent w/v solution of 2-naphthol in ethanol (95 per cent); an orange-red colour develops.
Nalidixic Acid Tablets

Nalidixic Acid Tablets contain not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of nalidixic acid, C12H12N2O3.
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel HF254. Mobile phase. A mixture of 70 volumes of ethanol (95 per cent), 20 volumes of dichloromethane and 10 volumes of 5 M ammonia. Test solution (a). Dissolve 0.2 g of the substance under examination in 10 ml of dichloromethane. Test solution (b). A 0.1 per cent w/v solution of the substance under examination in dichloromethane. Reference solution (a). A 0.002 per cent w/v solution of the substance under examination in dichloromethane.
Identification
To a quantity of the powdered tablets containing 1 g of Nalidixic Acid add 50 ml of chloroform, shake for 15 minutes, filter and evaporate the filtrate to dryness. The residue, after drying at 105, complies with the following tests. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with nalidixic acid RS or with the reference spectrum of nalidixic acid. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.0005 per cent w/v solution in 0.1 M sodium hydroxide shows
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IP 2007
absorption maxima at about 258 nm and 334 nm; ratio of the absorbance at about 258 nm to that at about 334 nm, 2.2 to 2.4.
Nalorphine Hydrochloride contains not less than 97.0 per cent and not more than 103.0 per cent of C19H21NO3,HCl, calculated on the dried basis. Description. A white or almost white, crystalline powder; odourless. It slowly darkens on exposure to air and light.
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel HF254. Mobile phase. A mixture of 70 volumes of ethanol (95 per cent), 20 volumes of dichloromethane and 10 volumes of 5 M ammonia. Test solution. Shake a quantity of the powdered tablets containing 0.1 g of Nalidixic Acid with 50 ml of chloroform for 15 minutes, filter, evaporate the filtrate to dryness and dissolve the residue in 5 ml of chloroform. Reference solution. Dilute 1 volume of the test solution to 200 volumes with chloroform. Apply to the plate 10 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Other Tests. Complies with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 0.1 g of Nalidixic Acid, add 150 ml of 0.1 M sodium hydroxide, shake for 3 minutes, dilute to 200.0 ml with 0.1 M sodium hydroxide, mix and allow to stand for 15 minutes. Dilute 2.0 ml of the solution to 100.0 ml with water and measure the absorbance of the resulting solution at the maximum at about 334 nm (2.4.7), using 0.1 M sodium hydroxide as the blank. Calculate the content of C12H12N2O3 taking 494 as the specific absorbance at 334 nm. Storage. Store protected from light and moisture.
Identification
Test A may be omitted if tests B, C, D and E are carried out. Tests C and D may be omitted if tests A, B and E are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with nalorphine hydrochloride RS. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.01 per cent w/v solution in 0.1 M sodium hydroxide shows an absorption maximum only at about 298 nm; absorbance at about 298 nm, about 0.6. C. To 10 ml of a 2 per cent w/v solution add 0.05 ml of dilute ammonia solution; a white precipitate soluble in sodium hydroxide solution is produced. D. Dissolve 2 mg in 2 ml of water, add 0.15 ml of potassium ferricyanide solution containing, in each ml, 0.05 ml of ferric chloride solution; a deep bluish green colour is produced immediately. E. Gives reaction A of chlorides (2.3.1).
Tests
Melting range (2.4.21). 260 to 263. Acidity. Dissolve 0.2 g in 10 ml of freshly boiled and cooled water and titrate with 0.02 M sodium hydroxide using methyl red solution as indicator; not more than 0.2 ml of 0.02 M sodium hydroxide is required to change the colour of the solution. Specific optical rotation (2.4.22). 122 to 125, determined in a 2.0 per cent w/v solution. Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 100 at a pressure not exceeding 0.7 kPa for 2 hours.
Nalorphine Hydrochloride
HO
O H HO N , HCl CH2
C19H21NO3,HCl
Mol. Wt. 347.8
Nalorphine Hydrochloride is 17-allyl-7,8-didehydro-4,5epoxymorphinan-3,6-diol hydrochloride.
Assay. Weigh accurately about 25 mg and dissolve in sufficient water to produce 250 ml. Measure the absorbance of the resulting solution at the maximum at about 285 nm (2.4.7). Calculate the content of C19H21NO3,HCl from the absorbance obtained by repeating the operation with nalorphine hydrochloride RS in place of the substance under examination.
806
IP 2007
NANDROLONE DECANOATE
Storage. Store protected from light and moisture.
Nandrolone Decanoate
O
Nalorphine Injection
Nalorphine Hydrochloride Injection
Nalorphine Injection is a sterile solution of Nalorphine Hydrochloride in Water for Injections containing suitable buffering agents. Nalorphine Injection contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of nalorphine hydrochloride, C19H21NO3,HCl.
H H O
H3C O H H
CH2(CH2)7CH3
C28H44O3
Mol. Wt. 428.7
Nandrolone Decanoate is 3-oxo-4-estren-17-yl decanoate.
Identification
A. To a volume containing 50 mg of Nalorphine Hydrochloride add dilute ammonia solution until the solution is alkaline and extract with 25 ml of a mixture of 1 volume of ethanol (95 per cent) and 3 volumes of chloroform and evaporate the extract to dryness. Dry the residue at a pressure not exceeding 2 kPa. The residue complies with the following test. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with nalorphine hydrochloride RS. B. To a volume containing 0.1 g of Nalorphine Hydrochloride add 0.05 ml of dilute ammonia solution; a white precipitate soluble in sodium hydroxide solution is produced. C. Gives reaction A of chlorides (2.3.1).
Nandrolone Decanoate contains not less than 97.0 per cent and not more than 103.0 per cent of C28H44O3, calculated on the dried basis. Description. A white to creamy-off white, crystalline powder; odour, faint and characteristic.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with nandrolone decanoate RS or with the reference spectrum of nandrolone decanoate. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.001 per cent w/v solution in ethanol (95 per cent) shows an absorption maximum only at about 239 nm; absorbance at about 239 nm, about 0.41. C. Dissolve 25 mg in 1 ml of methanol, add 2 ml of semicarbazide acetate solution, heat under a reflux condenser for 30 minutes and cool; the precipitate, after recrystallisation from ethanol (95 per cent), melts at about 175 (2.4.21).
Tests
pH (2.4.24). 6.0 to 7.5. Other Tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. Transfer an accurately measured volume containing about 10 mg of Nalorphine Hydrochloride to a separating funnel, add 1 ml of dilute hydrochloric acid and dilute to 10 ml with water. Extract with five successive quantities, each of 5 ml, of chloroform, allowing the layers to separate before drawing off each chloroform extract and discard the chloroform extracts. Transfer the aqueous layer to a 100-ml volumetric flask with the aid of small quantities of water and dilute to volume with water. Measure the absorbance of the resulting solution at the maximum at about 285 nm (2.4.7). Calculate the content of C19H21NO3,HCl from the absorbance obtained by repeating the operation with nalorphine hydrochloride RS. Storage. Store protected from light.
Tests
Specific optical rotation (2.4.22). +32.0 to +36.0, determined in a 2.0 per cent w/v solution in dioxan. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 70 volumes of heptane and 30 volumes of acetone. Test solution. Dissolve 0.1 g of the substance under examination in 10 ml of chloroform. Reference solution (a). A 0.005 per cent w/v solution of the substance under examination in chloroform. Reference solution (b). A 0.01 per cent w/v solution of nandrolone RS in chloroform.
807
NANDROLONE DECANOATE INJECTION
IP 2007
Apply to the plate 5 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. In the chromatogram obtained with the test solution any spot corresponding to nandrolone is not more intense than the spot in the chromatogram obtained with reference solution (b) and any other secondary spot is not more intense than the spot in the chromatogram obtained with reference solution (a). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying over phosphorus pentoxide at a pressure not exceeding 0.7 kPa for 4 hours. Assay. Weigh accurately about 10 mg and dissolve in sufficient ethanol (95 per cent) to produce 100.0 ml. Dilute 5.0 ml to 50.0 ml with ethanol (95 per cent) and measure the absorbance of the resulting solution at the maximum at about 239 nm (2.4.7). Calculate the content of C28H44O3 taking 407 as the specific absorbance at 239 nm. Storage. Store protected from light and moisture.
Tests
Other Tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. To an accurately measured volume containing about 0.1 g of Nandrolone Decanoate add sufficient chloroform to produce 100.0 ml. Dilute 3.0 ml of the solution to 50.0 ml with chloroform. To 5.0 ml of this solution add 10 ml of isoniazid solution and sufficient methanol to produce 20.0 ml. Allow to stand for 45 minutes and measure the absorbance of the resulting solution at the maximum at about 380 nm (2.4.7), using as the blank 5 ml of chloroform treated in the same manner. Calculate the content of C28H44O3 from the absorbance obtained by repeating the operation using a suitable quantity of nandrolone RS. 1 mg of C18H26O2 is equivalent to 1.562 mg of C28H44O3. Storage. Store protected from light.
Nandrolone Phenylpropionate
Nandrolone Phenpropionate
O H3C O H H O H H
Nandrolone Decanoate Injection

Nandrolone Decanoate Injection is a sterile solution of Nandrolone Decanoate in Ethyl Oleate or other suitable ester, in a suitable fixed oil or in any mixture of these. Nandrolone Decanoate Injection contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of nandrolone decanoate, C28H44O3.
Identification
Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 70 volumes of heptane and 30 volumes of acetone. Test solution. Dilute a suitable volume of the injection with carbon tetrachloride to give a solution containing 0.5 percent w/v solution of Nandrolone Decanoate. Reference solution. A 0.5 per cent w/v solution of nandrolone decanoate RS in carbon tetrachloride. Apply to the plate 5 l of each solution. After development, dry the plate in air until the odour of solvent is no longer detectable, spray with a 10 per cent v/v solution of sulphuric acid in ethanol (95 per cent), heat at 105 for 30 minutes and examine in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution. Ignore any subsidiary spots due to the vehicle.
C27H34O3
Mol.Wt. 406.6
Nandrolone Phenylpropionate is 3-oxo-4-estren-17-yl 3phenylpropionate. Nandrolone Phenylpropionate contains not less than 97.0 per cent and not more than 103.0 per cent of C27H34O3, calculated on the dried basis. Description. A white to creamy-white, crystalline powder; odour, characteristic.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with nandrolone phenylpropionate RS or with the reference spectrum of nandrolone phenylpropionate. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.001 per cent w/v solution in ethanol (95 per cent) shows an absorption maximum only at about 240 nm; absorbance at about 240 nm, about 0.43.
808
IP 2007
NANDROLONE PHENYLPROPIONATE INJECTION
C. Dissolve 25 mg in 1 ml of methanol, add 2 ml of semicarbazide acetate solution, heat under a reflux condenser for 30 minutes and cool; the precipitate, after recrystallisation from ethanol (95 per cent) melts at about 182 (2.4.21).
Identification
Dissolve a volume of the injection containing 50 mg of Nandrolone Phenylpropionate in 8 ml of light petroleum (40 to 60) and extract with three 8-ml quantities of a mixture of 7 volumes of glacial acetic acid and 3 volumes of water. Wash the combined extracts with 10 ml of light petroleum (40 to 60), dilute with water until the solution becomes turbid, allow to stand for 2 hours in ice and filter. The precipitate, after washing with water and drying over phosphorus pentoxide at a pressure not exceeding 0.7 kPa, complies with the following test. Determine by thin-layer chromatography (2.4.17), using a silica gel GF254 precoated plate the surface of which has been modified by chemically-bonded octadecylsilyl groups. Mobile phase. A mixture of 20 volumes of water, 40 volumes of acetonitrile and 60 volumes of propan-2-ol. Test solution. A 0.5 per cent w/v solution of the dried precipitate in chloroform. Reference solution (a). A 0.5 per cent w/v solution of nandrolone phenylpropionate RS in chloroform. Reference solution (b). A mixture of equal volumes of the test solution and the reference solution. Apply to the plate 5 l of each solution. After development, dry the plate in air until the solvent has evaporated and heat it at 100 for 10 minutes. Allow to cool and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with reference solution (a). The principal spot in the chromatogram obtained with reference solution (b) appears as a single spot.
Tests
Specific optical rotation (2.4.22). +48.0 to +51.0, determined in a 1.0 per cent w/v solution in dioxan. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 70 volumes of heptane and 30 volumes of acetone. Test solution. Dissolve 0.1 g of the substance under examination in 100 ml of chloroform. Reference solution (a). A 0.005 per cent w/v solution of the substance under examination in chloroform. Reference solution (b). A 0.01 per cent w/v solution of nandrolone RS in chloroform. Apply to the plate 5 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. In the chromatogram obtained with the test solution any spot corresponding to nandrolone is not more intense than the spot in the chromatogram obtained with reference solution (b) and any other secondary spot is not more intense than the spot in the chromatogram obtained with reference solution (a). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19) Not more than 0.5 per cent, determined on 1.0 g by drying over phosphorus pentoxide at a pressure not exceeding 0.7 kPa for 4 hours. Assay. Weigh accurately about 10 mg, dissolve in sufficient ethanol to produce 100.0 ml, dilute 5.0 ml to 50.0 ml with ethanol and measure the absorbance of the resulting solution at the maximum at about 240 nm (2.4.7). Calculate the content of C27H34O3 taking 430 as the specific absorbance at 240 nm. Storage. Store protected from light.
Tests
Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. To an accurately measured volume containing about 0.1 g of Nandrolone Phenylpropionate add sufficient chloroform to produce 100.0 ml. Dilute 3.0 ml of this solution to 50.0 ml with chloroform. To 5.0 ml of the resulting solution add 10 ml of isoniazid solution and sufficient methanol to produce 20.0 ml. Allow to stand for 45 minutes and measure the absorbance of the solution at the maximum at about 380 nm (2.4.7), using as blank 5 ml of chloroform treated in the same manner. Calculate the content of C27H34O3 from the absorbance obtained from a 0.006 per cent w/v solution of nandrolone phenylpropionate RS treated in the same manner. Storage. Store protected from light. Labelling. The label states that the preparation is for intramuscular injection only.
Nandrolone Phenylpropionate Injection

Nandrolone Phenylpropionate Injection is a sterile solution of Nandrolone Phenylpropionate in Ethyl Oleate or other suitable ester, in a suitable fixed oil or in a mixture of these. Nandrolone Phenylpropionate Injection contains not less than 92.5 per cent and not more than 107.5 per cent of the stated amount of nandrolone phenylpropionate, C27H34O3.
809
NAPHAZOLINE NITRATE
IP 2007
Naphazoline Nitrate
N N H
Test solution. Dissolve 0.2 g of the substance under examination in 10 ml of methanol. Reference solution. A solution containing 2 per cent w/v of naphazoline nitrate RS and 0.01 per cent w/v of naphthylacetylethylenediamine hydrochloride RS.
, HNO3
C4HI4N2,HNO3
Mol. Wt. 273.3
Naphazoline Nitrate is 2-(1-napthylmethyl)-2-imidazoline nitrate. Naphazoline Nitrate contains not less than 99.0 per cent and not more than 101.0 per cent of C4HI4N2,HNO3 calculated on the dried basis. Description. A white or almost white. crystalline powder.
Apply to the plate 10 l of each solution. After development, dry the plate at 105 for 5 minutes, spray with a 0.5 per cent w/v solution of ninhydrin in methanol and heat at 105 for 10 minutes. Any spot corresponding to naphthylacetylethylenediamine hydrochloride in the chromatogram obtained with the test solution is not more intense than the corresponding spot in the chromatogram obtained with the reference solution. The test is not valid unless the chromatogram obtained with the reference solution shows two clearly separated spots. Chlorides (2.3.12). 15.0 ml of 1.0 per cent w/v solution in carbon dioxide-free water complies with the limit test for chlorides (375 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105 for 3 hours. Assay. Weigh accurately about 0.2 g, dissolve in 30 ml of anhydrous glacial acetic acid. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.02733 g of C4HI4N2,HNO3. Storage. Store protected from light.
Identification
Test A may be omitted if tests B, C and D are carried out. Tests B and C may be omitted if tests A and D are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with naphazoline nitrate RS. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.002 per cent w/v solution in 0.01 M hydrochloric acid shows absorption maxima at about 270 nm, 280 nm, 287 nm and 291 nm; absorbances at these maxima are about 0.43, 0.50, 0.35 and 0.34 respectively. C. Dissolve about 0.5 mg in 1 m1 of methanol, add 0.5 ml of a freshly prepared 5 per cent w/v solution of sodium nitroprusside and 0.5 ml of a 2 per cent w/v solution of sodium hydroxide, allow to stand for 10 minutes and add 1 ml of a 8 per cent w/v solution of sodium bicarbonate; a violet colour is produced. D. Dissolve about 10 mg in 5 ml of water, add 0.2 g of magnesium oxide, shake mechanically for 30 minutes. add 10 ml of chloroform and shake vigorously. Allow to stand, separate the chloroform layer, filter and evaporate the aqueous layer to dryness. The residue gives reaction A for nitrates (2.3.1).
Nelfinavir Mesylate
CH3 O HO
S N H
O N OH H
H N
CH3 CH3 CH3 H
, CH3SO3H
Tests
Appearance of solution. A 1.0 per cent w/v solution in carbon dioxide-free water is clear (2.4.1) and colourless (2.4.1). pH (2.4.24). 5.0 to 6.5, determined in a 1.0 per cent w/v solution. Naphthylacetylethylenediamine. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 100 volumes of methanol and 1.5 volumes of strong ammonia solution.
C32H45N3O4S,CH4O3S
Mol. Wt. 663.9
Nelfinavir Mesylate is (3S,4aS,8aS)-N-(tert-butyldecahydro2-[(2R,3R)-3-(3-hydroxy-o-toluamido)-hydroxy-4(phenylthio)butyl]isoquinoline-3-carboxamide methyl sulphonate.
810
IP 2007
NELFINAVIR MESTYLATE ORAL POWDER
Nelfinavir Mesylate contains not less than 98.0 per cent and not more than 101.0 per cent of C32H45N3O 4S,CH4O3S, calculated on the anhydrous basis. Description. A white or almost white powder.
1 ml of 0.1 M sodium hydroxide is equivalent to 0.00961 g of CH3SO3H. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Water (2.3.43). Not more than 3.0 per cent, determined on 0.5 g. Assay. Determine by liquid chromatography (2.4.14). Test solution. A 0.01 per cent w/v solution of the substance under examination in the mobile phase. Reference solution. A 0.01 per cent w/v solution of nelfinavir mesylate RS in the mobile phase. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a filtered and degassed mixture of 45 volumes of acetonitrile, 20 volumes of methanol and 35 volumes of a buffer prepared by dissolving 4.0 g of sodium dihydrogen phosphate in 1000 ml of water, to which 1 ml of dimethylamine solution and 1 g of sodium octanesulphonate are added and mixed to dissolve, flow rate. 1 ml per minute, spectrophotometer set at 215 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the column efficiency determined from the nelfinavir peak is not less than 5000 theoretical plates, the tailing factor is not more than 2.0 and the relative standard deviation for replicate injections is not more than 2.0 per cent. Separately inject the test solution and the reference solution and measure the responses for the principal peak. Calculate the content of C32H45N3O4S,CH4O3S. Storage. Store protected from light.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with nelfinavir mesylate RS or with the reference spectrum of nelfinavir mesylate. B. In the Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the peak in the chromatogram obtained with the reference solution.
Tests
Specific optical rotation (2.4.22). 105 to 120, determined in a 1.0 per cent w/v solution in methanol. Related substances. Determine by liquid chromatography (2.4.14), using the chromatographic system described in the Assay. Test solution. A 0.1 per cent w/v solution of the substance under examination in the mobile phase. Reference solution (a). A 0.001 per cent w/v solution of the substance under examination in the mobile phase. Reference solution (b). A 0.01 per cent w/v solution of methanesulphonic acid in the mobile phase. Inject reference solution (a). The test is not valid unless the column efficiency determined from the nelfinavir peak is not less than 4000 theoretical plates and the tailing factor is not more than 2.0. Separately inject reference solution (b) and record the chromatograms. Separately inject the test solution and continue the chromatography for at least three times the retention time of the principal peak. In the chromatogram obtained with the test solution, the area of any peak other than the principal peak is not greater than half of the area of the principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent) and the sum of the areas of all such peaks is not greater than the area of the principal peak in the chromatogram obtained with reference solution (a) (1.0 per cent). Ignore any peak due to methanesulphonic acid corresponding to the retention time of the principal peak in the chromatogram obtained with reference solution (b). Methanesulphonic acid. 13.5 per cent to 15.5 per cent w/w, calculated on the anhydrous basis, determined by the following method. Weigh accurately about 0.6 g, dissolve in 50 ml of dimethylformamide and titrate with 0.1 M sodium hydroxide, determining the end-point potentiometrically (2.4.25). Carry out a blank titration.
Nelfinavir Mesylate Oral Powder

Nelfinavir Mesylate Oral Powder contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of nelfinavir, C32H45N3O4S.
Identification
In the Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the peak in the chromatogram obtained with the reference solution.
811
NELFINAVIR TABLETS
IP 2007
Tests
Dissolution (2.5.2). Apparatus. No 1 Medium. 900 ml of 0.1 M hydrochloric acid. Speed and time. 75 rpm and 45 minutes. Withdraw a suitable volume of the medium and filter. Determine by liquid chromatography (2.4.14). Test solution. Use the filtrate and, if necessary, dilute with the dissolution medium. Reference solution. A 0.065 per cent w/v solution of nelfinavir mesylate RS in methanol. Dilute 10 ml of the solution to 100 ml with the dissolution medium. Use the chromatographic system described under Assay. Inject the test solution and the reference solution. D. Not less than 75 per cent of the stated amount of C32H45N3O4S. Related substances. Determine by liquid chromatography (2.4.14). Test solution. Weigh accurately a quantity of the oral powder containing 50 mg of Nelfinavir Mesylate, disperse in 10 ml of methanol, dilute to 50 ml with the mobile phase and filter. Reference solution (a). Dissolve 10 mg of nelfinavir mesylate RS in 2 ml of methanol and dilute to 10 ml with the mobile phase. Reference solution (b). Dilute 1 ml of reference solution (a) to 100 ml with the mobile phase. Chromatographic system a stainless steel column 15 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), column temperature 45, mobile phase: a mixture of 28 volumes of a buffer solution prepared by dissolving 4.88 g of anhydrous sodium dihydrogen phosphate in 1000 ml of water, adjusting the pH to 3.4 with phosphoric acid and filtering, 27 volumes of acetonitrile, 20 volumes of methanol and 25 volumes of water. Adjust the pH to 4.8 with 0.1 M sodium hydroxide or orthophosphoric acid. flow rate. 1 ml per minute, spectrophotometer set at 220 nm, a 10 l loop injector. Inject the reference solution (a). The test is not valid unless the tailing factor is not more than 2.0 and the column efficiency in not less than 4000 theoretical plates. Inject the test solution and reference solution (b). In the chromatogram obtained with the test solution, the area of any secondary peak is not more than the area of the peak in the chromatogram obtained with the reference solution (b)
(1.0 per cent) and the sum of areas of all the secondary peaks is not more than twice the area of the peak in the chromatogram obtained with the reference solution (b) (2.0 per cent). Water (2.3.43). Not more than 12.0 per cent, determined on 0.5 g. Assay. Determine by liquid chromatography (2.4.14). Solvent mixture. 30 volumes of water and 70 volumes of methanol. Test solution. Weigh accurately a quantity of the powder containing 50 mg of Nelfinavir Mestlate, disperse in 50 ml of 0.1 M hydrochloric acid, dilute to 250.0 ml with the solvent mixture and filter. Reference solution. Dissolve 10 mg of nelfinavir mesylate RS in 10 ml of 0.1 M hydrochloric acid and dilute to 50.0 ml with the solvent mixture. Chromatographic system a stainless steel column 15 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), column temperature 40, mobile phase: a mixture of 35 volumes of a buffer solution prepared by dissolving 4 g of sodium dihydrogen phosphate dihydrate and 1g of 1-octane sulphonic acid sodium salt into 1000 ml of water, adding 1ml of dimethylamine and filtering, 45 volumes acetonitrile and 20 volumes of methanol, flow rate. 2 ml per minute, spectrophotometer set at 220 nm, a 10 l loop injector. Inject the reference solution. The test is not valid unless the tailing factor is not more than 2.0, the column efficiency in not less than 2000 theoretical plates and the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject the test solution and the reference solution. Calculate the content of C32H45N3O4S in the oral powder. Storage. Store protected from moisture, at a temperature not exceeding 30. Labelling. The label states the strength in terms of the equivalent amount of nelfinavir.
Nelfinavir Tablets
Nelfinavir Mesylate Tablets
Nelfinavir Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of nelfinavir mesylate, C32H45N3O4S,CH4O3S.
Identification
A. Shake a quantity of the powdered tablets containing about 0.1 g of Nelfinavir Mesylate with 80 ml of methanol for
812
IP 2007
NEOMYCIN SULPHATE
10 minutes, add sufficient methanol to produce 100 ml, mix and filter. Dilute 5 ml of the filtrate to 100 ml with methanol. When examined in the range 200 nm to 300 nm the resulting solution shows an absorption maximum only at about 254 nm (2.4.7). B. In the Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the peak in the chromatogram obtained with the reference solution.
Inject separately the diluent (10 ml of methanol diluted to 50 ml with the mobile phase) and the test solution and continue the chromatography for 4 times the retention time of the principal peak. Examine the diluent chromatogram for any extraneous peaks and ignore the corresponding peaks observed in the chromatogram obtained with the test solution. Any secondary peak observed in the chromatogram obtained with the test solution should not be more than 1.0 per cent and the sum of the areas of all the secondary peaks should not be more than 2.0 per cent when calculated by percentage area normalisation. Inhibit integration of peak due to methanesulphonic acid. Other tests. Complies with the tests stated under Tablets. Assay. Determine by liquid chromatography (2.4.14). Test solution. Weigh accurately a quantity of the powdered tablets containing about 200 mg of Nelfinavir Mesylate, add about 20 ml of methanol, mix with the aid of ultrasound for 10 minutes and dilute to 100.0 ml with the mobile phase. Filter through a membrane filter disc with an average pore diameter not greater than 1.0 m, rejecting the first few ml of the filtrate. Further dilute 5.0 ml of the filtrate to 100.0 ml with the mobile phase. Reference solution. Weigh accurately about 50 mg of nelfinavir mesylate RS, add about 10 ml of methanol, mix with the aid of ultrasound to dissolve and dilute to 50.0 ml with the mobile phase. Dilute 5.0 ml of this solution to 50.0 ml with the mobile phase. Use the chromatographic system described in the test for Related substances. Inject the reference solution. The test is not valid unless the column efficiency determined from the nelfinavir mesylate peak is not less than 5000 theoretical plates, the tailing factor is not more than 2.0 and the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject separately the test solution and the reference solution and measure the responses for the major peak. Calculate the content of C32H45N3O4S,CH4O3S in the tablets. Storage. Store protected from light.
Tests
Dissolution (2.5.2). Apparatus. No 1 Medium. 900 ml of 0.01 M hydrochloric acid. Speed and time. 50 rpm and 30 minutes. Withdraw a suitable volume of the medium and filter promptly through a membrane filter disc with an average pore diameter not greater than 1.0 m. Reject the first few ml of the filtrate and dilute a suitable volume of the filtrate with the same solvent. Measure the absorbance of the resulting solution at the maximum at about 250 nm (2.4.7). Calculate the content of C32H45N3O4S,CH4O3S from the absorbance of a solution of known concentration of nelfinavir mesylate RS. D. Not less than 75 per cent of the stated amount of C32H45N3O4S, CH4O3S. Related substances. Determine by liquid chromatography (2.4.14). Test solution. Weigh accurately a quantity of the powdered tablets containing about 100 mg of Nelfinavir Mesylate, add about 20 ml of methanol, mix with the aid of ultrasound for 10 minutes and dilute to 100 ml with the mobile phase. Reference solution. Weigh accurately about 10 mg of nelfinavir mesylate RS, add about 10 ml of methanol, shake for 10 minutes and dilute to 50 ml with the mobile phase. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica particles or ceramic microparticles (5 m), mobile phase: a filtered and degassed mixture of 45 volumes of acetonitrile, 20 volumes of methanol and 35 volumes of a buffer prepared by dissolving 4.0 g of sodium dihydrogen phosphate in 1000 ml of water, to which are added 1 ml of dimethylamine solution and 1 g of sodium octanesulphonate and mixing to dissolve, flow rate. 1 ml per minute, spectrophotometer set at 215 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the column efficiency determined from the nelfinavir mesylate peak is not less than 4000 theoretical plates and the tailing factor is not more than 2.0.
Neomycin Sulphate
Neomycin Sulphate is a mixture of the sulphates of substances obtained by the growth of certain selected strains of Streptomyces fradiae. Neomycin Sulphate has a potency of not less than 600 Units per mg, calculated on the dried basis. Description. A white or yellowish-white powder; odourless or almost odourless; hygroscopic.
813
NEOMYCIN EYE DROPS
IP 2007
Identification
A. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel H. Mobile phase. A freshly prepared 3.85 per cent w/v solution of ammonium acetate. Test solution. Dissolve 0.2 g of the substance under examination in 10 ml of water. Reference solution. A 2.0 per cent w/v solution of neomycin sulphate RS in water. Apply to the plate 1 l of each solution. After development, dry the plate in air for 10 minutes, heat at 100 for 1 hour and spray with a 0.1 per cent w/v solution of ninhydrin in 1-butanol saturated with water. Heat again at 100 for 5 minutes. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution. B. Dissolve about 10 mg in 5 ml of water, add 0.1 ml of pyridine and 2 ml of a 0.1 per cent w/v solution of ninhydrin and heat on a water-bath at a temperature of about 70 for 10 minutes; a deep violet colour is produced. C. A 5 per cent w/v solution gives the reactions of sulphates (2.3.1).
Mobile phase. A mixture of 80 volumes of a 20 per cent w/v solution of sodium chloride and 20 volumes of methanol. Test solution. Dissolve 40 mg of the substance under examination in water and dilute to 5 ml with the same solvent. Reference solution (a). Dissolve 30 mg of framycetin sulphate RS in water and dilute to 25 ml with the same solvent. Reference solution (b). Dilute 5 ml of reference solution (a) to 25 ml with water. Reference solution (c). Dissolve 40 mg of neomycin sulphate RS in water and dilute to 5 ml with the same solvent. Apply to the plate as 5-mm bands 5 l of each solution. Dry the bands; allow the mobile phase to rise at least 12 cm. Dry the plate at 100 to 105 for 10 minutes. Spray the plate with ethanolic ninhydrin solution and heat at 100 to 105 for 10 minutes. In the chromatogram obtained with the test solution the principal band corresponds to the principal band in the chromatogram obtained with reference solution (c) and the band due to neomycin C with an Rf value slightly less than that of the principal band is not more intense than the band obtained with reference solution (a) (15 per cent) but is more intense than the band in the chromatogram obtained with reference solution (b) (3 per cent). The test is not valid unless in the chromatogram obtained with reference solution (c) a band appears with an Rf value slightly less than that of the principal band. Sulphated ash (2.3.18). Not more than 1.0 per cent. Loss on drying (2.4.19). Not more than 8.0 per cent, determined on 0.5 g by drying in an oven at 60 over phosphorus pentoxide at a pressure not exceeding 0.7 kPa for 3 hours. Assay. Determine by the microbiological assay of antibiotics, Method A (2.2.10). Storage. Store protected from light and moisture. Labelling. The label states the strength in terms of Units of neomycin per mg.
Tests
pH (2.4.24). 5.0 to 7.5, determined in a 1.0 per cent w/v solution. Specific optical rotation (2.4.22).+53.5 to +59.0, determined in a 10.0 per cent w/v solution. Neamine. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel H. Mbile phase. A mixture of 30 volumes of methanol, 20 volumes of strong ammonia solution and 10 volumes of dichloromethane. Test solution. Dissolve 0.25 g of the substance under examination in 10 ml of water. Reference solution. A 0.05 per cent w/v solution of neamine RS in water. Apply to the plate as 5-mm bands 5 l of each solution. Dry the bands; allow the mobile phase to rise at least 8 cm. Dry the plate in a current of warm air, heat at 110 for 10 minutes, spray the plate with ninhydrin and stannous chloride reagent and heat at 110 for 15 minutes. Spray the plate again with the same reagent and heat at 110 for 15 minutes. Any band corresponding to neamine in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Neomycin C. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel of a suitable grade.
Neomycin Eye Drops

Neomycin Sulphate Eye Drops
Neomycin Sulphate Eye Drops are a sterile solution of Neomycin Sulphate in Purified Water. Neomycin Sulphate Eye Drops contain not less than 90.0 per cent and not more than 115.0 per cent w/v of the stated amount of neomycin sulphate.
Identification
Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel.
814
IP 2007
NEOMYCIN EYE OINTMENT
Mobile phase. A mixture of 60 volumes of methanol, 40 volumes of strong ammonia solution and 20 volumes of chloroform. Test solution. Dilute if necessary a volume of the eye drops to produce a solution containing 0.5 per cent w/v of Neomycin Sulphate in water. Reference solution (a). A 0.5 per cent w/v solution of neomycin sulphate RS in water. Reference solution (b). A mixture of equal volumes of the eye drops and reference solution (a). Apply to the plate 1 l of each solution. After development, dry the plate in air, spray with a 1 per cent w/v solution of ninhydrin in 1-butanol and heat at 105 for 2 minutes. The principal red spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with reference solution (a) and the principal red spot in the chromatogram obtained with reference solution (b) appears as a single spot.
Chromatographic system a stainless steel column 20 cm x 4.6 mm, packed with porous silica particles (5 m) (such as Nucleosil 100-5), mobile phase: a mixture of 97 ml of tetrahydrofuran, 1.0 ml of water and 0.5 ml of glacial acetic acid diluted with sufficient of a 2.0 per cent v/v solution of ethanol in ethanol-free chloroform to produce 250 ml, flow rate. 1.6 ml per minute, spectrophotometer set at 350 nm, a 10 l loop injector. If necessary the tetrahydrofuran and water content of the mobile phase may be adjusted so that the chromatogram obtained with the reference solution shows resolution similar to that in the specimen chromatogram supplied with framycetin sulphate RS. The mobile phase should be passed through the column for several hours before the solutions are injected. Continue the chromatography for 1.4 times the retention time of the peak due to neomycin B. The column efficiency, determined using the peak due to Neomycin B in the chromatogram obtained with the test solution, should be not less than 13,000 theoretical plates. In the chromatogram obtained with the test solution the area of the peak corresponding to neomycin C is not less than 3.0 per cent and not more than 15.0 per cent of sum of the areas of the peaks corresponding to Neomycin B and Neomycin C. Other Tests. Complies with the tests stated under Eye Drops. Assay. Measure accurately a quantity containing 5 mg of Neomycin Sulphate and dilute to 50.0 ml with sterile phosphate buffer pH 8.0 and mix. Dilute 10.0 ml of the resulting solution to 100.0 ml with the same solvent. Determine by the microbiological assay of antibiotics, Method A (2.2.10) The upper fiducial limit of error is not less than 90.0 per cent and the lower fiducial limit of error is not more than 115.0 per cent of the stated number of Units per ml. Storage. Store protected from light. Labelling. The strength is stated in terms of percentage w/v as well as the number of Units per ml.
Tests
Neamine. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel H. Mobile phase. A mixture of 30 volumes of methanol, 20 volumes of strong ammonia solution and 10 volumes of dichloromethane. Test solution. A volume of the eye drops containing 5 g (3.5 Units). Reference solution. The same volume of water containing 0.1 g of neamine RS. Apply to the plate each solution. After development, dry the plate in a current of warm air, heat at 110 for 10 minutes, spray the plate with ninhydrin and stannous chloride reagent and heat at 110 for 15 minutes. Spray the plate again with the same reagent and heat at 110 for 15 minutes. Any spot corresponding to neamine in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Neomycin C. Determine by liquid chromatography (2.4.14). Test solution. Dilute the eye drops with 0.02 M borax to contain 1 mg (700 Units) per ml. To 0.5 ml of the diluted solution add 1.5 ml of a freshly prepared 2 per cent w/v solution of 1-fluoro-2,4-dinitrobenzene in methanol, dilute to 25 ml with the mobile phase, allow to stand and use the clear lower layer. Reference solution. Add 1.5 ml of the 1-fluoro-2,4dinitrobenzene solution to 0.5 ml of a 0.1 per cent w/v solution of neomycin sulphate RS in 0.02 M borax, heat in a waterbath at 60 for 1 hour and cool; dilute the solution to 25 ml with the mobile phase, allow to stand and use the clear lower layer.
Neomycin Eye Ointment

Neomycin Sulphate Eye Ointment
Neomycin Sulphate Eye Ointment is a sterile preparation containing Neomycin Sulphate in a suitable basis. Neomycin Sulphate Eye Ointment contains not less than 90.0 per cent and not more than 115.0 per cent of the stated amount of neomycin sulphate.
815
NEOMYCIN EYE OINTMENT
IP 2007
Identification
Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel. Mobile phase. A mixture of 60 volumes of methanol, 40 volumes of strong ammonia solution and 20 volumes of chloroform. Test solution. Disperse a quantity of the eye ointment containing 20 mg of Neomycin Sulphate in 20 ml of chloroform, extract with 5 ml of water and use the aqueous extract. Reference solution (a). A 0.4 per cent w/v solution of neomycin sulphate RS in water. Reference solution (b). A mixture of equal volumes of test solution and reference solution (a). Apply to the plate 1 l of each solution. After development, dry the plate in air, spray with a 1 per cent w/v solution of ninhydrin in 1-butanol and heat at 105 for 2 minutes. The principal red spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with reference solution (a) and the principal red spot in the chromatogram obtained with reference solution (b) appears as a single spot.
cent w/v solution of 1-fluoro-2,4-dinitrobenzene in methanol, heat on a water-bath at 60 for 1 hour and cool. Dilute the resulting solution to 25 ml with the mobile phase, allow to stand and use the clear lower layer. Reference solution. Add 1.5 ml of the 1-fluoro-2,4dinitrobenzene solution to 0.5 ml of a 0.1 per cent w/v solution of neomycin sulphate RS in 0.02 M borax and proceed as for the test solution. Chromatographic system a stainless steel column 20 cm x 4.6 mm, packed with porous silica particles (5 m), mobile phase: 97 ml of tetrahydrofuran, 1.0 ml of water and 0.5 ml of glacial acetic acid with sufficient of a 2.0 per cent v/v solution of ethanol in ethanol-free chloroform to produce 250 ml, flow rate. 1.6 ml per minute, spectrophotometer set at 350 nm, a 10 l loop injector. If necessary the tetrahydrofuran and water content of the mobile phase may be adjusted so that the chromatogram obtained with reference solution shows resolution similar to that in the specimen chromatogram supplied with framycetin sulphate RS. The mobile phase should be passed through the column for several hours before the solutions are injected. Continue the chromatography for 1.4 times the retention time of the peak due to neomycin B. The column efficiency, determined using the peak due to Neomycin B in the chromatogram obtained with the test solution, should be not less than 13,000 theoretical plates. In the chromatogram obtained with the test solution the area of the peak corresponding to neomycin C is not less than 3.0 per cent and not more than 15.0 per cent of the sum of the areas of the peaks corresponding to Neomycin B and Neomycin C. Other Tests. Complies with the tests stated under Eye Ointments. Assay. Weigh accurately a quantity containing 5 mg of Neomycin Sulphate, dissolve in 25 ml of chloroform, extract with four quantities, each of 20 ml, of sterile phosphate buffer pH 8.0, combine the extracts and add sufficient of the buffer solution to produce 100.0 ml. Carry out the microbiological assay of antibiotics, Method A (2.2.10). The upper fiducial limit of error is not less than 90.0 per cent and the lower fiducial limit of error is not more than 115.0 per cent of the stated number of Units per g. Storage. Store protected from light. Labelling. The strength is stated in terms of percentage w/v as well as the number of Units per ml.
Tests
Neamine. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel H. Mobile phase. A mixture of 30 volumes of methanol, 20 volumes of strong ammonia solution and 10 volumes of dichloromethane. Test solution. Disperse a quantity of the eye ointment containing 20 mg of Neomycin Sulphate in 20 ml of chloroform, shake gently with 8 ml of water, allow the layers to separate and use the aqueous layer. Reference solution. A 0.005 per cent w/v solution of neamine RS in water. Apply to the plate 2 l of each solution. After development, dry the plate in a current of warm air, heat at 110 for 10 minutes, spray with ninhydrin and stannous chloride reagent and heat at 110 for 15 minutes. Spray the plate again with the same reagent and heat at 110 for 15 minutes. Any spot corresponding to neamine in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Neomycin C. Determine by liquid chromatography (2.4.17) Test solution. Disperse a quantity of the eye ointment containing 5 mg of Neomycin Sulphate in 20 ml of light petroleum (120 to 160), add 5 ml of 0.02 M borax, shake, separate the aqueous layer and centrifuge. To 0.5 ml of the separated aqueous layer add 1.5 ml of a freshly prepared 2 per
816
IP 2007
NEOSTIGMINE TABLETS
Neostigmine Bromide
CH3 H3C N CH3 O O N CH3 CH3 Br
measured immediately after preparation, not more than 0.25 (2.4.7). Sulphates (2.3.17). 0.75 g complies with the limit test for sulphates (200 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 1.0 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.5 g, dissolve in 20 ml of anhydrous glacial acetic acid, add 5 ml of acetic anhydride. Titrate with 0.1 M perchloric acid, using crystal violet solution as indicator. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.03032 g of C12H19BrN2O2. Storage. Store protected from light and moisture.
C12H19BrN2O2
Mol. Wt. 303.2
Neostigmine Bromide is 3-(dimethylcarbamoyloxy) trimethylanilinium bromide. Neostigmine Bromide contains not less than 98.0 per cent and not more than 101.0 per cent of C12H19BrN2O2, calculated on the dried basis. Description. Colourless crystals or a white, crystalline powder; odourless; hygroscopic.
Neostigmine Tablets
Neostigmine Bromide Tablets
Neostigmine Bromide Tablets contain not less than 92.5 per cent and not more than 107.5 per cent of the stated amount of neostigmine bromide, C12H19BrN2O2.
Identification
Test A may be omitted if tests B, C, D and E are carried out. Tests B, C and D may be omitted if tests A and E are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with neostigmine bromide RS. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.02 per cent w/v solution in 0.5 M sulphuric acid shows absorption maxima at about 260 nm and 266 nm. C. Warm about 50 mg with 1 ml of dilute sodium hydroxide solution; an odour of dimethylamine develops slowly. D. Warm about 50 mg with 0.4 g of potassium hydroxide and 2 ml of ethanol (95 per cent) on a water-bath for 3 minutes, replacing the evaporated ethanol. Cool, add 2 ml of dilute diazobenzenesulphonic acid solution; an orange-red colour is produced. E. Gives the reactions of bromides (2.3.1).
Identification
Triturate a quantity of the powdered tablets containing about 0.3 g of Neostigmine Bromide with three quantities, each of 5 ml of ether and discard the ether. Macerate the residue with several quantities, each of 10 ml of ethanol (95 per cent), filtering after each maceration. Evaporate the combined filtrates on a water-bath and dry the residue at 105 for 1 hour. The residue melts at about 167, with decomposition. The residue complies with the following tests. A. Warm about 50 mg with 0.4 g of potassium hydroxide and 2 ml of ethanol (95 per cent) on a water-bath for 3 minutes, replacing the evaporated ethanol. Cool, add 2 ml of dilute diazobenzenesulphonic acid solution; an orange-red colour is produced. B. Gives the reactions of bromides (2.3.1).
Tests
Appearance of solution. A 0.5 per cent w/v solution is clear (2.4.1), and colourless (2.4.1). Acidity. Dissolve 0.2 g in 20 ml of carbon dioxide-free water and titrate to pH 7.0 with 0.02 M sodium hydroxide (carbonatefree); not more than 0.1 ml is required. 3-Hydroxytrimethylanilinium bromide. Dissolve 50 mg in a mixture of 1 ml of sodium carbonate solution and 9 ml of water. Absorbance of the resulting solution at about 294 nm,
Tests
Other Tests. Complies with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 0.15 g of Neostigmine Bromide, transfer to a semi-micro ammonia-distillation apparatus, add 20 ml of a 50 per cent w/v solution of sodium hydroxide and 0.5 ml of a 2 per cent w/v solution of 2-octanol in liquid paraffin. Pass a current of steam through the mixture, collect the distillate in 50 ml of 0.01 M sulphuric acid until the
817
NEOSTIGMINE METHYLSULPHATE
IP 2007
volume is about 200 ml and titrate the excess of acid with 0.02 M sodium hydroxide using methyl red solution as indicator. Repeat the operation without the substance under examination. The difference between the titrations represents the amount of sulphuric acid required to neutralise the dimethylamine produced. 1 ml of 0.01 M sulphuric acid is equivalent to 0.006064 g of C12H19BrN2O2. Storage. Store protected from light and moisture.
phthalein solution; the solution is colourless. Add 0.3 ml of 0.01 M sodium hydroxide; the solution becomes red. Add 0.4 ml of 0.01 M hydrochloric acid; the solution becomes colourless. Add 0.1 ml of methyl red solution; the solution becomes red or yellowish-red. 3-Hydroxytrimethylanilinium methyl sulphate. Dissolve 50 mg in a mixture of 1 ml of sodium carbonate solution and 9 ml of water. Absorbance of the resulting solution at about 294 nm, measured immediately after preparation, not more than 0.20 (2.4.7). Chlorides (2.3.12). 1.0 g complies with the limit test for chlorides (250 ppm).
Neostigmine Methylsulphate
C13H22N2O6S Mol. Wt. 334.4 Neostigmine Methylsulphate is 3-(dimethylcarbamoyloxy)trimethylanilinium methyl sulphate. Neostigmine Methylsulphate contains not less than 98.5 per cent and not more than 101.0 per cent of C13H22N2O6S, calculated on the dried basis. Description. Colourless crystals or a white, crystalline powder; hygroscopic.
Sulphates (2.3.17). 0.75 g complies with the limit test for sulphates (200 ppm). Loss on drying (2.4.19). Not more than 1.0 per cent, determined on 1.0 g by drying in an oven at 105. Sulphated ash (2.3.18). Not more than 0.1 per cent. Assay. Weigh accurately about 0.3 g and dissolve in 150 ml of water. Add 100 ml of 2 M sodium hydroxide, distill and collect the distillate in 50 ml of a 4 per cent w/v solution of boric acid until a total volume of 250 ml is reached. Titrate the distillate with 0.1 M hydrochloric acid using 0.25 ml of methyl redmethylene blue solution as indicator. Repeat the operation without the substance under examination. The difference between the titrations represents the amount of hydrochloric acid required. 1 ml of 0.1 M hydrochloric acid is equivalent to 0.03344 g of C13H22N2O6S. Storage. Store protected from light and moisture.
Identification
Test A may be omitted if tests B, C and D are carried out. Tests B, C and D may be omitted if test A is carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with neostigmine methylsulphate RS or with the reference spectrum of neostigmine methylsulphate. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.05 per cent w/v solution in 0.5 M sulphuric acid shows absorption maxima, at about 261 nm and 267 nm. The ratio of the absorbance at the maximum at about 267 nm to that at the maximum at 261 nm is 0.84 to 0.87. C. Dissolve 0.1 g in 5 ml of distilled water and add 1 ml of a 6 per cent w/v solution of barium chloride; no precipitate is produced. Add 2 ml of hydrochloric acid and heat in a waterbath for 10 minutes; a white precipitate is produced. D. Warm about 50 mg with 0.4 g of potassium hydroxide and 2 ml of ethanol (95 per cent) on a water-bath for 3 minutes, replacing the evaporated ethanol. Cool, add 2 ml of dilute diazobenzenesulphonic acid solution; an orange-red colour is produced.
Neostigmine Injection
Neostigmine Methylsulphate Injection
Neostigmine Injection is a sterile solution of Neostigmine Methylsulphate in Water for Injections. Neostigmine Injection contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of neostigmine methylsulphate, C13H22N2O6S.
Identification
A. Dilute, if necessary, a volume of the injection containing 2.5 mg of Neostigmine Methylsulphate to 5 ml with water, shake with three quantities, each of 10 ml, of ether and discard the ether extracts. When examined in the range 230 nm to 360 nm (2.4.7), a 2 cm layer of the resulting solution shows absorption maxima at about 260 nm and 267 nm.
Tests
Appearance of solution. A 5.0 per cent w/v solution in distilled water is clear (2.4.1), and colourless (2.4.1). Acidity or alkalinity. To 4.0 ml of a 5.0 per cent w/v solution in distilled water add 6.0 ml of water and 0.1 ml of phenol-
818
IP 2007
NEVIRAPINE
B. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 50 volumes of chloroform, 35 volumes of methanol, 10 volumes of formic acid and 5 volumes of water. Test solution. Dilute the injection under examination, if necessary, with water to produce a solution containing 0.05 per cent w/v of Neostigmine Methylsulphate. Reference solution (a). A 0.05 per cent w/v solution of neostigmine methylsulphate RS in water. Reference solution (b). A mixture of equal volumes of the test solution and reference solution (a). Apply to the plate 10 l of each solution. After development, dry the plate in air, spray with dilute potassium iodobismuthate solution. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with reference solution (a). The principal spot in the chromatogram obtained with reference solution (b) appears as a single, compact spot. C. To 1 ml add 0.5 ml of sodium hydroxide solution and evaporate to dryness on a water-bath. Heat quickly in an oilbath to about 250 and maintain at this temperature for about 30 seconds. Cool, dissolve the residue in 1 ml of water, cool in ice water and add 1 ml of diazobenzenesulphonic acid solution; an orange-red colour is produced.
spectrophotometer set at 215 nm, a 10 l loop injector.
In the chromatogram obtained with reference solution (b) the principal peak has a retention time of about 6.8 minutes (neostigmine methylsulphate) and there is a peak with a relative retention time of about 0.5 ((3-hydroxy) trimethylanilinium methylsulphate). In the chromatogram obtained with the test solution, the area of any secondary peak with a retention time corresponding to that of the peak due to (3-hydroxy)trimethylanilinium methylsulphate in the chromatogram obtained with reference solution (b) is not greater than the area of the principal peak in the chromatogram obtained with reference solution (a) (1 per cent). Other Tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. Dilute an accurately measured volume containing about 25 mg of Neostigmine Methylsulphate to 50.0 ml with water. Measure the absorbance of the resulting solution at the maximum at about 260 nm (2.4.7). Calculate the content of C13H22N2O6S taking 14.35 as the specific absorbance at 260 nm. Storage. Store protected from light.
Nevirapine
H 3C O HN N
Tests
pH. (2.4.24) 4.5 to 6.5. 3-Hydroxy trimethylanilinium methyl sulphate. Determine by liquid chromatography (2.4.14). Test solution. Dilute the injection if necessary, with water to contain a 0.05 per cent w/v solution of Neostigmine Methylsulphate. Reference solution (a). Dilute 1 volume of the test solution to 100 volumes with water. Reference solution (b). Add 0.05 ml of 5 M sodium hydroxide to 1 ml of the test solution and allow to stand for 5 minutes. Add 0.1 ml of 5 M hydrochloric acid and use immediately. Chromatographic system a stainless steel column 25 cm 4.6 mm packed with octadecylsilane chemically bonded to porous silica particles (5 m) (such as Lichrosphere 60 RP-select B), mobile phase: 0.0015 M solution of sodium heptanesulphonate in a mixture of 15 volumes of acetonitrile and 85 volumes of 0.05 M potassium dihydrogen orthophosphate adjusted to pH 3.0 with orthophosphoric acid, flow rate. of 1.1 ml per minute, C15H14N4O
N
Mol. Wt. 266.3
Nevirapine is 11-cyclopropyl-5,11-dihydro-4-methy-6Hdipyrido[3,2-b:2,3-e][1,4]diazepin-6-one. Nevirapine contains not less than 98.0 per cent and not more than 102.0 per cent of C15H14N4O, calculated on the dried basis. Description. A white or almost white crystalline powder.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with nevirapine RS or with the reference spectrum of nevirapine. B. In the Assay, the principal peak in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution.
819
NEVIRAPINE TABLETS
IP 2007
Tests
Related substances. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 0.1 g of the substance under examination in 100 ml of methanol. Reference solution. Dilute 1 ml of the test solution to 100 ml with methanol. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica or ceramic microparticles (5 m), mobile phase: a filtered and degassed mixture of 20 volumes of methanol, 20 volumes of acetonitrile and 60 volumes of a buffer prepared by dissolving 12.0 g of sodium dihydrogen phosphate in about 800 ml of water, adjusting the pH to 3.0 with phosphoric acid and diluting to 1000.0 ml with water, flow rate. 1.2 ml per minute, spectrophotometer set at 230 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the column efficiency determined from the nevirapine peak is not less than 5000 theoretical plates and the tailing factor is not more than 2.0. Separately inject the test solution and the reference solution. In the chromatogram obtained with the test solution, the area of any peak other than the principal peak is not greater than half of the area of the principal peak in the chromatogram obtained with the reference solution (0.5 per cent) and the sum of the areas of all such peaks is not greater than the area of the principal peak in the chromatogram obtained with the reference solution (1.0 per cent). Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Sulphated ash (2.3.18). Not more than 0.2 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying at 105 for 3 hours. Assay: Determine by liquid chromatography (2.4.14). Test solution. A 0.005 per cent w/v solution of the substance under examination in methanol. Reference solution. A 0.005 per cent w/v solution of nevirapine RS in methanol. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica or ceramic microparticles (5 m), mobile phase: a filtered and degassed mixture of 20 volumes of methanol, 20 volumes of acetonitrile
and 60 volumes of a buffer prepared by dissolving 12.0 g of sodium dihydrogen phosphate in about 800 ml of water, adjusting the pH to 3.0 with orthophosphoric acid and diluting to 1000.0 ml with water, flow rate. 1.2 ml per minute, spectrophotometer set at 230 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the column efficiency determined from the nevirapine peak is not less than 3000 theoretical plates, the tailing factor is not more than 2.0 and the relative standard deviation for the replicate injections is not more than 2.0 per cent. Separately inject the test solution and the reference solution and measure the responses for the principal peak. Calculate the content of C15H14N4O. Storage. Store protected from moisture.
Nevirapine Tablets
Nevirapine Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of nevirapine, C15H14N4O.
Identification
A. When examined in the range 250 nm to 450 nm (2.4.7) a 0.001 per cent w/v solution in the mobile phase described under Assay, shows an absorption maximum at about 230 nm. B. In the Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the peak in the chromatogram obtained with the reference solution. Dissolution (2.5.2). Apparatus. No 1. Medium. 900 ml of 0.1 M hydrochloric acid. Speed and time. 50 rpm and 45 minutes. Withdraw a suitable volume of the medium and filter through a membrane filter disc with an average pore diameter not greater than 1.0 m, rejecting the first few ml of the filtrate. Measure the absorbance of the resulting solution, at the maximum at about 313 nm (2.4.7). Calculate the content of C15H14N4O from the absorbance obtained from a solution of known concentration of nevirapine RS in 0.1 M hydrochloric acid. D. Not less than 70 per cent of the stated amount of C15H14N4O. Related substances. Determine by liquid chromatography (2.4.14). Test solution. Shake a quantity of the powdered tablets with a suitable quantity of the mobile phase to obtain a mixture
820
IP 2007
NEVIRAPINE ORAL SUSPENSION
containing 0.05 per cent w/v of Nevirapine and filter through a membrane filter disc with an average diameter not exceeding 1.0 m, rejecting the first few ml of the filtrate. Reference solution. A 0.05 per cent w/v solution of nevirapine RS in the mobile phase. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica or ceramic microparticles (5 m), mobile phase: a filtered and degassed mixture of 20 volumes of methanol, 20 volumes of acetonitrile and 60 volumes of a buffer prepared by dissolving 12.0 g of sodium dihydrogen phosphate in about 800 ml of water, adjusting the pH to 3.0 with orthophosphoric acid and diluting to 1000.0 ml with water, flow rate. 1.2 ml per minute, spectrophotometer set at 230 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the column efficiency determined from the nevirapine peak is not less than 7500 theoretical plates and the tailing factor is not more than 1.5 and the relative standard deviation for replicate injections is not more than 2 per cent. Inject the test solution and continue the chromatography for at least five times the retention time of the principal peak. Determine the amount of related substances by the area normalisation method. Any individual impurity is not more than 1.0 per cent and the sum of all impurities is not more than 2.0 per cent. Other tests. Comply with the tests stated under Tablets. Assay. Determine by liquid chromatography (2.4.14). Test solution. Weigh and powder 20 tablets. Shake a quantity of powder containing about 100 mg of Nevirapine with sufficient of the mobile phase to obtain a mixture containing 0.05 per cent w/v of Nevirapine. Mix and filter through a membrane filter disc with an average pore diameter not greater than 1.0 m, rejecting the first few ml of the filtrate. Reference solution. A 0.05 per cent w/v solution of nevirapine RS in the mobile phase. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica or ceramic microparticles (5 m), mobile phase: a filtered and degassed mixture of 20 volumes of methanol, 20 volumes of acetonitrile and 60 volumes of a buffer prepared by dissolving 12.0 g of sodium dihydrogen phosphate in about 800 ml of water, adjusting the pH to 3.0 with phosphoric acid and diluting to 1000.0 ml with water,
flow rate. 1.2 ml per minute, spectrophotometer set at 230 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the column efficiency determined from the nevirapine peak is not less than 7500 theoretical plates, the tailing factor is not more than 1.5 and the relative standard deviation for replicate injections is not more than 2.0 per cent. Separately inject the test solution and the reference solution and measure the responses for the principal peak. Calculate the content of C15H14N4O in the tablets. Storage. Store protected from moisture at a temperature not exceeding 30.
Nevirapine Oral Suspension

Nevirapine Oral Suspension is a suspension of Nevirapine in a suitable flavoured vehicle. Nevirapine Oral Suspension contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of nevirapine, C15H14N4O.
Identification
A. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 40 volumes of 1-butanol, 30 volumes of heptane, 30 volumes of acetone and 10 volumes of strong ammonia solution. Test solution. Dilute the preparation under examination with methanol to obtain a solution containing 1 mg of Nevirapine per ml. Reference solution. A 0.1 per cent w/v solution of nevirapine RS in a mixture of 75 volumes of methanol and 25 volumes of water. Apply to the plate 10 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution. B. In the Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the peak in the chromatogram obtained with the reference solution.
Tests
pH (2.4.24). 5.0 to 7.0. Related substances. Determine by liquid chromatography (2.4.14).
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Test solution. To an accurately measured volume of the preparation under examination containing about 25 mg of Nevirapine add about 10 ml of methanol, mix with the aid of ultrasound for 10 minutes, dilute to 50 ml with water, mix and filter. Reference solution. Weigh accurately about 25 mg of nevirapine RS, add about 10 ml of methanol, mix with the aid of ultrasound to dissolve and dilute to 50 ml with water. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octylsilyl silica gel for chromatography (5 m)(such as Hypersil C8), mobile phase: filtered and degassed gradient mixtures of methanol and 0.1 M ammonium acetate, flow rate. 1 ml per minute, a linear gradient programme using the conditions given below, spectrophotometer set at 270 nm, a 20 l loop injector. Time 0.1 M ammonium acetate Methanol (in min.) (per cent v/v) (per cent v/v) 0 95 5 5 95 5 25 20 80 30 20 80 31 95 5 40 95 5 Inject the reference solution. The test is not valid unless the column efficiency determined from the nevirapine peak is not less than 3000 theoretical plates and the tailing factor is not more than 2.0. Inject separately the diluent (dilute 10 ml of methanol to 50 ml with water) and the test solution. Examine the diluent chromatogram for any extraneous peaks and ignore the corresponding peaks observed in the chromatogram obtained with the test solution. Ignore any peaks due to preservatives also. Any secondary peak observed in the chromatogram obtained with the test solution should not be more than 1.0 per cent and the sum of the areas of all the secondary peaks should not be more than 2.0 per cent when calculated by percentage area normalisation. Other tests. Comply with the tests stated under Oral Liquids. Assay. Determine by liquid chromatography (2.4.14) Test solution. Weigh accurately a quantity of the preparation under examination containing 25 mg of Nevirapine, add about 10 ml of methanol, mix with the aid of ultrasound for 10 minutes, dilute to 50.0 ml with water, mix and filter. Further dilute 10.0 ml of the filtrate to 25.0 ml with water.
Reference solution. Weigh accurately about 50 mg of nevirapine RS, add about 20 ml of methanol, mix with the aid of ultrasound to dissolve and dilute to 100.0 ml with water. Dilute 10.0 ml of this solution to 25.0 ml with water. Use the chromatographic system described under the test for Related substances. Inject the reference solution. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject separately the test solution and the reference solution and measure the responses for the principal peak. Determine the weight per ml of the oral suspension (2.4.29) and calculate the content of C15H14N4O weight in volume. Storage. Store protected from light.
Niclosamide
Anhydrous Niclosamide
Cl OH O N H Cl NO2
C13H8Cl2N2O4
Mol. Wt. 327.1
Niclosamide is 2,5-dichloro-4-nitrosalicylanilide. Niclosamide contains not less than 98.0 per cent and not more than 101.0 per cent of C13H8Cl2N2O4, calculated on the dried basis. Description. A yellowish white to yellowish, fine crystals or powder; odourless.
Identification
Test A may be omitted if tests B and C are carried out. Tests B and C may be omitted if test A is carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with niclosamide RS or with the reference spectrum of niclosamide. B. Heat 50 mg with 5 ml of 1 M hydrochloric acid and 0.1 g of zinc powder in a water-bath for 10 minutes, cool and filter. To the filtrate add 1 ml of a 0.5 per cent w/v solution of sodium nitrite and allow to stand for 3 minutes. Add 2 ml of a 2 per cent w/v solution of ammonium sulphamate, shake, allow to
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NICLOSAMIDE TABLETS
stand for 3 minutes and add 2 ml of a 0.5 per cent w/v solution of N- (1-naphthyl) ethylenediamine dihydrochloride; a violet colour is produced. C. Heat the substance under examination on a copper wire in a non-luminous flame; a green colour is imparted to the flame.
Niclosamide Tablets
Niclosamide Tablets contain not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of niclosamide, C 13 H 8 Cl 2 N 2O 4 . The tablets may contain sweetening and flavouring agents.
Tests
Chlorides (2.3.12). To 2.0 g add a mixture of 40 ml of water and 1.2 ml of 5 M acetic acid, boil for 2 minutes, cool and filter; 10 ml of the filtrate diluted to 15 ml with water complies with the limit test for chlorides (500 ppm). 2-Chloro-4-nitroaniline. Not more than 100 ppm, determined by the following method. Boil 0.25 g with 5 ml of methanol, cool, add 45 ml of 1 M hydrochloric acid, heat to boiling, cool, filter and dilute the filtrate to 50.0 ml with 1 M hydrochloric acid. To 10.0 ml of this solution add 0.5 ml of a 0.5 per cent w/v solution of sodium nitrite and allow to stand for 3 minutes. Add 1.0 ml of a 2 per cent w/v solution of ammonium sulphamate, shake, allow to stand for 3 minutes and add 1.0 ml of a 0.5 per cent w/v solution of N- (1-naphthyl) ethylenediamine dihydrochloride. Any pinkish violet colour produced is not more intense than that obtained in a solution prepared at the same time and in the same manner using 10.0 ml of a solution prepared by diluting 2.0 ml of a 0.00050 per cent w/v solution of 2-chloro-4-nitroaniline in methanol to 20 ml with 1 M hydrochloric acid and beginning at the words add 0.5 ml of a 0.5 per cent w/v solution of sodium nitrite...... 5-Chlorosalicylic acid. Not more than 60 ppm, determined by the following method. Boil 1.0 g with 15 ml of water for 2 minutes, cool, filter through a membrane filter (pore size 0.45 m), wash the filter and dilute the combined filtrate and washings to 20 ml with water (solution A). Dissolve 30 mg of 5-chlorosalicylic acid in 20 ml of methanol and add sufficient water to produce 100.0 ml. Dilute 1.0 ml of this solution to 100.0 ml with water (solution B). To 10.0 ml of each of solutions A and B add separately 0.1 ml of ferric chloride solution; any violet colour produced in solution A is not more intense than that obtained in solution B. Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105 for 4 hours. Assay. Weigh accurately about 0.3 g, dissolve in 80 ml of a mixture of equal volumes of acetone and methanol. Titrate with 0.1 M tetrabutylammonium hydroxide, determining the end-point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M tetrabutylammonium hydroxide is equivalent to 0.03271 g of C13H8Cl2N2O4. Storage. Store protected from light and moisture.
Identification
Heat a quantity of the powdered tablets containing 0.5 g of Niclosamide with 25 ml of hot ethanol (95 per cent), filter while hot and evaporate the filtrate to dryness on a waterbath. The residue complies with the following tests. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with niclosamide RS or with the reference spectrum of niclosamide. B. Heat 50 mg with 5 ml of 1 M hydrochloric acid and 0.1 g of zinc powder in a water-bath for 10 minutes, cool and filter. To the filtrate add 1 ml of a 0.5 per cent w/v solution of sodium nitrite and allow to stand for 3 minutes. Add 2 ml of a 2 per cent w/v solution of ammonium sulphamate, shake, allow to stand for 3 minutes and add 2 ml of a 0.5 per cent w/v solution of N- (1-naphthyl) ethylenediamine dihydrochloride; a violet colour is produced.
Tests
2-Chloro-4-nitroaniline. Not more than 100 ppm, Boil a quantity of the powdered tablets containing 0.1 g of Niclosamide with 20 ml of methanol and 20 ml of a solution in methanol containing 10 g of 2-chloro-4-nitroaniline, cool, add 45 ml of 1 M hydrochloric acid, heat to boiling, cool, filter and dilute the filtrate to 50.0 ml with 1 M hydrochloric acid. To 10.0 ml of this solution add 0.5 ml of a 0.5 per cent w/v solution of sodium nitrite and allow to stand for 3 minutes. Add 1.0 ml of a 2 per cent w/v solution of ammonium sulphamate, shake, allow to stand for 3 minutes and add 1.0 ml of a 0.5 per cent w/v solution of N- (1-naphthyl) ethylenediamine dihydrochloride. Any pinkish violet colour produced is not more intense than that obtained in a solution prepared at the same time and in the same manner using 10.0 ml of a solution prepared by diluting 2.0 ml of a 0.0005 per cent w/v solution of 2-chloro-4-nitroaniline in methanol to 20.0 ml with 1 M hydrochloric acid and beginning at the words add 0.5 ml of a 0.5 per cent w/v solution of sodium nitrite...... 5-Chlorosalicylic acid. Boil a quantity of the powdered tablets containing 0.5 g of Niclosamide with 10 ml of water for 2 minutes, cool, filter and to the filtrate add 0.2 ml of ferric chloride solution; no red or violet colour is produced. Disintegration. The test does not apply. Other Tests. Comply with the tests stated under Tablets.
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Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powdered tablets containing about 0.3 g of Niclosamide dissolved in 60 ml of dimethylformamide. Titrate with 0.1 M tetrabutylammonium hydroxide, determining the end-point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M tetrabutylammonium hydroxide is equivalent to 0.03271 g of C13H8Cl2N2O4. Storage. Store protected from light and moisture. Labelling. The label states that the tablets should be chewed thoroughly before swallowing.
Mobile phase. A mixture of 48 volumes of chloroform, 45 volumes of ethanol and 4 volumes of water. Test solution. Dissolve 0.8 g of the substance under examination in 10 ml of ethanol (50 per cent). Reference solution. A 0.02 per cent w/v solution of the substance under examination in ethanol (50 per cent). Apply to the plate 5 l of each solution. Allow the mobile phase to rise 10 cm. Dry the plate in air and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Heavy metals (2.3.13). Dissolve 0.67 g in 10 ml of water, 7.5 ml of 1 M hydrochloric acid and sufficient water to produce 25 ml. The solution complies with the limit test for heavy metals, Method A (30 ppm).
Nicotinamide
Niacinamide
N NH2 O
Chlorides (2.3.12). 1.0 g complies with the limit test for chlorides (250 ppm). Sulphates (2.3.17). 1.2 g complies with the limit test for sulphates (125 ppm). Mol. Wt. 122.1 Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying over phosphorus pentoxide at a pressure of 1.5 to 2.7 kPa for 18 hours. Assay. Weigh accurately about 0.25 g, dissolve in 20 ml of anhydrous glacial acetic acid, heating slightly if necessary. Add 5 ml of acetic anhydride. Titrate with 0.1 M perchloric acid, using crystal violet solution as indicator. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.01221 g of C6H6N2O. Storage. Store protected from moisture.
C6H6N2O Nicotinamide is pyridine-3-carboxamide.
Nicotinamide contains not less than 99.0 per cent and not more than 101.0 per cent of C6H6N2O, calculated on the dried basis. Description. Colourless crystals or a white, crystalline powder; odour, faint and characteristic.
Identification
Test A may be omitted if tests B, C and D are carried out. Tests B, C and D may be omitted if test A is carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with nicotinamide RS or with the reference spectrum of nicotinamide. B. Heat about 5 mg in a dry tube; pyridine is evolved. C. Boil 0.1 g with 1 ml of dilute sodium hydroxide solution; ammonia is evolved. D. To 2 ml of a 0.1 per cent w/v solution add 6 ml of cyanogen bromide solution and 1 ml of a 2.5 per cent w/v solution of aniline; a golden yellow colour develops.
Nicotinamide Tablets
Niacinamide Tablets
Nicotinamide Tablets contain not less than 92.5 per cent and not more than 107.5 per cent of the stated amount of nicotinamide, C6H6N2O.
Identification
Shake a quantity of the powdered tablets containing 0.2 g of Nicotinamide with 50 ml of ethanol for 15 minutes, filter and evaporate the filtrate to dryness on a water-bath. The residue complies with the following tests. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with nicotinamide RS or with the reference spectrum of nicotinamide.
Tests
Appearance of solution. A 5.0 per cent w/v solution in carbon dioxide-free water is clear (2.4.1), and not more intensely coloured than reference solution BYS7 (2.4.1). pH (2.4.24). 6.0 to 7.5, determined in a 5.0 per cent w/v solution. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254.
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NICLOTINIC ACID
B. Boil 0.1 g with 1 ml of dilute sodium hydroxide solution; ammonia is evolved. C. To 2 ml of a 0.1 per cent w/v solution add 6 ml of cyanogen bromide solution and 1 ml of a 2.5 per cent w/v solution of aniline; a golden yellow colour develops. D. When examined in the range 230 nm to 360 nm (2.4.7), the solution obtained in the Assay shows an absorption maximum only at about 262 nm and two shoulders at about 258 nm and 269 nm.
Nicotinic Acid contains not less than 99.5 per cent and not more than 100.5 per cent of C6H5NO2, calculated on the dried basis. Description. A white or creamy-white, crystalline powder.
Identification
Test A may be omitted if tests B, C and D are carried out. Tests B, C and D may be omitted if test A is carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with nicotinic acid RS or with the reference spectrum of nicotinic acid. B. Heat a small quantity with twice its weight of soda lime; pyridine is evolved. C. Dissolve about 50 mg in 20 ml of water, neutralise to litmus paper with 0.1 M sodium hydroxide, add 3 ml of copper sulphate solution; a blue precipitate is gradually produced. D. To 2 ml of a 0.1 per cent w/v solution add 6 ml of cyanogen bromide solution and 1 ml of a 2.5 per cent w/v solution of aniline; a golden yellow colour is produced.
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 48 volumes of chloroform, 45 volumes of ethanol and 4 volumes of water. Test solution. Shake a quantity of the powdered tablets containing 0.1 g of Nicotinamide with 15 ml of ethanol for 15 minutes, filter, evaporate to dryness on a water-bath and dissolve the residue as completely as possible in 1 ml of ethanol. Reference solution. Dilute 1 volume of the test solution to 400 volumes with ethanol Apply to the plate 5 l of each solution. Allow the mobile phase to rise 10 cm. Dry the plate in air and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Other Tests. Comply with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 50 mg of Nicotinamide, shake with 50 ml of ethanol (95 per cent) for 15 minutes and dilute to 100.0 ml with ethanol (95 per cent). Mix, filter, dilute 5.0 ml of the filtrate to 100.0 ml with ethanol (95 per cent) and measure the absorbance of the resulting solution at the maximum at about 262 nm (2.4.7). Calculate the content of C6H6N2O taking 241 as the specific absorbance at 262 nm. Storage. Store protected from light and moisture.
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 85 volumes of 1-propanol, 10 volumes of anhydrous formic acid and 5 volumes of water. Test solution. Dissolve 0.2 g of the substance under examination in 10 ml of water. Warm slightly, if necessary. Reference solution. A 0.01 per cent w/v solution of the substance under examination in water. Apply to the plate 5 l of each solution. After development, dry the plate at 105 for 10 minutes and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Heavy metals (2.3.13). Mix 1.0 g with 1.5 ml of dilute hydrochloric acid and sufficient water to produce 25 ml, heat gently and cool to room temperature. The resulting solution complies with the limit test for heavy metals, Method B (20 ppm). Chlorides (2.3.12). 1.0 g complies with the limit test for chlorides (250 ppm).
Nicotinic Acid
Niacin
N COOH
Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 1.0 per cent, determined on 1.0 g by drying in an oven at 105 for 1 hour. Mol. Wt. 123.1 Assay. Weigh accurately about 0.25 g, dissolve in 50 ml of carbon dioxide-free water and titrate with 0.1 M sodium
C6H5NO2 Nicotinic Acid is pyridine-3-carboxylic acid.
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IP 2007
hydroxide using phenol red solution as indicator. Repeat the operation without the substance under examination. The difference between the titrations represents the amount of sodium hydroxide required. 1 ml of 0.1 M sodium hydroxide is equivalent to 0.01231 g of C6H5NO2. Storage. Store protected from light and moisture.
stand for 15 minutes, swirling occasionally, and then shake for 10 minutes. Filter through a plug of cotton and wash the filter with ethanol (95 per cent). Add 50 ml of carbon dioxidefree water and titrate with 0.1 M sodium hydroxide using phenol red solution as indicator. 1 ml of 0.1 M sodium hydroxide is equivalent to 0.01231 g of C6H5NO2. Storage. Store protected from light and moisture.
Nicotinic Tablets
Niacin Acid Tablets
Nicotinic Acid Tablets contain not less than 92.5 per cent and not more than 107.5 per cent of the stated amount of nicotinic acid, C6H5NO2.
Nicoumalone
Acenocoumarol
O O NO2
Identification
A. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 48 volumes of chloroform, 45 volumes of ethanol (95 per cent) and 8 volumes of water. Test solution. Shake a quantity of the powdered tablets containing 50 mg of Nicotinic Acid with 50 ml of hot ethanol (95 per cent), filter and allow the filtrate to cool. Reference solution. A 0.1 per cent w/v solution of nicotinic acid RS in ethanol (95 per cent). Apply to the plate 5 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution. B. Triturate a quantity of the powdered tablets containing 50 mg of Nicotinic Acid with 10 ml of water and filter. To 2 ml of the filtrate add 6 ml of cyanogen bromide solution and 1 ml of a 2.5 per cent w/v solution of aniline; a golden yellow precipitate is produced. C. Shake a quantity of the powdered tablets containing 0.1 g of Nicotinic Acid with ethanol (95 per cent), filter and evaporate the filtrate to dryness. Add to the residue 10 mg of citric acid and 0.15 ml of acetic anhydride and heat on a water-bath; a reddish-violet colour is produced. C19H15NO6
OH O CH3
Mol. Wt. 353.3
Nicoumalone is (RS)-4-hydroxy-3-[1-(4-nitrophenyl)-3oxobutyl]coumarin. Nicoumalone contains not less than 98.5 per cent and not more than 100.5 per cent of C19H15NO6, calculated on the dried basis. Description. A white to brownish-white powder; odourless or almost odourless.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with nicoumalone RS or with the reference spectrum of nicoumalone. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.001 per cent w/v solution in a mixture of 9 volumes of methanol and 1 volume of 1 M hydrochloric acid shows absorption maxima at about 283 nm and 306 nm; absorbances at the maxima, about 0.65 and about 0.52, respectively. C. Heat 50 mg with 2.5 ml of glacial acetic acid, 0.5 ml of hydrochloric acid and 0.2 g of zinc powder on a water-bath for 5 minutes, cool and filter. To the filtrate add 0.05 ml of sodium nitrite solution and add the mixture to 10 ml of a 1 per cent w/v solution of 2-naphthol containing 3 ml of 5 M sodium hydroxide; a bright red precipitate is produced.
Tests
Other Tests. Comply with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 0.25 g of Nicotinic Acid, add 40 ml of hot ethanol (95 per cent), previously neutralised to phenolphthalein solution, and shake. Allow to
Tests
Appearance of solution. A 2.0 per cent w/v solution in acetone is clear (2.4.1).
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B. A 2.0 per cent w/v solution in 0.1 M sodium hydroxide is clear (2.4.1), and yellow. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 50 volumes of chloroform, 50 volumes of cyclohexane and 20 volumes of glacial acetic acid. Test solution. Dissolve 0.2 g of the substance under examination in 10 ml of acetone. Reference solution. A 0.002 per cent w/v solution of the substance under examination in acetone. Apply to the plate 20 l of each solution. After development, dry the plate in air and immediately examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.75 g, dissolve in 50 ml of acetone and titrate with 0.1 M sodium hydroxide using bromothymol blue solution as indicator. Repeat the operation without the substance under examination. The difference between the titrations represents the amount of sodium hydroxide required. 1 ml of 0.1 M sodium hydroxide is equivalent to 0.03533 g of C19H15NO6. Storage. Store protected from light.
On the residue determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with nicoumalone RS or with the reference spectrum of nicoumalone. B. When examined in the range 230 nm to 360 nm (2.4.7), the final solution obtained in the Assay shows absorption maxima at about 283 nm and 306 nm. C. Heat 50 mg of the residue obtained in test A, with 2.5 ml of glacial acetic acid, 0.5 ml of hydrochloric acid and 0.2 g of zinc powder on a water-bath for 5 minutes, cool and filter. To the filtrate add 0.05 ml of sodium nitrite solution and add the mixture to 10 ml of a 1 per cent w/v solution of 2-naphthol containing 3 ml of 5 M sodium hydroxide; a bright red precipitate is produced.
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 50 volumes of chloroform, 50 volumes of cyclohexane and 20 volumes of glacial acetic acid. Test solution. Shake a quantity of the powdered tablets containing 20 mg of Nicoumalone with 5 ml of acetone, centrifuge and use the supernatant liquid. Reference solution. Dilute 1 volume of the test solution to 200 volumes with acetone. Apply to the plate 20 l of each solution. After development, dry the plate in air and immediately examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Uniformity of content. Comply with the test stated under Tablets. Finely crush one tablet, add 30 ml of methanol, stir the mixture for 30 minutes and filter through sintered glass, washing the residue with three quantities, each of 15 ml, of methanol. To the combined filtrate and washings add 10 ml of 1 M hydrochloric acid and sufficient methanol to produce 100.0 ml. If necessary, dilute further with a solvent prepared by diluting 1 volume of 1 M hydrochloric acid to 10 volumes with methanol to produce a solution containing about 0.001 per cent w/v solution of Nicoumalone. Measure the absorbance of the resulting solution at the maximum at about 306 nm (2.4.7). Calculate the content of C19H15NO6 taking 521 as the specific absorbance at 306 nm. Other Tests. Comply with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 10 mg of
Nicoumalone Tablets
Acenocoumarol Tablets
Nicoumalone Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of nicoumalone, C19H15NO6.
Identification
A. Heat a quantity of the powdered tablets containing 50 mg of Nicoumalone with 30 ml of acetone under a reflux condenser for 5 minutes, filter and wash the residue with two quantities, each of 10 ml, of acetone. Evaporate the combined filtrate and washings to 5 ml, add water dropwise until the solution becomes turbid, heat on a water-bath until the solution is clear and allow to stand. Filter, wash the crystals with a mixture of equal volumes of acetone and water and dry at 100 at a pressure of 2 kPa for 30 minutes.
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NIFEDIPINE
IP 2007
Nicoumalone, add 30 ml of methanol, stir the mixture for 30 minutes and filter through sintered-glass, washing the residue with three quantities, each of 15 ml, of methanol. To the combined filtrate and washings add 10 ml of 1 M hydrochloric acid and sufficient methanol to produce 100.0 ml. Dilute 5.0 ml of this solution to 50.0 ml with a solvent prepared by diluting 1 volume of 1 M hydrochloric acid to 10 volumes with methanol and measure the absorbance of the resulting solution at the maximum at about 306 nm (2.4.7). Calculate the content of C19H15NO6 taking 521 as the specific absorbance at 306 nm. Storage. Store protected from light and moisture.
the peak due to nifedipine in the chromatogram obtained with the reference solution. C. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 60 volumes of cyclohexane and 40 volumes of ethyl acetate. Test solution. Dissolve 0.1 g of the substance under examination in 100 ml of methanol. Reference solution. A 0.1 per cent w/v solution of nifedipine RS in methanol. Apply to the plate 5 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution. D. To 25 mg add 10 ml of a mixture of 5 volumes of ethanol (95 per cent), 3.5 volumes of water and 1.5 volumes of hydrochloric acid and dissolve with gentle heating. Add 0.5 g of granulated zinc and allow to stand for 5 minutes, swirling occasionally. Filter, add 5 ml of a 1 per cent w/v solution of sodium nitrite to the filtrate and allow to stand for 2 minutes. Add 2 ml of a 5 per cent w/v solution of ammonium sulphamate, shake vigorously with care and add 2 ml of a 0.5 per cent w/v solution of N-(1- naphthyl) ethylenediamine dihydrochloride; an intense red colour develops which persists for more than 5 minutes.
Nifedipine
H3C H3CO O H N CH3 OCH3 O NO2
C17H18N2O6
Mol. Wt. 346.3
Nifedipine is dimethyl 1,4-dihydro-2,6-dimethyl-4-(2nitrophenyl)pyridine-3,5-dicarboxylate. Nifedipine contains not less than 98.0 per cent and not more than 102.0 per cent of C17H18N2O6, calculated on the dried basis. Description. A yellow, crystalline powder; readily affected by exposure to light. NOTE Nifedipine, when exposed to daylight and certain wavelengths of artificial light, readily converts to a nitrosophenyl derivative. Exposure to ultraviolet light leads to the formation of a nitrophenyl derivative. Perform the tests and assay in the dark or under long-wavelength light (greater than 420 nm). Use low-actinic glassware.
Tests
Related substances. Determine by liquid chromatography (2.4.14) Test solution. Dissolve 0.2 g of the substance under examination in 20 ml of methanol and dilute to 50 ml with the mobile phase. Reference solution (a). Dissolve an accurately weighed quantity of nifedipine RS in sufficient methanol to produce a 1.0 per cent w/v solution and dilute quantitatively with the mobile phase to obtain a 0.4 per cent w/v solution. Reference solution (b). A 0.04 per cent w/v solution of dimethyl-2,6-dimethyl-4-(2-nitrophenyl)pyridine-3,5dicaboxylate RS (nitrophenylpyridine analogue) in methanol. Reference solution (c). A 0.04 per cent w/v solution of dimethyl-2,6-dimethyl-4-(2-nitrosophenyl)pyridine-3,5dicarboxylate RS (nitrosophenylpyridine analogue) in methanol. Reference solution (d). Mix 1 volume of each of reference solutions (b) and (c) and 0.1 volume of the test solution, dilute to 10 volumes with the mobile phase and then dilute 2 volumes of the resulting solution to 10 volumes with the mobile phase.
Identification
Test A may be omitted if tests B, C and D are carried out. Tests B, C and D may be omitted if test A is carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with nifedipine RS or with the reference spectrum of nifedipine. B. In the test for Related substances, the principal peak in the chromatogram obtained with the test solution corresponds to
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IP 2007
NIFEDIPINE CAPSULES
Chromatographic system a stainless steel column 15 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: 55 volumes of water, 36 volumes of methanol and 9 volumes of acetonitrile, flow rate. 1 ml per minute, spectrophotometer set at 235 nm, a 20 l loop injector. Inject reference solution (d). The peaks appear in the order nitrophenylpyridine analogue, nitrosophenylpyridine analogue and nifedipine, which has a retention time of about 15.5 minutes. The test is not valid unless, in the chromatogram obtained with reference solution (d), (a) the resolution factor between the peaks due to the nitrophenylpyridine analogue and the nitrosophenylpyridine analogue is greater than 1.5, (b) the resolution between the peaks due to the nitrosophenylpyridine analogue and nifedipine is greater than 1.5, and (c) the height of the peak due to the nitrophenylpyridine analogue is at least 20 per cent of the full-scale deflection. Inject the test solution and reference solutions (a) and (d) and record the chromatograms for twice the retention time of nifedipine. In the chromatogram obtained with the test solution no secondary peak other than any peaks corresponding to the nitrophenylpyridine analogue and the nitrosophenylpyridine analogue has an area greater than that of the peak due to nifedipine in the chromatogram obtained with reference solution (d) and the areas of any peaks corresponding to the nitrophenylpyridine analogue and the nitrosophenylpyridine analogue are not greater than the areas of the corresponding peaks in the chromatogram obtained with reference solution (d). The total amount of related substances is not greater than 0.3 per cent. Ignore any peak with an area less than 10 per cent of the area of the peak due to nifedipine in the chromatogram obtained with reference solution (d). Heavy metals (2.3.13). 2.0 g complies with limit test for heavy metals, Method B (10 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105 for 2 hours. Assay. Weigh accurately about 0.13 g, dissolve in a mixture of 25 ml of 2-methyl-2-propanol and 25 ml of 1 M perchloric acid and titrate with 0.1 M ceric ammonium sulphate, using 0.1 ml of ferroin solution as indicator until the pink colour is discharged, titrating slowly towards the end-point. Carry out a blank titration. 1 ml of 0.1 M ceric ammonium sulphate is equivalent to 0.01732 g of C17H18N2O6. Storage. Store protected from light.
Nifedipine Capsules
Nifedipine Capsules contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of nifedipine, C17H18N2O6. NOTE Nifedipine, when exposed to daylight and certain wavelengths of artificial light, readily converts to a nitrosophenyl derivative. Exposure to ultraviolet light leads to the formation of a nitrophenyl derivative. Perform the tests and assay in the dark or under long-wavelength light (greater than 420 nm). Use low-actinic glassware.
Identification
Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of equal volumes of ethyl acetate and cyclohexane. Test solution. Transfer a quantity of the contents of the capsules containing 30 mg of Nifedipine into a centrifuge tube containing 0.1 M sodium hydroxide, add 25 ml of dichloromethane, stopper the tube and shake gently for 1 hour. Centrifuge for 10 minutes at 2000 to 2500 rpm. Remove the supernatant aqueous layer by aspiration with a syringe and transfer 5 ml of the clarified lower layer to a suitable vial. Reference solution (a). A 0.12 per cent w/v solution of nifedipine RS in dichloromethane. Reference solution (b). A mixture of equal volumes of test solution and reference solution (a). Apply to the plate 500 l of each solution as bands 20 mm by 3 mm. After development, dry the plate in air until the solvent is not detectable and immediately examine in ultraviolet light at 254 nm. The principal band, appearing as a dark blue band, in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution. Spray with a solution prepared in the following manner. Dissolve 3 g of bismuth subnitrate and 30 g of potassium iodide in 10 ml of 3 M hydrochloric acid and dilute with water to 100 ml; dilute 10 ml to 100 ml with 0.3 M hydrochloric acid. In the chromatogram obtained with test solution the principal band, appearing as a compact light orange band against a yellow background, corresponds to that in the chromatogram obtained with reference solution (a). The band obtained with reference solution (b) appears as a single band under both visualisation procedures.
Tests
Uniformity of content. Comply with the test stated under Capsules. Transfer the contents of a capsule quantitatively to a 200-ml volumetric flask with the aid of methanol, dilute to volume
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NIFEDIPINE SUSTAINED-RELEASE TABLETS
IP 2007
with methanol and mix. Complete the Assay beginning at the words Measure the absorbance.... and calculate the content of C17H18N2O6 in the capsule. Other Tests. Comply with the tests stated under Capsules. Assay. Transfer the contents of 5 capsules containing about 50 mg of Nifedipine quantitatively to a 200-ml volumetric flask with the aid of small quantities of methanol. Dilute to volume with methanol and mix. To 20.0 ml add sufficient methanol to produce 100.0 ml and mix. Measure the absorbance of the resulting solution at the maximum at about 350 nm (2.4.7). Calculate the content of C17H18N2O6 in the capsules from the absorbance obtained by repeating the operation with a 0.005 per cent w/v solution of nifedipine RS in methanol. Storage. Store protected from light.
with the reference solution. Spray with a solution prepared in the following manner. Dissolve 3 g of bismuth subnitrate and 30 g of potassium iodide in 10 ml of 3 M hydrochloric acid and dilute to 100 ml with water; dilute 10 ml of this solution to 100 ml with 0.3 M hydrochloric acid. In the chromatogram obtained with the test solution the principal band, appearing as a compact light orange band against a yellow background, corresponds to that in the chromatogram obtained with reference solution.
Tests
Dissolution (2.5.2) A. Apparatus No. 1 Medium. 900 ml of 0.1 M hydrochloric acid Speed and time. 150 rpm and 120 minutes. Withdraw a suitable volume of the medium and filter. Measure the absorbance of the filtrate, suitably diluted with the dissolution medium, if necessary, at the maximum at about 340 nm (2.4.7). Calculate the content of C17H18N2O6 in the medium from the absorbance obtained from a solution of known concentration of nifedipine RS in the same medium. D. Not less than 25 per cent and not more than 45 per cent of the stated amount of C17H18N2O6. B. Apparatus No. 1 Medium. 900 ml of phosphate buffer pH 6.8 Speed and time. 150 rpm and 6 hours. Withdraw a suitable volume of the medium and filter. Measure the absorbance of the filtrate, suitably diluted with the dissolution medium, if necessary, at the maximum at about 340 nm (2.4.7). Calculate the content of C17H18N2O6 in the medium from the absorbance obtained from a solution of known concentration of nifedipine RS in the same medium. D. Not less than 60 per cent of the stated amount of C17H18N2O6. Other tests. Comply with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 25 mg of Nifedipine, disperse in methanol, shake and dilute to 100.0 ml with methanol, filter. Dilute 20.0 ml of the filtrate to 100.0 ml with methanol. Measure the absorbance of the resulting solution at the maximum at about 350 nm (2.4.7). Calculate the content of C17H18N2O6 from the absorbance obtained with a 0.005 per cent w/v solution of nifedipine RS in methanol. Storage. Store protected from light and moisture.
Nifedipine Sustained-release Tablets

Nifedipine Sustained-release Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of nifedipine, C17H18N2O6. NOTE - Nifedipine, when exposed to daylight and certain wavelengths of artificial light, readily converts to a nitrosophenyl derivative. Exposure to ultraviolet light leads to the formation of a nitrophenyl derivative. Perform the tests and the assay in the dark or under long-wavelength light (greater than 420 nm). Use low-actinic glassware.
Identification
Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of equal volumes of ethyl acetate and cyclohexane. Test solution. Transfer a quantity of the contents of the capsules containing 30 mg of Nifedipine into a centrifuge tube containing 0.1 M sodium hydroxide, add 25 ml of dichloromethane, stopper the tube and shake gently for 1 hour. Centrifuge for 10 minutes at 2000 rpm to 2500 rpm. Remove the supernatant aqueous layer by aspiration with a syringe and use 5 ml of the clarified lower layer. Reference solution. A 0.12 per cent w/v solution of nifedipine RS in dichloromethane. Apply to the plate 500 l of each solution as bands 20 mm by 3 mm. After development, dry the plate in air until the odour of the solvent is not detectable and immediately examine in ultraviolet light at 254 nm. The principal band, appearing as a dark blue band, in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained
830
IP 2007
NIKETHAMIDE
Nifedipine Tablets
Nifedipine Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of nifedipine, C17H18N2O6. The tablets may be coated. NOTE Nifedipine, when exposed to daylight and certain wavelengths of artificial light, readily converts to a nitrosophenyl derivative. Exposure to ultraviolet light leads to the formation of a nitrophenyl derivative. Perform the tests and assay in the dark or under long-wavelength light (greater than 420 nm). Use low-actinic glassware.
Assay beginning at the words Measure the absorbance.... and calculate the content of C17H18N2O6 in the tablet. Other Tests. Comply with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing 50 mg of Nifedipine into a 200-ml volumetric flask. Dissolve with the aid of 50 ml of methanol. Dilute to volume with methanol, mix and filter. Dilute 20 ml of the filtrate to 100 ml with methanol and mix. Measure the absorbance of the resulting solution at the maximum at about 350 nm (2.4.7). Calculate the content of C17H18N2O6 from the absorbance obtained by repeating the operation with a 0.005 per cent w/v solution of nifedipine RS in methanol. Storage. Store protected from light and moisture.
Identification
Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of equal volumes of ethyl acetate and cyclohexane. Test solution. Transfer a quantity of the powdered tablets containing 30 mg of Nifedipine to a centrifuge tube containing 20 ml of 0.1 M sodium hydroxide, add 25 ml of dichloromethane, stopper the tube and shake gently for 1 hour. Centrifuge for 10 minutes at 2000 to 2500 rpm. Remove the supernatant aqueous layer by aspiration with a syringe and transfer 5.0 ml of the clarified lower layer to a suitable vial. Reference solution (a). A 0.12 per cent w/v solution of nifedipine RS in dichloromethane. Reference solution (b). A mixture of equal volumes of the test solution and reference solution (a). Apply to the plate 500 l of each solution as bands 20 mm by 3 mm. After development, dry the plate in air until the solvent is not detectable and immediately examine in ultraviolet light at 254 nm. The principal band, appearing as a dark blue band, in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with reference solution (a). Spray with a solution prepared in the following manner. Dissolve 3 g of bismuth subnitrate and 30 g of potassium iodide in 10 ml of 3 M hydrochloric acid and dilute with water to 100 ml; dilute 10 ml to 100 ml with 0.3 M hydrochloric acid. In the chromatogram obtained with the test solution the principal band, appearing as a compact light orange band against a yellow background, corresponds to that in the chromatogram obtained with reference solution (a). The band obtained with reference solution (b) appears as a single band under both visualisation procedures.
Nikethamide
N N O CH3 CH3
C10H14N2O
Mol. Wt. 178.2
Nikethamide is N,N-diethylpyridine-3-carboxamide. Nikethamide contains not less than 99.0 per cent and not more than 101.0 per cent of C10H14N2O, calculated on the anhydrous basis. Description. A colourless or slightly yellowish, oily liquid or crystalline mass; odour, slight and characteristic.
Identification
Test A may be omitted if tests B, C and D are carried out. Tests C and D may be omitted if tests A and B are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with nikethamide RS or with the reference spectrum of nikethamide. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.002 per cent w/v solution shows an absorption maximum only at about 263 nm; absorbance at about 263 nm, about 0.57. C. Heat 0.1 g with 1 ml of 2 M sodium hydroxide; diethylamine, recognisable by its odour, is evolved progressively; the fumes turn red litmus paper blue. D. To 2 ml of a 0.1 per cent w/v solution add 2 ml of cyanogen bromide solution and 3 ml of a 2.5 per cent w/v solution of aniline and mix; a yellow colour is produced.
Tests
Uniformity of content. Comply with the test stated under Tablets Shake one tablet with methanol in a 200-ml volumetric flask, dilute to volume with methanol, mix and filter. Complete the
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NIKETHAMIDE INJETION
IP 2007
Tests
Appearance of solution. The substance, in liquid form or liquefied by gentle heating, is clear (2.4.1), and not more intensely coloured than reference solution YS5 (2.4.1). pH (2.4.24). 6.0 to 7.8, determined in a 25.0 per cent w/v solution. Congealing temperature (2.4.10). 23 to 24.5. Refractive index (2.4.27). 1.522 to1.526. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 75 volumes of chloroform and 25 volumes of 1-propanol. Test solution. Dissolve 0.4 g of the substance under examination in 10 ml of methanol. Reference solution (a). A 0.04 per cent w/v solution of ethylnicotinamide RS in methanol. Reference solution (b). A 0.004 per cent w/v solution of ethylnicotinamide RS in methanol. Apply to the plate 10 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. Any spot corresponding to ethylnicotinamide in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (a) and any other secondary spot is not more intense than the spot in the chromatogram obtained with reference solution (b). Heavy metals (2.3.13). 2.0 g complies with limit test for heavy metals, Method B (10 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Water (2.3.43). Not more than 0.3 per cent, determined on 2.0 g. Assay. Weigh accurately about 0.15 g, dissolve in 20 ml of anhydrous glacial acetic acid and 5 ml of acetic anhydride. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.01782 g of C10H14N2O. Storge. Store protected from light.
Identification
A. Make 1 ml alkaline with 5 M sodium hydroxide, extract with 5 ml of dichloromethane and evaporate the solvent. On the residue determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with nikethamide RS or with the reference spectrum of nikethamide. B. Gives a voluminous precipitate with alkaline potassium mercuri-iodide solution and a greyish-brown flocculent precipitate with tannic acid solution. Gives no precipitate with iodine solution or with potassium mercuri-iodide solution. C. Heat 1 ml with 0.2 g of sodium hydroxide; diethylamine, recognisable by its odour, is evolved.
Tests
pH (2.4.24). 6.0 to 8.0. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 75 volumes of chloroform and 25 volumes of 1-propanol. Test solution. Dilute 1 ml of the injection to 5 ml with methanol. Reference solution (a). A 0.05 per cent w/v solution of ethylnicotinamide RS in methanol. Reference solution (b). A 0.005 per cent w/v solution of ethylnicotinamide RS in methanol. Apply to the plate 10 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. Any spot corresponding to ethylnicotinamide in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (a) and any other secondary spot is not more intense than the spot in the chromatogram obtained with reference solution (b). Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. Dilute 5.0 ml to 500.0 ml with water. To 5.0 ml of the solution add 5 ml of 1 M hydrochloric acid and sufficient water to produce 500.0 ml. Measure the absorbance of the resulting solution at the maximum at about 263 nm (2.4.7). Calculate the content of C10H14N2O taking 282 as the specific absorbance at 263 nm. Storage. Store protected from light, in single dose containers.
Nikethamide Injection
Nikethamide Injection is a sterile solution containing 25 per cent w/v solution of Nikethamide in Water for Injections. Nikethamide Injection contains not less than 24.0 per cent and not more than 26.0 per cent w/v solution of nikethamide, C10H14N2O.
832
IP 2007
NITRAZEPAM TABLETS
Nitrazepam
O HN N O2N
Test solution. Dissolve 0.2 g of the substance under examination in 10 ml of acetone. Reference solution. A 0.002 per cent w/v solution of the substance under examination in acetone. Apply to the plate 10 l of each solution. Allow the mobile phase to rise 12 cm. Dry the plate in air and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Mol. Wt. 281.3 Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105 for 4 hours. Assay. Weigh accurately about 0.25 g, dissolve in 50 ml of acetic anhydride. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.02813 g of C15H11N3O3. Storage. Store protected from light and moisture.
C15H11N3O3
Nitrazepam is 1,3-dihydro-7-nitro-5-phenyl-2H-1,4benzodiazepin-2-one. Nitrazepam contains not less than 99.0 per cent and not more than 101.0 per cent C15H11N3O3, calculated on the dried basis. Description. A yellow, crystalline powder; odourless or almost odourless.
Identification
Test A may be omitted if tests B, C and D are carried out. Tests B, C and D may be omitted if test A is carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with nitrazepam RS or with the reference spectrum of nitrazepam. B. Carry out the following procedure in subdued light. When examined in the range 230 nm to 360 nm (2.4.7), a freshly prepared 0.0005 per cent w/v solution in a 0.5 per cent w/v solution of sulphuric acid in methanol shows an absorption maximum only at about 280 nm; absorbance at about 280 nm, about 0.45. C. Dissolve 10 mg in 1 ml of methanol, warming if necessary, and add 0.05 ml of 2 M sodium hydroxide; an intense yellow colour is produced. D. Dissolve 20 mg in a mixture of 5 ml of hydrochloric acid and 10 ml of water, boil for 5 minutes, cool and add 2 ml of a 0.1 per cent w/v solution of sodium nitrite. Allow to stand for 1 minute, add 1 ml of a 0.5 per cent w/v solution of sulphamic acid, mix, allow to stand for 1 minute, add 1 ml of a 0.1 per cent w/v solution of N-(1-naphthyl)ethylenediamine dihydrochloride; a red colour is produced.
Nitrazepam Tablets
Nitrazepam Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of nitrazepam, C15H11N3O3.
Identification
Carry out the following procedure in subdued light. A. When examined in the range 230 nm to 360 nm (2.4.7), the final solution obtained in the Assay shows an absorption maximum only at about 280 nm. B. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 100 volumes of chloroform and 10 volumes of methanol. Test solution. Shake a quantity of the powdered tablets with sufficient methanol to produce a solution containing 5 mg of Nitrazepam per ml, allow to settle and decant the supernatant liquid. Reference solution. A 0.5 per cent w/v solution of nitrazepam RS in methanol. Apply to the plate 2 l of each solution. After development, dry the plate in air, spray it with ethanolic sulphuric acid (10 per cent v/v), heat at 105 for 10 minutes and examine in
Tests
Related substances and decomposition products. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 85 volumes of nitromethane and 15 volumes of ethyl acetate.
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NITROFURANTOIN
IP 2007
ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution. C. To a quantity of the powdered tablets containing 5 mg of Nitrazepam add 5 ml of hydrochloric acid and 10 ml of water, heat on a water-bath for 15 minutes, filter and cool. To the clear filtrate add 1 ml of a 0.1 per cent w/v solution of sodium nitrite, allow to stand for 3 minutes and add 1 ml of a 0.5 per cent w/v solution of sulphamic acid. Allow to stand for 3 minutes and add 1 ml of a 0.1 per cent w/v solution of N-(1naphthyl) ethylenediamine dihydrochloride; a red colour is produced.
light. Add 70 ml of a 0.5 per cent v/v solution of hydrochloric acid in methanol, shake for 15 minutes protected from light, add sufficient of the hydrochloric acid solution to produce 100.0 ml and filter. Dilute 10.0 ml of the filtrate to 100.0 ml with the same solvent and measure the absorbance of the resulting solution immediately at the maximum at about 280 nm (2.4.7). Calculate the content of C15H11N3O3 taking 910 as the specific absorbance at 280 nm. Storage. Store protected from light and moisture.
Nitrofurantoin
O O 2N O H C N N O NH
Tests
Related substances and decomposition products. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 40 volumes of nitromethane, 40 volumes of toluene and 20 volumes of chloroform. Test solution. Shake a quantity of the powdered tablets containing 40 mg of Nitrazepam with 25 ml of chloroform, filter, carefully evaporate the filtrate to dryness and dissolve the residue in 2 ml of chloroform. Reference solution. Dilute 1 volume of the test solution to 200 volumes with chloroform. Apply to the plate 5 l of each solution. Allow the mobile phase to rise 12 cm. Dry the plate in air and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Uniformity of content. Comply with the test stated under Tablets. NOTE Carry out the following procedure in subdued light. Powder 1 tablet, add 5 ml of water, mix and allow to stand for 15 minutes protected from light. Add 70 ml of a 0.5 per cent v/v solution of hydrochloric acid in methanol, shake for 15 minutes protected from light, add sufficient of the hydrochloric acid solution to produce 100.0 ml and filter. Dilute 10.0 ml of the filtrate with sufficient of the hydrochloric acid solution to produce a solution containing 0.0005 per cent w/v solution of Nitrazepam. Measure the absorbance of the resulting solution immediately at the maximum at about 280 nm (2.4.7). Calculate the content of C15H11N3O3 in the tablet taking 910 as the specific absorbance at 280 nm. Other Tests. Comply with the tests stated under Tablets. Assay. Carry out the following procedure in subdued light. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 5 mg of Nitrazepam, add 5 ml of water, mix and allow to stand for 15 minutes protected from C8H6N4O5 C8H6N4O5,H2O
Mol. Wt. 238.2 (anhydrous) Mol. Wt. 256.2 (hydrous)
Nitrofurantoin is 1-(5-nitrofurfurylideneamino)imidazolidine2,4-dione. It is anhydrous or contains one molecule of water of hydration. Nitrofurantoin contains not less than 98.0 per cent and not more than 102.0 per cent of C8H6N4O5, calculated on the dried basis. Description. Lemon yellow crystals or a crystalline powder; odourless or almost odourless.
Identification
Carry out the following test in subdued light. A. When examined in the range 230 nm to 400 nm (2.4.7), the final solution obtained in the Assay shows absorption maxima at about 266 nm and 367 nm; the ratio of the absorbance at the maximum at about 367 nm to that at the maximum at about 266 nm is 1.36 to 1.42. B. To 1 ml of a 0.1 per cent w/v solution in dimethylformamide add 0.1 ml of 0.5 M ethanolic potassium hydroxide; a brown colour develops.
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel HF254. Mobile phase. A mixture of 90 volumes of nitromethane and 10 volumes of methanol. Test solution. Dissolve 0.25 g of the substance under examination in minimum volume of dimethylformamide and dilute to 10 ml with acetone.
834
IP 2007
NITROFURAZONE
Reference solution. Dilute 1 volume of the test solution to 100 volumes with acetone. Apply to the plate 10 l of each solution. After development, dry the plate in air, heat at 105 for 5 minutes and examine in ultraviolet light at 254 nm. Spray with phenylhydrazine hydrochloride solution and heat the plate at 105 for further 10 minutes. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution by both methods of visualisation. Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). 5.0 to 7.1 per cent (hydrous form) and not more than 1.0 per cent (anhydrous form), determined on 1.0 g by drying in an oven at 105. Assay. Carry out the following procedure in subdued light. Weigh accurately about 75 mg and dissolve in 25.0 ml of dimethylformamide, add sufficient water to produce 500.0 ml and mix. Dilute 5.0 ml to 100.0 ml with a solution containing 1.8 per cent w/v solution of sodium acetate and 0.14 per cent v/v of glacial acetic acid. Measure the absorbance of the resulting solution at the maximum at about 367 nm (2.4.7), using as the blank the sodium acetate-acetic acid solution. Calculate the content of C8H6N4O5 taking 765 as the specific absorbance at 367 nm. Storage. Store protected from light and moisture. Labelling. The label states whether the material is anhydrous or hydrous.
Reference solution. Dilute 1 volume of the test solution to 100 volumes with acetone. Apply to the plate 10 l of each solution. After development, dry the plate in air, heat at 105 for 5 minutes and examine in ultraviolet light at 254 nm. Spray with phenylhydrazine hydrochloride solution and heat the plate at 105 for further 10 minutes. Any secondary spot in the chromatogram obtained with the test solution, is not more intense than the spot in the chromatogram obtained with the reference solution by both methods of visualisation. Other Tests. Comply with the tests stated under Tablets. Assay. Carry out the following procedure in subdued light. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 0.15 g of Nitrofurantoin, add 50.0 ml of dimethylformamide, shake for 5 minutes, add sufficient water to produce 1000.0 ml and mix. Dilute 5.0 ml to 100.0 ml with a solution containing 1.8 per cent w/v solution of sodium acetate and 0.14 per cent v/v of glacial acetic acid. Measure the absorbance of the resulting solution at the maximum at about 367 nm (2.4.7), using as the blank the sodium acetate-acetic acid solution. Calculate the content of C8H6N4O5 taking 765 as the specific absorbance at 367 nm. Storage. Store protected from light and moisture.
Nitrofurazone
Nitofural
H N O
Nitrofurantoin Tablets
Nitrofurantoin Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of nitrofurantoin, C8H6N4O5.
O2N O
H C
NH2
Identification
Carry out the following procedure in subdued light. A. When examined in the range 230 nm to 400 nm (2.4.7), the final solution obtained in the Assay shows absorption maxima at about 266 nm and 367 nm.
C6H6N4O4
Mol. Wt. 198.1
Nitrofurazone is 5-nitro-2-furaldehyde semicarbazone. Nitrofurazone contains not less than 97.0 per cent and not more than 103.0 per cent of C6H6N4O4, calculated on the dried basis. Description. A yellow to brownish-yellow, crystalline powder; odourless or almost odourless.
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel HF254. Mobile phase. A mixture of 90 volumes of nitromethane and 10 volumes of methanol. Test solution. Shake a quantity of the powdered tablets containing 0.1 g of Nitrofurantoin with 10 ml of a mixture of 9 volumes of acetone and 1 volume of dimethylformamide and filter.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with nitrofurazone RS or with the reference spectrum of nitrofurazone. B. Dissolve 1 mg in 1 ml of dimethylformamide and add 0.05 ml of 1 M ethanolic potassium hydroxide; a ruby red colour is produced.
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NITROUS OXIDE
IP 2007
Tests
pH (2.4.24). 5.0 to 7.0, determined in the filtrate obtained by shaking 1.0 g with 100 ml of carbon dioxide-free water and filtering. Related substances. Carry out the following procedure in subdued light. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 95 volumes of toluene and 5 volumes of dioxan. Test solution. Dissolve 0.1 g of the substance under examination in 10 ml of a mixture of equal volumes of acetone and dimethylformamide. Reference solution (a). A 0.002 per cent w/v solution of 5-nitrofurfurylidene azine RS in a mixture of equal volumes of acetone and dimethylformamide. Reference solution (b). A 0.01 per cent w/v solution of nitrofurfural diacetate RS in a mixture of equal volumes of acetone and dimethylformamide. Apply to the plate 10 l of each solution. After development, dry the plate in air, heat it at 105 for 5 minutes and spray with phenylhydrazine hydrochloride solution. In the chromatogram obtained with the test solution any spots corresponding to 5-nitrofurfurylidene azine and nitrofurfural diacetate are not more intense than the spots in the chromatograms obtained with reference solutions (a) and (b) respectively. Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Carry out the following procedure in subdued light. Weigh accurately about 60 mg, add 20.0 ml of dimethylformamide, swirl to dissolve and add sufficient water to produce 500.0 ml. Dilute 5.0 ml of the solution to 100.0 ml with water and mix. Measure the absorbance of the resulting solution at the maximum at about 375 nm (2.4.7). Calculate the content of C6H6N4O4 taking 822 as the specific absorbance at 375 nm. Storage. Store protected from light and moisture.
less than 6 hours before carrying out the tests Keep the cylinder in the vertical position with the outlet valve uppermost and deliver the gas at a rate of 4 litres per hour, unless otherwise directed. The test for Carbon monoxide should be carried out on the first portion of gas drawn from the cylinder and that for Nitric oxide and nitrogen dioxide immediately thereafter. Description. A colourless gas; odourless.
Identification
A. A glowing splinter of wood bursts into flame on contact with the gas. B. Shake with alkaline pyrogallol solution; the gas being examined is not absorbed and the solution does not become brown.
Tests
Acidity or alkalinity. Use hermetically-closed, flat-bottomed, glass cylinders with dimensions such that 50 ml of liquid reaches a height of 12 to 14 cm, fitted with an outlet tube and with an inlet tube with an orifice of 1 mm in internal diameter reaching to within 2 mm of the bottom of the cylinder. For solution (1) pass 2.0 litres of the gas under examination through a mixture of 0.1 ml of 0.01 M hydrochloric acid and 50 ml of carbon dioxide-free water. For solution (2) use 50 ml of carbon dioxide-free water. For solution (3) add 0.2 ml of 0.01 M hydrochloric acid to 50 ml of carbon dioxide-free water. To each solution add 0.1 ml of a 0.02 per cent w/v solution of methyl red in ethanol (70 per cent). The intensity of the colour of solution (1) is between those of solutions (2) and (3). Arsine and phosphine. Through a mercuric chloride paper attached to a glass tube as in the limit test for arsenic (2.3.10), pass 2.0 litres of the gas; no visible stain is produced. Carbon dioxide. Not more than 300 ppm v/v determined by the following method. Use the apparatus described in the test for Acidity or alkalinity. Pass 1.0 litre through 50 ml of clear barium hydroxide solution. Any turbidity produced in the resulting solution is not more than that obtained in a reference solution prepared at the same time by adding 1 ml of a 0.11 per cent w/v solution of sodium bicarbonate in carbon dioxide-free water to 50 ml of barium hydroxide solution. Carbon monoxide. Not more than 10 ppm v/v, determined by the following method. Connect in series a U-tube containing silica gel impregnated with chromium trioxide, a drechsel bottle containing 100 ml of a 40 per cent w/v solution of potassium hydroxide, a U-tube containing pellets of potassium hydroxide, a U-tube containing phosphorus pentoxide dispersed on previously granulated, fused pumice, a tube containing iodine pentoxide in granules, previously dried at 200 and kept at a temperature of 120, packed in 1-cm
Nitrous Oxide
N2O Mol. Wt. 44.0 Nitrous Oxide contains not less than 98.0 per cent v/v of N2O. NOTE Carry out the following tests on a full cylinder from which no gas has been withdrawn. The cylinder from which the gas is taken should be kept at room temperature for not
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IP 2007
NORADRENALINE BITARTRATE
columns separated by 1-cm columns of glass wool giving an effective length of 5 cm, and a flask containing 2.0 ml of 1 M potassium iodide and 0.15 ml of starch solution. Flush the apparatus with 5.0 litres of carbon dioxide-free air and, if necessary, discharge the blue colour in the iodide solution by adding a small quantity of freshly prepared 0.002 M sodium thiosulphate. Continue flushing until not more than 0.045 ml of 0.002 M sodium thiosulphate is required after passing 5.0 litres of carbon dioxide-free air. Pass 5.0 litres of the gas under examination through the apparatus and flush the last traces of liberated iodine into the reaction flask by passing through the apparatus 1.0 litre of carbon monoxidefree air. Titrate the liberated iodine with 0.002 M sodium thiosulphate. Carry out a blank titration under the same conditions, using 5.0 litres of carbon dioxide-free air. The difference between the volumes of 0.002 M sodium thiosulphate used in the two titrations is not greater than 1.0 ml. Halogens and hydrogen sulphide. Pass a volume containing 1.0 litre measured at 25 and at 101.3 kPa through a mixture of 100 ml of water and 1 ml of silver nitrate solution; neither opalescence nor darkening is produced. Nitric oxide and nitrogen dioxide. Not more than 2 ppm v/v in both the liquid and gaseous phases, determined by the following method. Use two of the cylinders described in the test for Acidity or alkalinity connected in series. Examine separately both the liquid and gaseous phases of the gas under examination. To obtain the liquid phase invert the cylinder. The liquid vaporises on leaving the valve. For solution A dissolve 1 g of sulphanilic acid in a mixture of 10 ml of glacial acetic acid and 180 ml of water. For solution B dissolve 0.2 g of N-(1-naphthyl)ethylenediamine dihydrochloride in 10 ml of a 50 per cent v/v solution of glacial acetic acid, heating gently, and dilute to 200 ml with water. Mix 9 volumes of solution A with 1 volume of solution B (reagent A). In the first cylinder place 15 ml of a solution containing 2.5 per cent w/v solution of potassium permanganate and 1.2 per cent v/v of sulphuric acid (96 per cent). Place 20 ml of reagent A in the second cylinder and connect the outlet tube of the first cylinder to the inlet tube of the second cylinder. Pass 2.5 litres of the gas under examination through the reagents at a rate of 15 litres per hour. Prepare a reference solution by adding 0.25 ml of a 0.00616 per cent w/v solution of sodium nitrite to 20 ml of reagent A. Allow both the sample and reference solutions to stand for 10 minutes. For both liquid and gaseous phases, any red colour in the sample solution is not more intense than that in the reference solution. Oxidising substances. Pass a volume containing 2.0 litres measured at 25 and at 101.3 kPa through a freshly prepared
solution of 0.5 g of soluble starch and 0.5 g of potassium iodide in 100 ml of water containing 0.05 ml of glacial acetic acid; the colour of the liquid is not changed. Water. Pass a measured quantity at a rate of 6 litres per hour through an absorption tube containing magnesium perchlorate; the increase in weight of the tube does not exceed 2 mg per litre of gas, the initial and final weighings of the tube being made when the air in it has been displaced by the nitrous oxide. Assay. Carry out the assay of nitrous oxide (2.3.32), using 100 ml of the gas under examination. Use a cylinder of the gas under examination from which at least 1 per cent w/w of the contents have been removed. Storage. Store under pressure in metal cylinders of the type conforming to the appropriate safety regulations and at a temperature not exceeding 37. Labelling. The cylinder is painted blue and carries a label stating Nitrous Oxide. In addition, Nitrous Oxide or the symbol N2O should be stencilled in paint on the shoulder of the cylinder.
Noradrenaline Bitartrate
Noradrenaline Acid Tartrate; Levarterenol Bitartrate; Norepinephrine Bitartrate
OH NH2 HO OH
C8H11NO3,C4H6O6,H2O
H OH , HOOC COOH , H2O H OH

Mol. Wt. 337.3
Noradrenaline Bitartrate is (R)-2-amino-1-(3,4dihydroxyphenyl)ethanol hydrogen (2R,3R)-tartrate monohydrate. Noradrenaline Bitartrate contains not less than 98.5 per cent and not more than 101.0 per cent of C8H11NO3,C4H6O6, calculated on the anhydrous basis. Description. A white or almost white, crystalline powder; odourless. It gradually darkens on exposure to air and light.
Identification
Test A may be omitted if tests B, C, D, E and F are carried out. Tests C, D, E may be omitted if tests A, B and F are carried out. A. Dissolve 0.2 g in 2 ml of water containing about 10 mg of sodium sulphite and add sufficient dilute ammonia solution to give an alkaline reaction. Keep the mixture at about 4 for 1 hour and filter.
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NORADRENALINE BITARTRATE INJECTION
IP 2007
On the residue (residue R) determine by infrared absorption spectrophotometry (2,4,6). Compare the spectrum with that obtained with noradrenaline acid tartrate RS treated in the same manner. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.005 per cent w/v solution in 0.01 M hydrochloric acid shows an absorption maximum at about 279 nm; absorbance at about 279 nm, about 0.40. C. Wash residue R obtained in test A with three quantities, each of 2 ml, of water, followed by 5 ml of ethanol (95 per cent) and 5 ml of ether and dry the precipitate under pressure of 1.5 to 2.5 kPa for 3 hours. The specific optical rotation (2.4.22), determined in a 2.0 per cent w/v solution of the dried precipitate in 0.5 M hydrochloric acid is 44 to 48. D. To 1 ml of a 1 per cent w/v solution, add 0.05 ml of ferric chloride solution; an intense green colour is produced. Add, drop by drop, sodium bicarbonate solution; the colour changes to blue and then red. E. To 1 ml of a 0.1 per cent w/v solution add 10 ml of phthalate buffer pH 3.6, add 1 ml of 0.05 M iodine, set aside for 5 minutes and add 2 ml of 0.1 M sodium thiosulphate; not more than a faint red colour is produced. Repeat the test using buffer solution pH 6.6 instead of phthalate buffer pH 3.6; a strong reddish violet colour is produced (distinction from adrenaline and isoprenaline). F. The filtrate obtained in test A gives the reactions of tartrates (2.3.1).
Apply to the plate 6 l of each of test solution, reference solutions (a) and (b) and 12 l of reference solution (c) as bands 20 mm by 2 mm. Allow the applied bands to dry in air, spray them with a saturated solution of sodium bicarbonate, allow to dry in air and spray the bands twice with acetic anhydride, drying between the two sprayings. Heat the plate at 50 for 90 minutes and develop the chromatograms. After removal of the plate, allow it to dry in air and spray with a freshly prepared mixture of 8 volumes of methanol, 2 volumes of ethylenediamine and 2 volumes of a 0.5 per cent w/v solution of potassium ferricyanide. Dry the plate at 60 for 10 minutes and examine in ultraviolet light at 254 and 365 nm. In the chromatogram obtained with the test solution any band with a slightly higher Rf value than the principal band is not more intense than the corresponding band in the chromatogram obtained with reference solution (b). The chromatogram obtained with reference solution (c) shows a clearly separated band corresponding to the most intense band in the chromatogram obtained with reference solution (a) at a higher Rf value than the most intense band. Noradrenalone. Absorbance of a 0.2 per cent w/v solution in 0.01 M hydrochloric acid at 310 nm, not more than 0.40 (2.4.7). Sulphated ash (2.3.18). Not more than 0.1 per cent. Water (2.3.43). 4.5 to 5.8 per cent, determined on 0.5 g. Assay. Weigh accurately about 0.6 g, dissolve in 50 ml of anhydrous glacial acetic acid, warming if necessary.Titrate with 0.1 M perchloric acid, using crystal violet solution as indicator, until a bluish green colour is obtained. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.03193 g of C8H11NO3,C4H6O6. Storage. Store protected from moisture.
Tests
Appearance of solution. A freshly prepared 2.0 per cent w/v solution is clear (2.4.1), and not more intensely coloured than reference solution BYS5 (2.4.1). pH (2.4.24). 3.5 to 5.0, determined in a 1.0 per cent w/v solution. Melting range (2.4.21). 100 to 106, with decomposition. Adrenaline. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 50 volumes of acetone, 50 volumes of dichloromethane and 0.5 volume of anhydrous formic acid. Prepare the following solutions immediately before use. Test solution. Dissolve 0.25g of the substance under examination in 10 ml of water. Reference solution (a). A 0.125 per cent w/v solution of adrenaline tartrate RS in water. Reference solution (b). A 0.025 per cent w/v solution of adrenaline tartrate RS in water. Reference solution (c). A mixture of equal volumes of the test solution and reference solution (b).
Noradrenaline Bitartrate Injection

Noradrenaline Acid Tartrate Injection; Noradrenaline Injection; Levarterenol Bitartrate Injection; Norepinephrine Bitartrate Injection
Noradrenaline Bitartrate Injection is a sterile solution of Noradrenaline Bitartrate. It is prepared by diluting Sterile Noradrenaline Concentrate to 250 times its volume with Sodium Chloride and Dextrose Injection or with Dextrose Injection (5 per cent w/v) immediately before use. Noradrenaline Bitartrate Injection contains in 1 ml 8 g of Noradrenaline Bitartrate equivalent to approximately 4 g of noradrenaline.
Tests
Other Tests. Complies with the tests stated under Parenteral Preparations (Injections).
838
IP 2007
NORETHISTERONE
Sterile Noradrenaline Concentrate Sterile Noradrenaline Concentrate is a sterile, isotonic solution containing 0.2 per cent w/v of Noradrenaline Bitartrate in Water for Injections. Sterile Noradrenaline Concentrate contains not less than 0.18 per cent and not more than 0.23 per cent w/v of noradrenaline bitartrate, C8H11NO3,C4H6O6,H2O.
Description. A white or yellowish-white, crystalline powder; odourless.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with norethisterone RS or with the reference spectrum of norethisterone. B. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Solvent mixture. A mixture of 90 volumes of acetone and 10 volumes of formamide. Mobile phase. A mixture of 40 volumes of hexane and 10 volumes of dioxan. Test solution. Dissolve 10 mg of the substance under examination in 10 ml of chloroform. Reference solution. Dissolve 10 mg of norethisterone RS in 10 ml of chloroform. Place the dry plate in a tank containing a shallow layer of the solvent mixture, allow the solvent mixture to ascend to the top, remove the plate from the tank and allow the solvent to evaporate. Use within 2 hours, with the flow of the mobile phase in the direction in which the aforementioned treatment was done. Apply to the plate 2 l of each solution. Allow the mobile phase to rise 12 cm. Dry the plate in a current of warm air, allow the solvent to evaporate, heat at 120 for 15 minutes and spray the hot plate with ethanolic sulphuric acid (20 per cent v/v). Heat at 120 for a further 10 minutes, allow to cool and examine in daylight and in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution corresponds to and exhibits fluorescence similar to that in the chromatogram obtained with the reference solution. C. Dissolve about 2 mg in 2 ml of ethanol (95 per cent) and add 1 ml of a 1 per cent w/v solution of butylated hydroxytoluene in ethanol (95 per cent) and 2 ml of 1 M sodium hydroxide. Heat in a water-bath for 30 minutes and cool; a yellowish pink colour is produced.
Identification
Mix 0.5 ml with 10 ml of phthalate buffer pH 3.6, add 1 ml of 0.05 M iodine, allow to stand for 5 minutes and add 2 ml of 0.1 M sodium thiosulphate; not more than a very faint red colour is produced. Repeat the test using phosphate buffer pH 6.6 instead of phthalate buffer pH 3.6; a strong reddish violet colour is produced.
Tests
pH (2.4.24). 3.0 to 4.6. Other Tests. Complies with the tests stated under Parenteral Preparations (Concentrated Solutions for Injection). Assay. Dilute 5.0 ml to 200.0 ml with water and measure the absorbance of the resulting solution at the maximum at about 279 nm (2.4.7). Calculate the content of C8H11NO3,C4H6O6,H2O taking 80 as the specific absorbance at 279 nm. Storage. Store protected from light, in single dose containers. Labelling. The label states (1) Sterile Noradrenaline Concentrate; (2) that 1 volume of the solution diluted to 250 volumes with Sodium Chloride and Dextrose Injection or with Dextrose Injection (5 per cent w/v) produces Noradrenaline Bitartrate Injection, which must be used immediately after preparation; (3) that if the solution is brown it should not be used.
Norethisterone
Norethindrone
H3C OH C CH H H O
C20H26O2 Mol. Wt. 298.4 Norethisterone is 17-hydroxy-19-nor-17-pregn-4-en-20-yn3-one. Norethisterone contains not less than 98.0 per cent and not more than 102.0 per cent of C20H26O2, calculated on the dried basis.
Tests
Appearance of solution. Dissolve 0.2 g in sufficient dioxan to produce 10 ml (solution A). The solution is clear (2.4.1), and not more intensely coloured than reference solution YS6 (2.4.1). Specific optical rotation (2.4.22). 33.0 to 37.0, determined in a solution prepared by diluting 5.0 ml of solution A to 10.0 ml with dioxan. Light absorption. Dissolve 10 mg in sufficient ethanol (95 per cent) to produce 100 ml, dilute 10 ml of the solution to 100 ml with methanol (98 per cent). Absorbance of the
839
NORETHISTERONE TABLETS
IP 2007
resulting solution at the maximum at about 240 nm, 0.55 to 0.59 (2.4.7). Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 90 volumes of chloroform and 10 volumes of acetone. Test solution. Dissolve 0.5 g of the substance under examination in 100 ml of the mobile phase. Reference solution (a). A 0.0025 per cent w/v solution of the substance under examination in the mobile phase. Reference solution (b). A solution containing 0.025 per cent w/v each of the substance under examination and ethisterone RS in the mobile phase. Apply to the plate 10 l of each solution. After development, dry the plate in air, spray with ethanolic sulphuric acid (20 per cent v/v), heat at 105 for 5 minutes and examine in ultraviolet light at 365 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (a). The chromatogram obtained with reference solution (b) shows two clearly separated spots of equal intensities. Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105 for 3 hours. Assay. Weigh accurately about 0.4 g, dissolve in 40 ml of tetrahydrofuran, add 10 ml of a 10 per cent w/v solution of silver nitrate and titrate with 0.1 M sodium hydroxide using 2 ml of bromocresol green solution as indicator. Repeat the operation without the substance under examination. The difference between the titrations represents the amount of sodium hydroxide required. 1 ml of 0.1 M sodium hydroxide is equivalent to 0.02984 g of C20H26O2. Storage. Store protected from light and moisture.
each of 5 ml, of light petroleum (60 to 80) and discard the washings. Extract the residue with 15 ml of chloroform, evaporate the extract to dryness and recrystallise from aqueous methanol. The residue complies with the following test. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Solvent mixture. A mixture of 90 volumes of acetone and 10 volumes of 1,2-propanediol. Mobile phase. A mixture of 40 volumes of cyclohexane and 10 volumes of toluene. Test solution. Dissolve 25 mg of the substance under examination in 10 ml of the solvent mixture. Reference solution (a). Dissolve 25 mg of norethisterone RS in 10 ml of the solvent mixture. Reference solution (b). Mix equal volumes of the test solution and reference solution (a). Place the dry plate in a tank containing a shallow layer of the solvent mixture, allow the solvent mixture to ascend to the top, remove the plate from the tank and allow the solvent to evaporate. Use within 2 hours, with the flow of the mobile phase in the direction in which the aforementioned treatment was done. Apply to the plate 2 l of each solution. Allow the mobile phase to rise 12 cm. Dry the plate in a current of warm air, allow the solvent to evaporate, heat at 120 for 15 minutes and spray the hot plate with ethanolic sulphuric acid (20 per cent v/v). Heat at 120 for a further 10 minutes, allow to cool and examine in daylight and in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with reference solution (a). The principal spot in the chromatogram obtained with reference solution (b) appears as a single, compact spot.
Tests
Uniformity of content. Comply with the test stated under Tablets.
Norethisterone Tablets
Norethindrone Tablets
Norethisterone Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of norethisterone, C20H26O2.
Identification
Place a quantity of the powdered tablets containing 25 mg of Norethisterone on a small filter, wash with three quantities,
Powder one tablet and warm with about 75 ml of ethanol (95 per cent) with stirring. Cool, transfer to a 100-ml volumetric flask and dilute to volume with ethanol (95 per cent). Centrifuge a few ml of the mixture until a clear supernatant liquid is obtained. Dilute 10.0 ml of the supernatant liquid to 50.0 ml with ethanol (95 per cent) and mix. Measure the absorbance of the resulting solution at the maximum at about 240 nm (2.4.7). Calculate the content of C20H26O2 in the tablet taking 570 as the specific absorbance at 240 nm. Other Tests. Comply with the tests stated under Tablets.
840
IP 2007
NORFLOXACIN
Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 0.2 g of Norethisterone and transfer to a glass column closed at the bottom with a small piece of absorbent cotton. The glass column consists of a piece of glass tubing (150 mm x 10 mm) tapered at the bottom and sealed at the top to another piece of glass tubing (150 mm x 25 mm). Place a small piece of absorbent cotton on top of the powder, pass 200 ml of light petroleum (60 to 80) through the column and discard the effluent. Extract the residue with 200 ml of chloroform, evaporate the chloroform from the extract and dry the residue at 105 for 2 hours. Allow to cool, dissolve in 40 ml of tetrahydrofuran, add 10 ml of a 10 per cent w/v solution of silver nitrate. Titrate with 0.1 M sodium hydroxide, determining the end-point potentiometrically (2.4.25). Repeat the operation without the substance under examination. The difference between the titrations represents the amount of sodium hydroxide required. 1 ml of 0.1 M sodium hydroxide is equivalent to 0.02984 g of C20H26O2. Storage. Store protected from light and moisture.
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G, previously washed with methanol and dried. Mobile phase. A mixture of 40 volumes of dichloromethane, 40 volumes of methanol, 20 volumes of toluene, 14 volumes of diethylamine and 8 volumes of water. Test solution. Dissolve 0.8 g of the substance under examination in 100 ml of a mixture of equal volumes of methanol and dichloromethane. Reference solution. Dissolve 4.0 mg of norfloxacin RS in 1 ml of glacial acetic acid, add 4 ml of methanol and mix; dilute 1 ml of the solution with 9 ml of a mixture of equal volumes of methanol and dichloromethane (reference solution A). Dilute a portion of reference solution A with an equal volume of the methanol-dichloromethane mixture (reference solution B). Apply separately to the plate spots of the three solutions in quantities indicated below. For spot 1 use 5 l of the test solution; for spots 2, 3 and 4 use 1 l, 1.5 l and 2 l respectively of reference solution A; for spot 5 use 5 l of reference solution B. Place the plate in a paper-lined chamber previously equilibrated with the mobile phase and allow the solvent front to move about nine-tenths of the length of the plate. After development, dry the plate in air and examine in ultraviolet light at 254 nm and 365 nm. Compare the intensities of any secondary spots in the chromatogram obtained with the test solution with those of the principal spots (2), (3), (4) and (5). The sum of the intensities of secondary spots obtained with the test solution is not more than 0.5 per cent of impurities. (The spots (2) (3) (4) and (5) represent 0.2 per cent, 0.3 per cent, 0.4 per cent and 0.5 per cent respectively of impurities). Heavy metals (2.3.13). 1.33 g complies with the limit test for heavy metals, Method B (15 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent, determined on 1.0 g in a platinum crucible. Loss on drying (2.4.19). Not more than 1.0 per cent, determined on 0.5 g by drying in an oven at 100 at a pressure not exceeding 0.7 kPa. Assay. Weigh accurately about 0.3 g and dissolve in 100 ml of anhydrous glacial acetic acid. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.4.25) and using a suitable anhydrous electrode system. (The electrode system may be rendered anhydrous by filling the electrode with 0.1 M lithium perchlorate in acetic anhydride after removing any aqueous solution contained in it). Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.03193 g of C16H18FN3O3.
Norfloxacin
HN N F O N COOH CH3
C16H18FN3O3
Mol. Wt. 319.3
Norfloxacin is 1-ethyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4dihydroquinoline-3-carboxylic acid. Norfloxacin contains not less than 99.0 per cent and not more than 101.0 per cent of C16H18FN3O3, calculated on the dried basis. Description. A white to pale yellow, crystalline powder.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with norfloxacin RS or with the reference spectrum of norfloxacin. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.0005 per cent w/v solution in 0.1 M sodium hydroxide shows an absorption maximum at about 273 nm.
841
NORFLOXACIN EYE DROPS
IP 2007
Norfloxacin Tablets
Norfloxacin Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of norfloxacin, C16H18FN3O3. The tablets may be coated.
Norfloxacin Eye Drops

Norfloxacin Eye Drops are a sterile solution of Norfloxacin in Purified water. Norfloxacin Eye Drops contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of norfloxacin, C16H18FN3O3.
Identification
A. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 40 volumes of chloroform, 40 volumes of methanol, 20 volumes of toluene, 14 volumes of diethylamine and 8 volumes of water. Test solution. Shake a quantity of the finely powdered tablets containing 75 mg of Norfloxacin with 50 ml of a mixture of equal volumes of acidified methanol (containing 0.9 per cent v/v of hydrochloric acid) and dichloromethane, centrifuge and use the clear supernatant solution. Reference solution. A 0.15 per cent w/v solution of norfloxacin RS in the same solvent mixture. Apply to the plate 50 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution. B. In the Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the peak due to norfloxacin in the chromatogram obtained with the reference solution.
Identification
Tests
pH (2.4.24). 4.6 to 5.5. Other tests. Comply with the tests stated under Eye Drops. Assay. Determine by liquid chromatography (2.4.14). Test solution. Dilute a suitable volume of the eye drops with a 0.1 per cent v/v solution of orthophosphoric acid to produce a solution containing 0.005 per cent w/v of Norfloxacin. Reference solution. A 0.005 per cent w/v solution of norfloxacin RS in 0.1 per cent v/v solution of orthophosphoric acid. Chromatographic system a stainless steel column 30 cm x 3.9 mm, packed with octadecylsilyl silica gel (10 m) (such as Bondapack C18), column temerature 50, mobile phase: a mixture of 300 volumes of methanol and 700 volumes of 0.1 per cent v/v orthophosphoric acid, flow rate. 2 ml per minute, spectrophotometer set at 280 nm, a 20 l loop injector. Precondition the column using 0.01 M anhydrous sodium dihydrogen orthophosphate, adjusted to pH 4.0 with orthophosphoric acid, at a flow rate of 0.5 ml per minute for 8 hours. Equilibrate the column with the mobile phase for about 30 minutes before starting the chromatography. Inject the reference solution. The test is not valid unless the tailing factor is not more than 2.0. The relative standard deviation for replicate injections is not more than 2.0 per cent. Inject alternately the test solution and the reference solution. Calculate the content of C16H18FN3O3 in the eye drops. Storage. Store protected from light.
Tests
Dissolution (2.5.2). Apparatus. No 1 Medium. 750 ml of acetate buffer pH 4.0. Speed and time. 50 rpm and 30 minutes. Withdraw a suitable volume of the medium and filter. Measure the absorbance of the filtrate, suitably diluted with acetate buffer pH 4.0, if necessary, at the maximum at about 278 nm (2.4.7). Concomitantly measure the absorbance of a solution of known concentration of norfloxacin RS in the same medium. Calculate the total content of C16H18FN3O3 in the medium. D. Not less than 70 per cent of the stated amount of C16H18FN3O3. Other Tests. Comply with the tests stated under Tablets. Assay. Determine by liquid chromatography (2.4.14). Test solution. Weigh and powder 20 tablets. Add 80 ml of the mobile phase to an accurately weighed quantity of the powdered tablets containing about 100 mg of Norfloxacin, mix with the aid of ultrasound for 10 minutes, dilute with a 0.1 per
842
IP 2007
NORGESTREL AND ETHINYLOESTRADIOL TABLETS
cent v/v solution of phosphoric acid to 200.0 ml and mix. Dilute 10.0 ml of this solution to 25.0 ml with the mobile phase, mix and use the resulting solution after filtration through a filter with porosity of not more than 0.1 m. Reference solution. A 0.02 per cent w/v solution of norfloxacin RS in the mobile phase. Chromatographic system a stainless steel column 30 cm x 3.9 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: 85 volumes of a 0.1 per cent v/v solution of phosphoric acid and 15 volumes of acetonitrile, temperature. column 40 1, after preconditioning with degassed 0.01 M sodium dihydrogen phosphate adjusted to pH 4.0 with phosphoric acid flowing at a rate of 0.5 ml per minute for 8 hours, flow rate. 2 ml per minute, spectrophotometer set at 275 nm, a 20 l loop injector. Inject the test solution and the reference solution. The assay is not valid unless the capacity factor is not less than 2, the column efficiency is not less than 1500 theoretical plates, the tailing factor for the norfloxacin peak is not more than 2.0 and the relative standard deviation for replicate injections is not more than 2.0 per cent. Calculate the content of C16H18FN3O3 in the tablets. Storage. Store protected from light and moisture.
C. Melting range (2.4.21). 205 to 212, but the range between beginning and end of melting does not exceed 4.
Tests
Specific optical rotation (2.4.22). 0.1 to +0.1, determined in a 5.0 per cent w/v solution in chloroform. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 80 volumes of dichloromethane and 20 volumes of ethyl acetate. Test solution. Dissolve 0.2 g of the substance under examination in 10 ml of chloroform. Reference solution (a). A 0.01 per cent w/v solution of the substance under examination in chloroform. Reference solution (b). A 0.004 per cent w/v solution of the substance under examination in chloroform. Apply to the plate 10 l of each solution. After development, dry the plate in air and spray with phosphomolybdic acid solution. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (a) and not more than two such spots are more intense than the spot in the chromatogram obtained with reference solution (b). Sulphated ash. (2.3.18) Not more than 0.3 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105 for 5 hours. Assay. Weigh accurately about 0.1 g, dissolve in sufficient ethanol (95 per cent) to produce 100.0 ml, dilute stepwise with ethanol (95 per cent) to obtain a solution containing 0.001 per cent w/v of Levonorgestrel and measure the absorbance of the resulting solution at the maximum at about 241 nm, (2.4.7). Calculate the content of C21H28O2 from the absorbance obtained with a 0.001 per cent w/v solution of norgestrel RS in ethanol (95 per cent). Storage. Store protected from moisture.
Norgestrel
H3 C H H O H H OH C CH
C21H28O2
Mol. Wt. 312.5
Norgestrel is rac-13-ethyl-17-hydroxy-18,19-dinor-17pregn-4-en-20-yn-3-one. Description. A white or almost white, crystalline powder; practically odourless.
Norgestrel and Ethinyloestradiol Tablets

Norgestrel and Ethinyloestradiol Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amounts of norgestrel, C21H28O 2 and ethinyloestradiol, C20H24O2. The tablets may be film-coated.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with norgestrel RS. B. When examined in the range 220 nm to 360 nm (2.4.7), a 0.001 per cent w/v solution in methanol shows an absorption maximum only at about 240 nm.
Identification
Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G.
843
NORTRIPTYLINE HYDROCHLORIDE
IP 2007
Mobile phase. A mixture of 96 volumes of dichloromethane and 4 volumes of ethanol (95 per cent). Test solution. Powder 20 tablets finely, triturate with 20 ml of dichloromethane, allow the solids to sediment and use the clear supernatant liquid. Reference solution. A solution containing 0.06 per cent w/v solution of norgestrel RS and 0.006 per cent w/v solution of ethinyloestradiol RS. Apply to the plate 40 l of each solution. After development, dry the plate in air, spray with ethanolic sulphuric acid (80 per cent v/v), heat at 110 for 10 minutes and examine in ultraviolet light at 365 nm. The principal spots in the chromatogram obtained with the test solution correspond to the spots for norgestrel (red fluorescence) and ethinyloestradiol (orange-yellow fluorescence) in the chromatogram obtained with the reference solution.
mobile phase: a mixture of 35 volumes of acetonitrile, 15 volumes of methanol and 45 volumes of water, flow rate. 1 to1.5 ml per minute, spectrophotometer set at 215 nm, a 20 l loop injector. Inject the reference solution. The resolution between the two major peaks is not less 2.5 and the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject the test solution and the reference solution. The relative retention times are about 0.7 for ethinyloestradiol and about 1.0 for norgestrel. Calculate the contents of norgestrel, C 21 H 28 O 2 , and ethinyloestradiol, C20H24O2 in the tablets. Storage. Store protected from light.
Tests
Uniformity of content. Comply with the test stated under Tablets. Carry out the procedure described under Assay but using the following test solution. Test solution. Add 2.0 ml of methanol (70 per cent) and 2.0 ml of a 0.00002 per cent w/v solution of diphenyl in methanol (70 per cent) (internal standard solution) to one tablet, shake for 20 minutes, centrifuge, filter the supernatant liquid through a membrane filter with a pore size of not more than 0.2 mm and use the filtrate. Calculate the contents of norgestrel C 21 H 28 O 2 , and ethinyloestradiol, C20H24O2, in the tablet. Other Tests. Comply with the tests stated under Tablets. Assay. Determine by liquid chromatography (2.4.14) using the chromatographic system described under Uniformity of content. Test solution. Weigh and powder 20 tablets. To a quantity of the powder equivalent to one tablet add 2.0 ml of methanol (70 per cent) and 2.0 ml of a 0.00002 per cent w/v solution of diphenyl in methanol (70 per cent) (internal standard solution), shake for 20 minutes, centrifuge, filter the supernatant liquid through a membrane filter with a pore size of not more than 0.2 mm and use the filtrate. Reference solution. A solution in methanol (70 per cent ) containing 0.15 mg per ml of norgestrel RS and 0.015 mg per ml of ethinyloestradiol RS. Take 2.0 ml of this solution and add 2.0 ml of a 0.00002 per cent w/v solution of diphenyl in methanol (70 per cent) and use the resulting solution. Chromatographic system a stainless steel column 15 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 to 7 m),
Nortriptyline Hydrochloride
, HCl N H CH3
C19H21N,HCl
Mol. Wt. 299.8
Nortriptyline Hydrochloride is 3-(10,11-dihydro-5H-dibenzo [a,d]cyclohept-5-ylidene)propyl(methyl)amine hydrochloride. Nortriptyline Hydrochloride contains not less than 98.0 per cent and not more than 101.5 per cent of C19H21N,HCl, calculated on the dried basis. Description. A white to off-white powder; odour slight and characteristic.
Identification
Test A may be omitted if tests B, C and D are carried out. Tests B and C may be omitted if tests A and, D are carried out. A. Dissolve 0.1 g in 10 ml of water, make alkaline with 1 M sodium hydroxide, extract with 5 ml of chloroform and evaporate to dryness using a current of nitrogen. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with nortriptyline hyrdochloride RS treated in the same manner or with the reference spectrum of nortriptyline. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.001 per cent w/v solution in methanol shows an absorption maximum only at about 239 nm; absorbance at about 239 nm, about 0.48.
844
IP 2007
NORTRIPTYLINE TABLETS
C. To about 50 mg dissolved in 3 ml of warm water, add 1 drop of a 2.5 per cent w/v solution of quinhydrone in methanol; a red colour is produced after a few minutes (distinction from amitriptyline). D. Gives the reactions of chlorides (2.3.1).
Identification
A. Shake a quantity of the powdered tablets containing about 5 mg of nortriptyline with 20 ml of methanol and filter. To 1 ml of the filtrate add 1 ml of a 2.5 per cent w/v solution of sodium bicarbonate, 1 ml of a 2 per cent w/v solution of sodium periodate and 1 ml of a 0.3 per cent w/v solution of potassium permanganate. Allow to stand for 15 minutes, acidify with 1 M sulphuric acid and extract with 10 ml of 2,2,4trimethylpentane. When examined in the range 230 nm to 360 nm (2.4.7), the resulting trimethylpentane solution shows an absorption maximum only at about 265 nm. B. Triturate a quantity of the powdered tablets containing 0.1 g of nortriptyline with 10 ml of chloroform, filter and evaporate the filtrate to a low volume. Add ether until a turbidity is produced and allow to stand. Dissolve 50 mg of the precipitate in 3 ml of warm water, cool and add 1 drop of a 2.5 per cent w/v solution of quinhydrone in methanol; a red colour is produced after a few minutes (distinction from amitriptyline).
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 85 volumes of cyclohexane, 15 volumes of ethyl acetate and 3 volumes of diethylamine. Test solution. Dissolve 0.2 g of the substance under examination in 10 ml of ethanol (95 per cent) prepared in subdued light. Reference solution. A 0.001 per cent w/v solution of dibenzosuberone RS in ethanol (95 per cent) prepared in subdued light. Apply to the plate 5 l of each solution. Allow the mobile phase to rise 14 cm in an unsaturated tank protected from light. Dry the plate in air, spray with a freshly prepared solution of sulphuric acid containing 4 per cent v/v of formaldehyde solution and examine immediately in ultraviolet light at 365 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Heavy metals (2.3.13). 2.0 g complies with the limit test for heavy metals, Method B (10 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105 for 3 hours. Assay. Weigh accurately about 0.25 g and dissolve in 25 ml of anhydrous glacial acetic acid, warm slightly, if necessary, to effect solution. Cool, add 5 ml of mercuric acetate solution. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.02998 g of C19H21N,HCl. Storage. Store protected from light and moisture.
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 85 volumes of cyclohexane, 15 volumes of ethyl acetate and 3 volumes of diethylamine. Test solution. Extract a quantity of the powdered tablets containing 20 mg of nortriptyline with 5 ml of a mixture of 9 volumes of ethanol (95 per cent) and 1 volume of 2 M hydrochloric acid, centrifuge and use the supernatant liquid. Reference solution. A 0.001 per cent w/v solution of dibenzosuberone RS in ethanol (95 per cent) prepared in subdued light. Apply to the plate 5 l of each solution. Allow the mobile phase to rise 14 cm in an unsaturated tank protected from light. Dry the plate in air, spray with a freshly prepared solution of sulphuric acid containing 4 per cent v/v of formaldehyde solution and examine immediately in ultraviolet light at 365 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Uniformity of content (For tablets containing 10 mg or less). Comply with the test stated under Tablets. Determine by liquid chromatography (2.4.14) Test solution. Powder one tablet, add 2.5 ml of water, shake vigorously to completely disperse the tablet, add 5 ml of methanol and shake for 30 minutes. Add sufficient water to produce 10 ml, centrifuge and use the clear supernatant liquid.
Nortriptyline Tablets
Nortriptyline HydrochlorideTablets
Nortriptyline Tablets contain less than 90.0 per cent and not more than 110.0 per cent of the stated amount of nortriptyline, C19H21N. The tablets are coated.
845
NOSCAPINE
IP 2007
Reference solution. A 0.01 per cent w/v solution of nortriptyline hydrochloride RS in methanol (50 per cent). Follow the procedure given in the Assay. Calculate the content of C20H23N in the tablet. Other Tests. Comply with the tests stated under Tablets. Assay. Determine by liquid chromatography (2.4.14). Test solution. Shake vigorously 20 tablets with 50 ml of water until the tablets disintegrate completely, add 100.0 ml of methanol and shake for 30 minutes. Add sufficient water to produce 200.0 ml, filter and dilute a volume of the filtrate containing about 25 mg of nortriptyline to 100.0 ml with methanol (50 per cent). Reference solution. A 0.025 per cent w/v solution of nortriptyline hydrochloride RS in methanol (50 per cent). Chromatographic system a stainless steel column 20 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (10 m), mobile phase: a 0.56 per cent w/v solution of sodium hexanesulphonate in a mixture of equal volumes of water and acetonitrile adjusted to pH 4.5 with glacial acetic acid, flow rate. 2 ml per minute, spectrophotometer set at 239 nm, a 20 l loop injector. Inject the test solution and the reference solution. Calculate the content of C19H21N in the tablets. Storage. Store protected from light and moisture.
Noscapine contains not less than 98.5 per cent and not more than 100.5 per cent of C22H23NO7, calculated on the dried basis. Description. Colourless crystals or a white crystalline powder.
Identification
Test A may be omitted if tests B, C and D are carried out. Tests B, C and D may be omitted if test A is carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with noscapine RS or with the reference spectrum of noscapine. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.005 per cent w/v solution in methanol shows absorption maxima at about 291 nm and 310 nm; ratio of absorbance at the maximum at about 310 nm to that at the maximum at about 291 nm, 1.2 to 1.3. C. To 0.1 g in a porcelain dish add a few drops of sulphuric acid and stir; a greenish-yellow solution is formed which on warming becomes red and finally violet. D. Dissolve 50 mg in 5 ml of 5 M hydrochloric acid, add 10 ml of a mixture of equal volumes of ethanol (95 per cent) and a saturated solution of sodium acetate, mix and allow to stand for about 3 minutes; shining crystals separate.
Tests
Appearance of solution. A 2.0 per cent w/v solution in acetone examined immediately after preparation is clear (2.4.1), and not more intensely coloured than reference solution YS6 (2.4.1). Specific optical rotation (2.4.22). +42.0 to +48.0, determined at 20 in a solution prepared by dissolving 0.5 g in sufficient 0.1 M hydrochloric acid to produce 25.0 ml. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G.
Noscapine
Narcotine
O O OCH3 H O H3CO OCH3 O N CH3 H
Mobile phase. A mixture of 60 volumes of acetone, 60 volumes of toluene, 9 volumes of ethanol (95 per cent) and 3 volumes of strong ammonia solution. Test solution. Dissolve 0.25 g of the substance under examination in 10 ml of acetone. Reference solution. A 0.0125 per cent w/v solution of the substance under examination in acetone. Apply to the plate 10 l of each solution. After development, dry the plate in air, spray with dilute potassium iodobismuthate solution. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Morphine. Dissolve 0.1 g in 10 ml of 0.1 M hydrochloric acid. To 1 ml of the resulting solution add a mixture of 1 ml of 846
C22H23NO7
Mol. Wt. 413.4
Noscapine is (3S)-6,7-dimethoxy-3-[(5R)-5,6,7,8-tetrahydro4-methoxy-6-methyl-1,3-dioxolo[4,5-g]isoquinolin-5yl]phthalide, an alkaloid obtained from opium.
IP 2007
NOVOBIOCIN SODIUM
potassium ferricyanide solution, 0.05 ml of ferric chloride test solution and 4 ml of water; no blue or dark green colour develops within 1 minute. Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 1.0 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.35 g, dissolve in 40 ml of anhydrous glacial acetic acid, warming gently. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.04134 g of C22H23NO7. Storage. Store protected from light and moisture.
Assay. Weigh accurately a quantity containing 60 mg of Noscapine, add 20 ml of water, 2 g of sodium chloride and 2 ml of 5 M sodium hydroxide and extract with successive quantities of 50, 50, 25 and 25 ml of ether. Combine the extracts, wash with three quantities, each of 5 ml, of water and evaporate to dryness. To the residue add 50.0 ml of 0.1 M hydrochloric acid, warm on a water-bath to dissolve and to remove any traces of ether and dilute to 100.0 ml with water. Dilute 3.0 ml to 50.0 ml with water and measure the absorbance of the resulting solution at the maximum at about 310 nm (2.4.7). Calculate the content of C22H23NO7 taking 90.7 as the specific absorbance at 310 nm. Determine the weight per ml of the linctus (2.4.29), and calculate the content of C22H23NO7, weight in volume. Storage. Store protected from light and moisture.
Noscapine Linctus
Narcotine Linctus
Noscapine Linctus is a solution of Noscapine in a suitable flavoured vehicle. It may contain up to 1 per cent w/v solution of Citric Acid. Noscapine Linctus contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of noscapine, C22H23NO7.
Novobiocin Sodium
H3CO CH3 O O CH3 O OH O CH3 O O O CH3 CH3 OH
H3 C
N ONa H
C31H35N2NaO11
Mol. Wt. 634.6
Identification
To a quantity containing 60 mg of Noscapine add 20 ml of water, 2 g of sodium chloride and 2 ml of 5 M sodium hydroxide. Extract with successive quantities of 50, 50, 25 and 25 ml of ether. Combine the extracts, wash with three quantities, each of 5 ml, of water and evaporate to dryness. Dissolve the residue in 20 ml of chloroform. Wash with three quantities, each of 20 ml, of water, dry the chloroform layer with anhydrous sodium sulphate, filter and evaporate the solvent. If necessary, induce crystallisation by scratching with a glass rod. The crystals comply with the following tests. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with noscapine RS or with the reference spectrum of noscapine. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.005 per cent w/v solution in methanol shows absorption maxima at about 291 nm and 310 nm; ratio of absorbance at the maximum at about 310 nm to that at the maximum at about 291 nm, 1.2 to 1.3.
Novobiocin Sodium is the monosodium salt of novobiocin, N-[7-{3-O-(aminocarbonyl)-6-deoxy-5-C-methyl-4-O-methyl-L-lyxo-hexopyranosyl}-4-hydroxy-8-methyl-2-oxo-2H-1benzopyran-3-yl]-4-hydroxy-3-(3-methyl-2butenyl)benzamide, an antimicrobial substance produced by the growth of certain strains of Streptomyces niveus or related organisms or by other means. Novobiocin Sodium contains the equivalent of not less than 850 g of novobiocin per mg, calculated on the dried basis. Description. A white or yellowish white, crystalline powder.
Identification
A. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 75 volumes of chloroform, 25 volumes of methanol and 1 volume of strong ammonia solution. Test solution. Dissolve a quantity of the substance under examination in methanol so as to obtain a solution containing 0.1 per cent w/v solution of novobiocin. Reference solution. A 0.1 per cent w/v solution of novobiocin RS in methanol.
Tests
Other tests. Complies with the tests stated under Oral Liquids.
847
NYSTATIN
IP 2007
Apply to the plate 1 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.001 per cent w/v solution in a 0.4 per cent w/v solution of potassium hydroxide shows an absorption maximum only at about 307 nm. C. The residue obtained by igniting it gives the tests for sodium salts (2.3.1).
Identification
A. When examined in the range 220 nm to 360 nm (2.4.7), the final solution obtained in the test for Light absorption shows absorption maxima at about 230 nm, 291 nm, 305 nm and 319 nm. The ratios of the absorbances at the maxima at about 291 nm and about 319 nm to the absorbance at the maximum at about 305 nm are 0.61 to 0.73 and 0.83 to 0.96, respectively. Use as the blank a solution prepared in the same manner without the substance under examination. B. Shake 30 mg with 5 ml of water for 2 minutes, add 2 ml of sodium molybdotungstophosphate solution, and allow to stand for 1 hour; the green colour produced is darker than that produced by repeating the test without the substance under examination. C. Shake 30 mg with 5 ml of water for 2 minutes, add 2 ml of a solution prepared by dissolving 0.1 g of pyrogallol in 100 ml of decolorised magenta solution, heat on a water-bath until a dark pink colour is produced, cool and allow to stand for 1 hour; the pink colour is retained. D. To 2 mg add 0.1 ml of hydrochloric acid; a brown colour is produced. E. To 2 mg add 0.1 ml of sulphuric acid; a brown colour is produced which becomes violet on standing.
Tests
pH (2.4.24). 6.6 to 8.5, determined in a 2.5 per cent w/v solution. Specific optical rotation (2.4.22). 50.0 to 58.0, determined in a 5.0 per cent w/v solution in methanol containing 1 per cent v/v of hydrochloric acid. Loss on drying (2.4.19). Not more than 6 per cent, determined on 0.2 g by drying in an oven at 60 over phosphorus pentoxide at a pressure not exceeding 0.7 kPa for 3 hours. Assay. Determine by the microbiological assay of antibiotics, Method A (2.2.10), and express the results in g of novobiocin per mg. Novobiocin Sodium intended for use in the manufacture of Parenteral Preparations complies with the following additional requirements. Bacterial endotoxins (2.2.3). Not more than 0.7 Endotoxin units per mg. Sterility. Complies with the test for sterility (2.2.11). Storage. Store protected from light and moisture at a temperature not exceeding 30. If it is intended for use in the manufacture of parenteral preparations, the container should be sterile and sealed so as to exclude micro-organisms. Labelling. The label states whether or not the contents are intended for use in the manufacture of parenteral preparations.
Tests
pH (2.4.24). 6.5 to 8.0, determined in a 3.0 per cent w/v suspension in water. Light absorption (2.4.7). Dissolve 0.1 g in a mixture of 5.0 ml of glacial acetic acid and 50 ml of methanol, add sufficient methanol to produce 100.0 ml and dilute 1.0 ml of the resulting solution to 100.0 ml with methanol. Absorbance of the resulting solution, measured within 30 minutes of preparation, at the maximum at about 305 nm, not less than 0.60. Use as the blank a solution prepared in the same manner without the substance under examination. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm).
Nystatin
Nystatin is an antifungal substance produced by the growth of certain strains of Streptomyces noursei or by any other means. It consists mainly of polyenes, the principal component being nystatin A1. Nystatin has a potency of not less than 4400 Units per mg, calculated on the dried basis. Description. A yellow to slightly brown powder; odour, characteristic; hygroscopic.
Sulphated ash (2.3.18). Not more than 3.5 per cent. Loss on drying (2.4.19). Not more than 5.0 per cent, determined on 1.0 g by drying in an oven at 60 at a pressure not exceeding 0.1 kPa for 3 hours. Assay. Protect the solution from light throughout the assay. Weigh accurately about 75 mg and dissolve in sufficient dimethylformamide to produce 50.0 ml; dilute 10.0 ml of the resulting solution to 200.0 ml with buffer solution No 4 (2.2.10). Determine by the microbiological assay of antibiotics (2.2.10).
848
IP 2007
NYSTATIN TABLETS
Nystatin intended for oral administration complies with the following additional requirement. Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity, using a quantity containing not less than 600 Units suspended in not more than 0.5 ml of a 0.5 per cent w/v solution of acacia and injecting the suspension intraperitoneally. Storage. Store protected from light and moisture. Labelling. The label states the strength in terms of the number of Units of Nystatin per mg.
Nystatin Pessaries
Nystatin Vaginal Tablets
Nystatin Pessaries contain not less than 90.0 per cent and not more than 130.0 per cent of the stated number of Units of nystatin.
Identification
Extract a quantity of the powdered pessaries containing 300,000 Units with a mixture of 50 ml of methanol and 5 ml of glacial acetic acid, add sufficient methanol to produce 100 ml and filter. Dilute 1 ml of the filtrate to 100 ml with methanol. The resulting solution complies with the follwing test. Disperse a quantity containing 25,000 Units in 10 ml of chloroform, add 40 ml of methanol and shake. Filter and dilute 1 ml of the filtrate to 25 ml with methanol. When examined in the range 230 nm to 360 nm (2.4.7), the resulting solution shows absorption maxima at about 291 nm, 305 nm and 319 nm. The ratios of the absorbances at the maxima at about 291 nm and about 319 nm to the absorbance at the maximum at about 305 nm are 0.61 to 0.73 and 0.83 to 0.96, respectively. Use as the blank a solution prepared exactly in the same manner without the substance under examination.
Nystatin Ointment
Nystatin Ointment is a dispersion of Nystatin in microfine powder in a suitable ointment basis. Nystatin Ointment contains not less than 90.0 per cent and not more than 130.0 per cent of the stated number of Units of nystatin.
Identification
Disperse a quantity containing 25,000 Units in 10 ml of chloroform, add 40 ml of methanol and shake. Filter and dilute 1 ml of the filtrate to 25 ml with methanol. When examined in the range 230 nm to 360 nm (2.4.7), the resulting solution shows absorption maxima at about 291 nm, 305 nm and 319 nm. The ratios of the absorbances at the maxima at about 291 nm and about 319 nm to the absorbance at the maximum at about 305 nm are 0.61 to 0.73 and 0.83 to 0.96, respectively. Use as the blank a solution prepared exactly in the same manner without the substance under examination.
Tests
Other Tests. Comply with the tests stated under Pessaries. Loss on drying (2.4.19). Not more than 5 per cent, determined on 1.0 g of the powdered pessaries by drying in an oven at 60 at a pressure not exceeding 0.7 kPa for 3 hours. Assay. Protect the solution from light throughout the assay. Weigh and powder 20 pessaries. Weigh accurately a quantity of the powder containing 200,000 Units and shake with 50.0 ml of dimethylformamide for 1 hour. Centrifuge, dilute 10.0 ml of the clear, supernatant liquid to 200.0 ml with buffer solution No 4 (2.2.10). Determine by the microbiological assay of antibiotics (2.2.10). Storage. Store protected from light and moisture. Labelling. The label states the strength in terms of the number of Units of Nystatin.
Tests
Other tests. Complies with the tests stated under Ointments. Assay. Protect the solution from light throughout the assay. Weigh accurately a quantity containing 400,000 Units, disperse in 20 ml of ether in a stoppered flask, add 70 ml of dimethylformamide, shake for a few minutes, add 10 ml of water, shake vigorously for a few minutes and add sufficient dimethylformamide to produce 100.0 ml. Mix well, filter and dilute 10.0 ml of the filtrate to 100.0 ml with buffer solution No 4 (2.2.10). Determine by the microbiological assay of antibiotics (2.2.10). Storage. Store protected from moisture. Labelling. The label states the strength in terms of the number of Units of Nystatin per g.
Nystatin Tablets
Nystatin Tablets contain not less than 90.0 per cent and not more than 130.0 per cent of the stated number of Units of nystatin. The tablets are coated.
849
NYSTATIN TABLETS
IP 2007
Identification
Extract a quantity of the powdered tablets containing 300,000 Units with a mixture of 50 ml of methanol and 5 ml of glacial acetic acid, add sufficient methanol to produce 100 ml and filter. Dilute 1 ml of the filtrate to 100 ml with methanol. The resulting solution complies with the follwing test. When examined in the range 230 nm to 360 nm (2.4.7), the solution shows absorption maxima at about 291 nm, 305 nm and 319 nm. The ratios of the absorbances at the maxima at about 291 nm and about 319 nm to the absorbance at the maximum at about 305 nm are 0.61 to 0.73 and 0.83 to 0.96, respectively. Use as the blank a solution prepared exactly in the same manner without the substance under examination.
tablets fail to disintegrate, wash them rapidly by immersion in water and continue the test using phosphate buffer pH 6.8; the tablets then disintegrate within a further 30 minutes. Loss on drying (2.4.19). Not more than 5.0 per cent, determined on 1.0 g of the powdered tablets by drying in an oven at 60 at a pressure not exceeding 0.7 kPa for 3 hours. Other Tests. Comply with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing 200,000 Units and shake with 50.0 ml of dimethylformamide for 1 hour. Centrifuge, dilute 10.0 ml of the clear, supernatant liquid to 200.0 ml with buffer solution No 4 (2.2.10). Determine by the microbiological assay of antibiotics (2.2.10). Storage. Store protected from moisture. Labelling. The label states the strength in terms of the number of Units of Nystatin.
Tests
Disintegration (2.5.1). 30 minutes, but using a 0.6 per cent v/v solution of hydrochloric acid in place of water. If the
850
MONOGRAPHS
O
Oestradiol Benzoate Oestradiol Injection Ofloxacin Ofloxacin Infusion Ofloxacin Opthalmic Solution Ofloxacin Tablets Olanzapine Olanzapine Tablets Oleic Acid Omeprazole Omeprazole Capsules Oral Rehydration Salts Ormeloxifen Hydrochloride Ormeloxifen Hydrochloride Tablets Orphenadrine Citrate Orphenadrine Hydrochloride Orphenadrine Tablets Oseltamivir Phosphate Oseltamivir Capsules Oseltamivir Oral Suspension Oxazepam Oxazepam Tablets Oxprenolol Hydrochloride Oxprenolol Tablets Oxygen Oxygen 93 Per Cent Oxyphenbutazone Oxyphenbutazone Tablets Oxytetracycline Dihydrate Oxytetracycline Injection
851
.... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... ....
MONOGRAPHS
Oxytetracycline Hydrochloride Oxytetracycline Capsules Oxytetracycline Eye Ointment Oxytetracycline Hydrochloride Injection Oxytocin Oxytocin Injection Oxytocin Nasal Solution
.... .... .... .... .... .... ....
852
IP 2007
OESTRADIOL INJECTION
Oestradiol Benzoate
H3C H O O H H OH
Test solution (b). Dissolve 0.1 g of the substance under examination in 100 ml of the same solvent mixture. Reference solution (a). A 0.02 per cent w/v solution of the substance under examination in the same solvent mixture. Reference solution (b). A 0.1 per cent w/v solution of oestradiol benzoate RS in the same solvent mixture. Apply to the plate 5 l of each solution. After development, dry the plate in air until the odour of the solvent is no longer detectable, heat at 110 for 10 minutes, spray the plate while hot with ethanolic sulphuric acid (20 per cent), heat again at 110 for 10 minutes and examine in ultraviolet light at 365 nm. Any secondary spot in the chromatogram obtained with test solution (a) is not more intense than the spot in the chromatogram obtained with reference solution (a). Sulphated ash (2.3.18). Not more than 0.2 per cent, determined on 0.5 g. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 0.5 g by drying in an oven at 105 for 3 hours. Assay. Weigh accurately about 25 mg, dissolve in sufficient ethanol (95 per cent) to produce 250.0 ml. Dilute 10.0 ml to 100.0 ml with ethanol (95 per cent) and measure the absorbance of the resulting solution at the maximum at about 231 nm (2.4.7). Calculate the content of C25H28O3 taking 500 as the specific absorbance at 231 nm. Storage. Store protected from light and moisture.
C25H28O3
Mol. Wt. 376.5
Oestradiol Benzoate is 17-hydroxyestra-1,3,5(10)-trien-3-yl benzoate. Oestradiol Benzoate contains not less than 97.0 per cent and not more than 103.0 per cent of C25H28O3, calculated on the dried basis. Description. Colourless crystals or a white, crystalline powder; odourless.
Identification
Test A may be omitted if tests B and C are carried out. Test C may be omitted if tests A and B are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with oestradiol benzoate RS or with the reference spectrum of oestradiol benzoate. B. In the test for Related substances, the principal spot in the chromatogram obtained with test solution (b) corresponds to that in the chromatogram obtained with reference solution (b) when examined in daylight and in ultraviolet light at 365 nm. C. To about 1 mg add 0.5 ml of a 5 per cent w/v solution of ammonium molybdate in sulphuric acid; a yellowish green colour develops which exhibits an intense green fluorescence when examined in ultraviolet light at 365 nm. Add 1 ml of sulphuric acid and 9 ml of water; the solution becomes pink with a yellowish fluorescence.
Oestradiol Injection
Oestradiol Benzoate Injection
Oestradiol Injection is a sterile solution of Oestradiol Benzoate in Ethyl Oleate or other suitable ester, in a suitable fixed oil or in any mixture of these. It may contain suitable alcohols. Oestradiol Benzoate Injection contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of oestradiol benzoate, C25H28O3.
Tests
Specific optical rotation (2.4.22). +57.0 to +63.0, determined in a 1.0 per cent w/v solution in dioxan. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 90 volumes of toluene and 10 volumes of ethanol (95 per cent). Test solution (a). Dissolve 0.2 g of the substance under examination in 10 ml of a mixture of 90 volumes of chloroform and 10 volumes of methanol.
Identification
Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 80 volumes of toluene and 20 volumes of ethyl acetate. Test solution. Add 10 ml of 2,2,4-trimethylpentane to a volume of the injection containing 2 mg of Oestradiol Benzoate and extract with three quantities, each of 10 ml, of ethanol (70 per cent). Wash the combined extracts with 15 ml of 2,2,4trimethylpentane, evaporate the ethanolic extract to dryness
853
OFLOXACIN
IP 2007
using a rotary evaporator and dissolve the residue in 2 ml of chloroform. Reference solution. A 0.1 per cent w/v solution of oestradiol benzoate RS in chloroform. Apply to the plate 5 l of each solution. After development, dry the plate in air, spray with ethanolic sulphuric acid (20 per cent), heat at 105 for 10 minutes and examine in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution.
Ofloxacin
H 3C N N F O O N COOH CH3
C18H20FN3O4
Mol. Wt. 361.4
Tests
Other Tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. Determine by liquid chromatography (2.4.14). Test solution (a). Dilute an accurately measured quantity of the injection containing about 1 mg of Oestradiol Benzoate to 10.0 ml with a mixture of 90 volumes of cyclohexane and 10 volumes of dioxan. Test solution (b). Add 1 ml of a solution prepared by dissolving 15 mg of 4-hydroxybenzaldehyde (internal standard) in 10.0 ml of dioxan, adding sufficient cyclohexane to produce 100.0 ml (solution A), to an accurately measured quantity of the injection containing about 1 mg of Oestradiol Benzoate and dilute to 10.0 ml with sufficient of a mixture of 90 volumes of cyclohexane and 10 volumes of dioxan. Reference solution. Add 10 ml of solution A to 10 mg of oestradiol benzoate RS, accurately weighed, and dilute to 100.0 ml with the same solvent mixture. Chromatographic system a stainless steel column 30 cm x 4 mm, packed with porous silica particles (10 m), mobile phase: 90 volumes of cyclohexane and 10 volumes of dioxan, flow rate. 2 ml per minute, spectrophotometer set at 254 nm, a 20 l loop injector. Inject test solutions (a), (b) and the reference solution. The assay is not valid unless the resolution between the peaks due to benzyl alcohol (if present) and oestradiol benzoate and between the peaks due to oestradiol benzoate and the internal standard is more than 1.5. Calculate the content of C25H28O3 in the injection. Storage. Store protected from light. Labelling. The label states (1) the nature and composition of the solvent; (2) that it is meant for intramuscular injection only; (3) that any solid matter that may have separated on standing should be redissolved by warming before use.
Ofloxacin is (RS)-9-fluoro-3-methyl-10-(4-methylpiperazin-1yl)-7-oxo-2,3-dihydro-7H-pyrido[1,2,3,-de]-1,4-benzoazeine6-carboxylic acid Ofloxacin contains not less than 98.5 per cent and not more than of 101.5 per cent of C18H20FN3O4, calculated on the dried basis. Description. A pale yellow or bright yellow, crystalline powder.
Identification
Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with ofloxacin RS.
Tests
Light absorption. Absorbance at 440 nm (2.4.7), of 0.5 per cent w/v solution in 0.1 M hydrochloric acid is not more than 0.25. Related substances. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 10 mg of the substance under examination in 10 ml of methanol. Reference solution (a). A 0.1 per cent w/v solution of ofloxacin RS in methanol. Reference solution (b). Dilute 1 ml of reference solution (a) to 100 ml with methanol. Chromatographic system a stainless steel column 10 cm x 4.6 mm packed with octadecylsilyl silica gel for chromatography (5 m), mobile phase: a mixture of 10 volumes of acetonitrile and 90 volumes of phosphate buffer pH 2.4 prepared by dissolving 27.2 g of monobasic potassium phosphate in 1000 ml of water, adjust the pH to 2.4 with orthophosphoric acid, flow rate. 2 ml per minute, spectrophotometer set at 294 nm, a 10 l loop injector. Inject reference solution (b). The test is not valid unless the column efficiency in not less than 1400 theoretical plates. Inject the test solution and reference solution (b). Run the chromatogram three times of the principal peak. In the
854
IP 2007
OFLOXACIN OPHTHALMIC SOLUTION
chromatogram obtained with the test solution, the area of any secondary peak is not more than 0.5 times the area of the peak in the chromatogram obtained with reference solution (b) (0.5 per cent) and the sum of areas of all the secondary peaks is not more than the area of the peak in the chromatogram obtained with the reference solution (b) (1.0 per cent). Heavy Metals (2.3.13). 2.0 g complies with the limit test for heavy metals, Method C (10 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.2 per cent, determined on 1.0 g by drying in an oven at 105 for 4 hours. Assay. Weigh accurately about 0.3 g, dissolve in 100 ml anhydrous glacial acetic acid. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.4.25). Carry out a blank titration. 1 ml 0.1 M perchloric acid is equivalent of 0.03614 g of C18H20FN3O4. Storage. Store protected from light and moisture.
mobile phase: a mixture of 80 volumes of buffer solution prepared by dissolving 6.8 g of potassium dihydrogen orthophosphate and 0.47 g sodium 1-hexane sulphonate in 1000 ml of water, add 1 ml of triethylamine and adjust the pH to 3.0 with orthophosphoric acid and 20 volumes of acetonitrile, flow rate. 1 ml per minute, spectrophotometer set at 294 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the tailing factor is not more than 2.0 and the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject the test solution and the reference solution. Calculate the content of C18H20FN3O4 in infusion.
Ofloxacin Ophthalmic Solution

Ofloxacin Ophthalmic Solution is a sterile aqueous solution of Ofloxacin. Ofloxacin Ophthalmic Solution contain not less than 90.0 per cent and more than 110.0 per cent of the stated amount of ofloxacin, C18H20FN3O4.
Ofloxacin Infusion
Ofloxacin Infusion contain Ofloxacin in water for injection. Ofloxacin Infusion contain not less than 90.0 per cent and not more than 120.0 per cent of the stated amount of ofloxacin, C18H20FN3O4.
Identification
Identification
Tests
pH (2.4.24). 6.0 to 7.2. Sterility (2.2.11). Comply with the test for sterility. Assay. Determine by liquid chromatography (2.4.14). Solvent mixture. Phosphate buffer pH 7.25 prepared by dissolving 3.56 g of disodium hydrogen phosphate in 1000 ml water, adjusted pH to 7.25 using orthophosphoric acid or sodium hydroxide solution. Test solution. Measure accurately a volume of Opthalmic Solution containing 30 mg of Ofloxacin in 100.0 ml of solvent mixture. Dilute 1.0 ml of the solution to 10.0 with solvent mixture. Reference solution. A 0.003 per cent w/v solution of ofloxacin RS in solvent mixture. Chromatographic system a stainless steel column 15 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5 m) (such as TSK GEL), mobile phase: a mixture of 20 volumes of acetonitrile, 80 volumes of phosphate buffer pH 7.25 prepared by
Tests
pH (2.4.24). 3.8 to 5.8 Other tests. Complies with the tests stated under Parenteral Preparation (Infusions). Assay. Determine by liquid chromatography (2.4.14). NOTE Protect the solutions from the light. Test solution. Measure accurately a volume containing 50 mg of Ofloxacin in 100.0 ml with mobile phase. Dilute 5.0 ml of the solution to 50.0 ml with mobile phase. Reference solution. A 0.005 per cent w/v solution of ofloxacin RS in mobile phase. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5 m),
855
OFLOXACIN TABLETS
IP 2007
dissolving 2.54 g of tetrabutyl ammonium hydrogen sulphate and 3.56 g of disodium hydrogen phosphate in 1000 ml water, flow rate. 1.5 ml per minute, spectrophotometer set at 254 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject the test solution and the reference solution. Calculate the content of C18H20FN3O4. Storage. Store protected from light.
Reference solution (b). Dilute 1 ml of reference solution (a) to 100 ml with methanol. Chromatographic system a stainless steel column 10 cm x 4.6 mm packed with octadecylsilyl silica gel for chromatography (5 m), mobile phase: a mixture of 10 volumes of acetonitrile and 90 volumes of phosphate buffer pH 2.4 prepared by dissolving 27.2 g of monobasic potassium phosphate in 1000 ml of water, adjust the pH to 2.4 with orthophosphoric acid, flow rate. 2 ml per minute, spectrophotometer set at 294 nm, a 10 l loop injector. Inject reference solution (a). The test is not valid unless the column efficiency in not less than 1400 theoretical plates.
Ofloxacin Tablets
Ofloxacin Tablets contain Ofloxacin. Ofloxacin Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of ofloxacin, C18H20FN3O4.
Identification
In the Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the peak in the chromatogram obtained with the reference solution (a).
Inject the test solution and reference solution (b). Run the chromatogram three times of the principal peak. In the chromatogram obtained with the test solution, the area of any secondary peak is not more than the area of the peak in the chromatogram obtained with reference solution (b) (1.0 per cent) and the sum of areas of all the secondary peaks is not more than twice the area of the peak in the chromatogram obtained with the reference solution (b) (2.0 per cent). Other tests. Comply with the tests stated under the Tablets. Assay. Determine by liquid chromatography (2.4.14). Test solution. Weigh and powder 20 tablets. Weigh accurately a quantity of powdered tablet containing 25 mg of Ofloxacin, disperse in 60 ml of methanol and dilute to 100 ml with methanol and filter. Reference solution. A 0.025 per cent w/v solution of ofloxacin RS in methanol. Chromatographic system a stainless steel column 15 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 92 volumes of buffer solution prepared by dissolving 27.2 g of potassium dihydrogen phosphate in 1000 ml of water and adjust the pH to 2.4 with orthophosphoric acid and 8 volumes of acetonitrile, flow rate. 2 ml per minute, spectrophotometer set at 294 nm, a 10 l loop injector. Inject the reference solution. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject the test solution and the reference solution. Calculate the content of C18H20FN3O4. Storage. Store protected from light and moisture.
Tests
Dissolution (2.5.2). Apparatus. No 1 Medium. 900 ml of 0.1 M hydrochloride acid. Speed and time. 50 rpm for 30 minutes. Withdraw a suitable volume of the medium and filter. Measure the absorbance of the filtrate, suitably diluted with the medium if necessary, at the maximum at about 294 nm (2.4.7). Calculate the content of C18H20FN3O4 in the medium from the absorbance obtained from a solution of known concentration of ofloxacin RS in the same medium. D. Not less than 75 per cent of the stated amount of C18H20FN3O4. Related substances. Determine by liquid chromatography (2.4.14). Test solution. Weigh and powder 20 tablets. Weigh accurately a quantity of powdered tablet containing 100 mg of Ofloxacin, disperse in 60 ml of methanol and dilute to 100 ml with methanol and filter. Reference solution (a). A 0.1 per cent w/v solution of ofloxacin RS in methanol.
856
IP 2007
OLANZAPINE TABLETS
Olanzapine
CH3 N N N N H
C17H20N4S
CH3
spectrophotometer set at 230 nm, a 10 l loop injector. Time Mobile phase A Mobile phase B (in min.) ( per cent v/v) ( per cent v/v) 0 100 0 20 63 37 30 45 55 32 100 0 38 100 0 Inject reference solution (b). The test is not valid unless the tailing factor is not more than 2.0 and the column efficiency in not less than 2000 theoretical plates. Inject the test solution and reference solution (b). Run the chromatogram for three times, the principal peak due to olanzapine. In the chromatogram obtained with the test solution, the area of any secondary peak is not more than 0.5 times the area of the peak in the chromatogram obtained with reference solution (b) (0.5 per cent) and the sum of areas of all the secondary peaks is not more than twice the area of the peak in the chromatogram obtained with the reference solution (b) (2.0 per cent). Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Water (2.3.43). Not more than 1.0 per cent, determined on 1.0 g. Assay. Weigh accurately about 0.2 g, dissolve in 40 ml of glacial acetic acid. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.01562 g of C17H20N4S. Storage. Store protected from light and moisture, at a temperature not exceeding 30.
Mol. Wt. 312.4
Olanzapine is 2-methyl-4-(4-methyl-1-piperazinyl)-10Hthieno[2,3-b][1,5]benzodiazepine Olanzapine contains not less than 98.0 per cent and not more than 102.0 per cent of C17H20N4S, calculated on the anhydrous basis. Description. A yellow crystalline powder.
Identification
Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with olanzapine RS or with the reference spectrum of olanzapine.
Tests
Related substances. Determine by liquid chromatography (2.4.14). Solvent mixture. 40 volumes of water and 60 volumes of acetonitrile. Test solution. Dissolve 50 mg of the substance under examination in 25 ml of solvent mixture. Reference solution (a). A 0.2 per cent w/v solution of olanzapine RS in solvent mixture. Reference solution (b). Dilute 1 ml of reference solution (a) to 100 ml with solvent mixture. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5 m), column temperature 40, mobile phase: A. a mixture of 80 volumes of buffer solution pH 6.8 prepared by dissolving 4.825 g of sodium dihydrogen orthophosphate monohydrate in 1000 ml of water, adjust pH to 6.8 with 10 per cent w/v of sodium hydroxide and 20 volumes of acetonitrile, B. acetonitrile, flow rate. 1.2 ml per minute,
Olanzapine Tablets
Olanzapine Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of olanzapine, C17H20N4S.
Identification
857
OLEIC ACID
IP 2007
Tests
Dissolution (2.5.2). Apparatus. No 1 Medium. 900 ml of 0.01 M hydrochloric acid. Speed and time. 50 rpm for 45 minutes. Withdraw a suitable volume of the medium and filter. Determine by liquid chromatography (2.4.14). NOTE Protect all the solutions from light. Test solution. Use the filtrate, if necessary dilute with dissolution medium. Reference solution. Weigh 16 mg of olanzapine RS, dissolve in about 2.5 ml of acetonitrile and dilute to 25 ml with 0.01 M hydrochloroc acid.Dilute suitabely to get 0.0016 per cent w/v in dissolution medium. Chromatographic system as described under Assay, using 50 l loop injector. Inject the reference solution . The test is not valid unless the tailing factor is not more than 2.0. The column efficiency in not less than 2500 theoretical plates. Inject the test solution and the reference solution. Calculate the content of C17H20N4S. D. Not less than 70 per cent of the stated amount of C17H20N4S. Other tests. Comply with the tests stated under Tablets. Assay. Determine by liquid chromatography (2.4.14). Test solution. Take 10 intact tablets to a suitable volumetric flask, disperse in acetonitrile and dilute with 0.01 M hydrochloroc acid to get final concentration of 0.01 per cent w/v of Olanzapine. Reference solution. Weigh 10 mg of olanzapine RS, dissolve in about 25 ml of acetonitrile and dilute to 100 ml with 0.01 M hydrochloroc acid. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with packed with octadecylsilyl silica gel for chromatography (5 m), mobile phase: a mixture of 70 volumes of buffer solution prepared by dissolving 3 g of ammonium dihydrogen orthophosphate in 900 ml water, add 2 ml of triethylamine and dilute to 1000 ml with water. Adjust pH to 2.5 with orthophosphoric acid and 30 volumes of methanol, flow rate. 1 ml per minute, spectrophotometer set at 220 nm, a 10 l loop injector. Inject the reference solution. The test is not valid unless the tailing factor is not more than 2.0, the column efficiency in not
less than 2500 theoretical plates. The relative standard deviation for replicate injections is not more than 2 per cent. Inject the test solution and the reference solution. Calculate the content of C17H20N4S. Storage. Store protected from light and moisture, at a temperature not exceeding 25.
Oleic Acid
Oleic Acid consists mainly of (Z)-octadec-9-enoic acid, C18H34O2, together with varying amounts of saturated and other unsaturated fatty acids and is obtained by the hydrolysis of fats or fixed oils and separation of the liquid acids by expression or other suitable means. It may contain a suitable antioxidant. Description. A clear, yellowish to pale brown, oily liquid; odour, characteristic. On exposure to air it darkens in colour and the odour becomes more pronounced.
Identification
A.To 1 ml add 1 ml of ethanol (95 per cent); the solution is clear. It turns orange or red on addition of 0.1 ml of methyl orange solution. B.Take a mixture of 1 ml of nitric acid and 1 ml of water in a test-tube with an internal diameter of about 12.5 mm and add 1 ml of the substance under examination on the surface of the mixture. Introduce 0.5 g of copper turnings into the lower layer and allow to stand under a hood for 4 hours; the upper layer solidifies. C.Complies with the test for Iodine value (2.3.28).
Tests
Weight per ml (2.4.29). 0.889 g to 0.895 g. Peroxide value (2.3.35). Not more than 10.0. Acid value (2.3.23). 195 to 202, determined on 0.5 g. Iodine value (2.3.28). 85 to 95, determined by Method A. Water-soluble acids. Shake 5 ml with 5 ml of water for 2 minutes, allow the liquids to separate and filter the aqueous layer through paper moistened with water. To the filtrate add 0.05 ml of methyl orange solution; the liquid does not become red. Neutral fats and mineral oils. Boil 1 ml with 5 ml of 0.5 M sodium carbonate and 25 ml of water in a large flask. The solution, while still hot, is not more opalescent than opalescence standard OS2 (2.4.1).
858
IP 2007
OMEPRAZOLE
Congealing point. Dry about 10 ml by heating to 110 with frequent stirring, transfer to a test-tube about 20 mm in diameter, cool and when at 15 immerse the tube in a suitable water-bath so that the cooling takes place at the rate of 2 per minute. Stir the sample with a thermometer; it does not become cloudy until the temperature has fallen to 10. On further cooling it congeals to a white solid or semi-solid mass at about 4. Sulphated ash (2.3.18). Not more than 0.1 per cent. Storage. Store protected from light and moisture in a refrigerator (8 to 15). Labelling. The label states (1) where applicable, that it is used for external use only; (2) the name and concentration of any added antioxidant.
Tests
Appearance of solution. A 2.0 per cent w/v solution in dichloromethane is clear (2.4.1). Light absorption (2.4.7). Absorbances of a freshly prepared 2.0 per cent w/v solution in dichloromethane at 400 nm, 500 nm and 600 nm are not more than 0.25, 0.10 and 0.10 respectively. Related substances. A. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 50 volumes of benzene, 30 volumes of ethyl acetate and 10 volumes of methanol. Test solution. Dissolve 0.4 g of the substance under examination in 100 ml of ethanol. Reference solution (a). A 0.4 per cent w/v solution of omeprazole RS in ethanol.
Omeprazole
H N H3CO
C17H19N3O3S
Reference solution (b). A 0.004 per cent w/v solution of omeprazole RS in ethanol.
O S
CH3 OCH3 CH3

Mol. Wt. 345.4
Reference solution (c). A 0.002 per cent w/v solution of omeprazole RS in ethanol. Apply to the plate 10 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm immediately. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (c) and the total intensity of all such spots in the chromatogram obtained with the test solution is not more than the intensity of the spot obtained with reference solution (b). B. In the Assay, the sum of the areas of all the secondary peaks is not greater than 1.5 per cent of the total area of all peaks. Heavy metals (2.3.13). The residue obtained in the test for Sulphated ash, complies with the limit test for heavy metals, Method B (20 ppm). Sulphated ash (2.3.18). Not more than 0.2 per cent. Loss on drying (2.4.19). Not more than 0.2 per cent, determined on 1.0 g by drying in an oven at 60 at a pressure not exceeding 0.7 kPa for 4 hours. Assay. Determine by liquid chromatography (2.4.14). Test solution. A 0.005 per cent w/v solution of the substance under examination in the mobile phase. Reference solution. A 0.005 per cent w/v solution of omeprazole RS in the mobile phase. Chromatographic system a stainless steel column 30 cm x 3.9 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 65 volumes of phosphate buffer pH 7.4 and 35 volumes of acetonitrile,
Omeprazole is 5-methoxy-2-[[(4-methoxy-3,5dimethylpyridin-2-yl)methyl]sulfinyl] -1H-benzimidazole. Omeprazole contains not less than 99.0 per cent and not more than 101.0 per cent of C17H19N3O3S, calculated on the dried basis. NOTE Perform the tests and assay in subdued light and use low-actinic glassware. Description. A white or almost white powder.
Identification
Test A may be omitted if tests B and C are carried out. Tests B and C may be omitted if test A is carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with omeprazole RS or with the reference spectrum of omeprazole. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.002 per cent w/v solution in 0.1 M sodium hydroxide shows absorption maxima at about 276 nm and 305 nm; the ratio of the absorbance at about 305 nm to that at about 276 nm, 1.6 to 1.8. C. In the test for Related substances, the principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with reference solution (a).
859
OMEPRAZOLE CAPSULES
IP 2007
flow rate. 1 ml per minute, spectrophotometer set at 302 nm, a 20 l loop injector. Inject the reference solution. Repeat the procedure at least five times and measure the peak responses of the peak due to omeprazole. The relative standard deviation of the replicate injections is not more than 2.0 per cent. Calculate the content of C17H19N3O3S. Storage. Store protected from light and moisture in a refrigerator (2 to 8). NOTE A combination of elevated temperatures (37-50) and high humidity degrades Omeprazole. It rapidly degrades under acidic conditions.
for 2 hours, drain the solution slowly without losing any granules. Transfer them quantitatively to a 100-ml volumetric flask, add 20 ml of 0.1 M sodium hydroxide and mix with the aid of ultrasound. Dilute to volume with 0.1 M sodium hydroxide, centrifuge about 15 ml for 5 minutes and dilute 5.0 ml of the clear supernatant liquid to 50.0 ml with the mobile phase. Using the resulting solution as the test solution, carry out the determination as described in the Assay. Calculate the content of C17H19N3O3S in the supernatant liquid. Calculate the percentage of omeprazole released in the acid medium by subtracting the content of C17H19N3O3S in the test solution from the total content of omeprazole determined in the Assay. Not more than 15 per cent of the stated amount of C17H19N3O3S is dissolved in 2 hours. B. Apparatus. No 1
Omeprazole Capsules
Omeprazole Capsules contain enteric-coated granules of Omeprazole. Omeprazole Capsules contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of omeprazole, C17H19N3O3S. NOTE Perform the tests and assay in subdued light and use low-actinic glassware.
Medium. 900 ml of phosphate buffer pH 6.8, Speed and time. 100 rpm and 45 minutes. Tap the granules from a capsule slightly with a glass rod to make them settle to the bottom. Rotate the paddle at 100 rpm for 45 minutes and filter the solution. Using the filtered medium as the test solution, carry out the determination as described in the Assay. Calculate the content of C17H19N3O3S in the medium. D. Not less than 70 per cent of the stated amount of C17H19N3O3S. Other Tests. Comply with the tests stated under Capsules. Loss on drying (2.4.19). Not more than 3.0 per cent, determined on 0.5 g of the contents of the capsules by drying in an oven at 60 at a pressure not exceeding 0.7 kPa for 4 hours. Assay. Determine by liquid chromatography (2.4.14). Test solution. Mix the contents of 20 capsules. Weigh and transfer the granules containing about 20 mg of Omeprazole to a 100-ml volumetric flask, add 20 ml of 0.1 M sodium hydroxide, mix with the aid of ultrasound and dilute to volume with 0.1 M sodium hydroxide. Centrifuge for 5 minutes and dilute 5.0 ml of the clear supernatant liquid to 50.0 ml with the mobile phase. Reference solution. Take 20 mg of omeprazole RS in a dry, stoppered test-tube, add 20.0 ml of 0.1 M sodium hydroxide, shake vigorously for 5 minutes and dilute 1.0 ml of the solution with the mobile phase to produce 50.0 ml. Chromatographic system a stainless steel column 30 cm x 3.9 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 65 volumes of phosphate buffer pH 7.4 and 35 volumes of acetonitrile, flow rate. 1 ml per minute, spectrophotometer set at 302 nm, a 20 l loop injector.
Identification
A.To a quantity of the contents of the capsules containing 50 mg of Omeprazole in a 100-ml volumetric flask add about 70 ml of 0.1 M sodium hydroxide. Mix in an ultrasonic bath for about 5 minutes and heat on a water-bath for 10 minutes. Cool, make up to volume with 0.1 M sodium hydroxide and filter. Dilute 2 ml of the filtrate to 100 ml with 0.1 M sodium hydroxide. When examined in the range 230 nm to 360 nm (2.4.7), the resulting solution shows absorption maxima at about 276 nm and 305 nm. B. In the Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the peak due to omeprazole in the chromatogram obtained with the reference solution.
Tests
Dissolution (2.5.2). A. Apparatus. No 1 Medium. 900 ml of 0.1 M hydrochloric acid, Speed and time. 100 rpm and 2 hours. Tap the granules from a capsule slightly with a glass rod to make them settle to the bottom. Rotate the paddle at 100 rpm
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IP 2007
ORAL REHYDRATION SALTS
Inject the reference solution. Repeat the procedure at least five times and measure the peak responses of the peak due to omeprazole. The relative standard deviation of the replicate injections is not more than 2.0 per cent. Calculate the content of C17H19N3O3S in the capsules. Storage. Store protected from light and moisture.
Oral Rehydration Salts contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of Dextrose (anhydrous) or Dextrose Monohydrate (as appropriate) and of the requisite amounts of sodium, Na, potassium, K, chloride, Cl, and citrate, C6H5O7, calculated from the stated amounts of the relevant constituents. Description. A white to creamy-white, amorphous or crystalline powder; odourless.
Oral Rehydration Salts

ORS Powder
Oral Rehydration Salts are dry, homogeneously mixed powders containing Dextrose, Sodium Chloride, Potassium Chloride and either Sodium Bicarbonate or Sodium Citrate for use in oral rehydration therapy after being dissolved in the requisite amount of water. They may contain suitable flavouring agents and, where necessary, suitable flow agents in the minimum quantity required to achieve a satisfactory product but may not contain artificial sweetening agents like mono- and/or polysaccharides. If saccharin/saccharin sodium or aspartame is used in preparations meant for paediatric use, the concentration of saccharin should be such that its daily intake is not more than 5 mg/kg of body weight and that of aspartame should be such that its daily intake is not more than 40 mg/kg of body weight. Strength. A formulation of reduced osmolarity (given below) recommended by the World Health Organization (WHO) for the Diarrhoeal Diseases Control Programme, and of the United Nations Childrens Fund (UNICEF). Composition of the formulation in terms of the amount, in g, to be dissolved in sufficient water to produce 1000 ml. Sodium Chloride 2.6 Dextrose (anhydrous) 13.5 or Dextrose Monohydrate 14.85 Potassium Chloride 1.5 Sodium Citrate 2.9 The molar concentrations of sodium, potassium, chloride and citrate ions in terms of millimoles per litre are given below: mmol/l Sodium 75 Potassium 20 Chloride 65 Citrate 10 Dextrose 75 The total osmolar concentration of the solution in terms of mOsmol per litre is 245.
Identification
A. When heated, melting and charring occurs and an odour of burnt sugar is produced. B. Add a few drops of the solution prepared as directed in the label to 5 ml of potassium cupri-tartrate solution; a copious red precipitate is produced on boiling. C. Gives reaction A of sodium salts, reaction A of potassium salts and reaction A of chlorides (2.3.1). D. A quantity containing about 50 mg of citric acid gives the reactions of citrates (2.3.1).
Tests
Uniformity of weight. Comply with the test for contents of packaged dosage forms (2.5.6). Seal test (only for sachets). Loosely bundle 10 sachets with a rubber band and submerge the bundle under water in a vacuum desiccator maintained at a pressure not exceeding 18 kPa for one minute. Examine the bundle for any fine stream of bubbles. Re-establish normal pressure and open the bundle. No penetration of water is observed in any sachet. Other Tests. Comply with the tests stated under Oral Powders. Assay. Carry out the following assays on the well-mixed contents of an individual sachet or on a suitable sample from the well-mixed contents of a bulk container. Where the amount in an individual sachet is insufficient to carry out all the assays, take a separate sachet for the Assay for citrate and for the Assay for dextrose. For the Assays for total sodium, for potassium and for total chloride weigh accurately about 8.0 g and dissolve in sufficient water to produce 500.0 ml (solution A). For total sodium Dilute a suitable volume of solution A with a sufficient volume of a solution of strontium chloride such that the final solution contains a 1500- to 2000-fold excess of strontium ions and determine by Method A for atomic absorption spectrophotometry (2.4.2), measuring at 589 nm and using sodium solution AAS, suitably diluted with the strontium chloride solution, for the standard solutions or, alternately by Method A for flame photometry (2.4.4). 1 g of Sodium Chloride, and of Sodium Citrate is equivalent to 0.3934 and 0.2345 g of Na respectively.
861
ORMELOXIFEN HYDROCHLORIDE
IP 2007
For potassium Dilute a suitable volume of solution A with a sufficient volume of solution of strontium chloride such that the final solution contains a 1500- to 2000-fold excess of strontium ions and determine by Method A for atomic absorption spectrophotometry (2.4.2), measuring at 767 nm and using potassium solution AAS, suitably diluted with the strontium chloride solution, for the standard solutions or, alternatively by Method A for flame photometry (2.4.4). 1 g of Potassium Chloride is equivalent to 0.5245 g of K. For total chloride Titrate 50 ml of solution A with 0.1 M silver nitrate using potassium chromate solution as indicator. 1 ml of 0.1 M silver nitrate is equivalent to 0.003545 g of Cl. 1 g of Sodium Chloride, and of Potassium Chloride is equivalent to 0.6066 and 0.4756 g of Cl respectively. For citrate Weigh accurately about 2.0 g and dissolve in 50 ml of anhydrous glacial acetic acid by heating at about 50. Cool, allow to stand for 10 minutes. Titrate with 0.1 M perchloric acid, using 1-naphtholbenzein solution as indicator. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.006303 g of C6H5O7. 1 g of Sodium Citrate is equivalent to 0.6430 g of C6H5O7. For dextrose Weigh accurately about 7.5 g, dissolve in 40 ml of water, add 0.5 ml of dilute ammonia solution, and dilute to 50 ml with water. Mix well, allow to stand for 30 minutes, filter the solution, if turbid, and measure the optical rotation in a 2-dm tube (2.4.22). The observed rotation, in degrees, multiplied by 0.9477 and 1.0424 represents the weight in g of C6H12O6 and C6H12O6,H2O respectively, as appropriate, in the weight taken for the Assay. Storage. Store protected from moisture in sachets, preferably made of aluminum foil, containing sufficient powder for a single dose or for a days treatment or for use in hospitals, in bulk containers containing sufficient quantity to produce a volume of solution appropriate to the daily requirements of the hospital concerned. Labelling. The label states (1) for sachets, the total weights, in g, of each constituent; (2) for bulk containers, the weights, in g, of each constituent in a stated quantity, in g, of the oral powder; (3) the molar concentration in millimoles per litre of sodium, potassium, chloride and citrate ions, and of dextrose as well as the total osmolar concentration in mOsmol per litre of the solution prepared from the oral powder; (4) the total weight of the contents of the container; (5) the directions for use; (6) that any portion of the solution prepared from the oral powder that remains unused for 24 hours after preparation should be discarded; (7) the storage conditions. g, of the oral powder.
Ormeloxifen Hydrochloride
Centchroman Hydrochloride; Centchroman
H3CO O CH3 CH3
, HCl
C30H35NO3,HCl
Mol. Wt. 493.5
Ormeloxifen Hydrochloride is trans-7-methoxy-2,2-dimethyl3-phenyl-4[4-(2-pyrrolidinoethoxy)phenyl]chroman hydrochloride. Ormeloxifen Hydrochloride contains not less than 98.5 per cent and not more than 101.5 per cent of C30H35NO3,HCl, calculated on the dried basis. Description. A white to off-white powder.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with ormeloxifen hydrochloride RS or with the reference spectrum of ormeloxifen. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.01 per cent w/v solution in methanol shows absorption maxima at about 278 and 282 nm. C. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 90 volumes of chloroform and 10 volumes of methanol. Test solution. Dissolve 0.25 g of the substance under examination in 100 ml of chloroform. Reference solution. A 0.25 per cent w/v solution of ormeloxifen hydrochloride RS in chloroform. Apply to the plate 2 l of each solution. After development, dry the plate in air, until the odour of the solvents is no longer detectable and spray with a 0.3 per cent w/v solution of potassium permanganate. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution. D. To 1 ml of a 2 per cent w/v solution in ethanol (95 per cent) add 1 ml of a saturated solution of picric acid in water, stir
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IP 2007
ORMELOXIFEN HYDROCHLORIDE TABLETS
well and set aside for 5 minutes. The yellow precipitate obtained after washing with water and drying at 60 for 4 hours melts at 212 to 218 (2.4.21).
Tests
Total basic substances. Weigh accurately 0.5 g, dissolve in 25 ml of anhydrous glacial acetic acid, add 10 ml of a 5 per cent w/v solution of mercuric acetate in anhydrous glacial acetic acid. Titrate with 0.1 M perchloric acid, using crystal violet solution as indicator to a bluish green end-point. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.04935 g of C30H35NO3,HCl. cis-Isomer. Not more than 1.5 per cent of the total content of hydrochlorides of trans-and cis-isomers determined in the Assay. Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 3.5 per cent, determined on 1.0 g by drying in an oven at 105 for 4 hours. Assay. Determine by liquid chromatography (2.4.14). Test solution. A 0.004 per cent w/v solution of the substance under examination in the mobile phase. Reference solution. A solution containing 0.004 per cent w/v of trans-ormeloxifen hydrochloride RS and 0.0006 per cent w/v of cis-ormeloxifen hydrochloride RS in the mobile phase. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: 80 volumes of acetonitrile and 20 volumes of water containing 0.04 per cent w/v solution of tetramethylammonium hydroxide adjusted to pH 7.6 with phosphoric acid, flow rate. 1.5 ml per minute, spectrophotometer set at 280 nm, a 20 l loop injector. Inject the reference solution and use an attenuation such that the response for the principal peak due to trans-ormeloxifen hydrochloride is more than 50 per cent of the full-scale deflection of the recorder. Make a total of six injections. When the chromatograms are recorded under the conditions described above, trans-ormeloxifen hydrochloride is eluted before cis-ormeloxifen hydrochloride.The test is not valid unless the relative standard deviation of six replicate injections is not greater than 6 per cent and the resolution between the peaks due to cis-ormeloxifen hydrochloride and transormeloxifen hydrochloride is greater than 1. If necessary, adjust the proportions and the flow rate of the mobile phase to obtain proper resolution. Inject a suitable volume of test solution and record the response. From the average peak areas of the
six replicate analyses calculate the content of trans-ormeloxifen hydrochloride and cis-ormeloxifen hydrochloride in the substance under examination by comparing with the peak responses for trans-ormeloxifen hydrochloride and cisormeloxifen hydrochloride respectively obtained with reference solution. Storage. Store protected from moisture.
Ormeloxifen Hydrochloride Tablets

Centchroman Hydrochloride Tablets; Centchroman Tablets
Ormeloxifen Hydrochloride Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of Ormeloxifen hydrochloride, C30H35NO3,HCl.
Identification
Shake a quantity of the powdered tablets containing 0.1 g of Ormeloxifen Hydrochloride with 10 ml of chloroform, filter and evaporate the filtrate to dryness at a pressure not exceeding 0.7 kPa. The residue complies with the following tests. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with ormeloxifen hydrochloride RS or with the reference spectrum of ormeloxifen. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.01 per cent w/v solution in methanol shows absorption maxima at about 278 and 282 nm. C. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 90 volumes of chloroform and 10 volumes of methanol. Test solution. Dissolve 0.25 g of the substance under examination in 100 ml of chloroform. Reference solution. A 0.25 per cent w/v solution of ormeloxifen hydrochloride RS in chloroform. Apply to the plate 2 l of each solution. After development, dry the plate in air, until the odour of the solvents is no longer detectable and spray with a 0.3 per cent w/v solution of potassium permanganate. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution.
Tests
Dissolution (2.5.2). Apparatus. No 2 Medium. 900 ml of water.
863
ORPHENADRINE CITRATE
IP 2007
Speed and time. 100 rpm and 45 minutes. Withdraw a suitable volume of the medium and filter. Measure the absorbance of the filtrate, suitably diluted if necessary, at the maximum at about 278 nm (2.4.7). Calculate the content of C30H35NO3,HCl in the medium from the absorbance obtained from a solution of known concentration of Ormeloxifen hydrochloride RS in water. D. Not less than 70 per cent of the stated amount of C30H35NO3,HCl. cis-Isomer. Not more than 1.5 per cent of the total content of hydrochlorides of trans-and cis-isomers determined in the Assay. Other Tests. Comply with the tests stated under Tablets. Assay. Determine by liquid chromatography (2.4.17). Test solution. Weigh and powder 20 tablets. Shake a quantity of the powder containing 0.1 g of Ormeloxifen Hydrochloride with three quantities, each of 5 ml, of methanol, centrifuge each extract, dilute the combined extracts to 25 ml with methanol and then dilute 250 l of the resulting solution to 25 ml with the mobile phase. Reference solution. A solution containing 0.004 per cent w/v of trans-ormeloxifen hydrochloride RS and 0.0006 per cent w/v of cis-ormeloxifen hydrochloride RS in the mobile phase. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: 80 volumes of acetonitrile and 20 volumes of water containing 0.04 per cent w/v solution of tetramethylammonium hydroxide adjusted to pH 7.6 with phosphoric acid, flow rate. 1.5 ml per minute, spectrophotometer set at 280 nm, a 20 l loop injector. Inject the reference solution and use an attenuation such that the response for the principal peak due to trans-ormeloxifen hydrochloride is more than 50 per cent of full-scale deflection of the recorder. Make a total of six injections. When the chromatograms are recorded under the conditions described above, trans-ormeloxifen hydrochloride is eluted before cisormeloxifen hydrochloride.The test is not valid unless the relative standard deviation of six replicate injections is not greater than 6 per cent and the resolution between the peaks due to cis-ormeloxifen hydrochloride and trans-ormeloxifen hydrochloride is greater than 1.If necessary, adjust the proportions and the flow rate of the mobile phase to obtain proper resolution. Inject a suitable volume of test solution and record the response. From the average peak areas of the six replicate analyses calculate the content of trans-ormeloxifen
hydrochloride and cis-ormeloxifen hydrochloride in the substance under examination. Storage. Store protected from moisture.
Orphenadrine Citrate
CH3 N CH3 ,
CH3 O
HO HOOC
COOH COOH
C18H23NO,C6H807
Mol.461.5
Orphenadrine Citrate is (RS)-dimethyl[2-(2methylbenzhydryloxy)ethyl]amine dihydrogen citrate. Orphenadrine Citrate contains not less than 98.5 per cent and not more than 101.0 per cent of C18H23NO, C6H807, calculated on the dried basis. Description. A white or almost white, crystalline powder; odourless or almost odourless.
Identification
Test A may be omitted if tests B, C, D and E are carried out. Tests B, C and D may be omitted if tests A and E are carried out. A. Determine by infrared absorption spectrophotometry,(2.4.6). Compare the spectrum with that obtained with orphenadrine citrate RS. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.06 per cent w/v solution in ethanol (95 per cent) shows absorption maxima at 258 nm, 264 nm and 271 nm; absorbances at the maxima are about 0.68, 0.72 and 0.47 respectively. C. Dissolve 50 mg in 10 ml of ethanol (50 per cent), add 10 ml of picric acid solution and allow to stand. The precipitate. after recrystallisation from ethanol (95 per cent), melts at about 89 or at about 107 (2.4.21). D. Dissolve 5 mg in 2 ml of sulphuric acid; an orange-red colour is produced. E. To 1 g add 10 ml water and 2 ml of 5 M sodium hydroxide, shake with two quantities, each of 10 mI, of chloroform and discard the chloroform. Heat the aqueous solution to boiling with an excess of mercuric sulphate solution, filter if necessary and boil the resulting solution with 0.2 ml of dilute potassium permanganate solution; the solution is decolorised and a white precipitate is produced.
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IP 2007
ORPHENADRINE HYDROCHLORIDE
Tests
Quaternary ammonium salt. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 50 volumes of 2-propanol, 30 volumes of butyl acetate. 15 volumes of water and 5 volumes of strong ammonia solution. Test solution. Dissolve 0.1 g of the substance under examination in 10 ml methanol. Reference solution. A 0.005 per cent w/v solution of ethyldimethyl [2-(2-methylbenzhydryloxy)ethyl ]ammonium chloride RS. Apply to the plate 5 l of each solution. After development, dry the plate in air and spray with dilute potossium iodobismuthate solution. Any spot corresponding to ethyldimethyl [2-(2-methylbenzhydryloxy) ethyl] ammonium chloride in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Secondary amine. Determine by thin-layer chromatography (2.4.17) coating the plate with silica gel GF254. Mobile phase. A mixture of 96 volumes of 1-butanol and 4 volumes of strong ammonia solution. Test solution. Dissolve 0.4 g of the substance under examination in 10 ml methanol. Reference solution. A 0.02 per cent w/v solution of methyl [2(2-methylbenzhydryloxy)ethyl]amine hydrochloride RS. Apply to the plate 10 l of each solution. After development, dry in air and examine in ultra-violet light at 254 nm. Spray the plate with dilute potassium iodobismuthate solution and examine again. By each method of visualisation any spot corresponding to methyl [2-(2-methylbenzhydryloxy) ethyl]amine hydrochloride in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Heavy Metals (2.3.13). 2.0 g complies with the limit test for heavy metals, Method B (10 ppm). Use 2 ml of lead standard solution (10 ppm Pb) to prepare the standard. Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.3.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105 for 3 hours. Assay. Weigh accurately about I.0 g dissolve in 30 ml of anhydrous glacial acetic acid. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.04615 g of C18H23NO, C6H807. Storage. Store protected from light.
Orphenadrine Hydrochloride
C18H23NO,HCl Mol. Wt.305.8 Orphenadrine Hydrochloride is (RS)-dimethyl [2(2methylbenzhydryloxy) ethyl]amine hydrochloride. Orphenadrine Hydrochloride contains not less than 98.5 per cent and not more than 101.0 per cent of C18H23NO,HCl. calculated on the dried basis. Description. A white or almost white, crystalline powder; odourless or almost odourless.
Identification
Tests A and F may be omitted if tests B, C, D and E are carried out. Tests B, C and D may be omitted if tests A, E and F are carried out. A. Determine by infrared absorption spectrophotometry,(2.4.6). Compare the spectrum with that obtained with orphenadrine hydrochloride RS. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.06 per cent w/v solution in ethanol (95 per cent) shows three absorption maxima. at about 258 nm,264 nm and 271 nm; absorbances at the maxima, about 1.07, 1.13 and 0.73 respectively. C. Dissolve about 5 mg in 2 ml of sulphuric acid; an orangered colour is produced. D. Dissolve about 50 mg in 10 ml of ethanol (95per cent), add 10 ml of picric acid solution and allow to stand. The precipitate, after recrystallisation from ethanol (95 per cent) melts at about 89 or at about 107 (2.4.21).
Tests
Quaternary ammonium salt. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 50 volumes of 2-propanol, 30 volumes of butyl acetate. 15 volumes of water and 5 volumes of strong ammonia solution. Test solution. Dissolve 0.1 g of the substance under examination in 10 of methanol. Reference solution. A 0.005 per cent w/v solution of ethyldimethyl [2-(2-methylbenzhydryloxy) ethyl] ammonium chloride RS. Apply to the plate 5 l of each solution. After development, dry the plate in air and spray with dilute potossium iodobismuthate solution. Any spot corresponding to ethyldimethyl [2-(2-methylbenzhydryloxy) ethyl] ammonium chloride in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution.
865
ORPHENADRINE TABLETS
IP 2007
Secondary amine. Determine by thin-layer chromatography (2.4.17) coating the plate with silica gel GF254. Mobile phase. A mixture of 96 volumes of l-butanol and 4 volumes of strong ammonia solution. Test solution. Dissolve 0.4 g of the substance under examination in 10 methanol. Reference solution. A 0.02 per cent w/v solution of methyl [2(2-methylbenzhydryloxy)ethyl]amine hydrochloride RS. Apply to the plate 10 l of each solution. After development, dry in air and examine in ultra-violet light at 254 nm. Spray the plate with dilute potassium iodobismuthate solution and examine again. By each method of visualisation any spot corresponding to methyl [2-(2-methylbenzhydryloxy) ethyl]amine hydrochloride in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Heavy Metals (2.3.13). 2.0 g complies with the limit test for heavy metals, Method B (10 ppm). Use 2 ml of lead standard solution (10 ppm Pb) to prepare the standard. Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.3.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105 for 3 hours. Assay. Weigh accurately about 1.0 g, dissolve in 20 ml of anhydrous glacial acetic acid, add 20 ml of mercuric acetate solution and titrate with 0.1 M perchloric acid using 1-naphtholbenzein solution as indicator. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.03058 g of C18H23NO, HCl. Storage. Store protected from light.
B. Dissolve 50 mg in 10 ml of ethanol (50 per cent), add 10 ml of picric acid solution and allow to stand. The precipitate. after recrystallisation from ethanol (95 per cent), melts at about 89 or at about 107 (2.4.21). C. A 5 per cent w/v solution gives reaction A of chlorides (2.3.1).
Tests
Other tests. Comply with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 70 mg Orphenadrine Hydrochloride and dissolve as completely as possible in a mixture of 5 ml of water and 5 ml of 2 M hydrochloric acid. Without delay extract with four quantities, each of 15 ml, of chloroform, filter the combined extracts and evaporate to about 20 ml. Add 30 ml of anhydrous glacial acetic and 2 ml of mercuric acetate solution and titrate with 0.02 M perchloric acid determining the end point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of O.02 M perchloric acid is equivalent to 0.006117 g of C18H23NO,HCl. Storage. Store protected from light and moisture.
Oseltamivir Phosphate
H3C H3C H N O O H2N O O CH3 , H3PO4
H3C
Orphenadrine Tablets
Orphenadrine Hydrochloride Tablets
Orphenadrine Tablets contain not less than 92.5 per cent and not more than 107.5 per cent of the stated amount of orphenadrine. hydrochloride, C18H23NO,HCl.
C16H28N2O4, H3PO4
Mol. Wt. 410.4
Oseltamivir Phosphate is phosphoric acid salt of ethyl (3R,4R,5S)-4-(acetylamino)-5-amino-3-(1-ethylpropoxy)-1cyclohexene-1-carboxylate. Oseltamivir Phosphate contain not less than 98.0 per cent and not more than 102.0 per cent of C16H28N2O4 .H3PO4, calculated on the dried basis. Description. A white to off-white powder.
Identification
Extract a quantity of the powdered tablets containing about 0.15 g of Orphenadrine Hydrochloride with chloroform, filter and evaporate the filtrate to dryness. The residue complies with the following tests. A. Dissolve 5 mg in 2 ml of sulphuric acid; an orange-red colour is produced.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with oseltamivir phosphate RS.
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IP 2007
OSELTAMIVIR CAPSULES
B. In the Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the peak in the chromatogram obtained with the reference solution.
Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1 g by drying in an oven at 105. Assay. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 50 mg of the substance under examination in 50.0 ml of the mobile phase. Dilute 10.0 ml of the solution to 50.0 ml with the mobile phase. Reference solution. A 0.02 per cent w/v solution of oseltamivir phosphate RS in the mobile phase. Chromatographic system a stainless steel column 15 cm x 4.6 mm, packed with octylsilane bonded to porous silica (5 m) (such as YMC pack PRO C8), column temperature 50, mobile phase: a mixture of 66 volumes of buffer solution prepared by dissolving 6.8 g of anhydrous monobasic potassium phosphate in 1000 ml of water, adjusting the pH to 6.0 with dilute sodium hydroxide, 24.5 volumes of methanol and 23.5 volumes of acetonitrile, flow rate. 1 ml per minute, spectrophotometer set at 207 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the tailing factor is not more than 2.0, the column efficiency in not less than 2000 theoretical plates and the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject the test solution and the reference solution. Calculate the content of C16H28N2O4,H3PO4. Storage. Store protected from light and moisture.
Tests
Specific optical rotation (2.4.22). - 42.0 to - 48.0, determined in a 1.0 per cent w/v solution in methanol. Related substances. Determine by liquid chromatography (2.4.14). NOTE Prepare the solutions immediately before use. Test solution. Dissolve 25 mg of the substance under examination in 25 ml of the mobile phase. Reference solution (a). A 0.1 per cent w/v solution of oseltamivir phosphate RS in the mobile phase. Reference solution (b). Dilute 1 ml of reference solution (a) to 100 ml with the mobile phase. Chromatographic system a stainless steel column 15 cm x 4.6 mm, packed with octylsilane bonded to porous silica (5 m) (such as YMC pack PRO C8), column temperature 50, mobile phase: a mixture of 66 volumes of a buffer solution prepared by dissolving 6.8 g of anhydrous monobasic potassium phosphate in 1000 ml of water and adjusting the pH to 6.0 with dilute sodium hydroxide, 24.5 volumes of methanol and 23.5 volumes of acetonitrile, flow rate. 1 ml per minute, spectrophotometer set at 207 nm, a 20 l loop injector. Inject reference solution (a). The test is not valid unless the column efficiency is not less than 2000 theoretical plates and the tailing factor is not more than 2.0. Inject the test solution and reference solution (b). In the chromatogram obtained with the test solution, the area of any secondary peak is not more than 0.5 time the area of the peak in the chromatogram obtained with the reference solution (b) (0.5 per cent) and the sum of all the secondary peaks is not more than the area of the peak in the chromatogram obtained with the reference solution (b) (1.0 per cent). Phosphoric acid. 23.4 to 24.4 per cent, calculated on a dried basis. Weigh accurately about 0.2 g and dissolve in 40 ml of distilled water. Titrate with 0.1 M sodium hydroxide, determining the end-point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M sodium hydroxide is equivalent to 0.0049 g of phosphoric acid. Heavy metals (2.3.13). 1 g complies with the limit test for heavy metals, Method A (20 ppm).
Oseltamivir Capsules
Oseltamivir Phosphate Capsules
Oseltamivir Capsules contain Oseltamivir Phosphate. Oseltamivir Capsules contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of oseltamivir, C16H28N2O4.
Identification
Tests
Dissolution (2.5.2). Apparatus. No. 1 Medium. 900 ml of 0.1 M hydrochloric acid. Speed and time. 50 rpm and 45 minutes
867
OSELTAMIVIR ORAL SUSPENSION
IP 2007
Withdraw a suitable volume of the medium and filter. Determine by liquid chromatography (2.4.14). Test solution. Use the filtrate. Reference solution. Dissolve 50 mg of oseltamivir phosphate RS in 20 ml of the mobile phase and dilute to 100 ml with the dissolution medium. Dilute 5 ml of the solution to 25 ml with the dissolution medium. Use the chromatographic system described under Assay. D. Not less than 80 per cent of the stated amount of C16H28N2O4. Related substances. Determine by liquid chromatography (2.4.14). NOTE Prepare the solutions immediately before use. Test solution. Weigh accurately a quantity of the mixed contents of 20 capsules containing about 50 mg of oseltamivir, disperse in 50 ml of mobile phase and filter. Reference solution (a). A 0.1 per cent w/v solution of oseltamivir phosphate RS in the mobile phase. Reference solution (b). Dilute 1 ml of reference solution to 100 ml with the mobile phase. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octylsilane bonded to porous silica (5 m) (such as YMC pack PRO C8), mobile phase: a mixture of 66 volumes of a buffer solution prepared by dissolving 6.8 g of anhydrous monobasic potassium phosphate in 1000 ml of water, adjusting the pH to 6.0 with dilute sodium hydroxide, 24.5 volumes of methanol and 23.5 volumes of acetonitrile, flow rate. 1.5 ml per minute, spectrophotometer set at 207 nm, a 20 l loop injector. Inject reference solution (a). The test is not valid unless the column efficiency is not less than 2000 theoretical plates and the tailing factor is not more than 2.0. Inject the test solution and reference solution (b). In the chromatogram obtained with the test solution, the area of any secondary peak is not more than the area of the peak in the chromatogram obtained with the reference solution (b) (1.0 per cent) and the sum of the areas of all the secondary peaks is not more than twice the area of the peak in the chromatogram obtained with the reference solution (b) (2.0 per cent). Other tests. Comply with the tests stated under Capsules. Water (2.3.43). Not more than 5.0 per cent, determined on 0.5 g. Assay. Determine by liquid chromatography (2.4.14). NOTE Prepare the solutions immediately before use.
Test solution. Weigh accurately a quantity of the mixed contents of 20 capsules containing about 200 mg of oseltamivir, disperse in 100.0 ml of the mobile phase and filter. Dilute 5.0 ml of the solution to 50.0 ml with the mobile phase. Reference solution. A 0.02 per cent w/v solution of oseltamivir phosphate RS in the mobile phase. Chromatographic system a stainless steel column 15 cm x 4.6 mm, packed with octylsilane bonded to porous silica (5 m), mobile phase: a mixture of 66 volumes of a buffer solution prepared by dissolving 6.8 g of anhydrous monobasic potassium phosphate in 1000 ml of water and adjusting the pH to 6.0 with dilute sodium hydroxide, 24.5 volumes of methanol and 23.5 volumes of acetonitrile, flow rate. 1.2 ml per minute, spectrophotometer set at 207 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the tailing factor is not more than 2.0, the column efficiency in not less than 2000 theoretical plates and the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject the test solution and the reference solution. Calculate the content of C16H28N2O4. Storage. Store protected from moisture, at a temperature not exceeding 30. Labelling. The label states the strength in terms of the equivalent amount of oseltamivir.
Oseltamivir Oral Suspension

Oseltamivir Phosphate Oral Suspension
Oseltamivir Oral Suspension is a mixture consisting of Oseltamivir Phosphate with buffering agents and other excipients. It contains a suitable flavouring agent. It is filled in a sealed container. The suspension is constituted by dispersing the contents of the sealed container in the specified volume of water just before issue. Oseltamivir Oral Suspension contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of oseltamivir C16H28N2O4. The contents of the sealed container comply with the following requirement. Water (2.3.43). Not more than 2.5 per cent, determined on 1.0 g. The constituted suspension complies with the requirements stated under Oral liquids and with the following requirements.
868
IP 2007
OXAZEPAM
Identification
A. In the Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the peak in the chromatogram obtained with reference solution (b). B. To 2 ml of the reconstituted suspension, add 2 ml of dilute nitric acid and 4 ml of a 10 per cent w/v solution of ammonium molybdate and warm the solution. A bright yellow precipitate is formed.
Test solution. Weigh accurately a quantity of the suspension containing 60 mg of oseltamivir, dissolve in 250.0 ml of the mobile phase and filter. Dilute 10.0 ml of the solution to 25.0 ml with the mobile phase and filter. Reference solution. A 0.0125 per cent w/v solution of oseltamivir phosphate RS in the mobile phase. Chromatographic system a stainless steel column 15 cm x 4.6 mm, packed with octylsilane bonded to porous silica (5 m), mobile phase: a mixture of 60 volumes of a buffer solution prepared by dissolving 6.8 g of potassium dihydrogen phosphate in 1000 ml of water and adjusting the pH to 6.0 with dilute sodium hydroxide, 24.5 volumes of methanol and 23.5 volumes of acetonitrile, flow rate. 1.2 ml per minute, spectrophotometer set at 207 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the tailing factor is not more than 2.0, the column efficiency in not less than 2000 theoretical plates and the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject the test solution and the reference solution. Calculate the content of C16H28N2O4. Storage. Store protected from moisture, at a temperature not exceeding 30. Labelling. The label states (1) the quantity of active ingredient in terms of the equivalent amount of oseltamivir; (b) the temperature of storage and the period during which the constituted suspension may be expected to be satisfactory for use.
Tests
pH (2.4.24). 3.0 to 5.0. Related substances. Determine by liquid chromatography (2.4.14). NOTE Prepare the solutions immediately before use. Test solution. Weigh accurately a quantity of the suspension containing 25 mg of oseltamivir, dissolve in 25 ml of the mobile phase and filter. Reference solution (a). A 0.1 per cent w/v solution of oseltamivir phosphate RS in the mobile phase. Reference solution (b). Dilute 1 ml of reference solution (a) to 100 ml with the mobile phase. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octylsilane bonded to porous silica (5 m), column temperature 35, mobile phase: 70 volumes of a buffer solution prepared by dissolving 6.8 g of potassium dihydrogen phosphate in 1000 ml of water and adjusting the pH to 6.0 with dilute sodium hydroxide, 15 volumes of methanol and 15 volumes of acetonitrile, flow rate. 1.5 ml per minute, spectrophotometer set at 207 nm, a 20 l loop injector. Inject reference solution (a). The test is not valid unless the column efficiency is not less than 2000 theoretical plates and the tailing factor is not more than 2.0. Inject the test solution and reference solution (b). In the chromatogram obtained with the test solution, the area of any secondary peak is not more than the area of the peak in the chromatogram obtained with the reference solution (b) (1.0 per cent) and the sum of areas of all the secondary peaks is not more than 3 times the area of the peak in the chromatogram obtained with the reference solution (b) (3.0 per cent). Other tests. Complies with the tests stated under Oral liquids. Assay. Determine by liquid chromatography (2.4.14). NOTE Prepare the solutions immediately before use.
Oxazepam
H N Cl N O OH
C15HIICIN2O2
Mol. Wt. 286.7
Oxazepam is 7-chloro-3-hydroxy-5-phenyl-1,3-dihydro-2H1,4-benzodiazepin-2-one. Oxazepam contains not less than 98.5 per cent and not more than 101.0 per cent of C15HIICIN2O2, calculated on the dried basis.
869
OXAZEPAM TABLETS
IP 2007
Description. A white or almost white, crystalline powder.
Reference solution (c). Dilute 1 ml of reference solution (a) to 100 ml with acetone. Reference solution (d). Dilute 5 ml of solution (d) to 10 ml with acetone. Apply to the plate 20 l of each solution. Allow the mobile phase to rise 17 cm in the same direction as the washing with methanol. Dry in air and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with test solution (a) is not more intense than the spot in thechromatogram obtained with reference solution (c) and at most one such spot is more intense than the spot in the chromatogram obtained with reference solution (d). The test is not valid unless the chromatogram obtained with reference solution (b) shows two clearly separated spots. Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.3.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105 at a pressure not exceeding 0.7 kPa. Assay. Weigh accurately about 0.25 g and dissolve in a mixture of 10 ml of anhydrous glacial acetic acid and 90 ml of acetic anhydride and titrate with 0.1 M perchloric acid determining the end point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.02867 g of Cl5HIICIN2O2. Storage. Store protected from light and moisture.
Identification
Test A may be omitted if tests B, C and D are carried out. Tests B and D may be omitted if tests A and C are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with oxazepam RS. B. Prepare the solutions immediately before use, protected from light. Dissolve about 20 mg in sufficient ethanol (95 per cent) to produce 100 ml. Dilute 10 ml of the solution to 50 ml with ethanol (95 per cent) (solution A). Dilute 10 ml of solution A to 100 ml with ethanol (95 per cent) (solution B). When examined in the range 230 nm to 360 nm (2.4.7), solution A shows an absorption maximum at about 316 nm. When examined in the range 220 nm to 250 nm, solution B shows an absorption maximum at about 229 nm; absorbance at about 229 nm, 1.220 to 1.300. C. In the test for Related substances, the principal spot in the chromatogram obtained with test solution (b) corresponds to that in the chromatogram obtained with reference solution (b). D. Dissolve about 20 mg in a mixture of 5 ml of hydrochloric acid and 10 ml of water. Heat to boiling for 5 minutes and cool. Add 2 ml of a 0.1 per cent w/v solution of sodium nitrite and allow to stand for 1 minute. Add 1 ml of a 0.5 per cent w/v solution of sulphamic acid, mix and allow to stand for 1 minute. Add 1 ml of 0.1 per cent w/v solution of a N- (1-naphthyl) ethylenediamine dihydrochloride; a red colour is produced.
Oxazepam Tablets
Oxazepam Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of oxazepam, CI5H11CIN2O2.
Tests
Related substances: Carry out the test protected from light. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Wash the plate with methanol before use. Mobile phase. A mixture of 100 volumes of dichloromethane and 10 volumes of methanol. Test solution (a). Dissolve 50 mg of the substance under examination in sufficient acetone to produce 10 ml. Test solution (b). Dilute 2 ml of test solution (a) to 10 ml with acetone. Reference solution (a). Dissolve 10 mg of oxazepam RS in sufficient acetone to produce 10 ml. Reference solution (b). Dissolve 10 mg each of oxazepam RS and bromazepam RS in sufficient acetone to produce 10 ml.
Identification
A. Extract a quantity of the powdered tablets containing 20 mg of Oxazepam with 25 ml of chloroform, filter, evaporate to dryness and dry the residue at 60 at a pressure not exceeding 0.7 kPa. On the residue, determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with oxazepam RS or with the reference spectrum of oxazepam. B. When examined in the range 210 nm to 360 nm (2.4.7), the solution obtained in the Assay shows absorption maxima at about 230 nm and 316 nm.
Tests
Related substances. Carry out the test protected from light.
870
IP 2007
OXPRENOLOL HYDROCHLORIDE
Determine by thin-layer chromatography (2.4.17) coating the plate with silica gel GF 254. Wash the plate with methanol before use. Mobile phase. A mixture of 100 volumes of dichloromethane and 10 volumes of methanol. Test solution. Shake a quantity of powdered tablets containing 30 mg of Oxazepam with 6 ml of acetone and centrifuge. Reference solution (a). Dilute 1 volume of the test solution to 100 volumes with acetone. Reference solution (b). Dilute 1 volume of the test solution to 500 volumes with acetone. Reference solution (c). A solution containing 0.1 per cent w/v each of oxazepam RS and bromazepam RS. Apply to the plate 20 l of each of the solutions. Allow the mobile phase to rise 17 cm in the same direction as in the washing with methanol. Dry in air and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (a) (1 per cent) and not more than one such spot is more intense than the spot in the chromatogram obtained with reference solution (b) (0.2 per cent). The test is not valid unless the chromatogram obtained with reference solution (c) shows two clearly separated spots. Other tests. Comply with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 25 mg of Oxazepam and shake with 150 mI of ethanol (95 per cent) for 30 minutes. Add sufficient ethanol (95 per cent) to produce 250.0 ml and centrifuge. Dilute 5.0 ml of the supernatant liquid to 100.0 ml with the same solvent and measure the absorbance of the resulting solution at the maximum at about 230 nm (2.4.7). Calculate the content of CI5HI1CIN2O2 taking 1250 as the specific absorbance at 230 nm. Storage. Store protected from light at a temperature not exceeding 30.
Oxprenolol Hydrochloride contains not less than 98.5 per cent and not more than 101.5 per cent of C15H23NO3,HCl, calculated on the dried basis. Description. A white or almost white, crystalline powder.
Identification
Test A may be omitted if tests B and C are carried out. Test B may be omitted if tests A and C are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with oxprenolol RS or with the reference spectrum of oxprenolol. B. In the test for Related substances, the principal spot in the chromatogram obtained with test solution (b) corresponds to that in the chromatogram obtained with reference solution (c). C. Gives reaction A of chlorides (2.3.1).
Tests
Appearance of solution. A 10.0 per cent w/v solution in carbon dioxide-free water is clear (2.4.1), and not more intensely coloured than reference solution GYS6 (2.4.1). pH (2.4.24). 4.5 to 6.0, determined in a freshly prepared 10.0 per cent w/v solution. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 88 volumes of chloroform, 12 volumes of methanol and 2 volumes of strong ammonia solution. Test solution (a). Dissolve 0.5 g of the substance under examination in 10 ml of a mixture of 90 volumes of chloroform and 10 volumes of methanol. Test solution (b). Dissolve 0.5 g of the substance under examination in 100 ml of a mixture of 90 volumes of chloroform and 10 volumes of methanol. Reference solution (a). A 0.02 per cent w/v solution of the substance under examination in a mixture of 90 volumes of chloroform and 10 volumes of methanol. Reference solution (b). A 0.01 per cent w/v solution of the substance under examination in a mixture of 90 volumes of chloroform and 10 volumes of methanol. Reference solution (c). A 0.5 per cent w/v solution of oxprenolol hydrochloride RS in a mixture of 90 volumes of chloroform and 10 volumes of methanol.
Oxprenolol Hydrochloride
OH O O
C15H23NO3,HCl
H N
CH3 , HCl
CH2 CH3
Mol. Wt. 301.8
Oxprenolol Hydrochloride is (RS)-1-(2-allyloxyphenoxy)-3isopropylaminopropan-2-ol hydrochloride.
Apply to the plate 2 l of each solution. Allow the mobile phase to rise 13 cm. Dry the plate in warm air for 10 minutes, allow to cool, spray with anisaldehyde solution, heat at 105 for 10 minutes and examine in daylight. Any secondary spot in the chromatogram obtained with test solution (a) is not more intense than the spot in the chromatogram obtained
871
OXPRENOLOL TABLETS
IP 2007
with reference solution (a) and not more than one such spot is more intense than the spot in the chromatogram obtained with reference solution (b). Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 60 over phosphorus pentoxide at a pressure of 1.5 to 2.5 kPa for 6 hours. Assay. Weigh accurately about 0.25 g, dissolve in 60 ml of anhydrous glacial acetic acid, add 5 ml of mercuric acetate solution. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.4.25).Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.03018 g of C15H23NO3,HCl. Storage. Store protected from light and moisture.
Mobile phase. A mixture of 88 volumes of chloroform, 12 volumes of methanol and 2 volumes of strong ammonia solution. Test solution. Extract a quantity of the powdered tablets containing 0.25 g of Oxprenolol Hydrochloride with 5 ml of water, centrifuge and use the supernatant liquid. Reference solution. Dilute 1 volume of the test solution to 200 volumes with water. Apply to the plate 2 l of each solution. Allow the mobile phase to rise 13 cm. Dry the plate in warm air for 10 minutes, allow to cool, spray with anisaldehyde solution, heat at 105 for 10 minutes and examine in daylight. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Other Tests. Comply with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 20 mg of Oxprenolol Hydrochloride, add 25 ml of 0.1 M hydrochloric acid and sufficient water to produce 250.0 ml. Mix with the aid of ultrasound for 5 minutes, shake for 15 minutes and filter. Measure the absorbance of the resulting solution at the maximum at about 273 nm (2.4.7). Calculate the content of C15H23NO3,HCl taking 74.5 as the specific absorbance at 273 nm. Storage. Store protected from moisture.
Oxprenolol Tablets
Oxprenolol Hydrochloride Tablets
Oxprenolol Hydrochloride Tablets contain not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of oxprenolol hydrochloride, C15H23NO3,HCl. The tablets are coated.
Identification
A.To a quantity of the powdered tablets containing 50 mg of Oxprenolol Hydrochloride add 10 ml of water and 2 ml of dilute sodium hydroxide solution, extract with 10 ml of chloroform and reserve the aqueous layer for test C. Dry the chloroform extract over anhydrous sodium sulphate, filter and evaporate the filtrate to dryness. On the residue, determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with oxprenolol hydrochloride RS or with the reference spectrum of oxprenolol hydrochloride. B. When examined in the range 230 nm to 360 nm (2.4.7), the solution obtained in the Assay shows an absorption maximum only at about 273 nm. C. The aqueous layer obtained in test A gives reaction A of chlorides (2.3.1). D. The residue obtained in test A melts at about 76 (2.4.21).
Oxygen
O2 Mol. Wt. 32.0 Oxygen contains not less than 99.0 per cent v/v of O2. This monograph applies to oxygen for medicinal use only. Description. A colourless gas; odourless.
Identification
A. A glowing splinter of wood bursts into flame on contact with the gas. B. Shake with alkaline pyrogallol solution; the gas under examination is absorbed and the solution becoming dark brown. C. When tested as described under Assay, not more than 1.0 ml of gas remains.
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G.
Tests
Carbon dioxide. Not more than 300 ppm v/v, determined by using a carbon dioxide detector tube (2.1.1).
872
IP 2007
OXYPHENBUTAZONE
Carbon monoxide. Not more than 5 ppm v/v, determined by using a carbon monoxide detector tube (2.1.1). Water vapour. Not more than 67 ppm v/v, determined by using a water vapour detector tube (2.1.1). Assay (2.3.33). Use 100 ml of the gas under examination and place spirals of freshly cleaned copper wire and 125 ml of ammonia buffer pH 10.9 in the pipette. The volume of the residual gas in the burette is not more than 1.0 ml. Storage. Store under pressure in metal cylinders of the type conforming to the appropriate safety regulations. Valves and taps should not be lubricated with oil or grease. Labelling. The shoulder of the metal cylinder should be painted white and the remainder should be painted black. The cylinder should carry a label stating Oxygen. In addition, Oxygen or the symbol O2 should be stencilled in paint on the shoulder of the cylinder.
compounds and must not be treated with any compound that will be irritating to the respiratory tract when the Oxygen 93 Per Cent is used. Labelling. Label each outlet Oxygen 93 Per Cent, when it is piped directly from the collecting tank to the point of use. If it is stored in cylinders, reduce the pressure by means of a regulator. Measure the gases with a gas volume meter downstream from the detector tube in order to minimise contamination or change of the specimens. The shoulder of the cylinder should be painted white and the remainder should be painted black. The cylinder should carry a label stating Oxygen 93 Per Cent and For medicinal use.
Oxyphenbutazone
OH
Oxygen 93 Per Cent

Oxygen 93 Per Cent contains not less than 90.0 per cent and not more than 96.0 per cent, v/v of O2, the remainder consisting mostly of argon and nitrogen. It is produced from air by the molecular sieve process. Description. A colourless gas; odourless.
O H3 C N N O , H2O
Identification
A. A glowing splinter of wood bursts into flame on contact with the gas. B. Shake with alkaline pyrogallol solution; the gas under examination is absorbed and the solution becomes dark brown. C. When tested as described under Assay, not more than 10.0 ml and not less than 4.0 ml of gas remain.
C19H20N2O3,H2O
Mol. Wt. 342.4
Oxyphenbutazone is (RS)-4-butyl-1-(4-hydroxyphenyl)-2phenylpyrazolidine-3,5-dione monohydrate. Oxyphenbutazone contains not less than 98.0 per cent and not more than 101.0 per cent of C19H20N2O3, calculated on the anhydrous basis. Description. A white to yellowish white, crystalline powder; almost odourless.
Tests
Carbon dioxide. Not more than 300 ppm v/v, determined by using a carbon dioxide detector tube (2.1.1). Carbon monoxide. Not more than 5 ppm v/v, determined by using a carbon monoxide detector tube (2.1.1). Assay (2.3.33). Use 100 ml of the gas under examination and place spirals of freshly cleaned copper wire and 125 ml of ammonia buffer pH 10.9 in the pipette. The volume of the residual gas in the burette is not more than 10.0 ml and not less than 4.0 ml. Storage. Store in cylinders or in a low pressure collecting tank. Containers used for Oxygen 93 Per Cent must not be treated with any toxic, sleep-inducing or narcosis-producing
Identification
Test A may be omitted if tests B, C and D are carried out. Tests B and D may be omitted if tests A and C are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with oxyphenbutazone RS or with the reference spectrum of oxyphenbutazone. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.001 per cent w/v solution in 0.01 M sodium hydroxide shows an absorption maximum at about 254 nm; absorbance at about 254 nm, 0.71 to 0.77. C. To 2 ml of a 1 per cent w/v solution in ethanol (95 per cent) add 2 ml of a 0.1 per cent w/v solution of 2,6-dichloroquinone-
873
OXYPHENBUTAZONE TABLETS
IP 2007
4-chlorimide in ethanol (95 per cent) and 1 ml of sodium carbonate solution; an intense green colour is produced. D. To 0.1 g add 1 ml of glacial acetic acid and 2 ml of hydrochloric acid and boil under a reflux condenser for 30 minutes. Cool, add 10 ml of water and filter. To the filtrate add 3 ml of a 0.7 per cent w/v solution of sodium nitrite; a yellow colour develops. To 1 ml of the resulting solution add a solution of 10 mg of 2-naphthol in 5 ml of sodium carbonate solution; an orange-red precipitate is produced.
Oxyphenbutazone Tablets
Oxyphenbutazone Tablets contain not less than 92.5 per cent and not more than 107.5 per cent of the stated amount of oxyphenbutazone, C19H20N2O3,H2O. The tablets are coated.
Identification
Extract a quantity of the powdered tablets containing 0.3 g of Oxyphenbutazone with 60 ml of acetone, filter and evaporate the filtrate to dryness. The residue complies with the following tests. A. When examined in the range 230 nm to 360 nm (2.4.7), a 0.001 per cent w/v solution in 0.01 M sodium hydroxide shows an absorption maximum at about 254 nm; absorbance at about 254 nm, 0.71 to 0.77. B. To 2 ml of a 1 per cent w/v solution in ethanol (95 per cent) add 2 ml of a 0.1 per cent w/v solution of 2,6-dichloroquinone4-chlorimide in ethanol (95 per cent) and 1 ml of sodium carbonate solution; an intense green colour is produced. C. To 0.1 g add 1 ml of glacial acetic acid and 2 ml of hydrochloric acid and boil under a reflux condenser for 30 minutes. Cool, add 10 ml of water and filter. To the filtrate add 3 ml of a 0.7 per cent w/v solution of sodium nitrite; a yellow colour develops. To 1 ml of the resulting solution add a solution of 10 mg of 2-naphthol in 5 ml of sodium carbonate solution; an orange-red precipitate is produced.
Tests
Appearance of solution. To 0.5 g add a mixture of 12 ml of 1 M sodium hydroxide and 8 ml of a 7.5 per cent w/v solution of glycine, shake for 1 minute and maintain at 25 for exactly 60 minutes. The solution is clear (2.4.1). Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel HF254. Mobile phase. A 0.02 per cent w/v solution of butylated hydroxytoluene in a mixture of 80 volumes of chloroform and 20 volumes of glacial acetic acid. Test solution. Dissolve 0.1 g of the substance under examination in 5 ml of a solution containing 0.02 per cent w/v of butylated hydroxytoluene in ethanol. Reference solution. Dilute 1 ml of the test solution to 200 ml with the ethanolic butylated hydroxytoluene solution. To prepare the plate, allow the mobile phase to rise 4 cm. Dry the plate in a current of cold air for 1 minute. Without delay and under a current of nitrogen, apply separately to the plate 5 l of each solution prepared immediately before use. Develop immediately, allowing the mobile phase to rise 10 cm. Dry the plate in a current of cold air for 15 minutes and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Water (2.3.43). 5.0 to 6.0 per cent, determined on 0.5 g. Assay. Weigh accurately about 0.5 g, dissolve in 50 ml of acetone and titrate with 0.1 M sodium hydroxide, using bromothymol blue solution as indicator and continuing the titration until the blue colour persists for at least 15 seconds. Carry out a blank titration. 1 ml of 0.1 M sodium hydroxide is equivalent to 0.03244 g of C19H20N2O3. Storage. Store protected from light.
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel HF254. Mobile phase. A 0.02 per cent w/v solution of butylated hydroxytoluene in a mixture of 80 volumes of chloroform and 20 volumes of glacial acetic acid. Test solution. Shake a quantity of the powdered tablets containing 0.2 g of Oxyphenbutazone with 10 ml of a solution containing 0.02 per cent w/v of butylated hydroxytoluene in ethanol for 15 minutes and centrifuge. Use the supernatant liquid. Reference solution. Dilute 1 ml of the test solution to 200 ml with the ethanolic butylated hydroxytoluene solution. To prepare the plate, allow the mobile phase to rise 4 cm. Dry the plate in a current of cold air for 1 minute. Without delay and under a current of nitrogen, apply separately to the plate 5 l of each solution prepared immediately before use. Develop immediately, allowing the mobile phase to rise 10 cm. Dry the plate in a current of cold air for 15 minutes and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution.
874
IP 2007
OXYTETRAXYCLINE DIHYDRATE
Dissolution (2.5.2). Apparatus. No 1 Medium. 900 ml of phosphate buffer pH 7.6. Speed and time. 100 rpm and 30 minutes. Withdraw a suitable volume of the medium and filter promptly through a membrane filter disc with an average pore diameter not greater than 1.0 m. Reject the first few ml of the filtrate and dilute a suitable volume of the filtrate with the same solvent. Measure the absorbance of the resulting solution at the maximum at about 262 nm (2.4.7). Calculate the content of C19H20N2O3,H2O in the medium form the absorbance obtained from a solution of known concentration of oxyphenbutazone RS in the same medium. D. Not less than 60 per cent of the stated amount of C19H20N2O3,H2O. Other Tests. Comply with the tests stated under Tablets. Assay. Weigh 20 tablets and reduce to a fine powder. Weigh accurately a quantity of the powder containing about 0.5 g of Oxyphenbutazone and extract successively with 30, 10 and 10 ml of warm acetone. Filter the combined extracts, cool. Titrate with 0.1 M sodium hydroxide, using bromothymol blue solution as indicator and continuing the titration until the blue colour persists for at least 15 seconds. Carry out a blank titration. 1 ml of 0.1 M sodium hydroxide is equivalent to 0.03424 g of C19H20N2O3,H2O. Storage. Store protected from moisture.
Oxytetracycline has a potency not less than 900 g of C22H24N2O9, per mg, calculated on the anhydrous basis. Description. A tan yellow or light yellow (with or without a greenish tinge), crystalline powder; odourless.
Identification
A. Determine by thin-layer chromatography (2.4.17), coating the plate with the substance prepared by mixing 25 g of silica gel G with 50 ml of a mixture of 2.5 ml of glycerin and 47.5 ml of 0.1 M disodium edetate previously adjusted to pH 7.0 with dilute ammonia solution. After spreading the plate, allow it to stand at room temperature till it is dry (70 to 90 minutes). Mobile phase. The lower layer formed after shaking 200 ml of a mixture of 2 volumes of ethyl acetate, 2 volumes of chloroform and 1 volume of acetone with 25 ml of 0.1 M disodium edetate previously adjusted to pH 7.0 with dilute ammonia solution. Test solution. Dissolve 0.05 g of the substance under examination in 100 ml of methanol. Reference solution (a). A 0.05 per cent w/v solution of oxytetracycline RS in methanol. Reference solution (b). A solution containing 0.05 per cent w/v each of demethylchlortetracycline hydrochloride RS, oxytetracycline hydrochloride RS and tetracycline hydrochloride RS in methanol. Apply to the plate 1 l of each solution, freshly prepared. After development, dry the plate in air, expose to the vapours of ammonia and examine in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows three clearly separated spots. B. To about 0.5 mg add 2 ml of sulphuric acid; a red colour is produced. Add the solution to 1 ml of water; the colour changes to yellow.
Oxytetracycline Dihydrate
Oxytetracycline
OH O OH O OH O NH2 H H H3C OH OH N CH3 H3C
C22H24N2O9,2H2O
OH
, 2H2O
C. Dissolve about 2 mg in 5 ml of a 1 per cent w/v solution of sodium carbonate and add 2 ml of diazotised sulphanilic acid solution; an orange-red to brownish-red colour is produced.
Mol. Wt. 496.5
Tests
pH (2.4.24). 4.5 to 7.5, determined in a 1.0 per cent w/v suspension in freshly boiled and cooled water. Specific optical rotation (2.4.22). 203 to 216, determined at 20 in a 1.0 per cent w/v solution in 0.1 M hydrochloric acid, after allowing the solution to stand protected from light for 30 minutes before measurement.
Oxytetracycline is (4S,4aR,5S,5aR,6S,12aS)-4-dimethyl amino-1,4,4a,5,5a,6,11,12a-octahydro-3,5,6,10,12,12ahexahydroxy-6-methyl-1,11-dioxonaphthacene-2carboxamide, a substance produced by the growth of certain strains of Streptomyces rimosus or obtained by any other means. It contains a variable quantity of water.
875
OXYTETRAXYCLINE DIHYDRATE
IP 2007
Light absorption. Absorbance of a 0.002 per cent w/v solution in buffer solution pH 2.0 at the maximum at about 353 nm, 0.58 to 0.62 (2.4.7). Light-absorbing impurities. A. Dissolve 20 mg in sufficient of a mixture of 1 volume of 1 M hydrochloric acid and 99 volumes of methanol to produce 10 ml. Absorbance of the resulting solution at about 430 nm, when measured within 1 hour of preparing the solution, not more than 0.25, calculated on the anhydrous basis (2.4.7). B. Dissolve 0.1 g in sufficient of a mixture of 1 volume of 1 M hydrochloric acid and 99 volumes of methanol to produce 10 ml. Absorbance of the resulting solution at about 490 nm, when measured within 1 hour of preparing the solution, not more than 0.20, calculated on the anhydrous basis (2.4.7). Related substances. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 20 mg of the substance under examination in 25 ml of 0.01 M hydrochloric acid. Reference solution (a). Dissolve 20 mg of oxytetracycline RS in 25 ml of 0.01 M hydrochloric acid. Reference solution (b). Dissolve 20 mg of 4-epioxytetracycline RS in 25 ml of 0.01 M hydrochloric acid. Reference solution (c). Dissolve 20 mg of tetracycline hydrochloride RS in 25 ml of 0.01 M hydrochloric acid. Reference solution (d). Dilute a mixture of 1.5 ml of reference solution (a), 1.0 ml of reference solution (b) and 3.0 ml of reference solution (c) to 25 ml with 0.01 M hydrochloric acid. Reference solution (e). Dilute a mixture of 1.0 ml of reference solution (b) and 4.0 ml of reference solution (c) to 200 ml with 0.01 M hydrochloric acid. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with styrene- divinylbenzene co-polymer (8 to 10 m), column. temperature 60, mobile phase: add 60 g of 2-methyl-2-propanol to a volumetric flask with the aid of 200 ml of water, add 60 ml of 0.33 M phosphate buffer pH 7.5, 50 ml of a 1.0 per cent w/v solution of tetrabutylammonium hydrogen sulphate previously adjusted to pH 7.5 with 2 M sodium hydroxide and 10 ml of a 0.04 per cent w/v solution of disodium edetate previously adjusted to pH 7.5 with 2 M sodium hydroxide and dilute to 1000.0 ml with water, flow rate. 1 ml per minute, spectrophotometer set at 254 nm, a 20 l loop injector.
Adjust the sensitivity so that the heights of the peaks in the chromatogram obtained with reference solution (d) are at least 50 per cent of the full-scale deflection of the recorder. The test is not valid unless (a) the resolution between the first peak (4-epioxytetracycline) and the second peak (oxytetracycline) is at least 4.0 (b) the resolution between the second peak and the third peak (tetracycline) is at least 5.0 (the content of 2-methyl-2-propanol in the mobile phase may be adjusted if necessary) and (c) the symmetry factor for the second peak is at most 1.25. Inject reference solution (a) six times. The test is not valid unless the relative standard deviation of the area of the peak due to oxytetracycline is not greater than 1.0 per cent. Inject the test solution and reference solution (e). In the chromatogram obtained with the test solution the area of any peak corresponding to 4-epioxytetracycline or tetracycline is not greater than the area of the corresponding peak in the chromatogram obtained with reference solution (e). In the chromatogram obtained with the test solution the area of any peak appearing on the tail of the principal peak is not greater than 4.0 times that of the peak due to 4-epioxytetracycline in the chromatogram obtained with reference solution (e). Heavy metals (2.3.13). 0.4 g complies with limit the test for heavy metals, Method B (50 ppm). Sulphated ash (2.3.18). Not more than 0.5 per cent. Water (2.3.43). 4.0 to 9.0 per cent, determined on 0.5 g. Assay. Determine by the microbiological assay of antibiotics, Method A or B (2.2.10), and express the results in g of oxytetracycline, C22H24N2O9, per mg. Oxytetracycline intended for use in the manufacture of parenteral preparations without a further procedure for the removal of bacterial endotoxins complies with the following additional requirement. Bacterial endotoxins (2.2.3). Not more than 0.4 Endotoxin Unit per mg of Oxytetracycline. Oxytetracycline intended for use in the manufacture of parenteral preparations without a further sterilisation procedure complies with the following additional requirement. Sterility. Complies with the test for sterility (2.2.11). Storage. Store protected from light and moisture. If it is intended for use in the manufacture of parenteral preparations, the container should be sterile and sealed so as to exclude micro-organisms. Labelling. The label states whether or not the contents are intended for use in the manufacture of parenteral preparations.
876
IP 2007
OXYTETRAXYCLINE HYDROCHLORIDE
Oxytetracycline Injection
Oxytetracycline Injection is a sterile solution of oxytetracycline with or without one or more suitable buffering agents, anaesthetics, preservatives, antioxidants, complexing agents and solvents. Oxytetracycline Injection contains not less than 90.0 per cent and not more than 120.0 per cent of the stated amount of anhydrous oxytetracycline, C22H24N2O9. Description. A clear, yellow to tan yellow solution. It may have a greenish tinge.
Other Tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. Determine by the microbiological assay of antibiotics, Method A or B (2.2.10), and express the result in mg of oxytetracycline, C22H24N2O9, per ml. Storage. Store protected from light. Labelling. The label states (1) the strength in mg of anhydrous oxytetracycline per ml; (2) that the contents are to be used for intramuscular use only; (3) the names of any preservatives used.
Identification
A. Determine by thin-layer chromatography (2.4.17), coating the plate with the substance prepared by mixing 25 g of silica gel G with 50 ml of a mixture of 2.5 ml of glycerin and 47.5 ml of 0.1 M disodium edetate previously adjusted to pH 7.0 with dilute ammonia solution. After spreading the plate, allow it to stand at room temperature till it is dry (70 to 90 minutes). Mobile phase. The lower layer formed after shaking 200 ml of a mixture of 2 volumes of ethyl acetate, 2 volumes of chloroform and 1 volume of acetone with 25 ml of 0.1 M disodium edetate previously adjusted to pH 7.0 with dilute ammonia solution. Test solution. Shake a quantity containing 10 mg of anhydrous oxytetracycline with 20 ml of methanol, centrifuging if necessary and use the clear supernatant liquid. Reference solution (a). A 0.05 per cent w/v solution of oxytetracycline hydrochloride RS in methanol. Reference solution (b). A solution containing 0.05 per cent w/v each of demethylchlortetracycline hydrochloride RS, oxytetracycline hydrochloride RS and tetracycline hydrochloride RS in methanol. Apply to the plate 1 l of each solution, freshly prepared. After development, dry the plate in air, expose to the vapours of ammonia and examine in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows three clearly separated spots. B. Add 0.1 ml to 2 ml of sulphuric acid; a red colour is produced. Add the solution to 1 ml of water; the colour changes to yellow.
Oxytetracycline Hydrochloride
C22H24N2O9,HCl Mol. Wt. 496.9 Oxytetracycline Hydrochloride is (4S,4aR,5S,5aR,6S, 12aS)4-dimethylamino-1,4,4a,5,5a,6,11,12a-octahydro-3,5, 6,10,12,12a-hexahydroxy-6-methyl-1,11-dioxonaphthacene-2carboxamide hydrochloride, a substance produced by the growth of certain strains of Streptomyces rimosus or obtained by any other means. Description. A pale yellow, crystalline powder; odourless; hygroscopic. Oxytetracycline Hydrochloride has a potency not less than 835 g of C22H24N2O9, per mg, calculated on the anhydrous basis.
Identification
A. Determine by thin-layer chromatography (2.4.17), coating the plate with the substance prepared by mixing 25 g of silica gel G with 50 ml of a mixture of 2.5 ml of glycerin and 47.5 ml of 0.1 M disodium edetate previously adjusted to pH 7.0 with dilute ammonia solution. After spreading the plate, allow it to stand at room temperature till it is dry (70 to 90 minutes). Mobile phase. The lower layer formed after shaking 200 ml of a mixture of 2 volumes of ethyl acetate, 2 volumes of chloroform and 1 volume of acetone with 25 ml of 0.1 M disodium edetate previously adjusted to pH 7.0 with dilute ammonia solution. Test solution. Dissolve 0.05 g of the substance under examination in 100 ml of methanol. Reference solution (a). A 0.05 per cent w/v solution of oxytetracycline hydrochloride RS in methanol. Reference solution (b). A solution containing 0.05 per cent w/v each of demethylchlortetracycline hydrochloride RS, oxytetracycline hydrochloride RS and tetracycline hydrochloride RS in methanol. Apply to the plate 1 l of each solution, freshly prepared. After development, dry the plate in air, expose to the vapours
Tests
pH (2.4.24). 8.0 to 9.0. Bacterial endotoxins (2.2.3). Not more than 0.4 Endotoxin Unit per mg of Oxytetracycline.
877
OXYTETRAXYCLINE HYDROCHLORIDE
IP 2007
of ammonia and examine in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows three clearly separated spots. B. To about 0.5 mg add 2 ml of sulphuric acid; a red colour is produced. Add the solution to 1 ml of water; the colour changes to yellow. C. Dissolve about 2 mg in 5 ml of a 1 per cent w/v solution of sodium carbonate and add 2 ml of diazotised sulphanilic acid solution; an orange-red to brownish-red colour is produced. D. A 5 per cent w/v solution gives the reactions of chlorides (2.3.1).
Reference solution (e). Dissolve 20 mg of b -apooxytetracycline RS in 20 ml of 0.01 M sodium hydroxide and dilute to 250 ml with 0.01 M hydrochloric acid. Reference solution (f). Dilute a mixture of 1.5 ml of reference solution (a), 1.0 ml of reference solution (b), 3.0 ml each of reference solutions (c) (d) and (e) to 25 ml with 0.01 M hydrochloric acid. Reference solution (g). Dilute a mixture of 1.0 ml of reference solution (b), 4.0 ml of reference solution (c) and 40.0 ml of reference solution (e) to 200 ml with 0.01 M hydrochloric acid. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with styrene-divinylbenzene co-polymer (8 to 10 m), column. temperature 60, mobile phase: transfer separately 30 g (for mobile phase A) or 100 g (for mobile phase B) of 2-methyl-2-propanol to volumetric flasks with the aid of 200 ml of water; to each flask add 60 ml of 0.33 M phosphate buffer pH 7.5, 50 ml of a 1.0 per cent w/v solution of tetrabutylammonium hydrogen sulphate previously adjusted to pH 7.5 with 2 M sodium hydroxide and 10 ml of a 0.04 per cent w/v solution of disodium edetate previously adjusted to pH 7.5 with 2 M sodium hydroxide and dilute each solution to 1000.0 ml with water., flow rate. 1 ml per minute, spectrophotometer set at 254 nm, a 20 l loop injector. Carry out a one-step gradient elution in the following manner. Pump a mixture containing 30 volumes of mobile phase B and 70 volumes of mobile phase A for 15 minutes, then pump a mixture containing 30 volumes of mobile phase A and 70 volumes of mobile phase B for 15 minutes and finally equilibrate with the first mixture. Adjust the sensitivity so that the heights of the peaks in the chromatogram obtained with reference solution (f) are at least 50 per cent of full-scale deflection of the recorder. The test is not valid unless, in the chromatogram obtained with reference solution (f), (a) the resolution between the first peak (4-epioxytetracycline) and the second peak (oxytetracycline) is at least 4.0, (b) the resolution between the second peak and the third peak (tetracycline) is at least 5.0, (c) the resolution between the fourth peak (a -apo-oxytetracycline) and the fifth peak (b-apo-oxytetracycline) is at least 3.5, and (d) the symmetry factor of the second peak is at most 1.25. If necessary adjust the proportions of the mobile phases used to produce the one-step gradient elution. Adjust the timeprogramme for the one-step gradient elution if necessary. Inject reference solution (a) six times. The test is not valid if the relative standard deviation of the area of the peak due to
Tests
pH (2.4.24). 2.0 to 3.0, determined in a 1.0 per cent w/v solution. Specific optical rotation (2.4.22). 188 to 200, determined at 20 in a 1 per cent w/v solution in 0.1 M hydrochloric acid. Light absorption (2.4.7). Absorbance of a 0.002 per cent w/v solution in chloride buffer solution pH 2.0 at the maximum at about 353 nm, 0.54 to 0.58. Light-absorbing impurities. A. Dissolve 20 mg in sufficient of a mixture of 1 volume of 1 M hydrochloric acid and 99 volumes of methanol to produce 10 ml. Absorbance of the resulting solution at about 430 nm, when measured within 1 hour of preparing the solution, not more than 0.50, calculated on the anhydrous basis (2.4.7). B. Dissolve 0.1 g in sufficient of a mixture of 1 volume of 1 M hydrochloric acid and 99 volumes of methanol to produce 10 ml. Absorbance of the resulting solution at about 490 nm, when measured within 1 hour of preparing the solution, not more than 0.20, calculated on the anhydrous basis (2.4.7). Related substances. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 20 mg of the substance under examination in 25 ml of 0.01 M hydrochloric acid. Reference solution (a). Dissolve 20 mg of oxytetracycline RS in 25 ml of 0.01 M hydrochloric acid. Reference solution (b). Dissolve 20 mg of 4-epioxytetracycline RS in 25 ml of 0.01 M hydrochloric acid. Reference solution (c). Dissolve 20 mg of tetracycline hydrochloride RS in 25 ml of 0.01 M hydrochloric acid. Reference solution (d). Dissolve 20 mg of a-apooxytetracycline RS in 20 ml of 0.01 M sodium hydroxide and dilute to 250 ml with 0.01 M hydrochloric acid.
878
IP 2007
OXYTETRAXYCLINE CAPSULES
oxytetracycline is greater than 1.0 per cent. If necessary, adjust the integrator parameters. Inject the test solution and reference solution (g). In the chromatogram obtained with the test solution the area of any peak corresponding to 4-epioxytetracycline or tetracycline is not greater than the area of the corresponding peak in the chromatogram obtained with reference solution (g) and the total area of the peaks corresponding to a -apo-oxytetracycline and to b -apo-oxytetracycline and any peak between the latter two is not greater than the area of the peak due to b -apooxytetracycline in the chromatogram obtained with reference solution (g). In the chromatogram obtained with the test solution the area of any peak appearing on the tail of the principal peak is not greater than 4.0 times that of the peak due to 4-epioxytetracycline in the chromatogram obtained with reference solution (g). Heavy metals (2.3.13). 0.4 g complies with the limit test for heavy metals, Method B (50 ppm). Sulphated ash (2.3.18). Not more than 0.5 per cent. Water (2.3.43). Not more than 2.0 per cent w/w, determined on 0.5 g. Assay. Determine by the microbiological assay of antibiotics, Method A or B (2.2.10), and express the result in g of oxytetracycline, C22H24N2O9, per mg. Oxytetracycline Hydrochloride intended for use in the manufacture of parenteral preparations without a further procedure for the removal of bacterial endotoxins complies with the following additional requirement. Bacterial endotoxins (2.2.3). Not more than 0.4 Endotoxin Unit per mg. Oxytetracycline Hydrochloride intended for use in the manufacture of parenteral preparations without a further sterilisation procedure complies with the following additional requirement. Sterility. Complies with test for sterility (2.2.11). Storage. Store protected from light and moisture. If it is intended for use in the manufacture of parenteral preparations, the container should be sterile and sealed so as to exclude micro-organisms. Labelling. The label states whether or not the contents are intended for use in the manufacture of parenteral preparations.
Identification
A. Determine by thin-layer chromatography (2.4.17), coating the plate with the substance prepared by mixing 25 g of silica gel G with 50 ml of a mixture of 2.5 ml of glycerin and 47.5 ml of 0.1 M disodium edetate previously adjusted to pH 7.0 with dilute ammonia solution. After spreading the plate, allow it to stand at room temperature till it is dry (70 to 90 minutes). Mobile phase. The lower layer formed after shaking 200 ml of a mixture of 2 volumes of ethyl acetate, 2 volumes of chloroform and 1 volume of acetone with 25 ml of 0.1 M disodium edetate previously adjusted to pH 7.0 with dilute ammonia solution. Test solution. Extract a quantity of the contents of the capsules containing 10 mg of Oxytetracycline Hydrochloride with 20 ml methanol, centrifuge and use the supernatant liquid. Reference solution (a). A 0.05 per cent w/v solution of oxytetracycline hydrochloride RS in methanol. Reference solution (b). A solution containing 0.05 per cent w/v each of demethylchlortetracycline hydrochloride RS, oxytetracycline hydrochloride RS and tetracycline hydrochloride RS in methanol. Apply to the plate 1 l of each solution, freshly prepared. After development, dry the plate in air, expose to the vapours of ammonia and examine in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows three clearly separated spots. B. To 0.5 mg of the contents of the capsules add 2 ml of sulphuric acid; a red colour is produced. Add the solution to 1 ml of water; the colour changes to yellow. C. Dissolve about 2 mg of the contents of the capsules in 5 ml of a 1 per cent w/v solution of sodium carbonate and add 2 ml of diazotised sulphanilic acid solution; a light brown colour is produced. D. The contents of the capsules give the reactions of chlorides (2.3.1).
Tests
Light-absorbing impurities. Dissolve a portion of the mixed contents of five capsules as completely as possible in sufficient of a mixture of 1 volume of 1 M hydrochloric acid and 99 volumes of methanol to produce two solutions of Oxytetracycline Hydrochloride containing (1) 0.2 per cent w/v and (2) 1.0 per cent w/v and filter each solution. Absorbance of the filtrate obtained from solution (1) at about 430 nm, when measured within 1 hour of preparing the solution, not greater
Oxytetracycline Capsules
Oxytetracycline Hydrochloride Capsules
Oxytetracycline Capsules contain not less than 95.0 per cent and not more than 110.0 per cent of the stated amount of oxytetracycline hydrochloride, C22H24N2O9,HCl.
879
OXYTETRAXYCLINE EYE OINTMENT
IP 2007
than 0.75 and of the filtrate obtained from solution (2) at about 490 nm, not more than 0.40 (2.4.7). Dissolution (2.5.2). Apparatus. No 1 Medium. 900 ml of 0.1 M hydrochloric acid. Speed and time. 100 rpm and 45 minutes. Withdraw a suitable volume of the medium and filter through a membrane filter disc with an average pore diameter not greater than 1.0 m. Measure the absorbance of the filtrate, suitably diluted if necessary, at the maximum at about 353 nm (2.4.7). Calculate the content of C22H24N2O9,HCl in the medium taking 282 as the specific absorbance at 353 nm. D. Not less than 75 per cent of the stated amount of C22H24N2O9,HCl. Loss on drying (2.4.19). Not more than 5.0 per cent, determined on 1.0 g of the mixed contents of the capsules by drying in an oven at 60 at a pressure not exceeding 0.7 kPa for 3 hours. Other Tests. Comply with the tests stated under Capsules. Assay. Weigh accurately a quantity of the mixed contents of 20 capsules containing about 0.25 g of Oxytetracycline Hydrochloride, add 250.0 ml of water, shake, filter. Determine by the microbiological assay of antibiotics, Method A or B (2.2.10), and express the results in mg of oxytetracycline hydrochloride per capsule taking each mg of oxytetracycline to be equivalent to 1.079 mg of oxytetracycline hydrochloride, C22H24N2O9,HCl. Storage. Store protected from light and moisture.
Test solution. A solution prepared by heating a quantity containing 20 mg of Oxytetracycline Hydrochloride with 20 ml of methanol for 20 minutes, cooling in ice, filtering, carefully evaporating the filtrate to dryness and dissolving the residue in 20 ml of methanol. Reference solution (a). A 0.05 per cent w/v solution of oxytetracycline hydrochloride RS in methanol. Reference solution (b). A solution containing 0.05 per cent w/v each of demethylchlortetracycline hydrochloride RS, oxytetracycline hydrochloride RS and tetracycline hydrochloride RS in methanol. Apply to the plate 1 l of each solution, freshly prepared. After development, dry the plate in air, expose to the vapours of ammonia and examine in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows three clearly separated spots.
Tests
Water (2.3.43). Not more than 1.0 per cent, determined on 0.5 g. Other Tests. Complies with the tests stated under Eye Ointments. Assay. Weigh accurately about 1.0 g and transfer to a separating funnel. Add 25 ml of peroxide-free ether, shake well and extract with five quantities, each of 20 ml, of 0.1 M hydrochloric acid. Combine the extracts and dilute to 200.0 ml with 0.1 M hydrochloric acid. Dilute a suitable volume of the resulting solution with buffer solution No 3 (2.2.10), to produce a solution containing 1 g of oxytetracycline per ml. Determine by the microbiological assay of antibiotics, Method B (2.2.10), and express the results as a percentage of oxytetracycline hydrochloride taking each mg of oxytetracycline to be equivalent to 1.079 mg of oxytetracycline hydrochloride, C22H24N2O9,HCl. Storage. Store protected from light and moisture.
Oxytetracycline Eye Ointment

Oxytetracycline Hydrochloride Eye Ointment
Oxytetracycline Eye Ointment contains not less than 90.0 per cent and not more than 115.0 per cent of the stated amount of oxytetracycline hydrochloride, C22H24N2O9,HCl.
Identification
Determine by thin-layer chromatography (2.4.17), coating the plate with the substance prepared by mixing 25 g of silica gel G with 50 ml of a mixture of 2.5 ml of glycerin and 47.5 ml of 0.1 M disodium edetate previously adjusted to pH 7.0 with dilute ammonia solution. After spreading the plate, allow it to stand at room temperature till it is dry (70 to 90 minutes). Mobile phase. The lower layer formed after shaking 200 ml of a mixture of 2 volumes of ethyl acetate, 2 volumes of chloroform and 1 volume of acetone with 25 ml of 0.1 M disodium edetate previously adjusted to pH 7.0 with dilute ammonia solution.
Oxytetracycline Hydrochloride Injection

Oxytetracycline Hydrochloride Injection is a sterile material consisting of Oxytetracycline Hydrochloride with or without buffering agents and other excipients. It is filled in a sealed container. The injection is constituted by dissolving the contents of the sealed container in the requisite amount of sterile Water for Injections, immediately before use.
880
IP 2007
OXYTOCIN
The constituted solution complies with the requirements for Clarity of solution and Particulate matter stated under Parenteral Preparations (Injections). Storage. The constituted solution may be used within three days of preparation when stored in a refrigerator (2 to 8). Oxytetracycline Hydrochloride Injection contains not less than 90.0 per cent and not more than 115.0 per cent of the stated amount of oxytetracycline, C22H24N2O9. The contents of the sealed container comply with the requirements stated under Parenteral Preparations (Powders for Injection) and with the following requirements. Description. A pale yellow, crystalline powder.
Tests
Appearance of solution. A 10.0 per cent w/v solution is clear (2.4.1) and yellow. pH (2.4.24). 2.0 to 3.0, determined in a 1.0 per cent w/v solution. Light-absorbing impurities. A. Dissolve 20 mg in sufficient of a mixture of 1 volume of 1 M hydrochloric acid and 99 volumes of methanol to produce 10 ml. Absorbance of the resulting solution at about 430 nm, when measured within 1 hour of preparing the solution, not more than 0.50, calculated on the anhydrous basis (2.4.7). B. Dissolve 0.1 g in sufficient of a mixture of 1 volume of 1 M hydrochloric acid and 99 volumes of methanol to produce 10 ml. Absorbance of the resulting solution at about 490 nm, when measured within 1 hour of preparing the solution, not more than 0.20, calculated on the anhydrous basis (2.4.7). Assay. Determine by the microbiological assay of antibiotics, method A or B (2.2.10), and express the result in g of oxytetracycline, C22H24N2O9, per mg. Bacterial endotoxins (2.2.3). Not more than 0.4 Endotoxin Unit per mg. Storage. Store protected from light and moisture. Labelling. The label states (1) the quantity of Oxytetracycline Hydrochloride contained in it in terms of the equivalent amount of oxytetracycline; (2) that the contents are to be used for intravenous injection only; (3) the names of the buffering agents used.
Identification
A. Determine by thin-layer chromatography (2.4.17), coating the plate with the substance prepared by mixing 25 g of silica gel G with 50 ml of a mixture of 2.5 ml of glycerin and 47.5 ml of 0.1 M disodium edetate previously adjusted to pH 7.0 with dilute ammonia solution. After spreading the plate, allow it to stand at room temperature till it is dry (70 to 90 minutes). Mobile phase. The lower layer formed after shaking 200 ml of a mixture of 2 volumes of ethyl acetate, 2 volumes of chloroform and 1 volume of acetone with 25 ml of 0.1 M disodium edetate previously adjusted to pH 7.0 with dilute ammonia solution. Test solution. Dissolve 0.05 g of the substance under examination in 100 ml of methanol. Reference solution (a). A 0.05 per cent w/v solution of oxytetracycline hydrochloride RS in methanol. Reference solution (b). A solution containing 0.05 per cent w/v each of demethylchlortetracycline hydrochloride RS, oxytetracycline hydrochloride RS and tetracycline hydrochloride RS in methanol. Apply to the plate 1 l of each solution, freshly prepared. After development, dry the plate in air, expose to the vapours of ammonia and examine in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows three clearly separated spots. B. To about 0.5 mg add 2 ml of sulphuric acid; a red colour is produced. Add the solution to 1 ml of water; the colour changes to yellow. C. Dissolve about 2 mg in 5 ml of a 1 per cent w/v solution of sodium carbonate and add 2 ml of diazotised sulphanilic acid solution; an orange-red to brownish-red colour is produced.
Oxytocin
Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-GlyNH2
C43H66N12O12S2
Mol. Wt.1007.2
Oxytocin is a cyclic nonapeptide hormone obtained by a process of fractionation from the posterior lobe of the pituitary gland of healthy oxen or other mammals or by synthesis that has the property of stimulating contraction of the uterus and milk ejection in receptive animals. It may be presented as a solid or as a solution in a solvent containing an appropriate antimicrobial preservative such as 0.2 per cent w/v solution of chlorbutol. If it is derived from animal species, Oxytocin contains not less than 90.0 per cent and not more than 111.0 per cent of the stated number of Units of oxytocic activity. If it is a synthetic product presented as a solid, it contains not less than 560 Units per mg, calculated with reference to the peptide content and when presented as a liquid, it contains not less than 150 Units per ml.
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Description. When presented as a solid, a white or almost white powder. When presented as a liquid, a clear colourless liquid.
Identification
Gives rise to an appropriate response when administered as directed under one of the methods described for Assay.
Method A. By depression of the blood pressure in chicken Anaesthetise a young healthy adult cockerel weighing 1.2 to 2.3 kg with an anaesthetic that will maintain a prolonged and constant high blood pressure. Expose the gluteus primus muscle in one thigh and cut and retract it to reveal the popliteal artery and crural vein. Cannulate the popliteal artery and record the blood pressure on a suitable recorder calibrated for use over a linear range. Cannulate the crural or brachial vein. Immediately before use prepare a solution of the Standard Preparation in saline solution so that the volume to be injected is between 0.1 ml and 0.5 ml. Record the blood pressure responses to the injection into the cannulated vein of two doses of this solution; the doses should be such as to produce clearly discriminated, precipitous, submaximal decreases in blood pressure; the required doses normally lie between 20 and 100 milliUnits. The interval between injections should be constant and lie between 3 and 10 minutes depending on the rate at which the blood pressure returns to normal. Immediately before use dilute the preparation being examined with saline solution so as to obtain responses similar to those obtained with the Standard Preparation. The ratio between the two doses of the preparation under examination should be the same as that between the two doses of the Standard Preparation and this ratio should be kept constant throughout the assay. The two doses of the Standard Preparation and the two doses of the preparation under examination should be given according to a randomised block or a Latin square design and at least six responses to each should be recorded. If the animal rapidly becomes insensitive to the repeated injections of the solutions another animal must be used. Measure all the responses and calculate the result of the assay by standard statistical methods. Method B. By contraction of the rat uterus Inject 100 g of oestradiol benzoate intramuscularly into a female rat weighing 120 to 200 g 18 to 24 hours before the assay. Immediately before the assay confirm by vaginal smear that the rat is in oestrus or preoestrus. Kill the rat and suspend one horn of the uterus in a bath containing a solution of the following composition. Composition (per cent w/v) Sodium chloride 0.662 Potassium chloride 0.045 Calcium chloride 0.007 Sodium bicarbonate 0.256 Disodium hydrogen phosphate 0.029 Sodium dihydrogen phosphate 0.003 Magnesium chloride 0.010 Dextrose 0.050
Tests
Peptide. 90.0 to 110.0 per cent of the stated amount of oxytocin, C43H66N12O12S2 expressed per mg for the solid, and in mg per ml for the liquid, Determine by liquid chromatography (2.4.14). Test solution. Dissolve 3.5 mg of the substance under examination in sufficient of a 1.56 per cent w/v solution of sodium dihydrogen phosphate to produce 10.0 ml or use the liquid preparation as appropriate. Reference solution. Dissolve 3.5 mg of oxytocin RS in sufficient of a 1.56 per cent w/v solution of sodium dihydrogen phosphate to produce 10.0 ml. Chromatographic system a stainless steel column 12 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: appropriate proportions of a 1.56 per cent w/v solution of sodium dihydrogen phosphate (mobile phase A) and a mixture of equal volumes of acetonitrile and water (mobile phase B), flow rate. 1 ml per minute, spectrophotometer set at 220 nm, a 20 l loop injector. Equilibrate the column with a mixture of 70 volumes of mobile phase A and 30 volumes of mobile phase B and record the chromatograms as follows. Operate by gradient elution increasing continuously and linearly the proportion of mobile phase B by 1.0 per cent v/v per minute for 30 minutes. Finally elute using the same mixture for 15 minutes to re-equilibrate the column. Calculate the content of the peptide, C43H66N12O12S2. Assay.The potency of oxytocin is determined by comparing its activity with that of the Standard Preparation of oxytocin under the conditions of a suitable method of assay. Standard Preparation The Standard Preparation is the 4th International Standard for Oxytocin, established in 1978, consisting of freeze-dried synthetic oxytocin peptide with human albumin and citric acid (supplied in ampoules containing 12.5 Units), or any other suitable preparation the potency of which has been determined in relation to the International Standard. NOTE Any of the following methods may be followed.
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OXYTOCIN
Maintain the bath at a temperature of 32 or at some other suitable temperature at which spontaneous contractions of the uterus are abolished and the preparation maintains its sensitivity. Oxygenate the solution with a mixture of 95 per cent of oxygen and 5 per cent of carbon dioxide and record the contractions of the muscle using a suitable instrument giving a linear response (for example an isotonic lever with a load not exceeding 2 g). Record the contractions produced by the addition to the bath of two doses of the Standard Preparation suitably diluted with the above solution. The doses should be such as to produce clearly discriminated, submaximal contractions; the required doses normally lie between 10 and 50 micro Units per ml of bath liquid. When maximal contraction has been reached, replace the bath liquid by a fresh solution. The doses should be added at regular intervals of 3 to 5 minutes depending upon the rate of recovery of the muscle. Dilute the preparation under examination so as to obtain responses on the addition of two doses similar to those obtained with the Standard Preparation. The ratio between the two doses of the preparation under examination should be the same as that between the two doses of the Standard Preparation and this ratio should be kept constant throughout the assay. The two doses of Standard Preparation and the two doses of the preparation under examination should be given according to a randomised block or a Latin square design and at least six responses to each should be recorded. Measure all the responses and calculate the result of the assay by standard statistical methods. Method C. By measurement of milk-ejection pressure in a lactating rat Select a lactating rat, in the third to twentyfirst day after parturition and weighing about 300 g, separate it from the litter and 30 to 60 minutes later anaesthetise (for example, by the intraperitoneal injection of a solution of Pentobarbitone Sodium). Tie the rat to an operating table, maintained at 37, by its hind legs leaving the front legs free. Cannulate the trachea with a short polyethylene tube of internal diameter about 2.5 mm in such a manner so as to ensure a free airway; apply artificial respiration only if necessary. Cannulate an external jugular or femoral vein with a polyethylene tube of internal diameter about 0.4 mm which is filled with saline solution and closed with a pin. Shave the skin surrounding the inguinal and abdominal teats and excise the tip of one teat, preferably the lower inguinal teat. Insert a polyethylene tube of internal diameter about 0.3 mm and external diameter about 0.6 mm, to a depth sufficient to obtain appropriate measurement of pressure (3 to 10 mm depth), into the primary teat duct which opens onto the cut surface and tie firmly in place with a ligature. Connect this cannula with a suitable strain gauge transducer (such as that used for recording arterial blood pressure in the rat) and fill
the whole system with a 3.8 per cent w/v solution of sodium citrate or saline solution containing 50 Units of heparin sodium per ml to prevent clotting of milk. After cannulation, inject a small volume (0.05 to 0.2 ml) of this solution into the teat duct through the transducer to clear the milk from the tip of the cannula. (This procedure may be repeated during the assay should obstruction arise from milk ejected into the cannula). Clamp the strain gauge so that a slight tension is applied to the teat and its natural alignment is preserved and connect the gauge to a potentiometric recorder adjusted to give full-scale deflection for an increase in milk-ejection pressure of about 5.3 kPa. Inject all solutions through the venous cannula using a 1-ml syringe graduated in 0.01 ml and wash them in with 0.2 ml of saline solution. Prepare a solution of the Standard Preparation and a solution of the preparation under examination in saline solution so that the volume to be injected is between 0.1 ml and 0.4 ml. Choose two doses of the Standard Preparation such that the increase in milk-ejection pressure is about 1.35 kPa for the lower dose and about 2.7 kPa for the higher dose. As an initial approximation, a lower dose of between 0.1 and 0.4 milliUnit and an upper dose of 1.5 to 2 times this amount may be tried. Choose two doses of the preparation under examination with the same inter-dose ratio, matching the effects of the doses of the Standard Preparation as closely as possible. Inject the four doses (two doses of the Standard Preparation and two doses of the preparation being examined) at intervals of 3 to 5 minutes. The two doses of Standard Preparation and the two doses of the preparation under examination should be given according to a randomised block or a Latin square design and at least four responses to each should be recorded. Measure all the responses and calculate the result of the assay by standard statistical methods. The estimated potency is not less than 90 per cent and not more than 111 per cent of the stated potency. The fiducial limits of error are not less than 80 per cent and not more than 125 per cent of the stated potency. Oxytocin of natural origin obtained by extraction and purification complies with the following additional requirement. Vasopressor activity. Not more than 0.5 Unit per 20 Units of oxytocic activity, when assayed by the biological assay for vasopressor activity described below. The vasopressor activity is estimated by comparing the activity of the preparation under examination with that of the Standard Preparation of arginine vasopressin under the conditions of a suitable method of assay. Standard Preparation The Standard Preparation is the Ist International Standard for Arginine vasopressin, established in 1978, consisting of
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freeze-dried synthetic arginine vasopressin peptide acetate with human albumin and citric acid (supplied in ampoules containing 8.20 Units), or another suitable preparation the potency of which has been determined in relation to that of the International Standard. Inject slowly into the tail vein of a male albino rat weighing about 300 g a solution of a suitable a-adrenoceptor blocking agent, for example 10 ml per kg of body weight of a solution prepared by dissolving 5 mg of phenoxybenzamine hydrochloride in 0.1 ml of ethanol (95 per cent), adding 0.05 ml of 1 M hydrochloric acid and diluting to 5 ml with saline solution. After 18 hours, anaesthetise the rat with an anaesthetic that will maintain a prolonged and uniform blood pressure. After 45 to 60 minutes, tie the rat on its back to the operating table by its hind legs. Cannulate the trachea with a short polyethylene tube of external diameter about 2.5 mm and dissect a carotid artery ready for cannulation. Then cannulate the femoral vein close to the inguinal ligament. Retract the abdominal muscles to expose the inguinal ligament. Retract the superficial pudendal vein to one side and dissect the femoral vein towards the inguinal ligament from the corresponding artery. When dissecting, a deep branch reaching the femoral vein must be found and tied off to prevent bleeding during cannulation. Tie a short polyethylene cannula of external diameter about 1 mm into the femoral vein by two ligatures and join by a short piece of flexible tubing to a 1-ml burette with an attached thistle funnel containing saline solution at about 37. Firmly fix a wet absorbent cotton swab to the thigh so as to cover the incision and cannula. At this stage inject through the venous cannula 200 Units of heparin, dissolved in saline solution, per 100 g of body weight. Then tie in a carotid cannula of external diameter about 1 mm and connect by a column of saline solution containing heparin with a suitable pressure measuring device such as a mercury manometer of internal diameter about 2 to 3 mm. The central and peripheral nervous system including both vagus and associated sympathetic nerves is left intact. No artificial respiration is necessary. Taking care that no air is injected, inject all solutions through the venous cannula by means of a 1-ml syringe graduated in 0.01 ml and wash in with 0.2 ml of saline solution from the burette. Dilute the extract of the Standard Preparation and the preparation under examination with saline solution so that the volume to be injected is between 0.1 ml and 0.5 ml. Choose two doses of the Standard Preparation such that the elevation of the blood pressure is about 4 kPa for the lower dose and about 7 kPa but always submaximal for the higher dose, the ratio of low to high dose being determined by the response and usually being 3 to 5. As an initial approximation doses of 3 and 5 milliUnits may be tried. Choose two doses of the preparation under examination with the same inter-dose
ratio, matching the effects of the dose of the Standard Preparation as closely as possible. Inject doses at intervals of 10 to 15 minutes. The two doses of the Standard Preparation and the two doses of the preparation under examination should be given in a randomised block or a Latin square design and four to five responses to each should be recorded. Measure all the responses and calculate the result of the assay by standard statistical methods. Oxytocin intended for use in the manufacture of parenteral preparations without a further procedure for the removal of bacterial endotoxins.complies with the following additional requirement. Bacterial endotoxins (2.2.3). Not more than 100 Endotoxin Units per 200 Units of oxytocin. Oxytocin intended for use in the manufacture of parenteral preparations without a further sterilisation procedure complies with the following additional requirement. Sterility (2.2.11). Complies with the test for sterility. Storage. Store protected from moisture. If the substance is intended for use in the manufacture of parenteral preparations, the container should be sterile, tamper-evident and sealed so as to exclude micro-organisms. Labelling. The label states (1) the number of Units of oxytocic activity per mg (for solid) or per ml (for liquid); (2) either the animal species from which it is obtained or whether it is synthetic, as appropriate; (3) whether or not the contents are intended for use in the manufacture of parenteral preparations.
Oxytocin Injection
Oxytocin Injection is a sterile solution of Oxytocin in Water for Injections. Oxytocin Injection contains not less than 90.0 per cent and not more than 110.0 per cent of the stated number of Units of oxytocin activity. Description. A clear, colourless liquid.
Identification
Gives rise to an appropriate response when administered as directed under one of the methods described for Assay.
Tests
pH (2.4.24). 3.5 to 4.5. Other Tests. Complies with the tests stated under Parenteral Preparations (Injections).
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IP 2007
OXYTOCIN INJECTION
Bacterial endotoxins (2.2.3). Not more than 100 Endotoxin Units per 200 Units of oxytocin. Assay.The potency of oxytocin is determined by comparing its activity with that of the Standard Preparation of oxytocin under the conditions of a suitable method of assay. Standard Preparation The Standard Preparation is the 4th International Standard for Oxytocin, established in 1978, consisting of freeze-dried synthetic oxytocin peptide with human albumin and citric acid (supplied in ampoules containing 12.5 Units), or any other suitable preparation the potency of which has been determined in relation to the International Standard. NOTE Any of the following methods may be followed. Method A. By depression of the blood pressure in chicken Anaesthetise a young healthy adult cockerel weighing 1.2 to 2.3 kg with an anaesthetic that will maintain a prolonged and constant high blood pressure. Expose the gluteus primus muscle in one thigh and cut and retract it to reveal the popliteal artery and crural vein. Cannulate the popliteal artery and record the blood pressure on a suitable recorder calibrated for use over a linear range. Cannulate the crural or brachial vein. Immediately before use prepare a solution of the Standard Preparation in saline solution so that the volume to be injected is between 0.1 ml and 0.5 ml. Record the blood pressure responses to the injection into the cannulated vein of two doses of this solution; the doses should be such as to produce clearly discriminated, precipitous, submaximal decreases in blood pressure; the required doses normally lie between 20 and 100 milliUnits. The interval between injections should be constant and lie between 3 and 10 minutes depending on the rate at which the blood pressure returns to normal. Immediately before use dilute the preparation being examined with saline solution so as to obtain responses similar to those obtained with the Standard Preparation. The ratio between the two doses of the preparation under examination should be the same as that between the two doses of the Standard Preparation and this ratio should be kept constant throughout the assay. The two doses of the Standard Preparation and the two doses of the preparation under examination should be given according to a randomised block or a Latin square design and at least six responses to each should be recorded. If the animal rapidly becomes insensitive to the repeated injections of the solutions another animal must be used. Measure all the responses and calculate the result of the assay by standard statistical methods. Method B. By contraction of the rat uterus Inject 100 g of oestradiol benzoate intramuscularly into a female rat weighing 120 to 200 g 18 to 24 hours before the assay. Immediately before the assay confirm by vaginal smear that the rat is in
oestrus or preoestrus. Kill the rat and suspend one horn of the uterus in a bath containing a solution of the following composition. Composition (per cent w/v) Sodium chloride 0.662 Potassium chloride 0.045 Calcium chloride 0.007 Sodium bicarbonate 0.256 Disodium hydrogen phosphate 0.029 Sodium dihydrogen phosphate 0.003 Magnesium chloride 0.010 Dextrose 0.050 Maintain the bath at a temperature of 32 or at some other suitable temperature at which spontaneous contractions of the uterus are abolished and the preparation maintains its sensitivity. Oxygenate the solution with a mixture of 95 per cent of oxygen and 5 per cent of carbon dioxide and record the contractions of the muscle using a suitable instrument giving a linear response (for example an isotonic lever with a load not exceeding 2 g). Record the contractions produced by the addition to the bath of two doses of the Standard Preparation suitably diluted with the above solution. The doses should be such as to produce clearly discriminated, submaximal contractions; the required doses normally lie between 10 and 50 micro Units per ml of bath liquid. When maximal contraction has been reached, replace the bath liquid by a fresh solution. The doses should be added at regular intervals of 3 to 5 minutes depending upon the rate of recovery of the muscle. Dilute the preparation being examined so as to obtain responses on the addition of two doses similar to those obtained with the Standard Preparation. The ratio between the two doses of the preparation under examination should be the same as that between the two doses of the Standard Preparation and this ratio should be kept constant throughout the assay. The two doses of Standard Preparation and the two doses of the preparation under examination should be given according to a randomised block or a Latin square design and at least six responses to each should be recorded. Measure all the responses and calculate the result of the assay by standard statistical methods. Method C. By measurement of milk-ejection pressure in a lactating rat Select a lactating rat, in the third to twentyfirst day after parturition and weighing about 300 g, separate it from the litter and 30 to 60 minutes later anaesthetise (for example, by the intraperitoneal injection of a solution of Pentobarbitone Sodium). Tie the rat to an operating table, maintained at 37, by its hind legs leaving the front legs free. Cannulate the trachea with a short polyethylene tube of
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internal diameter about 2.5 mm in such a manner so as to ensure a free airway; apply artificial respiration only if necessary. Cannulate an external jugular or femoral vein with a polyethylene tube of internal diameter about 0.4 mm which is filled with saline solution and closed with a pin. Shave the skin surrounding the inguinal and abdominal teats and excise the tip of one teat, preferably the lower inguinal teat. Insert a polyethylene tube of internal diameter about 0.3 mm and external diameter about 0.6 mm, to a depth sufficient to obtain appropriate measurement of pressure (3 to 10 mm depth), into the primary teat duct which opens onto the cut surface and tie firmly in place with a ligature. Connect this cannula with a suitable strain gauge transducer (such as that used for recording arterial blood pressure in the rat) and fill the whole system with a 3.8 per cent w/v solution of sodium citrate or saline solution containing 50 Units of heparin sodium per ml to prevent clotting of milk. After cannulation, inject a small volume (0.05 to 0.2 ml) of this solution into the teat duct through the transducer to clear the milk from the tip of the cannula. (This procedure may be repeated during the assay should obstruction arise from milk ejected into the cannula). Clamp the strain gauge so that a slight tension is applied to the teat and its natural alignment is preserved and connect the gauge to a potentiometric recorder adjusted to give full-scale deflection for an increase in milk-ejection pressure of about 5.3 kPa. Inject all solutions through the venous cannula using a 1-ml syringe graduated in 0.01 ml and wash them in with 0.2 ml of saline solution. Prepare a solution of the Standard Preparation and a solution of the preparation under examination in saline solution so that the volume to be injected is between 0.1 ml and 0.4 ml. Choose two doses of the Standard Preparation such that the increase in milk-ejection pressure is about 1.35 kPa for the lower dose and about 2.7 kPa for the higher dose. As an initial approximation, a lower dose of between 0.1 and 0.4 milliUnit and an upper dose of 1.5 to 2 times this amount may be tried. Choose two doses of the preparation under examination with the same inter-dose ratio, matching the effects of the doses of the Standard Preparation as closely as possible. Inject the four doses (two doses of the Standard Preparation and two doses of the preparation under examination at intervals of 3 to 5 minutes. The two doses of Standard Preparation and the two doses of the preparation under examination should be given according to a randomised block or a Latin square design and at least four responses to each should be recorded. Measure all the responses and calculate the result of the assay by standard statistical methods. The estimated potency is not less than 90 per cent and not more than 111 per cent of the stated potency. The fiducial limits of error are not less than 80 per cent and not more than 125 per cent of the stated potency.
Oxytocin Injection containing Oxytocin of natural origin obtained by extraction and purification complies with the following additional requirement. Vasopressor activity. Not more than 0.5 Unit per 20 Units of oxytocic activity, when assayed by the biological assay for vasopressor activity described below. The vasopressor activity is estimated by comparing the activity of the preparation under examination with that of the Standard Preparation of arginine vasopressin under the conditions of a suitable method of assay. Standard Preparation The Standard Preparation is the Ist International Standard for Arginine vasopressin, established in 1978, consisting of freeze-dried synthetic arginine vasopressin peptide acetate with human albumin and citric acid (supplied in ampoules containing 8.20 Units), or any other suitable preparation the potency of which has been determined in relation to that of the International Standard. Inject slowly into the tail vein of a male albino rat weighing about 300 g a solution of a suitable a-adrenoceptor blocking agent, for example 10 ml per kg of body weight of a solution prepared by dissolving 5 mg of phenoxybenzamine hydrochloride in 0.1 ml of ethanol (95 per cent), adding 0.05 ml of 1 M hydrochloric acid and diluting to 5 ml with saline solution. After 18 hours, anaesthetise the rat with an anaesthetic that will maintain a prolonged and uniform blood pressure. After 45 to 60 minutes, tie the rat on its back to the operating table by its hind legs. Cannulate the trachea with a short polyethylene tube of external diameter about 2.5 mm and dissect a carotid artery ready for cannulation. Then cannulate the femoral vein close to the inguinal ligament. Retract the abdominal muscles to expose the inguinal ligament. Retract the superficial pudendal vein to one side and dissect the femoral vein towards the inguinal ligament from the corresponding artery. When dissecting, a deep branch reaching the femoral vein must be found and tied off to prevent bleeding during cannulation. Tie a short polyethylene cannula of external diameter about 1 mm into the femoral vein by two ligatures and join by a short piece of flexible tubing to a 1-ml burette with an attached thistle funnel containing saline solution at about 37. Firmly fix a wet absorbent cotton swab to the thigh so as to cover the incision and cannula. At this stage inject through the venous cannula 200 Units of heparin, dissolved in saline solution, per 100 g of body weight. Then tie in a carotid cannula of external diameter about 1 mm and connect by a column of saline solution containing heparin with a suitable pressure measuring device such as a mercury manometer of internal diameter about 2 to 3 mm. The central and peripheral nervous system including both vagus and associated sympathetic nerves is left intact. No
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OXYTOCIN NASAL SOLUTION
artificial respiration is necessary. Taking care that no air is injected, inject all solutions through the venous cannula by means of a 1-ml syringe graduated in 0.01 ml and wash in with 0.2 ml of saline solution from the burette. Dilute the extract of the Standard Preparation and the preparation under examination with saline solution so that the volume to be injected is between 0.1 ml and 0.5 ml. Choose two doses of the Standard Preparation such that the elevation of the blood pressure is about 4 kPa for the lower dose and about 7 kPa but always submaximal for the higher dose, the ratio of low to high dose being determined by the response and usually being 3 to 5. As an initial approximation doses of 3 and 5 milliUnits may be tried. Choose two doses of the preparation under examination with the same inter-dose ratio, matching the effects of the dose of the Standard Preparation as closely as possible. Inject doses at intervals of 10 to 15 minutes. The two doses of the Standard Preparation and the two doses of the preparation under examination should be given in a randomised block or a Latin square design and four to five responses to each should be recorded. Measure all the responses and calculate the result of the assay by standard statistical methods. Storage. Store at temperature not exceeding 30. Do not freeze. Labelling. The label states (1) the number of Units of oxytocin activity per ml; (2) either the animal spieces from which it is obtained or whether it is synthetic, as appropriate.
synthetic oxytocin peptide with human albumin and citric acid (supplied in ampoules containing 12.5 Units), or any other suitable preparation the potency of which has been determined in relation to the International Standard. NOTE Any of the following methods may be followed. Method A. By depression of the blood pressure in chicken Anaesthetise a young healthy adult cockerel weighing 1.2 to 2.3 kg with an anaesthetic that will maintain a prolonged and constant high blood pressure. Expose the gluteus primus muscle in one thigh and cut and retract it to reveal the popliteal artery and crural vein. Cannulate the popliteal artery and record the blood pressure on a suitable recorder calibrated for use over a linear range. Cannulate the crural or brachial vein. Immediately before use prepare a solution of the Standard Preparation in saline solution so that the volume to be injected is between 0.1 ml and 0.5 ml. Record the blood pressure responses to the injection into the cannulated vein of two doses of this solution; the doses should be such as to produce clearly discriminated, precipitous, submaximal decreases in blood pressure; the required doses normally lie between 20 and 100 milliUnits. The interval between injections should be constant and lie between 3 and 10 minutes depending on the rate at which the blood pressure returns to normal. Immediately before use dilute the preparation being examined with saline solution so as to obtain responses similar to those obtained with the Standard Preparation. The ratio between the two doses of the preparation under examination should be the same as that between the two doses of the Standard Preparation and this ratio should be kept constant throughout the assay. The two doses of the Standard Preparation and the two doses of the preparation under examination should be given according to a randomised block or a Latin square design and at least six responses to each should be recorded. If the animal rapidly becomes insensitive to the repeated injections of the solutions another animal must be used. Measure all the responses and calculate the result of the assay by standard statistical methods. Method B. By contraction of the rat uterus Inject 100 g of oestradiol benzoate intramuscularly into a female rat weighing 120 to 200 g 18 to 24 hours before the assay. Immediately before the assay confirm by vaginal smear that the rat is in oestrus or preoestrus. Kill the rat and suspend one horn of the uterus in a bath containing a solution of the following composition. Sodium chloride Potassium chloride Calcium chloride Sodium bicarbonate Composition (per cent w/v) 0.662 0.045 0.007 0.256
Oxytocin Nasal Solution

Oxytocin Nasal Solution is a solution of Oxytocin in a suitable solvent containing an appropriate antimicrobial preservative. Oxytocin Nasal Solution contains not less than 85.0 per cent and not more than 120.0 per cent of the stated number of Units of oxytocic activity. Description. A clear, colourless solution.
Tests
pH (2.4.24). 3.5 to 4.5. Other Tests. Complies with the tests stated under Nasal Preparations. Assay.The potency of oxytocin is determined by comparing its activity with that of the Standard Preparation of oxytocin under the conditions of a suitable method of assay. Standard Preparation The Standard Preparation is the 4th International Standard for Oxytocin, established in 1978, consisting of freeze-dried
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Disodium hydrogen phosphate Sodium dihydrogen phosphate Magnesium chloride Dextrose
0.029 0.003 0.010 0.050
Maintain the bath at a temperature of 32 or at some other suitable temperature at which spontaneous contractions of the uterus are abolished and the preparation maintains its sensitivity. Oxygenate the solution with a mixture of 95 per cent of oxygen and 5 per cent of carbon dioxide and record the contractions of the muscle using a suitable instrument giving a linear response (for example an isotonic lever with a load not exceeding 2 g). Record the contractions produced by the addition to the bath of two doses of the Standard Preparation suitably diluted with the above solution. The doses should be such as to produce clearly discriminated, submaximal contractions; the required doses normally lie between 10 and 50 micro Units per ml of bath liquid. When maximal contraction has been reached, replace the bath liquid by a fresh solution. The doses should be added at regular intervals of 3 to 5 minutes depending upon the rate of recovery of the muscle. Dilute the preparation being examined so as to obtain responses on the addition of two doses similar to those obtained with the Standard Preparation. The ratio between the two doses of the preparation under examination should be the same as that between the two doses of the Standard Preparation and this ratio should be kept constant throughout the assay. The two doses of Standard Preparation and the two doses of the preparation under examination should be given according to a randomised block or a Latin square design and at least six responses to each should be recorded. Measure all the responses and calculate the result of the assay by standard statistical methods. Method C. By measurement of milk-ejection pressure in a lactating rat Select a lactating rat, in the third to twentyfirst day after parturition and weighing about 300 g, separate it from the litter and 30 to 60 minutes later anaesthetise (for example, by the intraperitoneal injection of a solution of Pentobarbitone Sodium). Tie the rat to an operating table, maintained at 37, by its hind legs leaving the front legs free. Cannulate the trachea with a short polyethylene tube of internal diameter about 2.5 mm in such a manner so as to ensure a free airway; apply artificial respiration only if necessary. Cannulate an external jugular or femoral vein with a polyethylene tube of internal diameter about 0.4 mm which is filled with saline solution and closed with a pin. Shave the skin surrounding the inguinal and abdominal teats and excise the tip of one teat, preferably the lower inguinal teat. Insert a polyethylene tube of internal diameter about 0.3 mm and external diameter about 0.6 mm, to a depth sufficient
to obtain appropriate measurement of pressure (3 to 10 mm depth), into the primary teat duct which opens onto the cut surface and tie firmly in place with a ligature. Connect this cannula with a suitable strain gauge transducer (such as that used for recording arterial blood pressure in the rat) and fill the whole system with a 3.8 per cent w/v solution of sodium citrate or saline solution containing 50 Units of heparin sodium per ml to prevent clotting of milk. After cannulation, inject a small volume (0.05 to 0.2 ml) of this solution into the teat duct through the transducer to clear the milk from the tip of the cannula. (This procedure may be repeated during the assay should obstruction arise from milk ejected into the cannula). Clamp the strain gauge so that a slight tension is applied to the teat and its natural alignment is preserved and connect the gauge to a potentiometric recorder adjusted to give full-scale deflection for an increase in milk-ejection pressure of about 5.3 kPa. Inject all solutions through the venous cannula using a 1-ml syringe graduated in 0.01 ml and wash them in with 0.2 ml of saline solution. Prepare a solution of the Standard Preparation and a solution of the preparation under examination in saline solution so that the volume to be injected is between 0.1 ml and 0.4 ml. Choose two doses of the Standard Preparation such that the increase in milk-ejection pressure is about 1.35 kPa for the lower dose and about 2.7 kPa for the higher dose. As an initial approximation, a lower dose of between 0.1 and 0.4 milliUnit and an upper dose of 1.5 to 2 times this amount may be tried. Choose two doses of the preparation under examination with the same inter-dose ratio, matching the effects of the doses of the Standard Preparation as closely as possible. Inject the four doses (two doses of the Standard Preparation and two doses of the preparation under examination at intervals of 3 to 5 minutes. The two doses of Standard Preparation and the two doses of the preparation under examination should be given according to a randomised block or a Latin square design and at least four responses to each should be recorded. Measure all the responses and calculate the result of the assay by standard statistical methods. The estimated potency is not less than 90 per cent and not more than 111 per cent of the stated potency. The fiducial limits of error are not less than 80 per cent and not more than 125 per cent of the stated potency. Oxytocin Nasal Solution containing Oxytocin of natural origin obtained by extraction and purification complies with the following additional requirement. Vasopressor activity. Not more than 0.5 Unit per 20 Units of oxytocic activity, when assayed by the biological assay for vasopressor activity described below. The vasopressor activity is estimated by comparing the activity of the preparation under examination with that of the Standard
888
IP 2007
Preparation of arginine vasopressin under the conditions of a suitable method of assay. Standard Preparation The Standard Preparation is the Ist International Standard for Arginine vasopressin, established in 1978, consisting of freeze-dried synthetic arginine vasopressin peptide acetate with human albumin and citric acid (supplied in ampoules containing 8.20 Units), or any other suitable preparation the potency of which has been determined in relation to that of the International Standard. Inject slowly into the tail vein of a male albino rat weighing about 300 g a solution of a suitable a-adrenoceptor blocking agent, for example 10 ml per kg of body weight of a solution prepared by dissolving 5 mg of phenoxybenzamine hydrochloride in 0.1 ml of ethanol (95 per cent), adding 0.05 ml of 1 M hydrochloric acid and diluting to 5 ml with saline solution. After 18 hours, anaesthetise the rat with an anaesthetic that will maintain a prolonged and uniform blood pressure. After 45 to 60 minutes, tie the rat on its back to the operating table by its hind legs. Cannulate the trachea with a short polyethylene tube of external diameter about 2.5 mm and dissect a carotid artery ready for cannulation. Then cannulate the femoral vein close to the inguinal ligament. Retract the abdominal muscles to expose the inguinal ligament. Retract the superficial pudendal vein to one side and dissect the femoral vein towards the inguinal ligament from the corresponding artery. When dissecting, a deep branch reaching the femoral vein must be found and tied off to prevent bleeding during cannulation. Tie a short polyethylene cannula of external diameter about 1 mm into the femoral vein by two ligatures and join by a short piece of flexible tubing to a 1-ml burette with an attached thistle funnel containing saline solution at about 37. Firmly fix a wet absorbent cotton swab to the thigh so as to cover the incision and cannula. At this stage inject through the venous cannula 200 Units of heparin, dissolved in saline solution, per 100 g of body weight. Then
tie in a carotid cannula of external diameter about 1 mm and connect by a column of saline solution containing heparin with a suitable pressure measuring device such as a mercury manometer of internal diameter about 2 to 3 mm. The central and peripheral nervous system including both vagus and associated sympathetic nerves is left intact. No artificial respiration is necessary. Taking care that no air is injected, inject all solutions through the venous cannula by means of a 1-ml syringe graduated in 0.01 ml and wash in with 0.2 ml of saline solution from the burette. Dilute the extract of the Standard Preparation and the preparation under examination with saline solution so that the volume to be injected is between 0.1 ml and 0.5 ml. Choose two doses of the Standard Preparation such that the elevation of the blood pressure is about 4 kPa for the lower dose and about 7 kPa but always submaximal for the higher dose, the ratio of low to high dose being determined by the response and usually being 3 to 5. As an initial approximation doses of 3 and 5 milliUnits may be tried. Choose two doses of the preparation under examination with the same inter-dose ratio, matching the effects of the dose of the Standard Preparation as closely as possible. Inject doses at intervals of 10 to 15 minutes. The two doses of the Standard Preparation and the two doses of the preparation under examination should be given in a randomised block or a Latin square design and four to five responses to each should be recorded. Measure all the responses and calculate the result of the assay by standard statistical methods. Storage. Store at a temperature not exceeding 30. Labelling. The label states (1) the number of Units of oxytocic activity per ml; (2) either the animal species from which it is obtained or whether it is synthetic, as appropriate; (3) that the preparation is intended for intranasal administration only.
889
MONOGRAPHS
P
Paclitaxel Paclitaxel Injection Pancreatin D-Panthenol Paracetamol Paracetamol Syrup Paracetamol Tablets Hard Paraffin Liquid Paraffin Light Liquid Paraffin Liquid Paraffin Emulsion White Soft Paraffin Yellow Soft Paraffin Paraffin Ointment Paraldehyde Penicillamine Penicillamine Tablets Diluted Pentaerythritol Tetranitrate Pentaerythritol Tetranitrate Tablets Pentamidine Isethionate Pentamidine Injection Pentazocine Pentazocine Hydrochloride Pentazocine Tablets Pentazocine Lactate Pentazocine Injection Pentobarbitone Sodium Pentobarbitone Tablets Pepsin Peritoneal Dialysis Solutions
891
.... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... ....
MONOGRAPHS
Pethidine Hydrochloride Pethidine Injection Pethidine Tablets Phenindamine Tartrate Phenindamine Tablets Phenindione Phenindione Tablets Pheniramine Maleate Pheniramine Injection Pheniramine Tablets Phenobarbitone Phenobarbitone Tablets Phenobarbitone Sodium Phenobarbitone Injection Phenobarbitone Sodium Tablets Phenol Phenolphthalein Phenoxymethylpenicillin Potassium Phenoxymethylpenicillin Potassium Tablets Phentolamine Mesylate Phentolamine Injection Phenylbutazone Phenylbutazone Tablets Phenylephrine Hydrochloride Phenylephrine Injection Phenylmercuric Acetate Phenylmercuric Nitrate Phenytoin Sodium Phenytoin Injection Phenytoin Tablets Pholcodine Pholcodine Linctus Phosphoric Acid
892
MONOGRAPHS
Physostigmine Salicylate Physostigmine Injection Pilocarpine Nitrate Pindolol Pindolol Tablets Piperazine Adipate Piperazine Adipate Tablets Piperazine Citrate Piperazine Citrate Syrup Piperazine Hydrate Piperazine Phosphate Piperazine Phosphate Tablets Piroxicam Piroxicam Capsules Plaster Of Paris Polyethylene Glycol 1500 Polyethylene Glycol 4000 Polyethylene Glycol 6000 Polysorbate 20 Polysorbate 80 Potassium Chloride Potassium Chloride And Dextrose Injection Potassium Chloride, Sodium Chloride And Dextrose Injection Potassium Citrate Potassium Clavulanate Potassium Clavulanate Diluted Potassium Iodide Potassium Permanganate Povidone Povidone-Iodine Povidone-Iodine Solution Pralidoxime Chloride
.... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... ....
893
MONOGRAPHS
Pralidoxime Chloride Injection Prazosin Hydrochloride Prazosin Tablets Prednisolone Prednisolone Tablets Prednisone Prednisone Tablets Prednisolone Sodium Phosphate Prednisolone Sodium Phosphate Injection Primaquine Phosphate Primaquine Tablets Probenecid Probenecid Tablets Procainamide Hydrochloride Procainamide Injection Procainamide Tablets Procaine Hydrochloride Procaine And Adrenaline Injection Procaine Penicillin Fortified Procaine Penicillin Injection Prochlorperazine Maleate Prochlorperazine Tablets Prochlorperazine Mesylate Prochlorperazine Injection Procyclidine Hydrochloride Procyclidine Tablets Proguanil Hydrochloride Proguanil Tablets Promethazine Hydrochlorid Promethazine Injection Promethazine Syrup Promethazine Tablets Promethazine Theoclate
894
.... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... ....
MONOGRAPHS
Promethazine Theoclate Tablets Propantheline Bromide Propantheline Tablets Propranolol Hydrochloride Propranolol Injection Propranolol Tablets Propyl Gallate Propylene Glycol Propylparaben Propylthiouracil Propylthiouracil Tablets Propyphenazone Protamine Sulphate Protamine Sulphate Injection Prothionamide Prothionamide Tablets Pseudoephedrine Hydrochloride Pseudoephedrine Syrup Pseudoephedrine Tablets Psoralen Pyrazinamide Pyrazinamide Tablets Pyridoxine Hydrochloride Pyridoxine Tablets Pyrimethamine Pyrimethamine And Sulphadoxine Tablets
.... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... ....
895
IP 2007
PACLITAXEL
Paclitaxel
Taxol
O H3C H3C NH O O OH O O CH3 CH3 OH H O O O O CH3 CH3 OH
C47H51NO14
Mol. Wt. 853.9
A taxane derivative first isolated from the bark of the Pacific yew tree, Taxus brevifolia. Paclitaxel contains not less than 97.0 per cent and not more than 102.0 per cent of C47H51NO14, calculated on the anhydrous basis. Description. A white or almost white powder. CAUTION Paclitaxel is potentially cytotoxic. Great care should be taken in handling the powder and preparing solutions.
mobile phase: A. a mixture of 60 volumes of water and 40 volumes of acetonitrile, B. acetonitrile flow rate. 1.2 ml per minute, a linear gradient programme using the conditions given below, spectrophotometer set at 227nm, a 10 l loop injector. Time Mobile phase A Mobile phase B (in min) (per cent v/v) (per cent v/v) 0 100 0 20 100 0 60 10 90 62 100 0 70 100 0 Inject reference solution (b). The test is not valid unless the resolution between the peak due to paclitaxel and 10-dactyl 7-epipactitaxyl is not less than 1.2 relative retention time for particles and 10-dactyl -7-epipactitaxyl is about 0.94. Inject the test solution and reference solution (a). In the chromatogram obtained with the test solution, the area of any secondary peak is not more than 0.5 times the area of the peak in the chromatogram obtained with the reference solution (a) (0.5 per cent) and the sum of areas of all the secondary peaks is not more than twice the area of the peak in the chromatogram obtained with the reference solution (a) (2.0 per cent). Heavy metals. (2.3.13). 1 g complies with the limit test for heavy metals, Method A ( 20ppm ). Loss on ignition (2.4.20). Not more than 0.2 per cent. Water (2.3.43). Not more than 4.0 per cent, determined on 0.1 g by coloumetry method. Bacterial endotoxins (2.2.3). Not more than 0.4 Endotoxin Unit per mg of paclitaxel. Microbial contamination (2.2.9). The total viable aerobic count does not exceed 100 cfu per g. It meets the requirements of the tests for the absence of Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella species, and Escherichia coli. Assay. Determine by liquid chromatography (2.4.14). Solvent mixture. Dissolve 200 l of glacial acetic acid in 1000 ml of methanol. Test solution. Dissolve 0.1 g of the substance under examination in 100.0 ml in solvent mixture. Reference solution. A 0.1 per cent w/v solution of paclitaxil RS in solvent mixture. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with pentafluoro phenyl groups chemically bonded to porous silica (5 m),
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with paciltaxel RS. B. In the Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the peak in the chromatogram obtained with the reference solution.
Tests
Specific optical rotation (2.4.22). 49.0 to 55.0, determined in 1.0 per cent w/v solution in methanol. Related substances. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 10 mg of substance under examination in 10 ml of acetonitrile. Reference solution (a). A 0.001 per cent w/v solution of paclitaxel RS in acetonitrile. Reference solution (b). A sloution containing 0.008 per cent w/v of 10 deacetyl -7-epipoelitoral and 0.1 per cent w/v of paclitaxel RS in acetonitrile. Chromatographic system a stainless steel column 15 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (3m). column temperature 35,
897
PACLITAXEL INJECTION
IP 2007
column temperature 35, mobile phase: a mixture of 11 volumes of water and 9 volumes of acetonitrile, filter. flow rate. 1.5 ml per minute, spectrophotometer set at 227nm, a 10 l loop injector. Inject the reference solution. The test is not valid unless the tailing factor is not more than 1.5. The relative standard deviation for replicate injections is not more than 2.0 per cent. Inject the test solution and reference solution. Calculate the content of C47H51NO14. Storage. Store protected from light, at a temperature not exceeding 25.
System suitability solution- A sloution containing 0.006 per cent w/v of 10 deacetyl -7-epipoelitoral and 0.12 per cent w/v of paclitaxel in acetonitrile. Chromatographic system a stainless steel column 15 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (3 m). column temperature 35, mobile phase: A. a mixture of 60 volumes of water and 40 volumes of acetonitrile, B. acetonitrile flow rate. 1.2 ml per minute, a linear gradient programme using the conditions given below, spectrophotometer set at 227nm, a 10 l loop injector. Time Mobile phase A Mobile phase B (in min) (per cent v/v) (per cent v/v) 0 100 0 26 100 0 66 17 83 67 100 0 Inject reference solution (b). The test is not valid unless the column efficiency is not less than 2000 theoretical plates and the tailing factor is not more than 2.0. Inject the test solution and reference solution (a). In the chromatogram obtained with the test solution, the area of any secondary peak is not more than 0.8 times the area of the peak in the chromatogram obtained with the reference solution (a) (0.8 per cent) and the sum of areas of all the secondary peaks is not more than twice the area of the peak in the chromatogram obtained with the reference solution (a) (2.0 per cent). Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Bacterial endotoxins (2.2.3). Not more than 0.67 Endotoxin Unit per mg of paclitaxel. Sterility (2.2.11). Complies with the test for sterility. Assay. Determine by liquid chromatography (2.4.14). Solvent mixture. Dissolve 200 l of glacial acetic acid in 1000 ml of methanol. Test solution. Accurately measure the volume of injection containing 6 mg of Paclitaxel and dissolve in 10 ml of solvent mixture. Reference solution. A 0.06 per cent w/v solution of paclitaxel RS in solvent mixture. Chromatographic system a stainless steel column 30 cm x 3.9 mm, packed with penta fluro phenyl group chemically bonded to porous silica (5 m),
Paclitaxel Injection
Paclitaxel Injection is a sterile solution of Paclitaxel suitable for dilution,for intravenous use. Paclitaxel Injection contains not less than 90.0 per cent and not more than 105.0 per cent of of the related amount of paclitaxel, C47H51NO14. Description. A clear colourless to slight yellow viscous solution.
Identification
A. In the Related substances, the major peak in the chromatogram obtained with test solution corresponds to the peak in the chromatogram obtained with reference solution. B. In the Assay, the principal peak in the chromatogram obtained with test solution corresponds to the peak in the chromatogram obtained with reference solution.
Tests
pH (2.4.24). 3.0 to 7.0, determined in a solution 10 per cent v/v solution in water. Light absorption. Absorbance of the injection at about 425 nm, not more than 0.01. Related substances. Determine by liquid chromatography (2.4.14). Test solution. Accurately measure the volume of injection containing 12 mg of Paclitaxel, dilute to 10 ml with acetonitrile. Reference solution (a). A 0.12 per cent w/v solution of paclitaxel RS in aceotnitrile. Reference solution (b). Dilute 1 ml of reference solution (a) to 100 ml with acetonitrile.
898
IP 2007
PANCREATIN
column temperature 35, mobile phase: a mixture of 11 volumes of water and 9 volumes of acetonitrile, flow rate. 1.5 ml per minute, spectrophotometer set at 227nm, a 10 l loop injector. Inject the reference solution. The test is not valid unless the tailing factor is not more than 1.5. The relative standard deviation for replicate injections is not more than 2.0 per cent. Inject the test solution and reference solution. Calculate the content of C47H51NO14. Storage. Store protected from light, at a temperature not exceeding 25.
Tests
Fat. Not more than 5.0 per cent, determined by the following method. Extract 1 g with light petroleum (40 to 60) for 3 hours in an apparatus for the continuous extraction of drugs (2.1.8), evaporate the extract and dry the residue at 105 for 2 hours. Microbial contamination (2.2.9). 1 g is free from Escherichia coli; 10 g is free from salmonellae. Loss on drying (2.4.19). Not more than 5.0 per cent, determined on 0.5 g by dying in an oven at 60 at a pressure not exceeding 0.7 kPa for 4 hours. Assay. For protease activity Weigh accurately 4.0 g of purified casein and dissolve in about 90 ml of water containing 3 ml of 1 M sodium hydroxide, adjust the pH of the solution to 8.7 and add sufficient water to produce 100.0 ml. Weigh accurately about 0.5 g of the substance under examination, triturate with water and add sufficient water to produce 300.0 ml to give the test solution. Dilute 15.0 ml of the casein solution with 30 ml of water, warm to 55 and add 10.0 ml of the unfiltered test solution. Heat rapidly to 55 and keep at this temperature for 20 minutes. Cool rapidly to room temperature. Dilute a further portion of 15.0 ml of the casein solution with 30 ml of water, add 10.0 ml of the unfiltered test solution, previously boiled and cooled, heat rapidly to 55 and keep at this temperature for 20 minutes. Cool to room temperature. To each solution add 0.75 ml of phenolphthalein solution and 10 ml of formaldehyde solution. Titrate each solution with 0.1 M sodium hydroxide until the colour of the solution matches that produced by mixing 10 ml of buffer solution pH 8.7 and 0.15 ml of phenolphthalein solution. The difference between the two titrations is not less than 4.5 ml. For lipase activity To 95 ml of water add 6.5 ml of triacetin and 0.2 ml of a 0.1 per cent w/v solution of bromocresol purple, neutralise with 0.5 M sodium hydroxide and add sufficient water to produce 110 ml. Place 50 ml of this solution in each of two large tubes 3 cm 20 cm A and B contained in a thermostat at 30. Insert in each tube a rubber stopper having two holes, one for the tip of a burette and the other for a short glass tube through which passes a thread operating a glass stirring coil. Stir the contents of the tube until they attain the temperature of the thermostat. Prepare a solution of 0.1 g of the substance under examination in 10.0 ml of water. To tube A add 1.0 ml of the solution, to tube B add 1.0 ml of the solution previously boiled. Adjust and maintain the pH of the solutions in the two tubes to 6.2 to 6.4 by the addition of 0.05 M sodium hydroxide dropwise, stirring frequently. After 30 minutes, the difference between the volumes of 0.05 M sodium hydroxide added to the two tubes is not less than 1.0 ml. For amylase activity Not less than 100 Units per g. Dissolve 0.1 g or a quantity containing 10 Units, accurately weighed, in
Pancreatin
Pancreatin is a preparation of mammalian pancreas containing protease, lipase and amylase activity. It may contain Sodium Chloride. Pancreatin contains not less than the minimum protease activity, amylase activity and lipase activity determined under the conditions of the Assay. Description. A white or buff-coloured, amorphous powder; odour, meaty and not unpleasant.
Identification
A. Triturate 0.5 g with 10 ml of water and adjust to pH 8.0 by the addition of 1 M sodium hydroxide using cresol red solution as indicator. Divide the resulting solution into two equal portions. Boil one portion [solution (1)] and leave the other untreated [solution (2)]. To each add a few shreds of congo red fibrin, warm to 39 1 and maintain at this temperature for 1 hour. Solution (2) is stained red and solution (1) is colourless or not more than slightly pink. B. Triturate 0.25 g with 10 ml of water and adjust to pH 8.0 by the addition of 1 M sodium hydroxide using cresol red solution as indicator. Divide the resulting solution into two equal portions. Boil one portion [solution (1)] and leave the other untreated [solution (2)]. Dissolve 0.1 g of soluble starch in 100 ml of boiling water, boil for 2 minutes, cool and dilute to 150 ml with water. Add solution (1) to half the starch mucilage and solution (2) to the remainder and maintain the mixtures at 39 1 for 5 minutes. To 1 ml of each mixture add 10 ml of iodinated potassium iodide solution. The liquid containing solution (2) retains the colour of the solution of iodine and the liquid containing solution (1) acquires an intense blue colour.
899
D-PANTHENOL
IP 2007
sufficient buffer solution pH 6.8 to produce 1000.0 ml. Filter if necessary (1 ml of the test solution should be capable of digesting about 10 mg of dry soluble maize or corn starch). Into each of six stoppered test-tubes add 5.0 ml of starch substrate without touching the sides of the test-tube. Place the test-tubes in a water-bath maintained at 40 0.1. When the temperature of the solution in the tubes has reached 40, add 0.35 ml, 0.4 ml, 0.45 ml, 0.5 ml, 0.55 ml and 0.6 ml of the test solution to each of the test-tubes marked 1 to 6 respectively and record the time of addition. Mix thoroughly and replace the tubes in the water-bath. After exactly 60 minutes remove the tubes and cool rapidly in cold water. Add to each tube 0.05 ml of 0.02 M iodine and mix well. Note the tube containing the lowest volume of test solution, which does not show a bluish or violet tinge (if there is doubt, warm the solution slightly, when the colour distinction is prominent). From this volume calculate the number of grams of dry soluble maize or corn starch digested by 1.0 g of the substance under examination. This represents the number of Units of amylase activity per g. Storage. Store protected from moisture. Labelling. The label states the name of any added substance.
Refractive index (2.4.27). 1.490 to 1.498, determined at 20. Heavy metals (2.3.13). 1.0 g dissolved in 25 ml of water complies with the limit test for heavy metals, Method A (20 ppm). Assay. Weigh accurately about 0.5 g and carry out Method A for the determination of nitrogen (2.3.30). 1 ml of 0.05 M sulphuric acid is equivalent to 0.001401 g of N. Storage. Store protected from moisture.
Paracetamol
Acetaminophen
O HN CH3
OH
C8H9NO2 Paracetamol is 4-hydroxyacetanilide. Mol. Wt. 151.2
D-Panthenol Pantothenol; Dextro-pantothenyl Alcohol

H OH N H OH
Mol. Wt. 205.3
Paracetamol contains not less than 99.0 per cent and not more than 101.0 per cent of C8H9NO2, calculated on the dried basis. Description. White crystals or a white, crystalline powder.
HO H3C CH3 O
C9H19NO4 dimethylbutanamide.
Identification
Test A may be omitted if tests B C and D are carried out. Tests B, C and D may be omitted if test A is carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with paracetamol RS or with the reference spectrum of paracetamol. B. Dissolve 50 mg in sufficient methanol to produce 100 ml. To 1 ml of this solution add 0.5 ml of 0.1 M hydrochloric acid and dilute to 100 ml with methanol. Protect the resulting solution from bright light and immediately measure the absorbance at the maximum at about 249 nm; absorbance at 249 nm, about 0.44 (2.4.7). C. Boil 0.1 g in 1 ml of hydrochloric acid for 3 minutes, add 10 ml of water and cool; no precipitate is produced. Add 0.05 ml of 0.0167 M potassium dichromate; a violet colour develops which does not turn red. D. Gives the reaction of acetyl groups (2.3.1).
D-Panthenol is (R)-2,4-dihydroxy-N-(3-hydroxypropyl)-3, 3-
D-Panthenol contains not less than 6.60 per cent and not more
than 6.95 per cent of nitrogen, N. Description. A clear, colourless or slightly yellow, viscous liquid; odourless.
Identification
Boil 50 mg with 5 ml of 0.1 M sodium hydroxide for 1 minute, cool and add 5 ml of 1 M hydrochloric acid and 0.1 ml of ferric chloride test solution; a deep yellow colour is produced.
Tests
pH (2.4.24). Not more than 10.5, determined in a 5.0 per cent w/v solution in carbon dioxide-free water. Specific optical rotation (2.4.22). +28.2 to +30.2, determined at 20 in a 5.0 per cent w/v solution.
Tests
4-Aminophenol. Dissolve 0.5 g in sufficient methanol (50 per cent) to produce 10 ml. Add 0.2 ml of freshly prepared alkaline
900
IP 2007
PARACETAMOL SYRUP
sodium nitroprusside solution, mix and allow to stand for 30 minutes. Any blue colour in the solution is not more intense than that in 10 ml of a solution prepared at the same time and in the same manner containing 0.5 g of 4-aminophenol-free paracetamol and 0.5 ml of a 0.005 per cent w/v solution of 4-aminophenol in methanol (50 per cent) (50 ppm). Related substances. Determine by thin layer chromatograpy (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 65 volumes of chloroform, 25 volumes of acetone and 10 volumes of toluene. Test solution (a). Transfer 1 g of the substance under examination, finely powdered, to a ground-glass stoppered 15-ml centrifuge tube, add 5 ml of peroxide-free ether, shake mechanically for 30 minutes and centrifuge at 1000 rpm for 15 minutes or until a clear supernatant liquid is obtained. Test solution (b). Dilute 1 ml of the test solution to 10 ml with ethanol (95 per cent). Reference solution (a). A 0.005 per cent w/v solution of 4-chloroacetanilide in ethanol (95 per cent). Reference solution (b). Dissolve 0.25 g of 4-chloroacetanilide and 0.1 g of the substance under examination in sufficient ethanol (95 per cent) to produce 100 ml. Apply to the plate 200 l of test solution (a) and 40 l of each of test solution (b) and reference solutions (a) and (b). After development, dry the plate in a current of warm air and examine in ultraviolet light at 254 nm. Any spot corresponding to 4-chloroacetanilide in the chromatogram obtained with test solution (a) is not more intense than the spot in the chromatogram obtained with reference solution (a). Any other secondary spot in the chromatogram obtained with test solution (b) is not more intense than the spot in the chromatogram obtained with reference solution (a). The chromatogram obtained with reference solution (b) shows two clearly separated spots, the spot corresponding to paracetamol having the lower Rf value. Heavy metals (2.3.13). 2.0 g complies with the limit test for heavy metals, Method B (10 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.5 g, dissolve in a mixture of 10 ml of water and 50 ml of 1 M sulphuric acid. Boil under a reflux condenser for 1 hour, cool and dilute to 100.0 ml with water. To 20.0 ml of the solution add 40 ml of water, 40 g of water in the form of ice, 15 ml of 2 M hydrochloric acid and 0.1 ml of ferroin solution and titrate with 0.1 M ceric ammonium sulphate until a yellow colour is produced. Carry out a blank titration.
1 ml of 0.1 M ceric ammonium sulphate is equivalent to 0.00756 g of C8H9No2. Storage. Store protected from light and moisture.
Paracetamol Syrup
Paracetamol Oral Solution; Acetaminophen Syrup
Paracetamol Syrup is a solution of Paracetamol in a suitable flavoured vehicle. Paracetamol Syrup contains not less than 95.0 per cent and not more than 105.0 per cent w/v solution of the stated amount of paracetamol, C8H9NO2.
Identification
Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 65 volumes of chloroform, 25 volumes of acetone, 10 volumes of toluene and 0.5 volumes of glacial acetic acid. Test solution. Dilute a volume containing 25 mg of Paracetamol to 10 ml with methanol and filter if necessary. Reference solution. A 0.25 per cent w/v solution of paracetamol RS in methanol Apply to the plate 10 l of each solution. After development, dry the plate in a current of warm air, examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution.
Tests
4-Aminophenol. Determine by liquid chromatography (2.4.14). Test solution. Shake 5 ml of the preparation under examination with 15 ml of the mobile phase, dilute to 25 ml with the mobile phase and filter if necessary. Reference solution. A 0.0025 per cent w/v solution of 4-aminophenol in the mobile phase. Chromatographic system a stainless steel column 20 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (10 m), mobile phase: 0.01 M sodium butanesulphonate in a mixture of 85 volumes of water, 15 volumes of methanol and 0.4 volume of formic acid, flow rate. of 2 ml per minute, spectrophotometer set at 272 nm, a 20 l loop injector. In the chromatogram obtained with the test solution the area of any peak corresponding to 4-aminophenol is not greater
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IP 2007
than the area of the peak in the chromatogram obtained with the reference solution. In the chromatogram obtained with the test solution peaks with a long retention time may occur due to preservatives in the preparations. Other tests. Complies with the tests stated under Oral Liquids. Assay. Determine by liquid chromatography (2.4.14). Test solution. Mix an accurately weighed quantity of the preparation under examination containing 25 mg of Paracetamol in 100 ml of the mobile phase, dilute to 200.0 ml with the mobile phase and filter if necessary. Reference Solution. A 0.0125 per cent w/v solution of paracetamol RS in the mobile phase. Chromatographic system a stainless steel column 20 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (10 m), mobile phase: 0.01 M sodium butanesulphonate in a mixture of 85 volumes of water, 15 volumes of methanol and 0.4 volume of formic acid, flow rate. of 2 ml per minute, spectrophotometer set at 243 nm, a 20 l loop injector. Determine the weight per ml of the syrup (2.4.29), and calculate the percentage content of C8H9NO2, weight in volume. Storage. Store protected from light and moisture.
Test solution. Shake a quantity of the powdered tablets containing 1 g of Paracetamol with 15 ml of methanol, dilute to 100 ml with water and filter. Reference solution. A 0.001 per cent w/v solution of 4-aminophenol in methanol (15 per cent). Chromatographic system a stainless steel column 20 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (10 m), mobile phase: 0.01 M sodium butanesulphonate in a mixture of 85 volumes of water, 15 volumes of methanol and 0.4 volume of formic acid, flow rate. 2 ml per minute, spectrophotometer set at 272 nm, a 20 l loop injector. In the chromatogram obtained with the test solution the area of any peak corresponding to 4-aminophenol is not greater than the area of the peak in the chromatogram obtained with the reference solution. In the chromatogram obtained with the test solution peaks with a long retention times may occur due to excipients. Related substances. Determine by thin layer chromatograpy (2.4.17), coating the plate with silica gel F254. Mobile phase. A mixture of 65 volumes of chloroform, 25 volumes of acetone and 10 volumes of toluene. Test solution (a). Transfer a quantity of the powdered tablets containing 1 g of Paracetamol to a ground-glass stoppered 15-ml centrifuge tube, add 5 ml of peroxide-free ether, shake mechanically for 30 minutes, centrifuge at 1000 rpm for 15 minutes or until a clear supernatant liquid is obtained and use the supernatant liquid. Test solution (b). Dilute 1 ml of the test solution to 10 ml with ethanol (95 per cent). Reference solution (a). A 0.005 per cent w/v solution of 4-chloroacetanilide in ethanol (95 per cent). Reference solution (b). Dissolve 0.25 g of 4-chloroacetanilide and 0.1 g of the substance under examination in sufficient ethanol (95 per cent) to produce 100 ml. Apply to the plate 200 l of test solution (a) and 40 l of each of test solution (b) and reference solutions (a) and (b). After development, dry the plate in a current of warm air and examine in ultraviolet light at 254 nm. Any spot corresponding to 4-chloroacetanilide in the chromatogram obtained with test solution (a) is not more intense than the spot in the chromatogram obtained with reference solution (a). Any other secondary spot in the chromatogram obtained with test solution (b) is not more intense than the spot in the chromatogram obtained with reference solution (a). The chromatogram obtained with reference solution (b) shows two
Paracetamol Tablets
Acetaminophen Tablets
Paracetamol Tablets contain not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of paracetamol, C8H9NO2.
Identification
Extract a quantity of the powdered tablets containing 0.5 g of Paracetamol with 20 ml of acetone, filter, evaporate the filtrate to dryness and dry at 105. The residue complies with the following tests. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with paracetamol RS or with the reference spectrum of paracetamol. B. Boil 0.1 g in 1 ml of hydrochloric acid for 3 minutes, add 10 ml of water and cool; no precipitate is produced. Add 0.05 ml of 0.0167 M potassium dichromate; a violet colour develops which does not turn red.
Tests
4-Aminophenol. Determine by liquid chromatography (2.4.14).
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LIQUID PARAFFIN
clearly separated spots, the spot corresponding to paracetamol having the lower Rf value. Dissolution (2.5.2). Apparatus. No 1 Medium. 900 ml of phosphate buffer pH 5.8 Speed and time. 50 rpm and 30 minutes. Withdraw a suitable volume of the medium and filter and dilute a suitable volume of the filtrate with the same solvent. Measure the absorbance of the resulting solution at the maximum at about 243 nm (2.4.7). Similarly measure the absorbance of a solution of known concentration of paracetamol RS. Calculate the content of C8H9NO2. D. Not less than 80 per cent of the stated amount of C8H9NO2. Other tests. Complies with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 0.15 g of Paracetamol, add 50 ml of 0.1 M sodium hydroxide, dilute with 100 ml of water, shake for 15 minutes and add sufficient water to produce 200.0 ml. Mix, filter and dilute 10.0 ml of the filtrate to 100.0 ml with water. To 10.0 ml of the resulting solution add 10 ml of 0.1 M sodium hydroxide, dilute to 100.0 ml with water and mix. Measure the absorbance of the resulting solution at the maximum at about 257 nm (2.4.7). Calculate the content of C8H9NO2 taking 715 as the specific absorbance at 257 nm. Storage. Store protected from light and moisture.
Liquid Paraffin
White Mineral Oil; Liquid Petrolatum
Liquid Paraffin is a purified mixture of liquid hydrocarbons obtained from petroleum to which not more than 10 ppm of tocopherol or of butylated hydroxytoluene may be added. Description. A transparent, colourless, oily liquid, free from fluorescence by daylight; odourless or almost odourless.
Tests
Weight per ml (2.4.29). 0.860 g to 0.904 g. Dynamic viscosity (2.4.28). 110 mPas to 230 mPas, determined at 20 1 by Method B. Acidity or alkalinity. To 10.0 g add 20 ml of boiling water, heat in a water-bath for 5 minutes, shake vigorously for 1 minute, cool, allow to separate and filter the aqueous layer. To 10 ml of the filtrate add 0.1 ml of phenolphthalein solution. The solution is colourless and not more than 0.1 ml of 0.1 M sodium hydroxide is required to change the colour of the solution to pink. Light absorption. When examined in the range 240 nm to 280 nm (2.4.7), a 2.0 per cent w/v solution in 2,2,4-trimethylpentane shows an absorption of not more than 0.1. Readily carbonisable substances. Place 5 ml in a dry, heatresistant glass-stoppered test-tube (125 mm x 18 mm) previously rinsed with chromic acid solution, then with water and dried. Add 5 ml of nitrogen-free sulphuric acid (containing 94.5 per cent to 95.5 per cent w/w of H2SO4), insert the stopper and shake as vigorously as possible in the longitudinal direction of the tube for 5 seconds. Loosen the stopper, immediately place the tube in a bath of boiling water, supporting it so as to prevent contact of the tube with the bottom or side of the bath and heat for 10 minutes. At the end of the second, fourth, sixth, and eighth minutes, remove the tube from the bath and shake as vigorously as possible in the longitudinal direction of the tube for 5 seconds. At the end of 10 minutes from the time the tube was placed in the bath remove the tube and allow to stand for 10 minutes. The lower acid layer is not more intensely coloured than a mixture of 3 ml of FCS, 1.5 ml of CCS and 0.5 ml of CSS (2.4.1), overlaid with 5 ml of liquid paraffin. If the sulphuric acid remains dispersed in the molten paraffin, the colour of the emulsion is not darker than that of the standard mixture when shaken vigorously. Solid paraffins. Place a suitable quantity, previously dried by heating at 100 for 2 hours and cooled in a desiccator over sulphuric acid, in a glass cylindrical vessel having an internal diameter of approximately 25 mm. Close the vessel and immerse in a mixture of ice and water; after 4 hours the liquid is sufficiently clear that a black line, 0.5 mm in width, held vertically behind the vessel is easily seen.
Hard Paraffin
Hard Paraffin is a purified mixture of solid hydrocarbons obtained from petroleum or from shale oil. Description. A white or colourless, translucent mass, frequently showing a crystalline structure; odourless even when freshly cut; slightly greasy to the touch. Burns with a luminous flame. When melted, the liquid is free from fluorescence by daylight.
Tests
Acidity or alkalinity. To 10.0 g add 20 ml of boiling water, heat in a water-bath for 5 minutes, shake vigorously for 1 minute, cool, allow to separate and filter the aqueous layer. To 10 ml of the filtrate add 0.1 ml of phenolphthalein solution. The solution is colourless and not more than 0.1 ml of 0.1 M sodium hydroxide is required to change the colour of the solution to pink. Congealing range (2.4.10). 50 to 65. Sulphated ash (2.3.18). Not more than 0.1 per cent.
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Sulphur compounds. Mix 4 ml with 2 ml of ethanol (95 per cent), and 2 drops of a clear, saturated solution of lead monoxide in sodium hydroxide solution and heat at 70 for 10 minutes with frequent shaking; the mixture remains colourless. Storage. Store protected from light.
dispersed in the molten paraffin, the colour of the emulsion is not darker than that of the standard mixture when shaken vigorously. Solid paraffins. Place a suitable quantity, previously dried by heating at 100 for 2 hours and cooled in a desiccator over sulphuric acid, in a glass cylindrical vessel having an internal diameter of approximately 25 mm. Close the vessel and immerse in a mixture of ice and water; after 4 hours the liquid is sufficiently clear that a black line, 0.5 mm in width, held vertically behind the vessel is easily seen. Sulphur compounds. Mix 4 ml with 2 ml of ethanol (95 per cent), and 2 drops of a clear, saturated solution of lead monoxide in sodium hydroxide solution and heat at 70 for 10 minutes with frequent shaking; the mixture remains colourless. Storage. Store protected from light.
Light Liquid Paraffin

Light Mineral Oil; Light Liquid Petrolatum
Light Liquid Paraffin is a purified mixture of liquid saturated hydrocarbons obtained from petroleum. It may contain a suitable stabiliser. Description. A transparent, colourless, oily liquid, free from fluorescence by daylight; almost odourless when cold.
Tests
Weight per ml (2.4.29). 0.820 g to 0.880 g. Dynamic viscosity (2.4.28). 25 mPa s to 80 mPa s, determined at 20 1 by Method B. Acidity or alkalinity. To 10.0 g add 20 ml of boiling water, heat in a water-bath for 5 minutes, shake vigorously for 1 minute, cool, allow to separate and filter the aqueous layer. To 10 ml of the filtrate add 0.1 ml of phenolphthalein solution. The solution is colourless and not more than 0.1 ml of 0.1 M sodium hydroxide is required to change the colour of the solution to pink. Light absorption. When examined in the range 240 nm to 280 nm (2.4.7), a 2.0 per cent w/v solution in 2,2,4-trimethylpentane shows an absorption of not more than 0.1. Readily carbonisable substances. Place 5 ml in a dry, heatresistant glass-stoppered test-tube (125 mm x 18 mm) previously rinsed with chromic acid solution, then with water and dried. Add 5 ml of nitrogen-free sulphuric acid (containing 94.5 per cent to 95.5 per cent w/w of H2SO4), insert the stopper and shake as vigorously as possible in the longitudinal direction of the tube for 5 seconds. Loosen the stopper, immediately place the tube in a bath of boiling water, supporting it so as to prevent contact of the tube with the bottom or side of the bath and heat for 10 minutes. At the end of the second, fourth, sixth, and eighth minutes, remove the tube from the bath and shake as vigorously ssas possible in the longitudinal direction of the tube for 5 seconds. At the end of 10 minutes from the time the tube was placed in the bath remove the tube and allow to stand for 10 minutes. The lower acid layer is not more intensely coloured than a mixture of 3 ml of FCS, 1.5 ml of CCS and 0.5 ml of CSS (2.4.1), overlaid with 5 ml of liquid paraffin. If the sulphuric acid remains
Liquid Paraffin Emulsion

Liquid Paraffin Oral Emulsion
Liquid Paraffin Emulsion is an oral emulsion of Liquid Paraffin in Purified Water. Liquid Paraffin Emulsion contains not less than 44.0 per cent and not more than 49.0 per cent w/w of liquid paraffin.
Tests
Other tests. Complies with the tests stated under Oral Liquids. Assay. Weigh accurately about 5.0 g, add 10 ml of water, extract with two quantities, each of 40 ml, of a mixture of 2 volumes of ethanol (95 per cent), 3 volumes of light petroleum (40 to 60) and 3 volumes of ether and then with 30 ml of a mixture of equal volumes of light petroleum (40 to 60) and ether. Wash the combined extracts with 15 ml of 0.5 M sodium hydroxide and then with 15 ml of water, evaporate the solvent, add 5 ml of acetone and evaporate again. Repeat the addition and evaporation of acetone until the residue is free from water, dry at 105 for 15 minutes and weigh. Storage. Store protected from moisture.
White Soft Paraffin

White Petroleum Jelly
White Soft Paraffin is a purified, semi-solid mixture of hydrocarbons obtained from petroleum and bleached. Description. A white, translucent, soft unctuous mass, retaining these characteristics on storage and when melted
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YELLOW SOFT PARAFFIN
and allowed to cool without stirring; not more than slightly fluorescent by daylight, even melted; odourless when rubbed on the skin.
Tests
Melting range (2.4.21). 38 to 56, determined by Method IV. Acidity or alkalinity. To 10.0 g add 20 ml of boiling water, heat in a water-bath for 5 minutes, shake vigorously for 1 minute, cool, allow to separate and filter the aqueous layer. To 10 ml of the filtrate add 0.1 ml of phenolphthalein solution. The solution is colourless and not more than 0.1 ml of 0.1 M sodium hydroxide is required to change the colour of the solution to pink. Light absorption (2.4.7). Absorbance of a 0.05 per cent w/v solution in 2,2,4-trimethylpentane at about 290 nm, not more than 0.5. Fixed oils, fats and resin. Digest 10 g with 50 ml of sodium hydroxide solution at 100 for 30 minutes and allow the aqueous layer to separate. On acidifying the aqueous layer with dilute sulphuric acid, no precipitate or oily matter is produced. Foreign organic matter. Volatilises when heated, without emitting an acrid odour. Consistency. 100 to 300, determined by the following method. Apparatus. The apparatus is essentially in agreement with IS 4887:1968 and comprises a penetrometer fitted with a polished cone-shaped metal plunger weighing 150 g having a detachable steel tip of the following dimensions. The tip of the cone has an angle of 30, the point being truncated to a diameter of 0.38 0.08 mm, the base of the tip is 8.38 0.13 mm in diameter and the length of the tip is 15 0.25 mm. The remaining portion of the cone has an angle of 90, is 28 to 29 mm in height, and has a maximum diameter of 65.1 mm at the base. The containers of the test are flat-bottomed metal or glass cylinders that are 102 6 mm in diameter and not less than 60 mm in height. Procedure. Melt a sufficient quantity at a temperature below 85 and pour into one or more of the containers filling to within 6 mm of the rim. Cool to 25 2.5 over a period of not less than 16 hours, protected from drafts. Two hours before the test, place the containers in a water-bath at 25 0.5. If the room temperature is below 23.5 or above 26.5, adjust the temperature of the cone to 25 0.5 by placing it in a waterbath. Without disturbing the surface of the substance under examination, place the container on the penetrometer table, and lower the cone until the tip just touches the top surface of the test substance at a spot 25 mm to 38 mm from the edge of the container. Adjust the zero setting and quickly release the plunger, then hold it free for 5 seconds. Secure the plunger
and read the total penetration from the scale. Make three or more trials, each so spaced that there is no overlapping of the areas of penetration. Where the penetration exceeds 20 mm, use a separate container of the test substance for each trial. Read the penetration to the nearest 0.1 mm. Calculate the average of the three or more readings and conduct further trials to a total of 10 if the individual results differ from the average by more than 3 per cent. The final average of the trials is not less than 10.0 mm and not more than 30.0 mm indicating a consistency value between 100 and 300. Sulphated ash (2.3.18). Not more than 0.1 per cent. Storage. Store protected from light and moisture.
Yellow Soft Paraffin

Yellow Petroleum Jelly
Yellow Soft Paraffin is a purified, semi-solid mixture of hydrocarbons obtained from petroleum. Description. A pale yellow to yellow, translucent, soft unctuous mass, retaining these characteristics on storage and when melted and allowed to cool without stirring; not more than slightly fluorescent by daylight, even melted; odourless when rubbed on the skin.
Tests
Melting range (2.4.21). 38 to 56, determined by Method IV. Light absorption (2.4.7). Absorbance of a 0.05 per cent w/v solution in 2,2,4-trimethylpentane at about 290 nm, not more than 0.75. Acidity or alkalinity. To 10.0 g add 20 ml of boiling water, heat in a water-bath for 5 minutes, shake vigorously for 1 minute, cool, allow to separate and filter the aqueous layer. To 10 ml of the filtrate add 0.1 ml of phenolphthalein solution. The solution is colourless and not more than 0.1 ml of 0.1 M sodium hydroxide is required to change the colour of the solution to pink. Fixed oils, fats and resin. Digest 10 g with 50 ml of sodium hydroxide solution at 100 for 30 minutes and allow the aqueous layer to separate. On acidifying the aqueous layer with dilute sulphuric acid, no precipitate or oily matter is produced. Foreign organic matter. Volatilises when heated, without emitting an acrid odour. Consistency. 100 to 300, determined by the following method. Apparatus. The apparatus is essentially in agreement with IS 4887.1968 and comprises a penetrometer fitted with a polished cone-shaped metal plunger weighing 150 g having a detachable
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steel tip of the following dimensions. The tip of the cone has an angle of 30, the point being truncated to a diameter of 0.38 0.08 mm, the base of the tip is 8.38 0.13 mm in diameter and the length of the tip is 15 0.25 mm. The remaining portion of the cone has an angle of 90, is 28 to 29 mm in height, and has a maximum diameter of 65.1 mm at the base. The containers of the test are flat-bottomed metal or glass cylinders that are 102 6 mm in diameter and not less than 60 mm in height. Procedure. Melt a sufficient quantity at a temperature below 85 and pour into one or more of the containers filling to within 6 mm of the rim. Cool to 25 2.5 over a period of not less than 16 hours, protected from drafts. Two hours before the test, place the containers in a water-bath at 25 0.5. If the room temperature is below 23.5 or above 26.5, adjust the temperature of the cone to 25 0.5 by placing it in a waterbath. Without disturbing the surface of the substance under examination, place the container on the penetrometer table, and lower the cone until the tip just touches the top surface of the test substance at a spot 25 mm to 38 mm from the edge of the container. Adjust the zero setting and quickly release the plunger, then hold it free for 5 seconds. Secure the plunger and read the total penetration from the scale. Make three or more trials, each so spaced that there is no overlapping of the areas of penetration. Where the penetration exceeds 20 mm, use a separate container of the test substance for each trial. Read the penetration to the nearest 0.1 mm. Calculate the average of the three or more readings and conduct further trials to a total of 10 if the individual results differ from the average by more than 3 per cent. The final average of the trials is not less than 10.0 mm and not more than 30.0 mm indicating a consistency value between 100 and 300. Sulphated ash (2.3.18). Not more than 0.1 per cent. Storage. Store protected from light and moisture.
Paraldehyde
H3C O O O CH3 CH3
C6H12O3
Mol. Wt. 132.7
Paraldehyde is 2,4,6-trimethyl-1,3,5-trioxane, the cyclic trimer of acetaldehyde. It may contain a suitable amount of antioxidant. Description. A colourless or slightly yellow, transparent liquid; odour, strong and characteristic. Solidifies at low temperature to form a crystalline mass.
Identification
A. Heat 5 ml with 0.1 ml of 1 M sulphuric acid; acetaldehyde, recognisable by its odour, is evolved. B. To 5 ml of a 10 per cent v/v solution add 5 ml of ammoniacal silver nitrate solution in a test-tube and heat on a water-bath; metallic silver is deposited as a mirror on the sides of the tube. C. A 10 per cent w/v solution in carbon dioxide-free water is clear (2.4.1), but becomes turbid on warming.
Tests
Congealing range (2.4.10). 10 to 13. Distillation range (2.4.8). Not more than 10 per cent distils below 123 and not less than 95 per cent distils below 126. Refractive index (2.4.27). 1.403 to 1.406. Relative density (2.4.29). 0.991 to 0.996. Acetaldehyde. Shake 5 ml with a mixture of 5 ml of ethanol (60 per cent), 5 ml of hydroxylamine hydrochloride reagent in ethanol (60 per cent) and 2 drops of methyl orange solution and titrate with 0.5 M sodium hydroxide to full yellow colour; not more than 0.8 ml of 0.5 M sodium hydroxide is required. Acidity. Mix 5 ml with 45 ml of carbon dioxide-free water and titrate with 0.1 M sodium hydroxide using phenolphthalein solution as indicator; not more than 1.5 ml is required. Chlorides. To 5 ml of a 1 per cent v/v solution add one drop of nitric acid and three drops of silver nitrate solution; no opalescence is produced immediately. Sulphates. To 5 ml of a 1 per cent v/v solution add one drop of hydrochloric acid and three drops of barium chloride solution; no turbidity is produced. Peroxides. In a stoppered vessel, dissolve 5 ml in sufficient of recently boiled and cooled water to produce 50 ml, add 5 ml of dilute sulphuric acid and 10 ml of potassium iodide solution.
Paraffin Ointment
White Beeswax 20 g Hard Paraffin 30 g Cetostearyl Alcohol 50 g White Soft Paraffin* 900 g *May be replaced by Yellow Soft Paraffin if other medicaments to be incorporated are coloured. Mix the ingredients, heat gently with stirring until homogeneous and stir until cold.
Tests
Paraffin Ointment complies with the tests stated under Ointments.
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Close the flask and set aside in the dark for 15 minutes. Titrate with 0.1 M sodium thiosulphate using starch solution as indicator; set aside for 5 minutes and, if necessary, complete the titration. Not more than 2.0 ml of 0.1 M sodium thiosulphate is required. Non-volatile matter. Heat 5 ml in a small dish on a water-bath and dry at 105 for 1 hour; the residue weighs not more than 3 mg (0.06 per cent w/v). Storage. Store protected from moisture, in complete darkness and at a temperature of 8 to 15. If solidified, the whole of the contents of the container should be liquified by warming before use. NOTE Do not use Paraldehyde if it has a brownish colour or an odour of acetic acid. Avoid contact with rubber and plastics. Labelling. The label states (1) the nature and the proportion of any antioxidant added; (2) that it may decompose on standing to form potentially harmful substances.
and expose to iodine vapour for 5 to 10 minutes. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution. B. Dissolve 0.5 g in a mixture of 0.5 ml of hydrochloric acid and 4 ml of warm acetone, cool in ice and scratch the inside of the tube with a glass rod to initiate crystallisation; a white precipitate is produced. Filter under vacuum, wash the precipitate with acetone and dry with suction. A 1 per cent w/v solution of the dried material is dextrorotatory. C. To 4 ml of a 1 per cent w/v solution add 2 ml of phosphotungstic acid solution and heat nearly to boiling; a blue colour is produced. D. In the test for Penicillamine disulphide, the principal peak in the chromatogram obtained with test solution (b) corresponds to the peak due to penicillamine in the chromatogram obtained with reference solution (a).
Tests
Appearance of solution. A 10.0 per cent w/v solution in carbon dioxide-free water (solution A) is clear (2.4.1), and not more intensely coloured than degree 6 of the appropriate range of reference solutions (2.4.1). pH (2.4.24). 4.5 to 5.5, determined in a 1.0 per cent w/v solution.
Penicillamine
D-Penicillamine
H3 C HS
CH3 O OH NH2 H
Specific optical rotation (2.4.22). 61.0 to 65.0, determined in a 5.0 per cent w/v solution in 1 M sodium hydroxide. Mol. Wt. 149.2 Heavy metals (2.3.13). 10 ml of solution A, complies with the limit test for heavy metals, Method D (20 ppm). Use lead standard solution (2 ppm Pb) to prepare the standard. Mercuric salts. Determine by atomic absorption spectrophotometry (2.4.2), using a solution prepared in the following manner. To 1.0 g of the substance under examination add 10 ml of water and 0.15 ml of perchloric acid and swirl until dissolution is complete. Add 1 ml of ammonium pyrrolidinedithiocarbamate solution that has been washed three times immediately before use, each time with an equal volume of 4-methyl-2-pentanone. Mix, add 2 ml of 4-methyl2-pentanone, shake for 1 minute, dilute to 25 ml with water, allow the layers to separate and use the 4-methyl-2-pentanone layer. Measure the absorbance at 254 nm using a mercury hollow-cathode lamp and an air-acetylene flame and setting the zero using a 4-methyl-2-pentanone layer obtained by repeating the procedure described above but omitting the substance under examination. For the standard solution dissolve 0.108 g of yellow mercuric oxide in the minimum volume of 2 M hydrochloric acid, add sufficient water to produce 1000.0 ml and treat suitable volumes in the same manner as the solution of the substance under examination (10 ppm). Penicillamine disulphide. Determine by liquid chromatography (2.4.14).
C5H11NO2S Penicillamine is 3-mercapto-D-valine.
Penicillamine contains not less than 98.0 per cent and not more than 101.0 per cent of C5H11NO2S, calculated on the dried basis. Description. A white or almost white, crystalline powder.
Identification
Test A may be omitted if test B, C and D carried out. Test D may be omitted if test A, B and C are carried out. A. Determine by thin layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 40 volumes of 1-butanol, 10 volumes of glacial acetic acid and 10 volumes of water. Test solution. Dissolve 0.25 g of the substance under examination in 100 ml of water. Reference solution. A 0.25 per cent w/v solution of penicillamine RS in water. Apply to the plate 2 l of each solution. Allow the mobile phase to rise 10 cm. Dry the plate at 105 for 5 to 10 minutes
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IP 2007
Test solution (a). Dissolve 40 mg of the substance under examination in 5 ml of the mobile phase, add 1 ml of a 0.0025 per cent w/v solution of sulphanilamide (internal standard) in the mobile phase and dilute to 10 ml with the mobile phase. Test solution (b). Dissolve 40 mg of the substance under examination in the mobile phase and dilute to 10 ml with the same solvent. Reference solution (a). A 0.4 per cent w/v solution of penicillamine RS in the mobile phase. Reference solution (b). Add 1 ml of test solution (a) to 1 ml of a 0.04 per cent w/v solution of penicillamine disulphide RS in the mobile phase and dilute to 10 ml with the mobile phase. Chromatographic system a stainless steel column 25 cm x 5 mm, packed with octylsilane bonded to porous silica (5 to 10 m), mobile phase: an equal volume of 0.2 per cent w/v solution of methanesulphonic acid and 0.01 per cent w/v solution of disodium edetate, flow rate. 2 ml per minute, spectrophotometer set at 220 nm, a 20 l loop injector. In the chromatogram obtained with test solution (a) the ratio of the area of any peak corresponding to penicillamine disulphide to the area of the peak due to the internal standard is not greater than the corresponding ratio in the chromatogram obtained with reference solution (b). Penicillin. Carry out the following procedure in a penicillinfree atmosphere and with equipment reserved for the test. Sterilise the equipment at 180 for 3 hours and the buffer solutions at 121 for 20 minutes before use. Liquefy a suitable nutrient medium such as that described below and inoculate at a suitable temperature with a culture of Micrococcus flavus (ATCC 9341) to give 5 x 104 microorganisms per ml or a quantity necessary to obtain the required sensitivity and formation of clearly defined inhibition zones of suitable diameter. Immediately pour the inoculated medium into five Petri dishes (10 cm in diameter) to give uniform layers 2 to 5 mm in depth. Alternatively, the medium may consist of two layers, only the upper layer being inoculated. Store the dishes so that no appreciable growth or death of microorganisms occurs before use and so that the surface of the medium is dry at the time of use. In each dish, place five stainless steel hollow cylinders (6 mm in diameter) on the surface of the medium evenly spaced on a circle with a radius of about 25 mm and concentric with the dish. For each dish, place in separate cylinders 0.15 ml of each of the following five solutions. For solution (1) dissolve 1.0 g of the substance under examination in 8 ml of phosphate buffer pH 2.5, add 8 ml of
ether and shake vigorously for 1 minute. Repeat the extraction and combine the ether layers. Add 8 ml of phosphate buffer pH 2.5, shake for 1 minute, allow to settle and separate the ether layer quantitatively, taking care to eliminate the aqueous phase completely. (Penicillin is unstable at pH 2.5; carry out the operations at this pH within 6 to 7 minutes). Add 8 ml of phosphate buffer pH 6.0, shake for 5 minutes, allow to settle, separate the aqueous layer and check that the pH is 6.0. For solution (2) add 20 l of penicillinase solution to 2 ml of solution (1) and incubate at 37 for 1 hour. For solution (3) dissolve 5 mg of benzylpenicillin sodium in 500 ml of phosphate buffer pH 6.0 and dilute 0.25 ml of this solution to 200 ml with phosphate buffer pH 2.5. Carry out the extraction procedure described under solution (1) using 8 ml of this solution and beginning at the words add 8 ml of ether.... For solution (4) add 20 ml of penicillinase solution to 2 ml of solution (3) and incubate at 37 for 1 hour. Prepare solution (5) in the same manner as solution (1) but omitting the substance under examination. Maintain the dishes at 30 for at least 24 hours. Measure the diameters of the zones of inhibition to within 0.1 mm. The test is not valid unless solution (3) gives a clear zone of inhibition and solutions (4) and (5) give no zones of inhibition. If solution (1) gives a zone of inhibition it is caused by penicillin provided solution (2) gives no zone of inhibition. If this is the case, the average diameter of the zones of inhibition given by solution (1) for the five Petri dishes is less than that given by solution (3) (0.1 ppm). Nutrient medium Peptone Yeast extract Meat extract Sodium chloride Agar Distilled water Adjust the pH to 5 g 1.5 g 1.5 g 3.5 g 15 g 1000 ml 6.0
Penillic acid. Absorbance of a 0.2 per cent w/v solution at about 268 nm, not more than 0.07 (2.4.7) (about 0.5 per cent). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 60 over phosphorus pentoxide at a pressure not exceeding 0.7 kPa. Assay. Dissolve 0.1 g in 30 ml of anhydrous glacial acetic acid. Titrate with 0.1 M perchloric acid, determining the endpoint potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.01492 g of C5H11NO2S. Storage. Store protected from moisture.
908
IP 2007
DILUTED PENTAERYTHRITOL TETRANITRATE
Penicillamine Tablets
D-Penicillamine Tablets
Penicillamine Tablets contain not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of penicillamine, C5H11NO2S. The tablets are coated.
In the chromatogram obtained with the test solution the area of any peak corresponding to penicillamine disulphide is not greater than the area of the principal peak in the chromatogram obtained with the reference solution (1.0 per cent). Other tests. Complies with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing 0.1 g of Penicillamine, dissolve as completely as possible in 50 ml of water and filter. Add to the filtrate 5 ml of 1 M sodium hydroxide and 0.2 ml of a 0.1 per cent w/v solution of dithizone in ethanol (95 per cent) and titrate with 0.02 M mercuric nitrate. 1 ml of 0.02 M mercuric nitrate is equivalent to 0.005968 g of C5H11NO2S. Storage. Store protected from moisture.
Identification
A. Shake a quantity of the powdered tablets containing 20 mg of Penicillamine with 4 ml of water and filter. Add to the filtrate 2 ml of phosphotungstic acid solution and allow to stand for 5 minutes; a blue colour is produced. B. Dissolve a quantity of the powdered tablets containing 10 mg of Penicillamine in 5 ml of water and add 0.3 ml of 5 M sodium hydroxide and 20 mg of ninhydrin; an intense blue or violet-blue colour is produced immediately.
Tests
Mercuric salts. Disperse a quantity of the powdered tablets containing 1 g of Penicillamine in 10 ml of water in a stoppered flask, add 0.2 ml of 9 M perchloric acid and swirl to dissolve. Add 1 ml of ammonium pyrrolidinedithiocarbamate solution, mix, add 2 ml of 4-methyl-2-pentanone, shake for 1 minute and add sufficient water to produce 25 ml. Determine by atomic absorption spectrophotometry (2.4.2), using a mercury hollowcathode lamp and an air-acetylene flame and setting the zero using a 4-methyl-2-pentanone layer obtained by repeating the procedure described above but omitting the substance under examination, measuring at 254 nm. Use mercury solution AAS, suitably diluted with water, for the standard solutions, adjusted to contain the same concentrations of 9 M perchloric acid, ammonium pyrrolidinedithiocarbamate solution and 4-methyl-2-pentanone as the solution under examination (40 ppm). Penicillamine disulphide. Determine by liquid chromatography (2.4.14). Test solution. Shake quantity of the powdered tablets containing 40 mg of Penicillamine with 10 ml of the mobile phase, filter and use the filtrate. Reference solution. A 0.004 per cent w/v solution of penicillamine disulphide RS in the mobile phase. Chromatographic system a stainless steel column 25 cm x 5 mm, packed with octylsilane bonded to porous silica (5 to 10 m), mobile phase: an equal volume of 0.2 per cent w/v solution of methanesulphonic acid and 0.01 per cent w/v solution of disodium edetate, flow rate. 2 ml per minute, spectrophotometer set at 220 nm, a 20 l loop injector.
Diluted Pentaerythritol Tetranitrate

O2 N O O O NO2 NO2
Mol. Wt. 316.1
O2N O
C5H8N4O12
Diluted Pentaerythritol Tetranitrate is a dry mixture of 2,2bis(hydroxymethyl)- propane1,3-diol tetranitrate with Lactose or Mannitol or a mixture of Lactose and Starch or any other suitable inert excipients which permit safe handling. Diluted Pentaerythritol Tetranitrate contains not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of pentaerythritol tetranitrate, C5H8N4O12. Description. A white or almost white, powder; odour, faint and mild.
Identification
A. Transfer a quantity containing 10 mg of pentaerythritol tetranitrate to a medium porosity sintered-glass filter, add 5 ml of dry acetone and collect the filtrate. Repeat with two further quantities, each of 5 ml, of dry acetone and evaporate the combined filtrate at a temperature not exceeding 60, with the aid of a gentle current of air, and dry the residue at 60 for 4 hours; the residue melts at 138 to 142 (2.4.21). B. Suspend 10 mg of the residue obtained in test A in a mixture of 2 ml of sulphuric acid and 1 ml of water; cool and carefully overlay with 3 ml of ferrous sulphate solution; a reddish brown colour is produced at the interface of the two liquids.
909
PENTAERYTHRITOL TETRANITRATE TABLETS
IP 2007
Tests
Heavy metals (2.3.13). 2.0 g complies with limit test for heavy metals, Method B (10 ppm). Assay. Weigh accurately a quantity containing about 50 mg of pentaerythritol tetranitrate and transfer to a 100-ml volumetric flask with the aid of about 30 ml of acetone. Add sufficient acetone to produce 50 ml and warm on a water-bath at a temperature not exceeding 60 and boil gently, with occasional swirling, for 5 minutes. Cool, dilute to volume with acetone and mix. Transfer a portion of the mixture to a glassstoppered centrifuge tube and centrifuge at 1500 rpm for 5 minutes. Transfer 1.0 ml of the supernatant solution to a 100-ml volumetric flask and evaporate at 35 with the aid of a current of air to dryness. To the residue add 1.0 ml of glacial acetic acid and swirl to dissolve. Add 2 ml of phenoldisulphonic acid solution, mix and allow to stand for 5 minutes. Add 25 ml of water and 10 ml of strong ammonia solution, cool, dilute to volume with water and mix. Measure the absorbance of the resulting solution at the maximum at about 409 nm (2.4.7), using water as the blank. Weigh accurately 0.13 g of potassium nitrate, previously dried at 105 for 4 hours, dissolve in 3 ml of water, dilute with sufficient glacial acetic acid to produce 200.0 ml and mix well. Using 1.0 ml of this solution repeat the procedure beginning at the words Add 2 ml of phenoldisulphonic acid solution,........ Calculate the content of C5H8N4O12 from the values of the absorbances so obtained. 1 ml of the potassium nitrate solution is equivalent to 0.000503 g of C5H8N4O12. Storage. Store protected from light and moisture. NOTE Undiluted pentaerythritol tetranitrate is a powerful explosive. It can be exploded with percussion or excessive heat. Great care and appropriate precautions should be taken in handling and only exceedingly small amounts should be isolated. Labelling. The label states the percentage content of pentaerythritol tetranitrate.
of dry acetone and collect the filtrate. Repeat with two further quantities, each of 5 ml, of dry acetone and evaporate the combined filtrate at a temperature not exceeding 60, with the aid of a gentle current of air, and dry the residue at 60 for 4 hours; the residue melts at 138 to 142 (2.4.21).
Tests
Uniformity of content. (For tablets containing 10 mg or less) Comply with the test stated under Tablets. Crush one tablet and transfer to a 50-ml volumetric flask with the aid of 15 ml of acetone. Add sufficient acetone to produce 25 ml, heat the mixture on a water-bath at a temperature not exceeding 60 and boil gently, with occasional swirling, for 5 minutes. Cool, dilute to volume with acetone and mix. Transfer a portion of the mixture to a glass-stoppered centrifuge tube and centrifuge at 1500 rpm for 5 minutes. Transfer 2.5 ml of the supernatant solution to a 100-ml volumetric flask and evaporate at 35 with the aid of a current of air to dryness. To the residue add 1.0 ml of glacial acetic acid and swirl to dissolve. Add 2 ml of phenoldisulphonic acid solution, mix and allow to stand for 5 minutes. Add 25 ml of water and 10 ml of strong ammonia solution, cool, dilute to volume with water and mix. Measure the absorbance of the resulting solution at the maximum at about 409 nm (2.4.7), using water as the blank. Weigh accurately 0.130 g of potassium nitrate, previously dried at 105 for 4 hours, dissolve in 3 ml of water, dilute with sufficient glacial acetic acid to produce 200.0 ml and mix well. Using 1.0 ml of this solution repeat the procedure beginning at the words Add 2 ml of phenoldisulphonic acid solution,........Calculate the content of C5H8N4O12 in the tablet. 1 ml of the potassium nitrate solution is equivalent to 0.000503 g of C5H8N4O12. Other tests. Complies with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 50 mg of pentaerythritol tetranitrate and transfer to a 100-ml volumetric flask with the aid of about 30 ml of acetone. Add sufficient acetone to produce 50 ml and warm on a water-bath at a temperature not exceeding 60 and boil gently, with occasional swirling, for 5 minutes. Cool, dilute to volume with acetone and mix. Transfer a portion of the mixture to a glass-stoppered centrifuge tube and centrifuge at 1500 rpm for 5 minutes. Transfer 1.0 ml of the supernatant solution to a 100-ml volumetric flask and evaporate at 35 with the aid of a current of air to dryness. To the residue add 1.0 ml of glacial acetic acid and swirl to dissolve. Add 2 ml of phenoldisulphonic acid solution, mix and allow to stand for 5 minutes. Add 25 ml of water and 10 ml of strong ammonia solution, cool, dilute to volume with water and mix. Measure the absorbance of the
Pentaerythritol Tetranitrate Tablets

Pentaerythritol Tetranitrate Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of pentaerythritol tetranitrate, C5H8N4O12.
Identification
A. Transfer a quantity containing 10 mg of pentaerythritol tetranitrate to a medium porosity sintered-glass filter, add 5 ml
910
IP 2007
PENTAMIDINE INJECTION
resulting solution at the maximum at about 409 nm (2.4.7), using water as the blank. Weigh accurately 0.13 g of potassium nitrate, previously dried at 105 for 4 hours, dissolve in 3 ml of water, dilute with sufficient glacial acetic acid to produce 200.0 ml and mix well. Using 1.0 ml of this solution repeat the procedure beginning at the words Add 2 ml of phenoldisulphonic acid solution,........ Calculate the content of C5H8N4O12 from the values of the absorbances so obtained. 1 ml of the potassium nitrate solution is equivalent to 0.000503 g of C5H8N4O12. Storage. Store protected from light and moisture. Labelling. The label states the strength in terms of the equivalent amount of pentaerythritol tetranitrate.
Tests
pH (2.4.24). 4.5 to 6.5, determined in a 5.0 per cent w/v solution. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. The upper layer obtained by shaking together 50 volumes of water, 40 volumes of 1-butanol and 10 volumes of glacial acetic acid. Test solution. Dissolve 0.5 g of the substance under examination in 10 ml of methanol. Reference solution. A 0.025 per cent w/v solution of the substance under examination in 100 ml of methanol. Activate the plate by heating at 105 for 1 hour, apply to the plate 10 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Ammonium isethionate. To 1.0 g in a test-tube (about 4 cm in diameter) add 10 ml of water and 20 ml of 1 M sodium hydroxide. Immediately attach a bung carrying a splash head and an aspirator tube (about 5 mm in diameter). Connect the splash head to two test-tubes in series, each containing 20 ml of 0.01 M sulphuric acid. Heat the tube containing the substance under examination in a water-bath at 45 to 50 and, maintaining this temperature, draw a current of air, previously passed through 1 M sulphuric acid, through the liquids in a series of tubes for 3 hours at such a rate that the bubbles are just too rapid to count. Titrate the combined solutions from the two absorption tubes with 0.02 M sodium hydroxide using methyl red-methylene blue solution as indicator; not less than 36.5 ml of 0.02 M sodium hydroxide is required. Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 4.0 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Dissolve 0.25 g in 50 ml of dimethylformamide. Titrate with 0.1 M tetrabutylammonium hydroxide, determining the end-point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M tetrabutylammonium hydroxide is equivalent to 0.02963 g of C19H24N4O2,2C2H6O4S. Storage. Store protected from moisture.
Pentamidine Isethionate
O H2N NH O NH2 . HO NH SO3H
2
C19H24N4O2,2C2H6O4S
Mol. Wt. 592.7
Pentamidine Isethionate is 4,4-[pentane-1,5diylbis(oxy)]dibenzamidine di(2-hydroxyethanesulphonate). Pentamidine Isethionate contains not less than 98.5 per cent and not more than 101.0 per cent of C19H24N4O2, 2C2H6O4S, calculated on the dried basis. Description. A white or almost white powder or crystals; odourless or almost odourless; hygroscopic.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with pentamidine isethionate RS or with the reference spectrum of pentamidine isethionate. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.001 per cent w/v solution in 0.01 M hydrochloric acid shows an absorption maximum only at about 262 nm; absorbance at about 262 nm, about 0.46). C. To 10 ml of a 0.05 per cent w/v solution add 1 ml of a 0.1 per cent w/v solution of glyoxal sodium bisulphite and 1 ml of a solution prepared by dissolving 4 g of boric acid in a mixture of 27 ml of 1 M sodium hydroxide and sufficient water to produce 100 ml. Heat on a water-bath for 10 minutes; a magenta colour is produced.
Pentamidine Injection
Pentamidine Isethionate Injection
Pentamidine Injection is a sterile material consisting of Pentamidine Isethionate with or without buffering agents and other excipients. It is filled in a sealed container.
911
PENTAZOCINE
IP 2007
The injection is constituted by dissolving the contents of the sealed container in the requisite amount of sterile Water for Injections, immediately before use. The constituted solution complies with the requirements for Clarity of solution and Particulate matter stated under Parenteral Preparations (Injections). Storage. The constituted solution should be used immediately after preparation but, in any case, within the period recommended by the manufacturer. Pentamidine Injection contains not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of pentamidine isethionate, C19H24N4O2, 2C2H6O4S. The contents of the sealed container comply with the requirements stated under Parenteral Preparations (Powders for Injection) and with the following requirements.
not more intense than the spot in the chromatogram obtained with the reference solution. Ammonium isethionate. To 1.0 g in a test-tube (about 4 cm in diameter) add 10 ml of water and 20 ml of 1 M sodium hydroxide. Immediately attach a bung carrying a splash head and an aspirator tube (about 5 mm in diameter). Connect the splash head to two test-tubes in series, each containing 20 ml of 0.01 M sulphuric acid. Heat the tube containing the substance under examination in a water-bath at 45 to 50 and, maintaining this temperature, draw a current of air, previously passed through 1 M sulphuric acid, through the liquids in a series of tubes for 3 hours at such a rate that the bubbles are just too rapid to count. Titrate the combined solutions from the two absorption tubes with 0.02 M sodium hydroxide using methyl red-methylene blue solution as indicator; not less than 36.5 ml of 0.02 M sodium hydroxide is required. Assay. Dissolve 0.25 g of the mixed contents of 10 containers in 50 ml of dimethylformamide. Titrate with 0.1 M tetrabutylammonium hydroxide, determining the end-point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M tetrabutylammonium hydroxide is equivalent to 0.02963 g of C19H24N4O2, 2C2H6O4S. Storage. Store in single dose containers.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with pentamidine isethionate RS or with the reference spectrum of pentamidine isethionate. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.001 per cent w/v solution in 0.01 M hydrochloric acid shows an absorption maximum only at about 262 nm; absorbance at about 262 nm, about 0.46. C. To 10 ml of a 0.05 per cent w/v solution add 1 ml of a 0.1 per cent w/v solution of glyoxal sodium bisulphite and 1 ml of a solution prepared by dissolving 4 g of boric acid in a mixture of 27 ml of 1 M sodium hydroxide and sufficient water to produce 100 ml. Heat on a water-bath for 10 minutes; a magenta colour is produced.
Pentazocine
HO
H3C
N CH3
CH3 CH3
Mol. Wt. 285.4
Tests
pH (2.4.24). 4.5 to 6.5, determined in a 5.0 per cent w/v solution. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. The upper layer obtained by shaking together 50 volumes of water, 40 volumes of 1-butanol and 10 volumes of glacial acetic acid. Test solution. Dissolve 0.5 g of the substance under examination in 10 ml of methanol. Reference solution. A 0.025 per cent w/v solution of the substance under examination in 100 ml of methanol. Activate the plate by heating at 105 for 1 hour, apply to the plate 10 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is C19H27NO
Pentazocine is (2RS,6RS,11RS)-6,11-dimethyl-3-(3-methylbut2-enyl)-1,2,3,4,5,6-hexahydro-2,6-methano-3-benzazocin-8-ol. Pentazocine contains not less than 98.0 per cent and not more than 101.0 per cent of C19H27NO, calculated on the dried basis. Description. A white or pale cream powder.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with pentazocine RS or with the reference spectrum of pentazocine. B. To 1 mg in a porcelain crucible add 0.5 ml of a solution of sulphuric acid containing 1 per cent w/v solution of
912
IP 2007
PENTAZOCINE HYDROCHLORIDE
ammonium molybdate; an intense blue colour is produced which changes to bluish green, green and finally, on standing, yellow. C. Dissolve 5 mg in 5 ml of sulphuric acid, add 0.05 ml of ferric chloride solution and mix; a yellow colour is produced which deepens slightly in intensity on warming. On the addition of 0.05 ml of nitric acid the yellow colour is unchanged.
Pentazocine Hydrochloride
HO , HCl H3C CH3
C19H27NO,HCl
CH3 CH3
Mol. Wt. 321.9
Tests
Light absorption (2.4.7). To 0.1 g add 20 ml of water and 10 ml of 1 M hydrochloric acid, shake to dissolve and add sufficient water to produce 100 ml. Dilute 10 ml to 100 ml with water. The absorbance of the resulting solution at the maximum at about 278 nm, 0.67 to 0.71. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel HF254. Mobile phase. A mixture of 94 volumes of chloroform, 3 volumes of 2-propylamine and 3 volumes of methanol. Test solution. Dissolve 0.2 g of the substance under examination in 10 ml of chloroform. Reference solution (a). A 0.02 per cent w/v solution of the substance under examination in chloroform. Reference solution (b). A 0.01 per cent w/v solution of the substance under examination in chloroform. Reference solution (c). A 0.005 per cent w/v solution of the substance under examination in chloroform. Apply to the plate 10 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. Heat the plate at 105 for 15 minutes, allow to cool, expose to iodine vapour and re-examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (a); not more than one such spot is more intense than the spot in the chromatogram obtained with reference solution (b) and not more than four such spots are more intense than the spot in the chromatogram obtained with reference solution (c). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 1.0 per cent, determined on 1.0 g by drying in an oven at 60 at a pressure not exceeding 0.7 kPa for 4 hours. Assay. Weigh accurately about 0.6 g, dissolve in 50 ml of anhydrous glacial acetic acid and titrate with 0.1 M perchloric acid, using crystal violet solution as indicator. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.02854 g of C19H27NO. Storage. Store protected from light and moisture.
Pentazocine Hydrochloride is (2RS,6RS,11RS)-6,11dimethyl-3-(3-methylbut-2-enyl)-1,2,3,4,5,6-hexahydro-2,6methano-3-benzazocin-8-ol hydrochloride. Pentazocine Hydrochloride contains not less than 98.0 per cent and not more than 101.0 per cent of C19H27NO,HCl, calculated on the dried basis. Description. A white or pale cream-coloured, crystalline powder; odourless. The material exhibits polymorphism.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with pentazocine hydrochloride RS or with the reference spectrum of pentazocine hydrochloride. B. To 1 mg in a porcelain crucible add 0.5 ml of a solution of sulphuric acid containing 1 per cent w/v solution of ammonium molybdate; an intense blue colour is produced which changes to bluish green, green and finally, on standing, yellow. C. Gives reaction A of chlorides (2.3.1).
Tests
pH (2.4.24). 4.0 to 6.0, determined in a 1.0 per cent w/v solution. Light absorption (2.4.7). Dissolve 0.1 g in 10 ml of 1 M hydrochloric acid and add sufficient water to produce 100 ml; dilute 10 ml to 100 ml with water. Absorbance of the resulting solution at the maximum at about 278 nm, 0.59 to 0.63. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel HF254. Mobile phase. A mixture of 94 volumes of chloroform, 3 volumes of 2-propylamine and 3 volumes of methanol. Test solution. Dissolve 0.2 g of the substance under examination in 10 ml of chloroform. Reference solution (a). A 0.02 per cent w/v solution of the substance under examination in chloroform.
913
PENTAZOCINE TABLETS
IP 2007
Reference solution (b). A 0.01 per cent w/v solution of the substance under examination in chloroform. Reference solution (c). A 0.005 per cent w/v solution of the substance under examination in chloroform. Apply to the plate 10 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. Heat the plate at 105 for 15 minutes, allow to cool, expose to iodine vapour and re-examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (a); not more than one such spot is more intense than the spot in the chromatogram obtained with reference solution (b) and not more than four such spots are more intense than the spot in the chromatogram obtained with reference solution (c). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 1.0 per cent, determined on 1.0 g by drying in an oven at 100 at a pressure not exceeding 0.7 kPa. Assay. Weigh accurately about 0.5 g, dissolve in 50 ml of anhydrous glacial acetic acid. Titrate with 0.1 M perchloric acid, using crystal violet solution as indicator. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.03219 g of C19H27NO,HCl. Storage. Store protected from light and moisture.
B. To a quantity of the powdered tablets containing 50 mg of Pentazocine Hydrochloride add 70 ml of water, shake for 15 minutes, add sufficient water to produce 100 ml and filter. To 10 ml of the filtrate add 10 ml of 1 M sodium hydroxide and sufficient water to produce 100 ml. When examined in the range 230 nm to 360 nm (2.4.7), the resulting solution shows absorption maxima at about 238 nm and 298 nm. C. Shake a quantity of the powdered tablets containing 25 mg of Pentazocine Hydrochloride with 5 ml of water and 0.5 ml of 2 M nitric acid for 1 minute and filter. The filtrate gives reaction A of chlorides (2.3.1).
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel HF254. Mobile phase. A mixture of 94 volumes of chloroform, 3 volumes of 2-propylamine and 3 volumes of methanol. Test solution. Shake a quantity of the powdered tablets containing 0.2 g of Pentazocine Hydrochloride with 10 ml of 0.1 M methanolic ammonia for 10 minutes, centrifuge and use the supernatant liquid. Reference solution (a). Dilute 1 volume of test solution to 100 volumes with the same solvent. Reference solution (b). Dilute 1 volume of test solution to 200 volumes with the same solvent. Reference solution (c). Dilute 1 volume of test solution to 400 volumes with the same solvent. Apply to the plate 10 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. Heat the plate at 105 for 15 minutes, allow to cool, expose to iodine vapour and re-examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (a), not more than one such spot is more intense than the spot in the chromatogram obtained with reference solution (b) and not more than four such spots are more intense than the spot in the chromatogram obtained with reference solution (c). Other tests. Complies with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 25 mg of Pentazocine Hydrochloride, shake with 100 ml of water for 15 minutes, add 2.5 ml of 1 M hydrochloric acid and sufficient water to produce 250.0 ml and filter. Measure the absorbance of the filtrate at the maximum at about 278 nm (2.4.7). Calculate the content of C19H27NO,HCl taking 61.2 as the specific absorbance at 278 nm. Storage. Store protected from light and moisture.
Pentazocine Tablets
Pentazocine Hydrochloride Tablets
Pentazocine Tablets contain not less than 92.5 per cent and not more than 107.5 per cent of the stated amount of pentazocine hydrochloride, C19H27NO,HCl.
Identification
A. Shake a quantity of the powdered tablets containing 0.1 g of Pentazocine Hydrochloride with 10 ml of water, filter, add 1 ml of 1 M sodium hydroxide and shake the resulting solution with 20 ml of chloroform. Wash the chloroform extract with 5 ml of water, dry over anhydrous sodium sulphate and filter. Evaporate the chloroform using a rotary evaporator and dry the oily residue at a temperature not exceeding 25 at a pressure of 2 kPa for 1 hour. On the residue, determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with pentazocine RS or with the reference spectrum of pentazocine.
914
IP 2007
PENTAZOCINE INJECTION
Pentazocine Lactate
HO COOH H3C CH3
C19H27NO,C3H6O3
Reference solution (b). A 0.01 per cent w/v solution of the substance under examination in chloroform. Reference solution (c). A 0.005 per cent w/v solution of the substance under examination in chloroform.
CH3 CH3
, H3C OH
Mol. Wt. 375.4
Pentazocine Lactate is (2RS,6RS,11RS)-6,11-dimethyl-3-(3methylbut-2-enyl)-1,2,3,4,5,6-hexahydro-2,6-methano-3benzazocin-8-ol lactate. Pentazocine Lactate contains not less than 98.5 per cent and not more than 101.0 per cent of C19H27NO,C3H6O3, calculated on the dried basis. Description. A white or pale cream-coloured, crystalline powder; odourless.
Apply to the plate 25 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. Heat the plate at 105 for 15 minutes, allow to cool, expose to iodine vapour and re-examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (a); not more than one such spot is more intense than the spot in the chromatogram obtained with reference solution (b) and not more than four such spots are more intense than the spot in the chromatogram obtained with reference solution (c). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 1.0 per cent, determined on 1.0 g by drying in an oven at 60 at a pressure not exceeding 0.7 kPa for 16 hours. Assay. Weigh accurately about 0.75 g of the substance under examination, dissolve in 50 ml of anhydrous glacial acetic acid. Titrate with 0.1 M perchloric acid, using crystal violet solution as indicator. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.03754 g of C19H27NO,C3H6O3. Storage. Store protected from light and moisture.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with pentazocine lactate RS or with the reference spectrum of pentazocine lactate. B. To 1 mg in a porcelain crucible add 0.5 ml of a solution of sulphuric acid containing 1 per cent w/v solution of ammonium molybdate; an intense blue colour is produced which changes to bluish green, green and finally, on standing, yellow. C. Gives reaction A of lactates (2.3.1).
Pentazocine Injection
Pentazocine Lactate Injection
Pentazocine Injection is a sterile solution in Water for Injections of either Pentazocine Lactate or pentazocine lactate prepared by the interaction of Pentazocine and Lactic Acid. Pentazocine Injection contains not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of pentazocine, C19H27NO. Description. A clear, colourless or almost colourless liquid.
Tests
pH (2.4.24). 5.5 to 6.5, determined in a 1.0 per cent w/v solution. Light absorption (2.4.7). Dissolve 0.1 g in 10 ml of 1 M hydrochloric acid and add sufficient water to produce 100 ml; dilute 10 ml to 100 ml with water. Absorbance of the resulting solution at the maximum at about 278 nm, 0.50 to 0.54. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel HF254. Mobile phase. A mixture of 94 volumes of chloroform, 3 volumes of 2-propylamine and 3 volumes of methanol. Test solution. Dissolve 0.2 g of the substance under examination in 10 ml of chloroform. Reference solution (a). A 0.02 per cent w/v solution of the substance under examination in chloroform.
Identification
A. To a volume containing 90 mg of pentazocine add 5 ml of 0.1 M sodium hydroxide and shake the resulting solution with 5 ml of chloroform. Wash the chloroform extract with 2 ml of water, dry over anhydrous sodium sulphate and filter. Evaporate the chloroform without applying heat and dry the oily residue at a temperature not exceeding 25 and at a pressure of 2 kPa for 1 hour.
915
PENTOBARBITONE SODIUM
IP 2007
On the residue, determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with pentazocine RS or with the reference spectrum of pentazocine (form B). B. Dilute a volume containing 30 mg of pentazocine to 100 ml with water. To 10 ml add 10 ml of 1 M sodium hydroxide and sufficient water to produce 100 ml. When examined in the range 230 nm to 360 nm (2.4.7), the solution shows absorption maxima, at about 238 nm and 298 nm. C. To a volume containing 30 mg of pentazocine add 2 ml of 0.1 M sodium hydroxide, extract with 2 ml of chloroform and evaporate 0.1 ml of the chloroform extract to dryness in a porcelain crucible. Add to the residue 0.5 ml of a 1 per cent w/v solution of ammonium molybdate in sulphuric acid; an intense blue colour is produced which changes to bluish green, green and finally, on standing, yellow.
100.0 ml. To 5.0 ml add 1 ml of 1 M hydrochloric acid and sufficient water to produce 100.0 ml. Measure the absorbance of the resulting solution at the maximum at about 278 nm (2.4.7). Calculate the content of C19H27NO, taking 69 as the specific absorbance at 278 nm. Storage. Store protected from light. Labelling. The label states the strength in terms of the equivalent amount of pentazocine in a suitable dose-volume.
Pentobarbitone Sodium
Soluble Pentobarbitone; Pentobarbital Sodium
H N N H3C O
Mol. Wt. 248.3
Tests
pH (2.4.24). 4.0 to 5.0. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel HF254. Mobile phase. A mixture of 94 volumes of chloroform, 3 volumes of 2-propylamine and 3 volumes of methanol. Test solution. Dilute a volume of the injection with sufficient ethanol (95 per cent) to produce a solution containing 2.0 per cent w/v solution of pentazocine. Reference solution (a). Dilute 1 volume of the test solution to 100 volumes with ethanol (95 per cent). Reference solution (b). Dilute 1 volume of the test solution to 200 volumes with ethanol (95 per cent). Reference solution (c). Dilute 1 volume of the test solution to 400 volumes with ethanol (95 per cent). Apply to the plate 10 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. Heat the plate at 105 for 15 minutes, allow to cool, expose to iodine vapour and re-examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (a), not more than one such spot is more intense than the spot in the chromatogram obtained with reference solution (b) and not more than four such spots are more intense than the spot in the chromatogram obtained with reference solution (c). Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. To an accurately measured volume containing about 0.15 g of pentazocine add sufficient water to produce C11H17N2NaO3
O H3C H3C
ONa
Pentobarbitone Sodium is sodium 5-ethyl-5-[(1RS)-1methylbutyl]barbiturate. Pentobarbitone Sodium contains not less than 99.0 per cent and not more than 101.5 per cent of C11H17N2NaO3, calculated on the dried basis. Description. A white, crystalline powder or granules; hygroscopic.
Identification
A. To 10 ml of a 10 per cent w/v solution add 5 ml of 2 M acetic acid; a white, crystalline precipitate is produced. Filter, wash the precipitate with water and dry at 105. Determine the melting point of the dried precipitate (2.4.21). Mix equal parts of the dried precipitate and phenobarbitone RS and determine the melting point (2.4.21). The difference between the melting points (which are about 131) is not greater than 2. B. Complies with the test for identification of barbiturates (2.3.1), using the dried precipitate obtained in test A for preparing the test solution. C. To 10 mg add 10 mg of vanillin and 2 ml of sulphuric acid, mix and heat on a water-bath for 2 minutes; a reddish-brown colour is produced. Cool and add 5 ml of ethanol; the colour changes to violet and then blue. D. Ignite 1 g; the residue gives reaction A of sodium salts (2.3.1).
916
IP 2007
PENTOBARBITONE TABLETS
Tests
Appearance of solution. Prepare freshly a 10.0 per cent w/v solution in carbon dioxide-free water (solution A). Solution A is clear (2.4.1). pH (2.4.24). 9.6 to 11.0, determined in solution A. Related substances. Complies with the test for related substances in barbiturates (2.3.4), but applying 10 l of each of the following solutions. Test solution. A 2.0 per cent w/v solution of the substance under examination. Reference solution. A 0.01 per cent w/v solution of the substance under examination. Do not ignore any spot remaining on the line of application. Free pentobarbitone. Not more than 3.5 per cent, determined by the following method. Weigh accurately about 2.0 g and dissolve in 75 ml of dimethylformamide, heating gently if necessary. Add 0.25 ml of a 1 per cent w/v solution of thymol blue in dimethylformamide and titrate with 0.1 M sodium methoxide to a blue end-point. Carry out a blank titration. 1 ml of 0.1 M sodium methoxide is equivalent to 0.02263 g of pentobarbitone. Isomer. Dissolve 0.3 g in 5 ml of a 5 per cent w/v solution of anhydrous sodium carbonate and add 0.3 g of 4-nitrobenzyl bromide dissolved in 10 ml of ethanol (95 per cent). Heat under a reflux condenser for 30 minutes, cool to 25 scratch the side of the vessel with a glass rod if necessary to induce crystallisation and filter. Wash the residue with five quantities, each of 5 ml, of water. Transfer the residue as completely as possible to a small flask, add 25 ml of ethanol (95 per cent) and heat under a reflux condenser for 10 minutes; the solid dissolves completely. Cool to 25 and scratch the side of the flask with a glass rod to induce crystallisation. Filter, wash the residue with two quantities, each of 5 ml, of water and dry at 105 for 30 minutes. The dried residue melts at 136 to 148 (2.4.21). Heavy metals (2.3.13). 0.67 g complies with the limit test for heavy metals, Method B (30 ppm). Loss on drying (2.4.19). Not more than 3.0 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.4 g, dissolve in 25 ml of a 12.75 per cent w/v solution of silver nitrate in pyridine and titrate with 0.1 M ethanolic sodium hydroxide using 0.5 ml of thymolphthalein solution as indicator, until a pure blue colour is obtained. Carry out a blank titration. 1 ml of 0.1 M ethanolic sodium hydroxide is equivalent to 0.02483 g of C11H17N2NaO3. Storage. Store protected from moisture.
Pentobarbitone Tablets
Pentobarbitone Sodium Tablets; Pentobarbital Sodium Tablets
Pentobarbitone Tablets contain not less than 92.5 per cent and not more than 107.5 per cent of the stated amount of pentobarbitone sodium, C11H17N2NaO3.
Identification
A. Shake a quantity of the powdered tablets containing 0.1 g of Pentobarbitone Sodium with 10 ml of a 10 per cent w/v solution of pyridine and filter. Add to the filtrate 1 ml of cupric sulphate with pyridine solution and set aside for 10 minutes; a reddish violet precipitate is produced. B. Shake a quantity of the powdered tablets containing 0.1 g of Pentobarbitone Sodium with 10 ml of water and filter. To the filtrate add 2 ml of hydrochloric acid; a white precipitate is produced (distinction from pentobarbitone). C. The residue obtained in the Assay melts at 127 to 130 (2.4.21). D. The powdered tablets, when moistened with hydrochloric acid and introduced on a platinum wire into a flame, impart a yellow colour to the flame.
Tests
Isomer. Dissolve a quantity of the powdered tablets containing 0.3 g of Pentobarbitone Sodium in 5 ml of a 5 per cent w/v solution of anhydrous sodium carbonate and add 0.3 g of 4-nitrobenzyl bromide dissolved in 10 ml of ethanol (95 per cent). Heat under a reflux condenser for 30 minutes, cool to 25 scratch the side of the vessel with a glass rod if necessary to induce crystallisation and filter. Wash the residue with five quantities, each of 5 ml, of water. Transfer the residue as completely as possible to a small flask, add 25 ml of ethanol (95 per cent) and heat under a reflux condenser for 10 minutes; filter the hot solution. Cool to 25 and scratch the side of the flask with a glass rod to induce crystallisation. Filter, wash the residue with two quantities, each of 5 ml, of water and dry at 105 for 30 minutes. The dried residue melts at 136 to 148 (2.4.21). Other tests. Complies with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 0.3 g of Pentobarbitone Sodium, dissolve as completely as possible in 10 ml of a 2 per cent w/v solution of sodium hydroxide, saturate with sodium chloride, acidify with hydrochloric acid and extract with successive quantities, each of 15 ml, of ether until complete extraction is effected. Wash the combined extracts with two quantities, each of 2 ml, of water and extract the combined washings with 10 ml of ether. Add the ether to
917
PEPSIN
IP 2007
the main ether layer, filter and wash the filter with ether. Evaporate the solvent and dry the residue to constant weight at 105. 1 g of residue is equivalent to 1.097 g of C11H17N2NaO3. Storage. Store protected from moisture.
Pepsin
Pepsin is obtained from the gastric mucosa of pigs, cattle or sheep. It contains gastric proteinases that are active in an acid medium, pH 1 to 5. It may contain a suitable diluent such as Lactose. Pepsin has an activity equivalent to its ability to digest not less than 3000 times its weight of coagulated egg albumin when determined by the method given under Assay. Description. A white or light buff-coloured, crystalline or amorphous powder or translucent scales; odour, faint and meaty but not rancid; hygroscopic.
65 ml of 1 M hydrochloric acid to 1000 ml with water, continue the trituration, dilute to 1000.0 ml with the acidified water and shake for 15 minutes. Prepare coagulated egg albumin by boiling fresh hen-eggs in water for 15 minutes, cooling rapidly to room temperature by immersion in cold water, separating the whites and rubbing through a no. 44 sieve. Reject the first portion that passes through the sieve and triturate 15.0 g of freshly prepared coagulated egg albumin with 50 ml of the acidified water ensuring that the particles of egg albumin are thoroughly disintegrated, add a further 50 ml of the acidified water and keep in a water-bath at 51 1 for 15 minutes. Add 20.0 ml of the prepared solution of the substance under examination and digest at 51 1 for 4 hours, shaking at intervals of 15 minutes. Centrifuge and decant off most of the clear supernatant liquid, wash the remainder into a 10-ml graduated cylinder and allow to stand for 30 minutes. The volume of the undissolved albumin is not more than 2 ml. Storage. Store protected from moisture.
Identification
A. Place 1 ml of congo red fibrin on a filter paper and wash until a colourless filtrate is obtained with a solution prepared by diluting 30 ml of 1 M hydrochloric acid to 1000 ml with water and adjusting the pH 1.5 to 1.7. Perforate the filter paper and wash the congo red fibrin through it with 20 ml of the same hydrochloric acid solution. Shake this suspension before use. Dissolve about 10 mg of the substance under examination in 2 ml of the hydrochloric acid solution and adjust the pH 1.5 to 1.7. Place 4 ml of the congo red fibrin suspension in each of two tubes. To one of the tubes add 1 ml of the solution of the substance under examination and to the other tube add 1 ml of water (control solution). Mix the contents of each tube and place in a water-bath at 25 with gentle shaking for 15 minutes; the control solution is colourless and the solution of the substance under examination is violet blue. B. The proteolytic activity of a solution in water is destroyed at once by boiling. It is destroyed by warming for 10 minutes at 40 at a pH of 8.0.
Peritoneal Dialysis Solutions

Intraperitoneal Dialysis Fluids
Peritoneal Dialysis Solutions are sterile preparations for intraperitoneal use containing electrolytes with a concentration close to the electrolytic composition of plasma. They contain dextrose in varying concentrations and/or other suitable osmotic agents. They do not contain antioxidants such as metabisulphite salts. Peritoneal Dialysis Solutions contain not less than 97.5 per cent and not more than 102.5 per cent of the stated amount of sodium, Na, not less than 95.0 per cent and not more than 105.0 per cent of the stated amounts of potassium, K, calcium, Ca, magnesium, Mg, chloride, Cl, acetate, C2H3O2, lactate, C3H5O3, sodium bicarbonate, NaHCO3, bicarbonate, HCO3 and dextrose, C6H12O6. Description. Clear, colourless or faintly straw-coloured solutions.
Tests
Microbial contamination (2.2.9). 1 g is free from Escherichia coli and 10 g is free from salmonellae. Sulphated ash (2.3.18). Not more than 5.0 per cent. Loss on drying (2.4.19). Not more than 5.0 per cent, determined on 1.0 g by drying in an oven at 60 at a pressure not exceeding 0.7 kPa for 4 hours. Assay. Weigh accurately 0.25 g, triturate with 1.0 g of sodium chloride, add slowly acidified water prepared by diluting
Identification
A. To 5 ml of the solution under examination, add 2 ml of dilute sodium hydroxide solution and 0.05 ml of potassium cupri-tartrate solution; the solution remains blue and clear. Heat to boiling; a copious red precipitate is formed. B. 20 ml gives reactions of chlorides, sodium salts, potassium salts and calcium salts (2.3.1). C. To 5 ml add 1 ml of hydrochloric acid in a test-tube fitted with a stopper and a bent tube, heat and collect a few ml of the distillate. The distillate gives reaction C of acetates (2.3.1).
918
IP 2007
PERITONEAL DIALYSIS SOLUTIONS
D. To 0.1 ml of titan yellow solution add 10 ml of water, 2 ml of the solution under examination and 1 ml of 1 M sodium hydroxide; a pink colour is produced if magnesium salts are present. E. Lactates and bicarbonates are identified together with the Assay for lactate and bicarbonate.
Tests
Appearance of solution. The solution under examination is clear (2.4.1), and not more intensely coloured than reference solution YS4 (2.4.1). pH (2.4.24). 4.5 to 6.5. If the solution contains bicarbonate, 6.5 to 8.0. 5-Hydroxymethylfurfural and Related substances. Dilute a volume containing 1.0 g of Dextrose to 250.0 ml with water and measure the absorbance of the resulting solution (2.4.7) at the maximum at about 284 nm; absorbance at about 284 nm, not more than 0.25. Aluminium. Adjust the pH of 400 ml of the solution under examination to pH 6.0 and add 10 ml of acetate buffer pH 6.0. Extract the resulting solution with successive quantities of 20, 20 and 10 ml of a 0.5 per cent w/v solution of 8-hydroxyquinoline in chloroform and dilute the combined extracts to 50.0 ml with chloroform. Use as the blank a mixture of 10 ml of acetate buffer pH 6.0 and 100 ml of water treated in the same manner and as the standard solution a mixture of 2.0 ml of aluminium standard solution (2 ppm Al), 10 ml of acetate buffer pH 6.0 and 90 ml of water treated in the same manner. Measure the fluorescence of the test solution (I1), of the standard solution (I2) and of the blank (I3), (2.4.5), using an excitation wavelength of 392 nm and a secondary filter with a transmission band centred at 518 nm, or a monochromator set to transmit at this wavelength. The fluorescence of the test solution (I1 I3) is not greater than that of the standard solution (I2 I3). Particulate contamination (2.5.9). Carry out the test using 50 ml of the solution under examination. The preparation meets the requirements of the test if it contains particles within the maximum limits shown below. Particle size in m (Equal to or larger than) 10 25 Maximum number of particles per ml 25 3
Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Bacterial endotoxins (2.2.3). Not more than 0.25 Endotoxin Unit per ml.
Pyrogens (2.2.8). Solutions for which a validated test for bacterial endotoxins cannot be carried out, comply with the test for pyrogens, injecting 10 ml of the solution per kg of the rabbits body weight. Sterility (2.2.11). Complies with the test for sterility. Assay. For sodium Dilute suitably with water and determine by Method A for flame photometry (2.4.4), or by Method A for atomic absorption spectrophotometry (2.4.2), measuring at 589 nm and using sodium solution FP, or sodium solution AAS respectively, suitably diluted with water for the standard solutions. For potassium Dilute suitably with water and determine by Method A for flame photometry (2.4.4), or by Method A for atomic absorption spectrophotometry (2.4.2), measuring at 767 nm and using potassium solution FP or potassium solution AAS respectively, suitably diluted with water for the standard solutions. For calcium Dilute suitably with water and determine by Method A for flame photometry (2.4.4), or by Method A for atomic absorption spectrophotometry (2.4.2), measuring at 422.7 nm and using calcium solution FP or calcium solution AAS respectively, suitably diluted with water for the standard solutions. For magnesium To 50.0 ml add 50 ml of water and 5 ml of strong ammonia-ammonium chloride solution and titrate with 0.005 M disodium edetate using 50 mg of eriochrome black T mixture as indicator. 1 ml of 0.005 M disodium edetate is equivalent to 0.1215 mg of Mg. For total chloride Dilute an accurately measured volume containing about 60 mg of chloride to 50.0 ml with water. Add 25.0 ml of 0.1 M silver nitrate and 2 ml of nitric acid. Filter, wash the precipitate with water slightly acidified with nitric acid and titrate the excess of silver nitrate with 0.1 M ammonium thiocyanate using ferric ammonium sulphate solution as indicator until a reddish yellow colour is produced. Carry out a blank titration. 1 ml of 0.1 M silver nitrate is equivalent to 0.003545 g of total chloride, calculated as Cl. For acetate (if present) Determine by liquid chromatography (2.4.14). Test solution. Dilute an accurately measured volume of the preparation under examination quantitatively with water to obtain a solution containing about 1.0 mg of acetate per ml. Reference solution. Dissolve an accurately weighed quantity of sodium acetate in water to obtain a solution having a known concentration of about 0.12 per cent w/v of Sodium Acetate. Chromatographic system a stainless steel column 30 cm x 7.8 mm, packed with a strong cation-exchange resin consisting of sulphonated
919
PERITONEAL DIALYSIS SOLUTIONS
IP 2007
cross-linked styrene-divinylbenzene copolymer in the hydrogen form (7 m), mobile phase: filtered and degassed 0.1 M sulphuric acid, flow rate. 0.8 ml per minute, column temperature. 60, spectrophotometer set at 210 nm, a 20 l loop injector.
Inject the reference solution and record the chromatograms. The test is not valid unless the tailing factor is not more than 2.0 and the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject separately the test and standard solutions and record the chromatograms. Measure the responses for the major peak and calculate the content of acetate in the preparation under examination. For lactate (if present) Determine by liquid chromatography (2.4.14). Test solution. Use the preparation under examination. Reference solution(a). Dissolve an accurately weighed quantity of sodium lactate RS in water to obtain a solution having a known concentration of about 2 mg per ml. Reference solution (b). Prepare a solution in water containing about 3 mg of anhydrous sodium acetate and 3 mg of sodium lactate RS per ml. Chromatographic system a stainless steel column 10 cm x 4.6 mm, packed with octadecylsilane chemically bonded to porous silica or ceramic microparticles 3 to 10 m, mobile phase: a filtered and degassed solution in water containing about 1 ml of formic acid and 1 ml of dicyclohexylamine per litre, flow rate. 1 ml per minute, spectrophotometer set at 210 nm, a 20 l loop injector. Inject separately reference solutions (a) and (b), and record the chromatograms. The test is not valid unless the resolution between the peaks due to acetate and lactate is not less than 2.0, the tailing factor for the analyte peak is not more than 2.0 and the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject separately the test solution and reference solution (a), and record the chromatograms. Measure the responses for the major peak and calculate the content of lactate in the preparation under examination. For sodium bicarbonate Titrate with 0.1 M hydrochloric acid a volume of the preparation under examination containing about 0.1 g of sodium bicarbonate, determining the end-point potentiometrically (2.4.25).
1 ml of 0.1 M hydrochloric acid is equivalent to 8.40 mg of NaHCO3. For lactate and bicarbonate Determine by liquid chromatography (2.4.14). Test solution. Use the preparation under examination. Reference solutions. Dissolve accurately weighed quantities of lactates and bicarbonates in order to obtain solutions having concentrations of about 90 per cent, 100 per cent and 110 per cent of the claim in 100 ml of water. Chromatographic system a stainless steel column 30 cm x 7.8 mm, packed with a cation-exchange resin (9 m), column. temperature 85, mobile phase: filtered and degassed 0.005 M sulphuric acid, flow rate. 0.6 ml per minute, differential refractometer detector, a 20 l loop injector. Inject separately the test solution and the reference solutions in duplicate, and record the chromatograms in the prescribed conditions. The peaks elute in the following order, lactates, then bicarbonates. Determine the concentration of lactates and bicarbonates in the test solution by interpolating the peak area for lactate and the peak height for bicarbonate from the linear regression curve obtained with the solutions prepared as reference solutions. For dextrose Transfer a volume of the preparation under examination containing about 25 mg of Dextrose to a 250-ml conical flask with a ground-glass neck and add 25.0 ml of cupri-citric solution. Add a few grains of pumice, fit a reflux condenser, heat so that boiling occurs within 2 minutes and boil for exactly 10 minutes. Cool and add 3 g of potassium iodide dissolved in 3 ml of water. Carefully add, in small amounts, 25 ml of a 25 per cent w/w solution of sulphuric acid. Titrate with 0.1 M sodium thiosulphate using starch solution as indicator. Carry out a blank titration using 25 ml of water. Calculate the content of anhydrous dextrose, C6H12O6, from the following Table. Volume of 0.1 M sodium thiosulphate consumed (ml) 8 9 10 11 12 13 14 15 16 Anhydrous dextrose (mg) 19.8 22.4 25.0 27.6 30.3 33.0 35.7 38.5 41.3
920
IP 2007
PETHIDINE HYDROCHLORIDE
Storage. Peritoneal Dialysis Solutions are supplied in rigid or semi-rigid plastic containers, in flexible plastic containers fitted with a special connecting device (these are generally filled to a volume below their nominal capacity and presented in closed protective envelopes) or in glass containers. Store at a temperature not exceeding 30. CAUTION Exposure to temperatures below 4 may cause crystallisation and separation of solid particles rendering the preparation unsuitable for use. Labelling. The label states (1) the formula of the solution for peritoneal dialysis, expressed in grams per litre and in millimoles per litre; (2) the total osmolar concentration in mOsmol per litre; (3) the nominal volume of the solution in the container; (4) that the solution is free from bacterial endotoxins, or where applicable, that it is apyrogenic; (5) that the solution is not to be used for intravenous infusion; (6) that any unused portion of the solution is to be discarded; (7) that the solution containing visible particles should not be used; (8) the storage conditions.
B. To 5 ml of a 1 per cent w/v solution add a few drops of potassium mercuri-iodide solution; a cream-coloured precipitate is produced. C. Dissolve 5 mg in 0.5 ml of water and add 0.1 ml of formaldehyde solution and 2 ml of sulphuric acid; an orangered colour is produced. D. Gives reaction A of chlorides (2.3.1).
Tests
Appearance of solution. A 2.0 per cent w/v solution in carbon dioxide-free water is clear (2.4.1), and colourless (2.4.1). Acidity or alkalinity. To 10 ml of a 2.0 per cent w/v solution in carbon dioxide-free water add 0.2 ml of methyl red solution and 0.2 ml of 0.01 M sodium hydroxide; the solution is yellow. Add 0.3 ml of 0.01 M hydrochloric acid; the solution is red. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with kieselguhr G. Mobile phase. The upper layer obtained by shaking together 100 volumes of light petroleum (50 to 70), 8 volumes of 2-phenoxyethanol and 1 volume of diethylamine. Test solution. Dissolve 0.1 g in 5 ml of water, add 0.5 ml of 10 M sodium hydroxide and 2 ml of ether and shake; allow the layers to separate and use the upper layer. Reference solution. Dilute 0.5 ml of the test solution to 50 ml with ether.
Pethidine Hydrochloride
Meperidine Hydrochloride
CH3 N O O CH3 , HCl
C15H21NO2,HCl
Mol. Wt. 283.8
Impregnate the dry plate by placing it in a closed tank containing a mixture of 90 volumes of acetone and 10 volumes of 2-phenoxyethanol so that the plate dips about 5 mm beneath the surface of the liquid, allowing the impregnating solvent to ascend at least 15 cm, removing the plate from the tank and drying in a current of air. Use immediately, with the flow of the mobile phase in the same direction as the impregnation. Apply to the plate 5 l of each solution. Allow the mobile phase to rise 12 cm. Dry the plate in air for 10 minutes, return the plate to the tank and repeat the development. Remove the plate, allow it to dry in air for 10 minutes and spray with a 0.2 per cent w/v solution of 2,7-dichlorofluorescein in methanol. Allow to stand for 5 minutes and spray with water until the background is white to pale yellow. Examine in daylight. The chromatograms show red or orange spots. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Examine without delay in ultraviolet light at 365 nm. The chromatograms show spots with intense yellow fluorescence. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution.
Pethidine Hydrochloride is ethyl 1-methyl-4phenylpiperidine-4-carboxylate hydrochloride. Pethidine Hydrochloride contains not less than 99.0 per cent and not more than 101.0 per cent of C15H21NO2,HCl, calculated on the dried basis. Description. Colourless crystals or a white, crystalline powder.
Identification
Test A may be omitted if tests B, C and D are carried out. Tests B and C may be omitted if tests A and D are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with pethidine hydrochloride RS or with the reference spectrum of pethidine hydrochloride.
921
PETHIDINE INJECTIONS
IP 2007
Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.5 g, dissolve in 30 ml of anhydrous glacial acetic acid, add 12 ml of mercuric acetate solution. Titrate with 0.1 M perchloric acid, using crystal violet solution as indicator. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.02838 g of C15H21NO2,HCl. Storage. Store protected from light and moisture.
necessary to 5 ml with water, with 0.5 ml of 5 M sodium hydroxide and 2 ml of ether. Reference solution. Dilute 0.5 ml of the test solution to 50 ml with ether. Impregnate the dry plate by placing it in a closed tank containing a mixture of 90 volumes of acetone and 10 volumes of 2-phenoxyethanol so that the plate dips about 5 mm beneath the surface of the liquid, allowing the impregnating solvent to ascend at least 15 cm, removing the plate from the tank and drying in a current of air. Use immediately, with the flow of the mobile phase in the same direction as the impregnation. Apply to the plate 5 l of each solution. Allow the mobile phase to rise 12 cm. Dry the plate in air for 10 minutes, return the plate to the tank and repeat the development. Remove the plate, allow it to dry in air for 10 minutes and spray with a 0.2 per cent w/v solution of 2,7-dichlorofluorescein in methanol. Allow to stand for 5 minutes and spray with water until the background is white to pale yellow. Examine in daylight. The chromatograms show red or orange spots. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Examine without delay in ultraviolet light at 365 nm. The chromatograms show spots with intense yellow fluorescence. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. Dilute an accurately measured volume containing about 0.1 g of Pethidine Hydrochloride with 40 ml of water, add 1 ml of 5 M sodium hydroxide and extract immediately with successive quantities of 25, 10 and 10 ml of chloroform. Wash each extract with the same 15 ml of water, filter into a dry flask and combine the extracts (which should be clear and free from droplets of water). Titrate with 0.02 M perchloric acid, using 0.15 ml of 1-naphtholbenzein solution as indicator. Carry out a blank titration. 1 ml of 0.02 M perchloric acid is equivalent to 0.005676 g of C15H21NO2,HCl. Storage. Store protected from light.
Pethidine Injection
Pethidine Hydrochloride Injection; Meperidine Hydrochloride Injection
Pethidine Injection is a sterile solution of Pethidine Hydrochloride in Water for Injections. Pethidine Injection contains not less than 95.0 per cent and not more than 105.0 per cent of pethidine hydrochloride, C15H21NO2,HCl.
Identification
A. To a volume containing 50 mg of Pethidine Hydrochloride add sufficient 1 M sodium hydroxide to make strongly alkaline to litmus paper and extract with two quantities, each of 10 ml, of chloroform. Wash the combined extracts with 5 ml of water, dry over anhydrous sodium sulphate, filter and evaporate the filtrate to dryness. Remove the last traces of chloroform by drying the residual oil at 60 at a pressure not exceeding 0.7 kPa. On the oily residue, determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with pethidine hydrochloride RS or with the reference spectrum of pethidine. B. To 0.5 ml add 0.1 ml of formaldehyde solution and 2 ml of sulphuric acid; an orange-red colour is produced. C. Gives the reactions of chlorides (2.3.1).
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with kieselguhr G. Mobile phase. The upper layer obtained by shaking together 100 volumes of light petroleum (50 to 70), 8 volumes of 2-phenoxyethanol and 1 volume of diethylamine. Test solution. The upper layer obtained by shaking a volume containing 0.1 g of Pethidine Hydrochloride, diluted if
Pethidine Tablets
Pethidine Hydrochloride Tablets; Meperidine Hydrochloride Tablets
Pethidine Tablets contain not less than 92.5 per cent and not more than 107.5 per cent of the stated amount of pethidine hydrochloride, C15H21NO2,HCl.
922
IP 2007
PHENINDAMINE TARTRATE
Identification
A. Shake a quantity of the powdered tablets containing 50 mg of Pethidine Hydrochloride with 20 ml of chloroform, filter, evaporate the filtrate to dryness and dry the residue at a pressure of 2 kPa. On the residue, determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with pethidine hydrochloride RS or with the reference spectrum of pethidine hydrochloride. B. Shake a quantity of the powdered tablets containing 0.2 g of Pethidine Hydrochloride with 20 ml of water and filter. To 5 ml of the filtrate add 10 ml of picric acid solution. The crystals so obtained, after washing with water and drying, melt at about 190 (2.4.21).
intense than the spot in the chromatogram obtained with the reference solution. Other tests. Comply with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 0.3 g of Pethidine Hydrochloride in 40 ml of water, add 2 ml of 5 M sodium hydroxide and extract immediately with successive quantities of 25, 10 and 10 ml of chloroform. Wash each extract with the same 15 ml of water and filter into a dry flask. Combine the extracts (which should be clear and free from droplets of water). Titrate with 0.05 M perchloric acid, using 0.15 ml of 1-naphtholbenzein solution as indicator. Carry out a blank titration. 1 ml of 0.05 M perchloric acid is equivalent to 0.01419 g of C15H21NO2,HCl. Storage. Store protected from light and moisture.
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with kieselguhr G. Mobile phase. The upper layer obtained by shaking together 100 volumes of light petroleum (50 to 70), 8 volumes of 2-phenoxyethanol and 1 volume of diethylamine. Test solution. The upper layer obtained by shaking a quantity of the powdered tablets containing 0.1 g of Pethidine Hydrochloride with 5 ml of water, filtering, shaking the filtrate with 0.5 ml of 5 M sodium hydroxide and 2 ml of ether and allowing the layers to separate. Reference solution. Dilute 0.5 ml of the test solution to 50 ml with ether. Impregnate the dry plate by placing it in a closed tank containing a mixture of 90 volumes of acetone and 10 volumes of 2-phenoxyethanol so that the plate dips about 5 mm beneath the surface of the liquid, allowing the impregnating solvent to ascend at least 15 cm, removing the plate from the tank and drying in a current of air. Use immediately, with the flow of the mobile phase in the same direction as the impregnation. Apply to the plate 5 l of each solution. Allow the mobile phase to rise 12 cm. Dry the plate in air for 10 minutes, return the plate to the tank and repeat the development. Remove the plate, allow it to dry in air for 10 minutes and spray with a 0.2 per cent w/v solution of 2,7-dichlorofluorescein in methanol. Allow to stand for 5 minutes and spray with water until the background is white to pale yellow. Examine in daylight. The chromatograms show red or orange spots. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Examine without delay in ultraviolet light at 365 nm. The chromatograms show spots with intense yellow fluorescence. Any secondary spot in the chromatogram obtained with the test solution is not more
Phenindamine Tartrate
H , N CH3 HOOC
OH COOH H OH
Mol. Wt. 411.5
C19H19N,C4H6O6
Phenindamine Tartrate is (RS)-2,3,4,9-tetrahydro-2-methyl-9phenyl-1H-indeno[2,1-c]pyridine (2R,3R)-tartrate. Phenindamine Tartrate contains not less than 98.5 per cent and not more than 101.0 per cent of C19H19N, C4H6O6, calculated on the dried basis. Description. A white or almost white voluminous powder; odourless or almost odourless.
Identification
A. When examined in the range 230 nm to 360 nm (2.4.7), a 0.004 per cent w/v solution shows an absorption maximum only at about 259 nm; absorbance at about 259 nm, about 0.88. B. Dissolve 25 mg in 5 ml of sulphuric acid; an orange-brown colour is produced which is discharged when the solution is carefully diluted with 20 ml of water. C. Dissolve 0.5 g in 15 ml of hot water, add a slight excess of 5 M sodium hydroxide, filter and neutralise the filtrate to litmus paper with 2 M hydrochloric acid. The solution gives reaction B of tartrates (2.3.1).
923
PHENINDAMINE TABLETS
IP 2007
D. Melting range (2.4.21). 160 to 162, when heated to 163 it re-solidifies and melts again at about 168, with decomposition.
Tests
pH (2.4.24). 3.4 to 3.9, determined in a 1.0 per cent w/v solution. Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 1.0 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.4 g, dissolve in 40 ml of warm water, cool, add 10 ml of dilute sodium carbonate solution and extract with successive quantities of 25, 10 and 10 ml of chloroform, washing each extract with the same 15 ml of water and filtering into a dry flask. Combine the extracts (which should be clear and free from droplets of water). Titrate with 0.05 M perchloric acid, using oracet blue B solution as indicator. Carry out a blank titration. 1 ml of 0.05 M perchloric acid is equivalent to 0.02057 g of C19H19N, C4H6O6. Storage. Store protected from light and moisture.
accurately measured quantity of the filtrate containing about 0.2 g of Phenindamine Tartrate add 10 ml of 5 M sodium hydroxide, and extract with 25 ml and then with three quantities, each of 10 ml, of chloroform, washing each extract with the same 15 ml of water and filtering in a dry flask. Combine the extracts (which should be clear and free from droplets of water).Titrate with 0.02 M perchloric acid, using oracet blue B solution as indicator. Carry out a blank titration. 1 ml of 0.02 M perchloric acid is equivalent to 0.008229 g of C19H19N, C4H6O6. Storage. Store protected from light and moisture.
Phenindione
O
O
C15H10O2 Phenindione is 2-phenylindane-1,3-dione. Mol. Wt. 222.2
Phenindamine Tablets
Phenindamine Tartrate Tablets
Phenindamine Tablets contain not less than 92.5 per cent and not more than 107.5 per cent of the stated amount of phenindamine tartrate, C19H19N, C4H6O6. The tablets are coated.
Phenindione contains not less than 98.0 per cent and not more than 100.5 per cent of C15H10O2, calculated on the dried basis. Description. Soft, white or creamy-white crystals; almost odourless.
Identification
A. Shake a quantity of the powdered tablets containing 20 mg of Phenindamine Tartrate with 100 ml of water, dilute 10 ml to 100 ml with water and filter; absorbance of the filtrate at the maximum at about 259 nm, about 0.44 (2.4.7). B. To a portion of the finely powdered tablets containing about 50 mg of Phenindamine Tartrate, add 5 ml of water and 3 ml of dilute ammonia solution and extract the liberated base with two successive quantities, each of 10 ml, of ether. Wash the combined ether extracts with 2 ml of water and evaporate to dryness. Dissolve 25 mg of the residue in 5 ml of sulphuric acid; an orange-brown colour is produced which is discharged when the solution is carefully diluted with 20 ml of water.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with phenindione RS. B. Dissolve 0.1 g in 30 ml of ethanol (95 per cent) with the aid of heat, cool and add sufficient ethanol (95 per cent) to produce 50 ml. Dilute 10 ml of this solution to 250 ml with 0.1 M sodium hydroxide and further dilute 5 ml to 100 ml with 0.1 M sodium hydroxide. When examined in the range 230 nm and 360 nm (2.4.7), the solution shows absorption maxima at about 278 nm and at about 330 nm; absorbance at about 278 nm, about 0.55 and at about 330 nm, about 0.16. C. To 1 g add 50 ml of ethanol (95 per cent) and 0.5 ml of aniline, heat gently under a reflux condenser for 3 hours, cool in ice and filter. The residue, after washing with 2 ml of ethanol (95 per cent) and recrystallisation from chloroform, melts at about 225 (2.4.21).
Tests
Other tests. Complies with the tests stated under Tablets. Assay. Weigh and digest 20 tablets with 70 ml of water and 5 ml of dilute hydrochloric acid until completely disintegrated, filter and wash the residue with 20 ml of water. Dilute the combined filtrate and washings to 100.0 ml with water. To an
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254.
924
IP 2007
PHENINDIONE TABLETS
Mobile phase. A 0.02 per cent w/v solution of butylated hydroxytoluene in a mixture of 80 volumes of toluene, 20 volumes of ethyl acetate and 4 volumes of glacial acetic acid. Test solution. Dissolve 0.1 g of the substance under examination in 10 ml of dichloromethane. Reference solution (a). A 0.02 per cent w/v solution of the substance under examination in dichloromethane. Reference solution (b). A 0.005 per cent w/v solution of the substance under examination in dichloromethane. Apply to the plate 10 l of each solution. Allow the mobile phase to rise 4 cm. Dry the plate in warm air and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (a) and not more than one such spot is more intense than the spot in the chromatogram obtained with reference solution (b). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 1.0 per cent, determined on 1.0 g by drying in oven at 105 for 2 hours. Assay. Weigh accurately about 0.3 g, add 50 ml of ethanol (95 per cent) and warm until solution is effected. Cool to room temperature, add 10 ml of a 10 per cent v/v solution of bromine in ethanol (95 per cent) and allow to stand for 10 minutes, shaking occasionally. Add 1 g of 2-naphthol and shake until the colour of the bromine is discharged. Remove any vapour of bromine in the flask with a current of air, add 50 ml of water and 10 ml of dilute potassium iodide solution and titrate the liberated iodine with 0.1 M sodium thiosulphate using starch solution as indicator. 1 ml of 0.1 M sodium thiosulphate is equivalent to 0.01111 g of C15H10O2. Storage. Store protected from moisture.
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with phenindione RS. B. Dissolve 0.1 g in 30 ml of ethanol (95 per cent) with the aid of heat, cool and add sufficient ethanol (95 per cent) to produce 50 ml. Dilute 10 ml of this solution to 250 ml with 0.1 M sodium hydroxide and further dilute 5 ml to 100 ml with 0.1 M sodium hydroxide. When examined in the range 230 nm to 360 nm (2.4.7), the solution shows absorption maxima at about 278 nm and 330 nm; absorbance at about 278. about 0.55 and at about 330 nm, about 0.16. C. To 50 mg add 1 ml of sulphuric acid; a deep blue to violet solution is produced. On dilution with water the solution becomes colourless and a white precipitate is produced.
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A 0.02 per cent w/v solution of butylated hydroxytoluene in a mixture of 80 volumes of toluene, 20 volumes of ethyl acetate and 4 volumes of glacial acetic acid. Test solution. Shake a quantity of the powdered tablets containing 50 mg of Phenindione with 15 ml of dichloromethane, filter, evaporate the filtrate to dryness and dissolve the residue in 5 ml of dichloromethane. Reference solution (a). Dilute 1 volume of the test solution to 50 volumes with dichloromethane. Reference solution (b). Dilute 1 volume of the test solution to 200 volumes with dichloromethane. Apply to the plate 10 l of each solution. Allow the mobile phase to rise 4 cm. Dry the plate in warm air and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (a) and not more than one such spot is more intense than the spot in the chromatogram obtained with reference solution (b). Uniformity of content. (For tablets containing 50 mg or less) Comply with the test stated under Tablets. Place one tablet in 50 ml of 0.1 M sodium hydroxide, dissolve completely by shaking gently, add a further 100 ml of 0.1 M sodium hydroxide and shake for 1 hour. Dilute to 250.0 ml with 0.1 M sodium hydroxide, filter and dilute a portion of the filtrate with sufficient 0.1 M sodium hydroxide to produce a solution containing 4 g of Phenindione per ml. Measure the absorbance of the resulting solution at the maximum at about 278 nm (2.4.7). Calculate the content of C15H10O2 taking 1310 as the specific absorbance at 278 nm.
Phenindione Tablets
Phenindione Tablets contain not less than 92.5 per cent and not more than 107.5 per cent of the stated amount of phenindione, C15H10O2.
Identification
Shake a quantity of the powdered tablets containing 0.2 g of Phenindione with 50 ml of chloroform, filter and evaporate the filtrate to dryness. Recrystallise the residue from ethanol (95 per cent). The crystals complies with the following tests.
925
PHENIRAMINE MALEATE
IP 2007
Other tests. Complies with the tests stated under Tablets. Assay. Weigh and powder 20 Tablets. Weigh accurately a quantity of the powder containing about 50 mg of Phenindione and shake with 150 ml of 0.1 M sodium hydroxide for 1 hour, add sufficient 0.1 M sodium hydroxide to produce 250.0 ml, filter and dilute 5.0 ml of the filtrate to 250.0 ml with 0.1 M sodium hydroxide. Measure the absorbance of the resulting solution at the maximum at about 278 nm (2.4.7). Calculate the content of C15H10O2 taking 1310 as the specific absorbance at 278 nm. Storage. Store protected from moisture.
add 5 ml of water; a yellow colour is produced. To 2 ml of the solution add 3 ml of a 50 per cent w/v solution of ammonium acetate, previously cooled in ice; a pink colour is produced which persists for at least 10 minutes in the cooled solution.
Tests
pH (2.4.24). 4.5 to 5.5, determined in a 1.0 per cent w/v solution. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 50 volumes of cyclohexane, 40 volumes of chloroform and 10 volumes of diethylamine. Test solution. Dissolve 0.2 g of the substance under examination in 10 ml of methanol. Reference solution (a). A 0.02 per cent w/v solution of the substance under examination in methanol. Reference solution (b). A 0.004 per cent w/v solution of the substance under examination in methanol. Apply to the plate 10 l of each solution. After development, dry the plate in air, spray with dilute potassium iodobismuthate solution. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (a) and not more than one such spot is more intense than the spot in the chromatogram obtained with reference solution (b). Heavy metals (2.3.13). Dissolve 1.0 g in 10 ml of water and add 2 ml of acetic acid and sufficient water to produce 25 ml. The resulting solution complies with the limit test for heavy metals, Method A (20 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 60 at a pressure not exceeding 0.7 kPa. Assay. Weigh accurately about 0.4 g, dissolve in 20 ml of anhydrous glacial acetic acid. Titrate with 0.1 M perchloric acid, using 1- naphtholbenzein solution as indicator. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.01782 g of C16H20N2,C4H4O4. Storage. Store protected from light and moisture.
Pheniramine Maleate
COOH N N CH3 CH3 , COOH

Mol. Wt. 356.4
C16H20N2,C4H4O4
Pheniramine Maleate is (3RS)-N,N-dimethyl-3-phenyl-3(pyridin-2-yl)propan-1-amine hydrogen maleate. Pheniramine Maleate contains not less than 99.0 per cent and not more than 101.0 per cent of C16H20N2,C4H4O4, calculated on the dried basis. Description. A white or almost white, crystalline powder; odourless or almost odourless.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with pheniramine maleate RS or with the reference spectrum of pheniramine maleate. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.002 per cent w/v solution in 0.1 M hydrochloric acid shows an inflection at about 262 nm; absorbance at about 265 nm, about 0.42. C. Dissolve 0.25 g in 5 ml of water, add 2 ml of strong ammonia solution and extract with three quantities, each of 5 ml, of chloroform. Evaporate the aqueous extract to dryness, add 0.2 ml of 1 M sulphuric acid and 5 ml of water, extract with four quantities, each of 25 ml, of ether and evaporate the combined ether extracts to dryness in a current of warm air. To the residue add 50 mg of resorcinol and 1 ml of sulphuric acid, heat in a water-bath for 2 minutes, shake well, heat in a water-bath for a further 30 minutes and cool in ice. Carefully
Pheniramine Injection
Pheniramine Maleate Injection
Pheniramine Injection is a sterile solution of Pheniramine Maleate in Water for Injections.
926
IP 2007
PHENIRAMINE TABLETS
Pheniramine Injection contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of pheniramine maleate, C16H20N2, C4H4O4.
Pheniramine Tablets
Pheniramine Maleate Tablets
Pheniramine Tablets contain not less than 92.5 per cent and not more than 107.5 per cent of the stated amount of pheniramine maleate, C16H20N2, C4H4O4.
Identification
Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 50 volumes of cyclohexane, 40 volumes of chloroform and 10 volumes of diethylamine. Test solution. Evaporate an appropriate volume of the injection to dryness in a current of nitrogen using the minimum amount of heat, dissolve the residue in sufficient chloroform to produce a solution containing 2.0 per cent w/v solution of Pheniramine Maleate and centrifuge. Reference solution. A 2.0 per cent w/v solution of pheniramine maleate RS in chloroform. Apply to the plate 10 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. The two principal spots in the chromatogram obtained with the test solution correspond to those in the chromatogram obtained with the reference solution. Spray the plate with dilute potassium iodobismuthate solution. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution.
Identification
Boil a quantity of the powdered tablets containing about 0.5 g of Pheniramine Maleate with 150 ml of acetone under a reflux condenser for about 45 minutes. Filter and evaporate the filtrate to dryness on a water-bath. The residue complies with the following tests. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with pheniramine maleate RS or with the reference spectrum of pheniramine maleate. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.002 per cent w/v solution in 0.1 M hydrochloric acid shows an inflection at about 262 nm; absorbance at about 265 nm, about 0.42. C. Dissolve 0.25 g in 5 ml of water, add 2 ml of strong ammonia solution and extract with three quantities, each of 5 ml, of chloroform. Evaporate the aqueous extract to dryness, add 0.2 ml of 1 M sulphuric acid and 5 ml of water, extract with four quantities, each of 25 ml, of ether and evaporate the combined ether extracts to dryness in a current of warm air. To the residue add 50 mg of resorcinol and 1 ml of sulphuric acid, heat in a water-bath for 2 minutes, shake well, heat in a water-bath for a further 30 minutes and cool in ice. Carefully add 5 ml of water; a yellow colour is produced. To 2 ml of the solution add 3 ml of a 50 per cent w/v solution of ammonium acetate, previously cooled in ice; a pink colour is produced which persists for at least 10 minutes in the cooled solution.
Tests
pH (2.4.24). 4.5 to 5.5. Related substances. Determine by the method described under the Identification test using as the reference solution a solution prepared by diluting 1 volume of the test solution to 500 volumes with chloroform. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. To an accurately measured volume containing about 0.11 g of Pheniramine Maleate add sufficient water to produce 50.0 ml and mix well. To 20.0 ml add sufficient 1 M sodium hydroxide to make the solution just alkaline to litmus paper, add 2 ml in excess and extract with two quantities, each of 50 ml, of ether. Wash each ether extract in succession with 20, 20 and 5 ml of 0.1 M hydrochloric acid, dilute the combined extracts to 100.0 ml with 0.1 M hydrochloric acid and mix. Dilute 5.0 ml to 100.0 ml with 0.1 M hydrochloric acid and measure the absorbance of the resulting solution at the maximum at about 265 nm (2.4.7), using 0.1 M hydrochloric acid as the blank. Calculate the content of C16H20N2, C4H4O4 taking 210 as the specific absorbance at 265 nm. Storage. Store protected from light.
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 50 volumes of cyclohexane, 40 volumes of chloroform and 10 volumes of diethylamine. Test solution. Shake a quantity of the powdered tablets containing 20 mg of Pheniramine Maleate with 10 ml of methanol, centrifuge and use the supernatant liquid. Reference solution (a). Dilute 1 volume of the test solution to 100 volumes with methanol. Reference solution (b). Dilute 1 volume of reference solution (a) to 20 volumes with methanol. Apply to the plate 10 l of each solution. After development, dry the plate in air, spray with dilute potassium iodobismuthate solution. Any secondary spot in the
927
PHENOBARBITONE
IP 2007
chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (a) and not more than one such spot is more intense than the spot in the chromatogram obtained with reference solution (b). Other tests. Complies with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 45 mg of Pheniramine Maleate, shake with 20 ml of 0.1 M hydrochloric acid, centrifuge and transfer the supernatant liquid to a 100-ml volumetric flask. Repeat the extraction with three further quantities, each of 20 ml, of 0.1 M hydrochloric acid. Combine the extracts and add sufficient 0.1 M hydrochloric acid to produce 100.0 ml. Mix and dilute 5.0 ml to 100.0 ml with 0.1 M hydrochloric acid; measure the absorbance of the resulting solution at the maximum at about 265 nm (2.4.7), using 0.1 M hydrochloric acid as the blank. Calculate the content of C16H20N2,C4H4O4 taking 210 as the specific absorbance at 265 nm. Storage. Store protected from light and moisture.
difference between the melting points, which are about 175, is not greater than 2. C. Complies with the test for identification of barbiturates (2.3.2). D. Dissolve about 20 mg in 5 ml of ethanol, add a drop of cobalt chloride solution and a drop of dilute ammonia solution; a violet colour is produced. E. Gives the reaction of non-nitrogen substituted barbiturates (2.3.1).
Tests
Appearance of solution. A 10.0 per cent w/v solution in a mixture of 20 volumes of 2 M sodium hydroxide and 30 volumes of water is clear (2.4.1), and not more intensely coloured than reference solution YS6 (2.4.1). Acidity. Mix 1.0 g with 50 ml of water, boil for 2 minutes, allow to cool, filter and adjust the volume to 50 ml. To 10 ml of the filtrate add 0.15 ml of methyl red solution; not more than 0.1 ml of 0.1 M sodium hydroxide is required to change the colour of the solution from orange-yellow to pure yellow. Related substances. Complies with the test for related substances in barbiturates (2.3.4). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 1.0 per cent w/w, determined on 1.0 g by drying in an oven at 105 for 2 hours. Assay. Weigh accurately about 0.1 g, dissolve in 5 ml of pyridine, add 0.25 ml of thymolphthalein solution and 10 ml of silver nitrate-pyridine reagent and titrate with 0.1 M ethanolic sodium hydroxide until a pure blue colour is obtained. Repeat the operation without the substance under examination. The difference between the titrations represents the amount of sodium hydroxide required. 1 ml of 0.1 M ethanolic sodium hydroxide is equivalent to 0.01161 g of C12H12N2O3. Storage. Store protected from moisture.
Phenobarbitone
Phenobarbital
O H3C NH O
C12H12N2O3 Mol. Wt. 232.2
H N
Phenobarbitone is 5-ethyl-5-phenylbarbituric acid. Phenobarbitone contains not less than 99.0 per cent and not more than 101.0 per cent of C12H12N2O3, calculated on the dried basis. Description. Colourless crystals or a white, crystalline powder; odourless.
Phenobarbitone Sodium
Phenobarbital Sodium; Soluble Phenobarbitone; Soluble Phenobarbital
O H3C N O
C12H11N2NaO3 Mol. Wt. 254.2
Identification
Test A may be omitted if tests B, C, D and E are carried out. Tests C, D and E may be omitted if tests A and B are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with phenobarbitone RS or with the reference spectrum of phenobarbitone. B. Determine the melting point (2.4.21) of the substance under examination and of a mixture of equal quantities of the substance under examination and phenobarbitone RS. The
H N
ONa
928
IP 2007
PHENOBARBITONE INJECTION
Phenobarbital Sodium; Soluble Phenobarbitone; Soluble Phenobarbital. Phenobarbitone Sodium contains not less than 99.0 per cent and not more than 101.0 per cent of C12H11N2NaO3, calculated on the dried basis. Description. A white powder or crystalline granules or flaky crystals; hygroscopic.
Test solution. A 1 per cent w/v solution of the substance under examination in ethanol (50 per cent). Reference solution. A 0.005 per cent w/v solution of the substance under examination in ethanol (50 per cent). Loss on drying (2.4.19). Not more than 7.0 per cent, determined on 0.5 g by drying in an oven at 150 for 4 hours. Assay. Weigh accurately about 0.15 g, dissolve in 2 ml of water and add 8 ml of 0.05 M sulphuric acid. Heat to boiling and cool. Add 30 ml of methanol and shake until dissolution is complete. Titrate with 0.1 M sodium hydroxide, determining the end-point potentiometrically (2.4.25). After the first inflection, stop the addition of the sodium hydroxide, add 10 ml of pyridine, mix and continue the titration until the second inflection is reached. The difference between the volumes represents the amount of sodium hydroxide required. 1 ml of 0.1 M sodium hydroxide is equivalent to 0.02542 g of C12H11N2NaO3. Storage. Store protected from moisture.
Identification
Test A may be omitted if tests B, C, D, E and F are carried out. Tests C, D and E may be omitted if tests A, B and F are carried out. A. Dissolve 0.2 g in 20 ml of ethanol (50 per cent), acidify with dilute hydrochloric acid and extract with 50 ml of ether. Wash the ether layer with 10 ml of water, dry over anhydrous sodium sulphate and filter. Evaporate the filtrate to dryness and dry the residue at 105. On the residue, determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with phenobarbitone RS or with the reference spectrum of phenobarbitone. B. Determine the melting point (2.4.21), of the residue obtained in test A and of a mixture of equal quantities of the residue and phenobarbitone RS. The difference between the melting points, which are about 175, is not greater than 2. C. Complies with the test for identification of barbiturates (2.3.2), but using the following solutions. Test solution. A 0.1 per cent w/v solution of the substance under examination in ethanol (50 per cent). Reference solution. A 0.09 per cent w/v solution of phenobarbitone RS in ethanol (50 per cent). D. 1 g dissolves completely in 20 ml of ethanol (90 per cent) (distinction from barbitone sodium). E. Gives the reaction of non-nitrogen substituted barbiturates (2.3.1). F. Ignite about 0.1 g; the residue gives the reactions of sodium salts (2.3.1).
Phenobarbitone Injection
Phenobarbital Sodium Injection; Phenobarbitone Sodium Injection; Soluble Phenobarbitone Injection
Phenobarbitone Injection is a sterile solution of Phenobarbitone Sodium in a mixture of nine volumes of Propylene Glycol and one volume of Water for Injections. Phenobarbitone Injection contains not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of phenobarbitone sodium, C12H11N2NaO3.
Identification
To a volume containing 1 g of Phenobarbitone Sodium add 15 ml of water if necessary, make slightly acidic with 1 M sulphuric acid and filter. The residue, after washing with water and drying at 105, complies with the following tests. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with phenobarbitone RS or with the reference spectrum of phenobarbitone. B. Dissolve 50 mg in 2 ml of a 0.2 per cent w/v solution of cobalt acetate in methanol, warm, add 50 mg of powdered borax and heat to boiling; a bluish violet colour is produced.
Tests
Appearance of solution. A 10.0 per cent w/v solution in ethanol (50 per cent) is clear (2.4.1), and not more intensely coloured than reference solution YS7 (2.4.1). pH (2.4.24). Not more than 10.2, determined in a 10.0 per cent w/v solution. Related substances. Complies with the test for related substances in barbiturates (2.3.4), but using the following solutions.
Tests
pH (2.4.24).10.0 to 11.0.
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PHENOBARBITONE SODIUM TABLETSS
IP 2007
Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. Weigh accurately about 2.0 g, add 30 ml of water and 3 g of sodium carbonate, stir to dissolve and titrate with 0.1 M silver nitrate until a distinct turbidity is observed when viewed against a black background, the solution being stirred vigorously throughout the titration. 1 ml of 0.1 M silver nitrate is equivalent to 0.02542 g of C12H11N2NaO3. Determine the weight per ml of the injection (2.4.29) and calculate the percentage weight in volume of C12H11N2NaO3. Storage. Store in single dose containers. Labelling. The label states that the injection should not be used if the solution is discoloured or if it contains a precipitate.
Tests
Other tests. Complies with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 0.3 g of Phenobarbitone Sodium, dissolve as completely as possible in 10 ml of a 2 per cent w/v solution of sodium hydroxide, saturate with sodium chloride, acidify with hydrochloric acid and extract with successive quantities, each of 15 ml, of ether until complete extraction is effected. Wash the combined extracts with two quantities, each of 2 ml, of water and extract the combined washings with 10 ml of ether. Add the ether to the main ether layer and dry the residue to constant weight at 105. 1 g of the residue is equivalent to 1.095 g of C12H11N2NaO3. Storage. Store protected from moisture.
Phenobarbitone Sodium Tablets

Phenobarbital Sodium Tablets; Soluble Phenobarbitone Tablets; Soluble Phenobarbital Tablets
Phenobarbitone Sodium Tablets contain not less than 92.5 per cent and not more than 107.5 per cent of the stated amount of phenobarbitone sodium, C12H11N2NaO3.
Phenobarbitone Tablets
Phenobarbital Tablets
Phenobarbitone Tablets contain not less than 92.5 per cent and not more than 107.5 per cent of the stated amount of phenobarbitone, C12H12N2O3.
Identification
Extract a quantity of the powdered tablets containing about 0.5 g of Phenobarbitone with 50 ml of ether, filter through anhydrous sodium sulphate and evaporate the ether to dryness on a water-bath. The residue complies with the following tests. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with phenobarbitone RS or with the reference spectrum of phenobarbitone. B. Dissolve 50 mg in 2 ml of a 0.2 per cent w/v solution of cobaltous acetate in methanol, warm, add 50 mg of powdered borax and heat to boiling; a bluish violet colour is produced.
Identification
A. Heat 0.1 g of the residue obtained in Assay on a water-bath with 15 ml of ethanol (25 per cent) until dissolved, filter while hot and allow to cool. Filter through a sintered-glass crucible, wash with a small quantity of ethanol (25 per cent) and dry at 105. Heat in a sealed tube at 105 for 1 hour. On the residue, determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with phenobarbitone RS or with the reference spectrum of phenobarbitone. B. The residue obtained in test A melts at about 175 (2.4.21). C. Dissolve 50 mg of the residue obtained in Assay in 2 ml of a 0.2 per cent w/v solution of cobaltous acetate in methanol, warm, add 50 mg of powdered borax and heat to boiling; a bluish violet colour is produced. D. Triturate a quantity of the powdered tablets containing 0.2 g of Phenobarbitone Sodium with 5 ml of water and filter; the filtrate is alkaline to litmus solution and yields a white precipitate on the addition of dilute hydrochloric acid. E. The powdered tablets, when moistened with hydrochloric acid and introduced on a platinum wire into a flame, impart a yellow colour to the flame.
Tests
Disintegration (2.5.1). 30 minutes Other tests. Complies with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 0.3 g of Phenobarbitone and extract in a continuous extraction apparatus (2.1.8) with ether until complete extraction is effected. Remove the ether and dry the residue, which is C12H12N2O3, to constant weight at 105. Storage. Store protected from moisture.
930
IP 2007
PHENOLPHTHALEIN
Phenol
Carbolic acid
OH
under examination. The difference between the titrations represents the amount of bromine required. 1 ml of 0.05 M bromine is equivalent to 0.001569 g of C6H6O. Storage. Store protected from light and moisture.
Phenolphthalein
C6H6O Mol. Wt. 94.1
O O
Phenol contains not less than 99.0 per cent and not more than 100.5 per cent of C6H6O. Description. Colourless or faintly pink or faintly yellowish crystals or crystalline masses; odour, characteristic; deliquescent.
OH HO
C20H14O4 Mol. Wt. 318.3
Identification
A. 0.5 g dissolves completely in 2 ml of strong ammonia solution. Dilute this solution to about 100 ml with water and to 2 ml of the resulting solution add 0.05 ml of sodium hypochlorite solution (3 per cent Cl); a blue colour develops which becomes progressively more intense. B. Dissolve 1.0 g in sufficient water to produce 15 ml (solution A) and to 1 ml add 10 ml of water and 0.1 ml of ferric chloride solution; a violet colour is produced which disappears on the addition of 5 ml of 2-propanol. C. To 1 ml of solution A add 10 ml of water and 1 ml of bromine solution; a pale yellow precipitate is produced.
Phenolphthalein is 3,3-bis(4-hydroxyphenyl)phthalide. Phenolphthalein contains not less than 98.0 per cent and not more than 102.0 per cent of C20H14O4, calculated on the dried basis. Description. A white or yellowish white, crystalline or amorphous powder; odourless or almost odourless.
Identification
A. Dissolves in dilute solutions of alkali hydroxides and in hot solutions of alkali carbonates forming a red solution which is decolorised by dilute acids. B. Melting range (2.4.21). 258 to 263.
Tests
Appearance of solution. Solution A is clear (2.4.1), and not more intensely coloured than reference solution BS6 (2.4.1). Acidity. To 2 ml of solution A add 0.05 ml of methyl orange solution; the solution is yellow. Freezing point (2.4.11). Not less than 39.5. Non-volatile matter. Not more than 0.05 per cent, when 5.0 g is volatilised on a water-bath and dried to constant weight at 105. Assay. Weigh accurately about 0.5 g and dissolve in sufficient water to produce 250.0 ml. Transfer 25.0 ml to a ground-glassstoppered flask, add 50.0 ml of 0.05 M bromine and 5 ml of hydrochloric acid, stopper, allow to stand for 30 minutes, swirling occasionally, and allow to stand for a further 15 minutes. Add 5 ml of a 20 per cent w/v solution of potassium iodide, shake and titrate with 0.1 M sodium thiosulphate until a faint yellow colour remains. Add 0.5 ml of starch solution and 10 ml of chloroform and continue the titration with vigorous shaking. Repeat the operation without the substance
Tests
Heavy metals (2.3.13). Heat 5 g with 50 ml of dilute hydrochloric acid on a water-bath for 5 min and filter. Evaporate the filtrate almost to dryness and dissolve the residue in 50 ml of water. 12 ml of this solution complies with the limit test for heavy metals, Method D (20 ppm). Use 10 ml of lead standard solution (2 ppm Pb) to prepare the standard. Fluoran. 0.5 g dissolves completely in a mixture of 4 ml of 1 M sodium hydroxide and 50 ml of water. Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 1.0 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.1 g, dissolve in 100.0 ml of ethanol (95 per cent), dilute 5.0 ml of this solution to 50.0 ml with ethanol (95 per cent) and evaporate 5.0 ml of the resulting
931
PHENOXYMETHYLPENICILLIN POTASSIUM
IP 2007
solution to dryness on a water-bath. Dissolve the residue in sufficient glycine buffer pH 11.3 to produce 100.0 ml, mix and immediately measure the absorbance of the resulting solution at the maximum at about 555 nm (2.4.7). Calculate the content of C20H14O4 taking 1055 as the specific absorbance at 555 nm. Storage. Store protected from moisture.
Phenoxyacetic acid. (Not more than 0.5 per cent). Determine by liquid chromatography (2.4.14). Diluent. pH 6.6.phosphate buffer. Test solution. Weigh accurately a suitable quantity of the substance under examination, dissolve in the diluent and dilute to obtain a solution having a known concentration of about 20.0 mg per ml. (Use this solution on the day prepared). Reference solution. Weigh accurately a suitable quantity of phenoxyacetic acid, dissolve in the diluent and dilute to obtain a solution having a known concentration of about 0.1 mg per ml. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilane chemically bonded to porous silica (5 m), mobile phase: a mixture of 65 volumes of water, 35 volumes of acetonitrile and 1 volume of glacial acetic acid, flow rate. 1 ml per minute, spectrophotometer set at 254 nm, a 20 l loop injector. Inject the reference solution. The tailing factor is not more than 1.5 and the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject alternately the test solution and the reference solution. Calculate the content of phenoxyacetic acid. Limit of p-hydroxyphenoxymethylpenicillin. Not more than 5.0 per cent. Using the chromatograms obtained with the test solution in the Assay, calculate the content of p-hydroxyphenoxymethylpenicillin from the peak response of p-hydroxyphenoxymethylpenicillin and the sum of the peak responses of p-hydroxyphenoxymethylpenicillin and phenoxymethylpenicillin. Water (2.3.43). Not more than 1.0 per cent, determined on 1.0 g. Assay. Determine by liquid chromatography (2.4.14). Test solution. Dissolve about 125 mg of the substance under examination in the mobile phase and dilute to 50.0 ml with the mobile phase. Reference solution (a). Dissolve an accurately weighed quantity of phenoxymethylpenicillin potassium RS in the mobile phase and dilute to obtain a solution having a known concentration of about 2.5 mg per ml. Reference solution (b). A solution in the mobile phase containing 0.25 per cent w/v each of benzylpenicillin potassium and phenoxymethylpenicillin potassium.
Phenoxymethylpenicillin Potassium
Penicillin V Potassium
O H COOK CH3 N CH3
O O
N S H H H
C16H17KN2O5S
Mol. Wt. 388.5
Phenoxymethylpenicillin Potassium is potassium (6R)-6-(2phenoxyacetamido)penicillinate, produced by the growth of certain strains of Penicillium notatum or related organisms on a culture medium containing an appropriate precursor, or obtained by any other means. Phenoxymethylpenicillin Potassium contains not less than 86.0 per cent of total penicillins C16H17N2O5S, calculated on the anhydrous basis. Description. A white, crystalline powder.
Identification
Test A may be omitted if tests B and C are carried out. Test B may be omitted if tests A and C are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with phenoxymethylpenicillin potassium RS. B. Gives reaction B of penicillins and cephalosporins (2.3.1). C. Gives reaction A of potassium salts (2.3.1).
Tests
pH (2.4.24). 5.5 to 7.5, determined in a 0.5 per cent w/v solution. Specific optical rotation (2.4.22). +215.0 to +230.0, determined in a 1.0 per cent w/v solution in carbon dioxide-free water. Light absorption (2.4.7). Absorbance of a 0.1 per cent w/v solution in 0.1 M sodium hydroxide at the maximum at about 306 nm, not more than 0.33. Absorbance of a 0.02 per cent w/v solution in 0.1M sodium hydroxide at the maximum at about 274 nm, not less than 0.50.
932
IP 2007
PHENOXYMETHYLPENICILLIN POTASSIUM TABLETS
Chromatographic system a stainless steel column 30 cm x 4 mm, packed with octadecylsilane chemically bonded to porous silica (3 to 10 m), mobile phase: a mixture of 650 volumes of water, 350 volumes of acetonitrile and 5.75 volumes of glacial acetic acid, flow rate. I ml per minute, spectrophotometer set at 254 nm, a 10 l loop injector. Inject reference solution (b).The relative retention times are about 0.8 for benzylpenicillin and 1.0 for phenoxymethylpenicillin. The column efficiency determined from the phenoxymethylpenicillin peak is not less than 1800 theoretical plates and the resolution between benzylpenicillin and phenoxymethylpenicillin is not less than 3.0. Inject reference solution (a). The relative standard deviation for replicate injections is not more than 1.0 per cent. Inject the test solution and reference solution (a). Record the chromatograms and measure the responses for the phenoxymethylpenicillin peak and any p-hydroxyphenoxymethylpenicillin peak with a retention time of about 0.4 relative to that of the main phenoxymethylpenicillin peak. Calculate the content of C16H17N2O5S, from the sum of the peak responses of the p-hydroxyphenoxymethylpenicillin and phenoxymethylpenicillin peaks in the chromatograms obtained with the test solution and reference solution (a). Storage. Store protected from moisture.
add 1.0 ml of penicillinase solution; the solution changes rapidly to red. C. Ignite 0.5 g of the powdered tablets, add 5 ml of 2 M hydrochloric acid, boil, cool and filter. The filtrate gives reaction B of potassium salts (2.3.1).
Tests
Dissolution (2.5.2). Apparatus. No 1 Medium. 900 ml of water Speed and time. 50 rpm and 45 minutes. Withdraw a suitable volume of the medium and filter. Measure the absorbance of the filtrate, suitably diluted if necessary, at the maximum at about 268 nm (2.4.7). At the same time measure the absorbance of a solution of known concentration of phenoxymethylpenicillin potassium RS at the maximum at about 268 nm. Calculate the content of C16H18N2O5S, in the medium. D. Not less than 75 per cent of the stated amount of C16H18N2O5S. Other tests. Complies with the tests stated under Tablets. Assay. Determine by liquid chromatography (2.4.14). Test solution. Weigh and finely powder 20 tablets. Dissolve an accurately weighed quantity of the powder containing about 0.25 g of phenoxymethylpenicillin in the mobile phase by shaking for 5 minutes and dilute to 100.0 ml with the mobile phase. Filter through a 0.5 m or finer filter and use the filtrate. Reference solution (a). Dissolve an accurately weighed quantity of phenoxymethylpenicillin potassium RS in the mobile phase and dilute to obtain a solution having a known concentration of about 2.5 mg per ml. Reference solution (b). A solution in the mobile phase containing 0.25 per cent w/v each of benzylpenicillin potassium and phenoxymethylpenicillin potassium. Chromatographic system a stainless steel column 30 cm x 4 mm, packed with octadecylsilane chemically bonded to porous silica, mobile phase: a mixture of 650 volumes of water, 350 volumes of acetonitrile and 5.75 volumes of glacial acetic acid, flow rate. 1 ml per minute, spectrophotometer set at 254 nm, a 10 l loop injector. Inject reference solution (b).The relative retention times are about 0.8 for benzylpenicillin and 1.0 for phenoxymethylpenicillin. The column efficiency determined from the phenoxymethylpenicillin peak is not less than 1800 theoretical plates and the resolution between the benzylpenicillin and phenoxymethylpenicillin peaks is not less than 3.0.
Phenoxymethylpenicillin Potassium Tablets

Phenoxymethylpenicillin Tablets; Penicillin V Potassium Tablets; Penicillin V Tablets
Phenoxymethylpenicillin Potassium Tablets contain not less than 92.5 per cent and not more than 107.5 per cent of the stated amount of phenoxymethylpenicillin, C16H18N2O5S.
Identification
A. Shake a quantity of the powdered tablets containing 80 mg of phenoxymethylpenicillin with water, dilute to 250 ml with water and filter. When examined between 230 and 360 nm (2.4.7), the filtrate shows absorption maxima at about 268 nm and 274 nm and a minimum at about 272 nm. B. Shake a quantity of the powdered tablets containing 10 mg of phenoxymethylpenicillin with 10 ml of water, filter and add 0.5 ml of neutral red solution. Add sufficient 0.01 M sodium hydroxide to produce a permanent orange colour and then
933
PHENOTOLAMINE MESYLATE
IP 2007
Inject reference solution (b). The relative standard deviation for replicate injections is not more than 1.0 per cent. Inject the test solution and reference solution (a). Record the chromatograms and measure the responses for the phenoxymethylpenicillin peak and any p-hydroxyphenoxymethylpenicillin peak with a retention time of about 0.4 relative to that of the main phenoxymethylpenicillin peak. Calculate the content of C16H17N2O5S, from the sum of the peak responses of the p-hydroxyphenoxymethylpenicillin and phenoxymethylpenicillin peaks in the chromatograms obtained with the test solution and reference solution (a) Storage. Store protected from moisture. Labelling. The label states the strength in terms of the equivalent amount of phenoxymethylpenicillin.
D. Mix 50 mg with 0.2 g of powdered sodium hydroxide, heat to fusion and continue the heating for a few seconds longer. Cool, add 0.5 ml of water and a slight excess of 2 M hydrochloric acid and warm; sulphur dioxide is evolved, which turns moistened starch iodate paper blue.
Tests
Acidity or alkalinity. Dissolve 0.1 g in 10 ml of carbon dioxidefree water and add 0.1 ml of methyl red solution. The solution is not red and not more than 0.05 ml of 0.1 M sodium hydroxide is required to change the colour of the solution. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 85 volumes of 2-butanone, 15 volumes of acetone and 5 volumes of strong ammonia solution. Test solution. Dissolve 0.2 g of the substance under examination in 10 ml of ethanol (95 per cent). Reference solution. A 0.01 per cent w/v solution of the substance under examination in ethanol (95 per cent). Apply to the plate 10 l of each solution. After development, dry the plate in air, spray with dilute potassium iodobismuthate solution. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.3 g and dissolve in 100 ml of anhydrous 2-propanol with the aid of ultrasound if necessary. Titrate with 0.1 M tetrabutylammonium hydroxide in 2-propanol. Determine the end-point potentiometrically (2.4.25), using a glass electrode and a calomel electrode containing a saturated solution of tetramethylammonium chloride in 2-propanol. Carry out a blank titration. 1 ml of 0.1 M tetrabutylammonium hydroxide is equivalent to 0.03775 g of C17H19N3O, CH4O3S. Storage. Store protected from light and moisture.
Phentolamine Mesylate
OH CH3 N N HN
C17H19N3O,CH4O3S Mol. Wt. 377.5
, CH3SO3H
Phentolamine Mesylate is 3-[[4,5-dihydro-1H-imidazol-2yl)methyl](4-methylphenyl)aminophenol methanesulphonate. Phentolamine Mesylate contains not less than 99.0 per cent and not more than 100.5 per cent of C17H19N3O, CH4O3S, calculated on the dried basis.
Identification
Test A may be omitted if tests B, C and D are carried out. Tests B, C and D may be omitted if test A is carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with phentolamine mesylate RS or with the reference spectrum of phentolamine mesylate. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.002 per cent w/v solution shows an absorption maximum only at about 278 nm; absorbance at about 278 nm, about 0.5. C. Dissolve 0.5 g in 5 ml of ethanol (95 per cent) and 5 ml of 0.1 M hydrochloric acid and add 2 ml of a 0.5 per cent w/v solution of ammonium metavanadate; a light green precipitate is produced.
Phentolamine Injection
Phentolamine Mesylate Injection
Phentolamine Injection is a sterile solution of Phentolamine Mesylate in Water for Injections containing Dextrose.
934
IP 2007
PHENYLBUTAZONE
Phentolamine Injection contains not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of phentolamine mesylate, C17H19N3O, CH4O3S.
B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.001 per cent w/v solution in 0.01 M sodium hydroxide shows an absorption maximum at about 264 nm; absorbance at about 264 nm, about 0.66. C. To 0.1 g add 1 ml of glacial acetic acid and 2 ml of hydrochloric acid and heat under a reflux condenser for 30 minutes. Cool, add 10 ml of water and filter. Add to the filtrate 3 ml of 0.1 M sodium nitrite; a yellow colour is produced. To 1 ml of this solution add a solution containing 10 mg of 2-naphthol in 5 ml of sodium carbonate solution; a reddish brown to reddish violet precipitate is produced.
Identification
The residue obtained in the Assay melts at about 138 (2.4.21).
Tests
pH (2.4.24). 3.5 to 5.0. Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. Dilute an accurately measured volume containing about 0.1 g of Phentolamine Mesylate to 40 ml with water, add 20 ml of a 20 per cent w/v solution of trichloroacetic acid, allow to stand for 3 hours, filter, wash the residue with two quantities, each of 5 ml, of water and dry to constant weight at 105. 1 g of the residue is equivalent to 0.8487 g of C17H19N3O, CH4O3S. Storage. Store protected from light.
Tests
Appearance of solution (2.4.1). Dissolve 1.0 g in 20 ml of 2 M sodium hydroxide and keep the solution at 25 for 3 hours. The solution is clear. Acidity or alkalinity. Heat to boiling 1.0 g with 50 ml of water, cool with shaking in a stoppered vessel and filter. To 25 ml of the filtrate add 0.5 ml of phenolphthalein solution; the solution is colourless and not more than 0.5 ml of 0.01 M sodium hydroxide is required to change the colour of the solution. Add 0.6 ml of 0.01 M hydrochloric acid and 0.1 ml of methyl red solution; the solution is red or orange. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 50 volumes of chloroform, 40 volumes of cyclohexane and 10 volumes of glacial acetic acid.
Phenylbutazone
O H3C
N O
Mol. Wt. 308.4
Prepare the following solutions immediately before use. Test solution. Dissolve 0.2 g of the substance under examination in a solution containing 0.02 per cent w/v of butylated hydroxytoluene in a mixture of equal volumes of chloroform and ethanol and dilute to 5 ml with the same solvent mixture. Reference solution. Dilute 1 ml of the test solution to 200 ml with the same solvent mixture. Before use, spray the plate evenly with a 2 per cent w/v solution of sodium metabisulphite until thoroughly wet, dry the plate in air for 15 minutes, heat at 120 for 30 minutes and allow to cool. Apply separately to the plate 5 l of each solution. After development, dry the plate in a current of warm air for 10 minutes and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent.
C19H20N2O2
Phenylbutazone is 4-butyl-1,2-diphenylpyrazolidine-3,5dione. Phenylbutazone contains not less than 98.0 per cent and not more than 100.5 per cent of C19H20N2O2, calculated on the dried basis. Description. A white or almost white, crystalline powder; odourless.
Identification
Test A may be omitted if tests B and C are carried out. Tests B and C may be omitted if test A is carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with phenylbutazone RS or with the reference spectrum of phenylbutazone.
935
PHENYLBUTAZONE TABLETS
IP 2007
Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 80 over phosphorous pentoxide at a pressure of 1.5 to 2.5 kPa for 4 hours. Assay. Weigh accurately about 0.5 g, dissolve in 25 ml of acetone and titrate with 0.1 M sodium hydroxide, using 0.5 ml of bromothymol blue solution as indicator and continuing the titration until the blue colour persists for at least 15 seconds. Carry out a blank titration. 1 ml of 0.1 M sodium hydroxide is equivalent to 0.03084 g of C19H20N2O2. Storage. Store protected from moisture.
Reference solution. Dilute 1 ml of the test solution to 200 ml with the same solvent mixture. Before use, spray the plate evenly with a 2 per cent w/v solution of sodium metabisulphite until thoroughly wet, dry the plate in air for 15 minutes, heat at 120 for 30 minutes and allow to cool. Apply to the plate 5 l of each solution. After development, dry the plate in a current of warm air for 10 minutes and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Other tests. Complies with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 0.5 g of Phenylbutazone and shake vigorously with 150 ml of 0.1 M sodium hydroxide for 45 minutes. Add sufficient 0.1 M sodium hydroxide to produce 250.0 ml, mix and filter, rejecting the first 20 ml of the filtrate. To 5.0 ml of the filtrate add 50 ml of water and 4 ml of hydrochloric acid and extract with three quantities, each of 30 ml, of ether. Combine the ether extracts and extract with three quantities, each of 30 ml, of 0.1 M sodium hydroxide. Combine the aqueous extracts, pass nitrogen through the solution to remove the residual ether and add sufficient 0.1 M sodium hydroxide to produce 100.0 ml and mix well. To 10.0 ml of this solution add sufficient 0.1 M sodium hydroxide to produce 100.0 ml and measure the absorbance of the resulting solution at the maximum at about 264 nm (2.4.7), using 0.1 M sodium hydroxide as the blank. Calculate the content of C19H20N2O2 from the absorbance obtained by carrying out the Assay simultaneously on 0.5 g of phenylbutazone RS in place of the substance under examination. Storage. Store protected from moisture.
Phenylbutazone Tablets
Phenylbutazone Tablets contain not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of phenylbutazone, C19H20N2O2. The tablets are coated.
Identification
Extract a quantity of the powdered tablets containing 0.2 g of Phenylbutazone with 40 ml of warm acetone, filter and evaporate the filtrate to dryness. The residue complies with the following tests. A. When examined in the range 230 nm to 360 nm (2.4.7), a 0.001 per cent w/v solution in 0.01 M sodium hydroxide shows an absorption maximum at about 264 nm; absorbance at about 264 nm, about 0.66. B. To 0.1 g add 1 ml of glacial acetic acid and 2 ml of hydrochloric acid and heat under a reflux condenser for 30 minutes. Cool, add 10 ml of water and filter. Add to the filtrate 3 ml of 0.1 M sodium nitrite; a yellow colour is produced. To 1 ml of this solution add a solution containing 10 mg of 2-naphthol in 5 ml of sodium carbonate solution; a reddish brown to reddish violet precipitate is produced.
Phenylephrine Hydrochloride
HO H OH H N CH3
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 50 volumes of chloroform, 40 volumes of cyclohexane and 10 volumes of glacial acetic acid. Prepare the following solutions immediately before use. Test solution. Shake a quantity of the powdered tablets containing 0.4 g of Phenylbutazone with 10 ml of a solution containing 0.02 per cent w/v of butylated hydroxytoluene in a mixture of equal volumes of chloroform and ethanol for 15 minutes and centrifuge. Use the supernatant liquid.
, HCl
C9H13NO2,HCl
Mol. Wt. 203.7
Phenylephrine Hydrochloride is (R)-1-(3-hydroxyphenyl)-2methylaminoethanol hydrochloride. Phenylephrine Hydrochloride contains not less than 98.5 per cent and not more than 101.0 per cent of C9H13NO2, HCl, calculated on the dried basis. Description. A white or almost white, crystalline powder.
936
IP 2007
PHENYLEPHRINE INJECTION
Identification
Test A may be omitted if tests B, C and D are carried out. Tests B and C may be omitted if tests A and D are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with phenylephrine hydrochloride RS or with the reference spectrum of phenylephrine hydrochloride. B. Dissolve about 10 mg in 1 ml of water and add 0.05 ml of cupric sulphate solution and 1 ml of 5 M sodium hydroxide; a violet colour is produced. Add 1 ml of ether and shake; the ether layer remains colourless. C. Dissolve 0.3 g in 3 ml of water, add 1 ml of 6 M ammonia and initiate crystallisation by scratching the side of the tube with a glass rod. The melting range of the crystals, after washing with iced water and drying at 105 for 2 hours, is 171 to 176 (2.4.21). D. Gives reaction A of chlorides (2.3.1).
Sulphates (2.3.17). 15 ml of solution A complies with the limit test for sulphates (500 ppm). Phenones. To 10 ml of solution A add sufficient 0.01 M hydrochloric acid to produce 50 ml. Absorbance of the resulting solution at the maximum at about 310 nm, not more than 0.20 (2.4.7). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 1.0 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.15 g, dissolve in a mixture of 0.5 ml of 0.1 M hydrochloric acid and 80 ml of ethanol (95 per cent) and titrate with 0.1 M ethanolic sodium hydroxide determining the end-point potentiometrically (2.4.25). Record the volume added between the two inflections. 1 ml of 0.1 M ethanolic sodium hydroxide is equivalent to 0.02037 g of C9H13NO2,HCl. Storage. Store protected from light and moisture.
Tests
Appearance of solution. Dissolve 2.0 g in 100 ml of carbon dioxide-free water prepared from distilled water (solution A). Solution A is clear (2.4.1), and colourless (2.4.1). Acidity or alkalinity. To 10 ml of solution A add 0.1 ml of methyl red solution and 0.2 ml of 0.01 M sodium hydroxide. The solution is yellow and not more than 0.4 ml of 0.01 M hydrochloric acid is required to change the colour of the solution to red. Specific optical rotation (2.4.22). 43.0 to 47.0, determined in solution A. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel H. Mobile phase. A mixture of 80 volumes of 2-propanol, 15 volumes of 10 M ammonia and 5 volumes of chloroform. Test solution. Dissolve 0.2 g of the substance under examination in 10 ml of methanol. Reference solution (a). A 0.01 per cent w/v solution of the substance under examination in methanol. Reference solution (b). A 0.004 per cent w/v solution of the substance under examination in methanol. Apply to the plate 5 l of each solution. After development, dry the plate in cold air, spray with ninhydrin solution, heat at 105 for 10 minutes and examine in daylight. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (a) and not more than two such spots are more intense than the spot in the chromatogram obtained with reference solution (b).
Phenylephrine Injection
Phenylephrine Hydrochloride Injection
Phenylephrine Injection is a sterile solution of Phenylephrine Hydrochloride in Water for Injections. Phenylephrine Injection contains not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of phenylephrine hydrochloride, C9H13NO2,HCl.
Identification
A. In the test for Related substances, the principal spot in the chromatogram obtained with test solution (b) corresponds to that in the chromatogram obtained with reference solution (b). B. To a volume containing 10 mg of Phenylephrine Hydrochloride add, if necessary, sufficient water to produce 1 ml and then add 0.05 ml of cupric sulphate solution and 1 ml of 5 M sodium hydroxide; a violet colour is produced. Add 1 ml of ether and shake; the ether layer remains colourless. C. Gives reaction A of chlorides (2.3.1).
Tests
pH (2.4.24). 4.5 to 6.5. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel H. Mobile phase. A mixture of 80 volumes of 2-propanol, 15 volumes of 10 M ammonia and 5 volumes of chloroform. Test solution (a) Evaporate a volume of the injection containing 20 mg of Phenylephrine Hydrochloride to dryness and dissolve the residue in 1 ml of methanol.
937
PHENYLMERCURIC ACETATE
IP 2007
Test solution (b). Dilute 1 volume of test solution (a) to 200 volumes with methanol. Reference solution (a). Dilute 1 volume of test solution (b) to 2.5 volumes with methanol. Reference solution (b). A 0.01 per cent w/v solution of phenylephrine hydrochloride RS in methanol. Apply to the plate 5 l of each solution. After development, dry the plate in cold air, spray with ninhydrin solution, heat at 105 for 10 minutes and examine in daylight. Any secondary spot in the chromatogram obtained with test solution (a) is not more intense than the spot in the chromatogram obtained with test solution (b) and not more than two such spots are more intense than the spot in the chromatogram obtained with reference solution (a). Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. To an accurately measured volume containing about 50 mg of Phenylephrine Hydrochloride add sufficient 0.5 M sulphuric acid to produce 100.0 ml. Dilute 10.0 ml of this solution to 100.0 ml with 0.5 M sulphuric acid and measure the absorbance of the resulting solution at the maximum at about 273 nm (2.4.7). Calculate the content of C9H13NO2,HCl taking 90 as the specific absorbance at 273 nm. Storage. Store protected from light.
a white precipitate is formed which darkens slowly on boiling and allowing to stand.
Tests
Mercuric salts and heavy metals. Heat about 100 mg with 15 ml of water, cool and filter. To the filtrate add a few drops of a freshly prepared 10 per cent w/v solution of sodium sulphide; the precipitate formed shows no immediate colour. Polymercurated benzene compounds. Shake 2.0 g with 100 ml of acetone. Filter, wash the residue with successive portions of acetone until a total of 50 ml is used, dry the residue at 105 for 1 hour and weigh. The weight of the residue does not exceed 30 mg (1.5 per cent). Sulphated ash (2.3.18). Not more than 0.2 per cent. Assay. Weigh accurately about 0.4 g, transfer to a 100-ml flask, add 15 ml of water, 5 ml of formic acid and 1 g of zinc dust and reflux for 30 minutes. Cool, filter and wash the filter paper and the amalgam with water until the washings are no longer acidic to litmus. Dissolve the amalgam in 40 ml of 8 M nitric acid. Heat on a water-bath for 3 minutes and add 0.5 g urea and sufficient 0.02 M potassium permanganate to produce a permanent pink colour. Cool and add hydrogen peroxide solution to decolorise the solution. Add 1 ml of ferric ammonium sulphate solution and titrate with 0.1 M ammonium thiocyanate. Repeat the operation without the substance under examination. The difference between the titrations represents the amount of ammonium thiocyanate required. 1 ml of 0.1 M ammonium thiocyanate is equivalent to 0.01684 g of C8H8HgO2. Storage. Store protected from light and moisture.
Phenylmercuric Acetate
O Hg O CH3
C8H8HgO2
Mol. Wt. 336.7
Phenylmercuric Nitrate
Phenylmercuric Nitrate is a mixture of phenylmercuric nitrate, C6H5HgNO3 and phenylmercuric hydroxide, C6H5HgOH. Phenylmercuric Nitrate contains not less than 62.5 per cent and not more than 63.5 per cent of mercury, Hg, calculated on the dried basis. Description. A white or pale yellow powder.
Phenylmercuric Acetate is (acetato)phenylmercury. Phenylmercuric Acetate contains not less than 98.0 per cent and not more than 100.5 per cent of C8H8HgO2. Description. A white to creamy white, crystalline powder, or small white prisms or leaflets.
Identification
A. To 100 mg add 0.5 ml of nitric acid, warm gently until a dark brown colour is produced and dilute with water to 10 ml; the characteristic odour of nitrobenzene is produced. B. To 100 mg add 0.5 ml of sulphuric acid and 1 ml of ethanol (95 per cent) and warm; the characteristic odour of ethyl acetate is produced. C. To 5 ml of a saturated solution in water, add a few drops of a freshly prepared 10 per cent w/v solution of sodium sulphide;
Identification
A. To 0.1 g add 3 ml of sulphuric acid; the mixture becomes yellow and the characteristic odour of nitrobenzene is produced. B. To 0.1 g add 45 ml of water and heat to boiling with shaking. Cool, filter and add sufficient water to produce 50 ml (solution A). To 1 ml of solution A add 1 ml of 2 M hydrochloric acid; a white, flocculent precipitate is produced.
938
IP 2007
PHENYTOIN SODIUM
C. To 5 ml of solution A add 8 ml of water and 0.1 ml of a freshly prepared 10 per cent w/v solution of sodium sulphide; a white precipitate is formed which darkens slowly on boiling and allowing to stand. D. To 5 ml of solution A add 1 ml of 2 M hydrochloric acid, 2 ml of dichloromethane and 0.2 ml of dithizone solution and shake; the lower layer is orange-yellow. E. Solution A gives reaction of nitrates (2.3.1).
Description. A white powder; odourless; somewhat hygroscopic.
Identification
Test A may be omitted if tests B, C and D are carried out. Tests B and C may be omitted if tests A and D are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with phenytoin sodium RS or with the reference spectrum of phenytoin sodium. B. Dissolve 0.25 g in 5 ml of water and acidify with dilute hydrochloric acid; a white precipitate is produced. C. Dissolve 0.1 g in 10 ml of a 10 per cent w/v solution of pyridine, add 1 ml of cupric sulphate with pyridine solution and allow to stand for 10 minutes; a blue precipitate is produced. D. Incinerate 0.1 g; the residue after neutralisation with hydrochloric acid and addition of 2 ml of water gives the reactions of sodium salts (2.3.1).
Tests
Appearance of solution. Solution A is colourless (2.4.1). Inorganic mercuric compounds. To a 10 ml of solution A add 2 ml of potassium iodide solution and 3 ml of 2 M hydrochloric acid and filter; the filtrate is colourless. Wash the precipitate with 2 ml of water, combine the filtrate and washings and add 2 ml of 2 M sodium hydroxide and sufficient water to produce 20 ml. 12 ml of the solution complies with the limit test for heavy metals, Method A (2.3.13). Use lead standard solution (1 ppm Pb) to prepare the standard (0.1 per cent). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 1.0 per cent, determined on 1.0 g by drying over phosphorus pentoxide at a pressure of 1.5 to 2.5 kPa for 24 hours. Assay. Weigh accurately about 0.2 g and dissolve in a mixture of 90 ml of water and 10 ml of nitric acid. Add 2 ml of ferric ammonium sulphate solution and titrate with 0.1 M ammonium thiocyanate until a persistent reddish yellow colour is obtained. Repeat the operation without the substance under examination. The difference between the titrations represents the amount of ammonium thiocyanate required. 1 ml of 0.1 M ammonium thiocyanate is equivalent to 0.02006 g of Hg. Storage. Store protected from light and moisture.
Tests
Appearance of solution. Suspend 1.0 g in 5 ml of water and dilute to 20 ml with 0.1 M sodium hydroxide; the solution is clear (2.4.1), and not more intensely coloured than reference solution BYS6 (2.4.1). Free phenytoin. Dissolve 0.3 g in 10 ml of a mixture of equal volumes of pyridine and water and add 0.5 ml of dilute phenolphthalein solution and 3 ml of silver nitrate-pyridine reagent. Not more than 1.0 ml of 0.1 M sodium hydroxide is required to change the colour of the solution to pink. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 45 volumes of chloroform, 45 volumes of 2-propanol and 10 volumes of strong ammonia solution. Test solution. Dissolve 0.4 g of the substance under examination in 10 ml of methanol. Reference solution (a). A 0.04 per cent w/v solution of the substance under examination in methanol.
Phenytoin Sodium
Diphenylhydantoin Sodium
H C6H5 N C6H5 N O
C15H11N2NaO2
ONa
Reference solution (b). A 0.02 per cent w/v solution of benzophenone in methanol. Apply to the plate 10 l of each solution. After development, dry the plate at 80 for 5 minutes and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (a) and any spot corresponding to benzophenone is not more intense than the spot in the chromatogram obtained with reference solution (b).
Mol. Wt. 274.3
Phenytoin Sodium is 4-oxo-5,5-diphenyl-2-imidazolidin-2-olate Phenytoin Sodium contains not less than 98.0 per cent and not more than 101.0 per cent of C15H11N2NaO2, calculated on the anhydrous basis.
939
PHENYTOIN INJECTION
IP 2007
Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Water (2.3.43). Not more than 3.0 per cent, determined on 1.0 g. Assay. Weigh accurately about 0.18 g, suspend in 2 ml of water, add 8 ml of 0.05 M sulphuric acid and heat gently for 1 minute. Add 30 ml of methanol, cool. Titrate with 0.1 M sodium hydroxide, determining the end-point potentiometrically (2.4.25). After the first inflection, stop the addition of sodium hydroxide, add 5 ml of silver nitrate solution in pyridine, mix and continue the titration until a second inflection is reached. Record the volume of 0.1 M sodium hydroxide added between the two inflections. 1 ml of 0.1 M sodium hydroxide is equivalent to 0.02743 g of C15H11N2NaO2. Storage. Store protected from moisture.
B. Dissolve 0.25 g in 5 ml of water and acidify with dilute hydrochloric acid; a white precipitate is produced. C. Dissolve 0.1 g in 10 ml of a 10 per cent w/v solution of pyridine, add 1 ml of cupric sulphate with pyridine solution and allow to stand for 10 minutes; a blue precipitate is produced. D. Incinerate 0.1 g; the residue after neutralisation with hydrochloric acid and addition of 2 ml of water gives the reactions of sodium salts (2.3.1).
Tests
Appearance of solution. Suspend 1.0 g in 5 ml of water and dilute to 20 ml with 0.1 M sodium hydroxide; the solution is clear (2.4.1), and not more intensely coloured than reference solution BYS6 (2.4.1). Completeness of solution. The contents dissolve in the quantity of the solvent recommended on the label and give a clear solution. pH (2.4.24). 10.0 to 12.0, determined in a 5.0 per cent w/v solution in the stated solvent. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 45 volumes of chloroform, 45 volumes of 2-propanol and 10 volumes of strong ammonia solution. Test solution. Dissolve 0.4 g of the substance under examination in 10 ml of methanol. Reference solution (a). A 0.04 per cent w/v solution of the substance under examination in methanol. Reference solution (b). A 0.02 per cent w/v solution of benzophenone in methanol. Apply to the plate 10 l of each solution. After development, dry the plate at 80 for 5 minutes and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (a) and any spot corresponding to benzophenone is not more intense than the spot in the chromatogram obtained with reference solution (b). Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Water (2.3.43). Not more than 3.0 per cent, determined on 1.0 g. Assay. Weigh accurately about 0.18 g of the mixed contents of 10 containers, suspend in 2 ml of water, add 8 ml of 0.05 M sulphuric acid and heat gently for 1 minute. Add 30 ml of methanol, cool. Titrate with 0.1 M sodium hydroxide,
Phenytoin Injection
Phenytoin Sodium Injection; Diphenylhydantoin Sodium injection
Phenytoin Injection is a sterile material consisting of Phenytoin Sodium with or without buffering agents and other excipients. It is filled in a sealed container. The injection is constituted by dissolving the contents of the sealed container in the requisite amount of sterile Water for Injection or other suitable solvent, immediately before use. The constituted solution complies with the requirements for Clarity of solution and Particulate matter stated under Parenteral Preparations (Injections). Storage. The constituted solution should be used immediately after preparation but, in any case, within the period recommended by the manufacturer. Phenytoin Injection contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of phenytoin sodium, C15H11N2NaO2. Description. A white powder; odourless; somewhat hygroscopic. The contents of the sealed container comply with the requirements stated under Parenteral Preparations (Powders for Injection) and with the following requirements.
Identification
Test A may be omitted if tests B, C and D are carried out. Tests B and C may be omitted if tests A and D are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with phenytoin sodium RS or with the reference spectrum of phenytoin sodium.
940
IP 2007
PHOLCODINE
determining the end-point potentiometrically (2.4.25). After the first inflection, stop the addition of sodium hydroxide, add 5 ml of silver nitrate solution in pyridine, mix and continue the titration until a second inflection is reached. Record the volume of 0.1 M sodium hydroxide added between the two inflections. 1 ml of 0.1 M sodium hydroxide is equivalent to 0.02743 g of C15H11N2NaO2. Storage. Store protected from moisture at a temperature not exceeding 30. Labelling. The label states (1) the quantity of Phenytoin Sodium contained in it; (2) the directions for preparing the Injection.
Reference solution. A 0.01 per cent w/v solution of benzophenone in methanol. Apply to the plate 5 l of each solution. Allow the mobile phase to rise 12 cm. Dry the plate in air and examine in ultraviolet light at 254 nm. In the chromatogram obtained with the test solution any spot corresponding to benzophenone is not more intense than the spot in the chromatogram obtained with the reference solution. Other tests. Complies with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 0.25 g of Phenytoin Sodium, shake with 40 ml of 0.01 M sodium hydroxide for 5 minutes and add sufficient 0.01 M sodium hydroxide to produce 50.0 ml. Centrifuge, acidify 25.0 ml of the clear liquid with 10 ml of 0.1 M hydrochloric acid and extract successively with 50, 40, 25 and 25 ml of ether. Wash the combined extracts with 10 ml of water, evaporate to dryness and dry the residue at 105. Dissolve in 50 ml of anhydrous pyridine and titrate with 0.1 M tetrabutylammonium hydroxide, using 0.3 per cent w/v solution of thymol blue in pyridine as indicator and taking care to prevent absorption of carbon dioxide from the atmosphere. Carry out a blank titration. 1 ml of 0.1 M tetrabutylammonium hydroxide is equivalent to 0.02743 g of C15H11N2NaO2. Storage. Store protected from moisture.
Phenytoin Tablets
Phenytoin Sodium Tablets; Diphenylhydantoin Sodium Tablets
Phenytoin Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of phenytoin sodium, C15H11N2NaO2. The tablets are coated.
Identification
A. Shake a quantity of the powdered tablets containing 0.1 g of Phenytoin Sodium with 20 ml of water, filter, acidify with dilute hydrochloric acid and extract with 10 ml of chloroform. Wash the chloroform extract with water, dry with anhydrous sodium sulphate and evaporate to dryness and dry the residue at 105. The residue complies with the following test. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with phenytoin sodium RS treated in the same manner or with the reference spectrum of phenytoin. B. Triturate a quantity of the powdered tablets containing 0.5 g of Phenytoin Sodium with 10 ml of water and filter. Acidify with dilute hydrochloric acid; a white precipitate is produced. C. The powdered tablets, when moistened with hydrochloric acid and introduced on a platinum wire into a flame, impart a yellow colour to the flame.
Pholcodine
N O O H HO
C23H30N2O4,H2O Mol. Wt. 416.5
, H2O NCH3
Pholcodine is (5R,6S)-4,5-epoxy-N-methyl-3-(2morpholinoethoxy)morphin-7-en-6-ol monohydrate. Pholcodine contains not less than 98.0 per cent and not more than 101.0 per cent of C23H30N2O4, calculated on the dried basis. Description. A white or almost white, crystalline powder.
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 75 volumes of hexane and 30 volumes of dioxan. Test solution. Shake a quantity of the powdered tablets containing 0.1 g of Phenytoin Sodium with 5 ml of methanol, warm on a water-bath with shaking and filter.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with pholcodine RS or with the reference spectrum of pholcodine.
941
PHOLCODINE LINCTUS
IP 2007
B. To 10 ml of a 0.1 per cent w/v solution add 75 ml of water and 10 ml of 1 M sodium hydroxide and dilute to 100 ml with water. When examined in the range 230 nm and 360 nm (2.4.7), the resulting solution shows an absorption maximum at about 284 nm; absorbance at about 284 nm, 0.36 to 0.39. C. Dissolve 50 mg in 1 ml of sulphuric acid and add 0.05 ml of a 10 per cent w/v solution of ammonium molybdate; a pale blue colour is produced. Warm gently; the colour changes to deep blue. Add 0.05 ml of 2 M nitric acid; the colour changes to brownish red.
1 ml of 0.1 M perchloric acid is equivalent to 0.01993 g of C23H30N2O4. Storage. Store protected from moisture.
Pholcodine Linctus
Pholcodine Linctus is a solution containing 0.1 per cent w/v solution of Pholcodine and 1 per cent w/v solution of Citric Acid Monohydrate in a suitable flavoured vehicle. Pholcodine Linctus contains not less than 0.090 per cent and not more than 0.110 per cent w/v of pholcodine, C23H30N2O4.
Tests
Specific optical rotation (2.4.22). 94.0 to 98.0, determined at 20 in a 2.0 per cent w/v solution in ethanol (95 per cent). Morphine. Dissolve 0.1 g in sufficient of 0.1 M hydrochloric acid to produce 5 ml, add 2 ml of a 1 per cent w/v solution of sodium nitrite, allow to stand for 15 minutes and add 3 ml of 6 M ammonia. The solution is not more intensely coloured than reference solution BS4 (2.4.1). Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 70 volumes of ethanol (95 per cent), 70 volumes of toluene, 65 volumes of acetone and 5 volumes of strong ammonia solution. Test solution. Dissolve 0.25 g of the substance under examination in 10 ml of chloroform. Reference solution (a). A 0.025 per cent w/v solution of the substance under examination in chloroform. Reference solution (b). A 0.0125 per cent w/v solution of the substance under examination in chloroform. Apply to the plate 10 l of each solution. After development, dry the plate in air, spray with dilute potassium iodobismuthate solution. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (a) and not more than one such spot of higher Rf value than the principal spot is more intense than the spot in the chromatogram obtained with reference solution (b). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). 3.9 to 4.5 per cent, determined on 0.5 g by drying in an oven at 105. Assay. Weigh accurately about 0.18 g, dissolve in 50 ml of anhydrous glacial acetic acid, warming gently. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically at the second inflection (2.4.25). Carry out a blank titration.
Identification
To 20 ml add 20 ml of water, make alkaline to litmus paper with 5 M ammonia, extract with two quantities, each of 20 ml, of chloroform, washing each extract with 5 ml of water, dry the combined extracts with anhydrous sodium sulphate, filter and evaporate to dryness. If necessary, add 0.1 ml of ether and scratch the sides of the vessel with a glass rod to induce crystallisation. The crystals, dried at a pressure not exceeding 2 kPa, comply with the following tests. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with pholcodine RS or with the reference spectrum of pholcodine. B. When examined in the range 230 nm and 360 nm (2.4.7), a 0.01 per cent w/v solution in 0.01 M sodium hydroxide shows an absorption maximum only at about 284 nm. C. To a portion of the crystals add 0.05 ml of nitric acid and mix; a yellow colour is produced. D. Dissolve the remainder of the crystals in 1 ml of sulphuric acid and add 0.05 ml of ammonium molybdate-sulphuric acid solution; a pale blue colour is produced. Warm gently; the colour changes to deep blue. Add 0.05 ml of 2 M nitric acid; the colour changes to brownish red.
Tests
Other tests. Complies with the tests stated under Oral Liquids. Assay. Weigh accurately about 50 g and add sufficient 5 M ammonia to make the solution alkaline to litmus paper, extract with four quantities, each of 25 ml, of chloroform, washing each extract with the same 5 ml of water. Combine the extracts and evaporate until the volume is reduced to 15 ml. Titrate with 0.02 M perchloric acid, using quinaldine red solution as indicator. Carry out a blank titration. 1 ml of 0.02 M perchloric acid is equivalent to 0.004165 g of C23H30N2O4,H2O. Determine the weight per ml of the linctus (2.4.29), and calculate the content of C23H30N2O4,H2O, weight in volume.
942
IP 2007
PHYSOSTIGMINE SALICYLATE
Storage. Store protected from light. Labelling. The label states the strength in terms of the equivalent amount of pholcodine.
heat on a water-bath for 5 minutes; the appearance of the solution does not change. Assay. Weigh accurately about 1.0 g, add a solution of 10 g of sodium chloride in 30 ml of water and titrate with1 M sodium hydroxide using dilute phenolphthalein solution as indicator. 1 ml of 1 M sodium hydroxide is equivalent to 0.04900 g of H3PO4. Storage. Store protected from moisture, in glass containers.
Phosphoric Acid
Orthophosphoric Acid; Concentrated Phosphoric Acid
H3PO4 Mol. Wt. 98.0 Phosphoric acid contains not less than 84.0 per cent w/w and not more than 90.0 per cent w/w of H3PO4. Description. A clear, colourless, syrupy liquid; corrosive. When kept at a low temperature it may solidify, producing a mass of colourless crystals which do not melt until the temperature reaches 28.
Physostigmine Salicylate
Eserine Salicylate
H3C N H CH3 N , CH3 O H3C NH
C15H21N3O2,C7H6O3 Mol. Wt. 413.5
COOH OH
Identification
A. Dilute with water; the solution is strongly acidic. B. Dilute 10.0 g to 150 ml with water (solution A). Neutralise with 2 M sodium hydroxide; the resulting solution gives the reactions of phosphates (2.3.1).
Tests
Appearance of solution. Solution A is clear (2.4.1), and colourless (2.4.1). Arsenic (2.3.10). Dissolve 5.0 g in 50 ml of water and add 10 ml of stannated hydrochloric acid; the resulting solution complies with the limit test for arsenic (2 ppm). Heavy metals (2.3.13). Dilute 1.2 ml with 10 ml of water, neutralise with dilute ammonia solution, add sufficient dilute acetic acid to render the solution acidic and then dilute to 25 ml with water. The resulting solution complies with the limit test for heavy metals, Method A (10 ppm). Iron (2.3.14). 10 ml of solution A complies with the limit test for iron (60 ppm). Chlorides (2.3.12). 3 ml of solution A complies with the limit test for chlorides (50 ppm). Sulphates (2.3.17). 20 ml of solution A complies with the limit test for sulphates (100 ppm). Alkali phosphates. To 1.7 g in a graduated cylinder add 6 ml of ether and 2 ml of ethanol (95 per cent); no turbidity is produced. Aluminium and calcium. To 1.7 g add 10 ml of water and 8 ml of dilute ammonia solution; the solution remains clear. Hypophosphorus acid and phosphorous acid. To 5 ml of solution A add 2 ml of a 1.7 per cent w/v solution of silver nitrate and
Physostigmine Salicylate is (3aS,8aR)-1,2,3,3a,8,8ahexahydro-1,3a,8-trimethylpyrrolo[2,3-b]indol-5-yl methylcarbamate salicylate. Physostigmine Salicylate contains not less than 98.5 per cent and not more than 101.0 per cent of C15H21N3O2, C7H6O3, calculated on the dried basis. Description. A colourless or faintly yellow crystals, turning red gradually under the action of air and light and rapidly in presence of moisture; odourless.
Identification
Test A may be omitted if tests B, C and D are carried out. Test C may be omitted if tests A, B and D are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with physostigmine salicylate RS. B. In the test for Related substances, the principal spot in the chromatogram obtained with test solution (b) corresponds to that in the chromatogram obtained with reference solution (a). C. To a 1 per cent w/v solution add 1 M sodium hydroxide; a white precipitate which turns pink is produced. The precipitate dissolves in an excess of the reagent, producing a red solution. D. A 0.9 per cent w/v solution in carbon dioxide-free water gives reaction A of salicylates (2.3.1).
943
PHYSOSTIGMINE INJECTIONS
IP 2007
Tests
Appearance of solution. Dissolve 0.9 g without heating in 95 ml of carbon dioxide-free water prepared from distilled water, and dilute to 100 ml with the same solvent (solution A). The solution, examined immediately after preparation, is clear (2.4.1), and colourless (2.4.1). pH (2.4.24). 5.1 to 5.9, determined in solution A immediately after preparation. Specific optical rotation (2.4.22). 90.0 to 94.0, determined in solution A immediately after preparation. Eseridine. To 5 ml of solution A, examined immediately after preparation, add a few crystals of potassium iodate and a drop of 2 M hydrochloric acid and 2 ml of chloroform and shake; the chloroform layer does not turn violet within 1 minute. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 100 volumes of cyclohexane, 23 volumes of 2-propanol and 2 volumes of strong ammonia solution. Test solution (a). Dissolve 0.2 g of the substance under examination in 10 ml of ethanol (95 per cent). Test solution (b). Dissolve 0.1 g of the substance under examination in 100 ml of ethanol (95 per cent). Reference solution (a). A 0.1 per cent w/v solution of physostigmine salicylate RS in ethanol (95 per cent). Reference solution (b). A 0.01 per cent w/v solution of physostigmine salicylate RS in ethanol (95 per cent). Apply to the plate 20 l of each solution. After development, dry the plate in cold air, carry out a second chromatographic development in same direction, dry the plate in air and spray with freshly prepared acetic potassium iodobismuthate solution and then with hydrogen peroxide solution (10 vol). Examine the plate within 2 minutes. Any secondary spot in the chromatogram obtained with test solution (a) is not more intense than the spot in the chromatogram obtained with reference solution (b). Sulphates (2.3.17). 15 ml of solution A complies with the limit test for sulphates (0.1 per cent). Sulphated ash (2.3.18). Not more than 0.1 per cent, determined on the residue obtained in the test for Loss on drying. Loss on drying (2.4.19). Not more than 1.0 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.35 g, dissolve in 50 ml of a mixture of equal volumes of chloroform and anhydrous glacial acetic acid. Titrate with 0.1 M perchloric acid, determining
the end-point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.04135 g of C15H21N3O2,C7H6O3. Storage. Store protected from light and moisture.
Physostigmine Injection
Physostigmine Salicylate Injection; Eserine Salicylate Injection
Physostigmine Injection is a sterile solution of Physostigmine Salicylate in Water for Injections. Physostigmine Injection contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of physostigmine salicylate, C15H21N3O2, C7H6O3.
Identification
A. Warm a volume containing 3 mg of Physostigmine Salicylate with 0.3 ml of 5 M ammonia; a yellowish red solution is produced which on evaporation gives a bluish residue. B. The residue obtained in test A dissolves in ethanol (95 per cent) producing a blue solution which, on the addition of 6 M acetic acid, appears blue by transmitted light and exhibits a red fluorescence which intensifies on dilution with water. C. The residue obtained in test A dissolves in sulphuric acid producing a green solution which, on the gradual addition of ethanol (95 per cent), changes to red but reverts to green when the ethanol is evaporated.
Tests
pH (2.4.24). 4.0 to 6.0. Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. Transfer an accurately measured volume containing 30 mg of Physostigmine Salicylate to a separator, add about 0.25 g of sodium bicarbonate and extract with six quantities, each of 15 ml, of chloroform. Filter the combined chloroform extracts through about 10 g of anhydrous sodium sulphate. Add 25 ml of anhydrous glacial acetic acid to the filtrate. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.01 M perchloric acid is equivalent to 0.004135 g of C15H21N3O2, C7H6O3. Storage. Store protected from light, in single dose containers. The injection should not be used if it is more than slightly discoloured.
944
IP 2007
PINDOLOL
Pilocarpine Nitrate
N N H3C
C11H16N2O2, HNO3
Test solution (b). Dissolve 0.1 g of the substance under examination in 100 ml of water.
O CH3 , HNO3
Reference solution (a). A 0.1 per cent w/v solution of pilocarpine nitrate RS in water. Reference solution (b). A 0.03 per cent w/v solution of pilocarpine nitrate RS in water.
Mol. Wt. 271.3
Pilocarpine Nitrate is (3S,4R)-3-ethyl-4-[(1-methyl-1Himidazol-5-yl)methyl]dihydrofuran-2(3H)-one nitrate. Pilocarpine Nitrate contains not less than 98.5 per cent and not more than 101.0 per cent of C11H16N2O2, HNO3, calculated on the dried basis. Description. Colourless crystals or a white, crystalline powder; odourless; sensitive to light.
Apply to the plate 10 l of each solution. After development, dry the plate at 105 for 10 minutes, cool and spray with potassium iodobismuthate solution. Any secondary spot in the chromatogram obtained with test solution (a) is not more intense than the spot in the chromatogram obtained with reference solution (b). Other alkaloids. To a 1 per cent w/v solution add dilute ammonia solution; no turbidity is produced. To a 1 per cent w/v solution add a few drops of potassium dichromate solution; no turbidity is produced. Chlorides (2.3.12). 25 ml of a 10.0 per cent w/v solution complies with the limit test for chlorides (100 ppm). Iron (2.3.14). 20 ml of a 10.0 per cent w/v solution complies with the limit test for iron (20 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent, determined on 0.5 g. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.25 g, dissolve in 30 ml of anhydrous glacial acetic acid. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.02713 g of C11H16N2O2, HNO3. Storage. Store protected from light and moisture.
Identification
Test A may be omitted if tests B, C and D are carried out. Tests B and C may be omitted if tests A and D are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with pilocarpine nitrate RS. B. In the test for Related substances, the principal spot in the chromatogram obtained with test solution (b) corresponds to that in the chromatogram obtained with reference solution (a). C. Dissolve about 10 mg in 2 ml of water, add 2 drops of a 5 per cent w/v solution of potassium dichromate, 1 ml of hydrogen peroxide solution (10 vol) and 2 ml of chloroform and shake; the chloroform layer turns violet. D. Gives reaction A of nitrates (2.3.1).
Tests
Appearance of solution. A 5.0 per cent w/v solution in carbon dioxide-free water is clear (2.4.1), and not more intensely coloured than reference solution YS6 (2.4.1). pH (2.4.24). 3.5 to 4.5, determined in a 5.0 per cent w/v solution prepared immediately before use in carbon dioxide-free water. Specific optical rotation (2.4.22). +79.5 to +83.0, determined in a 5.0 per cent w/v solution in carbon dioxide-free water, prepared immediately before use. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 85 volumes of chloroform, 14 volumes of methanol and 1 volume of strong ammonia solution. Test solution (a). Dissolve 0.3 g of the substance under examination in 10 ml of water.
Pindolol
OH HN O H N CH3 CH3
C14H20N2O2 Mol. wt. 248.3
Pindolol is (RS)-1-indol-4-yloxy-3-isopropylaminopropan-2ol. Pindolol contains not less than 99.0 per cent and not more than 101.0 per cent of C14H20N2O2, calculated on the dried basis. Description. A white or almost white, crystalline powder.
945
PINDOLOL TABLETS
IP 2007
Identification
Test A may be omitted if tests B and C are carried out. Test B and C may be omitted if test A is carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with pindolol RS. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.002 per cent w/v solution in a 0.085 per cent v/v solution of hydrochloric acid in methanol shows absorption maxima at about 264 nm and at about 287 nm, and a shoulder at about 275 nm, absorbances at about 264 nm, 0.66 to 0.70 and at about 287 nm, 0.34 to 0.38. C. In the test for Related substances, the principal spot in the chromatogram obtained with test solution (b) corresponds to that in the chromatogram obtained with reference solution (a).
Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method C (20 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.2 g, dissolve in 80 ml of methanol. Titrate with 0.1 M hydrochloric acid, determining the end point potentiometrically (2.4.25). 1 ml of 0.1 M hydrochloric acid is equivalent to 0.02483 g of C14H20N2O2. Storage. Store protected from light and moisture.
Tests
Appearance of solution. A 5.0 per cent w/v solution in dilute acetic acid is clear (2.4.1), and not more intensely coloured than reference solution BYS4 (2.4.1). Related substances. Determine as rapidly as possible, protected from light. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A freshly prepared mixture of 50 volumes of ethyl acetate, 50 volumes of methanol and 4 volumes of strong ammonia solution. Prepare the following solutions immediately before use. Test solution (a). Dissolve 0.2 g of the substance under examination in 10 ml of a mixture of 99 volumes of methanol and 1 volume of anhydrous glacial acetic acid. Test solution (b). Dissolve 0.2 g of the substance under examination in 100 ml of a mixture of 99 volumes of methanol and 1 volume of anhydrous glacial acetic acid. Reference solution (a). A 0.2 per cent w/v solution of pindolol RS in a mixture of 99 volumes of methanol and 1 volume of anhydrous glacial acetic acid. Reference solution (b). A 0.006 per cent w/v solution of pindolol RS in a mixture of 99 volumes of methanol and 1 volume of anhydrous glacial acetic acid. Apply to the plate 5 l of each solution. Allow the mobile phase to rise 10 cm. Dry the plate immediately in a current of cold air and examine in ultraviolet at 254 nm without delay. Any secondary spot in the chromatogram obtained with test solution (a) is not more intense than the spot in the chromatogram obtained with reference solution (b) (0.3 per cent).
Pindolol Tablets
Pindolol Tablets contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of pindolol, C14H20N2O2.
Identification
A. Shake a quantity of the powdered tablets containing 50 mg of Pindolol with 80 ml of ether for 30 minutes, filter and dry the extract with anhydrous sodium sulphate. Filter the extract, remove the ether using a rotary evaporator and dry the residue over phosphorus pentoxide at 110 at a pressure not exceeding 2 kPa for 1 hour. The residue complies with the following test. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with pindolol RS. B. When examined in the range 230 nm to 360 nm (2.4.7), the final solution obtained in the Assay shows absorption maxima at about 264 nm and 287 nm. C. Shake a quantity of the powdered tablets containing 20 mg of Pindolol with 5 ml of a mixture of 99 volumes of methanol and 1 volume of glacial acetic acid for 45 minutes Centrifuge and dilute 1 ml of the supernatant liquid to 50 ml with the acetic acid-methanol mixture. To 2 ml of this solution add 1 ml of dimethylaminobenzaldehyde solution; a violet-blue colour is produced.
Tests
Related substances. Determine as rapidly as possible, protected from light. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A freshly prepared mixture of 50 volumes of ethyl acetate, 50 volumes of methanol and 4 volumes of strong ammonia solution.
946
IP 2007
PIPERAZINE ADIPATE
Test solution. Shake a quantity of the powdered tablets containing 20 mg of Pindolol with 5 ml of a mixture of 99 volumes of methanol and 1 volume of glacial acetic acid for 15 minutes, centrifuge and apply the supernatant liquid to the plate as the last solution. Reference solution (a). Dilute 1 volume of the test solution to 10 volumes with the methanol-acetic acid mixture and further dilute 7 volumes of the solution to 100 volumes with the same solvent mixture. Reference solution (b). Dilute 1 volume of the test solution to 10 volumes with the methanol-acetic acid mixture and further dilute 3 volumes of this solution to 100 volumes with the same solvent mixture. Apply to the plate 10 l of each solution. Allow the mobile phase to rise 10 cm. Dry the plate in air, spray immediately with dimethylaminobenzaldehyde solution and warm at 50 for 20 minutes. Any spot with Rf value of about 0.1 in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (a) (0.7 per cent). Any other secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (b) (0.3 per cent). Ignore any spot remaining on the line of application. Other tests. Comply with the tests stated under tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powdered tablets containing about 90 mg of Pindolol and shake with 100.0 ml of methanol for 45 minutes. Centrifuge and dilute 15.0 ml of the supernatant liquid to 100.0 ml with methanol and measure the absorbance of the resulting solution at the maximum at about 264 nm (2.4.7). Calculate the content of C14H20N2O2 taking 338 as the specific absorbance at 264 nm. Storage. Store protected from light.
Identification
Test A may be omitted if tests B, C and D are carried out. Tests B and C may be omitted if tests A and D are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with piperazine adipate RS or with the reference spectrum of piperazine adipate. B. In the test for Related substances, examine the plate after spraying with both the ninhydrin and iodine solutions. The principal spot in the chromatogram obtained with test solution (b) corresponds to that in the chromatogram obtained with reference solution (a). C. Dissolve 0.1 g in 5 ml of water, add 0.5 g of sodium bicarbonate, 0.5 ml of a freshly prepared 5.0 per cent w/v solution of potassium ferricyanide and 0.1 ml of mercury, shake vigorously for 1 minute and allow to stand for 20 minutes; a reddish colour slowly develops.
Tests
Appearance of solution. A 5.0 per cent w/v solution is clear (2.4.1), and not more intensely coloured than reference solution BS8 (2.4.1). pH (2.4.24). 5.0 to 6.0, determined in a 5.0 per cent w/v solution. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A freshly prepared mixture of 80 volumes of acetone and 20 volumes of strong ammonia solution. Test solution (a). Dissolve 1 g of the substance under examination in 10 ml of a mixture of 3 volumes of strong ammonia solution and 2 volumes of ethanol. Test solution (b). Dissolve 1 g of the substance under examination in 100 ml of the same solvent mixture. Reference solution (a). A 1 per cent w/v solution of piperazine adipate RS in the same solvent mixture.
Piperazine Adipate
H N N H
C4H10N2, C6H10O4 Mol. Wt. 232.3
Reference solution (b). A 0.025 per cent w/v solution of ethylenediamine in the same solvent mixture.
HOOC
COOH
Reference solution (c). A 0.025 per cent w/v solution of triethylenediamine in the same solvent mixture. Reference solution (d). A solution containing 0.025 per cent w/v of triethylenediamine and 1 per cent w/v of the substance under examination in the same solvent mixture. . Apply to the plate 5 l of each solution. After development, dry the plate at 105, spray with a 0.3 per cent w/v solution of ninhydrin in a mixture of 3 volumes of anhydrous acetic acid and 100 volumes of 1-butanol and then with a 0.15 per cent w/v solution of ninhydrin in ethanol and dry at 105 for 10
Piperazine Adipate contains not less than 98.0 per cent and not more than 101.0 per cent of C4H10N2, C6H10O4, calculated on the anhydrous basis. Description. A white, crystalline powder.
947
PIPERAZINE ADIPATE TABLETS
IP 2007
minutes. Any secondary spot in the chromatogram obtained with test solution (a) is not more intense than the spot in the chromatogram obtained with reference solution (b). Spray the plate with 0.05 M iodine and allow to stand for about 10 minutes. Any spot corresponding to triethylenediamine in the chromatogram obtained with test solution (a) is not more intense than the spot in the chromatogram obtained with reference solution (c). The test is not valid unless the chromatogram obtained with reference solution (d) shows two separated spots. Ignore any spot remaining on the line of application. Heavy metals (2.3.13). Dissolve 1.0 g in 20 ml of water, 0.5 ml of 0.1 M hydrochloric acid and add sufficient water to produce 25 ml. The resulting solution complies with the limit test for heavy metals, Method A (20 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Water (2.3.43). Not more than 0.5 per cent, determined on 1.0 g. Assay. Weigh accurately about 0.2 g, dissolve in 10 ml of anhydrous glacial acetic acid with gentle heating and dilute to 70 ml with the same solvent. Titrate with 0.1 M perchloric acid, using 0.25 ml of 1-naphtholbenzein solution as indicator and titrating until the colour of the solution changes from brownish yellow to green. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.01161 g of C4H10N2, C6H10O4. Storage. Store protected from moisture.
Tests
Other tests. Comply with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 0.2 g of Piperazine Hydrate, shake with 10 ml of water for 1 hour, filter and wash the residue with two quantities, each of 10 ml, of water. To the combined extract and washings add 5 ml of 1 M sulphuric acid and 50 ml of picric acid solution, bring to boil, allow to stand for 1 hour and filter through a sintered-glass crucible (porosity No. 4) and wash the residue with successive quantities, each of 10 ml, of a mixture of equal volumes of a saturated solution of picric acid and water until the washings are free from sulphate. Wash with five quantities, each of 10 ml, of ethanol and dry to constant weight at 105. 1 g of the residue is equivalent to 0.3567 g of C4H10N2, 6H2O. Storage. Store protected from moisture. Labelling. The label states the strength in terms of the equivalent amount of piperazine hydrate.
Piperazine Citrate
H N , N H
HO HOOC
COOH COOH
2
(C4H10N2)3,2C6H8O7
Mol. Wt. 642.7 (anhydrous)
Piperazine Adipate Tablets

Piperazine Adipate Tablets contain not less than 92.5 per cent and not more than 107.5 per cent of the stated amount of piperazine hydrate, C4H10N2, 6H2O.
Piperazine Citrate is a salt of piperazine with citric acid containing a variable amount of water of crystallisation. Piperazine Citrate contains not less than 98.0 per cent and not more than 101.0 per cent of (C4H10N2)3, 2C6H8O7, calculated on the anhydrous basis. Description. A white, granular powder; almost odourless.
Identification
A. Extract a quantity of the powdered tablets containing 1 g of Piperazine Hydrate with 20 ml of water and filter. Dilute 1 ml of the filtrate to 5 ml with water, add 0.5 g of sodium bicarbonate, 0.5 ml of a freshly prepared 5.0 per cent w/v solution of potassium ferricyanide and 0.1 ml of mercury, shake vigorously for 1 minute and allow to stand for 20 minutes; a reddish colour slowly develops. B. To 10 ml of the filtrate obtained in test A add 5 ml of hydrochloric acid and extract with three quantities, each of 10 ml, of ether, evaporate the combined ether extracts to dryness; the residue, after washing with a small volume of water and drying at 105, melts at about 152 (2.4.21).
Identification
Test A may be omitted if tests B and C are carried out. Test B may be omitted if tests A and C are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with piperazine citrate RS or with the reference spectrum of piperazine citrate. B. In the test for Related substances, examine the plate after spraying with both the ninhydrin solutions. The principal spot in the chromatogram obtained with test solution (b) corresponds to that in the chromatogram obtained with reference solution (a).
948
IP 2007
PIPERAZINE CITRATE SYRUP
C. A 10 per cent w/v solution gives the reactions of citrates (2.3.1).
Tests
Appearance of solution. A 5.0 per cent w/v solution is clear (2.4.1), and not more intensely coloured than reference solution BS8 (2.4.1). pH (2.4.24). 5.0 to 6.0, determined in a 5.0 per cent w/v solution. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A freshly prepared mixture of 80 volumes of acetone and 20 volumes of strong ammonia solution. Test solution (a). Dissolve 1 g of the substance under examination in 10 ml of a mixture of 3 volumes of strong ammonia solution and 2 volumes of ethanol. Test solution (b). Dissolve 1 g of the substance under examination in 100 ml of the same solvent mixture. Reference solution (a). A 1 per cent w/v solution of piperazine citrate RS in the same solvent mixture. Reference solution (b). A 0.025 per cent w/v solution of ethylenediamine in the same solvent mixture. Reference solution (c). A 0.025 per cent w/v solution of triethylenediamine in the same solvent mixture. Reference solution (d). A solution containing 0.025 per cent w/v of triethylenediamine and 1 per cent w/v of the substance under examination in the same solvent mixture. Apply to the plate 5 l of each solution. After development, dry the plate at 105, spray with a 0.3 per cent w/v solution of ninhydrin in a mixture of 3 volumes of anhydrous acetic acid and 100 volumes of 1-butanol and then with a 0.15 per cent w/v solution of ninhydrin in ethanol and dry at 105 for 10 minutes. Any secondary spot in the chromatogram obtained with test solution (a) is not more intense than the spot in the chromatogram obtained with reference solution (b). Spray the plate with 0.05 M iodine and allow to stand for about 10 minutes. Any spot corresponding to triethylenediamine in the chromatogram obtained with test solution (a) is not more intense than the spot in the chromatogram obtained with reference solution (c). The test is not valid unless the chromatogram obtained with reference solution (d) shows two separated spots. Ignore any spot remaining on the line of application. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method A (20 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Water (2.4.19). 10.0 to 14.0 per cent, determined on 0.3 g. Assay. Weigh accurately about 0.2 g, dissolve in 10 ml of anhydrous glacial acetic acid with gentle heating and dilute
to 70 ml with the same solvent. Titrate with 0.1 M perchloric acid, using 0.25 ml of 1-naphtholbenzein solution as indicator and titrating until the colour of the solution changes from brownish yellow to green. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.01071 g of (C4H10N2)3, 2C6H8O7. Storage. Store protected from light and moisture.
Piperazine Citrate Syrup

Piperazine Citrate Oral Solution; Piperazine Citrate Elixir Piperazine Citrate Syrup is a solution of Piperazine Citrate in a suitable flavoured Vehicle. Piperazine Citrate Syrup contains the equivalent of not less than 92.5 per cent and not more than 107.5 per cent of the stated amount of piperazine hydrate, C4H10N2, 6H2O.
Identification
A. To 1 ml add 5 ml of 2 M hydrochloric acid and, with stirring, 1 ml of a freshly prepared 50 per cent w/v solution of sodium nitrite, cool in ice for 15 minutes, induce crystallisation, wash the crystalline precipitate with water and dry at 105. The crystals melt at about 159 (2.4.21). B. Warm 10 ml with activated charcoal and filter. Boil a portion of the filtrate with an excess of mercuric sulphate solution, filter, boil the filtrate and add 0.25 ml of dilute potassium permanganate solution; the permanganate solution is decolorised and a white precipitate is produced. C. Acidify a portion of the filtrate obtained in test B with 1 M sulphuric acid, add 0.25 ml of dilute potassium permanganate solution, warm until the colour is discharged and add an excess of bromine water; a white precipitate is produced either immediately or on cooling.
Tests
Other tests. Complies with the tests stated under Oral Liquids. Assay. Weigh accurately a quantity containing about 0.2 g of Piperazine Hydrate, add 3.5 ml of 0.5 M sulphuric acid and 10 ml of water, add 100 ml of picric acid solution, heat on a water-bath for 15 minutes, allow to stand for 1 hour and filter through a sintered-glass crucible (porosity No. 4). Wash the residue with successive quantities, each of 10 ml, of a mixture of equal volumes of a saturated solution of picric acid and water until the washings are free from sulphate. Wash the residue with five quantities, each of 10 ml, of ethanol and dry to constant weight at 105. 1 g of the residue is equivalent to 0.3567 g of C4H10N2, 6H2O.
949
PIPERAZINE HYDRATE
IP 2007
Determine the weight per ml of the syrup (2.4.29), and calculate the content of C4H10N2, 6H2O, weight in volume. Storage. Store protected from light. Labelling. The label states the strength in terms of the equivalent amount of piperazine hydrate.
Test solution (a). Dissolve 1 g of the substance under examination in 10 ml of a mixture of 3 volumes of strong ammonia solution and 2 volumes of ethanol. Test solution (b). Dissolve 1 g of the substance under examination in 100 ml of the same solvent mixture. Reference solution (a). A 1 per cent w/v solution of piperazine hydrate RS in the same solvent mixture. Reference solution (b). A 0.025 per cent w/v solution of ethylenediamine in the same solvent mixture. Reference solution (c). A 0.025 per cent w/v solution of triethylenediamine in the same solvent mixture.
Piperazine Hydrate
H N , 6H2O N H
C4H10N2, 6H2O Mol. Wt. 194.2
Reference solution (d). A solution containing 0.025 per cent w/v of triethylenediamine and 1 per cent w/v of the substance under examination in the same solvent mixture. Apply to the plate 5 l of each solution. After development, dry the plate at 105, spray with a 0.3 per cent w/v solution of ninhydrin in a mixture of 3 volumes of anhydrous acetic acid and 100 volumes of 1-butanol and then with a 0.15 per cent w/v solution of ninhydrin in ethanol and dry at 105 for 10 minutes. Any secondary spot in the chromatogram obtained with test solution (a) is not more intense than the spot in the chromatogram obtained with reference solution (b). Spray the plate with 0.05 M iodine and allow to stand for about 10 minutes. Any spot corresponding to triethylenediamine in the chromatogram obtained with test solution (a) is not more intense than the spot in the chromatogram obtained with reference solution (c). The test is not valid unless the chromatogram obtained with reference solution (d) shows two separated spots. Ignore any spot remaining on the line of application. Heavy metals (2.3.13).1.0 g complies with the limit test for heavy metals, Method A (20 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Assay. Weigh accurately about 0.2 g, dissolve in 10 ml of anhydrous glacial acetic acid with gentle heating and dilute to 70 ml with the same solvent. Titrate with 0.1 M perchloric acid, using 0.25 ml of 1-naphtholbenzein solution as indicator and titrating until the colour of the solution changes from brownish yellow to green. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.009705 g of C4H10N2, 6H2O. Storage. Store protected from light and moisture.
Piperazine Hydrate contains not less than 98.0 per cent and not more than 101.0 per cent of C4H10N2, 6H2O. Description. Colourless, glassy deliquescent crystals.
Identification
Test A may be omitted if tests B, C and D are carried out. Tests B and C may be omitted if tests A and D are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with piperazine hydrate RS. B. In the test for Related substances, examine the plate after spraying with both the ninhydrin solutions. The principal spot in the chromatogram obtained with test solution (b) corresponds to that in the chromatogram obtained with reference solution (a). C. Dissolve 0.2 g in 5 ml of dilute hydrochloric acid, add 0.5 g of sodium nitrite and heat to boiling. Cool in ice for 15 minutes, scratching the walls of the container with a glass rod and filter. The crystals, after washing with 10 ml of ice-cold water and drying at 105, melt at about 159 (2.4.21).
Tests
Appearance of solution. A 5.0 per cent w/v solution in carbon dioxide-free water is clear (2.4.1), and not more intensely coloured than reference solution BS8 (2.4.1). pH (2.4.24). 10.5 to 12.0, determined in a 5.0 per cent w/v solution in carbon dioxide-free water. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A freshly prepared mixture of 80 volumes of acetone and 20 volumes of strong ammonia solution.
Piperazine Phosphate
C4H10N2, H3PO4, H2O Mol. Wt. 202.2 Piperazine Phosphate contains not less than 98.5 per cent and not more than 100.5 per cent of C4H10N2, H3PO4, calculated on the anhydrous basis.
950
IP 2007
PIROXICAM
Description. A white, crystalline powder; odourless or almost odourless.
Identification
A. Dissolve 0.2 g in 5 ml of dilute hydrochloric acid, add 0.5 g of sodium nitrite and heat to boiling. Cool in ice for 15 minutes, scratching the walls of the container with a glass rod and filter. The crystals, after washing with 10 ml of icecold water and drying at 105, melt at about 159 (2.4.21). B. Dissolve 0.1 g in 5 ml of water, add 0.5 g of sodium bicarbonate, 0.5 ml of a freshly prepared 5.0 per cent w/v solution of potassium ferricyanide and 0.1 ml of mercury, shake vigorously for 1 minute and allow to stand for 20 minutes; a reddish colour slowly develops. C. Gives the reactions of phosphates (2.3.1).
A. To 4 ml of the filtrate, add 1 ml of hydrochloric acid, 0.5 g of sodium nitrite and heat to boiling. Cool in ice for 15 minutes, scratching the walls of the container with a glass rod and filter. The crystals, after washing with 10 ml of ice-cold water and drying at 105, melt at about 159 (2.4.21). B. Dilute 1 ml of the filtrate to 5 ml with water, add 0.5 g of sodium bicarbonate, 0.5 ml of a freshly prepared 5.0 per cent w/v solution of potassium ferricyanide and 0.1 ml of mercury, shake vigorously for 1 minute and allow to stand for 20 minutes; a reddish colour slowly develops. C. Gives the reactions of phosphates (2.3.1).
Tests
Disintegration. The test does not apply to Piperazine Phosphate Tablets intended to be chewed before swallowing. Other tests. Comply with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 0.15 g of Piperazine Phosphate, shake with 10 ml of water for 1 hour, filter and wash the residue with two quantities, each of 10 ml, of water. To the combined extract and washings add 5 ml of 1 M sulphuric acid and 50 ml of picric acid solution, boil, allow the mixture to stand for several hours and filter through a sintered glass crucible (porosity No. 4). Wash the residue with successive quantities, each of 10 ml, of a mixture of equal volumes of a saturated solution of picric acid and water until the washings are free from sulphate. Wash the residue with five quantities, each of 10 ml, of ethanol and dry to constant weight at 105. 1 g of the residue is equivalent to 0.3714 g of C4H10N2, H3PO4, H2O. Storage. Store protected from moisture. Labelling. The label states, where applicable, that the tablets are to be chewed before swallowing.
Tests
pH (2.4.24). 6.0 to 6.5, determined in a 1.0 per cent w/v solution. Heavy metals (2.3.13). Dissolve 1.0 g in 20 ml of 2 M acetic acid and add sufficient water to produce 25 ml. The solution complies with the limit test for heavy metals, Method A (20 ppm). Water (2.3.43). 8.0 to 9.5 per cent, determined on 0.25 g. Assay. Weigh accurately about 0.2 g, dissolve in a mixture of 3.5 ml of 0.5 M sulphuric acid and 10 ml of water. Add 100 ml of picric acid solution, heat on a water-bath for 15 minutes and allow to stand for 1 hour. Filter through a sintered-glass crucible (porosity No. 4) and wash the residue with successive quantities, each of 10 ml, of a mixture of equal volumes of a saturated solution of picric acid and water until the washings are free from sulphate. Wash the residue with five quantities, each of 10 ml, of ethanol and dry to constant weight at 105. 1 g of the residue is equivalent to 0.3382 g of C4H10N2, H3PO4. Storage. Store protected from moisture.
Piperazine Phosphate Tablets

If the tablets are intended to be chewed before swallowing they may contain suitable flavouring agents. Piperazine Phosphate Tablets contain not less than 92.5 per cent and not more than 107.5 per cent of the stated amount of piperazine phosphate, C4H10N2, H3PO4, H2O.
Piroxicam
O S O N CH3 H N N
OH O
Identification
Extract a quantity of the powdered tablets containing 1 g of Piperazine Phosphate with 20 ml of water and filter. The filtrate complies with the following tests. C15H13N3O4S Mol. Wt. 331.4 Piroxicam is 4-hydroxy-2-methyl-N-(2-pyridyl)-2H-1,2benzothiazine-3-carboxamide-1,1-dioxide.
951
PIROXICAM CAPSULES
IP 2007
Piroxicam contains not less than 97.0 per cent and not more than 103.0 per cent of C15H 13N 3O 4S, calculated on the anhydrous basis. Description. An off-white to light tan or light yellow powder; odourless.
water and 5.35 g of sodium phosphate in 100 ml of water to 1000 ml with water, flow rate. 1.2 ml per minute, spectrophotometer set at 254 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject alternately the test solution and the reference solution. Calculate the content of C15H13N3O4S. Storage. Store protected from light and moisture.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with piroxicam RS or with the reference spectrum of piroxicam. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.001 per cent w/v solution in 0.01 M methanolic hydrochloric acid shows absorption maxima at about 242 nm and 334 nm and a minimum at about 270 nm; absorbance at about 334 nm, about 0.87. C. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 95 volumes of toluene and 5 volumes of acetic acid. Test solution. Dissolve 0.1 g of the substance under examination in 100 ml of a mixture of equal volumes of chloroform and methanol. Reference solution. A 0.1 per cent w/v solution of piroxicam RS in a mixture of equal volumes of chloroform and methanol. Apply to the plate 20 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution.
Piroxicam Capsules
Piroxicam Capsules contain not less than 92.5 per cent and not more than 107.5 per cent of the stated amount of piroxicam, C15H13N3O4S.
Identification
Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 95 volumes of toluene and 5 volumes of acetic acid. Test solution. Dissolve a portion of the contents of the capsules in sufficient of a mixture of equal volumes of chloroform and methanol to obtain a solution containing about 0.1 per cent w/v of Piroxicam. Shake for 10 minutes, filter and use the filtrate. Reference solution. A 0.1 per cent w/v solution of piroxicam RS in a mixture of equal volumes of chloroform and methanol. Apply to the plate 20 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution.
Tests
Heavy metals (2.3.13). 0.4 g complies with the limit test for heavy metals, Method B (50 ppm). Sulphated ash (2.3.18). Not more than 0.3 per cent. Water (2.3.43). Not more than 0.5 per cent , determined on 2.0 g. Assay. Determine by liquid chromatography (2.4.14). Test solution. A 0,005 per cent w/v solution of the substance under examination in 0.1 M methanolic hydrochloric acid. Reference solution. A 0.005 per cent w/v solution of piroxicam RS in 0.1 M methanolic hydrochloric acid. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 45 volumes of methanol and 55 volumes of a buffer solution prepared by diluting a mixture of 7.72 g of anhydrous citric acid in 400 ml of
Tests
Dissolution (2.5.2). Apparatus No. 2 Medium. 900 ml of 0.1 M hydrochloric acid Speed and time. 100 rpm and 45 minutes. Withdraw 10 ml of the medium and filter. Measure the absorbance of the filtrate (2.4.7), suitably diluted if necessary, at the maximum at about 242 nm. Calculate the content of C15H13N3O4S, in the medium taking 352 as the specific absorbance at 242 nm.
952
IP 2007
POLYETHYLENE GLYCOL 1500
D. Not less than 75 per cent of the stated amount of C15H13N3O4S. Uniformity of content. (For capsules containing 10 mg or less) Comply with the test stated under Capsules. Test solution. Dissolve the contents of a capsule in 100.0 ml of 0.1 M methanolic hydrochloric acid and filter. Dilute further if necessary. Determine by liquid chromatography (2.4.14) using the chromatographic system and the reference solution described under Assay. Calculate the content of C15H13N3O4S in the capsule. Water (2.3.43). Not more than 8.0 per cent, determined on 0.25 g. Other tests. Comply with the tests stated under Capsules. Assay. Determine by liquid chromatography (2.4.14). Test solution. Dissolve an accurately weighed quantity of the mixed contents of the capsules containing about 50 mg of Piroxicam in 100.0 ml of 0.1 M methanolic hydrochloric acid. Further dilute 1.0 ml of the solution to 10.0 ml with the same solvent. Reference solution. A 0.005 per cent w/v solution of piroxicam RS in 0.1 M methanolic hydrochloric acid. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 45 volumes of methanol and 55 volumes of a buffer solution prepared by diluting a mixture of 7.72 g of anhydrous citric acid in 400 ml of water and 5.35 g of sodium phosphate in 100 ml of water to 1000 ml with water, flow rate. 1.2 ml per minute, spectrophotometer set at 254 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject alternately the test solution and the reference solution. Calculate the content of C15H13N3O4S in the capsules. Storage. Store protected from light and moisture.
is substantially converted into the hemihydrate, CaSO4,H2O, with minimum production of the anhydrous phases of calcium sulphate. It may contain suitable accelerators or decelerators. Description. A white or almost white powder; odourless or almost odourless; hygroscopic.
Identification
Gives the reactions of calcium salts and of sulphates (2.3.1).
Tests
pH (2.4.24). 6.5 to 9.0, determined in a 20.0 per cent w/v slurry in water. Acid insoluble matter. Dissolve 0.5 g in 30 ml of a mixture of 1 volume of hydrochloric acid and 2 volumes of water and evaporate to dryness in a dish on a water-bath. Heat for 2 hours at 120 and again add 20 ml of the acid mixture. Warm for a few minutes and filter. Wash the residue with warm water until free from chlorides, dry, ignite and weigh. The residue weighs not more than 5 mg (1.0 per cent). Setting properties. 20 g mixed with 10 ml of water at 15 to 20 in a cylindrical mould about 2.4 cm in diameter sets in not less than 4 minutes and not more than 6 minutes. The mass thus formed, after standing for 3 hours, possesses sufficient hardness to resist pressure of the fingers at the edges, which retain their sharpness of outline and do not crumble under pressure. Loss on ignition (2.4.20). 4.5 to 8.0 per cent, determined by igniting to constant weight at red heat. Storage. Store protected from moisture
Polyethylene Glycol 1500

Macrogol 1500
Polyethylene Glycol 1500 is a mixture of the polycondensation products of ethylene oxide and water obtained under controlled conditions. It is represented by the formula HOCH2[CH2OCH2]nCH2OH, where n is between 28 and 36. Description. A white or creamy white, soft, wax-like solid; odour, faint and characteristic.
Tests
Appearance of solution (2.4.1). A 25.0 per cent w/v solution is not more intensely coloured than reference solution BYS6. pH (2.4.24). 4.0 to 7.0, determined in a 5.0 per cent w/v solution. Freezing point (2.4.11). 42 to 46. Hydroxyl value (2.3.27). 70 to 86, determined on 5.0 g. Viscosity (2.4.28). 25 mm2s1 to 32 mm2s1, determined at 100 by Method A using a U-tube viscometer (size D).
Plaster Of Paris
Dried Calcium Sulphate; Exsiccated Calcium Sulphate
CaSO4, H2O Mol. Wt. 145.2 Plaster of Paris is prepared by heating powdered gypsum, CaSO4,H2O, at about 150 in a controlled manner such that it
953
POLYETHYLENE GLYCOL 4000
IP 2007
Arsenic (2.3.10). Mix 3.3 g with 3 g of anhydrous sodium carbonate, add 10 ml of bromine solution and mix thoroughly. Evaporate to dryness on a water-bath, gently ignite and dissolve the cooled residue in 16 ml of brominated hydrochloric acid and 45 ml of water. Remove the excess of bromine with 2 ml of stannous chloride solution AsT. The resulting solution complies with the limit test for arsenic (3 ppm). Heavy metals (2.3.13). Dissolve 4.0 g in 5 ml of a 1.0 per cent w/v solution of hydrochloric acid and sufficient water to produce 25 ml. The solution complies with the limit test for heavy metals, Method A (5 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Storage. Store protected from moisture.

Macrogol 6000
Polyethylene Glycol 6000 is a mixture of the polycondensation products of ethylene oxide and water obtained under controlled conditions. It is represented by the formula HOCH2[CH2OCH2]nCH2OH, where n is between 112 and 158. Description. A white to creamy white, wax-like solid or flakes; odour, faint and characteristic.
Tests
Appearance of solution (2.4.1). A 15.0 per cent w/v solution is not more intensely coloured than reference solution BYS6. pH (2.4.24). 4.5 to 7.5, determined in a 5.0 per cent w/v solution. Freezing point (2.4.11). 56 to 60. Viscosity (2.4.28). 250 mm2s1 to 390 mm2s1, determined at 100 by Method A using a U-tube viscometer (size F). Arsenic (2.3.10). Mix 3.3 g with 3 g of anhydrous sodium carbonate, add 10 ml of bromine solution and mix thoroughly. Evaporate to dryness on a water-bath, gently ignite and dissolve the cooled residue in 16 ml of brominated hydrochloric acid and 45 ml of water. Remove the excess of bromine with 2 ml of stannous chloride solution AsT. The resulting solution complies with the limit test for arsenic (3 ppm). Heavy metals (2.3.13). Dissolve 4.0 g in 5 ml of a 1.0 per cent w/v solution of hydrochloric acid and sufficient water to produce 25 ml. The resulting solution complies with the limit test for heavy metals, Method A (5 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Storage. Store protected from moisture.

Macrogol 4000
Polyethylene Glycol 4000 is a mixture of the polycondensation products of ethylene oxide and water obtained under controlled conditions. It is represented by the formula HOCH2[CH2OCH2]nCH2OH, where n is between 69 and 84. Description. A creamy white, hard, wax-like solid, powder or flakes; odour, faint and characteristic.
Tests
Appearance of solution (2.4.1). A 20.0 per cent w/v solution is not more intensely coloured than reference solution BYS6. pH (2.4.24). 4.5 to 7.5, determined in a 5.0 per cent w/v solution. Freezing point (2.4.11). 53 to 56. Hydroxyl value (2.3.27). 30 to 36, determined on 20.0 g. Viscosity (2.4.28). 76 mm2s1 to 110 mm2s1, determined at 100 by Method A using a U-tube viscometer (size E). Arsenic (2.3.10). Mix 3.3 g with 3 g of anhydrous sodium carbonate, add 10 ml of bromine solution and mix thoroughly. Evaporate to dryness on a water-bath, gently ignite and dissolve the cooled residue in 16 ml of brominated hydrochloric acid and 45 ml of water. Remove the excess of bromine with 2 ml of stannous chloride solution AsT. The resulting solution complies with the limit test for arsenic (3 ppm). Heavy metals (2.3.13). Dissolve 4.0 g in 5 ml of a 1.0 per cent w/v solution of hydrochloric acid and sufficient water to produce 25 ml. The solution complies with the limit test for heavy metals, Method A (5 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Storage. Store protected from moisture.
Polysorbate 20
Polyoxyethylene 20 Sorbitan Monolaurate
HO(H2CH2CO)w O (OCH2CH2)z O w + x + y + z = 20 (OCH2CH2)xOH (OCH2 CH2)yOH O C11H23
Polyoxyethylene 20 Sorbitan Monolaurate Polysorbate 20 is a mixture of partial lauric esters of D-glucitol and its anhydrides copolymerised with approximately 20 moles
954
IP 2007
POLYSORBATE 80
of ethylene oxide for each mole of D-glucitol and its anhydrides. The lauric acid used for the esterification may contain other fatty acids. Description. A clear or slightly opalescent, oily, yellowish or brownish yellow liquid; odour, faint and characteristic.
Polysorbate 80
Polyoxyethylene 80 Sorbitan Monooleate
HO(H2CH2CO)w (OCH2CH2)xOH (OCH2CH2)yOH O (OCH2CH2)z w+x+y+z = 20 O O C17H33
Identification
A. Dissolve 0.5 g in water at about 50 and dilute to 10 ml with the same solvent; the solution produces a copious foam on shaking. Add 0.5 g of sodium chloride and heat to boiling; the resulting cloudiness disappears during cooling to about 50. B. Dissolve 0.1 g in 5 ml of chloroform, add 0.1 g of potassium thiocyanate and 0.1 g of cobalt nitrate and stir with a glass rod; a blue colour is produced.
Polysorbate 80 is a mixture of partial oleic esters of D-glucitol and its anhydrides copolymerised with approximately 20 moles of ethylene oxide for each mole of D-glucitol and its anhydrides. Description. A clear or almost clear, oily, yellowish or brownish yellow liquid; odour, faint and characteristic.
Tests
pH (2.4.24). 5.0 to 7.0, determined in a 5.0 per cent w/v solution in carbon dioxide-free water. Acid value (2.3.23). Not more than 2.0, determined on 5.0 g. Hydroxyl value (2.3.27). 96 to 108, determined on 2.0 g. Saponification value (2.3.37). 40 to 50, using 15 ml of 0.5 M ethanolic potassium hydroxide and diluting with 50 ml of water before carrying out the titration. Iodine value (2.3.28). Not more than 5.0, determined by the iodine bromide method. Heavy metals (2.3.13). 2.0 g complies with the limit test for heavy metals, Method B (10 ppm). Reducing impurities. Dissolve 2.0 g in 25 ml of hot water, add 25 ml of 1 M sulphuric acid and 0.1 ml of ferroin solution and titrate with 0.01 M ceric ammonium sulphate shaking continuously until the colour changes from red to greenish blue persists for 30 seconds. Carry out a blank titration. Not more than 2.0 ml of 0.01 M ceric ammonium sulphate is required. Sulphated ash. Not more than 0.2 per cent, determined by the following method. Weigh accurately about 2.0 g in a silica or platinum crucible, add 0.5 ml of sulphuric acid and heat on a water-bath for 2 hours. Carefully ignite at a low temperature until thoroughly charred. Add to the carbonised mass 2 ml of nitric acid and 0.25 ml of sulphuric acid, cautiously heat until white fumes are evolved and then ignite at 600 until the carbon is completely burnt off. Allow to cool, weigh and repeat the operation for periods of 15 minutes until two successive weighings do not differ by more than 0.5 mg. Water (2.3.43). Not more than 3.0 per cent , determined on 1.0 g. Storage. Store protected from light and moisture.
Identification
A. Dissolve 0.5 g in water at about 50 and dilute to 10 ml with the same solvent; the solution produces a copious foam on shaking. Add 0.5 g of sodium chloride and heat to boiling; the resulting cloudiness disappears during cooling to about 50. B. Dissolve 0.1 g in 5 ml of chloroform, add 0.1 g of potassium thiocyanate and 0.1 g of cobalt nitrate and stir with a glass rod; a blue colour is produced. C. To 2 ml of a 5 per cent w/v solution add 0.5 ml of bromine water; the bromine is decolourised.
Tests
pH (2.4.24). 6.0 to 8.0, determined in a 5.0 per cent w/v solution in carbon dioxide-free water. Acid value (2.3.23). Not more than 2.0, determined on 5 g. Hydroxyl value (2.3.27). 65 to 80, determined on 2.0 g. Saponification value (2.3.37). 45 to 55, using 15 ml of 0.5 M ethanolic potassium hydroxide and diluting with 50 ml of water before carrying out the titration. Iodine value (2.3.28). 18 to 24, determined by the iodine bromide method. Reducing impurities. Dissolve 2.0 g in 25 ml of hot water, add 25 ml of 1 M sulphuric acid and 0.1 ml of ferroin solution and titrate with 0.01 M ceric ammonium sulphate shaking continuously until the colour changes from red to greenish blue persists for 30 seconds. Carry out a blank titration. Not more than 5.0 ml of 0.01 M ceric ammonium sulphate is required. Heavy metals (2.3.13). 2.0 g complies with the limit test for heavy metals, Method B (10 ppm).
955
POTASSIUM CHLORIDE
IP 2007
Sulphated ash. Not more than 0.2 per cent, determined by the following method. Weigh accurately about 2.0 g in a silica or platinum crucible, add 0.5 ml of sulphuric acid and heat on a water-bath for 2 hours. Carefully ignite at a low temperature until thoroughly charred. Add to the carbonised mass 2 ml of nitric acid and 0.25 ml of sulphuric acid, cautiously heat until white fumes are evolved and then ignite at 600 until the carbon is completely burnt off. Allow to cool, weigh and repeat the operation for periods of 15 minutes until two successive weighings do not differ by more than 0.5 mg. Water (2.3.43). Not more than 3.0 per cent, determined on 1.0 g. Storage. Store protected from light and moisture.
Iron (2.3.14). 20 ml of solution A complies with the limit test for iron (20 ppm). Bromides. Dilute 1.0 ml of solution A to 50.0 ml with water. To 5.0 ml of the solution add 2.0 ml of phenol red reagent and 1.0 ml of a 0.01 per cent solution of chloramine T and mix immediately. After exactly 2 minutes, add 0.15 ml of 0.1 M sodium thiosulphate, mix and dilute to 10.0 ml with water. The absorbance of the solution measured at about 590 nm (2.4.7), using water as the blank, is not more than that of a standard prepared at the same time and in the same manner, using 5.0 ml of a 0.0003 per cent w/v solution of potassium bromide (0.1 per cent). Iodides. Moisten 5 g by adding dropwise, a solution freshly prepared by mixing 25 ml of iodide-free starch solution, 2 ml of 0.5 M sulphuric acid, 0.15 ml of sodium nitrite solution and 25 ml of water and examine the mixture in daylight; the substance shows no blue colour after 5 minutes. Sulphates (2.3.17). 0.5 g complies with the limit test for sulphates (300 ppm). Loss on drying (2.4.19). Not more than 1.0 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.15 g, dissolve in 50 ml of water and titrate with 0.1 M silver nitrate using potassium chromate solution as indicator. 1 ml of 0.1 M silver nitrate is equivalent to 0.007455 g of KCl. Potassium Chloride intended for use in the manufacture of dialysis solutions complies with the following additional requirement. Aluminium. Dissolve 4.0 g in 100 ml of water and add 10 ml of acetate buffer pH 6.0. Extract with successive quantities of 20, 20 and 10 ml of a 0.5 per cent w/v solution of 8-hydroxyquinoline in chloroform and dilute the combined extracts to 50 ml with chloroform. Use as the standard solution a mixture of 2 ml of aluminium standard solution (2 ppm Al), 10 ml of acetate buffer pH 6.0 and 98 ml of water treated in the same manner and as the blank solution a mixture of 10 ml of acetate buffer pH 6.0 and 100 ml of water treated in the same manner. Measure the fluorescence of the test and standard solutions (2.4.5), using an excitation wavelength of about 392 nm and emission wavelength of about 518 nm and setting the instrument to zero with the blank solution in each case. The fluorescence of the test is not greater than that of the standard solution (1 ppm). Potassium chloride intended for use in the manufacture of Parenteral Preparations or for the preparation of haemodialysis solution complies with the following additional requirement. Sodium. Not more than 0.1 per cent, determined by atomic absorption spectrophotometry (2.4.2), using a 1.0 per cent
Potassium Chloride
KCl Mol. Wt. 74.6 Potassium Chloride contains not less than 99.0 per cent and not more than 100.5 per cent of KCl, calculated on the dried basis. Description. Colourless crystals or a white, crystalline powder; odourless.
Identification
Dissolve 10 g in 100 ml of carbon dioxide-free water prepared from distilled water (solution A). The solution gives the reactions of potassium salts and of chlorides (2.3.1).
Tests
Appearance of solution. Solution A is clear (2.4.1), and colourless (2.4.1). Acidity or alkalinity. 5.0 g dissolved in 50 ml of carbon dioxidefree water requires not more than 0.5 ml of 0.01 M sodium hydroxide or 0.01 M hydrochloric acid for neutralisation to bromothymol blue solution. Arsenic (2.3.10). Dissolve 10.0 g in 50 ml of water and add 10 ml of stannated hydrochloric acid AsT. The resulting solution complies with the limit test for arsenic (1 ppm). Barium. To 5 ml of solution A add 5 ml of water and 1 ml of 1 M sulphuric acid; the solution, after not less than 15 minutes, is not more opalescent than a mixture of 5 ml of solution A and 6 ml of water. Heavy metals (2.3.13). Dissolve 2.0 g in 10 ml of water to which are added 2 ml of dilute acetic acid and 13 ml of water. The solution complies with the limit test for heavy metals, Method A (10 ppm). Calcium and magnesium. Dissolve 1 g in 20 ml of water, add 1 ml of dilute ammonia solution and 1 ml of sodium phosphate solution; the solution remains clear.
956
IP 2007
POTASSIUM CHLORIDE, SODIUM CHLORIDE AND DEXTROSE INJECTION
w/v solution and measuring at 589 nm. Use sodium solution AAS, suitably diluted with water, for the standard solutions. Storage. Store protected from moisture. Labelling. The label states whether or not the material is suitable for use in the manufacture of Parenteral Preparations or for the preparation of haemodialysis or dialysis solutions.
0.1 M silver nitrate using potassium chromate solution as indicator. 1 ml of 0.1 M silver nitrate is equivalent to 0.007455 g of KCl. For dextrose To an accurately measured volume containing 2 g to 5 g of Dextrose, add 0.2 ml of 05 M ammonia and sufficient water to produce 100.0 ml. Mix well, allow to stand for 30 minutes and measure the optical rotation in a 2-dm tube (2.4.22). The observed rotation in degrees multiplied by 0.9477 represents the weight, in g, of dextrose, C6H12O6, in the volume taken for assay. Storage. Store in single dose containers. Labelling. The label states (1) the strength as the percentages w/v of Potassium Chloride and Dextrose; (2) the total osmolar concentration in mOsmol per litre; (3) where the contents are less than 100 ml, or where the label states that the Injection is not for direct use but is to be diluted before use, the label alternatively may state the total osmolar concentration in mOsmol per ml; (4) the content of potassium in millimoles; (5) that the injection containing visible particles in the solution should not be used.
Potassium Chloride And Dextrose Injection

Potassium Chloride in Dextrose Injection; Potassium Chloride and Dextrose Intravenous Infusion; Potassium Chloride and Glucose Intravenous Infusion Potassium Chloride and Dextrose Injection is a sterile solution of Potassium Chloride and Dextrose in Water for Injections. Potassium Chloride and Dextrose Injection contains not less than 95.0 per cent and not more than 110.0 per cent of the stated amount of potassium chloride, KCl, and not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of dextrose, C6H12O6. It contains no antimicrobial agent. Description. A clear, colourless or faintly straw-coloured solution.
Potassium Chloride, Sodium Chloride And Dextrose Injection

Potassium Chloride in Dextrose and Sodium Chloride Injection; Potassium Chloride, Sodium Chloride and Dextrose Intravenous Infusion; Potassium Chloride, Sodium Chloride and Glucose Intravenous Infusion
Potassium Chloride, Sodium Chloride and Dextrose Injection is a sterile solution of Potassium Chloride, Sodium Chloride and Dextrose in Water for Injections. Potassium Chloride, Sodium Chloride and Dextrose Injection contains not less than 95.0 per cent and not more than 110.0 per cent of the stated amounts of sodium, Na, potassium, K, and chloride, Cl, and not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of dextrose, C6H12O6. It contains no antimicrobial agent.
Identification
A. To 1 ml add 0.05 ml of potassium cupri-tartrate solution; the solution remains blue and clear. Heat to boiling, a copious red precipitate is formed. B. Gives reaction B of potassium salts and reaction A of chlorides (2.3.1).
Tests
pH (2.4.24). 3.5 to 6.5. 5-Hydroxymethylfurfural and Related substances. Dilute a volume containing 1.0 g of Dextrose to 500.0 ml with water and measure the absorbance of the resulting solution at the maximum at about 284 nm, absorbance at about 284 nm (2.4.7); not more than 0.25. Heavy metals (2.3.13). Evaporate a volume containing 4.0 g of Dextrose to 10 ml and add 2 ml of dilute acetic acid and sufficient water to produce 25 ml. The solution complies with the limit test for heavy metals, Method A (5 ppm). Bacterial endotoxins (2.2.3). Not more than 0.5 Endotoxin Unit per ml. Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. For potassium chloride Titrate an accurately measured volume containing 0.1 g of Potassium Chloride with
Identification
A. To 1 ml add 0.05 ml of potassium cupri-tartrate solution; the solution remains blue and clear. Heat to boiling, a copious red precipitate is formed. B. Gives reaction A of potassium salts, reaction B of sodium salts and reaction A of chlorides (2.3.1)
Tests
pH (2.4.24). 3.5 to 6.5, determined on a portion diluted with water, if necessary, to a concentration of not more than 5.0 per cent of dextrose.
957
POTASSIUM CITRATE
IP 2007
5-Hydroxymethylfurfural and Related substances. Dilute a volume containing 1.0 g of Dextrose to 500.0 ml with water and measure the absorbance of the resulting solution at the maximum at about 284 nm, absorbance at about 284 nm (2.4.7); not more than 0.25. Heavy metals (2.3.13). Evaporate a volume containing 4.0 g of Dextrose to 10 ml and add 2 ml of dilute acetic acid and sufficient water to produce 25 ml. The solution complies with the limit test for heavy metals, Method A (5 ppm). Bacterial endotoxins (2.2.3). Not more than 0.5 Endotoxin Unit per ml. Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. For sodium Dilute appropriately with water and determine by Method A for flame photometry (2.4.4), or by Method A for atomic absorption spectrophotometry (2.4.2), measuring at 589 nm and using sodium solution FP or sodium solution AAS respectively, suitably diluted with water for the standard solutions. For potassium Dilute appropriately with water and determine by Method A for flame photometry (2.4.4), or by Method A for atomic absorption spectrophotometry (2.4.2), measuring at 767 nm and using potassium solution FP or potassium solution AAS respectively, suitably diluted with water for the standard solutions. For total chlorides To 20.0 ml add 30 ml of water, 50.0 ml of 0.1 M silver nitrate and 2 ml of nitric acid. Filter, wash the precipitate with water slightly acidified with nitric acid and titrate the excess of silver nitrate with 0.1 M ammonium thiocyanate using ferric ammonium sulphate solution as indicator until a reddish yellow colour is produced. Carry out a blank titration. 1 ml of 0.1 M silver nitrate is equivalent to 0.003545 g of total chloride, calculated as Cl. For dextrose To an accurately measured volume containing 2 g to 5 g of Dextrose, add 0.2 ml of 5 M ammonia and sufficient water to produce 100.0 ml. Mix well, allow to stand for 30 minutes and measure the optical rotation in a 2-dm tube (2.4.22). The observed rotation in degrees multiplied by 0.9477 represents the weight, in g, of dextrose, C6H12O6, in the volume taken for assay. Storage. Store in single dose containers. Labelling. The label states (1) the strength as the percentages w/v of Potassium Chloride, Sodium Chloride and Dextrose; (2) the total osmolar concentration in mOsmol per litre; (3) where the contents are less than 100 ml, or where the label states that the Injection is not for direct use but is to be diluted before use, the label alternatively may state the total osmolar concentration in mOsmol per ml; (4) the content of potassium,
sodium and chloride in millimoles; (5) that the injection containing visible particles in the solution should not be used.
Potassium Citrate
HO KOOC
C6H5K3O7,H2O
COOK COOK ,H2O

Mol. Wt. 324.4
Potassium citrate is tripotassium 2-hydroxypropane-1,2,3tricarboxylate monohydrate. Potassium Citrate contains not less than 99.0 per cent and not more than 101.0 per cent of C6H5K3O7, calculated on the anhydrous basis. Description. White, granular crystals or a crystalline powder; odourless; hygroscopic.
Identification
A. Dissolve 10 g in 100 ml of carbon dioxide-free water prepared from distilled water (solution A). 0.5 ml of solution A gives the reactions of potassium salts (2.3.1). B. To 1 ml of solution A add 4 ml of water; the solution gives the reactions of citrates (2.3.1).
Tests
Appearance of solution. Solution A is clear (2.4.1), and colourless (2.4.1). Acidity or alkalinity. To 10 ml of solution A add 0.1 ml of dilute phenolphthalein solution; not more than 0.2 ml of 0.1 M hydrochloric acid or 0.1 M sodium hydroxide is required to change the colour of the solution. Arsenic (2.3.10). Dissolve 5.0 g in 50 ml of water and add 15 ml of stannated hydrochloric acid. The resulting solution complies with the limit test for arsenic (2 ppm). Heavy metals (2.3.13). 2.0 g complies with the limit test for heavy metals, Method A (10 ppm). Sodium. Not more than 0.3 per cent, determined by atomic absorption spectrophotometry (2.4.2), Method I I, measuring at 589 nm. Prepare the test solution by addition of 1 ml of 2 M hydrochloric acid to 10.0 ml of solution A and diluting to 100.0 ml with distilled water. Use sodium solution AAS, suitably diluted with distilled water, for the standard solutions. Chlorides (2.3.12). Dilute 25.0 ml of solution A to 35 ml with water. The resulting solution complies with the limit test for chlorides (100 ppm).
958
IP 2007
POTASSIUM CLAVULANATE
Oxalate. Dissolve 0.5 g in 4 ml of water, add 3 ml of hydrochloric acid and 1 g of granulated zinc and heat on a water-bath for 1 minute. Allow to stand for 2 minutes, decant into 0.25 ml of a 1 per cent w/v solution of phenylhydrazine hydrochloride, heat to boiling, cool rapidly, add an equal volume of hydrochloric acid and 0.25 ml of potassium ferricyanide solution, shake and allow to stand for 30 minutes. Any pink colour produced is not more intense than that obtained by treating 4.0 ml of a 0.005 per cent w/v solution of oxalic acid at the same time and in the same manner (300 ppm, calculated as anhydrous oxalic acid). Sulphates (2.3.17). To 10.0 ml of solution A add 2 ml of 7 M hydrochloric acid and dilute to 15 ml with distilled water; the solution complies with the limit test for sulphates (150 ppm). Readily carbonisable substances. Heat 0.20 g, in powder, with 10 ml of sulphuric acid (96 per cent w/w) in a water-bath at 90 1 for 60 minutes and cool rapidly. The solution is not more intensely coloured than reference solution YS2 or GYS2 (2.4.1). Water (2.3.43). 4.0 to 7.0 per cent, determined on 0.5 g, titration being done after stirring for 15 minutes subsequent to addition of the substance under examination. Assay. Weigh accurately about 0.2 g, dissolve in 20 ml of anhydrous glacial acetic acid, heat to about 50, allow to cool. Titrate with 0.1 M perchloric acid, using 0.25 ml of 1-naphtholbenzein solution as indicator and titrating until a green colour is obtained. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.01021 g of C6H5K3O7. Storage. Store protected from moisture.
Identification
A. In the Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the peak in the chromatogram obtained with reference solution (a). B. Gives reaction A of potassium salts (2.3.1).
Tests
pH (2.4.24). 5.5 to 8.0, determined in 1.0 per cent w/v solution. Related substances. Determine by liquid chromatography (2.4.14). NOTE Prepare the solutions immediately before use. Test solution Dissolve 0.25 g of the substance under examination in mobile phase A and dilute to 25 ml with mobile phase A. Reference solution (a). Dilute 1 ml of the test solution to 100 ml with mobile phase A. Reference solution (b). A solution containing 0.01 per cent w/ v each of lithium clavulanate RS and amoxycillin trihydrate RS in mobile phase A. Chromatographic system a stainless steel column 10 cm x 4.6 mm, packed with octadecylsilyl silica gel (5 m), column temperature 40, mobile phase: A. a 0.78 per cent w/v solution of sodium dihydrogen phosphate adjusted to pH 4.0 with phosphoric acid and filtered through a 0.5 m filter, B. a mixture of equal volumes of mobile phase A and methanol, a linear gradient programme using the conditions given below, flow rate. 1 ml per minute, spectrophotometer set at 230 nm, a 20 l loop injector. Time (min) 04 4 15 15 18 18 24 24 39 Mobile phase A (per cent v/v) 100 100 50 50 50 100 100 Mobile phase B (per cent v/v) 0 0 50 50 50 0 0
Potassium Clavulanate
O O N O H OK H OH
C8H8KNO5
Mol. Wt. 237.3
Potassium Clavulanate is potassium (Z)-(2R,5R)-3-(2hydroxyethylidene)-7-oxo-4-oxa-1-azabicyclo[3.2.0]heptane2-carboxylate. Potassium Clavulanate contains not less than 96.5 per cent and not more than 102.0 per cent of potassium clavulanate, C8H8KNO5, calculated on the anhydrous basis. Description. A white to off white, crystalline hygroscopic powder.
Inject reference solution (b). The resolution between clavulanate (first peak) and amoxycillin (second peak ) is not less than 13. Inject the test solution and reference solution (b). The area of any individual impurity peak obtained is not more than the area of the principal peak in the chromatogram obtained with
959
POTASSIUM CLAVULANATE DILUTED
IP 2007
reference solution (a) (1.0 per cent). The sum of all impurity peaks obtained is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (a) (2.0 per cent). Ignore any peaks with an area 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.05 per cent). Water (2.3.43). Not more than 1.5 per cent, determined on 0.5 g. Assay. Determine by liquid chromatography (2.4.14). NOTE Prepare the solutions immediately before use. Test solution. Dissolve 50.0 mg of the substance under examination in a 0.41 per cent w/v solution of sodium acetate previously adjusted to pH 6.0 with glacial acetic acid, and dilute to 50.0 ml with the same solution. Reference solution (a). A 0.1 per cent w/v solution of lithium clavulanate RS in a 0.41 per cent w/v solution of sodium acetate previously adjusted to pH 6.0 with glacial acetic acid. Reference solution (b). A solution containing 0.1 per cent w/ v each of lithium clavulanate RS and amoxycillin trihydrate RS in a 0.41 per cent w/v solution of sodium acetate previously adjusted to pH 6.0 with glacial acetic acid. Chromatographic system a stainless steel column 30 cm x 4.6 mm, packed with octadecylsilyl silica gel (5 m), mobile phase: a mixture of 5 volumes of methanol and 95 volumes of a 1.5 per cent w/v solution of sodium dihydrogen phosphate previously adjusted to pH 4.0 with dilute phosphoric acid, flow rate. 1 ml per minute, spectrophotometer set at 230 nm, a 10 l loop injector. Inject reference solution (b). The resolution between clavulanate (first peak) and amoxycillin (second peak) is not less than 3.5. Inject alternately the test solution and reference solution (a). Calculate the content of C8H8KNO5. 1 mg of clavulanate (C8H9NO5) is equivalent to 1.191 mg of C8H8KNO5. Potassium Clavulanate intended for use in the manufacture of Parenteral Preparations without a further procedure for the removal of bacterial endotoxins complies with the following additional requirement. Bacterial endotoxins (2.2.3). Not more than 0.03 Endotoxin Unit per mg of potassium clavulanate. Potassium Clavulanate intended for use in the manufacture of Parenteral Preparations without a further sterilisation procedure complies with the following additional requirement.
Sterility (2.2.11). Complies with the test for sterility. Storage. Store protected from moisture. Labelling. The label states whether it is intended for use in the manufacture of parenteral preparations.
Potassium Clavulanate Diluted

C8H8KNO5 Mol. Wt. 237.3 Potassium Clavulanate Diluted is a dry mixture of Potassium Clavulanate and Microcrystalline Cellulose, or Silica, colloidal anhydrous or Silica, colloidal hydrated. Potassium Clavulanate Diluted contain not less than 91.2 per cent and not more than 107.1 per cent of the stated amount of potassium clavulanate, C8H8KNO5. Description. A white or almost white powder, hygroscopic.
Identification
A. In the Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the peak in the chromatogram obtained with reference solution (a). B. Gives reaction A of potassium salts (2.3.1). C. Depending on the diluent used, carry out one of the relevant identification tests given below. (i) On a watch-glass place a quantity of the substance under examination corresponding to 20 mg of cellulose and disperse in 4 ml of iodinated zinc chloride solution. The powder becomes violet-blue in colour. (ii) It gives the reaction of silicates (2.3.1).
Tests
pH (2.4.24) 4.8 to 8.0, determined on a quantity of the substance under examination containing 0.2 g of potassium clavulanate dissolved in 20 ml of carbon dioxide-free water. Light absorption. When examined in the range 230 nm to 360 nm (2.4.7), a 0.1 per cent w/v solution in 0.1 M phosphate buffer solution pH 7.0 shows an absorption maximum only at about 278 nm; absorbance at about 278 nm, not more than 0.40. Related substances. Determine by liquid chromatography (2.4.14). Test solution. Dissolve a quantity of the substance under examination containing 0.25 g of potassium clavulanate in 5 ml of mobile phase A, dilute to 25.0 ml with mobile phase A and filter.
960
IP 2007
POTASSIUM IODIDE
Reference solution (a). Dilute 1.0 ml of the test solution to 100.0 ml with mobile phase A. Reference solution (b). Dissolve 10 mg of amoxycillin trihydrate RS in 1 ml of the test solution and dilute to 100 ml with mobile phase A. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilyl silica gel (5 m), mobile phase: A. a 0.78 per cent w/v solution of sodium dihydrogen phosphate with the pH adjusted to 4.0 with dilute phosphoric acid, B. a mixture of equal volumes of mobile phase A and methanol, flow rate. 1 ml per minute, a linear gradient programme using the conditions given below, spectrophotometer set at 230 nm, a 20 l loop injector. Time Mobile Mobile Comment phase A phase B (min) (per cent v/v) (per cent v/v) 0 100 0 Isocratic 4 100 0 begin linear gradient 15 50 50 18 50 50 end chromatogram, return to 100A 24 100 0 end equilibration, begin next chromatogram Inject reference solution (b). The resolution between the clavulanate (first peak) and amoxycillin (second peak) is not less than 13. Inject alternatively the test solution and reference solution (a). Any individual impurity is not more than 1.0 per cent and the sum of all impurities found is not more than 2.0 per cent. Water (2.3.43). Not more than 2.5 per cent, determined on 1.0 g. Assay. Determine by liquid chromatography (2.4.14). Test solution. Dissolve a quantity of the substance under examination containing about 50.0 mg of potassium clavulanate in a 0.4 per cent w/v solution of sodium acetate with the pH previously adjusted to 6.0 with glacial acetic acid, dilute to 50.0 ml with the same solution and filter. Reference solution (a). Dissolve 50.0 mg of lithium clavulanate RS in a 0.4 per cent w/v solution of sodium acetate with the pH previously adjusted to 6.0 with glacial acetic acid and dilute to 50.0 ml with the same solution.
Reference solution (b). Dissolve 10 mg of amoxycillin trihydrate RS in 10 ml of reference solution (a). Chromatographic system a stainless steel column 03 cm 4.6 mm, packed with octadecylsilane bonded to porous silica (10 m), mobile phase: a mixture of 5 volumes of methanol and 95 volumes of a 1.5 per cent w/v solution of sodium dihydrogen phosphate with the pH previously adjusted to 4.0 with dilute phosphoric acid, flow rate. 1 ml per minute, spectrophotometer set at 230 nm, a 20 l loop injector. Inject reference solution (b). The resolution between the clavulanate peak and amoxycillin peak is not less than 3.5. Inject alternately the test solution and reference solution (a). Calculate the content of C8H8KNO5. 1 mg of clavulanate (C8H9NO5) is equivalent to 1.191 mg of C8H8KNO5. Storage. Store protected from moisture. Labelling. The label states the percentage contents of potassium clavulanate and the diluent used to prepare the mixture.
Potassium Iodide
KI Mol. Wt. 166.0 Potassium Iodide contains not less than 99.0 per cent and not more than 100.5 per cent of KI, calculated on the dried basis. Description. Colourless crystals or a white powder; odourless.
Identification
Dissolve 10 g in 100 ml of carbon dioxide-free water (solution A). The solution gives the reactions of potassium salts, and of iodides (2.3.1).
Tests
Appearance of solution. Solution A is clear (2.4.1), and colourless (2.4.1). Alkalinity. To 10 ml of solution A add 0.2 ml of 0.01 M sulphuric acid; no colour is produced on addition of a drop of phenolphthalein solution. Arsenic (2.3.10). Dissolve 5.0 g in 50 ml of water and 12 ml of stannated hydrochloric acid AsT. The resulting solution complies with the limit test for arsenic (2 ppm).
961
POTASSIUM PERMANGANATE
IP 2007
Heavy metals (2.3.13). 2.0 g complies with the limit test for heavy metals, Method A (10 ppm). Iron (2.3.14). Solution A complies with the limit test for iron (20 ppm). Barium. Dissolve 0.5 g in 10 ml of water and add 1 ml of dilute sulphuric acid; no turbidity develops within one minute. Cyanide. Warm 5 ml of Solution A, add one drop of ferrous sulphate solution and 0.5 ml of sodium hydroxide solution and acidify with hydrochloric acid; no blue colour is produced. Iodate. Dissolve 0.5 g in 10 ml of carbon dioxide-free water and add 0.15 ml of dilute sulphuric acid and a drop of iodidefree starch solution; no blue colour is produced within 2 minutes. Sulphates (2.3.17). 1.0 g dissolved in 15 ml of water complies with the limit test for sulphates (150 ppm). Thiosulphate. Dissolve 1 g in 10 ml of water, add 0.1 ml of starch solution and 0.1 ml of 0.005 M iodine; a blue colour is produced. Loss on drying (2.4.19). Not more than 1.0 per cent, determined on 1.0 g of the powdered substance by drying in an oven at 105 for 3 hours. Assay. Weigh accurately about 0.35 g, dissolve in about 10 ml of water, add 35 ml of hydrochloric acid and 5 ml of chloroform. Titrate with 0.05 M potassium iodate until the purple colour of iodine disappears from the chloroform. Add the last portion of the iodate solution dropwise and agitate vigorously and continuously. Allow to stand for 5 minutes. If any colour develops in the chloroform layer continue the titration until the chloroform is decolorised. 1 ml of 0.05 M potassium iodate is equivalent to 0.0166 g of KI. Storage. Store protected from light and moisture.
B. Heated to redness, it decrepitates, evolves oxygen and leaves a black residue which with water forms potassium hydroxide solution; the resulting solution when neutralised with dilute hydrochloric acid gives the reactions of potassium salts (2.3.1).
Tests
Appearance of solution. Dissolve 1.5 g in 50 ml of distilled water, heat on a water-bath and add gradually 6 ml of ethanol (95 per cent), cool, dilute to 60 ml with distilled water and filter. The filtrate (solution A) is colourless (2.4.1). Chlorides (2.3.12). 40 ml of solution A complies with the limit test for chlorides (250 ppm). Sulphates (2.3.17). 10 ml of solution A complies with the limit test for sulphates (600 ppm). Water-insoluble matter. Dissolve 0.5 g in 50 ml of water, heat to boiling, filter through a tared, sintered-glass filter (porosity No. 4) and wash with water until the filtrate is colourless. The weight of the residue, dried at 105 to constant weight, is not more than 5 mg (1.0 per cent). Assay. Weigh accurately about 0.3 g, dissolve in sufficient water to produce 100.0 ml. To 20.0 ml add 20 ml of water, 1 g of potassium iodide and 10 ml of 2 M hydrochloric acid and titrate the liberated iodine with 0.1 M sodium thiosulphate using starch solution, added towards the end of the titration, as indicator. 1 ml of 0.1 M sodium thiosulphate is equivalent to 0.003160 g of KMnO4. Storage. Store protected from moisture. CAUTION Great care should be taken in handling potassium permanganate as dangerous explosions are liable to occur if it is brought into contact with organic or other readily oxidisable substances, either in solution or in the dry condition.
Potassium Permanganate
KMnO4 Mol. Wt. 158.0 Potassium Permanganate contains not less than 99.0 per cent and not more than 100.5 per cent of KMnO4. Description. A dark purple or brownish black, granular powder or dark purple or almost black slender, prismatic crystals, having a metallic lustre; odourless. It decomposes on contact with certain organic substances.
Povidone
Polyvinylpyrrolidone; Polyvidone
CH CH2 N O n
(C6H9NO)n Mol. Wt. (111.2)n
Identification
A. A solution in water acidified with sulphuric acid and heated to 70 is decolorised by hydrogen peroxide solution (20 vol).
962
IP 2007
POTASSIUM PERMANGANATE
Povidone is poly(2-oxopyrrolidin-1-ylethylene) and consists of linear polymers of 1-vinylpyrrolidin-2-one. The different types of Povidone are characterised by their viscosity in solution, expressed as K-value, in the range 10 to 95. Povidone with a nominal K-value of 15 or less has a K-value of not less than 85.0 per cent and not more than 115.0 per cent of the declared value. The K-value of povidone with a nominal K-value of more than 15, or a nominal K-value range with an average of more than 15, is not less than 90.0 per cent and not more than 107.0 per cent of the declared value or of the average of the declared range. It contains not less than 11.5 per cent and not more than 12.8 per cent of nitrogen, N, calculated on the anhydrous basis. Description. A white or yellowish white powder or flakes; odourless or almost odourless; hygroscopic.
not more intense than that obtained by treating in the same manner a mixture of 2 ml of the test solution obtained above and 10 ml of the 20 ml of solution obtained by repeating the procedure using 2 ml of lead standard solution (10 ppm Pb) in place of the substance under examination, adding 0.5 g of magnesium oxide in a silica crucible and continuing as described above beginning at the words Ignite to dull red heat.... (10 ppm). Aldehydes. Boil 20.0 g in 180 ml of a 25 per cent v/v solution of sulphuric acid in a ground-glass-stoppered flask under a reflux condenser for 45 minutes and allow to cool. Fit a distillation head, distil and collect 60 ml of the distillate in 20 ml of a 7.0 per cent w/v solution of hydroxylamine hydrochloride, previously adjusted to pH 3.1 using 1 M sodium hydroxide, and cooled in ice. Titrate with 0.1 M sodium hydroxide to pH 3.1. Carry out a blank titration. Not more than 9.1 ml of 0.1 M sodium hydroxide is required (0.2 per cent, calculated as acetaldehyde, C2H4O). Vinylpyrrolidone. Dissolve 10.0 g in 80 ml of water and add 1 g of sodium acetate. Titrate with 0.05 M iodine until a persistent colour is obtained and add a further 3.0 ml of the iodine solution. Allow to stand for 10 minutes and titrate the excess of iodine with 0.1 M sodium thiosulphate using 3 ml of starch solution, added towards the end of the titration, as indicator. Repeat the operation without the substance under examination using the same total volume of 0.05M iodine. The difference between the titrations represents the amount of iodine consumed by the vinylpyrrolidone monomer that may be present. Not more than 3.6 ml of 0.05 M iodine is required (0.2 per cent). Sulphated ash (2.3.18). Not more than 0.1 per cent. Water (2.3.43). Not more than 5.0 per cent determined on 0.5 g. K-value. For Povidone with a stated K-value of 18 or less, prepare a 5.0 per cent w/v solution. For Povidone with a declared K-value of more than 18, prepare a 1.0 per cent w/v solution. Allow the solution to stand for 1 hour and carry out Method B for the determination of viscosity (2.4.28), at 25 0.2 using a size no. 1 viscometer with a minimum flow time of 100 seconds. Calculate the K-value (Ko) from the expression
= 1.5 log z 1 0.15 + 0.003 c + 300 c log z + (c + 1.5 c log z) 2 0.15 c + 0.003 c 2
Identification
Test A may be omitted if tests B, C and D are carried out. Tests B and C may be omitted if tests A and D are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with povidone RS. B. Add 2.5 g in small portions to a suitable volume of carbon dioxide-free water, stirring with a magnetic stirrer, and dilute to 25 ml with the same solvent (solution A). To 0.4 ml of solution A add 10 ml of water, 5 ml of 2 M hydrochloric acid and 2 ml of potassium dichromate solution; an orange-yellow precipitate is produced. C. To 1 ml of solution A add 0.2 ml of dimethylaminobenzaldehyde reagent and 0.1 ml of sulphuric acid; a pink colour is produced. D. To 0.1 ml of solution A add 5 ml of water and 0.2 ml of 0.05 M iodine; a red colour is produced.
Tests
Appearance of solution. Solution A is clear (2.4.1), and not more intensely coloured than reference solution BS6 or BYS6 (2.4.1). Heavy metals. Mix 2.0 g with 0.5 g of magnesium oxide in a silica crucible. Ignite to dull red heat until a homogeneous white or greyish white mass is produced. Heat at 800 for about 1 hour, dissolve the residue using two quantities, each of 5 ml, of 5 M hydrochloric acid, add 0.1 ml of phenolphthalein solution and strong ammonia solution until a pink colour is produced. Cool, add glacial acetic acid until the solution is decolorised and add a further 0.5 ml. Filter if necessary and dilute the solution to 20 ml with water. To 12 ml of the resulting solution add 2 ml of acetate buffer pH 3.5, mix, add 1.2 ml of thioacetamide reagent, mix immediately and allow to stand for 2 minutes. Any brown colour produced is
where c is the percentage concentration w/v of the substance under examination, calculated on the anhydrous basis, and z is the viscosity of the solution relative to that of water. Nitrogen (2.3.30). Follow Method C, using 0.3 g, accurately weighed and 11 ml of nitrogen-free sulphuric acid. For complete destruction of organic matter repeat the addition of hydrogen peroxide (10 vol) (usually 3 to 6 times) until a clear, light-green solution is obtained, then heat for a further 4 hours.
963
POVIDONE-IODINE
IP 2007
Storage. Store protected from moisture. Labelling. The label states the viscosity in terms of a K-value or K-range.
on the dried basis, subtract the percentage of available iodine determined in the Assay and obtain the percentage of iodide. Sulphated ash (2.3.18). Not more than 0.2 per cent. Loss on drying (2.4.19). Not more than 8.0 per cent, determined on 0.5 g by drying in an oven at 105.
Povidone-Iodine
CH CH2 N O n
,xI
Assay. Weigh accurately about 3 g, transfer to a beaker and add 200 ml of water. Cover the beaker and stir with a mechanical stirrer at room temperature for not more than 1 hour to dissolve as completely as possible. Titrate immediately thereafter with 0.1 M sodium thiosulphate using 3 ml of starch solution, added towards the end of the titration, as indicator. 1 ml of 0.1 M sodium thiosulphate is equivalent to 0.01269 g of I. Storage. Store protected from light.
Povidone-Iodine is a complex produced by interaction between iodine and poly(2-oxopyrrolidin-1-ylethylene). Povidone-Iodine contains not less than 9.0 per cent and not more than 12.0 per cent of available iodine, I, calculated on the dried basis. Description. A yellowish brown amorphous powder; odour, slight and characteristic of iodine.
Povidone-Iodine Solution
Povidone-Iodine Solution is a solution of Povidone-Iodine in Purified Water. It may contain a small amount of Ethanol. Povidone-Iodine Solution contains not less than 85.0 per cent and not more than 120.0 per cent of the stated amount of iodine, I. Description. A deep brown liquid; odour, characteristic of iodine.
Identification
A. Add 0.05 ml of a 10 per cent w/v solution to a mixture of 1 ml of starch solution and 9 ml of water; a deep blue colour is produced. B. Spread 1 ml of a 10 per cent w/v solution over an area of about 20 cm x 20 cm on a glass plate and allow it to dry in air at room temperature and in an atmosphere of low humidity overnight; a brown, dry, non-smearing film is formed which dissolves readily in water.
Identification
A. Dilute 1 ml to 20 ml with water and add 1 ml of the resulting solution to a mixture of 1 ml of starch solution and 9 ml of water; a deep blue colour is produced. B. Transfer 10 ml to a small flask and cover the mouth of the flask with a filter paper soaked in starch solution; no blue colour is produced on the paper within 60 seconds. C. Dilute 20 ml to 100 ml with water. To 10 ml add dropwise 0.1 M sodium thiosulphate until the colour is just discharged. To 5 ml of the resulting solution add 5 ml of 2 M hydrochloric acid and 2 ml of potassium dichromate solution; a red precipitate is produced.
Tests
Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Nitrogen (2.3.30). 9.5 to 11.5 per cent, calculated on the dried basis, determined on about 0.3 g, by Method A. Iodide. Not more than 6.6 per cent, calculated on the dried basis, determined by the following method. Weigh accurately about 0.5 g and dissolve in 100 ml of water. Add sufficient sodium bisulphite solution until the colour of iodine is discharged. Add 25.0 ml of 0.1 M silver nitrate and 10 ml of nitric acid and mix. Titrate with 0.1 M ammonium thiocyanate, using ferric ammonium sulphate solution as indicator. Repeat the procedure without the substance under examination. The difference between the titrations represents the amount of silver nitrate required. 1 ml of 0.1 M silver nitrate is equivalent to 0.01269 g of I. From the percentage of total iodine, calculated
Tests
pH (2.4.24). 3.0 to 6.5. Ethanol (if present) (2.3.45). 90.0 to 110.0 per cent of the stated amount of C2H5OH, determined by Method A after decolorising the solution with just sufficient quantity of a 10 per cent w/v solution of sodium thiosulphate and treating with a few drops of dilute sodium hydroxide solution. Assay. To an accurately measured volume containing about 50 mg of iodine add sufficient water to produce not less than
964
IP 2007
PRALIDOXIME CHLORIDE INJECTION
30 ml and titrate immediately with 0.02 M sodium thiosulphate, using 3 ml of starch solution, added towards the end of the titration, as indicator. Carry out a blank titration. 1 ml of 0.02 M sodium thiosulphate is equivalent to 0.002538 g of I. Storage. Store protected from light. Labelling. The label states the quantities of iodine and ethanol (if present) as percentages w/v.
nonaethoxyethanol and titrate with 0.1 M silver nitrate, determining the end-point potentiometrically (2.4.25). 1 ml of 0.1 M silver nitrate is equivalent to 0.003545 g of Cl. Sulphated ash (2.3.18). Not more than 0.5 per cent. Loss on drying (2.4.19). Not more than 2.0 per cent, determined on 1.0 g by drying in an oven at 105 for 3 hours. Assay. Weigh accurately about 0.5 g, dissolve in sufficient water to produce 250.0 ml and mix. Dilute 5.0 ml of this solution to 100.0 ml with water. Transfer 5.0 ml to a 50-ml volumetric flask, dilute to about 40 ml with water, add 5.0 ml of 1 M sodium hydroxide and dilute to volume with water. Within 10 minutes of the addition of the alkali, measure the absorbance of the solution at the maximum at about 332 nm (2.4.7), using a solution of 5.0 ml of 1 M sodium hydroxide diluted with water to 50.0 ml as the blank. Calculate the content of C7H9ClN2O from the absorbance obtained by carrying out the assay simultaneously using about 0.5 g, accurately weighed, of pralidoxime chloride RS. Storage. Store protected from moisture.
Pralidoxime Chloride
CH3 N
NOH
Cl
Mol. Wt. 172.6
C7H9ClN2O
Pralidoxime Chloride is 2-hydroxyiminomethyl-1methylpyridinium chloride. Pralidoxime Chloride contains not less than 97.0 per cent and not more than 103.0 per cent of C7H9ClN2O, calculated on the dried basis. Description. A white to pale yellow, crystalline powder; odourless.
Pralidoxime Chloride Injection

Pralidoxime Chloride Injection is a sterile material consisting of Pralidoxime Chloride with or without buffering agents and other excipients. It is filled in a sealed container. The injection is constituted by dissolving the contents of the sealed container in the requisite amount of sterile Water for Injections, immediately before use. The constituted solution complies with the requirements for Clarity of solution and Particulate matter stated under Parenteral Preparations (Injections). Storage. The constituted solution should be used immediately after preparation but, in any case, within the period recommended by the manufacturer. Pralidoxime Chloride Injection contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of pralidoxime chloride, C7H9ClN2O. Description. A white to pale yellow powder. The contents of the sealed container comply with the requirements stated under Parenteral Preparations (Powders for Injection) and with the following requirements.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with pralidoxime chloride RS or with the reference spectrum of pralidoxime chloride. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.0005 per cent w/v solution in 0.1 M hydrochloric acid shows absorption maxima at about 242 nm and 292 nm and in 0.1 M sodium hydroxide, a maximum at about 332 nm. C. To 0.1 ml of a 20 per cent w/v solution add 1 ml of a 0.6 per cent w/v solution of ferric chloride; an amber-brown colour is produced. D. Gives the reactions of chlorides (2.3.1).
Tests
Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method A (20 ppm). Chloride content. 20.2 to 20.8 per cent, calculated on the dried basis, determined by the following method. Weigh accurately about 0.3 g, dissolve in about 150 ml of water, add 20 ml of glacial acetic acid and 10 drops of (4-tert-octylphenoxy)
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with pralidoxime chloride RS or with the reference spectrum of pralidoxime chloride.
965
PRAZOSIN HYDROCHLORIDE
IP 2007
B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.0005 per cent w/v solution in 0.1 M hydrochloric acid shows absorption maxima at about 242 nm and 292 nm and in 0.1 M sodium hydroxide, a maximum at about 332 nm. C. To 0.1 ml of a 20 per cent w/v solution add 1 ml of a 0.6 per cent w/v solution of ferric chloride; an amber-brown colour is produced. D. Gives the reactions of chlorides (2.3.1).
Description. A white or almost white powder; odourless or almost odourless.
Identification
Test A may be omitted if tests B, C and D are carried out. Tests B and C may be omitted if tests A and D are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with prazosin hydrochloride RS. B. Prepare a 0.05 per cent w/v solution of the substance under examination in a 0.1 per cent v/v solution of hydrochloric acid in methanol. Dilute separately 1 ml and 5 ml of this solution to 100 ml with the same acid solution (solutions A and B respectively). When examined in the range 220 nm to 280 nm (2.4.7), solution A shows an absorption maximum at about 247 nm; absorbance at the maximum, 0.66 to 0.70. When examined in the range 280 nm to 400 nm, solution B exhibits two maxima at about 330 nm and 343 nm; absorbances at the two maxima, 0.65 to 0.70 and 0.60 to 0.66 respectively. C. In the test for Related substances, the principal spot in the chromatogram obtained with test solution (a) corresponds to that in the chromatogram obtained with reference solution (b). D. A 0.1 per cent w/v solution gives the reactions of chlorides (2.3.1).
Tests
pH (2.4.24). 3.5 to 4.5, determined in a 5.0 per cent w/v solution. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method A (20 ppm). Bacterial endotoxins (2.2.3). Not more than 0.1 Endotoxin Unit per mg of pralidoxime chloride. Assay. Weigh accurately about 0.5 g, dissolve in sufficient water to produce 250.0 ml and mix. Dilute 5.0 ml of this solution to 100.0 ml with water. Transfer 5.0 ml to a 50-ml volumetric flask, dilute to about 40 ml with water, add 5.0 ml of 1 M sodium hydroxide and dilute to volume with water. Within 10 minutes of the addition of the alkali, measure the absorbance of the solution at the maximum at about 332 nm (2.4.7), using a solution of 5.0 ml of 1 M sodium hydroxide diluted with water to 50.0 ml as the blank. Calculate the content of C7H9ClN2O from the absorbance obtained by carrying out the assay simultaneously using about 0.5 g, accurately weighed, of pralidoxime chloride RS. Labelling. The label states the period within which the constituted injection should be used.
Tests
Iron. To 1.0 g add dropwise about 1.5 ml of nitric acid, heat cautiously on a water-bath until fumes are no longer evolved. Ignite by slowly raising the temperature from 150 to 1000, maintaining the final temperature for 1 hour. Cool, dissolve the residue in 20 ml of 2 M hydrochloric acid, evaporate to about 5 ml, dilute to 25 ml with 2 M hydrochloric acid and examine the resulting solution by atomic absorption spectrophotometry (2.4.2), measuring at 248 nm using an iron hollow-cathode light as a source of radiation, an air-acetylene flame and iron standard solution (8 ppm Fe), diluted as necessary with water to prepare the standard solutions (100 ppm). Reserve about 10 ml of the solution for the Nickel test. Nickel. Examine the final solution prepared in the test for Iron by atomic absorption spectrophotometry (2.4.2), measuring at 232 nm using a nickel hollow-cathode light as a source of radiation, an air-acetylene flame and using nickel standard solution (10 ppm Ni), diluted as necessary with water to prepare the standard solutions (50 ppm). Related substances. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 50.0 mg of the substance under examination in the mobile phase and dilute to 50 ml with the mobile phase.
Prazosin Hydrochloride
O N H3CO H3CO N N NH2
C19H21N5O4, HCl Mol. Wt. 419.9
O , HCl
Prazosin Hydrochloride is 2-[4-(2-furoyl)piperazin-1-yl]-6,7dimethoxyquinazolin-4-ylamine hydrochloride. Prazosin Hydrochloride contains not less than 98.5 per cent and not more than 101.0 per cent of C19H21N5O4, HCl, calculated on the anhydrous basis.
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IP 2007
PRAZOSIN TABLETS
Reference solution (a). Dilute 1 ml of the test solution to 100 ml with the mobile phase. Further dilute 1 ml of the solution to 10 ml with the mobile phase. Reference solution (b). Dissolve 8 mg of metoclopramide hydrochloride RS in 1 ml of the test solution and dilute to 25 ml with the mobile phase. Further dilute 1 ml of the solution to 10 ml with the mobile phase. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilyl silica gel (5 m), mobile phase: a mixture of 50 volumes of methanol and 50 volumes of a solution containing 3.484 g per litre of sodium pentanesulphonate and 3.64 g per litre of tetramethylammonium hydroxide adjusted to pH 5.0 with glacial acetic acid, flow rate.1 ml per minute, spectrophotometer set at 254 nm, a 20 l loop injector. Inject reference solution (b). The retention times are: prazocine about 9 minutes and metoclopramide about 5 minutes.The test is not valid unless the resolution between the peaks corresponding to prazocine and metoclopramide is at least 8. Inject the test solution and reference solution (a). Continue the chromatography of the test solution for 6 times the retention time of the principal peak. Any secondary peak obtained with the test solution is not more than twice the area of the principal peak in the chromatogram obtained with reference solution (a) (0.2 per cent).The sum of all the impurities is not more than five times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent). Ignore any peak with an area 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.05 per cent). Heavy metals (2.3.13). Determine on the residue obtained in the test for Sulphated ash. Dissolve in sufficient 2 M nitric acid to produce 50 ml. The resulting solution complies with the limit test for heavy metals, Method B (50 ppm). Sulphated ash (2.3.18). Not more than 0.2 per cent. Water (2.3.43). Not more than 0.5 per cent w/w, using 2.0 g dissolved in a mixture of equal volumes of dichloromethane and methanol. Assay. Weigh accurately about 0.35 g, dissolve in 20 ml of anhydrous formic acid and 30 ml of acetic anhydride. Titrate with 0.1 M perchloric acid, determining the end point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.04199 g of C19H21N504, HCl. Storage. Store protected from light.
Prazosin Tablets
Prazosin Hydrochloride Tablets
Prazosin Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of prazosin, C19H21N5O4. The tablets may contain permitted colours.
Identification
Shake a quantity of the powdered tablets containing about 10 mg of prazosin with a mixture of 10 ml of dichloromethane and 10 ml of 0.05 M potassium hydroxide, filter the organic layer through cotton, evaporate to dryness and dry the residue at 60 at a pressure not exceeding 2 kPa at 60 for 2 hours. On the residue determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with prazosin RS or with the reference spectrum of prazosin.
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel HF254. Mobile phase. A mixture of 95 volumes of ethyl acetate and 5 volumes of diethylamine. Test solution. Shake a quantity of the powdered tablets containing 5 mg of prazosin with 2 ml of a mixture of 10 volumes of dichloromethane, 10 volumes of methanol and 1 volume of diethylamine, centrifuge and pass the supernatant liquid through a 0.5 m PTFE (Polytetrafluoroethylene) filter. Reference solution (a). Dilute 1 volume of test solution to 200 volumes with the same solvent mixture Reference solution (b). Dilute 2 volumes of reference solution (a) to 5 volumes with the same solvent mixture. Apply to the plate 20 l of each solution. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (a) and not more than two such spots are more intense than the spot in the chromatogram obtained with reference solution (b). Uniformity of content. Comply with the tests stated under Tablets. Determine by liquid chromatography (2.4.14). Test solution. Shake one tablet for 1 hour in a suitable volume of a mixture of 96 volumes of methanol, 2 volumes of glacial acetic acid and 2 volumes of water to produce a solution containing 0.002 per cent w/v solution of prazosin, centrifuge and use the supernatant liquid. Reference solution. A 0.0022 per cent w/v solution of prazosin hydrochloride RS in the same solvent mixture.
967
PREDNISOLONE
IP 2007
Chromatographic system a stainless steel column 20 cm x 4 mm, packed with porous silica particles, (5 m), mobile phase: 0.01 per cent w/v solution of diethylamine in a mixture of 96 volumes of methanol, 2 volumes of glacial acetic acid and 2 volumes of water, flow rate. 1 ml per minute, spectrophotometer set at 254 nm, a 20 l loop injector. Calculate the content of C19H21N5O4 in the tablet. Other tests. Comply with the tests stated under Tablets. Assay. Determine by liquid chromatography (2.4.14). Test solution. Weigh and powder 20 tablets. Shake a quantity of the powdered tablets containing about 2 mg of prazosin with 100 ml in a mixture of 96 volumes of methanol, 2 volumes of glacial acetic acid and 2 volumes of water for 30 minutes, centrifuge and use the supernatant liquid. Reference solution. A 0.0022 per cent w/v solution of prazosin hydrochloride RS in a mixture of 96 volumes of methanol, 2 volumes of glacial acetic acid and 2 volumes of water. The chromatographic conditions as described under Uniformity of content may be used. Calculate the content of C19H21N5O4 in the tablets. Storage. Store protected from light. Labelling. The label states the strength in terms of the equivalent amount of prazosin.
Identification
Test A may be omitted if tests B and C are carried out. Test C may be omitted if tests A and B are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with prednisolone RS or with the reference spectrum of prednisolone. B. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Solvent mixture. A mixture of 90 volumes of acetone and 10 volumes of formamide. Mobile phase. Chloroform. Test solution. Dissolve 25 mg of the substance under examination in 10 ml of the solvent mixture. Reference solution (a). Dissolve 25 mg of prednisolone RS in 10 ml of the solvent mixture. Reference solution (b). Mix equal volumes of the test solution and reference solution (a). Place the dry plate in a tank containing a shallow layer of the solvent mixture, allow the solvent mixture to ascend to the top, remove the plate from the tank and allow the solvent to evaporate. Use within 2 hours, with the flow of the mobile phase in the direction in which the aforementioned treatment was done. Apply to the plate 2 l of each solution. Allow the mobile phase to rise 12 cm. Dry the plate in a current of warm air, allow the solvent to evaporate, heat at 120 for 15 minutes and spray the hot plate with ethanolic sulphuric acid (20 per cent v/v). Heat at 120 for a further 10 minutes, allow to cool and examine in daylight and in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with reference solution (a). The principal spot in the chromatogram obtained with reference solution (b) appears as a single, compact spot. A. C. Dissolve 2 mg in 2 ml of sulphuric acid by shaking and allow to stand for 5 minutes; an intense red colour is produced with a reddish brown fluorescence when examined in ultraviolet light at 365 nm. Pour the solution into 10 ml of water and mix; the colour fades and there is a yellow fluorescence under ultra-violet light (365 nm).
Prednisolone
O H3C H H O
C21H28O5 Mol. Wt. 360.5
HO H3C
OH OH
Prednisolone is 11,17,21-trihydroxypregna-1,4-diene-3,20dione. Prednisolone contains not less than 96.0 per cent and not more than 104.0 per cent of C21H28O5, calculated on the dried basis. Description. A white or almost white, crystalline powder; hygroscopic.
Tests
Specific optical rotation (2.4.22). +96.0 to +102, determined in a 1.0 per cent w/v solution in dioxan. Light absorption (2.4.7). Absorbance of a 0.001 per cent w/v solution in ethanol (95 per cent) at the maximum at about 240 nm, 0.40 to 0.43.
968
IP 2007
PREDNISOLONE TABLETS
Related substances. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 25 mg of the substance under examination in 2 ml of tetrahydrofuran and dilute to 10ml with water. Reference solution (a). Dissolve 2 mg of prednisolone RS and 2 mg of hydrocortisone RS in the mobile phase and dilute to 100 ml with the mobile phase. Reference solution (b). Dilute 1 ml of the test solution to 100 ml with the mobile phase. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with base deactivated end-capped octadecylsilyl silica gel (5 m), column temperature. 45, mobile phase: a mixture of 220 ml of tetrahydrofuran and 700 ml of water, allowed to equilibrate, diluted to 1000 ml with water and mixed again, flow rate. 1 ml per minute, spectrophotometer set at 254 nm, a 20 l loop injector. Equilibrate the column with the mobile phase for about 30 minutes. Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with reference solution (b) is not less than 50 per cent of the full scale of the recorder. Inject reference solution (a). The retention times are: prednisolone, about 14 minutes and hydrocortisone about 15.5 minutes. The test is not valid unless the resolution between the peaks corresponding to prednisolone and hydrocortisone is at least 2.2. If necessary, adjust the concentration of tetrahydrofuran in the mobile phase. Inject separately the solvent mixture of the test solution as a blank, the test solution and reference solution (b). Continue the chromatography of the test solution for 4.5 times the retention time of the principal peak. In the chromatogram obtained with the test solution, the area of any peak other than the principal peak, is not greater than half the area of the principal peak in the chromatogram obtained with reference solution (b) (1 per cent) and not more than one such peak has an area greater than half the area of the principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent); the sum of the areas of all the peaks other than the principal peak is not greater than 2.0 times the area of the principal peak in the chromatogram obtained with reference solution (b) (2 per cent). Ignore any peak obtained with the blank run and any peak with an area less than 0.05 times that of the principal peak in the chromatogram obtained with reference solution (b).
Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 1.0 per cent, determined on 1.0 g by drying in an oven at 105 for 3 hours. Assay. Weigh accurately about 0.1 g and dissolve in sufficient ethanol to produce 100.0 ml. Dilute 2.0 ml of this solution to 100.0 ml with ethanol. Measure the absorbance of the resulting solution at the maximum at about 243.5 nm. Calculate the content of C21H28O5 taking 415 as the specific absorbance at 243.5 nm. Storage. Store protected from light and moisture.
Prednisolone Tablets
Prednisolone Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of prednisolone, C21H28O5.
Identification
Extract a quantity of the powdered tablets containing 30 mg of Prednisolone with 10 ml of chloroform, filter and evaporate the filtrate to dryness. The residue complies with the following tests. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with prednisolone RS or with the reference spectrum of prednisolone. B. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Solvent mixture. A mixture of 90 volumes of acetone and 10 volumes of formamide. Mobile phase. Chloroform. Test solution. Dissolve 25 mg of the residue in 10 ml of the solvent mixture. Reference solution (a). Dissolve 25 mg of prednisolone RS in 10 ml of the solvent mixture. Reference solution (b). Mix equal volumes of the test solution and reference solution (a). Place the dry plate in a tank containing a shallow layer of the solvent mixture, allow the solvent mixture to ascend to the top, remove the plate from the tank and allow the solvent to evaporate. Use within 2 hours, with the flow of the mobile phase in the direction in which the aforementioned treatment was done. Apply to the plate 2 l of each solution. Allow the mobile phase to rise 12 cm. Dry the plate in a current of warm air, allow the solvent to evaporate, heat at 120 for 15 minutes and spray the hot plate with ethanolic sulphuric acid (20 per cent v/v).
969
PREDNISOLONE TABLETS
IP 2007
Heat at 120 for a further 10 minutes, allow to cool and examine in daylight and in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with reference solution (a). The principal spot in the chromatogram obtained with reference solution (b) appears as a single, compact spot.
Tests
Related substances. Determine by liquid chromatography (2.4.14). Test solution. Shake a quantity of the powdered tablets containing 10 mg of Prednisolone with 25 ml of methanol for 10 minutes and mix with the aid of ultrasound for 2 minutes; filter the extract (Whatman GF/F is suitable), wash the filter with two 10-ml quantities of methanol, combine the filtrate and washings and evaporate to dryness using a rotary evaporator and a warm water-bath, dissolve the residue in 10 ml of tetrahydrofuran and dilute to 20 ml with water. Reference solution (a). Dissolve 2 mg of prednisolone RS and 2 mg of hydrocortisone RS in the mobile phase and dilute to 100 ml with the mobile phase. Reference solution (b). Dilute 1ml of the test solution to 100 ml with a 50 per cent v/v solution of tetrahydrofuran. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilyl silica gel (5 m), column temperature. 45, mobile phase: a mixture of 220 ml of tetrahydrofuran and 700 ml of water, allowed to equilibrate, diluted to 1000 ml with water and mixed again, flow rate. 1 ml per minute, spectrophotometer set at 254 nm, a 20 l loop injector. Equilibrate the column with the mobile phase for about 30 minutes. Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with reference solution (b) is not less than 50 per cent of the full scale of the recorder. Inject reference solution (a). The retention times are: prednisolone, about 14 minutes and hydrocortisone about 15.5 minutes. The test is not valid unless the resolution between the peaks corresponding to prednisolone and hydrocortisone is at least 2.2. If necessary, adjust the concentration of tetrahydrofuran in the mobile phase. Inject separately the solvent mixture of the test solution as a blank, the test solution and reference solution (b). Continue the chromatography of the test solution for 4.5 times the
retention time of the principal peak. In the chromatogram obtained with the test solution, the area of any peak other than the principal peak, is not greater than half the area of the principal peak in the chromatogram obtained with reference solution (b) (1 per cent) and the sum of the areas of all the peaks other than the principal peak is not greater than three times the area of the principal peak in the chromatogram obtained with reference solution (b) (3 per cent). Ignore any peak obtained with the blank run and any peak with an area less than 0.05 times that of the principal peak in the chromatogram obtained with reference solution (b) (0.05 per cent) and any peak with a retention time of 3 minutes or less. Uniformity of content. (For tablets containing 10 mg or less) Comply with the test stated under Tablets. Powder one tablet, add 50 ml of ethanol (95 per cent), shake for 30 minutes, add sufficient ethanol (95 per cent) to produce 100.0 ml. Centrifuge and pipette a suitable volume of the supernatant liquid containing 0.5 mg of Prednisolone and dilute to 50.0 ml with ethanol (95 per cent). Measure the absorbance of the resulting solution at the maximum at about 240 nm (2.4.7). Calculate the content of C21H28O5 taking 415 specific absorbance at 240 nm. Dissolution (2.5.2). Apparatus. No 1 Medium. 900 ml of water Speed and time. 50 rpm and 30 minutes. Withdraw a suitable volume of the medium, filter and dilute a suitable volume of the filtrate with the same solvent. Measure the absorbance of the filtrate at the maximum at about 240 nm (2.4.7). Calculate the content of C21H28O5 in the medium from the absorbance obtained from a solution of known concentration of prednisolone RS. D. Not less than 70 per cent of the stated amount of C21H28O5. Other tests. Comply with the tests stated under Tablets. Assay. Determine by liquid chromatography (2.4.14). Test solution. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 5 mg of Prednisolone, add 58 ml of methanol, shake for 10 minutes and add sufficient water to produce 100.0 ml. Mix well and filter. Reference solution (a). A solution containing 0.005 per cent w/v of prednisolone RS and 0.0075 per cent w/v of dexamethasone (internal standard) in a mixture of 58 volumes of methanol and 42 volumes of water. Reference solution (b). Prepare in the same manner as the test solution but adding 10 ml of a 0.075 per cent w/v solution of dexamethasone in methanol and 48 ml of methanol in place of 58 ml of methanol.
970
IP 2007
PREDNISOLONE SODIUM PHOSPHATE
Chromatographic system a stainless steel column 20 cm x 4.6 mm, packed with octadecylsilyl silica gel (5 m), mobile phase: a mixture of 42 volumes of water and 58 volumes of methanol, flow rate. 1 ml per minute, spectrophotometer set at 254 nm, a 20 l loop injector. The assay is not valid unless the resolution factor between the peaks due to prednisolone and dexamethasone is greater than 2.5 and the column efficiency, determined using the peak due to prednisolone in the chromatogram obtained with the test solution is greater than 15,000 theoretical plates per metre. Calculate the content of C21H28O5 in the tablets. Storage. Store protected from light.
paper with dilute ammonia solution. The solution complies with reaction A of sodium salts and reaction B of phosphates (2.3.1).
Tests
pH (2.4.24). 7.5 to 10.5 determined in 1.0 per cent w/v solution. Specific optical rotation (2.4.22). +95.0 to +102.0, determined in a 1.0 per cent w/v solution in a mixture of 9 volumes of phosphate buffer pH 7.0 and 1 volume of carbon dioxide-free water. Related substances. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 62.5 mg of the substance under examination in the mobile phase and dilute to 25.0 ml with the mobile phase. Reference solution (a). Dissolve 25 mg of prednisolone sodium phosphate RS and 25 mg of prednisolone RS in the mobile phase and dilute to 25.0 ml with the same solvent. Dilute 1.0 ml of the solution to 25.0 ml with the mobile phase. Reference solution (b). Dilute 1.0 ml of the test solution to 50.0 ml with the mobile phase. Chromatographic system a stainless steel column 15 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica or ceramic microparticles (5 m), mobile phase: weigh 1.360 g of potassium dihydrogen phosphate and 0.6 g of hexylamine, mix and allow to stand for 10 minutes and then dissolve in 185 ml of water, add 65 ml of acetonitrile, mix and filter through a 0.45 m filter, flow rate. 1 ml per minute, spectrophotometer set at 254 nm, a 20 l loop injector. Inject reference solution (b). Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with reference solution (b) is 70 per cent to 90 per cent of the full scale of the recorder. Equilibrate the column with the mobile phase for about 30 minutes. Inject reference solution (a). The retention times are: prednisolone sodium phosphate, about 6.5 minutes and prednisolone, about 8.5 minutes. The test is not valid unless the resolution between the peaks due to prednisolone sodium phosphate and prednisolone is at least 4.5; if this resolution is not achieved, increase the concentration of acetonitrile or increase the concentration of water in the mobile phase. Inject separately the test solution and reference solution (b). Continue the chromatography for three times the retention
Prednisolone Sodium Phosphate

O HO H3C H O
C21H27Na2O8P Mol. Wt. 484.4
H3C H H
O O P ONa OH ONa
Predisolone Sodium Phosphate is 11,17, 21trihydroxypregna-1,4-diene-3,20-dione-21(dihydrogenphosphate)disodium salt. Prednisolone Sodium Phosphate contains not less than 96.0 per cent and not more than 102.0 per cent of C21H27Na2O8P, calculated on the dried basis.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with prednisolone sodium phosphate RS. If the spectra obtained in the solid state show differences, dissolve the substance under examination and the reference substance separately in the minimum volume of ethanol (95 per cent) evaporate to dryness on a water-bath and record the spectra again using the residues. B. To about 40 mg add 2 ml of sulphuric acid and heat gently until white fumes are evolved. Add nitric acid dropwise, continue the heating until the solution is almost colourless and cool. Add 2 ml of water, heat until white fumes are again evolved, cool, add 10 ml of water and neutralise to red litmus
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PREDNISOLONE SODIUM PHOSPHATE INJECTION
IP 2007
time of the principal peak. In the chromatogram obtained with the test solution, the area of any peak other than that from the principal peak is not greater than the area of the principal peak in the chromatogram obtained with reference solution (b) (2 per cent) and not more than one such peak has an area greater than half the area of the principal peak in the chromatogram obtained with reference solution (b) (1 per cent); the sum of the areas of all the peaks other than the principal peak is not greater than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (b) (3 per cent). Ignore any peak due to the solvent and any peak with an area less than 0.025 times the area of the principal peak in the chromatogram obtained with reference solution (b). Inorganic phosphate. Not more than 1.0 per cent of phosphate, PO4. Dissolve 50 mg in sufficient water to produce 100 ml. To 10 ml of the resulting solution add 5 ml of molybdovanadic reagent, mix and allow to stand for 5 minutes. Any yellow colour in the solution is not more intense than that produced in a standard prepared at the same time in the same manner using 10 ml of phosphate standard solution (5 ppm PO4). Water (2.3.43). Not more than 6.5 per cent, determined on 1.0 g. Assay. Weigh accurately about 0.1 g, dissolve in sufficient water to produce 100.0 ml and mix. Dilute 5.0 ml of the resulting solution to 250.0 ml with water. Measure the absorbance at the maximum at about 247 nm (2.4.7). Calculate the content of C21H27Na2O8P taking 312 as the specific absorbance at 247 nm. Storage. Store protected from light.
Tests
pH (2.4.24). 7.0 to 8.0. Bacterial endotoxins (2.2.3). Not more than 5.0 Endotoxin Units per mg of prednisolone phosphate. Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. Determine by liquid chromatography (2.4.14). Test solution. Dilute an accurately measured volume of the injection to obtain a solution containing 0.001 per cent w/v of prednisolone phosphate. Reference solution (a). Weigh accurately about 10 mg of prednisolone sodium phosphate RS, dissolve in sufficient water to produce 100.0 ml (solution A) and dilute 10.0 ml of the solution to 100.0 ml with water. Reference solution (b). Add 10 ml of a 0.01 per cent w/v solution of betamethasone sodium phosphate RS in water to 10 ml of solution A and dilute to 100 ml with water. Chromatographic system a stainless steel column 20 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica or ceramic microparticles (10 m) (such as Spherisorb ODS 1), mobile phase: a mixture of 45 volumes of methanol and 55 volumes of citro-phosphate buffer pH 5.0, flow rate. 2.0 ml per minute, spectrophotometer set at 247 nm, a 10 l loop injector. Inject reference solution (b). The test is not valid unless the resolution between the peaks due to betamethasone sodium phosphate and prednisolone sodium phosphate is at least 2.5. Inject alternatively the test solution and reference solution (a). Calculate the content of C21H29O8P in the injection. Storage. Store protected from light, in a single-dose or in multidose containers.
Prednisolone Sodium Phosphate Injection

Prednisolone Sodium Phosphate Injection is a sterile solution of Prednisolone Sodium Phosphate in Water for Injections. Prednisolone Sodium Phosphate Injection contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of prednisolone phosphate, C21H29O8P. Description. A clear, colourless liquid.
Prednisone
O H3C H H O
C21H26O5 Mol. Wt. 358.4 Prednisone is 17,21-dihydroxypregna-1,4-diene-3,11,20trione.
Identification
A. In the Assay, the chromatogram obtained with the test solution corresponds to the peak due to prednisolone sodium phosphate in the chromatogram obtained with reference solution (a). B. To a volume containing 0.2 mg of Prednisolone Sodium Phosphate slowly add 1 ml of sulphuric acid and allow to stand for 2 minutes. A deep red colour is produced.
O H3C
OH OH
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IP 2007
PREDNISOLONE SODIUM PHOSPHATE INJECTION
Prednisone contains not less than 96.0 per cent and not more than 104.0 per cent of C21H26O5, calculated on the dried basis. Description. A white or almost white, crystalline powder; odourless.
Tests
Specific optical rotation (2.4.22). +167 to +175, determined in a 1.0 per cent w/v solution in dioxan. Light absorption (2.4.7). Absorbance of a 0.001 per cent w/v solution in methanol at the maximum at about 240 nm, 0.40 to 0.43; the ratio of the absorbance at the maximum at about 240 nm to that at about 263 nm, 1.85 to 2.05. Related substances. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 25 mg of the substance under examination in methanol and dilute to 10 ml with the same solvent. Reference solution (a). Dissolve 2.0 mg of prednisolone RS and 2.0 mg of prednisone RS in methanol and dilute to 100 ml with the same solvent. Reference solution (b). Dilute 1 ml of the test solution to 100 ml with methanol. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilyl silica gel (5 m), column temperature. 45, mobile phase: A. a mixture of 100 ml of acetonitrile, 200 ml of methanol and 650 ml of water, allowed to equilibrate, diluted to 1000 ml with water and mixed again, B. acetonitrile, flow rate. 2.5 ml per minute, a linear gradient programme using the conditions given below, spectrophotometer set at 254 nm, a 20 l loop injector. Time Mobile Mobile Comment Phase A Phase B (min) (per cent v/v) (per cent v/v) 0 100 0 Isocratic 25 100 0 begin linear gradient 40 40 60 end chromatogram, change to 100B 41 0 100 being treatment with B 46 47 52=0 0 100 100 100 0 0 end treatment, return to 100A begin equilibration with A end equilibration, begin next chromatogram
Identification
Tests A and B may be omitted if tests C and D are carried out. Tests C and D may be omitted if tests A and B are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with prednisone RS or with the reference spectrum of prednisone. B. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Solvent mixture. A mixture of 90 volumes of acetone and 10 volumes of formamide. Mobile phase. Chloroform. Test solution. Dissolve 25 mg of the substance under examination in 10 ml of the solvent mixture. Reference solution (a). Dissolve 25 mg of prednisone RS in 10 ml of the solvent mixture. Reference solution (b). Mix equal volumes of the test solution and reference solution (a). Place the dry plate in a tank containing a shallow layer of the solvent mixture, allow the solvent mixture to ascend to the top, remove the plate from the tank and allow the solvent to evaporate. Use within 2 hours, with the flow of the mobile phase in the direction in which the aforementioned treatment was done. Apply to the plate 2 l of each solution. Allow the mobile phase to rise 12 cm. Dry the plate in a current of warm air, allow the solvent to evaporate, heat at 120 for 15 minutes and spray the hot plate with ethanolic sulphuric acid (20 per cent v/v). Heat at 120 for a further 10 minutes, allow to cool and examine in daylight and in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with reference solution (a). The principal spot in the chromatogram obtained with reference solution (b) appears as a single, compact spot. C. Dissolve 2 mg in 2 ml of sulphuric acid and allow to stand for 5 minutes; an orange colour is produced within 5 minutes, which exhibits a blue fluorescence in ultraviolet light at 365 nm. Pour the solution into 10 ml of water; the colour changes first to yellow and then fades gradually but the blue fluorescence in ultraviolet light remains. D. Dissolve 1 mg in 1 ml of ethanol (95 per cent), evaporate to dryness at a pressure not exceeding 0.7 kPa, add 5 ml of 1 M sodium hydroxide and heat at 70 for 30 minutes; not more than a slight yellow colour is produced (distinction from cortisone acetate).
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PREDNISONE TABLETS
IP 2007
Equilibrate the column with the mobile phase B for at least 30 minutes and then with mobile phase A for 5 minutes. For subsequent chromatograms, use the conditions described from 40.0 to 52.0 minutes. Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with reference solution (b) is not less than 50 per cent of the full scale of the recorder. Inject reference solution (a). The retention times are: prednisone, about 19 minutes and prednisolone about 23 minutes. The test is not valid unless the resolution between the peaks corresponding to prednisone and prednisolone is at least 2.7. If necessary, adjust the concentration of acetonitrile in mobile phase A. Inject separately methanol as a blank, the test solution and reference solution (b). In the chromatogram obtained with the test solution: the area of any peak other than the principal peak is not greater than 0.25 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.25 per cent); the sum of the areas of all the peaks, apart from the principal peak, is not greater than 0.75 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.75 per cent). Ignore any peak due to the blank run and any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (b). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 1.0 per cent, determined on 1.0 g by drying in an oven at 105 for 3 hours. Assay. Weigh accurately about 0.1 g and dissolve in sufficient ethanol to produce 100.0 ml. Dilute 2.0 ml of this solution to 100.0 ml with ethanol. Measure the absorbance of the resulting solution at the maximum at about 238 nm. Calculate the content of C21H26O5 taking 425 as the specific absorbance at 238 nm. Storage. Store protected from light.
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with prednisone RS or with the reference spectrum of prednisone. B. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Solvent mixture. A mixture of 90 volumes of acetone and 10 volumes of formamide. Mobile phase. Chloroform. Test solution. Dissolve 25 mg of the residue in 10 ml of the solvent mixture. Reference solution (a). Dissolve 25 mg of prednisone RS in 10 ml of the solvent mixture. Reference solution (b). Mix equal volumes of the test solution and reference solution (a). Place the dry plate in a tank containing a shallow layer of the solvent mixture, allow the solvent mixture to ascend to the top, remove the plate from the tank and allow the solvent to evaporate. Use within 2 hours, with the flow of the mobile phase in the direction in which the aforementioned treatment was done. Apply to the plate 2 l of each solution. Allow the mobile phase to rise 12 cm. Dry the plate in a current of warm air, allow the solvent to evaporate, heat at 120 for 15 minutes and spray the hot plate with ethanolic sulphuric acid (20 per cent v/v). Heat at 120 for a further 10 minutes, allow to cool and examine in daylight and in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with reference solution (a). The principal spot in the chromatogram obtained with reference solution (b) appears as a single, compact spot.
Tests
Related substances. Determine by liquid chromatography (2.4.14). Test solution. Shake a quantity of the powdered tablets containing 25 mg of Prednisone with 10 ml of methanol for 10 minutes, mix with the aid of ultrasound for 2 minutes and filter the extract (Whatman GF/F is suitable). Reference solution (a). Dissolve 2.0 mg of prednisolone RS and 2.0 mg of prednisone RS in methanol and dilute to 100 ml with the same solvent. Reference solution (b). Dilute 1 ml of the test solution to 100 ml with methanol. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilyl silica gel (5 m),
Prednisone Tablets
Prednisone Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of prednisone, C21H26O5.
Identification
Shake a quantity of the powdered tablets containing 30 mg of Prednisone with 10 ml of chloroform, filter and evaporate the filtrate to dryness. The residue complies with the following tests.
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IP 2007
PREDNISONE TABLETS
column temperature. 45, mobile phase: A. a mixture of 100 ml of acetonitrile, 200 ml of methanol and 650 ml of water, allowed to equilibrate, diluted to 1000 ml with water and mixed again, B. acetonitrile, flow rate. 2.5 ml per minute, a linear gradient programme using the conditions given below, spectrophotometer set at 254 nm, a 20 l loop injector. Time Mobile Mobile Comment Phase A Phase B (min) (per cent v/v) (per cent v/v) 0 100 0 Isocratic 25 100 0 begin linear gradient 40 40 60 end chromatogram, change to 100B 41 0 100 being treatment with B 46 0 100 end treatment, return to 100A 47 100 0 begin equilibration with A 52=0 100 0 end equilibration, begin next chromatogram Equilibrate the column with the mobile phase B for at least 30 minutes and then with mobile phase A for 5 minutes. For subsequent chromatograms use the conditions described from 40.0 to 52.0 minutes. Adjust the sensitivity of the system so that the height of the principal peak in the chromatogram obtained with reference solution (b) is not less than 50 per cent of the full scale of the recorder. Inject reference solution (a). The retention times are: prednisone, about 19 minutes and prednisolone about 23 minutes. The test is not valid unless the resolution between the peaks corresponding to prednisone and prednisolone is at least 2.7. If necessary, adjust the concentration of acetonitrile in mobile phase A. Inject separately methanol as a blank, the test solution and reference solution (b). In the chromatogram obtained with the test solution: the area of any peak other than the principal peak is not greater than 0.25 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.25 per cent); the sum of the areas of all the peaks, apart from the principal peak, is not greater than 0.75 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.75 per cent). Ignore any peak due to
the blank run and any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (b). Uniformity of content. Comply with the test stated under Tablets. Powder one tablet, add 50 ml of ethanol (95 per cent), shake for 30 minutes, add sufficient ethanol (95 per cent) to produce 100.0 ml. Centrifuge and pipette a suitable volume of the supernatant liquid equivalent to 0.5 mg of Prednisone and dilute to 50.0 ml with ethanol (95 per cent). Measure the absorbance of the resulting solution at the maximum at about 240 nm (2.4.7). Calculate the content of C21H26O5 taking 415 as specific absorbance at 240 nm. Dissolution (2.5.2). Apparatus. No 1 Medium. 900 ml of water Speed and time. 50 rpm and 30 minutes. Withdraw a suitable volume of the medium and filter. Measure the absorbance of the filtrate at the maximum at about 240 nm (2.4.7). Calculate the content of C21H26O5 in the medium from the absorbance obtained from a solution of known concentration of prednisone RS. D. Not less than 70 per cent of the stated amount of C21H26O5. Other tests. Comply with the tests stated under Tablets. Assay. Determine by liquid chromatography (2.4.14). Internal standard solution. Dissolve an accurately weighed quantity of acetanilide in a 50 per cent v/v solution of methanol to obtain a solution having a known concentration of 0.11 mg per ml. Test solution. Weigh and powder 20 tablets. To an accurately weighed quantity of the powdered tablets containing about 20 mg of Prednisone add 5 ml of water, mix with the aid of ultrasound for 1 minute, add 50 ml of methanol and mix with the aid of ultrasound for I minute. Dilute with water to 100.0 ml and mix. To 5.0 ml of this solution add 5.0 ml of internal standard solution and dilute to 50.0 ml with a 50 per cent v/v solution of methanol and mix. Filter through a 5 m filter and discard the first 20 ml of the filtrate. Reference solution. Weigh accurately a suitable quantity of prednisone RS and dissolve in a 50 per cent v/v solution of methanol to obtain a solution having a concentration of about 0.2 mg per ml. To 5.0 ml of this solution add 5.0 ml of internal standard solution and dilute to 50.0 ml with a 50 per cent v/v solution of methanol. Chromatographic system a stainless steel column 25 cm x 4 mm, packed with octadecylsilane chemically bonded to porous silica particles (3 to 10 m),
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PRIMAQUINE PHOSPHATE
IP 2007
mobile phase: a suitable filtered mixture of 688 volumes of water, 250 volumes of peroxide-free tetrahydrofuran and 62 volumes of methanol such that at a flow rate of 1 ml per minute the retention times of prednisone and acetanilide are about 8 and 6 minutes respectively, spectrophotometer set at 254 nm, a 10 l loop injector. Inject the reference solution. Adjust the operating parameters such that the peak obtained is about 50 per cent of the full scale. The relative standard deviation for replicate injections is not more than 2.0 per cent and the resolution between prednisone and the internal standard is not less than 3.0. Inject alternately the test solution and the reference solution. Calculate the content of C21H26O5 in the tablets. Storage. Store protected from light.
310 nm, the resulting solution shows absorption maxima at about 225 nm, 265 nm and 282 nm; absorbances at the maxima are 0.74 to 0.77, 0.50 to 0.53 and 0.50 to 0.52, respectively. C. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel F254. Mobile phase. A mixture of 60 volumes of chloroform, 40 volumes of methanol and 1 volume of strong ammonia solution. Test solution. Dissolve 0.2 g in 5 ml of water, dilute to 10 ml with methanol and dilute 1 volume of the resulting solution to 10 volumes with methanol (50 per cent). Reference solution. Dissolve 20 mg of primaquine phosphate RS in 5 ml of water, dilute to 10 ml with methanol. Apply to the plate 5 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution. D. Dissolve 50 mg in 5 ml of water, add 2 ml of 2 M sodium hydroxide and shake with two quantities, each of 5 ml, of chloroform. The aqueous layer, acidified by the addition of nitric acid, gives reaction B of phosphates (2.3.1).
Primaquine Phosphate
CH3 HN N H3CO
C15H21N3O,2H3PO4 Mol. Wt. 455.3
NH2 , 2H3PO4
Tests
pH (2.4.24). 2.5 to 3.5, determined in a 1.0 per cent w/v solution. Related substances. Determine by liquid chromatography (2.4.14). Test solution. Add 0.2 ml of strong ammonia solution to 1 ml of a 1.0 per cent w/v solution of the substance under examination, shake with 10 ml of the mobile phase and use the clear, lower layer. Reference solution (a). Dilute 3 ml of the test solution to 100 ml with the mobile phase. Reference solution (b). Dilute 1 ml of the test solution to 10 ml with the mobile phase and further dilute 1 ml of the resulting solution to 50 ml with the same solvent. Reference solution (c). Add 0.2 ml of strong ammonia solution to 1 ml of a 1.0 per cent w/v solution of the primaquine phosphate RS, shake with 10 ml of the mobile phase and use the clear, lower layer. Chromatographic system a stainless steel column 20 cm x 4.6 mm, packed with porous silica particles (10 m), mobile phase: a mixture of 45 volumes of chloroform, 45 volumes of hexane, 10 volumes of methanol and 0.1 volume of strong ammonia solution, flow rate. 3 ml per minute,
Primaquine Phosphate is (RS)-8-(4-amino-1methylbutylamino)-6-methoxyquinoline diphosphate. Primaquine Phosphate contains not less than 98.5 per cent and not more than 101.5 per cent of C15H21N3O,2H3PO4, calculated on the dried basis. Description. An orange, crystalline powder; odourless or almost odourless.
Identification
Test A may be omitted if tests B, C and D are carried out. Tests B and C may be omitted if tests A and D are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with primaquine phosphate RS or with the reference spectrum of primaquine phosphate. B. When examined in the range 300 nm to 450 nm (2.4.7), a 0.015 per cent w/v solution in 0.01 M hydrochloric acid shows absorption maxima at about 332 nm and 415 nm; absorbance at about 332 nm, about 0.68 to 0.78 and at about 415 nm, 0.41 to 0.53. Dilute 5 ml of the solution to 50 ml with 0.01 M hydrochloric acid. When examined in the range 215 nm to
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IP 2007
PROBENECID
spectrophotometer set at 261 nm, a 20 l loop injector. The test is not valid unless in the chromatogram obtained with reference solution (c) there is a peak just before the principal peak with an area of about 6 per cent of that of the principal peak and the resolution between these peaks is not less than 2.0. Inject each solution and record the chromatograms for at least twice the retention time of primaquine. The sum of the areas of any secondary peaks in the chromatogram obtained with the test solution is not greater than the area of the principal peak in the chromatogram obtained with reference solution (a). Ignore any peak the area of which is less than that of the principal peak in the chromatogram obtained with reference solution (b). Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.2 g, dissolve in 40 ml of anhydrous glacial acetic acid with gentle heating. Titrate with 0.1 M perchloric acid, determining the end point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.02277 g of C15H21N3O,2H3PO4. Storage. Store protected from light and moisture.
Tests
Uniformity of content. Comply with the tests stated under Tablets. Transfer one tablet into a suitable container, add 5 ml of hydrochloric acid, disperse the tablet in about 25 g of crushed ice and add sufficient water to produce 50.0 ml. Carry out the nitrite titration (2.3.31), using 0.01 M sodium nitrite as titrant. Carry out a blank titration. 1 ml of 0.01 M sodium nitrite is equivalent to 0.002594 g of C15H21N3O. Other tests. Comply with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 0.15 g of primaquine, dissolve in 20 ml of water, add 5 ml of 2 M sodium hydroxide and extract with four quantities, each of 25 ml, of chloroform. Combine the chloroform extracts and evaporate to a volume of about 10 ml. Add 40 ml of anhydrous glacial acetic acid, Titrate with 0.1 M perchloric acid, determining the end point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.01297 g of C15H21N3O. Storage. Store protected from moisture. Labelling. The label states the strength in terms of the equivalent amount of primaquine.
Primaquine Tablets
Primaquine Phosphate Tablets
Primaquine Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of primaquine, C15H21N3O. The tablets are coated.
Probenecid
H3C H 3C N S O O COOH
Identification
A. Dissolve a quantity of the powdered tablets containing 60 mg of primaquine in a mixture of 10 ml of water and 2 ml of 2 M sodium hydroxide and extract with two quantities, each of 20 ml, of chloroform. Wash the chloroform extracts with water, dry over anhydrous sodium sulphate, evaporate to dryness and dissolve the residue in 2 ml of chloroform. The residue complies with the following test. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with primaquine phosphate RS or with the reference spectrum of primaquine. B. Extract a quantity of the powdered tablets containing 25 mg of primaquine with 10 ml of water and filter. To 2 ml of the filtrate add 3 ml of water and 1 ml of a 5 per cent w/v solution of ceric ammonium sulphate in 2 M nitric acid; a deep violet colour is produced immediately. C13H19NO4S
Mol. Wt. 285.5
Probenecid is 4-(dipropylsulphamoyl)benzoic acid. Probenecid contains not less than 99.0 per cent and not more than 101.0 per cent of C13H19NO4S, calculated on the dried basis. Description. A white or almost white, crystalline powder. Identification Test A may be omitted if tests B and C are carried out. Tests B and C may be omitted if test A is carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with probenecid RS or with the reference spectrum of probenecid.
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PROBENECID TABLETS
IP 2007
B. When examined in the range 210 nm to 360 nm (2.4.7), a 0.001 per cent w/v solution in a mixture of 9 volumes of ethanol (95 per cent) and 1 volume of 0.1 M hydrochloric acid shows absorption maxima at about 223 nm and 248 nm; absorbance at about 248 nm is about 0.33. C. Dissolve 0.2 g in about 0.6 ml of 2 M ammonia and add 3 ml of 1.7 per cent w/v solution of silver nitrate; a white precipitate is produced which is soluble in an excess of dilute ammonia solution.
Probenecid Tablets
Probenecid Tablets contain not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of probenecid, C13H19NO4S.
Identification
A.Triturate a quantity of the powdered tablets containing 0.5 g of Probenecid with ethanol (95 per cent), filter and concentrate the filtrate by evaporation on a water-bath. Cool, filter and recrystallise the residue from ethanol (50 per cent). The residue complies with the following test. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with probenecid RS or with the reference spectrum of probenecid. B. When examined in the range 210 nm to 360 nm (2.4.7), the final solution obtained in the Assay shows absorption maxima at about 225 nm and 248 nm. C. The residue obtained in test A melts at about 199 (2.4.21).
Tests
Appearance of solution. A 10.0 per cent w/v solution in 2 M sodium hydroxide is clear (2.4.1), and not more intensely coloured than reference solution YS6 (2.4.1). Acidity. Add 2.0 g to 100 ml of water, heat on a water-bath for 30 minutes, cool, filter and dilute with water to 100.0 ml. Titrate 50.0 ml of the solution with 0.1 M sodium hydroxide using phenolphthalein solution as indicator. Not more than 0.5 ml of 0.1 M sodium hydroxide is required to change the colour of the solution. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 55 volumes of toluene, 20 volumes of di-isopropyl ether, 15 volumes of chloroform and 10 volumes of glacial acetic acid. Test solution. Dissolve 0.1 g of the substance under examination in 10 ml of acetone. Reference solution. A 0.005 per cent w/v solution of the substance under examination in acetone. Apply to the plate 20 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Heavy metals (2.3.13).1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.5 g and dissolve in 100 ml of ethanol (95 per cent), shaking well and heating gently if necessary. Cool and titrate with 0.1 M sodium hydroxide, using bromothymol blue solution as indicator. Carry out a blank titration. 1 ml of 0.1 M sodium hydroxide is equivalent to 0.02854 g of C13H19NO4S. Storage. Store protected from moisture.
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 55 volumes of toluene, 20 volumes of di-isopropyl ether, 15 volumes of chloroform and 10 volumes of glacial acetic acid. Test solution. Extract a quantity of the powdered tablets containing 0.2 g of Probenecid with 20 ml of acetone, filter and use the filtrate. Reference solution. Dilute 1 ml of the test solution to 200 ml with acetone. Apply to the plate 20 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Dissolution (2.5.2). Apparatus. No 1 Medium. 900 ml of phosphate buffer pH 7.6 Speed and time. 50 rpm and 30 minutes. Withdraw a suitable volume of the medium and filter, Dilute 4.0 ml of the filtrate to 100.0 ml with 0.1 M sodium hydroxide. Measure the absorbance of the resulting solution at the maximum at about 244 nm (2.4.7). Similarly measure the absorbance of a solution of a known concentration of probenecid RS. Calculate the content of C13H19O4S. D. Not less than 80 per cent of the stated amount of C13H19NO3S.
978
IP 2007
PROCAINAMIDE INJECTION
Other tests. Comply with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 0.2 g of Probencid, add 200 ml of ethanol (95 per cent) and 5 ml of 1 M hydrochloric acid, heat on a water-bath at 70 for 30 minutes, shaking occasionally. Cool, add sufficient ethanol (95 per cent) to produce 250.0 ml and filter. To 5.0 ml of the filtrate add 5 ml of 0.1 M hydrochloric acid, dilute to 250.0 ml with ethanol (95 per cent) and measure the absorbance of the resulting solution at the maximum at about 248 nm (2.4.7). Calculate the content of C13H19NO4S taking 332 as specific absorbance at 248 nm. Storage. Store protected from moisture.
Tests
Appearance of solution. A 10.0 per cent w/v solution in carbon dioxide-free water is clear (2.4.1), and not more intensely coloured than reference solution BS6 (2.4.1). pH (2.4.24). 5.6 to 6.3, determined in a 10.0 per cent w/v solution. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 60 volumes of 1-butanol, 30 volumes of water and 15 volumes of glacial acetic acid. Test solution. Dissolve 0.1 g of the substance under examination in 10 ml of ethanol (95 per cent). Reference solution. A 0.005 per cent w/v solution of the substance under examination in ethanol (95 per cent). Apply to the plate 5 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution.
Procainamide Hydrochloride
O N H H2N
C13H21N3O,HCl
CH3 N CH3 . HCl

Mol. Wt. 271.8
Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.25 g, dissolve in 75 ml of water and 10 ml of hydrochloric acid and carry out the nitrite titration (2.3.31). 1 ml of 0.1 M sodium nitrite is equivalent to 0.02718 g of C13H21N3O, HCl. Storage. Store protected from light.
Procainamide Hydrochloride is 4-amino-N-[2-(diethylamino) ethyl)benzamide hydrochloride. Procainamide Hydrochloride contains not less than 98.0 per cent and not more than 101.0 per cent of C13H21N3O,HCl, calculated on the dried basis. Description. A white or very slightly yellow, crystalline powder; hygroscopic.
Identification
Test A may be omitted if tests B, C and D are carried out. Tests B, C and D may be omitted if test A is carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with procainamide hydrochloride RS or with the reference spectrum of procainamide hydrochloride. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.001 per cent w/v solution in 0.1 M sodium hydroxide shows an absorption maximum at about 273 nm; absorbance at about 273 nm, 0.58 to 0.61. C. Dilute 1 ml of a solution, prepared by dissolving 2.5 g in sufficient carbon dioxide-free water to produce 25 ml, to 2 ml with water. 1 ml of this solution gives the reaction of primary aromatic amines (2.3.1). D. A 2 per cent w/v solution gives reaction A of chlorides (2.3.1).
Procainamide Injection
Procainamide Hydrochloride Injection
Procainamide Injection is a sterile solution of Procainamide Hydrochloride in Water for Injections. It may contain Sodium Metabisulphite as a stabilising agent. Procainamide Injection contains not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of procainamide hydrochloride, C13H21N3O, HCl.
Identification
A. Dilute a volume containing 0.25 g of Procainamide Hydrochloride to 25 ml with water, make alkaline with 5 M sodium hydroxide and extract with two quantities, each of 5 ml, of chloroform. Filter the combined extracts through anhydrous sodium sulphate, evaporate the filtrate to dryness
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PROCAINAMIDE TABLETS
IP 2007
using a rotatory evaporator and dissolve the residue in 5 ml of chloroform. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with procainamide hydrochloride RS or with the reference spectrum of procainamide. B. Dilute a suitable volume of the injection with water to produce a solution containing 0.0005 per cent w/v of Procainamide Hydrochloride. Absorbance of the resulting solution at about 280 nm, about 0.30 (2.4.7). C. Gives the reactions of chlorides (2.3.1).
quantities, each of 5 ml, of chloroform. Filter the combined extracts through anhydrous sodium sulphate, evaporate the filtrate to dryness using a rotatory evaporator and dissolve the residue in 5 ml of chloroform. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with procainamide hydrochloride RS or with the reference spectrum of procainamide. B. Triturate a quantity of the powdered tablets containing 1 g of Procainamide Hydrochloride with 10 ml of water and filter. The filtrate gives the reactions of chlorides (2.3.1).
Tests
pH (2.4.24). 4.0 to 5.5. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 60 volumes of 1-butanol, 30 volumes of water and 15 volumes of glacial acetic acid. Test solution. Dilute a volume of the injection containing 0.1 g of Procainamide Hydrochloride to 5 ml with methanol. Reference solution. Dilute 1 volume of the test solution to 100 volumes with methanol. Apply to the plate 5 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. To a volume containing about 0.25 g of Procainamide Hydrochloride add 45 ml of 6 M hydrochloric acid and boil for 1 minute. Carry out the nitrite titration (2.3.31). 1 ml of 0.1 M sodium nitrite is equivalent to 0.02718 g of C13H21N3O, HCl. Storage. Store protected from light.
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 60 volumes of 1-butanol, 30 volumes of water and 15 volumes of glacial acetic acid. Test solution. Shake a quantity of the powdered tablets containing 0.4 g of Procainamide Hydrochloride with 20 ml of methanol (90 per cent) for 15 minutes and filter. Reference solution. Dilute 1 volume of the test solution to 100 volumes with methanol. Apply to the plate 5 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Other tests. Comply with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 0.25 g of Procainamide Hydrochloride, add 100 ml of 6 M hydrochloric acid, shake for 15 minutes and boil for 1 minute. Carry out the nitrite titration (2.3.31). 1 ml of 0.1 M sodium nitrite is equivalent to 0.02718 g of C13H21N3O, HCl. Storage. Store protected from light and moisture.
Procainamide Tablets
Procainamide Hydrochloride Tablets
Procainamide Tablets contain not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of procainamide hydrochloride, C13H21N3O,HCl.
Procaine Hydrochloride
O O H2N
C13H20N2O2,HCl Mol. Wt. 272.8 Procaine Hydrochloride is 2-(diethylamino)ethyl 4aminobenzoate hydrochloride.
CH3 N CH3 , HCl
Identification
A. Shake a quantity of the powdered tablets containing 0.25 g of Procainamide Hydrochloride with 25 ml of water, make alkaline with 5 M sodium hydroxide and extract with two
980
IP 2007
PROCAINE AND ADRENALINE INJECTION
Procaine Hydrochloride contains not less than 99.0 per cent and not more than 101.0 per cent of C13H20N2O2,HCl, calculated on the dried basis. Description. Colourless crystals or a white, crystalline powder; odourless.
Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.3 g, dissolve in 50 ml of 2 M hydrochloric acid, add 3 g of potassium bromide, cool in ice and titrate slowly with 0.1 M sodium nitrite, stirring constantly. Determine the end point potentiometrically (2.4.25). 1 ml of 0.1 M sodium nitrite is equivalent to 0.02728 g of C13H20N2O2, HCl. Storage. Store protected from light.
Identification
Test A may be omitted if tests B, C, D and E are carried out. Tests B, C and D may be omitted if tests A and E are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with procaine hydrochloride RS or with the reference spectrum of procaine hydrochloride. B. To 0.2 ml of a 5 per cent w/v solution add 2 ml of water and 0.5 ml of 1 M sulphuric acid, shake and add 1 ml of a 0.1 per cent w/v solution of potassium permanganate; the colour is immediately discharged. C. To about 5 mg add 0.5 ml of fuming nitric acid, evaporate to dryness on a water-bath, cool, dissolve the residue in 5 ml of acetone and add 1 ml of 0.1 M ethanolic potassium hydroxide; only a brownish red colour develops. D. Dilute 1 ml of a 5 per cent w/v solution to 100 ml with water; 2 ml of the solution gives the reaction of primary aromatic amines (2.3.1). E. Gives reaction A of chlorides (2.3.1).
Procaine and Adrenaline Injection

Procaine Hydrochloride and Adrenaline Bitartrate Injection; Procaine Hydrochloride and Epinephrine Bitartrate Injection
Procaine and Adrenaline Injection is a sterile solution of Procaine Hydrochloride and Adrenaline Bitartrate in Water for Injections. Procaine and Adrenaline Injection contains not less than 1.9 per cent and not more than 2.1 per cent w/v of procaine hydrochloride, C13H20N2O2, HCl and the equivalent of not less than 0.00175 per cent and not more than 0.00225 per cent w/v of adrenaline, C9H13NO3. Description. A clear, colourless solution.
Tests
Appearance of solution. A 5.0 per cent w/v solution in carbon dioxide-free water is clear (2.4.1), and colourless (2.4.1). pH (2.4.24). 5.0 to 6.5, determined in a 2.0 per cent w/v solution. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 80 volumes of dibutyl ether, 16 volumes of n-hexane and 4 volumes of glacial acetic acid. Test solution. A 10 per cent w/v solution of the substance under examination in water. Reference solution. A 0.005 per cent w/v solution of 4-aminobenzoic acid in water. Apply to the plate 5 l of each solution. After development, dry the plate at 105 for 10 minutes and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. The principal spot remains on the line of application. Heavy metals (2.3.13). 2.0 g complies with the limit test for heavy metals, Method A (10 ppm).
Identification
A. To 5 ml add 5 ml of water and 10 ml of picric acid solution, shake gently and set aside for 1 hour; the crystalline precipitate, after washing with water and drying at 100, melts at about 134 (2.4.21). B. To 5 ml add 1 ml of hydrochloric acid, cool to 0, add 5 ml of sodium nitrite solution and pour the mixture into 2 ml of 2-naphthol solution containing 1 g of sodium acetate; an orange-red colour is produced. C. To 10 ml add 4 ml of disodium hydrogen phosphate solution and sufficient 0.1 M iodine to produce a distinct brown colour. Add 0.1 M sodium thiosulphate to remove the excess of iodine; a pink colour is produced.
Tests
pH (2.4.24). 3.0 to 5.5. Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. For procaine hydrochloride To 10.0 ml of the injection add 0.5 g of sodium carbonate and extract with three
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PROCAINE PENICILLIN
IP 2007
quantities, each of 20 ml, of a mixture of 1 volume of 2-propanol and 3 volumes of chloroform until complete extraction of procaine is effected. Shake the combined extracts with 5 ml of water, wash the water with the solvent mixture and add the washing to the combined extracts. Shake the combined extracts and washings with 10.0 ml of 0.1 M hydrochloric acid, separate the acid layer, wash the combined extracts and washings with 5 ml of water, add the aqueous extract to the separated acid layer and titrate with 0.1 M sodium hydroxide, using methyl red-methylene blue solution as indicator. 1 ml of 0.1 M hydrochloric acid is equivalent to 0.02728 g of C13H20N2O2,HCl. For adrenaline To 10.0 ml of the injection add 20 mg of sodium metabisulphite, 0.1 ml of ferrous sulphate-citrate solution, 1 ml of glycine buffer solution and mix. Allow to stand for 10 minutes, extract with 10 ml of ether, allow to separate, reject the ether and measure the absorbance of a 4-cm layer of the solution at about 540 nm (2.4.7). Calculate the content of adrenaline, C9H13NO3, from a reference curve prepared by treating suitable aliquots of a solution of adrenaline bitartrate RS in the same manner. 1 mg of adrenaline bitartrate is equivalent to 0.0005497 g of C9H13NO3. Storage. Store protected from light. Labelling. The label states the strength as Procaine Hydrochloride, 2 per cent w/v; Adrenaline, 0.002 per cent w/v.
penicillin RS or with the reference spectrum of procaine penicillin. B. In the Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the peak in the chromatogram obtained with the reference solution (a). C. A turbid solution of 0.1 g in 2 ml of 2 M hydrochloric acid gives the reaction of primary aromatic amines (2.3.1).
Tests
pH (2.4.24). 5.0 to 7.5, determined in a solution prepared by shaking 50 mg in sufficient carbon dioxide-free water to produce 15 ml until dissolution is complete. Specific optical rotation (2.4.22). +165 to +180, determined in a solution prepared by dissolving 0.25 g in sufficient of a mixture of 3 volumes of acetone and 2 volumes of water to produce 25 ml. Water (2.3.43). 2.8 to 4.2 per cent, determined on 0.5 g. Assay. Determine the contents of benzyl penicillin and procaine by liquid chromatography, (2.4.14). Test solution. Weigh accurately 70 mg of the substance under examination and dissolve in 30 ml of the mobile phase with the aid of ultrasound for about 2 minutes. Dilute to 50.0 ml with the mobile phase. Reference solution (a). Weigh accurately about 56 mg of benzyl penicillin potassium RS and 40 mg of procaine hydrochloride RS and dissolve in 25 ml of the mobile phase with the aid of ultrasound for about 2 minutes. Dilute to 50.0 ml with the mobile phase. Reference solution (b). Mix 1 volume of a 0.24 per cent w/v solution of phenoxymethylpenicillin potassium RS in the mobile phase with 3 volumes of reference solution (a).
Procaine Penicillin
O O H2N N CH3 CH3 , O O H N N H H H COOH CH3 S CH3 , H2O
C13H20N2O2,C16H18N2O4S,H2O
Mol. Wt. 588.7
Procaine Penicillin is 2-diethylaminoethyl 4-aminobenzoate (6R)-6-(2-phenylacetamido)penicillanate monohydrate. Procaine Penicillin contains not less than 51 per cent and not more than 59.6 per cent of penicillin G, calculated as C16H18N2O4S, and not less than 37.5 per cent and not more than 43.0 per cent of procaine, C13H20N2O2, both calculated on the anhydrous basis. Description. A white, crystalline powder.
Chromatographic system a stainless steel column 30 cm x 4 mm, packed with octadecylsilyl silica gel (10 m), mobile phase: a mixture of 50 volumes of a solution prepared by mixing 14 g of potassium dihydrogen phosphate in 6.5 g of tetrabutylammonium hydroxide (40 per cent) and adjusting the pH to 7.0 with 1 M potassium hydroxide or dilute phosphoric acid, 25 volumes of acetonitrile and 25 volumes of water, flow rate. 1 ml per minute, spectrophotometer set at 235 nm, a 20 l loop injector. Inject reference solution (b). The resolution between benzyl penicillin potassium and phenoxymethylpenicillin potassium is not less than 2. The relative retention time of procaine with reference to benzyl penicillin is about 2.2. Inject the test solution and reference solution (a). Record the peak responses of the main peaks of benzyl penicillin and
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with procaine
982
IP 2007
FORTIFIED PROCAINE PENICILLIN INJECTION
procaine. Calculate the content of benzyl penicillin, C16H18N2O4S and the content of procaine, C13H20N2O2 using the factor of 0.866 for converting the content of procaine hydrochloride to that of procaine. Procaine Penicillin intended for use in the manufacture of Parenteral Preparations without a further procedure for the removal of bacterial endotoxins complies with the following additional requirement. Bacterial endotoxins (2.2.3). Not more than 0.10 Endotoxin Unit per mg. Procaine Penicillin intended for use in the manufacture of Parenteral Preparations without a further sterilisation procedure complies with the following additional requirement. Sterility. Complies with the test for sterility (2.2.11). Storage. Store protected from moisture. If the material is intended for use in the manufacture of parenteral preparations, the container should be sterile and sealed so as to exclude micro-organisms. Labelling. The label states (1) where applicable, that the contents are free from bacterial endotoxins; (2) where applicable, that the contents are sterile.
The contents of the sealed container comply with the tests stated under Parenteral Preparations (Powders for Injection) and with the following requirements.
Identification
A. Dissolve 10 mg in 10 ml of water and add 0.5 ml of neutral red solution. Add sufficient 0.01 M sodium hydroxide to give a permanent orange colour and then add 1 ml of penicillinase solution; a red colour is produced rapidly. B. In the Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the peak in the chromatogram obtained with the reference solution. C. Give the reaction of primary aromatic amines (2.3.1), producing a bright, orange-red precipitate.
Tests
Water (2.3.43). Not more than 3.5 per cent, determined on 0.5 g. Assay. Determine by liquid chromatography (2.4.14). Test solution. Weigh accurately a quantity of the mixed contents of 10 containers equivalent to about 70 mg of procaine penicillin and dissolve in 30 ml of the mobile phase with the aid of ultrasound for about 2 minutes. Dilute to 50.0 ml with the mobile phase. Reference solution (a). Weigh accurately about 56 mg of benzylpenicillin potassium RS and 40 mg of procaine hydrochloride RS and dissolve in 25 ml of the mobile phase with the aid of ultrasound for about 2 minutes. Dilute to 50.0 ml with the mobile phase. Reference solution (b). Mix 1 volume of a 0.24 per cent w/v solution of phenoxymethylpenicillin potassium RS in the mobile phase with 3 volumes of reference solution (a). Chromatographic system a stainless steel column 30 cm x 4 mm, packed with octadecylsilyl silica gel (10 m), mobile phase: a mixture of 50 volumes of a solution prepared by mixing 14 g of potassium dihydrogen phosphate in 6.5 g of tetrabutyl ammonium hydroxide (40 per cent) and adjusting the pH to 7.0 with 1 M potassium hydroxide or dilute phosphoric acid, 25 volumes of acetonitrile and 25 volumes of water, flow rate. 1 ml per minute, spectrophotometer set at 235 nm, a 20 l loop injector. Inject reference solution (b). The resolution between benzylpenicillin potassium and phenoxymethylpenicillin potassium is not less than 2. The relative retention time of procaine with reference to benzylpenicillin is about 2.2.
Fortified Procaine Penicillin Injection

Procaine Penicillin with Benzylpenicillin Injection
Fortified Procaine Penicillin Injection is a sterile mixture of five parts of Procaine Penicillin and one part of Benzylpenicillin Potassium or Benzylpenicillin Sodium, together with suitable dispersing and buffering agents. It is filled in a sealed container. The injection is constituted by suspending the contents of the sealed container in the requisite amount of sterile Water for Injections, immediately before use. Storage. The constituted injection should be used within 24 hours (4 days if a buffering agent is present) of preparation when stored at a temperature not exceeding 20 or within 7 days (14 days if a buffering agent is present) when stored at a temperature between 2 and 8. Fortified Procaine Penicillin Injection contains a quantity of total penicillins calculated as C16H17N2NaO4S and equivalent to not less than 60.0 per cent and not more than 74.0 per cent of the stated amounts of procaine penicillin and benzylpenicillin potassium or benzylpenicillin sodium and a quantity of procaine, C13H20N2O2, equivalent to not less than 36.0 per cent and not more than 44.0 per cent of the stated amount of procaine penicillin. Description. A white or almost white powder.
983
PROCHLORPERAZINE MALEATE
IP 2007
Inject the test solution and reference solution (a). Record the peak responses of the main peaks of benzyl penicillin and procaine. Calculate the total penicillin content as benzylpenicillin, C16H18N2O4S and the content of procaine, C13H20N2O2 using the factor of 0.866 for converting the content of procaine hydrochloride to that of procaine. Labelling. The label states (1) the quantities of Procaine Penicillin and Benzylpenicillin Potassium or Benzylpenicillin Sodium contained in it; (2) the names of any added dispersing and buffering agents; (3) for intramuscular injection only.
C. Complies with the test for identification of phenothiazines (2.3.3). Test solution. A solution containing 0.1 per cent w/v of the substance under examination in a mixture of equal volumes of methanol and chloroform. Reference solution. A 0.1 per cent w/v solution of prochlorperazine maleate RS in the same solvent mixture Apply 4 l of each solution. D. Triturate 0.2 g with a mixture of 3 ml of water and 1 ml of 10 M sodium hydroxide and shake with three quantities, each of 5 ml, of ether. To 0.1 ml of the aqueous layer add a solution of 10 mg of resorcinol in 3 ml of sulphuric acid and heat in a water-bath for 15 minutes; no colour develops. To the remainder of the aqueous layer add 2 ml of bromine solution, heat in a water-bath for 15 minutes, then heat to boiling and cool. To 0.1 ml of the solution add a solution of 10 mg of resorcinol in 3 ml of sulphuric acid and heat in a water-bath for 15 minutes; a blue colour develops.
Prochlorperazine Maleate
COOH , N Cl
C20H24ClN3S,2C4H4O4
N S
N CH3
COOH
2
Tests
pH (2.4.24). 3.0 to 4.0, determined in a freshly prepared saturated solution. Related substances. Complies with the test for related substances in phenothiazines (2.3.5), using mobile phase A. Prepare the test solution immediately before use. Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 1.0 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.2 g, in powder, dissolve in 50 ml of anhydrous glacial acetic acid, heating gently on a water-bath, allow to cool. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.03031 g of C20H24ClN3S, 2C4H4O4. Storage. Store protected from light and moisture.
Mol. Wt. 606.1
Prochlorperazine Maleate is 2-chloro-10-[3-(4methylpiperazin-1-yl)propyl]phenothiazine di(hydrogen maleate). Prochlorperazine Maleate contains not less than 98.0 per cent and not more than 101.0 per cent of C20H24ClN3S, 2C4H4O4, calculated on the dried basis. Description. A white or pale yellow, crystalline powder; practically odourless.
Identification
Test A may be omitted if tests B, C and D are carried out. Tests B, C and D may be omitted if test A is carried out. A.To 20 mg add 5 ml of water and 1 ml of 1 M sodium hydoxide, shake and extract with 10 ml of ether. Wash the ether extract with 5 ml of water, dry with anhydrous sodium sulphate, evaporate to dryness and dissolve the residue in 0.2 ml of chloroform. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with prochlorperazine maleate RS or with the reference spectrum of prochlorperazine. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.001 per cent w/v solution in ethanol (95 per cent) containing 0.01 per cent v/v of strong ammonia solution shows an absorption maximum at about 258 nm and a less well-defined maximum at about 313 nm; absorbance at about 258 nm, about 0.6.
Prochlorperazine Tablets
Prochlorperazine Maleate Tablets
Prochlorperazine Tablets contain not less than 92.5 per cent and not more than 107.5 per cent of the stated amount of prochlorperazine maleate, C20H24ClN3S, 2C4H4O4.
Identification
A.To a quantity of the powdered tablets containing 40 mg of Prochlorperazine Maleate add 10 ml of water and 2 ml of 1 M
984
IP 2007
PROCHLORPERAZINE MESYLATE
sodium hydroxide, shake and extract with 15 ml of ether. Wash the ether with 5 ml of water, dry with anhydrous sodium sulphate, evaporate to dryness and dissolve the residue in 0.4 ml of chloroform. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with prochlorperazine maleate RS or with the reference spectrum of prochlorperazine. B. To a quantity of the powdered tablets containing 5 mg of Prochlorperazine Maleate add 5 ml of sulphuric acid and allow to stand for 5 minutes; a red colour is produced. C. Shake a quantity of the powdered tablets containing 0.2 g of Prochlorperazine Maleate with 2 ml of water and 1 ml of 5 M sodium hydroxide, mix and extract with three quantities, each of 10 ml, of ether. Dry the combined extracts with anhydrous sodium sulphate, filter, evaporate the filtrate to dryness and dissolve the residue in 10 ml of methanol and add a solution of 0.15 g of picric acid in 10 ml of methanol. The precipitate, after washing with a small quantity of methanol, melts at about 255, with decomposition (2.4.21).
Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 25 mg of Prochlorperazine Maleate and extract with three quantities, each of 10 ml, of ethanol containing 1 per cent v/v of strong ammonia solution. Filter the extracts and to the combined filtrates add sufficient ethanol to produce 100.0 ml. Dilute 10.0 ml to 50.0 ml with ethanol, dilute 10.0 ml of this solution to 50.0 ml with ethanol and measure the absorbance of the resulting solution at the maximum at about 258 nm (2.4.7). Calculate the content of C20H24ClN3S, 2C4H4O4 taking 620 as the specific absorbance at 258 nm. Storage. Store protected from light and moisture.
Prochlorperazine Mesylate
N S Cl
C20H24ClN3S,2CH4SO3
N N CH3
, 2CH3SO3H
Tests
Related substances. Comply with the test for related substances in phenothiazines (2.3.5), using mobile phase A. Prepare the following solutions freshly. Test solution. Extract a quantity of the powdered tablets containing 0.1 g of Prochlorperazine Maleate with 10 ml of methanol containing 0.5 per cent v/v of strong ammonia solution and filter. Reference solution. Dilute 1 volume of the test solution to 200 volumes with the same solvent. Apply to the plate 20 l of each solution. Uniformity of content. (For tablets containing 10 mg or less) Comply with the test stated under Tablets. Protect the solutions from light throughout the Assay. Crush one tablet and extract with three quantities, each of 10 ml, of ethanol containing 1 per cent v/v of strong ammonia solution. Filter the extracts and to the combined filtrates add sufficient ethanol to produce 50.0 ml. Dilute 10.0 ml of this solution to 100.0 ml with ethanol. Dilute further with ethanol, if necessary, to give a final solution containing 10 g of Prochlorperazine Maleate per ml and measure the absorbance of the resulting solution at the maximum at about 258 nm (2.4.7). Calculate the content of C20H24ClN3S, 2C4H4O4 taking 620 as the specific absorbance at 258 nm. Other tests. Comply with the tests stated under Tablets. Assay. Protect the solutions from light throughout the Assay.
Mol. Wt. 566.2
Prochlorperazine Mesylate is 2-chloro-10-[3-(4methylpiperazin-1-yl)propyl]phenothiazine di(methanesulphonate). Prochlorperazine Mesylate contains not less than 98.0 per cent and not more than 101.0 per cent of C20H24ClN3S,2CH4SO3, calculated on the dried basis. Description. A white or almost white powder; odourless.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with prochlorperazine mesylate RS or with the reference spectrum of prochlorperazine mesylate. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.001 per cent w/v solution in ethanol containing 0.01 per cent v/v of strong ammonia solution shows an absorption maximum at about 258 nm and a less well-defined maximum at about 313 nm; absorbance at about 258 nm, about 0.6. C. Dissolve 5 mg in 2 ml of sulphuric acid and allow to stand for 5 minutes; a red colour is produced. D. Mix 50 mg with 0.2 g of powdered sodium hydroxide, heat to fusion and continue the heating for a few seconds longer. Cool, add 0.5 ml of water and a slight excess of 2 M hydrochloric acid and warm; sulphur dioxide is evolved which turns moistened starch iodate paper blue.
985
PROCHLORPERAZINE INJECTION
IP 2007
Tests
pH (2.4.24). 2.0 to 3.0, determined in a 2.0 per cent w/v solution. Related substances. Complies with the test for related substances in phenothiazines (2.3.5), using mobile phase A. Test solution. Dissolve the substance under examination in methanol containing 0.5 per cent v/v of strong ammonia solution. Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 1.0 per cent, determined on 1.0 g by drying in an oven at 100 at a pressure not exceeding 0.7 kPa. Assay. Weigh accurately about 0.5 g, dissolve in 10 ml of water, add 5 ml of 1 M sodium hydroxide and extract with successive quantities of 50, 25, 25 and 25 ml of ether. Wash the combined ether extracts with 5 ml of water, shake the washings with 5 ml of ether, add the ether to the combined ether extracts and evaporate to dryness. Add 2 ml of ethanol, evaporate to dryness. Titrate with 0.1 M perchloric acid, using 1-naphtholbenzein solution as indicator. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.02831 g of C20H24ClN3S, 2CH4SO3. Storage. Store protected from light and moisture.
B. To a volume containing 5 mg of Prochlorperazine Mesylate add carefully 2 ml of sulphuric acid and allow to stand for 5 minutes; a red colour is produced. Tests pH (2.4.24). 5.5 to 6.5. Related substances. Carry out the test for related substances in phenothiazines (2.3.5), using mobile phase A. Test solution. Use the injection under examination. Reference solution (a). Dilute 1 volume of the test solution to 40 volumes with methanol containing 0.5 per cent v/v of strong ammonia solution immediately before use. Reference solution (b). Dilute 1 volume of the test solution to 200 volumes with the methanol-ammonia mixture immediately before use. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (a) and not more than one such spot is more intense than the spot in the chromatogram obtained with reference solution (b). Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. Protect the solutions from light throughout the Assay. To an accurately measured volume containing about 25 mg of Prochlorperazine Mesylate add sufficient ethanol containing 0.01 per cent v/v of strong ammonia solution to produce 200.0 ml. Dilute 5.0 ml of this solution to 100.0 ml with the ammoniacal ethanol and measure the absorbance of the resulting solution at the maximum at about 258 nm (2.4.7). Calculate the content of C20H24ClN3S, 2CH4SO3 taking 635 as the specific absorbance at 258 nm. Storage. Store protected from light.
Prochlorperazine Injection
Prochlorperazine Mesylate Injection
Prochlorperazine Injection is a sterile solution of Prochlorperazine Mesylate in Water for Injections free from dissolved air. Prochlorperazine Injection contains not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of prochlorperazine mesylate, C20H24ClN3S,2CH4SO3.
Procyclidine Hydrochloride
Identification
A. To a volume containing 0.1 g of Prochlorperazine Mesylate add 20 ml of water and 2 ml of 10 M sodium hydroxide. Shake and extract with 25 ml of ether. Wash the ether layer with two quantities, each of 5 ml, of water, dry with anhydrous sodium sulphate and evaporate to dryness. Dissolve the residue in 1 ml of chloroform. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with prochlorperazine mesylate RS treated in the same manner or with the reference spectrum of prochlorperazine. C19H29NO,HCI
OH N
, HCl
Mol. wt. 323.9
Procyclidine Hydrochloride is (RS)-1-cyclohexyl-1-phenyl-3pyrrolidin-1-ylpropan-1-ol hydrochloride.
986
IP 2007
PROCYCLIDINE TABLETS
Procyclidine Hydrochloride contains not less than 99.0 per cent and not more than 101.0 per cent of C19H29NO,HCI, calculated on the dried basis. Description. A white, crystalline powder, odourless or almost odourless.
ether, shake with anhydrous sodium sulphate and filter. Evaporate the filtrate and dissolve the residue in 1 ml of ether. Reference solution (a). Prepare in the same manner as the test solution but using 20 ml of a 0.5 per cent w/v solution of the substance under examination and omitting the addition of the internal standard solution. Reference solution (b). Prepare in the same manner as the test solution but using 20 ml of a 0.5 per cent w/v solution of the substance under examination. Chromatographic system a glass column 1.5 m x 4 mm, packed with acid-washed, silanised diatomaceous support (such as HP chromosorb W) coated with 10 per cent w/w of modified polyethylene glycol 20M (such as SP-1000) and 2 per cent w/w of potassium hydroxide, temperature : column. 240, inlet port and detector at 240, flow rate. 30 ml per minute of the carrier gas. The ratio of the sum of the areas of any secondary peaks to the area of the peak due to the internal standard in the chromatogram obtained with reference solution (b) is not more than the ratio of the area of the principal peak to the area of the internal standard peak in the chromatogram obtained with the test solution. Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.7 g, dissolve in 75 ml of anhydrous glacial acetic acid, warm if necessary to effect solution and cool. Add 10 ml of mercuric acetate solution. Titrate with 0.1 M perchloric acid, using crystal violet solution as indicator. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.03239 g of C19H29NO,HCI. Storage. Store protected from moisture.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with procyclidine hydrochloride RS. B. Dissolve 0.25 g in 10 ml of water, make alkaline with 5 M ammonia and extract with three quantities, each of 10 ml, of ether. Dry the combined ethereal extracts over anhydrous sodium sulphate, filter, remove the ether and scratch the residue with a glass rod to induce solidification. The residue melts at about 85 (2.4.21). C. Gives the reactions of chlorides (2.3.1).
Tests
pH (2.4.24). 4.5 to 6.5, determined in a 1.0 per cent w/v solution. Related substances. A. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 100 volumes of ether and 1 volume of strong ammonia solution. Test solution. Dissolve 0.2 g of the substance under examination in 10 ml of chloroform. Reference solution (a). A 0.004 per cent w/v solution of 1-phenyl-3-pyrrolidinopropan-1-one hydrochloride RS in chloroform. Reference solution (b). A 0.01 per cent w/v solution of the substance under examination in chloroform. Apply to the plate 5 l of each solution. After development, dry the plate at 105 for 15 minutes and examine in ultraviolet light at 254 nm. Any spot corresponding to 1-phenyl-3pyrrolidinopropan-1-hydrochloride-1-one in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (a). Spray the plate with dilute potassium iodobismuthate solution. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (b). B. Determine by gas chromatography (2.4.13). Test solution. Add 5 ml of 1.25 M sodium hydroxide to 20 ml of a 0.015 per cent w/v solution of the substance under examination and mix. Extract with two quantities, each of 20 ml, of ether, add to the combined extracts 5 ml of a 0.06 per cent w/v solution of triphenylethylene (internal standard) in
Procyclidine Tablets
Procyclidine Hydrochloride Tablets
Procyclidine Tablets contain not than 90.0 per cent and not more than 110.0 per cent of the stated amount of procyclidine hydrochloride, C19H29NO,HCI.
Identification
A. Dissolve a quantity of the powdered tablets containing about 25 mg of Procyclidine Hydrochloride in 10 ml of water,
987
PROGUANIL HYDROCHLORIDE
IP 2007
shake with 20 ml of ether and discard the ether layer. Make the aqueous layer alkaline with 2 M sodium hydroxide and extract with two quantities, each of 20 ml, of ether. Wash the combined ether extracts with two quantities, each of 10 ml, of water, dry by shaking with anhydrous sodium sulphate, filter and evaporate the filtrate to dryness. It necessary, induce crystallization by scratching with a glass rod. On the residue, determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with procyclidine hydrochloride RS. B. The powdered tablets give the reactions of chlorides (2.3.1).
0.5 ml of water and 4.5 ml of a 0.025 per cent w/v solution of bromocresol purple in the same manner beginning at the words extract with 20 ml of chloroform .. Calculate the content of C19H29NO,HCI from the absorbance obtained by repeating the operation using procyclidine hydrochloride RS in place of the powdered tablets. Storage. Store protected from moisture.
Proguanil Hydrochloride
Chloroguanide Hydrochloride
Cl N H
C11H16CIN5, HCl
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 100 volumes of ether and 1 volume of strong ammonia solution. Test solution. Shake a quantity of the powdered tablets containing 25 mg of Procyclidine Hydrochloride with 5 ml of chloroform and filter. Reference solution (a). A 0.001 per cent w/v solution of 1-phenyl-3-pyrrolidinopropan-1-one hydrochloride RS in chloroform. Reference solution (b). Dilute 1 volume of test solution to 200 volumes with chloroform. Apply to the plate 20 l of each solution. After development, dry the plate at 105 for 15 minutes and examine in ultraviolet light at 254 nm. Any spot corresponding to 1-phenyl-3pyrrolidinopropan-1-one in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (a) (0.2 per cent). Spray the plate with dilute potassium iodobismuthate solution. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (b) (0.5 per cent). Ignore any spot due to excipients on the line of application. Other tests. Comply with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 2.5 mg of Procyclidine Hydrochloride, transfer to a 100-ml volumetric flask, add 10.0 ml of water and mix, and dilute to volume with a 0.025 per cent w/v solution of bromocresol purple in 2 per cent v/v solution of glacial acetic acid. Allow the undissolved particles to settle. Transfer 5.0 ml of the supernatant solution to a separating funnel, extract with 20.0 ml of chloroform and filter the extract, discarding the first 5 ml of the filtrate. Measure the absorbance of the filtrate at the maximum at about 405 nm (2.4.7), using as the blank a solution prepared by treating
NH N H
NH N H
CH3 CH3 , HCl
Mol. Wt. 290.2
Proguanil Hydrochloride is 1-(4-chlorophenyl)-5isopropylbiguanide hydrochloride. Proguanil Hydrochloride contains not less than 99.0 per cent and not more than 101.0 per cent of C11H16ClN5, HCl, calculated on the dried basis. Description. A white, crystalline powder; odourless.
Identification
Test A may be omitted if tests B, C and D are carried out. Tests B and C may be omitted if tests A and D are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with proguanil hydrochloride RS or with the reference spectrum of proguanil hydrochloride. B. To 10 ml of a saturated solution add 0.25 ml of potassium ferrocyanide solution; a white precipitate is produced which dissolves on addition of a few drops of dilute nitric acid. C. Dissolve 5 mg in 5 ml of a warm 1.0 per cent w/v solution of cetrimide and add 1 ml of 5 M sodium hydroxide and 1 ml of bromine solution; a deep red colour is produced. D. Gives the reactions of chlorides (2.3.1).
Tests
Acidity or alkalinity. To 35 ml of water maintained at 60 to 65 add 0.2 ml of methyl red solution, neutralise with 0.01 M sodium hydroxide or 0.01 M hydrochloric acid, add 0.4 g of the substance under examination and stir until dissolved. The resulting solution is not acidic and requires for neutralisation not more than 0.2 ml of 0.01 M hydrochloric acid.
988
IP 2007
PROMETHAZINE HYDROCHLORIDE
4-Chloroaniline. Dissolve 0.1 g in 1 ml of 2 M hydrochloric acid, add sufficient water to produce 20 ml, cool to 5, add 1 ml of 0.05 M sodium nitrite, allow to stand at 5 for 5 minutes, add 2 ml of a 5 per cent w/v solution of ammonium sulphamate and allow to stand for 10 minutes. Add 2 ml of a 0.1 per cent w/v solution of N-(1-naphthyl)ethylenediamine dihydrochloride, dilute to 50 ml with water and allow to stand for 30 minutes. Any magenta colour produced is not more intense than that obtained by treating in the same manner and at the same time 20 ml of a solution containing 1.25 g of 4-chloroaniline. Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.15 g, dissolve in 25 ml of anhydrous glacial acetic acid and 10 ml of mercuric acetate solution. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.01451 g of C11H16ClN5, HCl. Storage. Store protected from light and moisture.
Tests
4-Chloroaniline. To a quantity of the powdered tablets containing about 0.1 g of Proguanil Hydrochloride add 5 ml of ethanol (95 per cent) and shake for 10 minutes. Add 2.5 ml of 2 M hydrochloric acid and 15 ml of water, mix and filter through a wetted filter paper, washing the filter with 5 ml of water. Cool to 5, add 1 ml of 0.05 M sodium nitrite, allow to stand at 5 for 5 minutes, add 2 ml of a 5 per cent w/v solution of ammonium sulphamate and allow to stand for 10 minutes. Add 2 ml of a 0.1 per cent w/v solution of N-(1-naphthyl)ethylenediamine dihydrochloride, dilute to 50 ml with water and allow to stand for 30 minutes. Any magenta colour produced is not more intense than that obtained by treating in the same manner and at the same time 20 ml of a solution containing 1.25 g of 4-chloroaniline. Other tests. Comply with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 0.1 g of Proguanil Hydrochloride, add 5 ml of water and warm on a water-bath with stirring until a smooth paste is obtained. Add 50 ml of water, continue warming for 10 minutes, cool, add sufficient water to produce 100.0 ml and filter. Dilute 10.0 ml of the filtrate to 100.0 ml with water and to 10.0 ml of the resulting solution add 70 ml of water, 5 ml of a 20 per cent w/v solution of cetrimide and 1 ml of 2-propanol. Adjust the temperature of the solution to 20 and add 2 ml of alkaline sodium hypobromite solution and sufficient water to produce 100.0 ml. Allow to stand at 20 for 25 minutes and measure the absorbance of the resulting solution at the maximum at about 480 nm (2.4.7). Calculate the content of C11H16ClN5,HCl from the absorbance obtained by repeating the operation using 10 ml of a 0.01 per cent w/v solution of proguanil hydrochloride RS beginning at the words add 70 ml of water,..... Storage. Store protected from light and moisture.
Proguanil Tablets
Proguanil Hydrochloride Tablets; Chloroguanide Hydrochloride Tablets
Proguanil Tablets contain not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of proguanil hydrochloride, C11H16ClN5, HCl.
Identification
A. Boil a quantity of the powdered tablets containing 0.5 g of Proguanil Hydrochloride with 5 ml of dilute hydrochloric acid, cool and filter. To the filtrate add a slight excess of sodium hydroxide solution, extract with 30 ml of ether and evaporate the ethereal extract; the residue, after drying at 105, melts at about 131 (2.4.21). Dissolve the residue obtained in test A in the minimum quantity of dilute hydrochloric acid; the solution diluted with water to about 40 ml and neutralised if necessary, with cautious addition of dilute ammonia solution, complies with tests the following tests. B. To 10 ml add 0.25 ml of potassium ferrocyanide solution; a white precipitate is produced which dissolves on addition of a few drops of dilute nitric acid. C. To 0.5 ml add 5 ml of a warm 1.0 per cent w/v solution of cetrimide and add 1 ml of 5 M sodium hydroxide and 1 ml of bromine solution; a deep red colour is produced.
Promethazine Hydrochloride
CH3 N CH3 CH3
N S
, HCl
C17H20N2S,HCl
Mol. Wt. 320.9
Promethazine Hydrochloride is (RS)-dimethyl(2phenothiazin-10-ylpropyl)amine hydrochloride.
989
PROMETHAZINE INJECTION
IP 2007
Promethazine Hydrochloride contains not less than 98.5 per cent and not more than 101.0 per cent of C17H20N2S, HCl, calculated on the dried basis. Description. A white or faintly yellowish, crystalline powder.
Assay. Weigh accurately about 0.25 g, dissolve in a mixture of 5 ml of 0.01 M hydrochloric acid and 50 ml of ethanol (95 per cent) and titrate with 0.1 M sodium hydroxide, determining the end-point potentiometrically (2.4.25). Record the volume added between the two inflections. 1 ml of 0.1 M sodium hydroxide is equivalent to 0.03209 g of C17H20N2S, HCl. Storage. Store protected from light and moisture.
Identification
Test A may be omitted if tests B, C and D are carried out. Tests B, C and D may be omitted if test A is carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with promethazine hydrochloride RS or with the reference spectrum of promethazine hydrochloride. B. Complies with the test for identification of phenothiazines (2.3.3). C. Dissolve 0.1 g in 3 ml of water and add 1 ml of nitric acid dropwise; a precipitate is produced which dissolves rapidly to give a red solution which becomes orange and then yellow. Heat the solution to boiling; it becomes orange and an orangered precipitate is produced. D. Gives reaction B of chlorides (2.3.1).
Promethazine Injection
Promethazine Hydrochloride Injection
Promethazine Injection is a sterile solution of Promethazine Hydrochloride in Water for Injections free from dissolved air. It may contain suitable stabilising agents. Promethazine Injection contains not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of promethazine hydrochloride, C17H20N2S, HCl.
Identification
A. To a volume containing 0.1 g of Promethazine Hydrochloride add 20 ml of water and 2 ml of 10 M sodium hydroxide. Shake and extract the mixture with 25 ml of ether. Wash the ether layer with two quantities, each of 5 ml, of water, dry with anhydrous sodium sulphate and evaporate to dryness. Dissolve the residue in 1 ml of chloroform. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with promethazine hydrochloride RS treated in the same manner or with the reference spectrum of promethazine. B. To a volume containing 0.2 g of Promethazine Hydrochloride add sufficient potassium carbonate to saturate the solution, extract with two quantities, each of 10 ml, of ether and evaporate the combined extracts to dryness. Dissolve the residue in 2 ml of methanol and pour into a solution of 0.4 g of picric acid in 10 ml of methanol, previously warmed to about 50. Cool, scratch the sides of the tube to induce crystallisation, allow to stand for 3 to 4 hours and filter. The residue, after washing with methanol and drying, melts at about 160 (2.4.21). C. To a volume containing 5 mg of Promethazine Hydrochloride add carefully 2 ml of sulphuric acid and allow to stand for 5 minutes; a red colour is produced.
Tests
pH (2.4.24). 4.0 to 5.0, determined in a 10.0 per cent w/v solution prepared immediately before use. Related substances. Carry out the test for related substances in phenothiazines (2.3.5), protected from bright light using mobile phase B. Prepare the following solutions immediately before use. Test solution. Dissolve 0.2 g of the substance under examination in 10 ml of a mixture of 95 volumes of methanol and 5 volumes of diethylamine. Reference solution (a). A 0.01 per cent w/v solution of the substance under examination in the same solvent mixture. Reference solution (b). A 0.02 per cent w/v solution of isopromethazine hydrochloride RS in the same solvent mixture.. Apply to the plate 10 l of each solution. Any spot corresponding to isopromethazine in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (b) and any other secondary spot is not more intense than the spot in the chromatogram obtained with reference solution (a). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105.
Tests
pH (2.4.24). 5.0 to 6.0. Related substances. Carry out the test for related substances in phenothiazines (2.3.5), using mobile phase B.
990
IP 2007
PROMETHAZINE TABLETS
Test solution. Dilute a volume of the injection with a mixture of 95 volumes of methanol and 5 volumes of diethylamine to contain 1 per cent w/v of Promethazine Hydrochloride. Reference solution (a). Dilute 1 volume of the test solution to 40 volumes with the same solvent mixture. Reference solution (b). Dilute 1 volume of the test solution to 200 volumes with the same solvent mixture. Reference solution (c). A 0.01 per cent w/v solution of isopromethazine hydrochloride RS in the same solvent mixture. Apply separately to the plate 10 l of each solution. Any spot corresponding to isopromethazine in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (c). Any other secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (a) and not more than one such spot is more intense than the spot in the chromatogram obtained with reference solution (b). Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. Carry out the following procedure protected from light. To an accurately measured volume containing about 25 mg of Promethazine Hydrochloride add sufficient 0.01 M hydrochloric acid to produce 100.0 ml. Dilute 10.0 ml to 100.0 ml with 0.01 M hydrochloric acid, dilute 10.0 ml of this solution to 50.0 ml with 0.01 M hydrochloric acid and measure the absorbance of the resulting solution at the maximum at about 249 nm (2.4.7). Calculate the content of C17H20N2S, HCl taking 910 as the specific absorbance at 249 nm. Storage. Store protected from light.
Test solution. A solution prepared by diluting a suitable volume of the syrup with water to contain 0.08 per cent w/v of Promethazine Hydrochloride. Apply 5 l to the plate.
Tests
Other tests. Complies with the tests stated under Oral Liquids. Assay. Carry out the following procedure protected from light. Weigh accurately a quantity containing about 10 mg of Promethazine Hydrochloride, add 25 ml of water and 5 ml of a 5 per cent w/v solution of sodium hydroxide. Extract the mixture with two quantities, each of 50 ml, of chloroform, shaking vigorously for 1 minute each time, evaporate the combined extracts to dryness at about 30 at a pressure of 2 kPa and dissolve the residue in sufficient 0.1 M hydrochloric acid to produce 50.0 ml (solution A). Dilute 10 ml of solution A to 50.0 ml with water (solution B). To a further 10 ml of solution A add 5 ml of peroxyacetic acid solution, allow to stand for 10 minutes and add sufficient water to produce 50.0 ml (solution C). Measure the absorbance of solution C at the maximum at about 336 nm (2.4.7), using solution B as the blank and measure the absorbance of solution B at the same wavelength using water as the blank. Repeat the procedure using a 0.02 per cent w/v solution of promethazine hydrochloride RS in 0.1 M hydrochloric acid in place of solution A and beginning at the words Dilute 10 ml of....... Determine the weight per ml of the syrup (2.4.29), and calculate the content of C17H20N2S, HCl, weight in volume. The result is not valid unless the absorbance of solution B is less than 0.10. Storage. Store protected from light and moisture.
Promethazine Tablets
Promethazine Hydrochloride Tablets
Promethazine Tablets contain not less than 92.5 per cent and not more than 107.5 per cent of the stated amount of promethazine hydrochloride, C17H20N2S, HCl. The tablets are coated.
Promethazine Syrup
Promethazine Hydrochloride Syrup; Promethazine Hydrochloride Oral Solution; Promethazine Oral Solution
Promethazine Syrup is a solution containing Promethazine Hydrochloride in a suitable flavoured vehicle. Promethazine Syrup contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of promethazine hydrochloride, C17H20N2S, HCl.
Identification
A. To a quantity of the powdered tablets containing 40 mg of Promethazine Hydrochloride add 10 ml of water and 2 ml of 1 M sodium hydroxide, shake and extract with 15 ml of ether. Wash the ether extract with 5 ml of water, dry with anhydrous sodium sulphate, evaporate to dryness and dissolve the residue in 0.4 ml of chloroform. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with promethazine
Identification
Carry out the method for identification of phenothiazines (2.3.3).
991
PROMETHAZINE THEOCLATE
IP 2007
hydrochloride RS treated in the same manner or with the reference spectrum of promethazine. B. Dissolve a quantity of the powdered tablets containing 0.2 g of Promethazine Hydrochloride in 2 ml of water, filter, add sufficient potassium carbonate to saturate the solution, extract with two quantities, each of 10 ml, of ether and evaporate the combined extracts to dryness. Dissolve the residue in 2 ml of methanol and pour into a solution of 0.4 g of picric acid in 10 ml of methanol, previously warmed to about 50. Cool, scratch the sides of the tube to induce crystallisation, allow to stand for 3 to 4 hours and filter. The residue, after washing with methanol and drying, melts at about 160 (2.4.21). C. To a quantity of the powdered tablets containing 5 mg of Promethazine Hydrochloride add 5 ml of sulphuric acid and allow to stand for 5 minutes; a red colour is produced.
Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 50 mg of Promethazine Hydrochloride with 10 ml of 2 M hydrochloric acid and add 200 ml of water. Shake for 15 minutes, add sufficient water to produce 500.0 ml and centrifuge about 50 ml of the mixture. To 5.0 ml of the clear supernatant liquid add 10 ml of 0.1 M hydrochloric acid and sufficient water to produce 100.0 ml. Measure the absorbance of the resulting solution at the maximum at about 249 nm (2.4.7). Calculate the content of C17H20N2S, HCl taking 910 as the specific absorbance at 249 nm. Storage. Store protected from light and moisture.
Promethazine Theoclate
O CH3 H H3C N N N CH3 , N CH3 O N CH3
Tests
Related substances. Carry out the test for related substances in phenothiazines (2.3.5), using mobile phase B. Test solution. A solution freshly prepared by extracting a quantity of the powdered tablets containing 0.1 g of Promethazine Hydrochloride with 10 ml of a mixture of 95 volumes of methanol and 5 volumes of diethylamine and filtering. Reference solution (a). Dilute 1 volume of the test solution to 40 volumes with the same solvent. Reference solution (b). Dilute 1 volume of the test solution to 200 volumes with the same solvent. Reference solution (c). A 0.01 per cent w/v solution of isopromethazine hydrochloride RS in the same solvent. Applying to the plate 10 l of each solution. Any spot corresponding to isopromethazine in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (c). Any other secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (a) and not more than one such spot is more intense than the spot in the chromatogram obtained with reference solution (b). Uniformity of content. (For tablets containing 10 mg or less) Comply with the test stated under Tablets. Crush one tablet, add 1 ml of dilute hydrochloric acid and 30 ml of water and shake for 15 minutes. Add sufficient water to produce 50.0 ml and centrifuge. Complete the procedure described in the Assay beginning at the words To 5.0 ml of the clear supernatant liquid..... Other tests. Comply with the tests stated under Tablets. Assay. Carry out the following procedure protected from light.
N S
Cl
C17H20N2S,C7H7ClN4O2
Mol. Wt. 499.0
Promethazine Theoclate is the (RS)-dimethyl(2phenothiazin-10-ylpropyl)amine salt of 8chlorotheophylline. Promethazine Theoclate contains not less than 98.0 per cent and not more than 101.0 per cent of C17H20N2S, C7H7ClN4O2, calculated on the dried basis. Description. A white or almost white powder; odourless or almost odourless.
Identification
Test A may be omitted if tests B, C, D and E are carried out. Tests B, C and D may be omitted if tests A and E are carried out. A. Shake 0.15 g with 2.5 ml of water, add 1 ml of 5 M ammonia and extract with 30 ml of ether. Wash the ether extract with 10 ml of water, dry with anhydrous sodium sulphate and evaporate to dryness. Dissolve the residue in 1 ml of chloroform. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with promethazine RS or with the reference spectrum of promethazine. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.0007 per cent w/v solution in ethanol containing 0.01 per cent v/v of strong ammonia solution shows an absorption maximum at about 255 nm; absorbance at about 0.53.
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PROMETHAZINE THEOCLATE TABLETS
C. Dissolve 5 mg in 2 ml of sulphuric acid and allow to stand for 5 minutes; a red colour is produced. D. Shake 0.4 g with 10 ml of water, add 4 ml of 5 M ammonia, shake with two quantities, each of 30 ml, of ether and add 4 ml of hydrochloric acid to the aqueous solution; a white precipitate is produced. Filter, wash with water and dry at 105. Dissolve 10 mg of the residue in 1 ml of hydrochloric acid, add 0.1 g of potassium chlorate and evaporate to dryness; a reddish residue remains which becomes purple on exposure to the vapour of ammonia. E. Fuse 50 mg of the residue obtained in test D with 0.5 g of anhydrous sodium carbonate, boil the residue with 5 ml of water, acidify to litmus paper with nitric acid and filter. The filtrate gives reaction A of chlorides (2.3.1).
Promethazine Theoclate Tablets

Promethazine Theoclate Tablets contain not less than 92.5 per cent and not more than 107.5 per cent of the stated amount of promethazine theoclate, C17H20N2S, C7H7ClN4O2.
Identification
A. To a quantity of the powdered tablets containing 40 mg of Promethazine Theoclate add 10 ml of water and 2 ml of 1 M sodium hydroxide, shake and extract with 15 ml of ether. Wash the ether extract with 5 ml of water, dry with anhydrous sodium sulphate, evaporate to dryness and dissolve the residue in 0.4 ml of chloroform. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with promethazine theoclate RS treated in the same manner or with the reference spectrum of promethazine. B. Dissolve a quantity of the powdered tablets containing 0.2 g of Promethazine Theoclate in 2 ml of water, filter, add sufficient potassium carbonate to saturate the solution, extract with two quantities, each of 10 ml, of ether and evaporate the combined extracts to dryness. Dissolve the residue in 2 ml of methanol and pour into a solution of 0.4 g of picric acid in 10 ml of methanol, previously warmed to about 50. Cool, scratch the sides of the tube to induce crystallisation, allow to stand for 3 to 4 hours and filter. The residue, after washing with methanol and drying, melts at about 160 (2.4.21). C. To a quantity of the powdered tablets containing 5 mg of Promethazine Theoclate add 5 ml of sulphuric acid and allow to stand for 5 minutes; a red colour is produced. D. Extract a quantity of the powdered tablets containing 0.2 g of Promethazine Theoclate with chloroform, filter and evaporate the filtrate to dryness. Shake the residue with 10 ml of water, add 4 ml of 5 M ammonia and extract with two quantities, each of 30 ml, of ether. Wash the combined extracts with 10 ml of water. Combine the aqueous layer and washings, add 4 ml of hydrochloric acid; a white precipitate is produced. Filter, wash the residue with water and dry at 105. Dissolve 10 mg of the residue in 1 ml of hydrochloric acid, add 0.1 g of potassium chlorate and evaporate to dryness; a reddish residue remains which becomes purple on exposure to the vapour of ammonia.
Tests
Chlorides (2.3.12). Shake 1.5 g with 50 ml of water for 2 minutes and filter. 25 ml of the filtrate complies with the limit test for chlorides (330 ppm). Related substances. Carry out the test for related substances in phenothiazines (2.3.5), protected from bright light, using mobile phase B. Prepare the following solutions immediately before use. Test solution. Dissolve 0.2 g of the substance under examination in 10 ml of a mixture of 95 volumes of methanol and 5 volumes of diethylamine. Reference solution (a). A 0.01 per cent w/v solution of the substance under examination in the same solvent mixture. Reference solution (b). A 0.02 per cent w/v solution of isopromethazine hydrochloride RS in the same solvent mixture. Apply to the plate 10 l of each solution. Any spot corresponding to isopromethazine in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (b) and any other secondary spot is not more intense than the spot in the chromatogram obtained with reference solution (a). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 1.0 g and dissolve in 200 ml of acetone. Titrate with 0.1 M perchloric acid, using 3 ml of a saturated solution of methyl orange in acetone as indicator. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.04990 g of C17H20N2S,C7H7ClN4O2. Storage. Store protected from light and moisture.
Tests
Related substances. Carry out the test for related substances in phenothiazines (2.3.5), using mobile phase B. Test solution. A solution freshly prepared by extracting a quantity of the powdered tablets containing 0.1 g of Promethazine Theoclate with 10 ml of a mixture of 95 volumes of methanol and 5 volumes of diethylamine and filtering.
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PROPANTHELINE BROMIDE
IP 2007
Reference solution (a). Dilute 1 volume of the test solution to 40 volumes with the same solvent mixture. Reference solution (b). Dilute 1 volume of the test solution to 200 volumes with the same solvent mixture. Reference solution (c). A 0.01 per cent w/v solution of isopromethazine hydrochloride RS in the same solvent mixture. Apply to the plate 10 l of each solution. Any spot corresponding to isopromethazine in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (c). Any other secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (a) and not more than one such spot is more intense than the spot in the chromatogram obtained with reference solution (b). Other tests. Comply with the tests stated under Tablets. Assay. Carry out the following procedure protected from light. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 50 mg of Promethazine Theoclate, add 5 ml of water and 1 ml of strong ammonia solution and allow to stand for 5 minutes. Add 50 ml of ethanol, shake for 5 minutes and filter, washing the residue with five quantities, each of 5 ml, of ethanol. Add sufficient ethanol to the filtrate to produce 100.0 ml. Dilute 10.0 ml to 100.0 ml with ethanol and dilute 10.0 ml of this solution to 100.0 ml with ethanol. Measure the absorbance of the resulting solution at the maximum at about 255 nm (2.4.7). Calculate the content of C17H20N2S, C7H7ClN4O2 taking 755 as the specific absorbance at 255 nm. Storage. Store protected from light and moisture.
Description. White or yellowish white crystals or powder; odourless; slightly hygroscopic.
Identification
Test A may be omitted if tests B, C, D and E are carried out. Tests B and C may be omitted if tests A, D and E are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with propantheline bromide RS or with the reference spectrum of propantheline bromide. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.006 per cent w/v solution in methanol shows absorption maxima at about 246 nm and 282 nm; absorbance at about 246 nm, about 0.7 and at about 282 nm, about 0.37. C. Dissolve 0.2 g in 15 ml of water, add 1 ml of 10 M sodium hydroxide, boil for 2 minutes, cool slightly, add 7.5 ml of 2 M hydrochloric acid, cool and filter. Wash the residue with water, recrystallise from ethanol (50 per cent) and dry at 105 for 1 hour. Dissolve about 10 mg of the crystals so obtained in 5 ml of sulphuric acid (96 per cent w/w); an intense yellow colour is produced which fluoresces strongly in ultraviolet light at 365 nm. D. In the test for Related substances, the principal spot in the chromatogram obtained with test solution (b) corresponds to that in the chromatogram obtained with reference solution (b). E. Gives the reactions of bromides (2.3.1).
Tests
Appearance of solution. A 3.0 per cent w/v solution is clear (2.4.1). Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254.
Propantheline Bromide
O O O H3C N H3C CH3 CH3 CH3 Br
Mobile phase. A mixture of 140 volumes of 1,2-dichloroethane, 60 volumes of methanol, 2.5 volumes of anhydrous formic acid and 2.5 volumes of water. Test solution (a). Dissolve 0.1 g of the substance under examination in 10 ml of chloroform. Test solution (b). Dissolve 25 mg of the substance under examination in 100 ml of chloroform.
C23H30BrNO3
Mol. Wt. 448.4
Propantheline Bromide is N-methyl-N,N-bis(1-methylethyl)2-[(9H-xanthen-9-ylcarbonyl)oxy]ethanaminium bromide. Propantheline Bromide contains not less than 98.0 per cent and not more than 102.0 per cent of C23H30BrNO3, calculated on the dried basis.
Reference solution (a). A 0.005 per cent w/v solution of the substance under examination in chloroform. Reference solution (b). A 0.025 per cent w/v solution of propantheline bromide RS in chloroform. Apply to the plate 10 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm.
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Any secondary spot in the chromatogram obtained with test solution (a) is not more intense than the spot in the chromatogram obtained with reference solution (a). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 1.0 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.4 g, dissolve in a mixture of 50 ml of acetic anhydride and 7 ml of mercuric acetate solution. Titrate with 0.1 M perchloric acid, using crystal violet solution as the indicator. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.04484 g of C23H30BrNO3. Storage. Store protected from moisture.
25.0 ml to 100.0 ml with 0.1M sodium hydroxide. The absorbance of the resulting solution at the maximum at about 248 nm is not more than 0.31 (2.4.7). Other tests. Comply with the tests stated under Tablets. Assay. Weigh and powder 20 tablets or more if necessary. Weigh accurately a quantity of the powder containing about 0.5 g of Propantheline Bromide, place it on a sintered-glass filter, add 10 ml of peroxide-free ether, dried over sodium and distilled before use, stir and filter. Repeat the extraction with four quantities, each of 10 ml, of ether and reserve the combined ether extracts for the test for Xanthanoic acid. Extract the residue on the filter with four quantities, each of 10 ml, of chloroform, evaporate the combined chloroform extracts to about 10 ml, add 10 ml of mercuric acetate solution. Titrate with 0.1 M perchloric acid, using crystal violet solution as indicator. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.04484 g of C23H30BrNO3. Storage. Store protected from moisture.
Propantheline Tablets
Propantheline Bromide Tablets
Propantheline Tablets contain not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of propantheline bromide, C23H30BrNO3. The tablets are coated.
Identification
Triturate a quantity of the powdered tablets containing 75 mg of Propantheline Bromide with 10 ml of chloroform, filter, evaporate the chloroform and stir the residue with 5 ml of ether until it solidifies. The solid complies with the following tests. A. Dissolve 60 mg in 2 ml of water, add 2 ml of 5 M sodium hydroxide, boil for 2 minutes, cool slightly, acidify with 2 M hydrochloric acid, heat to boiling, add ethanol (95 per cent) dropwise until the precipitate just dissolves, cool and filter. The residue, after washing with water, recrystallising from ethanol (50 per cent) and drying at 105 for 1 hour, melts at about 215 (2.4.21). B. To 10 mg of the crystals obtained in test A add 5 ml of sulphuric acid (96 per cent w/w); an intense yellow colour is produced which fluoresces strongly in ultraviolet light at 365 nm. C. Gives the reactions of bromides (2.3.1).
Propranolol Hydrochloride
OH O H N
CH3 , HCl CH3
C16H21NO2,HCl
Mol. Wt. 295.8
Propranolol Hydrochloride is (2RS)-1-[(1methylethyl)amino]-3-(naphthalen-1-yloxy)propan-2-ol hydrochloride. Propranolol Hydrochloride contains not less than 99.0 per cent and not more than 101.0 per cent of C16H21NO2, HCl, calculated on the dried basis. Description. A white or almost white powder.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with propranolol hydrochloride RS or with the reference spectrum of propranolol hydrochloride. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.002 per cent w/v solution in methanol shows absorption maxima at about 290 nm, 306 nm and 319 nm. C. Gives reaction A of chlorides (2.3.1).
Tests
Xanthanoic acid. Shake the combined ether extracts reserved in the Assay with two quantities, each of 30 ml, of 0.1 M sodium hydroxide containing 1.5 per cent w/v solution of sodium chloride. Remove the ether from the combined aqueous extracts by heating on a water-bath, add sufficient 0.1 M sodium hydroxide to produce 100.0 ml and dilute
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Tests
Appearance of solution. A 10.0 per cent w/v solution in methanol is clear (2.4.1), and not more intensely coloured than degree 6 of the appropriate range of reference solutions (2.4.1). pH (2.4.24). 5.0 to 6.0, determined in a 1.0 per cent w/v solution. Specific optical rotation (2.4.22). 1.0 to +1.0, determined in a 4.0 per cent w/v solution. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 90 volumes of toluene and 10 volumes of methanol. Test solution. Dissolve 1.0 g of the substance under examination in 10 ml of methanol. Reference solution. Dissolve 20.0 mg of the substance under examination in 100 ml of methanol. Apply to the plate 10 l of each solution. After development, dry the plate in air, spray with anisaldehyde solution and heat at 105 for 15 minutes. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.25 g, dissolve in 25 ml of ethanol (95 per cent) and titrate with 0.1 M sodium hydroxide, determining the end-point potentiometrically (2.4.25). 1 ml of 0.1 M sodium hydroxide is equivalent to 0.02958 g of C16H21NO2, HCl. Storage. Store protected from moisture.
extracts with water until the washings are free from alkali, dry with anhydrous sodium sulphate, filter, evaporate the filtrate to dryness and dry the residue at 50 at a pressure of 2 kPa for 1 hour. On the residue determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with propranolol hydrochloride RS treated in the same manner or with the reference spectrum of propranolol. B. When examined in the range 230 nm to 360 nm (2.4.7), the solution obtained in the Assay shows absorption maxima at about 290 nm, 306 nm and 319 nm.
Tests
pH (2.4.24). 3.0 to 3.5. Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. To an accurately measured volume containing about 2 mg of Propranolol Hydrochloride add sufficient methanol to produce 100.0 ml and measure the absorbance of the resulting solution at the maximum at about 290 nm (2.4.7). Calculate the content of C 16H 21NO 2, HCl taking 206 as the specific absorbance at 290 nm. Storage. Store protected from light, in a single dose containers.
Propranolol Tablets
Propranolol Hydrochloride Tablets
Propranolol Tablets contain not less than 92.5 per cent and not more than 107.5 per cent of the stated amount of propranolol hydrochloride, C16H21NO2, HCl.
Identification
A.Suspend a quantity of the powdered tablets containing 0.1 g of Propranolol Hydrochloride in 20 ml of water, filter, make the filtrate alkaline with 1 M sodium hydroxide and extract with three quantities, each of 10 ml, of ether. Wash the combined extracts with water until the washings are free from alkali, dry with anhydrous sodium sulphate, filter, evaporate the filtrate to dryness and dry the residue at 50 at a pressure of 2 kPa for 1 hour. On the residue, determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with propranolol hydrochloride RS treated in t he same manner or with the reference spectrum of propranolol. B. When examined in the range 230 nm to 360 nm (2.4.7), the final solution obtained in the Assay shows absorption maxima at about 290 nm, 306 nm and 319 nm.
Propranolol Injection
Propranolol Hydrochloride Injection
Propranolol Injection is a sterile solution of Propranolol Hydrochloride in Water for Injections containing Citric Acid. Propranolol Injection contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of propranolol hydrochloride, C16H21NO2, HCl.
Identification
A. Make alkaline with 1 M sodium hydroxide a volume containing 10 mg of Propranolol Hydrochloride and extract with three quantities, each of 5 ml, of ether. Wash the combined
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PROPYLENE GLYCOL
Tests
Uniformity of content. (For tablets containing 10 mg or less) Comply with the test stated under Tablets. Transfer one tablet to a 100-ml volumetric flask, add 5 ml of dilute hydrochloric acid and allow to stand, swirling occasionally, until it is disintegrated. Add about 70 ml of methanol and shake well for about 1 minute. Dilute to volume with methanol, mix and centrifuge an aliquot of the solution. Dilute a suitable volume of the clear solution with methanol to produce a solution containing 20 g of Propranolol Hydrochloride per ml. Measure the absorbance of the resulting solution at the maximum at about 290 nm (2.4.7), using methanol as the blank. Calculate the content of C16H21NO2, HCl taking 206 as the specific absorbance at 290 nm. Dissolution (2.5.2). Apparatus. No 1 Medium. 900 ml of 0.1 M hydrochloric acid Speed and time. 100 rpm and 45 minutes. Withdraw a suitable volume of the medium and filter. Dilute a suitable volume of the filtrate with the same solvent. Measure the absorbance of the resulting solution at the maximum at about 290 nm (2.4.7). Calculate the content of C16H21NO2,HCl in the medium taking 206 as the specific absorbance at 290 nm. D. Not less than 75 per cent of the stated amount of C16H21NO2, HCl. Other tests. Comply with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 20 mg of Propranolol Hydrochloride and shake with 20 ml of water for 10 minutes. Add 50 ml of methanol, shake for a further 10 minutes, add sufficient methanol to produce 100.0 ml and filter. Dilute 10.0 ml of the filtrate to 50.0 ml with methanol and measure the absorbance of the resulting solution at the maximum at about 290 nm (2.4.7). Calculate the content of C16H21NO2, HCl taking 206 as the specific absorbance at 290 nm. Storage. Store protected from light and moisture.
Description. A white or creamy white, crystalline powder; odourless or almost odourless.
Identification
A. When examined in the range 230 nm to 360 nm (2.4.7), a 0.001 per cent w/v solution in methanol shows an absorption maximum only at about 275 nm; absorption at about 275 nm, about 0.49. B. Dissolve 10 mg in 50 ml of water, cool and add 5 ml of dilute ammonia solution; a red colour is produced which becomes brown on standing and is restored on shaking. C. Dissolve 5 mg in 50 ml of water and add 0.05 ml of ferric chloride test solution; a bluish black colour is produced.
Tests
Chlorides (2.3.12). Shake 1.0 g with 50 ml of water for 5 minutes and filter. 25 ml of the filtrate complies with the limit test for chlorides (500 ppm). Sulphates (2.3.17). Shake 0.8 g with 100 ml of water for 5 minutes and filter. 15 ml of the filtrate complies with the limit test for sulphates (0.12 per cent). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 1.0 per cent, determined on 1.0 g by drying in an oven at 105. Storage. Store protected from light and moisture, in nonmetallic containers.
Propylene Glycol
1, 2-Propanediol
OH H3 C OH
C3H8O2 Propylene Glycol is (RS)-propane-1,2-diol.
Mol. Wt. 75.1
Propyl Gallate
O HO HO OH
C10H12O5 Mol. Wt. 212.2
Description. A clear, colourless, viscous liquid; practically odourless; hygroscopic.
CH3
Identification
A. To 0.5 ml of a 0.01 per cent w/v solution, cooled in ice, add 5 ml of a cooled mixture of 10 ml of water and 90 ml of sulphuric acid. Heat for 10 minutes on a water-bath at 70, cool and add 0.2 ml of a 3 per cent w/v solution of ninhydrin in a 2.5 per cent w/v solution of sodium metabisulphite; a violet colour slowly appears.
Propyl Gallate is propyl 3,4,5-trihydroxybenzoate.
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PROPYLPARABEN
IP 2007
B. Heat 0.15 ml with 0.1 g of boric acid; a pleasant odour develops. C. Add 1 ml to 0.5 g of potassium bisulphate and heat gently; a fruity odour develops and when the solution is heated to dryness, no sharp, acrid smell of acrolein is perceptible.
Tests
Appearance of solution. The substance under examination is clear (2.4.1), and colourless (2.4.1). Acidity. Mix 10 ml with 40 ml of water and add 0.1 ml of bromothymol blue solution. The solution is greenish yellow and not more than 0.05 ml of 0.1 M sodium hydroxide is required to change the colour to blue. Boiling range (2.4.8). 184 to 189. Relative density (2.4.29). 1.035 to 1.040. Refractive index (2.4.27). 1.431 to 1.433. Heavy metals (2.3.13). Dilute 3 ml to 12 ml with water. The resulting solution complies with the limit test for heavy metals, Method D (5 ppm). Use lead standard solution (1 ppm Pb) to prepare the standard. Oxidising substances. To 10 ml add 5 ml of water, 2 ml of potassium iodide solution and 2 ml of 1 M sulphuric acid and allow to stand in a ground-glass-stoppered flask protected from light for 15 minutes. Titrate the liberated iodine with 0.05 M sodium thiosulphate using 1 ml of starch solution, added towards the end of the titration, as indicator. Not more than 0.2 ml of 0.05 M sodium thiosulphate is required. Reducing substances. Mix 1 ml with 1 ml of 6 M ammonia and heat in a water-bath at 60 for 5 minutes; the solution is not yellow. Immediately add 0.15 ml of 0.1 M silver nitrate; the solution does not change its appearance within 5 minutes. Ethylene glycol and diethylene glycol. Determine by gas chromatography (2.4.13). Test solution. Dissolve 2 g of the substance under examination in sufficient ethanol (95 per cent) to produce 100 ml. Reference solution. Dissolve 2 g of the substance under examination, 0.02 g of ethylene glycol and 0.02 g of diethylene glycol in ethanol (95 per cent) and dilute to 100 ml with the same solvent. Chromatographic system a glass column 1.5 m x 3 mm, packed with 12 per cent Sorbitol on untreated siliceous earth (such as Chromosorb W-NAW (SINS), temperature: column: 165, inlet port and detector at 260, flow rate. 30 ml per minute of the carrier gas.
Inject 3 l or other suitable volume of the test solution. Record the chromatograms adjusting the sensitivity so that the height of the peak due to propylene glycol is more than 50 per cent of the full-scale deflection in the chromatograms. Inject the same volume of reference solution and record the chromatograms. The order of elution is propylene glycol, ethylene glycol and diethylene glycol. The test is not valid unless in the chromatogram obtained with the reference solution, the resolution between the peaks due to propylene glycol (first peak) and ethylene glycol (second peak) is not less than 1.0. No peaks corresponding to ethylene glycol and diethylene glycol are obtained in the chromatogram obtained with the test solution. Sulphated ash (2.3.18). Not more than 0.01 per cent w/w, determined by the following method. Heat 50 g until it burns, and ignite. Allow to cool, moisten the residue with sulphuric acid and ignite; repeat the operations. Water (2.3.43). Not more than 0.2 per cent, determined on 5.0 g. Storage. Store protected from moisture.
Propylparaben
Propyl Hydroxybenzoate
O O HO
C10H12O3 Propylparaben is propyl 4-hydroxybenzoate. Propylparaben contains not less than 99.0 per cent and not more than 101.0 per cent of C10H12O3, calculated on the dried basis. Description. A white, crystalline powder; odourless. Mol. Wt. 180.2
CH3
Identification
A. When examined in the range 230 nm to 360 nm (2.4.7), a 0.0005 per cent w/v solution in ethanol (95 per cent) shows an absorption maximum at about 258 nm; absorption at 258 nm, 0.44 to 0.47. B. To about 10 mg in a test-tube add 1 ml of sodium carbonate solution, heat to boiling for 30 seconds and cool (solution A). To a further 10 mg in a test-tube add 1 ml of sodium carbonate solution; the substance partly dissolves (solution B). Add at
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PROPYLTHIOURACIL
the same time to each of the solutions A and B 5 ml of aminophenazone solution and 1 ml of potassium ferricyanide solution and mix. Solution B is yellow to orange-brown. Solution A is orange to red and the colour is clearly more intense than any similar colour that may be obtained with solution B.
Tests
Appearance of solution. A 10.0 per cent w/v solution in ethanol (95 per cent) is clear (2.4.1), and not more intensely coloured than reference solution BYS6 (2.4.1). Acidity. Dissolve 1.0 g in sufficient ethanol (95 per cent) to produce 10 ml. To 2 ml of the solution add 3 ml of ethanol (95 per cent), 5 ml of carbon dioxide-free water and 0.1 ml of bromocresol green solution. Not more than 0.1 ml of 0.1 M sodium hydroxide is required to change the colour of the solution. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel HF254. Mobile phase. A mixture of 88 volumes of dichloromethane, 10 volumes of ethyl acetate and 2 volumes of anhydrous formic acid. Test solution. Dissolve 0.2 g of the substance under examination in 10 ml of methanol. Reference solution. A 0.02 per cent w/v solution of the substance under examination in methanol. Apply to the plate 5 l of each solution. After development, dry the plate in a current of hot air and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Chlorides (2.3.12). Heat 2.0 g with 100 ml of water, cool, add sufficient water to restore the original volume, and filter. 25 ml of the filtrate complies with the limit test for chlorides (500 ppm). Sulphates.To 10 ml of the filtrate obtained in the test for Chlorides add 0.15 ml of dilute hydrochloric acid and 0.1 ml of barium chloride solution; no turbidity is produced within 10 minutes. Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying over silica gel for 5 hours. Assay. Weigh accurately about 80 mg, transfer to a glassstoppered flask, add 25 ml of 2 M sodium hydroxide and boil gently under a reflux condenser for 30 minutes. Cool and add 25.0 ml of 0.0333 M potassium bromate, 5 ml of a 12.5 per cent w/v solution of potassium bromide and 40 ml of glacial acetic acid, cool in ice, add 10 ml of hydrochloric acid, immediately
stopper the flask and allow to stand for 15 minutes. Add 15 ml of potassium iodide solution, mix and titrate the liberated iodine with 0.1 M sodium thiosulphate using 2 ml of starch solution, added towards the end of the titration, as indicator. Repeat the operation without the substance under examination. The difference between the titrations represents the amount of potassium bromate required. The volume of 0.0333 M potassium bromate is equivalent to half of the volume of 0.1 M sodium thiosulphate required for the titration. 1 ml of 0.0333 M potassium bromate is equivalent to 0.006007 g of C10H12O3. Storage. Store protected from moisture.
Propylthiouracil
H3C H N S NH O
C7H10N2OS Mol. Wt. 170.2
Propylthiouracil is 2,3-dihydro-6-propyl-2-thioxopyrimidin4(1H)-one. Propylthiouracil contains not less than 98.0 per cent and not more than 100.5 per cent of C7H10N2OS, calculated on the dried basis. Description. A white or pale cream-coloured crystals or crystalline powder; odourless.
Identification
Test A may be omitted if tests B and C are carried out. Tests B and C may be omitted if test A is carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with propylthiouracil RS or with the reference spectrum of propylthiouracil. B. Examine the chromatograms obtained in the test for Related substances in ultraviolet light at 254 nm before exposure of the plate to iodine vapour. The principal spot in the chromatogram obtained with test solution (b) corresponds to that in the chromatogram obtained with reference solution (b). C. To about 20 mg add 8 ml of bromine water and shake for a few minutes. Boil until the mixture is decolorised, allow to cool and filter. Add 2 ml of barium chloride solution; a white
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PROPYLTHIOURACIL TABLETS
IP 2007
precipitate is produced. Add 5 ml of 2 M sodium hydroxide; the precipitate does not become violet.
Propylthiouracil Tablets
Propylthiouracil Tablets contain not less than 92.5 per cent and not more than 107.5 per cent of the stated amount of propylthiouracil, C7H10N2OS.
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 100 volumes of chloroform, 12 volumes of 2-propanol and 0.2 volume of glacial acetic acid. Test solution (a). Dissolve 0.1 g of the substance under examination in 10 ml of methanol. Test solution (b). Dissolve 0.1 g of the substance under examination in 100 ml of methanol. Reference solution (a). A 0.01 per cent w/v solution of the substance under examination in methanol. Reference solution (b). A 0.1 per cent w/v solution of propylthiouracil RS in methanol. Reference solution (c). A 0.0005 per cent w/v solution of thiourea in methanol. Apply to the plate 10 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. Expose the plate to iodine vapour for 10 minutes. By both methods of visualisation, any spot corresponding to thiourea in the chromatogram obtained with test solution (a) is not more intense than the spot in the chromatogram obtained with reference solution (c) and any other secondary spot is not more intense than the spot in the chromatogram obtained with reference solution (a). Arsenic (2.3.10). Dissolve 2.0 g in 50 ml of water and add 10 ml of stannated hydrochloric acid AsT. The resulting solution complies with the limit test for arsenic (5 ppm). Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method C (20 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in oven at 105. Assay. Weigh accurately about 0.3 g, add 30 ml of water and 30.0 ml (n1 ml) of 0.1 M sodium hydroxide, boil and shake until solution is complete. Add 50 ml of 0.1 M silver nitrate with stirring, boil gently for 5 minutes, cool and titrate with 0.1 M sodium hydroxide, determining the end-point potentiometrically (2.4.25) (n2 ml). Record the total volume (n1 + n2 ml) of 0.1 M sodium hydroxide added. 1 ml of 0.1 M sodium hydroxide is equivalent to 0.008511 g of C7H10N2OS. Storage. Store protected from light and moisture.
Identification
A. Shake a quantity of the powdered tablets containing 50 mg of Propylthiouracil with 20 ml of methanol for 10 minutes, filter and evaporate to dryness. On the residue, determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with propylthiouracil RS or with the reference spectrum of propylthiouracil. B. Shake a quantity of the powdered tablets containing 50 mg of Propylthiouracil with 60 ml of methanol for 20 minutes, dilute to 100 ml with methanol and filter. Dilute 5 ml of the filtrate to 250 ml with methanol. When examined in the range 230 nm to 360 nm (2.4.7), the resulting solution shows an absorption maximum only at about 274 nm. C. Extract a quantity of the powdered tablets in a continuous extraction apparatus (2.1.8) with ether and evaporate the solution to dryness. The residue, after drying at 105, melts at about 219 (2.4.21).
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 100 volumes of chloroform, 12 volumes of 2-propanol and 0.2 volume of glacial acetic acid. Test solution. Shake a quantity of the powdered tablets containing 50 mg of Propylthiouracil with 5 ml of methanol for 15 minutes, filter and use the filtrate. Reference solution (a). Dilute 1 volume of the test solution to 100 volumes with methanol. Reference solution (b). A 0.001 per cent w/v solution of thiourea in methanol. Apply to the plate 10 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. Expose the plate to iodine vapour for 10 minutes. By both methods of visualisation, any spot corresponding to thiourea in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (b) and any other secondary spot is not more intense than the spot in the chromatogram obtained with reference solution (a). Other tests. Comply with the tests stated under Tablets
1000
IP 2007
PROPYPHENAZONE
Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 0.15 g of Propylthiouracil, dissolve in a mixture of 20 ml of 0.1 M sodium hydroxide and 75 ml of water with the aid of gentle heat. Cool, add 4 g of sodium acetate, just acidify the solution to litmus paper with 6 M acetic acid, add 0.5 ml of a freshly prepared 0.5 per cent w/v solution of 1,5-diphenylcarbazone in ethanol (95 per cent) and titrate with 0.02 M mercuric nitrate until a pinkish violet colour persists for 2 to 3 minutes. 1 ml of 0.02 M mercuric nitrate is equivalent to 0.006808 g of C7H10N2OS. Storage. Store protected from light and moisture.
produce 50 ml (solution A). To 1 ml of solution A add 0.1 ml of ferric chloride solution; a brownish red colour is produced which becomes yellow on addition of 1 ml of 2 M hydrochloric acid.
Tests
Appearance of solution. Solution A is clear (2.4.1), and colourless (2.4.1). Acidity or alkalinity. To 10 ml of solution A add 0.1 ml of phenolphthalein solution; the solution is colourless. Add 0.2 ml of 0.01 M sodium hydroxide; the solution is pink. Add 0.4 ml of 0.01 M hydrochloric acid; the solution becomes colourless. Add 0.2 ml of methyl red solution; the solution is orange or red. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel HF254. Mobile phase. A mixture of 45 volumes of cyclohexane, 45 volumes of ethyl acetate and 10 volumes of 1-butanol. Test solution (a). An 8 per cent w/v solution of the substance under examination in methanol.
Propyphenazone
O H3C
CH3 CH3
Test solution (b). A 1.6 per cent w/v solution of the substance under examination in methanol. Reference solution (a). A 0.016 per cent w/v solution of the substance under examination in methanol. Mol. Wt. 230.3 Reference solution (b). A 1.6 per cent w/v solution of propyphenazone RS in methanol. Apply to the plate 5 l of each solution. After development, dry the plate in a current of hot air for 15 minutes and examine in ultraviolet light at 254 nm. Spray the plate with a mixture of equal volumes of potassium ferricyanide solution and ferric chloride solution. By both methods of visualisation, any secondary spot in the chromatogram obtained with test solution (a) is not more intense than the spot in the chromatogram obtained with reference solution (a). Arsenic (2.3.10). Mix 1.0 g with 10 ml of a 2 per cent w/v solution of magnesium nitrate in ethanol in a silica or platinum dish, evaporate on a water-bath and heat gradually in order to incinerate. If the material remains incompletely carbonised, moisten with a small quantity of nitric acid and ignite again. Cool, add 3 ml of hydrochloric acid and heat on a water-bath to dissolve the residue. The resulting solution complies with the limit test for arsenic (10 ppm). Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (10 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 60 over phosphorus pentoxide at a pressure of 1.5 to 2.5 kPa for 4 hours.
CH3
C14H18N2O
Propyphenazone is 4-isopropyl-2,3-dimethyl-1-phenyl-3pyrazolin-5-one. Propyphenazone contains not less than 99.0 per cent and not more than 101.0 per cent of C14H18N2O, calculated on the dried basis. Description. A white or slightly yellowish, crystalline powder; odourless.
Identification
Test A may be omitted if tests B and C are carried out. Tests B and C may be omitted if test A is carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with propyphenazone RS or with the reference spectrum of propyphenazone. B. In the test for Related substances, the principal spot in the chromatogram obtained with test solution (b) corresponds to that in the chromatogram obtained with reference solution (b). C. Dissolve 2 g in sufficient of a mixture of equal volumes of ethanol (95 per cent) and carbon dioxide-free water to
1001
PROTAMINE SULPHATE
IP 2007
Assay. Weigh accurately about 0.2 g of the dried material, dissolve in 30 ml of anhydrous glacial acetic acid. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.02303 g of C14H18N2O. Storage. Store protected from moisture.
Iron (2.3.14). Dissolve 2.0 g in water with the aid of heat and dilute to 20 ml with water. The resulting solution complies with the limit test for iron (20 ppm). Mercury. Add 20 ml of a mixture of equal volumes of nitric acid and sulphuric acid to 2.0 g in a 250-ml flask fitted with a ground-glass stopper, boil under a reflux condenser for 1 hour, cool and carefully dilute with water. Boil until nitrous fumes are no longer evolved. Cool, carefully dilute the solution to 200 ml with water, mix and filter. Transfer 50 ml of the filtrate to a separating funnel. Shake with successive small quantities of chloroform until the chloroform layer remains colourless. To the aqueous layer add 25 ml of 1 M sulphuric acid, 115 ml of water and 10 ml of a 20 per cent w/v solution of hydroxylamine hydrochloride. Titrate with dithizone solution; after each addition, shake the mixture 20 times and towards the endpoint of the titration allow to separate and discard the chloroform layer. Titrate until a greenish blue colour is produced. Calculate the content of mercury using the equivalent of mercury in g per ml of titrant determined in the standardisation of the dithizone solution (10 ppm). Nitrogen (2.3.30). 21.0 to 26.0 per cent, calculated on the dried basis, determined by Method C. Sulphates. 16 to 24 per cent, determined by the following method. Dissolve 0.15 g in 15 ml of water in a beaker, add 5 ml of 2 M hydrochloric acid and heat to boiling. Slowly add to the boiling solution 10 ml of barium chloride solution. Cover and heat on a water-bath for 1 hour. Filter and wash the precipitate several times with small quantities of hot water. Dry and ignite the residue to constant weight at 600. 1 g of the residue is equivalent to 0.4117 g of SO4. Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity, using 0.5 mg dissolved in 0.5 ml of water for injections. Loss on drying (2.4.19). Not more than 5.0 per cent, determined on 1.0 g by drying in an oven at 105 for 3 hours. Assay. Prepare solutions of the substance under examination in water containing (1) 0.015 per cent w/v (2) 0.01 per cent w/v and (3) 0.005 per cent w/v. Titrate each of the solutions in duplicate with a 174 IU per ml or a suitable dilution of heparin sodium RS using the following procedure. Introduce an accurately measured volume of the solution to be titrated, for example 1.5 ml, into the cell of a suitable spectrophotometer, set the instrument at a suitable wavelength (none is critical) in the visible range and add the titrant in small volumes until there is a sharp increase in the absorbance. Note the volume of titrant added. Carry out three independent assays. For each individual titration, calculate the number of Units of heparin titrated per mg of the substance under examination. Calculate the result of the assay as the average of the 18 values. Test the linearity of the response by standard statistical methods. The assay is
Protamine Sulphate
Protamine Sulphate is a purified mixture of the sulphates of basic peptides prepared from the sperm or mature testes of suitable species of fish. It binds with heparin in solution, inhibiting its anticoagulant activity. It is prepared in conditions designed to minimise the degree of Microbial contamination. Each mg of Protamine Sulphate precipitates not less than 100 Units of heparin sodium RS, calculated on the dried basis. Description. A white or almost white powder; hygroscopic.
Identification
A. Produces a precipitate under the conditions of the Assay. B. Dissolve 0.2 g in 5 ml of water and dilute to 10 ml with the same solvent (solution A). To 0.5 ml of solution A add 4.5 ml of water, 1 ml of a 10 per cent w/v solution of sodium hydroxide and 1 ml of a 0.02 per cent w/v solution of 1-naphthol and mix. Cool to 5 and add 0.5 ml of alkaline sodium hypobromite solution; an intense red colour is produced. C. Heat 2 ml of solution A in a water-bath at 60, add 0.1 ml of mercuric sulphate solution and mix; no precipitate is produced. Cool the mixture in ice; a white precipitate is produced. D. Gives reaction A of sulphates (2.3.1).
Tests
Appearance of solution. To 2.5 ml of solution A add 7.5 ml of water. The resulting solution is not more opalescent than opalescence standard OS2 (2.4.1), and not more intensely coloured than reference solution BYS6 (2.4.1). Specific optical rotation (2.4.22). 65.0 to 85.0, determined at 20 in a 1.0 per cent w/v solution in 0.1 M hydrochloric acid. Light absorption (2.4.7). Dilute 2.5 ml of solution A to 5.0 ml with water. Absorbance of the resulting solution at 260 to 280 nm is not more than 0.1. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm).
1002
IP 2007
PROTHIONAMIDE
not valid unless the standard deviations calculated for the results obtained with each test solution are less than 5 per cent of the average result. Protamine Sulphate intended for use in the manufacture of Parenteral Preparations without a further appropriate procedure for the removal of bacterial endotoxins complies with the following additional requirement. Bacterial endotoxins (2.2.3). Not more than 7.0 Endotoxin Units per mg of protamine sulphate. Protamine Sulphate intended for use in the manufacture of Parenteral Preparations without a further appropriate sterilisation procedure complies with the following additional requirement. Sterility. Complies with the test for sterility (2.2.11). Storage. Store protected from moisture. If it is intended for use in the manufacture of Parenteral Preparations, the container should be sterile and sealed so as to exclude micro-organisms. Labelling. The label states whether or not the contents are intended for use in the manufacture of Parenteral Preparations.
Light absorption. Dilute the injection, if necessary, with water to produce a solution containing 1 per cent w/v solution of Protamine Sulphate. Absorbance of the resulting solution at 260 to 280 nm, not more than 0.1 (2.4.7). Bacterial endotoxins (2.2.3). Not more than 7.0 Endotoxin Units per mg of protamine sulphate. Abnormal toxicity. Complies with the test for abnormal toxicity (2.2.1), using a volume containing 0.5 mg of Protamine Sulphate. Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. Prepare solutions of the substance under examination in water containing (1) 0.015 per cent w/v (2) 0.01 per cent w/v and (3) 0.005 per cent w/v. Titrate each of the solutions in duplicate with a 174 IU per ml or a suitable dilution of heparin sodium RS using the following procedure. Introduce an accurately measured volume of the solution to be titrated, for example 1.5 ml, into the cell of a suitable spectrophotometer, set the instrument at a suitable wavelength (none is critical) in the visible range and add the titrant in small volumes until there is a sharp increase in the absorbance. Note the volume of titrant added. Carry out three independent assays. For each individual titration, calculate the number of Units of heparin titrated per mg of the substance under examination. Calculate the result of the assay as the average of the 18 values. Test the linearity of the response by standard statistical methods. The assay is not valid unless the standard deviations calculated for the results obtained with each test solution are less than 5 per cent of the average result. Storage. Store protected from light, in single dose containers. Labelling. The label states (1) that the dose is calculated from the results of determinations of the amount required to produce an acceptable blood-clotting time in the patient; (2) the approximate number of Units of heparin activity 1 ml is capable of neutralising.
Protamine Sulphate Injection

Protamine Sulphate Injection is a sterile solution of Protamine Sulphate in Water for Injections. Protamine Sulphate Injection contains not less than 80.0 per cent of the stated amount of protamine sulphate.
Identification
A. Produces a precipitate under the conditions of the Assay. B. Dilute a suitable volume with water to give a solution containing 0.2 per cent w/v solution of Protamine Sulphate. To 5 ml of this solution add 1 ml of a 10 per cent w/v solution of sodium hydroxide and 1 ml of a 0.02 per cent w/v solution of 1-naphthol and mix. Cool to 5 and add 0.5 ml of alkaline sodium hypobromite solution; an intense red colour is produced. C. Heat 2 ml in a water-bath at 60, add 0.1 ml of mercuric sulphate solution and mix; no precipitate is produced. Cool the mixture in ice; a white precipitate is produced. D. Gives reaction A of sulphates (2.3.1).
Prothionamide
N CH3
Tests
pH (2.4.24). 2.5 to 3.5. Optical rotation (2.4.22). 0.52 to 0.68, determined in a solution prepared by diluting the injection with 0.5 M hydrochloric acid so as to contain 0.8 per cent w/v of Protamine Sulphate.
S
C9H12N2S
NH2
Mol. wt. 180.3
Prothionamide is 2-propyl-4-pyridinecarbothioamide.
1003
PROTHIONAMIDE TABLETS
IP 2007
Prothionamide contains not less than 99.0 per cent and not more than 101.0 per cent of C8H10N2S ,calculated on the dried basis. Description. A yellow, crystalline powder.
peak in the chromatogram obtained with the reference solution (1.0 per cent). Heavy Metals (2.3.13). 2.0 g complies with the limit test for heavy metals, Method B (10 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105 for 3 hours. Assay. Determine by liquid chromatography (2.4.14) as described under Related substances using the following solutions. Test solution. Dissolve 50.0 mg of the substance under examination in the mobile phase and dilute to 100.0 ml with the mobile phase. Dilute 5.0 ml of this solution to a 50.0 ml with the mobile phase. Reference solution. A 0.05 per cent w/v solution of prothionamide RS in the mobile phase. Dilute 5.0 ml of the solution to 50.0 ml with the mobile phase. Inject the reference solution. The tailing factor is not more than 2.0, the column efficiency in not less than 5000 theoretical plates and the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject alternatively the test solution and the reference solution. Calculate the content of C9H12N2S. Storage. Store protected from light and moisture.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with prothionamide RS. B. When examined in the range 230 nm to 350 nm (2.4.7), a 0.002 per cent w/v solution in ethanol (95 per cent) shows an absorption maximum only at about 291 nm. The absorbance at 291 nm is about 0.78.
Tests
Acidity. Dissolve 2.0 g in 20 ml of methanol, heating to about 50, and add 20 ml of water. Cool slightly, shake until crystallisation occurs, if any and allow to cool to room temperature. Add 60 ml of water and titrate with 0.1 M sodium hydroxide using 0.2 ml of cresol red solution as indicator. Not more than 0.2 ml of 0.1 M sodium hydroxide is required to change the colour of the indicator to red. Related substances. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 50 mg of the substance under examination in the mobile phase and dilute to 100 ml with the mobile phase. Reference solution. A 0.025 per cent w/v solution of prothionamide RS in the mobile phase. Dilute 1 ml of the solution to 100 ml with the mobile phase. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: 60 volumes of a buffer solution prepared by mixing 2.0 ml of triethylamine with 1000 ml of water, adjusting the pH to 6.0 with dilute orthophosphoric acid and 40 volumes of acetonitrile, flow rate.1 ml per minute, spectrophotometer set at 290 nm, a 20 l loop injector. Inject the reference solution.The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject alternatively the test solution and the reference solution. In the chromatogram obtained with the test solution the area of any individual impurity peak is not more than the area of the principal peak in the chromatogram obtained with the reference solution (0.5 per cent) and the sum of the areas of all such peaks is not more than twice the area of the principal
Prothionamide Tablets
Prothionamide Tablets contain not less than 90.0 per cent not more than 110.0 per cent of the stated amount of prothionamide, C9H12N2S. The tablets may be coated.
Identification
A. Extract a quantity of the powdered tablets containing 25 mg of Prothionamide with 5 ml of methanol, filter and evaporate the filtrate to dryness. The residue complies with the following test. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with prothionamide RS. B. In the Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the peak in the chromatogram obtained with the reference solution.
Tests
Dissolution (2.5.2). Apparatus. No 2 Medium. 900 ml 0.1 M hydrochloric acid.
1004
IP 2007
PSEUDOEPHEDRINE HYDROCHLORIDE
Speed and time. 100 rpm and 30 minutes. Withdraw a suitable volume of the medium and filter. Measure the absorbance of the filtered solution, suitably diluted with the dissolution medium if necessary, at the maximum at about 290 nm (2.4.7). Calculate the content of C9H12N2S in the medium from the absorbance obtained from a solution of known concentration of prothionamide RS in the same medium. D. Not less than 75 per cent of the stated amount of C9H12N2S. Related substances. Determine by liquid chromatography (2.4.14) as described under Assay using the following solutions. Test solution. Weigh accurately a quantity of the powdered tablets containing 50 mg of Prothionamide disperse in the mobile phase, shake, dilute to 100 ml with the mobile phase and filter. Reference solution. A solution containing 0.025 per cent w/v of prothionamide RS in the mobile phase. Dilute 1 ml of the solution to 100 ml with the mobile phase. Inject the reference solution. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent Inject alternatively the test solution and the reference solution. In the chromatogram obtained with the test solution the area of any individual impurity peak is not more than the area of the peak in the chromatogram obtained with the reference solution (0.5 per cent) and the sum of the areas of all such impurities is not more than twice the area of the peak in the chromatogram obtained with the reference solution (1.0 per cent). Other tests. Comply with the test stated under the Tablets. Assay. Determine by liquid chromatography (2.4.14). Test solution. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 50 mg of Prothionamide, disperse in the mobile phase, shake and dilute to 100.0 ml with the mobile phase. Dilute 5.0 ml of the resulting solution to 50.0 ml with the mobile phase. Reference solution. A 0.05 per cent w/v solution of prothionamide RS in the mobile phase. Dilute 5.0 ml of the solution to 50.0 ml with the mobile phase. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 60 volumes of a buffer solution prepared by mixing 2.0 ml of triethylamine with 1000 ml of water and adjusting the pH to 6.0 with dilute orthophosphoric acid, and 40 volumes of acetonitrile, flow rate. 1 ml per minute, spectrophotometer set at 290 nm,
a 20 l loop injector. Inject the reference solution. The test is not valid unless the tailing factor is not more than 2.0 the column efficiency is not less than 5000 theoretical plates and the relative standard deviation for replicate injections is not more than 2.0 per cent Inject alternatively the test solution and the reference solution. Calculate the content of C9H12N2S in the tablets. Storage. Store protected from light and moisture.
Pseudoephedrine Hydrochloride
H OH CH3 H
C10H15NO,HCl
NHCH3
, HCl
Mol. Wt. 201.7
Pseudoephedrine Hydrochloride is (1S,2S)-2-methylamino1-phenylpropan-1-ol hydrochloride. Pseudoephedrine Hydrochloride contains not less than 99.0 per cent and not more than 101.0 per cent of C10H15NO, HCl, calculated on the dried basis. Description. A white, crystalline powder; odourless or almost odourless.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with pseudoephedrine hydrochloride RS or with the reference spectrum of pseudoephedrine hydrochloride. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.1 per cent w/v solution shows absorption maxima at about 251 nm, 257 nm and 263 nm; absorbances at the maxima, about 0.75, about 0.98 and about 0.78, respectively. C. A 5 per cent w/v solution gives the reactions of chlorides (2.3.1).
Tests
Appearance of solution. A 5.0 per cent w/v solution is not more than very faintly opalescent and colourless (2.4.1). pH (2.4.24). 4.6 to 6.0, determined in a 5.0 per cent w/v solution. Specific optical rotation (2.4.22). +61.0 to +62.5, determined in a 5.0 per cent w/v solution using a 2-dm tube. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G.
1005
PSEUDOEPHEDRINE SYRUP
IP 2007
Mobile phase. A mixture of 40 volumes of butyl acetate, 20 volumes of acetone, 20 volumes of 1-butanol, 10 volumes of 5 M ammonia and 10 volumes of methanol. Test solution. A 10 per cent w/v solution of the substance under examination in ethanol (95 per cent). Reference solution. A 0.1 per cent w/v solution of the substance under examination in ethanol (95 per cent). Apply to the plate 10 l of each solution. After development, dry the plate in a current of warm air, spray with a solution containing 0.3 g of ninhydrin in a mixture of 100 ml 1-butanol and 3 ml of glacial acetic acid and heat at 120 for 20 minutes. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Ignore any yellow spot near the line of application. Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105 for 3 hours. Assay. Weigh accurately about 0.5 g, dissolve in a mixture of 50 ml of anhydrous glacial acetic acid and 10 ml of mercuric acetate solution. Titrate with 0.1 M perchloric acid, using crystal violet solution as indicator. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.02017 g of C10H15NO, HCl. Storage. Store protected from light and moisture.
the saame manner or with the reference spectrum of pseudoephedrine. B. The residue obtained in test A melts at about 118 (2.4.21). C. Dissolve 50 mg of the residue obtained in test A in 10 ml of 0.1 M hydrochloric acid; it is dextro-rotatory.
Tests
Other tests. Complies with the tests stated under Oral Liquids. Assay. Determine by liquid chromatography (2.4.14). Test solution. To an accurately measured volume of the syrup containing about 0.12 g of Pseudoephedrine Hydrochloride, add 50 ml of 0.1 M hydrochloric acid mix, add sufficient 0.1 M hydrochloric acid to produce 100.0 ml, filter and use the filtrate. Reference solution. A 0.12 per cent w/v solution of pseudoephedrine hydrochloride RS in 0.1 M hydrochloric acid. Chromatographic system a stainless steel column 25 cm x 4.2 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 85 volumes of ethanol and 15 volumes of a 0.4 per cent w/v solution of ammonium acetate, flow rate. 1.5 ml per minute, spectrophotometer set at 254 nm, a 20 l loop injector. Inject the reference solution and record the chromatogram. The test is not valid unless the relative standard deviation is not more than 2.0 per cent and the tailing factor is not more than 1.5. Inject alternately the test solution and the reference solution. Determine the weight per ml of the syrup (2.4.29), and calculate the content of C10H15NO, HCl weight in volume. Storage. Store protected from light and moisture.
Pseudoephedrine Syrup
Pseudoephedrine Hydrochloride Syrup
Pseudoephedrine Syrup is a solution of Pseudoephedrine Hydrochloride in a suitable flavoured vehicle. Pseudoephedrine Syrup contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of pseudoephedrine hydrochloride, C10H15NO, HCl.
Identification
A. Shake a quantity of the syrup containing 120 mg of Pseudoephedrine Hydrochloride with two quantities, each of 30 ml, of ether, and discard the ether layer. Add 4 ml of 1 M sodium hydroxide to the aqueous layer and extract with two quantities, each of 10 ml, of ether. Dry the combined ether extracts with anhydrous sodium sulphate, filter and evaporate the filtrate to dryness. On the residue, determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with pseudoephedrine hydrochloride RS treated in
Pseudoephedrine Tablets
Pseudoephedrine Hydrochloride Tablets
Pseudoephedrine Tablets contain not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of pseudoephedrine hydrochloride, C10H15NO, HCl. The tablets may be coated.
Identification
A. Shake a quantity of the powdered tablets containing 60 mg of Pseudoephedrine Hydrochloride with 10 ml of water and
1006
IP 2007
PSORALEN
filter. Shake the filtrate with 10 ml of ether, discard the ether layer. Add 1 ml of 1 M sodium hydroxide to the aqueous layer and extract with two quantities, each of 10 ml, of ether. Dry the combined ether extracts with anhydrous sodium sulphate, filter and evaporate the filtrate to dryness. On the residue, determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with pseudoephedrine hydrochloride RS treated in the same manner or with the reference spectrum of pseudoephedrine. B. In the test for Related substances, the principal spot in the chromatogram obtained with test solution (b) corresponds to that in the chromatogram obtained with reference solution (b).
Reference solution. A 0.12 per cent w/v solution of pseudoephedrine hydrochloride RS in methanol (50 per cent). Chromatographic system a stainless steel column 20 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (10 m), mobile phase: 0.005 M dioctyl sodium sulphosuccinate in a mixture of 65 volumes of methanol, 35 volumes of water and 1 volume of glacial acetic acid, flow rate. 1.5 ml per minute, spectrophotometer set at 258 nm, a 20 l loop injector. Inject alternately the test solution and the reference solution. Calculate the content of C10H15NO,HCl in the tablets. Storage. Store protected from light and moisture.
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 40 volumes of butyl acetate, 20 volumes of acetone, 20 volumes of 1-butanol, 10 volumes of 5 M ammonia and 10 volumes of methanol. Test solution (a). Add 25 ml of methanol to a quantity of the powdered tablets containing 0.5 g of Pseudoephedrine Hydrochloride, shake for 5 minutes, filter, wash the filter with methanol and evaporate the combined filtrate and washings to dryness. Dissolve the residue as completely as possible in 5 ml of methanol, centrifuge and use the supernatant liquid. Test solution (b). Dilute 1 volume of the test solution to 10 volumes with methanol. Reference solution (a). Dilute 1 volume of the test solution to 100 volumes with methanol. Reference solution (b). A 1.0 per cent w/v solution of pseudoephedrine hydrochloride RS in methanol. Apply to the plate 10 l of each solution. After development, dry the plate in a current of warm air, spray with a solution containing 0.3 g of ninhydrin in a mixture of 100 ml 1-butanol and 3 ml of glacial acetic acid and heat at 120 for 20 minutes. Any secondary spot in the chromatogram obtained with test solution (a) is not more intense than the spot in the chromatogram obtained with reference solution (a). Ignore any yellow spot near the line of application. Other tests. Comply with the tests stated under Tablets. Assay. Determine by liquid chromatography (2.4.14). Test solution. Weigh and powder 20 tablets. To a quantity of the powdered tablets containing about 0.12 g of Pseudoephedrine Hydrochloride, add 50 ml of 0.1 M hydrochloric acid mix with the aid of ultrasound for 15 minutes, add sufficient methanol to produce 100.0 ml, filter and use the filtrate.
Psoralen
O O O
C11H6O3
Mol. Wt. 186.1
Psoralen is 7H-furo[3,2-g][1]benzopyran-7-one, obtained from the fruits of Psoralea carylifolia Linn. (Fam. Leguminosae) and from the leaves of Ficus carica (Fam. Urticaceae) or prepared by synthesis. Psoralen contains not less than 95.0 per cent and not more than 101.0 per cent of C11H6O3, calculated on the dried basis. Description. Colourless needles; odourless.
Identification
A. Dissolve 1 mg in 5 ml of ethanol (95 per cent) and add 15 ml of a mixture containing 43 volumes of water, 5 volumes of acetic acid and 3 volumes of propylene glycol; a blue fluorescence is visible in ultraviolet light at 365 nm. B. Dissolve 1 mg in 2 ml of ethanol (95 per cent) and add 0.1 ml of 0.1 M sodium hydroxide; a yellow fluorescence is visible in ultraviolet light at 365 nm.
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 90 volumes of benzene and 10 volumes of ethyl acetate. Test solution. Dissolve 0.2 g of the substance under examination in 10 ml of chloroform.
1007
PYRAZINAMIDE
IP 2007
Reference solution. Dilute 1 volume of the test solution to 100 volumes with chloroform. Apply to the plate 5 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 365 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 1.0 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.1 g and dissolve in sufficient methanol to produce 100.0 ml. Dilute 2.0 ml of this solution to 100.0 ml with methanol and measure the absorbance of the resulting solution at the maximum at about 247 nm (2.4.7). Calculate the content of C11H6O3 from the absorbance obtained by repeating the operation using a final solution of 20 g of psoralen RS per ml in methanol in place of the substance under examination. Storage. Store protected from light and moisture.
range 230 nm to 290 nm, the solution shows an absorption maximum at about 268 nm; absorbance at about 268 nm, between 0.64 and 0.68. C. Boil 20 mg with 5 ml of sodium hydroxide solution; ammonia, recognisable by its odour, is evolved.
Tests
Appearance of solution. A 1.0 per cent w/v solution in carbon dioxide-free water (solution B) is clear (2.4.1), and colourless (2.4.1). Acidity or alkalinity. To 25 ml of solution B add 0.05 ml of phenolphthalein solution and 0.2 ml of 0.01 M sodium hydroxide; the solution is red. Add 1 ml of 0.01 M hydrochloric acid; the solution is colourless. Add 0.15 ml of methyl red solution; the solution is red. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 60 volumes of 1-butanol, 20 volumes of glacial acetic acid and 20 volumes of water. Test solution. Dissolve 0.1 g of the substance under examination in 10 ml of a mixture of 90 volumes of chloroform and 10 volumes of methanol. Reference solution. A 0.002 per cent w/v solution of the substance under examination in a mixture of 90 volumes of chloroform and 10 volumes of methanol.
Pyrazinamide
O N N
C5H5N3O Pyrazinamide is pyrazine-2-carboxamide. Pyrazinamide contains not less than 99.0 per cent and not more than 100.5 per cent of C5H5N3O, calculated on the anhydrous basis. Description. A white or almost white, crystalline powder; odourless or almost odourless. Mol. Wt. 123.1
NH2
Apply to the plate 20 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Heavy metals (2.3.13). 2.0 g complies with the limit test for heavy metals, Method B (10 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Water (2.4.19). Not more than 0.5 per cent, determined on 5.0 g. Assay. Weigh accurately about 0.3 g and transfer to the flask of an ammonia distillation apparatus. Add 200 ml of water and 75 ml of sodium hydroxide solution. Boil gently for 20 minutes, collecting the distillate in 50.0 ml of 0.05 M sulphuric acid. Boil vigorously to complete the distillation of the ammonia and titrate the excess of acid with 0.1 M sodium hydroxide, using methyl red solution as indicator. Repeat the operation without the substance under examination. The difference between the titrations represents the amount of acid required to neutralise the ammonia formed. 1 ml of 0.05 M sulphuric acid is equivalent to 0.01231 g of C5H5N3O. Storage. Store protected from moisture.
Identification
Test A may be omitted if tests B and C are carried out. Tests B and C may be omitted if test A is carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with pyrazinamide RS or with the reference spectrum of pyrazinamide. B. Dissolve 50 mg in water and dilute to 100 ml with the same solvent (solution A). Dilute 1 ml of solution A to 10 ml. When examined in the range 290 nm to 360 nm (2.4.7), the resulting solution shows an absorption maximum at about 310 nm. Dilute 2 ml of solution A to 100 ml with water. When examined in the
1008
IP 2007
PYRIDOXINE HYDROCHLORIDE
Pyrazinamide Tablets
Pyrazinamide Tablets contain not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of pyrazinamide, C5H5N3O.
Identification
A. Shake a quantity of the powdered tablets containing 0.25 g of Pyrazinamide with 20 ml of ethanol, filter, evaporate the filtrate to dryness and dry the residue at 105 for 30 minutes. On the residue, determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with pyrazinamide RS or with the reference spectrum of pyrazinamide. B. Shake a quantity of the powdered tablets containing 50 mg of Pyrazinamide with 50 ml of water, dilute to 100 ml with water and filter (solution A). Dilute 1 ml of solution A to 10 ml with water. When examined in the range 290 nm to 360 nm (2.4.7), the resulting solution shows an absorption maximum at about 310 nm. Dilute 2 ml of solution A to 100 ml with water. When examined in the range 230 nm to 290 nm, the resulting solution shows an absorption maximum at about 268 nm; absorbance at 268 nm, between 0.64 and 0.68. C. Boil a quantity of the powdered tablets containing 20 mg of Pyrazinamide with 5 ml of sodium hydroxide solution; ammonia, recognisable by its odour, is evolved.
occasionally, mix with the aid of ultrasound for 10 minutes and dilute to 500.0 ml with water. Filter and discard the first 20 ml of the filtrate. Dilute 5.0 ml of the filtrate to 100.0 ml with water and measure the absorbance of the resulting solution at the maximum at about 268 nm (2.4.7). Calculate the content of C5H5N3O taking 650 as the specific absorbance at 268 nm. Storage. Store protected from moisture.
Pyridoxine Hydrochloride
Vitamin B6
H3C HO OH
C8H11NO3,HCl Mol. Wt. 205.6
N OH , HCl
Pyridoxine Hydrochloride is 5-hydroxy-6-methylpyridine3,4-dimethanol hydrochloride. Pyridoxine Hydrochloride contains not less than 99.0 per cent and not more than 101.0 per cent of C8H11NO3,HCl, calculated on the dried basis. Description. A white or almost white, crystalline powder; odourless.
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 60 volumes of 1-butanol, 20 volumes of glacial acetic acid and 20 volumes of water. Test solution. Shake a quantity of the powdered tablets containing 0.1 g of Pyrazinamide with 50 ml of a mixture of 90 volumes of chloroform and 10 volumes of methanol, filter, evaporate to dryness and dissolve the residue in sufficient of the same solvent mixture to produce 10 ml. Reference solution. Dilute 1 volume of the test solution to 500 volumes with a mixture of 90 volumes of chloroform and 10 volumes of methanol. Apply to the plate 20 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Other tests. Comply with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 0.1 g of Pyrazinamide, add 200 ml of water, allow to stand for 10 minutes, swirling
Identification
Test A may be omitted if tests B, C and D are carried out. Tests B may be omitted if tests A, C and D are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with pyridoxine hydrochloride RS or with the reference spectrum of pyridoxine hydrochloride. B. When examined in the range 250 nm to 360 nm (2.4.7), a 0.001 per cent w/v solution in 0.1 M hydrochloric acid shows an absorption maximum at 288 nm to 296 nm; absorbance at the maximum, 0.420 to 0.445. A solution prepared by diluting 1 ml of a 0.1 per cent w/v solution in 0.1 M hydrochloric acid to 100 ml with 0.025 M standard phosphate buffer shows absorption maxima at 248 nm to 256 nm and at 320 nm to 327 nm; absorbances at the maxima, 0.175 to 0.195 and 0.345 to 0.365, respectively. C. In the test for Related substances, the principal spot in the chromatogram obtained with test solution (b) corresponds to that in the chromatogram obtained with reference solution (b).
1009
PYRIDOXINE TABLETS
IP 2007
D. A 5 per cent w/v solution gives reaction A of chlorides (2.3.1).
Pyridoxine Tablets
Pyridoxine Hydrochloride Tablets
Pyridoxine Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of pyridoxine hydrochloride, C8H11NO3, HCl.
Tests
Appearance of solution. A 5.0 per cent w/v solution is clear (2.4.1), and not more intensely coloured than reference solution YS7 (2.4.1). pH (2.4.24). 2.4 to 3.0, determined in a 5.0 per cent w/v solution. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 65 volumes of acetone, 13 volumes of dichloromethane, 13 volumes of tetrahydrofuran and 9 volumes of strong ammonia solution. Test solution (a). A 10 per cent w/v solution of the substance under examination in water. Test solution (b). A 1 per cent w/v solution of the substance under examination water. Reference solution (a). A 0.025 per cent w/v solution of the substance under examination in water. Reference solution (b). A 1 per cent w/v solution of pyridoxine hydrochloride RS in water. Apply to the plate 2 l of each solution. After development, dry the plate in air and spray with a 5 per cent w/v solution of sodium carbonate in a mixture of 70 volumes of water and 30 volumes of ethanol (95 per cent). Dry in a current of air, spray with a 0.1 per cent w/v solution of 2,6-dichloroquinone4-chloroimide in ethanol (95 per cent) and examine immediately. Any secondary spot in the chromatogram obtained with test solution (a) is not more intense than the spot in the chromatogram obtained with reference solution (a). Ignore any spots remaining on the line of application. Heavy metals (2.3.13). 12 ml of a 5.0 per cent w/v solution complies with the limit test for heavy metals, Method D (20 ppm). Use lead standard solution (1 ppm Pb) to prepare the standard. Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.15 g, dissolve in a mixture of 5 ml of anhydrous glacial acetic acid and 6 ml of mercuric acetate solution. Titrate with 0.1 M perchloric acid, using crystal violet solution as indicator, until a green colour is produced. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.02056 g of C8H11NO3, HCl. Storage. Store protected from moisture.
Identification
A. Shake a quantity of the powdered tablets containing 20 mg of Pyridoxine Hydrochloride with 50 ml of 0.025 M standard phosphate buffer for 15 minutes and dilute to 100 ml with the same solvent. Mix, filter and dilute 5 ml of the filtrate to 100 ml with the same solvent. When examined in the range 230 nm to 360 nm (2.4.7), the resulting solution exhibits two maxima, at about 254 nm and 324 nm. B. Triturate a quantity of the powdered tablets containing 20 mg of Pyridoxine Hydrochloride with 50 ml of water and allow to stand for 20 minutes. To 1 ml of the supernatant liquid add 10 ml of a 5 per cent w/v solution of sodium acetate, 1 ml of water and 1 ml of a 0.5 per cent w/v solution of 2,6-dichloroquinone-4-chloroimide in ethanol (95 per cent) and shake; a blue colour is produced which fades rapidly and becomes brown. Repeat the operation but adding 1 ml of a 0.3 per cent w/v solution of boric acid in place of 1 ml of water; no blue colour is produced.
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 65 volumes of acetone, 13 volumes of dichloromethane, 13 volumes of tetrahydrofuran and 9 volumes of strong ammonia solution. Test solution. Shake a quantity of the powdered tablets containing 40 mg of Pyridoxine Hydrochloride with 10 ml of water for 15 minutes, filter and use the filtrate. Reference solution. Dilute 1 ml of test solution to 200 ml with water. Apply to the plate 10 l of each solution. After development, dry the plate in air and spray with a 5 per cent w/v solution of sodium carbonate in a mixture of 70 volumes of water and 30 volumes of ethanol (95 per cent). Dry it in a current of air, spray with a 0.1 per cent w/v solution of 2,6-dichloroquinone4-chloroimide in ethanol (95 per cent) and examine immediately. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Uniformity of content. Comply with the test stated under Tablets. Powder one tablet, add 50 ml of 0.1 M hydrochloric acid and heat on a water-bath for 15 minutes, swirling occasionally.
1010
IP 2007
PYRIMETHAMINE
Cool, dilute to 100.0 ml with 0.1 M hydrochloric acid, filter, discarding the first 20 ml of the filtrate. If necessary, dilute quantitatively and stepwise with 0.1 M hydrochloric acid to produce a solution containing 10 g of the pyridoxine hydrochloride per ml and measure the absorbance of the resulting solution at the maximum at about 290 nm (2.4.7). Calculate the content of C8H11NO3, HCl taking 430 as the specific absorbance at 290 nm. Other tests. Comply with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 20 mg of Pyridoxine Hydrochloride, add 50 ml of 0.1 M hydrochloric acid and heat on a water-bath for 15 minutes, swirling occasionally. Cool, dilute to 100.0 ml with 0.1 M hydrochloric acid and filter, discarding the first 20 ml of the filtrate. Dilute 5.0 ml of the filtrate to 100.0 ml with 0.1 M hydrochloric acid and measure the absorbance of the resulting solution at the maximum at about 290 nm (2.4.7). Calculate the content of C8H11NO3, HCl taking 430 as the specific absorbance at 290 nm. Storage. Store protected from light and moisture.
C. Dissolve 0.14 g in sufficient ethanol to produce 100 ml, dilute 10 ml of this solution to 100 ml with 0.1 M hydrochloric acid and dilute 10 ml of the solution to 100 ml with the same solvent. When examined in the range 230 nm to 360 nm (2.4.7), the resulting solution shows an absorption maximum at about 272 nm; absorbance at about 272 nm, 0.43 to 0.46.
Tests
Appearance of solution. Dissolve 0.25 g in sufficient of a mixture of 3 volumes of dichloromethane and 1 volume of methanol to produce 10 ml. The resulting solution is clear (2.4.1), and not more intensely coloured than reference solution BYS6 (2.4.1). Acidity or alkalinity. Shake 1.0 g with 50 ml of water for 2 minutes and filter (solution A). To 10 ml of solution A add 0.05 ml of phenolphthalein solution; the solution is colourless and not more than 0.2 ml of 0.01 M sodium hydroxide is required to change the colour to pink. Add 0.4 ml of 0.01 M hydrochloric acid and 0.05 ml of methyl red solution; the solution is red or orange. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 76 volumes of toluene, 12 volumes of glacial acetic acid, 8 volumes of 1-propanol and 4 volumes of chloroform.
Pyrimethamine
H3C N N Cl
C12H13ClN4
NH2
Prepare the following solutions immediately before use. Test solution (a). Dissolve 0.1 g of the substance under examination in 10 ml of a mixture of 90 volumes of chloroform and 10 volumes of methanol. Mol. Wt. 248.7 Test solution (b). Dissolve 0.1 g of the substance under examination in 100 ml of the same solvent mixture. Reference solution (a). A 0.0025 per cent w/v solution of the substance under examination in the same solvent mixture. Reference solution (b). A 0.1 per cent w/v solution of pyrimethamine RS in the same solvent mixture. Apply to the plate 20 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with test solution (a) is not more intense than the spot in the chromatogram obtained with reference solution (a). Sulphates (2.3.17). 25 ml of Solution A complies with the limit test for sulphates. Use a mixture of 5.0 ml of sulphate standard solution (10 ppm SO4) and 10 ml of distilled water to prepare the standard (100 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105 for 4 hours.
NH2
Pyrimethamine is 5-(4-chlorophenyl)-6-ethylpyrimidine-2,4diamine. Pyrimethamine contains not less than 99.0 per cent and not more than 101.0 per cent of C12H13ClN4, calculated on the dried basis. Description. Colourless crystals or an almost white, crystalline powder; odourless.
Identification
Test A may be omitted if tests B and C are carried out. Tests B and C may be omitted if test A is carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with pyrimethamine RS or with the reference spectrum of pyrimethamine. B. In the test for Related substances, the principal spot in the chromatogram obtained with test solution (b) corresponds to that in the chromatogram obtained with reference solution (b).
1011
PYRIMETHAMINE AND SULPHADOXINE TABLETS
IP 2007
Assay. Weigh accurately about 0.2 g, dissolve in 25 ml of anhydrous glacial acetic acid, heating gently, cool. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.02487 g of C12H13ClN4. Storage. Store protected from light and moisture.
2.0 ml of solution A prepared by dissolving 0.1 g of phenacetin (internal standard) in 100 ml of acetonitrile and sufficient of the mobile phase to produce 50.0 ml. Reference solution. Weigh accurately about 25 mg of pyrimethamine RS and 500 mg of sulphadoxine RS, add 35 ml of acetonitrile and sufficient of the mobile phase to produce 100.0 ml and mix. To 25.0 ml add 2.0 ml of solution A and add sufficient of the mobile phase to produce 50.0 ml and mix well. Chromatographic system a stainless steel column 30 cm x 4 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 4 volumes of a 1 per cent v/v solution of glacial acetic acid and 1 volume of acetonitrile, flow rate. 2 ml per minute, spectrophotometer set at 254 nm, a 20 l loop injector. Inject the reference solution. The relative standard deviation for replicate injections is not more than 2.0 percent. Inject alternately the test solution and the reference solution. The relative retention times should be about 1.3 for pyrimethamine, 1.0 for phenacetin and 0.7 for sulphadoxine. Calculate the content of C12H13ClN4 and the content of C12H14N4O4S in the tablets. Storage. Store protected from light and moisture.
Pyrimethamine and Sulphadoxine Tablets

Pyrimethamine and Sulphadoxine Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amounts of pyrimethamine, C12H13ClN4, and of sulphadoxine, C12H14N4O4S.
Identification
Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 4 volumes of chloroform, 4 volumes of n-heptane, 1 volume of glacial acetic acid and 4 volumes of a mixture of 1 volume of methanol and 19 volumes of ethanol. Test solution. Shake a quantity of the powdered tablets containing 25 mg of Pyrimethamine with 50 ml of a 2 per cent w/v solution of strong ammonia solution in methanol. Reference solution (a). A 0.05 per cent w/v solution of pyrimethamine RS in methanol. Reference solution (b). A 1.0 per cent w/v solution of sulphadoxine RS in methanol. Apply to the plate 10 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. One of the principal spots in the chromatogram obtained with the test solution corresponds to the spot in the chromatogram obtained with reference solution (a) and the other corresponds to that in the chromatogram obtained with reference solution (b).
Tests
Other tests. Complies with the tests stated under Tablets. Assay. Determine by liquid chromatography (2.4.14). Test solution. Weigh accurately a quantity of the powdered tablets containing about 25 mg of Pyrimethamine and 500 mg of Sulphadoxine and shake with 35 ml of acetonitrile for 30 minutes in a 100-ml volumetric flask. Dilute to volume with the mobile phase, mix and filter. To 25.0 ml of the filtrate add
1012
MONOGRAPHS
Q
Quinalbarbitone Sodium Quinalbarbitone Tablets Quinidine Sulphate Quinidine Tablets Quinine Bisulphate Quinine Bisulphate Tablets Quinine Dihydrochloride Quinine Dihydrochloride Injection Quinine Sulphate Quinine Tablets Quiniodochlor Quiniodochlor Tablets .... .... .... .... .... .... .... .... .... .... .... ....
1013
IP 2007
QUINALBARBITONE SODIUM
Quinalbarbitone Sodium
Quinalbarbital Sodium; Secobarbital Sodium; Secobarbitone Sodium; Soluble Quinalbarbitone
O H2C H3C H3C
C12H17N2NaO3
Tests
Appearance of solution. A freshly prepared 10.0 per cent w/v solution in ethanol (95 per cent) is clear (2.4.1), and not more intensely coloured than reference solution YS7 (2.4.1). pH (2.4.24). Not more than 11.0, determined on a 10.0 per cent w/v solution. Heavy metals (2.3.13). 0.67 g dissolved in a mixture of 5 ml of 1 M sodium hydroxide and 20 ml of water complies with the limit test for heavy metals, Method C (30 ppm). Mol.Wt.260.3 Related substances. Complies with the test for related substances in barbiturates (2.3.4). Loss on drying (2.4.19). Not more than 3.0 per cent, determined on 0.5 g by drying in an oven at 105. Assay. Weigh accurately about 0.5 g, dissolve in 10 ml of ethanol, add 10 ml of silver nitrate-pyridine reagent and titrate with 0.1 M ethanolic sodium hydroxide using 0.5 ml of thymolphthalein solution as indicator, until a pure blue colour is obtained. Carry out a blank titration. 1 ml of 0.1 M ethanolic sodium hydroxide is equivalent to 0.02603 g of C12H17N2NaO3. Storage. Store protected from moisture.
H N N O
ONa
Quinalbarbitone Sodium is sodium (RS)-5-allyl-5-(1methylbutyl)barbiturate. Quinalbarbitone Sodium contains not less than 98.5 per cent and not more than 102.0 per cent of C12H17N2NaO3, calculated on the dried basis. Description. A white powder; odourless; hygroscopic.
Identification
Test A may be omitted if tests B, C, D and E are carried out. Tests B, C and D may be omitted if tests A and E are carried out. A. To 10 ml of a 10.0 per cent w/v solution in ethanol (95 per cent) add 120 ml of water and 5 ml of 2 M acetic acid, stir vigorously, add 200 ml of water and boil until the precipitate dissolves and no oily particles remain on the surface of the liquid. Allow to cool until a haziness begins to appear in the solution, induce crystallisation, if necessary and allow the solution to stand for 12 hours. Wash the crystals with three quantities, each of 10 ml, of water and dry the residue at 80. On the residue determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with quinalbarbitone RS treated in the same manner. B. To 0.5 g add 5 ml of sodium carbonate solution and 10 ml of nitrobenzyl chloride solution. Heat for 30 minutes on a waterbath under reflux and allow to stand for 1 hour. Filter, wash the precipitate successively with 10 ml of dilute sodium hydroxide solution and 50 ml of water; recrystallise from a mixture of equal volumes of chloroform and ethanol (95 per cent) and dry at 105. The crystals melt at about 156 (2.4.21) C. Complies with the test for identification of barbiturates (2.3.2). D. Gives the reaction of non-nitrogen substituted barbiturates (2.3.1). E. Ignite 1 g; the residue gives the reactions of sodium salts (2.3.1).
Quinalbarbitone Tablets
Quinalbarbital Sodium Tablets; Quinalbarbitone Sodium Tablets; Secobarbital Sodium Tablets; Secobarbitone Sodium Tablets
Quinalbarbitone Tablets contain not less than 92.5 per cent and not more than 107.5 per cent of the stated amount of quinalbarbitone sodium, C12H17N2NaO3. The tablets are coated.
Identification
A. Shake a quantity of the powdered tablets containing about 0.5 g of Quinalbarbitone Sodium with 10 ml of water and filter. To 10 ml of the filtrate add 5 ml of sodium carbonate solution and 10 ml of nitrobenzyl chloride solution. Heat for 30 minutes on a water-bath under reflux and allow to stand for 1 hour. Filter, wash the precipitate successively with 10 ml of dilute sodium hydroxide solution and 50 ml of water; recrystallise from a mixture of equal volumes of chloroform and ethanol (95 per cent) and dry at 105. The crystals melt at about 156 (2.4.21) B. Triturate a quantity of the powdered tablets containing 0.5 g of Quinalbarbitone Sodium with 10 ml of water, filter, acidify the filtrate with acetic acid; oily drops are formed which may eventually crystallise.
1015
IP 2007
C. The powdered tablets give the reactions of sodium salts (2.3.1).
Mobile phase. A mixture of 60 volumes of toluene, 36 volumes of ether and 15 volumes of diethylamine. Test solution. Dissolve 1 g of the substance under examination in 100 ml of methanol. Reference solution (a). A 1 per cent w/v solution of quinidine sulphate RS in methanol. Reference solution (b). A 1 per cent w/v solution of each of quinidine sulphate RS and quinine sulphate RS in methanol. Apply to the plate 4 l of each solution. After development, dry the plate in air for 15 minutes and repeat the development. Dry the plate at 105 for 30 minutes, allow to cool and spray with potassium iodoplatinate solution. The principal spot in the chromatogram obtained with the test solution corresponds to the spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows two clearly separated spots. B. To 5 ml of a 0.1 per cent w/v solution add 0.2 ml of bromine solution and 1 ml of dilute ammonia solution; an emeraldgreen colour is produced. C. To a 0.5 per cent w/v solution add an equal volume of dilute sulphuric acid; a strong blue fluorescence is produced. D. To 5 ml of a 1 per cent w/v solution add 1 ml of silver nitrate solution and stir with a glass rod; after a short interval, a white precipitate soluble in nitric acid is produced (distinction from many other alkaloids).
Tests
Other tests. Comply with the tests stated under Tablets. Assay. Weigh and digest 20 tablets with 50 ml of water until completely disintegrated and not more than a small residue remains. Add 5 ml of 1 M sodium hydroxide, filter and wash the residue with sufficient water to produce 100.0 ml. Extract a volume of the solution containing 0.5 g of Quinalbarbitone Sodium with two quantities, each of 10 ml, of ether, washing each ether extract with the same 3 ml of water. Add the water to the aqueous liquid, acidify with hydrochloric acid and extract with successive quantities, each of 15 ml, of ether until complete extraction is effected. Wash the combined extracts with two quantities, each of 2 ml, of water and extract the combined washings with 10 ml of ether. Add the ether to the main ether layer, filter and wash the filter with ether. Evaporate the solvent and dry the residue to constant weight at 50. 1 g of residue is equivalent to 1.092 g of C12H17N2NaO3. Storage. Store protected from moisture.
Quinidine Sulphate
Quinidine Bisulphate
OCH3 HO H N N H H
2
H H CH2 , H2SO4, 2H2O
E. A 1 per cent w/v solution gives the reactions of sulphates (2.3.1).
Tests
Appearance of solution. A 2.0 per cent w/v solution in 0.1 M hydrochloric acid is clear (2.4.1), and not more intensely coloured than reference solution GYS4 (2.4.1). Mol. Wt. 783.0 pH (2.4.24). 6.0 to 6.8, determined in a 1.0 per cent w/v solution. Specific optical rotation (2.4.22). +275 to +290, determined in a 2.0 per cent w/v solution in 0.1 M hydrochloric acid. Dihydroquinidine sulphate. Not more than 15.0 per cent, calculated on the dried basis and determined by the following method. Dissolve 0.2 g in 20 ml of water, add 0.5 g of potassium bromide and 15 ml of 2 M hydrochloric acid. Titrate slowly with 0.0167 M potassium bromate using methyl red solution as indicator until a yellow colour is obtained. Add a solution of 0.5 g of potassium iodide in 200 ml of water and stopper the flask immediately. Allow to stand in the dark for 5 minutes and titrate with 0.1 M sodium thiosulphate using starch solution, added towards the end of the titration, as indicator. Repeat the operation without the substance under examination.
(C20H24N2O2)2,H2SO4,2H2O
Quinidine Sulphate is (8R,9S)-6-methoxycinchonan-9-ol sulphate dihydrate. The alkaloid is obtained from the bark of various species of Cinchona and from Remijia pedunculata Fluckiger (Fam. Rubiaceae) or prepared from quinine. Quinidine Sulphate contains not less than 99.0 per cent and not more than 101.5 per cent of alkaloid monosulphates, calculated as (C20H24N2O2)2,H2SO4 on the dried basis. Description. A white or almost white, crystalline powder or needle-like crystals; odourless.
Identification
A. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G.
1016
IP 2007
1 ml of 0.0167 M potassium bromate is equivalent to 0.01867 g of (C20H24N2O2)2, H2SO4. Calculate the content of dihydroquinidine sulphate by subtracting the result from the assay result. Other cinchona alkaloids. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 20 mg of the substance under examination in 5 ml of the mobile phase. Heat gently, if necessary to dissolve the powder as completely as possible, cool, dilute to 10 ml with the mobile phase and mix. Reference solution (a). Dissolve 20 mg of quinine sulphate RS, with gentle heating if necessary, in 5 ml of the mobile phase and dilute to 10 ml with the mobile phase. Reference solution (b). Prepare in the same manner as reference solution (a) but using quinidine sulphate RS in place of quinine sulphate RS. Reference solution (c). Mix equal volumes of reference solutions (a) and (b). Reference solution (d). Dilute 1 volume of reference solution (a) to 10 volumes with the mobile phase and dilute 1 volume of the resulting solution to 50 volumes with the mobile phase. Reference solution (e). A solution containing 0.1 per cent w/v of thiourea in the mobile phase. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5 m) (such as Hypersil ODS 5 m), mobile phase: a solution prepared by dissolving 6.8 g of potassium dihydrogen orthophosphate and 3.0 g of hexylamine in 700 ml of water, adjusting the pH to 2.8 with 1 M orthophosphoric acid, adding 60 ml of acetonitrile and diluting to 1000 ml with water, flow rate. 1.5 ml per minute, spectrophotometer set at 250 nm for reference solution (e) and 316 nm for the other solutions, a 10 l loop injector. Inject separately reference solutions (b) and (e). If necessary, adjust the concentration of acetonitrile in the mobile phase so that in the chromatogram obtained with reference solution (b) the capacity factor of the peak due to quinidine is 3.5 to 4.5, V0 (the distance along the baseline between the point of injection and the perpendicular dropped from the maximum of the peak of an unretained component) being calculated from the peak due to thiourea in the chromatogram obtained with reference solution (e). Inject reference solutions (a), (b), (c) and (d). The chromatogram obtained with reference solution (a) shows a principal peak due to quinine and a peak due to dihydroquinine
with a retention time relative to quinine of about 1.4. The chromatogram obtained with reference solution (b) shows a principal peak due to quinidine and a peak due to dihydroquinidine, with a retention time relative to quinidine of about 1.2. The chromatogram obtained with reference solution (c) shows four peaks due to quinine, dihydroquinine, quinidine and dihydroquinidine which are identified by comparison of their retention times with those of the corresponding peaks in the chromatograms obtained with reference solutions (a) and (b). The test is not valid unless (a) in the chromatogram obtained with reference solution (c) the resolution between the peaks due to quinine and quinidine is at least 1.5 and the resolution between the peaks due to dihydroquinidine and quinine is at least 1.0 and (b) the signal-to-noise ratio of the principal peak in the chromatogram obtained with reference solution (d) is at least 5. Inject the test solution and allow the chromatography to proceed for 2.5 times the retention time of the principal peak. Calculate the percentage content of related substances by normalisation, ignoring any peaks the areas of which are less than that of the peak in the chromatogram obtained with reference solution (d) (0.2 per cent). The content of dihydroquinidine is not greater than 15 per cent, the content of any related substance eluting before quinidine is not greater than 5 per cent and the content of any other related substance is not greater than 2.5 per cent. Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). 3.0 per cent to 5.0 per cent, determined on 1.0 g by drying in an oven at 130. Assay. Weigh accurately about 0.2 g, dissolve in a mixture of 10 ml of chloroform and 20 ml of acetic anhydride. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.02490 g of (C20H24N2O2)2,H2SO4. Storage. Store protected from light.
Quinidine Tablets
Quinidine Sulphate Tablets
Quinidine Tablets contain not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of quinidine sulphate, (C20H24N2O2)2,H2SO4,2H2O.
Identification
1017
IP 2007
Mobile phase. A mixture of 80 volumes of toluene, 20 volumes of acetone and 10 volumes of diethylamine. Test solution. Extract a quantity of the powdered tablets containing 0.1 g of Quinidine Sulphate with 10 ml of a mixture of 2 volumes of chloroform and 1 volume of ethanol (95 per cent) and filter. Reference solution. 1.0 per cent w/v solution of quinidine sulphate RS in a mixture of 2 volumes of chloroform and 1 volume of ethanol (95 per cent). Apply to the plate 2 l of each solution. After development, dry the plate in air and spray with 0.05 M ethanolic sulphuric acid and then with dilute potassium iodobismuthate solution. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution. B. Extract a quantity of the powdered tablets containing 0.1 g of Quinidine Sulphate with 20 ml of water and filter. The filtrate (solution A) is dextro-rotatory. C. To 1 ml of solution A add 4 ml of water, 2 or 3 drops of bromine solution and 1 ml of dilute ammonia solution; an emerald green colour is produced. D. Solution A gives the reactions of sulphates (2.3.1).
Reference solution (b). Prepare in the same manner as reference solution (a) but using quinidine sulphate RS in place of quinine sulphate RS. Reference solution (c). Mix equal volumes of reference solutions (a) and (b). Reference solution (d). Dilute 1 volume of reference solution (a) to 10 volumes with the mobile phase and dilute 1 volume of the resulting solution to 50 volumes with the mobile phase. Reference solution (e). A solution containing 0.1 per cent w/v of thiourea in the mobile phase. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5 m) (such as Hypersil ODS 5 m), mobile phase: a solution prepared by dissolving 6.8 g of potassium dihydrogen orthophosphate and 3.0 g of hexylamine in 700 ml of water, adjusting the pH to 2.8 with 1 M orthophosphoric acid, adding 60 ml of acetonitrile and diluting to 1000 ml with water, flow rate. 1.5 ml per minute, spectrophotometer set at 250 nm for reference solution (e) and 316 nm for the other solutions, a 10 l loop injector. Inject separately reference solutions (b) and (e). If necessary, adjust the concentration of acetonitrile in the mobile phase so that in the chromatogram obtained with reference solution (b) the capacity factor of the peak due to quinidine is 3.5 to 4.5, V0 (the distance along the baseline between the point of injection and the perpendicular dropped from the maximum of the peak of an unretained component) being calculated from the peak due to thiourea in the chromatogram obtained with reference solution (e). Inject reference solutions (a), (b), (c) and (d). The chromatogram obtained with reference solution (a) shows a principal peak due to quinine and a peak due to dihydroquinine with a retention time relative to quinine of about 1.4. The chromatogram obtained with reference solution (b) shows a principal peak due to quinidine and a peak due to dihydroquinidine, with a retention time relative to quinidine of about 1.2. The chromatogram obtained with reference solution (c) shows four peaks due to quinine, dihydroquinine, quinidine and dihydroquinidine which are identified by comparison of their retention times with those of the corresponding peaks in the chromatograms obtained with reference solutions (a) and (b). The test is not valid unless (a) in the chromatogram obtained with reference solution (c) the resolution between the peaks due to quinine and quinidine is at least 1.5 and the resolution between the peaks due to dihydroquinidine and quinine is at least 1.0 and (b) the signal-to-noise ratio of the principal peak
Tests
Dissolution (2.5.2). Apparatus. No 2 Medium. 900 ml of 0.1 M hydrochloric acid Speed and time. 100 rpm and 30 minutes. Withdraw a suitable volume of the medium and filter. Measure the absorbance of the filtrate, suitably diluted if necessary, at the maximum at about 248 nm (2.4.7). Calculate the content of (C20H24N2O2)2,H2SO4,2H2O in the medium from the absorbance obtained from a solution of known concentration of quinidine sulphate RS. D. Not less than 70 per cent of the stated amount of (C20H24N2O2)2,H2SO4,2H2O. Other cinchona alkaloids. Determine by liquid chromatography (2.4.14). Test solution. Mix a quantity of the powdered tablets containing 50 mg of Quinidine Sulphate with 20 ml of the mobile phase. Heat gently to dissolve the powder as completely as possible, cool, dilute to 25 ml with the mobile phase and filter, discarding the first few ml of filtrate. Reference solution (a). Dissolve 20 mg of quinine sulphate RS, with gentle heating if necessary, in 5 ml of the mobile phase and dilute to 10 ml with the mobile phase.
1018
IP 2007
in the chromatogram obtained with reference solution (d) is at least 5. Inject the test solution and allow the chromatography to proceed for 2.5 times the retention time of the principal peak. Calculate the percentage content of related substances by normalisation, ignoring any peaks the areas of which are less than that of the peak in the chromatogram obtained with reference solution (d) (0.2 per cent). The content of dihydroquinidine is not greater than 15 per cent, the content of any related substance eluting before quinidine is not greater than 5 per cent and the content of any other related substance is not greater than 2.5 per cent. Other tests. Comply with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 0.4 g of Quinidine Sulphate, dissolve as completely as possible in 40 ml of acetic anhydride with the aid of heat and cool. Titrate with 0.1 M perchloric acid, using crystal violet solution as indicator. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.02610 g of (C20H24N2O2)2,H2SO4,2H2O. Storage. Store protected from light.
Mobile phase. A mixture of 60 volumes of toluene, 36 volumes of ether and 15 volumes of diethylamine. Test solution. Dissolve 1 g of the substance under examination in 100 ml methanol. Reference solution (a). A 1 per cent w/v solution of quinine sulphate RS in methanol. Reference solution (b). A 1 per cent w/v solution of each of quinidine sulphate RS and quinine sulphate RS in methanol. Apply to the plate 4 l of each solution. After development, dry the plate in air for 15 minutes and repeat the development. Dry the plate at 105 for 30 minutes, allow to cool and spray with potassium iodoplatinate solution. The principal spot in the chromatogram obtained with the test solution corresponds to the spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows two clearly separated spots. B. A 5 per cent w/v solution has a blue fluorescence. C. A 5 per cent w/v solution gives the reactions of sulphates (2.3.1).
Tests
pH (2.4.24). 2.8 to 3.4, determined in a 1.0 per cent w/v solution.
Quinine Bisulphate
Quinine Acid Sulphate
OCH3 HO H N N H H
Mol. Wt. 548.6
Specific optical rotation (2.4.22). 208 to 216, determined at 20 in a 3.0 per cent w/v solution in 0.1 M hydrochloric acid. Other cinchona alkaloids. Determine by liquid chromatography (2.4.14).
H H CH2 , H2SO4, 7H2O
Test solution. Dissolve 20 mg of the substance under examination in 5 ml of the mobile phase. Heat gently, if necessary to dissolve the powder as completely as possible, cool, dilute to 10 ml with the mobile phase and mix. Reference solution (a). Dissolve 20 mg of quinine sulphate RS, with gentle heating if necessary, in 5 ml of the mobile phase and dilute to 10 ml with the mobile phase. Reference solution (b). Prepare in the same manner as reference solution (a) but using quinidine sulphate RS in place of quinine sulphate RS. Reference solution (c). Mix equal volumes of reference solutions (a) and (b). Reference solution (d). Dilute 1 volume of reference solution (a) to 10 volumes with the mobile phase and dilute 1 volume of the resulting solution to 50 volumes with the mobile phase. Reference solution (e). A solution containing 0.1 per cent w/v of thiourea in the mobile phase.
C20H24N2O2,H2SO4,7H2O
Quinine Bisulphate is (8S,9R)-6-methoxycinchonan-9-ol hydrogen sulphate heptahydrate. The alkaloid is obtained from the bark of various species of Cinchona. Quinine Bisulphate contains not less than 98.5 per cent and not more than 101.5 per cent of alkaloid hydrogen sulphates, calculated as C20H24N2O2,H2SO4 on the anhydrous basis. Description. Colourless or faintly yellow crystals or a white or faintly yellow, crystalline powder; efflorescent in dry air.
Identification
1019
IP 2007
Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5 m) (such as Hypersil ODS 5 m), mobile phase: a solution prepared by dissolving 6.8 g of potassium dihydrogen orthophosphate and 3.0 g of hexylamine in 700 ml of water, adjusting the pH to 2.8 with 1 M orthophosphoric acid, adding 60 ml of acetonitrile and diluting to 1000 ml with water, flow rate. 1.5 ml per minute, spectrophotometer set at 250 nm for reference solution (e) and 316 nm for the other solutions, a 10 l loop injector. Inject separately reference solutions (b) and (e). If necessary, adjust the concentration of acetonitrile in the mobile phase so that in the chromatogram obtained with reference solution (b) the capacity factor of the peak due to quinidine is 3.5 to 4.5, V0 (the distance along the baseline between the point of injection and the perpendicular dropped from the maximum of the peak of an unretained component) being calculated from the peak due to thiourea in the chromatogram obtained with reference solution (e). Inject reference solutions (a), (b), (c) and (d). The chromatogram obtained with reference solution (a) shows a principal peak due to quinine and a peak due to dihydroquinine with a retention time relative to quinine of about 1.4. The chromatogram obtained with reference solution (b) shows a principal peak due to quinidine and a peak due to dihydroquinidine, with a retention time relative to quinidine of about 1.2. The chromatogram obtained with reference solution (c) shows four peaks due to quinine, dihydroquinine, quinidine and dihydroquinidine which are identified by comparison of their retention times with those of the corresponding peaks in the chromatograms obtained with reference solutions (a) and (b). The test is not valid unless (a) in the chromatogram obtained with reference solution (c) the resolution between the peaks due to quinine and quinidine is at least 1.5 and the resolution between the peaks due to dihydroquinidine and quinine is at least 1.0 and (b) the signal-to-noise ratio of the principal peak in the chromatogram obtained with reference solution (d) is at least 5. Inject the test solution and allow the chromatography to proceed for 2.5 times the retention time of the principal peak. Calculate the percentage content of related substances by normalisation, ignoring any peaks the areas of which are less than that of the peak in the chromatogram obtained with reference solution (d) (0.2 per cent). The content of dihydroquinine is not greater than 10 per cent, the content of any related substance eluting before quinine is not greater than 5 per cent and the content of any other related substance is not greater than 2.5 per cent.
Titratable cation. 75.3 to 79.6 per cent, calculated on the anhydrous basis, determined by the following method. Titrate the combined aqueous solutions reserved in the Assay with 0.1 M hydrochloric acid using 0.1 ml of phenolphthalein solution as indicator. 1 ml of 0.1 M sodium hydroxide is equivalent to 0.01632 g of [C20H26N2O2]2+. Sulphated ash (2.3.18). Not more than 0.1 per cent. Water (2.3.43). 19.0 to 25.0 per cent, determined on 0.2 g. Assay. Weigh accurately about 0.45 g, dissolve in 15 ml of water, add 25 ml of 0.1 M sodium hydroxide and extract with three quantities, each of 25 ml, of chloroform. Wash each chloroform extract with the same 20 ml of water. Combine the aqueous solutions and reserve for the test for Titratable cation. Dry the chloroform extracts with anhydrous sodium sulphate, evaporate to dryness at a pressure of 2 kPa and dissolve the residue in 50 ml of anhydrous glacial acetic acid. Titrate with 0.1 M perchloric acid, using crystal violet solution as indicator. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.02113 g of C20H24N2O2,H2SO4. Storage. Store protected from light.
Quinine Bisulphate Tablets

Quinine Acid Sulphate Tablets
Quinine Bisulphate Tablets contain not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of quinine bisulphate, C20H24N2O2,H2SO4,7H2O. The tablets are coated.
Identification
A. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 80 volumes of toluene, 20 volumes of acetone and 10 volumes of diethylamine. Test solution. Extract a quantity of the powdered tablets containing 0.1 g of Quinine Bisulphate with 10 ml of a mixture of 2 volumes of chloroform and 1 volume of ethanol (95 per cent) and filter. Reference solution. A 1.0 per cent w/v solution of quinine sulphate in the same solvent mixture. Apply to the plate 2 l of each solution. After development, dry the plate in air and spray with 0.05 M ethanolic sulphuric acid and then with dilute potassium iodobismuthate solution. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution.
1020
IP 2007
B. Extract a quantity of the powdered tablets containing 0.1 g of Quinine Bisulphate with 20 ml of water and filter (solution A). To 5 ml of solution A add 0.2 ml of bromine solution and 1 ml of dilute ammonia solution; an emerald-green colour is produced. C. Solution A is laevo-rotatory. D. Solution A gives the reactions of sulphates (2.3.1).
hexylamine in 700 ml of water, adjusting the pH to 2.8 with 1 M orthophosphoric acid, adding 60 ml of acetonitrile and diluting to 1000 ml with water, flow rate. 1.5 ml per minute, spectrophotometer set at 250 nm for reference solution (e) and 316 nm for the other solutions, a 10 l loop injector. Inject separately reference solutions (b) and (e). If necessary, adjust the concentration of acetonitrile in the mobile phase so that in the chromatogram obtained with reference solution (b) the capacity factor of the peak due to quinidine is 3.5 to 4.5, V0 (the distance along the baseline between the point of injection and the perpendicular dropped from the maximum of the peak of an unretained component) being calculated from the peak due to thiourea in the chromatogram obtained with reference solution (e). Inject reference solutions (a), (b), (c) and (d). The chromatogram obtained with reference solution (a) shows a principal peak due to quinine and a peak due to dihydroquinine with a retention time relative to quinine of about 1.4. The chromatogram obtained with reference solution (b) shows a principal peak due to quinidine and a peak due to dihydroquinidine, with a retention time relative to quinidine of about 1.2. The chromatogram obtained with reference solution (c) shows four peaks due to quinine, dihydroquinine, quinidine and dihydroquinidine which are identified by comparison of their retention times with those of the corresponding peaks in the chromatograms obtained with reference solutions (a) and (b). The test is not valid unless (a) in the chromatogram obtained with reference solution (c) the resolution f between the peaks due to quinine and quinidine is at least 1.5 and the resolution between the peaks due to dihydroquinidine and quinine is at least 1.0 and (b) the signal-to-noise ratio of the principal peak in the chromatogram obtained with reference solution (d) is at least 5. Inject the test solution and allow the chromatography to proceed for 2.5 times the retention time of the principal peak. Calculate the percentage content of related substances by normalisation, ignoring any peaks the areas of which are less than that of the peak in the chromatogram obtained with reference solution (d) (0.2 per cent). The content of dihydroquinine is not greater than 10 per cent, the content of any related substance eluting before quinine is not greater than 5 per cent and the content of any other related substance is not greater than 2.5 per cent. Other tests. Comply with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 0.6 g of Quinine Bisulphate, dissolve as completely as possible in 40 ml of
Tests
Dissolution (2.5.2). Apparatus. No 2 Medium. 900 ml of 0.1 M hydrochloric acid Speed and time. 100 rpm and 45 minutes. Withdraw a suitable volume of the medium and filter. Measure the absorbance of the filtrate, suitably diluted if necessary, at the maximum at about 348 nm (2.4.7). Calculate the content of C20H24N2O2,H2SO4,7H2O in the medium taking 99 as the specific absorbance at 348 nm. D. Not less than 70 per cent of the stated amount of C20H24N2O2,H2SO4,7H2O. Other cinchona alkaloids. Determine by liquid chromatography (2.4.14). Test solution. Remove any coating from the tablets and mix a quantity of the powdered tablet cores containing 50 mg of Quinine Bisulphate with 20 ml of the mobile phase. Heat gently to dissolve the powder as completely as possible, cool, dilute to 25 ml with the mobile phase and filter, discarding the first few ml of the filtrate. Reference solution (a). Dissolve 20 mg of quinine sulphate RS, with gentle heating if necessary, in 5 ml of the mobile phase and dilute to 10 ml with the mobile phase. Reference solution (b). Prepare in the same manner as reference solution (a) but using quinidine sulphate RS in place of quinine sulphate RS. Reference solution (c). Mix equal volumes of reference solutions (a) and (b). Reference solution (d). Dilute 1 volume of reference solution (a) to 10 volumes with the mobile phase and dilute 1 volume of the resulting solution to 50 volumes with the mobile phase. Reference solution (e). A solution containing 0.1 per cent w/v of thiourea in the mobile phase. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5 m) (such as Hypersil ODS 5 m), mobile phase: a solution prepared by dissolving 6.8 g of potassium dihydrogen orthophosphate and 3.0 g of
1021
IP 2007
acetic anhydride with the aid of heat and cool. Titrate with 0.1 M perchloric acid, using crystal violet solution as indicator. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.05486 g of C20H24N2O2,H2SO4,7H2O. Storage. Store protected from light.
obtained with reference solution (b) shows two clearly separated spots. B. To a 0.5 per cent w/v solution add an equal volume of dilute sulphuric acid; a strong blue fluorescence is produced. C. A 1 per cent w/v solution gives the reactions of chlorides (2.3.1).
Tests
Quinine Dihydrochloride
Quinine Acid Hydrochloride
OCH3 HO H N N
C20H24N2O2,2HCl
Specific optical rotation (2.4.22). 223 to 229, determined in a 3.0 per cent w/v solution in 0.1 M hydrochloric acid. Barium. To 15 ml of a 2 per cent w/v solution add 1 ml of dilute sulphuric acid; the solution remains clear for at least 15 minutes.
H H H CH2 , 2HCl
Mol. Wt. 397.3
Quinine Dihydrochloride is the (8S,9R)-6 methoxycinchonan-9-ol dihydrochloride. The alkaloid is obtained from the bark of various species of Cinchona. Quinine Dihydrochloride contains not less than 99.0 per cent and not more than 101.5 per cent of alkaloid dihydrochlorides, calculated as C20H24N2O2,2HCl on the dried basis. Description. A white or almost white powder; odourless.
Dihydroquinine dihydrochloride. Not more than 10.0 per cent, calculated on the dried basis and determined by the following method. Dissolve 0.2 g in 20 ml of water, add 0.5 g of potassium bromide and 15 ml of 2 M hydrochloric acid. Titrate slowly with 0.0167 M potassium bromate using methyl red solution as indicator until a yellow colour is obtained. Add a solution of 0.5 g of potassium iodide in 200 ml of water and stopper the flask immediately. Allow to stand in the dark for 5 minutes and titrate with 0.1 M sodium thiosulphate using starch solution, added towards the end of the titration, as indicator. Repeat the operation without the substance under examination. 1 ml of 0.0167 M potassium bromate is equivalent to 0.01987 g of C20H24N2O2,2HCl. Calculate the content of dihydroquinine dihydrochloride by subtracting the result from the assay result. Other cinchona alkaloids. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 20 mg of the substance under examination in 5 ml of the mobile phase. Heat gently, if necessary to dissolve the powder as completely as possible, cool, dilute to 10 ml with the mobile phase and mix. Reference solution (a). Dissolve 20 mg of quinine sulphate RS, with gentle heating if necessary, in 5 ml of the mobile phase and dilute to 10 ml with the mobile phase. Reference solution (b). Prepare in the same manner as reference solution (a) but using quinidine sulphate RS in place of quinine sulphate RS. Reference solution (c). Mix equal volumes of reference solutions (a) and (b). Reference solution (d). Dilute 1 volume of reference solution (a) to 10 volumes with the mobile phase and dilute 1 volume of the resulting solution to 50 volumes with the mobile phase. Reference solution (e). A solution containing 0.1 per cent w/v of thiourea in the mobile phase.
Identification
A. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 60 volumes of toluene, 36 volumes of ether and 15 volumes of diethylamine. Test solution. Dissolve 1 g of the substance under examination in 100 ml of methanol. Reference solution (a). A 1 per cent w/v solution of quinidne sulphate RS in methanol. Reference solution (b). A 1 per cent w/v solution of each of quinidine sulphate RS and quinine sulphate RS in methanol. Apply to the plate 4 l of each solution. After development, dry the plate in air for 15 minutes and repeat the development. Dry the plate at 105 for 30 minutes, allow to cool and spray with potassium iodoplatinate solution. The principal spot in the chromatogram obtained with the test solution corresponds to the spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram
1022
IP 2007
Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5 m) (such as Hypersil ODS 5 m), mobile phase: a solution prepared by dissolving 6.8 g of potassium dihydrogen orthophosphate and 3.0 g of hexylamine in 700 ml of water, adjusting the pH to 2.8 with 1 M orthophosphoric acid, adding 60 ml of acetonitrile and diluting to 1000 ml with water flow rate. 1.5 ml per minute, spectrophotometer set at 250 nm for reference solution (e) and 316 nm for the other solutions, a 10 l loop injector. Inject separately reference solutions (b) and (e). If necessary, adjust the concentration of acetonitrile in the mobile phase so that in the chromatogram obtained with reference solution (b) the capacity factor of the peak due to quinidine is 3.5 to 4.5, V0 (the distance along the baseline between the point of injection and the perpendicular dropped from the maximum of the peak of an unretained component) being calculated from the peak due to thiourea in the chromatogram obtained with reference solution (e). Inject reference solutions (a), (b), (c) and (d). The chromatogram obtained with reference solution (a) shows a principal peak due to quinine and a peak due to dihydroquinine with a retention time relative to quinine of about 1.4. The chromatogram obtained with reference solution (b) shows a principal peak due to quinidine and a peak due to dihydroquinidine, with a retention time relative to quinidine of about 1.2. The chromatogram obtained with reference solution (c) shows four peaks due to quinine, dihydroquinine, quinidine and dihydroquinidine which are identified by comparison of their retention times with those of the corresponding peaks in the chromatograms obtained with reference solutions (a) and (b). The test is not valid unless (a) in the chromatogram obtained with reference solution (c) the resolution between the peaks due to quinine and quinidine is at least 1.5 and the resolution between the peaks due to dihydroquinidine and quinine is at least 1.0 and (b) the signal-to-noise ratio of the principal peak in the chromatogram obtained with reference solution (d) is at least 5. Inject the test solution and allow the chromatography to proceed for 2.5 times the retention time of the principal peak. Calculate the percentage content of related substances by normalisation, ignoring any peaks the areas of which are less than that of the peak in the chromatogram obtained with reference solution (d) (0.2 per cent). The content of dihydroquinine is not greater than 10 per cent, the content of any related substance eluting before quinine is not greater than 5 per cent and the content of any other related substance is not greater than 2.5 per cent.
Titratable cation. 79.7 to 84.2 per cent, calculated on the dried basis, determined by the following method. Weigh accurately about 0.4 g, dissolve in 10 ml of water, add 40 ml of methanol and titrate with 0.1 M sodium hydroxide using phenolphthalein solution as indicator. 1 ml of 0.1 M sodium hydroxide is equivalent to 0.01632 g of [C20H26N2O2]2+. Sulphates (2.3.17). 0.125 g complies with the limit test for sulphates ( 0.12 per cent). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 3.0 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.3 g, dissolve in 50 ml of anhydrous glacial acetic acid and add 20 ml of acetic anhydride and 10 ml of mercuric acetate solution. Titrate with 0.1 M perchloric acid, using crystal violet solution as indicator. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent of 0.01987 g of C20H24N2O2,2HCl. Storage. Store protected from light.
Quinine Dihydrochloride Injection

Quinine Acid Hydrochloride Injection
Quinine Dihydrochloride Injection is a sterile solution of Quinine Dihydrochloride in Water for Injections. Quinine Dihydrochloride Injection contains not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of quinine dihydrochloride, C20H24N2O2,2HCl. Description. A clear, almost colourless to light yellow solution.
Identification
A. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 80 volumes of toluene, 20 volumes of acetone and 10 volumes of diethylamine. Test solution. Extract a volume of the injection containing 0.1 g of Quinine Dihydrochloride Bisulphate with 10 ml of a mixture of 2 volumes of chloroform and 1 volume of ethanol (95 per cent) and filter. Reference solution (a). A 1 per cent w/v solution of quinine sulphate in the same solvent mixture. Reference solution (b). A solution of 1 per cent w/v of each of quinidine sulphate RS and quinine sulphate RS in the same solvent mixture. Apply to the plate 2 l of each solution. After development, dry the plate in air and spray with 0.05 M ethanolic sulphuric
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acid and then with dilute potassium iodobismuthate solution. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows two clearly separated spots. B. Dilute 1 ml to 20 ml with water, add 0.1 M sodium hydroxide dropwise until the solution becomed turbid. Add 1 or 2 drops of 0.05 M sulphuric acid to obtain a clear solution. To this solution add an equal volume of dilute sulphuric acid; a strong blue fluorescence is produced. C. Solution A gives the reactions of chlorides (2.3.1).
that in the chromatogram obtained with reference solution (b) the capacity factor of the peak due to quinidine is 3.5 to 4.5, V0 (the distance along the baseline between the point of injection and the perpendicular dropped from the maximum of the peak of an unretained component) being calculated from the peak due to thiourea in the chromatogram obtained with reference solution (e). Inject reference solutions (a), (b), (c) and (d). The chromatogram obtained with reference solution (a) shows a principal peak due to quinine and a peak due to dihydroquinine with a retention time relative to quinine of about 1.4. The chromatogram obtained with reference solution (b) shows a principal peak due to quinidine and a peak due to dihydroquinidine, with a retention time relative to quinidine of about 1.2. The chromatogram obtained with reference solution (c) shows four peaks due to quinine, dihydroquinine, quinidine and dihydroquinidine which are identified by comparison of their retention times with those of the corresponding peaks in the chromatograms obtained with reference solutions (a) and (b). The test is not valid unless (a) in the chromatogram obtained with reference solution (c) the resolution between the peaks due to quinine and quinidine is at least 1.5 and the resolution factor between the peaks due to dihydroquinidine and quinine is at least 1.0 and (b) the signal-to-noise ratio of the principal peak in the chromatogram obtained with reference solution (d) is at least 5. Inject the test solution and allow the chromatography to proceed for 2.5 times the retention time of the principal peak. Calculate the percentage content of related substances by normalisation, ignoring any peaks the areas of which are less than that of the peak in the chromatogram obtained with reference solution (d) (0.2 per cent). The content of dihydroquinine is not greater than 10 per cent, the content of any related substance eluting before quinine is not greater than 5 per cent and the content of any other related substance is not greater than 2.5 per cent. Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. To an accurately measured volume containing about 0.5 g of Quinine Dihydrochloride add 20 ml of water and 5 ml of sodium hydroxide solution. Extract with successive quantities, each of 10 ml, of chloroform until complete extraction of the alkaloid is effected, washing each extract with the same two quantities, each of 5 ml, of water. Remove the chloroform from the combined extracts, dissolve the residue in 50 ml of anhydrous glacial acetic acid and add 20 ml of acetic anhydride. Titrate with 0.1 M perchloric acid, using crystal violet solution as indicator. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.01987 g of C20H24N2O2,2HCl.
Tests
pH (2.4.24).1.5 to 3.0. Other cinchona alkaloids. Determine by liquid chromatography (2.4.14). Test solution. Dilute, if necessary a suitable volume of the injection with the mobile phase to produce a solution containing 0.2 per cent w/v of Quinine Dihydrochloride. Reference solution (a). Dissolve 20 mg of quinine sulphate RS, with gentle heating if necessary, in 5 ml of the mobile phase and dilute to 10 ml with the mobile phase. Reference solution (b). Prepare in the same manner as reference solution (a) but using quinidine sulphate RS in place of quinine sulphate RS. Reference solution (c). Mix equal volumes of reference solutions (a) and (b). Reference solution (d). Dilute 1 volume of reference solution (a) to 10 volumes with the mobile phase and dilute 1 volume of the resulting solution to 50 volumes with the mobile phase. Reference solution (e). A solution containing 0.1 per cent w/v of thiourea in the mobile phase. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5 m) (such as Hypersil ODS 5 m), mobile phase: a solution prepared by dissolving 6.8 g of potassium dihydrogen orthophosphate and 3.0 g of hexylamine in 700 ml of water, adjusting the pH to 2.8 with 1 M orthophosphoric acid, adding 60 ml of acetonitrile and diluting to 1000 ml with water, flow rate. 1.5 ml per minute, spectrophotometer set at 250 nm for reference solution (e) and 316 nm for the other solutions, a 10 l loop injector. Inject separately reference solutions (b) and (e). If necessary, adjust the concentration of acetonitrile in the mobile phase so
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Storage. Store protected from light. Labelling. The label states that the solution must be diluted to a strength not exceeding 30 mg per ml before administration and that care should be taken to ensure slow intravenous injection.
C. To a 0.5 per cent w/v solution add an equal volume of dilute sulphuric acid; a strong blue fluorescence is produced. D. To 5 ml of a 1 per cent w/v solution add 1 ml of silver nitrate solution and stir with a glass rod; after a short interval, a white precipitate soluble in nitric acid is produced (distinction from many other alkaloids). E. A 1 per cent w/v solution gives the reactions of sulphates (2.3.1).
Quinine Sulphate
OCH3 HO H N N H H
2
Tests
Appearance of solution. A 2.0 per cent w/v solution in 0.1 M hydrochloric acid is clear (2.4.1), and not more intensely coloured than reference solution GYS4 (2.4.1).
H CH2 , H2SO4, 2H2O
pH (2.4.24). 5.7 to 6.6, determined in a 1.0 per cent w/v suspension in water. Specific optical rotation (2.4.22). 237 to 245, determined in a 2.0 per cent w/v solution in 0.1 M hydrochloric acid.
(C20H24N2O2)2,H2SO4,2H2O
Mol. Wt.783.0
Other cinchona alkaloids. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 20 mg of the substance under examination in 5 ml of the mobile phase. Heat gently, if necessary to dissolve the powder as completely as possible, cool, dilute to 10 ml with the mobile phase and mix. Reference solution (a). Dissolve 20 mg of quinine sulphate RS, with gentle heating if necessary, in 5 ml of the mobile phase and dilute to 10 ml with the mobile phase. Reference solution (b). Prepare in the same manner as reference solution (a) but using quinidine sulphate RS in place of quinine sulphate RS. Reference solution (c). Mix equal volumes of reference solutions (a) and (b). Reference solution (d). Dilute 1 volume of reference solution (a) to 10 volumes with the mobile phase and dilute 1 volume of the resulting solution to 50 volumes with the mobile phase. Reference solution (e). A solution containing 0.1 per cent w/v of thiourea in the mobile phase. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5 m) (such as Hypersil ODS 5 m), mobile phase: a solution prepared by dissolving 6.8 g of potassium dihydrogen orthophosphate and 3.0 g of hexylamine in 700 ml of water, adjusting the pH to 2.8 with 1 M orthophosphoric acid, adding 60 ml of acetonitrile and diluting to 1000 ml with water, flow rate. 1.5 ml per minute, spectrophotometer set at 250 nm for reference solution (e) and 316 nm for the other solutions, a 10 l loop injector.
Quinine Sulphate is (8S,9R)-6-methoxycinchonan-9-ol sulphate dihydrate. The alkaloid is obtained from the bark of various species of Cinchona. Quinine Sulphate contains not less than 99.0 per cent and not more than 101.0 per cent of alkaloid monosulphates, calculated as (C20H24N2O2)2,H2SO4 on the dried basis. Description. White or almost white, needle-like crystals or a crystalline powder.
Identification
A. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 40 volumes of toluene, 24 volumes of ether and 10 volumes of diethylamine. Test solution. Dissolve 1 g of the substance under examination in 100 ml of methanol. Reference solution. A 1 per cent w/v solution of quinine sulphate RS in methanol. Apply to the plate 5 l of each solution. After development, dry the plate in air for 15 minutes and repeat the development. Dry the plate at 105 for 30 minutes, allow to cool and spray with potassium iodoplatinate solution. The principal spot in the chromatogram obtained with the test solution corresponds to the spot in the chromatogram obtained with the reference solution. B. To 5 ml of a 0.1 per cent w/v solution add 0.2 ml of bromine solution and 1 ml of dilute ammonia solution; an emeraldgreen colour is produced.
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Inject separately reference solutions (b) and (e). If necessary, adjust the concentration of acetonitrile in the mobile phase so that in the chromatogram obtained with reference solution (b) the capacity factor of the peak due to quinidine is 3.5 to 4.5, V0 (the distance along the baseline between the point of injection and the perpendicular dropped from the maximum of the peak of an unretained component) being calculated from the peak due to thiourea in the chromatogram obtained with reference solution (e). Inject reference solutions (a), (b), (c) and (d). The chromatogram obtained with reference solution (a) shows a principal peak due to quinine and a peak due to dihydroquinine with a retention time relative to quinine of about 1.4. The chromatogram obtained with reference solution (b) shows a principal peak due to quinidine and a peak due to dihydroquinidine, with a retention time relative to quinidine of about 1.2. The chromatogram obtained with reference solution (c) shows four peaks due to quinine, dihydroquinine, quinidine and dihydroquinidine which are identified by comparison of their retention times with those of the corresponding peaks in the chromatograms obtained with solutions (a) and (b). The test is not valid unless (a) in the chromatogram obtained with reference solution (c) the resolution between the peaks due to quinine and quinidine is at least 1.5 and the resolution between the peaks due to dihydroquinidine and quinine is at least 1.0 and (b) the signal-to-noise ratio of the principal peak in the chromatogram obtained with reference solution (d) is at least 5. Inject the test solution and allow the chromatography to proceed for 2.5 times the retention time of the principal peak. Calculate the percentage content of related substances by normalisation, ignoring any peaks the areas of which are less than that of the peak in the chromatogram obtained with reference solution (d) (0.2 per cent). The content of dihydroquinine is not greater than 10 per cent, the content of any related substance eluting before quinine is not greater than 5 per cent and the content of any other related substance is not greater than 2.5 per cent. Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). 3.0 to 5.0 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.2 g, dissolve in a mixture of 10 ml of chloroform and add 20 ml of acetic anhydride. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.02490 g of (C20H24N2O2)2,H2SO4. Storage. Store protected from light.
Quinine Tablets
Quinine Sulphate Tablets
Quinine Sulphate Tablets contain not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of quinine sulphate, (C20H24N2O2)2,H2SO4,2H2O. The tablets are coated.
Identification
A. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 80 volumes of toluene, 20 volumes of acetone and 10 volumes of diethylamine. Test solution. Extract a quantity of the powdered tablets containing 0.1 g of Quinine Sulphate with 10 ml of a mixture of 2 volumes of chloroform and 1 volume of ethanol (95 per cent) and filter. Reference solution. A 1 per cent w/v solution of quinine sulphate in the same solvent mixture. Apply to the plate 2 l of each solution. After development, dry the plate in air and spray with 0.05 M ethanolic sulphuric acid and then with dilute potassium iodobismuthate solution. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution. B. Extract a quantity of the powdered tablets containing 0.1 g of Quinine Sulphate with 20 ml of water and filter (solution A). To 5 ml of solution A add 0.2 ml of bromine solution and 1 ml of dilute ammonia solution; an emerald-green colour is produced. C. Solution A is laevo-rotatory. D. Solution A gives the reactions of sulphates (2.3.1).
Tests
Dissolution (2.5.2). Apparatus. No 2 Medium. 900 ml of 0.1 M hydrochloric acid Speed and time. 100 rpm and 30 minutes. Withdraw a suitable volume of the medium and filter. Measure the absorbance of the filtrate, suitably diluted if necessary, at the maximum at about 348 nm (2.4.7). Calculate the content of C20H24N2O2, H2SO4, 2H2O in the medium from a solution of known concentration of quinine sulphate RS. D. Not less than 70 per cent of the stated amount of C20H24N2O2, H2SO4, 2H2O. Other cinchona alkaloids. Determine by liquid chromatography (2.4.14).
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IP 2007
Test solution. Remove any coating from the tablets and mix a quantity of the powdered tablet cores containing 50 mg of Quinine Sulphate with 20 ml of the mobile phase. Heat gently to dissolve the powder as completely as possible, cool, dilute to 25 ml with the mobile phase and filter, discarding the first few ml of the filtrate. Reference solution (a). Dissolve 20 mg of quinine sulphate RS, with gentle heating if necessary, in 5 ml of the mobile phase and dilute to 10 ml with the mobile phase. Reference solution (b). Prepare in the same manner as reference solution (a) but using quinidine sulphate RS in place of quinine sulphate RS. Reference solution (c). Mix equal volumes of reference solutions (a) and (b). Reference solution (d). Dilute 1 volume of reference solution (a) to 10 volumes with the mobile phase and dilute 1 volume of the resulting solution to 50 volumes with the mobile phase. Reference solution (e). A solution containing 0.1 per cent w/v of thiourea in the mobile phase. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5 m) (such as Hypersil ODS 5 m), mobile phase: a solution prepared by dissolving 6.8 g of potassium dihydrogen orthophosphate and 3.0 g of hexylamine in 700 ml of water, adjusting the pH to 2.8 with 1 M orthophosphoric acid, adding 60 ml of acetonitrile and diluting to 1000 ml with water, flow rate. 1.5 ml per minute, spectrophotometer set at 250 nm for reference solution (e) and 316 nm for the other solutions, a 10 l loop injector. Inject separately reference solutions (b) and (e). If necessary, adjust the concentration of acetonitrile in the mobile phase so that in the chromatogram obtained with reference solution (b) the capacity factor of the peak due to quinidine is 3.5 to 4.5, V0 (the distance along the baseline between the point of injection and the perpendicular dropped from the maximum of the peak of an unretained component) being calculated from the peak due to thiourea in the chromatogram obtained with reference solution (e). Inject reference solutions (a), (b), (c) and (d). The chromatogram obtained with reference solution (a) shows a principal peak due to quinine and a peak due to dihydroquinine with a retention time relative to quinine of about 1.4. The chromatogram obtained with reference solution (b) shows a principal peak due to quinidine and a peak due to dihydroquinidine, with a retention time relative to quinidine of about 1.2. The chromatogram obtained with reference solution (c) shows four peaks due to quinine, dihydroquinine,
quinidine and dihydroquinidine which are identified by comparison of their retention times with those of the corresponding peaks in the chromatograms obtained with reference solutions (a) and (b). The test is not valid unless (a) in the chromatogram obtained with reference solution (c) the resolution between the peaks due to quinine and quinidine is at least 1.5 and the resolution between the peaks due to dihydroquinidine and quinine is at least 1.0 and (b) the signal-to-noise ratio of the principal peak in the chromatogram obtained with reference solution (d) is at least 5. Inject the test solution and allow the chromatography to proceed for 2.5 times the retention time of the principal peak. Calculate the percentage content of related substances by normalisation, ignoring any peaks the areas of which are less than that of the peak in the chromatogram obtained with reference solution (d) (0.2 per cent). The content of dihydroquinine is not greater than 10 per cent, the content of any related substance eluting before quinine is not greater than 5 per cent and the content of any other related substance is not greater than 2.5 per cent. Other tests. Comply with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 0.4 g of Quinine Sulphate, dissolve as completely as possible in 40 ml of acetic anhydride with the aid of heat and cool. Filter, if necessary through Whatman No.1 filter paper and rinse with an additional 40 ml of acetic anhydride in small volumes. Titrate with 0.1 M perchloric acid, using crystal violet solution as indicator. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.02610 g of (C20H24N2O2)2,H2SO4,2H2O. Storage. Store protected from light.
Quiniodochlor
Clioquinol; Iodochlorhydroxyquinoline; Iodochlorhydroxyquin
OH I N
Cl
C9H5CIINO Mol. Wt. 305.5
Quiniodochlor is 5-chloro-7-iodoquinolin-8-ol.
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Quiniodochlor contains not less than 97.0 per cent and not more than 103.0 per cent of C9H5ClINO, calculated on the dried basis. Description. A yellowish white to brownish yellow powder; odour, faint and characteristic.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with quiniodochlor RS or with the reference spectrum of quiniodochlor. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.0005 per cent w/v solution in 3 M hydrochloric acid shows an absorption maximum at about 267 nm. C. Burn 20 mg by the oxygen-flask method (2.3.34), using 5 ml of 2 M sodium hydroxide as the absorbing liquid and dilute to 25 ml with water. To 5 ml add 1 ml of silver nitrate solution; a yellow precipitate is produced. Add 5 ml of 5 M ammonia, shake, filter and acidify the filtrate with nitric acid; a white precipitate is produced.
Reference solution (a). Add 0.5 ml of N,O-bis (trimethylsilyl)acetamide to a mixture of 0.1 g of the substance under examination and 0.5 ml of pyridine, mix, allow to stand for 15 minutes and add 5 ml of hexane. Reference solution (b). Treat a mixture of 0.1 g of the substance under examination and 0.5 ml of pyridine as described for the test solution. Chromatographic system a glass column 1.5 m x 4 mm, packed with silanised diatomaceous support (100 to 120 mesh) coated with 3 per cent w/w of methyl silicone gum, temperature: column.190, inlet port and detector. 240, flame ionisation detector, nitrogen as carrier gas. In the chromatogram obtained with the test solution the peaks following the solvent peak, in order of emergence, are due to (a) 5-chloro-8-hydroxyquinoline, (b) 5,7-dichloro-8-hydroxyquinoline, (c) the internal standard, (d) quiniodochlor and (e) 5,7-diiodo-8-hydroxyquinoline. In the chromatogram obtained with reference solution (b) calculate the content of 5-chloro-8-hydroxy-quinoline, 5,7dichloro-8-hydroxyquinoline and 5,7-diiodo-8hydroxyquinoline by reference to the corresponding peaks in the chromatogram obtained with the test solution. The total content of the named impurities does not exceed 3.0 per cent w/w, the content of any other impurity does not exceed 0.2 per cent w/w and the sum of the contents is not more than 4.0 per cent w/w. Sulphated ash (2.3.18). Not more than 0.2 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying over phosphorus pentoxide at a pressure not exceeding 0.7 kPa for 24 hours. Assay. Weigh accurately about 0.3 g and dissolve in 25 ml of anhydrous pyridine. Titrate with 0.1 M tetrabutylammonium hydroxide, determining the end-point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M tetrabutylammonium hydroxide is equivalent to 0.03055 g of C9H5ClINO. Storage. Store protected from light.
Tests
Acidity or alkalinity. Shake 0.5 g with 10 ml of water previously neutralised to phenolphthalein solution. The solution is colourless and not more than 0.05 ml of 0.1 M sodium hydroxide is required to change the colour of the solution to pink. Free iodine. Shake 1.0 g with a solution of 1 g of potassium iodide in 20 ml of water for 30 seconds, allow to stand for 5 minutes and filter. To 10 ml of the filtrate add 1 ml of 1 M sulphuric acid and 2 ml of chloroform and shake. Any colour in the chloroform layer is discharged on the addition of 0.1 ml of 0.005 M sodium thiosulphate. Halide ions. Shake 0.5 g with 25 ml of water for 1 minute and filter. To the filtrate add 0.5 ml of 2 M nitric acid and 0.5 ml of 0.1 M silver nitrate and allow to stand for 5 minutes. Any opalescence produced is not more intense than that obtained by adding 0.5 ml of 0.1 M silver nitrate to 25 ml of water containing 0.5 ml of 2 M nitric acid and 0.2 ml of 0.01 M hydrochloric acid and allowing to stand for 5 minutes. Related substances. Determine by gas chromatography (2.4.13). Test solution. Add 0.5 ml of N,O-bis (trimethylsilyl)acetamide to 0.5 ml of a solution in pyridine containing 0.4 per cent w/v of each of 5-chloro-8- hydroxyquinoline, 5,7-dichloro-8hydroxy- quinoline and 5-chloro-7-iodo-8-hydroxyquinoline and 0.04 per cent w/v of the substance under examination, mix, allow to stand for 15 minutes and add 5 ml of a 0.05 per cent w/v solution of dibutylphthalate (internal standard) in hexane.
Quiniodochlor Tablets
Clioquinol Tablets; Iodochlorhydroxyquinoline Tablets; Iodochlorhydroxyquin Tablets
Quiniodochlor Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of quiniodochlor, C9H5ClINO.
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IP 2007
Identification
Triturate a quantity of the powdered tablets containing about 250 mg of Quiniodochlor with 20 ml of acetone, filter and add 20 ml of water to the filtrate. Collect the precipitate formed on a filter and dry at 105. The residue complies with the following tests. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with quiniodochlor RS or with the reference spectrum of quiniodochlor. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.0005 per cent w/v solution in 3 M hydrochloric acid shows an absorption maximum at about 267 nm.
50.0 ml with 2-methoxyethanol. To 5.0 ml of this solution add 1 ml of water and sufficient of a mixture of 24 volumes of 2-methoxyethanol and 6 volumes of water to produce 50.0 ml. To 10.0 ml of the solution add 10 ml of 2-methoxyethanol and 2 ml of a solution prepared by dissolving 0.5 g of ferric chloride hexahydrate in 80 ml of 2-methoxyethanol and adding 0.1 ml of hydrochloric acid and sufficient 2-methoxyethanol to produce 100 ml. Dilute the solution to 25.0 ml with 2-methoxyethanol and measure the absorbance of the resulting solution at the maximum at about 650 nm (2.4.7), using as blank a solution prepared by treating 10 ml of the aqueous 2-methoxyethanol in the same manner beginning at the words add 10 ml of 2-methoxyethanol...... Calculate the content of C9H5ClINO from the absorbance obtained using 10.0 ml of a solution prepared in the following manner. Dissolve 0.125 g of quiniodochlor RS in sufficient 2methoxyethanol to produce 50.0 ml, warming to effect solution; add 1 ml of water to 5.0 ml of the solution and add sufficient of the mixture of 24 volumes of 2-methoxyethanol and 6 volumes of water to produce 50.0 ml. Using 10.0 ml of this solution repeat the operation beginning at the words add 10 ml of 2-methoxyethanol.... Storage. Store protected from light.
Tests
Disintegration (2.5.1). Maximum time, 30 minutes. Other tests. Comply with the tests stated under Tablets. Assay. Weigh and finely powder 20 tablets. Weigh accurately a quantity of the powder containing 0.125 g of Quiniodochlor, shake with 20 ml of hot 2-methoxyethanol, decant the hot supernatant liquid through a fine filter. Repeat the extraction with two further quantities, of 20 ml and 10 ml, of 2-methoxyethanol, combine the filtered extracts and dilute to
1029
MONOGRAPHS
R
Rabeprazole Sodium Rabeprazole Tablets Ramipril Ramipril Capsules Ramipril Tablets Ranitidine Hydrochloride Ranitidine Injection Ranitidine Tablets Purifid Rayon Reserpine Reserpine Injection Reserpine Tablets Riboflavine Riboflavine Sodium Phosphate Riboflavine Tablets Rifampicin Rifampicin Capsules Rifampicin Oral Suspension Rifampicin Tablets Rifampicin and Isoniazid Tablets Rifampicin, Isoniazid And Ethambutol Tablets Rifampicin, Isoniazid and Pyrazinamide Tablets Rifampicin, Isoniazid, Pyrazinamide And Ethambutol Tablets Ritonavir Ritonavir Capsules Ritonavir Tablets Rosiglitazone Maleate Rosiglitazone Tablets Rosuvastatin Calcium Rosuvastatin Tablets
1031
.... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... ....
MONOGRAPHS
Roxithromycin Roxithromycin Tablets
.... ....
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IP 2007
RABEPRAZOLE SODIUM
Rabeprazole Sodium
Na O N S N
C18H20N3O3S.Na
Water (2.3.43). Not more than 7.0 per cent, determined on 0.3 g. Assay. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 0.1 g of the substance under examination in 100.0 ml of mobile phase. Dilute 5.0 ml of the solution to 100.0 ml with the same solvent. Reference solution. A 0.005 per cent w/v solution of rabeprazole sodium RS in the mobile phase. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with octylsilane bonded to porous silica (5 m) (such as Hypersil keystone betabasic C8), column temperature 40, mobile phase: a mixture of 72 volumes of 0.1 M phosphate buffer pH 7.0 and 28 volumes of acetonitrile, flow rate. 1.4 ml per minute, spectrophotometer set at 282 nm, a 10 l loop injector. Inject the reference solution. The test is not valid unless the tailing factor is not more than 2.0 and the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject the test solution and the reference solution. Calculate the percentage content of C18H20N3O3S.Na. Storage. Store protected from light and moisture.
N O CH3
Mol. Wt. 381.4
OCH3
Rabeprazole sodium is 2-({[4-(3-methoxypropoxy)-3-methyl2-pyridinyl]methyl}sulphenyl)-1H-benzimidazole sodium. Rabeprazole sodium contains not less than 98.0 per cent and not more than 102.0 per cent of C18H20N3O3S.Na, calculated on the anhydrous basis. Description. A white to light yellow, crystalline powder, hygroscopic.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with rabeprazole sodium RS. B. A 10 per cent w/v solution in carbon dioxide-free water gives reaction of sodium (2.3.1).
Tests
Related substances. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 50 mg of the substance under examination in 100 ml with the mobile phase. Reference solution (a). A 0.05 per cent w/v solution of rabeprazole sodium RS in the mobile phase. Reference solution (b). Dilute 1 ml of reference solution (a) to 100 ml with mobile phase. Chromatographic system as described under Assay. Inject reference solution (a). The test is not valid unless the tailing factor is not more than 2.0 and the column efficiency in not less than 2000 theoretical plates. Inject the test solution and reference solution (b). Run the chromatogram three times of the principal peak. In the chromatogram obtained with the test solution, the area of any secondary peak is not more than 0.5 times the area of the peak in the chromatogram obtained with reference solution (b) (0.5 per cent) and the sum of areas of all the secondary peaks is not more than 1.5 times the area of the peak in the chromatogram obtained with the reference solution (b) (1.5 per cent). Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method A (20 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent.
Rabeprazole Tablets
Rabeprazole Sodium Tablets
Rabeprazole Tablets contain Rabeprazole Sodium. Rabeprazole Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of rabeprazole sodium, C18H20N3O3SNa.
Identification
Tests
Dissolution (2.5.2). Apparatus. No 1 Medium. 900 ml of 0.1 M hydrochloric acid. Speed and time. 50 rpm for 120 minutes. Replace the 0.1 M hydrochloric acid with phosphate buffer pH 7.4. Run the apparatus at 75 rpm for 45 minutes. Withdraw
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RAMIPRIL
IP 2007
a suitable volume of the medium and filter. Measure the absorbance of the filtered solution immediately, suitably diluted with phosphate buffer pH 10.4 if necessary, at the maximum at about 291 nm (2.4.7). Calculate the content of C18H20N3O3SNa in the medium from the absorbance obtained from a solution of known concentration of rabeprazole sodium RS, prepared by dissolving in minimum quantity of a mixture of 75 volumes of acetonitrile and 25 volumes of methanol and suitably diluted with phosphate buffer pH 10.4. D. Not less than 70 per cent of the stated amount of C18H20N3O3SNa. Related substances. Determine by liquid chromatography (2.4.14). Solvent mixture. 80 volumes of methanol, 20 volumes of water and 0.1 volume of diethylamine. Test solution. Weigh accurately a quantity of the powdered tablet containing 50 mg of Rabeprazole Sodium, disperse in 100 ml of solvent mixture and filter. Reference solution (a). A 0.05 per cent w/v solution of rabeprazole sodium RS in the solvent mixture. Reference solution (b). Dilute 1 ml of reference solution (a) to 100 ml with solvent mixture. Chromatographic system as described under Assay. Inject reference solution (a). The test is not valid unless the tailing factor is not more than 2.0 and the column efficiency in not less than 2000 theoretical plates. Inject the test solution and reference solution (b). Run the chromatogram three times of the principal peak. In the chromatogram obtained with the test solution, the area of any secondary peak is not more than twice the area of the peak in the chromatogram obtained with reference solution (b) (2.0 per cent) and the sum of areas of all the secondary peaks is not more than 6 times the area of the peak in the chromatogram obtained with the reference solution (b) (6.0 per cent). Uniformity of content (For tablets containing 10 mg or less). Comply with the tests stated under Tablets. Disperse 1 tablet in sufficient 0.1 M sodium hydroxide to produce 0.0015 per cent w/v solution. Measure the absorbance of the resulting solution at the maximum at about 292 nm (2.4.7). Calculate the content of C18H20N3O3SNa from the absorbance obtained from same concentration of rabeprazole sodium RS in the same medium. Other tests. Comply with the tests stated under Tablets. Assay. Determine by liquid chromatography (2.4.14). Solvent mixture. 80 volumes of methanol, 20 volumes of water and 0.1 volume of diethylamine. Test solution. Weigh and powder 20 tablets. Weigh accurately a quantity of the powdered tablet containing 50 mg of
Rabeprazole Sodium, disperse in 20 ml of 0.1 M sodium hydroxide and dilute to 100.0 ml with solvent mixture, filter. Reference solution. Weigh accurately about 25 mg of rabeprazole sodium RS, dissolve in 10 ml of 0.1 M sodium hydroxide and dilute to 50.0 ml with solvent mixture. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 65 volumes of 0.15 per cent w/v solution of potassium dihydrogen phosphate previously adjusted pH to 6.0 with orthophosphoric acid and 35 volumes of acetonitrile, flow rate. 1 ml per minute, spectrophotometer set at 280 nm, a 10 l loop injector. Inject the reference solution. The test is not valid unless the tailing factor is not more than 2.0 and the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject the test solution and the reference solution. Calculate the content of C18H20N3O3SNa. Storage. Store protected from light and moisture.
Ramipril
C23H32N2O5 Mol. Wt. 416.5 Ramipril is (2S,3aS,6aS)-1-[(S)-2-[[(S)-1-(ethoxycarbonyl)-3phenylpropyl]amino]propanoyl] octahydrocyclopenta[b]pyrrole-2-carboxylic acid. Ramipril contains not less than 98.0 per cent and not more than 101.0 per cent of C23H32N2O5, calculated on the dried basis. Description. A white or almost white, crystalline powder.
Identification
Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with ramipril RS.
Tests
Appearance of solution. A 1.0 per cent w/v solution in methanol is clear (2.4.1) and colourless (2.4.1). Specific optical rotation (2.4.22). + 32.0 to + 38.0, determined in 1.0 per cent w/v solution in 0.1 M methanolic hydrochloric acid. Related substances. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 25 mg of the substance under examination in 25 ml of mobile phase B.
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RAMIPRIL CAPSULES
Reference solution (a). A 0.1 per cent w/v solution of ramipril RS in the mobile phase B. Reference solution (b). Dilute 1 ml of reference solution (a) to 100 ml with mobile phase B. Chromatographic system a stainless steel column 25 cm x 4.0 mm packed with octadecylsilyl bonded to porous silica (5 m), column temperature 65, mobile phase: A. dissolve 2.0 g of sodium perchlorate in a mixture of 0.5 ml of triethylamine and 800 ml of water; adjust pH to 3.6 with orthophosphoric acid and add 200 ml of acetonitrile, B. dissolve 2.0 g of sodium perchlorate in a mixture of 0.5 ml of triethylamine and 300 ml of water; adjust pH to 2.6 with orthophosphoric acid and add 700 ml of acetonitrile, a linear gradient programme using the conditions given below, flow rate. 1 ml per minute, spectrophotometer set at 210 nm, a 10 l loop injector. Time Mobile phase A Mobile phase B (in min.) (per cent v/v) (per cent v/v) 0 90 10 6 90 10 7 75 25 20 65 35 30 25 75 40 25 75 45 90 10 55 90 10 Inject reference solution (a). The test is not valid unless the tailing factor is not more than 2.0 and the column efficiency in not less than 2000 theoretical plates. Inject the test solution and reference solution (b). In the chromatogram obtained with the test solution, the area of any secondary peak is not more than 0.5 times the area of the peak in the chromatogram obtained with reference solution (b) (0.5 per cent) and the sum of areas of all the secondary peaks is not more than the area of the peak in the chromatogram obtained with the reference solution (b) (1.0 per cent). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.2 per cent, determined on 1.0 g by drying in an oven at 60, under vaccume, for 4 hours. Assay. Weigh accurately about 0.3 gm, dissolve in 25 ml of methanol and add 25 ml of water. Titrate with 0.1 M sodium hydroxide. Determine the end-point potentiometrically (2.4.25). Carry out a blank titration.
1 ml of 0.1 M sodium hydroxide is equivalent to 0.04165 gm of C23H32N2O5. Storage. Store protected from light.
Ramipril Capsules
Ramipril Capsules contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of ramipril, C23H32N2O5.
Identification
Shake a quantity of the content of the capsules containing 25 mg of Ramipril with 50 ml of acetone, centrifuge for 10 minutes, filter. Evaporate the filtrate to dryness at 60 for 3 hours. The residue complies with the following test. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with ramipril RS.
Tests
Dissolution (2.5.2). Apparatus. No 1 Medium. 500 ml of 0.1 M hydrochloric acid. Speed and time. 75 rpm and 45 minutes. Withdraw a suitable volume of the medium and filter. Determine by liquid chromatography (2.4.14). Test solution. Dilute the filtrate to get 0.00025 per cent w/v solution of Ramipril with 0.1 M hydrochloric acid. Reference solution. A 0.00025 per cent w/v solution of ramipril RS in 0.1 M hydrochloric acid. Chromatographic system as described under Assay. Inject the test solution and the reference solution. Calculate the content of C23H32N2O5. D. Not less than 70 per cent of the stated amount of C23H32N2O5. Uniformity of content (For capsules containing 10 mg or less). Comply with the test stated under Capsules. Determine by liquid chromatography (2.4.14). Test solution. Disperse one capsule in 100 ml of 0.1 M hydrochloric acid, sonicate for 15 minutes. Dilute if necessary to produce 0.025 per cent w/v solution of Ramipril in 0.1 M hydrochloric acid. Reference solution. A 0.025 per cent w/v solution of ramipril RS in 0.1 M hydrochloric acid. Chromatographic system as described under Assay.
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Inject the test solution and the reference solution. Calculate the content of C23H32N2O5. Assay. Determine by liquid chromatography (2.4.14). Test solution. Weigh a quantity of the content of capsules containing 25 mg of Ramipril, disperse in 100.0 ml of 0.1 M hydrochloric acid, mix and centrifuge. Reference solution. A 0.025 per cent w/v solution of ramipril RS in 0.1 M hydrochloric acid. Chromatographic system a stainless steel column 12.5 cm x 4.6 mm packed with octadecylsilyl bonded to porous silica (5 m), mobile phase: a mixture of 42 volumes of acetonitrile and 58 volumes of a solution containing 1.4 per cent w/ v solution of sodium perchlorate and 0.58 per cent w/ v solution of orthophosphoric acid adjusted to pH 2.5 with triethylamine, adjust the pH of the mixture to 2.1 with orthophosphoric acid, flow rate. 1 ml per minute, spectrophotometer set at 210 nm, a 50 l loop injector. Inject the reference solution. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject the test solution and the reference solution. Calculate the content of C23H32N2O5.
Determine by liquid chromatography (2.4.14). Test solution. Dilute the filtrate to get 0.00025 per cent w/v solution of Ramipril with 0.1 M hydrochloric acid. Reference solution. A 0.00025 per cent w/v solution of ramipril RS in 0.1 M hydrochloric acid. Chromatographic system as described under Assay. Inject the test solution and the reference solution. Calculate the content of C23H32N2O5. D. Not less than 70 per cent of the stated amount of C23H32N2O5. Uniformity of content (For tablets containing 10 mg or less). Comply with the test stated under Tablets. Determine by liquid chromatography (2.4.14), as described under Assay. Test solution. Take one tablet, add 5 ml of 0.1 M hydrochloric acid, sonicate for 10 minutes, dilute, if necessary, with sufficient 0.1 M hydrochloric acid to produce a solution containing 0.025 per cent w/v of Ramipril, centrifuge and use the supernatant liquid. Calculate the content of C23H32N2O5. Assay. Determine by liquid chromatography (2.4.14). Test solution. Weigh and powder 20 tablets. Weigh a quantity of powdered tablets containing 25 mg of Ramipril, disperse in 100.0 ml of 0.1 M hydrochloric acid and centrifuge. Reference solution. A 0.025 per cent w/v solution of ramipril RS in 0.1 M hydrochloric acid. Chromatographic system a stainless steel column 12.5 cm x 4.6 mm packed with octadecylsilyl bonded to porous silica (5 m), mobile phase: a mixture of 42 volumes of acetonitrile and 58 volumes of a solution containing 1.4 per cent w/v solution of sodium perchlorate and 0.58 per cent w/v solution of orthophosphoric acid adjusted to pH 2.5 with triethylamine, adjust the pH of the mixture to 2.1 with orthophosphoric acid, flow rate. 1 ml per minute, spectrophotometer set at 210 nm, a 50 l loop injector. Inject the reference solution. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject the test solution and the reference solution. Calculate the content of C23H32N2O5. Storage. Store protected from moisture. 1036
Ramipril Tablets
Ramipril Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of ramipril, C23H32N2O5.
Identification
Shake a quantity of the powdered tablets containing 25 mg of Ramipril with 50 ml of acetone, centrifuge for 10 minutes, filter. Evaporate the filtrate to dryness at 60 for 3 hours. The residue complies with the following test. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with ramipril RS.
Tests
Dissolution (2.5.2). Apparatus. No 1 Medium. 500 ml of 0.1 M hydrochloric acid. Speed and time. 75 rpm and 45 minutes. Withdraw a suitable volume of the medium and filter.
IP 2007
RANITIDINE HYDROCHLORIDE
Ranitidine Hydrochloride
H3C N CH3 O S H N CHNO2 NHCH3
Mol. Wt. 350.9
Reference solution (c). Dilute 30.0 ml of reference solution (a) to 100 ml with methanol.
, HCl
Reference solution (d). Dilute 5.0 ml of reference solution (a) to 100 ml with methanol. Reference solution (e). Weigh accurately a quantity of ranitidine hydrochloride related compound A RS in methanol, and dilute with methanol to obtain a solution containing a known concentration of about 0.127 per cent w/v. Reference solution (f). Weigh accurately a quantity of ranitidine hydrochloride related compound B RS in methanol, and dilute with methanol to obtain a solution containing a known concentration of about 0.1 per cent w/v. Apply to the plate 10 l of each solution except reference solution (e). Apply separately an additional 10 l of the test solution and on top of this application, apply 10 l of reference solution (e). After development, dry the plate in air and expose it to iodine vapours in a closed chamber until the spots are revealed. Any spot in the chromatogram obtained with the test solution corresponding to the principal spot in the chromatogram obtained with reference solution (f) is not more intense than that of the principal spot in the chromatogram obtained with reference solution (b) (0.5 per cent) and no other spot in the chromatogram obtained with the test solution is more intense than the principal spot in the chromatogram obtained with reference solution (c) (0.3 per cent). The sum of the intensities of all the secondary spots in the chromatogram obtained with the test solution does not exceed 1.0 per cent. The test is not valid unless the chromatogram obtained with the combined test solution and reference solution (e) shows two clearly separated principal spots and the chromatogram obtained with reference solution (d) shows a clearly visible spot. Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.75 per cent, determined on 1.0 g by drying in an oven at 60 at a pressure not exceeding 2.75 kPa for 3 hours. Assay. Determine by liquid chromatography (2.4.14) Test solution. A 0.0112 per cent w/v of the substance under examination in the mobile phase. Reference solution. 0.0112 per cent w/v of ranitidine hydrochloride RS in the mobile phase. Chromatographic system a stainless steel column 25 cm x 4.0 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 85 volumes of methanol and 15 volumes of 0.1 M ammonium acetate, flow rate. 2 ml per minute,
C13H22N4O3S,HCl
Ranitidine Hydrochloride is N-[2-[[[5-[(dimethylamino) methyl]furan-2-yl]methyl]thio]ethyl]-N-methyl-2nitroethene-1,1-diamine hydrochloride. Ranitidine Hydrochloride contains not less than 97.5 per cent and not more than 102.0 per cent of C13H22N4O3S, HCl, calculated on the dried basis. Description. A white to pale yellow, crystalline powder.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with ranitidine hydrochloride RS or with the reference spectrum of ranitidine hydrochloride. B. In the test for Related substances, the principal spot in the chromatogram obtained with test solution (b) corresponds to that in the chromatogram obtained with reference solution (a). C. A 5 per cent w/v solution gives the reactions of chlorides (2.3.1).
Tests
Appearance of solution. A 1.0 per cent w/v solution is clear (2.4.1), and not more intensely coloured than reference solution BYS5 (2.4.1). pH (2.4.24). 4.5 to 6.0, determined in a 1.0 per cent w/v solution in carbon dioxide-free water. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 25 volumes of ethyl acetate, 15 volumes of 2-propanol, 4 volumes of strong ammonia solution and 2 volumes of water. Test solution (a). Dissolve 0.22 g of the substance under examination in 10 ml of methanol. Test solution (b). Dilute 1 ml of test solution (a) to 100 ml with methanol. Reference solution (a). Weigh accurately a quantity of ranitidine hydrochloride RS in methanol, and dilute with methanol to obtain a solution containing a known concentration of about 0.022 per cent w/v. Reference solution (b). Dilute 10.0 ml of reference solution (a) to 20 ml with methanol.
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IP 2007
spectrophotometer set at 322 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject alternately the test solution and the reference solution. Calculate the content of C13H22N4O3S, HCl. Storage. Store protected from light and moisture.
Reference solution (a). Weigh accurately a quantity of ranitidine hydrochloride RS in water, and dilute with water to obtain a solution containing a known concentration of about 0.056 per cent w/v. Reference solution (b). Dilute 10.0 ml of reference solution (a) to 20 ml with water. Reference solution (c). Dilute 5.0 ml of reference solution (a) to 20 ml with water. Reference solution (d). Dilute 6.0 ml of reference solution (c) to 10 ml with water. Reference solution (e). Dilute 5.0 ml of reference solution (b) to 50 ml with water. Reference solution (f). Dilute 5 ml of reference solution (e) to 10 ml with water. Reference solution (g). A 0.127 per cent w/v of solution of ranitidine impurity A RS in methanol. Apply to the plate 10 l of each solution. Apply separately an additional 10 l of the test solution and on top of this application, apply 10 l of reference solution (g). After development, dry the plate in air and expose it to iodine vapours in a closed chamber until the spots are revealed. The major secondary spot in the chromatogram obtained with the test solution is not more intense than that of the principal spot in the chromatogram obtained with reference solution (a) (2.0 per cent) and no other secondary spot in the chromatogram obtained with the test solution is more intense than the principal spot in the chromatogram obtained with reference solution (b) (1.0 per cent). The sum of the intensities of all the secondary spots in the chromatogram obtained with the test solution does not exceed 5.0 per cent. The test is not valid unless the chromatogram obtained with the combined test solution and reference solution (g) shows two clearly separated principal spots and the chromatogram obtained with reference solution (f) shows a clearly visible spot. Other tests. Comply with the tests stated under Parenteral Preparations (Injections). Assay. Determine by liquid chromatography (2.4.14) Test solution. Dilute a volume of the injection containing 10.0 mg of ranitidine to 100.0 ml with the mobile phase. Reference solution. A 0.0112 per cent w/v solution of ranitidine hydrochloride RS in the mobile phase. Chromatographic system a stainless steel column 25 cm x 4.0 mm packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 85 volumes of methanol and 15 volumes of 0.1 M ammonium acetate, flow rate. 2 ml per minute,
Ranitidine Injection
Ranitidine Hydrochloride Injection
Ranitidine Injection is a sterile solution of Ranitidine Hydrochloride in Water for Injections and may be suitably buffered. Ranitidine Hydrochloride Injection contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of ranitidine, C13H22N4O3S.
Identification
A. To a volume of the injection containing 25 mg of ranitidine add 20 ml of methanol, mix and evaporate to dryness. Add 1 ml of light petroleum (60 to 80) to the resulting residue, scratch the side of the vessel with a glass rod to induce crystallisation, evaporate to dryness and dry the residue at 60 for 10 minutes.The residue complies with the following test. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with ranitidine hydrochloride RS or with the reference spectrum of ranitidine hydrochloride. B. In the Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the peak in the chromatogram obtained with the reference solution.
Tests
pH (2.4.24). 6.7 to 7.3, if the preparation is buffered; 4.5 to 7.0, if the preparation is unbuffered. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 25 volumes of ethyl acetate, 15 volumes of 2-propanol, 4 volumes of strong ammonia solution and 2 volumes of water. Test solution. Dilute suitably a volume of the injection with water to produce a solution containing the equivalent of 2.5 per cent w/v of ranitidine in methanol.
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RANITIDINE TABLETS
spectrophotometer set at 322 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject alternately the test solution and the reference solution. Calculate the content of C13H22N4O3S in the injection. Storage. Store protected from light. Labelling. The label states (1) the strength in terms of the equivalent amount of ranitidine; (2) where appropriate, that the injection is buffered.
methanol to obtain a solution containing a known concentration of about 0.022 per cent w/v. Reference solution (b). Dilute 10 ml of reference solution (a) to 20 ml with methanol. Reference solution (c). Dilute 30 ml of reference solution (a) to 100 ml with methanol. Reference solution (d). Dilute 5 ml of reference solution (a) to 50 ml with methanol. Reference solution (e). Dilute 5 ml of reference solution (a) to 100 ml with methanol. Reference solution (f). Weigh accurately a quantity of ranitidine hydrochloride related compound A RS in methanol, and dilute with methanol to obtain a solution containing a known concentration of about 0.127 per cent w/v. Apply to the plate 10 l of each solution. Apply separately an additional 10 l of the test solution and on top of this application, apply 10 l of reference solution (f). After development, dry the plate in air and expose it to iodine vapours in a closed chamber until the spots are revealed. Any spot in the chromatogram obtained with the test solution corresponding to the principal spot in the chromatogram obtained with reference solution (f) is not more intense than that of the principal spot in the chromatogram obtained with reference solution (b) (0.5 per cent) and no other spot in the chromatogram obtained with the test solution is more intense than the principal spot in the chromatogram obtained with reference solution (c) (0.3 per cent). The sum of the intensities of all the secondary spots in the chromatogram obtained with the test solution does not exceed 2.0 per cent. The test is not valid unless the chromatogram obtained with the combined test solution and reference solution (f) shows two clearly separated principal spots and the chromatogram obtained with reference solution (e) shows a clearly visible spot. Other tests. Comply with the tests stated under Tablets. Assay. Determine by liquid chromatography (2.4.14) Test solution. Weigh and powder 20 tablets. Shake 1.5 g of the powder with 400 ml of the mobile phase, dilute to 500.0 ml with the mobile phase, filter and dilute the filtrate with the mobile phase to obtain a solution containing the equivalent of 0.01 per cent w/v of ranitidine. Reference solution. A 0.0112 per cent w/v solution of ranitidine hydrochloride RS in the mobile phase. Chromatographic system a stainless steel column 25 cm x 4.0 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 85 volumes of methanol and 15 volumes of 0.1 M ammonium acetate,
Ranitidine Tablets
Ranitidine Hydrochloride Tablets
Ranitidine Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of the ranitidine, C13H22N4O3S. The tablets are coated.
Identification
A. Shake a quantity of the powdered tablets containing 25 mg of ranitidine with 5 ml of methanol for 5 minutes, filter and evaporate the filtrate to dryness. Add 1 ml of light petroleum (60 to 80) to the resulting residue, scratch the side of the vessel with a glass rod to induce crystallisation, evaporate to dryness and dry the residue at 60 for 10 minutes. The residue complies with the following test. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with ranitidine hydrochloride RS or with the reference spectrum of ranitidine hydrochloride. B. In the Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the peak in the chromatogram obtained with the reference solution.
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 25 volumes of ethyl acetate, 15 volumes of 2-propanol, 4 volumes of strong ammonia solution and 2 volumes of water. Test solution. Shake a quantity of the powdered tablets containing 0.45 g of ranitidine with 20 ml of methanol and filter. Reference solution (a). Weigh accurately a quantity of ranitidine hydrochloride RS in methanol, and dilute with
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flow rate. 2 ml per minute, spectrophotometer set at 322 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject alternately the test solution and the reference solution. Calculate the content of C13H22N4O3S in the tablets. Storage. Store protected from light and moisture. Labelling. The label states the strength in terms of the equivalent amount of ranitidine.
allow to macerate for 2 hours. Decant the solution, squeeze the residual liquid carefully from the sample with a glass rod mix and filter. The filtered extract is colourless. Compare the colour of the extract with water using identical tubes of colourless, transparent, neutral glass 12 mm in diameter measuring 2 ml. Compare the colours in diffused daylight, viewing horizontally against a white background. Acidity or alkalinity. To 25 ml of filtered extract obtained in Colour of extract, add 0.1 ml of dilute phenolphthalein solution; to another 25 ml add 0.05 ml of methyl orange solution. Neither solution shows a pink colour. Foreign fibres. When examined under a microscope, it is seen to consist exclusively of viscose fibres, except that occasionally a few isolated foreign fibres may be present. Fluorescence. Examine a layer about 5 mm in thickness under ultraviolet light at 365 nm. It displays only a slight, brownishviolet fluorescence and a few yellow particles. Not more than a few isolated fibres show an intense blue fluorescence. Absorbency A. Sinking time. Not more than 10 seconds, determined by the following method. Apparatus A dry, cylindrical copper wire basket, 80 mm high and 50 mm in diameter, fabricated from wire of diameter 0.4 mm and having a mesh aperture of 15 to 20 mm; the basket weighs 2.4 to 3.0 g. Method Weigh the basket to the nearest 10 mg. Take five samples, each of approximately 1 g, from different places in the material under examination, place loosely in the basket and weigh the packed basket to the nearest 10 mg. Hold the basket with its long axis in the horizontal position and drop it from a height of about 10 mm into water at 25o contained in a beaker at least 12 cm in diameter and filled to a depth of 10 cm. Measure with a stopwatch the time taken by the basket to sink below the surface of the water. Repeat the procedure on two further samples and calculate the average value. B. Water-holding capacity. Not less than 18.0 g per g, determined by the following method. After the sinking time has been recorded in test A, remove the basket from the water, allow it to drain for 30 seconds with its long axis in the horizontal position over the beaker, transfer it to a tared beaker and weigh to the nearest 10 mg. Calculate the weight of water retained by the sample. Repeat the procedure on two further samples and calculate the average value. Colouring matter. Slowly extract 10 g in a narrow percolator with ethanol (95 per cent) until 50 ml of extract is obtained. The extract is not more intensely coloured than reference
Purified Rayon
Viscose Fibre; absorbent viscose
Description. White or very slightly yellow, purified rayon is a fibrous form of bleached regenerated cellulose, which can be produced with a lustrous or matt appearance, and is soft to the touch. The fibres can be produced with average staple length between 32 mm to 80 mm, and are practically odourless.
Identification
A. When examined under a microscope in the dry state, or when mounted in ethanol (95 per cent) and water, the following characteristics are observed. They are usually of more or less uniform width. Many longitudinal parallel lines are distributed unequally over the width in the case of standard viscose fibres, but such lines are absent or very few in fibres produced through the zinc-free process. The ends are cut more or less straight. Matt fibres contain numerous granular particles of approximately 1 average diameter. B. Treat with iodinated zinc chloride solution; the fibres become violet. C. To 0.1 g add 10 ml of zinc chloride-formic acid solution, heat to 40 and allow to stand for 2 hours 30 minutes, shaking occasionally. The fibres dissolve completely except for the matt variety where titanium dioxide particles remain. D. Dissolve the residue obtained in the test for Sulphated ash in 5 ml of sulphuric acid with slight warming, allow to cool, and carefully add 0.2 ml of hydrogen peroxide solution (10 volumes). The solution does not undergo colour change in case of lustrous variety of fibre, but for matt variety an orangeyellow colour is obtained, the intensity of which depends on the quantity of titanium dioxide present.
Tests
Colour of extract. Take 15 g of material under examination in a suitable vessel, add 150 ml of water, close the vessel and
1040
IP 2007
RESERPINE
solution YS5 or GYS6, (2.4.1) or a solution prepared in the following manner. To 3.0 ml of CSS add 7.0 ml of a solution of hydrochloric acid containing 1 per cent w/v of hydrochloric acid and dilute 0.5 ml of the resulting solution to 10 ml with the same solution of hydrochloric acid. Ether-soluble substances. Not more than 0.5 per cent, determined by the following method. Extract 5 g with ether in a continuous extraction apparatus such as a Soxhlet apparatus, for 4 hours in such a way that the rate is at least four extractions per hour. Evaporate the ether and dry the residue to constant weight at 105. Water-soluble substances. Not more than 0.7 per cent, determined by the following method. Boil 5 g with 500 ml of water for 30 minutes, stirring frequently and replacing the water lost by evaporation. Decant the liquid into a beaker, squeeze the residual liquid from the material carefully with a glass rod, mix the liquids and filter the extract whilst hot. Evaporate 400 ml of the filtrate and dry the residue to constant weight at 105. Hydrogen sulphide. To 10 ml of the filtered extract obtained in the Colour of extract, add 1.9 ml of water, 0.15 ml of dilute acetic acid and 1 ml of lead acetate solution. After 2 minutes, the solution is not more intensely coloured than a reference solution prepared at the same time using 0.15 ml of dilute acetic acid, 1.2 ml of thioacetamide reagent, 1.7 ml of lead standard solution (10 ppm Pb) and 10 ml of filtered extract. Sulphated ash (2.3.18). Not more than 1.5 per cent. Loss on drying (2.4.19). Not more than 13.0 per cent, determined on 5 g by drying in an oven at 105o.
Description. White to slightly yellow small crystals or a crystalline powder which darkens slowly on exposure to light.
Identification
Test A may be omitted if tests B, C, D and E are carried out. Tests B, C, D and E may be omitted if test A is carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with reserpine RS or with the reference spectrum of reserpine. B. Dilute 1 ml of a 0.2 per cent w/v solution in chloroform to 100.0 ml with ethanol (95 per cent). When examined immediately after preparation, in the range 230 nm to 360 nm (2.4.7), the resulting solution shows an absorption maximum at about 268 nm; absorbance at about 268 nm, about 0.55. Over the range 288 nm to 295 nm, the spectrum exhibits a slight minimum and then a shoulder or a slight maximum; absorbance over this range, about 0.34. C. To about 1 mg add 0.1 ml of a 0.1 per cent w/v solution of sodium molybdate in sulphuric acid; a yellow colour is produced which changes to blue within 2 minutes. D. To 1 mg add 0.2 ml of a freshly prepared 1 per cent w/v solution of vanillin in hydrochloric acid; a pink colour develops within 2 minutes. E. Mix about 0.5 mg with 5 mg of 4-dimethylaminobenzaldehyde and 0.2 ml of glacial acetic acid and add 0.2 ml of sulphuric acid; a green colour is produced. Add 1 ml of glacial acetic acid; the colour changes to red.
Tests
Specific optical rotation (2.4.22). 116 to 128, determined in a solution prepared immediately before use by dissolving 0.25 g in sufficient chloroform to produce 25 ml. Oxidation products. Absorbance of a 0.02 per cent w/v solution in glacial acetic acid at about 388 nm, measured immediately after preparation, not more than 0.10 (2.4.7).
Reserpine
H3CO N H H N H O O C OCH3 OCH3 OCH3 OCH3
Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 0.5 g by drying in an oven over phosphorus pentoxide at 60 at a pressure not exceeding 0.7 kPa for 3 hours. Assay. For total alkaloids Weigh accurately about 0.5 g and dissolve in a mixture of 40 ml of anhydrous glacial acetic acid and 6 ml of acetic anhydride. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.06087 g of total alkaloids. For reserpine, C 33H 40N2O 9 Carry out the following procedure protected from light. Weigh accurately about
H H3COOC
C33H40N2O9
Mol. Wt. 608.7
Reserpine is methyl 11,17-dimethoxy-18-[(3,4,5trimethoxybenzoyl)oxy]-3,20-yohimbane-16-carboxylate. Reserpine contains not less than 99.0 per cent and not more than 101.0 per cent of total alkaloids and not less than 98.0 per cent and not more than 102.0 per cent of reserpine, C33H40N2O9, both calculated on the dried basis.
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RESERPINE INJECTION
IP 2007
25.0 mg, moisten with 2 ml of ethanol (95 per cent), add 2 ml of 0.25 M sulphuric acid and 10 ml of ethanol (95 per cent) and warm gently to dissolve. Cool, dilute to 100.0 ml with ethanol (95 per cent) and dilute 5.0 ml to 50.0 ml with the same solvent (solution A). Transfer 10.0 ml to a boiling tube, add 2 ml of 0.25 M sulphuric acid and 2 ml of a freshly prepared 0.3 per cent w/v solution of sodium nitrite, mix and heat in a water-bath at 55 for 35 minutes. Cool, add 1 ml of a freshly prepared 5 per cent w/v solution of sulphamic acid and dilute to 25.0 ml with ethanol (95 per cent). Measure the absorbance of the resulting solution at the maximum at about 388 nm (2.4.7), using as the blank a solution prepared by treating a further 10.0 ml of solution A in the same manner and at the same time but omitting the sodium nitrite solution. Calculate the content of C33H40N2O9 from the absorbance obtained by repeating the operation using reserpine RS in place of the substance under examination. Storage. Store protected from light.
D. To 1 mg add 0.2 ml of a freshly prepared 1 per cent w/v solution of vanillin in hydrochloric acid; a pink colour develops within 2 minutes. E. Mix about 0.5 mg with 5 mg of 4-dimethylaminobenzaldehyde and 0.2 ml of glacial acetic acid and add 0.2 ml of sulphuric acid; a green colour is produced. Add 1 ml of glacial acetic acid; the colour changes to red.
Tests
pH (2.4.24). 3.0 to 4.0. Other tests. Comply with the tests stated under Parenteral Preparations (Injections). Assay. Protect the solutions from light throughout the assay. Measure accurately a volume of the injection containing about 10 mg of Reserpine and dilute with a 2 per cent w/v solution of citric acid to 100.0 ml. Extract 10.0 ml of this solution for 2 minutes with three quantities, each of 15 ml, of chloroform. Wash the combined extracts with 10 ml of a 1 per cent w/v solution of sodium bicarbonate, add sufficient chloroform to produce 50.0 ml, mix and evaporate 10.0 ml to dryness on a water-bath. Dissolve the residue in 10 ml of ethanol (95 per cent), add 2 ml of 0.25 M sulphuric acid and 10 ml of ethanol (95 per cent) and warm gently to dissolve. Cool, dilute to 100.0 ml with ethanol (95 per cent) and dilute 5.0 ml to 50.0 ml with the same solvent (solution A). Transfer 10.0 ml to a boiling tube, add 2 ml of 0.25 M sulphuric acid and 2 ml of a freshly prepared 0.3 per cent w/v solution of sodium nitrite, mix and heat in a water-bath at 55 for 35 minutes. Cool, add 1 ml of a freshly prepared 5 per cent w/v solution of sulphamic acid and dilute to 25.0 ml with ethanol (95 per cent). Measure the absorbance of the resulting solution at the maximum at about 388 nm (2.4.7), using as the blank a solution prepared by treating a further 10.0 ml of solution A in the same manner and at the same time but omitting the sodium nitrite solution. Calculate the content of C33H40N2O9 from the absorbance obtained by repeating the operation using reserpine RS in place of the substance under examination. Storage. Store protected from light in single dose (or if stabilising agents are present, in multiple dose) containers.
Reserpine Injection
Reserpine Injection is a sterile solution of Reserpine in Water for Injections prepared with the aid of a suitable acid. It may contain suitable antioxidants. Reserpine Injection contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of reserpine, C33H40N2O9.
Identification
Extract a suitable volume of the injection containing 10 mg of Reserpine with 10 ml of chloroform and evaporate the chloroform layer to dryness. The residue complies with the following tests. Test A may be omitted if tests B, C, D and E are carried out. Tests B, C, D and E may be omitted if test A is carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with reserpine RS or with the reference spectrum of reserpine. B. Dilute 1 ml of a 0.2 per cent w/v solution in chloroform to 100 ml with ethanol (95 per cent). When examined immediately after preparation, in the range 230 nm to 360 nm (2.4.7), the resulting solution shows an absorption maximum at about 268 nm; absorbance at about 268 nm, about 0.55. Over the range 288 nm to 295 nm, the spectrum exhibits a slight minimum and then a shoulder or a slight maximum; absorbance over this range, about 0.34. C. To about 1 mg add 0.1 ml of a 0.1 per cent w/v solution of sodium molybdate in sulphuric acid; a yellow colour is produced which changes to blue within 2 minutes.
Reserpine Tablets
Reserpine Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of reserpine, C33H40N2O9.
Identification
A. Powder a few tablets and extract with chloroform. Evaporate the extract to dryness, add 0.1 ml of a 0.1 per cent w/v solution
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IP 2007
RIBOFLAVINE
of sodium molybdate in sulphuric acid; a yellow colour is produced which changes to blue within 2 minutes. B. Powder a few tablets and extract with chloroform. Evaporate the extract to dryness, add 0.2 ml of a freshly prepared 1 per cent w/v solution of vanillin in hydrochloric acid; a pink colour develops within 2 minutes.
Tests
Uniformity of content. Protect the solutions from light throughout the test. Comply with the test stated under Tablets. Powder one tablet, disperse in 10 ml of a 2 per cent w/v solution of citric acid and extract for 2 minutes with three quantities, each of 5 ml, of chloroform, filter the extracts through a plug of cotton moistened with chloroform. Wash the chloroform extracts with 10 ml of a 1 per cent w/v solution of sodium bicarbonate and evaporate the chloroform extracts to dryness on a water-bath. For tablets containing upto 250 g of Reserpine per tablet, dissolve the residue in 10.0 ml of ethanol (95 per cent). For tablets containing more than 250 g of Reserpine per tablet, dissolve the residue in a suitable volume of ethanol (95 per cent) to give a concentration of 250 g of Reserpine per 10.0 ml; add 2 ml of 0.25 M sulphuric acid and 2 ml of a freshly prepared 0.3 per cent w/v solution of sodium nitrite, mix and heat in a water-bath at 55 for 35 minutes. Cool, add 1 ml of a freshly prepared 5 per cent w/v solution of sulphamic acid and dilute to 25.0 ml with ethanol (95 per cent). Measure the absorbance of the resulting solution at the maximum at about 388 nm (2.4.7), using as the blank a solution prepared by treating a further 10.0 ml of solution A in the same manner and at the same time but omitting the sodium nitrite solution. Calculate the content of C33H40N2O9 in the tablet from the absorbance obtained by repeating the operation using reserpine RS in place of the substance under examination. Other tests. Comply with the tests stated under Tablets. Assay. Protect the solutions from light throughout the assay. Weigh accurately a quantity of the powdered tablets containing about 1 mg of Reserpine, add 10 ml of a 2 per cent w/v solution of citric acid and extract for 2 minutes with three quantities, each of 15 ml, of chloroform. Wash the combined extracts with 10 ml of a 1 per cent w/v solution of sodium bicarbonate, add sufficient chloroform to produce 50.0 ml and evaporate 10.0 ml to dryness on a water-bath. Dissolve the residue in 10 ml of ethanol (95 per cent) and add 2 ml of 0.25 M sulphuric acid and 10 ml of ethanol (95 per cent) and warm gently to dissolve. Cool, dilute to 100.0 ml with ethanol (95 per cent) and dilute 5.0 ml to 50.0 ml with the same solvent (solution A). Transfer 10.0 ml to a boiling tube, add 2 ml of 0.25 M sulphuric acid and 2 ml of a freshly prepared 0.3 per
cent w/v solution of sodium nitrite, mix and heat in a waterbath at 55 for 35 minutes. Cool, add 1 ml of a freshly prepared 5 per cent w/v solution of sulphamic acid and dilute to 25.0 ml with ethanol (95 per cent). Measure the absorbance of the resulting solution at the maximum at about 388 nm (2.4.7), using as the blank a solution prepared by treating a further 10.0 ml of solution A in the same manner and at the same time but omitting the sodium nitrite solution. Calculate the content of C33H40N2O9 from the absorbance obtained by repeating the operation using reserpine RS in place of the substance under examination. Storage. Store protected from light.
Riboflavine
Lactoflavin; Vitamin B2
CH2OH HO C H HO C H HO C H CH2 N N N O
C17H20N4O6 Mol. Wt. 376.4
H3C H3C
O NH
Riboflavine is 3,10-dihydro-7,8-dimethyl-10-[(2S,3S,4R)2,3,4,5-tetrahydroxypentyl]benzopteridine-2,4-dione. Riboflavine contains not less than 98.0 per cent and not more than 101.0 per cent of C17H20N4O6, calculated on the dried basis. Description. A yellow to orange-yellow, crystalline powder; odour, slight.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with riboflavine RS or with the reference spectrum of riboflavine. B. Dissolve about 1 mg in 100 ml of water. The solution has a pale greenish yellow colour by transmitted light and an intense yellowish green fluorescence by reflected light, which disappears on addition of mineral acids or alkalis.
Tests
pH (2.4.24). 5.5 to 7.2, determined in a saturated solution.
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RIBOFLAVINE SODIUM PHOSPHATE
IP 2007
Specific optical rotation (2.4.22). 115 to 135, determined in a 0.5 per cent w/v solution in carbonate-free 0.05 M sodium hydroxide. Measure the angle of rotation within 30 minutes of preparing the solution. Light absorption (2.4.7). Dilute a suitable volume of the final solution obtained in the Assay with an equal volume of water. When examined in the range 210 nm to 460 nm, the resulting solution exhibits maxima at about 223 nm, 267 nm, 373 nm and 444 nm; the ratio of the absorbance at the maximum at about 373 nm to that at about 267 nm, 0.31 to 0.33 and the ratio of the absorbance at the maximum at about 444 nm to that at about 267 nm, 0.36 to 0.39. Lumiflavine. Shake 25 mg with 10 ml of chloroform for 5 minutes and filter. The filtrate is not more intensely coloured than reference solution BYS6 (2.4.1). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 1.5 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Carry out the procedure in subdued light. Weigh accurately about 65 mg and transfer to an amber-glass 500-ml volumetric flask, suspend in 5 ml of water, ensuring that it is completely wetted. Dissolve in 5 ml of 2 M sodium hydroxide. As soon as dissolution is complete add 100 ml of water and 2.5 ml of glacial acetic acid and dilute to 500.0 ml with water. To 20.0 ml of this solution add 3.5 ml of a 1.4 per cent w/v solution of sodium acetate and dilute to 200.0 ml with water. Measure the absorbance of the resulting solution at the maximum at about 444 nm (2.4.7). Calculate the content of C17H20N4O6 taking 328 as the specific absorbance at 444 nm. Storage. Store protected from light.
Riboflavin Sodium Phosphate is monosodium 3,10-dihydro7,8-dimethyl-10-[(2S,3S,4R)-2,3,4-trihydroxypentyl] benzopteridine-2,4-dione 5-phosphate dihydrate. Riboflavine Sodium Phosphate contains the equivalent of not less than 73.0 per cent and not more than 79.0 per cent of C17H20N4O6, calculated on the dried basis. Description. A yellow to orange-yellow, crystalline powder; hygroscopic.
Identification
A. When examined in the range 230 nm to 360 nm (2.4.7), a 0.001 per cent w/v solution in phosphate buffer pH 7.0 shows an absorption maximum at about 267 nm; absorbance at about 267 nm, 0.58 to 0.64. B. Dissolve about 10 mg in sufficient 2 M sodium hydroxide to produce 100 ml, expose 1 ml to ultraviolet light at 254 nm for 5 minutes, add sufficient 5 M acetic acid to make the solution acidic to litmus paper and shake the mixture with 2 ml of dichloromethane; the lower layer exhibits a yellow fluorescence. C. To 0.5 g add 10 ml of nitric acid, evaporate the mixture to dryness on a water-bath, ignite the residue until the carbon is removed, dissolve the final residue in 5 ml of water and filter. The filtrate gives the reactions of sodium salts and reaction B of phosphates (2.4.1).
Tests
pH (2.4.24). 4.0 to 6.3, determined in a 2.0 per cent w/v solution. Specific optical rotation (2.4.22). +38.0 to +42.0, determined in a 1.5 per cent w/v solution in 5 M hydrochloric acid. Heavy metals (2.3.13). To 2.0 g in a silica crucible add 2 ml of nitric acid dropwise followed by 0.25 ml of sulphuric acid. Heat cautiously until white fumes are evolved and ignite. Extract the cooled residue with two quantities, each of 2 ml, of hydrochloric acid and evaporate the extracts to dryness. Dissolve the residue in 2 ml of 2 M acetic acid and dilute to 20 ml with water. 12 ml of the solution complies with the limit test for heavy metals, Method D (10 ppm). Use 1.0 ml of lead standard solution (10 ppm Pb) to prepare the standard. Lumiflavine. Shake 35 mg with 10 ml of dichloromethane for 5 minutes and filter. The filtrate is not more intensely coloured than reference solution BYS6 (2.4.1).
Riboflavine Sodium Phosphate

Riboflavine-5-phosphate (Sodium Salt); Vitamin B2 Sodium Phosphate
O H2C O P ONa HO C H OH HO C H HO C H CH2 N N O N O NH
H3C H3C
, 2H2O
C17H20N4NaO9P, 2H2O
Mol. Wt. 514.4
Inorganic phosphate. Not more than 1.5 per cent, determined by the following method. Dissolve 0.1 g in sufficient water to produce 100 ml. Dilute 5 ml with 10 ml of water and add 5 ml of buffered cupric sulphate solution pH 4.0, 2 ml of a 3 per cent w/v solution of ammonium molybdate, 1 ml of a freshly prepared solution containing 2 per cent w/v of 4-methylaminophenol sulphate and 5 per cent w/v of sodium meta-
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IP 2007
RIFAMPICIN
bisulphite and 1 ml of a 3 per cent v/v solution of perchloric acid. Add sufficient water to produce 25 ml, mix and measure the absorbance of the resulting solution at the maximum at about 800 nm (2.4.7), within 15 minutes of its preparation, using as the blank a solution prepared in the same manner but omitting the substance under examination. The absorbance is not greater than that produced by repeating the operation using a solution prepared in the same manner using 15 ml of phosphate standard solution (5 ppm PO4) and beginning at the words add 5 ml of buffered cupric sulphate solution pH 4.0, Loss on drying (2.4.19). Not more than 8.0 per cent, determined on 0.5 g by drying in an oven at 100 at a pressure not exceeding 0.7 kPa for 5 hours. Assay. Carry out the procedure protected from light. Weigh accurately about 0.1 g, dissolve in 150 ml of water, add 2 ml of glacial acetic acid and dilute to 1000.0 ml with water. To 10.0 ml add 3.5 ml of a 1.4 per cent w/v solution of sodium acetate, dilute to 50.0 ml with water and measure the absorbance of the resulting solution at the maximum at about 444 nm (2.4.7). Calculate the percentage content of C17H20N4O6 taking 328 as the specific absorbance at 444 nm. Storage. Store protected from light.
Other tests. Comply with the tests stated under Tablets. Assay. Carry out the procedure in subdued light. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 10 mg of Riboflavine, add a mixture of 5 ml of glacial acetic acid and 100 ml of water and heat on a water-bath for 1 hour with occasional shaking. Dilute with 50 ml of water, cool, add 30 ml of 1 M sodium hydroxide with continuous stirring. Add sufficient water to produce 1000.0 ml, mix and filter, discarding the first few ml of the filtrate. Measure the absorbance of the filtrate at the maximum at about 444 nm (2.4.7). Calculate the content of C17H20N4O6 taking 328 as the specific absorbance at 444 nm. Storage. Store protected from light.
Rifampicin
Rifampin
CH3 CH3 HO H3C O OH CH3 CH3 OH H3C O OH CH3 O O NH OH CH3
Riboflavine Tablets
Lactoflavin Tablets; Vitamin B2 Tablets
Riboflavine Tablets contain not less than 90.0 per cent and not more than 115.0 per cent of the stated amount of riboflavine, C17H20N4O6.
O H3CO O
N N CH3
Identification
Shake a quantity of the powdered tablets containing 1 mg of Riboflavine with 100 ml of water and filter; the filtrate has a pale greenish yellow colour by transmitted light and an intense yellowish green fluorescence by reflected light, which disappears on addition of mineral acids or alkalis. C43H58N4O12
Mol. Wt. 823.0
Tests
Uniformity of content. Comply with the test stated under Tablets. Powder one tablet, add a mixture of 2.5 ml of glacial acetic acid and 50 ml of water and heat on a water-bath for 1 hour with occasional stirring. Dilute with 50 ml of water, add 15 ml of 1 M sodium hydroxide with continuous stirring and then sufficient water to produce a solution containing 10 g of Riboflavine per ml. Filter and discard the first few ml of the filtrate. On the clear filtrate carry out the Assay beginning at the words Measure the absorbance.....
Rifampicin is (12Z,14E,24E)-(2S,16S,17S,18R,19R,20R, 21S,22R,23S)-1,2-dihydro-5,6,9,17,19-pentahydroxy-23methoxy-2,4,12,16,18,20,22-heptamethyl-8-(4-methylpiperazin1-yliminomethyl)-1,11-dioxo-2,7-(epoxypentadeca-1,11,13trienimino)naphtho[2,1-b]furan-21-yl acetate. Rifampicin contains not less than 97.0 per cent and not more than 102.0 per cent of C43H58N4O12, calculated on the dried basis. Description. A brick-red to reddish brown, crystalline powder; practically odourless.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with rifampicin RS or with the reference spectrum of rifampicin.
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RIFAMPICIN CAPSULES
IP 2007
Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 1.0 per cent, determined on 1.0 g by drying in an oven at 80 at a pressure not exceeding 0.7 kPa for 4 hours. Assay. Determine by liquid chromatography (2.4.14). NOTE Prepare the solutions immediately before use. Solvent mixture. A mixture of 10 volumes of a 21.01 per cent w/v solution of citric acid, 23 volumes of a 13.61 per cent w/v solution of potassium dihydrogen phosphate, 77 volumes of a 17.42 per cent w/v solution of dipotassium hydrogen phosphate, 640 volumes of water and 250 volumes of acetonitrile. Test solution. Dissolve 20.0 mg of the substance under examination in 10.0 ml of acetonitrile and filter. Dilute 5.0 ml of the filtrate to 100.0 ml with the solvent mixture. Reference solution. A solution containing 0.2 per cent w/v of rifampicin RS in acetonitrile. Dilute 5.0 ml of this solution to 100.0 ml with the solvent mixture. Use the chromatographic system and the system suitability parameters described under the test for Related substances. Inject the reference solution. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject alternately the test solution and the reference solution. Calculate the content of C43H58N4O12. Storage. Store protected from light, in an atmosphere of nitrogen.
Tests
pH (2.4.24). 4.5 to 6.5, determined in a 1.0 per cent w/v suspension. Related substances. Determine by liquid chromatography (2.4.14). Solvent mixture. A mixture of 10 volumes of a 21.01 per cent w/v solution of citric acid, 23 volumes of a 13.61 per cent w/ v solution of potassium dihydrogen phosphate, 77 volumes of a 17.42 per cent w/v solution of dipotassium hydrogen phosphate, 640 volumes of water and 250 volumes of acetonitrile. Test solution. Weigh accurately a quantity of powder containing 20 mg of Rifampicin, add 10 ml of acetonitrile, shake and filter. Dilute 5 ml of the filtrate to 50 ml with the solvent mixture. Reference solution. A solution containing 0.02 per cent w/v of rifampicin quinone RS in acetonitrile. To 1 ml of the solution, add 1 ml of the test solution and dilute to 100 ml with the solvent mixture. Chromatographic system a stainless steel column 10 cm x 4.6 mm, packed with octylsilane bonded to porous silica (5 m), mobile phase: a mixture of 65 volumes of buffer solution pH 6.8 prepared by mixing 0.1 per cent w/v of phosphoric acid, 0.19 per cent w/v of sodium perchlorate, 0.59 per cent w/v of citric acid and 2.09 per cent w/v of potassium dihydrogen phosphate with water and adjusting the pH to 6.8 with dilute phosphoric acid, and 35 volumes of acetonitrile, flow rate. 1.5 ml per minute, spectrophotometer set at 254 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the resolution between the principal peaks is not less than 4. In the chromatogram obtained with the test solution the area of any peak due to rifampicin quinone is not more than 1.5 times the area of the principal peak in the chromatogram obtained with the reference solution (1.5 per cent); the area of any peak, other than the principal peak and the peak corresponding to rifampicin quinone, is not more than the area of the peak due to rifampicin in the chromatogram obtained with the reference solution (1.0 per cent) and the sum of the areas of any such peaks is not more than 3.5 times the area of the peak due to rifampicin in the chromatogram obtained with the reference solution (3.5 per cent ).
Rifampicin Capsules
Rifampin Capsules
Rifampicin Capsules contain not less than 92.5 per cent and not more than 107.5 per cent of the stated amount of rifampicin, C43H58N4O12.
Identification
A. Shake a quantity of the contents of the capsules containing 0.15 g of Rifampicin with 5 ml of chloroform, filter and evaporate the filtrate to dryness. The residue complies with the following test. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with rifampicin RS or with the reference spectrum of rifampicin.
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IP 2007
RIFAMPICIN CAPSULES
Tests
Related substances. Determine by liquid chromatography (2.4.14). Solvent mixture. A mixture of 10 volumes of a 21.01 per cent w/v solution of citric acid, 23 volumes of a 13.61 per cent w/v solution of potassium dihydrogen phosphate, 77 volumes of a 17.42 per cent w/v solution of dipotassium hydrogen phosphate, 640 volumes of water and 250 volumes of acetonitrile. Test solution. Shake a quantity of the contents of the capsules containing 200 mg of Rifampicin, with 100 ml of acetonitrile and filter. Dilute 5 ml of the filtrate to 50 ml with the solvent mixture. Reference solution (a). A solution containing 0.02 per cent w/v of rifampicin RS in acetonitrile. Dilute 1 ml of this solution to 100 ml with the solvent mixture. Reference solution (b). A solution containing 0.01 per cent w/v each of rifampicin RS and rifampicin quinone RS in acetonitrile. Dilute 5 ml of this solution to 50 ml with the solvent mixture. Chromatographic system a stainless steel column 10 cm x 4.6 mm, packed with octylsilane bonded to porous silica (5 m), column. temperature 30, mobile phase: a mixture of 65 volumes of buffer solution pH 6.8 prepared by mixing 0.1 per cent w/v of phosphoric acid, 0.19 per cent w/v of sodium perchlorate, 0.59 per cent w/v of citric acid and 2.09 per cent w/v of potassium dihydrogen phosphate with water and adjusting then pH to 6.8 with dilute phosphoric acid, and 35 volumes of acetonitrile, flow rate. 1.5 ml per minute, spectrophotometer set at 254 nm, a 20 l loop injector. Inject reference solution (b). The test is not valid unless the resolution between rifampicin and rifampicin quinone is not less than 4, the tailing factor is not more than 2.0 and the column efficiency is not less than 2000 theoretical plates. Inject the test solution and reference solution (a). The relative retention times are 1.0, 0.55, 1.25 and 2.61 for rifampicin, rifampicin quinone, rifampicin N-oxide and 3-formylrifamycin SV respectively. The response factors are 1.00, 1.19, 1.03 and 1.25 for rifampicin, rifampicin quinone, rifampicin N-oxide and 3-formylrifamycin SV respectively. In the chromatogram obtained with the test solution the area of any peak due to rifampicin quinone should not be more
than 4 times the area of the principal peak in the chromatogram obtained with the reference solution, the area of any peak due to rifampicin N-oxide should not be more than 1.5 times the area of the principal peak in the chromatogram obtained with the reference solution and the area of any peak due to 3-formylrifamycin SV should not be more than the area of the principal peak in the chromatogram obtained with the reference solution. In the chromatogram obtained with the test solution the area of any unknown peak should not be more than the area of the principal peak in the chromatogram obtained with the reference solution. Dissolution (2.5.2). Apparatus. No 2 Medium. 900 ml of 0.1 M hydrochloric acid Speed and time. 100 rpm and 45 minutes. Withdraw a suitable volume of the medium and filter promptly through a membrane filter disc having an average pore diameter not greater than 1.0 m, rejecting the first 1 ml of the filtrate. Dilute the filtrate, if necessary, with the same solvent. Measure the absorbance (2.4.7) of the resulting solution at the maximum at about 475 nm. Calculate the content of C43H58N4O12 in the medium from the absorbance obtained from a solution of known concentration of rifampicin RS. D. Not less than 75 per cent of the stated amount of C43H58N4O12. Other tests. Comply with the tests stated under Capsules. Assay. Determine by liquid chromatography (2.4.14). Test solution. Shake a quantity of the contents of the capsules containing about 300.0 mg of Rifampicin, with 200.0 ml of acetonitrile, filter. Dilute 10.0 ml of the filtrate to 50.0 ml with acetonitrile. Dilute 5.0 ml of this solution to 50.0 ml with the solvent mixture. Reference solution (a). A 0.15 per cent w/v solution of rifampicin RS in acetonitrile. Dilute 10.0 ml of this solution to 50.0 ml with acetonitrile. Dilute 5.0 ml of the resulting solution to 50.0 ml with the solvent mixture. Reference solution (b). A solution containing 0.01 per cent w/v each of rifampicin RS and rifampicin quinone RS in acetonitrile. Dilute 5ml of this solution to 50 ml with the solvent mixture. Use the chromatographic system and system suitability parameters, as described under the test for Related substances. Inject reference solution (a). The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject alternately the test solution and reference solution (a). Calculate the content of C43H58N4O12 in the capsules. Storage. Store protected from light and moisture.
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RIFAMPICIN ORAL SUSPENSION
IP 2007
Rifampicin Oral Suspension

Rifampicin oral suspension contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of rifampicin, C43H58N4O12.
Identification
A. To a quantity containing 0.1 g of Rifampicin add 30 ml of water and shake with two quantities, each of 50 ml, of chloroform. Dry the combined extracts with anhydrous sodium sulphate, filter and evaporate the filtrate to dryness at a temperature not exceeding 70. Wash the residue with 1 ml of ether and dry at 70. The residue complies with the following test. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with rifampicin RS or with the reference spectrum of rifampicin. B. In the Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the peak in the chromatogram obtained with the reference solution.
mobile phase: a mixture of 65 volumes of buffer solution pH 6.8 prepared by mixing 0.1 per cent w/v of phosphoric acid, 0.19 per cent w/v of sodium perchlorate, 0.59 per cent w/v of citric acid and 2.09 per cent w/v of potassium dihydrogen phosphate with water and adjusting the pH to 6.8 with dilute phosphoric acid, and 35 volumes of acetonitrile, flow rate. 1.5 ml per minute, spectrophotometer set at 254 nm, a 20 l loop injector. Inject reference solution (b). The test is not valid unless the resolution between rifampicin and rifampicin quinone is not less than 4; the tailing factor is not more than 2.0 and the column efficiency is not less than 2000 theoretical plates. Inject the test solution and reference solution (a). The relative retention times are 1.0, 0.55, 1.25, 1.51 and 2.61 for rifampicin, rifampicin quinone, rifampicin N-oxide, 3-formylrifamycin SV Isonicotinyl hydrazone and 3-formylrifamycin SV respectively. Multiply the areas of each known impurity by their response factor. The response factors are 1.00, 1.19, 1.03, 1.22 and 1.25 for rifampicin, rifampicin quinone, rifampicin N-oxide, Isonicotinyl hydrazone and 3-formylrifamycin SV respectively. In the chromatogram obtained with the test solution the area of any peak due to rifampicin quinone should not be more than 1.5 times the area of the principal peak in the chromatogram obtained with the reference solution, the area of any peak due to rifampicin N-oxide should not be more than the area of the principal peak in the chromatogram obtained with the reference solution and the area of any peak due to 3-formylrifamycin SV should not be more than 5.0 times the area of the principal peak in the chromatogram obtained with the reference solution. In the chromatogram obtained with the test solution the area of any unknown peak should not be more than the area of the principal peak in the chromatogram obtained with the reference solution. Other tests. Comply with the tests stated under Oral Suspensions. Assay. Determine by liquid chromatography (2.4.14). Test solution. Weigh accurately a quantity of the oral suspension containing about 150 mg of rifampicin and dilute to 100.0 ml with acetonitrile. Dilute 10.0 ml of this solution to 50.0 ml with acetonitrile. Dilute 10.0 ml of the resulting solution to 50.0 ml with the solvent mixture. Reference solution (a). A 0.15 per cent w/v solution of rifampicin RS in acetonitrile. Dilute 10.0 ml of the solution to 50.0 ml with acetonitrile. Dilute 10.0 ml of the resulting solution to 50.0 ml with the solvent mixture. Reference solution (b). A solution containing 0.01 per cent w/v of each of rifampicin RS and rifampicin quinone RS in
Tests
pH (2.4.24). 4.2 to 4.8. Related substances. Determine by liquid chromatography (2.4.14). Solvent mixture. A mixture of 10 volumes of a 21.01 per cent w/v solution of citric acid, 23 volumes of a 13.61 per cent w/v solution of potassium dihydrogen phosphate, 77 volumes of a 17.42 per cent w/v solution of dipotassium hydrogen phosphate, 640 volumes of water and 250 volumes of acetonitrile. Test solution. Add 5 ml of water to a quantity of the oral suspension containing 20 mg of Rifampicin and extract with four quantities, each of 10 ml, of dichloromethane, filter the combined extracts and evaporate to dryness at a temperature not exceeding 40. Dissolve the residue in 10 ml of acetonitrile. Dilute 5 ml of the resulting solution to 50 ml with the solvent mixture. Reference solution (a). A solution containing 0.02 per cent w/v of rifampicin RS in acetonitrile. Dilute 1 ml of this solution to a 100 ml with the solvent mixture. Reference solution (b). A solution containing 0.01 per cent w/v each of rifampicin RS and rifampicin quinone RS in acetonitrile. Dilute 5 ml of this solution to 50 ml with the solvent mixture. Chromatographic system a stainless steel column 10 cm x 4.6 mm, packed with octylsilane bonded to porous silica (5 m), column temperature 30,
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IP 2007
RIFAMPICIN TABLETS
acetonitrile. Dilute 5 ml of this solution to 50 ml with the solvent mixture. Use the chromatographic system and the system suitability parameters as described under the test for Related substances. Inject reference solution (a). The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject alternately the test solution and reference solution (a). Determine the weight per ml of the suspension (2.4.29) and calculate the content of C43H58N4O12 weight in volume. Storage. Store protected from light and moisture.
Reference solution (b). A solution containing 0.01 per cent w/v of each of rifampicin RS and rifampicin quinone RS in acetonitrile. Dilute 5 ml of this solution to 50 ml with the solvent mixture. Chromatographic system a stainless steel column 10 cm x 4.6 mm, packed with octylsilane bonded to porous silica (5 m), column. temperature 30, mobile phase: a mixture of 65 volumes of buffer solution pH 6.8 prepared by mixing 0.1 per cent w/v of phosphoric acid, 0.19 per cent w/v of sodium perchlorate, 0.59 per cent w/v of citric acid and 2.09 per cent w/v of potassium dihydrogen phosphate with water and adjusting the pH to 6.8 with dilute phosphoric acid, and 35 volumes of acetonitrile, flow rate. 1.5 ml per minute, spectrophotometer set at 254 nm, a 20 l loop injector. Inject reference solution (b). The test is not valid unless the resolution between rifampicin and rifampicin quinone is not less than 4, the tailing factor is not more than 2.0 and the column efficiency is not less than 2000 theoretical plates. Inject the test solution and reference solution (a). The relative retention times are 1.0, 0.55, 1.25 and 2.61 for rifampicin, rifampicin quinone, rifampicin N-oxide, and 3-formylrifamycin SV respectively. The response factors are 1.00, 1.19, 1.03 and 1.25 for rifampicin, rifampicin quinone, rifampicin N-oxide and 3-formylrifamycin SV respectively. In the chromatogram obtained with the test solution the area of any peak due to rifampicin quinone should not be more than 1.5 times the area of the principal peak in the chromatogram obtained with the reference solution, the area of any peak due to rifampicin N-oxide should not be more than the area of the principal peak in the chromatogram obtained with the reference solution and the area of any peak due to 3-formylrifamycin SV should not be more than 5.0 times the area of the principal peak in the chromatogram obtained with the reference solution. In the chromatogram obtained with the test solution the area of any unknown peak should not be more than the area of the principal peak in the chromatogram obtained with the reference solution. Dissolution (2.5.2). Apparatus. No 2 Medium. 900 ml of 0.1 M hydrochloric acid. Speed and time. 100 rpm and 45 minutes. Withdraw a suitable volume of the medium and filter promptly through a membrane filter disc having an average pore diameter not greater than 1.0 m, rejecting the first 1 ml of the filtrate. Dilute the filtrate, if necessary, with the same solvent. Measure the absorbance (2.4.7) of the resulting solution at the maximum
Rifampicin Tablets
Rifampin Tablets
Rifampicin Tablets contain not less than 92.5 per cent and not more than 107.5 per cent of the stated amount of rifampicin, C43H58N4O12.
Identification
A. Shake a quantity of the powdered tablets containing 0.15 g of Rifampicin with 5 ml of chloroform, filter and evaporate the filtrate to dryness. The residue complies with the following test. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with rifampicin RS or with the reference spectrum of rifampicin. B. In the Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the peak in the chromatogram obtained with the reference solution.
Tests
Related substances. Determine by liquid chromatography (2.4.14). Solvent mixture. A mixture of 10 volumes of a 21.01 per cent w/ v solution of citric acid, 23 volumes of a 13.61 per cent solution w/v of potassium dihydrogen phosphate, 77 volumes of a 17.42 per cent w/v solution of dipotassium hydrogen phosphate, 640 volumes of water and 250 volumes of acetonitrile. Test solution. Weigh accurately a quantity of the powdered tablets containing 200 mg of Rifampicin, dissolve in 100 ml of acetonitrile, filter. Dilute 5 ml of the filtrate to 50 ml with the solvent mixture. Reference solution (a). A solution containing 0.02 per cent w/v of rifampicin RS in acetonitrile. Dilute 1 ml of this solution to a 100 ml with the solvent mixture.
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RIFAMPICIN AND ISONIAZID TABLETS
IP 2007
at about 475 nm. Calculate the content of C43H58N4O12 in the medium from the absorbance obtained from a solution of known concentration of rifampicin RS. D. Not less than 75 per cent of the stated amount of C43H58N4O12. Other tests. Comply with the tests stated under Tablets. Assay. Determine by liquid chromatography (2.4.14). Test solution. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 300.0 mg of Rifampicin, dissolve in 200.0 ml of acetonitrile, filter. Dilute 10.0 ml of the filtrate to 50.0 ml with acetonitrile. Dilute 5.0 ml of this solution to 50.0 ml with the solvent mixture. Reference solution (a). A 0.15 per cent w/v solution of rifampicin RS in acetonitrile. Dilute 10.0 ml of this solution to 50.0 ml with acetonitrile. Dilute 5.0 ml of the resulting solution to 50.0 ml with the solvent mixture. Reference solution (b). A solution containing 0.01 per cent w/v each of rifampicin RS and rifampicin quinone RS in acetonitrile. Dilute 5.0 ml of this solution to 50.0 ml with the solvent mixture. Use the chromatographic system and system suitability parameters described under the test for Related substances. Inject reference solution (a). The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject alternately the test solution and reference solution (a). Calculate the content of C43H58N4O12 in the tablets. Storage. Store protected from light and moisture.
Solvent mixture. A mixture of 10 volumes of a 21.01 per cent w/v solution of citric acid, 23 volumes of a 13.61 per cent w/v solution of potassium dihydrogen phosphate, 77 volumes of a 17.42 per cent w/v solution of dipotassium hydrogen phosphate, 640 volumes of water and 250 volumes of acetonitrile. Test solution. Weigh accurately a quantity of the powdered tablets containing 200 mg of Rifampicin in 100 ml of acetonitrile and filter. Dilute 5 ml of the filtrate to 50 ml with the solvent mixture. Reference solution (a). Dissolve rifampicin RS in acetonitrile to obtain a solution containing 0.2 mg per ml. Dilute 1 ml of this solution to 100 ml with the solvent mixture. Reference solution (b). A solution containing 0.01 per cent w/v each of rifampicin RS and rifampicin quinone RS in acetonitrile. Dilute 5 ml of this solution to 50 ml with the solvent mixture. Chromatographic system a stainless steel column 10 cm x 4.6 mm, packed with octylsilane bonded to porous silica (5 m), column temperature 30, mobile phase: a mixture of 65 volumes of buffer solution pH 6.8 prepared by mixing 0.1 per cent w/v of phosphoric acid, 0.19 per cent w/v of sodium perchlorate, 0.59 per cent w/v of citric acid and 2.09 per cent w/v of potassium dihydrogen phosphate with water and adjusting the pH to 6.8 with dilute phosphoric acid, and 35 volumes of acetonitrile, flow rate. 1.5 ml per minute, spectrophotometer set at 254 nm, a 20 l loop injector. Inject reference solution (b). The test is not valid unless the resolution between rifampicin and rifampicin quinone is not less than 4, the tailing factor is not more than 2.0 and the column efficiency is not less than 2000 theoretical plates. Inject alternately the test solution and reference solution (a). The relative retention times are 1.0, 0.55, 1.25, 1.51 and 2.61 for rifampicin, rifampicin quinone, rifampicin N-oxide, 3-formylrifamycin SV isonicotinyl hydrazone and 3-formylrifamycin SV respectively. Multiply the area of each known impurity by its response factor. The response factors are 1.00, 1.19, 1.03, 1.22 and 1.25 for rifampicin, rifampicin quinone, rifampicin N-oxide, isonicotinyl hydrazone and 3-formylrifamycin SV respectively. In the chromatogram obtained with the test solution the area of any peak due to rifampicin quinone should not be more than 4 times the area of the principal peak in the chromatogram obtained with the reference solution (4 per cent), the area of any peak due to rifampicin N-oxide should not be more than 1.5 times the area of the principal peak in the chromatogram
Rifampicin and Isoniazid Tablets

Rifampin and Isonicotinylhydrazid Tablets
Rifampicin and Isoniazid Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amounts of rifampicin, C43H58N4O12 and isoniazid, C6H7N3O.
Identification
A. In the Assay, the chromatogram obtained with the test solution shows peaks that correspond to the peaks due to rifampicin RS and isoniazid RS in the chromatogram obtained with the reference solution.
Tests
Related substances. Determine by liquid chromatography (2.4.14).
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IP 2007
RIFAMPICIN AND ISONIAZID TABLETS
obtained with the reference solution (1.5 per cent), the area of any peaks due to 3-formylrifamycin SV and isonicotinyl hydrazone should not be more than 5 times the area of the principal peak in the chromatogram obtained with the reference solution (5 per cent) and the area of any peak due to 3-formylrifamycin SV should not be more than the area of the principal peak in the chromatogram obtained with the reference solution (1.0 per cent). In the chromatogram obtained with the test solution the area of any unknown peak should not be more than 1.5 times the area of the principal peak in the chromatogram obtained with the reference solution ( 1.5 per cent). Dissolution (2.5.2). Apparatus. No 2 Medium. 900 ml of 0.1 M hydrochloric acid. Speed and time. 100 rpm and 45 minutes. Withdraw a suitable volume of the medium and filter promptly through a membrane filter disc with an average pore diameter not greater than 0.8 m. Reject the first few ml of the filtrate and dilute a suitable volume of the filtrate with the medium. Reference stock solution. A solution containing 0.0165 per cent of rifampicin RS and 0.00825 per cent of isoniazid RS in the dissolution medium. For rifampicin Measure the absorbance of the sample solution and the reference stock solution suitably diluted with the dissolution medium at 475 nm (2.4.7). Calculate the content of C43H58N4O12 in the medium from the absorbance obtained from the reference stock solution. For isoniazid Determine by liquid chromatography (2.4.14). Use the reference stock solution and sample solution suitably diluted with a solution of 0.05 M potassium dihydrogen orthophosphate, adjust the pH to 6.2 with 0.1 M sodium hydroxide to obtain the reference solution and the test solution, respectively. Chromatographic system a stainless steel column 30 cm x 3.9 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 99 volumes of 0.05 M potassium dihydrogen phosphate and 1 volume of acetonitrile with the pH adjusted to 4.0 0.05 with 2 per cent w/v solution of phosphoric acid, flow rate. 1 ml per minute, spectrophotometer set at 254 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the tailing factor is not more than 2.0, the column efficiency in not less than 1500 theoretical plates and the relative standard deviation for replicate injections is not more than 2.0 per cent.
Inject alternately the test solution and the reference solution. Calculate the content of C6H7N3O in the dissolution medium. D. Not less than 75 per cent of the stated amounts of C43H58N4O12 and C6H7N3O. Other tests. Comply with the tests stated under Tablets. Assay. Determine by liquid chromatography (2.4.14). Solvent mixture. Dissolve 1.4 g of disodium hydrogen orthophosphate anhydrous in 1000 ml of water and adjust the pH to 6.8 with dilute phosphoric acid. Test solution. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 40 mg of Isoniazid, dissolve in 100.0 ml of methanol and dilute to 500.0 ml with the solvent mixture. Reference solution. A solution containing 0.08 per cent w/v of rifampicin RS and 0.04 per cent w/v of isoniazid RS in methanol. Dilute 10.0 ml of this solution to 50.0 ml with the solvent mixture. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), column. temperature 30, mobile phase: A. a mixture of 96 volumes of buffer solution pH 6.8 prepared by dissolving 1.4 g of disodium hydrogen orthophosphate anhydrous in 1000 ml of water and adjusting the pH to 6.8 0.05 with dilute phosphoric acid, and 4 volumes of acetronitrile. B. a mixture of 45 volumes of the buffer solution and 55 volumes of acetonitrile, flow rate. 1.5 ml per minute, a linear gradient programme using the conditions given below, spectrophotometer set at 238 nm, a 20 l loop injector. Time Mobile phase A Mobile phase B (in min.) (per cent v/v) (per cent v/v) 0 100 0 5 100 0 6 0 100 15 0 100 16 100 0 20 100 0 NOTE Saturate the column with mobile phase B for about 1 hour before injection. Inject the reference solution. The tailing factor is not more than 2.0 for rifampicin and isoniazid; the column efficiency for the isoniazid peak is not less than 3000 and for rifampicin not less than 25000 theoretical plates and the relative standard deviation for replicate injections is not more than 2.0 per cent.
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RIFAMPICIN, ISONIAZID AND ETHAMBUTOL TABLETS
IP 2007
Inject alternately the test solution and the reference solution. The retention times are about 1.0 for rifampicin and about 0.3 for isoniazid. Calculate the contents of C43H58N4O12 and C6H7N3O in the tablets. Storage. Store protected from moisture.
Rifampicin, Isoniazid and Ethambutol Tablets

Rifampin, Isonicotinylhydrazid and Ethambutol Hydrochloride Tablets
Rifampicin, Isoniazid and Ethambutol Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amounts of rifampicin, C43H58N4O12, isoniazid, C6H7N3O and ethambutol hydrochloride C10H24N2O2.2HCl.
Chromatographic system a stainless steel column 10 cm x 4.6 mm, packed with octylsilane bonded to porous silica (5 m), column. temperature 30, mobile phase: a mixture of 65 volumes of buffer solution pH 6.8 prepared by mixing 0.1 per cent w/v of phosphoric acid, 0.19 per cent w/v of sodium perchlorate, 0.59 per cent w/v of citric acid and 2.09 per cent w/v of potassium dihydrogen phosphate with water and adjusting the pH to 6.8 with dilute phosphoric acid, and 35 volumes of acetonitrile, flow rate. 1.5 ml per minute, spectrophotometer set at 254 nm, a 20 l loop injector. Inject reference solution (b). The test is not valid unless the resolution between rifampicin and rifampicin quinone is not less than 4, the tailing factor is not more than 2.0 and the column efficiency is not less than 2000 theoretical plates. Inject the test solution and reference solution (a). The relative retention times are 1.0, 0.55, 1.25, 1.51 and 2.61 for rifampicin, rifampicin quinone, rifampicin N-oxide, 3-formylrifamycin SV Isonicotinyl hydrazone and 3-formylrifamycin SV respectively. Multiply the areas of each known impurity by its response factor. The response factors are 1.00, 1.19, 1.03, 1.22 and 1.25 for rifampicin, rifampicin quinone, rifampicin N-oxide, Isonicotinyl hydrazone and 3-formylrifamycin SV respectively. In the chromatogram obtained with the test solution the area of any peak due to rifampicin quinone should not be more than 4 times the area of the principal peak in the chromatogram obtained with the reference solution (4 per cent), the area of any peak due to rifampicin N-oxide should not be more than 1.5 times the area of the principal peak in the chromatogram obtained with the reference solution (1.5 per cent), the area of any peak due to 3-formylrifamycin SV Isonicotinyl hydrazone should not be more than 5 times the area of the principal peak in the chromatogram obtained with the reference solution ( 5 per cent ) and the area of any peak due to 3-formylrifamycin SV should not be more than the area of the principal peak in the chromatogram obtained with the reference solution (1 per cent ). In the chromatogram obtained with the test solution the area of any unknown peak should not be more than 1.5 times the area of the principal peak in the chromatogram obtained with the reference solution (1.5 per cent). Dissolution (2.5.2). Apparatus. No 2 Medium. 900 ml of 0.1 M hydrochloric acid. Speed and time. 100 rpm and 45 minutes. Withdraw a suitable volume of the medium and filter promptly through a membrane filter disc with an average pore diameter not greater than 0.8 m. Reject the first few ml of the filtrate.
Identification
A. In the Assay for rifampicin and isoniazid, the principal peaks in the chromatogram obtained with the test solution correspond to the peaks in the chromatogram obtained with the reference solution. B. In the Assay for ethambutol hydrochloride the principal peak in the chromatogram obtained with the test solution corresponds to the peak in the chromatogram obtained with the reference solution.
Tests
Related substances. Determine by liquid chromatography (2.4.14). Solvent mixture. A mixture of 10 volumes of a 21.01 per cent w/v solution of citric acid, 23 volumes of a 13.61 per cent w/v solution of potassium dihydrogen phosphate, 77 volumes of a 17.42 per cent w/v solution of dipotassium hydrogen phosphate, 640 volumes of water and 250 volumes of acetonitrile. Test solution. Weigh accurately a quantity of the powdered tablets containing 200 mg of Rifampicin, dissolve in 100 ml of acetonitrile and filter. Dilute 5 ml of this solution to 50 ml with the solvent mixture. Reference solution (a). A solution containing 0.02 per cent w/v of rifampicin RS in acetonitrile. Dilute 1 ml of this solution to 100 ml with the solvent mixture. Reference solution (b). A solution containing 0.01 per cent w/v each of rifampicin RS and rifampicin quinone RS in acetonitrile. Dilute 5 ml of this solution to 50 ml with the solvent mixture.
1052
IP 2007
RIFAMPICIN, ISONIAZID AND ETHAMBUTOL TABLETS
Reference stock solution. A solution containing 0.0165 per cent w/v of rifampicin RS, 0.00825 per cent w/v of isoniazid RS and 0.031 per cent w/v of ethambutol hydrochloride RS in the dissolution medium. Keep this reference stock solution in the dissolution bath during the test run. For rifampicin Dilute the filtrate, if necessary, with the same solvent. Measure the absorbance (2.4.7) of the resulting solution at the maximum at about 475 nm. Calculate the content of C43H58N4O12 in the medium from the absorbance obtained from suitably diluted reference stock solution. For isoniazid Determine by liquid chromatography (2.4.14). Test solution. Suitably dilute the filtered medium with 0.05 M potassium dihydrogen orthophosphate with the pH adjusted to 6.2 with 0.1 M sodium hydroxide. Reference solution. Suitably dilute the reference stock solution with 0.05 M potassium dihydrogen orthophosphate with the pH adjusted to 6.2 with 0.1 M sodium hydroxide. Chromatographic system a stainless steel column 30 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 99 volumes of 0.05 M potassium dihydrogen phosphate and 1 volume of acetonitrile previously adjusted to pH 4.0 with a 2 per cent w/v solution of phosphoric acid, flow rate. 1 ml per minute, spectrophotometer set at 254 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the tailing factor is not more than 2.0, the column efficiency in not less than 1500 theoretical plates and the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject alternately the test solution and the reference solution. Calculate the content of C6H7N3O in the dissolution medium. For ethambutol hydrochloride Determine by liquid chromatography (2.4.14). Test solution. Suitably dilute the filtered medium with 0.05 M potassium dihydrogen orthophosphate adjusted to pH 6.2 with 0.1 M sodium hydroxide. Reference solution. Suitably dilute the reference stock solution with 0.05 M potassium dihydrogen orthophosphate with the pH adjusted to 6.2 with 0.1 M sodium hydroxide. Use the chromatographic system described under the Assay of ethambutol hydrochloride. Calculate the content of C10H24N2O2.2HCl in the dissolution medium. D. Not less than 75 per cent of the stated amounts of C43H58N4O12, C6H7N3O and C10H24N2O2.2HCl.
Other tests. Comply with the tests stated under Tablets. Assay. For rifampicin and isoniazid Determine by liquid chromatography (2.4.14). Test solution. Weigh and powder 20 tablets. Weigh accurately a quantity of the powdered tablets containing about 40 mg of Isoniazid dissolve in 100.0 ml of methanol, dilute to 500.0 ml with the diluent and mix. Reference solution. A solution containing 0.08 per cent w/v of rifampicin RS and 0.04 per cent w/v of isoniazid RS in methanol. Dilute 10.0 ml of this solution to 50.0 ml with the diluent. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), column. temperature 30, mobile phase: A. a mixture of 96 volumes of buffer solution pH 6.8 (diluent) prepared by dissolving 1.4 g of disodium hydrogen orthophosphate anhydrous in 1000 ml of water, and adjusting the pH to 6.8 0.05 with dilute phosphoroic acid, and 4 volumes of acetonitrile, B: a mixture of 45 volumes of the buffer solution and 55 volumes of acetonitrile, flow rate. 1.5 ml per minute, a linear gradient programme using the conditions given below, spectrophotometer set at 238 nm, a 20 l loop injector. Time Mobile phase A Mobile phase B (in min.) (per cent v/v) (per cent v/v) 0 100 0 5 100 0 6 0 100 15 0 100 16 100 0 22 100 0 NOTE Saturate the column with mobile phase B for about 1 hour. Inject the reference solution. The test is not valid unless the tailing factor is not less than 2.0 for rifampicin and isoniazid, the column efficiency determined from isoniazid peak is not less than 3000 and that from rifampicin is not less than 25000 theoretical plates respectively, and the relative standard deviation for replicate injections is not more than 2.0 per cent. The relative retention times are about 1.0 for rifampicin and about 0.3 for isoniazid. Calculate the contents of C43H58N4O12 and C6H7N3O in the tablets. For ethambutol hydrochloride Determine by liquid chromatography (2.4.14).
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RIFAMPICIN, ISONIAZID AND PYRAZINAMIDE TABLETS
IP 2007
Test solution. Weigh accurately a quantity of the powdered tablets containing about 60 mg of Ethambutol Hydrochloride and dissolve in 100.0 ml of the diluent. Reference solution. A 0.06 per cent w/v solution of ethambutol hydrochloride RS in the diluent. Chromatographic system a stainless steel column 15 cm x 4.6 mm, packed with nitrile groups chemically bonded to porous silica particles 5 (m) (such as Zorbax SB CN), mobile phase: a mixture of 50 volumes of acetonitrile, and 50 volumes of a buffer solution pH 7.0 prepared by dissolving 1 ml of triethylamine in 1000 ml of water and adjusting the pH to 7.0 with dilute phosphoric acid, flow rate. 1 ml per minute, spectrophotometer set at 200 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent, the tailing factor is not more than 3.0 and the column efficiency determined from Ethambutol hydrochloride peak is not less than 1500 theoretical plates. Inject alternately the test solution and the reference solution. Calculate the content of C10H24N2O2.2HCl in the tablets. Storage. Store protected from moisture.
phosphate, 640 volumes of water and 250 volumes of acetonitrile. Test solution. Weigh accurately a quantity of the powdered tablets containing about 200 mg of Rifampicin, dissolve in 100 ml of acetonitrile and filter. Dilute 5 ml of this solution to 50 ml with the solvent mixture. Reference solution (a). A solution containing 0.02 per cent w/v of rifampicin RS in acetonitrile. Dilute 1ml of this solution to a 100 ml with the solvent mixture. Reference solution (b). A solution containing 0.01 per cent w/v each of rifampicin RS and rifampicin quinone RS in acetonitrile. Dilute 5 ml of this solution to 50 ml with the solvent mixture. Chromatographic system a stainless steel column 10 cm x 4.6 mm, packed with octylsilane bonded to porous silica (5 m), column. temperature 30, mobile phase: a mixture of 65 volumes of buffer solution pH 6.8 prepared by mixing 0.1 per cent w/v of phosphoric acid, 0.19 per cent w/v of sodium perchlorate, 0.59 per cent w/v of citric acid and 2.09 per cent w/v of potassium dihydrogen phosphate with water and adjusting the pH to 6.8 with dilute phosphoric acid, and 35 volumes of acetonitrile, flow rate. 1.5 ml per minute, spectrophotometer set at 254 nm, a 20 l loop injector. Inject reference solution (b). The test is not valid unless the resolution between rifampicin and rifampicin quinone is not less than 4, the tailing factor is not more than 2.0 and the column efficiency is not less than 2000 theoretical plates. Inject the test solution and reference solution (a). The relative retention times are 1.0, 0.55, 1.25, 1.51 and 2.61 for rifampicin, rifampicin quinone, rifampicin N-oxide, 3-formylrifamycin SV Isonicotinyl hydrazone and 3-formylrifamycin SV respectively. Multiply the area of each known impurity by its response factor. The response factors are 1.00, 1.19, 1.03, 1.22 and 1.25 for rifampicin, rifampicin quinone, rifampicin N-oxide, Isonicotinyl hydrazone and 3-formylrifamycin SV respectively. In the chromatogram obtained with the test solution the area of any peak due to rifampicin quinone should not be more than 4 times the area of the principal peak in the chromatogram obtained with the reference solution (4 per cent), the area of any peak due to rifampicin N-oxide should not be more than 1.5 times the area of the principal peak in the chromatogram obtained with the reference solution (1.5 per cent), the area of any peak due to 3-formylrifamycin SV Isonicotinyl hydrazone should not be more than 5 times the area of the principal peak in the chromatogram obtained with the reference solution ( 5 per cent ) and the area of any peak due to 3-formylrifamycin SV should not be more than the area of the principal peak in
Rifampicin, Isoniazid and Pyrazinamide Tablets

Rifampin, Isonicotinylhydrazid and Pyrazinamide Tablets
Rifampicin, Isoniazid and Pyrazinamide Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amounts of rifampicin, C43H58N4O12, isoniazid, C6H7N3O and pyrazinamide C5H5N3O.
Identification
In the Assay of rifampicin, isoniazid and pyrazinamide, the principal peaks in the chromatogram obtained with the test solution correspond to the peaks in the chromatogram obtained with the reference solution.
Tests
Related substances. Determine by liquid chromatography (2.4.14). Solvent mixture. A mixture of 10 volumes of a 21.01 per cent w/v solution of citric acid, 23 volumes of a 13.61 per cent w/v solution of potassium dihydrogen phosphate, 77 volumes of 17.42 per cent w/v solution of dipotassium hydrogen
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IP 2007
RIFAMPICIN, ISONIAZID AND PYRAZINAMIDE TABLETS
the chromatogram obtained with the reference solution ( 1 per cent ). In the chromatogram obtained with the test solution the area of any unknown peak should not be more than 1.5 times the area of the principal peak in the chromatogram obtained with the reference solution (1.5 per cent). Dissolution (2.5.2). Apparatus. No 2 Medium. 900 ml of a solution containing 2 g of sodium chloride and 7.0 ml of hydrochloric acid in 1000 ml of water, with a pH of about 1.2. Speed and time. 100 rpm and 45 minutes. Withdraw a suitable volume of the medium and filter through a membrane filter disc with an average pore diameter not greater than 0.8 m. Reject the first few ml of the filtrate. Reference stock solution. A solution containing 0.0165 per cent w/v of rifampicin RS, 0.00825 per cent w/v of isoniazid RS and 0.04375 per cent w/v of pyrazinamide RS in the dissolution medium and filtered. Keep this reference stock solution in the dissolution bath during the test run. For rifampicin Dilute the filtrate, if necessary, with the same solvent. Measure the absorbance (2.4.7) of the resulting solution at the maximum at about 475 nm. Calculate the content of C43H58N4O12 in the medium from the absorbance obtained from suitably diluted reference stock solution. For isoniazid and pyrazinamide Determine by liquid chromatography (2.4.14). NOTE Use this solution within one hour from preparation. Test solution. To 15.0 ml of the filtered medium, add 15 ml of 1 M dibasic potassium phosphate, dilute to 100.0 ml with the mobile phase and mix. Reference solution (a). To 15.0 ml of the reference stock solution, add 15 ml of 1 M dibasic potassium phosphate, dilute to 100.0 ml with the mobile phase and mix. Reference solution (b). To 10.0 ml of a 0.0125 per cent w/v solution of isonicotinic acid in the dissolution medium, add 4.0 ml of the reference stock solution and 15 ml of 1 M dibasic potassium phosphate, dilute to 100.0 ml with the mobile phase and mix. Chromatographic system a stainless steel column 30 cm x 4.6 mm, packed with octylsilane bonded to porous silica (5 m), mobile phase: a mixture of 860 volumes of water, 100 volumes of 1 M monobasic potassium phosphate and 40 volumes of acetonitrile, flow rate. 1 ml per minute, spectrophotometer set at 254 nm, a 50 l loop injector. Inject the test solution and reference solutions (a) and (b). The relative retention times are about 0.7 for isonicotinic acid, 1.0 for pyrazinamide and 1.8 for isoniazid.
The test is not valid unless in the chromatogram obtained with reference solution (b) the resolution between isonicotinic acid and pyrazinamide is not more than 2.5 and between pyrazinamide and isoniazid is not more than 4.0. Calculate the contents of C6H7N3O and C5H5N3O in the medium. D. Not less than 75 per cent of the stated amounts of C43H58N4O12, C6H7N3O and C5H5N3O. Other tests. Comply with the tests stated under Tablets. Assay. For rifampicin, isoniazid and pyrazinamide Determine by liquid chromatography (2.4.14). Test solution. Weigh and powder 20 tablets. Weigh accurately a quantity of the powdered tablets containing about 40 mg of Isoniazid, dissolve in 100.0 ml of methanol, dilute to 500 ml with the solvent mixture and mix. Reference solution. A solution containing 0.08 per cent w/v of rifampicin RS, 0.04 per cent w/v of isoniazid RS and 0.2 per cent w/v of pyrazinamide RS in methanol. Dilute 10.0 ml of this solution to 50.0 ml with the solvent mixture. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), column. temperature 30, mobile phase: A. a mixture of 96 volumes of buffer solution pH 6.8 (diluent) prepared by dissolving 1.4 g of disodium hydrogen orthophosphate anhydrous in 1000 ml of water, adjusted to pH 6.8 0.05 with dilute phosphoroic acid and 4 volumes of acetonitrile, B. a mixture of 45 volumes of the buffer solution and 55 volumes of acetonitrile, flow rate. 1.5 ml per minute, a linear gradient programme using the conditions given below, spectrophotometer set at 238 nm, a 20 l loop injector. Time Mobile phase A Mobile phase B (in min.) (per cent v/v) (per cent v/v) 0 100 0 5 100 0 6 0 100 15 0 100 16 100 0 22 100 0 NOTE Saturate the column with mobile phase (B) for about 1 hour. Inject the test solution and the reference solution. The test is not valid unless the tailing factor is not less than 2.0 for rifampicin, isoniazid and pyrazinamide, the column efficiencies determined from Isoniazid, pyrazinamide and that from rifampicin are not less than 3000, 5000 and 25000 theoretical
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RIFAMPICIN, ISONIAZID, PYRAZINAMIDE AND ETHAMBUTOL TABLETS
IP 2007
plates respectively, and the relative standard deviation for replicate injections is not more than 2.0 per cent. The relative retention times are about 1.8 for rifampicin, about 0.7 for isoniazid and about 1.0 for pyrazinamide. Inject alternately the test solution and the reference solution. Calculate the contents of C43H58N4O12, C6H7N3O and C5H5N3O in the tablets. Storage. Store protected from moisture.
Rifampicin, Isoniazid, Pyrazinamide and Ethambutol Tablets

Rifampin, Isonicotinylhydrazid, Pyrazinamide and Ethambutol Hydrochloride Tablets
Rifampicin, Isoniazid, Pyrazinamide and Ethambutol Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amounts of rifampicin, C43H58N4O12, isoniazid, C 6 H 7 N 3 O, pyrazinamide, C 5 H 5 N 3 O and ethambutol hydrochloride, C10H24N2O2.2HCl.
acetonitrile. Dilute 5 ml of this solution to 50 ml with the solvent mixture. Chromatographic system a stainless steel column 10 cm x 4.6 mm, packed with octylsilane bonded to porous silica (5 m), column. temperature 30, mobile phase: a mixture of 65 volumes of buffer solution pH 6.8 prepared by mixing 0.1 per cent w/v of phosphoric acid, 0.19 per cent w/v of sodium perchlorate, 0.59 per cent w/v of citric acid and 2.09 per cent w/v of potassium dihydrogen phosphate with water, adjusting the pH to 6.8 with dilute phosphoric acid, and 35 volumes of acetonitrile, flow rate. 1.5 ml per minute, spectrophotometer set at 254 nm, a 20 l loop injector. Inject reference solution (b). The test is not valid unless the resolution between rifampicin and rifampicin quinone is not less than 4, the tailing factor is not more than 2.0 and the column efficiency is not less than 2000 theoretical plates. Inject the test solution and reference solution (a). The relative retention times are 1.0, 0.55, 1.25, 1.51 and 2.61 for rifampicin, rifampicin quinone, rifampicin N-oxide, 3-formylrifamycin SV Isonicotinyl hydrazone and 3-formylrifamycin SV respectively. Multiply the area of each known impurity by its response factor. The response factors are 1.00, 1.19, 1.03, 1.22 and 1.25 for rifampicin, rifampicin quinone, rifampicin N-oxide, Isonicotinyl hydrazone and 3-formylrifamycin SV respectively. In the chromatogram obtained with the test solution the area of any peak due to rifampicin quinone should not be more than 4 times the area of the principal peak in the chromatogram obtained with the reference solution (4 per cent), the area of any peak due to rifampicin N-oxide should not be more than 1.5 times the area of the principal peak in the chromatogram obtained with the reference solution (1.5 per cent), the area of any peak due to 3-formylrifamycin SV Isonicotinyl hydrazone should not be more than 5 times the area of the principal peak in the chromatogram obtained with the reference solution ( 5 per cent ) and the area of any peak due to 3-formylrifamycin SV should not be more than the area of the principal peak in the chromatogram obtained with the reference solution (1 per cent ). In the chromatogram obtained with the test solution the area of any unknown peak should not be more than 1.5 times the area of the principal peak in the chromatogram obtained with the reference solution (1.5 per cent). Dissolution (2.5.2). Apparatus. No 1 Medium. 900 ml of sodium phosphate buffer pH 6.8 prepared by dissolving 7 g of dibasic sodium phosphate anhydrous in 5000 ml of water and adjusting to pH 6.8 with phosphoric acid.
Identification
A. In the Assay for rifampicin, isoniazid and pyrazinamide, the principal peaks in the chromatogram obtained with the test solution correspond to the peaks in the chromatogram obtained with the reference solution. B. In the Assay for ethambutol hydrochloride, the principal peak in the chromatogram obtained with the test solution corresponds to the peak in the chromatogram obtained with the reference solution.
Tests
Related substances. Determine by liquid chromatography (2.4.14). Solvent mixture. A mixture of 10 volumes of a 21.01 per cent w/v solution of citric acid, 23 volumes of a 13.61 per cent w/v solution of potassium dihydrogen phosphate, 77 volumes of a 17.42 per cent w/v solution of dipotassium hydrogen phosphate, 640 volumes of water and 250 volumes of acetonitrile. Test solution. Weigh accurately a quantity of powdered tablets containing 200 mg of Rifampicin, dissolve in 100 ml of acetonitrile and filter. Dilute 5 ml of this solution to 50 ml with the solvent mixture. Reference solution (a). Dissolve 20 mg of rifampicin RS in 100 ml of acetonitrile. Dilute 1 ml of this solution to a 100 ml with the solvent mixture. Reference solution (b). A solution containing 0.01 per cent w/v each of rifampicin RS and rifampicin quinone RS in
1056
IP 2007
RITONAVIR
Speed and time. 100 rpm and 45 minutes. Withdraw a suitable volume of the medium and filter through a membrane filter disc with an average pore diameter not greater than 0.8 m. Reject the first few ml of the filtrate. Determine the amounts of rifampicin, C43H58N4O12, isoniazid, C6H7N3O, pyrazinamide, C5H5N3O and ethambutol hydrochloride, C10H24N2O2.2HCl by using the methods described in the Assay for rifampicin, isoniazid, pyrazinamide and ethambutol hydrochloride. D. Not less than 75 per cent of the stated amounts of C43H58N4O12, C6H7N3O, C5H5N3O and C10H24N2O2.2HCl. Other tests. Comply with the tests stated under Tablets. Assay. For rifampicin, isoniazid and pyrazinamide Determine by liquid chromatography (2.4.14). Test solution. Weigh and powder 20 tablets. Weigh accurately a quantity of the powdered tablets containing about 40 mg of Isoniazid, dissolve in 100 ml of methanol, dilute to 500 ml with the solvent mixture, mix and filter. Reference solution. A solution containing 0.08 per cent w/v of rifampicin RS, 0.04 per cent w/v of isoniazid RS and 0.2 per cent w/v of pyrazinamide RS in methanol. Dilute 10.0 ml of this solution to 50.0 ml with the solvent mixture. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), column. temperature 30, mobile phase: A. a mixture of 96 volumes of buffer solution pH 6.8 prepared by dissolving 1.4 g of disodium hydrogen orthophosphate anhydrous in 1000 ml of water, adjusting to pH 6.8 0.05 with dilute phosphoroic acid and 4 volumes of acetonitrile, B. a mixture of 45 volumes of the buffer solution and 55 volumes of acetonitrile, flow rate. 1.5 ml per minute, a linear gradient programme using the conditions given below, spectrophotometer set at 238 nm, a 20 l loop injector. Time Solution A Solution B (in min.) (per cent v/v) (per cent v/v) 0 100 0 5 100 0 6 0 100 15 0 100 16 100 0 22 100 0 Inject the reference solution. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent, the tailing factor is not more than 2.0 for rifampicin, isoniazid and pyrazinamide and the column
efficiencies for rifampicin, isoniazid and pyrazinamide are not less than 3000, 5000 and 25000 theoretical plates respectively. Inject alternately the test solution and the reference solution. The relative retention time for rifampicin is 1.8, for isoniazid, 0.7 and for pyrazinamide, 1.0. Calculate the contents of C43H58N4O12, C6H7N3O and C5H5N3O in the tablets. For ethambutol hydrochloride Determine by liquid chromatography (2.4.14). Test solution. Weigh accurately a quantity of the powdered tablets containing about 60 mg of Ethambutol Hydrochloride and dissolve in 100 ml of the solvent mixture. Reference solution. A 0.06 per cent w/v solution of ethambutol hydrochloride RS in the solvent mixture. Chromatographic system a stainless steel column 15 cm x 4.6 mm, packed with nitrile groups chemically bonded to porous silica particles (5 m), mobile phase: a mixture of 50 volumes of acetonitrile, and 50 volumes of buffer solution pH 7.0 (diluent) prepared by dissolving 1 ml of triethylamine in 1000 ml of water and adjusting the pH to 7.0 with dilute phosphoric acid, flow rate. 1 ml per minute, spectrophotometer set at 200 nm, a 50 l loop injector. Inject the reference solution. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent, the tailing factor is not more than 3.0 and the column efficiency determined from ethambutol hydrochloride peak is not less than 1500 theoretical plates. Calculate the content of C10H24N2O2.2HCl in the tablets. Storage. Store protected from moisture.
Ritonavir
CH3 H3C N S H3C O N N CH3 H CH3 H N O OH O N H O S N
C37H48N6O5S2
Mol. Wt. 721.0
1057
RITONAVIR CAPSULES
IP 2007
Ritonavir is (5S,8S,10S,11S)-10-hydroxy-2-methyl-5-(1methylethyl)-1-[2-(1-methylethyl)-4-thiazolyl]-3,6-dioxo-8,11bis(phenylmethyl)-2,4,7,12-tetraazatridecan-13-oic acid 5thiazolyl methyl ester. Ritonavir contains not less than 98.0 per cent and not more than 102.0 per cent of C37H48N 6O5S2, calculated on the anhydrous basis. Description. A white to off-white powder.
flow rate. 1 ml per minute, spectrophotometer set at 239 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the column efficiency determined from the ritonavir peak is not less than 1000 theoretical plates, the tailing factor is not more than 2.0, and the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject alternately the test solution and the reference solution.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with ritonavir RS or with the reference spectrum of ritonavir. B. In the Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the peak due to ritonavir in the chromatogram obtained with the reference solution. C. Melting point. 119 to 123 (2.4.21).
Calculate the content of C37H48N6O5S2. Storage. Store protected from light.
Ritonavir Capsules
Ritonavir Capsules contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of ritonavir C37H48N6O5S2.
Tests
Specific optical rotation (2.4.22). +7.0 to +10.5, determined in a 0.2 per cent w/v solution in methanol. Related substances. Determine by liquid chromatography (2.4.14), as described in the Assay and calculate the percentage of each impurity peak in the chromatogram obtained with the test solution. Not more than 0.5 per cent of any individual impurity and not more than 1.0 per cent of total impurities is found. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent Water (2.3.43). Not more than 0.5 per cent, determined on 2.0 g. Assay. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 25.0 mg of the substance under examination in sufficient of a mixture of 45 volumes of acetonitrile and 55 volumes of water to produce 100.0 ml. Reference solution. A 0.025 per cent w/v solution of ritonavir RS in a mixture of 45 volumes of acetonitrile and 55 volumes of water. Chromatographic system a stainless steel column 15 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 45 volumes of acetonitrile and 55 volumes of a buffer solution prepared by dissolving 3.4 g of sodium acetate and 0.94 g of sodium hexanesulphonate in 1000 ml of water and adjusting the pH to 4.0 with hydrochloric acid,
Identification
A. In the test for Assay, the principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution. B. When examined in the range 200 nm to 350 nm (2.4.7), a 0.001 per cent w/v solution in methanol shows absorption maxima at the same wavelengths as the reference solution.
Tests
Dissolution (2.5.2). Apparatus. No 1 Medium. 900 ml of 0.1 M hydrochloric acid with 25 mM polyoxyethylene 10 lauryl ether prepared by dissolving 15.65 g of polyoxyethylene 10 lauryl ether in 950 ml of water. Add 8.5 ml of hydrochloric acid and dilute to 1000 ml with water. Speed and time. 50 rpm and 90 minutes. Withdraw a suitable volume of the medium and filter. Determine by liquid chromatography (2.4.14). Test solution. Use the filtrate and if necessary, dilute with the dissolution medium. Reference solution. A 0.1 per cent w/v solution of ritonavir RS in methanol. Dilute 5 ml of the solution to 50 ml with the dissolution medium. Use the chromatographic system described under Assay. Inject the test solution and the reference solution. D. Not less than 70 per cent of the stated amount of C37H48N6O5S2.
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IP 2007
RITONAVIR TABLETS
Related substances. Determine by liquid chromatography (2.4.14). Solvent mixture. 40 volumes of 0.03M monobasic potassium phosphate buffer and 60 volumes of acetonitrile. Test solution. Weigh accurately a quantity of the mixed contents of 20 capsules containing about 25 mg of Ritonavir, dissolve in 50 ml of the solvent mixture and filter. Reference solution (a). A 0.05 per cent w/v solution of ritonavir RS in the solvent mixture. Reference solution (b). Dilute 1 ml of the solution to 100 ml with the solvent mixture. Chromatographic system a stainless steel column 15 cm x 4.6 mm, packed with butyl silane chemically bonded to porous silica (3 m) (such as ACE C4), mobile phase: A. a mixture of 69 volumes of 0.03 M monobasic potassium phosphate buffer prepared by dissolving 4.1 g of monobasic potassium phosphate in 1000 ml of water, 18 volumes of acetonitrile, 8 volumes of tetrahydrofuran and 5 volumes of n-butanol and filtering, B. a mixture of 40 volumes of 0.03 M monobasic potassium phosphate buffer, 47 volumes of acetonitrile, 8 volumes of tetrahydrofuran and 5 volumes of n-butanol, flow rate. 1 ml per minute, a linear gradient programme using the conditions given below, spectrophotometer set at 240 nm, a 50 l loop injector. Time mobile phase A mobile phase B (in min.) (per cent v/v) (per cent v/v) 0 100 0 60 100 0 120 0 100 155 100 0 Inject the reference solution (a). The test is not valid unless the tailing factor is not more than 2.0 and the column efficiency in not less than 5000 theoretical plates. Inject the test solution and reference solution (b). In the chromatogram obtained with the test solution, the area of any secondary peak is not more than 2.5 times the area of the peak in the chromatogram obtained with the reference solution (b) (2.5 per cent) and the sum of areas of all the secondary peaks is not more than 5 times the area of the peak in the chromatogram obtained with the reference solution (b) (5.0 per cent). Other tests. Comply with the tests stated under Capsules.
Assay. Determine by liquid chromatography (2.4.14). Test solution. Weigh accurately a quantity of the mixed contents of 20 capsules containing about 25 mg of Ritonavir, disperse in 100.0 ml of methanol and filter. Reference solution. A 0.025 per cent w/v solution of ritonavir RS in methanol. Chromatographic system a stainless steel column 5 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (3.5 m), column temperature 45, mobile phase: a mixture of 55 volumes of a buffer solution prepared by dissolving 3.4 g of sodium acetate trihydrate and 0.94 g of sodium 1-hexanesulphonate in 1000 ml of water and adjusting the pH to 4.0 with hydrochloric acid, and 45 volumes of acetonitrile, flow rate. 2.5 ml per minute, spectrophotometer set at 240 nm, a 10 l loop injector. Inject the reference solution. Run the chromatogram 1.5 times the retention time (about 3 minutes)of the principal peak The test is not valid unless the tailing factor is not more than 2.0, the column efficiency in not less than 2000 theoretical plates and the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject the test solution and the reference solution. Calculate the content of C37H48N6O5S2 in the capsules. Storage. Store protected from light in a refrigerator (2 to 8).
Ritonavir Tablets
Ritonavir Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of ritonavir, C37H48N6O5S2.
Identification
In the Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the principal peak in the chromatogram obtained with the reference solution.
Tests
Dissolution (2.5.2) Apparatus. No 1 Medium. 900 ml of 0.1 M hydrochloric acid. Speed and time. 100 rpm and 60 minutes. Withdraw a suitable volume of the medium and filter. Determine by liquid chromatography (2.4.14)
1059
ROSIGLITAZONE MALEATE
IP 2007
Test solution. The filtrate obtained as given above. Dilute the filtrate if necessary, with the dissolution medium. Reference solution. A 0.1 per cent w/v solution of ritonavir RS in methanol. Dilute 5 ml of the solution to 50 ml with the dissolution medium. Use the chromatographic system described under Assay. Inject the reference solution. The test is not valid unless the tailing factor not more than 2.0 and the relative standard deviation of replicate injections is not more than 2.0 per cent. Inject the test solution and the reference solution. Calculate the content of C37H48N6O5S2. D. Not less than 75 per cent of the stated amount of C37H48N6O5S2. Related substances. Determine by liquid chromatography (2.4.14). Solvent mixture. A mixture of 40 volumes of 0.03 M monobasic potassium phosphate and 60 volumes of acetonitrile. Test solution: Shake a quantity of the powdered tablets containing 25 mg of Ritonavir with 50 ml of the solvent mixture. Reference solution (a). A 0.05 per cent w/v solution of ritonavir RS in the solvent mixture. Reference solution (b). Dilute 1 ml of reference solution (a) to 100 ml with the solvent mixture. Chromatographic system a stainless steel column 15 cm x 4.6 mm, packed with silica gel consisting of porous spherical particles with chemically bonded with nitrile group (3m) (such as YMC C4), mobile phase: A. a mixture of 69 volumes of 0.03 M monobasic potassium phosphate solution, 18 volumes of acetonitrile, 8 volumes of tetrahydrofuran and 5 volumes of n- butanol, B. a mixture of 40 volumes of 0.03 M monobasic potassium phosphate solution, 47 volumes of acetonitrile, 8 volumes of tetrahydrofuran and 5 volumes of n-butanol, flow rate.1 ml per minute, a linesr gradient programme using the conditions given below, spectrophotometer set at 240 nm, a 100 l loop injector. Time Mobile phase A Mobile phase B (in min.) (per cent v/v) (per cent v/v) 0 100 0 60 100 0 120 0 100 121 100 0 155 100 0
Inject reference solution (a). The test is not valid unless the column efficiency is not less than 2000 theoretical plates and the tailing factor is not more than 2.0. Inject the test solution and reference solution (b). In the chromatogram obtained with the test solution, the area of any secondary peak is not more than 2.5 times the area of the peak in the chromatogram obtained with the reference solution (b) (2.5 per cent) and the sum of areas of all the secondary peaks is not more than 5 times the area of the peak in the chromatogram obtained with the reference solution (b) (5.0 per cent). Other tests. Comply with the tests stated under Tablets. Assay. Determine by liquid chromatography (2.4.14) Test solution. Weigh accurately a quantity of the powdered tablets containing 25 mg of Ritonavir and disperse in 100.0 ml of methanol. Reference solution. A 0.025 per cent w/v solution of ritonavir RS in methanol. Chromatographic system a stainless steel column 5 cm x 4.6 mm, packed with phenyl stationary phase bonded to porous silica (3m), column temperature 45, mobile phase: a mixture of 55 volumes of a buffer solution prepared by dissolving 3.4 g of sodium acetate trihydrate and 0.94 g of sodium 1-hexane sulphonate to 1000 ml with water and adjusting the pH to 4.0 with dilute hydrochloric acid, and 45 volumes of acetonitrile. flow rate. 2.5 ml per minute, spectrophotometer set at 239 nm, a 10 l loop injector. Inject the reference solution. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject the test solution and the reference solution. Calculate the content of C37H48N6O5S2 in the tablets. Storage. Store protected from moisture, at a temperature not exceeding 30.
Rosiglitazone Maleate
CH3 N N S O O NH O , COOH COOH
C18H19N3O3S,C4H4O4 Mol. Wt. 473.5 Rosiglitazone Maleate is ()-5-{p-[2-( Methyl-2-pyridylamino) ethoxy]benzyl}-2,4-thiazolidinedione maleate (1:1).
1060
IP 2007
ROSIGLITAZONE TABLETS
Rosiglitazone Maleate contains not less than 98.0 per cent and not more than 102.0 per cent of C18H19N3O3S,C4H4O4, calculated on the anhydrous basis. Description. A white to off-white crystalline powder.
to 3.0 with dilute phosphoric acid, 25 volumes of acetonitrile and 10 volumes of methanol, flow rate. 1 ml per minute, spectrophotometer set at 235 nm, a 10 l loop injector. Inject the reference solution. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject the test solution and the reference solution. Calculate the content of C18H19N3O3S,C4H4O4. Storage. Store protected from light.
Identification
Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with rosiglitazone maleate RS.
Tests
Related substances. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 25 mg of the substance under examination in 50 ml of mobile phase. Reference solution (a). A 0.05 per cent w/v solution of rosiglitazone maleate RS in the mobile phase. Reference solution (b). Dilute 1 ml of reference solution (a) to 100 ml with mobile phase. Chromatographic system as described under Assay. Inject reference solution (a). The test is not valid unless the tailing factor is not more than 2.0 and the column efficiency in not less than 2000 theoretical plates. Inject the test solution and reference solution (b). In the chromatogram obtained with the test solution, the area of any secondary peak is not more than 0.5 times the area of the peak in the chromatogram obtained with reference solution (b) (0.5 per cent) and the sum of areas of all the secondary peaks is not more than the area of the peak in the chromatogram obtained with the reference solution (b) (1.0 per cent). Heavy metals (2.3.13). 1 g complies with the limit test for heavy metals, Method B (20 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Water (2.3.43). Not more than 0.5 per cent, determined on 1.0 g. Assay. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 20 mg of the substance under examination in 100.0 ml of mobile phase. Reference solution. A 0.020 per cent w/v solution of rosiglitazone maleate RS in mobile phase. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 65 volumes of buffer solution prepared by dissolving 1.36 g of potassium dihydrogen orthophosphate in 1000 ml of water and adjust the pH
Rosiglitazone Tablets
Rosiglitazone Maleate Tablets Rosiglitazone Tablets contain Rosiglitazone Maleate. Rosiglitazone Tablets contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of rosiglitazone, C18H19N3O3S.
Identification
Tests
Dissolution (2.5.2). Apparatus. No 1 Medium. 900 ml of 0.1 M hydrochloric acid. Speed and time. 75 rpm for 30 minutes. Withdraw a suitable volume of the medium and filter promptly through a membrane filter disc having an average pore diameter not greater than 1.0 m, rejecting the first 1 ml of the filtrate. Dilute the filtrate, if necessary, with the same solvent. Measure the absorbance of the resulting solution at the maximum at about 318 nm (2.4.7). Similarly measure the absorbance of a standard solution of known concentration of rosiglitazone maleate RS and calculate the content of C18H19N3O3S. D. Not less than 70 per cent of the stated amount of C18H19N3O3S. Related substances. Determine by liquid chromatography (2.4.14). Test solution. Weigh accurately a quantity of powdered tablets containing 200 mg of Rosiglitazone, disperse in 100.0 ml of mobile phase. Centrifuge and use clear supernatant liquid.
1061
ROSUVASTATIN CALCIUM
IP 2007
Reference solution (a). A 0.2 per cent w/v solution of rosiglitazone maleate RS in the mobile phase. Reference solution (b). Dilute 1 ml of reference solution (a) to 100 ml with mobile phase. Chromatographic system as described under Assay. Inject reference solution (a). The test is not valid unless the tailing factor is not more than 2.0 and the column efficiency in not less than 2000 theoretical plates. Inject the test solution and reference solution (b). In the chromatogram obtained with the test solution, the area of any secondary peak is not more than the area of the peak in the chromatogram obtained with reference solution (b) (1.0 per cent) and the sum of areas of all the secondary peaks is not more than twice the area of the peak in the chromatogram obtained with the reference solution (b) (2.0 per cent). Uniformity of content. Comply with the tests stated under Tablets. Disperse 1 tablet in 0.1 M hydrochloric acid to produce 0.004 per cent w/v solution. Measure the absorbance of the resulting solution at the maximum at about 318 nm (2.4.7). Calculate the content of C18H19N3O3S from the absorbance obtained from same concentration of rosiglitazone maleate RS in the same medium. Other tests. Comply with the tests stated under Capsules. Assay. Determine by liquid chromatography (2.4.14). Test solution. Weigh and powder 20 tablets. Weigh accurately a quantity of powdered tablets containing 20 mg of Rosiglitazone, disperse in 100.0 ml of mobile phase. Centrifuge and use clear supernatant liquid. Reference solution. A 0.020 per cent w/v solution of rosiglitazone maleate RS in mobile phase. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with octadecysilane bonded to porous silica (5 m), mobile phase: a mixture of 65 volumes of 0.01 M potassium hydrogen phosphate adjusted to pH 3.0 with orthophosphoric acid, 25 volumes of acetonitrile and 10 volumes of methanol, flow rate. 1 ml per minute, spectrophotometer set at 235 nm, a 10 l loop injector. Inject the reference solution. The test is not valid unless the tailing factor is not more than 2.0, the column efficiency in not less than 4000 theoretical plates and the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject the test solution and the reference solution. Calculate the content of C18H19N3O3S. Storage. Store protected from light and moisture.
Rosuvastatin Calcium
F
OH OH O Ca++ H3C N N N SO2CH3 CH3 CH3

2
C22H27FN3O6S.Ca
Mol. Wt. 1001.1
Rosuvastatin Calcium is (E)-(3R,5S)-7-{4-(4-fluorophenyl)-6isopropyl-2-{methyl(methylsulphonyl amino)]pyrimidin-5yl}-3,5-dihydroxyhepten-6-oic acid calcium. Rosuvastatin Calcium contains not less than 98.0 per cent and not more than 102.0 per cent of C22H27FN3O6S.Ca. calculated on the dried basis. Description. An off- white to creamish white crystalline powder.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with rosuvastatin calcium RS. B. Dissolve 20 mg in 25 ml of methanol, add 2 drops of methyl red indicator neutralise with 6 M ammonium hydroxide. Add 3 M hydrochloric acid, dropwise until the solution is acidic to the indicator. Add ammonium oxalate, a white precipitate is obtained.
Tests
Related substances. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 50 mg of the substance under examination in 100 ml of the mobile phase. Reference solution (a). A 0.05 per cent w/v solution of rosuvastatin calcium RS in the mobile phase. Reference solution (b). Dilute 1 ml of reference solution (a) to 100 ml with mobile phase. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 50 volumes of 0.2 per cent w/v acetic acid in water, 25 volumes of acetonitrile and 25 volumes of methanol,
1062
IP 2007
ROSUVASTATIN TABLETS
flow rate. 1 ml per minute, spectrophotometer set at 248 nm, a 20 l loop injector. Inject reference solution (a). The test is not valid unless the tailing factor is not more than 2.0 and the column efficiency in not less than 2000 theoretical plates. Inject the test solution and reference solution (b). In the chromatogram obtained with the test solution, the area of any secondary peak is not more than 0.5 times the area of the peak in the chromatogram obtained with reference solution (b) (0.5 per cent) and the sum of areas of all the secondary peaks is not more than twice the area of the peak in the chromatogram obtained with the reference solution (b) (2.0 per cent). Water (2.3.43). Not more than 8.0 per cent, determined on 0.3 g. Assay. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 50 mg of the substance under examination in 100.0 ml of mobile phase. Dilute 5.0 ml of the solution to 25.0 ml with mobile phase, mix and filter. Reference solution. A 0.05 per cent w/v of rosuvastatin calcium RS in mobile phase. Dilute 5.0 ml of the solution to 25.0 ml with mobile phase. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 50 volumes of 0.2 per cent acetic acid in water, 25 volumes of acetonitrile and 25 volumes of methanol, filter and degas. flow rate. 1 ml per minute, spectrophotometer set at 248 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject the test solution and the reference solution. Calculate the content of C22H27FN3O6S.Ca. Storage. Store protected from light and moisture.
Identification
Tests
Dissolution (2.5.2). Apparatus. No 1 Medium. 900 ml of phosphate buffer pH 6.8, Speed and time. 50 rpm for 30 minutes. Withdraw a suitable volume of the medium and filter. Determine by liquid chromatography (2.4.14). Test solution. The filtrate obtained as given above. Reference solution. A 0.05 per cent w/v solution of rosuvastatin calcium RS in the mobile phase. Dilute 2 ml of the solution to 100 ml with Dissolution medium. Chromatographic system as described under Assay. Calculate the content of C22H27FN3O6S.Ca. D. Not less than 70 per cent of the stated amount of C22H27FN3O6S.Ca. Uniformity of content. Comply with the tests stated under Tablets. Determine by liquid chromatography (2.4.14), as described under Assay. Test solution. Disperse one tablet in 100 ml of the mobile phase. Dilute 5 ml of the solution to 10 ml with mobile phase and filter. Related substances. Determine by liquid chromatography (2.4.14). Test solution. Weigh and powder 20 tablets. Weigh accurately a quantity of powdered tablet containing 25 mg of Rosuvastatin, disperse in 50 ml of mobile phase and filter. Reference solution (a). A 0.05 per cent w/v solution of rosuvastatin calcium RS in the mobile phase. Reference solution (b). Dilute 1 ml of reference solution (a) to 100 ml with mobile phase. Chromatographic system as described under Assay. Inject reference solution (a). The test is not valid unless the tailing factor is not more than 2.0 and the column efficiency in not less than 2000 theoretical plates. Inject the test solution and reference solution (b). In the chromatogram obtained with the test solution, the area of any secondary peak is not more than 1.5 times the area of the peak in the chromatogram obtained with reference solution (b) (1.5 per cent) and the sum of areas of all the secondary peaks is
Rosuvastatin Tablets
Rosuvastatin Calcium Tablets
Rosuvastatin Tablets contain Rosuvastatin Calcium. Rosuvastatin Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of rosuvastatin calcium, C22H27FN3O6S.Ca.
1063
ROXITHROMYCIN
IP 2007
not more than 3.0 times the area of the peak in the chromatogram obtained with the reference solution (b) (3.0 per cent). Other tests. Comply with the tests stated under Tablets. Assay. Determine by liquid chromatography (2.4.14). Test solution. Weigh and powder 20 tablets. Weigh accurately a quantity of powdered tablet containing 25 mg of Rosuvastatin, disperse in 100.0 ml of mobile phase. Dilute 5.0 ml of the solution to 25.0 ml with mobile phase and filter. Reference solution. A 0.025 per cent w/v solution of rosuvastatin calcium RS in mobile phase. Dilute 5.0 ml of the solution to 25.0 ml with mobile phase. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5 m), column temperature 30, mobile phase: a mixture of 585 volumes of a buffer solution prepared by dissolving 1.54 g of ammonium acetate in 900 ml water, adjust pH to 4.0 with glacial acetic acid and dilute to 1000 ml with water, 360 volumes of acetonitrile and 50 volumes of tetrahydrofurone, flow rate. 1.5 ml per minute, spectrophotometer set at 248 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the column efficiency is not less than 2000 theoretical plates, the tailing factor is not more than 2.0 and the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject the test solution and the reference solution. Calculate the content of C22H27FN3O6S.Ca. Storage. Store protected from light and moisture. Labelling. The label states the strength in terms of the equivalent amount of Rosuvastatin.
Roxithromycin is (3R,4S,5S,6R,7R,9R,11S,12R,13S,14R)-4[(2,6-dideoxy-3-C-methyl-3-O-methyl--Lribohexopyranosyl)oxy]-14-ethyl-7,12,13-trihydroxy-10-[(E)[(2-methoxyethoxy)methoxy]imino]-3,5,7,9,11,13-hexamethyl6-[[3,4,6-trideoxy-3-(dimethylamino)--D-xylohexopyranosyl]oxy]oxacyclotetradecan-2-one; erythromycin 9-(E)-[O-[(2-methoxyethoxy)methyl]oxime]. Roxithromycin contains not less than 96.0 per cent and not more than 102.0 per cent of C41H76N2O15, calculated on the anhydrous basis. Description. A white, crystalline powder.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with roxithromycin RS. B. In the Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the peak in the chromatogram obtained with reference solution (a).
Tests
Appearance of solution. A 1.0 per cent w/v solution in methanol is clear (2.4.1) and colourless (2.4.1). Specific optical rotation (2.4.22). 93.0 to 96.0, determined in a 1.0 per cent w/v solution in acetone. Related substances. Determine by liquid chromatography (2.4.14). Solvent mixture. Mix 30 volumes of acetonitrile and 70 volumes of a 4.8 per cent w/v solution of ammonium dihydrogen phosphate and adjust the pH to 5.3 with dilute sodium hydroxide solution. Test solution. Dissolve 50.0 mg of the substance under examination in 25.0 ml of the solvent mixture.
Roxithromycin
N H3C HO H 3C H 3C O CH3 O OH H 3C O O O O CH3 OH CH3 HO O O OCH3 CH3 OH CH3 OCH3 CH3 N CH3 CH3
Reference solution (a). A 0.2 per cent w/v solution of roxithromycin RS in the solvent mixture. Reference solution (b). Dilute 1 ml of reference solution (a) to 100 ml with the solvent mixture. Reference solution (c). Dissolve 2 mg of erythromycin 9-(E)[O-[[(2-methoxyethoxy)methoxy]methyl]oxime] (impurity A) in 10 ml of reference solution (a). Further dilute 1 ml of this solution to 10 ml with reference solution (a). Reference solution (d). Dilute 1.0 ml of toluene to 100 ml with acetonitrile. Dilute 0.2 ml of this solution to 200 ml with the solvent mixture. Chromatographic system a stainless steel column 15 cm x 4.6 mm, packed with spherical end-capped octadecylsilyl silica gel for
C41H76N2O15
Mol. Wt. 837.0
1064
IP 2007
ROXITHROMYCIN TABLETS
chromatography (5 m), with a 10 nm pore size and a carbon loading of about 19 per cent, column temperature 15, mobile phase: A. a mixture of 26 volumes of acetonitrile and 74 volumes of a 5.97 per cent w/v solution of ammonium dihydrogen phosphate, adjusted to pH 4.3 with dilute sodium hydroxide solution, B. a mixture of 30 volumes of water and 70 volumes of acetonitrile, flow rate.1.1 ml per minute, a linear gradient programme using the conditions given below, spectrophotometer set at 205 nm, a 20 l loop injector. Time Mobile phase A Mobile phase B (in min.) (per cent v/v) (per cent v/v) 0 50 80 100 100 100 90 100 0 0 10 0
Sulphated ash (2.3.18). Not more than 0.1 per cent. Water (2.3.43). Not more than 3.0 per cent, determined on 0.2 g. Assay. Determine by liquid chromatography (2.4.14). Solvent mixture. Mix 30 volumes of acetonitrile and 70 volumes of a 4.8 per cent w/v solution of ammonium dihydrogen phosphate and adjust the pH to 5.3 with dilute sodium hydroxide solution. Test solution. Dissolve 50.0 mg of the substance under examination in 25.0 ml of the solvent mixture. Reference solution (a). A 0.2 per cent w/v solution of roxithromycin RS in the solvent mixture. Reference solution (b). Dissolve 2 mg of erythromycin 9-(E)[O-[[(2-methoxyethoxy)methoxy]methyl]oxime] (impurity A) in 10.0 ml of reference solution (a). Further dilute 1.0 ml of this solution to 10.0 ml with reference solution (a). Chromatographic system a stainless steel column 15 cm x 4.6 mm, packed with spherical end-capped octadecylsilyl silica gel for chromatography (5 m), with a 10 nm pore size and a carbon loading of about 19 per cent, column temperature 15, mobile phase: mix 307 volumes of acetonitrile and 693 volumes of a 4.91 per cent w/v solution of ammonium dihydrogen phosphate adjusted to pH 5.3 with dilute sodium hydroxide solution, flow rate.1.5 ml per minute, a linear gradient programme using the conditions given below, spectrophotometer set at 205 nm, a 20 l loop injector. Inject reference solution (b). The test is not valid unless the peak-to-valley ratio Hp / Hv is not less than 1.5 Inject alternately the test solution and reference solution (a) Calculate the content of C14H26N2O15. Storage. Store protected from light and moisture.
Inject reference solution (c). Relative retention time between roxithromycin and erythromycin 9-(E)-[O-[[(2-methoxyethoxy) methoxy]methyl] oxime] (impurity A) is about 1.15. The test is not valid unless the peak-to-valley ratio Hp / Hv is not more than 2.0, where Hp = height above the baseline of the peak due to impurity A and Hv = height above the baseline of the lowest point of the curve separating this peak from the peak due to roxithromycin. Inject the test solution and reference solutions (b) and (d). In the chromatogram obtained with the test solution the area of the impurity A peak obtained immediately after the peak obtained with roxithromycin is not more than the area of the principal peak in the chromatogram obtained with reference solution (b) (1.0 per cent). The area of any other individual impurity peak is not more than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent). The sum of the areas of all the peaks is not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (b) (3.0 per cent). Ignore any peak with an area 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.05 per cent). Ignore any peak due to toluene identified using reference solution (d). Heavy metals (2.3.13). Dissolve 2.0 g in a mixture of 15 volumes of water and 85 volumes of acetone and dilute to 20 ml with the same solvent mixture. 12 ml of this solution complies with the limit test for heavy metals, Method A (10 ppm). Use 1 ml of lead standard solution (10 ppm lead) prepared by diluting lead standard solution (100 ppm lead) with a mixture of 15 volumes of water and 85 volumes of acetone.
Roxithromycin Tablets
Roxithromycin Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of roxithromycin, C41H76N2O15.
Identification
1065
ROXITHROMYCIN TABLETS
IP 2007
Tests
Dissolution (2.5.2). Apprartus No. 1 Medium. 900 ml of phosphate buffer pH 6.0. Speed and time. 50 rpm and 45 minutes. Withdraw a suitable volume of the medium and filter. Determine by liquid chromatography (2.4.14) as given under the Assay using the following test solution. Test solution. The filtrate obtained as given above. Calculate the content of C41H76N2O15 in the medium. D. Not less than 70 per cent of the stated amount of C41H76N2O15. Other tests. Comply with the tests stated under Tablets. Assay. Determine by liquid chromatography (2.4.14). Solvent mixture. Mix 30 volumes of acetonitrile and 70 volumes of a 4.8 per cent w/v solution of ammonium dihydrogen phosphate and adjust the pH to 5.3 with dilute sodium hydroxide solution. Test solution. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 0.2 g of Roxithromycin, add 80 ml of the solvent mixture and mix. Dilute to 100.0 ml with the solvent mixture and filter. Reference solution (a). A 0.2 per cent w/v solution of roxithromycin RS in the solvent mixture. Reference solution (b). Dissolve 2 mg of erythromycin 9-(E)[O-[[(2-methoxyethoxy)methoxy]methyl]oxime] (impurity A) in 10 ml of reference solution (a). Further dilute 1 ml of this solution to 10 ml with reference solution (a).
Chromatographic system a stainless steel column 15 cm x 4.6 mm, packed with spherical end-capped octadecylsilyl silica gel (5 m), with a 10 nm pore size and a carbon loading of about 19 per cent, column temperature. 15, mobile phase: mix 307 volumes of acetonitrile and 693 volumes of a 4.91 per cent w/v solution of ammonium dihydrogen phosphate adjusted to pH 5.3 with dilute sodium hydroxide solution, flow rate. 1.5 ml per minute, spectrophotometer set at 205 nm, a 20 l loop injector. Inject reference solution (b). The relative retention time between roxithromycin and erythromycin -(E)-[O-[[(2methoxyethoxy)methoxy]methyl]oxime] (impurity A) is about 1.15. The test is not valid unless the peak-to-valley ratio Hp / Hv is not more than 1.5, where Hp = height above the baseline of the peak due to impurity A and Hv = height above the baseline of the lowest point of the curve separating this peak from the peak due to roxithromycin. Inject reference solution (a). The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject alternately the test solution and reference solution (a). Calculate the content of C41H76N2O15 in the tablets. Storage. Store protected from light and moisture. Labelling. If the tablets are dispersible, the label states that the tablets should be dispersed in water immediately before use.
1066
MONOGRAPHS
S
Saccharin Saccharin Sodium Salbutamol Salbutamol Inhalation Salbutamol Sulphate Salbutamol Injection Salbutamol Syrup Salbutamol Tablets Salicylic Acid Salmeterol Xinafoate Salmetrol and Fluticasone Propionate Inhalation Salmetrol and Fluticasone Propionate powder for Inhalation Saquinavir Saquinavir Mesylate Saquinavir Mesylate Tablets Sechidazole Sechidazole Tablets Colloidal Silicon Dioxide Silver Nitrate Sodium Acetate Sodium Alginate Sodium Aminosalicylate Sodium Aminosalicylate Tablets Sodium Ascorbate Sodium Aurothiomalate Sodium Aurothiomalate Injection Sodium Benzoate Sodium Bicarbonate Sodium Bicarbonate Injection Sodium Carbonate
1055
.... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... ....
MONOGRAPHS
Sodium Chloride ................................................................................................................. Sodium Chloride And Dextrose Injection ............................................................................. Sodium Chloride And Fructose Injection ............................................................................ Compound Sodium Chloride And Dextrose Injection .......................................................... Sodium Chloride Hypertonic Injection ................................................................................ Sodium Chloride Injection .................................................................................................. Compound Sodium Chloride Injection ............................................................................... Compound Sodium Chloride Solution ................................................................................ Sodium Chloride Irrigation Solution ...................................................................................... Sodium Citrate .................................................................................................................. Sodium Cromoglycate ....................................................................................................... Sodium Cromoglycate Powder for Inhalation .................................................................... Sodium Diatrizoate ............................................................................................................. Sodium Diatrizoate Injection .............................................................................................. Sodium Dihydrogen Phosphate Dihydrate ........................................................................... Sodium Fluoride ................................................................................................................. Sodium Formaldehyde Sulphoxylate .................................................................................... Sodium Fusidate ............................................................................................................... Sodium Fusidate Capsules ................................................................................................... Sodium Hydroxide .............................................................................................................. Sodium Lactate Injection .................................................................................................... Compound Sodium Lactate And Dextrose Injection ............................................................ Half Strength Compound Sodium Lactate And Dextrose Injection .................................... Modified Compound Sodium Lactate And Dextrose Injection .............................................. Compound Sodium Lactate Injection .................................................................................. Compound Sodium Lactate Solution For Irrigation ............................................................ Sodium Lauryl Sulphate ..................................................................................................... Sodium Metabisulphite ........................................................................................................ Sodium Methylparaben ...................................................................................................... Sodium Phosphate ............................................................................................................... Sodium Propylparaben ..................................................................................................... Sodium Salicylate ............................................................................................................... Sodium Starch Glycollate ....................................................................................................
1056
MONOGRAPHS
Sodium Stibogluconate Sodium Stibogluconate Injection Sodium Thiosulphate Sodium Thiosulphate Injection Sodium Valproate Sodium Valproate Oral Solution Sodium Valproate Tablets Sorbic Acid Sorbitol Sorbitol Solution (70 Per Cent) (Crystallising) Sorbitol Solution (70 Per Cent) (Non-Crystallising) Spironolactone Spironolactone Tablets Stavudine Stavudine Capsules Stavudine Oral Solution Stavudine And Lamivudine Tablets Stearic Acid Stearyl Alcohol Stilboestrol Stilboestrol Tablets Prepared Storax Streptokinase Streptokinase Injection Streptomycin Sulphate Streptomycin Injection Streptomycin Tablets Succinylcholine Chloride Succinylcholine Injection Sucrose Sulphacetamide Sodium Sulphacetamide Eye Drops Sulphadiazine
1057
.... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... ....
MONOGRAPHS
Sulphadiazine Tablets Sulphadimethoxine Sulphadimethoxine Tablets Sulphadimidine Sulphadimidine Sodium Sulphadimidine Injection Sulphadimidine Tablets Sulphadoxine Sulphafurazole Sulphafurazole Tablets Sulphalene Sulphamethizole Sulphamethoxazole Sulphaphenazole Sulphaphenazole Tablets Sulphobromophthalein Sodium Sulphobromophthalein Sodium Injection
.... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... ....
1058
IP 2007
SACCHARIN
Saccharin
O O S NH O
C7H5NO3S Mol. Wt. 183.2
Combine the lower layers, dry over anhydrous sodium sulphate and filter. Wah the filter and the sodium sulphate with 10 ml of dichloromethane. Combine the solution and the washings and evaporate almost to dryness in a water-bath at a temperature not exceeding 40. With a small quantity of dichloromethane transfer quantitatively the residue into a suitable 10-ml tube, evaporate to dryness in a current of nitrogen and dissolve the residue in 1.0 ml of the internal standard solution. Blank solution. Evaporate 200 ml of dichloromethane to dryness in a water-bath at a temperature not exceeding 40. Dissolve the residue in 1 ml of dichloromethane. Reference solution. Dissolve 20 mg of o-toluenesulphonamide and 20 mg of p-toluene sulphonamide in dichloromethane and dilute to 100 ml with the same solvent. Dilute 5 ml of the solution to 50 ml with dichloromethane. Evaporate 5 ml of the final solution to dryness in a current of nitrogen. Dissolve the residue in 1 ml of the internal standard solution. Chromatographic system a fused silica column 10 m x 0.53 mm packed with polymethylphenylsiloxane (film thickness 2 m), temperature: column.180, inlet port and detector. 250, flame ionization detector, flow rate. 10 ml per minute of nitrogen (carrier gas), split ratio. 1:2. Inject 1 l of each solution. The order of elution is o-toluenesulphonamide, p-toluenesulphonamide, caffeine. The test is not valid unless the resolution between the peaks due to o-toluenesulphonamide and p-toluenesulphonamide in the chromatogram obtained with the reference solution is not less than 1.5 and the chromatogram obtained with the blank solution does not show any peak with the same retention times as the internal standard, o-toluenesulphonamide and p-toluenesulphonamide. In the chromatogram obtained with the test solution the ratio of the area of the peak due to o-toluenesulphonamide to that of the internal standard is not greater than the corresponding ratio in the chromatogram obtained with the rference solution (10 ppm). In the chromatogram obtained with the test solution the ratio of the area of the peak due to p-toluenesulphonamide to that of the internal standard is not greater than the corresponding ratio in the chromatogram obtained with the reference solution (10 ppm). Arsenic (2.3.10). Mix 5.0 g with 3.0 g of anhydrous sodium carbonate, add 10 ml of bromine solution and mix thoroughly. Evaporate to dryness on a water-bath, gently ignite and add to the cooled residue a mixture of 16 ml of brominated
Saccharin is 1,2-benzisothiazol-3(2H)-one 1,1-dioxide. Saccharin contains not less than 98.0 per cent and not more than 101.0 per cent of C7H5NO3S, calculated on the dried basis. Description. White crystals or a white, crystalline powder; odourless or with a faint, aromatic odour.
Identification
Test A may be omitted if tests B, C and D are carried out. Tests B, C and D may be omitted if test A is carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with saccharin RS. B. Mix 20 mg with 20 mg of resorcinol, add 0.5 ml of sulphuric acid and heat over a small flame until a dark green colour is produced; allow to cool and add 10 ml of water and an excess of 2 M sodium hydroxide; a fluorescent green liquid is produced. C. Dissolve 0.1 g in 5 ml of a 10 per cent w/v solution of sodium hydroxide, evaporate to dryness and gently fuse the residue over a small flame until ammonia is no longer evolved. Allow to cool, dissolve in 20 ml of water, make the solution just acidic to litmus paper, filter and add 0.05 ml of ferric chloride solution; a violet colour is produced. D. A saturated solution is acidic to litmus paper.
Tests
Appearance of solution. Dissolve 5.0 g in 20 ml of a 20 per cent w/v solution of sodium acetate and dilute to 25 ml with the same solution; the solution is clear (2.4.1), and colourless (2.4.1). Related substances. Determine by gas chromatography (2.4.13). Internal standard solution. Dissolve 25 mg of caffeine in dichloromethane and dilute to 100 ml with the same solvent. Test solution. Suspend 10.0 g of the substance under examination in 20 ml of water and dissolve using about 5 ml of strong sodium hydroxide solution. If necessary, adjust the pH of the solution to 7.8 with 1 M sodium hydroxide or 1 M hydrochloric acid and dilute to 50 ml with water. Shake the solution with 4 quantities, each of 50 ml, of dichloromethane.
1059
SACCHARIN SODIUM
IP 2007
hydrochloric acid AsT and 5 ml of bromine solution. Add 40 ml of water and boil gently, adding sufficient bromine solution from time to time to maintain a slight excess. Filter and remove the excess of bromine with a sufficient quantity of stannous chloride solution AsT. The resulting solution complies with the limit test for arsenic (2 ppm). Heavy metals (2.3.13). 1.2 g complies with the limit test for heavy metals, Method D (20 ppm). Use lead standard solution (2 ppm Pb). Readily carbonisable substances. Dissolve 0.2 g in 5 ml of sulphuric acid (96 per cent w/w) and maintain at about 50 for 10 minutes. The solution is not more intensely coloured than reference solution BYS6 (2.4.1). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 1.0 per cent, determined on 1.0 g by drying in an oven at 105 for 4 hours. Assay. Weigh accurately about 0.5 g, dissolve in 75 ml of hot water, cool quickly and titrate with 0.1 M sodium hydroxide using phenolphthalein solution as indicator. 1 ml of 0.1 M sodium hydroxide is equivalent to 0.01832 g of C7H5NO3S. Storage. Store protected from moisture.
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with saccharin sodium RS. B. Dissolve 0.1 g in 5 ml of a 10 per cent w/v solution of sodium hydroxide, evaporate to dryness and gently fuse the residue over a small flame until ammonia is no longer evolved. Allow to cool, dissolve in 20 ml of water, make the solution just acidic to litmus paper, filter and add 0.05 ml of ferric chloride solution; a violet colour is produced. C. Mix 20 mg with 20 mg of resorcinol, add 0.5 ml of sulphuric acid and heat over a small flame until a dark green colour is produced; allow to cool and add 10 ml of water and an excess of 2 M sodium hydroxide; a fluorescent green liquid is produced. D. To 5 ml of a 10 per cent w/v solution add 3 ml of 2 M hydrochloric acid; a white precipitate is produced. The precipitate, after washing with water and drying at 105 melts at 226 to 230 (2.4.21). E. 0.5 ml of a 10 per cent w/v solution gives reaction B of sodium salts (2.3.1).
Tests
Appearance of solution. Solution A is clear (2.4.1), and colourless (2.4.1). Acidity or alkalinity. Dissolve 5.0 g in sufficient carbon dioxide-free water to produce 50 ml (solution A). To 10 ml of solution A add 10 ml of 0.005 M sulphuric acid, heat to boiling, cool and add 0.1 ml of phenolphthalein solution. Not less than 4.5 ml and not more than 5.5 ml of 0.01 M sodium hydroxide is required to change the colour of the solution to pink. Related substances. Determine by gas chromatography (2.4.13). Internal standard solution. Dissolve 25 mg of caffeine in dichloromethane and dilute to 100 ml with the same solvent.
Saccharin Sodium
Soluble Saccharin
O O S N Na O
C7H4NNaO3S Mol. Wt. 205.2
Saccharin Sodium is the sodium salt of 1,2-benzisothiazol3(2H)-3-one 1,1-dioxide. Saccharin Sodium contains not less than 99.0 per cent and not more than 101.0 per cent of C7H4NNaO3S, calculated on the anhydrous basis. Description. A white, crystalline powder or colourless crystals; efflorescent in dry air.
Identification
Test A may be omitted if tests B, C, D and E are carried out. Tests B, C and D may be omitted if tests A and E are carried out.
Test solution. Suspend 10.0 g of the substance under examination in 50 ml of water. If necessary, adjust the pH of the solution to 7.8 with 1 M sodium hydroxide or 1 M hydrochloric acid and dilute to 50 ml with water. Shake the solution with 4 quantities, each of 50 ml, of dichloromethane. Combine the lower layers, dry over anhydrous sodium sulphate and filter. Wah the filter and the sodium sulphate with 10 ml of dichloromethane. Combine the solution and the washings and evaporate almost to dryness in a water-bath at a temperature not exceeding 40. With a small quantity of dichloromethane transfer quantitatively the residue into a suitable 10-ml tube, evaporate to dryness in a current of nitrogen and dissolve the residue in 1.0 ml of the internal standard solution.
1060
IP 2007
SALBUTAMOL
Blank solution. Evaporate 200 ml of dichloromethane to dryness in a water-bath at a temperature not exceeding 40. Dissolve the residue in 1 ml of dichloromethane. Reference solution. Dissolve 20 mg of o-toluenesulphonamide and 20 mg of p-toluene sulphonamide in dichloromethane and dilute to 100 ml with the same solvent. Dilute 5 ml of this solution to 50 ml with dichloromethane. Evaporate 5 ml of the final solution to dryness in a current of nitrogen. Dissolve the residue in 1 ml of the internal standard solution. Chromatographic system a fused silica column 10 m x 0.53 mm packed with polymethylphenylsiloxane (film thickness 2 m), temperature: column.180, inlet port and detector. 250 flame ionization detector, flow rate. 10 ml per minute of nitrogen (carrier gas), split ratio. 1:2. Inject 1 l of each solution. The order of elution is o-toluenesulphonamide, p-toluenesulphonamide, caffeine. The test is not valid unless the resolution between the peaks due to o-toluenesulphonamide and p-toluenesulphonamide in the chromatogram obtained with the reference solution is not less than 1.5 and the chromatogram obtained with the blank solution does not show any peak with the same retention times as the internal standard, o-toluenesulphonamide and p-toluenesulphonamide. In the chromatogram obtained with the test solution the ratio of the area of the peak due to o-toluenesulphonamide to that of the internal standard is not greater than the corresponding ratio in the chromatogram obtained with the reference solution (10 ppm). In the chromatogram obtained with the test solution the ratio of the area of the peak due to p-toluenesulphonamide to that of the internal standard is not greater than the corresponding ratio in the chromatogram obtained with the rference solution (10 ppm). Arsenic (2.3.10). Mix 5.0 g with 3 g of anhydrous sodium carbonate, add 10 ml of bromine solution and mix thoroughly. Evaporate to dryness on a water-bath, gently ignite and add to the cooled residue a mixture of 16 ml of brominated hydrochloric acid AsT and 5 ml of bromine solution. Add 40 ml of water and boil gently, adding sufficient bromine solution from time to time to maintain a slight excess. Filter and remove the excess of bromine with a sufficient quantity of stannous chloride solution AsT. The resulting solution complies with the limit test for arsenic (2 ppm). Readily carbonisable substances. Dissolve 0.2 g in 5 ml of sulphuric acid (96 per cent w/w) and maintain at about 50
for 10 minutes. The solution is not more intensely coloured than reference solution BS6 (2.4.1). Heavy metals (2.3.13). 12 ml of solution A complies with the limit test for heavy metals, Method D (20 ppm). Use lead standard solution (2 ppm Pb). Water (2.3.43). Not more than 15.0 per cent, determined on 0.2 g. Assay. Weigh accurately about 0.15 g, dissolve in 50 ml of anhydrous glacial acetic acid, with slight heating if necessary. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.02052 g of C7H4NNaO3S. Storage. Store protected from moisture.
Salbutamol
Albuterol
OH H N H3 C OH
C13H21NO3 Mol. Wt. 239.3
CH3 CH3
HO
Salbutamol is (RS)-1-(4-hydroxy-3-hydroxymethylphenyl)-2(tert-butylamino)ethanol. Salbutamol contains not less than 98.0 per cent and not more than 101.0 per cent of C13H21NO3, calculated on the dried basis. Description. A white or almost white, crystalline powder.
Identification
Test A may be omitted if tests B, C and D are carried out. Tests B, C and D may be omitted if test A is carried out. A. Determine by infrared absorption spectrophotometry (2.4.6), Compare the spectrum with that obtained with salbutamol RS or with the reference spectrum of salbutamol. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.008 per cent w/v solution in 0.1 M hydrochloric acid shows an absorption maximum only at about 276 nm; absorbance at about 276 nm, about 0.53 to 0.60. C. In the test for Related substances, the principal spot in the chromatogram obtained with reference solution (a) corresponds to that in the chromatogram obtained with reference solution (b).
1061
SALBUTAMOL INHALATION
IP 2007
D. Dissolve 10 mg in 50 ml of a 2 per cent w/v solution of borax, add 1 ml of a 3 per cent w/v solution of 4-aminophenazone, 10 ml of a 2 per cent w/v solution of potassium ferricyanide and 10 ml of chloroform, shake and allow to separate; an orange-red colour is produced in the chloroform layer.
Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.2 g, dissolve in 30 ml of anhydrous glacial acetic acid. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.02393 g of C13H21NO3. Storage. Store protected from light.
Tests
Appearance of solution. A 2.0 per cent w/v solution in methanol is clear (2.4.1), and not more intensely coloured than reference solution BYS5 (2.4.1). Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 50 volumes of ethyl acetate, 30 volumes of 2-propanol, 16 volumes of water and 4 volumes of strong ammonia solution. Test solution. Dissolve 0.2 g of the substance under examination in 10 ml of methanol. Reference solution (a). A 0.01 per cent w/v solution of the substance under examination in methanol. Reference solution (b). A 0.01 per cent w/v solution of salbutamol RS in methanol. Apply to the plate 5 l of each solution. After development, dry the plate in air until the odour of the solvent is no longer detectable, place it for a few minutes in an atmosphere saturated with diethylamine and spray with diazotised sulphanilic acid solution. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (a). Boron. To 50 mg add 5 ml of a solution containing 1.3 per cent w/v of anhydrous sodium carbonate and 1.7 per cent w/v of potassium carbonate, evaporate to dryness on a water-bath and dry at 120. Ignite the residue rapidly until the organic matter has been destroyed, allow to cool, add 0.5 ml of water and 3.0 ml of a freshly prepared 0.125 per cent w/v solution of curcumin in glacial acetic acid. Evaporate to dryness and allow to cool. Add 3 ml of a mixture prepared by adding 5 ml of sulphuric acid, slowly and with stirring, to 5 ml of glacial acetic acid. Mix and allow to stand for 30 minutes. Add sufficient ethanol (95 per cent) to produce 100.0 ml, filter and measure the absorbance of the filtrate at 555 nm (2.4.7). Prepare a reference solution in the following manner. Dissolve 0.572 g of boric acid in 1000.0 ml of water. Dilute 1.0 ml to 100.0 ml with water. To 2.5 ml of this solution add 5 ml of a solution containing 1.3 per cent w/v of anhydrous sodium carbonate and 1.7 per cent w/v of potassium carbonate and treat this mixture in the same manner as described above beginning at the words Evaporate to dryness. The absorbance of the solution prepared from the substance under examination is not more than that of the reference solution (50 ppm).
Salbutamol Inhalation
Salbutamol Inhalation Aerosol; Albuterol Inhalation Aerosol
Salbutamol Inhalation is a suspension of microfine Salbutamol or Salbutamol Sulphate in a suitable liquid in a suitable pressurised container. It may contain suitable pharmaceutical aids such as surfactants, stabilising agents etc. Salbutamol Inhalation delivers not less than 80.0 per cent and not more than 120.0 per cent of the stated amount of salbutamol, C13H21NO3, per inhalation, by actuation of the valve.
Identification
A. Discharge the container a sufficient number of times into a mortar to obtain about 2 mg of salbutamol, grind the residue thoroughly with 0.1 g of potassium bromide, add a further 0.2 g of potassium bromide and mix thoroughly. On the resultant dispersion determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with salbutamol RS or with the reference spectrum of salbutamol. B. In the test for Related substances, the principal spot in the chromatogram obtained with reference solution (a) corresponds to that in the chromatogram obtained with reference solution (b).
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 50 volumes of ethyl acetate, 30 volumes of 2-propanol, 16 volumes of water and 4 volumes of strong ammonia solution. Test solution. Discharge the inhaler a sufficient number of times into a small, dry beaker to obtain 10 mg of Salbutamol and dissolve the residue in 0.5 ml of methanol.
1062
IP 2007
SALBUTAMOL SULPHATE
Reference solution (a). Dilute 1.0 volume of test solution (a) to 200 volumes with methanol. Reference solution (b). A 0.010 per cent w/v solution of salbutamol RS in methanol. Apply to the plate 5 l of each solution. After development, dry the plate in air until the odour of the solvent is no longer detectable, place it for a few minutes in an atmosphere saturated with diethylamine and spray with diazotised sulphanilic acid solution. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (a). Ignore any spot with an Rf value higher than 0.85. Other tests. Complies with the tests stated under Inhalation Preparations (Pressurised metered-dose Preparations). Follow the procedure described under Assay wherever the amount of active substance is to be determined in any test. Assay. Carry out the test for Content of active ingredient delivered per actuation stated under Inhalation Preparations (Pressurised metered-dose Preparations). Use 30 ml of ethanol for preparations containing salbutamol and 30 ml of a mixture of equal volumes of ethanol and water for preparations containing salbutamol sulphate and dilute the final solution and washings to 200.0 ml with ethanol. Dilute a suitable volume of this solution with ethanol to produce a solution containing 10 g of salbutamol per ml. To 20 ml of the solution in a separating funnel add 180 ml of water and in the following order, 4 ml of N,N-dimethyl-4-phenylenediamine sulphate solution and 4 ml of a freshly prepared 8 per cent w/ v solution of potassium ferricyanide. Mix, allow to stand for 15 minutes in subdued light and extract with two quantities, each of 10 ml, of chloroform. Filter the extracts through a plug of cotton wool, dilute to 25 ml with chloroform and measure the absorbance of the resulting solution at 605 nm (2.4.7). Calculate the content of C13H21NO3 in the solution from the absorbance obtained by repeating the operation using a suitable quantity of a 0.001 per cent w/v solution of salbutamol RS in ethanol. Calculate the amount of C13H21NO3 delivered per actuation of the valve. Determine the content of active ingredient a second and third time by repeating the procedure on the middle ten and on the last ten successive combined actuations of the valve. For each of the three determinations the average content of C13H21NO3 delivered per actuation of the valve meets the requirements. Storage. Store protected from light and moisture at a temperature not exceeding 30. Labelling. The label states whether the preparation contains Salbutamol or Salbutamol Sulphate.
When the active ingredient is Salbutamol Sulphate, the quantity is stated in terms of the equivalent amount of salbutamol.
Salbutamol Sulphate
Albuterol Sulphate
(C13H21NO3)2,H2SO4 Mol. Wt. 576.7 Salbutamol Sulphate is (RS)-1-(4-hydroxy-3-hydroxymethylphenyl)-2-(tert-butylamino)ethanol sulphate. Salbutamol Sulphate contains not less than 98.0 per cent and not more than 101.0 per cent of (C13H21NO3)2, H2SO4, calculated on the dried basis. Description. A white or almost white, crystalline powder.
Identification
Test A may be omitted if tests B, C and D are carried out. Tests B and C may be omitted if tests A and D are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6), Compare the spectrum with that obtained with salbutamol sulphate RS or with the reference spectrum of salbutamol sulphate. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.008 per cent w/v solution in 0.1 M hydrochloric acid shows an absorption maximum at about 276 nm; absorbance at about 276 nm, 0.44 to 0.51. C. In the test for Related substances, the principal spot in the chromatogram obtained with reference solution (a) corresponds to that in the chromatogram obtained with reference solution (b). D. Dissolve 10 mg in 50 ml of a 2 per cent w/v solution of borax, add 1 ml of a 3 per cent w/v solution of 4-aminophenazone, 10 ml of a 2 per cent w/v solution of potassium ferricyanide and 10 ml of chloroform, shake and allow to separate; an orange-red colour is produced in the chloroform layer. E. Gives reaction A of sulphates (2.3.1).
Tests
Appearance of solution. A 1.0 per cent w/v solution in carbon dioxide-free water is clear (2.4.1), and not more intensely coloured than reference solution BYS6 (2.4.1). Acidity or alkalinity. To 10 ml of a 1.0 per cent w/v solution in carbon dioxide-free water add 0.15 ml of methyl red solution and 0.2 ml of 0.01 M sodium hydroxide. The solution is yellow and not more than 0.4 ml of 0.01 M hydrochloric acid is required to change the colour of the solution to red.
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SALBUTAMOL INJECTION
IP 2007
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 50 volumes of ethyl acetate, 30 volumes of 2-propanol, 16 volumes of water and 4 volumes of strong ammonia solution. Test solution. Dissolve 0.2 g of the substance under examination in 10 ml of water. Reference solution (a). A 0.01 per cent w/v solution of the substance under examination in water. Reference solution (b). A 0.01 per cent w/v of salbutamol sulphate RS in water. Apply to the plate 5 l of each solution. After development, dry the plate in air until the odour of the solvent is no longer detectable, place it for a few minutes in an atmosphere saturated with diethylamine and spray with diazotised sulphanilic acid solution. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (a). Boron. To 50 mg add 5 ml of a solution containing 1.3 per cent w/v of anhydrous sodium carbonate and 1.7 per cent w/v of potassium carbonate, evaporate to dryness on a water-bath and dry at 120. Ignite the residue rapidly until the organic matter has been destroyed, allow to cool, add 0.5 ml of water and 3.0 ml of a freshly prepared 0.125 per cent w/v solution of curcumin in glacial acetic acid. Evaporate to dryness and allow to cool. Add 3 ml of a mixture prepared by adding 5 ml of sulphuric acid, slowly and with stirring, to 5 ml of glacial acetic acid. Mix and allow to stand for 30 minutes. Add sufficient ethanol (95 per cent) to produce 100.0 ml, filter and measure the absorbance of the filtrate at 555 nm (2.4.7). Prepare a reference solution in the following manner. Dissolve 0.572 g of boric acid in 1000.0 ml of water. Dilute 1.0 ml to 100.0 ml with water. To 2.5 ml of this solution add 5 ml of a solution containing 1.3 per cent w/v of anhydrous sodium carbonate and 1.7 per cent w/v of potassium carbonate and treat this mixture in the same manner as described above beginning at the words Evaporate to dryness......The absorbance of the solution prepared from the substance under examination is not more than that of the reference solution (50 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.4 g, dissolve in 5 ml of anhydrous formic acid, add 35 ml of anhydrous glacial acetic acid. Titrate with 0.1 M perchloric acid, determining the endpoint potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.05767 g of (C13H21NO3)2, H2SO4. Storage. Store protected from light.
Salbutamol Injection
Albuterol Sulphate Injection; Salbutamol Sulphate Injection
Salbutamol Injection is a sterile solution of Salbutamol Sulphate in Water for Injections containing suitable stabilising agents. Salbutamol Injection contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of salbutamol, C13H21NO3.
Identification
A. Dilute a volume with sufficient 0.1 M hydrochloric acid to produce a solution containing 0.008 per cent w/v of salbutamol. When examined in the range 230nm to 360 nm (2.4.7), the resulting solution shows an absorption maximum only at about 276 nm. B. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 50 volumes of ethyl acetate, 30 volumes of 2-propanol, 16 volumes of water and 4 volumes of strong ammonia solution. Test solution. Evaporate a suitable volume of the injection to dryness using a rotary evaporator, wash the residue with four quantities, each of 5 ml, of ethanol, filter, evaporate the filtrate to dryness and dissolve the residue in sufficient water to produce a solution containing the equivalent of 0.1 per cent w/v of salbutamol. Reference solution. A 0.12 per cent w/v solution of salbutamol sulphate RS in water. Apply to the plate 2 l of each solution. After development, dry the plate in air until the odour of solvent is no longer detectable, place for a few minutes in an atmosphere saturated with diethylamine and spray with diazotised nitroaniline solution. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution. C. Dilute a volume containing 0.5 mg of salbutamol to 50 ml with water, add 1 ml of dilute ammonia solution, 1 ml of a 3 per cent w/v solution of 4-aminophenazone, 10 ml of a 2 per cent w/v solution of potassium ferricyanide and 10 ml of chloroform. Shake and allow to separate; an orange-red colour is produced in the chloroform layer. D. A volume containing 1 mg of salbutamol gives the reactions of sulphates (2.3.1).
Tests
pH (2.4.24). 3.4 to 5.0.
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IP 2007
SALBUTAMOL TABLETS
Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. Dilute a volume, accurately measured, containing about 0.15 mg of salbutamol with sufficient water to produce 80 ml, add 4 ml of a 5 per cent w/v solution of sodium bicarbonate, 4 ml of N,N-dimethyl-4-phenylenediamine sulphate solution and 4 ml of a freshly prepared 8 per cent w/v solution of potassium ferricyanide. Mix, allow to stand for 15 minutes, protected from light. Extract with two quantities, each of 10 ml, of chloroform. Filter the extracts through a plug of cotton wool and dilute to 25.0 ml with chloroform. Measure the absorbance of the resulting solution at 605 nm (2.4.7). Calculate the content of C13H21NO3 from the absorbance obtained by repeating the operation using 10.0 ml of a 0.0018 per cent w/v solution of salbutamol sulphate. Storage. Store protected from light, in single dose containers in which the air has been displaced by nitrogen or other suitable inert gas. Labelling. The label states the strength in terms of the equivalent amount of salbutamol in a suitable dose-volume.
volumetric flask. Discard the ether extracts and dilute the aqueous solution with sufficient water to produce 250.0 ml. To 10.0 ml of this solution add sufficient water to produce 80 ml and add 4 ml of a 5 per cent w/v solution of sodium bicarbonate, 4 ml of N,N-dimethyl-4-phenylenediamine sulphate solution and 4 ml of a freshly prepared 8 per cent w/v solution of potassium ferricyanide. Mix, allow to stand for 15 minutes, protected from light. Extract with two quantities, each of 10 ml, of chloroform. Filter the extracts through a plug of cotton wool and dilute to 25.0 ml with chloroform. Measure the absorbance of the resulting solution at 605 nm (2.4.7). Calculate the content of C13H21NO3 from the absorbance obtained by repeating the operation using 10.0 ml of a 0.0018 per cent w/v solution of salbutamol sulphate. Storage. Store protected from light. Labelling. The label states the strength in terms of the equivalent amount of salbutamol in a suitable dose-volume.
Salbutamol Tablets
Albuterol Sulphate Tablets; Salbutamol Sulphate Tablets
Salbutamol Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of salbutamol, C13H21NO3.
Salbutamol Syrup
Albuterol Sulphate Syrup; Salbutamol Sulphate Syrup
Salbutamol Syrup contains Salbutamol Sulphate equivalent to not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of salbutamol, C13H21NO3.
Identification
A. Carry out the method described under Related substances applying separately to the plate 2 l of each of the following solutions. For the test solution shake a quantity of the powdered tablets containing 10 mg of salbutamol with 10 ml of methanol (80 per cent) and filter. The reference solution contains 0.12 per cent w/v of salbutamol sulphate RS. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution. B. Shake a quantity of the powdered tablets containing 8 mg of salbutamol with 50 ml of a 2 per cent w/v solution of borax, add 1 ml of a 3 per cent w/v solution of 4-aminophenazone, 10 ml of a 2 per cent w/v solution of potassium ferricyanide and 10 ml of chloroform. Shake and allow to separate; an orange-red colour is produced in the chloroform layer. C. Shake a quantity of the powdered tablets containing 4 mg of salbutamol with 10 ml of water and filter; the filtrate gives the reactions of sulphates (2.3.1).
Identification
A. To 5 ml add 50 ml of a 2 per cent w/v solution of borax, 1 ml of a 3 per cent w/v solution of 4-aminophenazone, 10 ml of a 2 per cent w/v solution of potassium ferricyanide and 10 ml of chloroform. Shake and allow to separate; an orange-red colour is produced in the chloroform layer. B. To 5 ml add sufficient 1 M sodium hydroxide to make the solution alkaline, add 1 ml of alkaline borate buffer pH 9.2 and 1 ml of a 0.04 per cent w/v solution of 2,6-dichloroquinone chlorimide in ethanol (95 per cent); a blue colour develops.
Tests
pH (2.4.24). 3.4 to 4.5. Other tests. Complies with the tests stated under Oral Liquids. Assay. To an accurately measured volume containing about 4 mg of salbutamol add 25 ml of 0.05 M sulphuric acid and extract with two quantities, each of 50 ml, of ether. Collect the aqueous layers into a 250-ml volumetric flask and combine the ether extracts. Wash the combined ether extracts with 50 ml of water and add the aqueous layer to the solution in the 250-ml
Tests
1065
SALICYLIC ACID
IP 2007
Mobile phase. A mixture of 50 volumes of ethyl acetate, 30 volumes of 2-propanol, 16 volumes of water and 4 volumes of strong ammonia solution. Test solution. Shake a quantity of the powdered tablets containing 10 mg of salbutamol with 1 ml of water for 15 minutes, centrifuge and use the supernatant liquid. Reference solution. A 0.006 per cent w/v solution of salbutamol sulphate RS in water. Apply to the plate 20 l of each solution. After development, dry the plate in air until the odour of the solvent is no longer detectable, place it for a few minutes in an atmosphere saturated with diethylamine and spray with diazotised sulphanilic acid solution. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Ignore any pink spot near the line of application. Uniformity of content. Comply with the test stated under Tablets. Determine by liquid chromatography (2.4.14). Test solution. Add 50 ml of water to one tablet, shake for 1 hour, add sufficient water to produce 100.0 ml, mix and centrifuge. Dilute further with water, if necessary, to produce a solution containing 0.002 per cent w/v of salbutamol. Reference solution (a). A 0.0024 per cent w/v of salbutamol sulphate RS in water. Reference solution (b). A 0.048 per cent w/v of 2-tertbutylamino-1-(4-hydroxy-3-methylphenyl) ethanol sulphate RS and 0.048 per cent w/v of salbutamol sulphate RS in methanol (10 per cent). Chromatographic system a stainless steel column 20 cm x 5 mm, packed with spherical particles of silica, 5 m in diameter, the surface of which has been modified with chemically-bonded nitrile groups (such as Spherisorb CN), mobile phase: a mixture of 65 volumes of water, 30 volumes of 0.05 M ammonium acetate and 5 volumes of 2-propanol, the pH of the mixture being adjusted to 4.5 with glacial acetic acid, flow rate. 2 ml per minute, spectrophotometer set at 276 nm, a 20 l loop injector. The test is not valid unless resolution between two principal peaks in the chromatogram obtained with reference solution (b) is at least 1.5. Calculate the content of C13H21NO3 in the tablet. Other tests. Comply with the tests stated under Tablets. Assay. Determine by liquid chromatography (2.4.14).
Test solution. Shake 10 tablets or a sufficient number of tablets containing 5.0 mg of salbutamol with about 60 ml of water for 1 hour, add sufficient water to produce 250.0 ml, mix and centrifuge 10 ml of the mixture and use the supernatant liquid. Reference solution (a). A 0.0024 per cent w/v of salbutamol sulphate RS in water.. Reference solution (b). A solution containing 0.0024 per cent w/v of 2-tert-butylamino-1-(4-hydroxy-3-methylphenyl) ethanol sulphate RS and 0.0024 per cent w/v of salbutamol sulphate RS in methanol (10 per cent). Follow the chromatographic procedure described under Uniformity of content. The test is not valid unless the resolution between the two principal peaks in the chromatogram obtained with reference solution (b) is at least 1.5. Calculate the content of C13H21NO3 in the tablets. Storage. Store protected from light. Labelling. The label states the strength in terms of the equivalent amount of salbutamol.
Salicylic Acid
COOH OH
C7H6O3 Salicylic Acid is 2-hydroxybenzoic acid.
Mol. Wt. 138.1
Salicylic Acid contains not less than 99.0 per cent and not more than 100.5 per cent of C7H6O3, calculated on the dried basis. Description. White or colourless, acicular crystals or a white, crystalline powder.
Identification
Test A may be omitted if tests B and C are carried out. Test B may be omitted if tests A and C are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with salicylic acid RS or with the reference spectrum of salicylic acid. B. Dissolve about 30 mg in 5 ml of 0.05 M sodium hydroxide, neutralise if necessary and dilute to 20 ml with water. 1 ml of the solution gives reaction A of salicylates (2.3.1). C. Melting point. 158 to 161 (2.4.21).
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IP 2007
SALMETEROL XINAFOATE
Tests
Appearance of solution. Dissolve 1.0 g in 10 ml of ethanol (95 per cent). The resulting solution is clear, and colourless (2.4.1). Heavy metals (2.3.13). Dissolve 2.0 g in 15 ml of ethanol (95 per cent) and add 5 ml of water. 12 ml of the resulting solution complies with the limit test for heavy metals, Method D (20 ppm). Use lead standard solution (100 ppm Pb) diluted with a mixture of 3 volumes of ethanol (95 per cent) and 1 volume of water to contain 2 g of Pb per ml to prepare the standard. Iron (2.3.14). Boil 12.0 g with 14 ml of dilute ammonia solution and 35 ml of water. Cool and adjust the pH 5.0 to 6.0 by the dropwise addition of dilute ammonia solution or dilute sulphuric acid and dilute to 50 ml with water, if necessary. Any pink colour produced is not more intense than that obtained by boiling 2.0 g with 1 ml of iron standard solution (20 ppm Fe), 2 ml of dilute ammonia solution and 45 ml of water, adjusting the pH 5.0 to 6.0 and diluting to 50 ml with water (2 ppm). Chlorides (2.3.12). Dissolve 5.0 g in 50 ml of boiling distilled water, cool and filter (solution A). 20 ml of solution A complies with the limit test for chlorides (125 ppm). Sulphates (2.3.17). 7.5 ml of solution A complies with the limit test for sulphates (200 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent, determined on 2.0 g. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying over phosphorus pentoxide in a desiccator. Assay. Weigh accurately about 0.3 g, dissolve in 50 ml of ethanol (95 per cent), add 20 ml of water and titrate with 0.1 M sodium hydroxide, using phenol red solution as indicator, until a reddish violet colour is obtained. 1 ml of 0.1 M sodium hydroxide is equivalent to 0.01381 g of C7H6O3. Storage. Store protected from light.
Salmeterol Xinafoate contains not less than 97.0 per cent and not more than 102.0 per cent of C36H45NO7, calculated on the anhydrous basis. Description. A white to off-white powder.
Identification
Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with salmeterol xinafoate RS or with the reference spectrum of salmeterol xinafoate.
Tests
Related substances. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 50 mg of the substance under examination in 50 ml of the mobile phase. Reference solution. A 0.002 per cent w/v solution of salmeterol xinafoate RS in the mobile phase. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 60 volumes of a buffer solution prepared by dissolving 0.0816 g of potassium dihydrogen phosphate in 1000 ml of water and adjusting the pH to 7.0 with triethylamine and 40 volumes of acetonitrile, flow rate. 1 ml per minute, spectrophotometer set at 220 nm, a 10 l loop injector. Inject the reference solution. The test is not valid unless the tailing factor is not more than 2.0 and the column efficiency in not less than 4500 theoretical plates. Inject the test solution. Any individual impurity is not more than 0.5 per cent and the sum of all the impurities found is not more than 1.0 per cent. Heavy metals (2.3.13). 1 g complies with limit test for heavy metals, Method B (20 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent.
Salmeterol Xinafoate
OH HO HO OH H N COOH O ,
Water (2.3.43). Not more than 0.5 per cent, determined on 0.5 g. Assay. Weigh accurately about 0.2 g and dissolve in 50 ml of glacial acetic acid. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.06038 g of C36H45NO7. Storage. Store protected from light. 1067
C25H37NO4, C11H8O3
Mol. Wt. 603.74
Salmeterol Xinafoate is (RS)-4-hydroxy--[[[6-(4phenylbutoxy)hexyl]amino]methyl]-1,3-benzenedimethanol 1-hydroxy-2-naphthoate.
SALMETEROL AND FLUTICASONE PROPIONATE INHALATION
IP 2007
Salmeterol and Fluticasone Propionate Inhalation

Salmeterol and Fluticasone Propionate Inhalation is a suspension of microfine Salmeterol Xinafoate and Fluticasone Propionate in a suitable liquid filled in a suitable pressurised container. It may contain suitable pharmaceutical aids such as surfactants, stabilizing agents. Salmeterol and Fluticasone Propionate Inhalation delivers not less than 80.0 per cent and not more than 120.0 per cent of the stated amounts of salmeterol, C25H37NO4 and fluticasone propionate, C25H31F3O5S, per inhalation by actuation of the valve.
mobile phase: a mixture of 45 volumes of a buffer solution prepared by dissolving 1.3 g of diammonium hydrogen orthophosphate to 1000 ml of water and adjusting the pH to 7.0 with orthophosphoric acid, 25 volumes of acetonitrile and 30 volumes of methanol, flow rate. 2 ml per minute, spectrophotometer set at 220 nm, inject 200 l. Inject reference solution(c). The test is not valid unless the column efficiency for salmeterol and fluticasone propionate peak is not less than 1000 and 2500 theoretical plates respectively and the tailing factor is not more than 2.0 for each peak and the relative standard deviation for replicate injections for each component is not more than 2.0 per cent. Inject the test solution and reference solution (c). Calculate the contents of C25H37NO4 and C25H31F3O5S in the solution and the contents of C25H37NO4 and C25H31F3O5S delivered per actuation of the valve. Determine the contents of the active ingredients a second and third time by repeating the procedure on the middle ten and on the last ten successive combined actuations of the valve. For each of the three determinations the average contents of C25H37NO4 and C25H31F3O5S delivered per actuation of the valve meet the requirements. Storage. Store protected from moisture at a temperature not exceeding 30. Labelling. The label states the amounts of active ingredients delivered per inhalation.
Identification
In the Assay, the principal peaks in the chromatogram obtained with test solution correspond to the peaks in the chromatogram obtained with reference solution (c).
Tests
Other tests. Comply with the tests stated under Inhalation Preparations (Pressurised Metered-dose Preparations). Follow the procedure described under Assay with suitable dilution of the reference solution wherever the amount of active substance is to be determined in any test. Assay. Carry out the test for Content of active ingredient delivered per actuation stated under Inhalation Preparations (Pressurised Metered-dose Preparations). Determine by liquid chromatography (2.4.14). Test solution. Prepare using the mobile phase as described under the test for Content of active ingredient delivered per actuation stated under Inhalation Preparations (Pressurised Metered-dose Preparations). Reference solution (a). A solution containing 0.5 mg of salmeterol per ml prepared by dissolving 10 mg of salmeterol xinafoate RS in 10 ml acetonitrile and adding sufficient of the mobile phase to produce 20 ml. Reference solution (b) .A solution containing 0.5 mg of fluticasone propionate per ml prepared by dissolving 10 mg of fluticasone propionate RS in 10 ml acetonitrile and adding sufficient of the mobile phase to produce 20 ml. Reference solution (c). Dilute suitable volumes of reference solution (a) and reference solution (b) with the mobile phase to obtain a solution containing 5 g of salmeterol and 50 g per ml of fluticasone propionate per ml. Chromatographic system a stainless steel column 15 cm x 3.9 mm, packed with octylsilyl silica gel (5 m), column temperature 40,
Salmeterol and Fluticasone Propionate Powder for Inhalation

Salmeterol and Fluticasone Propionate Powder for Inhalation consists of Fluticasone Propionate and Salmeterol Xinafoate in microfine powder either alone or admixed with Lactose in a pre-metered unit for use in a suitable powder inhaler. Salmeterol and Fluticasone Propionate Powder for Inhalation contains not less than 90.0 per cent and not more than 125.0 per cent of the stated amounts of salmeterol C25H37NO4 and fluticasone propionate, C25H31F3O5S per unit dose.
Identification
In the Assay, the principal peaks in the chromatogram obtained with the test solution correspond to the peaks in the chromatogram obtained with reference solution (c).
Tests
Other tests. Complies with the tests stated under the Inhalation Preparations (Powders for Inhalation).
1068
IP 2007
SAQUINAVIR
Follow the procedure described under Assay with suitable dilution of the reference solution wherever the amount of active substance is to be determined in any test. Assay. Determine by liquid chromatography (2.4.14). Test solution. To 10 intact capsules add 10 ml of water, and disperse with the aid of ultrasound till the shells get disintegrated. Add 60 ml of the mobile phase and mix further with the aid of ultrasound for 10 minutes with intermittent shaking. Add sufficient of the mobile phase to produce 100.0 ml. Dilute suitably with the mobile phase, if required, to get a final concentration of 5 g per ml of Salmeterol in the mobile phase. Reference solution (a). A solution containing 0.5 mg of salmeterol per ml prepared by dissolving 10 mg of salmeterol xinafoate RS in 10 ml acetonitrile and adding sufficient of the mobile phase to produce 20 ml. Reference solution (b). A solution containing 0.5 mg of fluticasone propionate per ml prepared by dissolving 10 mg of fluticasone propionate RS in 10 ml acetonitrile and adding sufficient of the mobile phase to produce 20 ml. Reference solution (c). Dilute suitable volumes of reference solution (a) and reference solution (b) with the mobile phase to obtain a solution containing 5 g of salmeterol and 50 g per ml of fluticasone propionate per ml. Chromatographic system a stainless steel column 15 cm x 3.9 mm, packed with octylsilyl silica gel (5 mm), column temperature 40, mobile phase: a mixture of 45 volumes of a buffer solution prepared by dissolving 1.3 g of diammonium hydrogen orthophosphate to 1000 ml of water and adjusting the pH to 7.0 with orthophosphoric acid, 25 volumes of acetonitrile and 30 volumes of methanol, flow rate. 2 ml per minute, spectrophotometer set at 220 nm, inject 200 l. Inject reference solution(c). The test is not valid unless the column efficiency determined from the salmeterol and fluticasone propionate peak is not less than 1000 and 2500 theoretical plates respectively, the tailing factor for each of salmeterol and fluticasone propionate peaks is not more than 2.0 and the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject the test solution and reference solution (c). Calculate the contents of C25H37NO4 and C25H31F3O5S per unit. Storage. Store protected from moisture, at temperature not exceeding 30. Labelling. The label states the quantities of active ingredients per pre-metered unit.
Saquinavir
N O O
H N
O N H NH2
O N OH H
H N
CH3 CH3 CH3 H
C38H50N6O5
Mol. Wt. 670.8
Saquinavir is (S)-N-[(S)--{(1R)-2-[(3S,4aS,8aS)-3-(tertbutylcarbamoyl)octahydro-2(1H)-isoquinolyl]-1hydroxyethyl}phenethyl]-2-quinaldamidosuccinamide. Saquinavir contains not less than 98.5 per cent and not more than 101.0 per cent of C38H50N6O5, calculated on the anhydrous basis. Description. A white crystalline powder.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6), Compare the spectrum with that obtained with saquinavir RS or with the reference spectrum of saquinavir. B. In the Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the peak due to saquinavir in the chromatogram obtained with reference solution (a). C. When examined in the range 220 nm to 350 nm (2.4.7), a 0.002 per cent w/v solution in methanol shows absorption maxima at about 239 nm and 290 nm.
Tests
Specific optical rotation (2.4.22). 50.0 to 55.0, determined in a 0.5 per cent w/v solution in methanol. Related substances. Determine by liquid chromatography (2.4.14), as described in the Assay using the test solution and reference solution (c). Inject reference solution (c). Calculate the amount of related substance by area normalisation method. In the chromatogram obtained with the test solution, the area of any peak other than the principal peak is not greater than the area of the principal peak obtained with reference solution (c) (0.1 per cent) and the sum of the areas of all such peaks is not greater than 5 times the area of the principal peak in the chromatogram obtained with reference solution (c) (0.5 per cent).
1069
SAQUINAVIR MESYLATE
IP 2007
Heavy metals (2.3.13). 2.0 g complies with the limit tests for heavy metals, Method B (10 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Water (2.3.43). Not more than 2.0 per cent, determined on 0.5 g. Assay. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 25.0 mg of the substance under examination in 100.0 ml of the mobile phase. Reference solution (a). A 0.025 per cent w/v solution of saquinavir RS in the mobile phase. Reference solution (b). Dissolve suitable quantities of saquinavir-related compound A RS and saquinavir RS in the mobile phase to obtain a solution containing 2 g per ml of saquinavir-related compound A and 0.25 mg per ml of saquinavir. Reference solution (c). A 0.000025 per cent w/v solution of saquinavir RS in the mobile phase. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 20 volumes of methanol, 50 volumes of acetonitrile and 30 volumes of a buffer prepared by dissolving 4 g of sodium dihydrogen phosphate in 1000.0 ml of water to which 1 ml of diethylamine and 1 g of sodium octane sulphonate has been added, flow rate. 1 ml per minute, spectrophotometer set at 210 nm, a 20 l loop injector. Inject reference solution (b). The relative retention times are about 0.89 for saquinavir-related compound A and about 1.0 for saquinavir. The test is not valid unless the resolution between the peaks due to saquinavir-related compound A and saquinavir is not less than 1.5, the column efficiency determined from the saquinavir peak is not less than 500 theoretical plates and the relative standard deviation for replicate injections is not more than 2.0 per cent. Separately inject the test solution and reference solution (a). Record the chromatograms up to three times the retention time of the principal peak and measure the responses for the principal peak. Calculate the content of C38H50N6O5. Storage. Store protected from light.
Saquinavir Mesylate
H N CH3 CH3 CH3 H , CH3SO3H
N O O
H N
O N H NH2
O N OH H
C38H50N6O5,CH4O3S
Mol. Wt. 767.0
Saquinavir mesylate is (S)-N-[(S)--{(1R)-2-[(3S,4aS,8aS)3-(tert-butylcarbamoyl)octahydro-2(1H)-isoquinolyl]-1hydroxyethyl}phenethyl]-2-quinaldamidosuccinamide methanesulphonate. Saquinavir Mesylate contains not less than 98.5 per cent and not more than 101.0 per cent of C38H50N6O5, CH4O3S, calculated on the anhydrous basis. Description. A white or almost white powder.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6), Compare the spectrum with that obtained with saquinavir mesylate RS or with the reference spectrum of saquinavir mesylate. B. In the Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the peak due to saquinavir mesylate in the chromatogram obtained with reference solution (a).
Tests
Specific optical rotation (2.4.22). 66.8 to 69.6, determined in a 0.5 per cent w/v solution in methanol at 436 nm. Related substances. Determine by liquid chromatography (2.4.14), as described in the Assay using the test solution and reference solution (c). Inject reference solution (c). Calculate the amount of related substances by area normalisation method. In the chromatogram obtained with the test solution, the area of any peak other than the principal peak is not greater than the area of the principal peak in the chromatogram obtained with reference solution (c) (0.1 per cent) and the sum of the areas of all such peaks is not greater than 5 times the area of the principal peak in the chromatogram obtained with reference solution (c) (0.5 per cent). Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Methanesulphonic acid. 11.9 to 13.1 per cent w/w, calculated on the anhydrous basis, determined by the following method.
1070
IP 2007
SAQUINAVIR MESYLATE TABLETS
Weigh accurately about 0.1 g of the substance under examination, dissolve in 50 ml of methanol. Titrate with 0.1 M sodium hydroxide, determining the end-point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M sodium hydroxide is equivalent to 0.00961 g of CH3SO3H. Sulphated ash (2.3.18). Not more than 0.1 per cent. Water (2.3.43). Not more than 1.0 per cent, determined on 0.5 g. Assay. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 25.0 mg of the substance under examination in 100.0 ml of the mobile phase. Reference solution (a). A 0.025 per cent w/v solution of saquinavir mesylate RS in the mobile phase. Reference solution (b). Dissolve suitable quantities of saquinavir-related compound A RS and saquinavir mesylate RS in the mobile phase to obtain a solution containing 2 g per ml of saquinavir- related compound A and 0.25 mg per ml of saquinavir mesylate. Reference solution (c). A 0.000025 per cent w/v solution of saquinavir mesylate RS in the mobile phase. NOTE Store the buffer solution protected from light. Make adjustments if necessary for system suitability. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 25 volumes of tetrahydrofuran, 5 volumes of acetonitrile and 17 volumes of a buffer prepared by mixing 10 ml of triethylamine with water to make 1000 ml and adjusting the pH of the solution to 2.5 with phosphoric acid, flow rate. 1 ml per minute, spectrophotometer set at 210 nm, a 20 l loop injector. Inject reference solution (b). The relative retention times are about 0.89 for saquinavir-related compound A and about 1.0 for saquinavir mesylate. The test is not valid unless the resolution between the peaks due to saquinavir related compound A and saquinavir mesylate is not less than 1.5, the column efficiency determined from the saquinavir mesylate peak is not less than 500 theoretical plates and the relative standard deviation for replicate injections is not more than 2.0 per cent. Separately inject the test solution and reference solution (a). Record the chromatograms upto three times the retention time of the principal peak and measure the responses for the principal peak. Calculate the content of C38H50N6O5, CH4O3S. Storage. Store protected from light.
Saquinavir Mesylate Tablets

Saquinavir Mesylate Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of saquinavir C38H50N6O5. The tablets may be coated. NOTE Perform the tests and assay using low-actinic glassware.
Identification
A. In the Assay, the principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution. B. When examined in the range 200 nm to 400 nm (2.4.7), 1 ml of a 0.1 per cent w/v solution in methanol diluted to 100 ml with citrate buffer pH 3.0 (see under Dissolution), shows absorption maxima at the same wavelengths as shown by the reference solution.
Tests
Dissolution (2.5.2). Apparatus. No 1 Medium. 900 ml of citrate buffer pH 3.0 prepared by dissolving 5.82 mg of anhydrous dibasic sodium phosphate and 16.7 mg of citiric acid monohydrate in 1000 ml of water and adjusting the pH to 3.0 with orthophosphoric acid. Speed and time. 50 rpm and 45 minutes. Withdraw a suitable volume of the medium and filter promptly. Dilute the filtrate, if necessary, with the medium. Measure the absorbance (2.4.7) of the resulting solution at the maximum at about 240 nm. Calculate the content of C38H50N6O5 in the medium from the absorbance obtained from a solution of known concentration of saquinavir mesylate RS. D. Not less than 75 per cent of the stated amount of C38H50N6O5. Related substances. Determine by liquid chromatography (2.4.14). Test solution. Weigh and powder 20 tablets Weigh accurately a quantity of the powder containing 100 mg of saquinavir in 100 ml of the mobile phase and filter. Reference solution (a). A 0.1 per cent w/v solution of saquinavir mesylate RS in the mobile phase. Reference solution (b). Dilute 1 ml of reference solution (a) to 100 ml with the mobile phase. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 14 volumes of triethylamine phosphate solution prepared by diluting 10 ml of
1071
SECNIDAZOLE
IP 2007
triethylamine to 1000 ml with water and adjusting the pH to 2.5 with orthophosphoric acid, 5 volumes of tetrahydrofuran and 1 volume of acetonitrile, flow rate. 1 ml per minute, spectrophotometer set at 210 nm, a 20 l loop injector. Inject reference solution (a). The test is not valid unless the column efficiency is not less than 3000 theoretical plates and the tailing factor is not more than 2.0. Inject the test solution and reference solution (b). Run the chromatogram for 5 times the retention time (about 12 minutes) of the principal peak. In the chromatogram obtained with the test solution, the area of any secondary peak is not more than 0.2 times the area of the peak in the chromatogram obtained with the reference solution (b) (0.2 per cent) and the sum of areas of all the secondary peaks is not more than the area of the peak in the chromatogram obtained with the reference solution (b) (1.0 per cent). Uniformity of content. Comply with the test stated under Tablets. Determine by liquid chromatography (2.4.14), as described under Assay. Test solution. Disperse one tablet in 500 ml of the mobile phase and filter. Dilute 5 ml of the filtrate to 20 ml with the mobile phase. Calculate the content of C38H50N6O5 in the tablet. Other tests. Comply with the tests stated under Tablets. Water (2.3.43). Not more than 6.0 per cent, determined on 0.5 g. Assay. Determine by liquid chromatography (2.4.14). Test solution. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing 100 mg of saquinavir, dissolve in 100.0 ml of mobile phase and filter. Dilute 5.0 ml of the filtrate to 20.0 ml with the mobile phase. Reference solution. A 0.1 per cent w/v solution of saquinavir mesylate RS in the mobile phase. Dilute 5.0 ml of the solution to 20.0 ml with the mobile phase. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 14 volumes of a solution prepared by diluting 10.0 ml of triethylamine to1000 ml with water, adjusting the pH to 2.5 with orthophosphoric acid and filtering, 5 volumes of tetrahydrofuran and 1 volume of acetonitrile. flow rate. 1.5 ml per minute, spectrophotometer set at 210 nm, a 20 l loop injector.
Inject the reference solution. The test is not valid unless the tailing factor is not more than 2.0, the column efficiency in not less than 2000 theoretical plates and the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject the test solution and the reference solution. Calculate the content of C38H50N6O5 in the tablets. Storage. Store protected from moisture. Labelling. The label states the strength in terms of the equivalent amount of saquinavir.
Secnidazole
OH CH3 O2N N N
C7H11N3O3 Mol. Wt. 185.2
CH3
Secnidazole is (RS)-1-(2-methyl-5-nitroimidazol-1-yl)propan2-ol. Secnidazole contains not less than 98.0 per cent and not more than 101.0 per cent of C6H9N3O3, calculated on the anhydrous basis. Description. A white to yellowish white, crystalline powder.
Identification
A. When examined in the range 230 nm to 360 nm (2.4.7), a 0.001 per cent w/v solution in 0.1 M hydrochloric acid shows an absorption maximum at about 277 nm, 0.325 to 0.355. B. Heat about 10 mg in a water-bath with 10 mg of zinc powder, 1 ml of water and 0.25 ml of 2 M hydrochloric acid for 5 minutes and cool. The solution gives the reaction of primary aromatic amines (2.3.1).
Tests
Appearance of solution. A 5 per cent w/v solution in 1 M hydrochloric acid is not more opalescent than opalescence standard OS2 (2.4.1) and not more intensely coloured than reference solution GYS4 (2.4.1). Related substances. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 50 mg of the substance under examination in 100.0 ml of the mobile phase.
1072
IP 2007
SECNIDAZOLE TABLETS
Reference solution (a). Dilute 1.0 ml of the test solution to 100.0 ml with mobile phase. Dilute 1.0 ml of the solution to 10.0 ml with the same solvent. Reference solution (b). A solution containing 0.005 per cent w/v each of 2-methyl-5-nitroimidazole RS and secnidazole RS in the mobile phase. Dilute 1.0 ml of the solution to 100.0 ml with the same solvent. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 35 volumes of methanol and 65 volumes of 0.14 per cent w/v solution of potassium dihydrogen phosphate, flow rate. 1 ml per minute, spectrophotometer set at 318 nm, a 10 l loop injector. Inject reference solution (b). The test is not valid unless the resolution between 2-methyl-4-nitroimidazole and secnidazole is not less than 1.5. Inject the test solution and reference solution (a). In the chromatogram obtained with the test solution, the area of any secondary peak is not more than 0.2 times the area of the peak in the chromatogram obtained with reference solution (a) (0.2 per cent) and the sum of areas of all the secondary peaks is not more than 0.5 times the area of the peak in the chromatogram obtained with the reference solution (b) (0.5 per cent). Disregard any peak which is 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.01 per cent). Heavy metals (2.3.13).1.0 g complies with the limit test for heavy metals, Method A (20 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Water (2.3.43). 4.0 to 5.0 per cent, determined on 0.4 g. Assay. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 25 mg of the substance under examination in 25.0 ml of the mobile phase. Dilute 5.0 ml of the solution to 100.0 ml with the same solvent. Reference solution. A 0.005 per cent w/v solution of secnidazole RS in mobile phase. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 35 volumes of methanol and 65 volumes of 0.14 per cent w/v solution of potassium dihydrogen phosphate, flow rate. 1 ml per minute, spectrophotometer set at 318 nm, a 20 l loop injector.
Inject the reference solution. The test is not valid unless the tailing factor is not more than 2.0 and the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject the test solution and the reference solution. Calculate the content of C7H11N3O3. Storage. Store protected from light and moisture.
Secnidazole Tablets
Secnidazole Tablets contain not less than 95.0 per cent and not more than 110.0 per cent of the stated amount of secnidazole, C7H11N3O3.
Identification
Tests
Dissolution (2.5.2). Apparatus. No 1 Medium. 900 ml of 0.1 M hydrochloric acid. Speed and time. 100 rpm and 30 minutes. Withdraw a suitable volume of the medium and filter promptly through a membrane filter disc having an average pore diameter not greater than 1.0 m, rejecting the first 1 ml of the filtrate. Dilute the filtrate, if necessary, with the same solvent. Measure the absorbance of the resulting solution at the maximum at about 277 nm (2.4.7). Calculate the content of C7H11N3O3 in the medium from the absorbance obtained from a solution of known concentration of secnidazole RS in the same medium. D. Not less than 80 per cent of the stated amount of C7H11N3O3. Related substances. Determine by liquid chromatography (2.4.14). Test solution. Weigh accurately a quantity of powdered tablets containing 50 mg of Secnidazole, disperse in 100 ml of mobile phase and filter. Reference solution (a). A 0.05 per cent w/v solution of secnidazole RS in the mobile phase. Reference solution (b). Dilute 1 ml of reference solution (a) to 100 ml with mobile phase. Chromatographic system as described under Assay. Inject reference solution (a). The test is not valid unless the tailing factor is not more than 2.0 and the column efficiency in not less than 2000 theoretical plates.
1073
COLLOIDAL SILICON DIOXIDE
IP 2007
Inject the test solution and reference solution (b). In the chromatogram obtained with the test solution, the area of any secondary peak is not more than the area of the peak in the chromatogram obtained with reference solution (b) (1.0 per cent) and the sum of areas of all the secondary peaks is not more than 2.0 times the area of the peak in the chromatogram obtained with the reference solution (b) (2.0 per cent). Water (2.3.43). Not more than 6.5 per cent, determined on 0.5 g. Assay. Determine by liquid chromatography (2.4.14). Test solution. Weigh and powder 20 tablets. Weigh accurately a quantity of powdered tablet containing 50 mg of Secnidazole, disperse in 100.0 ml of mobile phase and filter.. Dilute 5.0 ml of the filtrate to 50.0 ml with mobile phase. Reference solution. A 0.005 per cent w/v solution of secnidazole RS in mobile phase. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane chemically bonded to porous silica (5 m), mobile phase: a mixture of 85 volumes of 0.01 M potassium dihydrogen orthophosphate and 15 volumes of acetonitrile, flow rate. 1 ml per minute, spectrophotometer set at 228 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the column efficiency is not less than 2000 theoretical plates, the tailing factor is not more than 2.0 and the relative standard deviation of replicate injections is not more than 2.0 per cent. Inject the test solution and the reference solution. Calculate the content of C7H11N3O3. Storage. Store protected from light and moisture. Labelling. The label states the strength of secnidazole.
Identification
About 20 mg gives the reaction of silicates (2.3.1).
Tests
pH (2.4.24). 3.5 to 5.5, determined in a suspension of 1.0 g in 30 ml of carbon dioxide-free water. Arsenic (2.3.10). To 2.5 g contained in a round-bottomed flask add 50 ml of 3 M hydrochloric acid and heat under a reflux condenser for 30 minutes. Cool, filter with the aid of suction and transfer the filtrate to a 100-ml volumetric flask. Wash the filter with several portions of hot water and add the washings to the volumetric flask. Cool, dilute to volume with water and mix. To 50.0 ml of the solution add 3 ml of hydrochloric acid; the resulting solution complies with the limit test for arsenic (8 ppm). Heavy metals (2.3.13). Suspend 2.5 g in sufficient water to produce a semi-fluid slurry and dry at 140. When the dried substance is white, break up the mass using a glass rod, add 25 ml of 1 M hydrochloric acid, boil gently for 5 minutes, stirring frequently with the glass rod, centrifuge for 20 minutes and filter the supernatant liquid through a membrane filter. To the residue in the centrifuge tube add 3 ml of 2 M hydrochloric acid and 9 ml of water, boil, centrifuge for 20 minutes and filter the supernatant liquid through the same membrane filter. Wash the residue with small quantities of water, combine the filtrates and washings and dilute to 50.0 ml with water. To 20.0 ml of the solution add 50 mg of L-ascorbic acid and 1 ml of strong ammonia solution, neutralise with 2 M ammonia and dilute to 25 ml with water. 12 ml of the solution complies with the limit test for heavy metals, Method D (25 ppm). Use lead standard solution (1 ppm Pb) to prepare the standard. Chlorides (2.3.12). To 1.0 g add a mixture of 20 ml of 2 M nitric acid and 30 ml of water, heat on a water-bath for 15 minutes, shaking frequently, dilute to 50 ml with water if necessary, filter and cool. The filtrate complies with the limit test for chlorides (250 ppm). Loss on ignition (2.4.20). Not more than 5.0 per cent, determined on 0.2 g by igniting at 900 in a platinum crucible for 2 hours.
Colloidal Silicon Dioxide

Colloidal Anhydrous Silica
SiO2 Mol. Wt. 60.1 Colloidal Silicon Dioxide is a submicroscopic fumed silica prepared by the vapour-phase hydrolysis of a silicon compound. Colloidal Silicon Dioxide contains not less than 99.0 per cent and not more than 100.5 per cent of SiO2, calculated on the ignite basis. Description. A light, fine, white, amorphous powder. It has a particle size of about 15 nm.
Assay. To the residue obtained in the test for Loss on ignition add 0.2 ml of sulphuric acid and sufficient ethanol (95 per cent) to moisten the residue completely, add 6 ml of hydrofluoric acid and evaporate to dryness on a hot plate at 95 to 105, avoiding loss from sputtering. Wash the sides of the dish with 6 ml of hydrofluoric acid, evaporate to dryness in a well-ventilated hood, ignite at 1000, allow to cool in a desiccator and weigh. The difference between the weight of the final residue and that of the residue obtained in the test for Loss on ignition represents the amount of SiO2 in the amount of the substance taken for the test for Loss on ignition.
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IP 2007
SODIUM ACETATE
Storage. Store protected from light.
Identification
Dissolve 10.0 g in sufficient carbon dioxide-free water to produce 100.0 ml (solution A). 1 ml of solution A gives reaction B of acetates and reaction A of sodium salts (2.3.1). Mol. Wt. 169.9
Silver Nitrate
AgNO3 Silver Nitrate contains not less than 99.0 per cent and not more than 100.5 per cent of AgNO3. Description. Colourless crystals or a white, crystalline powder.
Tests
Appearance of solution. Solution A is clear (2.4.1), and colourless (2.4.1). pH (2.4.24). 7.5 to 9.0, determined in a 5.0 per cent w/v solution.
Identification
A. Gives the reactions of silver salts (2.3.1). B. 10 mg gives reaction A of nitrates (2.3.1).
Arsenic (2.3.10). Dissolve 5.0 g in 50 ml of water and add 15 ml of stannated hydrochloric acid AsT. The resulting solution complies with the limit test for arsenic (2 ppm). Calcium and magnesium. Not more than 50 ppm, calculated as Ca, determined by the following method. Mix 200 ml of water with 10 ml of ammonia buffer pH 10.0, 0.1 g of mordant black 11 mixture and 2 ml of 0.05 M zinc chloride. Add dropwise 0.02 M disodium edetate until the colour changes from violet to blue. To this solution add 10 g of the substance under examination, shake to dissolve and titrate with 0.02 M disodium edetate until the blue colour is restored. Not more than 0.65 ml of 0.02 M disodium edetate is required. Heavy metals (2.3.13).12 ml of solution A complies with the limit test for heavy metals, Method D (10 ppm). Iron (2.3.14). 20 ml of solution A complies with the limit test for iron (20 ppm). Chlorides (2.3.12). 10 ml of solution A complies with the limit test for chlorides (250 ppm). Sulphates (2.3.17). 15 ml of solution A complies with the limit test for sulphates (225 ppm). Reducing substances. Dissolve 1.0 g in 100 ml of boiling water, add 5 ml of 1 M sulphuric acid and 0.5 ml of 0.002 M potassium permanganate, mix and boil gently for 5 minutes; the pink colour is not completely discharged. Loss on drying (2.4.19). 39.0 to 40.5 per cent, determined on 0.2 g by drying in an oven at 130. Place the substance under examination in the oven while the oven is still cold. Assay. Weigh accurately about 0.25 g, dissolve in 50 ml of anhydrous glacial acetic acid, add 5 ml of acetic anhydride, mix and allow to stand for 30 minutes. Titrate with 0.1 M perchloric acid, using 0.3 ml of 1-naphtholbenzein solution as indicator, until a green colour is produced. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.00820 g of C2H3NaO2. Sodium Acetate intended for use in the preparation of dialysis solutions complies with the following additional requirement.
Tests
Appearance of solution. A 4.0 per cent w/v solution (solution A) is clear (2.4.1), and colourless (2.4.1). Acidity or alkalinity. To 2 ml of solution A add 0.1 ml of bromocresol green solution; the solution is blue. To 2 ml of this solution add 0.1 ml of phenol red solution; the solution is yellow. Aluminium, bismuth, copper and lead. Dissolve 1.0 g in a mixture of 4 ml of strong ammonia solution and 6 ml of water; the resulting solution is clear (2.4.1), and colourless (2.4.1). Foreign salts. Not more than 0.3 per cent, determined by the following method. To 30 ml of solution A add 7.5 ml of 2 M hydrochloric acid, shake vigorously, heat for 5 minutes on a water-bath, filter and evaporate 20 ml of the filtrate to dryness on a water-bath. Dry the residue at 105 and weigh. Assay. Weigh accurately about 0.3 g, dissolve in 50 ml of water, add 2 ml of 2 M nitric acid and 2 ml of ferric ammonium sulphate solution and titrate with 0.1 M ammonium thiocyanate until a reddish yellow colour is produced. 1 ml of 0.1 M ammonium thiocyanate is equivalent to 0.01699 g of AgNO3. Storage. Store protected from light and moisture, in nonmetallic containers.
Sodium Acetate
CH3COONa,3H20 C2H3NaO2, 3H2O Mol. Wt. 136.1 Sodium Acetate contains not less than 99.0 per cent and not more than 101.0 per cent of C2H3NaO2, calculated on the dried basis. Description. Colourless crystals or a white, crystalline powder.
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SODIUM ALGINATE
IP 2007
Aluminium. Dissolve 20 g in 100 ml of water and adjust to pH 6.0 by the addition of about 10 ml of 1 M hydrochloric acid. Extract with successive quantities of 20, 20 and 10 ml of a 0.5 per cent w/v solution of 8-hydroxyquinoline in chloroform and dilute the combined extracts to 50 ml with chloroform. Use as the standard solution a mixture of 0.4 ml of aluminium standard solution (2 ppm Al), 10 ml of acetate buffer pH 6.0 and 98 ml of water treated in the same manner and as the blank solution a mixture of 10 ml of acetate buffer pH 6.0 and 100 ml of water treated in the same manner. Measure the fluorescence (2.4.5) of the test solution and the standard solution, using an excitation wavelength of about 392 nm and emission wavelength of about 518 nm, and setting the instrument to zero with the blank solution in each case. The fluorescence of the test solution is not greater than that of the standard solution (0.2 ppm). Storage. Store protected from light. Labelling. The label states whether or not the material is intended for use in the manufacture of dialysis solutions.
Tests
Heavy metals (2.3.13). 0.5 g complies with the limit test for heavy metals, Method B (40 ppm), using nitric acid instead of sulphuric acid for wetting the sample. Chloride. Not more than 1.0 per cent, determined by the following method. Shake 2.5 g with 50 ml of 2 M nitric acid for 1 hour, dilute to 100 ml with 2M nitric acid and filter. To 50 ml of the filtrate add 10.0 ml of 0.1 M silver nitrate and 5 ml of toluene. Titrate with 0.1 M ammonium thiocyanate using 2 ml of ferric ammonium sulphate solution as indicator and shaking vigorously towards the end-point. 1 ml of 0.1 M silver nitrate is equivalent to 0.00355 g of Cl. Microbial contamination (2.2.9). 1 g is free from Escherichia coli; 10 g is free from salmonellae. Sulphated ash (2.3.18). 30.0 to 36.0 per cent, determined on 0.1 g and calculated on the dried basis. Loss on drying (2.4.19). Not more than 15.0 per cent, determined on 0.2 g by drying in an oven at 105 for 4 hours. Storage. Store protected from light.
Sodium Alginate
Sodium Polymannuronate
Sodium Alginate consists mainly of the sodium salt of alginic acid which is a mixture of polyuronic acids [(C6H8O6)n] composed of residues of D-mannuronic acid and L-guluronic acids and is obtained mainly from algae belonging to the order Phaeophyceae. Description. A cream-coloured to pale yellowish brown powder; almost odourless.
Sodium Aminosalicylate
Sodium PAS
COONa OH ,2H2O NH2
C7H6NNaO3.2H2O. Sodium Aminosalicylate is sodium 4-amino-2hydroxybenzoate dihydrate. Sodium Aminosalicylate contains not less than 99.0 per cent and not more than 101.0 per cent of C7H6NNaO3, calculated on the anhydrous basis. Description. A white to cream coloured crystalline powder. Mol. Wt. 211.2
Identification
A. Dissolve 0.2 g with shaking in 20 ml of water and to 5 ml of the resulting solution add 1 ml of calcium chloride solution; a voluminous gelatinous precipitate is produced. B. To 10 ml of the solution obtained in test A add 1 ml of 1 M sulphuric acid; a gelatinous mass is produced. C. To 5 mg add 5 ml of water, 1 ml of a freshly prepared 1 per cent w/v solution of naphthalene-1,3-diol in ethanol (95 per cent) and 5 ml of hydrochloric acid. Boil for 3 minutes, cool, add 5 ml of water and shake with 15 ml of di-isopropyl ether. The upper layer exhibits a deeper bluish red colour than the upper layer obtained by repeating the procedure without the substance under examination. D. The residue obtained in the test for Sulphated ash gives reaction A of sodium salts (2.3.1).
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with sodium aminosalicylate RS or with the reference spectrum of sodium aminosalicylate. B. A 5 per cent w/v solution complies with the tests for sodium salts (2.3.1).
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SODIUM ALGINATE
Tests
pH (2.4.24). 6.5 to 8.5, determined in a 2.0 per cent w/v solution. 3-aminophenol. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 50.0 mg of the substance under examination in 100 ml of the mobile phase. Reference solution (a). Dissolve 25.0 mg of 3-aminophenol RS in 100.0 ml of the mobile phase (solution A). Dilute 5.0 ml of the solution to 100.0 ml with the mobile phase. Dilute 5.0 ml of the resulting solution to 50.0 ml with the mobile phase. Reference solution (b). Dissolve 25.0 mg of sodium aminosalicylate RS in 100.0 ml of the mobile phase. Dilute 5.0 ml of this solution and 5 ml of solution A to 100.0 ml with the mobile phase. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilane bonded to porous base deactivated silica (5 m) (such as Hypersil BDS), mobile phase: dissolve 6.0 g of disodium hydrogen orthophosphate and 6.6 g of sodium dihydrogen orthophosphate dihydrate in 1600 ml with water, add 19 ml of tetra n-butyl ammonium hydroxide (20 per cent solution) and make the volume to 1700 ml with water and add 300 ml of methanol, flow rate. 1.5 ml per minute, spectrophotometer set at 280 nm, a 20 l loop injector. Inject reference solution (a). The relative standard deviation for replicate injections is not more than 5.0 per cent Inject reference solution (b). The test is not valid unless the resolution between m-aminophenol and sodium aminosalicylate is at least 5.0. Inject the test solution and reference solution (a). In the chromatogram obtained with the test solution, the area of the peak corresponding to 3-aminophenol is not more than the area of the corresponding peak in the chromatogram obtained with reference solution (a) ( 0.25 per cent). Hydrogen Sulphide and Sulphur dioxide. Dissolve 0.5 g in 5 ml of 1 M sodium hydroxide, add 6 ml of 3 M hydrochloric acid and stir vigorously. No odour of hydrogen sulphide or sulpher dioxide is perceptible, and not more than a faint odour of Amyl Alcohol is perceptible. A piece of moistened lead acetate paper held over the mixture does not become discoloured. Arsenic (2.3.10). Mix 5.0 g with 10 ml of bromine solution and evaporate to dryness on a water-bath, ignite gently, dissolve the cooled residue, ignoring any carbon, in 50 ml of water and 14 ml of brominated hydrochloric acid AsT and remove the
excess bromine with a few drops of stannous chloride solution AsT. The resulting solution complies with the limit test for arsenic (2 ppm). Heavy metals (2.3.13). 0.66 g complies with the limit test for heavy metals, Method B (30 ppm). Chlorides (2.3.12). Dissolve 1.0 g in 10 ml of water, add 3 ml of acetic acid and filter, dilute the filtrate to 50 ml with water. 10 ml of the solution complies with the limit test for chlorides (0.25 per cent). Sulphates (2.3.17). 0.1 g complies with the limit test for sulphates (0.15 per cent). Loss on drying (2.4.19). 16.0 to 17.5 per cent, determined on 0.5 g by drying in an oven at 105. Assay. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 50.0 mg of the substance under examination in 100.0 ml of the mobile phase. Dilute 5.0 ml of this solution to 100.0 ml with the mobile phase. Reference solution (a). Dissolve 50.0 mg of sodium aminosalicylate RS in 100.0 ml of the mobile phase (solution A). Dilute 5.0 ml of this solution to 100.0 ml with the mobile phase. Reference solution (b). Dissolve 25.0 mg of 3-aminophenol RS in 100.0 ml of the mobile phase. Dilute 5.0 ml of this solution and 5 ml of solution A to 100.0 ml with the mobile phase. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: dissolve 6.0 g of disodium hydrogen orthophosphate and 6.6 g of sodium dihydrogen orthophosphate dihydrate in 1600 ml with water, add 19 ml of tetra n-butyl ammonium hydroxide (20 per cent solution) and make the volume to 1700 ml with water and add 300 ml of methanol, flow rate. 1.5 ml per minute, spectrophotometer set at 254 nm, a 20 l loop injector. Inject reference solution (a). The tailing factor is not more than 2.0. The column efficiency in not less than 3000 theoretical plates. The relative standard deviation for replicate injections is not more than 5.0 per cent. Inject reference solution (b). The test is not valid unless the resolution between 3-aminophenol and sodium aminosalicylate is at least 5.0. Inject alternately the test solution and the reference solution. Calculate the content of C7H6NNaO3. Storage. Store protected from light and moisture.
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SODIUM AMINOSALICYLATE TABLETS
IP 2007
Sodium Aminosalicylate Tablets

Sodium PAS Tablets
Sodium aminosalicylate tablet contains not less than 95.0 per cent not more than 105.0 per cent of C7H6NNaO3.2H2O.
cent aqueous solution) and make the volume to 1700 ml with water, add 300 ml of methanol, mix well and degas, flow rate. 1.5 ml per minute, spectrophotometer set at 280 nm, a 20 l loop injector. Inject reference solution (a). The relative standard deviation for replicate injections is not more than 5.0 per cent. Inject reference solution (c). The resolution between 3-aminophenol and sodium aminosalicylate is not less than 5.0. Inject the test solution and reference solution (a). In the chromatogram obtained with the test solution, the area of the peak corresponding to 3-aminophenol is not more than the area of the peak in the chromatogram obtained with reference solution (a) ( 1.0 per cent). Dissolution (2.5.2). Apparatus. No 2 Medium. 900 ml water Speed and time.100 rpm for 45 minutes Withdraw a suitable volume of the medium and filter. Determine by liquid chromatography (2.4.14). Test solution. The filtrate diluted with the mobile phase to produce a 0.055 per cent w/v solution. Reference solution. A 0.055 per cent w/v solution of sodium aminosalicylate RS in the mobile phase. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: dissolve 6.0 g of disodium hydrogen orthophosphate and 6.6 g of sodium dihydrogen orthophosphate dihydrate in 1600 ml with water, add 19 ml of tetra n-butyl ammonium hydroxide (20 per cent aqueous solution) and make the volume to 1700 ml with water, add 300 ml of methanol, mix well and degas, flow rate. 1.5 ml per minute, spectrophotometer set at 254 nm, a 20 l loop injector. Inject the reference solution. The tailing factor is not more than 2.0 and the relative standard deviation for replicate injections is not more than 2.0 per cent Inject alternatively the test solution and the reference solution. Calculate the content of C7H6NNaO3.2H2O in the medium. D. Not less than 75 per cent of the stated amount of C7H6NNaO3.2H2O. Other tests. Comply with the tests stated under Tablets. Assay. Determine by liquid chromatography (2.4.14).
Identification
Digest a quantity of powdered tablets containing about 3.0 g of sodium aminosalicylate, with 40 ml of water, and filter. Add to the filtrate 15 ml of 1M acetic acid, and allow it to stand until precipitation has occurred. Collect the precipitate on a filter, wash well with water and dry at 105C for 30 min. The residue complies with the following tests. A. Place about 1 g of the residue in a small, round-bottom flask, and add 10 ml of acetic anhydride. Heat the flask on a steam bath for 30 minutes, and add 40 ml of water, mix, filter, cool, and allow to stand until diacetyl derivative crystallizes, wash well with water, and dry at 105 for 1 hour. The diacetyl derivative so obtained melts at 191 to 197. B. Shake 0.1 g of the residue with 10 ml of water, and filter. To 5 ml of the filtrate add 1 drop of ferric chloride solution. A violet colour is produced. C. In the Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the peak in the chromatogram obtained with the reference solution.
Tests
3-aminophenol. Determine by liquid chromatography (2.4.14). Test solution. Weigh and powder 20 tablets. Weigh a quantity of the powder containing about 50 mg of Sodium Aminosalicylate and dissolve in 100 ml of the mobile phase. Reference solution (a). A 0.024 per cent w/v solution of m-aminophenol RS in the mobile phase. Dilute 5 ml to 100 ml with the mobile phase. Dilute 5 ml of the resulting solution to 50 ml with the mobile phase. Reference solution (b). A 0.024 per cent w/v solution of sodium aminosalicylate RS in the mobile phase. Reference solution (c). Mix 5 ml each of reference solution (a) and reference solution (b) and dilute to 100 ml with the mobile phase. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilane bonded to porous base deactivated silica (5 m) (such as Hypersil BDS), mobile phase: dissolve 6.0 g of disodium hydrogen orthophosphate and 6.6 g of sodium dihydrogen orthophosphate dihydrate in 1600 ml with water, add 19 ml of tetra n-butyl ammonium hydroxide (20 per
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SODIUM AUROTHIOMALATE
Test solution. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 0.5 g of sodium aminosalicylate, add sufficient mobile phase to produce 100.0 ml and filter. Dilute 5.0 ml of this solution to 50.0 ml with the mobile phase. Reference solution. A 0.05 per cent w/v solution of sodium aminosalicylate RS in the mobile phase. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: dissolve 6.0 g of disodium hydrogen orthophosphate and 6.6 g of sodium dihydrogen orthophosphate dihydrate in 1600 ml with water, add 19 ml of tetra n-butyl ammonium hydroxide (20 per cent aqueous solution) and make the volume to 1700 ml with water, add 300 ml of methanol, mix well and degas, flow rate. 1.5 ml per minute, spectrophotometer set at 254 nm, a 20 l loop injector. Inject the reference solution. The tailing factor is not more than 2.0 and the relative standard deviation for replicate injections is not more than 2.0 per cent Inject alternately the test solution and the reference solution. Calculate the content of C7H6NNaO3. 2H2O in the tablets. Storage. Store protected from moisture.
A. Determine by infrared absorption spectrophotometry (2.4.6), Compare the spectrum with that obtained with sodium ascorbate RS. B. To 4 ml of a 2 per cent w/v solution add 1 ml of 0.1 M hydrochloric acid, add a few ml of 2,6-dichlorophenolindophenol solution; the solution is decolorised. C. To 4 ml of a 2 per cent w/v solution add 1 ml of 0.1 M hydrochloric acid, add 1 drop of a freshly prepared 5 per cent w/v solution of sodium nitroprusside and 2 ml of dilute sodium hydroxide solution. Add 0.6 ml of hydrochloric acid dropwise and stir; the yellow colour turns blue. D. A 2 per cent w/v solution gives the reactions of sodium salts (2.3.1).
Tests
Appearance of solution. A 5.0 per cent w/v solution in carbon dioxide-free water is clear (2.4.1) and not more intensely coloured than reference solution BYS7 (2.4.1). pH (2.4.24). 7.0 to 8.0, determined in a 10.0 per cent w/v solution. Specific optical rotation (2.4.22). +103 to +108, determined in a 10.0 per cent w/v solution. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Loss on drying (2.4.19). Not more than 0.25 per cent, determined on 1.0 g by drying over phosphorus pentoxide at a pressure not exceeding 0.7 kPa for 24 hours. Assay. Weigh accurately about 0.2 g, dissolve in a mixture of 100 ml of carbon dioxide-free water and 25 ml of 1 M sulphuric acid. Titrate immediately with 0.05 M iodine, using 1 ml of starch solution as indicator, until a persistent violet-blue colour is obtained. 1 ml of 0.05 M iodine is equivalent to 0.009905 g of C6H7NaO6. Storage. Store protected from light.
Sodium Ascorbate
CH2OH H COH O O NaO
C6H7NaO6
OH
Mol. Wt. 198.1
Sodium Aurothiomalate
Sodium Aurothiomalate consists mainly of the disodium salt of (aurothio) succinic acid. Sodium Aurothiomalate contains not less than 44.5 per cent and not more than 46.0 per cent of Au and not less than 10.8 per cent and not more than 11.5 per cent of Na, both calculated on the dried basis. Description. A fine, pale yellow powder; odour, slight; hygroscopic.
Sodium Ascorbate is L-ascorbic acid, monosodium salt. Sodium Ascorbate contains not less than 99.0 per cent and not more than 101.0 per cent of C6H7NaO6, calculated on the dried basis. Description. White or faintly yellow crystals or a crystalline powder; odourless or almost odourless. It darkens gradually on exposure to light.
Identification
Test A may be omitted if tests B, C and D are carried out. Test B and C may be omitted if tests A and D are carried out.
Identification
A. Ignite 0.1 g and dissolve a portion of the residue by warming with 2 ml of a mixture of 3 volumes of hydrochloric acid and
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SODIUM AUROTHIOMALATE INJECTION
IP 2007
1 volume of nitric acid and dilute to 20 ml with water (solution A). Reserve the remainder of the residue for use in test D. To 0.2 ml add 20 ml of water, boil, pour the boiling solution into 5 ml of stannous chloride solution and mix; a purple colour is produced. B. To 2 ml of solution A add 2 ml of hydrogen peroxide solution (20 vol) and 1 ml of 5 M sodium hydroxide; a precipitate is produced which appears brownish black by reflected light and bluish green by transmitted light. C. Solution A gives a black precipitate with hydrogen sulphide which is insoluble in 2 M hydrochloric acid but soluble in ammonium polysulphide solution. D. Extract a portion of the residue obtained in test A with 10 ml of 2 M hydrochloric acid. The solution, after neutralisation if necessary, gives the reactions of sodium salts and the reaction of sulphates (2.3.1).
Identification
A. To a volume equivalent to 20 mg of Sodium Aurothiomalate add 2 ml of hydrogen peroxide solution (100 vol) and 1 ml of 5 M sodium hydroxide and boil for 30 seconds; a colloidal precipitate is produced which appears bluish green by transmitted light. B. To a volume equivalent to 10 mg of Sodium Aurothiomalate add 0.1 ml of potassium cyanide solution and 0.1 ml of a 1 per cent w/v solution of sodium nitroprusside; a deep magenta colour is produced.
Tests
Appearance of solution. Dilute, if necessary, with water to give a solution containing 1.0 per cent w/v of Sodium Aurothiomalate. The colour of the solution is not more intense than that of a 0.02 per cent w/v solution of potassium ferricyanide. Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. To an accurately measured volume containing about 0.1 g of Sodium Aurothiomalate add 0.4 g of potassium bromide and 5 ml of nitric acid. Slowly evaporate the solution to dryness and continue heating until fumes cease to be evolved. Allow to cool, add 50 ml of water, warm, filter, wash the residue of gold, Au, with hot water, dry and ignite for 3 hours at a temperature not lower than 600. Storage. Store protected from light.
Tests
pH (2.4.24). 6.0 to 7.0, determined in a 10.0 per cent w/v solution. Stability. Dissolve 1.0 g in 10 ml of water, filter, seal in an ampoule, heat at 100 for 1 hour, cool and add sufficient water to produce 100 ml. The solution remains bright and is not more intensely coloured than a 0.01 per cent w/v solution of potassium ferricyanide. Loss on drying (2.4.19). Not more than 2.0 per cent, determined on 1.0 g by drying over phosphorus pentoxide at a pressure not exceeding 0.7 kPa for 24 hours. Assay. For Au Weigh accurately about 0.2 g, heat with 10 ml of sulphuric acid and continue to boil gently until a clear, pale yellow liquid is produced. Cool, add about 1 ml of nitric acid dropwise and boil again for 1 hour. Cool, dilute with 70 ml of water, boil for 5 minutes, filter, wash the residue of gold Au, with hot water, dry and ignite for 3 hours at a temperature not lower than 600. For Na Evaporate to dryness the filtrate and washings obtained in the Assay for Au, moisten with sulphuric acid and ignite for 3 hours at 600. 1 g of residue is equivalent to 0.3237 g of Na. Storage. Store protected from light.
Sodium Benzoate
COONa
C7H5NaO2
Mol. Wt. 144.1
Sodium Benzoate contains not less than 99.0 per cent and not more than 100.5 per cent of C7H5NaO2, calculated on the dried basis. Description. A white, crystalline or granular powder or flakes; odourless or with a faint odour; hygroscopic.
Sodium Aurothiomalate Injection

Sodium Aurothiomalate Injection is a sterile solution of Sodium Aurothiomalate in Water for Injection. Sodium Aurothiomalate Injection contains gold, Au, equivalent to not less than 42.3 per cent and not more than 48.3 per cent of the stated amount of sodium aurothiomalate.
Identification
A. To a 10 per cent w/v solution add ferric chloride test solution; a buff coloured precipitate is formed. Add dilute hydrochloric acid; a white crystalline precipitate is produced.
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IP 2007
SODIUM BICARBONATE
B. Gives the reactions of sodium salts and reactions B and C of benzoates (2.3.1).
Description. A white, crystalline powder or small, opaque, monoclinic crystals. It gradually forms sodium carbonate on heating in the dry state or in solution.
Tests Identification
Appearance of solution. A 10.0 per cent w/v solution in carbon dioxide-free water is clear (2.4.1), and not more intensely coloured than reference solution YS6 (2.4.1). Acidity or alkalinity. To 20 ml of a 5.0 per cent w/v solution in carbon dioxide-free water add 0.2 ml of phenolphthalein solution. Not more than 0.2 ml of 0.1 M hydrochloric acid or 0.2 ml of 0.1 M sodium hydroxide is required to change the colour of the solution. Arsenic (2.3.10). Mix 5.0 g with 10 ml of bromine solution and evaporate to dryness on a water-bath. Ignite gently, dissolve the cooled residue, ignoring any carbon, in 50 ml of water and 14 ml of brominated hydrochloric acid AsT, and remove the excess of bromine with 2 ml of stannous Chloride solution AsT. The resulting solution complies with the limit test for arsenic (2 ppm). Heavy metals (2.3.13). Dissolve 2.0 g in 45 ml of water and 5 ml of hydrochloric acid. 25 ml of the solution complies with the limit test for heavy metals, Method B (20 ppm). Chlorinated compounds. Dissolve 0.33 g in 5 ml of 0.5 M sodium carbonate, evaporate to dryness and heat the residue until completely charred, keeping the temperature below 400. Extract the residue with a mixture of 10 ml of water and 12 ml of dilute nitric acid and filter; the filtrate complies with the limit test for chlorides (2.3.12). Loss on drying (2.4.19). Not more than 2.0 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.25 g, dissolve in 20 ml of anhydrous glacial acetic acid, warming to 50 if necessary, cool. Titrate with 0.1 M perchloric acid, using 0.05 ml of 1-naphtholbenzein solution as indicator. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.01441 g of C7H5NaO2. Storage. Store protected from light. A. To 5 ml of a 5.0 per cent w/v solution in carbon dioxidefree water (solution A) add 0.1 ml of phenolphthalein solution; a pale pink colour is produced. On heating, a gas is evolved and the solution becomes red. B. Gives reaction A of bicarbonates (2.3.1). C. Solution A gives the reactions of sodium salts (2.3.1).
Tests
Appearance of solution. Solution A is clear (2.4.1), and colourless (2.4.1). Arsenic (2.3.10). Dissolve 5.0 g in 50 ml of water, add 15 ml of brominated hydrochloric acid AsT and remove the excess of bromine with a few drops of stannous chloride solution AsT. The resulting solution complies with the limit test for arsenic (2 ppm). Calcium. A 2.0 per cent w/v solution, when boiled for 5 minutes, is clear. Heavy metals (2.3.13). Mix 4.0 g with 5 ml of water and 18 ml of dilute hydrochloric acid, heat to boiling and maintain that temperature for 1 minute. Add 0.05 ml of phenolphthalein solution and sufficient 5 M ammonia dropwise to give the solution a faint pink colour, cool and dilute to 25 ml with water. The solution complies with the limit test for heavy metals, Method A (5 ppm). Iron (2.3.14). Dissolve 2.0 g in 20 ml of water and 4 ml of hydrochloric acid and dilute to 40 ml with water; the resulting solution complies with the limit test for iron (20 ppm). Carbonate. pH of solution A, when freshly prepared, not more than 8.6. Chlorides (2.3.12). 1.25 g dissolved in 15 ml of water and 2 ml of nitric acid complies with the limit test for chlorides (200 ppm). Sulphates (2.3.17). Suspend 1.0 g in 10 ml of distilled water, neutralise with hydrochloric acid and dilute to 15 ml with distilled water. The resulting solution complies with the limit test for sulphates (150 ppm). Assay. Weigh accurately about 1.5 g, dissolve in 50 ml of carbon dioxide-free water and titrate with 1 M hydrochloric acid using 0.2 ml of methyl orange solution as indicator.
Sodium Bicarbonate
Sodium Hydrogen Carbonate
NaHCO3 Mol. Wt. 84.0 Sodium Bicarbonate contains not less than 99.0 per cent and not more than 101.0 per cent of NaHCO3.
1 ml of 1 M hydrochloric acid is equivalent to 0.08401 g of NaHCO3. Storage. Store protected from moisture.
1081
SODIUM BICARBONATE INJECTION
IP 2007
Sodium Bicarbonate Injection

Sodium Bicarbonate Intravenous Infusion
Sodium Bicarbonate Injection is a sterile solution of Sodium Bicarbonate in Water for Injections. Sodium Bicarbonate Injection contains not less than 94.0 per cent and not more than 106.0 per cent of the stated amount of sodium bicarbonate, NaHCO3. Description. A clear, colourless solution.
Description. Anhydrous A white or almost white, slightly granular powder, hygroscopic. Monohydrate A white, crystalline powder or colourless crystals.
Identification
A. Dissolve 1 g in water and dilute to 10 ml with water; the solution is strongly alkaline. B. The solution prepared for test A gives reaction A of carbonates and reactions of sodium salts (2.3.1)
Identification
A. The residue on evaporation, when moistened with hydrochloric acid and introduced on a platinum wire into a flame, imparts a yellow colour to the flame. B. Gives reaction A of sodium salts and the reactions of bicarbonates (2.3.1).
Tests
Appearance of solution. Dissolve 2.0 g in 10 ml of water. The resulting solution is clear and not more intensely coloured than reference solution YS6, (2.4.1). Alkali hydroxides and bicarbonates. Dissolve 0.4 g in 20 ml of water, add 20 ml of barium chloride solution and filter. To 10 ml of the filtrate add 0.1 ml of phenolphthalein solution. The solution does not become red. Heat the remainder of the filtrate to boiling for 2 minutes. The solution remains clear. Heavy metals (2.3.13). Dissolve 2.0 g in portions in a mixture of 5 ml of hydrochloric acid and 25 ml of water. Heat the solution to boiling and cool. Add dilute sodium hydroxide solution until the solution is neutral. Dilute to 50 ml with water (solution A). 12 ml of the resulting solution complies with the limit test for heavy metals, Method A (50 ppm). Use lead standard solution (2 ppm Pb) for the standard. Iron (2.3.14). Dilute 5 ml of solution A to 10 ml with water. The solution complies with the limit test for iron (50 ppm). Chlorides (2.3.12). Dissolve 0.4 g in water, add 4 ml of dilute nitric acid and dilute to 15 ml with water. The solution complies with the limit test for chlorides (125 ppm). Sulphates (2.3.17). 15 ml of solution A complies with the limit test for sulphates, (250 ppm). Loss on drying (2.4.19). Not more than 1.0 per cent (for anhydrous form) or between 12.0 per cent and 15.0 per cent (for monohydrate form), determined on 2.0 g by drying in an oven at 300. Assay. Weigh accurately about 1.0 g, dissolve in 25 ml of water and titrate with 1 M hydrochloric acid using methyl orange solution as indicator. 1 ml of 1 M hydrochloric acid is equivalent to 0.05299 g of Na2CO3. Storage. Store in tightly-closed, non-metallic containers. Labelling. The label states whether the material is anhydrous or monohydrate.
Tests
pH (2.4.24). 7.0 to 8.5. Bacterial endotoxins (2.2.3). Not more than 0.5 Endotoxin Unit per ml. Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. Titrate a volume containing 1.5 g of Sodium Bicarbonate with 1 M hydrochloric acid using methyl orange solution as indicator. 1 ml of 1 M hydrochloric acid is equivalent to 0.08401 g of NaHCO3. Storage. Store in single dose containers. Labelling. The label states (1) the strength as the percentage w/v of Sodium Bicarbonate; (2) the approximate concentrations, in millimoles per litre, of the sodium ions and the bicarbonate ions; (3) that an injection containing visible particles in the solution should not be used.
Sodium Carbonate
Na2CO3 Na2CO3, H2O Mol. Wt. 106.0 (anhydrous) Mol.Wt.124.0 (monohydrate)
Sodium Carbonate is anhydrous or contains one molecule of water of hydration. Sodium Carbonate contains not less than 99.5 per cent and not more than 100.5 per cent of Na2CO3, calculated on the dried basis.
1082
IP 2007
SODIUM CHLORIDE
Sodium Chloride
NaCl Mol. Wt. 58.4 Sodium Chloride contains not less than 99.0 per cent and not more than 100.5 per cent of NaCl, calculated on the dried basis. Description. White or colourless crystals or a white crystalline powder.
Heavy metals (2.3.13). 4.0 g in 2 ml of dilute acetic acid and sufficient water to produce 25 ml. The solution complies with the limit test for heavy metals, Method A (5 ppm). Iodide. Moisten 5 g by adding dropwise, a solution freshly prepared by mixing 25 ml of iodide-free starch solution 2 ml of 0.5 M sulphuric acid, 0.15 ml of sodium nitrite solution and 25 ml of water and examine the mixture in daylight; the substance shows no blue colour after 5 minutes. Iron (2.3.14). 2.0 g dissolved in 20 ml of water complies with the limit test for iron (20 ppm). Sulphates (2.3.17). 2.5 ml of solution A diluted to 15 ml with water complies with the limit test for sulphates (300 ppm). Loss on drying (2.4.19). Not more than 1.0 per cent, determined on 1.0 g by drying in an oven at 105 for 3 hours. Assay. Weigh accurately about 0.1 g and dissolve in 50 ml of water in a glass-stoppered flask. Add 50.0 ml of 0.1 M silver nitrate, 5 ml of 2 M nitric acid and 2 ml of dibutyl phthalate, shake well and titrate with 0.1 M ammonium thiocyanate using 2 ml of ferric ammonium sulphate solution as indicator, until the colour becomes reddish yellow. 1 ml of 0.1 M silver nitrate is equivalent to 0.005844 g of NaCl. Sodium Chloride intended for use in the manufacture of parenteral preparations or in the manufacture of dialysis solutions complies with the following additional requirement. Potassium. Not more than 0.1 per cent, determined by flame photometry (2.4.4), using a 1.0 per cent w/v solution and measuring at 768 nm. Use suitable dilutions in water of potassium solution FP for the standard solution. Sodium Chloride intended for use in the preparation of dialysis solutions complies with the following additional requirement. Aluminium. Not more than 0.2 ppm, determined by the following method. Dissolve 20 g in 100 ml of water and add 10 ml of acetate buffer pH 6.0. Extract the resulting solution with successive quantities of 20, 20 and 10 ml of a 0.5 per cent w/v solution of 8-hydroxyquinoline in chloroform and dilute the combined extracts to 50 ml with chloroform. Use as the blank solution a mixture of 10 ml of acetate buffer pH 6.0 and 100 ml of water treated in the same manner and as the standard solution a mixture of 2 ml of aluminium standard solution (2 ppm Al), 10 ml of acetate buffer pH 6.0 and 90 ml of water treated in the same manner. Measure the fluorescence of the test solution and of the standard solution (2.4.5), using an excitation wavelength of 392 nm and a secondary filter with a transmission band centered at 518 nm, or a monochromator set to transmit at this wavelength, and setting the instrument to zero with the blank solution in each case. The fluorescence of the test solution is not greater than that of the standard solution. Storage. Store protected from light.
Identification
A. Gives the reactions of chlorides (2.3.1). B. A 20 per cent w/v solution in carbon dioxide-free water prepared from distilled water (solution A) gives the reactions of sodium salts (2.3.1).
Tests
Appearance of solution. Solution A is clear (2.4.1), and colourless (2.4.1). Acidity or alkalinity. To 20 ml of solution A add 0.1 ml of bromothymol blue solution; not more than 0.5 ml of 0.01 M hydrochloric acid or of 0.01 M sodium hydroxide is required to change the colour of the solution. Arsenic (2.3.10). Dissolve 10.0 g in 50 ml of water and 12 ml of stannated hydrochloric acid AsT. The resulting solution complies with the limit test for arsenic (1 ppm). Barium. Dissolve 2 g in 10 ml of water, and add 2 ml of dilute sulphuric acid; no turbidity is produced within 2 hours. Bromide. To 0.5 ml of solution A add 4.0 ml of water, 2.0 ml of phenol red reagent and 1.0 ml of 0.01 per cent w/v solution of chloramine T and mix immediately. After exactly 2 minutes, add 0.15 ml of 0.1 M sodium thiosulphate, mix and dilute to 10.0 ml with water. The absorbance of the solution measured at about 590 nm (2.4.7), using water as the blank, is not more than that of the standard solution prepared at the same time and in the same manner, using 5.0 ml of a 0.0003 per cent w/v solution of potassium bromide (100 ppm). Calcium and magnesium. Not more than 50 ppm, calculated as Ca, determined by the following method. Dissolve 20.0 g in 200 ml of water, and add 0.1 ml of hydrochloric acid, 5 ml of strong ammonia-ammonium chloride solution, 5 drops of eriochrome black T solution and titrate with 0.005 M disodium edetate to a blue end-point. 1 ml of 0.005 M disodium edetate is equivalent to 0.0002004 g of Ca. Ferrocyanide. Dissolve 2.0 g in 6 ml of water and add 0.5 ml of a mixture of 5 ml of a 1 per cent w/v solution of ferric ammonium sulphate in a 0.25 per cent w/v solution of sulphuric acid, and 95 ml of a 1 per cent w/v solution of ferrous sulphate; no blue colour is produced within 10 minutes.
1083
SODIUM CHLORIDE AND DEXTROSE INJECTION
IP 2007
Labelling. The label states whether or not the material is suitable for use in the manufacture of parenteral preparations or for the preparation of dialysis solutions.
multiplied by 0.9477 represents the weight, in g, of dextrose, C6H12O6, in the volume of the injection taken for assay. Storage. Store in single dose containers. On keeping, small solid particles may separate from a glass container. Labelling. The label states (1) the strength as the percentages w/v of Sodium Chloride and Dextrose; (2) that a solution containing visible particles must not be used. When the preparation is intended for intravenous infusion, the label states the approximate concentrations, in millimoles per litre, of the sodium and chloride ions and the number of grams per litre of dextrose, C6H12O6.
Sodium Chloride and Dextrose Injection

Sodium Chloride and Dextrose Intravenous Infusion; Sodium Chloride and Glucose Injection; Sodium Chloride and Glucose Intravenous Infusion
Sodium Chloride and Dextrose Injection is a sterile solution of Sodium Chloride and Dextrose in Water for Injections. Sodium Chloride and Dextrose Injection contains not less than 95.0 per cent and not more than 105.0 per cent of the stated amounts of sodium chloride, NaCl, and dextrose, C6H12O6. Description. A clear, colourless or faintly straw-coloured solution.
Sodium Chloride and Fructose Injection

Sodium Chloride and Fructose Intravenous Infusion; Sodium Chloride and Fructose Infusion; Fructose and Sodium Chloride Injection.
Sodium Chloride and Fructose Injection is a sterile solution of Sodium Chloride and Fructose in Water for Injections. Sodium Chloride and Fructose Injection contains not less than 95.0 per cent and not more than 105.0 per cent of the stated amounts of sodium chloride, NaCl, and fructose, C6H12O6. It contains no antimicrobial agent. Description. A clear, colourless or faintly straw-coloured solution.
Identification
A. To 1 ml add 0.05 ml of potassium cupri-tartrate solution; the solution remains blue and clear. Heat to boiling, a copious red precipitate is formed. B. Gives reaction B of sodium salts and reaction A of chlorides (2.3.1).
Tests
pH (2.4.24). 3.5 to 6.5. 5-Hydroxymethylfurfural and related substances. Dilute a volume containing 1.0 g of dextrose, C6H12O6, to 500 ml with water and measure the absorbance of the resulting solution at the maximum at about 284 nm (2.4.7); absorbance at about 284 nm, not more than 0.25. Bacterial endotoxins (2.2.3). Not more than 10 Endotoxin Units per g of dextrose. Other tests. Complies with the tests stated under Parenteral Preparations (Intravenous Infusions). Assay. For sodium chloride Titrate an accurately measured volume containing 0.1 g of Sodium Chloride with 0.1 M silver nitrate using potassium chromate solution as indicator. 1 ml of 0.1 M silver nitrate is equivalent to 0.005844 g of NaCl. For dextrose To an accurately measured volume containing 2 to 5 g of anhydrous dextrose, C6H12O6, add 0.2 ml of 5 M ammonia and sufficient water to produce 100.0 ml. Mix well, allow to stand for 30 minutes and measure the optical rotation in a 2-dm tube (2.4.22). The observed rotation in degrees
Identification
A. To 1 ml add 0.05 ml of potassium cupri-tartrate solution; the solution remains blue and clear. Heat to boiling, a copious red precipitate is formed. B. The solution prepared in the Assay is laevo-rotatory. C. Gives reaction B of sodium salts and reaction A of chlorides (2.3.1).
Tests
pH (2.4.24). 3.0 to 6.0. 5-Hydroxymethylfurfural and related substances. Dilute a volume containing 1.0 g of Fructose to 500.0 ml with water and measure the absorbance of the resulting solution at the maximum at about 284 nm (2.4.7); absorbance at about 284 nm, not more than 0.50. Heavy metals (2.3.13). Evaporate a volume containing 4.0 g of Fructose to 10 ml and add 2 ml of dilute acetic acid and sufficient water to produce 25 ml. The solution complies with the limit test for heavy metals, Method A (5 ppm).
1084
IP 2007
COMPOUND SODIUM CHLORIDE AND DEXTROSE INJECTION
Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Bacterial endotoxins (2.2.3). Not more than 0.5 Endotoxin Unit per ml. Assay. For sodium chloride Titrate an accurately measured volume containing about 0.1 g of Sodium Chloride with 0.1 M silver nitrate using potassium chromate solution as indicator. 1 ml of 0.1 M silver nitrate is equivalent to 0.005844 g of NaCl. For fructose To an accurately measured volume containing about 5.0 g of Fructose, add 0.2 ml of 6 M ammonia and sufficient water to produce 100.0 ml. Mix well, allow to stand for 30 minutes and measure the optical rotation in a 2-dm tube (2.4.22). The observed rotation in degrees multiplied by 0.5427 represents the weight, in g, of fructose, C6H12O6, in the volume taken for assay. Storage. Store in single dose containers. On keeping, small solid particles may separate from glass containers. Labelling. The label states (1) the strength as the percentages w/v of Sodium Chloride and Fructose; (2) when the preparation is intended for intravenous infusion, the approximate concentrations, in millimoles per litre, of the sodium and chloride ions and the number of grams per litre of fructose; (3) that a solution containing visible particles should not be used.
Identification
A. To 1 ml add 0.05 ml of potassium cupri-tartrate solution; the solution remains blue and clear. Heat to boiling, a copious red precipitate is formed. B. Gives reaction B of sodium salts and reaction A of chlorides (2.3.1). C. After evaporation to one half of its original volume, the solution gives reaction A of potassium salts and reaction B of calcium salts (2.3.1).
Tests
pH (2.4.24). 3.5 to 6.5. 5-Hydroxymethylfurfural and related substances. Dilute a volume containing 1.0 g of Dextrose to 500.0 ml with water and measure the absorbance of the resulting solution at the maximum at about 284 nm; absorbance at about 284 nm, not more than 0.25 (2.4.7). Bacterial endotoxins (2.2.3). Not more than 0.5 Endotoxin Unit per ml. Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. For sodium chloride Dilute suitably with water and determine by Method A for flame photometry (2.4.4), or by Method A for atomic absorption spectrophotometry (2.4.2), measuring at 589 nm and using sodium solution FP or sodium solution AAS respectively, suitably diluted with water for the standard solutions. 1 g of Na is equivalent to 2.542 g of NaCl. For potassium chloride Dilute suitably with water and determine by Method A for flame photometry (2.4.4), or by Method A for atomic absorption spectrophotometry (2.4.2), measuring at 767 nm and using potassium solution FP or potassium solution AAS respectively, suitably diluted with water for the standard solutions. 1 g of K is equivalent to 1.907 g of KCl. For calcium chloride To 50.0 ml add 5.0 ml of 0.01 M magnesium sulphate and 5 ml of ammonia buffer pH 10.9 and titrate with 0.01 M disodium edetate using eriochrome black T mixture as indicator. From the volume of 0.01 M disodium edetate required subtract the volume of 0.01 M magnesium sulphate added. 1 ml of the remainder of 0.01 M disodium edetate is equivalent to 0.00147 g of CaCl2, 2H2O. For total chloride To 20.0 ml add 30 ml of water, 50.0 ml of 0.1 M silver nitrate and 2 ml of nitric acid. Filter, wash the precipitate with water slightly acidified with nitric acid and titrate the excess of silver nitrate with 0.1 M ammonium
Compound Sodium Chloride and Dextrose Injection

Compound Sodium Chloride and Dextrose Intravenous Infusion
Compound Sodium Chloride and Dextrose Injection is a sterile solution containing 0.86 per cent w/v of Sodium Chloride, 0.03 per cent w/v of Potassium Chloride, 0.033 per cent w/v of Calcium Chloride and 5 per cent w/v of Dextrose in Water for Injections. Compound Sodium Chloride and Dextrose Injection contains not less than 0.82 per cent and not more than 0.90 per cent w/v of sodium chloride, NaCl, not less than 0.0285 per cent and not more than 0.0315 per cent w/v of potassium chloride, KCl, not less than 0.030 per cent and not more than 0.036 per cent w/v of calcium chloride, CaCl2,2H2O, and not less than 0.523 per cent and not more than 0.580 per cent w/v of chloride, Cl, and not less than 4.75 per cent and not more than 5.25 per cent w/v of dextrose, C6H12O6. It contains no antimicrobial agent. Description. A clear, colourless or faintly straw-coloured solution.
1085
SODIUM CHLORIDE HYPERTONIC INJECTION
IP 2007
thiocyanate using ferric ammonium sulphate solution as indicator until a reddish yellow colour is produced. Carry out a blank titration. 1 ml of 0.1 M silver nitrate is equivalent to 0.003545 g of total Chloride, calculated as Cl. For dextrose To an accurately measured volume containing 2 g to 5 g of Dextrose, add 0.2 ml of 5 M ammonia and sufficient water to produce 100.0 ml. Mix well, allow to stand for 30 minutes and measure the optical rotation in a 2-dm tube (2.4.22). The observed rotation in degrees multiplied by 0.9477 represents the weight, in g, of dextrose, C6H12O6, in the volume taken for assay. Storage. Store in single dose containers of glass or plastic at a temperature not exceeding 30. On keeping, small solid particles may separate from the solution in glass containers. Labelling. The label states (1) the strength as the percentages w/v of Sodium Chloride, Potassium Chloride, Calcium Chloride and Dextrose; (2) that the injection contains, in millimoles per litre, the following approximate amounts of the ions. sodium, 147.5, potassium, 4, calcium, 4.5, and Chloride, 156; (3) the total osmolar concentration in mOsmol per litre; (4) that the injection should not be used if it contains visible particles.
Other tests. Complies with the tests stated under Parenteral Preparations (Intravenous Infusions). Assay. To an accurately measured volume containing about 0.16 g of Sodium Chloride in a glass-stoppered flask add 50 ml of water and 50.0 ml of 0.1 M silver nitrate, 5 ml of 2 M nitric acid and 2 ml of dibutyl phthalate. Shake well and titrate with 0.1 M ammonium thiocyanate using 2 ml of ferric ammonium sulphate solution as indicator, until the colour becomes reddish yellow. 1 ml of 0.1 M silver nitrate is equivalent to 0.005844 g of NaCl. Storage. Store in single dose containers of glass or plastic. On keeping, small solid particles may separate from a glass container. Labelling. The label states (1) the strength as the percentage w/v of Sodium Chloride; (2) that a solution containing visible solid particles must not be used. When the preparation is intended for intravenous infusion, the label states that the injection contains approximately 270 millimoles each of sodium and chloride ions per litre.
Sodium Chloride Injection

Sodium Chloride Intravenous Infusion
Sodium Chloride Hypertonic Injection

Hypertonic Saline
Sodium Chloride Hypertonic Injection is a sterile 1.6 per cent w/v solution of Sodium Chloride in Water for Injections. It contains no antimicrobial agent. Sodium Chloride Hypertonic Injection contains not less than 1.52 per cent w/v and not more than 1.68 per cent w/v of sodium chloride, NaCl. Description. A clear, colourless solution.
Sodium Chloride Injection is a sterile 0.9 per cent w/v solution of Sodium Chloride in Water for Injections. It contains no antimicrobial agent. Sodium Chloride Injection contains not less than 0.85 per cent w/v and not more than 0.95 per cent w/v of sodium Chloride, NaCl. Description. A clear, colourless solution.
Identification
Gives the reactions of sodium salts and reaction A of chlorides (2.3.1).
Identification
Tests
pH (2.4.24). 4.5 to 7.0. Heavy metals (2.3.13). Evaporate 67 ml to about 20 ml, add 2 ml of dilute acetic acid and dilute to 25 ml with water. The solution complies with the limit test for heavy metals, Method A ( 0.3 ppm). Bacterial endotoxins (2.2.3). Not more than 0.5 Endotoxin Unit per ml. Other tests. Complies with the tests stated under Parenteral Preparations (Intravenous Infusions).
Tests
pH (2.4.24). 5.0 to 7.5. Heavy metals (2.3.13). Evaporate 40 ml to about 20 ml, add 2 ml of dilute acetic acid and dilute to 25 ml with water. The solution complies with the limit test for heavy metals, Method A (0.5 ppm). Bacterial endotoxins (2.2.3). Not more than 0.5 Endotoxin Unit per ml.
1086
IP 2007
COMPOUND SODIUM CHLORIDE SOLUTION
Assay. To an accurately measured volume containing about 0.16 g of Sodium Chloride in a glass-stoppered flask add 50 ml of water and 50.0 ml of 0.1 M silver nitrate, 5 ml of 2 M nitric acid and 2 ml of dibutyl phthalate. Shake well and titrate with 0.1 M ammonium thiocyanate using 2 ml of ferric ammonium sulphate solution as indicator, until the colour becomes reddish yellow. 1 ml of 0.1 M silver nitrate is equivalent to 0.005844 g of NaCl. Storage. Store in single dose containers of glass or plastic. On keeping, small solid particles may separate from a glass container. Labelling. The label states (1) the strength as the percentage w/v of Sodium Chloride; (2) that a solution containing visible solid particles must not be used. When the preparation is intended for intravenous infusion, the label states that the injection contains approximately 150 millimoles each of sodium and chloride ions per litre.
Other tests. Complies with the tests stated under Parenteral Preparations (Intravenous Infusions). Assay. For sodium chloride Dilute appropriately with water and determine by Method A for flame photometry (2.4.4), measuring at 589 nm or by Method A for atomic absorption spectrophotometry (2.4.2), using sodium solution FP, suitably diluted with water for the standard solutions. 1 g of Na is equivalent to 2.54 g of NaCl. For potassium Chloride Dilute appropriately with water and determine by Method A for flame photometry (2.4.4), measuring at 767 nm or by Method A for atomic absorption spectrophotometry (2.4.2), using potassium solution FP, suitably diluted with water for the standard solutions. 1 g of K is equivalent to 1.007 g of KCl. For Calcim Chloride To 50.0 ml add 5.0 ml of 0.01 M magnesium sulphate and 5 ml of ammonia buffer pH 10.9 and titrate with 0.01M disodium edetate using mordant black II mixture as indicator. From the volume of 0.01 M disodium edetate required subtract the volume of 0.01M magnesium sulphate added. 1 ml of the remainder of 0.01 M disodium edetate is equivalent to 0.00147 g of CaCl2, 2H2O. Storage. Store in single dose containers of glass or plastic. On keeping, small solid particles may separate from a glass container. Labelling. The label states (1) the strength as the percentages w/v of Sodium Chloride, Potassium Chloride and Calcium Chloride; (2) that a solution containing visible solid particles must not be used. When the preparation is intended for intravenous infusion, the label states that the Injection contains in millimoles per litre, the following approximate amounts of ions; sodium, 147.5; potassium, 4; calcium, 2.25 and chloride, 156.
Compound Sodium Chloride Injection

Ringers Injection
Compound Sodium Chloride Injection is a sterile solution containing 0.86 per cent w/v of Sodium Chloride, 0.03 per cent w/v of Potassium Chloride and 0.033 per cent w/v of Calcium Chloride in Water for Injections. It contains no antimicrobial agent. Compound Sodium Chloride Injection contains not less than 0.82 per cent w/v and not more than 0.90 per cent w/v of sodium chloride, NaCl, not less than 0.0285 per cent w/v and not more than 0.0315 per cent w/v of potassium chloride, KCl, and not less than 0.030 per cent w/v and not more than 0.036 per cent w/v of calcium chloride, CaCl2, 2H2O. Description. A clear, colourless solution.
Identification
Gives reaction B of sodium salts and reaction A of chlorides (2.3.1). When concentrated to one half of its original volume, it gives reaction A of potassium salts and reaction B of calcium salts (2.3.1).
Compound Sodium Chloride Solution

Ringers Solution
Compound Sodium Chloride Solution is a solution containing 0.86 per cent w/v of Sodium Chloride, 0.03 per cent w/v of Potassium Chloride and 0.033 per cent w/v of Calcium Chloride in Purified Water.The solution may be clarified by filtration. Compound Sodium Chloride Solution contains not less than 0.82 per cent w/v and not more than 0.90 per cent of sodium chloride, NaCl, not less than 0.025 per cent and not more than 0.035 per cent w/v of potassium chloride, KCl, and not less than 0.030 per cent and not more than 0.036 per cent w/v of calcium chloride, CaCl2, 2H2O.
Tests
pH (2.4.24). 5.0 to 7.5. Heavy metals (2.3.13). Evaporate 67 ml to about 20 ml, add 2 ml of dilute acetic acid and dilute to 25 ml with water. The solution complies with the limit test for heavy metals, Method A ( 0.3 ppm). Bacterial endotoxins (2.2.3). Not more than 0.5 Endotoxin Unit per ml.
1087
SODIUM CHLORIDE IRRIGATION SOLUTION
IP 2007
Description. A clear, colourless solution.
Identification
Gives reaction B of sodium salts and reaction A of chlorides (2.3.1). When concentrated to one half of its original volume, it gives reaction A of potassium salts and reaction B of calcium salts (2.3.1).
Sodium Chloride Irrigation Solution contains not less than 0.85 per cent w/v and not more than 0.95 per cent w/v of sodium Chloride, NaCl. It contains no antimicrobial agent. Description. A clear, colourless solution.
Identification
Tests
pH (2.4.24). 5.0 to 7.5. Heavy metals (2.3.13). Evaporate 67 ml to about 20 ml, add 2 ml of dilute acetic acid and dilute to 25 ml with water. The solution complies with the limit test for heavy metals, Method A ( 0.3 ppm). Assay. For sodium chloride Dilute appropriately with water and determine by Method A for flame photometry (2.4.4), measuring at 589 nm or by Method A for atomic absorption spectrophotometry (2.4.2), using sodium solution FP, suitably diluted with water for the standard solutions. 1 g of Na is equivalent to 2.54 g of NaCl. For potassium chloride Dilute appropriately with water and determine by Method A for flame photometry (2.4.4), measuring at 767 nm or by Method A for atomic absorption spectrophotometry (2.4.2), using potassium solution FP, suitably diluted with water for the standard solutions. 1 g of K is equivalent to 1.007 g of KCl. For calcium Chloride To 50.0 ml add 5.0 ml of 0.01 M magnesium sulphate and 5 ml of ammonia buffer pH 10.9 and titrate with 0.01 M disodium edetate using mordant black II mixture as indicator. From the volume of 0.01 M disodium edetate required subtract the volume of 0.01M magnesium sulphate added. 1 ml of the remainder of 0.01 M disodium edetate is equivalent to 0.00147 g of CaCl2, 2H2O. Sterility (2.2.11). Complies with the test for sterility. Storage. Store protected from light. Labelling. The label states the strength as the percentages w/v of Sodium Chloride, Potassium Chloride and Calcium Chloride. If the contents of the container are sterile, the label states (1) Sterile Compound Sodium Chloride Solution; (2) that the solution is not intended for injection.
Tests
pH (2.4.24). 4.5 to 7.0. Heavy metals (2.3.13). Evaporate 67 ml to about 20 ml, add 2 ml of dilute acetic acid and dilute to 25 ml with water. The solution complies with limit test for heavy metals, Method A ( 0.3 ppm). Bacterial endotoxins (2.2.3). Not more than 0.5 Endotoxin Unit per ml. Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. Titrate an accurately measured volume containing about 0.135 g of Sodium Chloride with 0.1 M silver nitrate using potassium chromate solution as indicator. 1 ml of 0.1 M silver nitrate is equivalent to 0.005844 g of NaCl. Storage. Store in single dose containers. The container may be designed to empty rapidly and may contain a volume of more than 1 litre. Labelling. The label states (1) the strength as the percentage w/v of Sodium Chloride; (2) that the solution should not be used if it contains visible particles; (3) For Irrigation only and Not for Injection; (4) that once the container is opened, the unused portion should be discarded.
Sodium Citrate
Trisodium Citrate
HO NaOOC COONa COONa ,2H2O
C6H5Na3O7,2H2O
Mol. Wt. 294.1
Sodium Citrate is trisodium 2-hydroxypropane-1,2,3tricarboxylate dihydrate. Sodium Citrate contains not less than 99.0 per cent and not more than 101.0 per cent of C6H5Na3O7, calculated on the anhydrous basis. Description. White, granular crystals or a white, crystalline powder; odourless; slightly deliquescent in moist air.
Sodium Chloride Irrigation Solution

Sodium Chloride Irrigation Solution is a sterile solution containing 0.9 per cent w/v of Sodium Chloride in Water for Injections.
1088
IP 2007
SODIUM CROMOGLYCATE
Identification
A. 10.0 per cent w/v solution in carbon dioxide-free water (solution A) gives the reactions of sodium salts and reaction A of citrates (2.3.1).
to cool. Titrate with 0.1 M perchloric acid, using 0.25 ml of 1-naphtholbenzein solution as indicator. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.008602 g of C6H5Na3O7. Storage. Store protected from light.
Tests
Appearance of solution. Solution A is clear (2.4.1), and colourless (2.4.1). Acidity or alkalinity. Titrate 20 ml of solution A with 0.05 M sulphuric acid or 0.1 M sodium hydroxide using thymol blue solution as indicator; not more than 0.5 ml of 0.05 M sulphuric acid or 0.1 M sodium hydroxide is required. Arsenic (2.3.10). Dissolve 5.0 g in 50 ml of water and add 15 ml of stannated hydrochloric acid AsT. The resulting solution complies with the limit test for arsenic (2 ppm). Heavy metals (2.3.13). Dissolve 2.0 g in 10 ml of water, 5 ml of dilute hydrochloric acid and sufficient water to produce 25 ml. The solution complies with the limit test for heavy metals, Method A ( 10 ppm). Chlorides (2.3.12). 25 ml of solution A complies with the limit test for chlorides (100 ppm). Oxalate. Dissolve 0.5 g in 4 ml of water, add 3 ml of hydrochloric acid and 1 g of zinc, in granules, and heat on a water-bath for 1 minute. Allow to stand for 2 minutes, decant the liquid into a test-tube containing 0.25 ml of a 1 per cent w/v solution of phenylhydrazine hydrochloride and heat to boiling. Cool rapidly, transfer to a graduated cylinder and add an equal volume of hydrochloric acid and 0.25 ml of potassium ferrocyanide solution. Shake and allow to stand for 30 minutes. Any pink colour produced is not more intense than that obtained by treating at the same time and in the same manner 4 ml of a 0.005 per cent w/v solution of oxalic acid (300 ppm, calculated as anhydrous oxalic acid). Sulphates (2.3.17). To 10 ml of solution A add 2 ml of 7 M hydrochloric acid and dilute to 15 ml with distilled water; the resulting solution complies with the limit test for sulphates (150 ppm). Tartrates. To a solution of 1 g in 2 ml of water in a test-tube, add 1 ml of a 10 per cent w/v solution of potassium acetate and 1 ml of 6 M acetic acid. Scratch the walls of the test-tube with a glass rod; no crystalline precipitate is formed. Readily carbonisable substances. Heat 0.2 g, in powder, with 10 ml of sulphuric acid (96 per cent w/w) in a water-bath at 90 1 for 1 hour and cool rapidly. The solution is not more intensely coloured than reference solution YS2 or GYS2 (2.4.1). Water (2.3.43). 11.0 to 13.0 per cent, determined on 0.3 g and after stirring for 15 minutes before titrating. Assay. Weigh accurately about 0.15 g and dissolve in 20 ml of anhydrous glacial acetic acid, warming to about 50. Allow
Sodium Cromoglycate
Sodium Cromoglicate
NaOOC O O O OH O O O COONa
C23H14Na2O11
Mol. Wt. 512.3
Sodium Cromoglycate is disodium 4,4-dioxo-5,5-(2hydroxytrimethylenedioxy)di(chromene-2-carboxylate). Sodium Cromoglycate contains not less than 98.0 per cent and not more than 101.0 per cent of C23H14Na2O11, calculated on the dried basis. Description. A white, crystalline powder; odourless; hygroscopic.
Identification
Test A may be omitted if tests B, C and D are carried out. Tests B and C may be omitted if tests A and D are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with sodium cromoglycate RS or with the reference spectrum of sodium cromoglycate. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.001 per cent w/v solution in phosphate buffer pH 7.4 shows absorption maxima at about 239 nm and 327 nm. The ratio of the absorbance at 327 nm to that at about 239 nm, 0.25 to 0.30. C. Dissolve about 5 mg in 0.5 ml of methanol, add 3 ml of a solution in methanol containing 0.5 per cent w/v of 4-aminophenazone and 1 per cent v/v of hydrochloric acid and allow to stand for 5 minutes; an intense yellow colour is produced. D. Gives reaction A of sodium salts (2.3.1).
Tests
Appearance of solution. A 2.0 per cent w/v solution in carbon dioxide-free water is not more opalescent than opalescence standard OS2 (2.4.1), and not more intensely coloured than reference solution BYS5 (2.4.1).
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SODIUM CROMOGLYCATE POWDER FOR INHALATION
IP 2007
Acidity or alkalinity. To 10 ml of a 2.0 per cent w/v solution in carbon dioxide-free water add 0.1 ml of phenolphthalein solution; the solution is colourless. Add 0.2 ml of 0.01 M sodium hydroxide; the solution is pink. Add 0.4 ml of 0.01 M hydrochloric acid; the solution is colourless. Add 0.25 ml of methyl red solution; the solution is red. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 50 volumes of ethyl acetate, 50 volumes of toluene and 5 volumes of glacial acetic acid. Test solution. A 2per cent w/v solution of the substance under examination in a mixture of 6 volumes of water, 4 volumes of tetrahydrofuran and 1 volume of acetone. Reference solution. A 0.01 per cent w/v of 1,3-bis(2-acetyl-3hydroxyphenoxy)-2-propanol RS in chloroform. Apply to the plate 10 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. The principal spot does not move from the line of application. Heavy metals (2.3.13). 2.0 g complies with the limit test for heavy metals, Method A (10 ppm). Oxalate. Dissolve 0.1 g in 20 ml of water, add 5.0 ml of iron salicylate solution and sufficient water to produce 50 ml. Measure the absorbance of the resulting solution at the maximum at about 480 nm (2.4.7). The absorbance is not less than that obtained by repeating the operation using 0.35 mg of oxalic acid in place of the substance under examination. Loss on drying (2.4.19). Not more than 10.0 per cent, determined on 0.5 g by drying over phosphorus pentoxide at 105 at a pressure of 0.3 kPa to 0.6 kPa. Assay. Weigh accurately about 0.2 g, dissolve in a mixture of 25 ml of ethane-1,2-diol and 5 ml of 2-propanol with the aid of heat and cool, add 30 ml of dioxan. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.02562 g of C23H14Na2O11. Storage. Store protected from moisture.
appropriately treated or Sodium Cromoglycate admixed with an approximately equal amount of Lactose. The contents of the capsules are in powder of a suitable fineness. Sodium Cromoglycate Powder for Inhalation contain not less than 100.0 per cent and not more than 120.0 per cent of the stated amount of sodium cromoglycate, C23H14Na2O11. Description. A white powder; hygroscopic.
Identification
A. Dissolve a suitable quantity in sufficient phosphate buffer pH 7.4 to produce a solution containing 0.001 per cent w/v of Sodium Cromoglycate. When examined in the range 230 nm to 360 nm, (2.4.7), the resulting solution shows absorption maxima at about 239 nm and 327 nm. B. To a quantity containing 0.1 g of Sodium Cromoglycate add 2 ml of water and 2 ml of 1.25 M sodium hydroxide and boil for 1 minute; a yellow colour is produced. Add 0.5 ml of diazobenzenesulphonic acid solution; a blood-red colour is produced. C. Gives reaction A of sodium salts (2.3.1). D. For capsules containing Lactose, the contents on heating with potassium cupri-tartrate solution give a copious red precipitate.
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 50 volumes of ethyl acetate, 50 volumes of toluene and 5 volumes of glacial acetic acid. Test solution. Dissolve a quantity containing 0.1 g of Sodium Cromoglycate in sufficient of a mixture of 6 volumes of water, 4 volumes of tetrahydrofuran that has been freed from stabiliser by passage through a column of suitable alumina and 1 volume of acetone to produce 5 ml and filter. Reference solution. Dilute 1 volume of the test solution to 200 volumes with the same solvent. Apply to the plate 10 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. The principal spot does not move from the line of application. Uniformity of weight. Weigh one capsule. Open without loss of shell material, remove the contents and weigh all parts of the shell; the difference between the weights represents the weight of the contents. Repeat the operation with a further 19
Sodium Cromoglycate Powder for Inhalation

Sodium Cromoglicate Powder for Inhalation; Sodium Cromoglycate Insufflation
Sodium Cromoglycate Powder for Inhalation consist of hard gelatin capsules containing either Sodium Cromoglycate
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IP 2007
SODIUM DIATRIZOATE
capsules and calculate the average weight of the contents of the 20 capsules. For preparations containing no Lactose the weight of the contents of each capsule does not deviate from the average weight by more than 25 per cent. For preparations containing Lactose, the weight of the contents of each capsule does not deviate from the average weight by more than 15 per cent except that, for two capsules, the weight of the contents may deviate by more than 25 per cent. Loss on drying (2.4.19). 9.0 to 18.0 per cent for capsules containing no Lactose and 5.5 to 10.0 per cent for capsules containing Lactose, determined on 0.5 g by drying in an oven at 105 at a pressure not exceeding 0.7 kPa. Assay. Weigh accurately a quantity of the mixed contents of 20 capsules containing about 0.1 g of Sodium Cromoglycate, dissolve in sufficient phosphate buffer pH 7.4 to produce 200.0 ml. Dilute 5.0 ml to 100.0 ml with phosphate buffer pH 7.4 and measure the absorbance of the resulting solution at 327 nm (2.4.7). Calculate the content of C23H14Na2O11 in a capsule of average content weight taking 164 as the specific absorbance at 327 nm. Storage. Store protected from light and moisture at a temperature not exceeding 30. Labelling. The label states (1) the strength in terms of the equivalent amount of Sodium Cromoglycate; (2) that the capsules are intended for use in an inhaler and are not to be swallowed; (3) where applicable, that the capsules contain Lactose.
Identification
Tests A and D may be omitted if tests B, C, E and F are carried out. Tests B, C and F may be omitted if tests A, D and E are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with sodium diatrizoate RS or with the reference spectrum of sodium diatrizoate. B. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 20 volumes of chloroform, 10 volumes of methanol and 2 volumes of strong ammonia solution. Test solution. Dissolve 0.1 g of the substance under examination in 100 ml of 0.08 per cent w/v solution of sodium hydroxide in methanol. Reference solution. A 0.1 per cent w/v of sodium diatrizoate RS in 0.08 per cent w/v solution of sodium hydroxide in methanol. Apply to the plate 10 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution. C. Heat 0.5 g in a crucible; violet vapours of iodine are evolved. D. To 20 mg add 5 ml of 1 M sodium hydroxide and boil gently under a reflux condenser for 10 minutes. Cool, add 5 ml of 2 M hydrochloric acid and cool in ice for 5 minutes. Add 4 ml of a 1 per cent w/v solution of sodium nitrite, cool in ice for 5 minutes, add 0.3 g of sulphamic acid, shake gently until effervescence ceases and add 2 ml of a 0.4 per cent w/v solution of N-(1-naphthyl) ethylenediamine dihydrochloride; an orange-red colour is produced. E. Heat 0.5 g with 1 ml of sulphuric acid on a water-bath until a pale violet solution is produced, add 2 ml of ethanol (95 per cent) and heat again; odour of ethyl acetate is produced.
Sodium Diatrizoate
Diatrizoate Sodium
COONa I N H
O H3C
I N H
O CH3
F. Gives the reactions of sodium salts (2.3.1).
Tests
Mol. Wt. 635.9 pH (2.4.24). 7.5 to 9.5, determined in a 50 per cent w/v solution. Free amine. Place 1.0 g in a 50-ml glass-stoppered volumetric flask, add 5 ml of water, 10 ml of 0.1 M sodium hydroxide and 25 ml of dimethyl sulphoxide. Stopper the flask, mix the contents gently and cool in ice, protected from light. After 5 minutes, slowly add 2 ml of hydrochloric acid, mix and allow to stand for 5 minutes. Add 2 ml of a 2 per cent w/v solution of sodium nitrite, mix and allow to stand for 5 minutes. Add 1 ml
C11H8I3N2NaO4
Sodium Diatrizoate is sodium 3,5-diacetamido-2,4,6triiodobenzoate. Sodium Diatrizoate contains not less than 98.0 per cent and not more than 101.0 per cent of C11H8I3N2NaO4, calculated on the anhydrous basis. Description. A white powder; odourless or almost odourless.
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SODIUM DIATRIZOATE INJECTION
IP 2007
of an 8 per cent w/v solution of sulphamic acid, mix and allow to stand for 5 minutes. Add 2 ml of a 0.1 per cent w/v solution of N-(1-naphthyl)ethylenediamine dihydrochloride in a 70 per cent w/v solution of 1,2-propanediol and mix. Remove the flask from the ice and allow to stand in water at 25 2 for 10 minutes, with occasional shaking. Add sufficient dimethyl sulphoxide to produce 50 ml and mix. Within 5 minutes, measure the absorbance of the resulting solution at the maximum at about 470 nm (2.4.7), using as the blank a solution prepared by treating 5 ml of water in the same manner; absorbance, not more than 0.40, calculated on the anhydrous basis. Free iodine and iodide. Dissolve 2.0 g in 24 ml of water taken in a 50-ml glass-stoppered centrifuge tube. Add 5 ml of toluene and 5 ml of 1 M sulphuric acid, shake well and centrifuge; no red colour appears in the toluene layer. To the mixture add 1 ml of a 2 per cent w/v solution of sodium nitrite, shake and centrifuge; any red colour in the toluene layer is not more intense than that produced in a solution prepared in the same manner using 2.0 ml of a 0.025 per cent w/v solution of potassium iodide and 22 ml of water (220 ppm). Heavy metals. Dissolve 1.0 g in 20.0 ml of water and 5 ml of 1 M sodium hydroxide, transfer the solution to a 50-ml Nessler cylinder, dilute with water to 40 ml and mix. Add 10 ml of sodium sulphide solution, shake and allow to stand for 5 minutes (20 ppm), the colour of the solution when viewed downward over a white surface is not more intense than that produced by treating 2.0 ml of lead standard solution (10 ppm Pb) in the same manner in place of the substance under examination. Water (2.3.43). 4.0 to 7.0 per cent, determined on 0.4 g. Assay. Weigh accurately about 0.4 g in a glass-stoppered conical flask, add 12 ml of 5 M sodium hydroxide and 1 g of zinc powder and boil under a reflux condenser for 30 minutes. Cool, rinse the condenser with 30 ml of water, filter through cotton and wash the flask and filter with two quantities, each of 20 ml, of water. To the combined filtrate and washings add 80 ml of hydrochloric acid, cool and titrate with 0.05 M potassium iodate until the dark brown colour becomes pale brown. Add 5 ml of chloroform and continue the titration, shaking well after each addition, until the chloroform becomes colourless. 1 ml of 0.05 M potassium iodate is equivalent to 0.02120 g of C11H8I3N2NaO4. Storage. Store protected from light.
amounts of suitable buffers, stabilisers and antimicrobial agents but the preparation intended for intravenous administration contains no antimicrobial preservative. Sodium Diatrizoate Injection contains not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of sodium diatrizoate, C11H8I3N2NaO4.
Identification
Evaporate a volume of the injection containing 1 g of Sodium Diatrizoate to dryness. The residue complies with following tests. A. Heat 0.5 g of residue in a crucible; violet vapours of iodine are evolved. B. To 20 mg of the residue add 5 ml of 1 M sodium hydroxide and boil gently under a reflux condenser for 10 minutes. Cool, add 5 ml of 2 M hydrochloric acid and cool in ice for 5 minutes. Add 4 ml of a 1 per cent w/v solution of sodium nitrite, cool in ice for 5 minutes, add 0.3 g of sulphamic acid, shake gently until effervescence ceases and add 2 ml of a 0.4 per cent w/v solution of N-(1-naphthyl) ethylenediamine dihydrochloride; an orange-red colour is produced. C. Heat 0.5 g of residue with 1 ml of sulphuric acid on a waterbath until a pale violet solution is produced, add 2 ml of ethanol (95 per cent) and heat again; odour of ethyl acetate is produced.
Tests
pH (2.4.24). 6.6 to 7.6. Free amine. To a volume containing 1.0 g of Sodium Diatrizoate in a 50-ml glass-stoppered volumetric flask, add 5 ml of water, 10 ml of 0.1 M sodium hydroxide and 25 ml of dimethyl sulphoxide. Stopper the flask, mix the contents gently and cool in ice, protected from light. After 5 minutes, slowly add 2 ml of hydrochloric acid, mix and allow to stand for 5 minutes. Add 2 ml of a 2 per cent w/v solution of sodium nitrite, mix and allow to stand for 5 minutes. Add 1 ml of an 8 per cent w/v solution of sulphamic acid, mix and allow to stand for 5 minutes. Add 2 ml of a 0.1 per cent w/v solution of N-(1naphthyl)ethylenediamine dihydrochloride in a 70 per cent w/v solution of 1,2-propanediol and mix. Remove the flask from the ice and allow to stand in water at 25 2 for 10 minutes, with occasional shaking. Add sufficient dimethyl sulphoxide to produce 50 ml and mix. Within 5 minutes, measure the absorbance of the resulting solution at the maximum at about 470 nm (2.4.7), using as the blank a solution prepared by treating 5 ml of water in the same manner; absorbance, not more than 0.30, calculated on the anhydrous basis. Free iodine and iodide. To a volume containing 2.0 g of Sodium Diatrizoate in 24 ml of water taken in a 50-ml glass-stoppered centrifuge tube add 5 ml of toluene and 5 ml of 1 M sulphuric
Sodium Diatrizoate Injection

Diatrizoate Sodium Injection
Sodium Diatrizoate Injection is a sterile solution of Sodium Diatrizoate in Water for Injections. It may contain small
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IP 2007
SODIUM DIHYDROGEN PHOSPHATE DIHYDRATE
acid, shake well and centrifuge; no red colour appears in the toluene layer. To the mixture add 1 ml of a 2 per cent w/v solution of sodium nitrite, shake and centrifuge; any red colour in the toluene layer is not more intense than that produced in a solution prepared in the same manner using 2.0 ml of a 0.025 per cent w/v solution of potassium iodide and 22 ml of water (220 ppm). Pyrogens (2.2.8). Complies with test for pyrogens, using per kg of the rabbits weight a volume containing 2.5 g of Sodium Diatrizoate. Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. Weigh accurately about 0.5 g in a glass-stoppered conical flask, add 12 ml of 5 M sodium hydroxide and 1 g of zinc powder and boil under a reflux condenser for 30 minutes. Cool, rinse the condenser with 30 ml of water, filter through cotton and wash the flask and filter with two quantities, each of 20 ml, of water. To the combined filtrate and washings add 80 ml of hydrochloric acid, cool and titrate with 0.05 M potassium iodate until the dark brown colour becomes pale brown. Add 5 ml of chloroform and continue the titration, shaking well after each addition, until the chloroform becomes colourless. 1 ml of 0.05 M potassium iodate is equivalent to 0.02120 g of C11H8I3N2NaO4. Storage. Store protected from light. Labelling. The label states (1) the concentration of the active ingredient; (2) whether the contents are intended for intravenous injection and, if so, that the unused portion remaining in the container after use must be discarded.
C. Solution A gives the reactions of phosphates (2.3.1).
Tests
Appearance of solution. Solution A is clear (2.4.1), and colourless (2.4.1). pH (2.4.24). 4.2 to 4.5, determined in a mixture of 5 ml of solution A and 5 ml of carbon dioxide-free water. Arsenic (2.3.10). Dissolve 0.5 g in 50 ml of water and add 10 ml of stannated hydrochloric acid AsT. The resulting solution complies with the limit test for arsenic (2 ppm). Heavy metals (2.3.13).12 ml of solution A complies with the limit test for heavy metals, Method D (10 ppm). Use lead standard solution (1 ppm Pb) to prepare the standard. Iron (2.3.14). 20 ml of solution A complies with the limit test for iron (20 ppm). Chlorides (2.3.12). 10 ml of solution A diluted to 20 ml with water complies with the limit test for chlorides (250 ppm) Sulphates (2.3.17). To 5 ml of solution A add 0.5 ml of hydrochloric acid and dilute to 15 ml with distilled water; the solution complies with the limit test for sulphates (300 ppm). Reducing substances. To 5 ml of solution A add 0.25 ml of 0.02 M potassium permanganate and 5 ml of 1 M sulphuric acid and heat in a water-bath for 5 minutes; the pink colour is not completely discharged. Disodium phosphate. Dilute 10 ml of solution A to 50 ml with water and titrate with 0.05 M sulphuric acid using bromocresol green solution as indicator; not more than 1 ml of 0.05 M sulphuric acid is required. Loss on drying (2.4.19). 21.5 to 24.0 per cent, determined on 0.25 g by drying in an oven at 130.
Sodium Dihydrogen Phosphate Dihydrate

Sodium Acid Phosphate
NaH2PO4,2H2O Mol. Wt. 156.0 Sodium Dihydrogen Phosphate Dihydrate contains not less than 98.0 per cent and not more than 100.5 per cent of NaH2PO4, calculated on the dried basis. Description. Colourless crystals or a white powder; odourless.
Assay. Weigh accurately about 2.5 g, dissolve in 40 ml of water and titrate with carbonate-free 1 M sodium hydroxide, determining the end-point potentiometrically (2.4.25). 1 ml of 1 M sodium hydroxide is equivalent to 0.120 g of NaH2PO4. Storage. Store protected from moisture.
Identification
A. Dissolve 10.0 g in sufficient carbon dioxide-free water to produce 100 ml (solution A). Solution A is faintly acid. B. Solution A neutralised with a 10 per cent w/v solution of potassium hydroxide gives reaction A of sodium salts (2.3.1).
Sodium Fluoride
NaF Mol. Wt. 41.9 Sodium Fluoride contains not less than 98.5 per cent and not more than 100.5 per cent of NaF, calculated on the dried basis. Description. A white powder or colourless crystals.
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SODIUM FORMALDEHYDE SULPHOXYLATE
IP 2007
Identification
A. Dissolve 2.5 g in sufficient carbon dioxide-free water without heating to produce 100 ml (solution A). To 2 ml of solution A add 0.5 ml of calcium chloride solution; a gelatinous white precipitate is produced which dissolves on adding 5 ml of ferric chloride solution. B. Add about 4 mg to a mixture of 0.1 ml of alizarin red S solution and 0.1 ml of zirconyl nitrate solution and mix; the colour changes to yellow. C. Gives reaction A of sodium salts (2.3.1).
Sodium Formaldehyde Sulphoxylate

O S OH ,2H2 O
Na O
CH3NaO3S,2H2O
Mol Wt. 154.1
Sodium Formaldehyde Sulphoxylate is monosodium hydroxymethane sulphinate dihydrate. It may contain a suitable stabilising agent such as sodium carbonate. Sodium Formaldehyde Sulphoxylate contains an amount of CH3NaO3S equivalent to not less than 45.0 per cent and not more than 55.0 per cent of SO2, calculated on the dried basis. Description. White crystals or hard white masses; odour, characteristic and garlic-like.
Tests
Appearance of solution. Solution A is clear (2.4.1), and colourless (2.4.1). Acidity or alkalinity. Dissolve 2.5 g of potassium nitrate in 40 ml of solution A, dilute to 50 ml with carbon dioxide-free water, cool to 0 and add 0.2 ml of dilute phenolphthalein solution. If the solution is colourless, not more than 1.0 ml of 0.1 M sodium hydroxide is required to produce a red colour that persists for not less than 15 seconds. If the solution is red, not more than 0.25 ml of 0.1 M hydrochloric acid is required to change the colour of the solution. Reserve the neutralised solution for the test for Fluorosilicate. Chlorides (2.3.12). 40 ml of solution A complies with the limit test for chlorides (250 ppm). Fluorosilicate. Heat to boiling the solution reserved in the test for Acidity or alkalinity and titrate while hot with 0.1 M sodium hydroxide until a red colour is produced. Not more than 1.5 ml of 0.1 M sodium hydroxide is required. Sulphates (2.3.17). Dissolve 0.25 g in 10 ml of a saturated solution of boric acid in distilled water and add 5 ml of distilled water and 0.6 ml of 7 M hydrochloric acid. The solution complies with the limit test for sulphates (200 ppm). Prepare the standard by mixing together 0.6 ml of 7 M hydrochloric acid, 5 ml of sulphate standard solution (10 ppm SO4) and 10 ml of a saturated solution of boric acid in distilled water. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 130 for 3 hours. Assay. Weigh accurately about 80 mg, add a mixture of 5 ml of acetic anhydride and 20 ml of anhydrous glacial acetic acid and heat to dissolve. Cool, add 20 ml of dioxan. Titrate with 0.1 M perchloric acid, using crystal violet solution as indicator, until a green colour is produced. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.004199 g of NaF. Storage. Store protected from moisture.
Identification
A. Dissolve about 4 g in 10 ml of water in a test-tube and add 1 ml of ammoniacal silver nitrate solution; metallic silver is produced either as a finely divided grey precipitate or as a bright metallic mirror on the inner surface of the tube. B. Add about 50 mg to a solution of 40 mg of salicylic acid in 5 ml of sulphuric acid and warm very gently; a permanent deep red colour develops.
Tests
Appearance of solution. A 5.0 per cent w/v solution in carbon dioxide-free water is clear (2.4.1), and colourless (2.4.1). Alkalinity. Dissolve 1.0 g in 50 ml of water and add 0.15 ml of dilute phenolphthalein solution; not more than 3.5 ml of 0.05 M sulphuric acid is required to change the colour of the solution. pH (2.4.24). 9.5 to 10.5, determined in a 2.0 per cent w/v solution in carbon dioxide-free water. Iron (2.3.14). Ignite 1.0 g, initially at a low temperature until thoroughly charred and finally at about 600, preferably in a muffle furnace, until all the carbon has been burnt off. Cool, dissolve the residue in 2 ml of hydrochloric acid and dilute to 50 ml with water. Add about 50 mg of ammonium persulphate and 5 ml of ammonium thiocyanate solution, mix and transfer to a Nessler cylinder. The red colour of the solution is not more intense than that of 1.0 ml of iron standard solution (10 ppm) treated in the same manner. Sulphides. Dissolve 6 g in 14 ml of water in a test-tube and wet a strip of lead acetate paper in the clear solution; no discolouration is evident within 5 minutes. Sodium sulphite. Not more than 5.0 per cent, calculated as
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IP 2007
SODIUM FUSIDATE
Na2SO3, determined by the following method. Transfer 4.0 ml of the solution obtained in the Assay to a flask, add 2 ml of formaldehyde solution and titrate with 0.1 M iodine that is used for the Assay, adding starch solution towards the end of the titration as indicator. Calculate the percentage of Na2SO3 from the expression 78.775(V2 V1)/W, where V1 and V2 are the volumes, in ml, of 0.1 M iodine consumed in this test and in the Assay respectively and W is the weight, in g, of the substance under examination taken for the Assay. Loss on drying (2.4.19). Not more than 27.0 per cent, determined on 0.5 g by drying in an oven at 105 for 3 hours. Assay. Weigh accurately about 1.0 g, dissolve in 25 ml of water, add sufficient water to produce 50.0 ml and mix. To 4.0 ml of this solution add 100 ml of water and titrate with 0.1 M iodine using 3 ml of starch solution, added towards the end of the titration, as indicator. 1 ml of 0.1 M iodine is equivalent to 0.001602 g of SO2. Storage. Store protected from light and moisture.
vigorously for 1 minute, allow to separate and filter the lower layer through absorbent cotton covered with anhydrous sodium sulphate. Repeat the extraction with two quantities, each of 5 ml, of chloroform, evaporate the combined extracts at a pressure of 2 kPa, dry the residue over phosphorus pentoxide at a pressure not exceeding 0.7 kPa for 2 hours and dissolve in 1 ml of chloroform IR. On the residue determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with fusidic acid RS or with the reference spectrum of fusidic acid. B. In the test for Related substances, the principal spot in the chromatogram obtained with the test solution (b) corresponds to that in the chromatogram obtained with reference solution (a). C. Ignite 1 g. The residue gives the reactions of sodium salts (2.3.1).
Tests
Appearance of solution. A 15.0 per cent w/v solution in carbon dioxide-free water is not more intensely coloured than reference solution BS6 (2.4.1). pH (2.4.24). 7.5 to 9.0, determined in a 1.25 per cent w/v solution. Specific optical rotation (2.4.22). +5.0 to +8.0, determined at 20 by dissolving 1.5 g in 25 ml of water, adding 0.1 ml of 5 M ammonia and diluting to 50 ml with water.
Sodium Fusidate
H3C CH3 COONa HO H3C H HO H3C
C31H47NaO6
H
CH 3
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 80 volumes of chloroform, 10 volumes of glacial acetic acid, 10 volumes of cyclohexane and 2.5 volumes of methanol. Test solution (a). Dissolve 0.2 g of the substance under examination in 10 ml of ethanol.
O CH3 O
CH3
H
Mol. Wt. 538.7
Sodium Fusidate is sodium (17Z)-16-acetoxy-3,11dihydroxyfusida-17(20),24-dien-21-oate, produced by the growth of certain strains of Fusidium coccineum or by any other means. Sodium Fusidate contains not less than 97.5 per cent and not more than 101.0 per cent of C31H47NaO6, calculated on the anhydrous basis. Description. A white or almost white, crystalline powder; slightly hygroscopic.
Test solution (b). Dissolve 0.2 g of the substance under examination in 100 ml of ethanol. Reference solution (a). A 0.24 per cent w/v solution of diethanolamine fusidate RS in ethanol. Reference solution (b). A 0.04 per cent w/v solution of diethanolamine fusidate RS in ethanol. Reference solution (c). A 0.04 per cent w/v solution of 3-ketofusidic acid RS in ethanol. Apply to the plate 5 l of each solution. After development, dry the plate at 110 for 10 minutes, spray with ethanolic sulphuric acid (10 per cent), dry at 110 for 10 minutes and examine in ultraviolet light at 365 nm. Any red secondary spot in the chromatogram obtained with test solution (a) is not more intense than the spot in the chromatogram obtained
Identification
A.Dissolve 0.1 g in 5 ml of water, add 5 ml of chloroform and 0.1 ml of a 10 per cent w/w solution of phosphoric acid, shake
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SODIUM FUSIDATE CAPSULES
IP 2007
with reference solution (b). Any yellow secondary spot in the chromatogram obtained with test solution (a) is not more intense than the spot in the chromatogram obtained with reference solution (c). Water (2.3.43). Not more than 2.0 per cent, determined on 0.5 g. Assay. Weigh accurately about 0.2 g and dissolve in a mixture of 15 ml of water and 20 ml of ethanol (95 per cent). Titrate with 0.1 M hydrochloric acid to pH 4.1, stirring continuously, determining the end-point potentiometrically (2.4.25). 1 ml of 0.1 M hydrochloric acid is equivalent to 0.05387 g of C31H47NaO6. Storage. Store protected from light and moisture.
Test solution (a). Extract a quantity of the contents of the capsules containing 50 mg of Sodium Fusidate with 5 ml of ethanol (95 per cent), centrifuge and use the supernatant liquid. Test solution (b). Dissolve 0.2 g of the substance under examination in 100 ml of ethanol. Reference solution (a). A 0.24 per cent w/v solution of diethanolamine fusidate RS in ethanol. Reference solution (b). A 0.04 per cent w/v solution of diethanolamine fusidate RS in ethanol. Reference solution (c). A 0.04 per cent w/v solution of 3ketofusidic acid RS in ethanol. Apply to the plate 5 l of each solution. After development, dry the plate at 110 for 10 minutes, spray with ethanolic sulphuric acid (10 per cent), dry at 110 for 10 minutes and examine in ultraviolet light (365 nm). Any red secondary spot in the chromatogram obtained with the test solution (a) is not more intense than the spot in the chromatogram obtained with reference solution (b). Any yellow secondary spot in the chromatogram obtained with the test solution (a) is not more intense than the spot in the chromatogram obtained with reference solution (c). Other tests. Complies with the tests stated under Capsules. Assay. Determine by liquid chromatography (2.4.14) Test solution. Dissolve a quantity of the mixed contents of 20 capsules containing about 25 mg of Sodium Fusidate in 20.0 ml of the mobile phase. Reference solution. A 0.15 per cent w/v solution of diethanolamine fusidate RS in the mobile phase. Chromatographic system a stainless steel column 20 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), (such as Lichrosorb RP 18), mobile phase: a mixture of 60 volumes of acetonitrile, 30 volumes of a 1 per cent v/v solution of glacial acetic acid and 10 volumes of methanol, flow rate. 1.5 ml per minute, spectrophotometer set at 235 nm, a 20 l loop injector. Inject alternately the test solution and the reference solution. The column efficiency, determined using the principal peak in the chromatogram obtained with the reference solution, should be not less than 14,000 theoretical plates per metre. Calculate the content of C31H47NaO6 in the capsules. Storage. Store protected from light and moisture.
Sodium Fusidate Capsules

Sodium Fusidate Capsules contain not less than 92.5 per cent and not more than 107.5 per cent of the stated amount of sodium fusidate, C31H47NaO6.
Ident ification
A.Dissolve a quantity of the contents of the capsules containing 0.1 g of Sodium Fusidate in 5 ml of water, add 5 ml of chloroform and 0.1 ml of a 10 per cent w/w solution of phosphoric acid, shake vigorously for 1 minute, allow to separate and filter the lower layer through absorbent cotton covered with anhydrous sodium sulphate. Repeat the extraction with two quantities, each of 5 ml, of chloroform, evaporate the combined extracts at a pressure of 2 kPa, dry the residue over phosphorus pentoxide at a pressure not exceeding 0.7 kPa for 2 hours and dissolve in 1 ml of chloroform IR. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with fusidic acid RS or with the reference spectrum of fusidic acid. B. In the test for Related substances, the principal spot in the chromatogram obtained with test solution (b) corresponds to that in the chromatogram obtained with reference solution (a). C. Ignite a portion of the contents of the capsules. The residue gives the reactions of sodium salts (2.3.1).
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 80 volumes of chloroform, 10 volumes of glacial acetic acid, 10 volumes of cyclohexane and 2.5 volumes of methanol.
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IP 2007
SODIUM LACTATE INJECTION
Sodium Hydroxide
Caustic Soda
NaOH Mol. Wt. 40.0 Sodium Hydroxide contains not less than 97.0 per cent and not more than 100.5 per cent of total alkali, calculated as NaOH. Description. White, crystalline masses supplied as sticks, pellets or slabs; deliquescent. Readily absorbs carbon dioxide. CAUTION Great care should be exercised in handling Sodium Hydroxide as it rapidly destroys tissues.
solution and titrate with 1 M hydrochloric acid. Add 0.3 ml of methyl orange solution and continue the titration with 1 M hydrochloric acid. 1 ml of 1 M hydrochloric acid used in the second part of the titration is equivalent to 0.0530 g of Na2CO3. 1 ml of 1 M hydrochloric acid used in the combined titrations is equivalent to 0.0400 g of total alkali, calculated as NaOH. Storage. Store protected from moisture, in non-metallic containers.
Identification
A. Carefully dissolve 5.0 g in 12 ml of distilled water, add 17 ml of 7 M hydrochloric acid, adjust the pH to 7.0 with 1 M hydrochloric acid and add sufficient distilled water to produce 50 ml (solution A). 2 ml of solution A gives reaction A of sodium salts (2.3.1). B. pH of a 0.01 per cent w/v solution, not less than 11.0 (2.4.24).
Sodium Lactate Injection

Sodium Lactate Intravenous Infusion
Sodium Lactate Injection is a sterile solution containing 1.85 per cent w/v of sodium lactate in Water for Injections. It is prepared from Lactic Acid with the aid of Sodium Hydroxide and sufficient Dilute Hydrochloric Acid to adjust the pH of the solution. Sodium Lactate Injection contains not less than 1.75 per cent and not more than 1.95 per cent w/v of sodium lactate, C3H5NaO3. Description. A clear, colourless solution.
Tests
Appearance of solution. A 10.0 per cent w/v solution is clear (2.4.1), and colourless (2.4.1). Arsenic (2.3.10). Dissolve 2.5 g in 50 ml of water, add 16 ml of brominated hydrochloric acid AsT, and remove the excess of bromine with a few drops of stannous chloride solution AsT. The resulting solution complies with the limit test for arsenic (4 ppm). Heavy metals (2.3.13). Dissolve 1.0 g in 5 ml of water and 10 ml of 3 M hydrochloric acid, heat to boiling, cool and dilute to 25 ml with water. The solution complies with the limit test for heavy metals, Method A ( 20 ppm). Iron (2.3.14). 20 ml of solution A complies with the limit test for iron (20 ppm). Carbonates. Not more than 2.0 per cent, calculated as Na2CO3, determined in the Assay. Chlorides (2.3.12). Dissolve 2.0 g in 5 ml of water, acidify with about 8 ml of nitric acid and dilute to 20 ml with water. The solution, without the addition of dilute nitric acid, complies with the limit test for chlorides (125 ppm). Sulphates (2.3.17). Dissolve 2.0 g in 6 ml of distilled water, adjust the pH to 7 with hydrochloric acid and dilute to 20 ml with distilled water. The resulting solution complies with the limit test for sulphates (75 ppm). Potassium. Acidify 2.5 ml of solution A with acetic acid and add 0.15 ml of sodium cobaltinitrite solution; no precipitate is formed. Assay. Weigh accurately about 2.0 g, dissolve in about 80 ml of carbon dioxide-free water, add 0.3 ml of phenolphthalein
Identification
A. When warmed with potassium permanganate, gives acetaldehyde, recognisable by its odour. B. The residue on evaporation, when moistened with hydrochloric acid and introduced on a platinum wire into a flame, imparts a yellow colour to the flame. C. Carry out reaction C of calcium salts (2.3.1); no white precipitate is produced.
Tests
pH (2.4.24). 5.0 to 7.0. Bacterial endotoxins (2.2.3). Not more than 2.0 Endotoxin Units per millimole. Other tests. Complies with the tests stated under Parenteral Preparations (Intravenous Infusions). Assay. Measure accurately 10 ml, evaporate to dryness in a platinum dish and ignite very gently until completely carbonised. Boil the residue with 25.0 ml of 0.05 M sulphuric acid, filter and wash thoroughly with hot water. Titrate the excess of acid in the combined filtrate and washings with 0.1 M sodium hydroxide using methyl orange solution as indicator. 1 ml of 0.05 M sulphuric acid is equivalent to 0.01121 g of C3H5NaO3.
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COMPOUND SODIUM LACTATE AND DEXTROSE INJECTION
IP 2007
Storage. Store in single dose containers of glass or plastic. On keeping, small solid particles may separate from the solution in glass containers. Labelling. The label states (1) that the Injection is one-sixth molar and contains, in one litre, approximately 167 millimoles each of sodium ions and of bicarbonate ions (as lactate); (2) that the injection should not be used if the solution contains visible solid particles.
D. Gives reaction C of calcium salts and reaction A of chlorides (2.3.1).
Tests
pH (2.4.24). 4.0 to 6.5. 5-Hydroxymethylfurfural and Related substances. Dilute a volume containing 1.0 g of Dextrose to 500.0 ml with water and measure the absorbance of the resulting solution at the maximum at about 284 nm; absorbance at about 284 nm, not more than 0.25 (2.4.7). Heavy metals (2.3.13). Evaporate a volume containing 4 g of dextrose to 10 ml and add 2 ml of dilute acetic acid and sufficient water to produce 25 ml. The solution complies with the limit test for heavy metals, Method A ( 5 ppm). Bacterial endotoxins (2.2.3). Not more than 0.5 Endotoxin Unit per ml. Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. For sodium Dilute suitably with water and determine by Method A for flame photometry (2.4.4), or by Method A for atomic absorption spectrophotometry (2.4.2), measuring at 589 nm and using sodium solution FP or sodium solution AAS respectively, suitably diluted with water for the standard solutions. For potassium Dilute suitably with water and determine by Method A for flame photometry (2.4.4), or by Method A for atomic absorption spectrophotometry (2.4.2), measuring at 767 nm and using potassium solution FP or potassium solution AAS respectively, suitably diluted with water for the standard solutions. For total chlorides To 20.0 ml add 30 ml of water, 50.0 ml of 0.1 M silver nitrate and 2 ml of nitric acid. Filter, wash the precipitate with water slightly acidified with nitric acid and titrate the excess of silver nitrate with 0.1 M ammonium thiocyanate using ferric ammonium sulphate solution as indicator until a reddish yellow colour is produced. Carry out a blank titration. 1 ml of 0.1 M silver nitrate is equivalent to 0.003545 g of total chloride, calculated as Cl. For calcium chloride To 50.0 ml add 5.0 ml of 0.01 M magnesium sulphate and 5 ml of ammonia buffer pH 10.9 and titrate with 0.01 M disodium edetate using eriochrome black T mixture as indicator. From the volume of 0.01 M disodium edetate required subtract the volume of 0.01 M magnesium sulphate added. 1 ml of the remainder of 0.01 M disodium edetate is equivalent to 0.00147 g of CaCl2, 2H2O. For lactate Determine by liquid chromatography (2.4.14).
Compound Sodium Lactate and Dextrose Injection

Compound Sodium Lactate with Dextrose Intravenous Infusion; Ringer-Lactate Solution with Dextrose for Injection; Hartmanns Solution with Dextrose for Injection.
Compound Sodium Lactate and Dextrose Injection is a sterile solution containing 0.24 per cent v/v of Lactic Acid (equivalent to 0.32 per cent w/v of sodium lactate) with 0.6 per cent w/v of Sodium Chloride, 0.04 per cent w/v of Potassium Chloride, 0.027 per cent w/v of Calcium Chloride and 5 per cent w/v of Dextrose in Water for Injections. Compound Sodium Lactate and Dextrose Injection contains not less than 0.27 per cent and not more than 0.32 per cent w/v of sodium, Na, not less than 0.019 per cent and not more than 0.022 per cent w/v of potassium, K, not less than 0.37 per cent and not more than 0.42 per cent w/v of total chloride, Cl, not less than 0.025 per cent and not more than 0.029 per cent w/v of calcium chloride, CaCl2,2H2O, and not less than 0.23 per cent and not more than 0.28 per cent w/v of lactate, calculated as C3H6O3, and not less than 4.50 per cent and not more than 5.25 per cent w/v of dextrose, C6H12O6. It contains no antimicrobial agent. Description. A clear, colourless or faintly straw-coloured solution.
Identification
A. To 1 ml add 0.05 ml of potassium cupri-tartrate solution; the solution remains blue and clear. Heat to boiling, a copious red precipitate is formed. B. When warmed with potassium permanganate gives acetaldehyde, recognisable by its odour. C. The residue on evaporation, when moistened with hydrochloric acid and introduced on a platinum wire into a flame imparts a yellow colour to the flame. When viewed through a suitable blue glass, the flame is tinged reddish purple.
1098
IP 2007
HALF STRENGTH COMPOUND SODIUM LACTATE AND DEXTROSE INJECTION
Test solution. The preparation under examination. Reference solution. A 0.28 per cent w/v solution of lithium lactate RS in the mobile phase. Chromatographic system a stainless steel column 20 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 90 volumes of water and 10 volumes of a 2 per cent v/v solution of octylamine in acetonitrile, the pH of which is adjusted to 7.0 with a 10 per cent v/v solution of phosphoric acid, flow rate. 2 ml per minute, spectrophotometer set at 210 nm, a 10 l loop injector. Inject separately the test solution and the reference solution and measure the responses for the major peak. Calculate the content of C3H6O3, in the injection. For dextrose To an accurately measured volume containing 2 g to 5 g of Dextrose, add 0.2 ml of 5 M ammonia and sufficient water to produce 100.0 ml. Mix well, allow to stand for 30 minutes and measure the optical rotation in a 2-dm tube (2.4.22). The observed rotation in degrees multiplied by 0.9477 represents the weight, in g, of dextrose, C6H12O6, in the volume taken for assay. Storage. Store in single dose containers of glass or plastic at a temperature not exceeding 30. On keeping, small particles may separate from the solution in glass containers. Labelling. The label states (1) that the injection contains, in millimoles per litre, the following approximate amounts of the ions. sodium, 131, potassium, 5, calcium, 2; bicarbonate (as lactate), 29 and Chloride, 111; (2) the total osmolar concentration in mOsmol per litre; (3) that the injection should not be used if it contains visible particles.
than 0.011 per cent w/v of potassium, K, not less than 0.185 per cent and not more than 0.210 per cent w/v of total chloride, Cl, not less than 0.0125 per cent and not more than 0.0145 per cent w/v of calcium chloride, CaCl2,2H2O, and not less than 0.115 per cent and not more than 0.140 per cent w/v of lactate, calculated as C3H6O3, and not less than 4.5 per cent and not more than 5.25 per cent w/v of dextrose, C6H12O6. It contains no antimicrobial agent. Description. A clear, colourless or faintly straw-coloured solution.
Identification
A. To 1 ml add 0.05 ml of potassium cupri-tartrate solution; the solution remains blue and clear. Heat to boiling, a copious red precipitate is formed. B. When warmed with potassium permanganate gives acetaldehyde, recognisable by its odour. C. The residue on evaporation, when moistened with hydrochloric acid and introduced on a platinum wire into a flame imparts a yellow colour to the flame. When viewed through a suitable blue glass, the flame is tinged reddish purple. D. Gives reaction C of calcium salts and reaction A of chlorides (2.3.1).
Tests
pH (2.4.24). 4.0 to 6.5. 5-Hydroxymethylfurfural and Related substances. Dilute a volume containing 1.0 g of Dextrose to 500.0 ml with water and measure the absorbance of the resulting solution at the maximum at about 284 nm; absorbance at about 284 nm, not more than 0.25 (2.4.7). Bacterial endotoxins (2.2.3). Not more than 0.5 Endotoxin Unit per ml. Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. For sodium Dilute suitably with water and determine by Method A for flame photometry (2.4.4), or by Method A for atomic absorption spectrophotometry (2.4.2), measuring at 589 nm and using sodium solution FP or sodium solution AAS respectively, suitably diluted with water for the standard solutions. For potassium Dilute suitably with water and determine by Method A for flame photometry (2.4.4), or by Method A for atomic absorption spectrophotometry (2.4.2), measuring at 767 nm and using potassium solution FP or potassium solution AAS respectively, suitably diluted with water for the standard solutions.
Half Strength Compound Sodium Lactate and Dextrose Injection

Half Strength Compound Sodium Lactate with Dextrose Intravenous Infusion; Half Strength Ringer-Lactate Solution with Dextrose Injection
Half Strength Compound Sodium Lactate and Dextrose Injection is a sterile solution containing 0.12 per cent v/v of Lactic Acid (equivalent to 0.16 per cent w/v of sodium lactate) with 0.3 per cent w/v of Sodium Chloride, 0.02 per cent w/v of Potassium Chloride, 0.0135 per cent w/v of Calcium Chloride and 5 per cent w/v of Dextrose in Water for Injections. Compound Sodium Lactate and Dextrose Injection contains not less than 0.135 per cent and not more than 0.16 per cent w/v of sodium, Na, not less than 0.0095 per cent and not more
1099
MODIFIED COMPOUND SODIUM LACTATE AND DEXTROSE INJECTION
IP 2007
For total chlorides To 20.0 ml add 30 ml of water, 50.0 ml of 0.1 M silver nitrate and 2 ml of nitric acid. Filter, wash the precipitate with water slightly acidified with nitric acid and titrate the excess of silver nitrate with 0.1 M ammonium thiocyanate using ferric ammonium sulphate solution as indicator until a reddish yellow colour is produced. Carry out a blank titration. 1 ml of 0.1 M silver nitrate is equivalent to 0.003545 g of total chloride, calculated as Cl. For calcium chloride To 50.0 ml add 5.0 ml of 0.01 M magnesium sulphate and 5 ml of ammonia buffer pH 10.9 and titrate with 0.01 M disodium edetate using eriochrome black T mixture as indicator. From the volume of 0.01 M disodium edetate required subtract the volume of 0.01 M magnesium sulphate added. 1 ml of the remainder of 0.01 M disodium edetate is equivalent to 0.00147 g of CaCl2, 2H2O. For lactate Determine by liquid chromatography (2.4.14). Test solution. The preparation under examination. Reference solution. A 0.28 per cent w/v solution of lithium lactate RS in the mobile phase. Chromatographic system a stainless steel column 20 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 90 volumes of water and 10 volumes of a 2 per cent v/v solution of octylamine in acetonitrile, the pH of which is adjusted to 7.0 with a 10 per cent v/v solution of phosphoric acid, flow rate. 2 ml per minute, spectrophotometer set at 210 nm, a 10 l loop injector. Inject separately the test solution and the reference solution and measure the responses for the major peak. Calculate the content of C3H6O3 in the injection. For dextrose To an accurately measured volume containing 2 g to 5 g of Dextrose, add 0.2 ml of 5 M ammonia and sufficient water to produce 100.0 ml. Mix well, allow to stand for 30 minutes and measure the optical rotation in a 2-dm tube (2.4.22). The observed rotation in degrees multiplied by 0.9477 represents the weight, in g, of dextrose, C6H12O6, in the volume taken for assay. Storage. Store in single dose containers of glass or plastic at a temperature not exceeding 30. On keeping, small particles may separate from the solution in glass containers. Labelling. The label states (1) that the injection contains, in millimoles per litre, the following approximate amounts of the ions. sodium, 65.5, potassium, 2.5, calcium, 1, bicarbonate (as lactate), 14.5, and chloride, 55.5; (2) the total osmolar
concentration in mOsmol per litre; (3) that the injection should not be used if it contains visible particles.
Modified Compound Sodium Lactate and Dextrose Injection

Modified Compound Sodium Lactate with Dextrose Intravenous Infusion; Modified Lactated Ringers and Dextrose Injection.
Modified Compound Sodium Lactate and Dextrose Injection is a sterile solution containing 0.048 per cent v/v of Lactic Acid (equivalent to 0.064 per cent w/v of sodium lactate) with 0.12 per cent w/v of Sodium Chloride, 0.008 per cent w/v of Potassium Chloride, 0.0054 per cent w/v of Calcium Chloride and 5 per cent w/v of Dextrose in Water for Injections. Modified Compound Sodium Lactate and Dextrose Injection contains not less than 0.054 per cent and not more than 0.064 per cent w/v of sodium, Na, not less than 0.0038 per cent and not more than 0.0044 per cent w/v of potassium, K, not less than 0.074 per cent and not more than 0.084 per cent w/v of total chloride, Cl, not less than 0.005 per cent and not more than 0.0058 per cent w/v of calcium chloride, CaCl2,2H2O, and not less than 0.046 per cent and not more than 0.056 per cent w/v of lactate, calculated as C3H6O3, and not less than 4.5 per cent and not more than 5.25 per cent w/v of dextrose, C6H12O6. It contains no antimicrobial agent. Description. A clear, colourless or faintly straw-coloured solution.
Identification
A. To 1 ml add 0.05 ml of potassium cupri-tartrate solution; the solution remains blue and clear. Heat to boiling, a copious red precipitate is formed. B. When warmed with potassium permanganate gives acetaldehyde, recognisable by its odour. C. The residue on evaporation, when moistened with hydrochloric acid and introduced on a platinum wire into a flame imparts a yellow colour to the flame. When viewed through a suitable blue glass, the flame is tinged reddish purple. D. Gives reaction C of calcium salts and reaction A of chlorides (2.3.1).
Tests
pH (2.4.24). 4.0 to 6.5. 5-Hydroxymethylfurfural and Related substances. Dilute a volume containing 1.0 g of Dextrose to 500.0 ml with water
1100
IP 2007
COMPOUND SODIUM LACTATE INJECTION
and measure the absorbance of the resulting solution at the maximum at about 284 nm; absorbance at about 284 nm (2.4.7), not more than 0.25. Bacterial endotoxins (2.2.3). Not more than 0.5 Endotoxin Unit per ml. Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. For sodium Dilute suitably with water and determine by Method A for flame photometry (2.4.4), or by Method A for atomic absorption spectrophotometry (2.4.2), measuring at 589 nm and using sodium solution FP or sodium solution AAS respectively, suitably diluted with water for the standard solutions. For potassium Dilute suitably with water and determine by Method A for flame photometry (2.4.4), or by Method A for atomic absorption spectrophotometry (2.4.2), measuring at 767 nm and using potassium solution FP or potassium solution AAS respectively, suitably diluted with water for the standard solutions. For total chlorides To 20.0 ml add 30 ml of water, 50.0 ml of 0.1 M silver nitrate and 2 ml of nitric acid. Filter, wash the precipitate with water slightly acidified with nitric acid and titrate the excess of silver nitrate with 0.1 M ammonium thiocyanate using ferric ammonium sulphate solution as indicator until a reddish yellow colour is produced. Carry out a blank titration. 1 ml of 0.1 M silver nitrate is equivalent to 0.003545 g of total chloride, calculated as Cl. For calcium chloride Evaporate 100.0 ml on a water-bath to 50 ml, add to this solution 5.0 ml of 0.01 M magnesium sulphate and 5 ml of ammonia buffer pH 10.9 and titrate with 0.01 M disodium edetate using eriochrome black T mixture as indicator. From the volume of 0.01 M disodium edetate required subtract the volume of 0.01 M magnesium sulphate added. 1 ml of the remainder of 0.01 M disodium edetate is equivalent to 0.00147 g of CaCl2, 2H2O. For lactate Determine by liquid chromatography (2.4.14). Test solution. The preparation under examination. Reference solution. A 0.28 per cent w/v solution of lithium lactate RS in the mobile phase. Chromatographic system a stainless steel column 20 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 90 volumes of water and 10 volumes of a 2 per cent v/v solution of octylamine in acetonitrile, the pH of which is adjusted to 7.0 with a 10 per cent v/v solution of phosphoric acid,
flow rate. 2 ml per minute, spectrophotometer set at 210 nm, a 10 l loop injector. Inject separately the test solution and the reference solution and measure the responses for the major peak. Calculate the content of C3H6O3 in the injection. For dextrose To an accurately measured volume containing 2 g to 5 g of Dextrose, add 0.2 ml of 5 M ammonia and sufficient water to produce 100.0 ml. Mix well, allow to stand for 30 minutes and determine the optical rotation in a 2-dm tube (2.4.22). The observed rotation in degrees multiplied by 0.9477 represents the weight, in g, of dextrose, C6H12O6, in the volume taken for assay. Storage. Store in single dose containers of glass or plastic at a temperature not exceeding 30. On keeping, small particles may separate from the solution in glass containers. Labelling. The label states (1) that the injection contains, in millimoles per litre, the following approximate amounts of the ions. sodium, 26.2, potassium, 1, calcium, 0.4, bicarbonate (as lactate), 5.8, and chloride, 22.2; (2) the total osmolar concentration in mOsmol per litre; (3) that the injection should not be used if it contains visible particles.
Compound Sodium Lactate Injection

Compound Sodium Lactate Intravenous Infusion; RingerLactate Solution for Injection; Hartmanns Solution for Injection
Compound Sodium Lactate Injection is a sterile solution containing 0.24 per cent v/v of Lactic Acid (equivalent to 0.32 per cent w/v of sodium lactate) with 0.6 per cent w/v of Sodium Chloride, 0.04 per cent w/v of Potassium Chloride and 0.027 per cent w/v of Calcium Chloride in Water for Injections. Compound Sodium Lactate Injection contains not less than 0.27 per cent and not more than 0.32 per cent w/v of sodium, Na, not less than 0.019 per cent and not more than 0.022 per cent w/v of potassium, K, not less than 0.37 per cent and not more than 0.42 per cent w/v of total chloride, Cl, not less than 0.025 per cent and not more than 0.029 per cent w/v of calcium chloride, CaCl2,2H2O, and not less than 0.23 per cent and not more than 0.28 per cent w/v of lactate, calculated as C3H6O3. Description. A clear, colourless solution.
Identification
A. When warmed with potassium permanganate gives acetaldehyde, recognisable by its odour. B. The residue on evaporation, when moistened with hydrochloric acid and introduced on a platinum wire into a
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COMPOUND SODIUM LACTATE SOLUTION FOR IRRIGATION
IP 2007
flame, imparts a yellow colour to the flame. When viewed through a suitable blue glass, the flame is tinged with reddish purple. C. Gives reaction C of calcium salts (2.3.1).
On keeping, small solid particles may separate from the solution in glass containers. Labelling. The label states (1) that the injection contains, in millimoles per litre, the following approximate amounts of the ions. sodium, 131, potassium, 5, calcium, 2, bicarbonate (as lactate), 29 and chloride, 111; (2) that the injection should not be used if the solution contains visible solid particles.
Tests
pH (2.4.24). 5.0 to 7.0. Bacterial endotoxins (2.2.3). Not more than 0.5 Endotoxin Unit per ml. Other tests. Complies with the tests stated under Parenteral Preparations (Intravenous Infusions). Assay. For sodium Dilute suitably with water and determine by Method A for flame photometry (2.4.4), or by atomic absorption spectrophotometry (2.4.2), measuring at 589 nm and using sodium solution FP or sodium solution AAS, suitably diluted for the standard solutions. For potassium Dilute suitably with water and determine by Method A for flame photometry (2.4.4), or by atomic absorption spectrophotometry (2.4.2), measuring at 767 nm and using potassium solution FP or potassium solution AAS, suitably diluted for the standard solutions. For total chlorides To 20 ml add 30 ml of water, 50.0 ml of 0.1 M silver nitrate and 2 ml of nitric acid, filter, wash the precipitate with water slightly acidified with nitric acid and titrate the excess of silver nitrate with 0.1 M ammonium thiocyanate using ferric ammonium sulphate solution as indicator. Carry out a blank titration. 1 ml of 0.1 M silver nitrate is equivalent to 0.003545 g of total chloride, calculated as Cl. For calcium chloride To 50 ml add 5.0 ml of 0.01 M magnesium sulphate and 5 ml of ammonia buffer pH 10.9 and titrate with 0.01 M disodium edetate using mordant black 11 mixture as indicator. From the volume of 0.01 M disodium edetate required subtract the volume of 0.01 M magnesium sulphate added. 1 ml of the remainder of 0.01 M disodium edetate is equivalent to 0.00147 g of CaCl2, 2H2O. For lactate, calculated as C3H6O3 Evaporate 50 ml to dryness in a platinum dish and ignite very gently until completely carbonised. Boil the residue with 25.0 ml of 0.05 M sulphuric acid, filter, and wash thoroughly with hot water.Titrate the excess of acid in the combined filtrate and washings with 0.1 M sodium hydroxide using methyl orange solution as indicator. 1 ml of 0.05 M sulphuric acid is equivalent to 0.009008 g of C3H6O3. Storage. Store in single dose containers of glass or plastic.
Compound Sodium Lactate Solution For Irrigation

Ringer-Lactate Solution for Irrigation; Hartmanns Solution for Irrigation
Compound Sodium Lactate Solution for Irrigation is a sterile solution containing 0.24 per cent v/v of Lactic Acid (equivalent to 0.32 per cent w/v of sodium lactate) with 0.6 per cent w/v of Sodium Chloride, 0.04 per cent w/v of Potassium Chloride and 0.027 per cent w/v of Calcium Chloride in Water for Injections. Compound Sodium Lactate Solution for Irrigation contains not less than 0.27 per cent and not more than 0.32 per cent w/v of sodium, Na, not less than 0.019 per cent and not more than 0.022 per cent w/v of potassium, K, not less than 0.37 per cent and not more than 0.42 per cent w/v of total chloride, Cl, not less than 0.025 per cent and not more than 0.029 per cent w/v of calcium chloride, CaCl2,2H2O, and not less than 0.23 per cent and not more than 0.28 per cent w/v of lactate, calculated as C3H6O3. It contains no antimicrobial agent. Description. A clear, colourless solution.
Identification
A. When warmed with potassium permanganate gives acetaldehyde, recognisable by its odour. B. The residue on evaporation, when moistened with hydrochloric acid and introduced on a platinum wire into a flame imparts a yellow colour to the flame. When viewed through a suitable blue glass, the flame is tinged reddish purple. C. Gives reaction C of calcium salts and reaction A of chlorides (2.3.1).
Tests
pH (2.4.24). 5.0 to 7.0. Bacterial endotoxins (2.2.3). Not more than 0.5 Endotoxin Unit per ml. Other tests. Complies with the tests stated under Parenteral Preparations (Injections).
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IP 2007
SODIUM LAURYL SULPHATE
Assay. For sodium Dilute suitably with water and determine by Method A for flame photometry (2.4.4), or by Method A for atomic absorption spectrophotometry (2.4.2), measuring at 589 nm and using sodium solution FP or sodium solution AAS respectively, suitably diluted with water for the standard solutions. For potassium Dilute suitably with water and determine by Method A for flame photometry (2.4.4), or by Method A for atomic absorption spectrophotometry (2.4.2), measuring at 767 nm and using potassium solution FP or potassium solution AAS respectively, suitably diluted with water for the standard solutions. For calcium chloride To 50.0 ml add 5.0 ml of 0.01 M magnesium sulphate and 5 ml of ammonia buffer pH 10.9 and titrate with 0.01 M disodium edetate using eriochrome black T mixture as indicator. From the volume of 0.01 M disodium edetate required subtract the volume of 0.01 M magnesium sulphate added. 1 ml of the remainder of 0.01 M disodium edetate is equivalent to 0.00147 g of CaCl2, 2H2O. For total chlorides To 20.0 ml add 30 ml of water, 50.0 ml of 0.1 M silver nitrate and 2 ml of nitric acid. Filter, wash the precipitate with water slightly acidified with nitric acid and titrate the excess of silver nitrate with 0.1 M ammonium thiocyanate using ferric ammonium sulphate solution as indicator until a reddish yellow colour is produced. Carry out a blank titration. 1 ml of 0.1 M silver nitrate is equivalent to 0.003545 g of total chloride, calculated as Cl. For lactate Determine by liquid chromatography (2.4.14). Test solution. The preparation under examination. Reference solution. A 0.28 per cent w/v solution of lithium lactate RS in the mobile phase. Chromatographic system a stainless steel column 20 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 90 volumes of water and 10 volumes of a 2 per cent v/v solution of octylamine in acetonitrile, the pH of which is adjusted to 7.0 with a 10 per cent v/v solution of phosphoric acid, flow rate. 2 ml per minute, spectrophotometer set at 210 nm, a 10 l loop injector. Inject separately the test solution and the reference solution and measure the responses for the major peak. Calculate the content of C3H6O3, in the injection. Storage. Store in single dose containers of glass or plastic. On keeping, small solid particles may separate from the solution
in glass containers. The container may be designed to empty rapidly and may contain a volume of more than one litre. Labelling. The label states (1) that the irrigation solution contains, in millimoles per litre, the following approximate amounts of the ions. sodium,131, potassium, 5, calcium, 2, bicarbonate (as lactate), 29, and chloride, 111; (2) that the solution should not be used if it contains visible particles; (3) For irrigation only and Not for injection; (4) that once the container is opened, the unused portion should be discarded.
Sodium Lauryl Sulphate

Sodium Lauryl Sulphate is a mixture of sodium alkyl sulphates consisting mainly of sodium dodecyl sulphate, CH3[CH2]10CH2OSO3Na. Sodium Lauryl Sulphate contains not less than 85.0 per cent of sodium alkyl sulphates, calculated as C12H25NaO4S. Description. A white or pale yellow powder or crystals.
Identification
A. A 1 per cent w/v solution, when shaken, produces plenty of foam. B. Mix 0.1 ml of a 1 per cent w/v solution with 0.1 ml of a 0.1 per cent w/v solution of methylene blue and 2 ml of 1 M sulphuric acid, add 2 ml of dichloromethane and shake; the dichloromethane layer is intensely blue. C. Mix about 10 mg with 10 ml of ethanol and heat to boiling on a water-bath, shaking frequently. Filter immediately and evaporate the ethanol. Dissolve the residue in 8 ml of water, add 3 ml of 2 M hydrochloric acid, evaporate the solution to half its volume and cool. Filter and to the filtrate add 1 ml of barium chloride solution; a white, crystalline precipitate is produced. D. Gives reaction B of sodium salts (2.3.1).
Tests
Alkalinity. Dissolve 1.0 g in 100 ml of carbon dioxide-free water and add 0.1 ml of phenol red solution. Not more than 0.5 ml of 0.1 M hydrochloric acid is required to change the colour of the solution. Non-esterified alcohols. Not more than 4 per cent, determined by the following method. Dissolve 10.0 g in 100 ml of water, add 100 ml of ethanol (95 per cent) and extract the solution with three quantities, each of 50 ml, of n-pentane, adding sodium chloride, if necessary, to promote separation of the two layers. Wash the combined organic layers with three quantities, each of 50 ml, of water. Dry the organic solution over anhydrous sodium sulphate, filter and evaporate on a
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SODIUM METABISULPHITE
IP 2007
water-bath until the odour of pentane is no longer detectable. Heat the residue at 105 for 15 minutes, cool and weigh. Sodium Chloride and Sodium Sulphate. Not more than a total of 8.0 per cent, determined by the following methods. For sodium chloride Dissolve 5.0 g in 50 ml of water, add 2 M nitric acid dropwise until the solution is neutral to litmus paper, add 2 ml of potassium chromate solution and titrate with 0.1 M silver nitrate. 1 ml of 0.1 M silver nitrate is equivalent to 0.005844 g of NaCl. For sodium sulphate Dissolve 0.5 g in 20 ml of water, warming gently if necessary, and add 1 ml of a 0.05 per cent w/v solution of dithizone in acetone. If the solution is red, add 1 M nitric acid, dropwise, until it becomes bluish green. Add 2 ml of dichloroacetic acid solution and 80 ml of acetone and titrate with 0.01 M lead nitrate until a permanent orangered colour is obtained. 1 ml of 0.01 M lead nitrate is equivalent to 0.001420 g of Na2SO4. Assay. Weigh accurately about 1.15 g, dissolve in sufficient water to produce 1000.0 ml, warming if necessary. To 20.0 ml add 15 ml of chloroform, 10 ml of dilute sulphuric acid and 1 ml of dimethyl yellow-oracet blue B solution and titrate with 0.004 M benzethonium chloride, shaking vigorously and allowing the layers to separate after each addition, until the chloroform layer acquires a permanent clear green colour. 1 ml of 0.004 M benzethonium chloride is equivalent to 0.001154 g of sodium alkyl sulphates, calculated as C12H25NaO4S. Storage. Store protected from moisture.
Tests
Acidity. A solution is acidic to phenol red solution. Arsenic (2.3.10). Mix 2.5 g in a porcelain dish with 10 ml of water, 1.25 g of potassium chlorate and 16 ml of hydrochloric acid and heat to expel chlorine, remove the last traces of chlorine with a few drops of stannous chloride solution AsT and add 35 ml of water. The resulting solution complies with the limit test for arsenic (4 ppm). Heavy metals (2.3.13). Dissolve 1.0 g in 10 ml of water, add 5 ml of hydrochloric acid and evaporate to dryness on a waterbath. Dissolve the residue in 25 ml of water containing 2 ml of dilute acetic acid. The solution complies with the limit test for heavy metals, Method A ( 20 ppm). Iron. To 0.5 g add 2 ml of hydrochloric acid and evaporate on a water-bath to dryness. Dissolve the residue in 2 ml of hydrochloric acid and 20 ml of water and add a few drops of bromine solution, cool, dilute with water to 25 ml, then add 50 mg of ammonium persulphate and 5 ml of ammonium thiocyanate solution. Any colour produced is not more intense than that obtained by adding 5 ml of ammonium thiocyanate solution to a mixture of iron standard solution (20 ppm Fe), 2 ml of hydrochloric acid, 50 mg of ammonium persulphate and sufficient water to produce 25 ml (40 ppm). Thiosulphate. Dissolve 1.0 g in 10 ml of 2 M hydrochloric acid and heat on a water-bath for 10 minutes; not more than a faint opalescence is produced. Assay. Weigh accurately about 0.1 g and dissolve in 50.0 ml 0.05 M iodine, add 1 ml of hydrochloric acid and titrate the excess of iodine with 0.1 M sodium thiosulphate using starch solution, added towards the end of the titration, as indicator. 1 ml of 0.05 M iodine is equivalent to 0.004753 g of Na2S2O5. Storage. Store protected from light and moisture. On exposure to air and moisture it is slowly oxidised to sulphate with disintegration of the crystals.
Sodium Metabisulphite
Sodium Pyrosulphite; Sodium Disulphite
Na2S2O5 Mol. Wt. 190.1 Sodium Metabisulphite may be prepared by saturating a solution of Sodium Hydroxide with sulphur dioxide and allowing crystallisation to occur. Sodium Metabisulphite contains not less than 95.0 per cent and not more than 100.5 per cent of Na2S2O5. Description. Colourless, prismatic crystals or a white or creamy white powder; odour, sulphurous.
Sodium Methylparaben
Sodium Methyl Hydroxybenzoate
O O CH3
Identification
NaO
A. Gives the reactions of sodium salts (2.3.1). B. A solution decolorises iodinated potassium iodide solution and the resulting solution gives the reactions of sulphates (2.3.1). C8H7NaO Mol. Wt. 174.1 Sodium Methylparaben is the sodium salt of methyl 4hydroxybenzoate.
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IP 2007
SODIUM PHOSPHATE
Sodium Methylparaben contains not less than 99.0 per cent and not more than 102.0 per cent of C8H7NaO3, calculated on the anhydrous basis. Description. A white, crystalline powder; odourless or almost odourless; hygroscopic.
Sodium Phosphate
Disodium Hydrogen Phosphate; Disodium Hydrogen Phosphate Dodecahydrate
Na2HPO4,12H2O Mol. Wt. 358.1 Sodium Phosphate contains not less than 98.0 per cent and not more than 101.0 per cent of Na2HPO4, calculated on the anhydrous basis. Description. Colourless, transparent crystals; very efflorescent.
Identification
A. Dissolve 0.5 g in 5 ml of water and acidify to litmus paper with hydrochloric acid; a white precipitate is produced. Wash the precipitate with water and dry. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with methylparaben RS. B. The residue on ignition gives the reactions of sodium salts (2.3.1).
Identification
A 10.0 per cent w/v solution in distilled water (solution A) gives the reactions of sodium salts and of phosphates (2.3.1).
Tests
Appearance of solution. Solution A is clear (2.4.1), and colourless (2.4.1). Arsenic (2.3.10). Dissolve 5.0 g in 50 ml of water and add 10 ml of stannated hydrochloric acid AsT. The resulting solution complies with the limit test for arsenic (2 ppm). Heavy metals (2.3.13). Dissolve 2.0 g in 10 ml of water, adding 4 ml of 1 M acetic acid and diluting to 25 ml with water. The solution complies with the limit test for heavy metals, Method A (10 ppm). Iron (2.3.14). 20 ml of solution A complies with the limit test for iron (20 ppm). Chlorides (2.3.10). To 10 ml of solution A add 10 ml of 2 M nitric acid and dilute to 20 ml with water. The resulting solution complies with the limit test for chlorides (250 ppm). Sulphates (2.3.17). To 2.5 ml of solution A add 2 ml of 2 M hydrochloric acid and dilute to 15 ml with distilled water. The resulting solution complies with the limit test for sulphates (600 ppm). Reducing substances. To 5 ml of solution A add 5 ml of 1 M sulphuric acid and 0.25 ml of 0.02 M potassium permanganate and heat on a water-bath for 5 minutes; the red colour is not completely discharged. Sodium dihydrogen phosphate. The value of the expression (n2 - 25)/(25 - n1), where n1 and n2 are the titres of 1 M sodium hydroxide obtained in the Assay, does not exceed 0.025. Water (2.3.43). 57.0 to 61.0 per cent, determined on 0.1 g dissolved in a mixture of 10 volumes of methanol and 40 volumes of dimethylformamide. Assay. Weigh accurately about 4.0 g (w), dissolve in 25 ml of water, add 25.0 ml of 1 M hydrochloric acid and titrate
Tests
Appearance of solution. A 10.0 per cent w/v solution in water is clear (2.4.1). pH (2.4.24). 9.5 to 10.5, determined in a 0.1 per cent w/v solution. Chlorides (2.3.10). Dissolve 1.0 g in 20 ml of water, add 0.2 ml of nitric acid and filter. 15 ml of the filtrate complies with the limit test for chlorides (330 ppm). Sulphates (2.3.17). Dissolve 0.5 g in 40 ml of water, add 3.5 ml of 2 M hydrochloric acid, dilute to 60 ml with water and filter. 15 ml of the filtrate complies with the limit test for sulphates (0.12 per cent). Water (2.3.43). Not more than 5.0 per cent, determined on 1.0 g. Assay. Weigh accurately about 0.1 g, gently boil under a reflux condenser with 25 ml of 1.25 M sodium hydroxide for 30 minutes. Allow to cool, add 25.0 ml of 0.0333 M potassium bromate, 5 ml of a 12.5 per cent w/v solution of potassium bromide and 10 ml of hydrochloric acid and immediately stopper the flask. Shake for 15 minutes and allow to stand for 15 minutes. Add 25 ml of dilute potassium iodide solution and shake vigorously. Titrate the liberated iodine with 0.1 M sodium thiosulphate using starch solution, added towards the end of the titration, as indicator. Repeat the operation without the substance under examination. The difference between the titrations represents the amount of potassium bromate required. The volume of 0.0333 M potassium bromate used is equivalent to half of the volume of 0.1 M sodium thiosulphate required for the titration. 1 ml of 0.0333 M potassium bromate is equivalent to 0.005804 g of C8H7NaO3. Storage. Store protected from moisture.
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SODIUM PROPYLPARABEN
IP 2007
potentiometrically with 1 M sodium hydroxide to the first inflection of the pH curve (n1 ml). Continue the titration until the second inflection of the curve is reached; the total volume of sodium hydroxide required is n2 ml. Calculate the content of Na2HPO4 from the expression 1420(25 - n1)/w (100 - d), where d is the percentage water content. Storage. Store protected from moisture.
15 ml of the filtrate complies with the limit test for sulphates (0.12 per cent). Water (2.3.43). Not more than 5.0 per cen, determined on 1.0 g. Assay. Weigh accurately about 0.1 g, gently boil under a reflux condenser with 25 ml of 1.25 M sodium hydroxide for 30 minutes. Allow to cool, add 25.0 ml of 0.0333 M potassium bromate, 5 ml of a 12.5 per cent w/v solution of potassium bromide and 10 ml of hydrochloric acid and immediately stopper the flask. Shake for 15 minutes and allow to stand for 15 minutes. Add 25 ml of dilute potassium iodide solution and shake vigorously. Titrate the liberated iodine with 0.1 M sodium thiosulphate using starch solution, added towards the end of the titration, as indicator. Repeat the operation without the substance under examination. The difference between the titrations represents the amount of potassium bromate required. The volume of 0.0333 M potassium bromate used is equivalent to half of the volume of 0.1 M sodium thiosulphate required for the titration. 1 ml of 0.0333 M potassium bromate is equivalent to 0.00674 g of C10H11NaO3. Storage. Store protected from moisture.
Sodium Propylparaben
Sodium Propyl Hydroxybenzoate
O O NaO
C10H11NaO3 Mol. Wt. 202.2
CH3
Sodium Propylparaben is the sodium salt of propyl 4hydroxybenzoate. Sodium Propylparaben contains not less than 99.0 per cent and not more than 102.0 per cent of C10H11NaO3, calculated on the anhydrous basis. Description. A white, crystalline powder; odourless or almost odourless; hygroscopic.
Sodium Salicylate
COONa OH
Identification
A. Dissolve 0.5 g in 5 ml of water and acidify to litmus paper with hydrochloric acid; a white precipitate is produced. Wash the precipitate with water and dry. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with propylparaben RS. B. The residue on ignition gives the reactions of sodium salts (2.3.1). C7H5NaO3 Mol. Wt. 160.1 Sodium Salicylate is sodium 2-hydroxybenzoate. Sodium Salicylate contains not less than 99.0 per cent and not more than 101.0 per cent of C7H5NaO3, calculated on the dried basis. Description. Colourless, small crystals or shiny flakes or a white, crystalline powder.
Tests
Appearance of solution. A 10.0 per cent w/v solution in water is clear (2.4.1). pH (2.4.24). 9.5 to 10.5, determined in a 0.1 per cent w/v solution. Chlorides (2.3.10). Dissolve 1.0 g in 20 ml of water, add 0.2 ml of nitric acid and filter. 15 ml of the filtrate complies with the limit test for chlorides (330 ppm). Sulphates (2.3.17). Dissolve 0.5 g in 40 ml of water, add 3.5 ml of 2 M hydrochloric acid, dilute to 60 ml with water and filter.
Identification
Test A may be omitted if tests B and C are carried out. Test B may be omitted if tests A and C are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with sodium salicylate RS or with the reference spectrum of sodium salicylate. B. A 10.0 per cent w/v solution in carbon dioxide-free water prepared from distilled water (solution A) gives the reactions of salicylates (2.3.1).
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IP 2007
SODIUM STARCH GLYCOLLATE
C. Gives reaction B of sodium salts (2.3.1).
Description. A very fine, white or off-white, free-flowing powder; odourless or almost odourless.
Tests
Appearance of solution. Solution A is clear (2.4.1), and not more intensely coloured than reference solution BYS6 (2.4.1). Acidity. To 20 ml of solution A add 0.1 ml of phenol red solution; the solution is yellow. Titrate with 0.01 M sodium hydroxide to a reddish violet colour; not more than 2.0 ml of 0.01 M sodium hydroxide is required. Arsenic (2.3.10). Mix 5.0 g with 10 ml of bromine solution and evaporate to dryness on a water-bath. Ignite gently, cool, dissolve the residue, ignoring any carbon, in 50 ml of water and 14 ml of brominated hydrochloric acid AsT and remove the excess of bromine with 2 ml of stannous chloride solution AsT. The resulting solution complies with the limit test for arsenic (2 ppm). Heavy metals (2.3.13). Dissolve 2.0 g in 46 ml of water, add with constant stirring 4 ml of dilute hydrochloric acid, filtering and using 25 ml of the filtrate. The solution complies with the limit test for heavy metals, Method A ( 20ppm). Chlorides (2.3.12). To 25 ml of solution A add 15 ml of water and 10 ml of 2 M nitric acid and filter. 25 ml of the filtrate complies with the limit test for chlorides (200 ppm). Sulphates (2.3.17). 2.5 ml of solution A diluted to 15 ml with distilled water complies with the limit test for sulphates (600 ppm). Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.15 g, dissolve in 30 ml of anhydrous glacial acetic acid. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.01601 g of C7H5NaO3. Storage. Store protected from light and moisture.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with sodium starch glycollate RS or with the reference spectrum of sodium starch glycollate. B. To 5 ml of a 2 per cent w/v dispersion in water add 0.05 ml of 0.005 M iodine; a dark blue colour is produced. C. The solution obtained in the test for Heavy metals gives the reactions of sodium salts (2.3.1).
Tests
pH (2.4.24). 5.5 to 7.5, determined in a 2.0 per cent w/v dispersion in carbon dioxide-free water. Heavy metals (2.3.13). To 4.0 g in a silica or platinum dish add 2 ml of a 50 per cent w/v solution of sulphuric acid, heat in a water-bath and then cautiously over a flame at about 600. Continue heating until all black particles have disappeared, allow to cool, add 0.1 ml of 1 M sulphuric acid, heat to ignition once again and allow to cool. Add 0.1 ml of 2 M ammonium carbonate, evaporate to dryness and cautiously ignite. To the residue add 5 ml of hydrochloric acid, evaporate to dryness on a water-bath and dissolve the residue in 100 ml of water. 25 ml of a solution complies with the limit test for heavy metals, Method A (20 ppm). Iron (2.3.14). 50 ml of the solution obtained in the test for Heavy metals complies with the limit test for iron (20 ppm). Sodium Chloride. Not more than 10.0 per cent, determined by the following method. To 1.0 g add 20.0 ml of 0.1 M silver nitrate and 30 ml of nitric acid and boil carefully for 30 minutes. Cool and add a sufficient volume of a saturated solution of potassium permanganate to change the colour of the solution to red. Discharge the colour by the dropwise addition of hydrogen peroxide solution (10 vol), add 3 ml of dibutyl phthalate and titrate with 0.1 M ammonium thiocyanate using ferric ammonium sulphate solution as indicator, shaking vigorously after each addition of titrant. Carry out a blank titration. 1 ml of 0.1 M silver nitrate is equivalent to 0.005844 g of NaCl. Sodium glycollate. To 0.2 g add 5 ml of glacial acetic acid, mix well and add 5 ml of water, stirring occasionally until solution is complete. Slowly add 50 ml of acetone with stirring and then add 1 g of sodium chloride. Filter, wash the residue with acetone and dilute the filtrate to 100 ml with acetone. Transfer 2 ml of this solution to an open flask, heat on a waterbath for exactly 20 minutes, cool, add 5 ml of naphthalenediol reagent and mix thoroughly. Add a further 15 ml of the same
Sodium Starch Glycollate

Sodium Carboxymethyl Starch
Sodium Starch Glycollate is the sodium salt of a poly-aglucopyranose in which some of the hydroxyl groups are in the form of carboxymethyl ether. Sodium Starch Glycollate contains not less than 2.8 per cent and not more than 4.5 per cent of sodium, Na, calculated on the material washed with Ethanol (95 per cent) and dried as described under Assay.
1107
SODIUM STIBOGLUCONATE
IP 2007
reagent, mix, cover the flask with aluminium foil and heat on a water-bath for 20 minutes. Cool and dilute to 25 ml with sulphuric acid. The absorbance (2.4.7) of the resulting solution at the maximum at about 540 nm using water as the blank, is not more than that of a solution prepared in the following manner. To 5 ml of a 0.062 per cent w/v solution of glycollic acid, previously dried at a pressure not exceeding 2 kPa for 16 hours, add 5 ml of glacial acetic acid, dilute to 100 ml with acetone and complete the procedure described above beginning at the words Transfer 2 ml.... (2.0 per cent). Microbial Contamination (2.2.9). 1.0 g is free from Escherichia coli and Salmonellae. Loss on drying (2.4.19). Not more than 10.0 per cent, determined on 0.5 g by drying in an oven at 105. Assay. Weigh accurately about 4.0 g, add 350 ml of a mixture of 4 volumes of ethanol (95 per cent) and 1 volume of water, add 0.25 ml of phenolphthalein solution and mix. Add 1 M sodium hydroxide dropwise until the colour of the suspension becomes faintly pink, shake for 30 minutes and decant through a sintered glass crucible. Repeat the extraction three times, or until a test for chloride ions is negative. Transfer the bulk of the residue to the crucible, wash the residue with ethanol (95 per cent) and dry at 110 to constant weight. Weigh accurately 0.5 g of the residue, add 80 ml of anhydrous glacial acetic acid, heat under a reflux condenser for 2 hours, cool. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.4.25). 1 ml of 0.1 M perchloric acid is equivalent to 0.0023 g of Na. Storage. Store protected from light and moisture.
Sodium Stibogluconate contains not less than 30.0 per cent and not more than 34.0 per cent of pentavalent antimony, calculated on the dried and methanol-free basis. Description. A colourless, mostly amorphous powder; odourless or almost odourless.
Identification
A. An aqueous solution is dextro-rotatory. B. Pass hydrogen sulphide into a 5 per cent w/v solution for several minutes; an orange precipitate is produced. C. When heated, it chars without melting, emitting an odour of burnt sugar and leaving a residue which gives the reactions of antimony compounds and the reactions of sodium salts (2.3.1).
Tests
pH (2.4.24). 5.0 to 5.6, determined in the solution obtained in the test for Stability of solution. Stability of solution. Heat a solution containing 10.0 per cent w/v of pentavalent antimony in an autoclave at 115.5 and at a pressure of 70 kPa for 30 minutes. The resulting solution is colourless or almost colourless. Trivalent antimony. Dissolve 2.0 g in 30 ml of water, add 15 ml of hydrochloric acid and titrate with 0.00833 M potassium bromate using methyl orange solution as indicator. Not more than 1.3 ml of 0.00833 M potassium bromate is required. Chlorides. Dissolve 2.5 g in 50 ml of water and add 2 ml of 2 M nitric acid and 75 ml of acetate buffer pH 5.0. Titrate with 0.1 M silver nitrate, determining the end-point potentiometrically (2.4.25). Not more than 3.0 ml of 0.1 M silver nitrate is required. Methanol. Not more than 2.0 per cent w/w, determined by gas chromatography (2.4.13). Test solution. Add 5 ml of water to 0.5 g of the substance under examination and mix with the aid of ultrasound until the solution is complete. Reference solution (a). Add 5 ml of a 0.2 per cent v/v solution of ethanol (internal standard) to 0.5 g of the substance under examination and mix with the aid of ultrasound until the solution is complete. Reference solution (b). Add 1 ml of a 1.0 per cent v/v solution of methanol to 5 ml of a 0.2 per cent v/v solution of the internal standard. Chromatographic system a glass column 1.5 m x 4 mm, packed with porous polymer beads (80 to 100 mesh), temperature: column. 130,
Sodium Stibogluconate
Sodium Antimony Gluconate
COONa COONa HC O O CH OH HO O CH HC O HC O Sb O Sb O CH HC OH HO CH H2C OH HO CH2
Sodium Stibogluconate is mainly the disodium salt of -oxybis[gluconato(3-)O2,O3,O4-hydroxo-antimony]. The method of manufacture is such as to ensure consistently controlled reaction stoichiometry in order to yield sodium stibogluconate that is satisfactory with regard to intrinsic toxicity.
1108
IP 2007
SODIUM THIOSULPHATE
Calculate the percentage w/w of methanol taking 0.792 g as its weight per ml at 20. Undue toxicity. Dissolve a suitable quantity of the substance under examination in water for injections to give a solution containing 28 mg of pentavalent antimony per ml. Inject intravenously 0.3 ml of the solution into each of 10 mice that have been deprived of food for not less than 17 hours. After injections allow the mice access to food and water. None of the mice dies within 24 hours. If one of the mice dies within 24 hours, repeat the test. None of the second group of mice dies within 24 hours. Loss on drying (2.4.19). Not more than 15.0 per cent, determined on 0.25 g by drying in an oven at 130 at a pressure not exceeding 0.7 kPa. Assay. Weigh accurately about 0.16 g, dissolve in 30 ml of hydrochloric acid, add 70 ml of phosphoric acid and stir carefully until completely mixed. Titrate with 0.05 M ferrous ammonium sulphate prepared using sulphuric acid (1 per cent), determining the end-point potentiometrically (2.4.25), using a platinum electrode and a silver-silver chloride reference electrode. 1 ml of 0.05 M ferric ammonium sulphate is equivalent to 0.003044 g of pentavalent antimony. Storage. Store protected from moisture.
Tests
pH (2.4.24). 5.0 to 5.6. Undue toxicity. Dilute a suitable volume of the injection under examination with sufficient water for injections to give a solution containing the equivalent of 28 mg of pentavalent antimony per ml. Inject intravenously 0.3 ml of the solution into each of 10 mice that have been deprived of food for not less than 17 hours. After injections allow the mice access to food and water. None of the mice dies within 24 hours. If one of the mice dies within 24 hours, repeat the test. None of the second group of mice dies within 24 hours. Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. To 1.0 ml, accurately measured, add 30 ml of hydrochloric acid and 70 ml of phosphoric acid and mix. Titrate with 0.05 M ferric ammonium sulphate prepared using sulphuric acid (1 per cent), determining the end-point potentiometrically (2.4.25), using a platinum electrode and a silver-silver chloride reference electrode. 1 ml of 0.05 M ferric ammonium sulphate is equivalent to 0.003044 g of pentavalent antimony. Labelling. The label states the strength in terms of the equivalent amount of pentavalent antimony per ml.
Sodium Thiosulphate Sodium Stibogluconate Injection

Sodium Antimony Gluconate Injection
Sodium Stibogluconate Injection is a sterile solution of Sodium Stibogluconate in Water for Injections. Injection in multiple dose containers must not contain phenol as preservative. Sodium Stibogluconate Injection contains not less than 9.5 per cent w/v and not more than 10.5 per cent w/v of pentavalent antimony, Sb. Description. A clear, colourless or faintly straw-coloured solution.
Sodium Hyposulphite
Na2S2O3,5H2O Mol. Wt. 248.2 Sodium Thiosulphate contains not less than 99.0 per cent and not more than 101.0 per cent of Na2S2O3,5H2O. Description. Colourless large crystals or a coarse, crystalline powder; odourless; deliquescent in moist air and effloresces in dry air at temperature above 33. It dissolves in its water of crystallisation at about 49.
Identification
A. To 0.5 ml of a 10.0 per cent w/v solution in carbon dioxidefree water (solution A) add 0.5 ml of water and 2 ml of 0.1 M silver nitrate; a white precipitate is produced which quickly becomes yellowish and finally black. B. To a portion of solution A add a few drops of iodine solution; the colour is discharged. C. Dilute 2.5 ml of solution A to 5 ml with water and add 1 ml of hydrochloric acid; a gas is evolved which turns starch-iodate paper blue and a precipitate of sulphur is produced. D. 1 ml of solution A gives reaction A of sodium salts (2.3.1).
Identification
A. It is dextro-rotatory. B. Dilute 2 ml to 10 ml with water and pass hydrogen sulphide through the solution; no immediate precipitate is produced. Continue to pass hydrogen sulphide through the solution for several minutes; an orange precipitate is produced. C. The residue obtained by evaporation to dryness, after incineration, gives the reactions of antimony compounds and the reactions of sodium salts (2.3.1).
1109
SODIUM THIOSULPHATE INJECTION
IP 2007
Tests
Appearance of solution. Solution A is clear (2.4.1), and colourless (2.4.1). pH (2.4.24). 6.0 to 8.4, determined in solution A. Arsenic (2.3.10). Dissolve 5.0 g in 50 ml of water and add 10 ml of stannated hydrochloric acid AsT. The resulting solution complies with the limit test for arsenic (2 ppm). Heavy metals (2.3.13). Dissolve 1.0 g in 10 ml of water. Slowly add 5 ml of dilute hydrochloric acid and evaporate the mixture to dryness on a water-bath. Gently boil the residue with 15 ml of water for 2 minutes and filter. Heat the filtrate to boiling, add sufficient bromine solution to the hot filtrate to produce a clear solution and add a slight excess of bromine solution. Boil the solution to expel the bromine completely, cool to room temperature and add a drop of phenolphthalein solution and sodium hydroxide solution until a slight pink colour is produced. Add 2 ml of dilute acetic acid and dilute with water to 25 ml. The solution complies with the limit test for heavy metals, Method A ( 20 ppm). Chlorides (2.3.12). To 12.5 ml of solution A add 15 ml of 2 M nitric acid, boil gently for 3 to 4 minutes, cool and filter. The filtrate complies with the limit test for chlorides (200 ppm). Sulphides. To 10 ml of solution A add 0.05 ml of a freshly prepared 5 per cent w/v solution of sodium nitroprusside; the solution does not become violet. Sulphates and sulphites (2.3.17). Dilute 2.5 ml of solution A to 10 ml with distilled water. To 3 ml of this solution add 2 ml of iodine solution and gradually add more iodine solution and dilute to 15 ml with distilled water. The resulting solution complies with the limit test for sulphates (0.2 per cent). Assay. Weigh accurately about 0.5 g, dissolve in 20 ml of water and titrate with 0.05 M iodine using starch solution, added towards the end of the titration, as indicator. 1 ml of 0.05 M iodine is equivalent to 0.02482 g of Na2S2O3,5H2O. Storage. Store protected from moisture.
Identification
A. To 0.5 ml of a 10.0 per cent w/v solution in carbon dioxidefree water (solution A) add 0.5 ml of water and 2 ml of 0.1 M silver nitrate; a white precipitate is produced which quickly becomes yellowish and finally black. B. To a portion of solution A add a few drops of iodine solution; the colour is discharged. C. Dilute 2.5 ml of solution A to 5 ml with water and add 1 ml of hydrochloric acid; a gas is evolved which turns starch-iodate paper blue and a precipitate of sulphur is produced. D. 1 ml of solution A gives reaction A of sodium salts (2.3.1).
Tests
pH (2.4.24). 7.0 to 9.0. Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. To an accurately measured volume containing about 0.5 g of Sodium Thiosulphate add about 20 ml of water and titrate with 0.05 M iodine, using 3 ml of starch solution, added towards the end of the titration, as indicator. 1 ml of 0.05 M iodine is equivalent to 0.02482 g of Na2S2O3,5H2O. Storage. Store in single dose containers.
Sodium Valproate
H3 C H3 C COONa
C8H15NaO2
Mol. Wt. 166.2
Sodium Valproate is sodium 2-propylpentanoate. Sodium Valproate contains not less than 98.5 per cent and not more than 101.0 per cent of C8H15NaO2, calculated on the dried basis. Description. A white or almost white, crystalline powder; hygroscopic.
Sodium Thiosulphate Injection

Sodium Hyposulphite Injection
Sodium Thiosulphate Injection is a sterile solution of Sodium Thiosulphate in Water for Injections. Sodium Thiosulphate Injection contains not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of sodium thiosulphate, Na2S2O3,5H2O. Description. A clear, colourless solution.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with sodium valproate RS or with the reference spectrum of sodium valproate. B. In the test for Related substances, the principal peak in the chromatogram obtained with test solution (b) corresponds to
1110
IP 2007
SODIUM VALPROATE ORAL SOLUTION
the peak in the chromatogram obtained with the reference solution. C. Dissolve 1.25 g in 20 ml of distilled water in a separating funnel, add 5 ml of 2 M nitric acid, shake and allow the mixture to stand for 12 hours; the lower layer (solution A) gives reactions of sodium salts (2.3.1).
inlet port at 200 and detector at 300, flow rate. 20 ml per minute of the carrier gas. Allow the chromatography to proceed for 2.5 times the retention time of valproic acid. Adjust the sensitivity so that the height of the peak due to the internal standard in the chromatogram obtained with test solution (a) is not less than 70 per cent of the full-scale deflection on the recorder. In the chromatogram obtained with test solution (c), the sum of the areas of any secondary peaks is not greater than the area of the peak due to the internal standard. Ignore any peaks the area of which is less than 1 per cent of the area of the peak due to the internal standard. The test is not valid unless the resolution between the peak due to the internal standard and the principal peak in the chromatogram obtained with test solution (a) is at least 12. Chlorides (2.3.12). Dissolve 1.25 g in 10 ml of water. The resulting solution complies with the limit test for chlorides (200 ppm). Sulphates (2.3.17). Solution A complies with the limit test for sulphates (200 ppm). Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B. Use 2 ml of lead standard solution (10 ppm) to prepare the standard (20 ppm). Loss on drying (2.4.19). Not more than 2.0 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.15 g and dissolve in 25 ml of anhydrous glacial acetic acid. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.01662 g of C8H15NaO2. Storage. Store protected from moisture.
Tests
Appearance of solution. A 20.0 per cent w/v solution is not more opalescent than opalescence standard OS2 (2.4.1), and is not more intensely coloured than reference solution YS6 (2.4.1). Acidity or alkalinity. To 10 ml of a 10.0 per cent w/v solution in carbon dioxide-free water add 0.1 ml of dilute phenolphthalein solution; not more than 0.75 ml of either 0.1 M sodium hydroxide or 0.1 M hydrochloric acid is required to change the colour of the solution. Related substances. Determine by gas chromatography (2.3.13). Test solution (a). Add 5 ml of 1 M sulphuric acid to 10 ml of a 0.04 per cent w/v solution of the substance under examination and shake with three quantities, each of 20 ml, of ether. Add 10 ml of a 0.02 per cent w/v solution of butyric acid (internal standard) in ether to the combined ether extracts, shake with anhydrous sodium sulphate, filter and evaporate the filtrate to a volume of about 10 ml at a temperature not exceeding 30 using a rotary evaporator. Test solution (b). Add 0.5 ml of 1 M sulphuric acid to 10 ml of a 0.04 per cent w/v solution of the substance under examination and shake with three quantities, each of 5 ml, of ether. Shake the combined ether extracts with anhydrous sodium sulphate, filter and evaporate the filtrate to a volume of about 10 ml at a temperature not exceeding 30 using a rotary evaporator. Test solution (c). Dissolve 0.5 g of the substance under examination in 10 ml of water, add 5 ml of 1 M sulphuric acid and treat as described for test solution (a) beginning at the words shake with three.... Reference solution. Prepare in the same manner as test solution (b) but using sodium valproate RS in place of the substance under examination. Chromatographic system a glass column 2.6 m x 2 mm, packed with silanised diatomaceous support (125 to 180 mesh) impregnated with 5 per cent w/w of polyethylene glycol 20,000 2nitroterephthalate and 1 per cent w/w of phosphoric acid, temperature. column.150 to 170 to obtain a retention time of about 12 minutes for valproic acid [the principal peak in test solution (b)],
Sodium Valproate Oral Solution

Sodium Valproate Elixir
Sodium Valproate Oral Solution is a solution of Sodium Valproate in a suitable flavoured vehicle. Sodium Valproate Oral Solution contains not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of sodium valproate, C8H15NaO2.
Identification
A. Shake a quantity containing about 0.25 g of Sodium Valproate with two quantities, each of 25 ml, of chloroform and discard the chloroform extracts. Add 10 ml of a saturated solution of sodium chloride and 10 ml of 2 M hydrochloric
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SODIUM VALPROATE TABLETS
IP 2007
acid, mix and shake with 25 ml of chloroform. Wash the chloroform layer with 5 ml of water, shake with anhydrous sodium sulphate, filter and evaporate to dryness. On the residue determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with valproic acid RS or with the reference spectrum of valproic acid. B. Shake a quantity containing about 0.25 g of Sodium Valproate with a mixture of 10 ml of a saturated solution of sodium chloride, 10 ml of 2 M hydrochloric acid and 25 ml of chloroform. Evaporate the chloroform layer to dryness, dissolve the residue in 2 ml of water, make just alkaline with 2 M sodium hydroxide and add 0.5 ml of a 10 per cent w/v solution of cobalt nitrate; a purple precipitate is produced which is soluble in dichloromethane.
ether and 1 volume of light petroleum (40 to 60), shake, allow to separate and reserve the ether layer. Shake the aqueous layer with a further 40 ml of the ether-light petroleum mixture and discard the aqueous layer. Shake each of the ether extracts with the same three quantities, each of 10 ml, of a saturated solution of sodium chloride, combine the extracts and evaporate to a volume of about 1 ml at a temperature not exceeding 30. Add 50 ml of ethanol (95 per cent), previously neutralised with 0.01 M sodium hydroxide using dilute phenolphthalein solution as indicator, and titrate with 0.1 M sodium hydroxide. 1 ml of 0.1 M sodium hydroxide is equivalent to 0.01662 g of C8H15NaO2. Determine the weight per ml of the preparation (2.4.29) and calculate the content of C8H15NaO2 weight in volume.
Tests
Related substances. Determine by gas chromatography (2.4.13). Test solution (a). Mix a quantity of the oral solution containing 0.50 g of Sodium Valproate with 10 ml of water, acidify with 2 M sulphuric acid and shake with three quantities, each of 20 ml, of dichloromethane. Wash the combined dichloromethane extracts with 10 ml of water, shake with anhydrous sodium sulphate, filter and evaporate the filtrate to a volume of about 10 ml at a temperature not exceeding 30 using a rotary evaporator. Test solution (b). Mix a quantity of the oral solution containing 0.5 g of Sodium Valproate with 10 ml of a 0.02 per cent w/v solution of octanoic acid (internal standard) in 0.1 M sodium hydroxide and continue as described for test solution (a) beginning at the words acidify with 2 M sulphuric acid.... Reference solution. A 0.02 per cent w/v solution of the internal standard in dichloromethane. Chromatographic system a glass column 1.5 m x 4 mm. packed with acid-washed, silanised diatomaceous support (80 to 180 mesh) coated with 15 per cent w/w of free fatty acid phase (such as Supelco FFAP 2-1063) and 1 per cent w/w of phosphoric acid, temperature. column. 170. In the chromatogram obtained with test solution (b) the sum of the areas of any secondary peaks is not greater than the area of the peak due to the internal standard. Other tests. Complies with the tests stated under Oral Liquids. Assay. Weigh accurately a quantity containing about 0.15 g of Sodium Valproate, add 50 ml of water, mix thoroughly and add 10 ml of saturated solution of sodium chloride, 10 ml of 2 M hydrochloric acid and 40 ml of a mixture of 2 volumes of
Sodium Valproate Tablets

Sodium Valproate Tablets contain not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of sodium valproate, C8H15NaO2.
Identification
A. Shake a quantity of the powdered tablets containing about 0.5 g of Sodium Valproate with 10 ml of water and centrifuge. Acidify 5 ml of the supernatant liquid with 2 M sulphuric acid, shake with 25 ml of chloroform and wash the chloroform layer with 5 ml of water. Dry by shaking with anhydrous sodium sulphate, filter and evaporate the chloroform. On the residue determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with valproic acid RS or with the reference spectrum of valproic acid. B. Shake a quantity of the powdered tablets containing about 0.25 g of Sodium Valproate with 3 ml of water and centrifuge. To 2 ml of the supernatant liquid add 0.5 ml of a 10 per cent w/v solution of cobalt nitrate; a purple precipitate is produced which is soluble in dichloromethane.
Tests
Related substances. Determine by gas chromatography (2.4.13). Test solution (a). Shake a quantity of the powdered tablets containing 0.50 g of Sodium Valproate with 10 ml of water, acidify with 2 M sulphuric acid and shake with three quantities, each of 20 ml, of dichloromethane. Wash the combined dichloromethane extracts with 10 ml of water, shake with anhydrous sodium sulphate, filter and evaporate the filtrate to a volume of about 10 ml at a temperature not exceeding 30 using a rotary evaporator.
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IP 2007
SORBIC ACID
Test solution (b). Shake a quantity of the powdered tablets containing 0.50 g of Sodium Valproate with 10 ml of a 0.020 per cent w/v solution of octanoic acid (internal standard) in 0.1 M sodium hydroxide and continue as described for test solution (a) beginning at the words acidify with 2 M sulphuric acid.... Reference solution. A 0.02 per cent w/v of the internal standard in dichloromethane. Chromatographic system a glass column 1.5 m x 4 mm, packed with acid-washed, silanised diatomaceous support (80 to 180 mesh) coated with 15 per cent w/w of free fatty acid phase (such as Supelco FFAP 2-1063) and 1 per cent w/w of phosphoric acid, temperature. column. 170, In the chromatogram obtained with test solution (b) the sum of the areas of any secondary peaks is not greater than the area of the peak due to the internal standard. Other tests. Comply with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 0.3 g of Sodium Valproate, add 70 ml of water, shake for 10 minutes, dilute to 100.0 ml with water and filter. To 50.0 ml of the filtrate add 10 ml of a 30 per cent w/v solution of sodium chloride and 10 ml of 2 M hydrochloric acid. Extract with 40 ml of a mixture of 2 volumes of ether and 1 volume of light petroleum (boiling range 40 to 60) and allow to separate. Shake the aqueous layer with a further 40 ml of the ether-light petroleum mixture. Shake each of the ether extracts with the same three quantities, each of 10 ml, of a saturated solution of sodium chloride, combine the ether extracts and evaporate to a volume of about 1 ml at a temperature not exceeding 30. Add 50 ml of ethanol (95 per cent), previously neutralised with 0.01 M sodium hydroxide using dilute phenolphthalein solution as indicator, and titrate with 0.1 M sodium hydroxide. 1 ml of 0.1 M sodium hydroxide is equivalent to 0.01662 g of C8H15NaO2.
Identification
Test A may be omitted if tests B and C are carried out. Tests B and C may be omitted if test A is carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with sorbic acid RS or with the reference spectrum of sorbic acid. B. Dissolve 50 mg in sufficient water to produce 250 ml and dilute 2 ml of this solution to 200 ml with 0.1 M hydrochloric acid. When examined in the range 230 nm to 360 nm (2.4.7), the resulting solution shows an absorption maximum at about 264 nm; absorbance at about 264 nm, 0.43 to 0.51. C. Dissolve 0.2 g in 2 ml of ethanol (95 per cent) and add 0.2 ml of bromine water; the solution is decolorised.
Tests
Appearance of solution. A 5.0 per cent w/v solution in ethanol (95 per cent) is clear (2.4.1), and colourless (2.4.1). Heavy metals (2.3.13). 2.0 g complies with the limit test for heavy metals, Method A (10 ppm). Aldehydes. Not more than 0.15 per cent, determined by the following method. Dissolve 1.0 g in a mixture of 50 ml of 2-propanol and 30 ml of water, adjust the pH of the solution to 4.0 with 0.1 M hydrochloric acid or 0.1 M sodium hydroxide and dilute to 100 ml with water. To 10 ml of the resulting solution add 1 ml of decolorised fuchsin solution and allow to stand for 30 minutes. Any colour produced is not more intense than that obtained in a solution prepared simultaneously by adding 1 ml of decolorised fuchsin solution to a mixture of 1.5 ml of acetaldehyde standard solution (100 ppm C2H4O), 4 ml of 2-propanol and 4.5 ml of water. Sulphated ash (2.3.18). Not more than 0.2 per cent. Water (2.3.43). Not more than 1.0 per cent, determined on 2.0 g. Assay. Weigh accurately about 0.2 g, dissolve in 20 ml of ethanol (95 per cent) and titrate with 0.1 M sodium hydroxide, using 0.2 ml of phenolphthalein solution as indicator, until a pink colour is produced.
Sorbic Acid
H3 C COOH
1 ml of 0.1 M sodium hydroxide is equivalent to 0.01121 g of C6H8O2. Mol. Wt. 112.1 Storage. Store protected from light and moisture.
C6H8O2 Sorbic Acid is (2E,4E)-hexa-2,4-dienoic acid.
Sorbic Acid contains not less than 99.0 per cent and not more than 101.0 per cent of C6H8O2, calculated on the anhydrous basis. 1113
SORBITOL
IP 2007
Sorbitol
D-Glucitol
after washing rapidly with 5 ml of a mixture of equal volumes of methanol and water and drying in a current of air, melts at about 175 (2.4.21).
H HO H H
CH2OH C OH C H C OH C OH CH2OH
Mol. Wt. 182.2
Tests
Appearance of solution. Solution A is clear (2.4.1), and colourless (2.4.1). Acidity or alkalinity. Dissolve 5.0 g in sufficient carbon dioxide-free water prepared from distilled water to produce 50 ml (solution A). To 10 ml of solution A add 10 ml of carbon dioxide-free water. To 10 ml of the resulting solution add 0.05 ml of phenolphthalein solution; not more than 0.2 ml of 0.01 M sodium hydroxide is required to change the colour of the solution to pink. To a further 10 ml of the solution add 0.05 ml of methyl red solution; Not more than 0.3 ml of 0.01 M hydrochloric acid is required to change the colour of the solution to red. Specific optical rotation (2.4.22). +4.0 to +7.0, determined in a solution prepared in the following manner. Dissolve a mixture of 5.0 g of the substance under examination and 6.4 g of borax in 40 ml of water, allow to stand for 1 hour, shaking occasionally, dilute to 50 ml with water and filter, if necessary. Arsenic (2.3.10). Dissolve 5.0 g in 50 ml of water and add 10 ml of stannated hydrochloric acid AsT. The resulting solution complies with the limit test for arsenic (2 ppm). Heavy metals (2.3.13). 2.0 g complies with the limit test for heavy metals, Method A (10 ppm). Chlorides (2.3.12). 5.0 g complies with the limit test for chlorides (50 ppm). Nickel. Dissolve 10.0 g in sufficient water to produce 20 ml, add 3 ml of bromine water and 2 ml of a 20 per cent w/v solution of citric acid, mix and add 10 ml of 6 M ammonia and 1 ml of a 1 per cent w/v solution of dimethylglyoxime in ethanol (95 per cent). Mix, dilute to 50 ml with water and allow to stand for 5 minutes; any colour produced is not more intense than that produced by treating in the same manner and at the same time 1.0 ml of nickel standard solution (10 ppm Ni) diluted to 20 ml with water (1 ppm). Sulphates (2.3.17). 12 ml of solution A complies with the limit test for sulphates (125 ppm). Reducing sugars. Dissolve 5.0 g in 3 ml of water with the aid of gentle heat, cool, add 20 ml of cupri-citric solution and a few glass beads, heat in such a manner that the solution boils in 4 minutes and continue boiling for a further 3 minutes. Cool rapidly and add 100 ml of a 2.4 per cent v/v solution of glacial acetic acid followed by 20.0 ml of 0.025 M iodine. Add, shaking continuously, 25 ml of a 6 per cent v/v solution of hydrochloric acid and, when any precipitate has redissolved, titrate the excess iodine with 0.05 M sodium thiosulphate
C6H14O6
Sorbitol is D-glucitol, a hexahydric alcohol related to glucose. Sorbitol contains not less than 98.0 per cent and not more than 101.0 per cent of C6H14O6, calculated on the anhydrous basis. Description. A white, crystalline powder; odourless.
Identification
A. Determine by thin-layer chromatography (2.4.17), coating the plate with a uniform 0.75-mm thick layer of the following mixture. Mix 0.1 g of carbomer with 110 ml of water and allow to stand, with gentle stirring, for 1 hour. Adjust to pH 7.0 by the gradual addition, with continuous shaking, of 2 M sodium hydroxide and add 30 g of silica gel H. Heat the plate at 110 for 1 hour, allow to cool and use immediately. Mobile phase. A mixture of 85 volumes of 2-propanol and 15 volumes of a 0.2 per cent w/v solution of boric acid. Test solution. Dissolve 0.25 g of the substance under examination in 100 ml of ethanol (95 per cent). Reference solution. A 0.25 per cent w/v solution of sorbitol RS in ethanol (95 per cent). Apply to the plate 2 l of each solution. After development, dry the plate at 100 to 105 for 15 minutes, allow to cool, spray with a 0.5 per cent w/v solution of potassium permanganate in 1 M sodium hydroxide and heat at 100 for 2 minutes. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution. B. Dissolve 50 mg in 3 ml of water, add 3 ml of catechol solution and pour the mixture into 6 ml of sulphuric acid; a pink colour is produced. C. Dissolve 5 g in 3 ml of water with the aid of gentle heat, cool, add 7 ml of methanol, 1 ml of benzaldehyde and 1 ml of hydrochloric acid, mix and shake continuously for 2 hours. Filter, dissolve the crystals in 20 ml of boiling sodium bicarbonate solution and allow to crystallise. The residue,
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IP 2007
SORBITOL SOLUTION (70 PER CENT) (CRYSTALLISING)
using 1 ml of starch solution, added towards the end of the titration, as indicator. Not less than 12.8 ml of 0.05 M sodium thiosulphate is required. Sulphated ash (2.3.18). Not more than 0.1 per cent. Water (2.3.43). Not more than 1.5 per cent, determined on 1.0 g. Assay. Weigh accurately about 0.4 g and dissolve in sufficient water to produce 100.0 ml. To 10.0 ml of the solution add 20 ml of a 2.14 per cent w/v solution of sodium periodate and 2 ml of 1 M sulphuric acid and heat on a water-bath for exactly 15 minutes. Cool, add 3 g of sodium bicarbonate, in small quantities, and 25.0 ml of 0.1 M sodium arsenite, mix, add 5 ml of a 20 per cent w/v solution of potassium iodide and allow to stand for 15 minutes. Titrate with 0.05 M iodine until the first trace of yellow colour appears. Repeat the operation without the substance under examination. The difference between the titrations represents the amount of iodine required. 1 ml of 0.05 M iodine is equivalent to 0.001822 g of C6H14O6. Sorbitol intended for use in the manufacture of parenteral preparations without a further appropriate procedure for removal of bacterial endotoxins complies with the following additional requirement. Bacterial endotoxins (2.2.3). Not more than 4.0 Endotoxin Units per g for parenteral preparations having a concentration of less than 10 per cent w/v of sorbitol and not more than 2.5 Endotoxin Units per g for parenteral preparations containing 10 per cent w/v or more of sorbitol. Storage. Store protected from moisture. Labelling. The label states whether or not the contents are intended for use in the manufacture of parenteral preparations.
B. Dry 1 g over phosphorus pentoxide at a pressure of 1.5 to 2.5 kPa. Heat 0.5 g of the residue with a mixture of 5 ml of acetic anhydride and 0.5 ml pyridine with the aid of heat and allow to stand for 10 minutes. Pour the mixture into 25 ml of water, allow to stand in ice for 2 hours and filter. The precipitate, after recrystallisation from a small volume of ethanol (95 per cent) and drying over phosphorus pentoxide at a pressure of 1.5 to 2.5 kPa, melts at about 100 (2.4.21). C. Determine by thin-layer chromatography (2.4.17), coating the plate with a uniform 0.75-mm thick layer of the following mixture. Mix 0.1 g of carbomer with 110 ml of water and allow to stand, with gentle stirring, for 1 hour. Adjust to pH 7.0 by the gradual addition, with continuous shaking, of 2 M sodium hydroxide and add 30 g of silica gel H. Heat the plate at 110 for 1 hour, allow to cool and use immediately. Mobile phase. A mixture of 85 volumes of 2-propanol and 15 volumes of a 0.2 per cent w/v solution of boric acid. Test solution. Dissolve 0.35 g of the substance under examination in 100 ml of ethanol (95 per cent). Reference solution. A 0.25 per cent w/v solution of sorbitol RS in ethanol (95 per cent). Apply to the plate 2 l of each solution. After development, dry the plate at 100 to 105 for 15 minutes, allow to cool, spray with a 0.5 per cent w/v solution of potassium permanganate in 1 M sodium hydroxide and heat at 100 for 2 minutes. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution.
Tests
Appearance of solution. A 14.0 per cent w/v solution in carbon dioxidefree water (solution A) is clear (2.4.1), and colourless (2.4.1). Acidity or alkalinity. To 10 ml of solution A add 0.05 ml of phenolphthalein solution; not more than 0.2 ml of 0.01 M sodium hydroxide is required to change the colour of the solution to pink. To a further 10 ml of the solution add 0.05 ml of methyl red solution. Not more than 0.3 ml of 0.01 M hydrochloric acid is required to change the colour of the solution to red. Refractive index (2.4.27). 1.457 to 1.462. Relative density (2.4.29). Not less than 1.290. Arsenic (2.3.10). Dissolve 5.0 g in 50 ml of water and add 10 ml of stannated hydrochloric acid AsT. The resulting solution complies with the limit test for arsenic (2 ppm). Heavy metals (2.3.13). 2.0 g complies with the limit test for heavy metals, Method A (10 ppm). Chlorides (2.3.12). 5.0 g complies with the limit test for chlorides (50 ppm).
Sorbitol Solution (70 Per Cent) (Crystallising)

Sorbitol (70 Per cent) (Crystallising)
Sorbitol Solution (70 per cent) (Crystallising) is an aqueous solution of hexitols. Sorbitol Solution ( 70 per cent) (Crystallising) contains not less than 68.0 per cent w/w and not more than 72.0 per cent w/w of hexitols, calculated as D-glucitol (C6H14O6). Description. A clear, colourless, syrupy liquid.
Identification
A. Dilute 7.0 g with 40 ml of water, add 6.4 g of borax, allow to stand for 1 hour, shaking occasionally, and dilute to 50.0 ml with water. Filter if necessary. The optical rotation of the resulting solution is 0 to +1.5 (2.4.22).
1115
SORBITOL SOLUTION (70 PER CENT) (NON-CRYSTALLISING)
IP 2007
Nickel. Dissolve 10.0 g in sufficient water to produce 20 ml, add 3 ml of bromine water and 2 ml of a 20 per cent w/v solution of citric acid, mix and add 10 ml of 6 M ammonia and 1 ml of a 1 per cent w/v solution of dimethylglyoxime in ethanol (95 per cent). Mix, dilute to 50 ml with water and allow to stand for 5 minutes; any colour produced is not more intense than that produced by treating in the same manner and at the same time 1.0 ml of nickel standard solution (10 ppm Ni) diluted to 20 ml with water (1 ppm). Sulphates (2.3.17). 12 ml of solution A complies with the limit test for sulphates (125 ppm). Reducing sugars. Dissolve 5.0 g in 3 ml of water with the aid of gentle heat, cool, add 20 ml of cupri-citric solution and a few glass beads, heat in such a manner that the solution boils in 4 minutes and continue boiling for a further 3 minutes. Cool rapidly and add 100 ml of a 2.4 per cent v/v solution of glacial acetic acid followed by 20.0 ml of 0.025 M iodine. Add, shaking continuously, 25 ml of a 6 per cent v/v solution of hydrochloric acid and, when any precipitate has redissolved, titrate the excess iodine with 0.05 M sodium thiosulphate using 1 ml of starch solution, added towards the end of the titration, as indicator. Not less than 12.8 ml of 0.05 M sodium thiosulphate is required. Sulphated ash (2.3.18). Not more than 0.1 per cent. Assay. Measure accurately a volume containing about 0.6 g of Sorbitol and dissolve in sufficient water to produce 100.0 ml. To 10.0 ml of the solution add 20 ml of a 2.14 per cent w/v solution of sodium periodate and 2 ml of 1 M sulphuric acid and heat on a water-bath for exactly 15 minutes. Cool, add 3 g of sodium bicarbonate, in small quantities, and 25.0 ml 0.1 M sodium arsenite, mix, add 5 ml of a 20 per cent w/v solution of potassium iodide and allow to stand for 15 minutes. Titrate with 0.05 M iodine until the first trace of yellow colour appears. Repeat the operation without the substance under examination. The difference between the titrations represents the amount of iodine required. 1 ml of 0.05 M iodine is equivalent to 0.001822 g of C6H14O6. Storage. Store protected from moisture.
cent w/w of solid matter and not less than 62.0 per cent w/w of polyols expressed as D-glucitol (C6H14O6). Description. A clear, colourless or faintly yellow, syrupy liquid; odourless.
Identification
A. Dilute 7.0 g with sufficient carbon dioxide-free water to produce 50 ml (solution A). To 3 ml of a freshly prepared 10 per cent w/v solution of catechol add 6 ml of sulphuric acid while cooling in ice. To 3 ml of the mixture add 0.3 ml of solution A and heat gently over a naked flame for 30 seconds; a pink colour is produced which becomes deep brownish red. B. Dry 1 g over phosphorus pentoxide at 80 at a pressure of 1.5 to 2.5 kPa. Dissolve 0.5 g of the residue in a mixture of 5 ml of acetic anhydride and 0.5 ml of pyridine with the aid of heat and allow to stand for 10 minutes. Pour the mixture into 25 ml of water, allow to stand in ice for 2 hours and filter. The residue, after recrystallisation from a small volume of ethanol (95 per cent) and drying over phosphorus pentoxide at a pressure of 1.5 to 2.5 kPa, melts at about 100 (2.4.21). C. Determine by thin-layer chromatography (2.4.17), coating the plate with a uniform 0.75-mm thick layer of the following mixture. Mix 0.1 g of carbomer with 110 ml of water and allow to stand, with gentle stirring, for 1 hour. Adjust to pH 7.0 by the gradual addition, with continuous shaking, of 2 M sodium hydroxide and add 30 g of silica gel H. Heat the plate at 110 for 1 hour, allow to cool and use immediately. Mobile phase. A mixture of 85 volumes of 2-propanol and 15 volumes of a 0.2 per cent w/v solution of boric acid. Test solution. Dissolve 0.35 g of the substance under examination in 100 ml of ethanol (95 per cent). Reference solution. A 0.25 per cent w/v solution of sorbitol RS in ethanol (95 per cent). Apply to the plate 2 l of each solution. After development, dry the plate at 100 to 105 for 15 minutes, allow to cool, spray with a 0.5 per cent w/v solution of potassium permanganate in 1 M sodium hydroxide and heat at 100 for 2 minutes. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution.
Sorbitol Solution (70 Per Cent) (NonCrystallising)

Sorbitol (70 per cent) (Non-crystallising)
Sorbitol Solution (70 per cent) (Non-crystallising) is an aqueous solution of hydrogenated, partly hydrolysed starch. Sorbitol Solution (70 per cent) (Non-crystallising) contains not less than 68.0 per cent w/w and not more than 72.0 per
Tests
Appearance of solution. Solution A is clear (2.4.1), and colourless (2.4.1). Acidity or alkalinity. To 10 ml of solution A add 10 ml of carbon dioxide-free water. To 10 ml of the resulting solution add 0.05 ml of phenolphthalein solution; not more than 0.2 ml of 0.01 M sodium hydroxide is required to change the colour of the solution to pink. To a further 10 ml of the solution add
1116
IP 2007
SPIRONOLACTONE
0.05 ml of methyl red solution; Not more than 0.3 ml of 0.01 M hydrochloric acid is required to change the colour of the solution to red. Optical rotation (2.4.22). +1.5 to + 3.5, determined in a solution prepared in the following manner. Dilute 7.0 g with 40 ml of water, add 6.4 g of borax, allow to stand for 1 hour, shaking occasionally, dilute to 50 ml with water and filter, if necessary. Refractive index (2.4.27). 1.455 to 1.465. Relative density (2.4.29). Not less than 1.285. Arsenic (2.3.10). Dissolve 5.0 g in 50 ml of water and add 10 ml of stannated hydrochloric acid AsT. The resulting solution complies with the limit test for arsenic (2 ppm). Heavy metals (2.3.13). 2.0 g complies with the limit test for heavy metals, Method A (10 ppm). Chlorides (2.3.12). 5.0 g complies with the limit test for chlorides (50 ppm). Nickel. Dissolve 10.0 g in sufficient water to produce 20 ml, add 3 ml of bromine water and 2 ml of a 20 per cent w/v solution of citric acid, mix and add 10 ml of 6 M ammonia and 1 ml of a 1 per cent w/v solution of dimethylglyoxime in ethanol (95 per cent). Mix, dilute to 50 ml with water and allow to stand for 5 minutes; any colour produced is not more intense than that produced by treating in the same manner and at the same time 1.0 ml of nickel standard solution (10 ppm Ni) diluted to 20 ml with water (1 ppm). Sulphates (2.3.17). 12 ml of solution A complies with the limit test for sulphates (125 ppm). Reducing sugars. Dissolve 5.0 g in 3 ml of water with the aid of gentle heat, cool, add 20 ml of cupri-citric solution and a few glass beads, heat in such a manner that the solution boils in 4 minutes and continue boiling for a further 3 minutes. Cool rapidly and add 100 ml of a 2.4 per cent v/v solution of glacial acetic acid followed by 20.0 ml of 0.025 M iodine. Add, shaking continuously, 25 ml of a 6 per cent v/v solution of hydrochloric acid and, when any precipitate has redissolved, titrate the excess iodine with 0.05 M sodium thiosulphate using 1 ml of starch solution, added towards the end of the titration, as indicator. Not less than 12.8 ml of 0.05 M sodium thiosulphate is required. Sulphated ash (2.3.18). Not more than 0.1 per cent. Assay. For solid matter Weigh accurately about 1.0 g and dry to constant weight over phosphorus pentoxide at 80 at a pressure of 1.5 to 2.5 kPa. Weigh the residue. For polyols Measure accurately a volume containing about 0.4 g of Sorbitol and dissolve in sufficient water to produce 100.0 ml. To 10.0 ml of the solution add 20 ml of a 2.14 per cent w/v solution of sodium periodate and 2 ml of 1 M sulphuric acid and heat on a water-bath for exactly 15 minutes. Cool,
add 3 g of sodium bicarbonate, in small quantities, and 25.0 ml of 0.1 M sodium arsenite, mix, add 5 ml of a 20 per cent w/v solution of potassium iodide and allow to stand for 15 minutes. Titrate with 0.05 M iodine until the first trace of yellow colour appears. Repeat the operation without the substance under examination. The difference between the titrations represents the amount of iodine required. 1 ml of 0.05 M iodine is equivalent to 0.001822 g of C6H14O6. Storage. Store protected from moisture.
Spironolactone
H3C H3C H O O H H S CH3
Mol. Wt. 416.6
O O
C24H32O4S
Spironolactone is 7-acetylthio-3-oxo-17-pregn-4-ene21,17-carbolactone. Spironolactone contains not less than 97.0 per cent and not more than 102.0 per cent of C24H32O4S, calculated on the dried basis. Description. A yellowish white to buff coloured powder; odourless or with a slight odour of thioacetic acid. It exhibits polymorphism.
Identification
Test A may be omitted if tests B and C are carried out. Test B may be omitted if tests A and C are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with spironolactone RS or with the reference spectrum of spironolactone. Examine the substances as 5 per cent w/v solutions in chloroform. B. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Solvent mixture. A mixture of 90 volumes of acetone and 10 volumes of 1,2-propanediol. Mobile phase. A mixture of 40 volumes of cyclohexane and 10 volumes of toluene.
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SPIRONOLACTONE TABLETS
IP 2007
Test solution. Dissolve 25 mg of the substance under examination in 10 ml of the solvent mixture. Reference solution (a). Dissolve 25 mg of spironolactone RS in 10 ml of the solvent mixture. Reference solution (b). Mix equal volumes of the test solution and reference solution (a). Place the dry plate in a tank containing a shallow layer of the solvent mixture, allow the solvent mixture to ascend to the top, remove the plate from the tank and allow the solvent to evaporate. Use within 2 hours, with the flow of the mobile phase in the direction in which the aforementioned treatment was done. Apply to the plate 2 l of each solution. Allow the mobile phase to rise 12 cm. Dry the plate in a current of warm air, allow the solvent to evaporate, heat at 120 for 15 minutes and spray the hot plate with ethanolic sulphuric acid (20 per cent v/v). Heat at 120 for a further 10 minutes, allow to cool and examine in daylight and in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with reference solution (a). The principal spot in the chromatogram obtained with reference solution (b) appears as a single, compact spot. C. Shake about 10 mg with 2 ml of sulphuric acid (50 per cent); an orange solution with an intense yellowish fluorescence is produced. Heat the solution gently; the colour becomes deep red and hydrogen sulphide is evolved which turns lead acetate paper black. Add the solution to 10 ml of water; a greenish yellow solution is produced which shows opalescence or a precipitate.
iodine and 0.1 ml of starch solution and mix; a blue colour develops. Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105 for 3 hours, Assay. Weigh accurately about 50 mg, dissolve in sufficient methanol to produce 250.0 ml, dilute 5.0 ml to 100.0 ml with methanol and measure the absorbance of the resulting solution at the maximum at about 238 nm (2.4.7). Calculate the content of C24H32O4S taking 470 as the specific absorbance at 238 nm. Storage. Store protected from light and moisture.
Spironolactone Tablets
Spironolactone Tablets contain not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of spironolactone, C24H32O4S.The tablets may contain added flavouring agents and may be coated.
Identification
A. Extract a quantity of the powdered tablets containing 0.125 g of Spironolactone with two quantities, each of 10 ml, of chloroform, filter, evaporate the combined filtrates to dryness and dissolve the residue in 2.5 ml of chloroform. On the resulting solution determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with spironolactone RS or with the reference spectrum of spironolactone. B. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Solvent mixture. A mixture of 90 volumes of acetone and 10 volumes of 1,2-propanediol. Mobile phase. A mixture of 40 volumes of cyclohexane and 10 volumes of toluene. Test solution. Extract a quantity of the powdered tablets containing 50 mg of Spironolactone with 10 ml of chloroform, filter and evaporate the filtrate to dryness. Dissolve 25 mg the residue in 10 ml of the solvent mixture. Reference solution (a). Dissolve 25 mg of spironolactone RS in 10 ml of the solvent mixture. Reference solution (b). Mix equal volumes of the test solution and reference solution (a). Place the dry plate in a tank containing a shallow layer of the solvent mixture, allow the solvent mixture to ascend to the
Tests
Specific optical rotation (2.4.22). 33.0 to 37.0, determined in a 1.0 per cent w/v solution in chloroform. Chromium. Mix 0.2 g with 1 g of potassium carbonate and 0.3 g of potassium nitrate in a platinum crucible, heat gently until fused and ignite at 600 to 650 until the carbon is removed. Cool, dissolve the residue as completely as possible in 10 ml of water with the aid of gentle heat, filter and dilute to 20 ml with water. To 10 ml of the solution add 0.5 g of urea and just acidify with sulphuric acid (14 per cent). When effervescence ceases add a further 1 ml of sulphuric acid (14 per cent), dilute to 20 ml with water and add 0.5 ml of diphenylcarbazide solution. Any colour produced is not more intense than that obtained by adding 1 ml of sulphuric acid (14 per cent) to 0.5 ml of a freshly prepared 0.00283 per cent w/v solution of potassium dichromate, diluting to 20 ml with water and adding 0.5 ml of diphenylcarbazide solution (50 ppm). Free mercapto compounds. Shake 2.0 g with 20 ml of water for 1 minute and filter. To 10 ml of the filtrate add 0.05 ml of 0.01 M
1118
IP 2007
STAVUDINE
top, remove the plate from the tank and allow the solvent to evaporate. Use within 2 hours, with the flow of the mobile phase in the direction in which the aforementioned treatment was done. Apply to the plate 2 l of each solution. Allow the mobile phase to rise 12 cm. Dry the plate in a current of warm air, allow the solvent to evaporate, heat at 120 for 15 minutes and spray the hot plate with ethanolic sulphuric acid (20 per cent v/v). Heat at 120 for a further 10 minutes, allow to cool and examine in daylight and in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with reference solution (a). The principal spot in the chromatogram obtained with reference solution (b) appears as a single, compact spot. C. Shake about 10 mg of the residue obtained in test B with 2 ml of sulphuric acid (50 per cent); an orange solution with an intense yellowish fluorescence is produced. Heat the solution gently; the colour becomes deep red and hydrogen sulphide is evolved which turns lead acetate paper black. Add the solution to 10 ml of water; a greenish yellow solution is produced which shows opalescence or a precipitate.
Stavudine
O HN O HO
C10H12N2O4 Mol. Wt. 224.2
CH3 N
Stavudine is 2',3',didehydro-3'-deoxythymidine. Stavudine contains not less than 98.0 per cent and not more than 102.0 per cent of C10H12N2O4, calculated on the dried basis. Description. A white or almost white powder.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with stavudine RS or with the reference spectrum of stavudine. B. In the Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the peak due to stavudine in the chromatogram obtained with reference solution (c). C. Melts at about 164 (2.4.21).
Tests
Dissolution (2.5.2). Apparatus. No 1 Medium. 1000 ml of 0.1 M hydrochloric acid containing 0.1 per cent w/v of sodium dodecyl sulphate. Speed and time. 75 rpm and 60 minutes. Withdraw a suitable volume of the medium, filter through a membrane filter disc with an average pore diameter not greater than 1.0 m. Measure the absorbance of the filtrate, suitably diluted if necessary, at the maximum at about 242 nm (2.4.7). Calculate the content of C24H32O4S in the medium taking 445 as the specific absorbance at 242 nm. D. Not less than 70.0 per cent of the stated amount of C24H32O4S. Other tests. Comply with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 25 mg of Spironolactone, add 100 ml of methanol and heat just to boiling, with swirling. Cool, add sufficient methanol to produce 250.0 ml, dilute 10.0 ml to 100.0 ml with methanol and measure the absorbance of the resulting solution at the maximum at about 238 nm (2.4.7). Calculate the content of C24H32O4S taking 470 as the specific absorbance at 238 nm. Storage. Store protected from light and moisture.
Tests
Specific optical rotation (2.4.22). 39.0 to 46.0, determined in a 0.7 per cent w/v solution in water. Related substances. Determine by liquid chromatography (2.4.14), as described in the Assay. Separately inject the test solution, reference solutions (a) and (b) and record the chromatograms for at least three times the retention time of the principal peak. In the chromatogram obtained with the test solution, the area of any peak corresponding to thymine is not greater than the area of the peak in the chromatogram obtained with reference solution (b) (1 per cent); the area of any peak corresponding to thymidine is not greater than the area of the corresponding peak in the chromatogram obtained with reference solution (a) (1 per cent); the area of any individual impurity peak other than thymine and -thymidine is not more than 0.5 per cent and the sum of the areas of all the impurity peaks other than thymine and -thymidine is not more than 1.0 per cent. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Sulphated ash (2.3.18). Not more than 0.3 per cent.
1119
STAVUDINE CAPSULES
IP 2007
Loss on drying (2.4.19). Not more than 1.0 per cent, determined on 1.0 g by drying at 105 for 3 hours. Assay. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 50.0 mg of the substance under examination in 100.0 ml of the mobile phase. Reference solution (a). A solution containing 0.05 per cent w/v of stavudine RS, 0.01 per cent w/v of thymine and 0.0005 per cent w/v of -thymidine in the mobile phase. Reference solution (b). A 0.0005 per cent w/v solution of thymine in the mobile phase. Reference solution (c). A 0.05 per cent w/v solution of stavudine RS in the mobile phase. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), column temperature 50, mobile phase: a mixture of 20 volumes of methanol and 80 volumes of water, flow rate. 1 ml per minute, spectrophotometer set at 265 nm, a 20 l loop injector. Inject separately reference solutions (a) and (c). The order of elution in the chromatogram obtained with reference solution (a) is thymine, -thymidine and stavudine with relative retention times of about 0.5, 0.7 and 1.0 respectively. The test is not valid unless the column efficiency determined from the stavudine peak in the chromatogram obtained with reference solution (a) is not less than 3000 theoretical plates, the tailing factor is not more than 1.5 and the relative standard deviation for replicate injections of reference solution (c) is not more than 2.0 per cent. Inject separately the test solution and reference solution (c) and measure the responses of the principal peak. Calculate the content of C10H12N2O4. Storage. Store protected from light and moisture.
When examined in the range 200 nm to 300 nm (2.4.7), the resulting solution shows an absorption maximum at about 265 nm. B. In the Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the peak due to stavudine in the chromatogram obtained with the reference solution.
Tests
Related substances. Determine by liquid chromatography (2.4.14). Test solution. Mix well the contents of 20 capsules and transfer an accurately weighed quantity containing about 50 mg of Stavudine to a 100-ml volumetric flask. Add about 60 ml of water, mix with the aid of ultrasound for 10 minutes, dilute to volume with water, mix and filter. Reference solution. Weigh 5 mg each of stavudine RS and thymine RS and transfer to a 25-ml volumetric flask. Add 5 ml of methanol, mix with the aid of ultrasound to dissolve and dilute to volume with water. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octylsilane bonded to porous silica (5 m), mobile phase: gradient mixtures of acetonitrile and 0.1 M ammonium acetate, flow rate. 1 ml per minute, a linear gradient programme using the conditions given below, spectrophotometer set at 270 nm, a 20 l loop injector. Time 0.1 M Ammonium acetate Acetonitrile (in min.) (per cent v/v) (per cent v/v) 0 95 5 5 95 5 25 20 80 30 20 80 31 95 5 35 95 5 Inject the reference solution. The test is not valid unless the column efficiency determined from the peaks of stavudine and thymine is not less than 7000 theoretical plates and the tailing factor is not more than 2.0. Inject separately water and the test solution. Examine the water chromatogram for any extraneous peaks and ignore the corresponding peaks observed in the chromatogram obtained with the test solution. Any secondary peak observed in the chromatogram obtained with the test solution corresponding to thymine is not more
Stavudine Capsules
Stavudine Capsules contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of stavudine, C10H12N2O4.
Identification
A.Shake a quantity of the mixed contents of the capsules containing about 50 mg of Stavudine with 80 ml of water for 10 minutes. Add sufficient water to produce 100 ml, mix and filter. Dilute 10 ml of the filtrate to 100 ml with water.
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IP 2007
STAVUDINE ORAL SOLUTION
than 3.0 per cent. Any other secondary peak observed in the chromatogram obtained with the test solution is not more than 0.5 per cent and the sum of the areas of all the secondary peaks is not more than 4.0 per cent when calculated by percentage area normalisation. Dissolution (2.5.2). Apparatus. No 1 Medium. 900 ml of 0.01 M hydrochloric acid Speed and time. 75 rpm and 45 minutes. Withdraw a suitable volume of the medium and filter. Determine by liquid chromatography (2.4.14). Test solution. The filtrate obtained as given above. Reference solution. In the case of capsules containing 30 mg of Stavudine, weigh accurately about 30 mg of stavudine RS and transfer to a 100-ml volumetric flask. Dissolve and dilute to volume with 0.01 M hydrochloric acid. Dilute 5.0 ml of this solution to 50.0 ml with 0.01 M hydrochloric acid; in the case of capsules containing 40 mg of stavudine, weigh accurately about 40 mg of stavudine RS and transfer to a 100-ml volumetric flask. Dissolve and dilute to volume with 0.01 M hydrochloric acid. Dilute 5.0 ml of this solution to 50.0 ml with 0.01 M hydrochloric acid. Chromatographic system a stainless steel column 15 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 85 volumes of water and 15 volumes of methanol, flow rate. 1.5 ml per minute, spectrophotometer set at 266 nm, a 20 l loop injector. Inject the reference solution and record the chromatograms for twice the retention time of stavudine. The test is not valid unless the column efficiency determined from the stavudine peak is not less than 3000 theoretical plates, the tailing factor is not more than 1.5 and the relative standard deviation for replicate injections is not more than 1.5 per cent Inject separately the test solution and the reference solution and measure the responses for the major peak. Calculate the content of C10H12N2O4 in the medium. D. Not less than 70 per cent of the stated amount of C10H12N2O4. Other tests. Comply with the tests stated under Capsules. Assay. Determine by liquid chromatography (2.4.14). Test solution. Weigh accurately a quantity of the mixed contents of 20 capsules containing about 50 mg of Stavudine and transfer to a 100-ml volumetric flask. Add about 60 ml of water, mix with the aid of ultrasound for 10 minutes, dilute to
volume with water, mix and filter. Further dilute 10.0 ml of the filtrate to 25.0 ml with water. Reference solution. Weigh accurately about 50 mg of stavudine RS and transfer to a 100-ml volumetric flask. Add about 60 ml of water, mix with the aid of ultrasound to dissolve and dilute to volume with water. Dilute 10.0 ml of this solution to 25.0 ml with water. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octylsilane bonded to porous silica (5 m), mobile phase: a mixture of 5 volumes of acetonitrile and 95 volumes of 0.1 M ammonium acetate, flow rate. 1 ml per minute, spectrophotometer set at 270 nm, a 20 l loop injector Inject the reference solution. The test is not valid unless the column efficiency determined from the stavudine peak is not less than 3000 theoretical plates, the tailing factor is not more than 2.0 and the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject separately the test solution and the reference solution and measure the responses for the major peak. Calculate the content of C10H12N2O4 in the capsules. Storage. Store protected from moisture.
Stavudine Oral Solution

Stavudine Oral Solution is a mixture consisting of Stavudine with buffering agents and other excipients. It contains a suitable flavouring agent. It is filled in a sealed container. The oral solution is constituted by dispersing the contents of the sealed container in the specified volume of water just before issue. Stavudine Oral Solution contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of stavudine C10H12N2O4. Storage. Store the constituted solution in a refrigerator (2 to 8). Discard any unused portion after 30 days of reconstitution. The contents of the sealed container comply with the requirements stated under Oral Liquids and with the following requirements.
Identification
In the Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the peak in the chromatogram obtained with reference solution (a).
1121
STAVUDINE AND LAMIVUDINE TABLETS
IP 2007
Tests
pH (2.4.24). 5.0 to 7.0, determined in the reconstituted solution. Related substances. Determine by liquid chromatography (2.4.14). NOTE Prepare the solutions immediately before use. Test solution. Weigh accurately a quantity of the reconstituted solution containing 10 mg of stavudine and dissolve in 20 ml of water. Reference solution (a). A 0.05 per cent w/v solution of stavudine RS in water. Reference solution (b). Dilute 1 ml of reference solution (a) to 100 ml with water. Reference solution (c). A solution containing 0.0125 per cent w/v each of thymine and thymidine in water. Dilute 2 ml of the solution to 100 ml of water. Use the chromatographic system described under Assay. Inject reference solution (a). The test is not valid unless the tailing factor is not more than 2.0 and the column efficiency in not less than 2000 theoretical plates. Inject reference solution (c). The test is not valid unless the resolution between thymine and thymidine peak is not less than 8.4. Inject the test solution and reference solution (b). Run the chromatogram for 4 times the retention time (about 9 minutes) of the principal peak. In the chromatogram obtained with the test solution, the area of any secondary peak is not more than 1.5 times the area of the peak in the chromatogram obtained with the reference solution (b) (1.5 per cent) and the sum of areas of all the secondary peaks is not more than 3 times the area of the peak in the chromatogram obtained with the reference solution (b) (3.0 per cent). Other tests. Complies with the tests stated under Oral Liquids. Water (2.3.43). Not more than 2.0 per cent, determined on 0.5 g. Assay. Determine by liquid chromatography (2.4.14). NOTE Prepare the solutions immediately before use. Test solution. Weigh accurately a quantity of the reconstituted solution containing 10 mg of Stavudine, disperse in sufficient water and diluted to 100.0 ml with water. Reference solution (a). A 0.01 per cent w/v solution of stavudine RS in water. Reference solution (b). A solution containing 0.0125 per cent w/v each of thymine and thymidine in water. Dilute 2 ml of the solution to 100 ml with water.
Chromatographic system a stainless steel column 3.3 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: A. 95 volumes of 25 mM ammonium acetate and 5 volumes of methanol, B. 50 volumes of 25 mM ammonium acetate and 50 volumes of methanol. flow rate. 1 ml per minute, a linear gradient programme using the conditions given below, spectrophotometer set at 268 nm, a 20 l loop injector. Time mobile phase A mobile phase B (in min.) (per cent v/v) (per cent v/v) 0 100 0 60 100 0 120 0 100 155 100 0 Inject reference solution (b). The test is not valid unless the resolution between thymine and thymidine is not less than 8.4. Inject reference solution (a). The test is not valid unless the tailing factor is not more than 2.0, the column efficiency in not less than 2000 theoretical plates and the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject the test solution and reference solution (a). Calculate the content of C10H12N2O4 in the oral solution. Storage. Store protected from moisture, at a temperature not exceeding 30.
Stavudine And Lamivudine Tablets

Stavudine and Lamivudine Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amounts of stavudine, C 10 H 12 N 2 O 4 and lamivudine, C8H11N3O3S. The tablets may be coated.
Identification
In the Assay, the two principal peaks in the chromatogram obtained with the test solution correspond to the peaks due to stavudine and lamivudine in the chromatogram obtained with the reference solution.
Tests
Related substances. Determine by liquid chromatography (2.4.14). Test solution. Weigh accurately a quantity of the powdered tablets containing about 100 mg of lamivudine and transfer to
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STAVUDINE AND LAMIVUDINE TABLETS
a 200-ml volumetric flask. Add about 100 ml of water, mix with the aid of ultrasound for 10 minutes with occasional shaking to obtain a uniform dispersion, cool to room temperature, dilute to volume with water and mix. Filter through a membrane filter disc with an average pore diameter not greater than 1.0 m, rejecting the first few ml of the filtrate. Reference solution (a). Weigh accurately about 65 mg of stavudine RS and 6.5 mg of thymine RS, and transfer to a 25-ml volumetric flask. Add 5 ml of methanol, sonicate to dissolve, dilute to volume with water and mix. Reference solution (b). Weigh accurately about 100 mg of lamivudine RS, transfer to a 200-ml volumetric flask, add about 100 ml of water and mix with the aid of ultrasound to dissolve. Add 10 ml of reference solution (a) to this solution and dilute to volume with water and mix. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octylsilane bonded to porous silica (5 m), mobile phase: gradient mixtures of acetonitrile and 0.1 M ammonium acetate, flow rate. 1 ml per minute, a linear gradient programme using the conditions given below, spectrophotometer set at 270 nm, a 20 l loop injector. Time 0.1 M Ammonium acetate Acetonitrile (in min.) (per cent v/v) (per cent v/v) 0 95 5 5 95 5 25 20 80 30 20 80 31 95 5 35 95 5 Inject separately reference solutions (b) and (c). The test is not valid unless the column efficiency determined from the thymine, stavudine and lamivudine peaks is not less than 3000 theoretical plates and the tailing factor for the same peaks is not more than 2.0. Inject separately water and the test solution. Examine the chromatogram obtained with water for any extraneous peaks and ignore the corresponding peaks observed in the chromatogram obtained with the test solution. Any secondary peak observed in the chromatogram obtained with the test solution corresponding to thymine is not more than 3.0 per cent. Any other secondary peak observed in the chromatogram obtained with the test solution is not more than 0.5 per cent and the sum of the areas of all the secondary peaks is not more than 4.0 per cent when calculated by percentage area normalisation.
Dissolution (2.5.2). Apparatus. No 1 Medium. 900 ml of 0.01 M hydrochloric acid Speed and time. 50 rpm and 45 minutes. Withdraw a suitable volume of the medium and filter. Determine by liquid chromatography (2.4.14). Test solution. The filtrate obtained as given above. Reference solution. In the case of tablets containing 30 mg of stavudine, weigh accurately about 33 mg of stavudine RS and 167 mg of lamivudine RS and transfer to a 100-ml volumetric flask. Add about 20 ml of methanol, sonicate to dissolve and dilute to volume with a solvent mixture of equal volumes of methanol and water (diluent). Dilute 5.0 ml of this solution to 50.0 ml with 0.01 M hydrochloric acid; in the case of tablets containing 40 mg of stavudine, weigh accurately about 44 mg of stavudine RS and 167 mg of lamivudine RS and transfer to a 100-ml volumetric flask. Add about 20 ml of methanol, mix with the aid of ultrasound to dissolve and dilute to volume with the diluent. Dilute 5.0 ml of this solution to 50.0 ml with 0.01 M hydrochloric acid. Chromatographic system a stainless steel column 15 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 35 volumes of methanol and 65 volumes of a buffer prepared by dissolving 0.68 g of potassium dihydrogen phosphate and 1.0 g of sodium octanesulphonate in 1000.0 ml of water to which 1 ml of triethylamine is added and the pH of which is adjusted to 2.5 with phosphoric acid, flow rate. 1 ml per minute, spectrophotometer set at 266 nm, a 10 l loop injector. Inject the reference solution. The test is not valid unless the column efficiency determined from the lamivudine peak is not less than 2000 theoretical plates, the tailing factor for the individual stavudine and lamivudine peaks is not more than 1.5 and the relative standard deviation for replicate injections for each of the peaks corresponding to stavudine and lamivudine is not more than 1.0 per cent. Inject separately the test solution and the reference solution and measure the responses for the major peaks due to stavudine and lamivudine. Calculate the contents of C10H12N2O4 and C8H11N3O3S in the medium. D. Not less than 70 per cent of the stated amounts of C10H12N2O4 and C8H11N3O3S. Other tests. Comply with the tests stated under Tablets. Assay. Determine by liquid chromatography (2.4.14).
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Test solution. Weigh and powder 20 tablets. Transfer an accurately weighed quantity of the powder containing about 150 mg of Lamivudine to a 100-ml volumetric flask, add about 20 ml of methanol and 50 ml of a mixture of equal volumes of water and methanol, mix with the aid of ultrasound for 5 minutes with occasional shaking to obtain a uniform dispersion. Cool to room temperature and dilute to volume with the same solvent mixture. Filter the solution through a membrane filter disc with an average pore diameter not greater than 1.0 m, rejecting the first few ml of the filtrate. Dilute suitably with the same solvent mixture to obtain a solution with a concentration of 0.15 mg per ml of lamivudine. Reference solution. A similarly prepared solution containing 0.015 per cent w/v of lamivudine RS and a concentration of stavudine RS similar to that of the concentration of stavudine in test solution. Use the chromatographic system described under Dissolution. Inject the reference solution. The test is not valid unless the column efficiency determined from the lamivudine peak is not less than 2000 theoretical plates, the tailing factor for the individual peaks due to lamivudine and stavudine is not more than 1.5 and the relative standard deviation for replicate injections for each of the peaks corresponding to stavudine and lamivudine is not more than 2.0 per cent. Inject separately the test solution and the reference solution and measure the responses for the major peaks. Calculate the contents of C10H12N2O4 and C8H11N3O3S in the tablets. Storage. Store protected from moisture.
principal peaks in the chromatogram obtained with reference solution (c).
Tests
Congealing temperature (2.4.10). Not lower than 54. Acid value (2.3.23). 200 to 212, determined on 1.0 g. Iodine value (2.3.28). Not more than 4.0 (iodine monochloride method). Mineral acid. Shake 5 g of the melted substance with an equal volume of hot water for 2 minutes, cool and filter. To the filtrate add 0.05 ml of methyl orange solution; no red colour is produced. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent, determined on 2.0 g. Assay. Determine by gas chromatography (2.4.13). Test solution. Add 5 ml of boron trifluoride solution to 0.1 g of the substance under examination and heat under a reflux condenser for 15 minutes, cool and transfer to a separating funnel with the aid of 10 ml of hexane. Add 10 ml of a saturated solution of sodium chloride and 10 ml of water, shake, discard the lower aqueous layer and dry the upper layer over anhydrous sodium sulphate. Reference solution (a). Add 5 ml of a 1 per cent w/v solution of nonadecanoic acid (internal standard) in toluene to a mixture of 50 mg of stearic acid RS and 50 mg of palmitic acid RS and evaporate to dryness. Add 5 ml of boron trifluoride solution and complete the procedure described for the test solution beginning at the words heat under a reflux condenser...... Reference solution (b). Add 5 ml of the internal standard solution to 0.1 g of the substance under examination, evaporate to dryness, add 5 ml of boron trifluoride solution and complete the procedure described for the test solution beginning at the words heat under a reflux condenser....... Reference solution (c). Prepare in the same manner as reference solution (a) but omitting the internal standard. Chromatographic system a glass column 1.5 m x 4 mm, packed with acid-washed, silanised diatomaceous support (100 to 120 mesh) coated with 15 per cent w/w of diethylene glycol succinate polyester, temperature: column 170, inlet port and detector at 240, flow rate. 30 ml per minute of the carrier gas. Calculate the content of C18H36O2, and C16H32O2. Storage. Store protected from moisture.
Stearic Acid
H3C COOH
Stearic Acid consists of a mixture of fatty acids, chiefly stearic acid (C18H36O2) and palmitic acid (C16H32O2). It may contain a suitable antioxidant. Stearic Acid contains not less than 40.0 per cent of C18H36O2 and the sum of the contents of C18H36O2 and C16H32O2 is not less than 90.0 per cent. Description. A white powder or white, greasy, flaky crystals or hard masses showing signs of crystallisation.
Identification
In the Assay, the chromatogram obtained with the test solution shows two principal peaks which correspond to the two
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STILBOESTROL
Stearyl Alcohol
Stearyl Alcohol is a mixture of solid alcohols consisting chiefly of 1-octadecanol, (C18H38O). Description. A white, unctuous mass or almost white flakes or granules; odour, faint and characteristic.
B. When examined in the range 230 nm to 450 nm (2.4.7), the irradiated solution prepared as directed in the Assay shows absorption maxima at about 292 nm and 418 nm. C. In the test for Mono- and di-methyl ethers, the principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with reference solution (a). D. Dissolve 0.5 mg in 0.2 ml of glacial acetic acid, add 1 ml of phosphoric acid and heat in a water-bath for 3 minutes; a deep yellow colour is produced which almost disappears on dilution with 3 ml of glacial acetic acid (distinction from dienoestrol).
Tests
Appearance of solution. Dissolve 0.5 g in 20 ml of ethanol (95 per cent) by heating to boiling and allow to cool. The solution is clear (2.4.1), and not more intensely coloured than reference solution BS6 (2.4.1). Melting range (2.4.21). 55 to 60. Acid value (2.3.23). Not more than 2.0. Hydroxyl value (2.3.27). 195 to 220. Iodine value (2.3.28). Not more than 2.0 (iodine bromide method), determined on 2.0 g dissolved in 25 ml of chloroform, warming if necessary to effect solution. Saponification value (2.3.37). Not more than 2.0, determined on 10.0 g. Storage. Store protected from moisture.
Tests
4,4'-Dihydroxystilben and related ethers. Absorbance of a 1.0 per cent w/v solution in ethanol at about 325 nm, not more than 0.50 (2.4.7). Mono- and di-methyl ethers. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel. Mobile phase. A mixture of 90 volumes of toluene and 10 volumes of diethylamine. Test solution. Dissolve 0.5 g of the substance under examination in 100 ml of ethanol (95 per cent). Reference solution (a). A 0.5 per cent w/v solution of stilboestrol RS in ethanol (95 per cent). Reference solution (b). A 0.05 per cent w/v solution of stilboestrol monomethyl ether RS in ethanol (95 per cent).
Stilboestrol
Diethylstilboestrol
H3C OH
Reference solution (c). A 0.05 per cent w/v solution of stilboestrol dimethyl ether RS in ethanol (95 per cent). Reference solution (d). A solution containing 0.25 per cent w/v each of dienoestrol RS and stilboestrol RS
HO
C18H20O2
CH3
Mol. Wt. 268.4
Stilboestrol is (E)-,-diethylstilbene-4,4'-diol. Stilboestrol contains not less than 97.0 per cent and not more than 101.0 per cent of C18H20O2, calculated on the dried basis. Description. A white or almost white, crystalline powder; odourless.
Identification
Test A may be omitted if tests B, C and D are carried out. Tests B and C may be omitted if tests A and D are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with stilboestrol RS or with the reference spectrum of stilboestrol.
Apply to the plate 1 l of each solution. After development, dry the plate in air, spray with ethanolic sulphuric acid (20 per cent) and heat at 120 for 10 minutes. Any secondary spots in the chromatogram obtained with the test solution corresponding to the mono-and di-methyl ethers of stilboestrol are not more intense than the spots in the chromatograms obtained with reference solutions (b) and (c) respectively. Stilboestrol sometimes produces two spots. The test is not valid unless the chromatogram obtained with reference solution (d) shows two clearly separated spots of approximately the same intensity. Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 20 mg, dissolve in sufficient ethanol to produce 100.0 ml and dilute 10.0 ml of this solution
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to 100.0 ml with ethanol. To 25.0 ml of the resulting solution add 25.0 ml of a solution prepared by dissolving 1 g of dipotassium hydrogen phosphate in 55 ml of water, transfer a portion of the mixture to a 1-cm closed quartz cell, place the cell 10 cm from a 15-watt short-wave, ultraviolet light of mercury lamp and irradiate for 10 minutes. Measure the absorbance of the irradiated solution at the maximum at about 418 nm (2.4.7). Calculate the content of C 18H20O2 from the absorbance obtained by repeating the operation using stilboestrol RS in place of the substance under examination. Storage. Store protected from light and moisture.
Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 5 mg of Stilboestrol, add 5 ml of ethanol, shake for 15 minutes, add sufficient ethanol to produce 100.0 ml and centrifuge. Dilute 20.0 ml of the clear, supernatant liquid to 50.0 ml with ethanol and mix. To 25.0 ml of the resulting solution, add 25.0 ml of a solution prepared by dissolving 1 g of dipotassium hydrogen phosphate in 55 ml of water, transfer a portion of the mixture to a 1-cm closed quartz cell, place the cell 10 cm from a 15-watt short-wave, ultraviolet light of mercury lamp and irradiate for 10 minutes. Measure the absorbance of the irradiated solution at 418 nm (2.4.7). Calculate the content of C18H20O2 in the tablet from the absorbance obtained by repeating the operation using stilboestrol RS in place of the substance under examination. Storage. Store protected from light and moisture.
Stilboestrol Tablets
Diethylstilboestrol Tablets
Stilboestrol Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of stilboestrol, C18H20O2. The tablets may be coated.
Prepared Storax
Storax
Prepared Storax is a balsam obtained from the wounded trunk of Liquidambar orientalis Miller (Fam.Hamameli-daceae) and subsequently purified by solution in Ethanol (95 per cent), filtration and evaporation of the solvent. Prepared Storax contains not less than 30.0 per cent of total balsamic acids calculated as cinnamic acid, C9H8O2, on the dried basis. Description. A brown, viscous substance, transparent in thin layers; odour, agreeable and balsamic.
Identification
A. When examined in the range 230 nm to 450 nm (2.4.7), the irradiated solution prepared as directed in the Assay shows absorption maxima at about 292 nm and 418 nm. B. Extract a quantity of the powdered tablets containing 3 mg of Stilboestrol with ether, filter and evaporate the filtrate to dryness. Dissolve 0.5 mg of the residue in 0.2 ml of glacial acetic acid, add 1 ml of phosphoric acid and heat in a waterbath for 3 minutes; a deep yellow colour is produced which almost disappears on dilution with 3 ml of glacial acetic acid (distinction from dienoestrol).
Tests
Uniformity of content. For tablets containing 10 mg or less Comply with the test stated under Tablets. Finely crush one tablet, add 10 ml of ethanol, shake for 30 minutes, add sufficient ethanol to produce 25.0 ml and centrifuge. Pipette an aliquot of the supernatant liquid containing 0.5 mg of Stilboestrol add 25.0 ml of a solution prepared by dissolving 1 g of dipotassium hydrogen phosphate in 55 ml of water, transfer a portion of the mixture to a 1-cm closed quartz cell, place the cell 10 cm from a 15-watt short-wave, ultraviolet light of mercury lamp and irradiate for 10 minutes. Measure the absorbance of the irradiated solution at the maximum at about 418 nm (2.4.7). Calculate the content of C18H20O2 in the tablet from the absorbance obtained by repeating the operation using stilboestrol RS in place of the substance under examination. Other tests. Comply with the tests stated under Tablets.
Identification
Shake 1 g with a 10 per cent w/v solution of potassium chromate and 1 ml of sulphuric acid; the odour of benzaldehyde is produced.
Tests
Acid value (2.3.23). 50 to 80, calculated on the dried basis. Ester value (2.3.26). 100 to 133, calculated on the dried basis. Saponification value (2.3.37). 170 to 200, calculated on the dried basis. Ethanol-soluble matter. Not less than 70 per cent, determined by the following method. Weigh accurately about 10.0 g in a beaker and heat at 105 for 30 minutes. Dissolve the residue in 100 ml of hot ethanol (95 per cent), filter through a tared sintered glass crucible, wash the residue with small amounts of hot ethanol (95 per cent) until the last washing is nearly colourless. Reserve the residue for the Ethanol-insoluble matter
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test. Combine the filtrate and the washings and evaporate at a temperature not exceeding 60. Dry the residue at 105 for 1 hour, cool and weigh. Ethanol-insoluble matter. Not more than 5 per cent, determined by the following method. Dry the residue obtained in the test for Ethanol-soluble matter at 105 for 1 hour, cool and weigh. Loss on drying (2.4.19). Not more than 5 per cent, determined on 1.0 g by drying in a thin layer over phosphorus pentoxide at 60 at a pressure not exceeding 2.7 kPa. Assay. Weigh accurately about 1.25 g and boil with 25 ml of dilute ethanolic potassium hydroxide solution under a reflux condenser for 1 hour. Remove the ethanol and digest the residue with 50 ml of hot water until diffused. Cool the liquid, add 150 ml of water and 1.5 g of magnesium sulphate dissolved in 50 ml of water. Mix thoroughly and set aside for 10 minutes. Filter, wash the residue on the filter with 20 ml of water, acidify the combined filtrate and washings with hydrochloric acid and extract with successive quantities of 50, 40, 30, 30 and 30 ml of ether. Combine the ether extracts and discard the aqueous portion. Extract with successive quantities of 20, 20, 10, 10 and 10 ml of sodium bicarbonate solution, washing each aqueous extract with the same 20 ml of ether. Discard the ether layers, acidify the combined aqueous extracts with hydrochloric acid and extract with successive quantities of 30, 20, 20 and 10 ml of chloroform, filtering each chloroform extract through a plug of cotton wool on which a layer of anhydrous sodium sulphate is placed. Evaporate the chloroform on a water-bath until about 10 ml remains and remove the remainder in a current of air stopping immediately when the last trace of solvent is removed. Dissolve the residue by warming with 10 ml of ethanol (95 per cent), previously neutralised to phenol red solution, cool and titrate with 0.1 M sodium hydroxide using phenol red solution as indicator. 1 ml of 0.1 M sodium hydroxide is equivalent to 0.01482 g of total balsamic acids, calculated as cinnamic acid, C9H8O2. Storage. Store protected from moisture.
Identification
A. Place 0.5 ml of citrated human, canine or rabbit plasma in a haemolysis tube maintained in a water-bath at 37. Add 0.1 ml of a solution of the substance under examination containing 10,000 Units per ml in citro-phosphate buffer pH 7.2 and 0.1 ml of a solution of thrombin containing 20 Units per ml in citro-phosphate buffer pH 7.2 and shake immediately; a clot forms and lyses within 30 minutes. Repeat the procedure using citrated bovine plasma; lysis does not occur within 1 hour. B. Dissolve 0.6 g of agar in 50.0 ml of mixed barbitone buffer pH 8.6, heating until a clear solution is obtained. Place glass plates (50 mm x 50 mm) that are free from traces of grease on a level surface. Apply to each plate 4 ml of the agar solution and allow to cool until set. Bore a hole 6 mm in diameter in the centre of the agar and an appropriate number of holes (not exceeding six) at distances of 11 mm from the central hole removing the residual agar by means of a cannula connected to a vacuum pump. Place a quantity of 80 l of goat or rabbit antistreptokinase serum containing 10,000 Units of antistreptokinase activity per ml in the central hole and 80 l of a solution of the substance under examination containing 125,000 Units of streptokinase activity per ml of each of the surrounding holes. Place the plates in a humidified tank for 24 hours. Only one precipitation arc is produced which is well-defined and localised between the application point of the serum and each hole containing the solution of the substance under examination.
Tests
pH (2.4.24). 6.8 to 7.5, determined on a solution prepared in freshly boiled and cooled water containing 5000 Units per ml. Streptodornase. Introduce 0.5 ml of a 0.1 per cent w/v solution of sodium deoxyribonucleate in imidazole buffer pH 6.5 into each of eight centrifuge tubes. To each of the first two tubes add 0.25 ml of imidazole buffer pH 6.5 and 0.25 ml of solution of the substance under examination in imidazole buffer pH 6.5 containing 150,000 Units per ml (solution A) followed immediately by 3.0 ml of 0.25 M perchloric acid. Mix the contents of each tube, centrifuge for 5 minutes at 3000 rpm and measure the absorbance of each of the supernatant liquids at about 260 nm (2.4.7), using as the blank a mixture of 1.0 ml of imidazole buffer pH 6.5 and 3.0 ml of 0.25 M perchloric acid. Calculate the sum of the two absorbances (A1). To each of the remaining six tubes add, respectively, 0.25, 0.25, 0.125, 0.125, 0 and 0 ml of imidazole buffer pH 6.5 followed by 0.25 ml of solution A and finally 0, 0, 0.125, 0.125, 0.25 and 0.25 ml respectively of a solution of the Standard Preparation containing 20 Units of streptodornase activity per ml in imidazole buffer pH 6.5. [The Standard Preparation is the 1st International Standard Preparation for Streptodornase,
Streptokinase
Streptokinase is a preparation of a protein obtained from culture filtrates of certain strains of Streptococcus haemolyticus group C. It has the property of combining with human plasminogen to form plasminogen activator and is purified to contain not less than 600 Units of Streptokinase activity per g of nitrogen before addition of any stabiliser or carrier. It usually contains a buffer and may be stabilised by the addition of suitable substances such as Human Albumin. Description. A white powder or a white, friable solid; hygroscopic.
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established in 1964, consisting of a freeze-dried mixture of streptodornase and streptokinase with lactose (supplied in ampoules containing 2400 Units of streptodornase activity), or another suitable preparation the activity of which has been determined in relation to the International Standard]. Mix the contents of each tube, incubate at 37 for 15 minutes and add to each tube 3.0 ml of 0.25 M perchloric acid. Mix the contents of each tube, centrifuge and measure the absorbance of each of the supernatant liquids at about 260 nm (2.4.7), using as the blank the mixture specified above. If the sum of the absorbances of the liquids in the third and fourth tubes is A2, that of the liquids in the fifth and six tubes is A3, and that of the liquids in the seventh and eighth tubes is (A4), (A1A2) is less than 0.5(A3+A4)A2. Streptolysin. Dissolve a quantity of the substance under examination containing 500,000 Units in 0.5 ml of a mixture of 90 volumes of saline solution and 10 volumes of citrophosphate buffer pH 7.2 in a haemolysis tube. Add 0.4 ml of a 2.3 per cent w/v solution of sodium thioglycollate and incubate in a water-bath at 37 for 10 minutes. Add 0.1 ml of a solution of a reference preparation of human antistreptolysin O containing 5 Units per ml and incubate at 37 for 5 minutes. Add 1 ml of rabbit erythrocyte suspension, continue the incubation for 30 minutes and centrifuge at about 1000 rpm. The absorbance of the supernatant liquid at about 550 nm (2.4.7), is not more than 1.5 times the absorbance obtained by repeating the above procedure using 0.5 ml of the mixture of saline solution and citro-phosphate buffer pH 7.2 in place of the solution containing the substance under examination. Loss on drying (2.4.19). Not more than 4.0 per cent, determined on 1.0 g by drying over phosphorus pentoxide at a pressure not exceeding 2.7 kPa for 24 hours. Assay. The potency of streptokinase is determined by comparing its ability to activate human plasminogen to form plasmin with that of the Standard Preparation. The plasmin generated is determined by measurement of the time taken to lyse a fibrin clot under the conditions of a suitable method of assay. Standard Preparation The Standard Preparation is the 2nd International Standard for Streptokinase, established in 1989, consisting of freezedried streptokinase (supplied in ampoules containing 700 Units of streptokinase activity), or another suitable preparation the activity of which has been determined in relation to the International reference preparation. Method Use citro-phosphate buffer pH 7.2 containing 3 per cent w/v of bovine serum albumin for the preparation of solutions and dilutions.
Prepare a solution of the Standard Preparation to contain 1000 Units of streptokinase activity per ml and prepare a solution of the preparation under examination expected to have the same concentration; keep the solutions in ice and use within 6 hours. Prepare three 1.5-fold serial dilutions of the solution of the Standard Preparation so that the longest clot-lysis time is less than 20 minutes and prepare three similar dilutions of the solution of the preparation under examination. Keep the solutions in ice and use within 1 hour. Using 24 tubes, 8 mm in diameter, label the tubes S1, S2, S3 for the dilutions of the Standard Preparation and T1, T2, T3 for the dilutions of the preparation under examination, allocating four tubes to each dilution. Place the tubes in ice. Into each tube introduce 0.2 ml of the appropriate dilution, 0.2 ml of citro-phosphate buffer pH 7.2 containing 3 per cent w/v of bovine serum albumin and 0.1 ml of a solution containing 20 Units of thrombin per ml. Place the tubes in a water-bath at 37 and allow to stand for 2 minutes to attain temperature equilibrium. Using an automatic pipette, introduce into the bottom of the first tube 0.5 ml of a 1 per cent w/v solution to human euglobulins ensuring mixing. At 5-second intervals introduce successively into the remaining tubes 0.5 ml of a 1 per cent w/ v solution of human euglobulins. Using a stop-watch, measure for each tube the time in seconds that elapses between the addition of the euglobulin and the lysis of the clot. Using the logarithms of the lysis times, calculate the result of the assay by standard statistical methods. The estimated potency is not less than 90 per cent and not more than 111 per cent of the stated potency. The fiducial limits of error are not less than 80 per cent and not more than 125 per cent of the stated potency. Streptokinase intended for use in the manufacture of parenteral preparations complies with the following additional requirements. Abnormal toxicity (2.2.1). Determine by Method A, using a solution containing 50,000 Units in 0.5 ml of water for injections administered in 15 to 20 seconds. Bacterial endotoxins (2.2.3). Dissolve the contents of the sealed container in water BET to give a solution containing 10,000 Units of Streptokinase per ml. Carry out the test on the resulting solution; the maximum allowable endotoxin concentration of the solution is 23.33 Units of endotoxin per ml. Carry out the test using the maximum valid dilution of the prepared solution calculated from the declared sensitively of the lysate used in the test. Sterility (2.2.11). Complies with the test for sterility. Storage. Store in sealed containers, protected from light. The containers should be sterile, tamper-evident and sealed so as to exclude micro-organisms. Under these conditions the contents may be expected to retain their potency for 2 years.
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STREPTOKINASE INJECTION
Labelling. The label states (1) the number of Units of streptokinase activity in the container; (2) the number of Units of streptokinase activity per mg, calculated with reference to the dried preparation; (3) the name and quantity of any added substances; (4) the storage conditions; (5) whether or not it is intended for use in the manufacture of parenteral preparations.
Only one precipitation arc is produced which is well-defined and localised between the application point of the serum and each hole containing the solution of the substance under examination.
Tests
pH (2.4.24). 6.8 to 7.5, determined on a freshly constituted injection containing 5000 Units per ml.
Streptokinase Injection
Streptokinase Injection is a sterile material consisting of Streptokinase with or without auxiliary agents. It is filled in a sealed container. The injection is constituted by dissolving the contents of the sealed container in the requisite amount of sterile Water for Injections, immediately before use. The constituted solution complies with the requirements for Clarity of solution and Particulate matter stated under Parenteral Preparations (Injections). Storage. The constituted solution should be used immediately after preparation but, in any case, within the period recommended by the manufacturer. The contents of the sealed container comply with the requirements stated under Parenteral Preparations (Powders for Injection) and with the following requirements.
Identification
A. Place 0.5 ml of citrated human, canine or rabbit plasma in a haemolysis tube maintained in a water-bath at 37. Add 0.1 ml of a solution of the contents of the container containing 10,000 Units per ml in citro-phosphate buffer pH 7.2 and 0.1 ml of a solution of thrombin containing 20 Units per ml in citro-phosphate buffer pH 7.2 and shake immediately; a clot forms and lyses within 30 minutes. Repeat the procedure using citrated bovine plasma; lysis does not occur within 1 hour. B. Dissolve 0.6 g of agar in 50.0 ml of mixed barbitone buffer pH 8.6, heating until a clear solution is obtained. Place glass plates (50 mm x 50 mm) that are free from traces of grease on a level surface. Apply to each plate 4 ml of the agar solution and allow to cool until set. Bore a hole 6 mm in diameter in the centre of the agar and an appropriate number of holes (not exceeding six) at distances of 11 mm from the central hole removing the residual agar by means of a cannula connected to a vacuum pump. Place a quantity of 80 l of goat or rabbit antistreptokinase serum containing 10,000 Units of antistreptokinase activity per ml in the central hole and 80 l of a solution of the contents of the container containing 125,000 Units of streptokinase activity per ml of each of the surrounding holes. Place the plates in a humidified tank for 24 hours.
Streptodornase. Introduce 0.5 ml of a 0.1 per cent w/v solution of sodium deoxyribonucleate in imidazole buffer pH 6.5 into each of eight centrifuge tubes. To each of the first two tubes add 0.25 ml of imidazole buffer pH 6.5 and 0.25 ml of solution of the contents of the container with the substance under examination in imidazole buffer pH 6.5 containing 150,000 Units per ml (solution A) followed immediately by 3.0 ml of 0.25 M perchloric acid. Mix the contents of each tube, centrifuge for 5 minutes at 3000 rpm and measure the absorbance of each of the supernatant liquids at about 260 nm (2.4.7), using as the blank a mixture of 1.0 ml of imidazole buffer pH 6.5 and 3.0 ml of 0.25 M perchloric acid. Calculate the sum of the two absorbances (A1). To each of the remaining six tubes add, respectively, 0.25, 0.25, 0.125, 0.125, 0 and 0 ml of imidazole buffer pH 6.5 followed by 0.25 ml of solution A and finally 0, 0, 0.125, 0.125, 0.25 and 0.25 ml respectively of a solution of the Standard Preparation containing 20 Units of streptodornase activity per ml in imidazole buffer pH 6.5. [The Standard Preparation is the 1st International Standard Preparation for Streptodornase, established in 1964, consisting of a freeze-dried mixture of streptodornase and streptokinase with lactose (supplied in ampoules containing 2400 Units of streptodornase activity), or another suitable preparation the activity of which has been determined in relation to the International Standard]. Mix the contents of each tube, incubate at 37o for 15 minutes and add to each tube 3.0 ml of 0.25 M perchloric acid. Mix the contents of each tube, centrifuge and measure the absorbance of each of the supernatant liquids at about 260 nm (2.4.7), using as the blank the mixture specified above. If the sum of the absorbances of the liquids in the third and fourth tubes is A2, that of the liquids in the fifth and six tubes is A3, and that of the liquids in the seventh and eighth tubes is (A4), (A1A2) is less than 0.5(A3+A4)A2. Streptolysin. Dissolve a quantity of the contents of the container containing 500,000 Units in 0.5 ml of a mixture of 90 volumes of saline solution and 10 volumes of citrophosphate buffer pH 7.2 in a haemolysis tube. Add 0.4 ml of a 2.3 per cent w/v solution of sodium thioglycollate and incubate in a water-bath at 37 for 10 minutes. Add 0.1 ml of a solution of a reference preparation of human antistreptolysin O containing 5 Units per ml and incubate at 37 for 5 minutes. Add 1 ml of rabbit erythrocyte suspension, continue the incubation for 30 minutes and centrifuge at about 1000 rpm.
1129
STREPTOMYCIN SULPHATE
IP 2007
The absorbance of the supernatant liquid at about 550 nm (2.4.7), is not more than 1.5 times the absorbance obtained by repeating the above procedure using 0.5 ml of the mixture of saline solution and citro-phosphate buffer pH 7.2 in place of the solution containing the substance under examination. Bacterial endotoxins (2.2.3). Dissolve the contents of the sealed container in water BET to give a solution containing 10,000 Units of Streptokinase per ml. Carry out the test on the resulting solution; the maximum allowable endotoxin concentration of the solution is 23.33 Units of endotoxin per ml. Carry out the test using the maximum valid dilution of the prepared solution calculated from the declared sensitively of the lysate used in the test. Assay. Determine on the mixed contents of ten containers. The potency of streptokinase is determined by comparing its ability to activate human plasminogen to form plasmin with that of the Standard Preparation. The plasmin generated is determined by measurement of the time taken to lyse a fibrin clot under the conditions of a suitable method of assay. Standard Preparation The Standard Preparation is the 2nd International Standard for Streptokinase, established in 1989, consisting of freezedried streptokinase (supplied in ampoules containing 700 Units of streptokinase activity), or another suitable preparation the activity of which has been determined in relation to the International reference preparation. Method Use citro-phosphate buffer pH 7.2 containing 3 per cent w/v of bovine serum albumin for the preparation of solutions and dilutions. Prepare a solution of the Standard Preparation to contain 1000 Units of streptokinase activity per ml and prepare a solution of the contents of the container expected to have the same concentration; keep the solutions in ice and use within 6 hours. Prepare three 1.5-fold serial dilutions of the solution of the Standard Preparation so that the longest clot-lysis time is less than 20 minutes and prepare three similar dilutions of the solution of the preparation under examination. Keep the solutions in ice and use within 1 hour. Using 24 tubes, 8 mm in diameter, label the tubes S1, S2, S3 for the dilutions of the Standard Preparation and T1, T2, T3 for the dilutions of the preparation under examination, allocating four tubes to each dilution. Place the tubes in ice. Into each tube introduce 0.2 ml of the appropriate dilution, 0.2 ml of citro-phosphate buffer pH 7.2 containing 3 per cent w/v of bovine serum albumin and 0.1 ml of a solution containing 20 Units of thrombin per ml. Place the tubes in a water-bath at 37 and allow to stand for 2 minutes to attain temperature equilibrium. Using an automatic pipette, introduce into the bottom of the first tube 0.5 ml of a
1 per cent w/v solution to human euglobulins ensuring mixing. At 5-second intervals introduce successively into the remaining tubes 0.5 ml of a 1 per cent w/v solution of human euglobulins. Using a stop-watch, measure for each tube the time in seconds that elapses between the addition of the euglobulin and the lysis of the clot. Using the logarithms of the lysis times, calculate the result of the assay by standard statistical methods. The estimated potency is not less than 90 per cent and not more than 111 per cent of the stated potency. The fiducial limits of error are not less than 80 per cent and not more than 125 per cent of the stated potency. Storage. Store in sealed containers, protected from light in a refrigerator (2 to 8). The containers should be sterile and sealed so as to exclude micro-organisms. Under these conditions the contents may be expected to retain their potency for 2 years. Labelling. The label states the total number of Units of streptokinase activity contained in it.
Streptomycin Sulphate
NH HO HO O CHO H3C OH OH HO O H3CHN OH

2
HN
NH2 OH
HN
NH2 NH , 3H2 SO4
(C21H39N7O12)2,3H2SO4
Mol. Wt. 1457.4
Streptomycin Sulphate is the sulphate of O-2-deoxy-2methylamino--L-glucopyranosyl-(12)-O-5-deoxy-3-Cformyl--L-lyxofuranosyl-(14)-N1,N3-diamidino-Dstreptamine, a substance produced by the growth of certain strains of Streptomyces griseus or obtained by any other means.
1130
IP 2007
STREPTOMYCIN SULPHATE
Streptomycin Sulphate has a potency equivalent to not less than 700 g and not more than 850 g of streptomycin per mg. It contains not less than 90.0 per cent of the stated amount of streptomycin, C21H39N7O12, calculated on the dried basis. Description. A white or almost white powder; odourless or with slight odour; hygroscopic.
E. Gives the reactions of sulphates (2.3.1).
Tests
Appearance of solution. A 25.0 per cent w/v solution in carbon dioxide-free water is not more intensely coloured than degree 3 of the appropriate range of reference solutions (2.4.1). The solution, after standing protected from light at a temperature of about 20 for 24 hours, is not more opalescent than opalescence standard OS2 (2.4.1). pH (2.4.24). 4.5 to 7.0, determined in a 25.0 per cent w/v solution. Sulphates. 18.0 to 21.5 per cent, calculated on the dried basis, when determined by the following method. Dissolve 0.25 g in 100 ml of water, adjust the pH to 11 with strong ammonia solution and add 10.0 ml of 0.1 M barium chloride and 0.5 mg of metalphthalein. Titrate the excess of barium chloride with 0.1 M disodium edetate, adding 50 ml of ethanol (95 per cent) when the colour of the solution begins to change and continuing the titration until the violet-blue colour disappears. 1 ml of 0.1 M barium chloride is equivalent to 0.009606 g of sulphate, SO4. Colorimetric test. Dissolve 0.1 g in sufficient water to produce 100 ml. To 5 ml, add 5 ml of 0.2 M sodium hydroxide and heat in a water-bath for exactly 10 minutes. Cool in ice for exactly 5 minutes, add 3 ml of a 1.5 per cent w/v solution of ferric ammonium sulphate in 0.25 M sulphuric acid and sufficient water to produce 25 ml and mix. Exactly 20 minutes after the addition of the ferric ammonium sulphate solution, measure the absorbance of a 2-cm layer of the solution at the maximum at about 525 nm (2.4.7), using as the blank a solution prepared in the same manner but omitting the substance under examination. The absorbance is not less than 90.0 per cent of that obtained by carrying out the procedure at the same time and in the same manner using streptomycin sulphate RS in place of the substance under examination, each absorbance being calculated on the dried basis. Streptomycin B. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 50 volumes of toluene, 25 volumes of glacial acetic acid and 25 volumes of methanol. Test solution. Dissolve 0.2 g of the substance under examination in 5 ml of a freshly prepared mixture of 97 volumes of methanol and 3 volumes of sulphuric acid, heat under a reflux condenser for 1 hour, cool, wash down the condenser with methanol and add sufficient methanol to produce 20 ml. Reference solution. Dissolve 36 mg of D-mannose in 5 ml of a freshly prepared mixture of 97 volumes of methanol and 3 volumes of sulphuric acid, heat under a reflux condenser for 1 hour, cool, wash down the condenser with methanol and add sufficient methanol to produce 50 ml. Dilute 5 ml of the
Identification
A. Determine by thin-layer chromatography (2.4.17). Prepare the plate by mixing 0.3 g of carbomer with 240 ml of water, allowing to stand with moderate stirring for 1 hour, adjusting the pH to 7.0 by the gradual addition with constant shaking of 2 M sodium hydroxide and adding 30 g of silica gel H. Spread a uniform layer of the resulting suspension 0.75 mm thick. Heat the plate at 110 for 1 hour, allow to cool and use immediately. Mobile phase. A 7 per cent w/v solution of potassium dihydrogen phosphate. Test solution. Dissolve 0.1 g of the substance under examination in 100 ml of water. Reference solution (a). A 0.1 per cent w/v of streptomycin sulphate RS in water. Reference solution (b). A solution containing 0.1 per cent w/v of streptomycin sulphate RS, 0.1 per cent w/v of neomycin sulphate RS and 0.1 per cent w/v of kanamycin monosulphate RS in water. Apply to the plate 10 l of each solution. Allow the mobile phase to rise 12 cm. Dry the plate in warm air, spray with a mixture of equal volumes of a 0.2 per cent w/v solution of naphthalene-1,3-diol in ethanol (95 per cent) and sulphuric acid (45 per cent) and heat at 150 for 5 to 10 minutes. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows three clearly separated spots. B. Dissolve 5 to 10 mg in 4 ml of water and add 1 ml of 1 M sodium hydroxide. Heat in a water-bath for 4 minutes. Add a slight excess of 2 M hydrochloric acid and 0.1 ml of a 10 per cent w/v solution of ferric chloride; a violet colour is produced. C. Dissolve 0.1 g in 2 ml of water and add 1 ml of dilute 1-naphthol solution and 2 ml of a mixture of equal volumes of dilute sodium hypochlorite solution and water; a red colour is produced. D. Dissolve 10 mg in 5 ml of water and add 1 ml of 1 M hydrochloric acid. Heat in a water-bath for 2 minutes. Add 2 ml of a 0.5 per cent w/v solution of 1-naphthol in 1 M sodium hydroxide and heat in a water-bath for 1 minute; a faint yellow colour is produced.
1131
STREPTOMYCIN INJECTION
IP 2007
resulting solution to 50 ml with methanol; this solution contains the equivalent of 0.03 per cent w/v of streptomycin B (1 mg of D-mannose is equivalent to 4.13 mg of streptomycin B). Apply to the plate 10 l of each solution. Allow the mobile phase to rise 13 to 15 cm. Dry the plate in air, and spray with a freshly prepared mixture of equal volumes of a 0.2 per cent w/v solution of naphthalene-1,3-diol in ethanol (95 per cent) and a 20 per cent v/v solution of sulphuric acid and heat at 110 for 5 minutes. Any spot in the chromatogram obtained with the test solution is not more intense than the corresponding spot in the chromatogram with the reference solution. Methanol. Determine by gas chromatography (2.4.13). Test solution. A 4 per cent w/v solution of the substance under examination in water. Reference solution. A 0.012 per cent w/v solution of methanol in water. Chromatographic system a glass column 1.5 to 2.0 m x 2 to 4 mm, packed with ethylvinylbenzene-divinylbenzene copolymer (150 to 180 m) porous polymer beads (such as Porapak Q), temperature: column 120 to 140, inlet port and detector at least 50 higher than that of the column, flow rate. 30 to 40 ml per minute of the carrier gas. The area of any peak corresponding to methanol in the chromatogram obtained with the test solution is not greater than that of the peak in the chromatogram obtained with reference solution (0.3 per cent). Sulphated ash (2.3.18). Not more than 1.0 per cent. Loss on drying (2.4.19). Not more than 7.0 per cent, determined on 1.0 g by drying over phosphorus pentoxide at 60 at a pressure not exceeding 0.1 kPa for 24 hours. Assay. Determine by the microbiological assay of antibiotics, Method A or B (2.2.10), and express the results in g of streptomycin per mg. Streptomycin Sulphate intended for use in the manufacture of parenteral preparations without a further appropriate procedure for removal of bacterial endotoxins complies with the following additional requirement. Bacterial endotoxins (2.2.3). Not more than 0.25 Endotoxin Unit per mg of streptomycin. Streptomycin Sulphate intended for use in the manufacture of parenteral preparations without a further appropriate sterilisation procedure complies with the following additional requirement. Sterility (2.2.11). Complies with the test for sterility.
Storage. Store protected from light and moisture. If it is intended for use in the manufacture of parenteral preparations the container should be sterile and sealed so as to exclude micro-organisms. Labelling. The label states (1) the equivalent weight of streptomycin contained in it; (2) whether or not the contents are intended for use in the manufacture of parenteral preparations; (3) the name and quantity of any added stabiliser.
Streptomycin Injection
Streptomycin Sulphate Injection
Streptomycin Injection is a sterile material consisting of Streptomycin Sulphate with or without auxiliary agents. It is filled in a sealed container. The injection is constituted by dissolving the contents of the sealed container in the requisite amount of sterile Water for Injections, immediately before use. The constituted solution complies with the requirements for Clarity of solution and Particulate matter stated under Parenteral Preparations (Injections). Storage. The constituted solution should be used immediately after preparation but, in any case, within the period recommended by the manufacturer. Streptomycin Injection contains not less than 90.0 per cent and not more than 115.0 per cent of the stated amount of streptomycin, C21H39N7O12, calculated on the dried basis. The contents of the sealed container comply with the requirements stated under Parenteral Preparations (Powders for Injection) and with the following requirements. Description. A white or almost white powder which yields a clear, colourless or faintly yellow coloured solution when dissolved in water.
Identification
A. Determine by thin-layer chromatography (2.4.17). Prepare the plate by mixing 0.3 g of carbomer with 240 ml of water, allowing to stand with moderate stirring for 1 hour, adjusting the pH to 7.0 by the gradual addition with constant shaking of 2 M sodium hydroxide and adding 30 g of silica gel H. Spread a uniform layer of the resulting suspension 0.75 mm thick. Heat the plate at 110 for 1 hour, allow to cool and use immediately. Mobile phase. A 7 per cent w/v solution of potassium dihydrogen phosphate. Test solution. Dissolve 0.1 g of the substance under examination in 100 ml of water. Reference solution (a). A 0.1 per cent w/v of streptomycin sulphate RS in water.
1132
IP 2007
SUCCINYLCHOLINE CHLORIDE
Reference solution (b). A solution containing 0.1 per cent w/v of streptomycin sulphate RS, 0.1 per cent w/v of neomycin sulphate RS and 0.1 per cent w/v of kanamycin monosulphate RS in water. Apply to the plate 10 l of each solution. Allow the mobile phase to rise 12 cm. Dry the plate in warm air, spray with a mixture of equal volumes of a 0.2 per cent w/v solution of naphthalene-1,3-diol in ethanol (95 per cent) and sulphuric acid (45 per cent) and heat at 150 for 5 to 10 minutes. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows three clearly separated spots. B. Dissolve 5 to 10 mg in 4 ml of water and add 1 ml of 1 M sodium hydroxide. Heat in a water-bath for 4 minutes. Add a slight excess of 2 M hydrochloric acid and 0.1 ml of a 10 per cent w/v solution of ferric chloride; a violet colour is produced. C. Dissolve 0.1 g in 2 ml of water and add 1 ml of dilute 1-naphthol solution and 2 ml of a mixture of equal volumes of dilute sodium hypochlorite solution and water; a red colour is produced. D. Dissolve 10 mg in 5 ml of water and add 1 ml of 1 M hydrochloric acid. Heat in a water-bath for 2 minutes. Add 2 ml of a 0.5 per cent w/v solution of 1-naphthol in 1 M sodium hydroxide and heat in a water-bath for 1 minute; a faint yellow colour is produced.
Identification
A. Triturate a quantity of the powdered tablets containing 0.2 g of streptomycin in a mixture of 2 ml of methanol and 0.1 ml of sulphuric acid, filter, if necessary, and allow to stand at about 25; crystals of streptidine sulphate separate in the course of 2 to 3 days. Dissolve the crystals in a solution of 0.1 g of picric acid in 10 ml of hot water and cool; the precipitate, after recrystallisation from hot water, melts at about 283 (2.4.21). B. Boil a small quantity of the powdered tablets containing 0.1 g of streptomycin with 5 ml of 1 M sodium hydroxide for a few minutes, add a slight excess of 2 M hydrochloric acid and 0.15 ml of a 10 per cent w/v solution of ferric chloride; a brilliant violet colour is produced.
Tests
Other tests. Comply with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 0.3 g of streptomycin and, triturate with 20 ml of buffer solution pH 8.0. Dilute to 100.0 ml with water. Determine by the microbiological assay of antibiotics, Method A or B (2.2.10). Storage. Store protected from moisture. Labelling. The label states the strength in terms of the equivalent amount of streptomycin.
Tests
pH (2.4.24). 4.5 to 7.0, determined in a 25.0 per cent w/v solution. Loss on drying (2.4.19). Not more than 7.0 per cent, determined on 1.0 g by drying in an oven at 60 over phosphorus pentoxide at a pressure not exceeding 0.1 kPa for 24 hours. Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. Determine on the mixed contents of ten containers by the microbiological assay of antibiotics, Method A or B (2.2.10). Bacterial endotoxins (2.2.3): Not more than 0.25 Endotoxin Unit per mg of streptomycin. Storage. Store protected from moisture. Labelling. The label states the strength in terms of the equivalent amount of streptomycin in a suitable dose-volume.
Succinylcholine Chloride
Suxamethonium Chloride
H3C H3 C N H3C O O O O CH3 N CH3 CH3
2Cl , 2H2 O
C14H30Cl2N2O4,2H2O
Mol. Wt. 397.3
Succinylcholine Chloride is 2,2'succinyldioxybis(ethyltrimethylammonium) dichloride dihydrate. Succinylcholine Chloride contains not less than 98.0 per cent and not more than 101.0 per cent of C14H30Cl2N2O4, calculated on the anhydrous basis. Description. A white or almost white, crystalline powder; almost odourless; hygroscopic.
Streptomycin Tablets
Streptomycin Sulphate Tablets Streptomycin Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of streptomycin, C21H39N7O12.
Identification
Test A may be omitted if tests B, C and D are carried out. Tests B and C may be omitted if tests A and D are carried out.
1133
SUCCINYLCHOLINE INJECTION
IP 2007
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with succinylcholine chloride RS or with the reference spectrum of succinylcholine chloride. B. Dissolve about 25 mg in 1 ml of water, add 0.1 ml of a 1 per cent w/v solution of cobalt chloride and 0.1 ml of potassium ferrocyanide solution; a green colour is produced. C. Dissolve 1 g in sufficient carbon dioxide-free water to produce 20 ml. To 1 ml of this solution add 9 ml of water, 10 ml of 1 M sulphuric acid and 30 ml of ammonium reineckate solution; a pink precipitate is produced. Allow to stand for 30 minutes, filter and wash with water, then with ethanol (95 per cent) and finally with ether. The residue, after drying at 80 melts at 180 to 185 (2.4.21). D. Gives the reactions of chlorides (2.3.1).
1 ml of 0.1 M perchloric acid is equivalent to 0.01807 g of C14H30Cl2N2O4. Storage. Store protected from light and moisture.
Succinylcholine Injection
Suxamethonium Chloride Injection
Succinylcholine Injection is a sterile solution of Succinylcholine Chloride in Water for Injections. Succinylcholine Injection contains not less than 90.0 per cent and not more than 107.5 per cent of the stated amount of succinylcholine chloride, C14H30Cl2N2O4,2H2O.
Tests
Appearance of solution. A 5.0 per cent w/v solution in carbon dioxide-free water (solution A) is clear (2.4.1). 4 ml of solution A diluted to 10 ml with water is colourless (2.4.1). pH (2,4,24). 4.0 to 5.0, determined in a 0.5 per cent w/v solution. Choline chloride. Determine by thin-layer chromatography (2.4.17), coating the plate with microcrystalline cellulose. Mobile phase. A mixture of 50 volumes of 1-butanol, 40 volumes of water and 10 volumes of anhydrous formic acid, shake for 10 minutes, allow to separate. Use the upper layer as the mobile phase. Test solution. Dissolve 0.4 g of the substance under examination in 10 ml of methanol. Reference solution. A solution containing 4 per cent w/v of succinylcholine chloride RS and 0.02 per cent w/v of choline chloride in methanol. Apply to the plate 5 l of each solution. After development, dry the plate in air, spray with potassium iodobismuthate solution. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot due to choline chloride in the chromatogram obtained with the reference solution. The test is not valid unless the chromatogram obtained with the reference solution shows two clearly separated spots. Sulphated ash (2.3.18). Not more than 0.1 per cent. Water (2.3.43). 8.0 to 10.0 per cent, determined on 0.3 g. Assay. Weigh accurately about 0.3 g, dissolve in 30 ml of anhydrous glacial acetic acid, add 30 ml of acetic anhydride and 10 ml of mercuric acetate solution. Titrate with 0.1 M perchloric acid, using crystal violet solution as indicator, until a bluish green colour is produced. Carry out a blank titration.
Identification
Dilute a volume containing 20 mg of Succinylcholine Chloride to 50 ml with water. To 0.5 ml add 2 ml of chloroform, 2 ml of a solution containing 0.16 per cent w/v of citric acid and 6.6 per cent w/v of disodium hydrogen phosphate and 0.1 ml of a solution containing 0.15 per cent w/v of each of bromothymol blue and anhydrous sodium carbonate. Shake for 2 minutes and allow to separate. The chloroform layer is yellow.
Tests
pH (2.4.28). 3.0 to 5.0. Hydrolysis products. The volume of 0.1 M sodium hydroxide required for the preliminary neutralisation in the Assay is not more than one tenth of the total volume of 0.1 M sodium hydroxide required for the preliminary neutralisation and the hydrolysis. Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. To an accurately measured volume containing about 0.25 g of Succinylcholine Chloride add 30 ml of carbon dioxidefree water and shake with five quantities, each of 25 ml, of ether. Wash the combined ether solutions with two quantities, each of 10 ml, of water and discard the ether. Shake the combined washings with two quantities, each of 10 ml, of ether, add the washings to the original aqueous solution and neutralise with 0.1 M sodium hydroxide using bromothymol blue solution as indicator. Add 25.0 ml of 0.1 M sodium hydroxide, heat under a reflux condenser for 40 minutes, allow to cool and titrate the excess of alkali with 0.1 M hydrochloric acid using bromothymol blue solution as indicator. Repeat the operation using 40 ml of carbon dioxide-free water beginning at the words Add 25.0 ml of 0.1 M sodium hydroxide...... The difference between the titrations represents the amount of sodium hydroxide required.
1134
IP 2007
SULPHACETAMIDE SODIUM
1 ml of 0.1 M sodium hydroxide is equivalent to 0.01987 g of C14H30Cl2N2O4,2H2O. Storage. Store protected from light. The injection should not be allowed to freeze.
Calcium. To 1 ml of solution A add 9 ml of water and 1 ml of ammonium oxalate solution; the solution remains clear for at least 1 minute. Heavy metals (2.3.13). Add 0.1 ml of dilute hydrochloric acid to 4 ml of solution A and dilute with sufficient water to produce 25 ml. The solution complies with the limit test for heavy metals, Method A (10 ppm). Sulphites. To 4 ml of solution A add sufficient water to produce 20 ml, add 0.05 ml of 0.1 M iodine and 0.05 ml of starch solution; a blue colour develops.
Sucrose
Refined Sugar
HO O OH OH HO O O OH HO OH
C12H22O11
Dextrins. To 2 ml of solution A add 8 ml of water, 0.05 ml of 2 M hydrochloric acid and 0.05 ml of 0.05 M iodine; the solution remains yellow or becomes faint bluish green. Glucose and invert sugar. Dissolve 20 g in sufficient water to make 100 ml and filter if necessary. Place 50 ml of the clear solution in a 250-ml beaker, add 50 ml of alkaline cupric tartarate solution, cover the beaker with a watch glass, heat the mixture at such a rate that it comes to a boil in approximately 4 minutes and continue boiling for exactly 2 minutes. Add immediately 100 ml of recently boiled and cooled water and collect the precipitated cuprous oxide on a tared sintered glass crucible. Wash the residue with the hot water, then with 10 ml of ethanol (95 per cent) and finally with 10 ml of ether. Dry at 105 for 1 hour; the weight of the cuprous oxide is not more than 112 mg. Colouring matter. A. To 100 ml of solution A in a groundglass-stoppered tube add 1 ml of dilute hypophosphorous acid and allow to stand for 1 hour; no unpleasant odour is detectable. B. Examine solution A in ultraviolet light at 365 nm. Any fluorescence is not more intense than that of a solution containing 0.4 mg of quinine sulphate in 0.005 M sulphuric acid. Sulphated ash (2.3.18). Not more than 0.1 per cent determined by dissolving 5.0 g in 5 ml of water, adding 2 ml of sulphuric acid, evaporating to dryness and igniting to constant weight. Storage. Store protected from light and moisture.
OH
Mol. Wt. 342.3
Sucrose is -D-fructofuranosyl--D-glucopyranoside. Description. An almost white or colourless crystals, dry crystalline powder; odourless; taste, sweet.
Identification
Dissolve 150.0 g in sufficient carbon dioxide-free water prepared from distilled water to produce 300 ml (solution A). Dilute 1 ml of solution A to 100 ml with water. To 5 ml of the solution add 2 ml of freshly prepared 2 M sodium hydroxide and 0.15 ml of a freshly prepared copper sulphate solution; the solution is clear and blue and remains so on boiling. To the hot solution add 4 ml of 2 M hydrochloric acid, heat to boiling and add 4 ml of 2 M sodium hydroxide; an orange precipitate is produced immediately.
Tests
Acidity or alkalinity. To 10 ml of solution A add 0.3 ml of phenolphthalein solution. The solution is colourless and not more than 0.6 ml of 0.01 M sodium hydroxide is required to change the colour of the solution to pink. Specific optical rotation (2.4.22). +65.9 to +67.0, determined in a 10 per cent w/v solution. Barium. To 10 ml of solution A add 1 ml of 1 M sulphuric acid. When examined immediately and after 1 hour any opalescence is not more intense than that of a mixture of 1 ml of distilled water and 10 ml of solution A.
Sulphacetamide Sodium
O O O S N CH3 ,H2O Na
Mol. Wt. 254.2
H2N
C8H9N2NaO3S,H2O
Sulphacetamide Sodium is the monohydrate of the sodium salt of N1-acetylsulphanilamide.
1135
SULPHACETAMIDE EYE DROPS
IP 2007
Sulphacetamide Sodium contains not less than 99.0 per cent and not more than 101.0 per cent of C8H9N2NaO3S, calculated on the anhydrous basis. Description. A white or yellowish white, crystalline powder; odourless.
Reference solution (b). A 0.025 per cent w/v solution of sulphanilamide in water. Reference solution (c). A 0.05 per cent w/v solution of sulphanilamide in the test solution. Apply to the plate 5 l of each solution. After development, dry the plate in air and spray with a freshly prepared 2 per cent w/v solution of dimethylaminobenzaldehyde in a mixture of 55 volumes of hydrochloric acid and 45 volumes of water. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (a) and not more than one such spot is more intense than the spot in the chromatogram obtained with reference solution (b). The chromatogram obtained with reference solution (c) shows two clearly separated spots. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Sulphates (2.3.17). Dissolve 1.5 g in sufficient distilled water to produce 25 ml, add 25 ml of 2 M acetic acid, shake for 30 minutes and filter. 25 ml of the filtrate complies with the limit test for sulphates (200 ppm). Water (2.3.43). 6.0 to 8.0 per cent, determined on 0.2 g. Assay. Weigh accurately about 0.25 g, dissolve in a mixture of 50 ml of water and 20 ml of 2 M hydrochloric acid, add 3 g of potassium bromide, cool in ice and carry out the nitrite titration (2.3.31). 1 ml of 0.1 M sodium nitrite is equivalent to 0.02362 g of C8H9N2NaO3S. Storage. Store protected from light and moisture.
Identification
Test A may be omitted if tests B, C, D, E and F are carried out. Tests B, C, D and E may be omitted if tests A and F are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with sulphacetamide sodium RS or with the reference spectrum of sulphacetamide sodium. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.001 per cent w/v solution in citro-phosphate buffer pH 7.0 shows an absorption maximum at about 255 nm; absorbance at about 255 nm, 0.66 to 0.72. C. Dissolve 1 g in 10 ml of water, add 6 ml of 2 M acetic acid and filter. Wash the precipitate with a small volume of water and dry at 105 for 4 hours. The melting range of the precipitate is 181 to 185 (2.4.21). D. Dissolve 0.1 g of the precipitate obtained in test C in 5 ml of ethanol (95 per cent), add 0.2 ml of sulphuric acid and heat; ethyl acetate, recognizable by its odour, is produced. E. Dissolve 1 mg of the precipitate obtained in test C in 5 ml of water with the aid of heat. The solution gives the reaction of primary aromatic amines (2.3.1), producing an orange-red precipitate. F. A 5 per cent w/v solution gives the reactions of sodium salts (2.3.1).
Tests
Appearance of solution. A 5.0 per cent w/v solution in carbon dioxide-free water is clear (2.4.1), and not more intensely coloured than reference solution GYS4 (2.4.1). pH (2.4.28). 8.0 to 9.5, determined in a 5.0 per cent w/v solution. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel HF254. Mobile phase. A mixture of 50 volumes of 1-butanol, 25 volumes of ethanol, 25 volumes of water and 10 volumes of strong ammonia solution. Test solution. A 10 per cent w/v solution of the substance under examination in water. Reference solution (a). A 0.05 per cent w/v solution of sulphanilamide in water.
Sulphacetamide Eye Drops

Sulphacetamide Sodium Eye Drops
Sulphacetamide Eye Drops are a sterile solution of Sulphacetamide Sodium in Purified Water. It may contain a suitable antimicrobial agent. Sulphacetamide Eye Drops contain not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of sulphacetamide sodium, C8H9N2NaO3S,H2O.
Identification
To a volume containing 0.5 g of Sulphacetamide Sodium add 6 ml of 5 M acetic acid, stirring constantly. Filter the precipitate, wash with water and dry at 105 for 4 hours. The residue complies with the following tests.
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SULPHADIAZINE
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with sulphacetamide RS or with the reference spectrum of sulphacetamide. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.001 per cent w/v solution in citro-phosphate buffer pH 7.0 shows an absorption maximum at about 255 nm; absorbance at about 255 nm, 0.66 to 0.72. C. Dissolve 10 mg in 2 ml of 2 M hydrochloric acid. The solution gives the reaction of primary aromatic amines (2.3.1).
Sulphadiazine
O O N S N N H H2N
C10H10N4O2S Mol. Wt. 250.3
Sulphadiazine is N1-(pyrimidin-2-yl)sulphanilamide. Sulphadiazine contains not less than 99.0 per cent and not more than 101.0 per cent of C10H10N4O2S, calculated on the dried basis. Description. White, yellowish white or pinkish white crystals or crystalline powder; almost odourless.
Tests
Appearance of solution. Dilute the eye drops, if necessary, to contain 10.0 per cent w/v of Sulphacetamide Sodium. The solution is not more intensely coloured than reference solution BYS4 (2.4.1). pH (2.4.24). 6.6 to 8.6. Related substances. Determine by thin-layer chromatography (2.4.14), coating the plate with silica gel HF254. Mobile phase. A mixture of 50 volumes of 1-butanol, 25 volumes of ethanol, 25 volumes of water and 10 volumes of strong ammonia solution. Test solution. Dilute a suitable volume with water to produce a solution containing 4 per cent w/v of Sulphacetamide Sodium. Reference solution. A 0.2 per cent w/v solution of sulphanilamide in water. Apply to the plate 5 l of each solution. After development, dry the plate in air and spray with a freshly prepared 2 per cent w/v solution of dimethylaminobenzaldehyde in a mixture of 55 volumes of hydrochloric acid and 45 volumes of water. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Other tests. Comply with the tests stated under Eye Drops. Assay. To an accurately measured volume containing about 0.5 g of Sulphacetamide Sodium add 75 ml of water and 10 ml of hydrochloric acid. Add 3 g of potassium bromide, cool in ice and carry out the nitrite titration (2.3.31). 1 ml of 0.1 M sodium nitrite is equivalent to 0.02542 g of C8H9N2NaO3S,H2O. Storage. Store protected from light and moisture. The Eye Drops should not be allowed to freeze. Labelling. The label states (1) the name and concentration of any antimicrobial agent used; (2) that it is not meant for injection; (3) that the solution should be used within one month of opening the container; (4) that the solution should not be used if it is dark brown in colour; (5) that it should not be allowed to freeze.
Identification
Test A may be omitted if tests B, C and D are carried out. Tests C and D may be omitted if tests A and B are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with sulphadiazine RS or with the reference spectrum of sulphadiazine. B. In the test for Related substances, the principal spot in the chromatogram obtained with test solution (b) corresponds to that in the chromatogram obtained with the reference solution. C. Dissolve about 5 mg in 10 ml of 1 M hydrochloric acid and dilute 1 ml of this solution to 10 ml with water. The solution, without further acidification, gives the reaction of primary aromatic amines (2.3.1). D. Heat 3 g in a test-tube inclined at an angle of 45 with the lower part immersed in a silicone oil-bath at about 270. It decomposes and a white or yellowish white sublimate is produced. The sublimate, after recrystallisation from toluene and drying at 100 melts at 123 to 127 (2.4.21).
Tests
Appearance of solution. Dissolve 0.8 g in 10 ml of 1 M sodium hydroxide. The solution is not more intensely coloured than reference solution YS5, BYS5 or GYS5 (2.4.1). Acidity. Heat 1.25 g of the finely powdered substance at about 70 with 25 ml of carbon dioxide-free water for 5 minutes. Cool for about 15 minutes in ice and filter. To 20 ml of the filtrate add 0.1 ml of bromothymol blue solution. Not more than 0.2 ml of 0.1 M sodium hydroxide is required to change the colour of the solution. Related substances (2.3.7). Complies with test C. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm).
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IP 2007
Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.2 g, dissolve in a mixture of 20 ml of 2 M hydrochloric acid and 50 ml of water. Add 3 g of potassium bromide, cool in ice and carry out the nitrite titration (2.3.31). 1 ml of 0.1 M sodium nitrite is equivalent to 0.02503 g of C10H10N4O2S. Storage. Store protected from light and moisture.
Dissolution (2.5.2). Apparatus. No 1 Medium. 900 ml of 0.1 M hydrochloric acid Speed and time. 100 rpm and 60 minutes. Withdraw a suitable volume of the sample and filter promptly through a membrane filter disc with an average pore diameter not greater than 1.0 mm. Reject the first few ml of the filtrate and dilute a suitable volume of the filtrate with the same solvent. Dilute suitably with 0.01 M sodium hydroxide. Measure the absorbances of the resulting solution and of a standard solution of sulphadiazine RS of similar concentration in the same medium at the maximum at about 254 nm (2.4.7). Calculate the content of C10H10N4O2S in the medium. D. Not less than 70.0 per cent of the stated amount of C10H10N4O2S. Other tests. Comply with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 0.5 g of Sulphadiazine and dissolve as completely as possible in a mixture of 50 ml of water and 10 ml of hydrochloric acid. Carry out the nitrite titration (2.3.31). 1 ml of 0.1 M sodium nitrite is equivalent to 0.02503 g of C10H10N4O2S. Storage. Store protected from light and moisture.
Sulphadiazine Tablets
Sulphadiazine Tablets contain not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of sulphadiazine, C10H10N4O2S.
Identification
A. Triturate a quantity of the powdered tablets containing 0.5 g Sulphadiazine with two successive quantities, each of 5 ml, of chloroform and reject the chloroform. Triturate the residue with 10 ml of dilute ammonia solution for 5 minutes, add 10 ml of water and filter. Warm the filtrate until most of the ammonia has been expelled, cool and acidify with acetic acid. Collect the precipitate, wash with water and dry at about 100; the residue melts at about 256, with decomposition (2.4.21). B. On the residue obtained in test A determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with sulphadiazine RS or with the reference spectrum of sulphadiazine. C. In the test for Related substances, the principal spot in the chromatogram obtained with test solution (b) corresponds to that in the chromatogram obtained with the reference solution.
Sulphadimethoxine
OCH3 O O S N H H2N
C12H14N4O4S
1
N N OCH3
Tests
Related substances (2.3.7). Complies with test C, but using the following solutions. Test solution (a). Extract a quantity of the powdered tablets containing 0.5 g of Sulphadiazine with 25 ml of a mixture of 90 volumes of methanol and 10 volumes of strong ammonia solution by shaking for 10 minutes, filter and use the filtrate. Test solution (b). Dilute 1 volume of test solution (a) to 5 volumes with a mixture of 24 volumes of methanol and 1 volume of strong ammonia solution. Test solution (c). Dilute 1 volume of test solution (a) to 200 volumes with the same solvent mixture. Reference solution. A 0.4 per cent w/v solution of sulphadiazine RS in the same solvent mixture.
Mol. Wt. 310.3
Sulphadimethoxine is N -(2,6-dimethoxypyrimidin-4-yl) sulphanilamide. Sulphadimethoxine contains not less than 99.0 per cent and not more than 101.0 per cent of C12H14N4O4S, calculated on the dried basis. Description. A white or creamy-white, crystalline powder.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with sulphadimethoxine RS or with the reference spectrum of sulphadimethoxine
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B. Dissolve about 0.1 g in 3 ml of 5 M sodium hydroxide and 50 ml of water and dilute to 100 ml with water. To 5 ml of the solution add 100 mg of phenol, heat to boiling, cool and add 0.5 ml of sodium hypochlorite solution and 0.15 ml of 5 M sodium hydroxide; a yellow colour is produced. C. Dissolve about 5 mg in 10 ml of 1 M hydrochloric acid and dilute 1 ml to 10 ml with water. The resulting solution, without further acidification, gives the reaction of primary aromatic amines (2.3.1). D. Suspend about 20 mg in 5 ml of water and add 5 M sodium hydroxide until completely dissolved. Add 0.2 ml of cupric sulphate solution; the solution turns yellow and a yellow precipitate is formed. E. Melting point (2.4.21). 197 to 204.
B. Dissolve about 0.1 g in 3 ml of 5 M sodium hydroxide and 50 ml of water and dilute to 100 ml with water. To 5 ml of the solution add 100 mg of phenol, heat to boiling, cool and add 0.5 ml of sodium hypochlorite solution and 0.15 ml of 5 M sodium hydroxide; a yellow colour is produced. C. Dissolve about 5 mg in 10 ml of 1 M hydrochloric acid and dilute 1 ml to 10 ml with water. The resulting solution, without further acidification, gives the reaction of primary aromatic amines (2.3.1). D. Suspend about 20 mg in 5 ml of water and add 5 M sodium hydroxide until completely dissolved. Add 0.2 ml of cupric sulphate solution; the solution turns yellow and a yellow precipitate is formed. E. Melting range (2.4.21). 197 to 204.
Tests
Related substances (2.3.7). Complies with test B. Heavy metals (2.3.1.3). Dissolve 1.0 g in 5 ml of 5 M sodium hydroxide and 20 ml of water. The solution complies with the limit test for heavy metals, Method A (20 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.3 g, dissolve in a mixture of 20 ml of 2 M hydrochloric acid and 50 ml of water. Add 3 g of potassium bromide, cool in ice and carry out the nitrite titration (2.3.31). 1 ml of 0.1 M sodium nitrite is equivalent to 0.03103 g of C12H14N4O4S. Storage. Store protected from light and moisture.
Tests
Related substances (2.3.7). Complies with test B, but using the following solutions. Test solution. Extract a quantity of the powdered tablets containing 0.25 g of Sulphadimethoxine with 100 ml of a mixture of 9 volumes of methanol and 1 volume of strong ammonia solution by shaking for 10 minutes, filter and use the filtrate. Reference solution. A 0.00125 per cent w/v solution of sulphanilamide. Other tests. Comply with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 0.3 g of Sulphadimethoxine, dissolve as completely as possible in a mixture of 50 ml of water and 10 ml of hydrochloric acid and carry out the nitrite titration (2.3.31). 1 ml of 0.1 M sodium nitrite is equivalent to 0.03103 g of C12H14N4O4S. Storage. Store protected from light and moisture.
Sulphadimethoxine Tablets
Sulphadimethoxine Tablets contain not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of sulphadimethoxine, C12H14N4O4S.
Sulphadimidine
CH3 O O N S N N H H2N
C12H14N4O2S
1
Identification
Triturate a quantity of the powdered tablets containing 0.5 g of Sulphadimethoxine with 5 ml of 0.5 M hydrochloric acid, filter and neutralise the filtrate to litmus paper with 0.5 M sodium hydroxide. The precipitate, after washing with water and drying at 105 complies with the following tests. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with sulphadimethoxine RS or with the reference spectrum of sulphadimethoxine
CH3
Mol. Wt. 278.3
Sulphadimidine is N -(4,6-dimethylpyrimidin-2-yl) sulphanilamide.
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SULPHADIMIDINE SODIUM
IP 2007
Sulphadimidine contains not less than 99.0 per cent and not more than 101.0 per cent of C12H14N4O2S, calculated on the dried basis. Description. White or almost white crystals or powder.
Sulphadimidine Sodium
C12H13N4NaO2S Mol. Wt. 300.3 Sulphadimidine Sodium is the sodium salt of N 1-(4,6dimethylpyrimidin-2-yl)sulphanilamide. Sulphadimidine Sodium contains not less than 98.0 per cent and not more than 101.0 per cent of C12H13N4NaO2S, calculated on the dried basis. Description. White or creamy white crystals or powder; odourless or almost odourless; hygroscopic.
Identification
Test A may be omitted if tests B, C and D are carried out. Tests C and D may be omitted if tests A and B are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with sulphadimidine RS or with the reference spectrum of sulphadimidine. B. In the test for Related substances, the principal spot in the chromatogram obtained with test solution (b) corresponds to that in the chromatogram obtained with the reference solution. C. Heat 3 g in a test-tube inclined at an angle of about 45 with the lower part immersed in a silicone oil-bath at about 270. It decomposes and a white or yellowish white sublimate is produced. The sublimate, after recrystallisation from toluene and drying at 100 melts at 150 to 154 (2.4.31). D. Dissolve about 5 mg in 10 ml of 1 M hydrochloric acid and dilute 1 ml of this solution to 10 ml with water. The solution, without further acidification, gives the reaction of primary aromatic amines (2.3.1).
Identification
A. Dissolve 0.1 g in 10 ml water, acidify with 1 M hydrochloric acid, filter, wash the precipitate with water and dry the residue at 105. On the residue determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with sulphadimidine RS or with the reference spectrum of sulphadimidine. B. Acidify a solution of 0.1 g in 5 ml of water with 6 M acetic acid. A precipitate is produced which, after washing with cold water and drying at 105, gives the reaction of primary aromatic amines (2.3.1), producing a bright orange-red precipitate. C. The washed and dried precipitate obtained in test B melts at about 198 (2.4.21). D. Incinerate 0.5 g. The residue, when moistened with hydrochloric acid and introduced on a platinum wire into the flame of a Bunsen burner, imparts a yellow colour to the flame.
Tests
Appearance of solution. A 5.0 per cent w/v solution in 1 M sodium hydroxide is not more intensely coloured than reference solution YS5, BYS5 or GYS5 (2.4.1). Acidity. Heat 1.25 g of the finely powdered substance at about 70 with 25 ml of carbon dioxide-free water for 5 minutes. Cool for about 15 minutes in ice and filter. To 20 ml of the filtrate add 0.1 ml of bromothymol blue solution. Not more than 0.2 ml of 0.1 M sodium hydroxide is required to change the colour of the solution. Related substances (2.3.7). Complies with test C. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.25 g, dissolve in 20 ml of 2 M hydrochloric acid and 50 ml of water, add 3 g of potassium bromide, cool in ice and carry out the nitrite titration (2.3.31). 1 ml of 0.1 M sodium nitrite is equivalent to 0.02783 g of C12H14N4O2S. Storage. Store protected from light and moisture.
Tests
Appearance of solution. A 33.3 per cent w/v solution is clear (2.4.1), and not more intensely coloured than reference solution YS4 (2.4.1). pH (2.4.24). 10.0 to 11.0, determined in a 10.0 per cent w/v solution. Related substances (2.3.7). Complies with test A, but using as the test solution a solution prepared by dissolving the substance under examination in 1 volume of strong ammonia solution and then diluting with 9 volumes of ethanol (95 per cent) to produce a 1.0 per cent w/v solution. Loss on drying (2.4.19). Not more than 2.0 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.3 g, dissolve in a mixture of 75 ml of water and 10 ml of hydrochloric acid, add 3 g of potassium bromide, cool in ice and carry out the nitrite titration (2.3.31). 1 ml of 0.1 M sodium nitrite is equivalent to 0.03003 g of C12H13N4NaO2S.
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SULPHADIMIDINE TABLETS
Add 3 g of potassium bromide, cool in ice and carry out the nitrite titration (2.3.31). 1 ml of 0.1 M sodium nitrite is equivalent to 0.03003 g of C12H13N4NaO2S. Storage. Store protected from light, in single dose containers. Labelling. When the injection is prepared from Sulphadimidine the strength is stated as the amount of sulphadimidine sodium in a suitable dose-volume.
Sulphadimidine Injection
Sulphadimidine Sodium Injection
Sulphadimidine Injection is a sterile solution of Sulphadimidine Sodium in Water for Injections free from dissolved air. It is prepared either from Sulphadimidine Sodium or by the interaction of Sulphadimidine and Sodium Hydroxide. Sulphadimidine Injection contains not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of sulphadimidine sodium, C12H13N4NaO2S.
Sulphadimidine Tablets
Sulphadimidine Tablets contain not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of sulphadimidine, C12H14N4O2S.
Identification
A. Acidify a volume containing 0.1 g of Sulphadimidine Sodium with 6 M acetic acid, filter, reserving the filtrate, wash the precipitate with water and dry at 105. On the residue determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with sulphadimidine RS or with the reference spectrum of sulphadimidine. B. The residue obtained in test A gives the reaction of primary aromatic amines (2.3.1). C. Evaporate the filtrate obtained in test A and incinerate. The residue, when moistened with hydrochloric acid and introduced on a platinum wire into the flame of a Bunsen burner, imparts a yellow colour to the flame.
Identification
A. Triturate a quantity of the powdered tablets containing 0.5 g of Sulphadimidine with two quantities, each of 5 ml, of chloroform and discard the chloroform. Triturate the residue with 10 ml of 5 M ammonia for 5 minutes, add 10 ml of water and filter. Warm the filtrate until most of the ammonia has been removed, cool, acidify with 6 M acetic acid, wash the precipitate with water and dry at 105. On the residue determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with sulphadimidine RS or with the reference spectrum of sulphadimidine. B. In the test for Related substances, the principal spot in the chromatogram obtained with test solution (b) corresponds to that in the chromatogram obtained with the reference solution. C. The residue obtained in test A gives the reaction of primary aromatic amines (2.3.1), producing a bright orange-red precipitate.
Tests
Appearance of solution. An injection containing 1.0 g of Sulphadimidine Sodium in 3 ml is not more intensely coloured than reference solution YS4 (2.4.1). pH (2.4.24). 10.0 to 11.0. Related substances (2.3.7). Complies with test A, using following solutions. Test solution. The injection under examination diluted with water to contain 0.2 per cent w/v of sulphadimidine sodium Reference solution. A 0.002 per cent w/v solution of sulphanilamide in a mixture of 1 volume of strong ammonia solution and 9 volumes of ethanol (95 per cent). Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. Dilute an accurately measured volume containing about 0.5 g of Sulphadimidine Sodium to 75 ml with water, add 10 ml of hydrochloric acid and pass air slowly through the solution until the odour of sulphur dioxide is no longer detectable and the vapours do not turn moistened starch iodate paper blue.
Tests
Related substances (2.3.7). Complies with test C, but using the following solutions. Test solution (a). Extract a quantity of the powdered tablets containing 0.5 g of Sulphadimidine with 25 ml of a mixture of 9 volumes of methanol and 1 volume of strong ammonia solution by shaking for 10 minutes, filter and use the filtrate. Test solution (b). Dilute 1 volume of test solution (a) to 5 volumes with a mixture of 24 volumes of methanol and 1 volume of strong ammonia solution. Test solution (c). Dilute 1 volume of test solution (a) to 200 volumes with the same solvent mixture. Reference solution. A 0.4 per cent w/v of sulphadimidine RS in the same solvent mixture.
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IP 2007
Other tests. Comply with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 0.25 g of Sulphadimidine, dissolve as completely as possible in a mixture of 50 ml of water and 10 ml of hydrochloric acid, add 3 g of potassium bromide, cool in ice and carry out the nitrite titration (2.3.31). 1 ml of 0.1 M sodium nitrite is equivalent to 0.02783 g of C12H14N4O2S. Storage. Store protected from light and moisture.
D. Dissolve about 5 mg in 10 ml of 1 M hydrochloric acid and dilute 1 ml to 10 ml with water. The resulting solution, without further acidification, gives the reaction of primary aromatic amines (2.3.1).
Tests
Appearance of solution. Dissolve 0.8 g in 10 ml of 1 M sodium hydroxide. The solution is not more intensely coloured than reference solution YS5, BYS5 or GYS5 (2.4.1). Acidity. Heat 1.25 g of the finely powdered substance at about 70 with 25 ml of carbon dioxide-free water for 5 minutes. Cool for about 15 minutes in ice and filter. To 20 ml of the filtrate add 0.1 ml of bromothymol blue solution. Not more than 0.2 ml of 0.1 M sodium hydroxide is required to change the colour of the solution. Related substances (2.3.7). Complies with test C, but using the following solution. Test solution (a). A 2 per cent w/v solution of the substance under examination in a mixture of 24 volumes of methanol and 1 volume of strong ammonia solution. Heavy metals (2.3.13).1.0 g complies with the limit test for heavy metals, Method B (20 ppm).
Sulphadoxine
Sulphormethoxine; Sulphoethomidine
OCH3 H3CO O O S N H H2N
C12H14N4O4S Mol. Wt. 310.3
N N
Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.25 g, dissolve in 50 ml of 2 M hydrochloric acid, add 3 g of potassium bromide, cool in ice and carry out the nitrite titration (2.3.31). 1 ml of 0.1 M sodium nitrite is equivalent to 0.03103 g of C12H14N4O4S. Storage. Store protected from light and moisture.
Sulphadoxine is N1-(5,6-dimethoxypyrimidin-4yl)sulphanilamide. Sulphadoxine contains not less than 99.0 per cent and not more than 101.0 per cent of C12H14N4O4S, calculated on the dried basis. Description. White or yellowish white crystals or crystalline powder.
Identification
Test A may be omitted if tests B, C and D are carried out. Tests B and D may be omitted if tests A and C are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with sulphadoxine RS or with the reference spectrum of sulphadoxine. B. In the test for Related substances, the principal spot in the chromatogram obtained with test solution (b) corresponds to that in the chromatogram obtained with the reference solution. C. Dissolve 0.5 g in 1 ml of sulphuric acid (40 per cent), heating gently to effect solution, and continue heating until a crystalline precipitate is produced. Allow to cool, add 10 ml of 2 M sodium hydroxide, cool again, add 25 ml of ether and shake for 5 minutes. Dry the upper layer over anhydrous sodium sulphate, filter and evaporate the solvent by heating on a water-bath. The residue melts either at 80 to 82 or at 90 to 92 (2.4.21).
Sulphafurazole
Sulfisoxazole
O O O N S CH3 N H CH3
Mol. Wt. 267.3
1
H2N
C11H13N3O3S
Sulphafurazole is N -(3,4-dimethylisoxazol-5yl)sulphanilamide. Sulphafurazole contains not less than 99.0 per cent and not more than 101.0 per cent of C11H13N3O3S, calculated on the dried basis.
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Description. White or yellowish white crystals or crystalline powder; odourless.
Reference solution (b). A 0.4 per cent w/v of sulphafurazole RS in a mixture of 1 volume of strong ammonia solution and 24 volumes of methanol. Apply to the plate 5 l of each solution. After development, dry the plate at 105 and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution (a) is not more intense than the spot in the chromatogram obtained with reference solution (a). Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.5 g, dissolve in 50 ml of dry acetone. Titrate with 0.1 M tetrabutylammonium hydroxide, using 0.4 per cent w/v solution of thymol blue in methanol as indicator. Carry out a blank titration. 1 ml of 0.1 M tetrabutylammonium hydroxide is equivalent to 0.02673 g of C11H13N3O3S. Storage. Store protected from light and moisture.
Identification
Test A may be omitted if tests B, C and D are carried out. Tests B and D may be omitted if tests A and C are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with sulphafurazole RS or with the reference spectrum of sulphafurazole. B. In the test for Related substances, the principal spot in the chromatogram obtained with test solution (b) corresponds to that in the chromatogram obtained with reference solution (b). C. Dissolve 0.5 g in 1 ml of sulphuric acid (40 per cent), heating gently to effect solution, and continue heating until a crystalline precipitate is produced. Allow to cool, add 10 ml of 2 M sodium hydroxide, cool again, add 25 ml of ether and shake for 5 minutes. Dry the upper layer over anhydrous sodium sulphate, filter and evaporate the solvent by heating on a water-bath. The residue melts at 119 to 123 (2.4.21). D. Dissolve about 5 mg in 10 ml of 1 M hydrochloric acid and dilute 1 ml to 10 ml with water. The resulting solution, without further acidification, gives the reaction of primary aromatic amines (2.3.1).
Tests
Appearance of solution. Dissolve 0.8 g in 10 ml of 1 M sodium hydroxide. The solution is not more intensely coloured than reference solution YS5, BYS5 or GYS5 (2.4.1). Acidity. Heat 1.25 g of the finely powdered substance at about 70 with 25 ml of carbon dioxide-free water for 5 minutes. Cool for about 15 minutes in ice and filter. To 20 ml of the filtrate add 0.1 ml of bromothymol blue solution. Not more than 0.2 ml of 0.1 M sodium hydroxide is required to change the colour of the solution. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 75 volumes of dichloromethane, 25 volumes of methanol and 1 volume of strong ammonia solution. Test solution (a). Dissolve 0.2 g of the substance under examination in 10 ml of a mixture of 1 volume of strong ammonia solution and 24 volumes of methanol. Test solution (b). Dissolve 0.4 g of the substance under examination in 100 ml of a mixture of 1 volume of strong ammonia solution and 24 volumes of methanol. Reference solution (a). Dissolve 10 mg of the substance under examination in 100 ml of a mixture of 1 volume of strong ammonia solution and 24 volumes of methanol.
Sulphafurazole Tablets
Sulphafurazole Tablets contain not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of sulphafurazole, C11H13N3O3S.
Identification
A. Shake a quantity of the powdered tablets containing 0.6 g of Sulphafurazole with 30 ml of acetone, filter and evaporate the filtrate to dryness; Dry the residue for 2 hours at 105. On the residue determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with sulphafurazole RS or with the reference spectrum of sulphafurazole. B. Dissolve 0.5 g of residue obtained in test A in 1 ml of sulphuric acid (40 per cent), heating gently to effect solution, and continue heating until a crystalline precipitate is produced. Allow to cool, add 10 ml of 2 M sodium hydroxide, cool again, add 25 ml of ether and shake for 5 minutes. Dry the upper layer over anhydrous sodium sulphate, filter and evaporate the solvent by heating on a water-bath. The residue melts at 119 to 123 (2.4.21). C. Extract a quantity of the powdered tablets containing 50 mg of Sulphafurazole with 2 ml of warm dilute hydrochloric acid and filter. The filtrate gives the reaction of primary aromatic amines (2.3.1).
1143
SULPHALENE
IP 2007
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 75 volumes of dichloromethane, 25 volumes of methanol and 1 volume of strong ammonia solution. Test solution (a). Extract a quantity of the powdered tablets containing 0.5 g of Sulphafurazole with 25 ml of a mixture of 90 volumes of methanol and 10 volumes of strong ammonia solution by shaking for 10 minutes, filter and use the filtrate. Test solution (b). Dissolve 0.4 g of the substance under examination in 100 ml of a mixture of 1 volume of strong ammonia solution and 24 volumes of methanol. Reference solution (a). Dissolve 10 mg of the substance under examination in 100 ml of a mixture of 1 volume of strong ammonia solution and 24 volumes of methanol. Reference solution (b). A 0.4 per cent w/v solution of sulphafurazole RS in a mixture of 1 volume of strong ammonia solution and 24 volumes of methanol. Apply to the plate 5 l of each solution. After development, dry the plate at 105 and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with test solution (a) is not more intense than the spot in the chromatogram obtained with reference solution (a). Other tests. Comply with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 0.5 g of Sulphafurazole, dissolve as completely as possible in 50 ml of dry acetone. Titrate with 0.1 M tetrabutylammonium hydroxide, using 0.4 per cent w/v solution of thymol blue in methanol as indicator. Carry out a blank titration. 1 ml of 0.1 M tetrabutylammonium hydroxide is equivalent to 0.02673 g of C11H13N3O3S. Storage. Store protected from light and moisture.
Sulphalene contains not less than 99.0 per cent and not more than 101.0 per cent of C11H12N4O3S, calculated on the dried basis. Description. A white or yellowish white powder; odourless or almost odourless.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with sulphalene RS or with the reference spectrum of sulphalene. B. Dissolve about 5 mg in 10 ml of 1 M hydrochloric acid and dilute 1 ml of this solution to 10 ml with water. The solution, without further acidification, gives the reaction of primary aromatic amines (2.3.1). C. Melting range (2.4.21). 175 to 178.
Tests
Acidity. Heat 1.25 g of the finely powdered substance at about 70 for 5 minutes with 25 ml of carbon dioxide-free water. Cool in ice for about 15 minutes and filter. To 20 ml of the filtrate add 0.1 ml of bromothymol blue solution. Not more than 0.2 ml of 0.1 M sodium hydroxide is required to change the colour of the solution. Related substances (2.3.7). Complies with test B. Heavy metals (2.3.13). Dissolve 1.0 g in 5 ml of 5 M sodium hydroxide and 20 ml of water. The solution complies with the limit test for heavy metals, Method A (20 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.3 g, dissolve in a mixture of 20 ml of 2 M hydrochloric acid and 50 ml of water. Add 3 g of potassium bromide, cool in ice and carry out the nitrite titration (2.3.31). 1 ml of 0.1 M sodium nitrite is equivalent to 0.02803 g of C11H12N4O3S. Storage. Store protected from light and moisture.
Sulphalene
Sulphamethopyrazine; Sulphamethoxypyrazine
H3CO O O S N H H2N
C11H12N4O3S Mol. Wt. 280.3 1 Sulphalene is N -(3-methoxypyrazin-2-yl)sulphanilamide.
Sulphamethizole
N N O O S N S H H2N
C9H10N4O2S2
1
N N
CH3
Mol. Wt. 270.3
Sulphamethizole is N -(5-methyl-1,3,4-thiadiazol-2yl)sulphanilamide.
1144
IP 2007
SULPHAMETHOXAZOLE
Sulphamethizole contains not less than 99.0 per cent and not more than 101.0 per cent of C9H10N4O2S2, calculated on the dried basis. Description. White or yellowish white crystals or crystalline powder; odourless.
Apply to the plate 2 l of each solution. After development, dry the plate at 105 and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with test solution (a) is not more intense than the spot in the chromatogram obtained with reference solution (a). Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.25 g, dissolve in 50 ml of 2 M hydrochloric acid, add 3 g of potassium bromide, cool in ice and carry out the nitrite titration (2.3.31). 1 ml of 0.1 M sodium nitrite is equivalent to 0.02703 g of C9H10N4O2S2. Storage. Store protected from light and moisture.
Identification
Test A may be omitted if tests B, C and D are carried out. Tests C and D may be omitted if tests A and B are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with sulphamethizole RS or with the reference spectrum of sulphamethizole. B. In the test for Related substances, the principal spot in the chromatogram obtained with test solution (b) corresponds to that in the chromatogram obtained with reference solution (b). C. Dissolve 50 mg in 4 ml of methanol and add 0.2 ml of a 4.0 per cent w/v solution of cupric acetate; a flocculent, yellowish green precipitate is produced which becomes dark green. D. Dissolve about 5 mg in 10 ml of 1 M hydrochloric acid and dilute 1 ml of this solution to 10 ml with water. The solution, without further acidification, gives the reaction of primary aromatic amines (2.3.1).
Sulphamethoxazole
CH3 O O S N H H2N
C10H11N3O3S Sulphamethoxazole is N -(5-methylisoxazol-3yl)sulphanilamide. Sulphamethoxazole contains not less than 99.0 per cent and not more than 101.0 per cent of C10H11N3O3S, calculated on the dried basis. Description. A white or almost white, crystalline powder; almost odourless.
1
Tests
Appearance of solution. Dissolve 0.8 g in 10 ml of 1 M sodium hydroxide. The solution is not more intensely coloured than reference solution YS5, BYS5 or GYS5 (2.4.1). Acidity. Heat 1.25 g of the finely powdered substance at about 70 with 25 ml of carbon dioxide-free water for 5 minutes. Cool for about 15 minutes in ice and filter. To 20 ml of the filtrate add 0.1 ml of bromothymol blue solution. Not more than 0.2 ml of 0.1 M sodium hydroxide is required to change the colour of the solution. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 80 volumes of chloroform and 15 volumes of methanol. Test solution (a). Dissolve 0.3 g of the substance under examination in 10 ml of acetone. Test solution (b). Dissolve 0.3 g of the substance under examination in 100 ml of acetone. Reference solution (a). Dissolve 15 mg of the substance under examination in 100 ml of acetone. Reference solution (b). A 0.3 per cent w/v solution of sulphamethizole RS in acetone.
Mol. Wt. 253.3
Identification
Test A may be omitted if tests B, C and D are carried out. Tests B and C may be omitted if tests A and D are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with sulphamethoxazole RS or with the reference spectrum of sulphamethoxazole. B. In the test for Related substances, the principal spot in the chromatogram obtained with test solution (b) corresponds to that in the chromatogram obtained with the reference solution. C. Dissolve about 5 mg in 10 ml of 1 M hydrochloric acid and dilute 1 ml to 10 ml with water. The resulting solution, without
1145
SULPHAPHENAZOLE
IP 2007
further acidification, gives the reaction of primary aromatic amines (2.3.1). D. Melting range (2.4.21). 169 to 172.
Identification
Test A may be omitted if tests B, C and D are carried out. Tests B and C may be omitted if tests A and D are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with sulphaphenazole RS. B. In the test for Related substances, the principal spot in the chromatogram obtained with test solution (b) corresponds to that in the chromatogram obtained with the reference solution. C. Dissolve about 5 mg in 10 ml of 1 M hydrochloric acid and dilute 1 ml of this solution to 10 ml with water. The solution, without further acidification, gives the reaction of primary aromatic amines (2.3.1). D. Melting range (2.4.21). 178 to 183.
Tests
Appearance of solution. Dissolve 0.8 g in 10 ml of 1 M sodium hydroxide. The solution is not more intensely coloured than reference solution YS5, BYS5 or GYS5 (2.4.1). Acidity. Heat 1.25 g of the finely powdered substance with 25 ml of carbon dioxide-free water at 70 for 5 minutes. Cool for about 15 minutes in ice and filter. To 20 ml of the filtrate add 0.1 ml of bromothymol blue solution. Not more than 0.3 ml of 0.1 M sodium hydroxide is required to change the colour of the solution. Related substances (2.3.7). Complies with test C, but using the following solution. Test solution (a). Dissolve 0.1 g of the substance under examination in sufficient of a mixture of 1 volume of strong ammonia solution and 24 volumes of methanol to produce 5 ml. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.2 g, dissolve in 50 ml of 2 M hydrochloric acid, add 3 g of potassium bromide, cool in ice and carry out the nitrite titration (2.3.31). 1 ml of 0.1 M sodium nitrite is equivalent to 0.02533 g of C10H11N3O3S. Storage. Store protected from light and moisture.
Tests
Appearance of solution. Dissolve 0.8 g in 10 ml of 1 M sodium hydroxide. The solution is not more intensely coloured than reference solution YS5, BYS5 or GYS5 (2.4.1). Acidity. Heat 1.25 g of the finely powdered substance at about 70 with 25 ml of carbon dioxide-free water for 5 minutes. Cool for about 15 minutes in ice and filter. To 20 ml of the filtrate add 0.1 ml of bromothymol blue solution. Not more than 0.2 ml of 0.1 M sodium hydroxide is required to change the colour of the solution. Related substances (2.3.7). Complies with test C. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Iron (2.3.14). Ignite 2.0 g with 1 g of anhydrous sodium carbonate, cool, dissolve the residue in 5 ml of hydrochloric acid and dilute to 35 ml with water. The resulting solution complies with the limit test for iron (20 ppm).
Sulphaphenazole
O O S N H H 2N N N
Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.2 g, dissolve in a mixture of 20 ml of 2 M hydrochloric acid and 50 ml of water. Add 3 g of potassium bromide, cool in ice and carry out the nitrite titration (2.3.31). 1 ml of 0.1 M sodium nitrite is equivalent to 0.03144 g of C15H14N4O2S. Storage. Store protected from light and moisture.
C15H14N4O2S
1
Mol. Wt. 314.4
Sulphaphenazole is N -(1-phenyl-1H pyrazol-5yl)sulphanilamide. Sulphaphenazole contains not less than 99.0 per cent and not more than 101.0 per cent of C15H14N4O2S, calculated on the dried basis.
1146
IP 2007
SULPHOBROMOPHTHALEIN SODIUM
Sulphaphenazole Tablets
Sulphaphenazole Tablets contain not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of sulphaphenazole, C15H14N4O2S.
mixture of 50 ml of water and 10 ml of hydrochloric acid, cool in ice and carry out the nitrite titration (2.3.31). 1 ml of 0.1 M sodium nitrite is equivalent to 0.03144 g of C15H14N4O2S. Storage. Store protected from light and moisture.
Identification
Mix a quantity of the powdered tablets containing about 1.5 g of Sulphaphenazole with 30 ml of acetone and heat under a reflux condenser for 5 minutes. Filter, evaporate the filtrate on a water-bath to a volume of about 10 ml, add 40 ml of water, heat for a further 15 minutes and cool in ice; a precipitate is formed which, after washing with water and drying at 105, complies with the following tests. Test A may be omitted if tests B, C and D are carried out. Tests B and C may be omitted if tests A and D are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with sulphaphenazole RS or with the reference spectrum of sulphaphenazole. B. In the test for Related substances, the principal spot in the chromatogram obtained with test solution (b) corresponds to that in the chromatogram obtained with the reference solution. C. Dissolve about 5 mg in 10 ml of 1 M hydrochloric acid and dilute 1 ml of this solution to 10 ml with water. The solution, without further acidification, gives the reaction of primary aromatic amines (2.3.1). D. Melting range (2.4.21). 178 to 183. C20H8Br4Na2O10S2
Sulphobromophthalein Sodium
Br Br O Br Br SO3Na OH SO3Na
Mol. Wt. 837.9
HO
Sulphobromophthalein Sodium is disodium 5,5' -(4,5,6,7tetrabromo-1,3-dihydro-3-oxo-isobenzofuran-1,1-diyl)bis(2hydroxybenzenesulphonate). Sulphobromophthalein Sodium contains not less than 7.4 per cent and not more than 8.2 per cent of sulphur, S, and not less than 36.0 per cent and not more than 39.0 per cent of bromine, Br, both calculated on the dried basis. Description. A white, crystalline powder; hygroscopic.
Tests
Related substances (2.3.7). Comply with test C, but using the following solutions. Test solution (a). Extract a quantity of the powdered tablets containing 0.5 g of Sulphaphenazole with 25 ml of a mixture of 90 volumes of methanol and 10 volumes of strong ammonia solution by shaking for 10 minutes, filter and use the filtrate. Test solution (b). Dilute 1 volume of test solution (a) to 5 volumes with a mixture of 24 volumes of methanol and 1 volume of strong ammonia solution. Test solution (c). Dilute 1 volume of test solution (a) to 200 volumes with the same solvent mixture. Reference solution. A 0.4 per cent w/v of sulphaphenazole RS in the same solvent mixture. Other tests. Comply with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 0.25 g of Sulphaphenazole, dissolve as completely as possible in a
Identification
A. Absorbance of a 0.0005 per cent w/v solution in 0.05 M sodium hydroxide at the maximum at about 580 nm, about 0.4 (2.4.7). B. Mix 0.1 g with 0.5 g of sodium carbonate and ignite until thoroughly charred. Cool, add 5 ml of hot water, heat for 5 minutes on a water-bath and filter; the filtrate gives the reactions of bromides (2.3.1). C. Gives reaction A of sodium salts (2.3.1).
Tests
Appearance of solution. A 2.0 per cent w/v solution in carbon dioxide-free water is clear (2.4.1), and colourless (2.4.1). Halide ions. To 5 ml of a 1.0 per cent w/v solution add 1 ml of dilute nitric acid and 1 ml of silver nitrate solution; not more than a slight opalescence is produced. Calcium. Ignite 5.0 g in a platinum dish until free from carbon, cool, add 1 ml of hydrochloric acid and 10 ml of water, heat
1147
SULPHOBROMOPHTHALEIN SODIUM INJECTION
IP 2007
on a water-bath for 5 minutes, add 1 g of ammonium sulphate and 4 ml of dilute ammonia solution and heat for a further 5 minutes. Transfer the contents of the dish to a flask with the aid of 50 ml of water, add 20 ml of strong ammonia solution, dilute to 100 ml with water, add 0.3 ml of sodium sulphide solution, 1 ml of potassium cyanide solution and 75 ml of ethanol (95 per cent) and titrate with 0.01 M disodium edetate using 0.1 g of a mixture of 99 parts of potassium nitrate and 1 part of methyl thymol blue as indicator until a faint grey colour is produced. Not more than 6.25 ml of 0.01 M disodium edetate is required. Sulphates. To 10 ml of a 0.2 per cent w/v solution add 0.25 ml of dilute hydrochloric acid, boil and add 1 ml of barium chloride solution; the hot solution remains clear for 2 minutes. Loss on drying (2.4.19). Not more than 5.0 per cent determined on 1.0 g by drying in an oven at 105. Assay. For sulphur Determine by the oxygen-flask method (2.3.34), burning 0.2 g in a 1000-ml glass-stoppered flask and a mixture of 30 ml of water and 0.5 ml of hydrogen peroxide solution (100 vol) as the absorbing liquid. When the process is complete, add 2 ml of hydrochloric acid, dilute to 250 ml with water, heat to boiling and slowly add 10 ml of barium chloride solution. Heat on a water-bath for 1 hour, filter, wash the precipitate with water, dry and ignite at a temperature of about 600 until, after further ignition, two successive weighings do not differ by more than 0.2 per cent. 1 g of residue is equivalent to 0.1374 g of sulphur, S. For bromine Determine by the oxygen-flask method (2.3.34), burning 0.2 g in a 1000-ml glass-stoppered flask and a mixture of 10 ml of 0.1 M sodium hydroxide, 0.5 ml of hydrogen peroxide solution (100 vol) and 10 ml of water as the absorbing liquid. When the process is complete, boil the solution for 5 minutes, cool, acidify with 2 M nitric acid, add 20.0 ml of 0.1 M silver nitrate, shake, and titrate the excess of silver nitrate with 0.1 M ammonium thiocyanate using ferric ammonium sulphate solution as indicator. 1 ml of 0.1 M silver nitrate is equivalent to 0.007991 g of bromine, Br. Storage. Store protected from moisture.
Sulphobromophthalein Sodium Injection

Sulphobromophthalein Sodium Injection is a sterile solution of Sulphobromophthalein Sodium in Water for Injections. Sulphobromophthalein Sodium Injection contains not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of sulphobromophthalein sodium, C20H8Br4Na2O10S2.
Identification
A. To 5 ml add 0.15 ml of 1 M sodium hydroxide; an intense purple colour is produced which disappears on the addition of an acid. B. To a volume containing 50 mg of Sulphobromophthalein Sodium add 0.25 g of sodium carbonate, evaporate to dryness and ignite. Cool the residue, add 2.5 ml of hot water, heat for 5 minutes on a water-bath and filter; the filtrate gives the reactions of bromides (2.3.1).
Tests
pH (2.4.24). 5.0 to 6.5, determined by using a suitable agarpotassium nitrate salt bridge. Bacterial endotoxins (2.2.3). Not more than 1.0 Endotoxin Unit per mg of Sulphobromophthalein Sodium. Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. To an accurately measured volume containing 0.1 g of Sulphobromophthalein Sodium add sufficient water to produce 500.0 ml and mix. To 5.0 ml of this solution add sufficient of a 1.0 per cent w/v solution of sodium carbonate to produce 200.0 ml and mix. Measure the absorbance of the resulting solution at the maximum at about 580 nm (2.4.7), using as the blank the sodium carbonate solution. Calculate the content of C 20 H 8 Br 4 Na 2 O 10 S 2 from the absorbance obtained by carrying out the determination simultaneously on 0.1 g, accurately weighed, of sulphobromophthalein sodium RS.
1148
MONOGRAPHS
T
Talc Tamoxifen Citrate Tamoxifen Tablets Tartaric Acid Tenofovir Disoproxil Fumartea Tenofovir Disoproxil Fumarate Tablets Tenofovir and Emtricitabine Tablets Terbutaline Sulphate Terbutaline Inhalation Terbutaline Injection Terbutaline Tablets Testosterone Propionate Testosterone Propionate Injection Tetracycline Tetracycline Hydrochloride Tetracycline Capsules Tetracycline Ointment Theophylline Theophylline Injection Thiabendazole Thiabendazole Tablets Thiacetazone Thiacetazone And Isoniazid Tablets Thiamine Hydrochloride Thiamine Injection Thiamine Tablets Thiamine Mononitrate Thiomersal Thiopentone Sodium Thiopentone Injection
1149
.... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... ....
MONOGRAPHS
Thiotepa Thiotepa Injection Thymol Thyroxine Sodium Thyroxine Tablets Timolol Maleate Timolol Eye Drops Timolol Tablets Tinidazole Tinidazole Tablets Tiotropium Bromide Monohydrate Tiotropium powder for Inhalation Titanium Dioxide Tizanidine Hydrochloride Tizanidine Tablets Tobramycin Tobramycin Injection Tocopheryl Acetate Tolbutamide Tolbutamide Tablets Topotecan Hydrochloride Topotecan Injection Triamcinolone Triamcinolone Tablets Triamcinolone Acetonide Triamcinolone Acetonide Injection Triamterene Triamterene Capsules Trifluoperazine Hydrochloride Trifluoperazine Injection Trifluoperazine Tablets Triflupromazine Hydrochloride Triflupromazine Injection
1150
.... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... ....
MONOGRAPHS
Triflupromazine Tablets Trimethoprim Trimethoprim and Sulphamethoxazole Oral Suspension Trimethoprim And Sulphamethoxazole Tablets Trimethoprim Tablets Triprolidine Hydrochloride Triprolidine Tablets Trisodium Edetate Concentrate for Injection Tropicamide Tropicamide Eye Drops Troxidone Troxidone Capsules Tubocurarine Chloride Tubocurarine Injection
.... .... .... .... .... .... .... .... .... .... .... .... .... ....
1151
IP 2007
TAMOXIFEN CITRATE
Talc
Purified Talc; Talcum
Talc is a powdered, selected natural hydrated magnesium silicate. It may contain varying amounts of aluminium and iron in forms insoluble in 1M sulphuric acid. Description. A white or almost white powder, free from grittiness; readily adheres to the skin; unctuous to the touch; odourless.
Organic compounds. The residue obtained in the test for Loss on drying is not more than slightly yellow or grey. Loss on drying (2.4.19). Not more than 1.0 per cent, determined on 1.0 g by drying in an oven at 180 for 1 hour. Storage. Store protected from moisture.
Tamoxifen Citrate
O CH3 N CH3 , H3C
Identification
A. When examined microscopically, shows irregular plates, the majority less than 50 m in length. The particles are not notably stained by a 0.1 per cent w/v solution of methylene blue in ethanol (95 per cent). B. Melt 0.5 g in a metal crucible with 1 g of potassium nitrate and 3 g of anhydrous sodium carbonate, add 20 ml of boiling water, mix and filter. Wash the residue with 50 ml of water. Mix the residue with a mixture of 0.5 ml of hydrochloric acid and 5 ml of water and filter. To the filtrate add 1 ml of 9 M ammonia and 1 ml of ammonium chloride solution and filter. To the filtrate add 1 ml of disodium hydrogen phosphate solution; a white, crystalline precipitate is produced. C. Gives the reaction of silicates (2.3.1).
HO HOOC
COOH COOH
C26H29NO,C6H8O7
Mol. Wt. 563.7
Tamoxifen Citrate is (Z)-2-[4-(1,2-diphenylbut-1-enyl)-1phenoxy]ethyldimethylamine citrate. Tamoxifen Citrate contains not less than 99.0 per cent and not more than 101.0 per cent of C26H29NO, C6H8O7, calculated on the dried basis. Description. A white or almost white, crystalline powder.
Tests
Acidity or alkalinity. Shake 5.0 g with 25 ml of carbon dioxidefree water for 1 minute, filter and add to the filtrate 0.5 ml of bromothymol blue solution; the solution is not acid and requires not more than 0.3 ml of 0.1 M hydrochloric acid to make it acid. Iron (2.3.14). Boil 4.0 g with 25 ml of water for 30 minutes, replacing the water lost by evaporation, and filter. The filtrate, after the addition of 5 ml of nitric acid, complies with the limit test for iron (10 ppm). Acid-soluble substances. Not more than 2.0 per cent, determined by the following method. Digest 2.0 g with 40 ml of dilute hydrochloric acid for 15 minutes, filter, evaporate the filtrate; to the residue add 0.1 ml of sulphuric acid and ignite to constant weight. Water-soluble substances. Shake 5.0 g with 25 ml of water for 1 minute, filter, evaporate the filtrate and dry to constant weight; the residue weighs not more than 10 mg. Carbonates. To 1 g add 20 ml of dilute hydrochloric acid; no effervescence is produced. Chlorides (2.3.12). Suspend 2.0 g in 10 ml of water, add 10 ml of 2 M nitric acid, shake for 15 minutes and filter. 10 ml of the filtrate complies with the limit test for chlorides (250 ppm).
Identification
Test A may be omitted if tests B, C and D are carried out. Tests C and D may be omitted if tests A and B are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with tamoxifen citrate RS or with the reference spectrum of tamoxifen citrate. B. When examined in the range 220 nm to 360 nm (2.4.7), a 0.002 per cent w/v solution in methanol shows absorption maxima at about 237 nm and 275 nm. C. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 90 volumes of toluene and 10 volumes of triethylamine. Test solution. Dissolve 0.1 g of the substance under examination in 10 ml of methanol. Reference solution. A 1 per cent w/v solution of tamoxifen citrate RS in methanol. Apply to the plate 5 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution.
1153
TAMOXIFEN TABLETS
IP 2007
D. To 10 mg add 4 ml of pyridine and 2 ml of acetic anhydride and shake; a yellow colour is produced. Heat in a water-bath for 2 minutes; a pink to red colour is produced.
Tests
E-Isomer and related substances. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 0.25 g of the substance under examination in 100 ml of a mixture of 60 volumes of acetonitrile, 25 volumes of water and 15 volumes of tetrahydrofuran. Reference solution (a). A 0.0025 per cent w/v solution of the substance under examination in a mixture of 60 volumes of acetonitrile, 25 volumes of water and 15 volumes of tetrahydrofuran. Reference solution (b). A 0.25 per cent w/v solution of tamoxifen citrate impurity standard RS in the same solvent mixture. Chromatographic system a stainless steel column 20 cm x 5 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 60 volumes of acetonitrile, 25 volumes of water, 15 volumes of tetrahydrofuran and 0.4 volume of strong ammonia solution, flow rate. 1.5 ml per minute, spectrophotometer set at 240 nm, a 20 l loop injector. In the chromatogram obtained with reference solution (b) a peak due to E-isomer appears immediately following the peak due to Z-tamoxifen. Adjust the sensitivity of the instrument so that the height of the peak due to E-isomer is about 15 per cent of the full-scale deflection on the recorder. Measure the height of the peak due to E-isomer by dropping a perpendicular from the top of the peak to a line drawn tangentially between the troughs on each side of the E-isomer peak or the trough between the E- and Z-isomer peaks and the baseline, whichever is appropriate. The test is not valid unless the height of the trough separating the peaks due to E- and Z-tamoxifen in the chromatogram obtained with reference solution (b) is less than 7 per cent of full-scale deflection on the recorder and the retention time of the principal peak is less than 30 minutes. (The retention time decreases with increasing concentration of ammonia in the mobile phase). The content of E-isomer in the substance under examination is not more than 1 per cent calculated from the declared content of E-isomer in tamoxifen citrate impurity standard RS. The area of any secondary peak in the chromatogram obtained with the test solution, other than a peak due to E-isomer, is not greater than half that of the peak due to tamoxifen citrate
in the chromatogram obtained with reference solution (a) and the sum of the areas of all such peaks is not greater than the area of the peak due to tamoxifen in the chromatogram obtained with reference solution (a). Ignore any peak with a retention time less than 2.5 minutes and any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (a). Heavy metals (2.3.13). 2.0 g complies with limit the test for heavy metals, Method B (10 ppm). Sulphated ash (2.3.18). Not more than 0.2 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 1.0 g, dissolve in 150 ml of anhydrous glacial acetic acid. Titrate with 0.1 M perchloric acid, using 1-naphtholbenzein solution as indicator. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.05636 g of C26H29NO, C6H8O7. Storage. Store protected from light and moisture.
Tamoxifen Tablets
Tamoxifen Citrate Tablets
Tamoxifen Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of tamoxifen, C26H29NO.
Identification
A. To a quantity of the powdered tablets containing 0.1 g of tamoxifen add 20 ml of water, warm, add 2 ml of 5 M sodium hydroxide and cool. Extract with two quantities, each of 10 ml, of ether, filtering after each extraction. Combine the ether extracts and evaporate to dryness in a stream of nitrogen at room temperature. Dry the residue at a pressure not exceeding 0.7 kPa for 30 minutes. On the residue, determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with tamoxifen citrate RS or with the reference spectrum of tamoxifen citrate. B. When examined in the range 220 nm to 360 nm (2.4.7), a 0.002 per cent w/v solution in methanol of the residue obtained in test A shows absorption maxima at about 237 nm and 275 nm. C. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254.
1154
IP 2007
TAMOXIFEN TABLETS
Mobile phase. A mixture of 100 volumes of ethyl acetate, 10 volumes of methanol and 1 volume of strong ammonia solution. Test solution. Shake a quantity of the powdered tablets containing 0.1 g tamoxifen with 10 ml of methanol, filter, evaporate the filtrate to dryness on a water-bath, dry the residue at 100 for 30 minutes and dissolve 10 mg of the residue in 10 ml of methanol. Reference solution. A 0.1 per cent w/v solution of tamoxifen citrate RS in methanol. Apply to the plate 10 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution. D. Dissolve 10 mg of the residue obtained in the preparation of the test solution in test C in 4 ml of pyridine, add 2 ml of acetic anhydride and heat on a water-bath for 2 minutes; a pink to red colour is produced.
cent of full-scale deflection on therecorder. Measure the height of the peak due to E-isomer by dropping a perpendicular from the top of the peak to a line drawn tangentially between the troughs on each side of the E-isomer peak or the trough between the E- and Z-isomer peaks and the baseline, whichever is appropriate. The test is not valid unless the height of the trough separating the peaks due to E- and Z-tamoxifen in the chromatogram obtained with reference solution (b) is less than 7 per cent of the full-scale deflection on the recorder and the retention time of the principal peak is less than 30 minutes. (The retention time decreases with increasing concentration of ammonia in the mobile phase). The content of E-isomer in the substance under examination is not more than 1 per cent calculated from the declared content of E-isomer in tamoxifen citrate impurity standard RS. The area of any secondary peak in the chromatogram obtained with the test solution, other than a peak due to E-isomer, is not greater than half that of the peak due to tamoxifen citrate in the chromatogram obtained with reference solution (a) and the sum of the areas of all such peaks is not greater than the area of the peak due to tamoxifen in the chromatogram obtained with reference solution (a). Ignore any peak with a retention time less than 2.5 minutes and any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (a). Uniformity of content. (For tablets containing equivalent of 10 mg or less) Comply with the test stated under Tablets. Crush one tablet and transfer to a 100-ml volumetric flask with the aid of 75 ml of methanol. Shake well for 5 minutes, add sufficient methanol to produce 100.0 ml and filter. Dilute 10.0 ml of the filtrate to 100.0 ml with methanol. Measure the absorbance of the resulting solution at the maximum at about 275 nm (2.4.7). Calculate the content of C26H29NO in the tablet taking 325 as the specific absorbance at 275 nm. Other tests. Comply with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 25 mg of tamoxifen, shake with 100 ml of methanol for 15 minutes, add sufficient methanol to produce 250.0 ml and filter. Dilute 10.0 ml of the filtrate to 100.0 ml with methanol and measure the absorbance of the resulting solution at the maximum at about 275 nm (2.4.7). Calculate the content of C26H29NO taking 325 as the specific absorbance at 275 nm. Storage. Store protected from light and moisture. Labelling. The label states the strength in terms of the equivalent amount of tamoxifen.
Tests
E-Isomer and Related substances. Determine by liquid chromatography (2.4.14). Test solution. Mix a quantity of the powdered tablets containing 60 mg of tamoxifen with 60 ml of a mixture of 60 volumes of acetonitrile, 25 volumes of water and 15 volumes of tetrahydrofuran with the aid of ultrasound for 5 minutes, dilute to 100 ml with the same solvent mixture and filter through Whatman No. 1 filter paper. Reference solution (a). Dilute 1.0 ml of the test solution to 100 ml with the same solvent mixture. Reference solution (b). A 0.1 per cent w/v solution of tamoxifen citrate impurity standard RS in the same solvent mixture. Chromatographic system a stainless steel column 20 cm x 5 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 60 volumes of acetonitrile, 25 volumes of water, 15 volumes of tetrahydrofuran and 0.4 volume of strong ammonia solution, flow rate. 1.5 ml per minute, spectrophotometer set at 240 nm, a 20 l loop injector. In the chromatogram obtained with reference solution (b) a peak due to E-isomer appears immediately following the peak due to Z-tamoxifen. Adjust the sensitivity of the instrument so that the height of the peak due to E-isomer is about 15 per
1155
TARTARIC ACID
IP 2007
Tartaric Acid
L-Tartaric Acid
Assay. Weigh accurately about 1.0 g, dissolve in 25 ml of water and titrate with 1 M sodium hydroxide using 0.5 ml of phenolphthalein solution as indicator.
H OH COOH H OH
1 ml of 1 M sodium hydroxide is equivalent to 0.07505 g of C4H6O6. Storage. Store protected from moisture.
HOOC
C4H6O6
Mol. Wt. 150.1
Tenofovir Disoproxil Fumarate

NH2 N N N N O CH3 O CH3 O P O O O CH3 O O O CH3 O CH3 H , HOOC H COOH
Tartaric Acid is (2R,3R)-2,3-dihydroxybutanedioic acid. Tartaric Acid contains not less than 99.5 per cent and not more than 101.0 per cent of C4H6O6, calculated on the dried basis. Description. Colourless crystals or a white or almost white crystalline powder.
Identification
A. Ignite a few mg; it gradually decomposes giving off an odour resembling that of burnt sugar (distinction from citric acid). B. A 10 per cent w/v solution in distilled water (solution A) is strongly acidic. C. Gives reactions A and B of tartrates (2.3.1).
C19H30N5O10P,C4H4O4
Mol. Wt. 635.5
Tenofovir Disoproxil Fumarate salt of bis(isopropyloxycarbonyloxymethyl ester of (R)-9-(2phosphonomethoxypropyl)adenine with fumaric acid. Tenofovir Disoproxil Fumarate contain not less than 97.0 per cent and not more than 102. 0 per cent of C19H30N5O10P,C4H4O4, calculated on the anhydrous basis. Description. A white to off- white crystalline powder.
Tests
Appearance of solution. Solution A is clear (2.4.1), and not more intensely coloured than reference solution YS6 (2.4.1). Specific optical rotation (2.4.22). +12.0 to +12.8, determined in a 20.0 per cent w/v solution. Arsenic (2.3.10). Dissolve 5.0 g in 50 ml of water and add 10 ml of stannated hydrochloric acid AsT. The resulting solution complies with the limit test for arsenic (2 ppm). Heavy metals (2.3.13). 2.0 g complies with the limit test for heavy metals, Method A (10 ppm). Chlorides (2.3.12). 20 ml of solution A complies with the limit test for chlorides (125 ppm). Sulphates (2.3.17). 10 ml of solution A complies with the limit test for sulphates (150 ppm). Oxalate. Neutralise 10 ml of solution A with dilute ammonia solution, add 0.1 ml of dilute acetic acid and 10 ml of calcium sulphate solution; no opalescence is produced within 20 minutes. Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.2 per cent, determined on 1.0 g by drying in an oven at 105.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with tenofovir disoproxil fumarate RS or with the reference spectrum of tenofovir disoproxil fumarate. B. In the Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the peak in the chromatogram obtained with the reference solution.
Tests
Related substances. Determine by liquid chromatography (2.4.14). NOTE Prepare the solutions immediately before use. Test solution. Dissolve 100 mg of the substance under examination in 50 ml of methanol. Reference solution (a). A 0.2 per cent w/v solution of tenofovir disoproxil fumarate RS in methanol. Reference solution (b). Dilute 1.0 ml of reference solution (a) to 100.0 ml with methanol. Reference solution (c). Dissolve 10 mg of the fumaric acid in 50 ml of methanol.
1156
IP 2007
TENOFOVIR DISOPROXIL FUMARATE TABLETS
Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m) (such as ODS 3V), column. temperature 30, mobile phase: A. 0.1 M ammonium acetate solution with the pH adjusted to 3.8 with glacial acetic acid, B. a mixture of 70 volumes of methanol and 30 volumes of acetonitrile, flow rate. 1.5 ml per minute, a linear gradient programme using the conditions given below, spectrophotometer set at 260 nm, a 10 l loop injector. Time mobile phase A mobile phase B (in min.) (per cent v/v) (per cent v/v) 0 95 5 10 50 50 25 50 50 50 20 80 60 95 5 65 95 5 Inject reference solution (a). The test is not valid unless the tailing factor is not more than 2.0 and the column efficiency in not less than 2000 theoretical plates. Inject the test solution, reference solution (b) and reference solution (c). In the chromatogram obtained with the test solution, the area of any secondary peak is not more than the area of the peak in the chromatogram obtained with the reference solution (b) (1.0 per cent) and the sum of all the secondary peaks is not more than 2.5 times the area of the peak in the chromatogram obtained with the reference solution (b) (2.5 per cent). Ignore any peak corresponding to the peak obtained in the chromatogram in the reference solution (c) and any peak having area less than 0.02 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.02 per cent). Fumaric Acid. 17.5 per cent to 19.0 per cent. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 25 mg of the substance under examination in 100 ml of the mobile phase. Reference solution. Dissolve 25 mg of the fumaric acid in 50 ml of the mob.ile phase. Dilute 10 ml of the solution to 100 ml with the mobile phase. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 40 volumes of acetonitrile and 60 volumes of a buffer solution prepared by adding
0.1 per cent v/v of triethylamine in 0.05M sodium dihydrogen phosphate with the pH adjusted to 2.3 with orthophosphoric acid and filtered, flow rate. 1 ml per minute, spectrophotometer set at 210 nm, a 10 l loop injector. Inject the reference solution. The test is not valid unless the tailing factor is not more than 2.0, the column efficiency in not less than 2000 theoretical plates and the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject the test solution and the reference solution. Calculate the content of fumaric acid. Water (2.3.43). Not more than 1.0 per cent, determined on 1.0 g. Assay. Determine by liquid chromatography (2.4.14). NOTE Prepare the solutions immediately before use and carry out the test protected from light. Test solution. Dissolve 50 mg of the substance under examination in 50.0 ml of the mobile phase. Dilute 5.0 ml of the solution to 50.0 ml with the mobile phase. Reference solution. A 0.1 per cent w/v solution of tenofovir disoproxil fumarate RS in the mobile phase. Dilute 5.0 ml of the solution to 50.0 ml with the mobile phase. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 40 volumes of acetonitrile and 60 volumes of a buffer solution prepared by adding 1 ml triethylamine to 0.05M sodium dihydrogen phosphate with the pH adjusted to 2.3 with orthophosphoric acid and filtered, flow rate. 1 ml per minute, spectrophotometer set at 260 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the tailing factor is not more than 2.0, the column efficiency in not less than 2000 theoretical plates and the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject the test solution and the reference solution. Calculate the content of C19H30N5O10P,C4H4O4. Storage. Store protected from light in a refrigerator (2 to 8).
Tenofovir Disoproxil Fumarate Tablets

Tenofovir Disoproxil Fumarate Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of tenofovir disoproxil fumarate, C19H30N5O10P,C4H4O4.
1157
TENOFOVIR DISOPROXIL FUMARATE TABLETS
IP 2007
Identification
A. In the Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the peak in the chromatogram obtained with the reference solution. B. When examined in the range 200 nm to 400 nm (2.4.7), a 0.001 per cent w/v solution in methanol shows an absorption maximum at the same wavelength as the reference solution.
Time (in min.) 0 10 25 50 60
mobile phase A (per cent v/v) 95 50 50 20 95
mobile phase B (per cent v/v) 5 50 50 80 5
Tests
Dissolution (2.5.2). Apparatus. No 1 Medium. 900 ml of 0.1 M hydrochloric acid. Speed and time. 50 rpm and 45 minutes Withdraw a suitable volume of the medium and filter promptly. Dilute the filtrate, if necessary, with the same solvent. Measure the absorbance (2.4.7) of the resulting solution at the maximum at about 260 nm. Calculate the content of C19H30N5O10P,C4H4O4 in the medium from the absorbance obtained from a solution of known concentration of tenofovir disoproxil fumarate RS. D. Not less than 80 per cent of the stated amount of C19H30N5O10P,C4H4O4. Related substances. Determine by liquid chromatography (2.4.14). NOTE Prepare the solutions immediately before use. Test solution. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing 100 mg of Tenofovir Disoproxil Fumarate and disperse in 50 ml of methanol and filter. Reference solution (a). A solution of tenofovir disoproxil fumarate RS containing 0.2 per cent of tenofovir disoproxil in methanol. Reference solution (b). Dilute 1 ml of reference solution (a) to 100 ml with methanol. Reference solution (c). Dissolve 10 mg of fumaric acid in 50 ml of methanol. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: A. dissolve 1.9 g of ammonium acetate in 1000 ml of water and adjust the pH to 3.8 with glacial acetic acid, B. methanol, flow rate. 1.5 ml per minute, a linear gradient programme using the conditions given below, spectrophotometer set at 260 nm, a 10 l loop injector.
Inject reference solution (a). The test is not valid unless the tailing factor is not more than 2.0 and the column efficiency in not less than 2000 theoretical plates. Inject the test solution, reference solutions (b) and (c). In the chromatogram obtained with the test solution, the area of any secondary peak is not more than 3.5 times the area of the peak in the chromatogram obtained with the reference solution (b) (3.5 per cent) and the sum of areas of all the secondary peaks is not more than 6 times the area of the peak in the chromatogram obtained with the reference solution (b) (6.0 per cent). Ignore the peak corresponding to the peak in the chromatogram obtained in the reference solution (c). Other tests. Comply with the tests stated under Tablets. Water (2.3.43). Not more than 5.0 per cent, determined on 0.5 g. Assay: Determine by liquid chromatography (2.4.14). NOTE Prepare the solutions immediately before use. Solvent mixture. Equal volumes of water and methanol. Test solution. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing 300 mg of Tenofovir Disoproxil Fumarate, dissolve in 100 ml of solvent mixture and filter. Dilute 5.0 ml of the filtrate to 50.0 ml with the mobile phase. Reference solution. A 0.120 per cent w/v solution of tenofovir disoproxil fumarate RS in the solvent mixture. Dilute 5.0 ml of the solution to 20.0 ml with the mobile phase. Chromatographic system a stainless steel column 15 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m) (such as Inertsil ODS 3V), mobile phase: a mixture of 60 volumes of a buffer solution prepared by dissolving 7.8 g of sodium dihydrogen orthophosphate in 1000 ml of water, adding 1 ml of triethylamine and adjusting the pH to 2.3 with orthophosphoric acid (10 per cent v/v), and 40 volumes of acetonitrile, flow rate. 1 ml per minute, spectrophotometer set at 260 nm, a 10 l loop injector. Inject the reference solution. The test is not valid unless the tailing factor is not more than 2.0, the column efficiency in not
1158
IP 2007
TENOFOVIR AND EMTRICITABINE TABLETS
less than 2000 theoretical plates and the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject the test solution and the reference solution. Calculate the content of C19H30N5O10P,C4H4O4 in the tablets. Storage. Store protected from moisture, at a temperature not exceeding 30.
disperse in 5 ml of methanol, dilute to 50 ml with mobile phase A and filter. Reference solution (a). A 0.1 per cent w/v solution of emtricitabine RS in mobile phase A. Reference solution (b). Dilute 1 ml of reference solution (a) to 100 ml with mobile phase A. Chromatographic system, a stainless steel column 25 cm x 4.6 mm, packed with octadecysilane bonded to porous silica (5m), column temperature 35, mobile phase: A. a mixture of 95 volumes of a buffer solution prepared by dissolving 1.9 g of ammonium acetate in 1000 ml of water, adjusted the pH to 3.8 with glacial acetic acid, and 5 volumes of methanol and filtered, B. a filtered mixture of 30 volumes of the buffer solution and 70 volumes of methanol, flow rate. 1 ml per minute, a linear gradient programme using the conditions given below, spectrophotometer set at 277 nm, a 20 l loop injector. Time Mobile phase A Mobile phase B (in min.) (per cent v/v) (per cent v/v) 0.01 100 0 30 100 0 35 0 100 55 0 100 60 100 0 75 100 0 Inject reference solution (a). The test is not valid unless the column efficiency is not less than 500 theoretical plates and the tailing factor is not more than 2.0. Inject the test solution and reference solution (b). In the chromatogram obtained with the test solution, the area of any secondary peak is not more than the area of the peak in the chromatogram obtained with the reference solution (b) (1.0 per cent) and the sum of areas of all the secondary peaks is not more than 3 times the area of the peak in the chromatogram obtained with the reference solution (b) (3.0 per cent). For Tenofovir Disoproxil Fumarate Test solution. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing 50 mg of Tenofovir Disoproxil Fumarate and disperse in 50 ml of methanol. Reference solution (a). A 0.1 per cent w/v solution of tenofovir disoproxil fumarate RS in methanol. Reference solution (b). Dilute 1 ml of reference solution (a) to 100 ml with methanol.
Tenofovir and Emtricitabine Tablets

Tenofovir Disoproxil Fumarate and Emtricitabine Tablets Tenofovir and Emtricitabine Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amounts of tenofovir disoproxil fumarate, C19H30N5O10P,C4H4O4 and emtricitabine, C8H10FN3O3S.
Identification
Tests
Dissolution (2.5.2). Apparatus. No 1 Medium. 900 ml of 0.1 M hydrochloric acid, Speed and time. 50 rpm and 45 minutes. Withdraw a suitable volume of the medium and filter. Determine by liquid chromatography (2.4.14) Test solution. The filtrate obtained as given above. Dilute the filtrate if necessary, with the dissolution medium. Reference solution. A solution containing 0.32 per cent w/v of tenofovir disoproxil fumarate RS and 0.24 per cent w/v of emtricitabine RS in methanol. Dilute 5 ml of the solution to 50 ml with the dissolution medium. Use the chromatographic system given under Assay. Inject the test solution and the reference solution. Calculate the contents of C 19 H 30 N 5 O 10 P.C 4 H 4 O 4 and C8H10FN3O3S. D. Not less than 75 per cent of the stated amounts of C19H30N5O10P.C4H4O4 and C8H10FN3O3S. Related substances. Determine by liquid chromatography (2.4.14). For Emtricitabine Test solution. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing 50 mg of Emtricitabine,
1159
TERBUTALINE SULPHATE
IP 2007
Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5m), mobile phase: A. a buffer solution prepared by dissolving 1.9 g of ammonium acetate in 1000 ml of water and adjusting the pH to 3.8 with glacial acetic acid, B. methanol, flow rate. 1.5 ml per minute, a linesr gradient programme using the conditions given below, spectrophotometer set at 260 nm, a 20 l loop injector. Time Mobile phase A Mobile phase B (in mins) (per cent) (per cent) 0 95 5 10 50 50 25 50 50 50 20 80 60 95 5 75 95 5 Inject reference solution (a). The test is not valid unless the column efficiency is not less than 2000 theoretical plates and the tailing factor is not more than 2.0. Inject the test solution and reference solution (b). In the chromatogram obtained with the test solution, the area of any secondary peak is not more than 3.5 times the area of the peak in the chromatogram obtained with the reference solution (b) (3.5 per cent) and the sum of areas of all the secondary peaks is not more than 6 times the area of the peak in the chromatogram obtained with the reference solution (b) (6.0 per cent). Other tests. Comply with the tests stated under the Tablets. Assay. Determine by liquid chromatography (2.4.14). Solvent mixture. 30 volumes of water and 70 volumes of methanol. Test solution. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing 100 mg of Tenofovir Disoproxil Fumarate, disperse in 10 ml of water and dilute to 250.0 ml with methanol. Dilute 5.0 ml of the solution to 25.0 ml with the solvent mixture. Reference solution. A solution containing 0.025 per cent w/v of emtricitabine RS and 0.04 per cent w/v of tenofovir disoproxil fumarate RS in methanol. Dilute 5.0 ml of the solution to 25.0 ml with the solvent mixture. Chromatographic system a stainless steel column 4.6 mm x 5 cm packed with octadecylsilane bonded to porous silica (3m), column temperature 40,
mobile phase: A. a buffer solution prepared by dissolving 1.35 g of monobasic potassium phosphate in 1000 ml of water and adjusting the pH to 3.0 with orthophosphoric acid, B. a mixture of 20 volumes of the buffer solution and 80 volumes of acetonitrile, flow rate. 1.5 ml per minute, a linear gradient programme using the conditions given below, spectrophotometer set at 260 nm, a 10 l loop injector. Time Mobile phase A Mobile phase B (in mins) (per cent) (per cent) 0 94 6 3 94 6 5 50 50 8 35 65 9 94 6 12 94 6 Inject the reference solution. The test is not valid unless the tailing factor is not more than 2.0, the column efficiency in not less than 2000 theoretical plates for the peak due to tenofovir disoproxil, 500 theoretical plates for the peak due to emtricitabine and the relative standard deviation for replicate injections is not more than 2.0 per cent for each component. Inject the test solution and the reference solution. Calculate the contents of C 19 H 30 N 5 O 10 P,C 4 H 4 O 4 and C8H10FN3O3S in the tablets. Storage. Store protected from moisture, at a temperature not exceeding 30.
Terbutaline Sulphate
OH HO H N
CH3 CH3 CH3

2
, H2SO4
OH
(C12H19NO3)2,H2SO4 Mol. Wt. 548.7
Terbutaline Sulphate is (RS)-2-(tert-butylamino)-1-(3,5dihydroxyphenyl)ethanol sulphate. Terbutaline Sulphate contains not less than 98.0 per cent and not more than 101.0 per cent of (C12H19NO3)2, H2SO4, calculated on the dried basis.
1160
IP 2007
TERBUTALINE INHALATION
Description. A white or almost white, crystalline powder; odourless or almost odourless.
Identification
Test A may be omitted if tests B, C and D are carried out. Tests B and C may be omitted if tests A and D are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with terbutaline sulphate RS or with the reference spectrum of terbutaline sulphate. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.007 per cent w/v solution in 0.1 M hydrochloric acid shows absorption maxima at about 276 nm and 280 nm, which may be fused; absorbance at both 276 nm and 280 nm, 0.46 to 0.49. C. In the test for Related substances, the principal spot in the chromatogram obtained with test solution (b) corresponds to that in the chromatogram obtained with reference solution (b). D. A 2.0 per cent w/v solution in carbon dioxide-free water gives reaction A of sulphates (2.3.1).
Wash the plate with the mobile phase and apply to the plate 20 l of each solution. After development, dry the plate in air, spray with dilute potassium permanganate solution. Any secondary spot in the chromatogram obtained with test solution (a) is not more intense than the spot in the chromatogram obtained with reference solution (a). Heavy metals (2.3.13). Mix 1.6 g with 0.6 g of anhydrous sodium sulphate and ignite without melting the sodium sulphate. Cool, add 3 ml of 2 M hydrochloric acid, boil and dilute to 50 ml with water. Cool and filter, 25 ml of the filtrate complies with the limit test for heavy metals, Method A (25 ppm). Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105 for 3 hours. Assay. Weigh accurately about 0.5 g, dissolve in 60 ml of anhydrous glacial acetic acid with the aid of heat. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2,4,25). Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.05486 g of (C12H19NO3)2, H2SO4. Storage. Store protected from light and moisture. Labelling. The label states (1) the number of metered doses in the container; (2) the amount of active ingredient delivered per inhalation; (3) that the container should be shaken before use each time; (4) a warning that the container is pressurised and must be kept away from heat and direct sunlight and that it must not be punctured, broken or incinerated even when apparently empty; (5) the warning Keep out of reach of children.
Tests
Appearance of solution. A 2.0 per cent w/v solution in carbon dioxide-free water is clear (2.4.1); absorbance of a 2-cm layer at 400 nm, not more than 0.11 (2.4.7). Acidity. Dissolve 0.2 g in 10 ml of carbon dioxide-free water and titrate with 0.01 M sodium hydroxide, using methyl red solution as indicator. Not more than 1.2 ml of 0.01 M sodium hydroxide is required to change the colour of the solution to yellow. tert-Butylamino-3,5-dihydroxyacetophenone. Absorbance of a 2 per cent w/v solution in 0.01 M hydrochloric acid at about 330 nm, not more than 0.50 (2.4.7). Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 90 volumes of aldehyde-free methanol, 10 volumes of water and 1.5 volumes of strong ammonia solution. Test solution (a). Dissolve 0.25 g of the substance under examination in 1 ml of water and add sufficient ethanol (95 per cent) to produce 10 ml. Test solution (b). Dilute 1 ml of test solution (a) to 5 ml with ethanol (95 per cent). Reference solution (a). Dilute 1 ml of test solution (a) to 200 ml with ethanol (95 per cent). Reference solution (b). Dissolve 25 mg of terbutaline sulphate RS in 0.5 ml of water and add sufficient ethanol (95 per cent) to produce 5 ml.
Terbutaline Inhalation
Terbutaline Sulphate Inhalation; Terbutaline Inhalation Aerosol; Terbutaline Sulphate Inhalation Aerosol Terbutaline Inhalation is a suspension of Terbutaline Sulphate, as a superfine powder, in a suitable liquid in a suitable pressurised container. It may contain suitable pharmaceutical aids such as surfactants, stabilising agents, etc. Terbutaline Inhalation delivers not less than 75.0 per cent and not more than 125.0 per cent of the stated amount of terbutaline sulphate (C12H19NO3)2,H2SO4, per inhalation, by actuation of the valve.
Identification
Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 65 volumes of 2-propanol, 25 volumes of cyclohexane and 5 volumes of formic acid.
1161
TERBUTALINE INJECTION
IP 2007
Test solution. Remove the actuator from the pressurised container, shake the container for about 30 seconds and place it in an inverted position in a small beaker containing 5 ml of water. Discharge a sufficient number of deliveries containing 5 mg of Terbutaline Sulphate, under the surface of the solvent. Reference solution. A 0.1 per cent w/v solution of terbutaline sulphate RS in water. Apply to the plate 2 l of each solution. After development, dry the plate in air, spray with a 2 per cent w/v solution of 4aminoantipyrine in methanol. Examine in daylight and in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution corresponds to the chromatogram obtained with reference solution.
Labelling. The label states the amount of active ingredient delivered per inhalation.
Terbutaline Injection
Terbutaline Sulphate Injection
Terbutaline Injection is a sterile solution of Terbutaline Sulphate in Water for Injections. Terbutaline Injection contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of terbutaline sulphate (C12H19NO3)2, H2SO4.
Tests
Other tests. Complies with the tests stated under Inhalation Preparations (Pressurised metered-dose Preparations). Follow the procedure described under Assay wherever the amount of active substance is to be determined in any test. Assay. Carry out the test for Content of active ingredient delivered per actuation stated under Inhalation Preparations (Pressurised metered-dose Preparations). Use 35 ml of water and finally dilute to 50.0 ml with water. Dilute a suitable volume of this solution with water to produce a solution containing 50 g of Terbutaline Sulphate per ml. To 10.0 ml of this solution in a 50-ml volumetric flask add 35 ml of buffer solution pH 9.5 and 1.0 ml of 4-aminoantipyrine solution. Mix, add 1.0 ml of potassium ferricyanide solution with vigorous swirling of the flask, dilute to volume with water and mix. Exactly 75 seconds after the addition of the potassium ferricyanide solution measure the absorbance of the resulting solution at the maximum at about 550 nm (2.4.7), using as the blank a solution prepared in the same manner using 10.0 ml of water in place of the solution of the substance under examination. Calculate the content of (C12H19NO3)2,H2SO4 in the solution from the absorbance obtained by repeating the operation using a solution containing 100 g of terbutaline sulphate RS in place of the solution of the substance under examination. Calculate the amount of (C12H19NO3)2,H2SO4 delivered per actuation of the valve. Determine the content of active ingredient a second and third time by repeating the procedure on the middle ten and on the last ten successive combined actuations of the valve. For each of the three determinations the average content of (C12H19NO3)2,H2SO4 delivered per actuation of the valve meets the requirements. Storage. Store protected from moisture at a temperature not exceeding 30.
Identification
Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 65 volumes of 2-propanol, 25 volumes of cyclohexane and 5 volumes of formic acid. Test solution. Use the injection. Reference solution. A 0.1 per cent w/v solution of terbutaline sulphate RS in saline solution. Apply to the plate 2 l of each solution. After development, dry the plate in air, spray with a 2 per cent w/v solution of 4-aminoantipyrine in methanol. Dry the plate in air and spray with a freshly prepared 8.0 per cent w/v solution of potassium ferricyanide in a mixture of 4 volumes of strong ammonia solution and 1 volume of water. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution.
Tests
pH (2.4.24). 3.0 to 5.0. Other tests. Complies with the tests stated under Parenteral preparations (Injections). Assay. Measure accurately a volume containing about 5 mg of Terbutaline Sulphate and add sufficient water to produce 50.0 ml. To 5.0 ml add 35 ml of a buffer solution prepared by dissolving 36.3 g of tris (hydroxymethyl) aminomethane in 900 ml of water, adjusting the pH to between 9.4 and 9.6 and adding sufficient water to produce 1000 ml. Add 1.0 ml of a freshly prepared 2.0 per cent w/v solution of 4-aminoantipyrine, mix and add 1.0 ml of a freshly prepared 8.0 per cent w/v solution of potassium ferricyanide with vigorous swirling and sufficient of the buffer solution to produce 50.0 ml. Exactly 75 seconds after the addition of the potassium ferricyanide solution measure the absorbance of the resulting solution at the maximum at about 550 nm (2.4.7), using water as the blank.
1162
IP 2007
TESTOSTERONE PROPIONATE
Calculate the content of (C12H 19NO 3)2, H2SO4 from the absorbance obtained by repeating the operation using a 0.01 per cent w/v solution of terbutaline sulphate RS and beginning at the words To 5.0 ml add 35 ml....... Storage. Store protected from light, in single dose containers. Labelling. The label states that the injection should not be used if the solution is discoloured.
Terbutaline Tablets
Terbutaline Sulphate Tablets
Terbutaline Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of terbutaline sulphate (C12H19NO3)2, H2SO4.
Powder one tablet, transfer to a 25-ml volumetric flask, add 15 ml of 0.01 M hydrochloric acid and shake for 10 minutes. Dilute to volume with 0.01 M hydrochloric acid and filter, rejecting the first 5 ml of the filtrate. Dilute, if necessary, a suitable volume of the filtrate with 0.01 M hydrochloric acid to produce a solution containing 0.01 per cent w/v solution of Terbutaline Sulphate. Carry out the method as described under Assay beginning at the words To 5.0 ml add 35 ml of a buffer solution..... Calculate the content of (C12H19NO3)2, H2SO4 in the tablet from the absorbance obtained by carrying out the Assay simultaneously using terbutaline sulphate RS. Other tests. Comply with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 5 mg of Terbutaline Sulphate, transfer to a 50-ml volumetric flask, add 30 ml of 0.01 M hydrochloric acid and shake for 10 minutes. Dilute to volume with 0.01 M hydrochloric acid and filter, rejecting the first 5 ml of the filtrate. To 5.0 ml add 35 ml of a buffer solution prepared by dissolving 36.3 g of tris (hydroxymethyl) aminomethane in 900 ml of water, adjusting the pH to between 9.4 and 9.6 and adding sufficient water to produce 1000 ml. Add 1.0 ml of a freshly prepared 2.0 per cent w/v solution of 4-aminoantipyrine, mix and add 1.0 ml of a freshly prepared 8.0 per cent w/v solution of potassium ferricyanide with vigorous swirling and sufficient of the buffer solution to produce 50.0 ml. Exactly 75 seconds after the addition of the potassium ferricyanide solution measure the absorbance of the resulting solution at the maximum at about 550 nm (2.4.7), using water as the blank. Calculate the content of (C12H19NO 3)2, H2SO 4 from the absorbance obtained by carrying out the Assay simultaneously using terbutaline sulphate RS. Storage. Store protected from light and moisture.
Identification
A. Shake a quantity of the powdered tablets containing 20 mg of Terbutaline Sulphate with 50 ml of 0.1 M sodium hydroxide for 10 minutes, dilute to 100 ml with 0.1 M sodium hydroxide and filter. Dilute 20 ml of the filtrate to 50 ml with 0.1 M sodium hydroxide. When examined in the range 230 nm to 360 nm (2.4.7), the resulting solution shows an absorption maximum at about 296 nm. B. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 65 volumes of 2-propanol, 25 volumes of cyclohexane and 5 volumes of formic acid. Test solution. Shake a quantity of the powdered tablets containing 10 mg of Terbutaline Sulphate with 4 ml of a mixture of equal volumes of ethanol (95 per cent) and water for 10 minutes, centrifuge and use the clear supernatant liquid. Reference solution (a). A 0.25 per cent w/v solution of terbutaline sulphate RS in water. Reference solution (b). A mixture of equal volumes of the test solution and reference solution (a). Apply to the plate 2 l of each solution. After development, dry the plate in air, allow to stand for a few minutes in an atmosphere saturated with diethylamine and spray with diazotised nitroaniline solution. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with reference solution (a) and the principal spot in the chromatogram obtained with reference solution (b) appears as a single compact spot.
Testosterone Propionate
O H 3C O H3C H O H H CH3
Tests
Uniformity of content. Comply with the test stated under Tablets
C22H32O3
Mol. Wt. 344.5
Testosterone Propionate is 3-oxoandrost-4-en-17-yl propionate.
1163
TESTOSTERONE PROPIONATE INJECTION
IP 2007
Testosterone Propionate contains not less than 97.0 per cent and not more than 103.0 per cent of C22H32O3, calculated on the dried basis. Description. A white or almost white powder or colourless crystals; odourless.
The test is not valid unless in the chromatogram obtained with reference solution (a) the resolution between the peaks due to testosterone and testosterone acetate is not less than 4.0. In the chromatogram obtained with the test solution the area of any peak other than the principal peak is not more than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent); the sum of the areas of any secondary peaks is not greater than the area of the principal peak in the chromatogram obtained with reference solution (b) (1.0 per cent). Ignore any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.05 per cent). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105 for 2 hours. Assay. Weigh accurately about 25 mg, dissolve in sufficient ethanol to produce 250.0 ml, mix, dilute 5.0 ml to 50.0 ml with the same solvent and measure the absorbance of the resulting solution at the maximum at about 241 nm (2.4.7). Calculate the content of C22H32O3 taking 490 as the specific absorbance at 241 nm. Storage. Store protected from light and moisture.
Identification
Test A may be omitted if tests B and C are carried out. Tests B and C may be omitted if test A is carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with testosterone propionate RS or with the reference spectrum of testosterone propionate. B. In the test for Related substances the principal peak in the chromatogram obtained with the test solution corresponds to the peak due to testosterone propionate in the chromatogram obtained with reference solution (a). C. Melting range. 119 to 123 (2.4.21).
Tests
Specific optical rotation (2.4.22). +83.0 to +90.0, determined in a 1.0 per cent w/v solution in ethanol. Related substances. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 50 mg of the substance under examination in methanol and dilute to 50 ml with the same solvent. Reference solution (a). Dissolve 2 mg of the substance under examination and 2 mg of testosterone acetate RS in methanol and dilute to 50 ml with the same solvent. Reference solution (b). Dilute 1 ml of the test solution to 100 ml with methanol. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 20 volumes of water and 80 volumes of methanol, flow rate. 1.5 ml per minute, spectrophotometer set at 254 nm, a 20 l loop injector. Inject the test solution and the reference solutions. Continue the chromatography for twice the retention time for testosterone propionate. The relative retention time of about four impurities with reference to testosterone propionate (retention time, about 9 minutes) range from 0.5 to about 1.4.
Testosterone Propionate Injection

Testosterone Propionate Injection is a sterile solution of Testosterone Propionate in Ethyl Oleate or any other suitable ester, in a suitable fixed oil or in any mixture of these. Testosterone Propionate Injection contains not less than 92.5 per cent and not more than 107.5 per cent of the stated amount of testosterone propionate, C22H32O3.
Identification
Dissolve a volume containing 50 mg of Testosterone Propionate in 8 ml of light petroleum (40 to 60) and extract with three quantities, each of 8 ml, of a mixture of 7 volumes of glacial acetic acid and 3 volumes of water. Wash the combined extracts with 10 ml of light petroleum (40 to 60), dilute with water until the solution becomes turbid, allow to stand for 2 hours in ice and filter. The precipitate, after washing with water and drying at 105, complies with the following tests. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with testosterone propionate RS or with the reference spectrum of testosterone propionate.
1164
IP 2007
TETRACYCLINE
B. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Solvent mixture. A mixture of 90 volumes of light petroleum (40 to 60) and 10 volumes of liquid paraffin. Mobile phase. A mixture of 30 volumes of water and 20 volumes of glacial acetic acid. Test solution. Dissolve 25 mg of the residue in 10 ml of the solvent mixture. Reference solution (a). A 0.25 per cent w/v solution of testosterone propionate RS in the solvent mixture. Reference solution (b). Mix equal volumes of the test solution and reference solution (a). Place the dry plate in a tank containing a shallow layer of the solvent mixture, allow the solvent mixture to ascend to the top, remove the plate from the tank and allow the solvent to evaporate. Use within 2 hours, with the flow of the mobile phase in the direction in which the aforementioned treatment was done. Apply to the plate 2 l of each solution. Allow the mobile phase to rise 12 cm. Dry the plate in a current of warm air, allow the solvent to evaporate, heat at 120 for 15 minutes and spray the hot plate with ethanolic sulphuric acid (20 per cent v/v). Heat at 120 for a further 10 minutes, allow to cool and examine in daylight and in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with reference solution (a). The principal spot in the chromatogram obtained with reference solution (b) appears as a single, compact spot.A.
Tetracycline
OH O HO O HO CONH2
H HO CH 3
C22H24N2O8
OH N(CH3)2
Mol. Wt. 444.5
Tetracycline is (4S,4aS,5aS,6S,12aS)-4-dimethylamino1,4,4a,5,5a,6,11,12a-octahydro-3,6,10,12,12a-pentahydroxy-6methyl-1,11-dioxonaphthacene-2-carboxamide. It contains a variable quantity of water. Tetracycline contains not less than 88.0 per cent of C22H24N2O8, calculated on the dried basis. Description. A yellow, crystalline powder.
Identification
A. In the test for Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the peak due to tetracycline hydrochloride in the chromatogram obtained with reference solution (a). B. To about 2 mg add 5 ml of sulphuric acid; a reddish violet colour develops. Add the solution to 2.5 ml of water; the colour changes to yellow. C. Dissolve about 10 mg in a mixture of 1 ml of 2 M nitric acid and 5 ml of water, shake well and add 1 ml of silver nitrate solution. Any opalescence produced is not more intense than that in a solution prepared in the same manner omitting the substance under examination.
Tests
Other tests. Complies with the tests stated under Parenteral preparations (Injections). Assay. To an accurately measured volume containing about 0.1 g of Testosterone Propionate add sufficient chloroform to produce 100.0 ml and mix. Dilute 3.0 ml to 50.0 ml with chloroform and to 5.0 ml of the solution add 10 ml of isoniazid solution and sufficient methanol to produce 20.0 ml. Allow to stand for 45 minutes and measure the absorbance of the resulting solution at the maximum at about 380 nm (2.4.7), using as the blank a solution prepared by treating 5 ml of chloroform in the same manner. Calculate the content of C 22H 32O3 from the absorbance obtained by repeating the operation using a 0.006 per cent w/v solution of testosterone propionate RS in chloroform and beginning at the words to 5.0 ml of the solution...... Storage. Store protected from light.
Tests
pH (2.4.24). 3.5 to 6.0, determined in a 1.0 per cent w/v suspension in carbon dioxide-free water. Specific optical rotation (2.4.22).260 to 280, determined at 20 in a 1.0 per cent w/v solution in 0.1 M hydrochloric acid. Related substances. Determine by liquid chromatography (2.4.14) Test solution. Dissolve 0.1 g of the substance under examination in 100 ml of a mixture of 68 ml of 0.1 M ammonium oxalate and 27 ml of dimethylformamide (diluting solvent). Reference solution (a). A 0.0025 per cent w/v solution of 4-epitetracycline hydrochloride RS in the diluting solvent. Reference solution (b). A solution containing 0.0025 per cent w/v of 4-epitetracycline hydrochloride RS and 0.01 per cent w/v of tetracycline hydrochloride RS in the diluting solvent.
1165
TETRACYCLINE HYDROCHLORIDE
IP 2007
Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octylsilane bonded to porous silica (5 to 10 m), mobile phase: a mixture of 680 ml of 0.1 M ammonium oxalate, 270 ml of dimethylformamide and 50 ml of 0.2 M dibasic ammonium phosphate, the pH of the mixture being adjusted, if necessary, to 7.6 to 7.7 with 3 M ammonia or 3 M phosphoric acid, flow rate. 2 ml per minute, spectrophotometer set at 280 nm, a 20 l loop injector. The test is not valid unless the resolution between the two principal peaks in the chromatogram obtained with reference solution (b) is not less than 1.5. In the chromatogram obtained with the test solution, the area of any peak corresponding to 4-epitetracycline is not greater than the area of the principal peak in the chromatogram obtained with reference solution (a) and the total area of all the peaks other than the principal peak is not greater than 5.0 per cent. Heavy metals (2.3.13). 0.5 g complies with the limit test for heavy metals, Method B (50 ppm). Use 2.5 ml of lead standard solution (10 ppm Pb) to prepare the standard. Sulphated ash (2.3.18). Not more than 0.5 per cent. Loss on drying (2.4.19). Not more than 13.0 per cent, determined on 0.5 g by drying in an oven at 105. Assay. Determine by liquid chromatography (2.4.14). Test solution. Weigh accurately about 50 mg of the substance under examination and dissolve in 100.0 ml of a mixture of 68 ml of 0.1 M ammonium oxalate and 27 ml of dimethylformamide (diluting solvent). Reference solution (a). A 0.05 per cent w/v solution of tetracycline hydrochloride RS in the diluting solvent. Reference solution (b). A solution containing 0.0025 per cent w/v of 4-epitetracycline hydrochloride RS and 0.01 per cent w/v of tetracycline hydrochloride RS in the diluting solvent. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octylsilane bonded to porous silica (5 to 10 m), mobile phase: a mixture of 680 ml of 0.1 M ammonium oxalate, 270 ml of dimethylformamide and 50 ml of 0.2 M dibasic ammonium phosphate, the pH of the mixture being adjusted, if necessary, to 7.6 to 7.7 with 3 M ammonia or 3 M phosphoric acid, flow rate. 1.5 ml per minute, spectrophotometer set at 280 nm, a 20 l loop injector. The test is not valid unless the resolution between the two principal peaks in the chromatogram obtained with reference solution (b) is not less than 1.5.
Calculate the content of C22H24N2O8. 1 mg of C22H24N2O8, HCl is equivalent to 0.92 mg of C22H24N2O8. Storage. Store protected from light and moisture.
Tetracycline Hydrochloride
OH O OH O HO CONH2
, HCl
H HO CH3
OH N(CH3)2
C22H24N2O8,HCl
Mol. Wt. 480.9
Tetracycline Hydrochloride is (4S,4aS,5aS,6S,12aS)-4dimethylamino-1,4,4a,5,5a,6,11,12a-octahydro-3,6,10,12,12apentahydroxy-6-methyl-1,11-dioxonaphthacene-2carboxamide hydrochloride. Tetracycline Hydrochloride contains not less than 95.0 per cent and not more than 100.5 per cent of C22H24N2O8, HCl, calculated on the dried basis. Description. A yellow, crystalline powder.
Identification
A. In the test for Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the peak due to tetracycline hydrochloride in the chromatogram obtained with reference solution (a). B. To about 2 mg add 5 ml of sulphuric acid; a reddish violet colour develops. Add the solution to 2.5 ml of water; the colour changes to yellow. C. Gives reaction A of chlorides (2.3.1).
Tests
pH (2.4.24). 1.8 to 3.0, determined in a 1.0 per cent w/v suspension in carbon dioxide-free water. Specific optical rotation (2.4.22). 239 to 255, determined at 20 in a 1.0 per cent w/v solution in 0.1 M hydrochloric acid. Related substances. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 0.1 g of the substance under examination in 100 ml of a mixture of 68 ml of 0.1 M ammonium oxalate and 27 ml of dimethylformamide (diluting solvent). Reference solution (a). A 0.0025 per cent w/v solution of 4-epitetracycline hydrochloride RS in the diluting solvent.
1166
IP 2007
TETRACYCLINE CAPSULES
Reference solution (b). A solution containing 0.0025 per cent w/v of 4-epitetracycline hydrochloride RS and 0.01 per cent w/v of tetracycline hydrochloride RS in the diluting solvent. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octylsilane bonded to porous silica (5 to 10 m), mobile phase: 680 ml of 0.1 M ammonium oxalate, 270 ml of dimethylformamide and 50 ml of 0.2 M dibasic ammonium phosphate, the pH of the mixture being adjusted, if necessary, to 7.6 to 7.7 with 3 M ammonia or 3 M phosphoric acid, flow rate. 2 ml per minute, spectrophotometer set at 280 nm, a 20 l loop injector. The test is not valid unless the resolution between the two principal peaks in the chromatogram obtained with reference solution (b) is not less than 1.5. In the chromatogram obtained with the test solution the area of any peak corresponding to 4-epitetracycline is not greater than the area of the principal peak in the chromatogram obtained with reference solution (a) and the total area of all peaks other than the principal peak is not greater than 5.0 per cent. Heavy metals (2.3.13). 0.5 g complies with the limit test for heavy metals, Method B (50 ppm). Use 2.5 ml of lead standard solution (10 ppm Pb) to prepare the standard. Sulphated ash (2.3.18). Not more than 0.5 per cent. Loss on drying (2.4.19). Not more than 2.0 per cent, determined on 1.0 g by drying in an oven at 60 over phosphorus pentoxide at a pressure not exceeding 0.7 kPa for 3 hours. Assay. Determine by liquid chromatography (2.4.14). Test solution. Weigh accurately about 50 mg of the substance under examination and dissolve in about 50 ml of the diluting solvent described in the test for related substances and further add sufficient diluting solvent to produce 100.0 ml. Reference solution (a). A 0.05 per cent w/v solution of tetracycline hydrochloride RS in the diluting solvent. Reference solution (b). A solution containing 0.0025 per cent w/v of 4-epitetracycline hydrochloride RS and 0.01 per cent w/v of tetracycline hydrochloride RS in the diluting solvent. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octylsilane bonded to porous silica (5 to 10 m), mobile phase: a mixture of 680 volumes of 0.1 M ammonium oxalate, 270 volumes of dimethylformamide and 50 volumes of 0.2 M dibasic ammonium phosphate, the pH of the mixture being adjusted, if necessary, to 7.6 to 7.7 with 3 M ammonia or 3 M phosphoric acid, flow rate. 1.5 ml per minute, spectrophotometer set at 280 nm, a 20 l loop injector.
The test is not valid unless the resolution between the two principal peaks in the chromatogram obtained with reference solution (b) is not less than 1.5. Calculate the content of C22H24N2O8. Tetracycline Hydrochloride intended for use in the manufacture of parenteral preparations without a further appropriate procedure for removal of bacterial endotoxins complies with the following additional requirement. Bacterial endotoxins (2.2.3). Not more than 0.5 Endotoxin Unit per mg. Tetracycline hydrochloride intended for use in the manufacture of parenteral preparations without a further appropriate sterilisation procedure complies with the following additional requirement. Sterility (2.2.11). Complies with the test for sterility. Storage. Store protected from light and moisture. If it is intended for use in the manufacture of parenteral preparations, the container should be sterile, tamper-evident and sealed so as to exclude microorganisms. Labelling. The label states whether or not the material is intended for use in the manufacture of parenteral preparations.
Tetracycline Capsules
Tetracycline Hydrochloride Capsules
Tetracycline Capsules contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of tetracycline hydrochloride, C22H24N2O8, HCl.
Identification
A. In the test for Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the peak due to tetracycline hydrochloride in the chromatogram obtained with reference solution (a). B. To a quantity of the mixed contents of 20 capsules containing about 10 mg of Tetracycline Hydrochloride add 20 ml of warm ethanol (95 per cent), allow to stand for 20 minutes, filter and evaporate the filtrate to dryness on a waterbath. To 0.5 mg of the residue add 2 ml of sulphuric acid; a reddish violet colour develops. Add the solution to 2.5 ml of water; the colour changes to yellow. C. The residue obtained in test B gives reaction A of chlorides (2.3.1).
Tests
1167
TETRACYCLINE ONITMENT
IP 2007
Test solution. Shake a quantity of the mixed contents of 20 capsules containing about 25 mg of Tetracycline Hydrochloride with 80 ml of 0.01 M methanolic hydrochloric acid for 10 minutes, dilute to 100 ml with the same solvent, mix and filter if necessary. Reference solution (a). A 0.002 per cent w/v solution of 4-epitetracycline hydrochloride RS in 0.01 M methanolic hydrochloric acid. Reference solution (b). A solution containing 0.0015 per cent w/v each of 4-epitetracycline hydrochloride RS and tetracycline hydrochloride RS in 0.01 M methanolic hydrochloric acid. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (10 m), column. temperature 40, mobile phase: a mixture of 5 volumes of dimethylformamide and 95 volumes of 0.1 M oxalic acid, the pH of the mixture being adjusted to 3.9 with triethylamine, flow rate. 2 ml per minute, spectrophotometer set at 280 nm, a 20 l loop injector. The test is not valid unless the resolution between the two principal peaks in the chromatogram obtained with reference solution (b) is not less than 2.0. In the chromatogram obtained with the test solution the area of any peak corresponding to 4-epitetracycline hydrochloride is not greater than the area of the principal peak in the chromatogram obtained with reference solution (a) and the total area of all the peaks other than the principal peak is not greater than 10.0 per cent. Dissolution (2.5.2). Apparatus. No 1 Medium. 900 ml of water freshly prepared by distillation. Speed and time. 75 rpm and 60 minutes. Withdraw a suitable volume of the medium and filter through a membrane filter disc having an average pore diameter not greater than 1.0 m, rejecting the first few ml of the filtrate. Measure the absorbance of the resulting solution, suitably diluted if necessary, at the maximum at about 276 nm (2.4.7). Calculate the content of C22H24N2O8, HCl in the medium from the absorbance obtained from a solution of known concentration of tetracycline hydrochloride. D. Not less than 70 per cent of the stated amount of C22H24N2O8, HCl. Loss on drying (2.4.19). Not more than 3.0 per cent, determined on 1.0 g of the contents of the capsules by drying in an oven at 60 at a pressure not exceeding 0.7 kPa for 3 hours. Other tests. Comply with the tests stated under Capsules.
Assay. Determine by liquid chromatography (2.4.14). Test solution. Weigh accurately a quantity of the mixed contents of 20 capsules containing about 50 mg of Tetracycline Hydrochloride, shake with about 50 ml of 0.01 M methanolic hydrochloric acid and dilute with the same solvent to produce 100.0 ml, mix and filter. Discard the first few ml of the filtrate. Reference solution (a). A 0.05 per cent w/v solution of tetracycline hydrochloride RS in 0.01 M methanolic hydrochloric acid. Reference solution (b). A solution containing 0.0025 per cent w/v of 4-epitetracycline hydrochloride RS and 0.01 per cent w/v of tetracycline hydrochloride RS in 0.01 M methanolic hydrochloric acid. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octylsilane bonded to porous silica (5 to10 m), mobile phase: a mixture of 68 volumes of 0.1 M ammonium oxalate and 27 volumes of dimethylformamide and 5 volumes of 0.2 M dibasic ammonium phosphate, the pH of the mixture being adjusted, if necessary, to 7.6 to 7.7 with 3 M ammonia or 3 M phosphoric acid, flow rate. 1.5 ml per minute, spectrophotometer set at 280 nm, a 20 l loop injector. The test is not valid unless the resolution between the two principal peaks in the chromatogram obtained with reference solution (b) is not less than 1.5. Calculate the content of C22H24N2O8, HCl in the capsules. Storage. Store protected from light and moisture.
Tetracycline Ointment
Tetracycline Hydrochloride Eye Ointment
Tetracycline Ointment contains not less than 90.0 per cent and not more than 115.0 per cent of the stated amount of tetracycline hydrochloride, C22H24N2O8, HCl.
Identification
In the test for Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the peak due to tetracycline hydrochloride in the chromatogram obtained with the reference solution.
Tests
Water (2.3.43). Not more than 0.5 per cent, determined on 2.0 g dissolved in a mixture of 2 volumes of carbon
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THEOPHYLLINE
tetrachloride, 2 volumes of chloroform and 1 volume of methanol. Other tests. Complies with the tests stated under Eye Ointments. Assay. Determine by liquid chromatography (2.4.14). Test solution. Weigh accurately a quantity containing about 25 mg of Tetracycline Hydrochloride and transfer to a separating funnel with the aid of 15 ml of cyclohexane. Add 15 ml of a mixture of 68 volumes of 0.1 M ammonium oxalate and 27 volumes of dimethylformamide (diluting solvent) and shake well. Collect the lower layer in a 50-ml volumetric flask. Repeat the extraction with two further quantities, each of 15 ml, of the diluting solvent, combining the extracts in the same 50-ml volumetric flask. Add sufficient diluting solvent to produce 50.0 ml, mix and filter. Reference solution. A 0.05 per cent w/v solution of tetracycline hydrochloride RS in the diluting solvent. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octylsilane bonded to porous silica (5 to10 m), mobile phase: a mixture of 68 volumes of 0.1 M ammonium oxalate and 27 volumes of dimethylformamide and 5 volumes of 0.2 M dibasic ammonium phosphate, the pH of the mixture being adjusted, if necessary, to 7.6 to 7.7 with 3 M ammonia or 3 M phosphoric acid, flow rate. 1.5 ml per minute, spectrophotometer set at 280 nm, a 20 l loop injector. Calculate the content of C22H24N2O8, HCl in the eye ointment. Storage. Store protected from moisture.
Description. A white, crystalline powder; odourless.
Identification
Test A may be omitted if tests B, C and D are carried out. Tests B and D may be omitted if tests A and C are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with theophylline RS or with the reference spectrum of theophylline. B. Dissolve about 10 mg in 10 ml of water, add 0.5 ml of a 5 per cent w/v solution of mercuric acetate and allow to stand; a white, crystalline precipitate is produced. C. The melting range, after drying at 105, 270 to 274 (2.4.21). D. Gives the reaction of xanthines (2.3.1).
Tests
Appearance of solution. Dissolve 0.5 g in 75 ml of carbon dioxide-free water with heating; the resulting solution (solution A), is clear (2.4.1), and colourless (2.4.1). Acidity. To 50 ml of solution A add 0.1 ml of methyl red solution. The solution is red and not more than 1.0 ml of 0.01 M sodium hydroxide is required to change the colour of the solution to yellow. Light absorption (2.4.7). Absorbance of a 0.001 per cent w/v solution in 0.1 M hydrochloric acid at the maximum at about 270 nm, not less than 0.53. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 40 volumes of 1-butanol, 30 volumes of acetone, 30 volumes of chloroform and 10 volumes of strong ammonia solution. Test solution. Dissolve 0.2 g of the substance under examination in 10 ml of a mixture of 60 volumes of chloroform and 40 volumes of methanol.
Theophylline
O H3C O N H N
Reference solution. Dissolve 10 mg of the substance under examination in 100 ml of a mixture of 60 volumes of chloroform and 40 volumes of methanol. Apply to the plate 10 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Heavy metals (2.3.13).1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent (for the anhydrous form) and 8.0 per cent to 9.5 per cent (for the
N N CH3
Mol. Wt. 180.2 (anhydrous) Mol. Wt. 198.2 (hydrate)
C7H8N4O2 C7H8N4O2,H2O
Theophylline is 1,3-dimethyl-3,7-dihydro-1H-purine-2,6dione. It contains one molecule of water or is anhydrous. Theophylline contains not less than 98.0 per cent and not more than 101.0 per cent of C7H8N4O2, calculated on the dried basis.
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THEOPHYLLINE INJECTION
IP 2007
hydrated form), determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.25 g, add 50 ml of water and gently warm the mixture on a water-bath until complete solution is effected. Cool, add 20.0 ml of 0.1 M silver nitrate and 1.0 ml of bromothymol solution and titrate with 0.1 M sodium hydroxide until a blue colour is obtained. 1 ml of 0.1 M sodium hydroxide is equivalent to 0.01802 g of C7H8N4O2. Storage. Store protected from moisture.
at a flow rate of about 3.5 ml per minute, discarding the first 50 ml of the eluate. Measure the absorbance of the eluate at 284 nm, using water as the blank; the absorbance is not more than 0.25 (2.4.7). Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. For theophylline Dilute a suitable volume of the injection with sufficient 0.1 M sodium hydroxide to produce a solution containing 0.008 per cent w/v of anhydrous theophylline. Measure the absorbance of the resulting solution at the maximum at about 274 nm (2.4.7), using as the blank a solution prepared in the same manner omitting the substance under examination. Calculate the content of C7H8N4O2 from the absorbance obtained by repeating the operation using theophylline RS in place of the substance under examination. For dextrose Transfer an accurately measured volume of the injection containing about 5 mg of Dextrose to a 100-ml volumetric flask, add 0.2 ml of 6 M ammonia, dilute to volume with water and mix. Determine the optical rotation of the resulting solution in a suitable polarimeter tube at 25 (2.4.22). The observed rotation, in degrees multiplied by 1.0425A, represents the weight, in g, of C6H12O6,H2O in the volume of the injection taken for the assay, where A is the ratio 200 divided by the length, in mm, of the polarimeter tube employed. Storage. Store protected from light, in single dose containers. Labelling. The label states the strength in terms of the amounts of anhydrous theophylline and Dextrose.
Theophylline Injection
Theophylline in Dextrose Injection
Theophylline Injection is a sterile solution of Theophylline and Dextrose in Water for Injections. Theophylline Injection contains not less than 92.5 per cent and not more than 107.5 per cent of the stated amount of anhydrous theophylline, C7H8N4O2, and not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of dextrose, C6H12O6, H2O.
Identification
A. When examined in the range 230 nm to 360 nm (2.4.7), the solution obtained in the Assay for theophylline shows an absorption maximum at about 274 nm. B. Add 0.2 ml of the injection to 5 ml of potassium cupritartrate solution and heat to boiling; a red to orange precipitate is formed.
Thiabendazole
Tiabendazole
H N N N
Tests
pH (2.4.24). 3.5 to 6.5. 5-Hydroxymethylfurfural and Related substances. Use a glass chromatographic column (66 cm 11 mm) with a sealed-in, coarse-porosity sintered disc or a glass wool plug and fitted with a stopcock. Mix 8 g of a 20- to 50-mesh styrenedivinylbenzene anion-exchange resin in the hydroxide form with 25 ml of water, allow to settle and decant the supernatant liquid until a slurry of resin remains. Pour the slurry into the column and allow to settle as a homogeneous bed having a bed volume of about 15 ml. Wash the resin bed at a flow rate of about 3 ml per minute with 100 ml of a 5.7 per cent w/v solution of ammonium carbonate followed by washing with water until the eluate has a pH of 7. Dilute an accurately measured volume of the injection containing 1.0 g of Dextrose, C6H12O6,H2O, to 250.0 ml with water. Pass this solution through the resin bed in the column
C10H7N3S
Mol. Wt. 201.3
Thiabendazole is 2-(1,3-thiazol-4-yl)-1H-benzimidazole. Thiabendazole contains not less than 98.0 per cent and not more than 101.0 per cent of C10H7N3S, calculated on the anhydrous basis. Description. A white or almost white, crystalline powder.
Identification
Test A may be omitted if tests B, C and D are carried out. Tests B, C and D may be omitted if test A is carried out.
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THIABENDAZOLE TABLETS
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with thiabendazole RS or with the reference spectrum of thiabendazole. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.0005 per cent w/v solution in 0.1 M hydrochloric acid shows absorption maxima at about 243 nm and 302 nm; ratio of the absorbance at the maximum at about 302 nm to that at about 243 nm, 1.8 to 2.1. C. In the test for Related substances, the principal spot in the chromatogram obtained with the test solution (b) corresponds to that in the chromatogram obtained with reference solution (c). D. Dissolve 5 mg in 5 ml of 0.1 M hydrochloric acid, add 3 mg of 4-phenylenediamine dihydrochloride and shake until dissolved. Add 0.1 g of zinc powder, mix, allow to stand for 2 minutes and add 10 ml of ferric ammonium sulphate solution; a deep blue or bluish violet colour is produced.
Sulphated ash (2.3.18). Not more than 0.2 per cent. Water (2.3.43). Not more than 0.5 per cent, determined on 1.0 g. Assay. Weigh accurately about 0.15 g, dissolve in 30 ml of anhydrous glacial acetic acid. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.02013 g of C10H7N3S. Storage. Store protected from light and moisture.
Thiabendazole Tablets
Tiabendazole Tablets
Thiabendazole Tablets contain not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of thiabendazole, C10H7N3S. The tablets may contain permitted colours.
Tests
Appearance of solution. Add 5 ml of methanol to 0.5 g in a flask fitted with a ground-glass stopper, stir for 5 minutes, with a magnetic stirrer and filter through a sintered-glass filter (1.6 m to 4 m). The solution is not more intensely coloured than reference solution BS6 (2.4.1). Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel HF254. Mobile phase. A mixture of 62.5 volumes of toluene, 25 volumes of glacial acetic acid, 10 volumes of acetone and 2.5 volumes of water. Test solution (a). Dissolve 0.1 g of the substance under examination in 10 ml of methanol. Test solution (b). Dilute 2 ml of test solution (a) to 20 ml with methanol. Reference solution (a). Dilute 1 ml of test solution (b) to 10 ml with methanol. Reference solution (b). Dilute 1 ml of test solution (b) to 25 ml with methanol. Reference solution (c). Dissolve 20 mg of thiabendazole RS in 20 ml of methanol. Apply to the plate 20 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with test solution (a) is not more intense than the spot in the chromatogram obtained with reference solution (a) and not more than one such spot is more intense than the spot in the chromatogram obtained with reference solution (b) Heavy metals (2.3.13). 2.0 g complies with the limit test for heavy metals, Method B (10 ppm).
Identification
A. When examined in the range 230 nm to 360 nm (2.4.7), the final solution obtained in the Assay shows an absorption maximum at about 302 nm. B. Dissolve a quantity of the powdered tablets containing 30 mg of Thiabendazole in 5 ml of 0.1 M hydrochloric acid, add 3 mg of 4-phenylenediamine dihydrochloride and shake until dissolved. Add 0.1 g of zinc powder, mix, allow to stand for 2 minutes and add 10 ml of ferric ammonium sulphate solution; a deep bluish violet colour is produced.
Tests
Disintegration. The test does not apply. Other tests. Comply with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 0.1 g of Thiabendazole, add 75 ml of 0.1 M hydrochloric acid, warm on a water-bath for 15 minutes, shaking occasionally, cool, dilute to 100.0 ml with 0.1 M hydrochloric acid and filter. Dilute 5.0 ml of the filtrate to 1000.0 ml with 0.1 M hydrochloric acid and measure the absorbance of the resulting solution at the maximum at about 302 nm (2.4.7). Calculate the content of C10H7N3S taking 1230 as the specific absorbance at 302 nm. Storage. Store protected from light and moisture. Labelling. The label states that the tablets should be chewed before swallowing.
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THIACETAZONE
IP 2007
Thiacetazone
O H3 C
C10H12N4OS Thiacetazone is 4-acetamidobenzaldehyde thiosemicarbazone. Thiacetazone contains not less than 98.0 per cent and not more than 102.0 per cent of C10H12N4OS, calculated on the dried basis. Description. Pale yellow crystals or a crystalline powder; almost odourless.
N N H
H N S
NH2
Reference solution. Dissolve with the aid of heat 0.016 g of 4-acetamidobenzalazine RS in 150 ml of methanol, cool and dilute to 200 ml with methanol and further dilute 5 ml of this solution to 50 ml with methanol. Apply to the plate 5 l of each solution. After development, dry the plate in air, spray with 2 M nitric acid and within 2 minutes examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Heavy metals (2.3.13). 2.0 g complies with the limit test for heavy metals, Method B ( 10 ppm). Sulphated ash (2.3.18). Not more than 0.2 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.1 g, dissolve in 60 ml of methanol by heating at 60 in a water-bath, add slowly 20 ml of hot methanolic silver nitrate solution, maintain the solution at 60 until the precipitate coagulates and leaves a clear supernatant liquid. Cool, filter through a sintered-glass crucible (porosity No. 4), wash the residue with methanol until the washings are free from silver nitrate and dry to constant weight at 105. 1 g of residue is equivalent to 0.4606 g of C10H12N4OS. Storage. Store protected from light and moisture.
Mol. Wt. 236.3
Identification
Test A may be omitted if tests B and C are carried out. Tests B and C may be omitted if test A is carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with thiacetazone RS or with the reference spectrum of thiacetazone. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.0003 per cent w/v solution in ethanol (95 per cent) shows an absorption maximum at about 328 nm absorbance at about 328 nm, about 0.55. C. Boil 10 mg with 5 ml of 1 M hydrochloric acid for 3 minutes, cool and add sufficient water to produce 200 ml. Mix 5 ml of this solution with 0.25 ml of sodium nitrite solution and add the mixture to 0.5 ml of 2-naphthol solution; a red colour is produced.
Thiacetazone And Isoniazid Tablets

Thiacetazone and Isoniazid Tablets contain one part by weight of Thiacetazone and two parts by weight of Isoniazid. Thiacetazone and Isoniazid Tablets contain not less than 92.5 per cent and not more than 107.5 per cent of the stated amounts of thiacetazone, C10H12N4OS, and isoniazid, C6H7N3O2.
Tests
Thiosemicarbazide. To 2.0 g, finely powdered, add sufficient water to produce 50 ml, shake, allow to stand for 1 hour with occasional shaking and filter, discarding the first few ml of the filtrate. Acidify 25 ml of the clear filtrate with dilute sulphuric acid, add 0.1 ml of o-phenanthroline-ferrous complex solution and titrate with 0.1 M ceric ammonium sulphate to a blue end-point which persists for 1 minute; not more than 0.8 ml is required. 4-acetamidobenzalazine. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. Ethyl acetate. Test solution. Dissolve 0.4 g of the substance under examination in 100 ml of methanol.
Identification
A. Extract a quantity of the powdered tablets containing about 30 mg of Thiacetazone with 70 ml of ethanol (95 per cent) with the aid of heat for 15 minutes with occasional shaking. Cool, dilute to 100 ml with ethanol (95 per cent) and filter. Dilute 1 ml to 100 ml with ethanol (95 per cent). The solution complies with the following test. When examined in the range 230 nm to 360 nm (2.4.7), a 0.0005 per cent w/v solution in 0.1 M hydrochloric acid shows absorption maxima at about 243 nm and 302 nm; ratio of the absorbance at the maximum at about 302 nm to that at about 243 nm, 1.8 to 2.1. B. Shake a quantity of the powdered tablets containing 1 mg of Isoniazid with 50 ml of ethanol (95 per cent) and filter. To
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THIAMINE HYDROCHLORIDE
5 ml of the filtrate add 0.1 g of borax and 5 ml of a 5 per cent w/v solution of 1-chloro-2,4-dinitrobenzene in ethanol (95 per cent), evaporate to dryness on a water-bath and continue heating for a further 10 minutes. To the residue add 10 ml of methanol and mix; a reddish purple colour is produced.
Thiamine Hydrochloride contains not less than 98.5 per cent and not more than 101.5 per cent of C12H17ClN4OS, HCl, calculated on the dried basis. Description. A white or almost white, crystalline powder or small colourless crystals; odour, slight and characteristic.
Tests
Disintegration (2.5.1). 30 minutes. Other tests. Comply with the tests stated under Tablets. Assay. For thiacetazone Weigh and finely powder 20 tablets. Weigh accurately a quantity of the powder containing about 30 mg of Thiacetazone, add 70 ml of ethanol (95 per cent) and heat on a water-bath for 15 minutes with intermittent shaking. Cool, add sufficient ethanol (95 per cent) to produce 100.0 ml and filter. To 1.0 ml of the filtrate add sufficient ethanol (95 per cent) to produce 100.0 ml and measure the absorbance of the resulting solution at the maximum at about 328 nm (2.4.7), using ethanol (95 per cent) as the blank. Calculate the content of C10H12N4OS from the absorbance obtained by repeating the operation using thiacetazone RS in place of the tablets under examination. For isoniazid Weigh accurately a quantity of the powdered tablets containing about 0.2 g of Isoniazid, dissolve as completely as possible in 100 ml of water and filter. Wash the residue with water, combine the filtrate and washings and dilute to 250.0 ml with water. To 50.0 ml of the resulting solution add 50 ml of water, 20 ml of hydrochloric acid and 0.2 g of potassium bromide. Titrate with 0.0167 M potassium bromate, determining the end-point potentiometrically (2.4.25). 1 ml of 0.0167 M potassium bromate is equivalent to 0.003429 g of C6H7N3O2. Storage. Store protected from light and moisture.
Identification
Test A may be omitted if tests B and C are carried out. Test B may be omitted if tests A and C are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with thiamine hydrochloride RS or with the reference spectrum of thiamine hydrochloride. B. Dissolve about 20 mg in 10 ml of water, add 1 ml of 2 M acetic acid and 1.6 ml of 1 M sodium hydroxide, heat on a water-bath for 30 minutes and allow to cool. Add 5 ml of 2 M sodium hydroxide, 10 ml of potassium ferricyanide solution and 10 ml of 1-butanol and shake vigorously for 2 minutes. The upper layer exhibits an intense light blue fluorescence, particularly in ultraviolet light at 365 nm. Repeat the test but adding 0.9 ml of 1 M sodium hydroxide and 0.2 g of sodium sulphite in place of the 1.6 ml of 1 M sodium hydroxide; practically no fluorescence is produced. C. Gives reaction A of chlorides (2.3.1).
Tests
Appearance of solution. A 5.0 per cent w/v solution is clear (2.4.1), and not more intensely coloured than reference solution YS7 or GYS6 (2.4.1). pH (2.4.24). 2.7 to 3.3, determined in a 2.5 per cent w/v solution. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method A (20 ppm). Nitrates. To 2 ml of a 2.0 per cent w/v solution add 2 ml of sulphuric acid, cool and superimpose 2 ml of ferrous sulphate solution; no brown ring is produced at the junction of the two layers. Sulphates (2.3.17). 0.5 g complies with the limit test for sulphates (300 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 5.0 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.15 g, dissolve in 5 ml of anhydrous formic acid, add 65 ml of anhydrous glacial acetic acid and 10 ml of mercuric acetate solution, with stirring. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.01686 g of C12H17ClN4OS, HCl.
Thiamine Hydrochloride
Aneurine Hydrochloride; Vitamin B1
NH2 N H3C N N S CH3 OH Cl, HCl
C12H17ClN4OS, HCl
Mol. Wt. 337.3
Thiamine Hydrochloride is 3-[(4-amino-2-methylpyrimidin-5yl)methyl]-5-(2-hydroxyethyl)-4-methylthiazolium chloride hydrochloride hydrate.
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THIAMINE INJECTION
IP 2007
Storage. Store protected from light and moisture, non-metallic containers.
Thiamine Injection
Thiamine Hydrochloride Injection; Aneurine Hydrochloride Injection; Vitamin B1 Injection
Thiamine Injection is a sterile solution of Thiamine Hydrochloride in Water for Injection. Thiamine Injection contains not less than 95.0 per cent and not more than 110.0 per cent of the stated amount of thiamine hydrochloride, C12H17ClN4OS, HCl.
mobile phase: a solution prepared by dissolving 1 g of sodium heptanesulphonate in a mixture of 180 ml of methanol and 10 ml of triethylamine, diluting to 1000 ml with water and adjusting the pH to 3.2 with phosphoric acid, flow rate. 2 ml per minute, spectrophotometer set at 244 nm, a 20 l loop injector. Calculate the content of C12H17ClN4OS. 1 mg of C12H17N5O4S is equivalent to 1.030 mg of C12H17ClN4OS, HCl. Storage. Store protected from light.
Identification
A. To a volume containing 20 mg of Thiamine Hydrochloride in 10 ml of water, add 1 ml of 2 M acetic acid and 1.6 ml of 1 M sodium hydroxide, heat on a water-bath for 30 minutes and allow to cool. Add 5 ml of 2 M sodium hydroxide, 10 ml of potassium ferricyanide solution and 10 ml of 1-butanol and shake vigorously for 2 minutes. The upper layer exhibits an intense light blue fluorescence, particularly in ultraviolet light at 365 nm. Repeat the test but adding 0.9 ml of 1 M sodium hydroxide and 0.2 g of sodium sulphite in place of the 1.6 ml of 1 M sodium hydroxide; practically no fluorescence is produced. B. To a mixture of 0.1 ml of nitrobenzene and 0.2 ml of sulphuric acid add a volume containing 5 mg of Thiamine Hydrochloride. Allow to stand for 10 minutes, cool in ice and add slowly with stirring 5 ml of water followed by 5 ml of 10 M sodium hydroxide. Add 5 ml of acetone and allow to stand; no violet colour is produced in the upper layer.
Thiamine Tablets
Thiamine Hydrochloride Tablets; Aneurine Hydrochloride Tablets; Vitamin B1 Tablets
Thiamine Tablets contain not less than 92.5 per cent and not more than 110.0 per cent of the stated amount of thiamine hydrochloride, C12H17ClN4OS, HCl.
Identification
A. Dissolve a quantity of the powdered tablets containing 20 mg of Thiamine Hydrochloride as completely as possible in 10 ml of water and 2 ml of 1 M acetic acid and filter. Add 5 ml of 2 M sodium hydroxide, 10 ml of potassium ferricyanide solution and 10 ml of 1-butanol and shake vigorously for 2 minutes. The upper layer exhibits an intense light blue fluorescence, particularly in ultraviolet light at 365 nm. Repeat the test but adding 0.9 ml of 1 M sodium hydroxide and 0.2 g of sodium sulphite in place of the 1.6 ml of 1 M sodium hydroxide; practically no fluorescence is produced. B. To a mixture of 0.1 ml of nitrobenzene and 0.2 ml of sulphuric acid add the powdered tablets containing 5 mg of Thiamine Hydrochloride. Allow to stand for 10 minutes, cool in ice and add slowly with stirring 5 ml of water followed by 5 ml of 10 M sodium hydroxide. Add 5 ml of acetone and allow to stand; no violet colour is produced in the upper layer. C. The powdered tablets give the reactions of chlorides (2.3.1).
Tests
pH (2.4.24). 2.5 to 4.5. Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. Determine by liquid chromatography (2.4.14). Test solution. Dilute a volume of the injection containing about 0.1 g of Thiamine Hydrochloride to 100.0 ml with 0.1 M hydrochloric acid and further dilute 5.0 ml to 100.0 ml with water. Reference solution. A 0.005 per cent w/v solution of thiamine mononitrate RS in 0.005 M hydrochloric acid. Chromatographic system a stainless steel column 10 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m),
Tests
Uniformity of content. (For tablets containing 10 mg or less) Comply with the test stated under Tablets. Finely crush one tablet, add 20 ml of ethanol (95 per cent), stir the mixture for 30 minutes and centrifuge. Repeat the extraction with three further quantities, each of 15 ml, of ethanol (95 per cent). Combine the extracts, add sufficient ethanol (95 per cent) to produce 100.0 ml and mix. Dilute a
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IP 2007
THIAMINE MONONITRATE
suitable volume of the resulting solution containing 1 mg of Thiamine Hydrochloride with sufficient ethanol (95 per cent) to produce 100.0 ml. Measure the absorbance of the resulting solution at the maximum at about 233 nm (2.4.7). Calculate the content of C12H17ClN4OS, HCl in the tablet taking 380 as the specific absorbance at 233 nm. Other tests. Comply with the tests stated under Tablets. Assay. Determine by liquid chromatography (2.4.14). Test solution. For tablets containing less than 10 mg of Thiamine Hydrochloride, add 5 ml of 0.1 M hydrochloric acid and 50 ml of water to a quantity of the powdered tablets containing 6 mg of Thiamine Hydrochloride, shake for 20 minutes, dilute to 100.0 ml with water and filter. For tablets containing 10 mg or more of Thiamine Hydrochloride, add 50 ml of 0.1 M hydrochloric acid and 500 ml of water to a quantity of the powdered tablets containing 60 mg of Thiamine Hydrochloride, shake for 20 minutes, dilute to 1000.0 ml with water and filter. Reference solution. A 0.006 per cent w/v solution of thiamine mononitrate RS in 0.005 M hydrochloric acid. Chromatographic system a stainless steel column 10 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a solution prepared by dissolving 1 g of sodium heptanesulphonate in a mixture of 180 ml of methanol and 10 ml of triethylamine, diluting to 1000 ml with water and adjusting the pH to 3.2 with phosphoric acid, flow rate. 2 ml per minute, spectrophotometer set at 244 nm, a 20 l loop injector. Calculate the content of C12H17ClN4OS in the tablets. 1 mg of C12H17N5O4S is equivalent to 1.030 mg of C12H17ClN4OS, HCl. Storage. Store protected from light and moisture in non-metallic containers.
Thiamine Mononitrate is 3-[(4-amino-2-methylpyrimidin-5yl)methyl]-5-(2-hydroxyethyl)-4-methylthiazolium nitrate. Thiamine Mononitrate contains not less than 98.0 per cent and not more than 102.0 per cent of C12H17N5O4S, calculated on the dried basis. Description. A white or almost white, crystalline powder or small, colourless crystals.
Identification
Test A may be omitted if tests B and C are carried out. Test B may be omitted if tests A and C are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with thiamine mononitrate RS or with the reference spectrum of thiamine mononitrate. B. Dissolve about 20 mg in 10 ml of water, add 1 ml of 2 M acetic acid and 1.6 ml of 1 M sodium hydroxide, heat on a water-bath for 30 minutes and allow to cool. Add 5 ml of 2 M sodium hydroxide, 10 ml of potassium ferricyanide solution and 10 ml of 1-butanol and shake vigorously for 2 minutes. The upper layer exhibits an intense light blue fluorescence, particularly in ultraviolet light at 365 nm. Repeat the test but adding 0.9 ml of 1 M sodium hydroxide and 0.2 g of sodium sulphite in place of the 1.6 ml of 1 M sodium hydroxide; practically no fluorescence is produced. C. Gives reaction A of nitrates (2.3.1).
Tests
Appearance of solution. A 2.0 per cent w/v solution in carbon dioxide-free water is clear (2.4.1), and not more intensely coloured than reference solution YS7 (2.4.1). pH (2.4.24). 6.8 to 7.6, determined in a 2.0 per cent w/v solution. Heavy metals (2.3.13). 1.0 g complies with limit test for heavy metals, Method A (20 ppm). Chlorides (2.3.12). 1.0 g complies with the limit test for chlorides (250 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 1.0 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.15 g, dissolve in 5 ml of anhydrous formic acid, add 70 ml of acetic anhydride. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.01637 g of C12H17N5O4S. Storage. Store protected from light and moisture, non-metallic containers.
Thiamine Mononitrate
Thiamine Nitrate
NH2 N H3 C N N S CH3 OH . NO 3
C12H17N5O4S
Mol. Wt. 327.4
1175
THIOMERSAL
IP 2007
Thiomersal
Thimerosal
SHgCH2CH3 COONa
expression 0.7019 (Ab - Aa - Ae)/(Aa + Ad - Ab - Ac), where the figure 0.7019 is a constant obtained from the formula
concentrat ion of HgCl 2 atomic weight of mercury molecular weight of HgCl 2 100
Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying over phosphorus pentoxide at a pressure not exceeding 0.7 kPa for 24 hours. C9H9HgNaO2S Mol. Wt. 404.8 Thiomerosal is the sodium salt of [(2-carboxyphenyl)thio] ethylmercury. Thiomersal contains not less than 97.0 per cent and not more than 101.0 per cent of C9H9HgNaO2S, calculated on the dried basis. Description. A light cream, crystalline powder; odour, slight and characteristic. Assay. Weigh accurately about 0.5 g, transfer to a 100-ml longnecked flask, add 5 ml of sulphuric acid and heat gently until charring occurs; continue to heat and add hydrogen peroxide solution (100 vol) dropwise, until the mixture is colourless. Dilute with water, evaporate until slight fuming occurs, dilute to 10 ml with water, cool and titrate with 0.1 M ammonium thiocyanate using ferric ammonium sulphate solution as indicator. 1 ml of 0.1 M ammonium thiocyanate is equivalent to 0.02024 g of C9H9HgNaO2S. Storage. Store protected from light and moisture.
Identification
A. Dissolve 0.1 g in 10 ml of water and add 2 ml of silver nitrate solution; a pale yellow precipitate is produced. B. Dissolve 0.5 g in 10 ml of water and add 2 ml of 2 M hydrochloric acid; a white precipitate is produced which, after washing with water and drying over phosphorus pentoxide at a pressure not exceeding 0.7 kPa, melts at about 110 (2.4.21).
Thiopentone Sodium
Thiopental Sodium
O H3C H3 C
C11H17N2NaO2S
Tests
pH (2.4.24). 6.0 to 8.0, determined in a 1.0 per cent w/v solution. Ether-soluble matter. Shake 0.5 g with 20 ml of ether for 10 minutes, filter and evaporate to dryness. The residue, after drying over phosphorus pentoxide at a pressure not exceeding 0.7 kPa for 24 hours, weighs not more than 3 mg. Inorganic mercury compounds. Not more than 0.7 per cent, determined by the following method. Protect the solutions from light throughout the procedure. Label five 10-ml volumetric flasks A, B, C, D and E. Place 5 ml of a 0.1 per cent w/v solution of the substance under examination in each of flasks A, B, C and D. To each of flasks C and D add 0.5 ml of a 0.0095 per cent w/v solution of mercuric chloride. Add sufficient water to flasks A and C to produce 10.0 ml and add sufficient of a freshly prepared 33.2 per cent w/v solution of potassium iodide to flasks B and D to produce 10.0 ml. Place 5 ml of the potassium iodide solution in flask E and add sufficient water to produce 10.0 ml. Measure the absorbances of each of the solutions (Aa, Ab, Ac, Ad, Ae) at about 323 nm (2.4.7), using water as the blank. Calculate the content of inorganic mercury compounds, expressed as Hg, in the substance under examination from the
H N N O CH3
SNa + Na2CO3
Mol. Wt. 264.3
Thiopentone Sodium is a mixture of sodium (RS)-5-ethyl-5(1-methylbutyl)-2-thiobarbiturate and anhydrous sodium carbonate. Thiopentone Sodium contains not less than 84.0 per cent and not more than 87.0 per cent of C11H18N2O2S and not less than 10.2 per cent and not more than 11.2 per cent of Na, both calculated on the dried basis. Description. A yellowish white powder; odour, faintly resembling garlic; hygroscopic.
Identification
Test A may be omitted if tests B, C, D and E are carried out. Tests B and D may be omitted if tests A, C and E are carried out. A. Acidify 10 ml of a 10 per cent w/v solution in carbon dioxidefree water with 2 M hydrochloric acid; the solution
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THIOPENTONE INJECTION
effervesces. Shake the solution with 20 ml of ether, separate the ether layer, wash with 10 ml of water and dry over anhydrous sodium sulphate. Filter, evaporate the filtrate to dryness and dry the residue at 105. On the residue, determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with thiopentone RS or with the reference spectrum of thiopentone. B. Complies with the test for identification of barbiturates (2.3.2), but using the following solutions. Test solution. A 0.1 per cent w/v solution of the substance under examination in water. Reference solution. Dissolve 85 mg of thiopentone RS in 10 ml of 2 M sodium hydroxide and dilute to 100 ml with water. C. Acidify 5 ml of a 5 per cent w/v solution with dilute acetic acid and filter. Wash the precipitate with water, recrystallise from water and dry at 70; the crystals melt at about 160 (2.4.21). D. Dissolve 1 mg of the crystals obtained in test A in 1 ml of 0.1 M sodium hydroxide. Add about 1 mg of sodium nitroprusside and, after 15 minutes, 1 ml of dilute hydrochloric acid; a reddish violet colour is produced. E. Gives the reactions of sodium salts (2.3.1).
methoxide. Titrate immediately with 0.1 M lithium methoxide, using 1 drop of a 0.2 per cent w/v solution of thymol blue in methanol as indicator, until a blue colour is obtained. Protect the solution from absorption of carbon dioxide during the titration. 1 ml of 0.1 M lithium methoxide is equivalent to 0.02423 g of C11H18N2O2S. For sodium Weigh accurately about 0.4 g, dissolve in 30 ml of water, add 1 drop of methyl red solution and titrate with 0.05 M sulphuric acid until the yellow colour changes to red. Boil gently for 2 minutes, cool and, if necessary, continue the titration with 0.05 M sulphuric acid until the red colour is restored. 1 ml of 0.05 M sulphuric acid is equivalent to 0.002299 g of Na. Storage. Store protected from light and moisture.
Thiopentone Injection
Thiopentone Sodium Injection; Thiopental Injection
Thiopentone Injection is a sterile material consisting of Thiopentone Sodium with or without auxiliary agents. It is filled in a sealed container. The injection is constituted by dissolving the contents of the sealed container in the requisite amount of sterile Water for Injections, immediately before use. The constituted solution complies with the requirements for Clarity of solution and Particulate matter stated under Parenteral Preparations (Injections). Storage. The constituted solution should be used immediately after preparation but, in any case, within the period recommended by the manufacturer. Thiopentone Injection contains thiopentone, C11H18N2O2S that is not less than 77.0 per cent and not more than 92.0 per cent and sodium, Na that is not less than 9.4 per cent and not more than 11.8 per cent of the stated amount of thiopentone sodium. The contents of the sealed container comply with the requirements stated under Parenteral Preparations (Powders for Injection) and with the following requirements. Description. A yellowish white powder; hygroscopic.
Tests
Appearance of solution. A 10.0 per cent w/v solution in carbon dioxide-free water (solution A) is clear (2.4.1), and not more intensely coloured than reference solution GYS3 (2.4.1). Related substances (2.3.4). Complies with the test, but using water as the solvent for the test solution and the reference solution. After development, examine the plate in ultraviolet light at 254 nm but do not spray it with the diphenylcarbazonemercury reagent. Chlorides (2.3.12). To 10 ml of solution A add 30 ml of water and 10 ml of 2 M nitric acid. Shake successively with three quantities, each of 25 ml, of ether, discard the ether layers and heat the aqueous solution on water-bath to remove any residual ether. 30 ml of the aqueous layer complies with the limit test for chlorides (330 ppm). Loss on drying (2.4.19). Not more than 2.5 per cent, determined on 0.5 g by drying in an oven at 100 at a pressure of 1.5 to 2.5 kPa for 4 hours. Assay. For thiopentone Weigh accurately about 0.15 g, dissolve in 5 ml of water, add 2 ml of 1 M sulphuric acid and extract with four quantities, each of 10 ml, of chloroform. Filter the combined chloroform extracts, evaporate the filtrate to dryness on a water-bath and dissolve the residue in 30 ml of dimethylformamide, previously neutralised with 0.1 M lithium
Identification
Test A may be omitted if tests B, C, D and E are carried out. Tests B and D may be omitted if tests A, C and E are carried out. A. Acidify 10 ml of a 10 per cent w/v solution in carbon dioxidefree water with 2 M hydrochloric acid; the solution
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THIOTEPA
IP 2007
effervesces. Shake the solution with 20 ml of ether, separate the ether layer, wash with 10 ml of water and dry over anhydrous sodium sulphate. Filter, evaporate the filtrate to dryness and dry the residue at 105. On the residue, determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with thiopentone RS or with the reference spectrum of thiopentone. B. Complies with the test for identification of barbiturates (2.3.2), but using the following solutions. Test solution. 0.1 per cent w/v solution of the substance under examination in water. Reference solution. Dissolve 85 mg of thiopentone RS in 10 ml of 2 M sodium hydroxide and dilute to 100 ml with water. C. Acidify 5 ml of a 5 per cent w/v solution with dilute acetic acid and filter. Wash the precipitate with water, recrystallise from water and dry at 70; the crystals melt at about 160 (2.4.21). D. Dissolve 1 mg of the crystals obtained in test A in 1 ml of 0.1 M sodium hydroxide. Add about 1 mg of sodium nitroprusside and, after 15 minutes, 1 ml of dilute hydrochloric acid; a reddish violet colour is produced. E. Gives the reactions of sodium salts (2.3.1).
1 ml of 0.1 M lithium methoxide is equivalent to 0.02423 g of C11H18N2O2S. For sodium Mixed the contents of 10 containers and weigh accurately about 0.4 g of the contents, dissolve in 30 ml of water, add 1 drop of methyl red solution and titrate with 0.05 M sulphuric acid until the yellow colour changes to red. Boil gently for 2 minutes, cool and, if necessary, continue the titration with 0.05 M sulphuric acid until the red colour is restored 1 ml of 0.05 M sulphuric acid is equivalent to 0.002299 g of Na. Storage. Store in single dose containers. Labelling. The label states the amount of active ingredient in terms of Thiopentone Sodium.
Thiotepa
S N P N N
C6H12N3PS Mol. Wt. 189.2
Thiotepa is tris(1-aziridinyl)phosphine sulphide. Thiotepa contains not less than 97.0 per cent and not more than 102.0 per cent of C6H12N3PS, calculated on the anhydrous basis. Description. Fine, white, crystalline flakes; odourless or almost odourless.
Tests
Appearance of solution. A 10.0 per cent w/v solution in carbon dioxide-free water (solution A) is clear (2.4.1), and not more intensely coloured than reference solution GYS3 (2.4.1). Related substances (2.3.4). Complies with the test, but using water as the solvent for the test solution and the reference solution. After development, examine the plate in ultraviolet light at 254 nm but do not spray it with the diphenylcarbazonemercury reagent. Loss on drying (2.4.19). Not more than 2.5 per cent, determined on 0.5 g by drying in an oven at 100 at a pressure of 1.5 to 2.5 kPa for 4 hours. Assay. For thiopentone Mixed the contents of 10 containers and weigh accurately about 0.15 g, of the contents, dissolve in 5 ml of water, add 2 ml of 1 M sulphuric acid and extract with four quantities, each of 10 ml, of chloroform. Filter the combined chloroform extracts, evaporate the filtrate to dryness on a water-bath and dissolve the residue in 30 ml of dimethylformamide, previously neutralised with 0.1 M lithium methoxide. Titrate immediately with 0.1 M lithium methoxide, using 1 drop of a 0.2 per cent w/v solution of thymol blue in methanol as indicator, until a blue colour is obtained. Protect the solution from absorption of carbon dioxide during the titration.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with thiotepa RS or with the reference spectrum of thiotepa. B. Determine on 20 mg by the oxygen-flask method (2.3.34), using 5 ml of 1.25 M sodium hydroxide as the absorbing liquid. When the process is complete, dilute to 25 ml with water (solution A). To 5 ml of solution A add 0.1 ml of hydrogen peroxide solution (100 vol) and 1 ml of 1 M hydrochloric acid, mix and add 0.05 ml of barium chloride solution; a turbidity is produced. C. To 2 ml of solution A add 40 ml of water and 4 ml of ammonium molybdate-sulphuric acid solution, mix, add 0.1 g of L-ascorbic acid and boil for 1 minute; a blue colour is produced.
Tests
Appearance of solution. A 2.0 per cent w/v solution is clear (2.4.1).
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IP 2007
THYMOL
Water (2.3.43). Not more than 2.0 per cent, determined on 1.2 g. Assay. Weigh accurately about 0.2 g into a stoppered flask, add 50 ml of a 20 per cent w/v solution of sodium thiosulphate and titrate immediately with 0.1 M hydrochloric acid, using methyl orange solution as indicator, until a faint red colour persists for 10 seconds. Stopper the flask, allow to stand for 30 minutes and titrate with 0.1 M sodium hydroxide using phenolphthalein solution as indicator. Subtract the volume of 0.1 M sodium hydroxide used from the volume of 0.1 M hydrochloric acid used. Repeat the operation without the substance under examination. The difference between the titrations represents the amount of hydrochloric acid required. 1 ml of 0.1 M hydrochloric acid is equivalent to 0.006307 g of C6H12N3PS. Storage. Store protected from light and moisture. At higher temperatures it tends to polymerise with loss of activity.
B. To 2 ml of solution A add 40 ml of water and 4 ml of ammonium molybdate-sulphuric acid solution, mix, add 0.1 g of L-ascorbic acid and boil for 1 minute; a blue colour is produced.
Tests
Appearance of solution. Dissolve a quantity containing 15 mg of Thiotepa in 4 ml of water; the solution is clear (2.4.1). pH (2.4.24). 5.5 to 7.5, determined in a 1.0 per cent w/v solution in carbon dioxide-free water. Bacterial endotoxins (2.2.3). Not more than 6.25 Endotoxin Units per mg of Thiotepa. Assay. Determine by liquid chromatography (2.4.14). Test solution. Dilute a suitable quantity of the injection containing 0.15 g of Thiotepa in 100 ml of water. Reference solution. A 0.15 per cent w/v solution of thiotepa RS in water. Chromatographic system a stainless steel column 20 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 70 volumes of water and 30 volumes of acetonitrile, flow rate. 1 ml per minute, spectrophotometer set at 215 nm, a 20 l loop injector. Calculate the content of C6H12N3PS in the injection. Storage. Store protected from light. If solid matter separates from the constituted injection, the solution should not be used.
Thiotepa Injection
Thiotepa Injection is a sterile material consisting of Thiotepa with or without auxiliary agents. It is filled in a sealed container. The injection is constituted by dissolving the contents of the sealed container in the requisite amount of sterile Water for Injections, immediately before use. The constituted solution complies with the requirements for Clarity of solution and Particulate matter stated under Parenteral Preparations (Injections). Storage. The constituted solution should be used immediately after preparation but, in any case, within the period recommended by the manufacturer. Thiotepa Injection contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of thiotepa, C6H12N3PS. The contents of the sealed container comply with the requirements stated under Parenteral Preparations (Powders for Injection) and with the following requirements. Description. A white powder.
Thymol
OH CH3 CH3 H3 C
C10H14O Thymol is 2-isopropyl-5-methylphenol. Thymol contains not less than 99.0 per cent and not more than 101.0 per cent of C10H14O. Description. Colourless crystals; odour, characteristic. Mol. Wt. 150.2
Identification
A. Determine on 20 mg by the oxygen-flask method (2.3.34), using 5 ml of 1.25 M sodium hydroxide as the absorbing liquid. When the process is complete, dilute to 25 ml with water (solution A). To 5 ml of solution A add 0.1 ml of hydrogen peroxide solution (100 vol) and 1 ml of 1 M hydrochloric acid, mix and add 0.05 ml of barium chloride solution; a turbidity is produced.
Identification
Test A may be omitted if tests B, C and D are carried out. Tests B, C and D may be omitted if test A is carried out.
1179
THYROXINE SODIUM
IP 2007
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with thymol RS or with the reference spectrum of thymol. B. Dissolve 0.2 g with heating in 2 ml of 2 M sodium hydroxide, add 0.2 ml of chloroform and heat on a water-bath; a violet colour develops. C. Dissolve about 2 mg in 1 ml of anhydrous acetic acid, add 0.15 ml of sulphuric acid and 0.05 ml of nitric acid; a bluish green colour develops. D. Melting range (2.4.21). 48 to 52.
area of the principal peak in the chromatogram obtained with reference solution (a). Ignore any peak with an area less than that of the principal peak in the chromatogram obtained with reference solution (c). Residue on evaporation. Evaporate 2.0 g on a water-bath and heat at 105 for 1 hour. The residue weighs not more than 1.0 mg (0.05 per cent). Assay. Weigh accurately about 0.1 g, transfer to an iodine flask and dissolve in 25 ml of 1 M sodium hydroxide. Add 20 ml of hot dilute hydrochloric acid and immediately titrate with 0.05 M bromine to within 1 to 2 ml of the calculated endpoint. Warm the solution to about 75, add 0.1 ml of methyl orange solution and shake vigorously. If the solution is red, continue the titration, dropwise and with shaking until the red colour is discharged. Repeat the alternate addition of 0.05 M bromine and methyl orange solution until the red colour is discharged after the addition of the methyl orange solution. 1 ml of 0.05 M bromine is equivalent to 0.003755 g of C10H14O. Storage. Store protected from light and moisture.
Tests
Appearance of solution. Dissolve 1.0 g in 10 ml of 2 M sodium hydroxide. The solution is not more opalescent than opalescence standard OS4 (2.4.1), and not more intensely coloured than reference solution RS6 (2.4.1). Acidity. To 1.0 g in a glass-stoppered flask add 20 ml of water, boil until dissolution is complete, cool, stopper the flask and shake vigorously for 1 minute. Add a few crystals of the substance under examination to induce crystallisation, shake vigorously for 1 minute and filter. To 5 ml of the filtrate add 0.05 ml of methyl red solution and 0.05 ml of 0.01 M sodium hydroxide; the solution is yellow. Related substances. Determine by gas chromatography (2.4.13). Test solution. Dissolve 0.1 g in sufficient ethanol (95 per cent) to produce 10 ml. Reference solution (a). Dilute 1 ml of the test solution to 100 ml with ethanol (95 per cent). Reference solution (b). Dilute 1 ml of reference solution (a) to 10 ml with ethanol (95 per cent). Reference solution (c). Dilute 5 ml of reference solution (b) to 10 ml with ethanol (95 per cent). Chromatographic system a glass column 4 m x 2 mm, packed with diatomaceous support (125 to 180 mesh) impregnated with a mixture suitable for the separation of free fatty acids (such as FFAP), temperature: column 80, inlet port at 250 and detector at 300, flow rate. 30 ml per minute of the carrier gas. Inject separately 1 l of each solution and, after 2 minutes, increase the temperature of the column to 240 at a rate of 8 per minute and maintain at this temperature for 15 minutes. In the chromatogram obtained with the test solution the sum of the areas of any secondary peaks is not greater than the
Thyroxine Sodium
Levothyroxine Sodium; L-Thyroxine Sodium
I H O I I OH
C15H10I4NNaO4,xH2O Mol. Wt. 798.9 (anhydrous)
COONa NH2 , xH2O
Thyroxine Sodium is sodium O4-(4-hydroxy-3,5diiodophenyl)-3,5-diiodo-L-tyrosinate and contains a variable quantity of water of crystallisation. Thyroxine Sodium contains not less than 97.0 per cent and not more than 101.0 per cent of C15H10I4NNaO4, calculated on the dried basis. Description. A white or slightly brownish yellow powder, slightly coloured, crystalline powder.
Identification
A. When examined in the range 230 nm to 360 nm (2.4.7), a 0.01 per cent w/v solution in 0.1 M sodium hydroxide shows
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IP 2007
THYROXINE TABLETS
an absorption maximum at about 325 nm; absorbance at about 325 nm, 0.73 to 0.79. B. In the test for Liothyronine, the principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with reference solution (b). C. To about 50 mg in a porcelain dish add a few drops of sulphuric acid (96 per cent w/w); violet vapors are evolved. D. To 20 mg add 2 ml of 1 M sulphuric acid. Heat on a waterbath followed by heating carefully over a naked flame, increasing the temperature to about 600. Continue ignition until most of the black particles have disappeared. Dissolve the residue in 2 ml of water; the solution gives reaction A of sodium salts (2.3.1).
the chromatogram obtained with reference solution (a) shows two clearly separated spots. Loss on drying (2.4.19). 6.0 to 12.0 per cent, determined on 0.5 g by drying in an oven at 105. Assay. Determine by liquid chromatography (2.4.14). Test solution. A 0.001 per cent w/v solution of the substance under examination in 0.1 M methanolic sodium hydroxide. Reference solution. A 0.001 per cent w/v solution of levothyroxine sodium RS in 0.1 M methanolic sodium hydroxide. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with silicagel consisting of porous spherical particles with chemically bonded nitrile groups (5 m), mobile phase: a mixture of 65 volumes of water, 0.2 volume of orthophosphoric acid and 35 volumes of acetonitrile, flow rate. 1 ml per minute, spectrophotometer set at 225 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject alternately the test solution and the reference solution. Calculate the content of C15H10I4NNaO4. Storage. Store protected from light and moisture.
Tests
Appearance of solution. Dissolve 0.5 g in 23 ml of a gently boiling mixture of 4 volumes of ethanol (95 per cent) and 1 volume of 1 M hydrochloric acid. Cool and dilute to 25 ml with the same mixture of solvents (solution A). The freshly prepared solution is not more intensely coloured than reference solution BYS3 (2.4.1). Specific optical rotation (2.4.22).+16.0 to +20.0, determined in solution A. Liothyronine. Determine by thin-layer chromatography (2.4.17), coating the plate with a slurry of 30 g of silica gel H in 60 ml of a 0.75 per cent w/v solution of soluble starch. Solvent mixture. 70 volumes of methanol and 5 volumes of strong ammonia solution. Mobile phase. A mixture of 55 volumes of ethyl acetate, 35 volumes of 2-propanol and 20 volumes of strong ammonia solution. Test solution. Dissolve 0.1 g of the substance under examination in 10 ml of the solvent mixture. Reference solution (a). A solution containing 1.0 per cent w/v of the substance under examination and 0.01 per cent w/v of the liothyronine RS in the solvent mixture. Reference solution (b). A 1 per cent w/v solution of levothyroxine sodium RS in the solvent mixture. Reference solution (c). A 0.01 per cent w/v solution of liothyronine RS in the solvent mixture. Apply to the plate 5 l of each solution. After development, dry the plate in air, spray lightly with ferric chlorideferricyanide-arsenite solution. Any spot corresponding to liothyronine in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (c). The test is not valid unless
Thyroxine Tablets
Thyroxine Sodium Tablets; Levothyroxine Tablets; Levothyroxine Sodium Tablets; L-Thyroxine Sodium Tablets
Thyroxine Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of anhydrous thyroxine sodium, C15H10I4NNaO4.
Identification
A. To a quantity of the powdered tablets containing 500 g of anhydrous thyroxine sodium add a mixture of 3 ml of ethanol (50 per cent) and 0.2 ml of hydrochloric acid, boil gently for 30 seconds, cool, filter, add 0.1 ml of a 10 per cent w/v solution of sodium nitrite and boil; a yellow colour is produced. Cool and make alkaline with 5 M ammonia; the solution becomes orange. B. In the Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the peak due
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TIMOLOL MALEATE
IP 2007
to thyroxine sodium in the chromatogram obtained with the reference solution.
Timolol Maleate
N N O
C13H24N4O3S,C4H4O4
Tests
Uniformity of content. Comply with the test stated under Tablets. using the following solutions. Test solution. Place one tablet in a 25-ml volumetric flask, add 10 ml of 0.1 M methanolic sodium hydroxide and shake for about 30 minutes. Dilute to volume with 0.1 M methanolic sodium hydroxide. Dilute further, if necessary, with 0.1 M methanolic sodium hydroxide to produce a solution containing 0.0002 per cent w/v solution of Thyroxine Sodium. Reference solution. A 0.0002 per cent w/v solution of levothyroxine sodium RS in 0.1 M methanolic sodium hydroxide. Follow the procedure described under Assay. Calculate the content of C15H10I4NNaO4 in the tablet. Other tests. Comply with the tests stated under Tablets. Assay. Determine by liquid chromatography (2.4.14). Test solution. Weigh accurately a quantity of the powdered tablets containing about 1000 g of Thyroxine Sodium, transfer to a 100-ml volumetric flask and add 40 ml of 0.1 M methanolic sodium hydroxide. Shake for about 30 minutes, mix well and dilute to volume with the same solvent, mix well and filter. Reference solution. A 0.001 per cent w/v solution of levothyroxine sodium RS in 0.1 M methanolic sodium hydroxide. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with a stationary phase consisting of porous silica particles (5 to 10 m) to which nitrile groups are chemically bonded, mobile phase: a mixture of 65 volumes of water, 0.2 volume of orthophosphoric acid and 35 volumes of acetonitrile, flow rate. 1 ml per minute, spectrophotometer set at 225 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject alternately the test solution and the reference solution. Calculate the content of C15H10I4NNaO4 in the tablets. Storage. Store protected from light and moisture. Labelling. The label states the strength in terms of the equivalent amount of anhydrous thyroxine sodium.
N O OH
H3 C N H
CH3 CH3
COOH , COOH
Mol. Wt. 432.5
Timolol Maleate is (S)-1-tert-butylamino-3-(4-morpholino1,2,5-thiadiazol-3-yloxy)propan-2-ol hydrogen maleate. Timolol Maleate contains not less than 98.5 per cent and not more than 101.0 per cent of C13H24N4O3S,C4H4O4, calculated on the dried basis. Description. A white or almost white, crystalline powder or colourless crystals.
Identification
Test A may be omitted if tests B and C are carried out. Tests B and C may be omitted if test A is carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with timolol maleate RS or with the reference spectrum of timolol maleate. B. In the test for Related substances, after exposure to iodine vapour, the principal spot in the chromatogram obtained with test solution (b) corresponds to that in the chromatogram obtained with reference solution (b). C. Triturate 0.1 g with a mixture of 1 ml of 2 M sodium hydroxide and 3 ml of water and shake with three quantities, each of 5 ml, of ether. To 0.1 ml of the aqueous layer add a solution of 10 mg of resorcinol in 3 ml of sulphuric acid and heat on a waterbath for 15 minutes; no violet-red colour is produced. Neutralise the remainder of the aqueous layer with 1 M sulphuric acid, add 1 ml of bromine water, heat on a water-bath for 15 minutes, then heat to boiling and cool. To 0.2 ml of this solution add a solution of 10 mg of resorcinol in 3 ml of sulphuric acid and heat on a water-bath for 15 minutes; a violet-red colour is produced. Add 0.2 ml of a 10 per cent w/v solution of potassium bromide and heat on a water-bath for 5 minutes; the colour changes to violet-blue.
Tests
Appearance of solution. A 2.0 per cent w/v solution in carbon dioxide-free water is clear (2.4.1), and not more intensely coloured than reference solution BS8 (2.4.1). pH (2.4.24). 3.8 to 4.3, determined in a 2.0 per cent w/v solution. Specific optical rotation (2.4.22). 5.7 to 6.2, determined in a 10.0 per cent w/v solution in M hydrochloric acid.
1182
IP 2007
TIMOLOL EYE DROPS
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 80 volumes of dichloromethane, 20 volumes of methanol and 1 volume of strong ammonia solution. Test solution (a). Dissolve 0.5 g of the substance under examination in 10 ml of methanol. Test solution (b). Dissolve 0.1 g of the substance under examination in 100 ml of methanol. Reference solution (a). A 0.02 per cent w/v solution of the substance under examination in methanol. Reference solution (b). A 0.1 per cent w/v solution of timolol maleate RS in methanol. Apply to the plate 10 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm and then expose to iodine vapour for 2 hours and examine in daylight. By both methods of visualisation, any secondary spot in the chromatogram obtained with test solution (a) is not more intense than the spot in the chromatogram obtained with reference solution (a). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.35 g, dissolve in 60 ml of anhydrous glacial acetic acid. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.04325 g of C13H24N4O3S, C4H4O4. Storage. Store protected from light and moisture.
quantities, each of 40 ml, of dichloromethane. Reserve the aqueous layer for test B. Dry the combined dichloromethane extracts with anhydrous sodium sulphate and evaporate to dryness. Dry the residue at 60 at a pressure of 2 kPa for 15 minutes. On the residue, determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with timolol RS or with the reference spectrum of timolol. B. Filter the aqueous layer reserved in test A and evaporate to about 1 ml. Add 1 ml of bromine solution, heat in a water-bath for 10 minutes, boil, cool and add 0.1 ml of the solution to a solution of 10 mg resorcinol in 3 ml of sulphuric acid; a bluish black colour is produced on heating in a water-bath for 15 minutes.
Tests
pH (2.4.24). 6.5 to 7.5. Related substances. Determine by liquid chromatography (2.4.14). Test solution. Use the undiluted eye drops. Reference solution (a). Dilute 1 volume of the eye drops to 250 volumes with the mobile phase. Reference solution (b). Dilute 1 volume of the eye drops to 500 volumes with the mobile phase. Reference solution (c). A 0.3 per cent w/v solution of maleic acid in the mobile phase. Chromatographic system a stainless steel column 20 cm x 4 mm, packed with octadecylsilane bonded to porous silica (10 m), mobile phase: a mixture of 57.5 volumes of methanol and 42.5 volumes of 0.02 M sodium octanesulphonate, adjusted to pH 3.0 with glacial acetic acid, flow rate. 2 ml per minute, spectrophotometer set at 295 nm, a 20 l loop injector. Record the chromatogram of the test solution for 4 times the retention time of the principal peak. In the chromatogram obtained with the test solution the area of any secondary peak, other than the peak corresponding to maleic acid, is not greater than the area of the peak in the chromatogram obtained with reference solution (a) and not more than two such peaks have an area greater than that of the peak obtained with reference solution (b). Other tests. Comply with the tests stated under Eye Drops. Assay. To a volume containing about 25 mg of timolol add water to produce 50.0 ml and mix. To 5.0 ml add 15 ml of
Timolol Eye Drops

Timolol Maleate Eye Drops
Timolol Eye Drops are a sterile solution of Timolol Maleate in Purified Water. They may contain suitable antimicrobial preservatives, buffering agents, stabilisers and suitable substances to increase the viscosity of the solution. Timolol Eye Drops contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of timolol, C13H24N4O3S.
Identification
A. Add a volume containing 50 mg of timolol to an equal volume of carbonate buffer pH 9.7 and extract with two
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TIMOLOL TABLETS
IP 2007
carbonate buffer pH 9.7 and extract with three quantities, each of 20 ml, and one quantity of 10 ml of toluene. Wash each extract successively with the same 10-ml volume of carbonate buffer pH 9.7. Combine the toluene extracts and extract with four quantities, each of 20 ml, of 0.05 M sulphuric acid. Combine the extracts, dilute to 100.0 ml, filter and measure the absorbance of the filtrate at the maximum at about 295 nm (2.4.7), using as the blank a solution prepared by treating 5.0 ml of water in the same manner beginning at the words add 15 ml of carbonate buffer pH 9.7.... Calculate the content of C13H24N4O3S taking 279 as the specific absorbance at 295 nm. Storage. Store protected from light and moisture. Labelling. The label states (1) the strength in terms of the equivalent amount of timolol; (2) the names and concentration of any added antimicrobial preservatives.
Test solution. Shake a quantity of the powdered tablets containing 0.1 g of Timolol Maleate with 10 ml of the mobile phase for 10 minutes and filter. Reference solution (a). Dilute 1 volume of the test solution to 250 volumes with the mobile phase. Reference solution (b). Dilute 1 volume of the test solution to 500 volumes with the mobile phase. Chromatographic system a stainless steel column 20 cm x 4 mm, packed with octadecylsilane bonded to porous silica (10 m), mobile phase: a mixture of 57.5 volumes of methanol and 42.5 volumes of 0.02 M sodium octanesulphonate, adjusted to pH 3.0 with glacial acetic acid, flow rate. 2 ml per minute, spectrophotometer set at 295 nm, a 20 l loop injector. Record the chromatogram of the test solution for 4 times the retention time of the principal peak. In the chromatogram obtained with the test solution the area of any secondary peak, other than the peak corresponding to maleic acid, is not greater than the area of the peak in the chromatogram obtained with reference solution (a) and not more than two such peaks have an area greater than that of the peak obtained with reference solution (b). Uniformity of content. Comply with the test stated under Tablets. To one tablet add 25.0 ml of 0.05 M sulphuric acid, shake for 20 minutes and complete the Assay beginning at the words and centrifuge..... Other tests. Comply with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 15 mg of Timolol Maleate, add 25.0 ml of 0.05 M sulphuric acid, shake for 20 minutes and centrifuge until clear. Add 5.0 ml of the resulting supernatant liquid to 15 ml of carbonate buffer pH 9.7 and extract with three quantities, each of 20 ml, and one quantity of 10 ml of toluene. Wash each extract successively with the same 10-ml volume of carbonate buffer pH 9.7, combine the toluene extracts and extract with four quantities, each of 20 ml, of 0.05 M sulphuric acid. Combine the acid extracts, dilute to 100.0 ml, filter and measure the absorbance of the filtrate at the maximum at about 295 nm (2.4.7), using as the blank a solution prepared by treating a mixture of 5 ml of water and 15 ml of carbonate buffer pH 9.7 in the same manner beginning at the words and extract with three quantities,... Calculate the content of C13H24N4O3S,C4H4O4 taking 204 as the specific absorbance at 295 nm. Storage. Store protected from moisture.
Timolol Tablets
Timolol Maleate Tablets
Timolol Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of timolol maleate, C13H24N4O3S,C4H4O4.
Identification
A.To a quantity of the powdered tablets containing 70 mg of Timolol Maleate add 20 ml of carbonate buffer pH 9.7 and extract with two quantities, each of 40 ml, of dichloromethane. Reserve the aqueous layer for test B. Dry the extracts with anhydrous sodium sulphate, evaporate to dryness using a rotary evaporator, dry the residue at 60 at a pressure of 2 kPa for 15 minutes. On the residue, determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with timolol RS or with the reference spectrum of timolol. B. Filter the aqueous layer reserved in test A and evaporate to about 1 ml. Add 1 ml of bromine water, heat on a water-bath for 15 minutes, then heat to boiling and cool. Add 0.1 ml of this solution to a solution of 10 mg of resorcinol in 3 ml of sulphuric acid and heat on a water-bath for 15 minutes; a bluish black colour is produced.
Tests
1184
IP 2007
TINIDAZOLE TABLETS
Tinidazole
NO2 N N CH3
C8H13N3O4S Mol. Wt. 247.3
test solution is not more intense than the spot in the chromatogram obtained with the reference solution.
O O S
Sulphated ash (2.3.18). Not more than 0.2 per cent.
CH3
Loss on drying (2.4.19). Not more than 2.0 per cent, determined on 1.0 g by drying in an oven at 105 for 4 hours. Assay. Weigh accurately about 0.5 g, dissolve in 30 ml of anhydrous glacial acetic acid. Titrate with 0.1 M perchloric acid, using 0.15 ml of nile blue A solution as indicator. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.02473 g of C8H13N3O4S. Storage. Store protected from light and moisture.
Tinidazole is 1-[2-(ethylsulphonyl)ethyl]-2-methyl-5nitroimidazole. Tinidazole contains not less than 98.0 per cent and not more than 100.5 per cent of C8H13N3O4S, calculated on the dried basis. Description. Pale yellow crystals or a crystalline powder; odour, slight and characteristic.
Tinidazole Tablets
Tinidazole Tablets contain not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of tinidazole, C8H13N3O4S. The tablets may be coated.
Identification
Test A may be omitted if tests B and C are carried out. Tests B and C may be omitted if test A is carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with tinidazole RS or with the reference spectrum of tinidazole. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.001 per cent w/v solution in methanol shows an absorption maximum at about 310 nm; absorbance at about 310 nm, about 0.35. C. To about 5 mg add 5 ml of 0.1 M hydrochloric acid, 50 mg of zinc powder, 4 ml of hydrochloric acid and allow to stand for 30 minutes. Add 4 ml of a 1 per cent w/v solution of vanillin, heat on a boiling water-bath for 20 minutes, allow to cool to room temperature and dilute to 20 ml with water; a greenish yellow colour is produced.
Identification
A. When examined in the range 230 nm to 360 nm (2.4.7), the final solution obtained in the Assay shows an absorption maximum at about 310 nm. Extract a quantity of the powdered tablets containing 0.1 g of Tinidazole with 10 ml of methanol, filter and evaporate the filtrate to dryness. The residue complies with the following tests. B. To about 5 mg add 5 ml of 0.1 M hydrochloric acid, 50 mg of zinc powder, 4 ml of hydrochloric acid and allow to stand for 30 minutes. Add 4 ml of a 1 per cent w/v solution of vanillin, heat on a boiling water-bath for 20 minutes, allow to cool to room temperature and dilute to 20 ml with water; a greenish yellow colour is produced. C. Melting range (2.4.21). 125 to 128.
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 95 volumes of ethyl acetate, 5 volumes of methanol and 5 volumes of diethylamine. Test solution. Dissolve 0.2 g of the substance under examination in 10 ml of a mixture of equal volumes of chloroform and methanol. Reference solution. A 0.02 per cent w/v solution of the substance under examination in a mixture of equal volumes of chloroform and methanol. Apply to the plate 10 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the
Tests
Other tests. Comply with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 0.15 g of Tinidazole, add 20 ml of methanol, shake well and add sufficient methanol to produce 100.0 ml. Mix well and filter. Dilute 10.0 ml of the solution to 100.0 ml with methanol and further dilute 10.0 ml of this solution to 100.0 ml with methanol. Measure the absorbance of the resulting solution at the maximum at about 310 nm (2.4.7). Calculate the content of C8H13N3O4S taking 356 as the specific absorbance at 310 nm.
1185
TITANIUM DIOXIDE
IP 2007
Titanium Dioxide
TiO2 Mol. Wt. 79.9 Titanium Dioxide contains not less than 98.0 per cent and not more than 100.5 per cent of TiO2. Description. A white or almost white, infusible powder; odourless.
Identification
A. When strongly heated it becomes pale yellow; the colour is discharged on cooling. B. To 0.5 g add 5 g of anhydrous sodium sulphate and 10 ml of water and mix. Add 10 ml of sulphuric acid and boil gently until clear; cool, add slowly 30 ml of a 25 per cent v/v solution of sulphuric acid and dilute with water to 100 ml (solution A). To 5 ml of solution A add 0.1 ml of strong hydrogen peroxide solution; an orange-red colour is produced. C. To 5 ml of solution A add 0.5 g of zinc, in granules; after 45 minutes a violet-blue colour is produced.
Tests
Appearance of solution. Solution A is not more opalescent than opalescence standard OS2 (2.4.1), and colourless (2.4.1). Acidity or alkalinity. Shake 5.0 g with 50 ml of carbon dioxidefree water for 5 minutes and centrifuge until a clear solution is obtained. To 10 ml of the solution add 0.1 ml of bromothymol blue solution. Not more than 1.0 ml of either 0.01 M hydrochloric acid or 0.01 M sodium hydroxide is required to change the colour of the solution. Water-soluble substances. Not more than 0.5 per cent, determined by the following method. Boil 10.0 g for 5 minutes with 150 ml of water containing 0.5 g of ammonium sulphate. Cool, dilute to 200 ml with water and filter until a clear solution is obtained. Evaporate 100 ml of the filtrate to dryness ignite and weigh. Arsenic (2.3.10). To 0.2 g in a 100-ml Kjeldahl flask add 2 g of anhydrous sodium sulphate, 7 ml of sulphuric acid and 5 ml of nitric acid. Heat gently until a clear solution is obtained, cool, add 10 ml of water, cool again and add 5 g of hydrazine reducing mixture and 10 ml of hydrochloric acid. Immediately attach an air condenser and distil into 15 ml of cooled water until a total volume of 30 ml is obtained. Rinse the condenser with 5 ml of water and dilute the combined distillate and rinsings to 40 ml with water. 20 ml of the resulting solution complies with the limit test for arsenic. Use a mixture of 0.5 ml of arsenic standard solution (1 ppm As) and 24.5 ml of water to prepare the standard (5 ppm).
Barium. Shake 20.0 g with 30 ml of hydrochloric acid, add 100 ml of distilled water and boil. Filter while hot through a hardened filter paper until a clear filtrate is obtained. Wash the filter with 60 ml of distilled water and dilute the combined filtrate and washings to 200 ml with distilled water. To 10 ml of this solution add 1 ml of 1 M sulphuric acid. After 30 minutes any opalescence is not more intense than that of a mixture of 10 ml of the test solution and 1 ml of distilled water. Heavy metals (2.3.13). Dilute 10 ml of the solution prepared in the test for Barium to 20 ml with water. 12 ml of the solution complies with the limit test for heavy metals, Method D (20 ppm). Iron. To 8 ml of solution A add 4 ml of water, mix and add 0.05 ml of bromine water, allow to stand for 5 minutes, remove the excess of bromine with a current of air and add 3 ml of potassium thiocyanate solution. Any colour in the solution is not more intense than that in a standard prepared at the same time and in the same manner using a mixture of 4 ml of iron standard solution (2 ppm Fe) and 8 ml of a 20 per cent w/v solution of sulphuric acid (200 ppm). Assay. Weigh accurately about 0.5 g, transfer to a 300-ml Kjeldahl flask, add 5 g of anhydrous sodium sulphate and 10 ml of water, mix and add 10 ml of sulphuric acid. Boil gently until clear, cool, add slowly 40 ml of cooled sulphuric acid (25 per cent), cool again and dilute with water to 100.0 ml (solution B). To 300 g of zinc, in granules, add 300 ml of a 2 per cent w/v solution of mercuric nitrate and 2 ml of nitric acid, shake for 10 minutes and wash with water. Pack the zinc amalgam into a glass tube (400 mm x 20 mm) fitted with a tap and a filter plate. Pass through the column, at a rate of about 3 ml per minute, 100 ml of 1 M sulphuric acid followed by 100 ml of water, ensuring that the amalgam is covered with liquid throughout. Pass slowly through the column, at a rate of about 3 ml per minute, 200 ml of 0.5 M sulphuric acid followed by 100 ml of water. Collect the combined eluates in a 500-ml conical flask containing 50 ml of a 15 per cent w/v solution of ferric ammonium sulphate in sulphuric acid (25 per cent) and titrate immediately with 0.1 M ceric ammonium nitrate using ferroin solution as indicator until a greenish colour is obtained (n1 ml). Pass slowly through the column 100 ml of 0.5 M sulphuric acid followed by 20.0 ml of solution B, wash with 100 ml of 0.5 M sulphuric acid followed by 100 ml of water. Collect the combined eluates in a 500-ml conical flask containing 50 ml of a 15 per cent w/v solution of ferric ammonium sulphate in sulphuric acid (25 per cent), rinse the lower end of the column with water and titrate immediately with 0.1 M ceric ammonium nitrate using ferroin solution as indicator until a greenish colour is obtained (n2 ml). Calculate the percentage content of TiO2 from the expression 3.99(n2 - n1)/w Where, w is the weight, in g, of the substance under examination used in the preparation of solution A.
1186
IP 2007
TIOTROPIUM POWDER FOR INHALATION
Storage. Store protected from moisture. Avoid contact with aluminium.
Tiotropium Bromide Monohydrate

H3C N CH3 O S O O
C19H22BrNO4S2, HCl
adjusting the pH to 5.5 with dilute acetic acid (10 per cent v/v), flow rate. 1 ml per minute, spectrophotometer set at 235 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the tailing factor is not more than 2.0 and the column efficiency in not less than 3000 theoretical plates. Inject the test solution. Any individual impurity is not more than 0.5 per cent and the sum of all the impurities is not more than 1.0 per cent.
Br, HCl S
Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Sulphated ash (2.3.18). Not more than 0.3 per cent. Water (2.3.43). 3.5 to 4.5 per cent, determined on 0.5 g.
OH
Mol. Wt. 508.9
Assay. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 20 mg of the substance under examination in 100.0 ml of the mobile phase. Dilute 5.0 ml of the resulting solution to 50.0 ml with the mobile phase. Reference solution. A 0.002 per cent w/v solution of tiotropium bromide monohydrate RS in the mobile phase. Chromatographic system a stainless steel column 25 cm 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 45 volumes of methanol and 55 volumes of a buffer solution prepared by dissolving 3.85 g of ammonium acetate in 1000 ml of water and adjusting the pH to 5.5 with dilute acetic acid ( 10 per cent v/v), flow rate. 1 ml per minute, spectrophotometer set at 235 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the tailing factor is not more than 2.0, the column efficiency in not less than 3000 theoretical plates and the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject the test solution and the reference solution. Calculate the content of C19H22NO4S2,Br. Storage. Store protected from light and moisture.
Tiotropium Bromide Monohydrochloride is 6,7-epoxy-3hydroxy-8-methyl-1H-5H-tropanium bromide hydrochloride. Tiotropium Bromide Monohydrate contains not less than 98.0 per cent and not more than 102.0 per cent of tiotropium bromide, C19H22NO4S2Br, calculated on the anhydrous basis. Description. A white to off-white powder.
Identification
A. Determine by infrared absorption spectrophotometry ( 2.4.6). Compare the spectrum with that obtained with tiotropium bromide monohydrate RS or with the reference spectrum of tiotropium bromide monohydrate. B. In the Assay, the principal peak in the chromatogram obtained with test solution corresponds to the principal peak in the chromatogram obtained with the reference solution.
Tests
Related substances. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 10 mg of the substance under examination in 100 ml of the mobile phase. Reference solution. A 0.002 per cent w/v solution of tiotropium bromide monohydrate RS in the mobile phase. Chromatographic system a stainless steel column 25 cm 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 45 volumes of methanol and 55 volumes of a buffer solution prepared by dissolving 3.85 g of ammonium acetate in 1000 ml of water and
Tiotropium Powder for Inhalation

Tiotropium Powder for Inhalation consists of Tiotropium Bromide in microfine powder either alone or admixed with Lactose in a pre-metered unit for use in a suitable powder inhaler.
1187
TIZANIDINE HYDROCHLORIDE
IP 2007
Tiotropium Powder for Inhalation contains not less than 90.0 per cent and not more than 125.0 per cent of the stated amount of tiotropium, C19H22NO4S2 per unit dose.
Tizanidine Hydrochloride
N S Cl N NH NH
C9H8ClN5S.HCl Mol. Wt. 290.2
Identification
N , HCl
Tests
Other tests. Complies with the tests stated under Inhalation Preparations (Powders for Inhalation). Follow the procedure described under Assay with suitable dilution of the reference solution wherever the amount of active substance is to be determined in any test. Assay. Determine by liquid chromatography (2.4.14). Solvent mixture. 90 volumes of 0.05 per cent v/v orthophosphoric acid and 10 volumes of acetonitrile. Test solution. To 10 intact capsules add about 70 ml of the solvent mixture and disperse with the aid of ultrasound for about 5 minutes with intermittent shaking. Add sufficient of the solvent mixture to produce 100.0 ml and dilute suitably, if required, to get a solution containing 1.8 g per ml of Tiotropium per ml. Reference solution. A solution containing 1.8 g per ml of tiotropium bromide RS per ml in the solvent mixture. Chromatographic system a stainless steel column 15 cm x 4.6 mm, packed with octadecylsilyl silica gel (5 mm), mobile phase: a mixture of 80 volumes of a buffer solution prepared by dissolving 2 ml triethylamine in 1000 ml of water and adjusting the pH to 2.5 with orthophosphoric acid, and 20 volumes of acetonitrile, flow rate. 2 ml per minute, spectrophotometer set at 237 nm, inject 200 l. Inject the reference solution. The test is not valid unless the column efficiency is not less than 4500 theoretical plates, the tailing factor is not more than 1.7 and the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject the test solution and the reference solution. Calculate the content of C19H22NO4S2 per unit. Storage. Store protected from moisture, at temperature not exceeding 30. Labelling. The label states the quantity of active ingredient per pre-metered unit.
Tizanidine Hydrochloride is 5-chloro-N-(2-imidazolin-2-yl)2,1,3-benzothiadiazol-4-yl-amine. Tizanidine Hydrochloride contains not less than 98.0 per cent and not more than 102.0 per cent of C9H8ClN5S.HCl, calculated on the dried basis. Description. A white to yellowish white, crystalline powder.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with tizanidine hydrochloride RS. B. In the Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the peak in the chromatogram obtained with the reference solution. C. Gives reaction A for chlorides (2.3.1).
Tests
pH (2.4.24). 3.5 to 5.3, determined on 5.0 per cent w/v solution in water. Related substances. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 50 mg of the substance under examination in 50.0 ml of the mobile phase. Reference solution (a). A 0.1 per cent w/v solution of tizanidine hydrochloride RS in the mobile phase. Reference solution (b). Dilute 1 ml of reference solution (a) to 100 ml with mobile phase. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5 m), column temperature 50, mobile phase: a mixture of 80 volumes of buffer solution prepared by dissolving 3.5 g of sodium-1-pentane sulphonate in 1000 ml of water, adjust the pH to 3.0 with 12 per cent orthophosphoric acid solution or 1 M sodium hydroxide and 20 volumes of acetonitrile,
1188
IP 2007
TIZANIDINE TABLETS
flow rate. 1 ml per minute, spectrophotometer set at 230 nm, a 10 l loop injector. Inject reference solution (a). The test is not valid unless the tailing factor is not more than 2.0 and the column efficiency in not less than 5000 theoretical plates. Inject the test solution and reference solution (b). In the chromatogram obtained with the test solution, the area of any secondary peak is not more than 0.5 times the area of the peak in the chromatogram obtained with reference solution (b) (0.5 per cent) and the sum of areas of all the secondary peaks is not more than the area of the peak in the chromatogram obtained with the reference solution (b) (1.0 per cent). Heavy metals (2.3.13). Dissolve 1.0 g in 20 ml of water. 12 ml of this solution complies with limit test for heavy metals, Method D (20 ppm). Total Chloride. 11.9 per cent to 12.5 per cent. Weigh accurately about 0.5 g and dissolve in 50 ml of water. Titrate with 0.1 M silver nitrate. Determine the end point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M silver nitrate is equivalent to 0.003545 g of chloride. Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1 g by drying in an oven at 105 for 3 hours. Assay. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 25 mg of the substance under examination in 50.0 ml of the mobile phase. Dilute 10.0 ml of the solution to 50.0 ml with the same solvent. Reference solution. A 0.01 per cent w/v solution of tizanidine hydrochloride RS in the mobile phase. Chromatographic system a stainless steel column 15 cm x 4.6 mm packed with octylsilane bonded to porous silica (5 m), mobile phase: a mixture of 50 volumes of a buffer solution prepared by dissolving 6.8 g of monobasic potassium phosphate in 1000 ml of water adjusting the pH to 7.5 with 5.3 M potassium hydroxide, and 50 volumes of acetonitrile, flow rate. 1 ml per minute, spectrophotometer set at 230 nm, a 10 l loop injector. Inject the reference solution. The test is not valid unless the tailing factor is not more than 2.0 and the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject the test solution and the reference solution. Calculate the content of C9H8ClN5S.HCl.
Tizanidine Tablets
Tizanidine Hydrochloride Tablets
Tizanidine Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of tizanidine, C9H8ClN5S.
Identification
Tests
Dissolution (2.5.2). Apparatus. No 1 Medium: 900 ml of 0.1 M hydrochloric acid. Speed and time. 50 rpm for 45 minutes. Withdraw a suitable volume of the medium and filter. Measure the absorbance of the filtrate, suitably diluted with dissolution medium if necessary, at the maximum at about 320 nm (2.4.7). Calculate the content of C9H8ClN5S in the medium from the absorbance obtained from a solution of known concentration of tizanidine Hydrochloride RS in the same medium. D. Not less than 70 per cent of the stated amount of C9H8ClN5S. Uniformity of content. Comply with the tests stated under Tablets. Determine by liquid chromatography (2.4.14), as described under Assay. Test solution. Crush one tablet and disperse in 50 ml phosphate buffer pH 6.6, dilute to 100.0 ml with acetonitrile and filter. Calculate the content of C9H8ClN5S. Other tests. Comply with the tests stated under Tablets. Assay. Determine by liquid chromatography (2.4.14). Test solution. Weigh and powder 20 tablets. Weigh accurately a quantity of the powdered tablet containing about 20 mg of Tizanidine, disperse in 50 ml phosphate buffer pH 6.6 and dilute to 100.0 ml with acetonitrile and filter. Reference solution. Weigh accurately 10 mg of tizanidine hydrochloride RS, dissolve in 25 ml phosphate buffer pH 6.6 and dilute to 50.0 ml with acetonitrile. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5 m),
1189
TOBRAMYCIN
IP 2007
mobile phase: a mixture of 80 volumes of phosphate buffer pH 6.6 and 20 volumes of acetonitrile, flow rate. 1 ml per minute, spectrophotometer set at 320 nm, a 20 l loop Injector. Inject the reference solution. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject the test solution and the reference solution. Calculate the content of C9H8ClN5S. Storage. Store protected from light and moisture. Labelling. The label states the strength in terms of the equivalent amount of Tizanidine.
Test solution. Dissolve 0.4 g of the substance under examination in 100 ml of water. Reference solution (a). A 0.4 per cent w/v solution of tobramycin RS in water. Reference solution (b). A solution containing 0.4 per cent w/v each of kanamycin sulphate RS, neomycin sulphate RS and tobramycin RS in water. Apply to the plate 5 l of each solution. After development, dry the plate in warm air, spray with a mixture of equal volumes of a 46 per cent w/v solution of sulphuric acid and a 0.2 per cent w/v solution of 1,3-naphthalenediol in ethanol (95 per cent) and heat at 105 for 10 minutes. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows three clearly separated principal spots. B. Dissolve about 5 mg in 5 ml of water, add 5 ml of a 0.1 per cent w/v solution of ninhydrin in ethanol (95 per cent) and heat in a water-bath for 3 minutes; a violet-blue colour develops.
Tobramycin
OH
NH2
HO H2N O
Tests
OH O
HO NH2 O H2N
pH (2.4.24). 9.0 to 11.0, determined in a 10.0 per cent w/v solution. Specific optical rotation (2.4.22). +138 to +148, determined in a 4.0 per cent w/v solution. 2-Methyl-1-propanol. Not more than 1.0 per cent w/w, determined by gas chromatography (2.4.13).
HO
NH2
C18H37N5O9
Mol. Wt. 467.3
Test solution (a). A 10 per cent w/v solution of the substance under examination in water. Test solution (b). A solution containing 10 per cent w/v of the substance under examination and 0.2 per cent v/v of 2-propanol (internal standard). Reference solution. A solution containing 0.1 per cent w/v of 2-methyl-1-propanol and 0.2 per cent v/v of the internal standard. Chromatographic system a glass column 1.5 m x 4 mm, packed with porous polymer beads (80 to 100 mesh) (such as Porapak Q), temperature. column 165, Calculate the percentage w/w of 2-methyl-1-propanol. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel H. Mobile phase. A mixture of equal volumes of strong ammonia solution, 2-butanone and ethanol (95 per cent).
Tobramycin is 6-O-(3-amino-3-deoxy--D-glucopyranosyl)2-deoxy-4-O-(2,6-diamino-2,3,6-trideoxy- -D-ribohexopyranosyl)-D-streptamine, an antimicrobial substance produced by Streptomyces tenebrarius or by any other means. Tobramycin has potency not less than 930 Units per mg, calculated on the anhydrous and 2-methyl-1-propanol-free basis. Description. A white or almost white powder.
Identification
A. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel H. Mobile phase. A mixture of 60 volumes of methanol, 40 volumes of strong ammonia solution and 20 volumes of chloroform.
1190
IP 2007
TOBRAMYCIN INJECTION
Test solution. Dissolve 0.8 g of the substance under examination in 100 ml of 0.02 M ammonia. Reference solution. A 0.008 per cent w/v solution of the substance under examination in 0.02 M ammonia. Apply to the plate 5 l of each solution. After development, dry the plate in warm air, heat at 110 for 10 minutes and spray the hot plate with a solution prepared immediately before use by diluting sodium hypochlorite solution (3 per cent Cl) with water to contain 0.5 per cent w/v of available chlorine. Dry in a current of cold air until a sprayed area of the plate below the line of application gives at most a very faint blue colour with a drop of potassium iodide and starch solution; avoid prolonged exposure to cold air. Spray the plate with potassium iodide and starch solution. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Sulphated ash (2.3.18). Not more than 0.3 per cent. Water (2.3.43). Not more than 8.0 per cent, determined on 0.3 g. Assay. Determine by the microbiological assay of antibiotics, Method B (2.2.10). Tobramycin intended for use in the manufacture of parenteral preparations without a further appropriate procedure for the removal of bacterial endotoxins complies with the following additional requirement. Bacterial endotoxins (2.2.3). Not more than 2.0 Endotoxin Units per mg of tobramycin. Tobramycin intended for use in the manufacture of parenteral preparations or eye drops without a further appropriate sterilisation procedure complies with the following additional requirements. Sterility (2.2.11). Complies with the test for sterility. Storage. Store protected from moisture. If the material is sterile, the container should be sterile, tamper-evident and sealed so as to exclude micro-organisms. Labelling. The label states (1) the number of Units per mg; (2) where applicable, that it is sterile; (3) where applicable, that it is free from bacterial endotoxins and depressor substances.
Tobramycin Injection contains not less than 90.0 per cent and not more than 120.0 per cent of the stated amount of tobramycin, C18H37N5O9. Description. A colourless solution.
Identification
A. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel H. Mobile phase. A mixture of 60 volumes of methanol, 40 volumes of strong ammonia solution and 20 volumes of chloroform. Test solution. Dilute a suitable volume of the injection with water to produce a solution containing 0.4 per cent w/v solution of tobramycin. Reference solution (a). A 0.4 per cent w/v solution of tobramycin RS in water. Reference solution (b). A solution containing 0.4 per cent w/v each of kanamycin sulphate RS, neomycin sulphate RS and tobramycin RS in water. Apply to the plate 5 l of each solution. After development, dry the plate in warm air, spray with a mixture of equal volumes of a 46 per cent w/v solution of sulphuric acid and a 0.2 per cent w/v solution of 1,3-naphthalenediol in ethanol (95 per cent) and heat at 105 for 10 minutes. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows three clearly separated principal spots. B. Gives the reactions of sulphates (2.3.1).
Tests
pH (2.4.24). 3.5 to 6.0. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel H. Mobile phase. A mixture of equal volumes of strong ammonia solution, 2-butanone and ethanol (95 per cent). Test solution. Dilute a suitable volume of the injection with 0.01 M ammonia to obtain a solution containing 40 mg of Tobramycin in 4 ml. Shake with 10 ml of ether and use the aqueous layer. Reference solution. A 0.008 per cent w/v solution of the substance under examination in 0.02 M ammonia. Apply to the plate 5 l of each solution. After development, dry the plate in warm air, heat at 110 for 10 minutes and spray the hot plate with a solution prepared immediately before use by diluting sodium hypochlorite solution (3 per cent Cl)
Tobramycin Injection
Tobramycin Sulphate Injection
Tobramycin Injection is a sterile solution of Tobramycin in Water for Injections containing sufficient Sulphuric Acid to adjust the pH to 3.5 to 6.0.
1191
TOCOPHERYL ACETATE
IP 2007
with water to contain 0.5 per cent w/v of available chlorine. Dry in a current of cold air until a sprayed area of the plate below the line of application gives at most a very faint blue colour with a drop of potassium iodide and starch solution; avoid prolonged exposure to cold air. Spray the plate with potassium iodide and starch solution. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Bacterial endotoxins (2.2.3). Not more than 2.0 Endotoxin Units per mg of tobramycin. Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. Determine by the microbiological assay of antibiotics, Method B (2.2.10). Calculate the content of tobramycin in the injection, taking each 1000 Units found to be equivalent to 1 mg of tobramycin. The upper fiducial limits of error is not less than 97.0 per cent and not more than 110.0 per cent of the stated potency.
B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.01 per cent w/v solution in ethanol exhibits a maximum at about 284 nm, a shoulder at about 278 nm and a minimum at about 254 nm. C. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel HF254. Mobile phase. A mixture of 80 volumes of cyclohexane and 20 volumes of ether. Test solution (a). Dissolve 0.5 g of the substance under examination in 100 ml of cyclohexane. Test solution (b). Dissolve 10 mg of the substance under examination in 2 ml of 5 M ethanolic sulphuric acid, heat on a water-bath for 5 minutes, cool, add 2 ml of water and 2 ml of cyclohexane and shake for 1 minute; use the upper layer. Reference solution (a). A 0.5 per cent w/v solution of a-tocopheryl acetate RS in cyclohexane. Reference solution (b). Prepare in the same manner as test solution (b) but using 10 mg of a -tocopheryl acetate RS in place of the substance under examination. Apply to the plate 10 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with test solution (a) corresponds to that in the chromatogram obtained with reference solution (a). There are two spots in each of the chromatograms obtained with test solution (b) and reference solution (b). The spots of higher Rf value are due to atocopheryl acetate and correspond to that in the chromatogram obtained with reference solution (a). The spots of lower Rf value are due to a-tocopheryl. Spray the plate with a mixture of 1 volume of hydrochloric acid, 4 volumes of a 0.25 per cent w/v solution of ferric chloride in ethanol (95 per cent) and 4 volumes of a 1.0 per cent w/v solution of 1,10-phenanthroline hydrochloride in ethanol (95 per cent). In the chromatograms obtained with test solution (b) and reference solution (b) the spot of lower Rf value a-tocopheryl is orange.
Tocopheryl Acetate
-Tocopheryl Acetate; -Tocopherol Acetate; Vitamin E Acetate
CH3 H3C O H3C O CH3 O CH3 CH3 CH3
CH3 CH3
C31H52O3
Mol. Wt. 472.8
Tocopheryl Acetate is (2RS,4'RS,8'RS)-6-acetoxy-2,5,7,8tetramethyl-2-(4', 8', 12'-trimethyltridecyl)chroman(all-rac-tocopherol acetate). Tocopheryl Acetate contains not less than 96.0 per cent and not more than 102.0 per cent of C31H52O3. Description. A clear, slightly greenish yellow, viscous, oily liquid.
Tests
Refractive index (2.4.27). 1.494 to 1.498, determined at 20. Acid value (2.3.23). Not more than 2.0, determined on 2.0 g. Free tocopherol. Not more than 2.0 per cent, determined by the following method. Weigh accurately about 0.5 g, dissolve in 100 ml of 0.25 M ethanolic sulphuric acid, add 20 ml of water and 0.1 ml of a 0.25 per cent w/v solution of diphenylamine in sulphuric acid and titrate with 0.01 M ceric ammonium nitrate until a blue colour is produced that persists for at least 5 seconds. Repeat the operation without the substance under examination. The difference between the titrations represents the amount of ceric ammonium nitrate required.
Identification
Test A may be omitted if tests B and C are carried out. Tests B and C may be omitted if test A is carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with a-tocopheryl acetate RS.
1192
IP 2007
TOLBUTAMIDE
1 ml of 0.01 M ceric ammonium nitrate is equivalent to 0.002154 g of tocopherol. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Assay. Weigh accurately about 0.3 g, dissolve in 25 ml of ethanol (95 per cent), add 20 ml of 2.5 M ethanolic sulphuric acid and heat on a water-bath under a reflux condenser for 3 hours. Cool, transfer the solution quantitatively to a 200-ml volumetric flask, rinse the apparatus with ethanol (95 per cent) and add the rinsings to the flask. Make up to volume with ethanol (95 per cent) and mix. To 25.0 ml of the resulting solution in a flask add 25 ml of 0.25 M ethanolic sulphuric acid, 10 ml of water and titrate with 0.01 M ceric ammonium nitrate using 0.1 ml of diphenylamine as indicator, until a blue colour persisting for at least 5 seconds is obtained. Repeat the operation without the substance under examination. The difference between the titrations represents the amount of ceric ammonium nitrate required. 1 ml of 0.01 M ceric ammonium nitrate is equivalent to 0.002364 g of C31H52O3. Storage. Store protected from light and moisture.
263 nm and 275 nm and a shoulder at about 268 nm. Dilute the solution with methanol to produce a 0.001 per cent w/v solution. When examined in the range 220 nm to 235 nm, the resulting solution shows an absorption maximum at about 228 nm; absorbance at about 228 nm, about 0.50. C. Boil 0.1 g with 8 ml of a 50 per cent v/v solution of sulphuric acid under a reflux condenser for 30 minutes. Make the solution strongly alkaline with sodium hydroxide solution and steam distil for 30 minutes, receiving the distillate in 20 ml of 0.1 M hydrochloric acid. To 1 ml of the solution containing the distillate add 0.1 g of sodium acetate and 10 ml of buffer solution pH 9.4. Cool in an ice-bath for 10 minutes, add 1 ml of diazotised nitroaniline solution, set aside for 20 minutes and add dropwise 1 ml of sodium hydroxide solution; an orangered colour is produced. D. Boil 0.1 g with 8 ml of a 50 per cent v/v solution of sulphuric acid under a reflux condenser for 30 minutes. Cool in an icebath; a crystalline precipitate of 4-toluenesulphonylamide is formed, which after recrystallisation from hot water and drying at 105 melts at 135 to 140 (2.4.21).
Tests
Appearance of solution. A 2.0 per cent w/v solution in 1 M sodium hydroxide is clear (2.4.1), and colourless (2.4.1). pH (2.4.24). 4.5 to 5.5, determined in a solution prepared by dissolving 2.0 g in 50 ml of carbon dioxide-free water by heating at 70 for 5 minutes, cooling rapidly and filtering.
Tolbutamide
O O O S N N H H H3C
C12H18N2O3S Mol. Wt. 270.4
CH3
Non-sulphonyl urea. Dissolve 0.5 g in 1 ml of dilute ammonia solution and 9 ml of water; not more than a faint opalescence is produced. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 90 volumes of chloroform, 8 volumes of methanol and 2 volumes of anhydrous formic acid. Test solution. Dissolve 0.5 g of the substance under examination in 10 ml of acetone. Reference solution (a). A 0.015 per cent w/v solution of 4-toluenesulphonamide in acetone. Reference solution (b). A mixture of equal volumes of the test solution and reference solution (a). Apply to the plate 5 l of each of test solution and reference solution (a) and 10 l of reference solution (b). After development, dry the plate in a current of warm air and heat at 110 for 10 minutes. While still hot, place the plate in a chromatographic tank with an evaporating dish containing a 5 per cent w/v solution of potassium permanganate, add an equal volume of hydrochloric acid and close the tank. Leave the plate in the tank for 2 minutes, then place it in a current of
Tolbutamide is 3-butyl-1-[(4-methylphenyl)sulphonyl]urea. Tolbutamide contains not less than 99.0 per cent and not more than 101.0 per cent of C12H18N2O3S, calculated on the dried basis. Description. A white, crystalline powder; almost odourless.
Identification
Test A may be omitted if tests B, C and D are carried out. Tests B, C and D may be omitted if test A is carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with tolbutamide RS or with the reference spectrum of tolbutamide. B. Dissolve 25 mg in sufficient methanol to produce 100.0 ml. When examined in the range 245 nm to 360 nm (2.4.7), the resulting solution shows absorption maxima at about 258 nm,
1193
TOLBUTAMIDE TABLETS
IP 2007
cold air until the excess of chlorine is removed and an area of coating below the line of application gives only a very faint blue colour with potassium iodide and starch solution; avoid prolonged exposure to cold air. Spray the plate with potassium iodide and starch solution and allow to stand for 5 minutes. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (a). The chromatogram obtained with reference solution (b) shows two clearly separated spots. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105 for 3 hours. Assay. Weigh accurately about 0.5 g and dissolve in a mixture of 40 ml of ethanol (95 per cent) and 20 ml of water. Titrate with 0.1 M sodium hydroxide using phenolphthalein solution as indicator. 1 ml of 0.1 M sodium hydroxide is equivalent to 0.02704 g of C12H18N2O3S. Storage. Store protected from moisture.
Mobile phase. A mixture of 90 volumes of chloroform, 8 volumes of methanol and 2 volumes of anhydrous formic acid. Test solution. Shake a quantity of the powdered tablets containing 0.5 g of Tolbutamide with 10 ml of acetone and filter. Reference solution (a). A 0.015 per cent w/v solution of 4-toluenesulphonamide in acetone. Reference solution (b). A mixture of equal volumes of the test solution and reference solution (a). Apply to the plate 5 l of each of test solution and reference solution (a) and 10 l of reference solution (b). After development, dry the plate in a current of warm air and heat at 110 for 10 minutes. While still hot, place the plate in a chromatographic tank with an evaporating dish containing a 5 per cent w/v solution of potassium permanganate, add an equal volume of hydrochloric acid and close the tank. Leave the plate in the tank for 2 minutes, then place it in a current of cold air until the excess of chlorine is removed and an area of coating below the line of application gives only a very faint blue colour with potassium iodide and starch solution; avoid prolonged exposure to cold air. Spray the plate with potassium iodide and starch solution and allow to stand for 5 minutes. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (a). The chromatogram obtained with reference solution (b) shows two clearly separated spots. Dissolution (2.5.2). Apparatus. No 1 Medium. 900 ml of a solution containing 2.04 per cent w/v of disodium hydrogen phosphate and 0.135 per cent w/v of potassium dihydrogen phosphate. Speed and time. 50 rpm and 30 minutes. Withdraw a suitable volume of the medium and filter. Measure the absorbance of the filtrate suitably diluted if necessary, at the maximum at about 228 nm (2.4.7). Calculate the content of C12H 18N 2O 3S in the medium taking 417 as the specific absorbance at 228 nm. D. Not less than 70 per cent of the stated amount of C12H18N2O3S. Other tests. Comply with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 0.5 g of Tolbutamide, add 50 ml of ethanol (95 per cent), previously neutralised to phenolphthalein solution, warm to dissolve, add 25 ml of water and titrate with 0.1 M sodium hydroxide using phenolphthalein solution as indicator.
Tolbutamide Tablets
Tolbutamide Tablets contain not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of tolbutamide, C12H18N2O3S.
Identification
Extract a quantity of the powdered tablets containing 1 g of Tolbutamide with 10 ml of chloroform, filter, evaporate the filtrate to dryness, scratching the sides of the vessel, if necessary, to induce crystallisation, and dry the residue at 105 for 30 minutes. The residue complies with the following tests. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with tolbutamide RS or with the reference spectrum of tolbutamide. B. Boil 0.1 g of residue with 8 ml of a 50 per cent v/v solution of sulphuric acid under a reflux condenser for 30 minutes. Cool in an ice-bath; a crystalline precipitate of 4-toluenesulphonylamide is formed, which after recrystallisation from hot water and drying at 105 melts at 135 to 140.
Tests
1194
IP 2007
TOPOTECAN HYDROCHLORIDE
1 ml of 0.1 M sodium hydroxide is equivalent to 0.02704 g of C12H18N2O3S. Storage. Store protected from moisture.
Weigh accurately about 0.5 g, dissolve in 10 ml of methanol, add 20 ml of water and 20 ml of glacial acetic acid. Titrate with 0.1 M silver nitrate solution using eosin yellow solution as indicator. Colour changes from orange to dark pink. 1 ml of 0.1 M silver nitrate is equivalent to 0.003545 g of Cl. Related substances. Determine by liquid chromatography (2.4.14). Solvent mixture. A mixture of 30 volumes of acetonitrile and 70 volumes of buffer solution prepared by diluting 1 ml of trifluoroacetic acid in 1000 ml of water.
Topotecan Hydrochloride
CH3 N CH3 HO N N H3C HO O O , HCl O
Test solution. Dissolve 40 mg of the substance under examination in 100 ml of solvent mixture. Reference solution (a). A 0.04 per cent w/v solution of topotecan hydrochloride RS in solvent mixture. Reference solution (b). Dilute 1 ml of the reference solution (a) to 100 ml with solvent mixture. Chromatographic system as described under Assay. Mol. Wt. 457.9 Inject reference solution (a). The test is not valid unless the column efficiency is not less than 2000 theoretical plates and the tailing factor is not more than 2.0. Inject the test solution and reference solution (b). In the chromatogram obtained with the test solution, the area of any secondary peak is not more than 0.15 times the area of the peak in the chromatogram obtained with the reference solution (b) (0.15 per cent) and the sum of areas of all the secondary peaks is not more than the area of the peak in the chromatogram obtained with the reference solution (b) (1.0 per cent). Heavy metals (2.3.13). 1.0 g complies with limit test for heavy metals, Method B (20 ppm). Test solution. Weigh accurately about 0.5 g of the substance under examination in 5 ml of dimethylsulphoxide. Pippet a 2 ml of this solution to a 10 ml head space vial,caped cup the vial. Reference solution (a). To 30 ml of dimethylsulphoxide, add 300 mg of methanol, 500 mg ethyl acetate, 500 mg isopropyl alchohol, 200 mg of acetone, 500 mg of ethanol and 200 mg of N,N-dimethyl formamide, diluted to 100 ml with dimethylsulphoxide. Reference solution (b). To 15 ml of dimethylsulphoxide, add 60 mg of chloroform, diluted to 100 ml with dimethylsulphoxide. Reference solution (c). Dilute 10 ml each of reference solutions (a) and (b) to 100 ml with dimethylsulphoxide.Pippet 2 ml of this solution to a head space vial. Chromatographic system a capillary column 30 m x 0.53 mm, packed with mega bore coated with a mixture of 6 per cent cyanopropylphenyl and 94 per cent dimethylpolysiloxane (3 m ),
C23H23N3O5.HCl
Topotecan Hydrochloride is (4S)-10[(dimethylamino)methyl]-4-ethyl-4,9-dihydroxy-1Hpyrano[3,4:6,7]indolozino[1,2-b]quinoline3,14(4H,12H)dione hydrochloride. Topotecan Hydrochloride contains not less than 98.0 per cent and not more than 102.0 per cent of C23H23N3O5.HCl, calculated on the anhydrous basis. Description. A light yellow to greenish yellow powder. CAUTION Topotecan Hydrochloride is cytotoxic; extra care required to prevent inhaling particles and exposing the skin to it.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with topotecan hydrochloride RS. B. In the Assay, the principal peak in the chromatogram of the test solution corresponds to the peak in the chromatogram obtained with the reference solution.
Tests
Appearance of solution. A 1.0 per cent w/v solution is clear (2.4.1) and not more intensely coloured than the reference solution GYS3 (2.4.1). pH (2.4.24). 3.5 to 4.5, determined in a 1.0 per cent w/v solution. Specific optical rotation (2.4.22). + 30 to 38, determined on 1.0 per cent w/v solution in methanol. Total Chloride. 7.6 per cent to 8.1 per cent.
1195
TOPOTECAN INJECTION
IP 2007
temperature: column. 35 for 5 minutes increase @ 5 per minute to 150 @ 20 per minute to 220 hold for 2 minutes, inlet port 180and detector. 260, Head space conditions Vial equilibrium temperature 90, loop temperature 100 , Transfer line 115, vial equilibrium 15 minutes, vial pressurisation 0.02 minute, sample loop fill 0.02 minute, loop equilibrium 0.05 minute sample injection 1 minute, vial pressure 10 psi. a flame ionisation detector, nitrogen as carrier gas. Inject 1 ml of the reference solution (c). The test is not valid unless the resolution between two adjacent peaks is not less than 1.0. Inject 1 ml of the test solution and reference solution (c). In the chromatogram obtained with test solution, the area of peaks due to methanol, ethyl acetate, isopropyl alcohol, acetone, ethanol, chloroform and N,N-dimethyl formamide is not more than the area of peaks obtained in the chromatogram due to the reference solution (c). Loss on ignition (2.4.20). Not more than 0.1 per cent. Water (2.3.43). Not more than 5.0 per cent, determined on 0.5 g. Bacterial endotoxins (2.2.3). Not more than 16 Endotoxin Unit per mg of topotecan hydrochloride. Microbial contamination (2.2.9). Total viable aerobic count, not more than 100 cfu per g. It also meets the requirements of the tests for the absence of Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella species, and Escherichia coli. Assay. Determine by liquid chromatography (2.4.14). Solvent mixture. A mixture of 30 volumes of acetonitrile and 70 volumes of buffer solution, prepared by diluting 1 ml of trifluoroacetic acid in 1000 ml of water. Test solution. Dissolve 10 mg of the substance under examination in 25.0 ml of solvent mixture. Reference solution. A 0.04 per cent w/v solution of topotecan hydrochloride RS in solvent mixture. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsylane bonded to porous silica (5 m), mobile phase: A. dissolve 1 ml of trifluoroacetic acid in 1000 ml of water, B. acetonitrile, flow rate. 1.2 ml per minute, a linear gradient programme using the conditions given below, spectrophotometer set at 260 nm, a 10 l loop injector.
Time ( in min) 0 28 38 43 48
Mobile phase A (per cent v/v) 85 70 70 85 85
Mobile phase B (per cent v/v) 15 30 30 15 15
Inject the reference solution. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject the test solution and reference solution. Calculate the content of C23H23N3O5.HCl. Storage. Store protected from light, at a temperature between 2 to 8.
Topotecan Injection
Topotecan Hydrochloride Injection
Topotecan Injection is a sterile, stabilised solution of Topotecan Hydrochloride in Water for Injection. Topotecan Injection contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of the topotecan, C23H23N3O5. Description. A clear, light yellow solution.
Identification
A. In the Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the peak in the chromatogram obtained with the reference solution. B. It gives the reaction of chlorides (2.3.1).
Tests
pH (2.4.24). 2.5 to 3.5. Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Bacterial endotoxins (2.2.3). Not more than 64.9 Endotoxin Unit per mg of topotecan. Sterility (2.2.11). Complies with the test for sterility. Assay. Determine by liquid chromatography (2.4.14). Test solution. Accurately measure the volume of injection containing 2 mg of Topotecan, diluted to 50.0 ml with mobile phase. Reference solution. Dissolve 10 mg of topotecan RS in 5 ml of water and dilute to 25.0 ml with mobile phase. Dilute 5.0 ml of the solution to 50.0 ml with mobile phase.
1196
IP 2007
TRIAMCINOLONE
Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 78 volumes of water, 22 volumes of acetonitrile and 1 volume of 1 M hydrochloric acid, flow rate. 0.7 ml per minute, spectrophotometer set at 254 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the relative standard deviation is not more than 2.0 per cent. Inject the test solution and the reference solution. Calculate the content of C23H23N3O5. Storage. Store protected from light, at a temperature not exceeding 2 to 8.
heat on a water-bath under a reflux condenser for 20 minutes; a pinkish lavender colour is produced.
Tests
Specific optical rotation (2.4.22). +65.0 to +72.0, determined in a 1.0 per cent w/v solution in dimethylformamide. Related substances. Determine by liquid chromatography (2.4.14). Prepare the following solutions immediately before use and protect from light. Test solution. Dissolve 25 mg of the substance under examination in a mixture of equal volumes of methanol and water and dilute to 10 ml with the same solvent mixture. Reference solution. Dilute 1 ml of the test solution to 100 ml with a mixture of equal volumes of methanol and water. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with base-deactivated end-capped octadecylsilyl silica gel (5 m), mobile phase: a mixture prepared by mixing 525 ml of methanol with 400 ml of water, allowing to equilibrate, adjusting the volume to 1000.0 ml with water and mixing again, flow rate. 1 ml per minute, spectrophotometer set at 238 nm, a 20 l loop injector. Inject the test solution and the reference solution. Continue the chromatography for 4.5 times the retention time of triamcinolone (about 11 minutes). In the chromatogram obtained with the test solution the area of any peak other than the principal peak is not more than the area of the principal peak in the chromatogram obtained with the reference solution (1 per cent); not more than two such peaks have an area greater than half the area of the principal peak in the chromatogram obtained with the reference solution (0.5 per cent); the sum of the areas of all the peaks other than the principal peak is not more than twice the area of the principal peak in the chromatogram obtained with the reference solution (2 per cent). Ignore any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with the reference solution (0.05 per cent). Heavy metals (2.3.13). 0.8 g complies with the limit test for heavy metals, Method B (25 ppm). Sulphated ash (2.3.18). Not more than 0.2 per cent. Loss on drying (2.4.19). Not more than 2.0 per cent, determined on 1.0 g by drying in an oven at 60 at a pressure not exceeding 0.7 kPa for 3 hours. Assay. Weigh accurately about 25 mg, dissolve in sufficient ethanol (95 per cent) to produce 100.0 ml and mix. Dilute
Triamcinolone
O H3C H F O
C21H27FO6 Triamcinolone is 9-fluoro-11,16,17,21tetrahydroxypregna-1,4-diene-3,20-dione. Triamcinolone contains not less than 97.0 per cent and not more than 103.0 per cent of C21H27FO6, calculated on the dried basis. Description. A white or almost white, crystalline powder; slightly hygroscopic. Mol. Wt. 394.4
HO H3C
OH OH OH
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with triamcinolone RS or with the reference spectrum of triamcinolone. B. When examined in the range 220 nm to 360 nm (2.4.7), a 0.002 per cent w/v solution in methanol shows an absorption maximum at about 238 nm; absorbance at about 238 nm, about 0.76. C. Dissolve 1 mg in 6 ml of ethanol (95 per cent), add 5 ml of a 1 per cent w/v solution of butylated hydroxytoluene in ethanol (95 per cent) and 5 ml of 1 M sodium hydroxide and
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2.0 ml to 50.0 ml with ethanol (95 per cent) and measure the absorbance of the resulting solution at the maximum at about 238 nm (2.4.7). Calculate the content of C21H27FO6 taking 380 as the specific absorbance at 238 nm. Storage. Store protected from light and moisture.
spectrophotometer set at 238 nm, a 20 l loop injector. Inject reference solution (b) and record the chromatogram for 4 times the retention time of the triamcinolone peak. The test is not valid unless the column efficiency determined from the principal peak in the chromatogram obtained with reference solution (a) is at least 10,000 theoretical plates per metre. In the chromatogram obtained with the test solution the area of any secondary peak is not greater than twice the area of the principal peak in the chromatogram obtained with reference solution (b) (2 per cent) and not more than one such peak has an area greater than that of the principal peak in the chromatogram obtained with reference solution (b) (1 per cent). The sum of the areas of any such peaks is not greater than the area of the principal peak in the chromatogram obtained with reference solution (a) (4 per cent). Uniformity of content. Comply with the test stated under Tablets. Crush one tablet to a fine powder, add 100 ml of ethanol (95 per cent) and shake for 10 minutes. Filter, dilute 2.0 ml of the filtrate with sufficient ethanol (95 per cent) to produce a solution containing 0.002 per cent w/v of Triamcinolone and measure the absorbance of the resulting solution at the maximum at about 238 nm (2.4.7). Calculate the content of C21H27FO6 in the tablet taking 380 as the specific absorbance at 238 nm. Other tests. Comply with the tests stated under Tablets. Assay. Determine by liquid chromatography (2.4.14). Test solution. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing 2.5 mg of Triamcinolone, shake with 5 ml of methanol and 20 ml of a mixture of 5 volumes of methanol and 3 volumes of water. Shake for 15 minutes, mix with the aid of ultrasound for 10 minutes, centrifuge and use the supernatant liquid. Reference solution (a). Prepare in the same manner as the test solution but add 5 ml of a 0.06 per cent w/v solution of testosterone (internal standard) in methanol (solution A) in place of the 5 ml of methanol. Reference solution (b). Add 5 ml of solution A to 5 ml of a 0.08 per cent w/v solution of triamcinolone RS in methanol and add 15 ml of methanol (50 per cent). Chromatographic system a stainless steel column 20 cm x 4 mm, packed with octadecylsilane bonded to porous silica (10 m), mobile phase: a mixture of 70 volumes of methanol, 30 volumes of water and 0.1 volume of glacial acetic acid,
Triamcinolone Tablets
Triamcinolone Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of triamcinolone, C21H27FO6.
Identification
A. Dissolve 1 mg of the residue obtained in the test for Related substances in 6 ml of ethanol (95 per cent), add 5 ml of a 1 per cent w/v solution of butylated hydroxytoluene in ethanol (95 per cent) and 5 ml of 1 M sodium hydroxide and heat on a water-bath under a reflux condenser for 20 minutes; a pinkish lavender colour is produced. B. In the Assay, the chromatogram obtained with the principal peak obtained with the test solution corresponds to the peak due to triamcinolone in the chromatogram obtained with reference solution (a).
Tests
Related substances. Determine by liquid chromatography (2.4.14). Test solution. Shake a quantity of the powdered tablets containing 15 mg of Triamcinolone with 15 ml of ethanol for 15 minutes, filter under reduced pressure through a fine filter paper (such as Whatman No 42) and evaporate the filtrate to dryness using a rotary evaporator. Reserve 1 mg of the residue for Identification test A. Dissolve the remainder of the residue in 15 ml of methanol. Reference solution (a). Dilute 4 ml of the test solution to 100 ml with methanol. Reference solution (b). Dilute 5 ml of the test solution to 50 ml with methanol. Dilute 5 ml of the resulting solution to 50 ml with the same solvent. Chromatographic system a stainless steel column 20 cm x 4.6 mm, packed with octadecylsilyl silica gel (5 m), mobile phase: a mixture of methanol and water, adjusted so that the retention time of triamcinolone is about 5 minutes (approximately equal volumes of methanol and water), flow rate. 2 ml per minute,
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flow rate. 2 ml per minute, spectrophotometer set at 238 nm, a 20 l loop injector. Calculate the content of C21H27FO6 in the tablets. Storage. Store protected from moisture.
evaporate. Use within 2 hours, with the flow of the mobile phase in the direction in which the aforementioned treatment was done. Apply to the plate 5 l of each solution. Allow the mobile phase to rise 12 cm. Dry the plate in a current of warm air, allow the solvent to evaporate, heat at 120 for 15 minutes and spray the hot plate with ethanolic sulphuric acid (20 per cent v/v). Heat at 120 for a further 10 minutes, allow to cool and examine in daylight and in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with reference solution (a). The principal spot in the chromatogram obtained with reference solution (b) appears as a single, compact spot. C. Heat 0.5 ml of chromic-sulphuric acid in a test-tube (5 cm x about 6 mm) in a naked flame until white fumes are evolved; the solution wets the sides of the tube readily and there is no greasiness. Add about 2 mg of the substance under examination and again heat in a naked flame until white fumes appear; the solution does not wet the sides of the tube and does not pour easily from the tube.
Triamcinolone Acetonide
HO H3C F O
C24H31FO6 Mol. Wt. 434.5
O H3C H H
OH O O
CH3 CH3
Triamcinolone Acetonide is 9-fluoro-11,21-dihydroxy16,17-isopropylidenedioxy-1,4-pregnadiene-3,20-dione. Triamcinolone Acetonide contains not less than 96.0 per cent and not more than 104.0 per cent of C24H31FO6, calculated on the anhydrous basis. Description. A white or almost white, crystalline powder; odourless or almost odourless.
Tests
Light absorption (2.4.7). When examined in the range 230 nm to 360 nm, a 0.001 per cent w/v solution in ethanol (95 per cent) shows an absorption maximum at about 239 nm; absorbance at about 239 nm, 0.34 to 0.37. Specific optical rotation (2,4,22). +100 to +107, determined in a 1.0 per cent w/v solution in dioxan. Related substances. Determine by liquid chromatography (2.4.14). Carry out the test protected from light. Test solution. Dissolve 25 mg of the substance under examination in 7 ml of methanol and dilute to 10 ml with water. Reference solution (a). Dissolve 2 mg of triamcinolone acetonide RS and 2 mg of triamcinolone in the mobile phase and dilute to 100 ml with the mobile phase. Reference solution (b). Dilute 1 ml of the test solution to 100 ml with the mobile phase. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilyl silica gel (5 m), mobile phase: a mixture prepared by mixing 525 ml of methanol with 400 ml of water, allowing to equilibrate, adjusting the volume to 1000.0 ml with water and mixing again, flow rate. 1.5 ml per minute, spectrophotometer set at 254 nm, a 20 l loop injector.
Identification
Test A may be omitted if tests B and C are carried out. Test C may be omitted if tests A and B are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with triamcinolone acetonide RS. B. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Solvent mixture. A mixture of 90 volumes of acetone and 10 volumes of formamide. Mobile phase. A mixture of 115 volumes of cyclohexane, 56 volumes of chloroform and 29 volumes of toluene. Test solution. Dissolve 25 mg of the substance under examination in 10 ml of the solvent mixture. Reference solution (a). Dissolve 25 mg of triamcinolone acetonide RS in 10 ml of the solvent mixture. Reference solution (b). Mix equal volumes of the test solution and reference solution (a). Place the dry plate in a tank containing a shallow layer of the solvent mixture, allow the solvent mixture to ascend to the top, remove the plate from the tank and allow the solvent to
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TRIAMCINOLONE ACETONIDE INJECTION
IP 2007
Equilibrate the column with the mobile phase for about 10 minutes. Inject reference solution (b). Adjust the sensitivity of the system so that the height of the principal peak is at least 50 per cent of the full scale of the recorder. Inject reference solution (a). The retention times are; triamcinolone, about 5 minutes and triamcinolone acetonide about 17 minutes. The test is not valid unless the resolution between the peaks corresponding to triamcinolone and triamcinolone acetonide is at least 15; if necessary, adjust the concentration of methanol in the mobile phase. Inject the test solution and reference solution (b). Continue the chromatography for 3.5 times the retention time of the principal peak in the chromatogram obtained with the test solution. In the chromatogram obtained with the test solution the area of any peak other than the principal peak is not more than 0.25 times the area of the principal peak in the chromatogram obtained with the reference solution (b) (0.25 per cent); the sum of the areas of all the peaks other than the principal peak is not greater than half the area of the principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent). Ignore any peak due to the solvent and any peak with an area less than 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (b). Sulphated ash (2.3.18). Not more than 0.2 per cent. Water (2.3.43). Not more than 2.0 per cent, determined on 0.2 g. Assay. Weigh accurately about 25 mg and dissolve in sufficient ethanol to produce 100.0 ml and mix. Dilute 5.0 ml to 100.0 ml with ethanol (95 per cent) and measure the absorbance of the resulting solution at the maximum at about 239 nm (2.4.7). Calculate the content of C24H31FO6 taking 354 as the specific absorbance at 239 nm. Storage. Store protected from light and moisture.
through a sintered-glass filter, wash the residue with four quantities, each of 5 ml of water and dry at 105 for 1 hour. The residue complies with the following tests. Test A may be omitted if tests B and C are carried out. Test C may be omitted if tests A and B are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with triamcinolone acetonide RS or with the reference spectrum of triamcinolone acetonide. B. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Solvent mixture. A mixture of 90 volumes of acetone and 10 volumes of formamide. Mobile phase. A mixture of 115 volumes of cyclohexane, 56 volumes of chloroform and 29 volumes of toluene. Test solution. Dissolve 25 mg of the substance under examination in 10 ml of the solvent mixture. Reference solution (a). Dissolve 25 mg of triamcinolone acetonide RS in 10 ml of the solvent mixture. Reference solution (b). Mix equal volumes of the test solution and reference solution (a). Place the dry plate in a tank containing a shallow layer of the solvent mixture, allow the solvent mixture to ascend to the top, remove the plate from the tank and allow the solvent to evaporate. Use within 2 hours, with the flow of the mobile phase in the direction in which the aforementioned treatment was done. Apply to the plate 5 l of each solution. Allow the mobile phase to rise 12 cm. Dry the plate in a current of warm air, allow the solvent to evaporate, heat at 120 for 15 minutes and spray the hot plate with ethanolic sulphuric acid (20 per cent v/v). Heat at 120 for a further 10 minutes, allow to cool and examine in daylight and in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with reference solution (a). The principal spot in the chromatogram obtained with reference solution (b) appears as a single, compact spot. C. Heat 0.5 ml of chromic-sulphuric acid in a test-tube (5 cm x about 6 mm) in a naked flame until white fumes are evolved; the solution wets the sides of the tube readily and there is no greasiness. Add about 2 mg of the substance under examination and again heat in a naked flame until white fumes appear; the solution does not wet the sides of the tube and does not pour easily from the tube.
Triamcinolone Acetonide Injection

Triamcinolone Acetonide Injection is a sterile suspension of Triamcinolone Acetonide in very fine particles in Water for Injections containing suitable dispersing agents. Triamcinolone Acetonide Injection contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of triamcinolone acetonide, C24H31FO6.
Identification
Extract a volume containing about 50 mg of Triamcinolone Acetonide with two quantities, each of 10 ml, of peroxide-free ether and discard the ether extracts. Filter the aqueous layer
Tests
pH (2.4.24). 5.0 to 7.5.
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Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. Determine by liquid chromatography (2.4.14). Test solution. Mix an accurately measured volume of the injection, diluted with methanol, with sufficient of a solution of prednisolone RS (internal standard) with methanol to obtain a final concentration of 0.02 per cent w/v of triamcinolone acetonide and 0.01 per cent w/v of prednisolone, centrifuge and use the clear supernatant liquid. Reference solution (a). A solution containing 0.02 per cent w/v of triamcinolone acetonide RS and 0.01 per cent w/v of prednisolone RS in methanol. Reference solution (b). Prepare in the same manner as test solution but omitting the internal standard. Chromatographic system a stainless steel column 25 cm x 4 mm, packed with octadecylsilane bonded to porous silica (10 m), mobile phase: a mixture of 56 volumes of methanol and 44 volumes of water, flow rate. 1.4 ml per minute, spectrophotometer set at 254 nm, a 20 l loop injector. Adjust the flow rate of the mobile phase such that the separation of the triamcinolone acetonide and internal standard is optimised with a retention time of about 15 minutes for triamcinolone acetonide. Calculate the content of C24H31FO6 in the injection. Storage. Store protected from light.
absorption maxima at about 262 nm and 360 nm, and a shoulder at about 285 nm. B. A 0.1 per cent w/v solution in anhydrous formic acid, when examined in ultraviolet light at 365 nm shows an intense blue fluorescence. Solutions in other acids also exhibit a blue fluorescence.
Tests
Acidity. Boil 1.0 g with 20 ml of water for 5 minutes, cool, filter and wash the filter with three quantities, each of 10 ml, of water. Combine the filtrate and washings and add 0.3 ml of phenolphthalein solution. Not more than 1.5 ml of 0.01 M sodium hydroxide is required to change the colour of the solution. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 90 volumes of ethyl acetate, 10 volumes of 18 M ammonia and 10 volumes of methanol. Test solution. Dissolve 0.1 g of the substance under examination in 20 ml of dimethyl sulphoxide and dilute 2 ml of the resulting solution to 50 ml with methanol. Reference solution. Dilute 1 volume of the test solution to 200 volumes with methanol. Apply to the plate 5 l of each solution. After development, dry the plate in air until the odour of solvent is no longer detectable and examine in ultraviolet light at 365 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 1.0 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.15 g, dissolve in 5 ml of anhydrous formic acid, add 100 ml of anhydrous glacial acetic acid. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.02533 g of C12H11N7. Storage. Store protected from light and moisture.
Triamterene
H2N N N N N NH2
C12H11N7 Mol. Wt. 253.3
NH2
Triamterene is 2,4,7-triamino-6-phenylpteridine. Triamterene contains not less than 99.0 per cent and not more than 101.0 per cent of C12H11N7, calculated on the dried basis. Description. A yellow, crystalline powder; odourless.
Identification
A. When examined in the range 250 nm to 380 nm (2.4.6), a 0.001 per cent w/v solution in a mixture of 9 volumes of ethanol (95 per cent) and 1 volume of 1 M hydrochloric acid shows
Triamterene Capsules
Triamterene Capsules contain not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of triamterene, C12H11N7.
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TRIFLUOPERAZINE HYDROCHLORIDE
IP 2007
Identification
The final solution obtained in the Assay has a bluish fluorescence and when examined in the range 250 nm to 380 nm (2.4.7), shows an absorption maximum at about 360 nm.
Trifluoperazine Hydrochloride contains not less than 99.0 per cent and not more than 101.0 per cent of C21H24F3N3S, 2HCl, calculated on the dried basis. Description. A white to pale yellow, crystalline powder; odourless or almost odourless; slightly hygroscopic. NOTE In the following procedures, the test and standard specimens and the solutions containing them should be protected by carrying out the tests without delay and in subdued light.
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 90 volumes of ethyl acetate, 10 volumes of 18 M ammonia and 10 volumes of methanol. Test solution. Dissolve a quantity of the contents of the capsules containing 0.1 g of Triamterene in sufficient dimethyl sulphoxide to produce 20 ml and dilute 2 ml to 50 ml with methanol. Reference solution. Dilute 1 volume of the test solution to 200 volumes with methanol. Apply to the plate 5 l of each solution. After development, dry the plate in air until the odour of solvent is no longer detectable and examine in ultraviolet light at 365 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Other tests. Comply with the tests stated under Capsules. Assay. Weigh accurately a quantity of the contents of 20 capsules containing about 0.1 g of Triamterene, dissolve in 50 ml of a mixture of equal volumes of glacial acetic acid and water with the aid of gentle heat, cool and add sufficient water to produce 500.0 ml. Dilute 5.0 ml of this solution to 100.0 ml with 1 M acetic acid and measure the absorbance of the resulting solution at the maximum at about 360 nm (2.4.7). Calculate the content of C 12H11N7 from the absorbance obtained by repeating the operation using triamterene RS in place of the contents of the capsules. Storage. Store protected from moisture.
Identification
A. Dissolve 20 mg in 10 ml of water, make the solution alkaline to litmus paper with 5 M sodium hydroxide and extract with two quantities, each of 20 ml, of light petroleum (60 to 80). Combine the extracts, wash with 10 ml of water, shake with 5 g of anhydrous sodium sulphate, filter and evaporate the filtrate carefully to dryness. On the residue, determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with trifluoperazine hydrochloride RS or with the reference spectrum of trifluoperazine hydrochloride. B. Complies with the test for identification of phenothiazines (2.3.3), using as reference solution a 0.2 per cent w/v solution of trifluoperazine hydrochloride RS in chloroform. C. When examined in the range 280 nm to 360 nm (2.4.7), a 0.01 per cent w/v solution in 0.1 M hydrochloric acid measured immediately after preparation shows an absorption maximum at about 305 nm. Dilute 10 ml of the solution to 100 ml with 0.1 M hydrochloric acid. When examined in the range 230 nm to 280 nm, the resulting solution shows an absorption maximum at about 256 nm; absorbance at about 256 nm, about 0.65. D. Dissolve 5 mg in 2 ml of sulphuric acid and allow to stand for 5 minutes; an orange colour is produced. E. A 10 per cent w/v solution gives the reactions of chlorides (2.3.1).
Trifluoperazine Hydrochloride
Tests
N S CF3
N N CH3 , 2HCl
Related substances. Complies with the test for related substances in phenothiazines (2.3.5), using mobile phase A. Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 1.0 per cent, determined on 1.0 g by drying in an oven at 105 at a pressure not exceeding 0.7 kPa. Assay. Weigh accurately about 0.4 g, dissolve in 50 ml of anhydrous glacial acetic acid add 10 ml of mercuric acetate
C21H24F3N3S,2HCl Mol. Wt. 480.4 Trifluoperazine hydrochloride is 10-[3-(4-methylpiperazin-1yl)propyl]-2-trifluoromethylphenothiazine dihydrochloride.
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IP 2007
TRIFLUOPERAZINE TABLETS
solution. Titrate with 0.1 M perchloric acid, using crystal violet solution as indicator. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.02402 g of C21H24F3N3S, 2HCl. Storage. Store protected from light and moisture.
Trifluoperazine Tablets
Trifluoperazine Hydrochloride Tablets
Trifluoperazine Tablets contain not less than 92.5 per cent and not more than 107.5 per cent of trifluoperazine, C21H24F3N3S. The tablets are coated. NOTE In the following procedures, the test and standard specimens and the solutions containing them should be protected by carrying out the tests without delay and in subdued light.
Trifluoperazine Injection
Trifluoperazine Hydrochloride Injection
Trifluoperazine Injection is a sterile solution of Trifluoperazine Hydrochloride in Water for Injections. Trifluoperazine Injection contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of trifluoperazine, C21H24F3N3S. Description. A clear, colourless solution. NOTE In the following procedures, the test and standard specimens and the solutions containing them should be protected by carrying out the tests without delay and in subdued light.
Identification
A. Shake a quantity of the powdered tablets containing 20 mg of trifluoperazine with 30 ml of 1 M hydrochloric acid for 10 minutes, filter, make the filtrate alkaline to litmus paper with 5 M sodium hydroxide and extract with two quantities, each of 20 ml, of light petroleum (60 to 80). Combine the extracts, wash with 10 ml of water, shake with 5 g of anhydrous sodium sulphate, filter and evaporate the filtrate carefully to dryness. On the residue, determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with trifluoperazine hydrochloride RS or with the reference spectrum of trifluoperazine hydrochloride. B. Extract a quantity of the powdered tablets containing 5 mg of trifluoperazine with 5 ml of acetone, filter and evaporate the filtrate to dryness. Add 2 ml of sulphuric acid to the residue and allow to stand for 5 minutes; an orange colour is produced.
Identification
When examined in the range 230 nm to 360 nm (2.4.7), the final solution obtained in Assay shows an absorption maximum at about 256 nm.
Tests Tests
pH (2.4.24). 4.0 to 5.0. Other tests. Complies with the tests stated under Parenteral preparations (Injections). Assay. To an accurately measured volume of the injection containing about 50 mg of trifluoperazine add 10 ml of 2 M sulphuric acid and extract with three quantities, each of 25 ml, of carbon tetrachloride. Discard the carbon tetrachloride extract after each extraction. Add 10 ml of strong ammonia solution and extract with five quantities, each of 50 ml, of cyclohexane. Extract the combined cyclohexane extracts with five quantities, each of 50 ml, of 0.1 M hydrochloric acid. Dilute the combined acid extracts to 500.0 ml and mix. Measure the absorbance of the resulting solution at the maximum at about 256 nm (2.4.7). Calculate the content of C21H24F3N3S taking 743 as the specific absorbance at 256 nm. Storage. Store protected from light. Labelling. The label states the strength in terms of the equivalent amount of trifluoperazine per ml. Uniformity of content. Comply with the test stated under Tablets. Place one tablet in a 100-ml volumetric flask, add 50 ml of a mixture of 19 volumes of water and 1 volume of hydrochloric acid, shake until the tablet has completely disintegrated, dilute to volume with the same mixture, mix and filter, rejecting the first few ml of the filtrate. Dilute suitably, if necessary with the same solvent mixture and measure the absorbance of the resulting solution at the maximum at about 256 nm (2.4.7). Calculate the content of C21H24F3N3S taking 743 as the specific absorbance at 256 nm. Other tests. Comply with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 5 mg of trifluoperazine, shake for 15 minutes with 400 ml of a mixture of 19 volumes of water and 1 volume of hydrochloric acid, dilute to 500.0 ml with the same solvent mixture, mix and filter. Measure the absorbance of the filtrate at the maximum at about 256 nm (2.4.7). Calculate the content of C21H24F3N3S taking 743 as the specific absorbance at 256 nm.
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TRIFLUPROMAZINE HYDROCHLORIDE
IP 2007
Storage. Store protected from light and moisture. Labelling. The label states the strength in terms of the equivalent amount of trifluoperazine.
D. A 10 per cent w/v solution gives the reactions of chlorides (2.3.1).
Tests
Related substances. Complies with the test for related substances in phenothiazines (2.3.5), using mobile phase A. Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105 for 2 hours.
Triflupromazine Hydrochloride
Flupromazine Hydrochloride
N S CF3
C18H19F3N2S, HCl
N CH3
CH3 , HCl
Assay. Weigh accurately about 0.8 g, dissolve in 50 ml of anhydrous glacial acetic acid add 10 ml of mercuric acetate solution. Titrate with 0.1 M perchloric acid, using crystal violet solution as indicator. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.03889 g of C18H19F3N2S, HCl.
Mol. Wt. 388.9
Triflupromazine Hydrochloride is 10-[3(dimethylamino)propyl)]-2-trifluoromethylphenothiazine hydrochloride. Triflupromazine Hydrochloride contains not less than 97.0 per cent and not more than 103.0 per cent of C18H19F3N2S, HCl, calculated on the dried basis. Description. A white to pale yellowish brown, crystalline powder; odour slight and characteristic. NOTE In the following procedures, the test and standard specimens and the solutions containing them should be protected by carrying out the tests without delay and in subdued light.
Triflupromazine Injection
Triflupromazine Hydrochloride Injection; Flupromazine Hydrochloride Injection; Flupromazine Injection
Triflupromazine Injection is a sterile solution of Triflupromazine Hydrochloride in Water for Injections. Triflupromazine Injection contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of triflupromazine hydrochloride, C18H19F3N2S, HCl. Description. A clear, almost colourless solution. NOTE In the following procedures, the test and standard specimens and the solutions containing them should be protected by carrying out the tests without delay and in subdued light.
Identification
A. Dissolve 20 mg in 10 ml of water, make the solution alkaline to litmus paper with 5 M sodium hydroxide and extract with two quantities, each of 20 ml, of light petroleum (60 to 80). Combine the extracts, wash with 10 ml of water, shake with 5 g of anhydrous sodium sulphate, filter and evaporate the filtrate carefully to dryness. On the residue, determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with triflupromazine hydrochloride RS or with the reference spectrum of triflupromazine hydrochloride. B. Complies with the test for identification of phenothiazines (2.3.3), using as reference solution a 0.2 per cent w/v solution of triflupromazine hydrochloride RS in chloroform. C. When examined in the range 230 nm to 360 nm (2.4.7), a 0.001 per cent w/v solution in 0.1 M hydrochloric acid shows an absorption maximum at about 256 nm and a less well-defined maximum at about 305 nm.
Identification
A. To an appropriate quantity of the injection add sufficient 0.1 M hydrochloric acid to produce a solution containing 0.001 per cent w/v solution of Triflupromazine Hydrochloride. When examined in the range 230 nm to 360 nm (2.4.7), the resulting solution shows an absorption maximum at about 256 nm. B. To a volume containing about 100 mg of Triflupromazine Hydrochloride add 5 ml of 8 M nitric acid and mix; a pink to amber colour develops which quickly turns dark brown and then changes to a clear solution having a yellow tint.
Tests
pH (2.4.24). 3.5 to 5.2.
1204
IP 2007
TRIMETHOPRIM
Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. To an accurately measured volume of the injection containing about 20 mg of Triflupromazine Hydrochloride add sufficient of a mixture of 19 volumes of water and 1 volume of hydrochloric acid to produce 100.0 ml and mix. Dilute 5.0 ml of the solution to 100.0 ml with the same mixture and mix. Measure the absorbance of the resulting solution at the maximum at about 256 nm (2.4.7). Calculate the content of C18H19F3N2S, HCl taking 700 as the specific absorbance at 256 nm. Storage. Store protected from light.
Comply with the test stated under Tablets. Crush one tablet and transfer to a 100-ml volumetric flask with the aid of a mixture of 19 volumes of water and 1 volume of hydrochloric acid. Shake well, dilute to volume with the same mixture and complete the procedure described under the Assay beginning at the words mix and filter,.... Calculate the content of C18H19F3N2S, HCl in the tablet. Other tests. Comply with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 50 mg of Triflupromazine Hydrochloride and shake for 15 minutes with 200 ml of a mixture of 19 volumes of water and 1 volume of hydrochloric acid. Dilute to 500.0 ml with the same solvent mixture, mix and filter, rejecting the first few ml of the filtrate. Dilute 10.0 ml to 100.0 ml with the same solvent mixture. Measure the absorbance of the filtrate at the maximum at about 256 nm (2.4.7). Calculate the content of C18H19F3N2S, HCl taking 700 as the specific absorbance at 256 nm. Storage. Store protected from light and moisture.
Triflupromazine Tablets
Triflupromazine Hydrochloride Tablets; Flupromazine Hydrochloride Tablets; Flupromazine Tablets
Triflupromazine Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of triflupromazine hydrochloride, C18H19F3N2S, HCl. NOTE In the following procedures, the test and standard specimens and the solutions containing them should be protected by carrying out the tests without delay and in subdued light.
Trimethoprim
NH2 N H2 N N OCH3 OCH3 OCH3
Identification
A. Shake a quantity of the powdered tablets containing 20 mg of Triflupromazine Hydrochloride with 30 ml of 1 M hydrochloric acid for 10 minutes, filter, make the filtrate alkaline to litmus paper with 5 M sodium hydroxide and extract with two quantities, each of 20 ml, of light petroleum (boiling range 60 to 80). Combine the extracts, wash with 10 ml of water, shake with 5 g of anhydrous sodium sulphate, filter and evaporate the filtrate carefully to dryness. On the residue, determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with triflupromazine hydrochloride RS or with the reference spectrum of triflupromazine hydrochloride. B. Extract a quantity of the powdered tablets containing 5 mg of Triflupromazine Hydrochloride with 5 ml of acetone, filter and evaporate the filtrate to dryness. When examined in the range 230 nm to 360 nm (2.4.7), a 0.001 per cent w/v solution in 0.1 M hydrochloric acid shows an absorption maximum at about 256 nm.
C14H18N4O3
Mol. Wt. 290.3
Trimethoprim is 5-(3,4,5-trimethoxybenzyl)pyrimidine-2,4diamine. Trimethoprim contains not less than 98.5 per cent and not more than 101.0 per cent of C14H18N4O3, calculated on the dried basis. Description. A white or yellowish white powder; odourless or almost odourless.
Identification
Test A may be omitted if tests B and C are carried out. Tests B and C may be omitted if test A is carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with trimethoprim RS or with the reference spectrum of trimethoprim. B. Dissolve 25 mg in 25 ml of ethanol (95 per cent) and dilute 2.0 ml to 100 ml with 0.1 M sodium hydroxide.
Tests
Uniformity of content. For tablets containing 10 mg or less.
1205
TRIMETHOPRIM AND SULPHAMETHOXAZOLE ORAL SUSPENSION
IP 2007
When examined in the range 230 nm to 360 nm (2.4.7), the resulting solution shows an absorption maximum at about 287 nm; absorbance at about 287 nm, about 0.49. C. Dissolve about 25 mg in 5 ml of 0.005 M sulphuric acid, with heating if necessary, add 2 ml of a 1.6 per cent w/v solution of potassium permanganate in 0.1 M sodium hydroxide. Heat to boiling and to the hot solution add 0.4 ml of formaldehyde solution. Mix, add 1 ml of 0.5 M sulphuric acid, mix and heat to boiling. Cool and filter. Add 2 ml of chloroform to the filtrate and shake vigorously. The chloroform layer exhibits a green fluorescence when examined in ultraviolet light at 365 nm.
Assay. Weigh accurately about 0.25 g, dissolve in 50 ml of anhydrous glacial acetic acid. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.02903 g of C14H18N4O3. Storage. Store protected from light.
Tests
Appearance of solution. A 5.0 per cent w/v solution in a mixture of 10 volumes of chloroform, 9 volumes of methanol and 2 volumes of water is not more intensely coloured than reference solution BYS7 (2.4.1). Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 85 volumes of ethyl acetate, 10 volumes of methanol, 5 volumes of water and 2 volumes of anhydrous formic acid. Test solution. Dissolve 0.4 g of the substance under examination in 10 ml of a mixture of 10 volumes of chloroform, 9 volumes of methanol and 2 volumes of water. Reference solution. A 0.008 per cent w/v solution of the substance under examination in a mixture of 10 volumes of chloroform, 9 volumes of methanol and 2 volumes of water. Apply to the plate 5 l of each solution. Allow the mobile phase to rise 17 cm. Dry the plate in a current of cold air for 5 minutes and examine in ultraviolet light at 254 nm. Place an evaporating dish containing a mixture of 2 volumes of a 1.5 per cent w/v solution of potassium permanganate, 1 volume of 7 M hydrochloric acid and 1 volume of water at the bottom of a closed tank and allow to stand for 15 minutes. Place the dried plate in the closed tank and expose to the chlorine vapour for 5 minutes. Remove the plate from the tank and remove the chlorine in a current of cold air until an area below the line of application does not give a blue colour on the addition of 0.05 ml of potassium iodide and starch solution. Spray the plate with potassium iodide and starch solution and examine in daylight. By both methods of visualisation any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 1.0 per cent, determined on 1.0 g by drying in an oven at 105.
Trimethoprim and Sulphamethoxazole Oral Suspension

Sulphamethoxazole and Trimethoprim Oral Suspension; Co-trimoxazole Oral Suspension; Co-trimoxazole Mixture
Trimethoprim and Sulphamethoxazole Oral Suspension is a suspension of Trimethoprim and Sulphamethoxazole in a suitable flavoured vehicle. It contains 5 parts of Sulphamethoxazole for 1 part, by weight, of Trimethoprim. Trimethoprim and Sulphamethoxazole Oral Suspension contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amounts of trimethoprim, C14H18N4O3, and sulphamethoxazole, C10H11N3O3S.
Identification
Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 20 volumes of chloroform, 2 volumes of methanol and 1 volume of dimethylformamide. Test solution. Add 20 ml of methanol (or a suitable volume of methanol to yield 0.4 per cent w/v solution of Trimethoprim) to 5 ml of the suspension, mix, shake with 10 g of anhydrous sodium sulphate, centrifuge and use the supernatant liquid. Reference solution (a). A 2.0 per cent w/v solution of sulphamethoxazole RS in methanol. Reference solution (b). A 0.4 per cent w/v solution of trimethoprim RS in methanol. Apply to the plate 5 l of each solution. After development, dry the plate in air, spray with dilute potassium iodobismuthate solution. One of the principal spots in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with reference solution (a) and the other corresponds to that in the chromatogram obtained with reference solution (b)
Tests
pH (2.4.24). 5.0 to 6.5. Other tests. Complies with the tests stated under Oral liquids.
1206
IP 2007
TRIMETHOPRIM AND SULPHAMETHOXAZOLE TABLETS
Assay. For trimethoprim Weigh accurately about 4 g, add 30 ml of 0.1 M sodium hydroxide, shake and extract with four quantities, each of 50 ml, of chloroform, washing each extract with the same two quantities, each of 10 ml, of 0.1 M sodium hydroxide. Reserve the combined aqueous solution and washings for the Assay for sulphamethoxazole. Extract the combined chloroform extracts with four quantities, each of 50 ml, of 1 M acetic acid. Wash the combined extracts with 5 ml of chloroform and dilute the aqueous extracts to 250.0 ml with 1 M acetic acid. To 10.0 ml of this solution add 10 ml of 1 M acetic acid and sufficient water to produce 100.0 ml, mix and measure the absorbance of the resulting solution at the maximum at about 271 nm (2.4.7). Calculate the content of C14H18N4O3 taking 204 as the specific absorbance at 271 nm. Determine the weight per ml of the suspension (2.4.29), and calculate the content of C14H18N4O3, weight in volume. For sulphamethoxazole Carry out the following procedure protected from light. Dilute the combined aqueous solution reserved in the Assay for trimethoprim to 250.0 ml with water, filter and dilute 5.0 ml of the filtrate to 200.0 ml with water (solution A). To 2.0 ml of solution A add 0.5 ml of 4 M hydrochloric acid and 1 ml of a 0.1 per cent w/v solution of sodium nitrite and allow to stand for 2 minutes. Add 1 ml of a 0.5 per cent w/v solution of ammonium sulphamate and allow to stand for 3 minutes. Add 1 ml of a 0.1 per cent w/v solution of N- (1-napthyl) ethylenediamine dihydrochloride and allow to stand for 10 minutes. Dilute the solution to 25.0 ml with water and measure the absorbance of the resulting solution at about 538 nm (2.4.7), using as the blank a solution prepared in the same manner but using 2 ml of water in place of solution A. Weigh accurately about 0.25 g of sulphamethoxazole RS, dissolve in 50 ml of 0.1 M sodium hydroxide and dilute to 250.0 ml with water. Dilute 5.0 ml of the resulting solution to 200.0 ml with water (solution B). Repeat the procedure using 2.0 ml of solution B and beginning at the words add 0.5 ml of 4 M hydrochloric acid....... Calculate the content of C10H11N3O3S from the values of the absorbances obtained. Calculate the content of C10H11N3O3S, weight in volume from the weight per ml determined in the Assay for trimethoprim. Storage. Store protected from light and moisture. The suspension should not be allowed to freeze. Labelling. The label states (1) the content of Trimethoprim and of Sulphamethoxazole in each 5 ml of the suspension; (2) that the contents should be shaken before use; (3) that a suspension containing 40 mg of Trimethoprim and 200 mg of Sulphamethoxazole in 5 ml is meant for paediatric use.
Trimethoprim And Sulphamethoxazole Tablet

Sulphamethoxazole and Trimethoprim Tablets; Cotrimoxazole Tablets
Trimethoprim and Sulphamethoxazole Tablets contain 5 parts of Sulphamethoxazole for 1 part, by weight, of Trimethoprim. Trimethoprim and Sulphamethoxazole Tablets contain not less than 92.5 per cent and not more than 107.5 per cent of the stated amounts of trimethoprim, C 14 H 18 N 4 O 3, and sulphamethoxazole, C10H11N3O3S.
Identification
A. To a quantity of the powdered tablets containing 50 mg of Trimethoprim add 30 ml of 0.1 M sodium hydroxide and extract with two quantities, each of 50 ml, of chloroform. Wash the combined chloroform extracts with two quantities, each of 10 ml, of 0.1 M sodium hydroxide and then with 10 ml of water. Combine the aqueous extract and washings (solution A) and reserve for test B. Shake with 5 g of anhydrous sodium sulphate, filter and evaporate to dryness using a rotary evaporator. On the residue, determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with trimethoprim RS or with the reference spectrum of trimethoprim. B. Filter solution A, add, dropwise, sufficient 2 M hydrochloric acid to the filtrate to make it just acidic and extract with 50 ml of ether. Wash the ether layer with 10 ml of water, shake with 5 g of anhydrous sodium sulphate, filter and evaporate the filtrate to dryness using a rotary evaporator. Dissolve the residue in the minimum volume of a 5 per cent w/v solution of sodium carbonate, add 1 M hydrochloric acid dropwise until precipitation is complete and filter. Wash the residue sparingly with water and dry at 105. On the residue, determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with sulphamethoxazole RS or with the reference spectrum of sulphamethoxazole. C. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 20 volumes of chloroform, 2 volumes of methanol and 1 volume of dimethylformamide. Test solution. Shake a quantity of the powdered tablets containing 0.4 g of Sulphamethoxazole with 20 ml of methanol and filter. Reference solution (a). A 2.0 per cent w/v solution of sulphamethoxazole RS in methanol.
1207
TRIMETHOPRIM TABLETS
IP 2007
Reference solution (b). A 0.4 per cent w/v solution of trimethoprim RS in methanol. Apply to the plate 5 l of each solution. After development, dry the plate in air, spray with dilute potassium iodobismuthate solution. One of the principal spots in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with reference solution (a) and the other corresponds to that in the chromatogram obtained with reference solution (b)
On the residue, determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with trimethoprim RS or with the reference spectrum of trimethoprim. B. Shake a quantity of the powdered tablets containing 0.1 g of Trimethoprim with 60 ml of 0.1 M hydrochloric acid for 20 minutes, add sufficient 0.1 M hydrochloric acid to produce 100 ml, filter and dilute 5 ml of the filtrate to 250 ml with 0.1 M sodium hydroxide. When examined in the range 230 nm to 360 nm (2.4.7), the resulting solution shows an absorption maximum at about 287 nm.
Tests
Other tests. Comply with the tests stated under Tablets. Assay. For trimethoprim Weigh accurately a quantity of the powdered tablets containing about 50 mg of Trimethoprim, add 30 ml of 0.1 M sodium hydroxide and extract with four quantities, each of 50 ml, of chloroform, washing each extract with the same two quantities, each of 10 ml, of 0.1 M sodium hydroxide. Combine the chloroform extracts and extract with four quantities, each of 50 ml, of 1 M acetic acid. Wash the combined extracts with 5 ml of chloroform and dilute the aqueous extracts to 250.0 ml with 1 M acetic acid. To 10.0 ml of the solution add 10 ml of 1 M acetic acid and sufficient water to produce 100.0 ml, mix and measure the absorbance of the resulting solution at the maximum at about 271 nm (2.4.7). Calculate the content of C14H18N4O3 taking 204 as the specific absorbance at 271 nm. For sulphamethoxazole Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 0.2 g of Sulphamethoxazole, dissolve as completely as possible in 60 ml of water and 10 ml of hydrochloric acid. Add 3 g of potassium bromide, cool in ice and carry out the nitrite titration (2.3.31). 1 ml of 0.1 M sodium nitrite is equivalent to 0.02533 g of C10H11N3O3S. Storage. Store protected from light and moisture. Labelling. The label states the quantities of Trimethoprim and of Sulphamethoxazole in each tablet.
Tests
Related substances. Determine by liquid chromatography (2.4.14). Test solution. Shake a quantity of the powdered tablets containing 0.2 g of Trimethoprim with 50 ml of the mobile phase, filter and use the filtrate. Reference solution. Dilute 1 volume of the test solution to 100 volumes with the mobile phase. Chromatographic system a stainless steel column 20 cm x 5 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: 0.14 per cent w/v solution of sodium perchlorate in methanol (60 per cent) adjusted to pH 3.1 with 0.1 M hydrochloric acid, flow rate. 1.3 ml per minute, spectrophotometer set at 280 nm, a 20 l loop injector. The column efficiency, determined using the peak due to trimethoprim in the chromatogram obtained with reference solution, should be at least 8,000 theoretical plates per metre. In the chromatogram obtained with the test solution the sum of the areas of any secondary peaks is not greater than the area of the principal peak in the chromatogram obtained with reference solution. Other tests. Comply with the tests stated under Tablets.
Trimethoprim Tablets
Trimethoprim Tablets contain not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of trimethoprim, C14H18N4O3.
Identification
A. Shake a quantity of the powdered tablets containing 0.1 g of Trimethoprim with 10 ml of chloroform, filter and evaporate the filtrate to dryness.
Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 0.1 g of Trimethoprim, add 100 ml of glacial acetic acid, shake for 20 minutes, dilute to 200.0 ml with glacial acetic acid and filter. To 5.0 ml of the filtrate add 15 ml of glacial acetic acid and dilute to 100.0 ml with water. Measure the absorbance of the resulting solution at the maximum at about 271 nm (2.4.7). Calculate the content of C14H18N4O3 taking 204 as the specific absorbance at 271 nm. Storage. Store protected from moisture.
1208
IP 2007
TRIPROLIDINE TABLETS
Triprolidine Hydrochloride
H N N , HCl , H2O CH3
the chromatogram obtained with the test solution any spot corresponding to Z-triprolidine is not more intense than the spot in the chromatogram obtained with reference solution (b) and any other secondary spot is not more intense than the spot in the chromatogram obtained with reference solution (a). Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Water (2.3.43). 4.0 to 6.0 per cent, determined on 0.4 g. Assay. Weigh accurately about 0.25 g and dissolve in a mixture of 50 ml of anhydrous glacial acetic acid and 0.5 ml of acetic anhydride. Titrate with 0.1 M perchloric acid, using crystal violet solution as indicator. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.01574 g of C19H22N2, HCl. Storage. Store protected from light and moisture.
C19H22N2,HCl,H2O
Mol. Wt. 332.9
Triprolidine Hydrochloride is (E)-2-(3-pyrrolidin-1-yl-1-(4tolyl)prop-1-enyl)pyridine hydrochloride monohydrate. Triprolidine Hydrochloride contains not less than 98.0 per cent and not more than 101.0 per cent of C19H22N2, HCl, calculated on the anhydrous basis. Description. A white, crystalline powder; almost odourless.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with triprolidine hydrochloride RS or with the reference spectrum of triprolidine hydrochloride. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.001 per cent w/v solution shows absorption maxima at about 230 nm and 276 nm; absorbance at about 230 nm, about 0.50 and at about 276 nm, about 0.25. C. When examined in the range 230 nm to 360 nm (2.4.7), a 0.002 per cent w/v solution in 0.05 M sulphuric acid shows an absorption maximum at about 290 nm; absorbance at about 290 nm, about 0.6. D. Dissolve 0.1 g in 2 ml of 2 M hydrochloric acid and add 0.5 ml of potassium mercuri-iodide solution; a pale yellow precipitate is produced. E. A 5 per cent w/v solution gives the reactions of chlorides (2.3.1).
Triprolidine Tablets
Triprolidine Hydrochloride Tablets
Triprolidine Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of triprolidine hydrochloride, C19H22N2,HCl,H2O.
Identification
A. Extract a quantity of the powdered tablets containing 10 mg of Triprolidine Hydrochloride with ether, filter, discard the ether extract and dry the residue. Extract the residue with chloroform, filter and evaporate the filtrate to dryness. Add 0.1 ml of ether, stir and allow to evaporate. On the residue, determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with triprolidine hydrochloride RS or with the reference spectrum of triprolidine hydrochloride. B. In the test for Related substances, the principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with reference solution (b).
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel HF254. Mobile phase. A mixture of equal volumes of 2-butanone and dimethylformamide. Test solution. Dissolve 0.1 g of the substance under examination in 10 ml of methanol. Reference solution (a). Dissolve 10 mg of the substance under examination in 100 ml of methanol. Reference solution (b). A 0.02 per cent w/v solution of Z-triprolidine RS in methanol. Apply to the plate 5 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. In
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel HF254. Mobile phase. A mixture of equal volumes of 2-butanone and dimethylformamide. Test solution. Extract a quantity of powdered tablets containing 10 mg of Triprolidine Hydrochloride with methanol, filter, evaporate to dryness and dissolve the residue in 1 ml of methanol.
1209
TRISODIUM EDETATE CONCENTRATE FOR INJECTION
IP 2007
Reference solution (a). A 0.02 per cent w/v solution of Z-triprolidine RS in methanol. Reference solution (b). A 1 per cent w/v solution of triprolidine hydrochloride RS in methanol. Reference solution (c). A 0.01 per cent w/v solution of triprolidine hydrochloride RS in methanol. Apply to the plate 5 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. In the chromatogram obtained with the test solution any spot corresponding to Z-triprolidine is not more intense than the spot in the chromatogram obtained with reference solution (a) and any other secondary spot is not more intense than the spot in the chromatogram obtained with reference solution (c). Uniformity of content. For tablets containing 10 mg or less. Comply with the test stated under Tablets. Powder one tablet, weigh accurately a quantity of the powder containing 7.5 mg of Triprolidine Hydrochloride and carry out the Assay beginning at the words add 15 ml of water..... Calculate the content of C19H22N2,HCl,H2O in the tablet. Other tests. Comply with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 7.5 mg of Triprolidine Hydrochloride, add 15 ml of water and 1 g of sodium chloride, shake for 2 to 3 minutes and add sufficient 5 M sodium hydroxide to make it alkaline. Extract with four quantities, each of 20 ml, of ether and wash the combined extracts with two quantities, each of 5 ml, of a mixture of equal volumes of a saturated solution of sodium chloride and water. Extract the ether solution with 20 ml of 0.1 M hydrochloric acid, wash the ether with two quantities, each of 5 ml, of water and add the washings to the acid extract. Heat on a water-bath for 30 minutes, cool and add sufficient water to produce 50.0 ml. Dilute 10.0 ml to 100.0 ml with 0.1 M hydrochloric acid. Measure the absorbance of the resulting solution at the maximum at about 290 nm (2.4.7). Calculate the content of C19H22N2, HCl, H2O taking 290 as the specific absorbance at 290 nm. Storage. Store protected from light and moisture.
Disodium Edetate and Sodium Hydroxide. It should be diluted with a suitable diluent in accordance with the manufacturers instructions. Trisodium Edetate Concentrate for Injection contains not less than 19.0 per cent w/v and not more than 21.0 per cent w/v solution of trisodium edetate, C10H13N2Na3O8. Description. A colourless solution.
Identification
A. Dissolve 2 g in 25 ml of water, add 6 ml of lead nitrate solution, shake and add 3 ml of potassium iodide solution; no yellow precipitate is produced. Make alkaline to red litmus paper with 2 M ammonia and add 5 ml of ammonium oxalate solution; no precipitate is produced. B. Dissolve 0.5 g in 10 ml of water, add 0.5 ml of a 10 per cent w/v solution of calcium chloride, make alkaline to red litmus paper with 2 M ammonia and add 3 ml of ammonium oxalate solution; no precipitate is produced. C. Evaporate to dryness and ignite. The residue gives the reactions of sodium salts (2.3.1).
Tests
pH (2.4.24). 7.0 to 8.0. Pyrogens (2.2.8). Complies with the test, using per kg of the rabbits weight 5 ml of a solution prepared in the following manner. To 1 volume of the injection add 2.5 volumes of calcium gluconate solution. Dilute the resulting solution with sufficient water for injections to give a final concentration of 5.0 per cent w/v solution of trisodium edetate. Other tests. Complies with the tests stated under Parenteral Preparations (Concentrated Solutions for Injection). Assay. Dilute 10.0 ml to 100.0 ml with water and use this solution to titrate a mixture of 25.0 ml of 0.05 M magnesium sulphate and 10 ml of ammonia buffer pH 10.9 using mordant black II mixed triturate as indicator. 1 ml of 0.05 M magnesium sulphate is equivalent to 0.01791 g of C10H13N2Na3O8. Storage. Store in hermetically-sealed, lead-free glass containers.
Trisodium Edetate Concentrate for Injection

Trisodium Edetate Concentrate for Injection is a sterile solution in Water for Injections containing 20 per cent w/v solution of trisodium edetate prepared by the interaction of
Labelling. The label states (1) the strength in terms of anhydrous trisodium edetate contained in a suitable dosevolume; (2) Trisodium Edetate Concentrate for Injection; (3) that the solution must be diluted with either Sodium Chloride Intravenous Infusion or Dextrose Intravenous Infusion before administration.
1210
IP 2007
TROPICAMIDE EYE DROPS
Tropicamide
N H3C N O
C17H20N2O2 Mol. Wt. 284.4
more than one such spot is more intense than the spot in the chromatogram obtained with reference solution (b).
OH
Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 80 at a pressure not exceeding 0.7 kPa for 4 hours. Assay. Weigh accurately about 0.5 g, dissolve in 80 ml of anhydrous glacial acetic acid. Titrate with 0.1 M perchloric acid, using 1-naphtholbenzein solution as indicator. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.02844 g of C17H20N2O2. Storage. Store protected from light and moisture.
Tropicamide is (RS)-N-ethyl-3-hydroxy-2-phenyl-N-(pyrid-4ylmethyl)propionamide Tropicamide contains not less than 99.0 per cent and not more than 101.0 per cent of C17H20N2O2, calculated on the dried basis. Description. A white or almost white, crystalline powder; odourless or almost odourless.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with tropicamide RS or with the reference spectrum of tropicamide. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.003 per cent w/v solution in 0.1 M hydrochloric acid shows an absorption maximum only at about 254 nm; absorbance at about 254 nm, about 0.54. C. Dissolve 5 mg in 3 ml of a mixture of 9 ml of acetic anhydride, 1 ml of 6 M acetic acid and 0.1 g of citric acid and heat on a water-bath for 5 to 10 minutes; a reddish yellow colour is produced.
Tropicamide Eye Drops

Tropicamide Eye Drops are a sterile solution of Tropicamide in Purified Water. They may contain stabilisers, suitable antimicrobial agents and suitable substances to increase the viscosity of the solution. Tropicamide Eye Drops contain not less than 95.0 per cent and not more than 110.0 per cent of the stated amount of tropicamide, C17H20N2O2.
Identification
A. Shake a volume containing 20 mg of Tropicamide with 10 ml of chloroform, dry the chloroform layer over anhydrous sodium sulphate, filter and evaporate the filtrate to dryness. Dissolve the residue in minimum quantity of chloroform, add dropwise to finely powdered potassium bromide IR, mix and dry at 60. On the residue determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with tropicamide RS or with the reference spectrum of tropicamide. B. When examined in the range 230 nm to 360 nm (2.4.7), the final solution obtained in the Assay shows an absorption maximum at about 254 nm.
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 95 volumes of chloroform, 5 volumes of methanol and 0.5 volume of strong ammonia solution. Test solution. Dissolve 0.1 g of the substance under examination in 10 ml of chloroform. Reference solution (a). A 0.005 per cent w/v solution of the substance under examination in chloroform. Reference solution (b). A 0.002 per cent w/v solution of the substance under examination in chloroform. Apply to the plate 20 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (a) and not
Tests
pH (2.4.24). 4.0 to 5.8. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 95 volumes of chloroform, 5 volumes of methanol and 0.5 volume of strong ammonia solution.
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TROXIDONE
IP 2007
Test solution. A volume of the eye drops containing 0.2 mg of Tropicamide. Reference solution (a). Dilute 1 volume of the eye drops to 200 volumes with chloroform. Reference solution (b). Dilute 1 volume of the eye drops to 500 volumes with chloroform. Apply to the plate 20 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (a) and not more than one such spot is more intense than the spot in the chromatogram obtained with reference solution (b). Other tests. Comply with the tests stated under Eye Drops. Assay. To a volume containing about 30 mg of Tropicamide add sufficient water to produce 100.0 ml. To 10.0 ml of the resulting solution add 2 ml of a 10 per cent w/v solution of anhydrous sodium carbonate and extract with four quantities, each of 20 ml, of chloroform. Wash the combined chloroform extracts with 25 ml of phosphate buffer pH 6.5. Wash the aqueous layer with 10 ml of chloroform, combine the chloroform extracts and shake with four quantities, each of 20 ml, of 0.5 M sulphuric acid. Combine the acid extracts, dilute to 100.0 ml with 0.5 M sulphuric acid and measure the absorbance of the resulting solution at the maximum at about 254 nm (2.4.7). Calculate the content of C17H20N2O2 taking 172 as the specific absorbance at 254 nm. Storage. Store in a refrigerator (8 to 15). It should not be allowed to freeze.
Identification
Test A may be omitted if tests B and C are carried out. Tests B and C may be omitted if test A is carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with troxidone RS. B. To 2 ml of a 5.0 per cent w/v solution in carbon dioxide-free water (solution A) add 1 ml of barium hydroxide solution; a white precipitate is produced which dissolves on the addition of 1 ml of 2 M hydrochloric acid. C. Dissolve 0.3 g in a mixture of 5 ml of ethanolic potassium hydroxide solution and 5 ml of ethanol (95 per cent) and allow to stand for 10 minutes. Add 0.05 ml of phenolphthalein solution and carefully add hydrochloric acid until the solution is neutral. Evaporate to dryness on a water-bath, shake the residue with four quantities, each of 5 ml, of ether, filter the combined ether extracts and evaporate the filtrate to dryness. The residue, after recrystallisation from 5 ml of toluene and drying, melts at about 80 (2.4.21).
Tests
Appearance of solution. Solution A is clear (2.4.1), and colourless (2.4.1). Acidity or alkalinity. To 10 ml of solution A add 0.1 ml of methyl red solution. Not more than 0.1 ml of 0.01 M hydrochloric acid or 0.01 M sodium hydroxide is required to change the colour of the solution. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying over anhydrous silica gel for 6 hours.
Troxidone
Trimethadione
H3 C H3 C O O N CH3 O
Assay. Determine by gas chromatography (2.4.13). Test solution. Weigh accurately 0.1 g of the substance under examination and dissolve in sufficient solutions prepared by dissolving 0.125 g of 1-decanol (internal standard) in sufficient ethanol to produce 25.0 ml (solution B). Reference solution. Dissolve 0.1 g of troxidone RS in sufficient solution B to produce 10.0 ml. Mol. Wt. 143.1 Chromatographic system a stainless steel column 0.75 m x 3 mm, packed with porous polymer beads (120 to 150 m), temperature: column 210, inlet port at 240 and detector at 270, flow rate. 30 ml per minute of the carrier gas. Inject 1 l of the test solution and the reference solution.
C6H9NO3
Troxidone is 3,5,5-trimethyloxazolidine-2,4-dione. Troxidone contains not less than 98.0 per cent and not more than 102.0 per cent of C6H9NO3, calculated on the dried basis. Description. Colourless or almost colourless crystals; odour, slightly camphoraceous.
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IP 2007
TUBOCURARINE CHLORIDE
Calculate the content of C6H9NO3. Storage. Store protected from light and moisture.
Reference solution. Dissolve 0.1 g of troxidone RS in sufficient solution B to produce 10.0 ml. Chromatographic system a stainless steel column 0.75 m x 3 mm, packed with porous polymer beads (120 to 150 m), temperature: column 210, inlet port at 240 and detector at 270, flow rate. 30 ml per minute of the carrier gas. Inject 1 l of the test solution and the reference solution. Calculate the content of C6H9NO3 in the capsules. Storage. Store protected from moisture.
Troxidone Capsules
Trimethadione Capsules
Troxidone Capsules contain not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of troxidone, C6H9NO3.
Identification
To a quantity of the contents of the capsules containing 1 g of Troxidone add 25 ml of ether, set aside in a stoppered flask for 20 minutes, decant the ether through a filter, and if an insoluble residue remains, digest it with another 10-ml portion of ether as before, and filter into the first ether filtrate. Evaporate the ether extracts to dryness with the aid of a current of air and dry the residue at a pressure of 2 kPa for 2 hours. The residue complies with the following tests. Test A may be omitted if tests B and C are carried out. Tests B and C may be omitted if test A is carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with troxidone RS or with the reference spectrum of troxidone. B. To 2 ml of a 5.0 per cent w/v solution in carbon dioxide-free water (solution A) add 1 ml of barium hydroxide solution; a white precipitate is produced which dissolves on the addition of 1 ml of 2 M hydrochloric acid. C. Dissolve 0.3 g in a mixture of 5 ml of ethanolic potassium hydroxide solution and 5 ml of ethanol (95 per cent) and allow to stand for 10 minutes. Add 0.05 ml of phenolphthalein solution and carefully add hydrochloric acid until the solution is neutral. Evaporate to dryness on a water-bath, shake the residue with four quantities, each of 5 ml, of ether, filter the combined ether extracts and evaporate the filtrate to dryness. The residue, after recrystallisation from 5 ml of toluene and drying, melts at about 80 (2.4.21).
Tubocurarine Chloride
H3 CO OH O
CH3 Cl, HCl, 5H2O
H3C H3C N
CH2 O OH OCH3
C37H41ClN2O6,HCl,5H2O
Mol. Wt. 771.7
Tubocurarine Chloride is 7,12-dihydroxy-6,6-dimethoxy2,2,2-trimethyltubocuraranium chloride hydrochloride pentahydrate. Tubocurarine Chloride contains not less than 98.0 per cent and not more than 102.0 per cent of C37H41ClN2O6,HCl, calculated on the anhydrous basis. Description. A white or yellowish white, crystalline powder.
Identification Tests
Other tests. Comply with the tests stated under Capsules. Assay. Determine by gas chromatography (2.4.13). Test solution. To a quantity of the contents of the capsules containing about 1.0 g of Troxidone add 25 ml of a 0.5 per cent w/v solution of 1-decanol (internal standard) in ethanol, shake for 30 minutes, add sufficient of internal standard solution to produce 100.0 ml, mix and centrifuge. Use the supernatant liquid. Test A may be omitted if tests B, C, D and E are carried out. Tests B, C and D may be omitted if tests A and E are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with tubocurarine chloride RS or with the reference spectrum of tubocurarine chloride. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.005 per cent w/v solution shows an absorption maximum at
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TUBOCURARINE INJECTION
IP 2007
about 280 nm and a minimum at about 255 nm, 0.56 to 0.62, calculated on the anhydrous basis. C. To 1 ml of a 2.5 per cent w/v solution add 0.2 ml of ferric chloride solution and heat in a water-bath for 1 minute; a green colour is produced; 1 ml of water treated in the same manner gives a brown colour. D. To 20 ml of a 0.05 per cent w/v solution add 0.2 ml of sulphuric acid and 2 ml of a 1 per cent w/v solution of potassium iodate, mix and warm on a water-bath for 30 minutes; a yellow colour is produced. E. Gives reaction A of chlorides and the reactions of alkaloids (2.3.1).
water and 2 volumes of ferric chloride solution, prepared immediately before use. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (a) and not more than one such spot is more intense than the spot in the chromatogram obtained with reference solution (b). Sulphated ash (2.3.18). Not more than 0.25 per cent, determined on 0.2 g. Water (2.3.43). 9.0 to 12.0 per cent, determined on 0.3 g. Assay. Weigh accurately about 25 mg, dissolve in sufficient water to produce 500.0 ml and measure the absorbance of the resulting solution at the maximum at about 280 nm (2.4.7). Calculate the content of C37H41ClN2O6,HCl from the absorbance obtained by repeating the operation using 25 mg, accurately weighed, of tubocurarine chloride RS in place of the substance under examination. Storage. Store protected from moisture.
Tests
Appearance of solution. Dissolve 0.5 g in sufficient carbon dioxide-free water to produce 50.0 ml (solution A). Solution A is clear (2.4.1), and not more intensely coloured than reference solution YS6 (2.4.1). pH (2.4.24). 4.0 to 6.0, determined in Solution A. Specific optical rotation (2.4.22). +210 to +222, determined in solution A 3 hours after preparation. Chloroform-soluble substances. Not more than 2 per cent, determined by the following method. Dissolve 0.25 g in 150 ml of water contained in a separating funnel with a grease-free stopcock. Add 5 ml of a saturated solution of sodium bicarbonate and extract with three quantities, each of 20 ml, of chloroform. Wash the combined chloroform extracts with 10 ml of water, filter the chloroform solution into a tared beaker and wash the filter with two successive quantities, each of 5 ml, of chloroform. Add the washings to the filtrate. Remove the chloroform on a water-bath, dry the residue at 105 for 1 hour, cool and weigh. The residue does not dissolve in 10 ml of water but dissolves on the addition of 1 ml of 2 M hydrochloric acid. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. The lower layer of a mixture of equal volumes of chloroform, methanol and a 12.5 per cent w/v solution of trichloroacetic acid. Test solution. Dissolve 0.25 g of the substance under examination in 10 ml of water. Reference solution (a). A 0.0375 per cent w/v solution of the substance under examination in water. Reference solution (b). A 0.01875 per cent w/v solution of the substance under examination in water. Apply to the plate 5 l of each solution. After development, dry the plate in a current of cold air and spray with a mixture of 1 volume of potassium ferricyanide solution, 1 volume of
Tubocurarine Injection
Tubocurarine Chloride Injection
Tubocurarine Injection is a sterile solution of Tubocurarine Chloride in Water for Injections. It may contain suitable buffering agents. Tubocurarine Injection contains not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of tubocurarine chloride, C37H41ClN2O6,HCl,5H2O. Description. A colourless or faintly coloured solution.
Identification
A. Mix a volume containing 15 mg of Tubocurarine Chloride with 5 ml of acetone, evaporate the liquid at a pressure of 2 kPa and add successive quantities of 2 ml of acetone, evaporating each quantity at a pressure of 2 kPa until a dry residue is obtained. On the residue, determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with tubocurarine chloride RS or with the reference spectrum of tubocurarine chloride. B. Dilute 1 ml to 30 ml with water. To 1 ml of the resulting solution add 0.5 ml of mercuric nitrate solution; a cherry-red colour slowly develops.
Tests
pH (2.4.24). 4.0 to 6.0.
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IP 2007
TUBOCURARINE INJECTION
Optical rotation (2.4.22).+0.172 to +0.206 for each mg of tubocurarine chloride, C37H41ClN2O6,HCl,5H2O per ml stated on the label. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. The lower layer of a mixture of equal volumes of chloroform, methanol and a 12.5 per cent w/v solution of trichloroacetic acid. Test solution. A volume of the injection containing 10 mg of Tubocurarine Chloride diluted to 1 ml. Reference solution (a). Dilute 3 volumes of test solution to 200 volumes with water. Reference solution (b). Dilute 1 volume of reference solution (a) to 2 volumes with water. Apply to the plate 10 l of each solution. After development, dry the plate in a current of cold air and spray with a mixture of
1 volume of potassium ferricyanide solution, 1 volume of water and 2 volumes of ferric chloride solution, prepared immediately before use. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (a) and not more than one such spot is more intense than the spot in the chromatogram obtained with reference solution (b). Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. Dilute a volume containing about 50 mg of Tubocurarine Chloride to 1000.0 ml with water and measure the absorbance of the resulting solution at the maximum at about 280 nm (2.4.7). Calculate the content of C37H41ClN2O6,HCl,5H2O taking 105 as the specific absorbance at 280 nm. Storage. Store protected from moisture.
1215
MONOGRAPHS
U
Undecenoic Acid Urea Urea Cream Urokinase .... .... .... ....
1217
IP 2007
UREA
Undecenoic Acid
Undecylenic Acid
H H2C COOH
1 ml of 0.5 M sodium hydroxide is equivalent to 0.09214 g of C11H20O2. Storage. Store protected from light and moisture.
Urea
O H2 N NH2
C11H20O2 Undecenoic Acid is 10-undecenoic acid.
Mol. Wt. 184.3
Undecenoic Acid contains not less than 97.0 per cent and not more than 102.0 per cent of C11H20O2. Description. A white or very pale yellow, crystalline mass or colourless or pale yellow liquid; odour, characteristic.
CH4N2O Urea is the diamide of carbonic acid.
Mol. Wt. 60.1
Identification
A. Dissolve 0.1 g in a mixture of 2 ml of 1 M sulphuric acid and 5 ml of glacial acetic acid and add dropwise 0.25 ml of potassium permanganate solution; the colour of the permanganate solution is discharged. B. Boil 2 g under a reflux condenser with 2 ml of freshly distilled aniline for 10 minutes, allow to cool, add 30 ml of ether and extract with three quantities, each of 20 ml, of 2 M hydrochloric acid and then with 20 ml of water. Evaporate the organic layer to dryness on a water-bath. The residue, after recrystallising twice from ethanol (70 per cent) and drying over phosphorus pentoxide at a pressure of 1.5 to 2.5 kPa for 3 hours melts at 66 to 68.
Urea contains not less than 99.0 per cent and not more than 101.0 per cent of CH4N2O, calculated on the dried basis. Description. A white, crystalline powder or transparent crystals; odourless or almost odourless, but may gradually develop a slight odour of ammonia upon long standing; slightly hygroscopic.
Identification
Test A may be omitted if tests B and C are carried out. Tests B and C may be omitted if test A is carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with urea RS or with the reference spectrum of urea. B. Heat 0.5 g in a test-tube; it liquefies, and ammonia is evolved which is recognised by its characteristic odour. Heat further until the liquid is turbid, cool and dissolve in 10 ml of water. Add 1 ml of a 10 per cent w/v solution of sodium hydroxide and 0.05 ml of copper sulphate solution; a reddish violet colour is produced. C. Dissolve 0.1 g in 1 ml of water and add 1 ml of nitric acid; a white, crystalline precipitate is produced.
Tests
Congealing range (2.4.10). 21 to 24. Refractive index (2.4.27). 1.447 to 1.450. Peroxide value (2.3.35). Not more than 10. Iodine value (2.3.28). 131 to 140. Fixed and mineral oils. Boil 1.0 g with 25 ml of water and 5 ml of sodium carbonate solution for 3 minutes. The hot solution is not more opalescent than opalescence standard OS2 (2.4.1). Water-soluble acids. Shake 2.0 g with 20 ml of warm water, allow to separate and filter the aqueous layer through a moistened filter paper. To 5 ml of the filtrate add 0.01 ml of dilute phenolphthalein solution. Not more than 0.1 ml of 0.1 M sodium hydroxide is required to change the colour of the solution. Heavy metals (2.3.13). 2.0 g complies with the limit test for heavy metals, Method B (10 ppm). Sulphated ash (2.3.18). Not more than 0.15 per cent. Assay. Weigh accurately about 0.75 g, dissolve in 10 ml of ethanol (95 per cent) and titrate with 0.5 M sodium hydroxide using 0.1 ml of dilute phenolphthalein solution as indicator.
Tests
Appearance of solution. Solution A is clear (2.4.1), and colourless (2.4.1). Alkalinity. To 10 ml of a 5.0 per cent w/v solution (solution A) add 0.1 ml of methyl red solution and 0.4 ml of 0.01 M hydrochloric acid; the resulting solution is red to orange. Biuret. Not more than 0.1 per cent, determined by the following method. To 10 ml of a 20 per cent w/v solution add 5 ml of water, 0.5 ml of a 0.5 per cent w/v solution of copper sulphate and 0.5 ml of 10 M sodium hydroxide and allow to stand for 5 minutes. Any reddish violet colour obtained is not more intense than that in a standard prepared at the same time and in the same manner using 10 ml of a 0.02 per cent w/v solution of biuret in place of the substance under examination.
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UREA CREAM
IP 2007
Ethanol-insoluble matter. Not more than 0.04 per cent, determined by the following method. Dissolve 5.0 g in 50 ml of warm ethanol (95 per cent), filter through a tared filter, wash the filter with 20 ml of warm ethanol (95 per cent) and dry at 105 for 1 hour. Heavy metals (2.3.13). Dissolve 1.0 g in 20 ml of water and 5 ml of 0.1 M hydrochloric acid. The solution complies with the limit test for heavy metals, Method A (20 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 1.0 per cent, determined on 1.0 g by drying in an oven at 105 for 1 hour. Assay. Weigh accurately about 0.5 g, dissolve in sufficient of a 10 per cent v/v solution of sulphuric acid to produce 100.0 ml and mix. Place 5.0 ml of the resulting solution in a long-necked flask, add 10 ml of sulphuric acid and heat gently until evolution of gas ceases. Boil gently for 10 minutes, cool, cautiously add 40 ml of water, cool again and place in a steamdistillation apparatus. Add 50 ml of 10 M sodium hydroxide and distil immediately by passing steam through the mixture. Continue the distillation for 1 hour, collecting the distillate in 40 ml of a 4 per cent w/v solution of boric acid. Titrate with 0.1 M hydrochloric acid, using 0.25 ml of methyl redmethylene blue solution as indicator. Carry out a blank titration. 1 ml of 0.1 M hydrochloric acid is equivalent to 0.003003 g of CH4N2O. Storage. Store protected from moisture.
Apply to the plate 10 l of each solution. After development, dry the plate in air, spray with a solution containing 0.5 per cent w/v solution of 4-dimethylaminobenzaldehyde and 0.5 per cent v/v of sulphuric acid in ethanol. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with reference solution (a). The chromatogram obtained with reference solution (b) shows a single, compact spot. B. To a quantity containing 0.1 g of Urea add 50 ml of water and heat until dispersed, cool in ice and filter through glass wool. Adjust the pH to 6.0 to 7.0 using 0.1 M hydrochloric acid or 0.1 M sodium hydroxide as necessary. To 5 ml add 5 ml of a 0.1 per cent w/v suspension of urease-active meal and allow to stand for 30 minutes in a stoppered flask at 37. When the resulting solution is heated in a water-bath a vapour is produced that turns moist red litmus paper blue.
Tests
Other tests. Comply with the tests stated under Creams. Assay. Weigh accurately a quantity containing about 42 mg of Urea, shake with 150 ml of hot water for 20 minutes, allow to cool and dilute to 500.0 ml with water. Filter through a fine glass microfibre filter paper or by any other means, transfer 1.0 ml of the filtrate to a 100-ml volumetric flask, add 2 ml of a 0.1 per cent w/v suspension of urease-active meal, stopper the flask and allow to stand for 15 minutes at 37. Immediately add 25 ml of a solution containing 12 g of sodium salicylate and 0.24 g of sodium nitroprusside in 200 ml and 25 ml of a solution prepared by diluting a volume of sodium hypochlorite solution containing 0.66 g of available chlorine with 0.2 M sodium hydroxide to 1000 ml. Mix well, allow to stand at 37 for 5 minutes and dilute to 100.0 ml with water. Measure the absorbance of the resulting solution at the maximum at about 665 nm (2.4.17), using as the blank a solution prepared in the same manner but using 1.0 ml of water in place of 1.0 ml of the filtrate. Calculate the content of CH4N2O from the absorbance obtained by using 42 mg of urea RS in place of the substance under examination. Storage. Store in accordance with the instructions of the manufacturer.
Urea Cream
Urea Cream contains Urea in a suitable basis. Urea Cream contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of urea, CH4N2O.
Identification
A. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. For the first development use 2,2,4trimethylpentane. Dry the plate in air. For the second development use a mixture of 99 volumes of ethanol and 1 volume of strong ammonia solution. Test solution. Disperse with heating a quantity of the cream containing 50 mg of Urea in 1 ml of water, cool, add 4 ml of acetone, mix and filter. Reference solution (a). Dissolve 50 mg of urea RS in 1 ml of water and add 4 ml of acetone. Reference solution (b). A mixture of equal volumes of the test solution and reference solution (a).
Urokinase
Urokinase is an enzyme, obtained from human urine that activates plasminogen. It consists of a mixture of low molecular weight and high molecular weight forms, the high molecular weight form being predominant. The molecular weights of the low and high molecular weight forms are 33,000 and 54,000 respectively.
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IP 2007
UROKINASE
It is prepared in conditions designed to minimise microbial and viral contamination. In particular, adequate measures, such as heating the substance in solution at 60 for 10 hours, are taken to inactivate viruses. Urokinase contains not less than 70,000 Units of urokinase activity per mg of protein. Description. A white or almost white, amorphous powder.
examination and multiplying the result by 6.25 to obtain the content of protein. Hepatitis-B surface antigen. Examine by a suitably sensitive method such as radio-immunoassay; hepatitis-B surface antigen is not detected. Abnormal toxicity (2.2.1). Complies with the test, using a solution containing 2,500 Units in 0.5 ml of saline solution. Thromboplastic contaminants. Dissolve suitable quantities of the substance under examination in barbitone buffer pH 7.4 to obtain solutions containing 5000, 2500, 1250, 625 and 312 Units per ml. Into each of six haemolysis tubes, 1 cm in internal diameter, place 0.1 ml of citrated rabbit plasma. Add 0.1 ml of one of each of the solutions of the substance under examination to each of five of the tubes and 0.1 ml of barbitone buffer pH 7.4 to the sixth (control). Incubate the six tubes at 25 0.5 for 5 minutes and then add 0.1 ml of a 0.3675 per cent w/v solution of calcium chloride. Using a stop-watch, measure the clotting time for each solution and the control. Plot the shortening of the recalcification time (control clotting time minus clotting time for each solution) against log concentration. Extrapolate the best-fitting straight line through the five points until it reaches the log concentration axis. The urokinase activity at the intersection point represents the limit concentration for clotting activity (zero clotting activity). The zero clotting activity is not less than 150 Units per ml. Vasoactive substances. Anesthetise a rabbit by intraperitoneal injection of 0.15 g of phenobarbitone sodium per kg of body weight. Dissolve in normal saline solution a sufficient quantity of the substance under examination to give a solution containing 40,000 Units per ml. Administer by intravenous infusion at a rate of 1 ml per minute a sufficient volume of the solution of the substance under examination such that the dose is 20,000 Units per kg of body weight. Measure the arterial pressure and heart rate at intervals of 15 minutes for 5 hours after the infusion. No significant and lasting alterations in arterial pressure or heart rate are produced, except those arising from the effects of the anaesthetic. Assay. The potency of urokinase is determined by comparing its ability to activate human plasminogen to form plasmin with that of the Standard Preparation. The plasmin generated is determined by measurement of the time taken to lyse a fibrin clot under the conditions of a suitable method of Assay. Standard Preparation The Standard Preparation is the 1st International Reference Preparation for Urokinase, human, established in 1968, consisting of partially purified freeze-dried urokinase from human urine with lactose (supplied in ampoules containing 4800 Units of urokinase activity) or another suitable preparation the activity of which has been determined in relation to the International Reference Preparation.
Identification
A. Place 0.5 ml of citrated human plasma and 0.5 ml of citrated bovine plasma in two separate haemolysis tubes maintained in a water-bath at 37. To each tube add 0.1 ml of a solution of the substance under examination containing 1000 Units per ml in phosphate buffer pH 7.4 and 0.1 ml of a solution of thrombin containing 20 Units per ml in phosphate buffer pH 7.4 and shake immediately; in both tubes, a clot forms and lyses within 30 minutes. B. Carry out a suitable immunodiffusion test.
Tests
Appearance of solution. A 0.1 per cent w/v solution in water is clear (2.4.1), and colourless (2.4.1). Molecular fractions. Determine by size-exclusion chromatography (2.4.16) Test solution. Dissolve 0.1 g of the substance under examination in 100 ml of 0.02 M phosphate buffer pH 8.0. Chromatographic system a column 90 cm x 16 mm, packed with a cross-linked dextran suitable for fractionation of proteins in the range of molecular weight from 4000 to 150,000 (such as Sephadex G-100), at 5, mobile phase: 1.75 per cent w/v solution of sodium chloride in 0.02 M phosphate buffer pH 8.0, flow rate. 6 ml per hour, spectrophotometer set at 280 nm. Apply 1 ml of the test solution to the column, rinse twice with 0.5-ml portions of the buffer and then carry out the elution. The eluate may be collected in 1-ml fractions. Plot the individual absorbances on a graph. Draw perpendicular lines towards the axis of the abscissae from the minima before the high molecular weight peak, between the high and the low molecular weight peaks, and after the low molecular weight peak. Combine separately the high and low molecular weight fractions and for each combined fraction determine the activity by the method described under Assay. The ratio of the activity in the combined high molecular weight fraction to that in the combined low molecular weight fraction is not less than 2.0. Total protein. Determine by Method C for the determination of nitrogen (2.3.30), using 10 mg of the substance under
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UROKINASE
IP 2007
Method Unless otherwise prescribed, use phosphate buffer pH 7.4 containing 3 per cent w/v solution of bovine albumin for the preparation of solutions and dilutions. Prepare a solution of the Standard Preparation containing 1000 Units of urokinase activity per ml and prepare a solution of the preparation under examination expected to have the same concentration; keep the solutions in ice and use within 6 hours. Prepare three 1.5-fold serial dilutions of the solution of the Standard Preparation so that the longest clot-lysis time is less than 20 minutes and the shortest clot-lysis time is greater than 3 minutes. Prepare three similar dilutions of the solution of the preparation under examination. Keep the solutions in ice and use within 1 hour. Using 24 tubes 8 mm in diameter, label the tubes S1, S2, S3 for the dilutions of the Standard Preparation and T1, T2, T3 for the dilutions of the preparation under examination, allocating four tubes to each dilution. Place the tubes in ice. Into each tube introduce 0.2 ml of the appropriate dilution, 0.2 ml of phosphate buffer pH 7.4 containing 3 per cent w/v solution of bovine albumin and 0.1 ml of a solution containing 20 Units of thrombin per ml. Place the tubes in a water-bath at 37 and allow to stand for 2 minutes to attain temperature equilibrium. Using an automatic pipette, introduce into the bottom of the first tube 0.5 ml of a 1.0 per cent w/v solution of bovine euglobulin fraction ensuring mixing. At 5-second intervals introduce successively
into the remaining tubes 0.5 ml of a 1.0 per cent w/v solution of bovine euglobulin fraction. Using a stop-watch, measure for each tube the time in seconds that elapses between the addition of the euglobulin and the lysis of the clot. Using the logarithms of the lysis times, calculate the result of the assay by standard statistical methods. The estimated potency is not less than 90.0 per cent and not more than 111.0 per cent of the stated potency. The fiducial limits of error are not less than 80 per cent and not more than 125 per cent of the stated potency. Urokinase intended for use in the manufacture of parenteral preparations or ophthalmic preparations complies with the following additional requirements. Bacterial endotoxins (2.2.3). Not more than 0.002 Endotoxin Unit per Unit of urokinase activity. Sterility (2.2.11). Complies with the test for sterility. Storage. Store protected from light in a refrigerator (2 to 8). The containers should be sterile, tamper-evident and sealed so as to exclude micro-organisms. Labelling. The label states (1) the number of Units of urokinase activity in the container; (2) the number of Units of urokinase activity per mg of protein; (3) the storage conditions; (4) whether or not it is intended for use in the manufacture of parenteral preparations or ophthalmic preparations.
1222
MONOGRAPHS
V
Vanillin Vasopressin Injection Verapamil Hydrochloride Verapamil Injection Verapamil Tablets Vinblastine Sulphate Vinblastine Injection Vincristine Sulphate Vincristine Injection Vinorelbine Tartrate Vinorelbine Tartrate Injection Vitamin A Concentrate Oil Vitamin A Concentrate Powder Water-Miscible Vitamin A Concentrate Vitamins A And D Capsules Concentrated Vitamin D Solution Concentrated Vitamins A And D Solution .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... .... ....
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VASOPRESSIN INJECTION
Vanillin
CHO
OCH3 OH
C8H8O3 Mol. Wt. 152.2
Vanillin is 4-hydroxy-3-methoxybenzaldehyde. Vanillin contains not less than 99.0 per cent and not more than 101.0 per cent of C8H8O3, calculated on the dried basis. Description. A white or slightly yellow powder or crystalline needles; odour, characteristic of vanilla.
Use an unsaturated tank and allow the mobile phase to rise 10 cm. Apply to the plate 5 l of each solution. After development, dry the plate in cold air and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with test solution (a) is not more intense than the spot in the chromatogram obtained with reference solution (a). Spray with dinitrophenylhydrazine-aceto-hydrochloric solution and examine in daylight. Any secondary spot in the chromatogram obtained with test solution (a) is not more intense than the spot in the chromatogram obtained with reference solution (a). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 1.0 per cent, determined on 1.0 g by drying over phosphorus pentoxide for 4 hours. Assay. Weigh accurately about 0.12 g, dissolve in 20 ml of ethanol (95 per cent), add 60 ml of carbon dioxide-free water. Titrate with 0.1 M sodium hydroxide, determining the endpoint potentiometrically (2.4.25). 1 ml of 0.1 M sodium hydroxide is equivalent to 0.01521 g of C8H8O3. Storage. Store protected from light and moisture.
Identification
Test A may be omitted if tests B, C and D are carried out. Tests B, C and D may be omitted if test A is carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with vanillin RS. B. In the test for Related substances, the principal spot in the chromatogram obtained with test solution (b) corresponds to that in the chromatogram obtained with reference solution (b). Examine the chromatograms in daylight after spraying. C. To 5 ml of a saturated solution add 0.2 ml of ferric chloride solution; a blue colour is produced. Heat to 80o; the solution becomes brown and a white precipitate is produced on cooling. D. Melting range (2.4.21). 81 to 84.
Vasopressin Injection
Vasopressin Injection is a sterile aqueous solution containing the water-soluble pressor principle obtained from the posterior lobe of the pituitary of healthy oxen or other mammals or by synthesis. Vasopressin Injection contains a pressor activity equivalent to not less than 90.0 per cent and not more than 110.0 per cent of the stated number of Units. Description. A clear, colourless or practically colourless liquid; odour, faint and characteristic.
Tests
Appearance of solution. A 5.0 per cent w/v solution in ethanol (95 per cent) is clear (2.4.1), and not more intensely coloured than reference solution BS6 (2.4.1). Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 98.5 volumes of dichloromethane, 1 volume of methanol and 0.5 volume of anhydrous acetic acid. Test solution (a). Dissolve 0.2 g of the substance under examination in 10 ml of methanol. Test solution (b). Dissolve 0.2 g of the substance under examination in 100 ml of methanol. Reference solution (a). Dissolve 10 mg of the substance under examination in 100 ml of methanol. Reference solution (b). A 0.2 per cent w/v solution of vanillin RS in methanol.
Identification
A. Inject into the vein of a mammal, anaesthetised by a general anaesthetic or by destruction of the brain; it causes a rise of blood pressure. B. Inject under the skin of a mammal and at the same time administer a volume of water by mouth; it causes a delay in the excretion of the water.
Tests
pH (2.4.24). 2.5 to 4.5. Oxytocin activity. Not more than 1.2 Units per 20 Units of vasopressor activity, determined by the biological assay of oxytocin.
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VERAPAMIL HYDROCHLORIDE
IP 2007
Vasopressor activity. Not more than 0.5 Unit per 20 Units of oxytocic activity, when assayed by the following method. The vasopressor activity is estimated by comparing the activity of the preparation under examination with that of the Standard Preparation of arginine vasopressin under the conditions of a suitable method of assay. Standard Preparation The Standard Preparation is the Ist International Standard for Arginine vasopressin, established in 1978, consisting of freeze-dried synthetic arginine vasopressin peptide acetate with human albumin and citric acid (supplied in ampoules containing 8.20 Units), or another suitable preparation the potency of which has been determined in relation to that of the International Standard. Suggested Method Inject slowly into the tail vein of a male albino rat weighing about 300 g a solution of a suitable a-adrenoceptor blocking agent, for example 10 ml per kg of body weight of a solution prepared by dissolving 5 mg of phenoxybenzamine hydrochloride in 0.1 ml of ethanol (95 per cent), adding 0.05 ml of 1 M hydrochloric acid and diluting to 5 ml with saline solution. After 18 hours, anaesthetise the rat with an anaesthetic that will maintain a prolonged and uniform blood pressure. After 45 to 60 minutes, tie the rat on its back to the operating table by its hind legs. Cannulate the trachea with a short polyethylene tube of external diameter about 2.5 mm and dissect a carotid artery ready for cannulation. Then cannulate the femoral vein close to the inguinal ligament. Retract the abdominal muscles to expose the inguinal ligament. Retract the superficial pudendal vein to one side and dissect the femoral vein towards the inguinal ligament from the corresponding artery. When dissecting, a deep branch reaching the femoral vein must be found and tied off to prevent bleeding during cannulation. Tie a short polyethylene cannula of external diameter about 1 mm into the femoral vein by two ligatures and join by a short piece of flexible tubing to a 1-ml burette with an attached thistle funnel containing saline solution at about 37. Firmly fix a wet absorbent cotton swab to the thigh so as to cover the incision and cannula. At this stage inject through the venous cannula 200 Units of heparin, dissolved in saline solution, per 100 g of body weight. Then tie in a carotid cannula of external diameter about 1 mm and connect by a column of saline solution containing heparin with a suitable pressure measuring device such as a mercury manometer of internal diameter about 2 to 3 mm. The central and peripheral nervous system including both vagus and associated sympathetic nerves is left intact. No artificial respiration is necessary. Taking care that no air is injected, inject all solutions through the venous cannula by means of a 1-ml syringe graduated in 0.01 ml and wash in with 0.2 ml of saline solution from the burette.
Dilute the extract of the Standard Preparation and the preparation under examination with saline solution so that the volume to be injected is 0.1 ml to 0.5 ml. Choose two doses of the Standard Preparation such that the elevation of the blood pressure is about 4 kPa for the lower dose and about 7 kPa but always submaximal for the higher dose, the ratio of low to high dose being determined by the response and usually being 3 to 5. As an initial approximation doses of 3 and 5 milliUnits may be tried. Choose two doses of the preparation under examination with the same inter-dose ratio, matching the effects of the dose of the Standard Preparation as closely as possible. Inject doses at intervals of 10 to 15 minutes. The two doses of the Standard Preparation and the two doses of the preparation under examination should be given in a randomised block or a Latin square design and four to five responses to each should be recorded. Measure all the responses and calculate the result of the assay by standard statistical methods. Storage. Store in a refrigerator (2 to 8). Labelling. The label states (1) the number of Units of the vasopressor activity per ml; (2) either the animal source of the vasopressin or that it is synthetic.
Verapamil Hydrochloride
Verapamil Chloride; Iproveratril Hydrochloride
C27H38N2O4, HCl,
Mol. Wt. 491.1
Verapamil Hydrochloride is (2RS)-2-(3,4-dimethoxyphenyl)5-[[2-(3,4-dimethoxyphenyl)ethyl](methyl)amino]-2-(1methylethyl)pentanenitrile hydrochloride. Verapamil Hydrochloride contains not less than 99.0 per cent and not more than 101.0 per cent of C27H38N2O4, HCl, calculated on the dried basis. Description. A white, crystalline powder.
Identification
Test A may be omitted if tests B, C and D are carried out. Tests B and C may be omitted if tests A and D are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with verapamil
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IP 2007
VERAPAMIL INJECTION
hydrochloride RS or with the reference spectrum of verapamil hydrochloride. B. When examined in the range 220 nm to 360 nm (2.4.7), a 0.002 per cent w/v solution in 0.01 M hydrochloric acid shows absorption maxima at about 229 nm and 278 nm and there may be a shoulder at about 282 nm. The ratio of the absorbance at the maximum at about 278 nm to that at the maximum at about 229 nm is 0.35 to 0.39. C. In test A for Related substances, the principal spot in the chromatogram obtained with test solution (b) corresponds to that in the chromatogram obtained with reference solution (a). D. Gives reaction B of chlorides (2.3.1).
with reference solution (c) is clearly visible. Ignore any spot remaining on the line of application. B. Carry out test A but using a mixture of 85 volumes of cyclohexane and 15 volumes of diethylamine as the mobile phase and applying separately to the plate 10 l of each of test solution (a), reference solutions (b) and (c) and heat at 110 for 90 minutes after the second development. Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.4 g, dissolve in 40 ml of anhydrous glacial acetic acid, add 6 ml of mercuric acetate solution. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.04911 g of C27H38N2O4, HCl. Storage. Store protected from light and moisture.
Tests
Appearance of solution. A 5.0 per cent w/v solution in carbon dioxide-free water prepared with the aid of gentle heat is clear (2.4.1), and colourless (2.4.1). pH (2.4.24). 4.5 to 6.0, determined in a 5.0 per cent w/v solution prepared with the aid of gentle heat. Related substances. A. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 70 volumes of toluene, 20 volumes of methanol, 5 volumes of glacial acetic acid and 5 volumes of acetone. Test solution (a). Dissolve 0.5 g of the substance under examination in 10 ml of chloroform. Test solution (b). Dilute 1 ml of test solution (a) to 100 ml with chloroform. Reference solution (a). A 0.05 per cent w/v solution of verapamil hydrochloride RS in chloroform. Reference solution (b). Dilute 5 ml of reference solution (a) to 100 ml with chloroform. Reference solution (c). A 0.001 per cent w/v solution of verapamil hydrochloride RS in chloroform. Apply to the plate 10 l of each solution. After development, dry the plate in air for 10 minutes and repeat the development. Dry the plate at 110 for 30 minutes and allow to stand until the odour of solvent is no longer detectable. Spray with a solution prepared by dissolving 5 g of ferric chloride hexahydrate and 2 g of iodine in sufficient of a mixture of equal volumes of acetone and a 20 per cent w/v solution of (+)-tartaric acid to produce 100 ml, applying a total of 15 to 20 ml of the reagent for a plate (20 cm x 20 cm), and examine immediately. Any secondary spot in the chromatogram obtained with test solution (a) is not more intense than the principal spot in the chromatogram obtained with reference solution (b) and not more than three such spots are more intense than the spot in the chromatogram obtained with reference solution (c). The test is not valid unless the spot in the chromatogram obtained
Verapamil Injection
Verapamil Hydrochloride Injection; Verapamil Chloride Injection; Iproveratril Hydrochloride Injection
Verapamil Injection is a sterile solution of Verapamil Hydrochloride in Water for Injections. Verapamil Injection contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of verapamil hydrochloride, C27H38N2O4, HCl.
Identification
A. Dilute a volume containing 10 mg of Verapamil Hydrochloride to 5 ml with 0.1 M hydrochloric acid, extract with 5 ml of ether, discard the ether extract and make the aqueous layer just alkaline with 2 M potassium carbonate. Extract with 5 ml of ether, filter the ether layer through anhydrous sodium sulphate and evaporate to dryness. The residue complies with the following test. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with verapamil hydrochloride RS treated in the same manner or with the reference spectrum of verapamil. B. To a volume containing 5 mg of Verapamil Hydrochloride add 0.2 ml of a 5 per cent w/v solution of mercuric chloride; a white precipitate is produced. C. To a volume containing 5 mg of Verapamil Hydrochloride add 0.5 ml of 3 M sulphuric acid and 0.2 ml of dilute potassium permanganate solution; a violet precipitate is produced which quickly dissolves to produce a very pale yellow solution.
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IP 2007
Tests
pH (2.4.24). 4.5 to 6.0. Related substances. A. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 70 volumes of toluene, 20 volumes of methanol, 5 volumes of glacial acetic acid and 5 volumes of acetone. Test solution. Evaporate a volume containing 5 mg of Verapamil Hydrochloride carefully to dryness on a water-bath in a current of nitrogen and dissolve the residue as completely as possible in 0.25 ml of chloroform. Reference solution. Dilute 1 volume of the test solution to 100 volumes with chloroform and dilute 1 volume of the resulting solution to 10 volumes with chloroform. Apply to the plate 30 l of each solution. After development, dry the plate in air for 10 minutes and repeat the development. Dry the plate at 110 for 30 minutes and allow to stand until the odour of solvent is no longer detectable. Spray with a solution prepared by dissolving 5 g of ferric chloride hexahydrate and 2 g of iodine in sufficient of a mixture of equal volumes of acetone and a 20 per cent w/v solution of (+)-tartaric acid to produce 100 ml, applying a total of 15 to 20 ml of the reagent for a plate (20 cm x 20 cm), and examine immediately. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the principal spot in the chromatogram obtained with the reference solution. Ignore any spot remaining on the line of application. B. Carry out test A but using a mixture of 85 volumes of cyclohexane and 15 volumes of diethylamine as the mobile phase and applying separately to the plate 30 l of each of the test solution and the reference solution. Other tests. Comply with the tests stated under Parenteral Preparations (Injections). Assay. Dilute an accurately measured volume containing 5 mg of Verapamil Hydrochloride to 100.0 ml with 0.01 M hydrochloric acid and measure the absorbance of the resulting solution at the maximum at about 278 nm (2.4.7). Calculate the content of C 27H38N2O4, HCl taking 118 as the specific absorbance at 278 nm. Storage. Store protected from light.
Identification
A. Shake a quantity of the powdered tablets containing 0.1 g of Verapamil Hydrochloride with 25 ml of 0.1 M hydrochloric acid, filter, extract the filtrate with 25 ml of ether, discard the ether extract and make the aqueous layer just alkaline with 2 M potassium carbonate. Extract with 25 ml of ether, filter the ether layer through anhydrous sodium sulphate and evaporate to dryness. The residue complies with the following tests. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with verapamil hydrochloride RS treated in the same manner or with the reference spectrum of verapamil. B. Shake a quantity of the powdered tablets containing 0.1 g of Verapamil Hydrochloride with 10 ml of dichloromethane, filter, evaporate the filtrate to dryness and dissolve the residue in 10 ml of water (solution A). To 2 ml of the resulting solution add 0.2 ml of a 5 per cent w/v solution of mercuric chloride; a white precipitate is produced. C. To 2 ml of solution A obtained in test B add 0.5 ml of 3 M sulphuric acid and 0.2 ml of dilute potassium permanganate solution; a violet precipitate is produced which quickly dissolves to produce a very pale yellow solution.
Tests
Related substances. A. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 70 volumes of toluene, 20 volumes of methanol, 5 volumes of glacial acetic acid and 5 volumes of acetone. Test solution. Shake a quantity of the powdered tablets containing 0.1 g of Verapamil Hydrochloride with 10 ml of dichloromethane, filter, wash the filter with a further 5 ml of dichloromethane, evaporate the combined filtrate and washings to dryness and dissolve the residue in 2 ml of chloroform. Reference solution. Dilute 1 volume of the test solution to 100 volumes with chloroform and dilute 1 volume of the resulting solution to 10 volumes with chloroform. Apply to the plate 10 l of each solution. After development, dry the plate in air for 10 minutes and repeat the development. Dry the plate at 110 for 30 minutes and allow to stand until the odour of solvent is no longer detectable. Spray with a solution prepared by dissolving 5 g of ferric chloride hexahydrate and 2 g of iodine in sufficient of a mixture of equal volumes of acetone and a 20 per cent w/v solution of (+)-tartaric acid to produce 100 ml, applying a total of 15 to 20 ml of the reagent for a plate (20 cm x 20 cm), and examine immediately. Any secondary spot in the chromatogram obtained with the test
Verapamil Tablets
Verapamil Hydrochloride Tablets; Verapamil Chloride Tablets; Iproveratril Hydrochloride Tablets
Verapamil Tablets contain not less than 92.5 per cent and not more than 107.5 per cent of the stated amount of verapamil hydrochloride, C27H38N2O4, HCl. The tablets may be coated.
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VINBLASTINE SULPHATE
solution is not more intense than the spot in the chromatogram obtained with the reference solution. Ignore any spot remaining on the line of application. B. Carry out test A but using a mixture of 85 volumes of cyclohexane and 15 volumes of diethylamine as the mobile phase and applying separately to the plate 10 l of each of the test solution and the reference solution and heat at 110 for 90 minutes after the second development. Other tests. Comply with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing 0.1 g of Verapamil Hydrochloride, shake with 150 ml of 0.1 M hydrochloric acid for 10 minutes, add sufficient 0.1 M hydrochloric acid to produce 200.0 ml and filter. Dilute 10.0 ml of the filtrate to 100.0 ml with water and measure the absorbance of the resulting solution at the maximum at about 278 nm (2.4.7). Calculate the content of C27H38N2O4, HCl taking 118 as the specific absorbance at 278 nm.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with vinblastine sulphate RS. B. In the Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the peak due to vinblastine sulphate in the chromatogram obtained with reference solution (b). C. A 10 per cent w/v solution gives the reaction of sulphates (2.3.1).
Tests
Appearance of solution. A 0.5 per cent w/v solution in carbon dioxide-free water is clear (2.4.1), and not more intensely coloured than reference solution YS7 (2.4.1). pH (2.4.24). 3.5 to 5.0, determined in a 0.15 per cent w/v solution. Related substances. In the Assay, the area of any secondary peak in the chromatogram obtained with the test solution is not greater than that of the principal peak in the chromatogram obtained with reference solution (c) and the sum of the areas of any such peaks is not greater than 2.5 times the area of the principal peak in the chromatogram obtained with reference solution (c). Ignore any peak with an area less than that of the peak in the chromatogram obtained with reference solution (d). Loss on drying (2.4.19). Not more than 15.0 per cent, determined by Method B, on an appropriately calibrated instrument using about 3.0 mg, accurately weighed. Heat the substance under examination at the rate of 5 per minute between ambient temperature and 200 in a current of nitrogen for chromatography with a flow rate of 40 ml per minute. From the thermogram, determine the accumulated loss in weight between ambient temperature and a point on the plateau before decomposition is indicated (at about 160). Assay. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 0.1 g of the substance under examination in 100 ml of water. Reference solution (a). A solution containing 0.1 per cent w/v each of vinblastine sulphate RS and vincristine sulphate RS in water. Reference solution (b). A 0.1 per cent w/v solution of vinblastine sulphate RS in water. Reference solution (c). A 0.002 per cent w/v solution of vinblastine sulphate RS in water. Reference solution (d). A 0.0001 per cent w/v solution of vinblastine sulphate RS in water. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octylsilane bonded to porous silica (5 m), (b) a guard
Vinblastine Sulphate
N OH CH3 N H H CH3 O COCH3 H O , H2SO4
N H H3COOC H3CO
N HO O CH3 CH3
C46H58N4O9,H2SO4
Mol. Wt. 909.1
Vinblastine Sulphate is methyl (3aR,4R,5S,5aR,10bR,13aR)4-acetoxy-3a-ethyl-9-[(5S,7R,9S)-5-ethyl-5-hydroxy-9methoxycarbonyl-1,4,5,6,7,8,9,10-octahydro-2H-3,7methanoazacycloundecino[(5,4-b)indol-9-yl]-6-formyl-5hydroxy-8-methoxy-3a,4,5,5a,6,11,12,13a-octahydro-1Hindolizino[8,1-cd]carbazole-5-carboxylate sulphate. Vinblastine Sulphate contains not less than 95.0 per cent and not more than 104.0 per cent of C46H58N4O9, H2SO4, calculated on the dried basis. Description. A white or yellowish, amorphous or crystalline powder; very hygroscopic. CAUTION Handle Vinblastine Sulphate with great care since it is a potent cytotoxic agent. Avoid contact with eyes; irritant to tissues.
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VINBLASTINE INJECTION
IP 2007
column packed with a suitable silica gel placed between the pump and the injection device, mobile phase: a mixture of 50 volumes of methanol, 38 volumes of a 1.5 per cent v/v solution of diethylamine adjusted to pH 7.5 with phosphoric acid and 12 volumes of acetonitrile, flow rate. 1 ml per minute, spectrophotometer set at 262 nm, a 10 l loop injector.
The constituted solution complies with the requirements for Clarity of solution and Particulate matter stated under Parenteral Preparations (Injections). Storage. The constituted solution should be used immediately after preparation but, in any case, within the period recommended by the manufacturer. Vinblastine Injection contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of vinblastine sulphate, C46H58N4O9,H2SO4. The contents of the sealed container comply with the requirements stated under Parenteral Preparations (Powders for Injection) and with the following requirements.
NOTE Store all solutions in ice before use. Inject each solution and record the chromatograms for 3 times the retention time of the peak due to vinblastine. The assay is not valid unless the resolution between the peaks due to vincristine and vinblastine in the chromatogram obtained with reference solution (a) is at least 4 and the signalto-noise ratio of the peak in the chromatogram obtained with reference solution (d) is at least 5. Calculate the percentage content of C46H58N4O9, H2SO4. Vinblastine Sulphate intended for use in the manufacture of parenteral preparations without a further appropriate procedure for the removal of bacterial endotoxins complies with the following additional requirement. Bacterial endotoxins (2.2.3). Not more than 10.0 Endotoxin Units per mg. Vinblastine Sulphate intended for use in the manufacture of parenteral preparations without a further appropriate sterilisation procedure complies with the following additional requirement. Sterility (2.2.11). Complies with the test for sterility. Storage. Store protected from light and moisture in a deep freezer (Below -18). If the material is intended for use in the manufacture of parenteral preparations, it should be stored in sterile, tamper-evident glass containers and sealed so as to exclude micro-organisms. Labelling. The label states whether or not the contents are suitable for use in the manufacture of parenteral preparations.
Identification
CAUTION Handle Vinblastine Injection with great care since it is a potent cytotoxic agent. Avoid contact with eyes; irritant to tissues. A. When examined in the range 230 nm to 360 nm (2.4.7), the solution obtained in the Assay shows an absorption maximum at about 267 nm. B. In the test for Related substances, the principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with reference solution (a). C. To 1 ml add 0.2 ml of a freshly prepared 1 per cent w/v solution of vanillin in hydrochloric acid; a pink colour is produced in about 1 minute (distinction from Vincristine Sulphate).
Tests
pH (2.4.24). 3.5 to 5.0, determined in a 0.15 per cent w/v solution of the dried contents. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 80 volumes of toluene, 40 volumes of chloroform and 6 volumes of diethylamine. Test solution. Dissolve a quantity of the contents of a container in sufficient methanol to produce a solution containing the equivalent of 1 per cent w/v of dried vinblastine sulphate. Reference solution (a). A 1 per cent w/v solution of vinblastine sulphate RS in methanol. Reference solution (b). A 0.02 per cent w/v solution of vincristine sulphate RS in methanol. Apply to the plate 5 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (b).
Vinblastine Injection
Vinblastine Sulphate Injection
Vinblastine Injection is a sterile material consisting of Vinblastine Sulphate with or without auxiliary substances. It is filled in a sealed container. The injection is constituted by dissolving the contents of the sealed container in the requisite amount of sterile 0.9 per cent w/v solution of Sodium Chloride, immediately before use.
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VINCRISTINE SULPHATE
Bacterial endotoxins (2.2.3). Not more than 10.0 Endotoxin Units per mg of the dried contents. Assay. Weigh the contents of 20 sealed containers. Weigh accurately a quantity of the mixed contents containing about 20 mg of dried vinblastine sulphate and dissolve it in 100.0 ml of methanol. Dilute 10.0 ml to 100.0 ml with methanol and measure the absorbance of the resulting solution at the maximum at about 267 nm (2.4.7). Calculate the content of C46H58N4O9,H2SO4 taking 185 as the specific absorbance at 267 nm. Storage. Store in sealed containers in a deep freezer (Below 18). Labelling. The label states (1) the strength in terms of the weight of dried vinblastine sulphate contained in it; (2) the names of auxiliary substances, if any; (3) that the contents are to be used by intravenous injection only; (4) the storage conditions.
B. In the Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the peak due to vincristine sulphate in the chromatogram obtained with reference solution (b). C. A 10 per cent w/v solution gives the reaction for sulphates (2.3.1).
Tests
Appearance of solution. A 0.5 per cent w/v in carbon dioxidefree water is clear (2.4.1), and not more intensely coloured than reference solution YS7 (2.4.1). pH (2.4.24). 3.5 to 4.5. Determined in a 0.1 per cent w/v solution. Related substances. In the Assay, the area of any secondary peak in the chromatogram obtained with the test solution is not greater than that of the principal peak in the chromatogram obtained with reference solution (c) and the sum of the areas of any such peaks is not greater than 2.5 times the area of the principal peak in the chromatogram obtained with reference solution (c). Ignore any peak with an area less than that of the peak in the chromatogram obtained with reference solution (d). Loss on drying (2.4.19). Not more than 12.0 per cent, determined by Method B, on an appropriately calibrated instrument using about 3.0 mg, accurately weighed. Heat the substance under examination at the rate of 5 per minute between ambient temperature and 200 in current of nitrogen for chromatography with a flow rate of 40 ml per minute. Assay. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 0.1 g of the substance under examination in 100 ml of water. Reference solution (a). A solution containing 0.1 per cent w/v each of vinblastine sulphate RS and vincristine sulphate RS in water. Reference solution (b). A 0.1 per cent w/v solution of vincristine sulphate RS in water. Reference solution (c). A 0.002 per cent w/v solution of vincristine sulphate RS in water. Reference solution (d). A 0.0001 per cent w/v solution of vincristine sulphate RS in water. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octylsilane bonded to porous silica (5 m), (b) a guard column packed with a suitable silica gel placed between the pump and the injection device, mobile phase: a mixture of 70 volumes of methanol and 30 volumes of a 1.5 per cent v/v solution of diethylamine adjusted to pH 7.5 with phosphoric acid, flow rate. 1 ml per minute,
Vincristine Sulphate
N OH CH3 N H H CH3 O COCH3 H O , H2SO4
N H H3COOC H3CO
N HO O CHO CH3
C46H56N4O10,H2SO4 Mol. Wt. 923.1 Vinblastine Sulphate is methyl (3aR,4R,5S,5aR,10bR,13aR) 4acetoxy-3a-ethyl-9-[(5S,7R,9S)-5-ethyl-5-hydroxy-9methoxycarbonyl-1,4,5,6,7,8,9,10-octahydro-2H-3,7methanoazacycloundecino[(5,4-b)indol-9-yl]-5-hydroxy- 8methoxy-6-methyl-3a,4,5,5a,6,11,12,13a-octahydro-1Hindolizino[8,1-cd]carbazole-5-carboxylate sulphate. Vincristine Sulphate contains not less than 95.0 per cent and not more than 104.0 per cent of C46H56N4O10,H2SO4, calculated on the dried basis. Description. A white to slightly yellowish, amorphous or crystalline powder; very hygroscopic. CAUTION Handle Vincristine Sulphate with great care since it is a potent cytotoxic agent. Avoid contact with eyes; irritant to tissues.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with vincristine sulphate RS.
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VINCRISTINE INJECTION
IP 2007
spectrophotometer set at 297 nm, a 10 l loop injector. NOTE Store all solutions in ice before use. Inject each solution and record the chromatograms for 3 times the retention time of the peak due to vincristine. The assay is not valid unless the resolution between the peaks due to vincristine and vinblastine in the chromatogram obtained with reference solution (a) is at least 4 and the signalto-noise ratio in the peak in the chromatogram obtained with reference solution (d) is at least 5. Calculate the content of C46H56N4O10,H2SO4. Vincristine Sulphate intended for use in the manufacture of parenteral preparations without a further appropriate procedure for the removal of bacterial endotoxins complies with the following additional requirement. Bacterial endotoxins (2.2.3). Not more than 62.5 Endotoxin Units per mg. Vincristine Sulphate intended for use in the manufacture of parenteral preparations without a further appropriate sterilisation procedure complies with the following additional requirement. Sterility (2.2.11). Complies with the test for sterility. Storage. Store protected from light in a deep freezer (Below 18). If the material is intended for use in the manufacture of parenteral preparations, it should be stored in sterile, tamperevident glass containers and sealed so as to exclude microorganisms. Labelling. The label states whether or not the contents are suitable for use in the manufacture of parenteral preparations.
Vincristine Injection contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of vincristine sulphate, C46H56N4O10,H2SO4. The contents of the sealed container comply with the requirements stated under Parenteral Preparations (Powders for Injection) and with the following requirements.
Identification
CAUTION Handle Vincristine Injection with great care since it is a potent cytotoxic agent. Avoid contact with eyes; irritant to tissues. A. When examined in the range 230 nm to 360 nm (2.4.7), the solution obtained in the Assay shows an absorption maximum at about 297 nm. B. In the test for Related substances, the principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with reference solution (b). C. Shake a quantity containing 1 mg of dried vincristine sulphate with 3 ml of chloroform, filter and wash the filter with 2 ml of chloroform. Reserve the residue for test D. Evaporate the combined chloroform solutions to dryness at 40. Add 0.2 ml of a freshly prepared 1 per cent w/v solution of vanillin in hydrochloric acid to the residue; an orange colour is produced in about 1 minute (distinction from vinblastine sulphate). D. Dissolve the residue reserved in test C in 1 ml of water. The solution complies with the following tests. Heat 0.5 ml with 1 ml of potassium cupri-tartrate solution; a copius precipitate of copper oxide is produced. Heat 0.5 ml with 0.3 ml of a 6.5 per cent w/v solution of cupric acetate in a 1 per cent v/v solution of glacial acetic acid; no precipitate is produced (distinction from fructose, glucose and galactose).
Vincristine Injection
Vincristine Sulphate Injection
Vincristine Injection is a sterile material consisting of Vincristine Sulphate with or without auxiliary substances. It is filled in a sealed container. The injection is constituted by dissolving the contents of the sealed container in the requisite amount of sterile 0.9 per cent w/v solution of Sodium Chloride, immediately before use. The constituted solution complies with the requirements for Clarity of solution and Particulate matter stated under Parenteral Preparations (Injections). Storage. The constituted solution should be used immediately after preparation but, in any case, within the period recommended by the manufacturer.
Tests
Appearance of solution. A solution prepared by dissolving the contents of a sealed container in 10 ml of carbon dioxidefree water is clear (2.4.1). pH (2.4.24). 3.5 to 5.0, determined in a solution containing 0.15 per cent w/v solution of the dried contents. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 80 volumes of toluene, 40 volumes of chloroform and 6 volumes of diethylamine. Test solution. Dissolve a quantity of the contents of a container in sufficient methanol (75 per cent) to produce a solution containing the equivalent of 1 per cent w/v of dried vincristine sulphate.
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IP 2007
VINORELBINE TARTRATE
Reference solution (a). A 0.02 per cent w/v solution of vinblastine sulphate RS in methanol. Reference solution (b). A 1 per cent w/v solution of vincristine sulphate RS in methanol. Apply to the plate 5 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (a). Bacterial endotoxins (2.2.3). Not more than 62.5 Endotoxin Units per mg of dried vincristine sulphate. Assay. Weigh the contents of 20 sealed containers. Weigh accurately a quantity of the mixed contents of the containers containing about 20 mg of dried vincristine sulphate and dissolve it in 100.0 ml of methanol. Dilute 10.0 ml to 100.0 ml with methanol and measure the absorbance of the resulting solution at the maximum at about 297 nm (2.4.7). Calculate the content of C46H56N4O10,H2SO4 taking 177 as the specific absorbance at 297 nm. Storage. Store in sealed containers in a deep freezer (Below 18). Labelling. The label states (1) the strength in terms of the weight of dried vincristine sulphate contained in it; (2) the names of auxilary substances, if any; (3) that the contents are to be used by intravenous injection only; (4) the storage conditions.
CAUTION Vinorelbine Tartrate is cytotoxic; extra care required to prevent inhaling particles and exposing the skin to it.
Identification
A. Dissolve 10 mg in 5 ml of water, add 0.5 ml of 5 M sodium hydroxide, and extract with 5 ml of methylene chloride. Filter the organic layer through anhydrous sodium sulphate, and evaporate to dryness. Determine by Infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with vinorelbine tartrate RS treated in the same manner. B. In the test for Related substances, the principal peak in the chromatogram of the test solution corresponds to that due to vinorelbine tartarate in the chromatogram obtained with the refrence solution. C. It gives the reactions for tartrate (2.3.1).
Tests
Appearance of solution. A 1.0 per cent w/v solution is clear (2.4.1). Light absorption (2.4.7). The absorbance of 1.0 per cent w/v solution, at about 420 nm is not more than 0.03. pH (2.4.24). 3.3 to 3.8, determined on a 1.0 per cent w/v solution. Related substances. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 35 mg of the substance under examination in 25 ml of mobile phase.
Vinorelbine Tartrate
N H N H O N H H3CO H3C NH O O OH OCH3 CH3 O CH3 CH3
Reference solution (a). A 0.14 per cent w/v solution of vinorelbine tartrate RS in mobile phase. Reference solution (b). Dilute 1 ml of the reference solution (a) to 100 ml with mobile phase. Chromatographic system as described under Assay.
H OH , 2 HOOC H COOH OH
OCH3
Inject reference solution (a). The test is not valid unless the column efficiency is not less than 2000 theoretical plates and the tailing factor is not more than 2.0. Inject the test solution and reference solution (b). In the chromatogram obtained with the test solution, the area of any secondary peak is not more than 0.3 times the area of the peak in the chromatogram obtained with the reference solution (b) (0.3 per cent) and the sum of areas of all the secondary peaks is not more than the area of the peak in the chromatogram obtained with the reference solution (b) (1.0 per cent). Ignore any secondary peak having area less than 0.1 per cent. Test solution. Weigh accurately about 0.2 g of the substance under examination, add 0.5 ml of water and 0.5 ml of N,Ndimethyl formamide in to a 10 ml head space vial and seal it.
C45H54N4O8, 2C4H6O6
Mol. Wt. 1079.11
Vinorelbine Tartrate is the ditartrate salt of vinorelbine, a semisynthetic Vinca alkaloid; structurally relate to vinblastine. Vinorelbine Tartrate contains not less than 98.0 per cent and not more than 102.0 per cent of C45H54N4O8.2C4H6O6, calculated on the anhydrous basis. Description. A white to yellow or light brown amorphous powder.
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VINORELBINE INJECTION
IP 2007
Reference solution (a). To 50 ml of N,N-dimethyl formamide, add 600 mg of methanol and 1000 mg of acetone, diluted to 100 ml with N,N-dimethyl formamide. Reference solution (b). To 50 ml of N,N-dimethyl formamide, add 2400 mg of dichloromethane, 2880 mg of tetrahydrofuran and 240 mg of chloroform, diluted to 100 ml with N,N-dimethyl formamide. Dilute 5 ml of the solution to 100 ml with N,Ndimethyl formamide. Reference solution (c). Dilute 10 ml each of reference solutions (a) and (b) to 50 ml with N,N-dimethyl formamide. Mix 0.5 ml of this solution with 0.5 ml of water in to a 10 ml head space vial and seal it. Chromatographic system a capillary column 30 m x 0.32 mm, coated with 1 per cent vinyl and 5 per cent phenylmethylpolysiloxane (0.5 m), temperature: column. 40 for 12 minutes increase @ 30 per minute to 220 hold for 5 minutes, inlet port 120and detector. 250, nitrogen as carrier gas with a flow rate 0.5 ml per min. Headspace conditions Sample oven temperature 75, Sample valve temperature 95 , Transfer line 100, vial equilibrium 30 minutes, vial Pressurisation 0.2 minute, sample loop fill 0.2 minute, loop equilibrium 0.05 minute sample injection 1 minute. a flame ionisation detector, Inject 1 ml of the reference solution (c). The test is not valid unless the resolution between two adjacent peaks is not less than 1.5. Inject 1 ml of the test solution and reference solution (c). In the chromatogram obtained with test solution, the area of peaks due to methanol, acetone, dichloromethane, chloroform and tetrahydrofuran is not more than the area of peaks obtained in the chromatogram due to reference solution (c). Sulphated ash (2.3.18). Not more than 0.1 per cent. Water (2.3.43). Not more than 4.0 per cent, determined on 0.1 g. Bacterial endotoxins (2.2.3). Not more than 1.5 Endotoxin Unit per mg of vinorelbine base. Microbial contamination (2.2.9). Total viable aerobic count, not more than 100 cfu per g, total combined molds and yeast does not exceed 50 cfu per g. It also meets the requirement of the tests for the absence of Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella species, Escherichia coli, Enterobacteria and Closteridia. Assay. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 35 mg of the substance under examination in 25.0 ml of mobile phase. Reference solution. A 0.14 per cent w/v solution of vinorelbine tartrate RS in mobile phase.
Chromatographic system a stainless steel column 15 cm x 3.9 mm, packed with octadecylsilane bonded to porous silica (5 m), column temperature 40, mobile phase: 62 volumes of methanol containing 1.22 g of sodium 1-decane sulphonate and 38 volumes of phosphate buffer solution, prepared by dissolving 6.9 g of monobasic sodium phosphate in 900 ml of water, adjust the pH to 4.2 with orthophosphoric acid and dilute to 1000 ml with water, flow rate. 1 ml per minute, spectrophotometer set at 267 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject the test solution and reference solution. Calculate the content of C45H54N4O8.2C4H6O6. Storage. Store protected from light, at a temperature not exceeding 25.
Vinorelbine Injection
Vinorelbine Tartrate Injection
Vinorelbine Injection is a sterile solution of vinorelbine tartrate in Water for Injection. Vinorelbine Injection contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of vinorelbine, C45H54N4O8. Description. A clear, colourless to pale yellow solution.
Identification
A. In the Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the peak in the chromatogram obtained with the reference solution. B. When examined in the range 220 nm to 380 nm (2.4.7), a solution containing 0.01 per cent w/v of Vinorelbine Tartrate, exhibits the maxima at about 267 nm.
Tests
pH (2.4.24). 3.0 to 3.8. Light absorption. The absorbance of the injection at about 420 nm (2.4.7), is not more than 0.06. Related substances. Determine by liquid chromatography (2.4.14). Test solution. Accurately measured volume of injection containing 10 mg of Vinorelbine Tartrate, dilute to 10 ml with mobile phase.
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IP 2007
VITAMIN A CONCENTRATE OIL
Reference solution (a). A 0.1 per cent w/v solution of vinorelbine tartrate RS in mobile phase. Reference solution (b). Dilute 1 ml of the reference solution (a) to 100 ml with mobile phase. Chromatographic system as described under Assay. Inject reference solution (a). The test is not valid unless the column efficiency is not less than 2000 theoretical plates and the tailing factor is not more than 2.0. Inject the test solution and reference solution (b). Run the chromatograms three times the retention time of the peak due to vinorelbine. In the chromatogram obtained with the test solution, the area of any secondary peak is not more than 0.2 times the area of the peak in the chromatogram obtained with the reference solution (b) (0.2 per cent) and the sum of areas of all the secondary peaks is not more than twice the area of the peak in the chromatogram obtained with the reference solution (b) (2.0 per cent). Other tests. Complies with the tests stated under Parenteral Preparation (Injections). Bacterial endotoxins (2.2.3). Not more than 3.0 Endotoxin Unit per mg of vinorelbine tartrate. Sterility (2.2.11). Complies with the test for sterility. Assay. Determine by liquid chromatography (2.4.14). Test solution. Accurately measured volume of injection containing 10 mg of Vinorelbine Tartrate, diluted to 100.0 ml with water. Reference solution. A 0.1 per cent w/v solution of vinorelbine tartrate RS in water. Chromatographic system a stainless steel column 15 cm x 3.9 mm, packed with octadecylsilane bonded to porous silica (5 m), column temperature 40, mobile phase: a mixture of 62 volumes of methanol containing 1.22 g of sodium1-decane sulphonate and 38 volumes of phosphate buffer solution prepared by dissolving 6.9 g of monobasic sodium phosphate in 900 ml of water adjusted the pH to 4.2 with orthophosphoric acid and dilute to 1000 ml with water, flow rate. 1 ml per minute, spectrophotometer set at 267 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the relative standard deviation is not more than 2.0 per cent. Inject the test solution and the reference solution. Calculate the content of C45H54N4O8. Storage. Store protected from light, in single-dose container.
Vitamin A Concentrate Oil

Synthetic Vitamin A Concentrate (Oily Form); Synthetic Retinol Concentrate (Oily Form)
Vitamin A Concentrate Oil consists of an ester or a mixture of esters of retinol (as acetate, propionate or palmitate) prepared by synthesis. It may be diluted with a suitable vegetable oil. It may contain suitable stabilising agents such as antioxidants. Vitamin A Concentrate Oil contains not less than 500,000 Units of Vitamin A per g, and not less than 95.0 per cent and not more than 110.0 per cent of the stated number of Units of Vitamin A per g. Description. A yellow to brownish yellow, oily liquid; odour, faint and characteristic.
Identification
A. When examined in the range 230 nm to 360 nm (2.4.7), a solution in 2-propanol containing 10 to 15 Units per ml shows an absorption maximum at about 325 nm to 327 nm. B. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 80 volumes of cyclohexane and 20 volumes of ether. Test solution. Prepare a solution containing 5 Units per l of the substance under examination in cyclohexane. Reference solution (a). Prepare a solution containing 5 Units per l of retinyl acetate RS in cyclohexane. Reference solution (b). Prepare a solution containing 5 Units per l of retinyl propionate RS in cyclohexane. Reference solution (c). Prepare a solution containing 5 Units per l of retinyl palmitate RS in cyclohexane. Apply to the plate 2 l of each solution. After development, dry the plate in air, spray with antimony trichloride solution. The principal spot or spots in the chromatogram obtained with the test solution correspond to one or more of the spots in the chromatograms obtained with reference solutions (a), (b) and (c). C. Dissolve a quantity containing 10 to 15 Units in 1 ml of chloroform and add 5 ml of antimony trichloride solution; a transient bright blue colour is produced immediately.
Tests
Acid value (2.3.23). Not more than 2.0, determined on 2.0 g. Peroxides. Add 0.3 g to 25.0 ml of a mixture of 6 volumes of toluene and 4 volumes of methanol (solution A). Add in a test-tube, in the following order, mixing after each addition, 0.3 ml of a 1.8 per cent w/v solution of ammonium thiocyanate,
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VITAMIN A CONCENTRATE POWDER
IP 2007
10.0 ml of methanol, 0.3 ml of ferrous sulphate solution and 15.0 ml of toluene and add 1.0 ml of solution A. The colour produced after 5 minutes is not more intense than that obtained in a solution prepared at the same time and in the same manner but using a solution prepared in the following manner in place of solution A. Add 1.0 ml of a 27.0 per cent w/v solution of ferric chloride hexahydrate to 99 ml of a mixture of 6 volumes of toluene and 4 volumes of methanol and dilute 2.0 ml to 100.0 ml with the same solvent mixture. Assay. Carry out the assay of vitamin A, Method A (2.3.41). Storage. Store protected from light, in well-filled containers. Once the container has been opened, its contents should be used as soon as possible; any part of the contents not used at once should be protected by an atmosphere of an inert gas. Labelling. The label states (1) the number of Units of Vitamin A per g; (2) the name of the ester or esters; (3) the name(s) of any added stabilising agent(s); (4) the method of restoring the solution if partial crystallisation has occurred.
A. When examined in the range 230 nm to 360 nm (2.4.7), a solution in 2-propanol containing 10 to 15 Units per ml shows an absorption maximum at about 325 nm to 327 nm. B. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 80 volumes of cyclohexane and 20 volumes of ether. Test solution. Evaporate 10 ml of solution A to dryness in a current of nitrogen and dissolve the residue in 0.5 ml of cyclohexane. Reference solution (a). Prepare a solution containing 5 Units per l of retinyl acetate RS in cyclohexane. Reference solution (b). Prepare 5 Units per l of retinyl propionate RS in cyclohexane. Reference solution (c). Prepare a solution containing 5 Units per l of retinyl palmitate RS in cyclohexane. Apply to the plate 2 l of each solution. After development, dry the plate in air, spray with antimony trichloride solution. The principal spot or spots in the chromatogram obtained with the test solution correspond to one or more of the spots in the chromatograms obtained with reference solutions (a), (b) and (c). C. Dilute 2 ml of solution A to 50 ml with n-pentane and evaporate 1 ml of the solution to dryness in a current of nitrogen. Dissolve the residue in 1 ml of chloroform and add 5 ml of antimony trichloride solution; a transient bright blue colour is produced immediately.
Vitamin A Concentrate Powder

Synthetic Vitamin A Concentrate (Powder Form); Synthetic Retinol Concentrate (Powder Form)
Vitamin A Concentrate Powder consists of an ester or a mixture of esters of retinol (as acetate, propionate or palmitate) prepared by synthesis and dispersed in a matrix of Gelatin, Acacia or any other suitable material. It may contain suitable stabilising agents such as antioxidants. Vitamin A Concentrate Powder contains not less than 250,000 Units of Vitamin A per g, and not less than 95.0 per cent and not more than 115.0 per cent of the stated number of Units of Vitamin A per g. Description. A yellowish powder usually in the form of pellets or particles of almost uniform size.
Tests
Related substances and degradation products. Using the relative absorbances obtained in the Assay, the ratio A300/ A325 is not more than 0.612 and the sum of the ratios A300/A325 and A350/A325 is not more than 1.054, where A300, A325 and A350 are the absorbances measured at about 300 nm, 325 nm and 350 nm respectively. Assay. Carry out the assay of vitamin A, Method B (2.3.41), adding 5 ml of water to the mixture for saponification, using 2-propanol as the blank and taking 0.612 as the maximum value of the ratio A300/A325. Storage. Store protected from light. Once the container has been opened, its contents should be used as soon as possible; any part of the contents not used at once should be protected by an atmosphere of an inert gas. Labelling. The label states (1) the number of Units of Vitamin A per g; (2) the name of the ester or esters; (3) the name of the principal excipient or excipients used; (4) the name of any added stabilising agents.
Identification
To a quantity containing 50,000 Units of Vitamin A add 1.5 ml of 2 M ammonia previously heated to 60 and heat in a waterbath at 60, shaking occasionally. After 10 minutes add 40 ml of ethanol, dilute to 200 ml with ether and shake. Allow to stand for a few minutes and use the supernatant liquid (solution A) for the following tests. Certain samples may not react sufficiently during the course of the above treatment. In such cases the volume of solution A used in the following tests should be increased which may be as much as 10-fold.
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IP 2007
VITAMIN A AND D CAPSULES
Water-Miscible Vitamin A Concentrate

Synthetic Vitamin A Concentrate (Water-dispersible Form); Synthetic Retinol Concentrate (Water-dispersible Form)
Water-miscible Vitamin A Concentrate consists of an ester or a mixture of esters of retinol (as acetate, propionate or palmitate) prepared by synthesis to which suitable solubilisers have been added. It may contain suitable stabilising agents such as antimicrobial preservatives and antioxidants. Water-miscible Vitamin A Concentrate contains not less than 100,000 Units of Vitamin A per g, and not less than 95.0 per cent and not more than 115.0 per cent of the stated number of Units of Vitamin A per g. Description. A yellow or yellowish liquid of variable opalescence and viscosity; odour, characteristic. Highly concentrated solutions may become cloudy at low temperatures or gel at room temperature.
in the chromatograms obtained with reference solutions (a), (b) and (c). C. Evaporate 0.1 ml of solution A to dryness in a current of nitrogen, dissolve the residue in 1 ml of chloroform and add 5 ml of antimony trichloride solution; a transient bright blue colour is produced immediately.
Tests
Water miscibility. Mix about 1 g with 10 ml of water previously heated to 50 and cool to 20. Immediately after cooling, a uniform, slightly opalescent and slightly yellow dispersion is obtained. Related substances and degradation products. Using the relative absorbances obtained in the Assay, the ratio A300/ A325 is not more than 0.618 and the sum of the ratios A300/A325 and A350/A325 is not more than 1.060, where A300, A325 and A350 are the absorbances measured at about 300 nm, 325 nm and 350 nm respectively. Assay. Carry out the assay of vitamin A, Method B (2.3.41), using 2-propanol as the blank and taking 0.618 as the maximum value of the ratio A300/A325. Storage. Store protected from light at the temperature stated on the label. Once the container has been opened, its contents should be used as soon as possible; any part of the contents not used at once should be protected by an atmosphere of an inert gas. Labelling. The label states (1) the number of Units of Vitamin A per g; (2) the name of the ester or esters; (3) the name of the principal excipient or excipients used; (4) the temperature at which it should be stored.
Identification
To a quantity containing about 10,000 Units of Vitamin A add 5 ml of water and homogenise. Add 5 ml of ethanol (95 per cent) and 20 ml of n-pentane and shake vigorously for 30 seconds. Allow to stand for a few minutes and use the supernatant liquid (solution A) for the following tests. A. Dilute solution A with sufficient 2-propanol so that the absorbance at the wavelength of maximum absorption is 0.3 to 0.7 (2.4.7). When examined in the range 230 nm to 360 nm (2.4.7), the solution shows an absorption maximum at 325 to 327 nm. B. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 80 volumes of cyclohexane and 20 volumes of ether. Test solution. Evaporate 10 ml of solution A to dryness in a current of nitrogen and dissolve the residue in 0.5 ml of cyclohexane. Reference solution (a). Prepare a solution containing 5 Units per l of retinyl acetate RS in cyclohexane. Reference solution (b). Prepare a solution containing 5 Units per l of retinyl propionate RS in cyclohexane. Reference solution (c). Prepare a solution containing 5 Units per l of retinyl palmitate RS in cyclohexane. Apply to the plate 2 l of each solution. After development, dry the plate in air, spray with antimony trichloride solution. The principal spot or spots in the chromatogram obtained with the test solution correspond to one or more of the spots
Vitamins A And D Capsules

Vitamins A and D Capsules contain Vitamin A Oil and a source of vitamin D such as Cholecalciferol or Ergocalciferol in an edible vegetable oil. Vitamins A and D Capsules contain not less than 90.0 per cent of the stated number of Units of vitamin A and vitamin D.
Tests
Other tests. Comply with the tests stated under Capsules. Assay. For vitamin A Weigh accurately a portion of the mixed contents of 20 capsules containing about 500 Units of Vitamin A and carry out the assay of vitamin A, Method A (2.3.41). For vitamin D Weigh accurately a portion of the mixed contents of 20 capsules containing about 5000 Units of vitamin D and carry out the assay of vitamin D (2.3.42).
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CONCENTRATED VITAMIN D SOLUTION
IP 2007
Storage. Store protected from light and moisture. Labelling. The label states the number of Units of vitamin A and vitamin D per capsule.
Labelling. The label states (1) the number of Units of vitamin D per g; (2) the storage conditions; (3) the nature and concentration of any stabilising agent added.
Concentrated Vitamin D Solution

Concentrated Vitamin D Solution is a solution of Cholecalciferol or Ergocalciferol in an edible vegetable oil. It may contain suitable stabilising agents such as antioxidants. Concentrated Vitamin D Solution contains not less than 10,000 Units of vitamin D and not less than 90.0 per cent of the stated number of Units of vitamin D. Description. A pale yellow to yellow, oily liquid; odour, faint but not rancid.
Concentrated Vitamins A And D Solution

Concentrated Vitamins A and D Solution is a solution of Vitamin A Oil and a source of vitamin D such as Cholecalciferol or Ergocalciferol in an edible vegetable oil. It may contain suitable stabilising agents such as antioxidants. Concentrated Vitamins A and D Solution contains not less than 50,000 Units of vitamin A and 5000 Units of vitamin D per g, and not less than 90.0 per cent and not more than 110.0 per cent of the stated number of Units of Vitamin A and vitamin D per g.
Identification
Dissolve a quantity containing about 1000 Units of vitamin D in 1 ml of chloroform and add 10 ml of antimony trichloride solution; a pinkish red colour appears at once.
Tests
Acid value (2.3.23). Not more than 2.5, determined on 2.0 g. Assay. For vitamin A Carry out the assay of vitamin A, Method A (2.3.41). For vitamin D Carry out the assay of vitamin D (2.3.42). Storage. Store protected from light and moisture. Labelling. The label states (1) the number of Units of vitamin A and vitamin D per gram; (2) the storage conditions.
Tests
Acid value (2.3.23). Not more than 2.5, determined on 2.0 g. Assay. Carry out the assay of vitamin D (2.3.42). Storage. Store protected from light, in well-filled containers at a temperature of 6 to 15. The contents of an opened container should be used as soon as possible.
1238
MONOGRAPHS
W
Warfarin Sodium Warfarin Sodium Clathrate Warfarin Tablets Purified Water Water For Injections Water For Injections in Bulk Sterile Water For Injections Wool Fat Hydrous Wool Fat .... .... .... .... .... .... .... .... ....
1239
IP 2007
WARFARIN SODIUM CLATHRATE
Warfarin Sodium
O O
maximum at about 385 nm, measured within 15 minutes of preparation, is not more than 0.20 (2.4.7). Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254.
NaO O
C19H15NaO4
CH3
Mobile phase. A mixture of 50 volumes of chloroform, 50 volumes of cyclohexane and 20 volumes of glacial acetic acid. Mol. Wt. 330.3 Test solution (a). Dissolve 0.2 g of the substance under examination in 10 ml of acetone. Test solution (b). Dissolve 0.4 g of the substance under examination in 100 ml of acetone. Reference solution (a). A 0.002 per cent w/v solution of the substance under examination in acetone. Reference solution (b). A 0.4 per cent w/v solution of warfarin sodium RS in acetone. Reference solution (c). A solution containing 0.1 per cent w/v of acenocoumarol RS and 0.2 per cent w/v of the substance under examination in acetone. Apply to the plate 20 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with test solution (a) is not more intense than the spot in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (c) shows two clearly separated spots and the chromatogram obtained with reference solution (a) shows a clearly visible spot. Water (2.3.43). Not more than 4.0 per cent, determined on 0.75 g. Assay. Weigh accurately about 0.1 g and dissolve in sufficient 0.01 M sodium hydroxide to produce 100.0 ml. Dilute 10.0 ml to 100.0 ml with 0.01 M sodium hydroxide and dilute 5.0 ml to 50.0 ml with 0.01 M sodium hydroxide. Measure the absorbance of the resulting solution at the maximum at about 308 nm (2.4.7). Calculate the content of C19H15NaO4 taking 431 as the specific absorbance at 308 nm. Storage. Store protected from light and moisture.
Warfarin Sodium is sodium 2-oxo-3-[(1RS)-3-oxo-1phenylbutyl]-2H-1-benzopyran-4-olate. Warfarin Sodium contains not less than 98.0 per cent and not more than 102.0 per cent of C19H15NaO4, calculated on the anhydrous basis. Description. A white powder; hygroscopic.
Identification
Test A may be omitted if tests B, C, D and E are carried out. Tests B and D may be omitted if tests A, C and E are carried out. A. Dissolve 1 g in 25 ml of water, add 2 ml of 2 M hydrochloric acid and filter. Wash the residue with water and dry over phosphorus pentoxide. On the residue, determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with warfarin sodium RS or with the reference spectrum of warfarin sodium. B. In the test for Related substances, the principal spot in the chromatogram obtained with test solution (b) corresponds to that in the chromatogram obtained with reference solution (b). C. Dissolve 1 g in 10 ml of water, add 5 ml of nitric acid and filter. To the filtrate add 2 ml of potassium dichromate solution, shake for 5 minutes and allow to stand for 20 minutes; the solution is not greenish blue when compared with a blank. D. The residue obtained in test A, after washing with water and drying at 105, melts at 159 to 163 (2.4.21). E. The filtrate obtained in test A gives the reactions of sodium salts (2.3.1).
Warfarin Sodium Clathrate

Warfarin Sodium Clathrate is a clathrate form of Warfarin Sodium consisting principally of Warfarin Sodium and Isopropyl Alcohol in a 2.1 molecular ratio. Warfarin Sodium Clathrate contains not less than 97.0 per cent and more than 102.0 per cent of C19H15NaO4 calculated on the anhydrous, isopropyl alcohol-free basis and not less than 8.0 per cent and not more than 8.5 per cent of isopropy alcohol.
Tests
Appearance of solution. A 5.0 per cent w/v solution is clear (2.4.1), and colourless (2.4.1). pH (2.4.24). 7.6 to 8.6, determined in a 1.0 per cent w/v solution. Phenolic ketones. Absorbance of a 12.5 per cent w/v solution in a 5.0 per cent w/v solution of sodium hydroxide at the
1241
WARFARIN SODIUM CLATHRATE
IP 2007
Description. A white crystalline powder; hygroscopic.
Identification
Test A may be omitted if test B, C, D and E are carried out. Tests B and D may be omitted if tests A, C and E are carried out. A. Dissolve about 1 g in 25 ml of water, add 2 ml of 2 M hydrochloric acid and filter. Wash the precipitate 5 to 6 times with water. Dry the residue over phosphorus pentoxide. On the residue, determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with warfarin sodium RS or with the reference spectrum of warfarin sodium. B. In the test for Related substances, the principal spot in the chromatogram obtained with test solution (b) corresponds to that in the chromatogram obtained with reference solution (b). C. Dissolve 1 g in 10 ml of water, add 5 ml of nitric acid and filter. To the filtrate add 2 ml of potassium dichromate solution, shake for 5 minutes and allow to stand for 20 minutes; the solution is not greenish-blue when compared with a blank. D. The residue obtained in test A, after washing with water and drying at 105 melts at 159 to 163. E. The filtrate obtained in test A gives the reactions of sodium salts (2.3.1).
Reference solution (c). A solution containing 0.1 per cent w/v of acenocomarol RS and 0.2 per cent w/v of the substance under examination in acetone. Apply to the plate 20 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with test solution (a) is not more intense than the spot in the chromatogram with reference solution (a). The test in not valid unless the chromatogram obtained with reference solution (c) show two clearly separated spots and the chromatogram obtained with reference solution (a) shows a clearly visible spot. Water (2.3.43). Not more than 4.0 per cent, determined on 0.75 g. Isopropyl alcohol. Determine by gas chromatography (2.4.13). Test solution. Dissolve 0.5 g of the substance under examination in sufficient water to produce 10 ml. Reference solution (a). A solution containing 5.0 per cent w/v of the substance under examination and 0.5 per cent v/v of propan-I-ol (internal standard). Reference solution (b). A solution containing 0.5 per cent v/v each of propan-2-ol and the internal standard. Chromatographic system a glass column 1.5 m x 4 mm, packed with porous polymer beads (125 to 150 mm) (such as Porapak Q), temperature: column 150, inlet port at 180 and detector at 200, flow rate. 40 ml per minute of the carrier gas. The column temperature may be varied so that the resolution, R, between propan-I-ol and propanol-2 ol is not less than 2.0, the tailing factor. T, for the propan-2-ol is not less than 2.0 the tailing factor, T, for the propan-2-ol peak is not more than 1.5 and the relative standard deviation of the ratio of the area due to the peak of propanol-2-ol to that due to propan-1-ol for five replicate injections of reference solution (b) is not more than 2.0 per cent. Calculate the content of isopropyl alcohol. Assay. Weigh accurately about 0.1 g and dissolve in sufficient 0.01 M sodium hydroxide to produce 100.0 ml. Dilute 10.0 ml to 100.0 ml with 0.01 M sodium hydroxide and dilute 5.0 ml to 50.0 ml with 0.01 M sodium hydroxide. Measure the absorbance of the resulting solution at the maximum at about 308 nm (2.4.7). Calculate the content of C19H15NaO4 taking 431 as the specific absorbance at 308 nm. Storage. Store protected from light and moisture.
Tests
Appearance of solution. A 5.0 per cent w/v solution is clear (2.4.1), and colourless (2.4.1). pH (2.4.24). 7.2 to 8.3, determined in a 1.0 per cent w/v solution. Phenolic ketones. Absorbance of a 12.5 per cent w/v solution in a 5.0 per cent w/v solution of sodium hydroxide at the maximum at about 385 nm, measured within 15 minutes of preparation, is not more than 0.20 (2.4.7). Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 50 volumes of chloroform, 50 volumes of cyclohexane and 20 volumes of glacial acetic acid. Test solution (a). Dissolve 0.2 g of the substance under examination in 10 ml of acetone. Test solution (b). Dissolve 0.4 g of the substance under examination in 100 ml of acetone. Reference solution (a). A 0.002 per cent w/v solution of the substance under examination in acetone. Reference solution (b). A 0.4 per cent w/v solution of warfarin sodium RS in acetone.
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IP 2007
WARFARIN TABLETS
Warfarin Tablets
Warfarin Sodium Tablets
Warfarin Tablets contain not less than 92.5 per cent and not more than 107.5 per cent of the stated amount of warfarin sodium, C19H15NaO4.
the chromatograms obtained with reference solutions (b) and (c) respectively and any other secondary spot is not more intense than the spot in the chromatogram obtained with reference solution (a). Uniformity of content. Comply with the test stated under Tablets. Determine by liquid chromatography (2.4.14). Test solution. Shake one tablet with 10 ml of 0.01 M sodium hydroxide for 15 minutes, add 10 ml of a 2 per cent v/v solution of glacial acetic acid in acetonitrile, centrifuge for 10 minutes and use the clear supernatant liquid. Chromatographic system a stainless steel column 10 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 55 volumes of acetonitrile, 45 volumes of water and 1 volume of glacial acetic acid, flow rate. 2 ml per minute, spectrophotometer set at 283 nm, a 20 l loop injector. Determine the content of C19H15NaO4 in the tablet. Dissolution (2.5.2). Apparatus. No 1 Medium. 900 ml of a 0.68 per cent w/v solution of potassium dihydrogen phosphate with the pH adjusted to 6.8 by the addition of 1 M sodium hydroxide Speed and time. 100 rpm and 45 minutes. For tablets containing 2 mg or less of warfarin sodium, use three tablets for each test; for tablets containing more than 2 mg of warfarin sodium, use a single tablet for each test. Withdraw a suitable volume of the medium and filter. Measure the absorbance of a layer of suitable thickness of the filtrate, suitably diluted if necessary, at the maxima at about 307 nm and 360 nm (2.4.7), and calculate the difference between the two absorbances (DA). Calculate the total content of warfarin sodium, C19H15NaO4, in the medium taking 428 as the specific absorbance value of DA. D. Not less than 70 per cent of the stated amount of C19H15NaO4. Other tests. Comply with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 20 mg of Warfarin Sodium and shake with 250.0 ml of 0.01 M sodium hydroxide for 15 minutes and filter. To 20.0 ml of the filtrate add 0.15 ml of hydrochloric acid and extract with three quantities, each of 15 ml, of chloroform. Extract the combined chloroform layers with three quantities, each of 20 ml, of 0.01 M sodium hydroxide. Dilute the combined aqueous layers to 100.0 ml with 0.01 M
Identification
A. Extract a quantity of the powdered tablets containing 0.1 g of Warfarin Sodium with 30 ml of water, add 0.1 ml of 2 M hydrochloric acid, filter, wash the precipitate with water and dry. Warm the residue gently with 3 ml of ethanol (95 per cent), filter and add the filtrate to 25 ml of water containing 0.1 ml of 2 M hydrochloric acid. Filter, wash the precipitate with water and dry it at 105. On the residue, determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with warfarin sodium RS or with the reference spectrum of warfarin sodium. B. The final residue obtained in test A melts at about 159 (2.4.21).
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 50 volumes of chloroform, 50 volumes of cyclohexane and 20 volumes of glacial acetic acid. Test solution. Shake a quantity of the powdered tablets containing 40 mg of Warfarin Sodium with 30 ml of water for 15 minutes, add 0.1 ml of hydrochloric acid and extract with three quantities, each of 10 ml, of chloroform, drying each extract with anhydrous sodium sulphate. Evaporate the combined extracts at a temperature not exceeding 40 and dissolve the residue in 2 ml of acetone. Reference solution (a). Dilute 1 volume of the test solution to 200 volumes with acetone. Reference solution (b). A 0.002 per cent w/v solution of (E)-4phenylbut-3-en-2-one in acetone. Reference solution (c). A 0.02 per cent w/v solution of 4-hydroxycoumarin in acetone. Apply to the plate 20 l of each solution. After development, dry the plate in air and examine immediately in visible light noting the position of any coloured spots and then examine in ultraviolet light at 254 nm, ignoring any spot that was noted in visible light. Any spots corresponding to (E)-4-phenylbut-3en-2-one and 4-hydroxycoumarin in the chromatogram obtained with the test solution are not more intense than the spots in
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PURIFIED WATER
IP 2007
sodium hydroxide, filter and measure the absorbance of the resulting solution at the maximum at about 307 nm (2.4.7). Calculate the content of C19H15NaO4 taking 431 as the specific absorbance at 307 nm. Storage. Store protected from light.
Purified Water
H2O Mol. Wt. 18.0 Purified Water is prepared by distillation, by means of ion exchange or by any other appropriate means from suitable potable water that complies with all relevant statutory regulations. During production and subsequent storage, it is recommended that adequate measures are taken to ensure that the microbial quality is controlled and monitored. Appropriate alert and action limits are set so as to detect adverse trends. Under controlled conditions, an appropriate action limit is a total viable count (2.2.9) of 100 micro-organisms per ml, determined by membrane filtration. In addition, the test for oxidisable substances (given below) is carried out. The adequacy of these measures may be determined by carrying out the test for conductivity (2.4.9) off-line or in-line. Description. A clear, colourless liquid; odourless and tasteless.
Nitrates. To 5 ml in a test-tube immersed in ice add 0.4 ml of a 10 per cent w/v solution of potassium chloride, 0.1 ml of diphenylamine solution and, dropwise with shaking, 5 ml of sulphuric acid. Transfer the tube to a water-bath at 50 and allow to stand for 15 minutes. Any blue colour in the solution is not more intense than that in a solution prepared at the same time and in the same manner using a mixture of 4.5 ml of nitrate-free water and 0.5 ml of nitrate standard solution (2 ppm NO3) (0.2 ppm). Sulphates (2.3.17). To 10 ml add 0.1 ml of 2 M hydrochloric acid and 0.1 ml of barium chloride solution. The appearance of the solution does not change for at least 1 hour. Oxidisable substances. To 100 ml add 10 ml of 1 M sulphuric acid and 0.1 ml of 0.02 M potassium permanganate and boil for 5 minutes; the solution remains faintly pink. Residue on evaporation. Evaporate 100 ml to dryness on a water-bath and dry to constant weight at 105. The residue weighs not more than 1 mg (0.001 per cent). Purified Water intended for use in the manufacture of dialysis solutions and also without a further procedure for the removal of bacterial endotoxins complies with the following additional requirements. Aluminium (2.3.8). Not more than 10 ppb, determined using the following solutions. Test solution. To 400 ml of the water under examination add 10 ml of acetate buffer solution pH 6.0 and 100 ml of distilled water. Reference solution. Mix 2 ml of aluminium standard solution (2 ppm Al), 10 ml of acetate buffer solution pH 6.0 and 98 ml of distilled water. Blank solution. Mix 10 ml of acetate buffer solution pH 6.0 and 100 ml of distilled water. Bacterial endotoxins (2.2.3). Not more than 0.25 Endotoxin Unit per ml. Storage. Store protected from light.
Tests
Acidity or alkalinity. To 10 ml, freshly boiled and cooled in a borosilicate glass flask, add 0.05 ml of methyl red solution; the resulting solution is not red. To 10 ml add 0.1 ml of bromothymol blue solution; the resulting solution is not blue. Ammonium. To 20 ml add 1 ml of alkaline potassium mercuriiodide solution, and allow to stand for 5 minutes. When viewed vertically the solution is not more intensely coloured than a solution prepared at the same time by adding 1 ml of alkaline potassium mercuri-iodide solution to a mixture of 4.0 ml of ammonium standard solution (1 ppm NH4) and 16.0 ml of ammonia-free water (0.2 ppm). Calcium and magnesium. To 100 ml add 2 ml of ammonia buffer pH 10.0, 50 mg of mordant black II mixture and 0.5 ml of 0.01 M disodium edetate; a pure blue colour is produced. Heavy metals (2.3.13). Evaporate 150 ml to 15 ml on a waterbath; 12 ml of the solution complies with the limit test for heavy metals, Method D (0.1 ppm). Use lead standard solution (1 ppm Pb) to prepare the standard. Chlorides (2.3.12). To 10 ml add 1 ml of 2 M nitric acid and 0.2 ml of 0.1 M silver nitrate; the appearance of the solution does not change for at least 15 minutes.
Water For Injections

H2O Mol. Wt. 18.0 Water for Injections is water intended for use in the preparations of medicines for parenteral administration when water is used as a vehicle (Water for Injections in bulk) and for dissolving or diluting substances or preparations for injectable preparations (Sterile Water for Injections).
1244
IP 2007
STERILE WATER FOR INJECTIONS
Water For Injections in Bulk

Production Water for Injections in bulk is obtained by distilling potable water or Purified Water from a neutral glass, quartz or suitable metal still fitted with an effective device for preventing the entrainment of droplets; the still must be suitably maintained to ensure the production of a pyrogenic water. The first portion of the distillate is discarded and the remainder is collected and stored in conditions designed to prevent the growth of microorganisms and to avoid any other contamination. During production and subsequent storage, it is recommended that adequate measures are taken to ensure that the microbial quality is controlled and monitored. Appropriate alert and action limits are set so as to detect adverse trends. The adequacy of these measures is determined by the following tests that may be done off-line or in-line. Total organic carbon (2.4.30). Not more than 0.5 mg per litre. Conductivity (2.4.9). Meets the requirements of the test. Description. A clear and colourless liquid; odourless and tasteless.
allow to stand for 15 minutes. Any blue colour in the solution is not more intense than that in a solution prepared at the same time and in the same manner using a mixture of 4.5 ml of nitrate-free water and 0.5 ml of nitrate standard solution (2 ppm NO3) (0.2 ppm). Sulphates (2.3.17). To 10 ml add 0.1 ml of 2 M hydrochloric acid and 0.1 ml of barium chloride solution. The appearance of the solution does not change for at least 1 hour. Aluminium (2.3.8) For water for injections intended for use in the manufacture of dialysis solutions. Not more than 10 ppb, determined using the following solutions. Test solution. To 400 ml of the water under examination add 10 ml of acetate buffer solution pH 6.0 and 100 ml of distilled water. Reference solution. Mix 2 ml of aluminium standard solution (2 ppm Al), 10 ml of acetate buffer solution pH 6.0 and 98 ml of distilled water. Blank solution. Mix 10 ml of acetate buffer solution pH 6.0 and 100 ml of distilled water. Bacterial endotoxins (2.2.3). Not more than 0.25 Endotoxin Unit per ml. Storage. Store protected from moisture in containers designed to prevent the growth of micro-organisms. Labelling. The label on the container in which the bulk has been distributed states that the contents have not been sterilised.
Tests
Acidity or alkalinity. To 10 ml, freshly boiled and cooled in a borosilicate glass flask, add 0.05 ml of methyl red solution; the resulting solution is not red. To 10 ml add 0.1 ml of bromothymol blue solution; the resulting solution is not blue. Ammonium. To 20 ml add 1 ml of alkaline potassium mercuriiodide solution and allow to stand for 5 minutes. When viewed vertically the solution is not more intensely coloured than a solution prepared at the same time by adding 1 ml of alkaline potassium mercuri-iodide solution to a solution containing 2.5 ml of dilute ammonium chloride solution and 7.5 ml of the liquid under examination. Calcium and magnesium. To 100 ml add 2 ml of ammonia buffer pH 10.0, 50 mg of mordant black II mixture and 0.5 ml of 0.01 M disodium edetate; a pure blue colour is produced. Heavy metals (2.3.13). Evaporate 150 ml to 15 ml on a waterbath. 12 ml of the solution complies with the limit test for heavy metals, Method D (0.1 ppm). Use lead standard solution (1 ppm Pb) to prepare the standard. Chlorides (2.3.12). To 10 ml add 1 ml of 2 M nitric acid and 0.2 ml of 0.1 M silver nitrate; the appearance of the solution does not change for at least 15 minutes. Nitrates. To 5 ml in a test-tube immersed in ice add 0.4 ml of a 10 per cent w/v solution of potassium chloride, 0.1 ml of diphenylamine solution and, dropwise with shaking, 5 ml of sulphuric acid. Transfer the tube to a water-bath at 50 and
Sterile Water For Injections

Sterile Water for Injections is Water for Injections in bulk that has been distributed in suitable containers of glass or any other material, sealed and sterilised by heat under conditions that ensure that the water complies with the test for bacterial endotoxins. It is free from any added substances. Each container contains a sufficient quantity of Water for Injections to permit the withdrawal of the nominal volume. Description. A clear, colourless liquid; odourless.
Tests
Appearance of solution. When examined in suitable conditions of visibility, it is clear (2.4.1) colourless (2.4.1) and practically free from suspended particles. Acidity or alkalinity. To 20 ml add 0.05 ml of phenol red solution. If the solution is yellow, it becomes red on the addition of 0.1 ml of 0.01 M sodium hydroxide; if red, it becomes yellow on the addition of 0.15 ml of 0.01 M hydrochloric acid.
1245
WOOL FAT
IP 2007
Ammonium. To 20 ml add 1 ml of alkaline potassium mercuriiodide solution and allow to stand for 5 minutes. When viewed vertically the solution is not more intensely coloured than a solution prepared at the same time by adding 1 ml of alkaline potassium mercuri-iodide solution to a solution containing 2.5 ml of dilute ammonium chloride solution and 7.5 ml of the liquid under examination. Calcium and magnesium. To 100 ml add 2 ml of ammonia buffer pH 10.0, 50 mg of mordant black II mixture and 0.5 ml of 0.01 M disodium edetate; a pure blue colour is produced. Heavy metals (2.3.13). Evaporate 150 ml to 15 ml on a waterbath. 12 ml of the solution complies with the limit test for heavy metals, Method D (0.1 ppm). Use lead standard solution (1 ppm Pb) to prepare the standard. Chlorides.To 10 ml add 1 ml of 2 M nitric acid and 0.2 ml of 0.1 M silver nitrate; the appearance of the solution does not change for at least 15 minutes. For containers with a nominal volume of 100 ml or less, 15 ml complies with the limit test for chlorides (2.3.12) (0.5 ppm), using a standard solution prepared by mixing 1.5 ml of chloride standard solution (5 ppm Cl) and 13.5 ml of water. Nitrates. To 5 ml in a test-tube immersed in ice add 0.4 ml of a 10 per cent w/v solution of potassium chloride, 0.1 ml of diphenylamine solution and, dropwise with shaking, 5 ml of sulphuric acid. Transfer the tube to a water-bath at 50 and allow to stand for 15 minutes. Any blue colour in the solution is not more intense than that in a solution prepared at the same time and in the same manner using a mixture of 4.5 ml of nitrate-free water and 0.5 ml of nitrate standard solution (2 ppm NO3) (0.2 ppm). Sulphates. To 10 ml add 0.1 ml of 2 M hydrochloric acid and 0.1 ml of barium chloride solution. The appearance of the solution does not change for at least 1 hour. Oxidisable substances. Boil 100 ml with 10 ml of 1 M sulphuric acid, add 0.4 ml of 0.02 M potassium permanganate (for Sterile Water for Injection in containers with fill volume of less than 50 ml) or 0.2 ml of 0.02 M potassium permanganate (for Sterile Water for Injection in containers with fill volume of 50 ml or more) and boil for 5 minutes. If a precipitate forms, cool in an ice-bath to room temperature and filter through a sintered glass filter (porosity No.3). The pink colour of the solution does not disappear completely. Residue on evaporation. Evaporate 100 ml to dryness on a water-bath and dry the residue to constant weight at 105. For containers with a nominal volume of 10 ml or less, the residue weighs not more than 4 mg (0.004 per cent) and for containers with a nominal volume greater than 10 ml, the residue weighs not more than 3 mg (0.003 per cent). Particulate contamination (2.5.9). Complies with the requirements of Method 1 or Method 2.
Bacterial endotoxins (2.2.3). Not more than 0.25 Endotoxin Unit per ml. Sterility (2.2.11). Complies with the test for sterility. Storage. Store in single dose containers of not larger than one litre size.
Wool Fat
Anhydrous Lanolin
Wool Fat is purified, anhydrous, waxy material obtained from the wool of sheep. It may contain Butylated Hydroxytoluene as an antioxidant. Description. A pale yellow, unctuous substance; odour, characteristic.
Identification
A. To a solution of 0.5 g in 5 ml of chloroform add 1 ml of acetic anhydride and 0.1 ml of sulphuric acid; a green colour develops. B. To a solution of 50 mg in 5 ml of chloroform add 5 ml of sulphuric acid and shake; a red colour is produced and a strong fluorescence appears in the lower layer.
Tests
Melting range (2.4.21). 34 to 44, determined by Method IV. To fill the metal cup, melt the substance under examination on a water-bath, cool to about 50, pour into the cup and allow to stand at 15 to 20 for 24 hours. Acid value (2.3.23). Not more than 1.0, determined on 5.0 g dissolved in 25 ml of the prescribed mixture of solvents. Peroxide value (2.3.35). Not more than 20. Saponification value. (2.3.37). 90 to 105. Heat for 4 hours. Water-absorption capacity. Weigh 10.0 g into a mortar. Add water in quantities of 0.2 to 0.5 ml from a burette and stir vigorously, incorporating all the water before proceeding to the next addition. The end-point is reached when visible droplets remain that cannot be incorporated; not less than 20 ml of water is absorbed. Water-soluble acidic or alkaline substances. Shake vigorously 5.0 g, previously melted on a water-bath, for 2 minutes with 75 ml of water previously heated to 90 to 95. Allow to cool and filter through filter paper previously washed with water. To 60 ml of the filtrate, which may not be clear, add 0.25 ml of bromothymol blue solution. Not more than 0.2 ml of 0.02 M hydrochloric acid or 0.15 ml of 0.02 M sodium hydroxide is required to change the colour of the solution.
1246
IP 2007
HYDROUS WOOL FAT
Water-soluble oxidisable substances. To 10 ml of the filtrate obtained in the test for Water-soluble acidic or alkaline substances add 1 ml of 1 M sulphuric acid and 0.1 ml of 0.02 M potassium permanganate; the solution is not completely decolorised within 10 minutes. Ammonia. To 10 ml of the filtrate obtained in the test for Watersoluble acidic or alkaline substances add 1 ml of 1 M sodium hydroxide and boil; the vapours do not turn red litmus paper blue. Chlorides. Boil 1.0 g with 20 ml of ethanol (90 per cent) under a reflux condenser for 5 minutes, cool, add 40 ml of water and 0.5 ml of nitric acid and filter. To the filtrate add 0.15 ml of a 1 per cent w/v solution of silver nitrate in ethanol (90 per cent). After 5 minutes, protected from light; any opalescence produced is not more intense than that obtained by adding 0.15 ml of a 1 per cent w/v solution of silver nitrate in ethanol (90 per cent) to a mixture of 0.2 ml of 0.02 M hydrochloric acid, 20 ml of ethanol (90 per cent), 40 ml of water and 0.5 ml of nitric acid (150 ppm). Paraffins. Prepare an alumina column 23 cm x 2 cm by adding a slurry of anhydrous aluminium oxide and light petroleum (40 to 60) to a glass tube fitted with a tap and containing the light petroleum; the tap and absorbent cotton plugs should be free from grease. Allow to settle and reduce the depth of the solvent above the column to about 4 cm. Dissolve 3.0 g of the substance under examination in 50 ml of warm light petroleum (40 to 60), cool, pass the solution through the column at a rate of 3 ml per minute and wash with 250 ml of the light petroleum. Distil the combined eluate and washings to low bulk, evaporate to dryness on a water-bath and heat the residue at 105 for periods of 10 minutes until the difference between two successive weighings is not greater than 1 mg; the residue weighs not more than 30 mg. Butylated hydroxytoluene (if present). Not more than 200 ppm, determined by gas chromatography (2.4.13). Test solution (a). A 10 per cent w/v solution of the substance under examination in carbon disulphide. Test solution (b). A solution containing 10 per cent w/v of the substance under examination and 0.002 per cent w/v of methyl n-decanoate (internal standard) in carbon disulphide. Reference solution. A solution containing 0.002 per cent w/v each of butylated hydroxytoluene and the internal standard. Chromatographic system a glass column 1.5 m x 4 mm, packed with silanised diatomaceous support (80 to 100 mesh) (such as Diatomite) impregnated with 10 per cent w/w of silicone gum rubber (methyl) (such as SE-30), temperature: column 150, inlet port at 180 and detector at 300,
flow rate. 40 ml per minute of the carrier gas. Calculate the content of butylated hydroxytoluene in the substance under examination from the heights or areas of the peaks due to butylated hydroxytoluene and the internal standard in the chromatograms obtained with test solution (b) and the reference solution. Sulphated ash (2.3.18). Not more than 0.15 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105 for 1 hour. Storage. Store protected from moisture. Labelling. The label states the proportion of any butylated hydroxytoluene present.
Hydrous Wool Fat

Hydrous Wool Fat is a mixture of 75 per cent w/w of Wool Fat and 25 per cent w/w of Purified Water. Description. A pale yellow, unctuous substance; odour, faint and characteristic. On heating, it separates at first into two layers; with continued heating with stirring, water is driven off and the residue which is transparent while warm, cools to form a yellowish, tenacious, soft mass.
Identification
A. To a solution of 0.5 g in 5 ml of chloroform add 1 ml of acetic anhydride and 0.1 ml of sulphuric acid; a green colour develops. B. To a solution of 50 mg in 5 ml of chloroform add 5 ml of sulphuric acid and shake; a red colour is produced and a strong fluorescence appears in the lower layer.
Tests
Melting range (2.4.21). 34 to 44, determined by Method IV. To fill the metal cup, melt the substance under examination on a water-bath, cool to about 50, pour into the cup and allow to stand at 15 to 20 for 24 hours. Acid value (2.3.23). Not more than 1.0, determined on 5.0 g dissolved in 25 ml of the prescribed mixture of solvents. Paraffins. Prepare an alumina column 23 cm x 2 cm by adding a slurry of anhydrous aluminium oxide and light petroleum (40 to 60) to a glass tube fitted with a tap and containing the light petroleum; the tap and absorbent cotton plugs should be free from grease. Allow to settle and reduce the depth of the solvent above the column to about 4 cm. Dissolve 3 g of the substance under examination in 50 ml of warm light petroleum (40 to 60), cool, pass the solution through the column at a rate of 3 ml per minute and wash with 250 ml of the
1247
HYDROUS WOOL FAT
IP 2007
light petroleum. Distil the combined eluate and washings to low bulk, evaporate to dryness on a water-bath and heat the residue at 105 for periods of 10 minutes until the difference between two successive weighings is not greater than 1 mg; the residue weighs not more than 30 mg. Peroxide value (2.3.35). Not more than 15. Saponification value (2.3.37). 67 to 79. Heat for 4 hours. Water-absorption capacity. Weigh 10.0 g of the residue obtained in the test for Wool fat content into a mortar. Add water in quantities of 0.2 to 0.5 ml from a burette and stir vigorously, incorporating all the water before proceeding to the next addition. The end-point is reached when visible droplets remain that cannot be incorporated; not less than 20 ml of water is absorbed. Water-soluble acidic or alkaline substances. Shake vigorously 6.7 g, previously melted on a water-bath, for 2 minutes with 75 ml of water previously heated to 90 to 95, Allow to cool and filter through filter paper previously washed with water. To 60 ml of the filtrate, which may not be clear, add 0.25 ml of bromothymol blue solution. Not more than 0.2 ml of 0.02 M hydrochloric acid or 0.15 ml of 0.02 M sodium hydroxide is required to change the colour of the solution. Water-soluble oxidisable substances. 10 ml of the filtrate obtained in the test for Water-soluble acidic or alkaline substances add 1 ml of 1 M sulphuric acid and 0.1 ml of 0.02 M potassium permanganate; the solution is not completely decolorised within 10 minutes.
Ammonia. To 10 ml of the filtrate obtained in the test for Watersoluble acidic or alkaline substances add 1 ml of 1 M sodium hydroxide and boil; the vapours do not turn red litmus paper blue. Chlorides. Boil 1.0 g with 20 ml of ethanol (90 per cent) under a reflux condenser for 5 minutes, cool, add 40 ml of water and 0.5 ml of nitric acid and filter. To the filtrate add 0.15 ml of a1 per cent w/v solution of silver nitrate in ethanol (90 per cent). After 5 minutes, protected from light, any opalescence produced is not more intense than that obtained by adding 0.15 ml of a 1 per cent w/v solution of silver nitrate in ethanol (90 per cent) to a mixture of 0.2 ml of 0.02 M hydrochloric acid, 20 ml of ethanol (90 per cent), 40 ml of water and 0.5 ml of nitric acid (150 ppm). Wool fat content. 72.5 to 77.5 per cent, determined by the following method. Weigh accurately about 30 g in a tared porcelain dish containing a glass rod, heat on a water-bath with continuous stirring to constant weight and weigh the residue. solution. Titrate with 0.1 M perchloric acid, using 1-naphtholbenzein solution as indicator. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.02808 g of C16H24N2, HCl. Storage. Store protected from light and moisture.
1248
MONOGRAPHS
X
Xylometazoline Hydrochloride Xylometazoline Nasal Drops Xylose .... .... ....
1249
IP 2007
XYLOMETAZOLINE NASAL DROPS
Xylometazoline Hydrochloride
H3C H3C CH3
C16H24N2,HCl
CH3 CH3 N , HCl
HN
Mol. Wt. 280.8
ninhydrin in a mixture of 100 ml of 1-butanol and 3 ml of glacial acetic acid. Heat at 100 for 10 minutes, allow to cool, and spray with dilute potassium iodobismuthate solution. Any spot corresponding to N-(2-aminoethyl)-4-tert-butyl2,6-xylyl- aceta0-mide in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Iron (2.4.14). Moisten the residue obtained in the test for Sulphated ash with 5 ml of hydrochloric acid, evaporate to dryness and dissolve in sufficient water to produce 50 ml. 10 ml of the resulting solution complies with the limit test for iron (50 ppm). Sulphates (2.3.17). 0. 75 g complies with the limit test for sulphates (200 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.5 g, dissolve in 50 ml of anhydrous glacial acetic acid, add 10 ml of mercuric acetate solution. Titrate with 0.1 M perchloric acid, using 1-naphtholbenzein solution as indicator. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.02808 g of C16H24N2, HCl. Storage. Store protected from light and moisture.
Xylometazoline Hydrochloride is 2-(4-tert-butyl-2,6dimethylbenzyl)-2-imidazoline hydrochloride. Xylometazoline Hydrochloride contains not less than 99.0 per cent and not more than 101.0 per cent of C16H24N2, HCl, calculated on the dried basis. Description. A white or almost white, crystalline powder; odourless or almost odourless.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with xylometazoline hydrochloride RS or with the reference spectrum of xylometazoline hydrochloride. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.05 per cent w/v solution in 0.1 M hydrochloric acid shows an absorption maximum at about 265 nm and a minimum at about 257 nm with two inflections at about 270 nm and 275 nm; absorbance at about 265 nm, about 0.5. C. To 1 ml of a 0.05 per cent w/v solution, add 0.2 ml of a 5 per cent w/v solution of sodium nitroprusside and 0.1 ml of 5 M sodium hydroxide, allow to stand for 10 minutes and add 2 ml of sodium bicarbonate solution; a violet colour is produced. D. Gives the reactions of chlorides (2.3.1).
Xylometazoline Nasal Drops

Xylometazoline Hydrochloride Nasal Drops
Xylometazoline Nasal Drops are a solution of Xylometazoline Hydrochloride in Purified Water. Xylometazoline Nasal Drops contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of xylometazoline hydrochloride, C16H24N2, HCl.
Tests
pH (2.4.24). 5.0 to 6.6, determined in a 5.0 per cent w/v solution. N-(2-Aminoethyl)-4-tert-butyl-2, 6-xylylacetamide. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel HF254. Mobile phase. A mixture of 200 volumes of methanol and 3 volumes of strong ammonia solution. Test solution. Dissolve 0.2 g of the substance under examination in 10 ml of methanol. Reference solution. A 0.01 per cent w/v solution of N-(2aminoethyl)-4-tert-butyl-2,6-xylylacetamide RS in methanol. Apply to the plate 5 l of each solution. After development, dry the plate in air, spray with a solution containing 0.3 g of
Identification
A. To a volume containing 50 mg of Xylometazoline Hydrochloride add 5 ml of 1 M sodium hydroxide, extract with 10 ml of dichloromethane, evaporate to dryness and dissolve the residue in 0.5 ml of dichloromethane. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with xylometazoline hydrochloride RS treated in the same manner or with the reference spectrum of xylometazoline. B. To a volume containing 0.5 mg of Xylometazoline Hydrochloride add 0.2 ml of a 5 per cent w/v solution of sodium
1251
XYLOSE
IP 2007
nitroprusside and 0.1 ml of 5 M sodium hydroxide, allow to stand for 10 minutes and add 1 ml of sodium bicarbonate solution; a violet colour is produced.
Tests
pH (2.4.24). 5.6 to 6.6. N-(2-Aminoethyl)-4-tert-butyl-2,6-xylylacetamide. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel HF254. Mobile phase. A mixture of 200 volumes of methanol and 3 volumes of strong ammonia solution. Test solution. Add a volume containing 10 mg of Xylometazoline Hydrochloride to 30 ml of water, add 5 ml of 5 M sodium hydroxide, mix, extract with three quantities, each of 20 ml, of dichloromethane, evaporate the combined extracts to dryness and dissolve the residue in 1 ml of dichloromethane. Reference solution. A 0.03 per cent w/v solution of N-(2aminoethyl)-4-tert-butyl-2,6-xylylacetamide RS in dichloromethane. Apply to the plate 5 l of each solution. After development, dry the plate in air, spray with a solution containing 0.3 g of ninhydrin in a mixture of 100 ml of 1-butanol and 3 ml of glacial acetic acid. Heat at 100 for 10 minutes, allow to cool, and spray with dilute potassium iodobismuthate solution. Any spot corresponding to N-(2-aminoethyl)-4-tert-butyl2,6-xylylacetamide in the chromatogram obtained with test solution is not more intense than the spot in the chromatogram obtained with reference solution. Other tests. Comply with the tests stated under Nasal Preparations. Assay. To a volume containing 10 mg of Xylometazoline Hydrochloride add 5 ml of water, 10 ml of 2 M hydrochloric acid and 10 ml of dichloromethane and shake for 1 minute. Discard the dichloromethane layer and repeat the extraction with two further quantities, each of 10 ml, of dichloromethane. Add to the aqueous extract 10 ml of 5 M sodium hydroxide and 10 ml of dichloromethane, shake for 1 minute and allow to separate. Filter the dichloromethane extract through glass wool and repeat the extraction with four further quantities, each of 10 ml, of dichloromethane. Evaporate the combined dichloromethane extracts almost to dryness on a water-bath, remove the final traces of solvent in a current of air and dissolve the residue in 10.0 ml of 0.01 M hydrochloric acid. To 2.0 ml of this solution add 3 ml of water, 2.5 ml of 1 M sodium hydroxide and 2.5 ml of a 5 per cent w/v solution of sodium nitroprusside, mix and allow to stand protected from light for 10 minutes. Add 10 ml of a freshly prepared 8.3 per cent w/v solution of sodium bicarbonate, dilute to 100.0 ml with water, allow to stand protected from light for 10 minutes and measure
the absorbance of the resulting solution at the maximum at about 560 nm (2.4.7), using as blank a solution prepared by treating 5 ml of water and 2.5 ml of 1 M sodium hydroxide in the same manner beginning at the words and 2.5 ml of a 5 per cent w/v solution of sodium nitroprusside,...... Calculate the content of C16H24N2, HCl from the absorbance obtained by repeating the operation using a 0.1 per cent w/v solution of xylometazoline hydrochloride RS in place of the nasal drops. Storage. Store protected from light and moisture.
Xylose
D-Xylose; D-Xylopyranose
O OH HO OH
C5H10O5 Mol. Wt. 150.1 Xylose contains not less than 98.0 per cent and not more than 102.0 per cent of C5H10O5, calculated on the dried basis. Description. Colourless needles or a white, crystalline powder.
OH
Identification
A. Determine by thin-layer chromatography (2.4.17), coating the plate with a suspension of silica gel G in a 0.3 per cent w/v solution of sodium acetate to form a uniform layer 0.5 mm thick. Mobile phase. A mixture of 70 volumes of glacial acetic acid, 60 volumes of chloroform and 10 volumes of water. Test solution. Dissolve 0.5 g of the substance under examination in 10 ml of water. Reference solution (a). A 5.0 per cent w/v solution of xylose RS in water. Reference solution (b). A mixture of equal volumes of the test solution and reference solution (a). Apply to the plate 2 l of each solution. Develop the plate in a continuous elution tank for about 4 hours. Dry the plate in warm air, spray with a solution in acetone containing 1 per cent w/v solution of diphenylamine, 1 per cent v/v of aniline and 1 per cent v/v of phosphoric acid and heat for 10 minutes at 130. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with reference solution (a). The principal spot in the chromatogram obtained with reference solution (b) appears as a single, compact spot.
1252
IP 2007
XYLOSE
B. When heated with potassium cupri-tartrate solution it produces a copious precipitate of cuprous oxide.
Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 100 at a pressure not exceeding 0.7 kPa. Assay. Carry out the following procedure keeping strict control of time between steps. Weigh accurately about 1.0 g of the substance under examination, dissolve in a saturated solution of benzoic acid in a 100-ml volumetric flask and dilute to volume with the same solvent. Dilute 1.0 ml of this solution to 100.0 ml with the same solvent. To 1.0 ml of the resulting solution, in two different test-tubes, add 5 ml of 4-bromoaniline solution into each tube and mix. Loosely stopper one tube, place in a water-bath maintained at 70 for 10 minutes, remove, cool rapidly to room temperature and mix. Keep the tube in the dark for 70 minutes and measure the absorbance at the maximum at about 520 nm (2.4.7), using the untreated solution in the second test tube as the blank. Simultaneously, carry out the operation using 1.0 ml of a 0.01 per cent w/v solution of xylose RS in a saturated solution of benzoic acid beginning at the words add 5 ml of 4-bromoaniline solution...... Calculate the content of C5H10O5. Storage. Store protected from moisture.
Tests
Appearance of solution. A 10.0 per cent w/v solution in carbon dioxide-free water is clear (2.4.1), and colourless (2.4.1). Acidity. Dissolve 5.0 g in 50 ml of carbon dioxide-free water. Not more than 0.2 ml of 0.1 M sodium hydroxide is required to neutralise the solution using dilute phenolphthalein solution as indicator. Specific optical rotation (2.4.22). +18.5 to +19.5, determined at 20 in a 10.0 per cent w/v solution containing 0.4 per cent v/v of 5 M ammonia. Arsenic (2.3.10). Dissolve 10.0 g in 50 ml of water and add 10 ml of stannated hydrochloric acid AsT. The resulting solution complies with the limit test for arsenic (1 ppm). Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method A (20 ppm). Iron (2.3.14). A solution of 4.0 g in 25 ml of water complies with the limit test for iron (10 ppm). Chlorides (2.3.12). Dissolve 3.0 g in 20 ml of water. 5 ml of the resulting solution complies with the limit test for chlorides (330 ppm).
1253
MONOGRAPHS
Z
Zidovudine Zidovudine Capsules Zidovudine Injection Zidovudine Oral Solution Zidovudine Tablets Zidovudine, Lamivudine And Nevirapine Tablets Zinc Chloride Zinc Oxide Zinc Oxide Cream Zinc Stearate Zinc Sulphate Zinc Sulphate Eye Drops Zinc Undecenoate Zinc Undecenoate Ointment .... .... .... .... .... .... .... .... .... .... .... .... .... ....
1255
IP 2007
ZIDOVUDINE
Zidovudine
O HN HO O O N3
C10H13N5O4 Mol. Wt. 267.2
CH3 N
spots in the chromatogram obtained with the reference solution. No secondary spot in the chromatogram obtained with the test solution is more intense than the principal spot in the chromatogram obtained with the reference solution (0.5 per cent). Spray the plate with a mixture of 0.5 g of carbazole in 95 ml of ethanol (95 per cent) and 5 ml of sulphuric acid, heat for 10 minutes at 120 and compare the intensities of any secondary spots observed in the chromatogram obtained with the test solution with those of the principal spots in the chromatogram obtained with the reference solution. No spot corresponding to triphenylmethanol (Rf value about 2.3 relative to the Rf of zidovudine) is more intense than the corresponding spot in the chromatogram obtained with the reference solution (0.5 per cent). No secondary spot in the chromatogram obtained with the test solution is more intense than the principal spot in the chromatogram obtained with the reference solution. B. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 0.1 g of the substance under examination in 100 ml of methanol. Reference solution (a). A 0.001 per cent w/v solution of zidovudine RS in methanol. Reference solution (b). A solution containing 0.1 per cent w/v of zidovudine RS and 0.001 per cent w/v each of zidovudine-related compound B RS and zidovudine- related compound C RS in methanol. Chromatographic system a stainless steel column 15 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: A. water B. methanol, flow rate. 1 ml per minute, a linear gradient programme using the conditions given below, spectrophotometer set at 265 nm, a 10 l loop injector. Time (in min.) 0 15 30 35 40 45 Water (per cent v/v) 90 35 35 5 5 90 Methanol (per cent v/v) 10 65 65 95 95 10
Zidovudine is 1-(3-azido-2,3-dideoxy--D-ribofuranosyl)-5methylpyrimidine-2,4(1H,3H)-dione. Zidovudine contains not less than 97.0 per cent and not more than 102.0 per cent of C10H13N5O4, calculated on the anhydrous basis. Description. A white or almost white powder.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with zidovudine RS or with the reference spectrum of zidovudine. B. In the Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the peak due to zidovudine in the chromatogram obtained with reference solution (a). C. Melting range (2.4.21). 122 to 125.
Tests
Specific optical rotation (2.4.22). +60.5 to +63.0, determined in a 1.0 per cent w/v solution in ethanol (95 per cent). Related substances. A. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 90 volumes of dichloromethane and 10 volumes of methanol. Test solution. Dissolve 0.2 g of the substance under examination in 10 ml of methanol. Reference solution. A solution containing 0.01 per cent w/v each of zidovudine RS and triphenylmethanol in methanol. Apply to the plate 10 l of each solution. Allow the mobile phase to rise to about three-fourths of the height of the plate. Dry the plate in air and examine in ultraviolet light at 254 nm. Any secondary spots observed in the chromatogram obtained with the test solution correspond to those of the principal
Inject reference solution (b). The test is not valid unless the resolution between the peaks due to zidovudine and
1257
ZIDOVUDINE CAPSULES
IP 2007
zidovudine-related compound B is not less than 1.5 and the tailing factor for zidovudine is not more than 1.5. Separately inject the test solution and reference solution (a). The area of the peak corresponding to zidovudine- related compound B is not greater than 1.0 per cent and of that to zidovudine-related compound C is not greater than 2.0 per cent. The sum of the percentages of related substances by tests A and B is not greater than 3.0 per cent. Heavy metals (2.3.13). 1.0 g complies with limit test for heavy metals, Method B ( 20 ppm). Sulphated ash (2.3.18). Not more than 0.25 per cent. Water (2.3.43). Not more than 1.0 per cent, determined on 0.5 g. Assay. Determine by liquid chromatography (2,4,14). Test solution. Weigh accurately about 100 mg of the substance under examination, dissolve in a suitable quantity of methanol in a 50-ml volumetric flask and make up to volume with the mobile phase. Dilute 5.0 ml of this solution to 50.0 ml with the mobile phase. Reference solution (a). Weigh accurately about 100 mg of zidovudine RS, dissolve in a suitable quantity of methanol in a 50-ml volumetric flask and make up to volume with the mobile phase (solution A). Dilute 5.0 ml of solution A to 50.0 ml with the mobile phase. Reference solution (b). Transfer 2.0 ml of a 0.005 per cent w/v solution of zidovudine-related compound B RS in the mobile phase to a 50-ml volumetric flask, add 5.0 ml of solution A and make up to volume with the mobile phase. Chromatographic system a stainless steel column 15 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 70 volumes of water and 30 volumes of methanol, flow rate. 1 ml per minute, spectrophotometer set at 265 nm, a 20 l loop injector. Inject reference solution (b). The test is not valid unless the resolution between the peaks due to zidovudine and zidovudine-related compound B is not less than 2.0. Inject reference solution (a). The relative standard deviation for replicate injections is not more than 2.0 per cent. Separately inject the test solution and reference solution (a) and measure the responses for the principal peak. Calculate the content of C10H13N5O4. Storage. Store protected from light.
Zidovudine Capsules
Zidovudine Capsules contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of zidovudine, C10H13N5O4.
Identification
A.Transfer a quantity of the mixed contents of the capsules containing about 15 mg of Zidovudine to a 100-ml volumetric flask. Add about 80 ml of a mixture of 75 volumes of methanol and 25 volumes of water, shake for 10 minutes and dilute to volume with the same solvent mixture, mix and filter. Dilute 10 ml of the filtrate to 100 ml with the same solvent mixture. When examined in the range 200 nm to 300 nm (2.4.7), the resulting solution shows an absorption maximum at about 265 nm. B. In the Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the peak due to zidovudine in the chromatogram obtained with reference solution (c).
Tests
Related substances. Determine by liquid chromatography (2.4.14). Test solution. Weigh accurately a quantity of the mixed contents of 20 capsules containing about 100 mg of Zidovudine and transfer to a 100-ml volumetric flask. Add about 60 ml of the mobile phase, mix with the aid of ultrasound for 10 minutes, dilute to volume with the mobile phase, mix and filter. Dilute 5.0 ml of the filtrate to 50.0 ml with the mobile phase. Reference solution (a). Weigh accurately about 100 mg of zidovudine RS and transfer to a 100-ml volumetric flask. Dissolve in about 60 ml of the mobile phase and dilute to volume with the mobile phase. Reference solution (b). Weigh accurately about 20 mg of thymine and transfer to a 200-ml volumetric flask, dissolve and make up to volume with methanol. Reference solution (c). Transfer 10.0 ml of the reference solution (a) and 2.0 ml of reference solution (b) to a 100-ml volumetric flask and make up to the volume with the mobile phase. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 20 volumes of methanol and 80 volumes of water, flow rate. 1 ml per minute, spectrophotometer set at 265 nm, a 10 l loop injector.
1258
IP 2007
ZIDOVUDINE INJECTION
Inject reference solution (c). The test is not valid unless the relative retention times are about 0.2 for thymine and 1.0 for zidovudine, the resolution between zidovudine and thymine is not less than 5.0, the tailing factor is not more than 2.0 and the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject separately the test solution and reference solution (c) and measure the responses for the thymine peak. The content of thymine in the capsules should not be more than 3.0 per cent. Dissolution (2.5.2). Apparatus. No 1 Medium. 900 ml of water Speed and time. 50 rpm and 45 minutes. Withdraw a suitable volume of the medium and filter promptly through a membrane filter disc having an average pore diameter not greater than 1.0 m, rejecting the first few ml of the filtrate and dilute a suitable volume of the filtrate, if necessary with water. Determine by liquid chromatography (2.4.14). Test solution. The filtrate obtained as given above. Reference solution. A known quantity of zidovudine RS is dissolved in 1 ml of methanol and suitably diluted with water to obtain a solution having a similar concentration as that of the test solution. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 20 volumes of methanol and 80 volumes of water, flow rate. 1 ml per minute, spectrophotometer set at 265 nm, a 10 l loop injector. Inject the reference solution. The tailing factor is not more than 2.0 for zidovudine peak and the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject the test solution and the reference solution and measure the peak responses of the major peak. Calculate the content of C10H13N5O4 in the medium. D. Not less than 75 per cent of the stated amount of C10H13N5O4. Other tests. Comply with the tests stated under Capsules. Assay. Determine by liquid chromatography (2.4.14), as described under Related substances. Inject separately the test solution and reference solution (c) and measure the responses for the principal peak. Calculate the content of C10H13N5O4 in the capsules.
Storage. Store protected from moisture.
Zidovudine Injection
Zidovudine Injection is a sterile solution of Zidovudine in Water for Injections. Zidovudine Injection contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of C10H13N5O4. Description. A clear, colourless solution.
Identification
A. When examined in the range 220 nm to 360 nm (2.4.7), a 0.0015 per cent w/v solution in a mixture of 75 volumes of methanol and 25 volumes of water shows absorption maxima similar to those obtained with a solution of zidovudine RS of the same concentration. B. In the Assay, the principal peak in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution.
Tests
pH (2.4.24). 3.5 to 7.0, in a mixture containing a volume of injection containing 150 mg of zidovudine and 5 ml of 0.12 M potassium chloride. Related substances. Determine by liquid chromatography (2.4.14). Test solution. Dilute an accurately measured volume of the injection containing 25 mg of Zidovudine, to 25 ml with methanol. Reference solution (a). A 0.1 per cent w/v solution of zidovudine RS in methanol. Reference solution (b). A solution containing 0.1 per cent w/ v of zidovudine RS and 0.001 per cent w/v of zidovudine related compound C (thymine). Chromatographic system a stainless steel column 25 cm 4.0 mm, packed with octadecylsilane bonded to porous silica ( 5 m), mobile phase: a mixture of 80 volumes of water and 20 volumes of methanol, flow rate. 1 ml per minute, spectrophotometer set at 265 nm, a 10 l loop injector. Inject reference solution (b). The test is not valid unless the resolution between zidovudine and thymine is not less than 4.0, the tailing factor is not more than 2.0 and the relative standard deviation for replicate injections is not more than 2.0 per cent.
1259
ZIDOVUDINE ORAL SOLUTION
IP 2007
Inject the test solution and reference solution (b). In the chromatogram obtained with the test solution the area of any peak corresponding to thymine is not greater than twice the area of the corresponding peak in the chromatogram obtained with reference solution (b) (2.0 per cent). Other tests. Complies with the tests stated under Parenteral preparations (Injections). Bacterial endotoxins (2.2.3). Not more than 1.0 Endotoxin Unit per mg of zidovudine. Assay. Determine by liquid chromatography (2.4.14), as given under the test for Related substances. Inject alternately the test solution and reference solution (a). Calculate the content of C10H13N5O4 in the injection. Storage. Store protected from light and moisture.
Tests
pH (2.4.24). 3.0 to 4.0, determined in a mixture containing a volume of the preparation under examination containing 150 mg of zidovudine and 5 ml of 0.12 M potassium chloride. Related substances. Determine by liquid chromatography (2.4.14). Test solution. Transfer an accurately measured volume of the preparation under examination containing about 100 mg of zidovudine to a 100-ml volumetric flask, dissolve and dilute to volume with the mobile phase and mix. Dilute 5.0 ml of this solution to 50.0 ml with the mobile phase. Reference solution (a). Weigh accurately about 100 mg of zidovudine RS and transfer to a 100-ml volumetric flask, add about 50 ml of the mobile phase, mix with the aid of ultrasound to dissolve, dilute to volume with the mobile phase and mix. Reference solution (b). Weigh accurately about 20 mg of thymine RS and transfer to a 200-ml volumetric flask, add about 150 ml of the mobile phase, mix with the aid of ultrasound to dissolve, dilute to volume with the mobile phase and mix. Reference solution (c). Transfer 10.0 ml of the reference solution (a) and 2.0 ml of reference solution (b) to a 100-ml volumetric flask and make up to the volume with the mobile phase. Chromatographic system a stainless steel column 12.5 cm x 4.0 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 90 volumes of 0.04 M sodium acetate, 9 volumes of methanol, 1 volume of acetonitrile and 0.2 volume of glacial acetic acid, flow rate. 1 ml per minute, spectrophotometer set at 240 nm, a 10 l loop injector. Inject reference solution (c). The test is not valid unless the relative retention times are about 0.12 for thymine and 1.0 for zidovudine, the resolution between zidovudine and thymine is not less than 4.0, the tailing factor is not more than 2.0 and the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject separately the test solution and reference solution (c) and measure the responses for the thymine peak. The content of thymine in the capsules should not be more than 3.0 per cent. Other tests. Comply with the tests stated under Oral Liquids. Assay. Determine by liquid chromatography (2.4.14), as described under Related substances. Inject separately the test solution and reference solution (c) and measure the responses for the major peak.
Zidovudine Oral Solution

Zidovudine Oral Solution is a solution of Zidovudine in a suitable flavoured vehicle. Zidovudine Oral Solution contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of zidovudine, C10H13N5O4.
Identification
A. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 40 volumes of 1-butanol, 30 volumes of heptane, 30 volumes of acetone and 10 volumes of strong ammonia solution. Test solution. Dilute the preparation under examination with methanol to obtain a solution containing 5 mg of zidovudine per ml. Reference solution. A 0.5 per cent w/v solution of zidovudine RS in a mixture of 75 volumes of methanol and 25 volumes of water. Apply to the plate 5 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution. B. In the Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the peak due to zidovudine in the chromatogram obtained with reference solution (c).
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IP 2007
ZIDOVUDINE TABLETS
Calculate the content of C10H13N5O4 in the preparation under examination. Storage. Store protected from light and moisture.
Zidovudine Tablets
Zidovudine Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of zidovudine, C10H13N5O4. The tablets may be coated.
Identification
A. In the Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the peak due to Zidovudine in the chromatogram obtained with reference solution (a). B. Remove the coating from a few tablets and crush them in a mortar so that no large pieces remain. On the powder determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with zidovudine RS or with the reference spectrum of zidovudine.
mobile phase: a mixture of 90 ml of methanol and 40 ml of acetonitrile and a buffer solution prepared by dissolving 3.0 g of sodium acetate and 3.0 g of sodium octanesulfonate in 900 ml of water and adjusting the pH to 5.3 with glacial acetic acid, flow rate. 1.3 ml per minute, spectrophotometer set at 265 nm, a 20 l loop injector. Inject reference solution (c). The test is not valid unless the resolution between zidovudine and the peak having relative retention time of 1.2 is not less than 2.5, the tailing factor for the zidovudine peak is not more than 2.0 and relative standard deviation for replicate injections is not more than 2.0 per cent. Separately inject the test solution and record the chromatograms for 3 times the retention time of zidovudine. The content of the impurity at a relative retention time of 0.17 compared to that of the zidovudine peak is not greater than 1.5 per cent and that of any other impurity is not greater than 0.2 per cent. The sum of all the impurities is not greater than 2.0 per cent. Dissolution (2.5.2). Apparatus. No 1 Medium. 900 ml of water Speed and time. 50 rpm and 30 minutes. Withdraw a suitable volume of the medium and filter promptly through a membrane filter disc having an average pore diameter not greater than 1.0 m, rejecting the first few ml of the filtrate and dilute a suitable volume of the filtrate, if necessary with water. Determine by liquid chromatography (2.4.14). Test solution. The filtrate obtained as given above. Reference solution. A known quantity of zidovudine RS is dissolved in 1 ml of methanol and suitably diluted with water to obtain a solution having a similar concentration as that of the test solution. Chromatographic system a stainless steel column 15 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 90 ml of methanol and 40 ml of acetonitrile and a buffer prepared by dissolving 3.0 g of sodium acetate and 3.0 g of sodium 1-octanesulphonate in 900 ml of water and adjusting the pH to 5.3 with glacial acetic acid, flow rate. 1.3 ml per minute, spectrophotometer set at 265 nm, a 10 l loop injector. Inject the reference solution. The tailing factor is not more than 2.0 for zidovudine peak and the relative standard deviation for replicate injections is not more than 2.0 per cent.
Tests
Related substances. Determine by liquid chromatography (2.4.14) Test solution. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 100 mg of Zidovudine and transfer to a 100-ml volumetric flask. Add about 60 ml of the mobile phase and mix with the aid of ultrasound for 10 minutes. Make up to volume with the mobile phase, mix and filter through a membrane filter disc with an average pore diameter not greater than 1.0 m, rejecting the first few ml of the filtrate. Further dilute 5.0 ml of the filtrate to 50.0 ml with the mobile phase. Reference solution (a). Weigh accurately about 100 mg of the zidovudine RS and transfer to a 100-ml volumetric flask. Dissolve in about 50 ml of the mobile phase and make up to volume with the mobile phase. Reference solution (b). Weigh accurately about 20 mg of thymine and transfer to a 200-ml volumetric flask, dissolve and make up to volume with methanol. Reference solution (c). Transfer 10.0 ml of reference solution (a) and 2.0 ml of reference solution (b) to a 100-ml volumetric flask and make up to volume with the mobile phase. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m),
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ZIDOVUDINE, LAMIVUDINE AND NEVIRAPINE TABLETS
IP 2007
Inject separately the test solution and the reference solution and measure the peak responses of the major peak. Calculate the content of C10H13N5O4 in the medium. D. Not less than 80 per cent of the stated amount of C10H13N5O4. Other tests. Comply with the tests stated under Tablets. Assay. Determine by liquid chromatography (2.4.14), as described under Related substances. Separately inject the test solution and reference solution (c) and measure the peak responses for the major peak. Calculate the content of C10H13N5O4 in the tablets. Storage. Store protected from light and moisture.
flow rate. 1 ml per minute, a linear gradient programme using the conditions given below, spectrophotometer set at 270 nm, a 20 l loop injector. Time 0.1 M ammonium acetate acetonitrile (in min.) (per cent v/v) (per cent v/v) 0 95 5 5 95 5 25 20 80 30 20 80 31 95 5 35 95 5 Inject the reference solution. The test is not valid unless the column efficiency determined from the zidovudine, lamivudine and nevirapine peaks is not less than 3000 theoretical plates and the tailing factor for the same peaks is not more than 2.0. Inject separately water and the test solution. Examine the chromatogram obtained with water for any extraneous peaks and ignore the corresponding peaks observed in the chromatogram obtained with the test solution. Any secondary peak observed in the chromatogram obtained with the test solution corresponding to a relative retention time of 0.35 should not be more than 1.0 per cent. Any other secondary peak observed in the chromatogram obtained with the test solution should not be more than 0.5 per cent and the sum of the areas of all the secondary peaks should not be more than 2.5 per cent when calculated by percentage area normalisation. Dissolution (2.5.2). Apparatus. No 1 Medium. 900 ml of 0.1 M hydrochloric acid Speed and time. 50 rpm and 30 minutes. Withdraw a suitable volume of the medium and filter promptly through a membrane filter disc having an average pore diameter not greater than 1.0 m, rejecting the first few ml of the filtrate and dilute a suitable volume of the filtrate, if necessary with water. Determine by liquid chromatography (2.4.14). Test solution. The filtrate obtained as given above. Reference solution. Weigh accurately about 300 mg of zidovudine RS, 150 mg of lamivudine RS and 200 mg of nevirapine RS and transfer to a 100-ml volumetric flask. Add about 20 ml of methanol, mix with the aid of ultrasound to dissolve and dilute to volume with a solvent mixture of equal volumes of methanol and water. Dilute 5.0 ml of this solution to 50.0 ml with 0.1 M hydrochloric acid.
Zidovudine, Lamivudine and Nevirapine Tablets

Zidovudine, Lamivudine and Nevirapine Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amounts of zidovudine, C10H 13N5O4, lamivudine, C8H11N3O3S and nevirapine, C15H14N4O. The tablets may be coated.
Identification
A. In the Assay, the three principal peaks in the chromatogram obtained with the test solution correspond to the peaks due to zidovudine, lamivudine and nevirapine in the chromatogram obtained with the reference solution.
Tests
Related substances. Determine by liquid chromatography (2.4.14). Test solution. Weigh accurately a quantity of the powdered tablets containing about 100 mg of Zidovudine and transfer to a 200-ml volumetric flask. Add about 150 ml of water, mix with the aid of ultrasound for 10 minutes, dilute to volume with water, mix and filter. Reference solution. Weigh accurately about 100 mg of zidovudine RS, 50 mg of lamivudine RS and 65 mg of nevirapine RS and transfer to a 200-ml volumetric flask. Add about 20 ml of methanol, mix with the aid of ultrasound to dissolve, dilute to volume with water and mix. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octylsilane bonded to porous silica (5 m) (such as Hypersil C8), mobile phase: A. 0.1 M ammonium acetate, B. acetonitrile,
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IP 2007
ZINC CHLORIDE
Chromatographic system a stainless steel column 15 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 35 volumes of methanol and 65 volumes of a buffer solution prepared by dissolving 0.68 g of potassium dihydrogen phosphate and 1.0 g of sodium octanesulphonate in 1000.0 ml of water to which 1 ml of triethylamine is added and the pH of which is adjusted to 2.5 with phosphoric acid, flow rate. 1 ml per minute, spectrophotometer set at 266 nm, a 10 l loop injector. Inject the reference solution. The tailing factor for the individual zidovudine, lamivudine and nevirapine peaks is not more than 2.0 and the relative standard deviation for replicate injections of all the analyte peaks is not more than 2.0 per cent. Inject the test solution and the reference solution and measure the peak responses of the major peaks due to zidovudine, lamivudine and nevirapine. Calculate the contents of C10H 13N5O4, C 8H11N 3O3S and C15H14N4O in the medium. D. Not less than 70 per cent of the stated amounts of C10H13N5O4, C8H11N3O3S and C15H14N4O. Other tests. Comply with the tests stated under Tablets. Assay. Determine by liquid chromatography (2.4.14). Test solution. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 100 mg of Zidovudine and transfer to a 200-ml volumetric flask. Add about 20 ml of methanol, mix with the aid of ultrasound for 10 minutes, dilute to volume with water, mix and filter. Further dilute 10.0 ml of the filtrate to 25.0 ml with water. Reference solution. Weigh accurately about 100 mg of zidovudine RS, 50 mg of lamivudine RS and 65 mg of nevirapine RS and transfer to a 200-ml volumetric flask. Add about 20 ml of methanol, mix with the aid of ultrasound to dissolve, dilute to volume with water and mix. Further dilute 10.0 ml of this solution to 25.0 ml with water. Follow the chromatographic procedure described under Dissolution. Inject the reference solution. The tailing factor for the individual peaks due to zidovudine, lamivudine and nevirapine is not more than 2.0 and the relative standard deviation for replicate injections of all the analyte peaks is not more than 2.0 per cent. Inject separately the test solution and the reference solution and measure the responses for the major peaks. Calculate the contents of C10H 13N5O4, C 8H11N 3O3S and C15H14N4O in the tablets.
Storage. Store protected from moisture.
Zinc Chloride
ZnCl2 Mol. Wt. 136.3 Zinc Chloride contains not less than 95.0 per cent and not more than 100.5 per cent of ZnCl2. Description. A white or practically white, crystalline powder; odourless; very deliquescent.
Identification
A. To 2 g add 38 ml of carbon dioxide-free water prepared from distilled water and add 2 M hydrochloric acid dropwise until solution is complete and dilute to 40 ml with carbon dioxide-free water prepared from distilled water (solution A). Solution A gives the reaction of zinc salts (2.3.1). B. A 5 per cent w/v solution in 2 M nitric acid gives the reactions of chlorides (2.3.1).
Tests
pH (2.4.24). 4.6 to 6.0, determined in a solution prepared by dissolving 1.0 g in 9 ml of freshly boiled and cooled water, ignoring any slight turbidity. Aluminium, calcium, heavy metals, iron and magnesium. To 8 ml of solution A add 2 ml of strong ammonia solution and shake; the solution is clear (2.4.1), and colourless (2.4.1). Add 1 ml of a 9 per cent w/v solution of disodium hydrogen phosphate; the resulting solution remains clear for at least 5 minutes. Add 0.2 ml of sodium sulphide solution; a white precipitate is produced and the supernatant liquid remains colourless. Ammonium salts. To 5 ml of a 10 per cent w/v solution add 1 M sodium hydroxide until the precipitate first formed is redissolved and then warm the solution; no odour of ammonia is perceptible. Oxychlorides. Dissolve 1.5 g in 1.5 ml of carbon dioxide-free water; the solution is not more opalescent than opalescence standard OS2 (2.4.1). Add 7.5 ml of ethanol (95 per cent); the solution may become cloudy within 10 minutes but becomes clear on the addition of 0.2 ml of 2 M hydrochloric acid. Sulphates (2.3.17). 15 ml of solution A complies with the limit test for sulphates (225 ppm). Assay. Weigh accurately about 3.0 g, dissolve in 125 ml of water, add 3 g of ammonium chloride and add sufficient water to produce 250.0 ml. To 25.0 ml of the resulting solution add 100 ml of water and 10 ml of strong ammonia-ammonium chloride solution. Titrate with 0.1 M disodium edetate, using
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ZINC OXIDE
IP 2007
eriochrome black T solution as indicator until a deep blue colour is obtained. 1 ml of 0.1 M disodium edetate is equivalent to 0.01363 g of ZnCl2. Storage. Store protected from moisture, in non-metallic containers.
Assay. Dissolve 0.15 g in 10 ml of 2 M acetic acid and dilute to 50 ml with water. To the resulting solution add about 50 mg of xylenol orange triturate and sufficient hexamine to produce violet-pink colour. Add a further 2 g of hexamine and titrate with 0.1 M disodium edetate until the solution becomes yellow. 1 ml of 0.1 M disodium edetate is equivalent to 0.008138 g of ZnO. Storage. Store protected from moisture.
Zinc Oxide
ZnO Mol. Wt. 81.4 Zinc Oxide contains not less than 99.0 per cent and not more than 100.5 per cent of ZnO, calculated on the ignited basis. Description. A soft, white or faintly yellowish white amorphous powder, free from grittiness. It gradually absorbs carbon dioxide from air.
Zinc Oxide Cream

Zinc Cream
Zinc Oxide Cream contains 32 per cent w/v of Zinc Oxide in a suitable water-in-oil emulsified base. Zinc Oxide Cream contains not less than 30.0 per cent and not more than 34.0 per cent w/w of zinc oxide, ZnO.
Identification
A. It becomes yellow when strongly heated; the yellow colour disappears on cooling. B. Dissolve 0.1 g in 1.5 ml of 2 M hydrochloric acid and dilute to 5 ml with water. The solution gives the reaction of zinc salts (2.3.1).
Identification
The residue obtained in the Assay is yellow when hot and white when cool.
Tests
Other tests. Complies with the tests stated under Creams. Assay. Weigh accurately about 0.5 g in a porcelain dish, heat gently over a small flame until the base is completely volatilised or charred. Increase the heat until all the carbon is removed. Dissolve the residue in 10 ml of 2 M acetic acid and add sufficient water to produce 50 ml. To the resulting solution add about 50 mg of xylenol orange triturate and sufficient hexamine to produce violet-pink colour. Add a further 2 g of hexamine and titrate with 0.1 M disodium edetate until the solution becomes yellow. 1 ml of 0.1 M disodium edetate is equivalent to 0.008138 g of ZnO.
Tests
Alkalinity. Shake 1.0 g with 10 ml of boiling water, add 0.1 ml of phenolphthalein solution and filter. If the filtrate is red, not more than 0.3 ml of 0.1 M hydrochloric acid is required to discharge the colour. Carbonate and substances insoluble in acids. Dissolve 1.0 g in 15 ml of 2 M hydrochloric acid; no effervescence is produced and the solution is not more opalescent than opalescence standard OS2 (2.4.1), and colourless (2.4.1). Arsenic (2.3.10). Dissolve 2.0 g in 15 ml of brominated hydrochloric acid AsT and 45 ml of water and remove the excess of bromine with a few drops of stannous chloride solution AsT. The resulting solution complies with the limit test for arsenic (5 ppm). Iron (2.3.14). Dissolve 0.1 g in a mixture of 5 ml of water and 1 ml of hydrochloric acid and dilute to 40 ml with water. The resulting solution complies with the limit test for iron (400 ppm). Use 0.5 ml of thioglycollic acid in the test. Lead. Dissolve 2 g in a mixture of 20 ml water and 5 ml of glacial acetic acid and add 0.25 ml of potassium chromate solution; the solution remains clear. Loss on ignition (2.4.20). Not more than 1.0 per cent, determined on 2.0 g by igniting at 500.
Zinc Stearate
(C17H35COO)2 Zn Mol. Wt. 632.3 Zinc Stearate consists mainly of zinc stearate but many contain variable proportions of zinc palmitate (C15H31COO)2 Zn, and zinc oleate (C17H33COO)2Zn. Zinc Stearate contains not less than 10.0 per cent and not more than 12.0 per cent of zinc, Zn. Description. A fine, white, bulky, amorphous powder, free from grittiness; odour, faint and characteristic.
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IP 2007
ZINC SULPHATE
Identification
A. To 5.0 g add 50 ml of ether and 40 ml of a 7.5 per cent v/v solution of nitric acid in distilled water and heat under a reflux condenser until dissolution is complete. Allow to cool, separate the aqueous layer and shake the ether layer with two quantities, each of 4 ml, of distilled water. Combine the washings with the aqueous layer, wash with 15 ml of ether and heat on a water-bath until ether is completely eliminated. Allow to cool and dilute to 50.0 ml with distilled water (solution A). Evaporate the ether layer to dryness and dry the residue at 105. The freezing point of the residue is not lower than 53 (2.4.11). B. Neutralise 5 ml of solution A to red litmus paper with 10 M sodium hydroxide. The solution gives the reactions of zinc salts (2.3.1).
Assay. Weigh accurately about 1.0 g and boil with 50 ml of 2 M acetic acid until the fatty acid layer which separates is clear, adding more water if necessary to maintain the original volume. Cool, filter and wash the filter and the flask thoroughly with water until the last washing is not acidic to blue litmus paper. To the combined filtrate and washings add about 50 mg of xylenol orange triturate and sufficient hexamine to produce violet-pink colour. Add a further 2 g of hexamine and titrate with 0.1 M disodium edetate until the colour changes to yellow. 1 ml of 0.1 M disodium edetate is equivalent to 0.00654 g of Zn.
Zinc Sulphate
ZnSO4, 7H2O Mol. Wt. 287.5 Zinc Sulphate contains not less than 99.0 per cent and not more than 104.0 per cent of ZnSO4, 7H2O. Description. Colourless, transparent crystals or a white, crystalline powder; odourless; efflorescent.
Tests
Appearance of solution. Solution A is not more intensely coloured than reference solution YS6 (2.4.1). Acidity or alkalinity. Shake 1.0 g with 5 ml of ethanol (95 per cent) and add 20 ml of carbon dioxide-free water and 0.1 ml of phenol red solution. Not more than 0.3 ml of 0.1 M hydrochloric acid or 0.1 ml of 0.1 M sodium hydroxide is required to change the colour of the solution. Alkalis and alkaline earths. Add 1.0 g to a mixture of 25 ml of water and 5 ml of hydrochloric acid, boil, filter immediately and wash with 25 ml of hot water. Add dilute ammonia solution to make the filtrate just alkaline and then add ammonium sulphide solution in excess to precipitate the zinc as zinc sulphide completely. Filter, add 0.5 ml of sulphuric acid to the filtrate, evaporate to dryness and ignite to constant weight; the residue weighs not more than 20 mg. Arsenic (2.3.10). Mix 5.0 g with 10 ml of bromine solution and evaporate to dryness on a water-bath. Ignite gently, dissolve the cooled residue, ignoring any carbon, in 50 ml of water and 14 ml of brominated hydrochloric acid AsT and remove the excess of bromine with 2 ml of stannous chloride solution AsT. The resulting solution complies with the limit test for arsenic (2 ppm). Heavy metals (2.3.13). Heat 5.0 g with 40 ml of 2 M acetic acid and allow to cool. Filter, wash the residue with two successive quantities, each of 5 ml, of warm water and dilute the combined filtrate and washings to 100.0 ml with water. 12 ml of the solution complies with the limit test for heavy metals, Method D (20 ppm). Use 1.0 ml of lead standard solution (10 ppm Pb) to prepare the standard. Chlorides (2.3.12). 10 ml of solution A complies with the limit test for chlorides (250 ppm). Sulphates (2.3.17). 2.5 ml of solution A complies with the limit test for sulphates (0.6 per cent).
Identification
Dissolve 2.5 g in sufficient carbon dioxide-free water to produce 50 ml (solution A). Solution A gives the reactions of zinc salts and sulphates (2.3.1).
Tests
Appearance of solution. Solution A is clear (2.4.1), and colourless (2.4.1). pH (2.4.24). 4.4 to 5.6, determined in solution A. Arsenic (2.3.10). Dissolve 1.0 g in 50 ml of water and add 10 ml of stannated hydrochloric acid AsT. The resulting solution complies with the limit test for arsenic (10 ppm). Iron (2.3.14). Dissolve 0.4 g in 20 ml of water. The resulting solution complies with the limit test for iron (100 ppm). Chlorides (2.3.12). 20 ml of solution A complies with the limit test for chlorides (250 ppm). Assay. Weigh accurately about 0.5 g and dissolve in 5 ml of 2 M acetic acid and dilute to 50 ml with water. To the resulting solution add about 50 mg of xylenol orange triturate and sufficient hexamine to produce violet-pink colour. Add a further 2 g of hexamine and titrate with 0.1 M disodium edetate until the colour changes to yellow. 1 ml of 0.1 M disodium edetate is equivalent to 0.02875 g of ZnSO4, 7H2O. Storage. Store protected from moisture, in non-metallic containers.
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ZINC SULPHATE EYE DROPS
IP 2007
Zinc Sulphate Eye Drops

Zinc Sulphate Eye Drops are a sterile solution containing 0.25 per cent w/v of Zinc Sulphate in Purified Water. Zinc Sulphate Eye Drops contain not less than 0.22 per cent and not more than 0.28 per cent w/v of zinc sulphate, ZnSO4, 7H2O. Description. A clear, colourless solution.
B. Dissolve 0.1 g in a mixture of 2 ml of 1 M sulphuric acid and 5 ml of glacial acetic acid and add, dropwise, 0.25 ml of potassium permanganate solution; the pink colour of permanganate is discharged. C. A mixture of 1 ml of the aqueous layer reserved in test A and 4 ml of water gives the reaction of zinc salts (2.3.1). D. Melting range (2.4.21). 115 to 121.
Tests Identification
Give the reactions of zinc salts and of sulphates (2.3.1). Alkalinity. Mix 1.0 g with 5 ml of ethanol (95 per cent) and 0.5 ml of phenol red solution, add 50 ml of carbon dioxidefree water and examine immediately; no reddish colour is produced. Alkalis and alkaline earths. Add 1.0 g to a mixture of 25 ml of water and 5 ml of hydrochloric acid, boil, filter immediately and wash with 25 ml of hot water. Add dilute ammonia solution to make the filtrate just alkaline and then add ammonium sulphate solution in excess to precipitate the zinc as zinc sulphide completely. Filter, add 0.5 ml of sulphuric acid to the filtrate, evaporate to dryness and ignite to constant weight; the residue weighs not more than 20 mg. Sulphates (2.3.17). To 0.25 g add a mixture of 10 ml of distilled water and 5 ml of 2 M hydrochloric acid. Cool, filter and dilute to 20 ml with distilled water. The resulting solution complies with the limit test for sulphates (600 ppm). Degree of unsaturation. Dissolve 0.1 g in a mixture of 5 ml of 2 M hydrochloric acid and 30 ml of glacial acetic acid and titrate with 0.05 M bromine using 0.05 ml of ethoxychrysoidine hydrochloride solution, added towards the end of the titration, as indicator. Not less than 9.1 ml and not more than 9.4 ml of 0.05 M bromine is required to discharge the red colour. Loss on drying (2.4.19). Not more than 1.5 per cent, determined on 0.5 g by drying in an oven at 105. Assay. Weigh accurately about 0.35 g, add 25 ml of 2 M acetic acid, heat to boiling, cool and dilute to 50 ml with water. Add about 50 mg of xylenol orange triturate and sufficient hexamine to produce a violet-pink colour. Add a further 2 g of hexamine and titrate with 0.1 M disodium edetate until the colour changes to yellow. 1 ml of 0.1 M disodium edetate is equivalent to 0.04319 g of C22H38O4Zn. Storage. Store protected from light and moisture.
Tests
Other tests. Comply with the tests stated under Eye Drops. Assay. To 5.0 ml add 50 ml of water and 5 ml of ammonia buffer pH 10.9 and titrate with 0.01 M disodium edetate using mordant black II solution as indicator. 1 ml of 0.01 M disodium edetate is equivalent to 0.002875 g of ZnSO4, 7H2O. Storage. Store in containers of glass or any other non-metallic material and sealed so as to exclude micro-organisms.
Zinc Undecenoate
CH2 O O
C22H38O4Zn
O Zn O
H2C
Mol. Wt. 431.9
Zinc Undecenoate is zinc di(undec-10-enoate). Zinc Undecenoate contains not less than 98.0 per cent and not more than 102.0 per cent of C22H38O4Zn, calculated on the dried basis. Description. A white or almost white, fine powder.
Identification
A. To 2.5 g add 10 ml of water and 10 ml of 1 M sulphuric acid and extract with two quantities, each of 10 ml, of ether. Reserve the aqueous layer for test C. Wash the combined ether extracts with water and evaporate to dryness. To the residue add 2 ml of freshly distilled aniline and boil under a reflux condenser for 10 minutes, cool and add 30 ml of ether. Extract with three quantities, each of 20 ml, of 2 M hydrochloric acid and then with 20 ml of water. Evaporate the ether extract to dryness on a water-bath. The residue, after recrystallising twice from ethanol (70 per cent) and drying at a pressure not exceeding 2 kPa for 3 hours, melts at about 67 (2.4.21).
Zinc Undecenoate Ointment

Zinc Undecylenate Ointment
Zinc Undecenoate Ointment contains 20 per cent w/v of Zinc Undecenoate in a suitable ointment basis.
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IP 2007
ZINC UNDECENOATE OINTMENT
Zinc Undecenoate Ointment contains not less than 18.0 per cent and not more than 22.0 per cent of zinc undecenoate, C22H38O4Zn, and not less than 4.5 per cent and not more than 5.5 per cent of free undecenoic acid, C11H20O2.
Tests
Other tests. Complies with the tests stated under Ointments. Assay. For zinc undecenoate Weigh accurately about 2.0 g, add 20 ml of dilute hydrochloric acid and boil under a reflux condenser for at least 20 minutes or until the fatty layer is clear. Filter while hot and wash the residue with hot water. Cool the combined filtrate and washings, neutralise to litmus paper with dilute ammonia solution, add 3 ml of dilute hydrochloric acid and 5 g of hexamine and titrate with 0.05 M disodium edetate using xylenol orange solution as indicator, until the colour changes to yellow. 1 ml of 0.05 M disodium edetate is equivalent to 0.02160 g of C22H38O4Zn. For free undecenoic acid Weigh accurately about 5.0 g, add 100 ml of dilute hydrochloric acid and heat to 70 with constant stirring. Cool and transfer to a separator with the aid of four quantities, each of 25 ml, of ether and add the rinsings to the mixture in the separator. Dilute the aqueous phase to
300 ml, saturate it with sodium chloride and shake the mixture. Transfer the aqueous layer to a second separator and extract with another 100 ml of ether. Wash the combined ether extracts with successive quantities, each of 10 ml, of water until the washings are free from chloride. Transfer the ether solution to a beaker and evaporate on a water-bath to about 5 ml. Add 20 ml of carbon tetrachloride, mix, transfer the mixture to a small separator and drain the carbon tetrachloride layer into a 100-ml volumetric flask. Rinse the beaker with three quantities, each of 5 ml, of carbon tetrachloride and transfer the rinsings to the volumetric flask, dilute to volume with carbon tetrachloride and mix. Evaporate 50.0 ml of the resulting solution to about 5 ml, add 100 ml of ethanol (95 per cent), previously neutralised, and 0.15 ml of phenolphthalein solution and titrate the total undecenoic acid with 0.1 M sodium hydroxide. 1 ml of 0.1 M sodium hydroxide is equivalent to 0.01843 g of C11H20O2. Calculate the content of free undecenoic acid from the difference between the total undecenoic acid and the undecenoic acid equivalent to the determined zinc undecenoate (the content of zinc undecenoate multiplied by 0.8533 gives the equivalent of undecenoic acid). Storage. Store protected from light and moisture.
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MONOGRAPHS
VACCINES AND IMMUNOSERA FOR HUMAN USE

General Requirements ..................................................................................................... Monographs ..................................................................................................................... Adsorbed Diphtheria, Tetanus and Hepatitis B (rDNA) Vaccine ........................................ Adsorbed Diphtheria, Tetanus and Pertussis (Acellular Component) and Haemophilus Type b Conjugate Vaccine ........................................................................ Adsorbed Diphtheria, Tetanus and Pertussis (Acellular Component) and Hepatitis B (rDNA) Vaccine ........................................................................................... Adsorbed Diphtheria, Tetanus and Pertussis (Acellular Component), Inactivated Poliomyelitis Vaccine and Haemophilus Type b Conjugate Vaccine ................. Adsorbed Diphtheria, Tetanus, Pertussis (Acellular Component) and Inactivated Poliomyelitis Vaccine ................................................................................... Adsorbed Diphtheria, Tetanus, Pertussis and Poliomyelitis (Inactivated) Vaccine .................... Adsorbed Diphtheria, Tetanus, Pertussis, Poliomyelitis (Inactivated) and Haemophilus Type b Conjugate Vaccine ........................................................................ Adsorbed Pertussis Vaccine (Acellular Component) Adsorbed Pertussis Vaccine (Acellular, Co-purified) ......................................................... .........................................................
Bacillus Calmette-Guerin Vaccine (Freeze-Dried) ............................................................... Diphtheria and Tetanus Vaccine (Adsorbed) ........................................................................ Diphtheria and Tetanus Vaccine (Adsorbed) for Adults and Adolescents .............................. Diphtheria, Tetanus and Pertussis Vaccine (Adsorbed) ..................................................... Diphtheria, Tetanus, Pertussis (Whole Cell), Hepatitis B (rDNA) and Haemophilus Type b Conjugate Vaccine (Adsorbed) .................................................... Diphtheria, Tetanus, Pertussis (Whole Cell) Hepatitis B (rDNA) Vaccine (Adsorbed) ................................................................................................... Diphtheria, Tetanus, Pertussis (Whole Cell) and Haemophilus Type b Conjugate Vaccine (Adsorbed) ...................................................................................... Diphtheria Vaccine (Adsorbed) ........................................................................................... Haemophilus Type b Conjugate Vaccine ............................................................................. Hepatitis A (Inactivated) and Hepatitis B (rDNA) Vaccine (Adsorbed) ................................. Hepatitis B Vaccine (rDNA) ............................................................................................. Inactivated Hepatitis A Vaccine (Adsorbed) ....................................................................... Inactivated Hepatitis B Vaccine ...........................................................................................
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MONOGRAPHS
Inactivated Influenza Vaccine (Split Virion) ........................................................................... Inactivated Influenza Vaccine (Surface Antigen) .................................................................... Inactivated Influenza Vaccine (Whole Virion) .................................................................... Japanese Encephalitis Vaccine (Human) .............................................................................. Measles and Rubella Vaccine (Live) ..................................................................................... Measles Vaccine (Live) ...................................................................................................... Measles, Mumps and Rubella Vaccine (Live) ....................................................................... Meningococcal Polysaccharide Vaccine .............................................................................. Mumps Vaccine (Live) ...................................................................................................... Pertussis Vaccine ................................................................................................................ Pneumococcal Polysaccharide Vaccine ................................................................................ Poliomyelitis Vaccine (Inactivated) ...................................................................................... Poliomyelitis Vaccine, Live (Oral) ....................................................................................... Rabies Vaccine, Human ...................................................................................................... Rubella Vaccine (Live) ....................................................................................................... Tetanus Vaccine (Adsorbed) ............................................................................................... Tick-borne Encephalitis Vaccine (Inactivated) ..................................................................... Tuberculin Purified Protein Derivative ................................................................................... Typhoid (Strain Ty 21a) Vaccine, Live (Oral) .................................................................... Typhoid Polysaccharide Vaccine ......................................................................................... Typhoid Vaccine ................................................................................................................ Typhoid Vaccine (Freeze Dried) ......................................................................................... Varicella Vaccine (Live) ....................................................................................................... Viper Venom ........................................................................................................................ Yellow Fever Vaccine (Live) ...............................................................................................
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IP 2007
VACCINES : GENERAL REQUIREMENTS
Vaccines : General Requirements

Vaccines are preparations of antigenic substances that are administered for the purpose of inducing in the recipient a specific and active immunity against the infective agent or toxin produced by it. Vaccines may contain living micro-organisms suitably treated to attenuate their virulence but retain their antigenic potency or they may consist of pathogenic organisms which have been killed or inactivated. Some vaccines consist of antigenic fractions or substances produced by the same pathogenic organisms but rendered harmless whilst retaining their antigenic efficiency. Vaccines may be prepared from one species only or from a mixture of two or more species. Vaccines may be prepared by the method described in the individual monographs or by the general methods given below or in any other manner provided the identity of the antigens is maintained and the vaccines are free from microbial contamination and extraneous agents. Suitable adjuvants may be added during the preparation but streptomycin, penicillin or other -lactam antibiotics may not be added at any stage of manufacture or in the final vaccine. A suitable bactericide may be added to sterile and inactivated vaccines. The final products are distributed aseptically into sterile containers which are then sealed to exclude extraneous micro-organisms. Unless otherwise indicated in the monograph, the final vaccine may be filled into single dose or multiple dose containers but vaccines in multiple dose containers must invariably contain a bactericide. Bacterial Vaccines. Bacterial vaccines are either sterile suspensions of live or killed bacteria or sterile extracts of derivatives of bacteria. They may be simple vaccines prepared from one species or may be mixed vaccines prepared by blending two or more simple vaccines from different species or strains. Bacterial vaccines may be prepared from cultures grown on suitable solid or liquid media. The whole culture or parts of it may be used in preparing the vaccine. The identity, antigenic potency and purity of each bacterial culture must be carefully controlled. Vaccines containing killed organisms may be prepared by killing the organisms by chemical or physical means provided the antigenic potency of the vaccine is preserved. Vaccines containing living bacteria may be prepared from strains which are avirulent for humans but which stimulate the production of antibodies active against pathogenic strains of the same species. The final vaccines must be free from any substance known to cause toxic, allergic or other undesirable immunological reactions in humans. Bacterial vaccines are suspensions of varying degrees of opacity in colourless or slightly coloured liquids or they may 1
be freeze-dried so that the water content is not more than 3.0 per cent w/w unless otherwise stated in the individual monograph. They may be standardized in terms of interopacity units or, where appropriate, by numbers of living or killed bacteria determined by direct cell count or by viable count. Bacterial toxoids. Bacterial toxoids are toxins or material derived therefrom, the toxicity of which has been reduced to a very low level or completely eliminated by chemical or physical means without destroying their immunizing potency. The toxins are obtained from selected strains of specific micro-organisms, grown in media free from ingredients known to cause toxic, allergic or other undesirable immunological reactions in humans. Toxoids produced by the action of formaldehyde are known as formol toxoids. Bacterial toxoids may be liquid or may be prepared by adsorbing on mineral carriers such as aluminium phosphate, aluminium hydroxide or any other suitable adsorbent; the adsorbed product may be separated, washed and suspended in a saline or other appropriate solution isotonic with blood. Bacterial toxoids are clear or slightly opalescent liquids, colourless or slightly yellow. Adsorbed toxoids may be white or greyish white suspensions or pale-yellow liquids with a sediment at the bottom of the container. Freeze-dried preparations are greyish white or yellowish white powders or pellets. Viral and rickettsial vaccines. Viral and rickettsial vaccines are suspensions of viruses or rickettsiae and are prepared from infected tissues or blood obtained from artificially infected animals, from cultures in fertile eggs, or from cell or tissue culture. Viral vaccines may be live or killed and they may be freeze-dried. Live vaccines are usually prepared using attenuated strains of the specific organisms. Killed vaccines may be inactivated by suitable chemical or physical means. Mixed Vaccines. Mixed vaccines are mixtures of two or more vaccines. A suitable antibacterial substance may be added to inactivated or live viral and rickettsial vaccines provided that it has no action against the specific organisms.
Production
General provisions. Requirements for production including in-process testing are included in individual monographs. Where justified and authorized, certain tests may be omitted where it can be demonstrated, for example by validation studies, that the production process consistently ensures compliance with the test. Unless otherwise justified and authorized, vaccines are produced using a seed-lot system. The methods of preparation are designed to maintain adequate immunogenic properties, to render the preparation harmless and to prevent contamination with extraneous agents.
VACCINES : GENERAL REQUIREMENTS
IP 2007
Unless otherwise justified and authorized, in the production of a final lot of vaccine, the number of passages of a virus, or the number of subcultures of a bacterium, from the master seed lot shall not exceed that used for production of the vaccine shown in clinical studies to be satisfactory with respect to safety and efficacy. Vaccines are as far as possible free from ingredients known to cause toxic, allergic or other undesirable reactions in man. Suitable additives, including stabilizers and adjuvants may be incorporated. Penicillin and streptomycin are neither used at any stage of production nor added to the final product; however, master seed lots prepared with media containing penicillin or streptomycin may, where justified and authorized, be used for production. Substrates for propagation. Substrates for propagation comply with the relevant requirements of the Pharmacopoeia or in the absence of such requirements with those of the competent authority. Processing of cell banks and subsequent cell cultures is done under aseptic conditions in an area where no other cells are being handled. Serum and trypsin used in the preparation of cell suspensions shall be shown to be free from extraneous agents. Seed lot. The strain of bacterium or virus used in a master seed lot is identified by historical records that include information on the origin of the strain and its subsequent manipulation. No micro-organism other than the seed strain shall be present in a seed lot. Culture media. Culture media are as far as possible free from ingredients known to cause toxic, allergic or other undesirable reactions in man; if inclusion of such ingredients is necessary, it shall be demonstrated that the amount present in the final lot is reduced to such a level as to render the product safe. Approved animal (but not human) serum may be used in the growth medium for cell cultures but the medium used for maintaining cell growth during virus multiplication shall not contain serum, unless otherwise stated. Cell culture media may contain a pH indicator such as phenol red and approved antibiotics at the lowest effective concentration although it is preferable to have a medium free from antibiotics during production. Propagation and harvest. The seed cultures are propagated and harvested under defined conditions. The purity of the harvest is verified by suitable tests as defined in the monograph. Control cells. For vaccines produced in cell cultures, control cells are maintained and tested as prescribed. In order to provide a valid control, these cells must be maintained in conditions that are rigorously identical with those used for the production cell cultures, including use of the same batches of media and media changes. 2
Control eggs. For live vaccines produced in eggs, control eggs are incubated and tested as prescribed in the monograph. Purification. Where applicable, validated purification procedures may be applied. Inactivation. Inactivated vaccines are produced using a validated inactivation process whose effectiveness and consistency have been demonstrated. Where there are recognised potential contaminants of a harvest, for example in vaccines produced in eggs from healthy, non-SPF flocks, the inactivation process is also validated with respect to the potential contaminants. A test for inactivation is carried out as soon as possible after the inactivation process, unless otherwise justified and authorised. Intermediates. Where applicable, the stability of intermediates in given storage conditions shall be evaluated and a period of validity established. Final bulk. The final bulk is prepared by aseptically blending the ingredients of the vaccine. Adsorbents. Vaccines may be adsorbed on aluminium hydroxide, aluminium phosphate, calcium phosphate or other suitable adsorbent; the adsorbents are prepared in special conditions which confer the appropriate physical form and adsorptive properties. Antimicrobial preservative. A suitable antimicrobial preservative may be included in sterile and inactivated vaccines and is invariably added if these preparations are issued in multidose containers, unless otherwise stated. If an antimicrobial preservative is used, it shall be shown that it does not impair the safety or efficacy of the vaccine and its effectiveness throughout the period of validity shall be demonstrated. Final lot. For vaccines for parenteral administration, the final lot is prepared by aseptically distributing the final bulk into sterile tamper-proof containers which, after freeze-drying where applicable, are closed so as to exclude contamination. For vaccines for administration by a non-parenteral route, the final lot is prepared by distributing the final bulk under suitable conditions into sterile, tamper-proof containers. Stability. Maintenance of potency of the final lot throughout the period of validity shall be demonstrated by validation studies; the loss of potency in the recommended storage conditions is assessed and excessive loss even within the limits of acceptable potency may indicate that the vaccine is unacceptable. Degree of adsorption. During development of an adsorbed vaccine, the degree of adsorption is evaluated as part of the consistency testing. A release specification for the degree of adsorption is established in the light of results found for batches used in clinical testing. From the stability data
IP 2007
ADSORBED DIPHTHERIA, TETANUS AND HEPATITIS B (rDNA) VACCINE
generated for the vaccine it must be shown that at the end of the period of validity the degree of adsorption will not be less than for batches used in clinical testing.
Adsorbed Diphtheria, Tetanus and Hepatitis B (rDNA) Vaccine

Diphtheria, Tetanus and Hepatitis B (rDNA) Vaccine (Adsorbed) is a combined vaccine composed of: diphtheria formol toxoid; tetanus formol toxoid; hepatitis B surface antigen (HBsAg); a mineral adsorbent such as aluminium hydroxide or hydrated aluminium phosphate. The formol toxoids are prepared from the toxins produced by the growth of Corynebacterium diphtheriae and Clostridium tetani, respectively. HBsAg is a component protein of hepatitis B virus; the antigen is obtained by recombinant DNA technology.
Tests
Vaccines, reconstituted where necessary, comply with the following requirements unless otherwise stated in the individual monograph. Phenol (If present) (2.3.36). Not more than 0.25 per cent w/v. Thiomersal (If present) (2.3.48). Between 0.005 per cent w/v and 0.02 per cent w/v. Free formaldehyde (If present) (2.3.20). Maximum 0.02 g/l. Aluminium (If present) (2.3.9). Not more than 1.25 mg per dose. Sterility (2.2.11). Unless otherwise stated all vaccines comply with tests for sterility, except that for living bacterial vaccines, growth of the organism from which the vaccine was prepared is permitted (sterility means abesence of becterial and fungal contaminants except where specified in the individual monograph). Abnormal toxicity (2.2.1). Unless otherwise stated, all vaccines comply with the test for abnormal toxicity, Method B. In vaccines containing phenol as preservative, the test in mice may be inappropriate. NOTE The statements given in this general chapter is intended to be read in conjunction with the monographs on the individual vaccine in this Pharmacopoeia which refer to preparations for human use; they do not necessarily apply to vaccines for use in veterinary practice. Storage. Liquid vaccines must be stored at a temperature between 2o and 8o and should not be allowed to freeze unless otherwise specified in the individual monograph. Freeze-dried preparations must be stored at temperatures below 20o or as specified in the individual monograph. At higher temperatures vaccines deteriorate rapidly. Labelling. The label states (1) for liquid vaccines, the total number of ml in the container and, for dried vaccines, the number of doses in the container; (2) unless otherwise indicated the minimum number of Units per dose or per ml or, for viral vaccines, the minimum viral titre; (3) the dose and route of administration; (4) the name and proportion of any antibacterial preservative or other auxiliary substances added to the vaccine; (5) the date after which the vaccine is not intended to be used; (6) the conditions under which it should be stored; (7) for dried vaccines, the liquid to be used for reconstitution and its volume; (8) that the vaccine should be used immediately after reconstitution; (9) unless otherwise directed, that the vaccine should be shaken well before use; (10) any contraindication to the use of the vaccine. 3
Production
General provisions The production method shall have been shown to yield consistently vaccines comparable with the vaccine of proven clinical efficacy and safety in man. The production method is validated to demonstrate that the product, if tested, would comply with the test for abnormal toxicity for antisera and vaccines, and with the following test for specific toxicity of the diphtheria and tetanus components: inject subcutaneously 5 times the single human dose stated on the label into each of 5 healthy guinea-pigs, each weighing between 250 and 350 g, that have not previously been treated with any material that will interfere with the test. If within 42 days of the injection any of the animals shows signs of or dies from diphtheria toxaemia or tetanus, the vaccine does not comply with the test. If more than 1 animal dies from nonspecific causes, repeat the test once; if more than 1 animal dies in the second test, the vaccine does not comply with the test. The content of bacterial endotoxins in the bulk purified diphtheria toxoid and tetanus toxoid is determined to monitor the purification procedure and to limit the amount in the final vaccine. For each component, the content of bacterial endotoxin is less than the limit approved for the particular vaccine and in any case the contents are such that the final vaccine contains less than 100 IU per single human dose. Reference vaccine(s) Provided valid assays can be performed, monocomponent reference vaccines may be used for the assays on the combined vaccine. If this is not possible because of interaction between the components of the combined vaccine or because of the difference in composition between monocomponent reference vaccine and the test vaccine, a batch of combined vaccine shown to be effective in clinical trials or a batch representative thereof is used as a reference vaccine. For the preparation of
ADSORBED DIPHTHERIA, TETANUS AND HEPATITIS B (rDNA) VACCINE
IP 2007
a representative batch, strict adherence to the production process used for the batch tested in clinical trials is necessary. The reference vaccine may be stabilised by a method that has been shown to have no effect on the assay procedure. Production of the components The production of the components complies with the requirements of the monographs on Diphtheria Vaccine (Adsorbed), Tetanus Vaccine (Adsorbed) and Hepatitis B Vaccine (rDNA). FINAL BULK VACCINE The final bulk vaccine is prepared by adsorption, separately or together, of suitable quantities of bulk purified diphtheria toxoid, tetanus toxoid and HBsAg onto a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate. Suitable antimicrobial preservatives may be added. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT Only a final lot that is satisfactory with respect to the test for osmolality and with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided the test for antimicrobial preservative and the assays for the diphtheria and tetanus components have been carried out with satisfactory results on the final bulk vaccine, they may be omitted on the final lot. Provided the content of free formaldehyde has been determined on the bulk purified antigens or on the final bulk and it has been shown that the content in the final lot will not exceed 0.2 g/l, the test for free formaldehyde may be omitted on the final lot. If an in vivo assay is used for the hepatitis B component, provided it has been carried out with satisfactory results on the final bulk vaccine, it may be omitted on the final lot. Osmolality (2.4.23). The osmolality of the vaccine is within the limits approved for the particular preparation.
vaccines, is given as an example. Dissolve in the vaccine under examination sufficient sodium citrate to give a 10 per cent w/v solution. Maintain at 37 for about 16 hours and centrifuge until a clear supernatant is obtained. The clear supernatant liquid reacts with a suitable diphtheria antitoxin, giving a precipitate. B. Tetanus toxoid is identified by a suitable immunochemical method (2.2.14). The following method, applicable to certain vaccines, is given as an example. The clear supernatant liquid obtained during identification test A reacts with a suitable tetanus antitoxin, giving a precipitate. C. The assay or, where applicable, the electrophoretic profile, serves also to identify the hepatitis B component of the vaccine.
Tests
Aluminium (2.3.9). Maximum 1.25 mg per single human dose, if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. Free formaldehyde (2.3.20). Maximum 0.2 g/l. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Complies with the test for sterility. Pyrogens (2.2.8). Complies with the test for pyrogens. Inject the equivalent of 1 human dose into each rabbit. Assay Diphtheria component Carry out one of the prescribed methods for the assay as stated under Diphtheria Vaccine (Adsorbed). The lower confidence limit (P = 0.95) of the estimated potency is not less than 30 IU per single human dose. Tetanus component Carry out one of the prescribed methods for the assay as stated under Tetanus Vaccine (Adsorbed). The lower confidence limit (P = 0.95) of the estimated potency is not less than 40 IU per single human dose. Hepatitis B component It complies with the assay of Hepatitis B Vaccine. Labelling. The label states (1) the minimum number of International Units of diphtheria and tetanus toxoid per single human dose; (2) the amount of HBsAg per single human dose; (3) the type of cells used for production of the HBsAg component; (4) where applicable, that the vaccine is intended for primary vaccination of children and is not necessarily 4
Identification
A. Diphtheria toxoid is identified by a suitable immunochemical method (2.2.14). The following method, applicable to certain
IP 2007
A. D. T. P. (ACELLULAR COMPONENT) AND HAEMOPHILUS TYPE B CONJUGATE VACCINE
suitable for reinforcing doses or for administration to adults; (5) the name and the amount of the adsorbent; (6) that the vaccine must be shaken before use; (7) that the vaccine is not to be frozen.
consistently the vaccines comparable with the vaccine of proven clinical efficacy and safety in man. If the vaccine is presented with the haemophilus component in a separate vial, as part of consistency studies the assays of the diphtheria, tetanus and pertussis components are carried out on a suitable number of batches of vaccine reconstituted for use. For subsequent routine control, the assays of these components may be carried out without mixing with the haemophilus component. The content of bacterial endotoxins in bulk purified diphtheria toxoid, tetanus toxoid, pertussis components and PRP conjugate is determined to monitor the purification procedure and to limit the amount in the final vaccine. For each component, the content of bacterial endotoxins is less than the limit approved for the particular vaccine; if the vaccine is presented with the haemophilus component in a separate container, the contents of the diphtheria, tetanus and pertussis antigens are in any case such that the final vial for these components contains less than 100 IU per single human dose. The production method is validated to demonstrate that the product, if tested, would comply with the test for abnormal toxicity for antisera and vaccines. During development studies and wherever revalidation is necessary, it shall be demonstrated by tests in animals that the vaccine induces a T-cell dependent B-cell immune response to PRP. Reference vaccine Provided valid assays can be performed, monocomponent reference vaccines may be used for the assays on the combined vaccine. If this is not possible because of interaction between the components of the combined vaccine or because of the difference in composition between monocomponent reference vaccine and the test vaccine, a batch of combined vaccine shown to be effective in clinical trials or a batch representative thereof is used as a reference vaccine. For the preparation of a representative batch, strict adherence to the production process used for the batch tested in clinical trials is necessary. The reference vaccine may be stabilised by a method that has been shown to have no effect on the assay procedure. Production of the components The production of the components complies with the tests of the monographs on Diphtheria Vaccine (Adsorbed), Tetanus Vaccine (Adsorbed), Pertussis Vaccine (Acellular Component, Adsorbed) and Haemophilus Type b Conjugate Vaccine. FINAL BULK VACCINE Different methods of preparation may be used: a final bulk vaccine may be prepared by adsorption, separately or together, of suitable quantities of bulk purified diphtheria toxoid, tetanus 5
Adsorbed Diphtheria, Tetanus, Pertussis (Acellular Component) and Haemophilus Type B Conjugate Vaccine
Diphtheria, Tetanus, Pertussis (Acellular Component) and Haemophilus Type b Conjugate Vaccine (Adsorbed) is a combined vaccine composed of: diphtheria formol toxoid; tetanus formol toxoid; individually purified antigenic components of Bordetella pertussis; polyribosylribitol phosphate (PRP) covalently bound to a carrier protein; a mineral absorbent such as aluminium hydroxide or hydrated aluminium phosphate. The product may be presented with the haemophilus type b component in a separate container, the contents of which are mixed with the other components immediately before use. The formol toxoids are prepared from the toxins produced by the growth of Corynebacterium diphtheriae and Clostridium tetani respectively. The vaccine contains either pertussis toxoid or a pertussistoxin-like protein free from toxic properties produced by expression of a genetically modified form of the corresponding gene. Pertussis toxoid is prepared from pertussis toxin by a method that renders the toxin harmless while maintaining adequate immunogenic properties and avoiding reversion to toxin. The acellular pertussis component may also contain filamentous haemagglutinin, pertactin (a 69 kDa outermembrane protein) and other defined components of B. pertussis such as fimbrial-2 and fimbrial-3 antigens. The latter two antigens may be copurified. The antigenic composition and characteristics are based on evidence of protection and freedom from unexpected reactions in the target group for which the vaccine is intended. PRP is a linear copolymer composed of repeated units of 3--D-ribofuranosyl-(11)-ribitol-5-phosphate [(C10H19O12P)n], with a defined molecular size and derived from a suitable strain of Haemophilus influenzae type b. The carrier protein, when conjugated to PRP, is capable of inducing a T-cell-dependent B-cell immune response to the polysaccharide.
Production
General provisions The production method shall have been shown to yield
A. D. T. P. (ACELLULAR COMPONENT) AND HAEMOPHILUS TYPE B CONJUGATE VACCINE
IP 2007
toxoid, acellular pertussis components and PRP conjugate onto a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate; or 2 final bulks may be prepared and filled separately, one containing the diphtheria, tetanus and pertussis components, the other the haemophilus component, which may be freeze-dried. Suitable antimicrobial preservatives may be added. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT Only a final lot that is satisfactory with respect to the test for osmolality shown below and with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided the tests for absence of residual pertussis toxin, irreversibility of pertussis toxoid and antimicrobial preservative and the assays have been carried out with satisfactory results on the final bulk vaccine, they may be omitted on the final lot. Provided the free formaldehyde content has been determined on the bulk purified antigens or the final bulk and it has been shown that the content in the final lot will not exceed 0.2 g/l, the test for free formaldehyde may be omitted on the final lot. Osmolality (2.4.23). The osmolality of the vaccine, reconstituted where applicable, is within the limits approved for the particular preparation. pH (2.4.24). The pH of the vaccine, reconstituted if necessary, is within the range approved for the particular product. Free PRP. Unbound PRP is determined after removal of the conjugate, for example by anion-exchange, size-exclusion or hydrophobic chromatography (2.4.16), ultrafiltration or other validated methods. The amount of free PRP is not greater than that approved for the particular product.
w/v solution. Maintain at 37 for about 16 h and centrifuge until a clear supernatant is obtained. The clear supernatant reacts with a suitable diphtheria antitoxin, giving a precipitate. B. Tetanus toxoid is identified by a suitable immunochemical method (2.2.14). The following method, applicable to certain vaccines, is given as an example. The clear s u p e r n a t a n t obtained as described in Identification test A reacts with a suitable tetanus antitoxin, giving a precipitate. C. The pertussis components are identified by a suitable immunochemical method (2.2.14). The following method, applicable to certain vaccines, is given as an example. The clear supernatant obtained as described in Identification test A reacts with a specific antisera to the pertussis components of the vaccine. D. The haemophilus component is identified by a suitable immunochemical method (2.2.14) for PRP.
Tests
If the product is presented with the haemophilus component in a separate container: the tests for absence of residual pertussis toxin, irreversibility of pertussis toxoid, aluminium, free formaldehyde, antimicrobial preservative and sterility are carried out on the container with the diphtheria, tetanus and pertussis components; the tests for PRP content, water (where applicable), sterility and pyrogens are carried out on the container with the haemophilus component. If the haemophilus component is freeze-dried, some tests may be carried out on the freeze-dried product rather than on the bulk conjugate where the freeze-drying process may affect the component under test. Absence of residual pertussis toxin and irreversibility of pertussis toxoid This test is not necessary for the product obtained by genetic modification. Use 3 groups each of not less than 5 histaminesensitive mice. Inject intraperitoneally into each mouse of the first group twice the single human dose of the vaccine stored at 2 to 8. Inject intraperitoneally into each mouse of the second group twice the single human dose of the vaccine incubated at 37 for 4 weeks. Inject diluent into the third group of mice. After 5 days, inject into each mouse 2 mg of histamine base intraperitoneally in a volume not exceeding 0.5 ml and observe for 24 h. The test is invalid if 1 or more control mice die following histamine challenge. The vaccine complies with the test if no animal in the first or second group dies following histamine challenge. If 1 mouse dies in either or both of the first and second groups, the test may be repeated with the same number of mice or with a greater number and the results of valid tests combined; the vaccine complies with the test if, in both of the groups given the vaccine, not more than 5.0 per cent of the total number of mice die following histamine challenge. 6
Identification
If the vaccine is presented with the haemophilus component in a separate vial: identification tests A, B and C are carried out using the vial containing the diphtheria, tetanus and pertussis components; identification test D is carried out on the vial containing the haemophilus components. A. Diphtheria toxoid is identified by a suitable immunochemical method (2.2.14). The following method, applicable to certain vaccines, is given as an example. Dissolve in the vaccine under examination sufficient sodium citrate to give a 10 per cent
IP 2007
A. D. T. AND P. (ACELLULAR COMPONENT) AND HEPATITIS B (rDNA) VACCINE
The histamine sensitivity of the strain of mice used is verified at suitable intervals as follows: inject intravenously threefold dilutions of a reference pertussis toxin preparation in phosphate-buffered saline solution containing 0.2 per cent w/v of gelatin and challenge with histamine as above; the strain is suitable if more than 50 per cent of the animals are sensitised by 50 ng of pertussis toxin and none of the control animals injected with only diluent and challenged similarly with histamine shows symptoms of sensitisation. PRP. Minimum 80.0 per cent of the amount of PRP stated on the label. PRP is determined either by assay of ribose (2.7.1) or phosphorus (2.7.1), by an immunochemical method (2.2.14) or by anion-exchange liquid chromatography with pulsedamperometric detection. Aluminium (2.3.9). Maximum 1.25 mg per single human dose, if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. Free formaldehyde (2.3.20 ). Maximum 0.2 g/l. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Water (2.3.43). Maximum 3.0 per cent for the freeze-dried haemophilus component. Sterility (2.2.11). Complies with the test for sterility. Pyrogens (2.2.8). Complies with the test for pyrogens. Inject per kg of the rabbits mass a quantity of the vaccine equivalent to: 1 mg of PRP for a vaccine with diphtheria toxoid or CRM 197 diphtheria protein as carrier; 0.1 mg of PRP for a vaccine with tetanus toxoid as carrier; 0.025 mg of PRP for a vaccine with OMP as carrier. Assay Diphtheria component Carry out one of the prescribed methods for the assay as stated under Diphtheria Vaccine (Adsorbed). The lower confidence limit (P = 0.95) of the estimated potency is not less than the minimum potency stated on the label. Unless otherwise justified and authorised, the minimum potency stated on the label is 30 IU per single human dose. Tetanus component Carry out one of the prescribed methods for the assay as stated under Tetanus Vaccine (Adsorbed). The lower confidence limit (P = 0.95) of the estimated potency is not less than 40 IU per single human dose. Pertussis component The vaccine complies with the assay as the stated Adsorbed Pertussis Vaccine (Acellular Component). 7
Labelling. The label states (1) the minimum number of International Units of diphtheria and tetanus toxoid per single human dose; (2) the names and amounts of the pertussis components per single human dose; (3) the number of micrograms of PRP per single human dose; (4) the type and nominal amount of carrier protein per single human dose; (5) where applicable, that the vaccine is intended for primary vaccination of children and is not necessarily suitable for reinforcing doses or for administration to adults; (6) the name and the amount of the adsorbent; (7) that the vaccine must be shaken before use; (8) that the vaccine is not to be frozen (9) where applicable, that the vaccine contains a pertussis toxinlike protein produced by genetic modification.
Adsorbed Diphtheria, Tetanus and Pertussis (Acellular Component) and Hepatitis B (rDNA) Vaccine
Diphtheria, Tetanus, Pertussis (Acellular Component) and Hepatitis B (rDNA) Vaccine (Adsorbed) is a combined vaccine composed of: diphtheria formol toxoid; tetanus formol toxoid; individually purified antigenic components of Bordetella pertussis; hepatitis B surface antigen; a mineral adsorbent such as aluminium hydroxide or hydrated aluminium phosphate. The formol toxoids are prepared from the toxins produced by the growth of Corynebacterium diphtheriae and Clostridium tetani, respectively. The vaccine contains either pertussis toxoid or a pertussistoxin-like protein free from toxic properties, produced by expression of a genetically modified form of the corresponding gene. Pertussis toxoid is prepared from pertussis toxin by a method that renders the latter harmless while maintaining adequate immunogenic properties and avoiding reversion to toxin. The vaccine may also contain filamentous haemagglutinin, pertactin (a 69 kDa outer-membrane protein) and other defined components of B. pertussis such as fimbrial2 and fimbrial-3 antigens. The latter 2 antigens may be copurified. The antigenic composition and characteristics are based on evidence of protection and freedom from unexpected reactions in the target group for which the vaccine is intended. Hepatitis B surface antigen is a component protein of hepatitis B virus; the antigen is obtained by recombinant DNA technology.
Production
General provisions The production method shall have been shown to yield consistently vaccines comparable with the vaccine of proven
A. D. T. AND P. (ACELLULAR COMPONENT) AND HEPATITIS B (rDNA) VACCINE
IP 2007
clinical efficacy and safety in man. The content of bacterial endotoxins in the bulk purified diphtheria toxoid, tetanus toxoid and pertussis components is determined to monitor the purification procedure and to limit the amount in the final vaccine. For each component, the content of bacterial endotoxins is less than the limit approved for the particular vaccine. Reference vaccine(s) Provided valid assays can be performed, monocomponent reference vaccines may be used for the assays on the combined vaccine. If this is not possible because of interaction between the components of the combined vaccine or because of the difference in composition between monocomponent reference vaccine and the test vaccine, a batch of combined vaccine shown to be effective in clinical trials or a batch representative thereof is used as a reference vaccine. For the preparation of a representative batch, strict adherence to the production process used for the batch tested in clinical trials is necessary. The reference vaccine may be stabilised by a method that has been shown to have no effect on the assay procedure. Production of the components The production of the components complies with the requirements of the monographs on Diphtheria Vaccine (Adsorbed), Tetanus Vaccine (Adsorbed), Pertussis Vaccine (Acellular Component, Adsorbed) and Hepatitis B Vaccine (rDNA). FINAL BULK VACCINE The final bulk vaccine is prepared by adsorption, separately or together, of suitable quantities of bulk purified diphtheria toxoid, tetanus toxoid, acellular pertussis components and hepatitis B surface antigen onto a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate. Suitable antimicrobial preservatives may be added. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT Only a final lot that is satisfactory with respect to the test for osmolality and with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. 8
Provided the tests for absence of residual pertussis toxin, irreversibility of pertussis toxoid and antimicrobial preservative and the assays for the diphtheria, tetanus and pertussis components have been carried out with satisfactory results on the final bulk vaccine, they may be omitted on the final lot. Provided the content of free formaldehyde has been determined on the bulk purified antigens or on the final bulk and it has been shown that the content in the final lot will not exceed 0.2 g/l, the test for free formaldehyde may be omitted on the final lot. If an in vivo assay is used for the hepatitis B component, provided it has been carried out with satisfactory results on the final bulk vaccine, it may be omitted on the final lot. Osmolality (2.2.23). The osmolality of the vaccine is within the limits approved for the particular preparation.
Identification
A. Diphtheria toxoid is identified by a suitable immunochemical method (2.2.14). The following method, applicable to certain vaccines, is given as an example. Dissolve in the vaccine under examination sufficient sodium citrate to give a 10 per cent w/v solution. Maintain at 37 for about 16 h and centrifuge until a clear supernatant is obtained. The clear supernatant reacts with a suitable diphtheria antitoxin, giving a precipitate. B. Tetanus toxoid is identified by a suitable immunochemical method (2.2.14). The following method, applicable to certain vaccines, is given as an example. The clear supernatant obtained as described in identification test A reacts with a suitable tetanus antitoxin, giving a precipitate. C. The pertussis components are identified by a suitable immunochemical method (2.2.14). The following method, applicable to certain vaccines, is given as an example. The clear supernatant obtained as described in identification test A reacts with a specific antisera to the pertussis components of the vaccine. D. The assay or, where applicable, the electrophoretic profile, serves also to identify the hepatitis B component of the vaccine.
Tests
Absence of residual pertussis toxin and irreversibility of pertussis toxoid This test is not necessary for the product obtained by genetic modification. Use 3 groups each of not less than 5 histaminesensitive mice. Inject intraperitoneally into each mouse of the first group twice the single human dose of the vaccine stored at 2 to 8. Inject intraperitoneally into each mouse of the second group twice the single human dose of the vaccine incubated at 37 for 4 weeks. Inject diluent into the third group of mice. After 5 days, inject into each mouse 2 mg of histamine
IP 2007
A. D. T. P. (ACELL. COMPONENT), INACTIVATED POLIOMYLITIS VACCINE AND HAEMOPHILUS TYPE B CONJUGATE
base intraperitoneally in a volume not exceeding 0.5 ml and observe for 24 h. The test is invalid if 1 or more control mice die following histamine challenge. The vaccine complies with the test if no animal in the first or second group dies following histamine challenge. If 1 mouse dies in either or both of the first and second groups, the test may be repeated with the same number of mice or with a greater number and the results of valid tests combined; the vaccine complies with the test if, in both of the groups given the vaccine, not more than 5 per cent of the total number of mice die following histamine challenge. The histamine sensitivity of the strain of mice used is verified at suitable intervals as follows: inject intravenously threefold dilutions of a reference pertussis toxin preparation in phosphate-buffered saline solution containing 0.2 per cent w/v of gelatin and challenge with histamine as above; the strain is suitable if more than 50.0 per cent of the animals are sensitised by 50 ng of pertussis toxin and none of the control animals injected with only diluent and challenged similarly with histamine shows symptoms of sensitisation. Aluminium (2.3.9). Maximum 1.25 mg per single human dose, if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. Free formaldehyde (2.3.20). Maximum 0.2 g/l. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility ( 2.2.11). Complies with the test for sterility. Pyrogens (2.2.8). Complies with the test for pyrogens. Inject the equivalent of 1 human dose into each rabbit. Assay Diphtheria component Carry out one of the prescribed methods for the assay as stated under Diphtheria Vaccine (Adsorbed). The lower confidence limit (P = 0.95) of the estimated potency is not less than the minimum potency stated on the label. Unless otherwise justified and authorised, the minimum potency stated on the label is 30 IU per single human dose. Tetanus component Carry out one of the prescribed methods for the assay as stated under Tetanus Vaccine (Adsorbed). The lower confidence limit (P = 0.95) of the estimated potency is not less than 40 IU per single human dose. Pertussis component 9
The vaccine complies with the assay as stated under Adsorbed Pertussis Vaccine (Acellular Component). Hepatitis B component The vaccine complies with the assay as stated under Hepatitis B Vaccine (rDNA). Labelling. The label states (1) the minimum number of International Units of diphtheria and tetanus toxoid per single human dose; (2) the names and amounts of the pertussis components per single human dose; (3) the amount of HBsAg per single human dose; (4) the type of cells used for production of the hepatitis B component; (5) where applicable, that the vaccine is intended for primary vaccination of children and is not necessarily suitable for reinforcing doses or for administration to adults; (6) the name and the amount of the adsorbent; (7) that the vaccine must be shaken before use; (8) that the vaccine is not to be frozen (9) where applicable, that the vaccine contains a pertussis toxin-like protein produced by genetic modification.
Adsorbed Diphtheria, Tetanus, Pertussis (Acellular Component), Inactivated Poliomyelitis Vaccine and Haemophilus Type b Conjugate Vaccine
Diphtheria, Tetanus, Pertussis (Acellular Component) and Haemophilus Type b Conjugate Vaccine (Adsorbed) is a combined vaccine composed of: diphtheria formol toxoid; tetanus formol toxoid; individually purified antigenic components of Bordetella pertussis; polyribosylribitol phosphate (PRP) covalently bound to a carrier protein; a mineral absorbent such as aluminium hydroxide or hydrated aluminium phosphate. The product may be presented with the haemophilus type b component in a separate container, the contents of which are mixed with the other components immediately before use. The formol toxoids are prepared from the toxins produced by the growth of Corynebacterium diphtheriae and Clostridium tetani respectively. The vaccine contains either pertussis toxoid or a pertussistoxin-like protein free from toxic properties produced by expression of a genetically modified form of the corresponding gene. Pertussis toxoid is prepared from pertussis toxin by a method that renders the toxin harmless while maintaining adequate immunogenic properties and avoiding reversion to toxin. The acellular pertussis component may also contain filamentous haemagglutinin, pertactin (a 69 kDa outer-
A. D. T. P. (ACELL. COMPONENT), INACTIVATED POLIOMYLITIS VACCINE AND HAEMOPHILUS TYPE B CONJUGATE IP 2007
membrane protein) and other defined components of B. pertussis such as fimbrial-2 and fimbrial-3 antigens. The latter 2 antigens may be co-purified. The antigenic composition and characteristics are based on evidence of protection and freedom from unexpected reactions in the target group for which the vaccine is intended. PRP is a linear copolymer composed of repeated units of 3--D-ribofuranosyl-(11)-ribitol-5-phosphate [(C10H19O12P)n], with a defined molecular size and derived from a suitable strain of Haemophilus influenzae type b. The carrier protein, when conjugated to PRP, is capable of inducing a Tcell-dependent B-cell immune response to the polysaccharide.
Reference vaccine(s) Provided valid assays can be performed, monocomponent reference vaccines may be used for the assays on the combined vaccine. If this is not possible because of interaction between the components of the combined vaccine or because of the difference in composition between monocomponent reference vaccine and the test vaccine, a batch of combined vaccine shown to be effective in clinical trials or a batch representative thereof is used as a reference vaccine. For the preparation of a representative batch, strict adherence to the production process used for the batch tested in clinical trials is necessary. The reference vaccine may be stabilised by a method that has been shown to have no effect on the assay procedure. Production of the components
Production
General provisions The production method shall have been shown to yield consistently vaccines comparable with the vaccine of proven clinical efficacy and safety in man. The content of bacterial endotoxins in bulk purified diphtheria toxoid, tetanus toxoid, pertussis components, purified, inactivated monovalent poliovirus harvests and bulk PRP conjugate is determined to monitor the purification procedure and to limit the amount in the final vaccine. For each component, the content of bacterial endotoxins is less than the limit approved for the particular vaccine and, in any case, the contents are such that the final vaccine contains less than 100 IU per single human dose. The production method is validated to demonstrate that the product, if tested, would comply with the following test. Inject subcutaneously 5 times the single human dose stated on the label into each of 5 healthy guinea-pigs, each weighing between 250 and 350 g, that have not previously been treated with any material that will interfere with the test. If within 42 days of the injection any of the animals shows signs of or dies from diphtheria, toxaemia or tetanus, the vaccine does not comply with the test. If more than 1 animal dies from nonspecific causes, repeat the test once; if more than 1 animal dies in the second test, the vaccine does not comply with the test. During development studies and wherever revalidation is necessary, it shall be demonstrated by tests in animals that the vaccine induces a T-cell dependent B-cell immune response to PRP. As part of consistency studies the assays of the diphtheria, tetanus, pertussis and poliomyelitis components are carried out on a suitable number of batches of vaccine reconstituted for use. For subsequent routine control, the assays of these components may be carried out without mixing with the haemophilus component. 10 The production of the components complies with the requirements of the monographs on Diphtheria Vaccine (Adsorbed), Tetanus Vaccine (Adsorbed), Pertussis Vaccine (Acellular Component, Adsorbed), Poliomyelitis Vaccine (Inactivated) and Haemophilus Type b Conjugate Vaccine. FINAL BULK VACCINE The final bulk of the diphtheria, tetanus, pertussis and poliomyelitis components is prepared by adsorption, separately or together, of suitable quantities of bulk purified diphtheria toxoid, bulk purified tetanus toxoid and bulk purified acellular pertussis components onto a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate and admixture of suitable quantities of purified, monovalent harvests of human polioviruses 1, 2 and 3 or a suitable quantity of a trivalent pool of such monovalent harvests. Suitable antimicrobial preservatives may be added. The final bulk of the haemophilus component is prepared by dilution of the bulk conjugate to the final concentration with a suitable diluent. A stabiliser may be added. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Bovine serum albumin. Determine on the poliomyelitis components by a suitable immunochemical method (2.2.14) during preparation of the final bulk vaccine, before addition of the adsorbent, the amount of bovine serum albumin is such that the content in the final vaccine will not be more than 50 ng per single human dose. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium.
IP 2007
A. D. T. P. (ACELL. COMPONENT), INACTIVATED POLIOMYLITIS VACCINE AND HAEMOPHILUS TYPE B CONJUGATE
FINAL LOT The final bulk of the haemophilus component is freeze-dried. Only a final lot that is satisfactory with respect to the test for osmolality shown below and with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided that the test for absence of residual pertussis toxin and irreversibility of pertussis toxoid, the test for antimicrobial preservative and the assay have been carried out with satisfactory results on the final bulk vaccine, they may be omitted on the final lot. Provided that the free formaldehyde content has been determined on the bulk purified antigens and the purified monovalent harvests or the trivalent pool of polioviruses or the final bulk and it has been shown that the content in the final lot will not exceed 0.2 g/l, the test for free formaldehyde may be omitted on the final lot. Provided that the in vivo assay for the poliomyelitis component has been carried out with satisfactory results on the final bulk vaccine, it may be omitted on the final lot. Osmolality (2.4.23). The osmolality of the vaccine, reconstituted where applicable, is within the limits approved for the particular preparation. Free PRP Unbound PRP is determined on the haemophilus component after removal of the conjugate, for example by anion-exchange, size-exclusion or hydrophobic chromatography (2.4.16), ultrafiltration or other validated methods. The amount of free PRP is not greater than that approved for the particular product.
immunochemical methods (2.2.14). The following method, applicable to certain vaccines, is given as an example. The clear supernatant obtained during identification test A reacts with specific antisera to the pertussis components of the vaccine. D. The vaccine is shown to contain human polioviruses 1, 2 and 3 by a suitable immunochemical method (2.2.14), such as determination of D-antigen by enzyme-linked mmunosorbent assay (ELISA). E. The haemophilus component is identified by a suitable immunochemical method (2.2.14) for PRP.
Tests
The tests for absence of residual pertussis toxin, irreversibility of pertussis toxoid, aluminium, free formaldehyde, antimicrobial preservative and sterility are carried out on the container with the diphtheria, tetanus, pertussis and poliomyelitis components; the tests for PRP content, water, sterility and pyrogens are carried out on the container with the haemophilus component. Some tests for the haemophilus component may be carried out on the freeze-dried product rather than on the bulk conjugate where the freeze-drying process may affect the component under test. Absence of residual pertussis toxin and irreversibility of pertussis toxoid This test is not necessary for the product obtained by genetic modification. Use 3 groups each of not fewer than 5 histaminesensitive mice. Inject intraperitoneally into each mouse of the first group twice the single human dose of the vaccine stored at 2 to 8. Inject intraperitoneally into each mouse of the second group twice the single human dose of the vaccine incubated at 37 for 4 weeks. Inject diluent into the third group of mice. After 5 days, inject into each mouse 2 mg of histamine base intraperitoneally in a volume not exceeding 0.5 ml and observe for 24 hours. The test is invalid if 1 or more control mice die following histamine challenge. The vaccine complies with the test if no animal in the first or second group dies following histamine challenge. If 1 mouse dies in either or both of the first and second groups, the test may be repeated with the same number of mice or with a greater number and the results of valid tests combined; the vaccine complies with the test if, in both of the groups given the vaccine, not more than 5 per cent of the total number of mice die following histamine challenge. The histamine sensitivity of the strain of mice used is verified at suitable intervals as follows: inject intravenously threefold dilutions of a reference pertussis toxin preparation in phosphate-buffered saline solution containing 0.2 per cent w/v of gelatin and challenge with histamine as above; the 11
Identification
Identification tests A, B, C and D are carried out using the vial containing the diphtheria, tetanus, pertussis and poliomyelitis components; identification test E is carried out on the vial containing the haemophilus component. A. Diphtheria toxoid is identified by a suitable immunochemical method (2.2.14). The following method, applicable to certain vaccines, is given as an example. Dissolve in the vaccine under examination sufficient sodium citrate to give a 10 per cent w/v solution. Maintain at 37 for about 16 hours and centrifuge until a clear supernatant is obtained. The clear supernatant reacts with a suitable diphtheria antitoxin, giving a precipitate. B. Tetanus toxoid is identified by a suitable immunochemical method (2.2.14). The following method, applicable to certain vaccines, is given as an example. The clear supernatant obtained during identification test A reacts with a suitable tetanus antitoxin, giving a precipitate. C. The pertussis components are identified by suitable
A. D. T. P. (ACELLULAR. COMPONENT) AND INACTIVATED POLIOMYLITIS VACCINE
IP 2007
strain is suitable if more than 50.0 per cent of the animals are sensitised by 50 ng of pertussis toxin and none of the control animals injected with only diluent and challenged similarly with histamine show symptoms of sensitisation. PRP. Minimum 80.0 per cent of the amount of PRP stated on the label. PRP is determined either by assay of ribose ( 2.7.1) or phosphorus ( 2.7.1), by an immunochemical method ( 2.2.14) or by anion-exchange liquid chromatography with pulsedamperometric detection. Aluminium (2.3.9). Maximum 1.25 mg per single human dose, if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. Free formaldehyde (2.3.20). Maximum 0.2 g/l. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Water (2.3.43). Maximum 3.0 per cent for the haemophilus component. Sterility (2.2.11 ). Complies with the test for sterility. Pyrogens (2.2.8). Complies with the test for pyrogens. Inject per kg of the rabbits mass a quantity of the vaccine equivalent to: 1 mg of PRP for a vaccine with diphtheria toxoid or CRM 197 diphtheria protein as carrier; 0.1 mg of PRP for a vaccine with tetanus toxoid as carrier; 0.025 mg of PRP for a vaccine with OMP as a carrier. Assay Diphtheria component Carry out one of the prescribed methods for the assay as stated under Diphtheria Vaccine (Adsorbed). Unless otherwise justified and authorised, the lower confidence limit (P = 0.95) of the estimated potency is not less than 30 IU per single human dose. Tetanus component Carry out one of the prescribed methods for the assay as stated under Tetanus Vaccine (Adsorbed). The lower confidence limit (P = 0.95) of the estimated potency is not less than 40 IU per single human dose. Pertussis component It complies with the assay as stated under Adsorbed Pertussis Vaccine (Acellular Component). Poliomyelitis component D-antigen content As a measure of consistency of production, determine the Dantigen content for human polioviruses 1, 2 and 3 by a suitable 12
immunochemical method (2.2.14) using a reference preparation calibrated in units of D-antigen. For each type, the content, expressed with reference to the amount of D-antigen stated on the label, is within the limits approved for the particular product. Poliomyelitis vaccine (inactivated) reference preparation is calibrated in Units and intended for use in the assay of D-antigen. The Unit and the International Unit are equivalent. In vivo test The vaccine complies with the in vivo assay as stated under Inactivated Poliomyelitis Vaccine. Labelling. The label states (1) the minimum number of International Units of diphtheria and tetanus toxoid per single human dose (2) the names and amounts of the pertussis components per single human dose; (3) the nominal amount of poliovirus of each type (1, 2 and 3), expressed in units of D-antigen per single human dose; (4) the type of cells used for production of the poliomyelitis component; (5) the number of micrograms of PRP per single human dose; (6) the type and nominal amount of carrier protein per single human dose; (7) where applicable, that the vaccine is intended for primary vaccination of children and is not necessarily suitable for reinforcing doses or for administration to adults; (8) the name and the amount of the adsorbent; (9) that the vaccine must be shaken before use; (10) that the vaccine is not to be frozen; (11) where applicable, that the vaccine contains a pertussis toxin-like protein produced by genetic modification.
Adsorbed Diphtheria, Tetanus, Pertussis (Acellular Component) and Inactivated Poliomyelitis Vaccine
Diphtheria, Tetanus, Pertussis (Acellular Component) and Poliomyelitis (Inactivated) Vaccine is a combined vaccine containing: diphtheria formol toxoid; tetanus formol toxoid; individually purified antigenic components of Bordetella pertussis; suitable strains of human polioviruses 1, 2 and 3 grown in suitable cell cultures and inactivated by a validated method; a mineral adsorbent such as aluminium hydroxide or hydrated aluminium phosphate. The formol toxoids are prepared from the toxins produced by the growth of Corynebacterium diphtheriae and Clostridium tetani respectively. The vaccine contains either pertussis toxoid or a pertussistoxin-like protein free from toxic properties produced by expression of a genetically modified form of the corresponding gene. Pertussis toxoid is prepared from pertussis toxin by a method that renders the toxin harmless while maintaining
IP 2007
adequate immunogenic properties and avoiding reversion to toxin. The vaccine may also contain filamentous haemagglutinin, pertactin (a 69 kDa outer-membrane protein) and other defined components of B. pertussis such as fimbrial2 and fimbrial-3 antigens. The latter 2 antigens may be copurified. The antigenic composition and characteristics are based on evidence of protection and freedom from unexpected reactions in the target group for which the vaccine is intended.
bulk purified diphtheria toxoid, tetanus toxoid, acellular pertussis components and admixture of suitable quantities of purified monovalent harvests of human polioviruses 1, 2 and 3 or a suitable quantity of a trivalent pool of such purified monovalent harvests. Suitable antimicrobial preservatives may be added. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Bovine serum albumin. Determine on the poliomyelitis components by a suitable immunochemical method (2.2.14) after virus harvest and before addition of the adsorbent in the preparation of the final bulk vaccine, the amount of bovine serum albumin is such that the content in the final vaccine will be not more than 50 ng per single human dose. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT Only a final lot that is satisfactory with respect to the test for osmolality and with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided the tests for absence of residual pertussis toxin, irreversibility of pertussis toxoid and antimicrobial preservative and the assays for the diphtheria, tetanus and pertussis components have been carried out with satisfactory results on the final bulk vaccine, they may be omitted on the final lot. Provided the free formaldehyde content has been determined on the bulk purified antigens or on the final bulk and it has been shown that the content in the final lot will not exceed 0.2 g/l, the test for free formaldehyde may be omitted on the final lot. Provided that the determination of D-antigen content has been carried out with satisfactory results during preparation of the final bulk before addition of the adsorbent, it may be omitted on the final lot. Provided that the in vivo assay for the poliomyelitis component has been carried out with satisfactory results on the final bulk vaccine, it may be omitted on the final lot. Osmolality (2.4.23). The osmolality of the vaccine is within the limits approved for the particular preparation.
Production
General provisions The production method shall have been shown to yield consistently vaccines comparable with the vaccine of proven clinical efficacy and safety in man. The production method is validated to demonstrate that the product, if tested, would comply with the test for abnormal toxicity for antisera and vaccines. The content of bacterial endotoxins in bulk purified diphtheria toxoid, tetanus toxoid, pertussis components and purified, inactivated monovalent poliovirus harvests is determined to monitor the purification procedure and to limit the amount in the final vaccine. For each component, the content of bacterial endotoxins is less than the limit approved for the particular vaccine and, in any case, the contents are such that the final vaccine contains less than 100 IU per single human dose. Reference vaccine(s) Provided valid assays can be performed, monocomponent reference vaccines may be used for the assays on the combined vaccine. If this is not possible because of interaction between the components of the combined vaccine or because of the difference in composition between monocomponent reference vaccine and the test vaccine, a batch of combined vaccine shown to be effective in clinical trials or a batch representative thereof is used as a reference vaccine. For the preparation of a representative batch, strict adherence to the production process used for the batch tested in clinical trials is necessary. The reference vaccine may be stabilised by a method that has been shown to have no effect on the assay procedure. Production of the components The production of the components complies with the requirements of the monographs on Diphtheria Vaccine (Adsorbed), Tetanus Vaccine (Adsorbed), Pertussis Vaccine (Acellular Component, Adsorbed) and Poliomyelitis Vaccine (Inactivated). FINAL BULK VACCINE The final bulk vaccine is prepared by adsorption onto a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate, separately or together, of suitable quantities of 13
Identification
A. Diphtheria toxoid is identified by a suitable immunochemical
IP 2007
method (2.2.14). The following method, applicable to certain vaccines, is given as an example. Dissolve in the vaccine under examination sufficient sodium citrate to give a 10 per cent w/v solution. Maintain at 37 for about 16 h and centrifuge until a clear supernatant is obtained. The clear supernatant reacts with a suitable diphtheria antitoxin, giving a precipitate. B. Tetanus toxoid is identified by a suitable immunochemical method (2.2.14). The following method, applicable to certain vaccines, is given as an example. The clear supernatant obtained as described in Identification test A reacts with a suitable tetanus antitoxin, giving a precipitate. C. The pertussis components are identified by a suitable immunochemical method (2.2.14). The following method, applicable to certain vaccines, is given as an example. The clear supernatant obtained as described in Identification test A reacts with a specific antisera to the pertussis components of the vaccine. D. The vaccine is shown to contain human polioviruses 1, 2 and 3 by a suitable immunochemical method (2.2.14) such as the determination of D-antigen by enzyme-linked immunosorbent assay (ELISA).
sensitised by 50 ng of pertussis toxin and none of the control animals injected with only diluent and challenged similarly with histamine shows symptoms of sensitisation. Aluminium (2.3.9). Maximum 1.25 mg per single human dose if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. Free formaldehyde (2.3.20). Maximum 0.2 g/l. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Complies with the test for sterility. Assay Diphtheria component Carry out one of the prescribed methods for the assay as stated under Diphtheria Vaccine (Adsorbed). The lower confidence limit (P = 0.95) of the estimated potency is not less than the minimum potency stated on the label. Unless otherwise justified and authorised, the minimum potency stated on the label is 30 IU per single human dose. Tetanus component Carry out one of the prescribed methods for the assay as stated under Tetanus Vaccine (Adsorbed). The lower confidence limit (P = 0.95) of the estimated potency is not less than 40 IU per single human dose. Pertussis component The vaccine complies with the assay as stated under Adsorbed Pertussis Vaccine (Acellular Component). Poliomyelitis component D-antigen content As a measure of consistency of production, determine the Dantigen content for human polioviruses 1, 2 and 3 by a suitable immunochemical method (2.2.14) following desorption using a reference preparation calibrated in units of D-antigen. For each type, the content, expressed with reference to the amount of D-antigen stated on the label, is within the limits approved for the particular product. Poliomyelitis vaccine (inactivated) reference preparation is calibrated in Units and intended for use in the assay of D-antigen. The Unit and the International Unit are equivalent. In vivo test The vaccine complies with the in vivo assay as stated under Inactivated Poliomyelitis Vaccine. 14
Tests
Absence of residual pertussis toxin and irreversibility of pertussis toxoid This test is not necessary for the product obtained by genetic modification. Use 3 groups each of not less than 5 histaminesensitive mice. Inject intraperitoneally into each mouse of the first group twice the single human dose of the vaccine stored at 2 to 8. Inject intraperitoneally into each mouse of the second group twice the single human dose of the vaccine incubated at 37 for 4 weeks. Inject diluent into the third group of mice. After 5 days, inject into each mouse 2 mg of histamine base intraperitoneally in a volume not exceeding 0.5 ml and observe for 24 hours. The test is invalid if 1 or more control mice die following histamine challenge. The vaccine complies with the test if no animal in the first or second group dies following histamine challenge. If 1 mouse dies in either or both of the first and second groups, the test may be repeated with the same number of mice or with a greater number and the results of valid tests combined; the vaccine complies with the test if, in both of the groups given the vaccine, not more than 5.0 per cent of the total number of mice die following histamine challenge. The histamine sensitivity of the strain of mice used is verified at suitable intervals as follows: inject intravenously threefold dilutions of a reference pertussis toxin preparation in phosphate-buffered saline solution containing 0.2 per cent w/v of gelatin and challenge with histamine as above; the strain is suitable if more than 50.0 per cent of the animals are
IP 2007
ADSORBED DIPHTHERIA TETANUS PERTUSSIS AND POLIOMYLITIS (INACTIVATED) VACCINE
Labelling. The label complies with the requirements stated under Vaccine and also states (1) the minimum number of International Units of diphtheria and tetanus toxoid per single human dose (2) the names and amounts of the pertussis components per single human dose; (3) the nominal amount of poliovirus of each type (1, 2 and 3), expressed in units of Dantigen per single human dose; (4) the type of cells used for production of the poliomyelitis component; (5) the number of micrograms of PRP per single human dose; (6) the type and nominal amount of carrier protein per single human dose; (7) where applicable, that the vaccine is intended for primary vaccination of children and is not necessarily suitable for reinforcing doses or for administration to adults; (8) the name and the amount of the adsorbent; (9) that the vaccine must be shaken before use; (10) that the vaccine is not to be frozen; (11) where applicable, that the vaccine contains a pertussis toxin-like protein produced by genetic modification.
specific causes, repeat the test once; if more than 1 animal dies in the second test, the vaccine does not comply with the test. Reference vaccine(s) Provided valid assays can be performed, monocomponent reference vaccines may be used for the assays on the combined vaccine. If this is not possible because of interaction between the components of the combined vaccine or because of the difference in composition between monocomponent reference vaccine and the test vaccine, a batch of combined vaccine shown to be effective in clinical trials or a batch representative thereof is used as a reference vaccine. For the preparation of a representative batch, strict adherence to the production process used for the batch tested in clinical trials is necessary. The reference vaccine may be stabilised by a method that has been shown to have no effect on the assay procedure. Production of the components
Adsorbed Diphtheria, Tetanus, Pertussis and Poliomyelitis (Inactivated) Vaccine

Diphtheria, Tetanus, Pertussis and Poliomyelitis (Inactivated) Vaccine (Adsorbed) is a combined vaccine containing: diphtheria formol toxoid; tetanus formol toxoid; an inactivated suspension of Bordetella pertussis; suitable strains of human polioviruses 1, 2 and 3 grown in suitable cell cultures and inactivated by a validated method; a mineral adsorbent such as aluminium hydroxide or hydrated aluminium phosphate. The formol toxoids are prepared from the toxins produced by the growth of Corynebacterium diphtheriae and Clostridium tetani respectively.
The production of the components complies with the requirements of the monographs on Diphtheria Vaccine (Adsorbed), Tetanus Vaccine (Adsorbed), Pertussis Vaccine (Adsorbed) and Poliomyelitis Vaccine (Inactivated). FINAL BULK VACCINE The final bulk vaccine is prepared by adsorption onto a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate, separately or together, of suitable quantities of bulk purified diphtheria toxoid and bulk purified tetanus toxoid and admixture of suitable quantities of an inactivated suspension of B. pertussis and purified monovalent harvests of human polioviruses 1, 2 and 3 or a suitable quantity of a trivalent pool of such purified monovalent harvests. Suitable antimicrobial preservatives may be added. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Specific toxicity Use not less than 5 healthy mice each weighing between 14 and 16 g for the vaccine group and for the saline control. Use mice of the same sex or distribute males and females equally between the groups. Allow the animals access to food and water for at least 2 hours before injection and during the test. Inject each mouse of the vaccine group intraperitoneally with 0.5 ml, containing a quantity of the vaccine equivalent to not less than half the single human dose. Inject each mouse of the control group with 0.5 ml of a 0.9 per cent sterile solution of sodium chloride, preferably containing the same amount of antimicrobial preservative as that injected with the vaccine. Weigh the groups of mice immediately before the injection and 72 hours and 7 days after the injection. The vaccine 15
Production
General provisions The production method shall have been shown to yield consistently the vaccines comparable with the vaccine of proven clinical efficacy and safety in man. The production method is validated to demonstrate that the product, if tested, would comply with the test for abnormal toxicity for antisera and vaccines, and with the following test for specific toxicity of the diphteria and tetanus components : inject subcutaneously 5 times the single human dose stated on the label into each of 5 healthy guinea-pigs, each weighing between 250 and 350 g, that have not previously been treated with any material that will interfere with the test. If within 42 days of the injection any of the animals shows signs of or dies from diphtheria toxaemia or tetanus, the vaccine does not comply with the test. If more than 1 animal dies from non-
ADSORBED DIPHTHERIA TETANUS PERTUSSIS AND POLIOMYLITIS (INACTIVATED) VACCINE
IP 2007
complies with the test if: (a) at the end of 72 hours the total mass of the group of vaccinated mice is not less than that preceding the injection; (b) at the end of 7 days the average increase in mass per vaccinated mouse is not less than 60 per cent of that per control mouse; and (c) not more than 5 per cent of the vaccinated mice die during the test. The test may be repeated and the results of the tests combined. Bovine serum albumin. Determine on the poliomyelitis components by a suitable immunochemical method (2.2.14) during preparation of the final bulk vaccine; before addition of the adsorbent, the amount of bovine serum albumin is such that the content in the final vaccine will be not more than 50 ng per single human dose. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT Only a final lot that is satisfactory with respect to the test for osmolality and with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided that the tests for specific toxicity and antimicrobial preservative, and the assays for the diphtheria, tetanus and pertussis components have been carried out with satisfactory results on the final bulk vaccine, they may be omitted on the final lot. Provided that the free formaldehyde content has been determined on the bulk purified antigens, the inactivated B. pertussis suspension and the purified monovalent harvests or the trivalent pool of polioviruses or on the final bulk and it has been shown that the content in the final lot will not exceed 0.2 g/l, the test for free formaldehyde may be omitted on the final lot. Provided that the in vivo assay for the poliomyelitis component has been carried out with satisfactory results on the final bulk vaccine, it may be omitted on the final lot. Osmolality (2.4.23). The osmolality of the vaccine is within the limits approved for the particular preparation.
reacts with a suitable diphtheria antitoxin, giving a precipitate. B. Tetanus toxoid is identified by a suitable immunochemical method (2.2.14). The following method, applicable to certain vaccines, is given as an example. The clear s u p e r n a t a n t obtained during identification test A reacts with a suitable tetanus antitoxin, giving a precipitate. C. The centrifugation residue obtained in identification A may be used. Other suitable methods for separating the bacteria from the adsorbent may also be used. Identify pertussis vaccine by agglutination of the bacteria from the resuspended precipitate by antisera specific to B. pertussis or by the assay of the pertussis component prescribed under Assay. D. The vaccine is shown to contain human polioviruses 1, 2 and 3 by a suitable immunochemical method (2.2.14) such as the determination of D-antigen by enzyme-linked immunosorbent assay (ELISA).
Tests
Aluminium (2.3.9). Maximum 1.25 mg per single human dose, if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. Free formaldehyde (2.3.20). Maximum 0.2 g/l. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Complies with the test for sterility. Assay Diphtheria component Carry out one of the prescribed methods for the assay as stated under Diphtheria Vaccine (Adsorbed). The lower confidence limit (P = 0.95) of the estimated potency is not less than 30 IU per single human dose. Tetanus component Carry out one of the prescribed methods for the assay as stated under Tetanus Vaccine (Adsorbed). If the test is carried out in guinea pigs, the lower confidence limit (P = 0.95) of the estimated potency is not less than 40 IU per single human dose; if the test is carried out in mice, the lower confidence limit (P = 0.95) of the estimated potency is not less than 60 IU per single human dose. Pertussis component Carry out the assay as stated under Pertussis Vaccine. The estimated potency is not less than 4 IU per single human 16
Identification
A. Diphtheria toxoid is identified by a suitable immunochemical method (2.2.14). The following method, applicable to certain vaccines, is given as an example. Dissolve in the vaccine under examination sufficient sodium citrate to give a 10 per cent w/v solution. Maintain at 37 for about 16 hours and centrifuge until a clear supernatant is obtained. The clear supernatant
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A. D., T., P., POLIOMYLITIS (INACTIVATED) AND HAEMOPHILUS TYPE B CONJUGATE VACCINE
dose and the lower confidence limit (P = 0.95) of the estimated potency is not less than 2 IU per single human dose. Poliomyelitis component D-antigen content As a measure of consistency of production, determine the Dantigen content for human polioviruses 1, 2 and 3 by a suitable immunochemical method (2.2.14) using a reference preparation calibrated in Units of D-antigen. For each type, the content, expressed with reference to the amount of D-antigen stated on the label, is within the limits approved for the particular product. Poliomyelitis vaccine (inactivated) reference preparation is calibrated in Units and is intended for use in the assay of D-antigen. The Unit and the IU are equivalent. In vivo test The vaccine complies with the in vivo assay as stated under Poliomyelitis Vaccine (Inactivated). Labelling. The label states (1) the minimum number of International Units of diphtheria and tetanus toxoid per single human dose; (2) the minimum number of International Units of pertussis vaccine per single human dose; (3) the nominal amount of poliovirus of each type (1, 2 and 3), expressed in units of D-antigen per single human dose; (4) the type of cells used for production of the poliomyelitis component; (5) where applicable, that the vaccine is intended for primary vaccination of children and is not necessarily suitable for reinforcing doses or for administration to adults; (6) the name and the amount of the adsorbent; (7) that the vaccine must be shaken before use; (8) that the vaccine is not to be frozen.
The formol toxoids are prepared from the toxins produced by the growth of Corynebacterium diphtheriae and Clostridium tetani respectively. PRP is a linear copolymer composed of repeated units of 3--D-ribofuranosyl-(11)-ribitol-5-phosphate [(C10H19O12P)n], with a defined molecular size and derived from a suitable strain of Haemophilus influenzae type b. The carrier protein, when conjugated to PRP, is capable of inducing a Tcell-dependent B-cell immune response to the polysaccharide.
Production
General provisions The production method shall have been shown to yield consistently vaccines comparable with the vaccine of proven clinical efficacy and safety in man. The production method is validated to demonstrate that the product, if tested, would comply with the test for abnormal toxicity for antisera and vaccines, and with the following test for specific toxicity of the diphtheria and tetanus components: inject subcutaneously 5 times the single human dose stated on the label into each of 5 healthy guinea-pigs, each weighing between 250 and 350 g, that have not previously been treated with any material that will interfere with the test. If within 42 days of the injection any of the animals shows signs of or dies from diphtheria toxaemia or tetanus, the vaccine does not comply with the test. If more than 1 animal dies from nonspecific causes, repeat the test once; if more than 1 animal dies in the second test, the vaccine does not comply with the test. During development studies and wherever revalidation is necessary, it shall be demonstrated by tests in animals that the vaccine induces a T-cell dependent B-cell immune response to PRP. As part of consistency studies the assays of the diphtheria, tetanus, pertussis and poliomyelitis components are carried out on a suitable number of batches of vaccine reconstituted for use. For subsequent routine control, the assays of these components may be carried out without mixing with the haemophilus component. Reference vaccine(s) Provided valid assays can be performed, monocomponent reference vaccines may be used for the assays on the combined vaccine. If this is not possible because of interaction between the components of the combined vaccine or because of the difference in composition between monocomponent reference vaccine and the test vaccine, a batch of combined vaccine shown to be effective in clinical trials or a batch representative thereof is used as a reference vaccine. For the preparation of a representative batch, strict adherence to the production process used for the batch tested in clinical trials is necessary. 17
Adsorbed Diphtheria, Tetanus, Pertussis, Poliomyelitis (Inactivated) and Haemophilus Type b Conjugate Vaccine
Diphtheria, Tetanus, Pertussis, Poliomyelitis (Inactivated) and Haemophilus Type b Conjugate Vaccine (Adsorbed) is a combined vaccine composed of: diphtheria formol toxoid; tetanus formol toxoid; an inactivated suspension of Bordetella pertussis; suitable strains of human polioviruses 1, 2 and 3 grown in suitable cell cultures and inactivated by a suitable method; polyribosylribitol phosphate (PRP) covalently bound to a carrier protein; a mineral adsorbent such as aluminium hydroxide or hydrated aluminium phosphate. The product is presented with the haemophilus component in a separate container, the contents of which are mixed with the other components immediately before use.
IP 2007
The reference vaccine may be stabilised by a method that has been shown to have no effect on the assay procedure. Production of components The production of the components complies with the requirements of the monographs on Diphtheria Vaccine (Adsorbed), Tetanus Vaccine (Adsorbed), Pertussis Vaccine (Adsorbed), Poliomyelitis Vaccine (Inactivated) and Haemophilus Type b Conjugate Vaccine. FINAL BULK VACCINE The final bulk of the diphtheria, tetanus, pertussis and poliomyelitis components is prepared by adsorption, separately or together, of suitable quantities of bulk purified diphtheria toxoid, and bulk purified tetanus toxoid onto a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate and admixture of suitable quantities of an inactivated suspension of B. pertussis and of purified, monovalent harvests of human polioviruses 1, 2 and 3 or a suitable quantity of a trivalent pool of such monovalent harvests. Suitable antimicrobial preservatives may be added. The final bulk of the haemophilus component is prepared by dilution of the bulk conjugate to the final concentration with a suitable diluent. A stabiliser may be added. Only final bulk that complies with the following requirements may be used in the preparation of the final lot. Specific toxicity Use not less than 5 healthy mice each weighing between 14 and 16 g, for the vaccine group and for the saline control. Use mice of the same sex or distribute males and females equally between the groups. Allow the animals access to food and water for at least 2 hours before injection and during the test. Inject each mouse of the vaccine group intraperitoneally with 0.5 ml, containing a quantity of the vaccine equivalent to not less than half the single human dose. Inject each mouse of the control group with 0.5 ml of a 0.9 per cent sterile solution of sodium chloride, preferably containing the same amount of antimicrobial preservative as that injected with the vaccine. Weigh the groups of mice immediately before the injection and 72 hours and 7 days after the injection. The vaccine complies with the test if (a) at the end of 72 hours the total mass of the group of vaccinated mice is not less than that preceding the injection; (b) at the end of 7 days the average increase in mass per vaccinated mouse is not less than 60 per cent of that per control mouse; and (c) not more than 5 per cent of the vaccinated mice die during the test. The test may be repeated and the results of the tests combined. Bovine serum albumin. Determine on the poliomyelitis components by a suitable immunochemical method (2.2.14) during preparation of the final bulk vaccine, before addition 18
of the adsorbent, the amount of bovine serum albumin is such that the content in the final vaccine will not be more than 50 ng per single human dose. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT The final bulk of the haemophilus component is freeze-dried. Only a final lot that is satisfactory with respect to the test for osmolality shown below and with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided that the tests for specific toxicity and antimicrobial preservative, and the assays for the diphtheria, tetanus and pertussis components have been carried out with satisfactory results on the final bulk vaccine, they may be omitted on the final lot. Provided that the free formaldehyde content has been determined on the bulk purified antigens, the inactivated B. pertussis suspension and the purified monovalent harvests or the trivalent pool of polioviruses or on the final bulk and it has been shown that the content in the final lot will not exceed 0.2 g/l, the test for free formaldehyde may be omitted on the final lot. Provided that the in vivo assay for the poliomyelitis component has been carried out with satisfactory results on the final bulk vaccine, it may be omitted on the final lot. Osmolality (2.4.23). The osmolality of the vaccine, reconstituted where applicable, is within the limits approved for the particular preparation. Free PRP Unbound PRP is determined on the haemophilus component after removal of the conjugate, for example by anion-exchange, size-exclusion or hydrophobic chromatography (2.4.16), ultrafiltration or other validated methods. The amount of free PRP is not greater than that approved for the particular product.
Identification
Identification tests A, B, C and D are carried out using the vial containing the diphtheria, tetanus, pertussis and poliomyelitis components; identification test E is carried out on the vial containing the haemophilus component. A. Diphtheria toxoid is identified by a suitable immunochemical method (2.2.14). The following method, applicable to certain
IP 2007
vaccines, is given as an example. Dissolve in the vaccine under examination sufficient sodium citrate to give a 10 per cent w/v solution. Maintain at 37 for about 16 hours and centrifuge until a clear supernatant is obtained. The clear supernatant reacts with a suitable diphtheria antitoxin, giving a precipitate. B. Tetanus toxoid is identified by a suitable immunochemical method (2.2.14). The following method, applicable to certain vaccines, is given as an example. The clear s u p e r n a t a n t obtained during identification test A reacts with a suitable tetanus antitoxin, giving a precipitate. C. The centrifugation residue obtained in identification A may be used. Other suitable methods for separating the bacteria from the adsorbent may also be used. Identify pertussis vaccine by agglutination of the bacteria from the resuspended precipitate by antisera specific to B. pertussis or by the assay of the pertussis component prescribed under Assay. D. The vaccine is shown to contain human polioviruses 1, 2 and 3 by a suitable immunochemical method (2.2.14), such as determination of D-antigen by enzymelinked immunosorbent assay (ELISA). E. The haemophilus component is identified by a suitable immunochemical method (2.2.14) for PRP.
Sterility (2.2.11). Complies with the test for sterility. Pyrogens (2.2.8). Complies with the test for pyrogens. Inject per kg of the rabbits mass a quantity of the vaccine equivalent to 1 mg of PRP for a vaccine with diphtheria toxoid or CRM 197 diphtheria protein as carrier; 0.1 mg of PRP for a vaccine with tetanus toxoid as carrier; 0.025 mg of PRP for a vaccine with OMP as carrier. Assay Diphtheria component Carry out one of the prescribed methods for the assay as stated under Diphtheria Vaccine (Adsorbed). The lower confidence limit (P = 0.95) of the estimated potency is not less than 30 IU per single human dose. Tetanus component Carry out one of the prescribed methods for the assay as stated under Tetanus Vaccine (Adsorbed). If the test is carried out in guinea-pigs, the lower confidence limit (P = 0.95) of the estimated potency is not less than 40 IU per single human dose; if the test is carried out in mice, the lower confidence limit (P = 0.95) of the estimated potency is not less than 60 IU per single human dose. Pertussis component Carry out the assay as stated under Pertussis Vaccine. The estimated potency is not less than 4 IU per single human dose and the lower confidence limit (P = 0.95) of the estimated potency is not less than 2 IU per single human dose. Poliomyelitis component D-antigen content As a measure of consistency of production, determine the Dantigen content for human polioviruses 1, 2 and 3 by a suitable immunochemical method (2.2.14) using a reference preparation calibrated in Units of D-antigen. For each type, the content, expressed with reference to the amount of D-antigen stated on the label, is within the limits approved for the particular product. Poliomyelitis vaccine (inactivated) reference preparation is calibrated in Units and intended for use in the assay of D-antigen. The Unit and the IU are equivalent. In vivo test The vaccine complies with the in vivo assay as stated under Poliomyelitis Vaccine (Inactivated). Labelling. The label states (1) the minimum number of International Units of diphtheria and tetanus toxoid per single human dose; (2) the minimum number of International Units of pertussis vaccine per single human dose; (3) the nominal amount of poliovirus of each type (1, 2 and 3), expressed in 19
Tests
The tests for specific toxicity, aluminium, free formaldehyde, antimicrobial preservative and sterility are carried out on the container with diphtheria, tetanus, pertussis and poliomyelitis components; the tests for PRP content, water, sterility and pyrogens are carried out on the container with the haemophilus component. Some tests for the haemophilus component may be carried out on the freeze-dried product rather than on the bulk conjugate where the freeze-drying process may affect the component under test. PRP. Minimum 80.0 per cent of the amount of PRP stated on the label. PRP is determined either by assay of ribose (2.7.1) or phosphorus (2.7.1), by an immunochemical method (2.2.14) or by anion-exchange liquid chromatography with pulsedamperometric detection. Aluminium (2.3.9). Maximum 1.25 mg per single human dose, if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. Free formaldehyde (2.3.20). Maximum 0.2 g/l. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Water (2.3.43). Maximum 3.0 per cent for the haemophilus component.
ADSORBED PERTUSSIS VACCINE (ACELLULAR COMPONENT)
IP 2007
Units of D-antigen per single human dose; (4) the type of cells used for production of the poliomyelitis component; (5) the number of micrograms of PRP per single human dose; (6) the type and nominal amount of carrier protein per single human dose; (7) where applicable, that the vaccine is intended for primary vaccination of children and is not necessarily suitable for reinforcing doses or for administration to adults; (8) the name and the amount of the adsorbent; (9) that the vaccine must be shaken before use; (9) that the vaccine is not to be frozen.
individual components that comply with the following requirements; after demonstration of consistency, the tests need not be applied routinely to each batch. Adenylate cyclase. Not more than 500 ng in the equivalent of 1 dose of the final vaccine, determined by immunoblot analysis or another suitable method. Tracheal cytotoxin. Not more than 2 pmol in the equivalent of 1 dose of the final vaccine, determined by a suitable method such as a biological assay or liquid chromatography (2.4.14). Absence of residual dermonecrotic toxin. Inject intradermally into each of 3 unweaned mice, in a volume of 0.1 ml, the amount of component or antigenic fraction equivalent to 1 dose of the final vaccine. Observe for 48 hours. No dermonecrotic reaction is demonstrable. Specific properties. The components of the vaccine are analysed by one or more of the methods shown below in order to determine their identity and specific properties (activity per unit amount of protein) in comparison with reference preparations. Pertussis toxin Chinese hamster ovary (CHO) cell-clustering effect and haemagglutination as in vitro methods; lymphocytosispromoting activity, histamine-sensitising activity and insulin secretory activity as in vivo methods. The toxin shows ADPribosyl transferase activity using transducin as the acceptor. Filamentous haemagglutinin Haemagglutination and inhibition by specific antibody. Pertactin, fimbrial-2 and fimbrial-3 antigens. Reactivity with specific antibody. Pertussis toxoid The toxoid induces in animals production of antibodies capable of inhibiting all the properties of pertussis toxin. PURIFIED COMPONENTS Production of each component is based on a seed-lot system. The seed cultures from which toxin is prepared are managed to conserve or where necessary restore toxinogenicity by deliberate selection. None of the media used at any stage contains blood or blood products of human origin. Media used for the preparation of seed lots and inocula may contain blood or blood products of animal origin. Pertussis toxin and, where applicable, filamentous haemagglutinin and pertactin are purified and, after appropriate characterisation, detoxified using suitable chemical reagents, by a method that avoids reversion of the toxoid to toxin, particularly on storage or exposure to heat. Other components such as fimbrial-2 and fimbrial-3 antigens are purified either 20
Adsorbed Pertussis Vaccine (Acellular Component)

Pertussis Vaccine (Acellular Component, Adsorbed) is a preparation of individually prepared and purified antigenic components of Bordetella pertussis adsorbed on a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate. The vaccine contains either pertussis toxoid or a pertussis toxin, like protein free from toxic properties, produced by expression of a genetically modified form of the corresponding gene. Pertussis toxoid is prepared from pertussis toxin by a method that renders the latter harmless while maintaining adequate immunogenic properties and avoiding reversion to toxin. The vaccine may also contain filamentous haemagglutinin, pertactin (a 69 kDa outer-membrane protein) and other defined components of B. pertussis such as fimbrial2 and fimbrial-3 antigens. The latter 2 antigens may be copurified. The antigenic composition and characteristics are based on evidence of protection and freedom from unexpected reactions in the target group for which the vaccine is intended.
Production
General provisions The production method shall have been shown to yield consistently the vaccines comparable with the vaccine of proven clinical efficacy and safety in man. Reference vaccine A batch of vaccine shown to be effective in clinical trials or a batch representative thereof is used as a reference vaccine. For the preparation of a representative batch, strict adherence to the production process used for the batch tested in clinical trials is necessary. The reference vaccine is preferably stabilised by a method that has been shown to have no significant effect on the assay procedure when the stabilised and non-stabilised batches are compared. CHARACTERISATION OF COMPONENTS During development of the vaccine, the production process shall be validated to demonstrate that it yields consistently
IP 2007
ADSORBED PERTUSSIS VACCINE (ACELLULAR COMPONENT)
separately or together, characterised and shown to be free from toxic substances. The purification procedure is validated to demonstrate appropriate clearance of substances used during culture or purification. The content of bacterial endotoxins is determined to monitor the purification procedure and to limit the amount in the final vaccine. The limits applied for the individual components are such that the final vaccine contains less than 100 IU per single human dose. Before detoxification, the purity of the components is determined by a suitable method such as polyacrylamide gel electrophoresis (PAGE) or liquid chromatography. SDS-PAGE or immunoblot analysis with specific monoclonal or polyclonal antibodies may be used to characterise subunits. Requirements are established for each individual product. Only purified components that comply with the following requirements may be used in the preparation of the final bulk vaccine. Sterility (2.2.11). Carry out the test for sterility using for each medium a quantity of purified component equivalent to not less than 100 doses. Absence of residual pertussis toxin This test is not necessary for the product obtained by genetic modification. Use a group of not fewer than 5 histaminesensitive mice each weighing between 18 and 26 g. Inject into each mouse the equivalent of 1 human dose intravenously or twice the human dose intraperitoneally, diluted to not more than 0.5 ml with phosphate-buffered saline solution containing 0.2 per cent w/v of gelatin. Inject diluent into a second group of control mice. After 5 days, inject 2 mg of histamine base intraperitoneally in a volume not exceeding 0.5 ml and observe for 24 hours. If no animal dies, the preparation complies with the test. The histamine sensitivity of the strain of mice used is verified at suitable intervals as follows: inject three-fold dilutions of a reference pertussis toxin preparation in phosphate-buffered saline solution containing 0.2 per cent w/v of gelatin and challenge with histamine as above; the strain is suitable if more than 50 per cent of the animals are sensitised by 50 ng of pertussis toxin and none of the control animals injected with only diluent and challenged similarly with histamine show symptoms of sensitisation. A validated test based on the clustering effect of the toxin for Chinese hamster ovary (CHO) cells may be used instead of the test on mice. Residual detoxifying agents and other reagents The content of residual detoxifying agents and other reagents is determined and shown to be below approved limits unless 21
validation of the process has demonstrated acceptable clearance. Antigen content Determine the antigen content by a suitable immunochemical method (2.2.14) and protein nitrogen by sulphuric acid digestion (2.2.30) or another suitable method. The ratio of antigen content to protein nitrogen is within the limits established for the product. FINAL BULK VACCINE The vaccine is prepared by adsorption of suitable quantities of purified components, separately or together, onto aluminium hydroxide or hydrated aluminium phosphate. A suitable antimicrobial preservative may be added. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT Only a final lot that is satisfactory with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided that the tests for absence of residual pertussis toxin and irreversibility of pertussis toxoid, antimicrobial preservative, free formaldehyde and the assay have been carried out with satisfactory results on the final bulk vaccine, these tests may be omitted on the final lot.
Identification
Subject the vaccine to a suitable desorption procedure such as the following: dissolve in the vaccine under examination sufficient sodium citrate to give a 10 per cent w/v solution; maintain at 37 for about 16 h and centrifuge until a clear supernatant liquid is obtained. Examined by a suitable immunochemical method (2.2.14), the clear supernatant liquid reacts with specific antisera to the components stated on the label.
Tests
Absence of residual pertussis toxin and irreversibility of pertussis toxoid This test is not necessary for the product obtained by genetic modification. Use 3 groups each of not fewer than 5 histaminesensitive mice. Inject intraperitoneally into the first group twice
IP 2007
the single human dose of the vaccine stored at 2 to 8. Inject intraperitoneally into the second group twice the single human dose of the vaccine incubated at 37 for 4 weeks. Inject diluent into the third group of mice. After 5 days, inject into each mouse 2 mg of histamine base intraperitoneally in a volume not exceeding 0.5 ml and observe for 24 hours. The test is invalid if 1 or more control mice die following histamine challenge. The vaccine complies with the test if no animal in the first or second group dies following histamine challenge. If 1 mouse dies in either or both of the first and second groups, the test may be repeated with the same number of mice or with a greater number and the results of valid tests combined; the vaccine complies with the test if, in both of the groups given the vaccine, not more than 5.0 per cent of the total number of mice die following histamine challenge. The histamine sensitivity of the strain of mice used is verified at suitable intervals as follows: inject intravenously threefold dilutions of a reference pertussis toxin preparation in phosphate-buffered saline solution containing 0.2 per cent w/v of gelatin and challenge with histamine as above; the strain is suitable if more than 50.0 per cent of the animals are sensitised by 50 ng of pertussis toxin and none of the control animals injected with only diluent and challenged similarly with histamine show symptoms of sensitisation. Aluminium (2.3.9). Maximum 1.25 mg per single human dose, if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. Free formaldehyde (2.3.20). Maximum 0.2 g/l. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Complies with the test for sterility. Assay The capacity of the vaccine to induce the formation of specific antibodies is compared with the same capacity of a reference preparation examined in parallel; antibodies are determined using suitable immunochemical methods (2.2.14) such as enzyme-linked immunosorbent assay (ELISA). The test on mice shown below uses a three-point model but, after validation, for routine testing a single-dilution method may be used. Requirement The capacity to induce antibodies is not significantly (P = 0.95) less than that of the reference vaccine. The following test model is given as an example of a method that has been found to be satisfactory. 22
Selection and distribution of test animals Use in the test healthy mice (for example, CD1 strain) of the same stock 4 to 8 weeks old. Distribute the animals in 6 groups of a number appropriate to the requirements of the assay. Use 3 dilutions of the vaccine under examination and 3 dilutions of a reference preparation and attribute each dilution to a group of mice. Inject intraperitoneally or subcutaneously into each mouse 0.5 ml of the dilution attributed to its group. Collection of serum samples 4 to 5 weeks after vaccination, bleed the mice individually under anaesthesia. Store the sera at -20 until tested for antibody content. Antibody determination Assay the individual sera for content of specific antibodies to each component using a validated method such as the ELISA test shown below. ELISA Microtitre plates (poly(vinyl chloride) or polystyrene as appropriate for the specific antigen) are coated with the purified antigen at a concentration of 100 ng per well. After washing, unreacted sites are blocked by incubating with a solution of bovine serum albumin and then washed. Two-fold dilutions of sera from mice immunised with test or reference vaccines are made on the plates. After incubation at 22 to 25 for 1 h, the plates are washed. A suitable solution of anti-mouse IgG enzyme conjugate is added to each well and incubated at 22 to 25 for 1 h. After washing, a substrate is added from which the bound enzyme conjugate liberates a chromophore which can be quantified by measurement of absorbance. The test conditions are designed to obtain a linear response for absorbance with respect to antibody content over the range of measurement used and absorbance values within the range 0.1 to 2.0. A reference antiserum of assigned potency is used in the test and serves as the basis for calculation of the antibody levels in test sera. A standardised control serum is also included in the test. The test is not valid if (a) the value found for the control serum differs by more than 2 standard deviations from the assigned value; (b) the confidence interval of the potency estimate is greater than 50.0 per cent to 200.0 per cent. Calculation The antibody titres in the sera of mice immunised with reference and test vaccines are calculated and from the values obtained the potency of the test vaccine in relation to the reference vaccine is calculated by the usual statistical methods. Labelling. The label states (1) the names and amounts of the components present in the vaccine; (2) where applicable, that
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ADSORBED PERTUSSIS VACCINE (ACELLULAR CO-PURIFIED)
the vaccine contains a pertussis toxin-like protein produced by genetic modification; (3) the name and amount of the adsorbent; (4) that the vaccine must be shaken before use; (5) that the vaccine is not to be frozen.
Tracheal cytotoxin. Not more than 2 pmol in the equivalent of 1 dose of the final vaccine, determined by a suitable method such as a biological assay or liquid chromatography (2.4.14). Absence of residual dermonecrotic toxin. Inject intradermally into each of 3 unweaned mice, in a volume of 0.1 ml, the amount of antigenic fraction equivalent to 1 dose of the final vaccine. Observe for 48 hours. No dermonecrotic reaction is demonstrable. Specific properties. The antigenic fraction is analyzed by one or more of the methods shown below in order to determine the identity and specific properties (activity per unit amount of protein) of its components in comparison with reference preparations. Pertussis toxin Chinese hamster ovary (CHO) cell-clustering effect and haemagglutination as in vitro methods; lymphocytosispromoting activity, histamine-sensitising activity and insulin secretory activity as in vivo methods. The toxin shows ADP-ribosyl transferase activity using transducin as the acceptor. Filamentous haemagglutinin Haemagglutination and inhibition by specific antibody. Pertactin, fimbrial-2 and fimbrial-3 antigens. Reactivity with specific antibody. Pertussis toxoid The toxoid induces in animals the production of antibodies capable of inhibiting all the properties of pertussis toxin. PURIFIED ANTIGENIC FRACTION Production of the antigenic fraction is based on a seed-lot system. The seed cultures are managed to conserve or, where necessary, restore toxinogenicity by deliberate selection. None of the media used at any stage contains blood or blood products of human origin. Media used for the preparation of seed batches and inocula may contain blood or blood products of animal origin. The antigenic fraction is purified and, after appropriate characterisation, detoxified using suitable reagents by a method that ensures minimal reversion of toxoid to toxin, particularly on or exposure to heat. The purification procedure is validated to demonstrate appropriate clearance of substances used during culture or purification. The content of bacterial endotoxins is determined to monitor the purification procedure and to limit the amount in the final vaccine. The limits applied are such that the final vaccine contains not more than 100 IU per single human dose. Before detoxification, the purity of the antigenic fraction is 23
Adsorbed Pertussis Vaccine (Acellular, Co-Purified)

Pertussis Vaccine (Acellular, Co-Purified, Adsorbed) is a preparation of antigenic components of Bordetella pertussis adsorbed on a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate. The vaccine contains an antigenic fraction purified without separation of the individual components. The antigenic fraction is treated by a method that transforms pertussis toxin to toxoid, rendering it harmless while maintaining adequate immunogenic properties of all the components and avoiding reversion to toxin. The antigenic fraction is composed of pertussis toxoid, filamentous haemagglutinin, pertactin (a 69 kDa outermembrane protein) and other defined components of B. pertussis such as fimbrial-2 and fimbrial-3 antigens. It may contain residual pertussis toxin up to a maximum level approved by the competent authority. The antigenic composition and characteristics are based on evidence of protection and freedom from unexpected reactions in the target group for which the vaccine is intended.
Production
General provisions. The production method shall have been shown to yield consistently vaccines comparable with the vaccine of proven clinical efficacy and safety in man. Reference vaccine. A batch of vaccine shown to be effective in clinical trials or a batch representative thereof is used as a reference vaccine. For the preparation of a representative batch, strict adherence to the production process used for the batch tested in clinical trials is necessary. The reference vaccine is preferably stabilised, by a method that has been shown to have no significant effect on the assay procedure when the stabilised and non-stabilised batches are compared. CHARACTERISATION OF COMPONENTS During development of the vaccine, the production process shall be validated to demonstrate that it yields consistently an antigenic fraction that complies with the following requirements; after demonstration of consistency, the tests need not be applied routinely to each batch. Adenylate cyclase. Not more than 500 ng in the equivalent of 1 dose of the final vaccine, determined by immunoblot analysis or another suitable method.
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determined by a suitable method such as polyacrylamide gel electrophoresis (PAGE) (2.4.12) or liquid chromatography (2.4.14). SDS-PAGE or immunoblot analysis with specific monoclonal or polyclonal antibodies may be used to characterise subunits. Requirements are established for each individual product. Only a purified antigenic fraction that complies with the following requirements may be used in the preparation of the final bulk vaccine. Sterility (2.2.11). Carry out the test for sterility using for each medium a quantity of purified antigenic fraction equivalent to not less than 100 doses of the final vaccine. Test for residual pertussis toxin. Use 3 groups of not fewer than 5 histamine-sensitive mice each weighing between 18 and 26 g. Using phosphate-buffered saline containing 0.2 per cent of gelatin, prepare a series of dilutions of the purified antigenic fraction that have been shown to yield a graded response and attribute each dilution to a separate group of mice. Inject intraperitoneally into each mouse the dilution attributed to its group. Inject diluent into a fourth group of control mice. After 5 days, inject intraperitoneally into each mouse 1 mg of histamine base in a volume not exceeding 0.5 ml. Record the number of animals that die within 24 h of histamine challenge. Calculate the weight or volume of a preparation that sensitises 50.0 per cent of the mice injected using a suitable statistical method such as probit analysis. The residual activity of pertussis toxin does not exceed that of batches shown to be safe in clinical studies. The histamine sensitivity of the strain of mice used is verified at suitable intervals as follows: inject threefold dilutions of a reference pertussis toxin preparation in phosphate-buffered saline solution containing 0.2 per cent w/v of gelatin and challenge with histamine as described above; the strain is suitable if more than 50 per cent of the animals are sensitised by 50 ng of pertussis toxin and none of the control animals injected with only diluent and challenged similarly with histamine show symptoms of sensitisation. A validated test based on the clustering effect of the toxin for Chinese hamster ovary (CHO) cells may be used instead of the test on mice. Residual detoxifying agents and other reagents. The content of residual detoxifying agents and other reagents is determined and shown to be below approved limits unless validation of the process has demonstrated acceptable clearance. Antigen content. Determine the complete quantitative antigen composition of the antigenic fraction by suitable immunochemical methods (2.2.14) and protein nitrogen by sulphuric acid digestion or another suitable method. The ratio of total antigen content to protein nitrogen is within the limits established for the product. 24
FINAL BULK VACCINE The vaccine is prepared by adsorption of a suitable quantity of the antigenic fraction onto aluminium hydroxide or hydrated aluminium phosphate. A suitable antimicrobial preservative may be added. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT Only a final lot that is satisfactory with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided that the tests for residual pertussis toxin, reversibility of toxoid, antimicrobial preservative, free formaldehyde and the assay have been carried out with satisfactory results on the final bulk vaccine, these tests may be omitted on the final lot.
Identification
Subject the vaccine to a suitable desorption procedure such as the following: dissolve in the vaccine under examination sufficient sodium citrate to give a 10 per cent w/v solution; maintain at 37 for about 16 h and centrifuge until a clear supernatant is obtained. Examine by a suitable immunochemical method (2.2.14), the clear supernatant reacts with specific antisera to the components in the vaccine.
Tests
Test for residual pertussis toxin. Use 3 groups of not fewer than 5 histamine-sensitive mice (see under Production) each weighing between 18 and 26 g. Using phosphate-buffered saline containing 0.2 per cent w/v of gelatin, prepare a series of dilutions of the vaccine under examination that have been shown to yield a graded response and attribute each dilution to a separate group of mice. Inject intraperitoneally into each mouse the dilution attributed to its group. Inject diluent into a fourth group of control mice. After 5 days, inject intraperitoneally into each mouse 1 mg of histamine base in a volume not exceeding 0.5 ml. Note the number of animals that die within 24 h of histamine challenge. Calculate the weight or volume of a preparation that sensitises 50 per cent of the mice injected using a suitable statistical method such as probit analysis. The residual activity of pertussis toxin does not exceed that of batches shown to be safe in clinical studies.
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BACILLUS CALMETTE-GUERIN VACCINE (FREEZE-DRIED)
Reversibility of toxoid. Carry out the test for residual pertussis toxin described above using the vaccine incubated at 37 for 4 weeks in parallel with a sample stored at 2 to 8. The degree of reversibility does not exceed that of batches shown to be safe in clinical studies. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Aluminium (2.3.9). Maximum 1.25 mg per single human dose, if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. Free formaldehyde (2.3.20). Maximum 0.2 g/l. Sterility (2.2.11). Complies with the test for sterility. Assay The vaccine complies with the assay as stated under Adsorbed Pertussis Vaccine (Acellular Component). Labelling. The label states (1) the names and amounts of the antigenic components present in the vaccine; (2) the maximum amount of residual pertussis toxin present in the vaccine; (3) the maximum degree of reversion of toxoid to toxin during the period of validity; (4) the name and amount of the adsorbent; (5) that the vaccine must be shaken before use; (6) that the vaccine is not to be frozen.
designed that all cultures and vaccines are protected from direct sunlight and from ultraviolet light at all stages of manufacture, testing and storage. Production of the vaccine is based on a seed-lot system. The production method shall have been shown to yield consistently BCG vaccines that induce adequate sensitivity to tuberculin in man, that have acceptable protective potency in animals and are safe. The vaccine is prepared from cultures which are derived from the master seed lot by as few subcultures as possible and in any case not more than 12 subcultures e.g. If the secondary seed lot is 4 culture passages removed from the primary seed lot, the no. of passages from the secondary seed lot must not exceed 8. The capacity of the working seed lot to induce sensitivity to tuberculin in guinea-pigs is demonstrated. If a bioluminescence test or other biochemical method is used instead of viable count, the method is validated against the viable count for each stage of the process at which it is used. SEED LOT The strain used to establish the master seed lot is chosen for and maintained to preserve its stability, its capacity to sensitise man and guinea-pigs to tuberculin and to protect animals against tuberculosis, and its relative absence of pathogenicity for man and laboratory animals. The strain used shall be identified by historical records that include information on its origin and subsequent manipulation. A suitable batch of vaccine is prepared from the first working seed lot and is reserved for use as the comparison/ in-house reference vaccine. When a new working seed lot is established, a suitable test for delayed hypersensitivity in guinea-pigs is carried out on a batch of vaccine prepared from the new working seed lot; the vaccine is shown to be not significantly different in activity from the comparison vaccine. Only a working seed lot that complies with the following requirements may be used for propagation.
Bacillus Calmette-Guerin Vaccine (Freeze-Dried)

Freeze-dried BCG Vaccine is a preparation of live bacteria derived from a culture of the bacillus of Calmette and Gurin (Mycobacterium bovis BCG) capacity of which to protect against tuberculosis has been established. Vaccine complies with the requirements stated under Vaccines with the following modifications.
Identification
The bacteria in the working seed lot are identified as Mycobacterium bovis BCG using microbiological techniques, which may be supplemented by molecular biology techniques (for example, nucleic acid amplification and restrictionfragment-length polymorphism). Sterility (2.2.11). Complies with the test for sterility, carried out using 10 ml for each medium. The working seed lot complies with the test for sterility except for the presence of mycobacteria. Virulent mycobacteria Examine the working seed lot as prescribed under Tests, using 10 guinea pigs. 25
Production
General provisions The production method is validated to demonstrate that the product, if tested, would comply with the tests for safety and efficacy. BCG vaccine shall be produced by a staff consisting of healthy persons who do not work with other infectious agents; in particular they shall not work with virulent strains of Mycobacterium tuberculosis, during the course of production cycle nor shall they be exposed to a known risk of tuberculosis infection. BCG vaccine is susceptible to sunlight: the procedures for the preparation of the vaccine shall be so
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PROPAGATION AND HARVEST The bacteria are grown in a suitable medium for not more than 21 days by surface or submerged culture. The culture medium shall contain no substances known to cause toxic or allergic reactions in human beings or to cause the bacteria to become virulent for guinea-pigs. The culture is harvested and suspended in a sterile liquid medium that protects the viability of the vaccine as determined by a suitable method of viable count. Test for purity. Purity is checked by acid fast staining. FINAL BULK VACCINE The final bulk vaccine is prepared from a single harvest or by pooling a number of single harvests. A stabiliser may be added; if the stabiliser interferes with the determination of bacterial concentration on the final bulk vaccine, the determination is carried out before addition of the stabiliser. Only final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Virulent mycobacteria. Examine as prescribed under Tests. Sterility (2.2.11). Complies with the test for sterility using 10 ml for each medium except for the presence of mycobacteria. Count of viable units Determine the number of viable units per ml by viable count on solid medium using a method suitable for the vaccine under examination or by determination of adenosine triphosphate by a bioluminescence reaction. Carry out the test in parallel on a reference preparation of the same strain. Bacterial concentration Determine the total bacterial concentration by a suitable method, either directly by determining the mass of the microorganisms, or indirectly by an opacity method that has been calibrated in relation to the mass of the organisms; if the bacterial concentration is determined before addition of a stabiliser, the concentration in the final bulk vaccine is established by calculation. The total bacterial concentration is within the limits approved for the particular product by National Regulatory Authority. The ratio of the count of viable units to the total bacterial concentration is not less than that approved for the particular product by National Regulatory Authority. FINAL LOT The final bulk vaccine is distributed into sterile containers and freeze-dried to a moisture content favourable to the stability of the vaccine; the containers are closed either under vacuum or under a gas that is not deleterious to the vaccine. 26
Except where the filled and closed containers are stored at a temperature of -20 or lower, the expiry date is not later than 4 years from the date of harvest. Only a final lot that complies with the following requirement for count of viable units and with each of the requirements given below under Identification, Tests and Assay may be released for use. Provided the test for virulent mycobacteria has been carried out with satisfactory results on the final bulk vaccine, it may be omitted on the final lot. Provided the test for excessive dermal reactivity has been carried out with satisfactory results on the working seed lot and on 5 consecutive final lots produced from it, the test may be omitted on the final lot. Count of viable units Determine the number of viable units per ml of the reconstituted vaccine by viable count on solid medium using a method suitable for the vaccine under examination or by determination of adenosine triphosphate by a bioluminescence reaction. The ratio of the count of viable units after freezedrying to that before is not less than that approved for the particular product.
Identification
BCG vaccine is identified by microscopic examination of the bacilli in stained smears demonstrating their acid-fast property and by the characteristic appearance of colonies grown on solid medium. Alternatively, molecular biology techniques (like nucleic acid amplification) may be used.
Tests
Virulent mycobacteria If this test is satisfactory at final bulk stage it can be omitted at the final lot. Inject subcutaneously or intramuscularly into each of 6 guineapigs, each weighing between 250 and 400 g and having received no treatment likely to interfere with the test, a quantity of vaccine equivalent to at least 50 human doses. Observe the animals for at least 42 days. At the end of this period, kill the guinea-pigs and examine by autopsy for signs of infection with tuberculosis, ignoring any minor reactions at the site of injection. Animals that die during the observation period are also examined for signs of tuberculosis. The vaccine complies with the test if none of the guinea-pigs shows signs of tuberculosis and if not more than one animal dies during the observation period. If 2 animals die during this period and autopsy does not reveal signs of tuberculosis repeat the test on 6 other guinea-pigs. The vaccine complies with the test if not more than one animal dies during the 42 days following the injection and autopsy does not reveal any sign of tuberculosis.
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DIPHTHERIA AND TETANUS VACCINE (ADSORBED)
Sterility (2.2.11). The reconstituted vaccine complies with the test for sterility except for the presence of mycobacteria. Excessive dermal reactivity Use 6 healthy white or pale-coloured guinea-pigs, each weighing not less than 250 g and having received no treatment likely to interfere with the test. Inject intradermally into each guinea-pig, according to a randomised plan, 0.1 ml of the reconstituted vaccine and of 2 tenfold serial dilutions of the vaccine and identical doses of the comparison vaccine. Observe the lesions formed at the site of the injection for 4 weeks. The vaccine complies with the test if the reaction it produces is not markedly different from that produced by the comparison vaccine. Temperature stability Maintain samples of the freeze-dried vaccine at 37 for 4 weeks. Determine the number of viable units in the heated vaccine and in unheated vaccine as described below. The number of viable units in the heated vaccine is not less than 20.0 per cent of that in unheated vaccine. Water (2.3.43). Not more than 3.0 per cent, determined by the semi-micro determination of water. Assay Determine the number of viable units in the reconstituted vaccine by viable count on solid medium or using a suitable validated biochemical method for the vaccine under examination. The number is within the range stated on the label. Determine the number of viable units in the comparison vaccine in parallel. Labelling. The label states (1) the minimum and maximum number of viable units per ml in the reconstituted vaccine; (2) that the vaccine must be protected from direct sunlight; (3) that the vaccine is to be used immediately after broaching the container; (4) the age group for which the vaccine is intended; (5) the dose for each age group; (6) follow instructions as mentioned in the product insert/leaflet.
Production
General provisions Bulk purified diphtheria and tetanus toxoids The bulk purified diphtheria and tetanus toxoids are prepared as described in the monographs on Diphtheria vaccine (adsorbed) and Tetanus vaccine (adsorbed) and comply with the requirements prescribed therein. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. Absence of toxin and irreversibility of toxoid Inject subcutaneously into each of 5 guinea-pigs at least 500 Lf of the non-incubated bulk purified toxoid in a volume of 1 ml, using the same buffer solution as for the final vaccine, without adsorbent. Animals that die shall be autopsied and examined for symptoms of diphtheria intoxication (red adrenals). The bulk purified toxoid shall pass the test if no guinea-pig shows symptoms of specific intoxication within six weeks of injection and if at least 80 per cent of the animals survive the test period. The guinea-pigs shall not have been used previously for experimental purposes. Alternatively, a cell-culture test system may be used; in this case, the sensitivity of the test shall have been demonstrated to be not less than that of the guinea-pig test, and the test procedures shall be approved by the National Regulatory Authority. Each bulk purified toxoid shall be tested to ensure that reversion to toxicity cannot take place on storage. The bulk purified toxoid shall be diluted in order to obtain the same concentration and chemical environment as that present in the final bulk vaccine, except for the presence of adjuvant. To determine whether reversion has occurred, diluted toxoids that have been stored at 37 for six weeks shall be tested. The test employed shall be approved by the National Regulatory Authority and should be sufficiently sensitive to detect very small amounts of toxin. No toxicity shall be detected. Intradermal tests in guinea-pigs and cell-culture tests both are considered to be suitable.
Diphtheria and Tetanus Vaccine (Adsorbed)

Diphtheria and Tetanus Vaccine (Adsorbed) is a preparation of diphtheria formol toxoid and tetanus formol toxoid adsorbed on mineral carrier. The formol toxoids are prepared from the toxins produced by the growth of Corynebacterium diphtheriae and Clostridium tetani, respectively. The specification for individual component used in formulation is referred in the text of individual monograph. 27
Antigenic purity Not less than 1500 Lf per mg of protein nitrogen for diphtheria toxoid and not less than 1000 Lf/mg of protein nitrogen for tetanus toxoid. FINAL BULK VACICNE The final bulk vaccine is prepared by adsorption of suitable quantities of bulk purified diphtheria toxoid and tetanus toxoid onto mineral carrier such as hydrated aluminium phosphate,
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aluminium hydroxide; the resulting mixture is approximately isotonic with blood. Suitable antimicrobial preservatives may be added. Antimicrobial preservatives of the phenolic type must not be used. Only final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot.
Only a final lot that is satisfactory with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided the tests for specific toxicity, free formaldehyde and antimicrobial preservative and the assay have been carried out with satisfactory results on the final bulk vaccine, they may be omitted on the final lot.
Identification
A. Dissolve sufficient sodium citrate in the vaccine under examination to give a 10 per cent w/v concentration. Maintain at 37o for about 16 hours and centrifuge. The clear supernatant reacts with a suitable diphtheria antitoxin and yields a precipitate. B. The clear supernatant obtained in test A reacts with a suitable tetanus antitoxin and yields a precipitate. pH (2.4.24). 6.0 to 7.0. Specific toxicity. Use 5 normal, healthy guinea-pigs weighing between 250 and 350 g which have been maintained for at least 1 week on a uniform, unrestricted diet, and have not been previously treated with any material that will interfere with the test. Weigh the animals separately and record their weights. Inject subcutaneously into each animal 5 times the dose stated on the label. Weigh all the animals at weekly intervals for 6 weeks. None of the animals shows any symptoms of diphtheria or tetanus toxaemia or dies from diphtheria within 42 days or loses weight at the end of the test. If more than one animal dies from non-specific causes or loses weight, repeat the test. If an animal dies or loses weight in the second test, the vaccine fails the test. Assay Diphtheria toxoid Complies with the test as stated under Diphtheria Vaccine (Adsorbed). Tetanus toxoid Complies with the test as stated under Tetanus Vaccine (Adsorbed). Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The amount is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Free formaldehyde (2.3.20). Maximum 0.2 g/l Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT The final bulk vaccine is filled and stored aseptically into sterile containers. The containers are closed so as to prevent contamination. 28
Identification
A. Diphtheria toxoid is identified by a suitable immunochemical method (2.2.14). Dissolve in the vaccine under examination by adding sufficient sodium citrate to give a 10 per cent w/v solution. Maintain at 37 for about 16 hours and centrifuge until a clear supernatant liquid is obtained. The clear supernatant reacts with a suitable diphtheria antitoxin, giving a precipitate or visible floccules. B. Tetanus toxoid is identified by a suitable immuno-chemical method (2.2.14). The clear supernatant liquid obtained during test A reacts with a suitable tetanus antitoxin, giving a precipitate or visible floccules.
Tests
Sterility (2.2.11). Complies with the test for sterility. Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity Aluminium (2.3.9.). Not more than 1.25 mg per single human dose when hydrated aluminium phosphate or aluminium hydroxide is used as the adsorbent. pH (2.4.24). The pH of the vaccine is within the range approved for the product (6.0 to 7.0). Free formaldehyde (2.3.20). Maximum 0.2 g/l. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the quantity stated on the label. Assay Diphtheria component Carry out one of the described methods for the assay of Diphtheria Vaccine (Adsorbed). Viz a) Intradermal challenge method, b) Lethal challenge method, c) Antibody induction method, d) Validated serological assay in guinea pigs or mice as approved by National Regulatory Authority. Tetanus component Carry out one of the described methods for the assay of Tetanus Vaccine (Adsorbed) Viz a) Antibody induction
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DIPHTHERIA AND TETANUS VACCINE (ADSORBED) FOR ADULTS AND ADOLESCENTS
method; b) Challenge method in guinea pigs/mice; c) Validated serological assay in guinea pigs or mice as approved by National Regulatory Authority. Labelling. The label states (1) the human dose; (2) the minimum Lf units per single human dose or the minimum International Units per single human dose if potency test done by challenge method; (3) the name and the amount of the adsorbent and preservative; (4) that the vaccine must be shaken before use; (5) that the vaccine is not to be frozen.
Specific toxicity Inject subcutaneously 5 times the single human dose stated on the label into each of 5 healthy guinea-pigs, each weighing between 250 and 350 g, that have not previously been treated with any material that will interfere with the test. If within 42 days of the injection any of the animals shows signs of or dies from diphtheria toxaemia or tetanus, the vaccine does not comply with the test. If more than 1 animal dies from nonspecific causes, repeat the test once; if more than 1 animal dies in the second test, the vaccine does not comply with the test. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable physicochemical method. The amount is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Free formaldehyde (2.3.20). Maximum 0.2 g/l. Sterility (2.2.11). Carry out the test for sterility using 10 ml for each medium. FINAL LOT The final bulk vaccine is distributed aseptically into sterile, tamper-proof containers. The containers are closed so as to prevent contamination. Only a final lot that is satisfactory with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided the tests for free formaldehyde and antimicrobial preservative and the assay have been carried out with satisfactory results on the final bulk vaccine, they may be omitted on the final lot.
Diphtheria and Tetanus Vaccine (Adsorbed) for Adults and Adolescents

Diphtheria and Tetanus Vaccine (Adsorbed) for Adults and Adolescents is a preparation of diphtheria formol toxoid and tetanus formol toxoid adsorbed on a mineral carrier. The formol toxoids are prepared from the toxins produced by the growth of Corynebacterium diphtheriae and Clostridium tetani, respectively.
Production
General provisions Bulk purified diphtheria and tetanus toxoids The bulk purified diphtheria and tetanus toxoids are prepared as described in the monographs on Diphtheria vaccine (adsorbed) and Tetanus vaccine (adsorbed) and comply with the requirements prescribed therein. FINAL BULK VACCINE The vaccine is prepared by adsorption of suitable quantities of bulk purified diphtheria toxoid and tetanus toxoid onto a mineral carrier such as hydrated aluminium phosphate or aluminium hydroxide. Suitable antimicrobial preservatives may be added. Certain antimicrobial preservatives, particularly those of the phenolic type, adversely affect the antigenic activity and must not be used. Only final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot.
Identification
A. Diphtheria toxoid is identified by a suitable immunochemical method (2.2.14). The following method, applicable to certain vaccines, is given as an example. Dissolve in the vaccine under examination sufficient sodium citrate to give a 10 per cent w/v solution. Maintain at 37 for about 16 hours and centrifuge until a clear supernatant is obtained. The clear supernatant reacts with a suitable diphtheria antitoxin, giving a precipitate. If a satisfactory result is not obtained with a vaccine adsorbed on aluminium hydroxide, carry out the test as follows. Centrifuge 15 ml of the vaccine under examination and suspend the residue in 5 ml of a freshly prepared mixture of 1 volume of a 56 g/l solution of sodium edetate and 49 volumes of a 90 g/l solution of disodium hydrogen phosphate. Maintain at 37 for not less than 6 hours and centrifuge. The clear supernatant reacts with a suitable diphtheria antitoxin, giving a precipitate. B. Tetanus toxoid is identified by a suitable immunochemical method (2.2.14). The following method, applicable to certain
Identification
A. Dissolve sufficient sodium citrate in the vaccine under examination to give a 10 per cent w/v concentration. Maintain at 37o for about 16 hours and centrifuge. The clear supernatant reacts with a suitable diphtheria antitoxin and yields a precipitate. B. The clear supernatant obtained in test A reacts with a suitable tetanus antitoxin and yields a precipitate. pH (2.4.24 ). 6.0 to 7.0. 29
DIPHTHERIA, TETANUS AND PERTUSSIS VACCINE (ADSORBED)
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vaccines, is given as an example. The clear supernatant obtained during identification test A reacts with a suitable tetanus antitoxin, giving a precipitate.
respectively. The Bordetella pertussis suspension is prepared by growth of suitable strains in an appropriate medium, under controlled conditions. The specification for individual component used in formulation is referred in the text of individual monograph.
Tests
Aluminium (2.3.9). Maximum 1.25 mg per single human dose. Free formaldehyde (2.3.20). Maximum 0.2 g/l. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable physicochemical method. The amount is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Complies with the test for sterility. pH (2.4.24). 6.0 to 7.0. Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity. Assay Diphtheria component Carry out the prescribed method for assay of Diphtheria Vaccine by lethal challenge method described in the assay of Diphtheria Vaccine (Adsorbed). The lower confidence limit (P = 0.95) of the estimated potency is not less than 2 IU per single human dose. Tetanus component Carry out one of the prescribed methods for the assay as stated under Tetanus Vaccine (Adsorbed). The lower confidence limit (P = 0.95) of the estimated potency is not less than 40 IU per single human dose. Labelling. The label states (1) the human dose; (2) the minimum number of International Units of each component per single human dose, if potency determined by challenge method or the minimum Lf units per single human dose if test done by antibody induction method; (3) the name and the amount of the adsorbent; (4) that the vaccine must be shaken before use; (5) that the vaccine is not to be frozen.
Production
General provisions The production method must be validated to demonstrate that the product if tested, would comply with the tests for safety as described under monographs of Diphtheria Vaccine, Tetanus Vaccine (Adsorbed) and Pertussis Vaccine. Bulk purified diphtheria and tetanus toxoids, bulk inactivated B. pertussis suspension The bulk purified diphtheria and tetanus toxoids and inactivated B. pertussis suspension are prepared as described in the monograph on Diphtheria Vaccine (Adsorbed), Tetanus Vaccine (Adsorbed) and Pertussis Vaccine respectively and comply with the respective requirements. FINAL BULK VACCINE The final bulk vaccine is prepared by adsorption of suitable quantities of bulk purified diphtheria toxoid and tetanus toxoid onto hydrated aluminium phosphate or aluminium hydroxide and admixture of an appropriate quantity of a suspension of inactivated B. pertussis. The B. pertussis concentration of the final bulk vaccine does not exceed that corresponding to an opacity of 20 I.U. per single human dose. If two or more strains of B. pertussis are used, the composition of consecutive lots of the final bulk vaccine shall be consistent with respect to the proportion of each strain as measured in opacity units. Suitable antimicrobial preservatives may be added to the bulk vaccine. Antimicrobial preservatives particularly those of phenolic type which affect the antigenic activity must not be used. Only final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 percent and not more than115.0 percent of the intended amount. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT The final bulk vaccine is filled and stored aseptically into sterile containers. The containers are closed so as to prevent contamination. 30
Diphtheria, Tetanus and Pertussis Vaccine (Adsorbed)

Diphtheria, Tetanus and Pertussis Vaccine (Adsorbed) is a preparation of diphtheria formol toxoid, tetanus formol toxoid adsorbed on mineral carrier and a suspension of killed Bordetella pertussis organisms. The formal toxoids are prepared from the toxins produced by the growth of Corynebacterium diphtheriae and Clostridium tetani,
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DIPHTHERIA, TETANUS AND PERTUSSIS VACCINE (ADSORBED)
Only a final lot that is satisfactory with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided the tests for specific toxicity of diphtheria, tetanus and pertussis components, free formaldehyde, antimicrobial preservative and the Assay have been carried out with satisfactory results on the final bulk vaccine, they may be omitted on the final lot.
diphtheria toxemia or tetanus, the vaccine does not comply with the test. If more than one animal dies from non-specific causes, repeat the test once; if more than one animal dies in the second test, the vaccine does not comply with the test. Pertussis component. Use not less than 10 healthy mice each weighing between 14 and 16 g for the vaccine group and for the saline control. Use mice of the same sex or distribute males and females equally between the groups. Allow the animals access to food and water for at least 2 hours before injection and during the test. Inject each mouse of the vaccine group intraperitoneally with 0.5 ml, containing a quantity of the vaccine equivalent to not less than half the single human dose. Inject each mouse of the control group with 0.5 ml of a 0.9 per cent sterile solution of sodium chloride, preferably containing the same amount of antimicrobial preservative as that injected with the vaccine. Weigh the mice groups immediately before the injection and 72 hours and 7 days after the injection. The vaccine complies with the test if: (a) at the end of 72 hours the total mass of the group of vaccinated mice is not less than that preceding the injection; (b) at the end of 7 days the average increase in mass per vaccinated mouse is not less than 60 per cent of that per control mouse; and (c) not more than 5 per cent of vaccinated mice should die during the test. The test may be repeated and the results of the tests combined. Aluminium (2.3.9). Not more than 1.25 mg per single human dose, when hydrated aluminium phosphate or aluminium hydroxide is used as the adsorbent. pH (2.4.24). 6.0 to 7.0. Free formaldehyde (2.3.20). Maximum 0.2 g/l. Antimicrobial preservative (2.2.2 ) Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not more than115.0 per cent of the intended amount. Assay Diphtheria component Carry out one of the methods for the assay as stated under Diphtheria Vaccine (Adsorbed). Tetanus component Carry out one of the methods for the assay as stated under Tetanus Vaccine (Adsorbed). Pertussis component Carry out the assay as stated under Pertussis Vaccine. Labelling. The label states (1) in case done by challenge method the minimum number of International Units; if units (as applicable for each component) per single human dose; 31
Identification
A. Diphtheria toxoid is identified by a suitable immunochemical method (2.2.14). Dissolve in the vaccine under examination by adding sufficient sodium citrate to give a 10 per cent w/v solution. Maintain at 37 for about 16 hours and centrifuge until a clear supernatant is obtained; reserve the precipitate for identification test C. The clear supernatant reacts with a suitable diphtheria antitoxin, giving a precipitate. B. Tetanus toxoid is identified by a suitable immuno-chemical method (2.2.14). The clear supernatant obtained during identification test A reacts with a suitable tetanus antitoxin, giving a precipitate. C. The pertussis component is identified by agglutination of the bacteria from the resuspended centrifugation residue (see identification test A; other suitable methods for separating the bacteria from the adsorbent may also be used) by antisera specific to B. pertussis or by the assay of the pertussis component.
Tests
Sterility (2.2.11). Complies with the test for sterility. Abnormal toxicity (2.2.1). Each final lot shall be tested for abnormal toxicity by injecting intraperitoneally one human dose , but not more than 0.25 ml into each of the five mice weighing between 17 to 22 g and at least one human dose but not more than 1.0 ml into each of the two guinea pigs weighing between 250 and 350 g. The preparation passes the test if none of the animals dies or shows signs of ill health in 7 days following the injection. If one of the animal dies or shows the signs of ill health, repeat the test. The preparation passes the test if none of the animals in the second group dies or shows signs of ill health in the time interval specified. Specific toxicity Diphtheria and tetanus components. Inject subcutaneously five times the single human dose stated on the label into each of five healthy guinea-pigs, each weighing between 250 and 350 g, that have not previously been treated with any material that will interfere with the test. The animals should be weighted every week and observations be made. If within 42 days of the injection any of the animals shows signs of or dies from
D., T., P., (WHOLE CELL), HEPATITIS B (rDNA) AND HAEMOPHILUS TYPE B CONJUGATE VACCINE (ADSORBED)
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(2) In case done by antibody induction method the minimum number of International Units per single human dose of pertussis component and minimum number of Lf of diphtheria toxoid and tetanus toxoid; (3) the name and the amount of the adsorbent and preservative; (4) that the vaccine must be shaken before use; (5) that the vaccine is not to be frozen; (6) for vaccine contained in single-dose containers where the space is too small to accommodate the full name of the vaccine, the abbreviation DTP may be used in the label and the container provided that the same code is also stated in the label on the package.
The final product contains a suitable antimicrobial preservative. The antigenic properties of the vaccine are adversely affected by the presence of certain antimicrobial preservatives particularly those of the phenolic type and some of the quaternary ammonium type and must not be used.
Production
General provisions The production method shall have been shown to yield consistently the vaccines comparable with the vaccine of proven clinical efficacy and safety in man. If the vaccine is presented with the haemophilus component in a separate vial, as part of consistency studies the assays of the diphtheria, tetanus, pertussis and hepatitis B are carried out on a suitable number of batches of vaccine reconstituted as for use. For subsequent routine control, the assays of these components may be carried out without mixing with the haemophilus component. The production method is validated to demonstrate that the product, if tested, would comply with the following test for specific toxicity of the diphtheria and tetanus component: inject subcutaneously 5 times the single human dose stated on the label into each of 5 healthy guinea pigs, each weighing between 250 and 350 g, that have not previously been treated with any material that will interfere with the test. If within 42 days of the injection any of the animals shows signs of or dies from diphtheria, toxemia or tetanus, the vaccine does not comply with the test. If more than 1 animal dies from nonspecific causes, repeat the test once; if more than 1 animal shows signs of or dies in the second test, the vaccine does not comply with the test. The stability of the final lot and the relevant intermediates is evaluated using one or more indicator tests. For the haemophilus component, such tests may include determination of molecular size, determination of free PRP in the conjugate and kinetics of depolymerisation. Taking account of the results of the stability testing, release requirements are set for these indicator tests to ensure that the vaccine will be satisfactory at the end of the period of validity. Reference vaccine(s) Provided valid assays can be performed, monocomponent reference vaccines may be used for the assays on the combined vaccine. If this is not possible because of interaction between the components of the combined vaccine or because of the difference in composition between monocomponent reference vaccine and the test vaccine, a batch of combined vaccine shown to be effective in clinical trials or a batch representative thereof is used as a reference vaccine. For the preparation of a representative batch, strict adherence to the production 32
Diphtheria, Tetanus, Pertussis (Whole Cell), Hepatitis B (rDNA) and Haemophilus Type b Conjugate Vaccine (Adsorbed)
Diphtheria, Tetanus, Pertussis (Whole cell), Hepatitis B (rDNA) and Haemophilus Type b Conjugate Vaccine (Adsorbed) is a combined vaccine composed of diphtheria formol toxoid containing not less than 1,500 Lf, (2.2.16) per mg of protein nitrogen, purified tetanus formol toxoid containing not less than 1,000 Lf, (2.2.16), per mg of protein nitrogen, hepatitis B surface antigen and haemophilus type b conjugated to suitable protein with a mineral adsorbent to which a suspension of killed Bordetella pertussis has been added. Mineral adsorbent is a suspension of hydrated aluminium hydroxide, aluminium phosphate or calcium phosphate, in saline solution or other appropriate solution isotonic with blood. The formol toxoids are prepared from the toxin produced by the growth of Corynebacterium diphtheriae and Clostridium tetani, respectively, in suitable media. The toxins are converted to toxoids by treatment with formaldehyde solution by methods which avoid reversibility of the toxoids. Hepatitis B surface antigen is a component protein of hepatitis B virus; the antigen is obtained by recombinant DNA technology. The polysaccharide, polyribosyl ribitol phosphate, PRP is a linear copolymer composed of repeated units of 3--Dribofuranosyl-(11)-ribitol-5-phosphate [(C10H19O12P)n], with a defined molecular size and derived from a suitable strain of Haemophilus influenzae type b. The carrier protein, when conjugated to PRP, is capable of inducing a T-cell dependent B-cell immune response to the polysaccharide. The product may be presented with the haemophilus component in a separate container, the contents of which are mixed with the other components immediately before or during use.
IP 2007
process used for the batch tested in clinical trials is necessary. The reference vaccine may be stabilized by a method that has been shown to have no effect on the assay procedure. Production of the components The production of the components complies with the requirements of the monographs on Diphtheria Vaccine (Adsorbed), Tetanus Vaccine (Adsorbed), Pertussis Vaccine, Hepatitis B Vaccine (rDNA) and Haemophilus Type b Conjugate Vaccine. FINAL BULK VACCINE Vaccine with all components in the same container The final bulk is prepared by adsorption, separately or together, of suitable quantities of bulk purified diphtheria toxoid, bulk purified tetanus toxoid, bulk purified hepatitis B surface antigen onto a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate, admixture of an appropriate quantity of a suspension of inactivated B. pertussis component and admixture of a suitable quantity of PRP conjugate; the resulting mixture is approximately isotonic with blood. The B. pertussis concentration of the final bulk vaccine does not exceed that corresponding to an opacity of 20 IU per single human dose. If 2 or more strains of B. pertussis are used, the composition of consecutive lots of the final bulk vaccine shall be consistent with respect to the proportion of each strain as measured in opacity units. Suitable antimicrobial preservatives may be added. Vaccine with the haemophilus component in a separate container The final bulk is prepared by adsorption, separately or together, of suitable quantities of bulk purified diphtheria toxoid, bulk purified tetanus toxoid, bulk purified hepatitis B surface antigen onto a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate, admixture of an appropriate quantity of a suspension of inactivated B. pertussis component and admixture of a suitable quantity of PRP conjugate; the resulting mixture is approximately isotonic with blood. The B. pertussis concentration of the final bulk vaccine does not exceed that corresponding to an opacity of 20 IU per single human dose. If 2 or more strains of B. pertussis are used, the composition of consecutive lots of the final bulk vaccine shall be consistent with respect to the proportion of each strain as measured in opacity units. The final bulk is filled separately. Suitable antimicrobial preservatives may be added. The final bulk of the haemophilus component is prepared by dilution of the bulk conjugate to the final concentration with a suitable diluent. A stabilizer may be added. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. 33
Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The amount is not less than 85.0 per cent and not greater than 115.0 per cent of the intended content. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT Only a final lot that is satisfactory with respect to the test for osmolality and with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided the tests for specific toxicity of diphtheria toxoid, tetanus toxoid and pertussis component and antimicrobial preservative and the assays for the diphtheria, tetanus and pertussis components have been carried out with satisfactory results on the final bulk vaccine, they may be omitted on the final lot. Provided the content of free formaldehyde has been determined on the bulk purified antigens or on the final bulk and it has been shown that the content in the final lot will not exceed 0.2 g/l, the test for free formaldehyde may be omitted on the final lot. If an in vivo assay is used for the hepatitis B component, provided it has been carried out with satisfactory results on the final bulk vaccine, it may be omitted on the final lot. Free PRP Unbound PRP is determined after removal of the conjugate, for example by anion exchange, size exclusion or hydrophobic chromatography (2.4.16), ultrafiltration or other validated methods. The amount of free PRP is not greater than that approved for the particular product. Osmolality (2.4.23). The osmolality of the vaccine is within the limits approved for the particular preparation. pH (2.4.24). 6.0 to 7.0. Description. Whitish turbid liquid in which the mineral carrier tends to settle down slowly on keeping.
Identification
Tests A, B, C, D and E may be omitted if test F is carried out. Test F may be omitted if tests A, B, C, D and E are carried out. A. Diphtheria toxoid. Dissolve sufficient sodium citrate in the vaccine under examination to give a 10 per cent w/v concentration. Maintain at 37o for about 16 hours and centrifuge. Reserve the residue for test C. The clear supernatant reacts with a suitable diphtheria antitoxin and yields a precipitate. B. Tetanus toxoid. The clear supernatant obtained in test A reacts with a suitable tetanus antitoxin and yields a precipitate. C. Pertussis component. To a suspension of the residue
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obtained in test A in saline solution add a suitable Bordetella pertussis antiserum; agglutination indicates presence of pertussis component. D. Hepatitis B surface antigen. The suspension of the residue obtained in test A gives a positive reactions when tested by suitable in-vitro assay. E. PRP. The suspension of the residue obtained in the test A gives a positive reaction when tested by a suitable immunochemical method for PRP. F. The vaccine confers an active immunity in mice and guineapigs when administered as directed in the test for Assay.
shows signs of ill health, repeat the test. The preparation passes the test if none of the animals in the second group dies or shows signs of ill health in the time interval specified. Pyrogens (2.2.8). This test is carried out for Haemophilus influenzae type b vaccine only if Haemophilus influenzae type b vaccine is presented as separate lyophilized vial. The vaccine complies with the test for pyrogens. Inject per kg of the rabbits mass a quantity of the vaccine equivalent to: 1 mg of PRP for a vaccine with diphtheria toxoid or CRM 197 diphtheria toxoid as carrier; 0.1 mg of PRP for a vaccine with tetanus toxoid as carrier protein; 0.025 mg of PRP for vaccine with OMP as carrier. Specific toxicity Diphtheria and tetanus components Complies with the test as stated under Diphtheria and Tetanus Vaccine (Adsorbed). Pertussis component Complies with the test as stated under Diphtheria, Tetanus and Pertussis Vaccine (Adsorbed). Assay Diphtheria toxoid (adsorbed) Complies with the test as stated under Diphtheria and Tetanus Vaccine (Adsorbed). Tetanus toxoid (adsorbed) Complies with the test as stated under assay of Tetanus Vaccine (Adsorbed). Pertussis vaccine Complies with the test as stated under Diphtheria, Tetanus and Pertussis Vaccine (Adsorbed). Hepatitis B surface antigen (adsorbed) Complies with the test as stated under Hepatitis B Vaccine (Adsorbed). Storage. When stored under the prescribed conditions the vaccine may be expected to retain potency for not less than 2 years from the date on which the potency test for the pertussis component was started. Labelling. The label states (1) the human dose; (2) Diphtheria and Tetanus components; (a) in case done by challenge method, the minimum number of International Units (as applicable for each component) per single human dose; (b) in case done by antibody induction, the minimum Lf units per single human dose; (3) pertussis component IU or IOU per single human dose; (4) hepatitis B component - mg HBsAg per single human dose; (5) haemophilus conjugate component 34
Tests
If the product is presented with the haemophilus component in a separate container; the tests for specific toxicity of diphtheria toxoid, tetanus toxoid and pertussis component, aluminium, free formaldehyde, antimicrobial preservative and sterility are carried out on the container with the diphtheria, tetanus, pertussis and hepatitis B components; the tests for PRP content, water (where applicable), sterility and pyrogens are carried out on the container with the haemophilus component. If the haemophilus component is freeze-dried, some tests may be carried out on the freeze-dried product rather than on the bulk conjugate where the freeze-drying process may affect the component under test. PRP. Not less than 80.0 per cent of the amount of PRP stated on the label. PRP is determined either by assay of ribose (2.7.1), or phosphorus (2.7.1), by an immunochemical method (2.2.14) or by anion exchange liquid chromatography with pulsed amperometric detection. Aluminium (2.3.9). Not more than 1.25 mg per single human dose, if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. Free formaldehyde (2.3.20). Maximum 0.2 g/l. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Complies with the test for sterility. Abnormal toxicity (2.2.1). Each final lot shall be tested for abnormal toxicity by injecting intraperitoneally one human dose, but not more than 0.25 ml into each of five mice weighing between 17 and 22 g and at least one human dose but not more than 1.0 ml into each of two guinea pigs weighing between 250 and 350 g. The preparation passes the test if none of the animals dies or shows signs of ill health in seven days following the injection. If one of the animals dies or
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DIPHTHERIA TETANUS PERTUSSIS (WHOLE CELL), HEPATITIS B (rDNA) VACCINE (ADSORBED)
- mg PRP per single human dose; (6) the type and nominal amount of carrier protein per single human dose; (7) the name and amount of adsorbent and added preservative; (8) that the vaccine must be shaken before use; (9) that the vaccine is not to be frozen.
from diphtheria, toxaemia or tetanus, the vaccine does not comply with the test. If more than 1 animal dies from nonspecific causes, repeat the test once; if more than 1 animal shows signs of or dies in the second test, the vaccine does not comply with the test. The stability of the final lot and the relevant intermediates is evaluated using one or more indicator tests. Taking account of the results of the stability testing, release requirements are set for these indicator tests to ensure that the vaccine will be satisfactory at the end of the period of validity. Reference vaccine(s) Provided valid assays can be performed, monocomponent reference vaccines may be used for the assays on the combined vaccine. If this is not possible because of interaction between the components of the combined vaccine or because of the difference in composition between monocomponent reference vaccine and the test vaccine, a batch of combined vaccine shown to be effective in clinical trials or a batch representative thereof is used as a reference vaccine. Production of the components The production of the components complies with the requirements of the monographs on Diphtheria Vaccine (Adsorbed), Tetanus Vaccine (Adsorbed), Pertussis Vaccine and Hepatitis B Vaccine (rDNA). FINAL BULK VACCINE The final bulk vaccine is prepared by adsorption, separately or together, of suitable quantities of bulk purified diphtheria toxoid, tetanus toxoid, pertussis components and hepatitis B surface antigen onto a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate. Suitable antimicrobial preservatives may be added. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The amount is not less than 85.0 per cent and not greater than 115.0 per cent of the intended content. pH (2.4.24). 6.0 to 7.0. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT Only a final lot that is satisfactory with respect to the test for osmolality and with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided the tests for specific toxicity of diphtheria toxoid, tetanus toxoid and pertussis component and antimicrobial preservative and the assays for the diphtheria, 35
Diphtheria, Tetanus, Pertussis (Whole Cell) and Hepatitis B (rDNA) Vaccine (Adsorbed)
Diphtheria, Tetanus, Pertussis and Hepatitis B (rDNA) Vaccine (Adsorbed) is a combined vaccine composed of diphtheria formol toxoid containing not less than 1,500 Limes flocculationis (Lf) (2.2.16), per mg of protein nitrogen, purified tetanus formol toxoid containing not less than 1,000 Lf ( 2.2.16), per mg of protein nitrogen and hepatitis B surface antigen with a mineral adsorbent to which a suspension of killed Bordetella pertussis has been added. Mineral adsorbent is a suspension of hydrated aluminium hydroxide, aluminium phosphate or calcium phosphate in saline solution or other appropriate solution isotonic with blood. The formol toxoids are prepared from the toxin produced by the growth of Corynebacterium diphtheriae and Clostridium tetani, respectively, in suitable media. The toxins are converted to toxoids by treatment with formaldehyde solution by methods, which avoid reversibility of the toxoids. Hepatitis B surface antigen is a component protein of hepatitis B virus; the antigen is obtained by recombinant DNA technology. The final product contains a suitable antimicrobial preservative. The antigenic properties of the vaccine are adversely affected by the presence of certain antimicrobial preservatives particularly those of the phenolic type and some of the quaternary ammonium type and must not be used.
Production
General provisions The production method shall have been shown to yield consistently the vaccines comparable with the vaccine of proven clinical efficacy and safety in man. The production method is validated to demonstrate that the product, if tested, would comply with the following test for specific toxicity of the diphtheria and tetanus component: inject subcutaneously 5 times the single human dose stated on the label into each of 5 healthy guinea pigs, each weighing between 250 and 350 g, that have not previously been treated with any material that will interfere with the test. If within 42 days of the injection any of the animals shows signs of or dies
DIPHTHERIA TETANUS PERTUSSIS (WHOLE CELL), HEPATITIS B (rDNA) VACCINE (ADSORBED)
IP 2007
tetanus and pertussis components have been carried out with satisfactory results on the final bulk vaccine, they may be omitted on the final lot. Provided the content of free formaldehyde has been determined on the bulk purified antigens or on the final bulk and it has been shown that the content in the final lot will not exceed 0.2 g/l, the test for free formaldehyde may be omitted on the final lot. If an in vivo assay is used for the hepatitis B component, provided it has been carried out with satisfactory results on the final bulk vaccine, it may be omitted on the final lot. Osmolality (2.4.23). The osmolality of the vaccine is within the limits approved for the particular preparation. Description. Whitish turbid liquid in which the mineral carrier tends to settle down slowly on keeping.
Abnormal toxicity (2.2.1). Each final lot shall be tested for abnormal toxicity by injecting intraperitoneally one human dose, but not more than 0.25 ml into each of five mice weighing between 17 and 22 g and at least one human dose but not more than 1.0 ml into each of two guinea pigs weighing between 250 and 350 g. The preparation passes the test if none of the animals dies or shows signs of ill health in seven days following the injection. If one of the animals dies or shows signs of ill health, repeat the test. The preparation passes the test if none of the animals in the second group dies or shows signs of ill health in the time interval specified. Specific toxicity Diphtheria and tetanus components Complies with the test as stated under Diphtheria and Tetanus Vaccine (Adsorbed). Pertussis component Complies with the test as stated under Diphtheria, Tetanus and Pertussis Vaccine (Adsorbed). Assay Diphtheria toxoid (adsorbed) Complies with the test as stated under Diphtheria and Tetanus Vaccine (Adsorbed). Tetanus toxoid (adsorbed) Complies with the test as stated under assay for Tetanus Vaccine (Adsorbed). Pertussis vaccine Complies with the test as stated under Diphtheria, Tetanus and Pertussis Vaccine (Adsorbed). Hepatitis B surface antigen (adsorbed) Complies with the test as stated under Hepatitis B Vaccine (Adsorbed). Storage. When stored under the prescribed conditions the vaccine may be expected to retain potency for not less than 2 years from the date on which the potency test for the pertussis component was started. Labelling. The label states (1) the human dose (ml); (2) diphtheria and tetanus components, (a) in case done by challenge method, the minimum number of International Units (as applicable for each component) per single human dose and (b) in case done by antibody induction, the minimum Lf units per single human dose; (3) pertussis component - IU or IOU per single human dose; (4) hepatitis B component - mg HBsAg per single human dose; (5) the name and amount of adsorbent and added preservative; (6) that the vaccine must be shaken before use; (7) that the vaccine is not to be frozen. 36
Identification
Tests A, B, C and D may be omitted if test E is carried out. Test E may be omitted if tests A, B C and D are carried out. A. Diphtheria toxoid. Dissolve sufficient sodium citrate in the vaccine under examination to give a 10 per cent w/v concentration. Maintain at 37o for about 16 hours and centrifuge. Reserve the residue for test C. The clear supernatant reacts with a suitable diphtheria antitoxin and yields a precipitate. B. Tetanus toxoid. The clear supernatant obtained in test A reacts with a suitable tetanus antitoxin and yields a precipitate. C. Pertussis component. To a suspension of the residue obtained in test A in saline solution add a suitable B. pertussis antiserum; agglutination indicates presence of pertussis component. D. Hepatitis B surface antigen. The suspension of the residue obtained in test A gives a positive reactions when tested by suitable in-vitro assay. E. The vaccine confers an active immunity in mice and guineapigs when administered as directed under Assay.
Tests
pH (2.4.24). 6.0 to 7.0. Aluminium (2.3.9). Not more than 1.25 mg per single human dose, if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. Free formaldehyde (2.3.20). Maximum 0.2 g/l. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Complies with the test for sterility.
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D., T., P., (WHOLE CELL) AND HAEMOPHILUS TYPE B CONJUGATE VACCINE (ADSORBED)
Diphtheria, Tetanus, Pertussis (Whole Cell) and Haemophilus Type b Conjugate Vaccine (Adsorbed)
Diphtheria, Tetanus, Pertussis and Haemophilus type b Conjugate Vaccine (Adsorbed) is a combined vaccine composed of diphtheria formol toxoid containing not less than 1,500 Limes flocculationis; (Lf), (2.2.16) per mg of protein nitrogen, purified tetanus formol toxoid containing not less than 1,000 Lf, (2.2.16), per mg of protein nitrogen, and Haemophilus type b conjugated to suitable protein with a mineral adsorbent to which a suspension of killed Bordetella pertussis has been added. The mineral adsorbent is a suspension of hydrated aluminium hydroxide, aluminium phosphate or calcium phosphate, in saline solution or other appropriate solution isotonic with blood. The formol toxoids are prepared from the toxin produced by the growth of Corynebacterium diphtheriae and Clostridium tetani, respectively in suitable media. The toxins are converted to toxoids by treatment with formaldehyde solution by methods which avoid reversibility of the toxoids. The polysaccharide, polyribosyl ribitol phosphate, PRP is a linear copolymer composed of repeated units of 3--D-ribofuranosyl-(11)-ribitol-5-phosphate [(C10H19O12P)n], with a defined molecular size and derived from a suitable strain of Haemophilus influenzae type b. The carrier protein, when conjugated to PRP, is capable of inducing a Tcell dependent B-cell immune response to the polysaccharide. The product may be presented with the haemophilus component in a separate container, the contents of which are mixed with the other components immediately before or during use. The final product contains a suitable antimicrobial preservative. The antigenic properties of the vaccine are adversely affected by the presence of certain antimicrobial preservatives particularly those of the phenolic type and some of the quaternary ammonium type must not be used.
The production method is validated to demonstrate that the product, if tested, would comply with the following test for specific toxicity of the diphtheria and tetanus component: inject subcutaneously 5 times the single human dose stated on the label into each of 5 healthy guinea pigs, each weighing between 250 and 350 g, that have not previously been treated with any material that will interfere with the test. If within 42 days of the injection any of the animals shows signs of or dies from diphtheria, toxemia or tetanus, the vaccine does not comply with the test. If more than 1 animal dies from nonspecific causes, repeat the test once; if more than 1 animal shows signs of or dies in the second test, the vaccine does not comply with the test. The stability of the final lot and the relevant intermediates is evaluated using one or more indicator tests. For the haemophilus component, such tests may include determination of molecular size, determination of free PRP in the conjugate and kinetics of depolymerisation. Taking account of the results of the stability testing, release requirements are set for these indicator tests to ensure that the vaccine will be satisfactory at the end of the period of validity. Reference vaccine(s) Provided valid assays can be performed, monocomponent reference vaccines may be used for the assays on the combined vaccine. If this is not possible because of interaction between the components of the combined vaccine or because of the difference in composition between monocomponent reference vaccine and the test vaccine, a batch of combined vaccine shown to be effective in clinical trials or a batch representative thereof is used as a reference vaccine. For the preparation of a representative batch, strict adherence to the production process used for the batch tested in clinical trials is necessary. The reference vaccine may be stabilized by a method that has been shown to have no effect on the assay procedure. Production of the components The production of the components complies with the requirements of the monographs on Diphtheria Vaccine (Adsorbed), Tetanus Vaccine (Adsorbed), Pertussis Vaccine (Whole Cell) and Haemophilus influenzae Type b Conjugate Vaccine. FINAL BULK VACCINE Vaccine with all components in the same container The final bulk is prepared by adsorption, separately or together, of suitable quantities of bulk purified diphtheria toxoid, bulk purified tetanus toxoid, onto a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate, admixture of an appropriate quantity of a suspension of inactivated B. pertussis component and admixture of a suitable 37
Production
General provisions The production method shall have been shown to yield consistently the vaccines comparable with the vaccine of proven clinical efficacy and safety in man. If the vaccine is presented with the haemophilus component in a separate vial, as part of consistency studies, the assays of the diphtheria, tetanus and pertussis are carried out on a suitable number of batches of vaccine reconstituted for use. For subsequent routine control, the assays of these components may be carried out without mixing with the haemophilus component.
D., T., P., (WHOLE CELL) AND HAEMOPHILUS TYPE CONJUGATE VACCINE (ADSORBED)
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quantity of PRP conjugate; the resulting mixture is approximately isotonic with blood. The B. pertussis concentration of the final bulk vaccine does not exceed that corresponding to an opacity of 20 IU per single human dose. If 2 or more strains of B. pertussis are used, the composition of consecutive lots of the final bulk vaccine shall be consistent with respect to the proportion of each strain as measured in opacity units. Suitable antimicrobial preservatives may be added. Vaccine with the haemophilus component in a separate container The final bulk is prepared by adsorption, separately or together, of suitable quantities of bulk purified diphtheria toxoid, bulk purified tetanus toxoid onto a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate, admixture of an appropriate quantity of a suspension of inactivated B. pertussis component and admixture of a suitable quantity of PRP conjugate; the resulting mixture is approximately isotonic with blood. The B. pertussis concentration of the final bulk vaccine does not exceed that corresponding to opacity of 20 IU per single human dose. If 2 or more strains of B. pertussis are used, the composition of consecutive lots of the final bulk vaccine shall be consistent with respect to the proportion of each strain as measured in opacity units. The final bulk is filled separately. Suitable antimicrobial preservatives may be added. The final bulk of the haemophilus component is prepared by dilution of the bulk conjugate to the final concentration with a suitable diluent. A stabilizer may be added. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The amount is not less than 85.0 per cent and not greater than 115.0 percent of the intended content. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT Where the haemophilus component is in a separate container, the final bulk of the haemophilus component is freeze-dried. Only a final lot that is satisfactory with respect to the test for osmolality and with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided the tests specific toxicity of diphtheria toxoid, tetanus toxoid and pertussis component and antimicrobial preservative and the assays for the diphtheria, tetanus and pertussis components have been carried out with satisfactory results on the final bulk vaccine, they may be omitted on the 38
final lot. Provided the content of free formaldehyde has been determined on the bulk purified antigens or on the final bulk and it has been shown that the content in the final lot will not exceed 0.2 g/l, the test for free formaldehyde may be omitted on the final lot. Free PRP. Unbound PRP is determined after removal of the conjugate, for example by anion exchange, size exclusion or hydrophobic chromatography (2.4.16), ultrafiltration or other validated methods. The amount of free PRP is not greater than approved for the particular product. Osmolality (2.4.23). The osmolality of the vaccine is within the limits approved for the particular preparation. pH (2.4.24). 6.0 to 7.0. Description. Whitish turbid liquid in which the mineral carrier tends to settle down slowly on keeping.
Production Identification
Tests A, B, C and D may be omitted if test E is carried out. Test E may be omitted if tests A, B, C and D are carried out. A. Diphtheria toxoid. Dissolve sufficient sodium citrate in the vaccine under examination to give a 10 per cent w/v concentration. Maintain at 37o for about 16 hours and centrifuge. Reserve the residue for test C. The clear supernatant reacts with a suitable diphtheria antitoxin and yields a precipitate. B. Tetanus toxoid. The clear supernatant obtained in test A reacts with a suitable tetanus antitoxin and yields a precipitate. C. Pertussis component. To a suspension of the residue obtained in test A in saline solution add a suitable Bordetella pertussis antiserum; agglutination indicates presence of pertussis component. D. PRP. The suspension of the residue obtained in test A gives a positive reaction when tested by a suitable immunochemical method for PRP. E. The vaccine confers an active immunity in mice and guinea pigs when administered as directed in the test for Potency.
Tests
If the product is presented with the haemophilus component in a separate container; the tests for specific toxicity of diphtheria toxoid, tetanus toxoid and pertussis component, aluminium, free formaldehyde, antimicrobial preservative and sterility are carried out on the container with the diphtheria, tetanus and pertussis components; the tests for PRP content, water (where applicable), sterility and pyrogens are carried out on the container with the haemophilus component.
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DIPHTHERIA VACCINE (ADSORBED)
If the haemophilus component is freeze-dried, some tests may be carried out on the freeze-dried product rather than on the bulk conjugate where the freeze-drying process may affect the component under test. PRP. Not less than 80.0 per cent of the amount of PRP stated on the label. PRP is determined either by assay of ribose (2.7.1) or phosphorus (2.7.1), by an immunochemical method (2.2.14) or by anion exchange liquid chromatography with pulsed amperometric detection. Aluminium (2.3.9). Maximum 1.25 mg per single human dose, if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. Free formaldehyde (2.3.20). Maximum 0.2 g/l. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Complies with the test for sterility. Abnormal toxicity (2.2.1). Each final lot shall be tested for abnormal toxicity by injecting intraperitoneally one human dose , but not more than 0.25 ml into each of the five mice weighing between 17 to 22 g and at least one human dose but not more than 1.0 ml into each of the two guinea pigs weighing between 250 and 350 g. The preparation passes the test if none of the animals dies or shows signs of ill health in 7 days following the injection . If one of the animal dies or shows the signs of ill health, repeat the test . The preparation passes the test if none of the animals in the second group dies or shows signs of ill health in the time interval specified. Pyrogens (2.2.8). This test is carried out for Haemophilus influenzae type b vaccine only if Haemophilus influenzae type b vaccine is presented as separate lyophilized vial. The vaccine complies with the test for pyrogens. Inject per kg of the rabbits mass a quantity of the vaccine equivalent to: 1 mg of PRP for a vaccine with diphtheria toxoid or CRM 197 diphtheria toxoid as carrier; 0.1 mg of PRP for a vaccine with tetanus toxoid as carrier protein; 0.025 mg of PRP for vaccine with OMP as carrier. Specific toxicity Diphtheria and tetanus components Complies with the test as stated under Diphtheria and Tetanus Vaccine (Adsorbed). Pertussis component Complies with the test as stated under Diphtheria, Tetanus and Pertussis Vaccine (Adsorbed). Assay Diphtheria toxoid (adsorbed) 39
Complies with the test as stated under Diphtheria and Tetanus Vaccine (Adsorbed). Tetanus toxoid (adsorbed) Complies with the test as stated under assay of Tetanus Vaccine (Adsorbed). Pertussis vaccine Complies with the test as stated under Diphtheria, Tetanus and Pertussis Vaccine (Adsorbed). Storage. When stored under the prescribed conditions the vaccine may be expected to retain potency for not less than 2 years from the date on which the potency test for the pertussis component was started. Labelling. The label states (1) the human dose (ml); (2) Diphtheria and Tetanus components (a) in case done by challenge method the minimum number of international units (as applicable for each component) per single human dose; (b) in case done by antibody induction method, the minimum Lf units per single human dose; (3) pertussis component IU or IOU per single human dose; (4) haemophilus conjugate component - g PRP per single human dose; (5) the type and nominal amount of carrier protein per single human dose; (6) the name and amount of adsorbent and added preservative; (7) that the vaccine must be shaken before use; (9) that the vaccine is not to be frozen.
Diphtheria Vaccine (Adsorbed)

Diphtheria Vaccine (Adsorbed) is a preparation of diphtheria formol toxoid with a mineral adsorbent. The formol toxoid is prepared from the toxin produced by the growth of Corynebacterium diphtheriae.
Production
General provisions The maximum number of Lf units per single human dose of diphtheria vaccine (adsorbed) is 30. Bulk purified toxoid For the production of diphtheria toxin, from which toxoid is prepared, seed cultures are managed in a defined seed-lot system in which toxinogenicity is conserved and, where necessary, restored by deliberate reselection. A highly toxinogenic strain of Corynebacterium diphtheriae with known origin and history is grown in a suitable liquid medium. At the end of cultivation, the purity of each culture is tested and contaminated cultures are discarded. Toxin-containing culture medium is separated aseptically from the bacterial mass
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as soon as possible. The toxin content (Lf per ml) is checked to monitor consistency of production. Single harvests may be pooled to prepare the bulk purified toxoid. The toxin is purified to remove components likely to cause adverse reactions in humans. The purified toxin is detoxified with formaldehyde by a method that avoids destruction of the immunogenic potency of the toxoid and reversion of the toxoid to toxin, particularly on exposure to heat. Alternatively, purification may be carried out after detoxification. Only bulk purified toxoid that complies with the following requirements may be used in the preparation of the final bulk vaccine. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. Absence of toxin and irreversibility of toxoid Inject subcutaneously into each of 5 guinea-pigs at least 500 Lf of the non-incubated bulk purified toxoid in a volume of 1 ml, using the same buffer solution as for the final vaccine, without adsorbent. Animals that die shall be autopsied and examined for symptoms of diphtheria intoxication (red adrenals). The bulk purified toxoid shall pass the test if no guinea-pig shows symptoms of specific intoxication within six weeks of injection and if at least 80 per cent of the animals survive the test period. The guinea-pigs shall not have been used previously for experimental purposes. Alternatively, a cell-culture test system may be used; in this case, the sensitivity of the test shall have been demonstrated to be not less than that of the guinea-pig test, and the test procedures shall be approved by the National Regulatory Authority. Each bulk purified toxoid shall be tested to ensure that reversion to toxicity cannot take place on storage. The bulk purified toxoid shall be diluted in order to obtain the same concentration and chemical environment as those present in the final bulk vaccine, except for the presence of adjuvant. To determine whether reversion has occurred, diluted toxoids that have been stored at 37 for six weeks shall be tested. The test employed shall be approved by the National Regulatory Authority and should be sufficiently sensitive to detect very small amounts of toxin. No toxicity shall be detected. Intradermal tests in guinea-pigs and cell-culture tests both are considered to be suitable. Cell culture method Using the same buffer solution as for the final vaccine, without adsorbent, prepare a solution of bulk purified toxoid at 100 Lf per ml. Divide the solution into 2 equal parts. Maintain 1 part at 5 3 and the other at 37 for 6 weeks. Carry out a test in Vero cells for active diphtheria toxin using 50 l per well of both samples. The sample should not contain antimicrobial 40
preservatives and detoxifying agents should be determined to be below the concentration toxic to Vero cells. Non-specific toxicity may be eliminated by dialysis. Use freshly trypsinised Vero cells at a suitable concentration, for example 2.5 105 per ml and a reference diphtheria toxin diluted in 100 Lf per ml diphtheria toxoid. A suitable reference diphtheria toxin will contain either not less than 100 LD50/ml or 67 to 133 lr/100 in 1 Lf and 25,000 to 50,000 minimal reacting doses for guinea-pig skin in 1 Lf (diphtheria toxin RP is suitable for use as the reference toxin). Dilute the toxin in 100 Lf/ml diphtheria toxoid to a suitable concentration, for example 2 10-4 Lf per ml. Prepare serial twofold dilutions of the diluted diphtheria toxin and use undiluted test samples (50 l per well). Distribute them in the wells of a sterile tissue culture plate containing a medium suitable for Vero cells. To ascertain that any cytotoxic effect noted is specific to diphtheria toxin, prepare in parallel dilutions where the toxin is neutralised by a suitable concentration of diphtheria antitoxin, for example 100 IU/ml. Include control wells without toxoid or toxin and with non-toxic toxoid at 100 Lf per ml on each plate to verify normal cell growth. Add cell suspension to each well, seal the plates and incubate at 37 for 5 to 6 days. Cytotoxic effect is judged to be present where there is complete metabolic inhibition of the Vero cells, indicated by the pH indicator of the medium. Confirm cytopathic effect by microscopic examination or suitable staining such as MTT dye. The test is invalid if 5 10-5 Lf per ml of reference diphtheria toxin in 100 Lf per ml toxoid has no cytotoxic effect on Vero cells or if the cytotoxic effect of this amount of toxin is not neutralised in the wells containing diphtheria antitoxin. The bulk purified toxoid complies with the test if no toxicity neutralisable by antitoxin is found in either sample. Antigenic purity. Not less than 1,500 Lf per mg of protein nitrogen. FINAL BULK VACCINE The final bulk vaccine is prepared by adsorption of a suitable quantity of bulk purified toxoid onto a mineral carrier such as hydrated aluminium phosphate or aluminium hydroxide; the resulting mixture is approximately isotonic with blood. Suitable antimicrobial preservatives may be added. Certain antimicrobial preservatives, particularly those of the phenolic type, adversely affect the antigenic activity and must not be used. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot.
Identification
Dissolve sufficient sodium citrate in the vaccine under examination to give a 10 per cent w/v concentration. Maintain at 37o for about 16 hours and centrifuge. The clear supernatant
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reacts with a suitable diphtheria antitoxin and yields a precipitate. pH (2.4.24). 6.0 to 7.0. Specific toxicity. Use 5 normal, healthy guinea-pigs weighing between 250 and 350 g, which have been maintained for at least 1 week on a uniform, unrestricted diet, and have not been previously treated with any material that will interfere with the test. Weigh the animals separately and record their weights. Inject subcutaneously into each animal 5 times the dose stated on the label. Weigh all the animals at weekly intervals for 6 weeks. None of the animals shows any symptoms of diphtheria or tetanus toxaemia or dies from diphtheria within 42 days or loses weight at the end of the test. If more than one animal dies from non-specific causes or loses weight, repeat the test. If an animal dies or loses weight in the second test, the vaccine fails the test. Potency. Determine by any of the methods of biological assay of Adsorbed Diphtheria Vaccine described. Biological assay of adsorbed diphtheria vaccine (a) Intradermal challenge method The potency of adsorbed diphtheria vaccine is determined by comparing the dose necessary to protect guinea-pigs against the erythrogenic effects of a range of intradermal injections of diphtheria toxin with the dose of the Standard preparation of adsorbed diphtheria toxoid necessary to give the same protection. For this comparison, the Standard preparation of adsorbed diphtheria toxoid and a suitable preparation of diphtheria toxin, for use as a challenge toxin, are required. Standard preparation The Standard preparation is International standard of Diphtheria toxoid, adsorbed, or another suitable preparation the potency of which has been determined in relation to the International Standard. Suggested method Test animals. Use white guinea-pigs, weighing between 250 and 350 g, from the same stock. Distribute the guinea-pigs into no fewer than six equal groups; use groups containing a number of animals sufficient to obtain results that fulfill the requirements for a valid Assay prescribed below. The guineapigs are all of the same sex or the males and females are distributed equally among the groups. If the challenge toxin to be used has not been shown to be stable or has not been adequately standardized, include five guinea-pigs as unvaccinated controls. Selection of the challenge toxin. Select a preparation of diphtheria toxin containing 67 to 133 Limes reactionis/100 (Lr/ 100) in Limes flocculationis (Lf) and 25,000 to 50,000 minimal reacting doses for guinea-pig skin in 1 Lf. If the challenge 41
toxin preparation has been shown to be stable, it is not necessary to verify the activity for every assay. Preparation of the challenge toxin solutions. Immediately prior to use, dilute the challenge toxin with a suitable diluent to obtain a challenge toxin solution containing about 512x10-4 Lf in 0.2 ml. Dilute a portion of this challenge toxin solution to give a series of five 4-fold dilutions. Determination of potency of the vaccine. Prepare in saline solution dilutions of the vaccine under examination and of the Standard preparation such that, for each, the dilutions form a series differing by not more than 2.5 fold steps and in which the dilutions of intermediate concentration, when injected subcutaneously in 1.0 ml volumes into guinea-pigs, result in an intradermal score of approximately three when the animals are challenged. Allocate the dilutions, one to each of the groups of guinea-pigs, and inject subcutaneously 1.0 ml of each dilution into each guinea-pig in the group to which that dilution is allocated. After 28 days shave both flanks of each guinea-pig and inject each animal intradermally with 0.2 ml of the challenge toxin solution and with 0.2 ml of each of the five dilutions thereof in such a way as to minimize interference between adjacent sites. If necessary, inject the unvaccinated control guinea-pigs with dilutions containing 8x10-5, 4x10-5, 2x10-5, 1x10-5 and 5x10-6 Lf of the challenge toxin. Examine all the injection sites 48 hours after injection of the challenge toxin and record the incidence of specific diphtheria erythema. Record also the number of sites free from such reactions as the intradermal challenge score. Tabulate the intradermal challenge scores for all the animals receiving the same dilution of vaccine and use those data with a suitable transformation, such as (score)2 or arcsin [(score/6)2], to obtain an estimate of the relative potency for each of the test preparations by parallel-line quantitative analysis. The test is not valid unless (a) for both the preparation under examination and the Standard preparation, the mean score obtained at the lowest dose level is more than three; (b) if applicable, the toxin dilution that contains 4x10-5 Lf gives a positive erythema in at least 80.0 per cent of the control guineapigs and the dilution that contains 2x10-5 Lf gives no reaction in at least 80 per cent of the guinea-pigs (if these criteria are not met a different toxin has to be selected); (c) the fiducial limits of the assay fall between 50.0 and 200.0 per cent of the estimated potency; (d) the statistical analysis shows no deviation from linearity and parallelism. The test may be repeated but when more than one test is performed the results of all valid tests must be combined in the estimate of potency. The lower fiducial limit of error of the estimated potency is not less than 30 Units per dose. (b) Lethal challenge method Test animals. Use healthy, white or light-coloured guinea-
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pigs from the same stock, weighing between 250 and 350 g. Distribute them into six groups of sixteen; and four groups of four. The guinea-pigs should all be of the same sex or the males and females should be distributed equally between the six groups of sixteen. Challenge toxin. Select a preparation of diphtheria toxin containing not less than 100 LD50 in 1.0 ml. Preparation of the challenge toxin solutions. Immediately prior to use, prepare from the challenge toxin by dilution in phosphate buffered saline pH 7.4, or normal saline a challenge toxin containing approximately 100 LD50 in 1.0 ml. Dilute portions of this challenge toxin solution to 2LD50, 1 LD50 and LD50 in the same solution Determination of potency of the vaccine. Prepare in saline solution three dilutions of the vaccine under examination and three dilutions of the Standard preparation such that for each, the dilutions form a series differing by not more than 2.5 fold steps and in which the dilutions of intermediate concentration, when injected subcutaneously in 1.0 ml volumes into guineapigs, protect approximately 50 per cent of the animals from the lethal effects of the subcutaneous injection of the quantity of diphtheria toxin prescribed for this test. Allocate the six dilutions, one to each of the six groups of sixteen guinea-pigs, and inject subcutaneously 1.0 ml of each dilution into each guinea-pig in the groups to which that dilution is allocated. After 28 days inject subcutaneously into each animal in the six groups of sixteen, 1.0 ml of the challenge toxin solution. Allocate the challenge toxin solution and the three dilutions made from it, one to each of the four groups of four guineapigs and inject subcutaneously 1.0 ml of each toxin solution into each guinea-pig in the group to which that solution is allocated. Examine the guinea pigs twice in a day, remove dead animals and kill the animals showing definite signs of diphtheria. Count the number of surviving animals 5 days later and calculate the potency of the vaccine under examination relative to the potency of the Standard preparation on the basis of the number of animals that survive in each of the six groups of sixteen, using appropriate statistical methods. The test is not valid unless (a) for the vaccine under examination and the Standard preparation the 50.0 per cent protective doses lie between the largest and smallest doses of the preparations given to the guinea-pigs; (b) the survivors among the four groups of guinea-pigs injected with the challenge toxin and its dilutions indicate that the challenge was approximately 100 LD50 and; (c) statistical analysis shows parallelism, linearity and a significant slope of the doseresponse lines. The test may be repeated any number of times but when more than one test is performed the results of all valid tests must be combined in the estimate of potency. If the lower limit of 95.0 per cent confidence interval of estimated potency is less than 30 IU per single human dose then the 42
limits of the 95.0 per cent confidence interval should be within 50 to 200 of the estimated potency. (c) Antibody induction method Inject subcutaneously on each of two occasions separated by an interval of not more than 4 weeks, one-fiftieth of the stated human dose diluted to 1 ml with saline solution, into each of 10 normal, healthy guinea-pigs weighing between 250 and 350 g. Not earlier than 2 weeks and not later than 3 weeks after the second injection, collect the serum from each animal and determine the antitoxin content of the serum of each animal, as described under Diphtheria Antitoxin or any other method approved by National Regulatory Authority. The geometric mean of the antitoxin contents shall be not less than 2.0 Units per ml with reference to the Diphtheria antitoxin standard. (d) Any other validated serological assay in guinea pigs or mice as approved by National Regulatory Authority Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The amount is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT The final bulk vaccine is distributed aseptically into sterile, tamper-proof containers. The containers are closed so as to prevent contamination. Only a final lot that is satisfactory with respect to each of the requirements given below under, Identification, Tests and Assay may be released for use. Provided the tests for specific toxicity, free formaldehyde and antimicrobial preservative and the assay have been carried out with satisfactory results on the final bulk vaccine, they may be omitted on the final lot.
Identification
Diphtheria toxoid is identified by a suitable immunochemical method. The following method, applicable to certain vaccines, is given as an example. Dissolve in the vaccine under examination sufficient sodium citrate to give a 10 per cent w/ v solution. Maintain at 37 for about 16 hours and centrifuge until a clear supernatant liquid is obtained. The clear supernatant liquid reacts with a suitable diphtheria antitoxin, giving a precipitate.
Tests
Aluminium (2.3.9). Maximum 1.25 mg per single human dose, if aluminium hydroxide or hydrated aluminium phosphate is used as the absorbent.
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HAEMOPHILUS TYPE B CONJUGATE
Free formaldehyde (2.3.20). Maximum 0.2 g/l. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Complies with the test for sterility. Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity. pH (2.4.24). 6.0 to 7.0. Assay Carry out one of the prescribed methods for the assay of Diphtheria Vaccine (Adsorbed). The lower confidence limit (P = 0.95) of the estimated potency is not less than 30 IU per single human dose. Labelling. The label states (1) the human dose; (2) the minimum Lf units per single human dose or the minimum International Units per single human dose if potency test done by challenge method; (3) the name and the amount of the adsorbent and preservative; (4) that the vaccine must be shaken before use; (5) that the vaccine is not to be frozen.
immunogenicity test on mice. Taking account of the results of the stability testing, release requirements are set for these indicator tests to ensure that the vaccine will be satisfactory at the end of the period of validity. SEED LOT The strain of H. influenzae type b used in preparing Haemophilus type b conjugate vaccine shall be identified by a record of its history, including the source from which it was obtained and the tests made to determine the characteristics of the strain. The strain shall have been shown to be capable of producing Type b polysaccharide. The production of PRP and of the carrier protein is based on defined seed lot systems. Master seed lot and working seed lot shall be properly characterized and defined. Cultures derived from the working seed shall have the same characteristics as of the master seed lot. The sample of culture of single harvests taken before killing shall be tested for contamination by examination of Gram-stained smears and by inoculation on suitable media. H. Influenzae Type b Polysaccharide (PRP) H. influenzae Type b is grown in a liquid medium that does not contain high-molecular-weight polysaccharides; if any ingredient of the medium contains blood-group substances, the process shall be validated to demonstrate that after the purification step they are no longer detectable. The culture may be inactivated. PRP is separated from the culture liquid and purified by a suitable method. Volatile matter, including water, In the purified polysaccharide is determined by methods such as thermogravimetry, Karl Fischer or any other suitable method. All chemical analysis shall be based on the dry weight of the polysaccharide, in its salt form. Only those pools of PRP that comply with the following requirements may be used in the preparation of the conjugate. The partially purified PRP shall be stored frozen at or below -20.
Haemophilus Type b Conjugate Vaccine

Haemophilus Type b Conjugate Vaccine is a liquid or freezedried preparation of a polysaccharide, derived from a suitable strain of Haemophilus influenzae type b, covalently bound to a carrier protein. The polysaccharide, polyribosylribitol phosphate, referred to as PRP, is a linear copolymer composed of repeated units of 3--D-ribofuronosyl-(11)-ribitol-5phosphate [(C10H19O12P)n], with a defined molecular size. The carrier protein, when conjugated to PRP, is capable of inducing a T-cell-dependent B-cell immune response to the polysaccharide.
Identification
The PRP is identified by an immunochemical method (2.2.14) or other suitable method (e.g. 1H or 13C NMR spectroscopy). Molecular size. The percentage of PRP eluted before a given K0 value or within a range of K0 values, is determined by gel filtration or high performance size-exclusion chromatography (HPSEC) (2.4.16), either alone or in combination with light scattering and refractive index detectors (e.g. multiple angle laser light scattering i.e. MALLS) or any other suitable method. An acceptable value is established for the particular product and each batch of PRP must be shown to comply with this limit. Limits for currently approved products, using the indicated stationary phases, are shown for information in 43
Production
General provisions The production method shall have been shown to yield consistently H. influenzae type b conjugate vaccines of adequate safety and immunogenicity in humans. The production method is validated to demonstrate that the product, if tested, would comply with the test for safety and efficacy of Vaccines. The stability of the final lot and relevant intermediates is evaluated using one or more indicator tests. Such tests may include determination of molecular size, determination of free PRP in the conjugate and the
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Table 1 - Specifications for different components of Haemophilus Type b Conjugate Vaccine. Polysaccharide Type of PRP Nominal amount per dose Type Carrier Protein Purity Nominal amount per dose 18 g Coupling method Cyanogen bromide activation of PRP Carbodiimidemediated coupling Reductive amination one(step method) or N-hydroxysuccinimide activation Thioether bond Conjugation Procedure
Polysaccharide 25 g (size reduced) K0 60.0 per cent: 0.6-0.7 Polysaccharide: 10 g PRP 50.0 per cent K0: 0.30 Polysaccharide 10 g (size reduced) Dp=15-35 or 10-35
Diphtheria toxoid
>1500 Lf per mg of protein nitrogen
Activated diphtheria toxoid (D-AH+), cyanogen bromide activated PRP ADH-activated PRP (PRP-cov.-AH) + tetanus toxoid + EDAC Direct coupling of PRP to CRM 197 (cyanoboro-hydride activated)
Tetanus toxoid
>1500 Lf per mg of protein nitrogen
20 g
CRM 197 diphtheria protein
>90.0 per cent diphtheria protein
25g
Polysaccharide (size-reduced) K0 0.3 0.6
15 g
Meningococcal group B outer membrane protein (OMP)
Outer membrane 125 or protein vesicles 250 g <8.0 per cent of lipopolysaccharide
PRP activation by CDI PRP-IM + BuA2 + BrAc = PRP-BuA2-BrAc + thioactivated OMP
Abbreviations: ADH = BrAc = BuA2 = CDl =
adipic acid dihydrazide bromoacetyl chloride butane-1, 4-diamide carbonyldi-imidazole
Dp EDAC IM Mw
= = = =
degree of polymerization l-ethyl-3-(3-dimethylaminopropyl) carbodimide imidazolium weight-average molecular weight.
Table 2 - Requirements on bulk conjugate Test Specifications Free Polysaccharide (PRP) Unbound protein Protein Carrier Tetanus toxoid CRM 197 <20.0 per cent <1.0 per cent, where applicable <25.0 per cent <1.0 per cent or <2.0 per cent, depending on the coupling method 0.3-0.7 50.0 per cent 0.3-0.6
Diphtheria toxoid <37.0 per cent <5.0 per cent, where applicable
OMP <15.0 per cent Not applicable
PRP to protein ratio Molecular size (K0): Crosslinked agarose for chromatography R Cross-linked agarose for chromatography R1
1.25-1.75 95.0 per cent <0.75
0.30-0.55 60.0 per cent <0.2
0.05-0.1 85.0 per cent < 0.25
0.6-0.7
85.0 per cent <0.5
44
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Tables 1 and 2. Where applicable, the molecular-size distribution is also determined after chemical modification of the polysaccharide. A validated determination of the degree of polymerization or of the weight-average molecular weight and the dispersion of molecular masses may be used instead of the determination of molecular size distribution. Ribose (2.7.1). Not less than 32.0 per cent, calculated with reference to the dried substance, as estimated by Bial reaction for pentose, using D-ribose as a standard or any other suitable assay. Phosphorus (2.7.1). 6.8 per cent to 9.0 per cent, calculated with reference to the dried substance. Protein (2.3.49). Not more than 1.0 per cent, calculated with reference to the dried substance. Use sufficient PRP to allow detection of 1 per cent of protein (e.g. a minimum of 1 mg of PRP). Nucleic acid (2.7.1). Not more than 1.0 per cent, calculated with reference to the dried substance by spectroscopy or any other suitable method. Bacterial endotoxins (2.2.3 ). Not more than 25 IU of endotoxin per microgram of PRP. Residual reagents. Where applicable, tests are carried out to determine residues of reagents used during inactivation and purification. An acceptable value for each reagent is established for the particular product and each batch of PRP must be shown to comply with this limit. Where validation studies have demonstrated removal of a residual reagent, the test on PRP may be omitted. Carrier protein The carrier protein is chosen in a way so that when the PRP is conjugated it is able to induce a T-cell-dependent immune response. Currently approved carrier proteins and coupling methods are listed for information in Table 1. The carrier proteins are produced by culture of suitable microorganisms; the bacterial purity of the culture is verified; the culture may be inactivated; the carrier protein is purified by a suitable method. Only a carrier protein that complies with the following requirements may be used in preparation of the conjugate.
Diphtheria toxoid. Diphtheria toxoid is produced as stated under Diphtheria Vaccine (Adsorbed) and complies with the requirements prescribed there for bulk purified toxoid. Tetanus toxoid. Tetanus toxoid is produced as stated under Tetanus Vaccine (Adsorbed) and complies with the requirements prescribed there for bulk purified toxoid except that the antigenic purity is not less than 1500 Lf per mg of protein nitrogen. Diphtheria protein CRM 197. Suitable tests are carried out, for validation or routinely, to demonstrate that the product is non-toxic. The protein obtained contains not less than 90.0 per cent of diphtheria CRM 197 protein, when prepared by liquid chromatography (2.4.14) or any other suitable method. The carrier protein shall be characterized by a suitable chemical or physicochemical method like SDS-PAGE, HPLC, isoelectric focusing, amino acid sequencing, circular dichroism, fluorescence spectroscopy, peptide mapping or mass spectroscopy, as appropriate. OMP (Meningococcal group B outer membrane protein complex) OMP complex of Neisseria meningitidis complies with the following requirements for lipopolysaccharide and pyrogens. Lipopolysaccharide. Not more than 8.0 per cent of lipopolysaccharide, determined by a suitable method. Pyrogens (2.2.8). Inject into each rabbit 0.25 g of OMP per kg body weight, for determining the pyrogenic effect. Bulk conjugate PRP is chemically modified to enable conjugation; it is usually partly depolymerised either before or during this procedure. Reactive functional groups or spacers may be introduced into the carrier protein or PRP prior to conjugation. The conjugate is obtained by the covalent binding of PRP and carrier protein. Where applicable, unreacted but potentially reactogenic functional groups are made unreactive by means of capping agents; the conjugate is purified to remove reagents. Where validation studies have demonstrated removal of a residual reagent (eg. CN, Br etc.), the test on bulk conjugate may be omitted. Only a bulk conjugate that complies with the following requirements may be used in preparation of the final bulk vaccine. For each test and for each particular product, limits of acceptance are established and each batch of conjugate must be shown to comply with these limits. Limits applied to currently approved products for some of these tests are listed for information in Table 2. PRP. The PRP content is determined by assay of phosphorus (2.7.1) or by assay of ribose (2.7.1) or by an immunochemical method (2.2.14) or by any suitable method. 45
Identification
The carrier protein is identified by a suitable immunochemical method (2.2.14). Sterility (2.2.11). Carry out the test for sterility using for each medium 10 ml or the equivalent of one hundred doses, whichever is less.
HEPATITIS A (INACTIVATED) AND HEPATITIS B (rDNA) VACCINE (ADSORBED)
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Protein (2.7.1). The protein content is determined by a suitable chemical method. PRP to protein ratio. Determine the ratio by calculation. Molecular size. Molecular-size distribution is determined by gel filtration or size-exclusion chromatography (2.4.16), using a gel matrix, appropriate to the expected size of the conjugate. Free PRP. Unbound PRP is determined after removal of the conjugate, for example by size-exclusion or hydrophobic chromatography (2.4.16), ultra-filtration or other validated methods. Free carrier protein. Free carrier protein is determined by a suitable method (which may include deriving the content by calculation from the results of other tests). The amount is within the limits approved for the particular product. Unreacted functional groups. No unreacted functional groups are detectable in the bulk conjugate unless process validation has shown that unreacted functional groups detectable at this stage are removed during the subsequent manufacturing process (for example, owing to short half-life). Residual reagents. Removal of residual reagents such as cyanide, EDAC (ethyldimethylaminopropylcarbodimide) and phenol is confirmed by suitable tests or by validation of the purification process. Sterility (2.2.11). Carry out the tests for sterility using for each medium 10 ml or the equivalent of one hundred doses, whichever is less. FINAL BULK VACCINE An adjuvant, an antimicrobial preservative and a stabilizer may be added to the bulk conjugate before dilution to the final concentration with a suitable diluent. Only a final bulk vaccine that complies with the following requirements may be used in preparation of the final lot. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT The final lots are filled in suitable containers, under stringent aseptic conditions. Only a final lot that is satisfactory with respect to each of the requirements given under Identification, Tests and Assay may be released for use. Provided the test for antimicrobial preservative has been carried out on the final bulk vaccine, it may be omitted on the final lot. 46
Identification
The vaccine is identified by a suitable immunochemical method (2.2.14) for PRP or the assay serves also to identify the vaccine.
Tests
Sterility (2.2.11). Complies with the test for sterility. Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity. Pyrogens (2.2.8). Complies with the test for pyrogens. Inject per kg of the rabbits mass a quantity of the vaccine equivalent to 1 g of PRP for a vaccine with diphtheria toxoid or CRM 197 diphtheria protein as carrier; 0.1 g of PRP for a vaccine with tetanus toxoid as carrier; 0.025 g of PRP for a vaccine with OMP as carrier. pH (2.4.24). The pH of the vaccine, reconstituted if necessary, is within the range approved for the product (6.5 to 7.5). Aluminium (2.3.9). When aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent, Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Water (2.3.43). Not more than 3.0 per cent. PRP. Not less than 80.0 per cent of the amount of PRP stated on the label as determined by ribose assay (2.7.1) or by phosphorus assay (2.7.1) or by an immunochemical method (2.2.14) or by any other suitable method like colorimetery or by anion exchange liquid chromatography (2.4.14) with pulsed amperometric detection. Free PRP. Unbound PRP is determined after removal of the conjugate for example by size-exclusion or hydrophobic chromatography (2.4.16), ultra filtration or other validated methods. Labelling. The label states (1) the number of micrograms of PRP per human dose; (2) the type and nominal amount of carrier protein per single human dose; (3) for vaccine contained in single-dose containers where the space is too small to accommodate the full name of the vaccine the abbreviation Hib may be used in the label on the container provided that the same code is also stated in the label on the package.
Hepatitis A (Inactivated) and Hepatitis B (rDNA) Vaccine (Adsorbed)

Hepatitis A (Inactivated) and Hepatitis B (rDNA) Vaccine (Adsorbed) is a suspension consisting of a suitable strain of hepatitis A virus, grown in cell cultures and inactivated by a
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HEPATITIS B (rDNA) VACCINE
validated method, and of hepatitis B surface antigen (HBsAg), a component protein of hepatitis B virus obtained by recombinant DNA technology; the antigens are adsorbed on a mineral carrier such as aluminium hydroxide or hydrated aluminium phosphate.
carried out with satisfactory results on the final bulk vaccine, it may be omitted on the final lot.
Identification
The vaccine is shown to contain hepatitis A virus antigen and hepatitis B surface antigen by suitable immunochemical methods (2.2.14), using specific antibodies or by the mouse immunogenicity tests described under assay.
Production
General provisions The two components are prepared as described in the monographs on Hepatitis A Vaccine (Inactivated, Adsorbed) and Hepatitis B Vaccine (rDNA) and comply with the requirements prescribed therein. The production method is validated to demonstrate that the product, if tested, would comply with the test for abnormal toxicity for antisera and vaccines. Reference preparation The reference preparation is part of a representative batch shown to be at least as immunogenic in animals as a batch that, in clinical studies in young, healthy adults, produces not less than 95.0 per cent seroconversion, corresponding to a level of neutralizing antibody recognized to be protective, after a full-course primary immunization. For hepatitis A, an antibody level not less than 20 mIU/ml determined by enzymelinked immunosorbent assay is recognized as being protective. For hepatitis B, antibody level not less than 10 mIU/ml against HBsAg is recognized as being protective. FINAL BULK VACCINE The final bulk vaccine is prepared from one or more inactivated harvests of hepatitis A virus and one or more batches of purified antigen of Hepatitis B (rDNA). Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT Only a final lot that complies with each of the requirements given below under Identification, Tests and Assay may be released for use. Provided that the tests for free formaldehyde (where applicable) and antimicrobial preservative content (where applicable) have been carried out on the final bulk vaccine with satisfactory results, they may be omitted on the final lot. If the assay of the hepatitis A and/or the hepatitis B component is carried out in vivo, then provided it has been 47
Tests
Aluminium (2.3.9). Maximum 1.25 mg per single human dose if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. Free formaldehyde (2.3.20). Maximum 0.2 g/l. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Complies with the test for sterility. Bacterial endotoxins (2.2.3). Less than 2 IU per human dose.
Assay
Hepatitis A component Complies with the assay as stated under Inactivated Hepatitis A Vaccine (Adsorbed). Hepatitis B component Complies with the assay as stated under Hepatitis B Vaccine (rDNA). Labelling. The label states (1) the amount of hepatitis A virus antigen and hepatitis B surface antigen per container; (2) the type of cells used for production of the vaccine; (3) the name and amount of the adsorbent used; (4) that the vaccine must be shaken before use; (5) that the vaccine must not be frozen.
Hepatitis B Vaccine (rDNA)

Hepatitis B Vaccine (rDNA) is a non-infectious preparation containing the purified major surface antigen of Hepatitis B virus (HBsAg). This preparation is white or almost white translucent liquid in which the mineral carrier tends to settle down slowly on keeping but is free from foreign particles/ floccules.
Production
General provisions The antigen is manufactured by recombinant DNA technology by culturing genetically engineered yeast cells or other suitable
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cell lines which carry the gene that codes for major surface antigen of the Hepatitis-B virus as approved by the competent authority. Several physico-chemical steps are employed to purify the Hepatitis-B surface antigen (HBsAg). The vaccine may contain the product of the S gene (major protein), a combination of the S gene and pre-S2 gene products (middle protein) or a combination of S gene, the pre-S2 gene, and preS1 gene products (large protein). The purity of the antigen is determined by comparison with a reference preparation using liquid chromatography or other suitable methods such as SDSPAGE with any suitable staining method. The purified antigen is finally adsorbed on aluminium hydroxide or aluminium phosphate. The method used for production of the vaccine must have been shown to yield a product consistently complying with the requirements for immunogenicity and safety. It must also have been shown to induce specific, protective antibodies in human beings. The production method must be validated to demonstrate that the product if tested, would comply with the tests for safety and efficacy. Reference preparation. A part of a batch shown to be at least as immunogenic as a batch that was used in clinical studies and approved by National Regulatory Authority and determined by any suitable method. For hepatitis B, antibody level not less than 10 mIU/ml against HBsAg is recognized as being protective.. Characterisation of the substance Development studies are carried out to characterize the antigen. The complete protein, lipid and carbohydrate structure of the antigen is established. The morphological characteristics of the antigen particles are established by electron microscopy. The buoyant density of the antigen particles is determined by a physico-chemical method, for example gradient centrifugation. The antigenic epitopes are characterized. The protein fraction of the antigen is characterized in terms of the primary structure (for example, by determination of the aminoacid composition, by partial amino-acid sequence analysis and by peptide mapping). PROPAGATION AND HARVEST Identity, microbial purity, plasmid retention and consistency of yield are determined at suitable production stages. If mammalian cells are used, tests for extraneous agents and mycoplasmas are performed in accordance with tests for extraneous agents in viral vaccines for human use. PURIFIED ANTIGEN Only a purified antigen that complies with the following requirements may be used in the preparation of the final bulk. 48
Total protein (2.3.49). The total protein is determined by a validated method. The content is within the limits approved for the specific product. Antigen content and identification. The quantity and specificity of HBsAg is determined in comparison with the International standard for HBsAg subtype ad or an in-house reference, by a suitable immunochemical method such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunoblot (preferably using a monoclonal antibody directed against a protective epitope) or single radial diffusion. The antigen/protein ratio is within the limits approved for the specific product. The molecular weight of the major band in a sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) under reduced conditions corresponds to the value expected from the gene sequence and possible glycosylation. Antigenic purity. The purity of the antigen is determined by comparison with a reference preparation using liquid chromatography or other suitable methods such as SDS-PAGE with staining. A suitable method is sensitive enough to detect a potential contaminant at a concentration of 1.0 per cent of total protein. Not less than 95.0 per cent of the total protein consists of hepatitis B surface antigen. Composition. The content of proteins, lipids, nucleic acids and carbohydrates is determined. Host-cell and vector-derived DNA (2.2.15). If mammalian cells are used for production, not more than 10 pg of DNA in the quantity of purified antigen equivalent to a single human dose of vaccine. Caesium. If a caesium salt is used during production, a test for residual caesium is carried out on the purified antigen. The content is within the limits approved for the specific product. Sterility (2.2.11). The purified antigen complies with the test for sterility, carried out using 10 ml for each medium. Additional tests on the purified antigen may be required depending on the production method used: for example, a test for residual animal serum where mammalian cells are used for production or tests for residual chemicals used during extraction and purification. FINAL BULK VACCINE An antimicrobial preservative and an adjuvant may be included in the vaccine. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount.
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Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT Only a final lot that complies with each of the requirements given below under Identification, Tests and Assay may be released for use. Provided that the tests for free formaldehyde, antimicrobial preservative content and the assay in animals, where applicable, have been carried out on the final bulk vaccine with satisfactory results, they may be omitted on the final lot.
the test. Use animals of the same sex in the test. Inject similar groups of animals with the reference preparation of HepatitisB vaccine (r DNA). One group of control animals remains unvaccinated but is injected intraperitoneally with the same volume of the diluent alone. Anaesthetize and bleed the animals 28 to 42 days later, keeping the individual sera separate. Assay the individual sera for specific HBsAg antibody concentration by a suitable immunochemical method such as ELISA or RIA. Calculate the result of the assay by standard statistical methods (5.7). From the distribution of reaction levels measured on all the sera in the unvaccinated (control group), the maximum reaction level that can be expected to occur in an unvaccinated animal for that particular assay is determined. Any response in the vaccinated animals that exceeds this level is by definition seroconversion. The percentage of animals showing seroconversion in each group is transformed (for example, probit transformation) and a parallel line model, using the log dose response curve, is applied to the data. The potency of the preparation under examination relative to the reference preparation is thus established. The test is not valid unless (a) for both the test and reference vaccine, the ED50 lies between the smallest and the largest doses given to animals ; (b) the statistical analysis shows no deviation from linearity or parallelism; (c) the fiducial limits of the estimated relative potency fall between 33.0 and 300.0 per cent of the estimated potency. Method B (In vitro) The potency of the vaccine under examination is determined by an in vitro method that has been validated against the biological test. Enzyme Linked Immunosorbent Assay (ELISA) using monoclonal antibodies specific for protection inducing epitopes of HBsAg have been shown to be suitable. Adequate number of dilutions and replicates of the vaccine under examination and the reference standard are employed in the assay. The data obtained is analyzed by a parallel-line model and may be suitably transformed for statistical evaluation. Commercially available kits for measuring HBsAg in vitro may be used provided they are validated to produce equally precise and accurate results. The test is not valid unless (a) the statistical analysis shows no deviation from linearity or parallelism; (b) the fiducial limits of the estimated relative potency fall between 80.0 and 125.0 per cent of the estimated potency. The acceptance criteria are approved for a given reference preparation by the National Regulatory Authority in the light of the validation data. Labelling. The label states (a) the amount of HBsAg per dose; (b) the type of cells used for production of the vaccine; (c) the 49
Identification
The assay or, where applicable, the electrophoretic profile, also serves to identify the vaccine.
Tests
Aluminium (2.3.9). Maximum 1.25 mg per single human dose, if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbant. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Complies with the test for sterility. Pyrogens (2.2.8). Complies with the test for pyrogens. Inject the equivalent of one human dose into each rabbit. A validated test for bacterial endotoxins may be used instead of the test for pyrogens. Assay. The vaccine complies with the assay of Hepatitis-B Vaccine (rDNA) described below. Potency. The upper fiducial limit (P = 0.95) of the estimated relative potency is not less than 1.0. Determine the potency either in animals (Method A) or by a validated in vitro procedure (Method B) described below: Method A (Biological) The potency of the vaccine under examination is determined in animals by comparing in given conditions its capacity to induce specific anti-HBsAg antibodies in mice or guinea-pigs with the same capacity as with the reference standard. Inject intraperitoneally not less than three doses of suitable dilutions of the vaccine under examination diluted with adjuvant used in the vaccine into groups of a suitable strain of mice, weighing between 15 and 20 g (about 5 weeks old), of either sex distributed randomly into several groups of mice. Healthy guinea pigs weighing between 300 and 350g (about 7 weeks old) that have not been previously treated with any material that will interfere with the test will also be suitable for
INACTIVATED HEPATITIS A VACCINE (ADSORBED)
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name and amount of the adjuvant; (d) that the vaccine must be shaken before use; (e) that the vaccine must not be frozen.
Virus concentration. The virus concentration of each master and working seed lot is determined to monitor consistency of production. Extraneous agents (2.7.3). The master and working seed lots comply with the requirements for seed lots for virus vaccines. In addition, if primary monkey cells have been used for isolation of the strain, measures are taken to ensure that the strain is not contaminated with simian viruses such as simian immunodeficiency virus and filoviruses. PROPAGATION AND HARVEST All processing of the cell bank and subsequent cell cultures is done under aseptic conditions in an area where no other cells are being handled. Animal serum (but not human serum) may be used in the cell culture media. Serum and trypsin used in the preparation of cell suspensions and media are shown to be free from extraneous agents. The cell culture media may contain a pH indicator, such as phenol red and approved antibiotics at the lowest effective concentration. Not less than 500 ml of the cell cultures employed for vaccine production is set aside as uninfected cell cultures (control cells). Multiple harvests from the same production cell culture may be pooled and considered as a single harvest. Only a single harvest that complies with the following requirements may be used in the preparation of the vaccine. When the determination of the ratio of virus concentration to antigen content has been carried out on a suitable number of single harvests to demonstrate consistency, it may subsequently be omitted as a routine test.
Inactivated Hepatitis A Vaccine (Adsorbed)

Hepatitis A Vaccine (Inactivated, Adsorbed) is a liquid preparation of a suitable strain of hepatitis A virus grown in cell cultures, inactivated by a validated method and adsorbed on a mineral carrier. The vaccine is an opalescent suspension. The vaccine complies with the monograph on Vaccines.
Production
Production of the vaccine is based on a virus seed-lot system and a cell-bank system. The production method shall have been shown to yield consistently vaccines that comply with the requirements for immunogenicity, safety and stability. The production method is validated to demonstrate that the product, if tested, would comply with the test for safety and efficacy. Unless otherwise justified and authorised, the virus in the final vaccine shall not have undergone more passages from the master seed lot than were used to prepare the vaccine shown in clinical studies to be satisfactory with respect to safety and efficacy. Reference preparation. A part of a batch shown to be at least as immunogenic as a batch that, in clinical studies in young healthy adults, produced not less than 95.0 per cent seroconversion, corresponding to a level of neutralising antibody accepted to be protective, after a full-course of primary immunisation is used as a reference preparation. An antibody level of 20 mIU /ml determined by enzyme-linked immunosorbent assay is recognised as being protective. Substrate for virus propagation The virus is propagated in a human diploid cell line or in a continuous cell line approved by the competent authority. SEED LOT The strain of hepatitis A virus used to prepare the master seed lot shall be identified by historical records that include information on the origin of the strain and its subsequent manipulation. Only a seed lot that complies with the following requirements may be used for virus propagation. Description. A clear, colourless or light coloured liquid.
Identification
The test for antigen content also serves to identify the single harvest. Sterility (2.2.11). The single harvest complies with the test for sterility, carried out using 10 ml for each medium. Mycoplasmas (2.7.4). The single harvest complies with the test for mycoplasmas carried out using 1ml for each medium. Control cells. The control cells of the production cell culture comply with a test for identity and the requirements for extraneous agents. Antigen content. Determine the hepatitis A antigen content by a suitable immunochemical method (2.2.14) to monitor production consistency; the content is within the limits approved for the particular product. Ratio of virus concentration to antigen content. The consistency of the ratio of the concentration of infectious virus, as determined by a suitable cell culture method, to antigen content is established by validation on a suitable number of single harvests. 50
Identification
Each master and working seed lot is identified as hepatitis A virus using specific antibodies.
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INACTIVATED HEPATITIS A VACCINE (ADSORBED)
PURIFICATION AND PURIFIED HARVEST The harvest, which may be a pool of several single harvests, is purified by validated methods. If continuous cell lines are used for production, the purification process shall have been shown to reduce consistently the level of host-cell DNA. Only a purified harvest that complies with the following requirements may be used in the preparation of the inactivated harvest. Virus concentration. The concentration of infective virus in the purified harvest is determined by a suitable cell culture method to monitor production consistency and as a starting point for monitoring the inactivation curve. Antigen:total protein ratio. Determine the hepatitis A virus antigen content by a suitable immunochemical method (2.2.14). Determine the total protein by a validated method. The ratio of hepatitis A virus antigen content to total protein content is within the limits approved for the particular product. Bovine serum albumin. Not more than 50 ng in the equivalent of a single human dose, determined by a suitable immunochemical method (2.2.14). Where appropriate in view of the manufacturing process, other suitable protein markers may be used to demonstrate effective purification. Residual host-cell DNA (2.2.15). If a continuous cell line is used for virus propagation, the content of residual host-cell DNA, determined using a suitable method is not greater than 100 pg in the equivalent of a single human dose. Residual chemicals. If chemical substances are used during the purification process, tests for these substances are carried out on the purified harvest (or on the inactivated harvest), unless validation of the process has demonstrated total clearance. The concentration must not exceed the limits approved for the particular product. INACTIVATION AND INACTIVATED HARVEST Several purified harvests may be pooled before inactivation. In order to avoid interference with the inactivation process, virus aggregation must be prevented or aggregates must be removed immediately before and/or during the inactivation process. The virus suspension is inactivated by a validated method; the method shall have been shown to be consistently capable of inactivating hepatitis A virus without destroying the antigenic and immunogenic activity; as part of the validation studies, an inactivation curve is plotted representing residual live virus concentration measured on at least three occasions (for example, on days 0, 1 and 2 of the inactivation process). If formaldehyde is used for inactivation, the presence of excess free formaldehyde is verified at the end of the inactivation process. Only an inactivated harvest that complies with the following requirements may be used in the preparation of the final bulk vaccine. 51
Inactivation. Carry out an amplification test for residual infectious hepatitis A virus by inoculating a quantity of the inactivated harvest equivalent to 5 per cent of the batch or if the harvest contains the equivalent of 30,000 doses or more, not less than 1,500 doses of vaccine into cell cultures of the same type as those used for production of the vaccine and incubating the cells for at least 28 days. Make two passages and at the end of incubation carry out a test of suitable sensitivity for residual infectious virus. No evidence of hepatitis A virus multiplication is found in the samples taken at the end of the inactivation process. Use infective virus inocula concurrently as positive controls to demonstrate cellular susceptibility and absence of interference. Sterility (2.2.11). The inactivated viral harvest complies with the test for sterility, carried out using 10 ml for each medium. Bacterial endotoxins (2.2.3). Not more than 2 IU of endotoxin in the equivalent of a single human dose. Antigen content. Determine the hepatitis A virus antigen content by a suitable immunochemical method (2.2.14). Residual chemicals. As stated under Purification and Purified Harvest. FINAL BULK VACCINE The final bulk vaccine is prepared from one or more inactivated harvests. Approved adjuvants, stabilisers and antimicrobial preservatives may be added. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT The final bulk vaccine is distributed aseptically into sterile containers. The containers are then closed so as to avoid contamination. Only a final lot that complies with each of the requirements given below under Identification, Tests and Assay may be released for use. Provided that the tests for free formaldehyde (where applicable) and antimicrobial preservative content (where applicable) and the assay have been carried out on the final bulk vaccine with satisfactory results, these tests may be omitted on the final lot. If the assay is carried out using mice or other animals, then provided it has been carried with satisfactory results on the final bulk vaccine, it may be omitted on the final lot.
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Identification
The vaccine is shown to contain hepatitis A virus antigen by a suitable immunochemical method using specific antibodies or by the mouse immunogenicity test described under Assay.
antibodies against hepatitis A virus by a suitable immunochemical method (2.2.14). Calculations. Carry out the calculations by the usual statistical methods (5.7) for an assay with a quantal response. From the distribution of reaction levels measured on all the sera in the unvaccinated group, determine the maximum reaction level that can be expected to occur in an unvaccinated animal for that particular assay. Any response in vaccinated animals that exceeds this level is by definition a seroconversion. Make a suitable transformation of the percentage of animals showing seroconversion in each group (for example, a probit transformation) and analyse the data according to a parallelline log dose-response model. Determine the potency of the test preparation relative to the reference preparation. Validity conditions. The test is not valid unless (a) for both the test and the reference vaccine, the ED50 lies between the smallest and the largest doses given to the animals; (b) the statistical analysis shows no significant deviation from linearity or parallelism; (c) the fiducial limits of the estimated relative potency fall between 33.0 and 300.0 per cent of the estimated potency. Potency. The upper fiducial limit (P = 0.95) of the estimated relative potency is not less than 1.0. In vitro assay Carry out an immunochemical determination of antigen content (2.2.14) with acceptance criteria validated against the in vivo test. The acceptance criteria are approved for a given reference preparation by the National Regulatory Authority in the light of the validation data. Labelling. The label states (1) the biological origin of the cells and; (2) the adjuvant used for the preparation of the vaccine.
Tests
Aluminium (2.3.9). Maximum 1.25 mg per single human dose, if hydrated aluminium phosphate or aluminium hydroxide is used as the adsorbent. Free formaldehyde (2.3.20). When formaldehyde has been used for inactivation, the vaccine complies with the test prescribed in Vaccines. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Complies with the test for sterility. Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity. Assay The vaccine complies with the assay of Hepatitis A vaccine. The assay is carried out either in vivo, by comparing in given condition the capacity to induce specific antibodies in mice with the same capacity of a reference preparation or in vitro by an immunochemical determination of antigen content (2.2.14). In vivo assay The test on mice shown below is given as an example of a method that has been found suitable for a given vaccine; other validated methods may also be used. Selection and distribution of the test animals. Healthy mice from the same stock, about 5 weeks old and from a strain shown to be suitable should be used in the test. Use animals of the same sex. Distribute the animals in at least seven equal groups of a number suitable for the requirements of the assay. Determination of potency of the vaccine under examination. Using a 0.9 per cent w/v solution of sodium chloride containing the aluminium adjuvant used for the vaccine, prepare at least three dilutions of the vaccine under examination and matching dilutions of the reference preparation. Allocate the dilutions one to each of the groups of animals and inject subcutaneously not more than 0.5 ml of each dilution into each animal in the group to which that dilution is allocated. Maintain a group of unvaccinated controls, injected subcutaneously with the same volume of diluent. After 28 to 32 days, anaesthetise and bleed all animals, keeping the individual sera separate. Assay the individual sera for specific 52
Inactivated Hepatitis B Vaccine

Inactivated Hepatitis B Vaccine is a non-infectious inactivated liquid preparation derived from the surface antigen of Hepatitis B virus (HbsAg). This preparation is white or almost white translucent liquid in which the mineral carrier tends to settle down slowly on keeping but is free from foreign particles / floccules.
Production
The antigen is harvested and purified from the plasma of human carriers of Hepatitis B virus. The surface antigen contains all the three antigen species (S, Pre-S1, Pre-S2). The individual donor plasma is shown by sensitive tests to be seronegative for HIV-I and HIV-2 and for HCV. The plasma pool is tested for freedom from adventitious viruses and blood borne
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transmissible pathogens by appropriate methods. The purified antigen is further inactivated by a validated method, usually with formalin or any other inactivating agent, to render the hepatitis B virus harmless. The preparation is also tested for the residual HBV DNA using a sensitive test approved by the competent authority and the level is shown to be less than 1 pg HBV DNA per 50 doses. The method used for production of the vaccine must have been shown to yield a product consistently complying with the requirements of immunogenicity, safety and stability. The production method must also be validated to demonstrate that the product, if tested, would comply with the tests for safety and efficacy. Reference preparation. A part of a batch shown to be at least as immunogenic as a batch that produced in clinical studies in young healthy adults not less than 95.0 per cent seroconversion, corresponding to a level of neutralizing antibody accepted to be protective (HbsAg antibody titre not less than 10 mIU/ml after a full course of primary immunization determined by enzyme-linked immunosorbent assay (ELISA) is used as a reference preparation. Characterisation of the substance Development studies are carried out to characterize the antigen. The complete protein, lipid and carbohydrate structure of the antigen is established. The morphological characteristics of the antigen particles are established by electron microscopy. The buoyant density of the antigen particles is determined by a physico-chemical method (2.4.29), for example gradient centrifugation. The antigenic epitopes are characterized. The protein fraction of the antigen is characterized in terms of the primary structure (for example, by determination of the aminoacid composition, by partial amino-acid sequence analysis and by peptide mapping). PROPAGATION AND HARVEST Identity, microbial purity, plasmid retention and consistency of yield are determined at suitable production stages. PURIFIED ANTIGEN Only a purified antigen that complies with the following requirements may be used in the preparation of the final bulk. Total protein (2.3.49). The total protein is determined by a validated method. The content is within the limits approved for the specific product. Antigen content and identification. The quantity and specificity of HBsAg is determined in comparison with the International standard for HBsAg subtype ad or an in-house reference, by a suitable immunochemical method such as radio immunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunoblot (preferably using a monoclonal antibody directed against a protective epitope) or single radial diffusion. 53
The antigen/protein ratio is within the limits approved for the specific product. The molecular weight of the major band in a sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) under reduced conditions corresponds to the value expected from the gene sequence and possible glycosylation. Antigenic purity. The purity of the antigen is determined by comparison with a reference preparation using liquid chromatography or other suitable methods such as SDS-PAGE with staining. A suitable method is sensitive enough to detect a potential contaminant at a concentration of 1.0 per cent of total protein. Not less than 95.0 per cent of the total protein consists of hepatitis B surface antigen. Composition. The content of proteins, lipids, nucleic acids and carbohydrates is determined. Sterility (2.2.11). The purified antigen complies with the test for sterility carried out using 10 ml for each medium. Additional tests on the purified antigen may be required depending on the production method used. FINAL BULK VACCINE An antimicrobial preservative and an adjuvant may be included in the vaccine. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical or physico-chemical method. The amount is not less than the 85.0 per cent and not greater than 115.0 per cent of that stated on the label. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT Only a final lot that complies with each of the requirements given below under Identification, Tests and Assay may be released for use. Provided that the tests for free formaldehyde, antimicrobial preservative content and the assay in animals, where applicable, have been carried out on the final bulk vaccine with satisfactory results, they may be omitted on the final lot.
Identification
The assay or, where applicable, the electrophoretic profile, also serves to identify the vaccine.
Tests
Aluminium (2.3.9). When hydrated aluminium phosphate or aluminium hydroxide is used as the adsorbent, the vaccine
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complies with the test prescribed in the monograph on Vaccines. Test for inactivating agent. The concentration of any inactivating agent remaining in the final vaccine shall be determined by methods approved by the competent authority. The concentration shall not exceed a specified upper limit. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Complies with the test for sterility. Pyrogens (2.2.8). Complies with the test for pyrogens. Inject the equivalent of one human dose into each rabbit. A validated test for bacterial endotoxins (2.2.3) may be used instead of the test for pyrogens. Assay The upper fiducial limit (P = 0.95) of the estimated relative potency is not less than 1.0. Determine the potency by method A (Biological) as described under Hepatitis-B vaccine (rDNA). Labelling. The label states (1) the amount of HBsAg per dose; (2) the name and amount of inactivating agent; (3) the name and amount of the adjuvant; (4) that the vaccine must be shaken before use; (5) that the vaccine must not be frozen.
recommends new strains corresponding to prevailing epidemiological evidence. The origin and passage history of virus strains shall be approved by the National Regulatory Authority. Substrate for virus propagation Influenza virus seed to be used in the production of vaccine is propagated in fertilised eggs from chicken flocks free from specified pathogens or in suitable cell cultures, such as chickembryo fibroblasts or chick kidney cells obtained from chicken flocks free from specified pathogens. For production, the virus of each strain is grown in the allantoic cavity of eggs derived from specific pathogen free flocks. SEED LOT The production of vaccine is based on a seed-lot system. Working seed lots represent not more than fifteen passages from the approved reassorted virus or the approved virus isolate. The final vaccine represents one passage from the working seed lot. The haemagglutinin and neuraminidase antigens of each seed lot are identified as originating from the correct strain of influenza virus by suitable methods. Only a working virus seed lot that complies with the following requirements may be used in the preparation of the monovalent pooled harvest. Sterility (2.2.11). Carry out the test for sterility using 10 ml for each medium. Mycoplasmas (2.7.4). Carry out the test for mycoplasmas using 10 ml. PROPAGATION AND HARVEST An antimicrobial agent may be added to the inoculum. After incubation at a controlled temperature, the allantoic fluids are harvested and combined to form a monovalent pooled harvest. An antimicrobial agent may be added at the time of harvest. At no stage in the production, penicillin or streptomycin is used. MONOVALENT POOLED HARVEST To limit the possibility of contamination, inactivation is initiated as soon as possible after preparation. The virus is inactivated by a method that has been demonstrated on three consecutive batches to be consistently effective for the manufacturer. The inactivation process shall have been shown to be capable of inactivating the influenza virus without destroying its antigenicity; the process should cause minimum alteration of the haemagglutinin and neuraminidase antigens. The inactivation process shall also have been shown to be capable of inactivating avian leucosis viruses and mycoplasmas. If the monovalent pooled harvest is stored after inactivation, it 54
Inactivated Influenza Vaccine (Split Virion)

Influenza Vaccine (Split Virion, Inactivated) is a sterile, aqueous suspension of a strain or strains of influenza virus, type A or B, or a mixture of strains of the two types grown individually in eggs derived from specific pathogen free flock or cell cultures, inactivated and treated so that the integrity of the virus particles has been disrupted without diminishing the antigenic properties of the haemagglutinin and neuraminidase antigens. The stated amount of haemagglutinin antigen for each strain present in the vaccine is 15 g per dose, unless clinical evidence supports the use of a different amount.
Production
The production method is validated to demonstrate that the product, if tested, would comply with the test for safety and efficacy. Choice of vaccine strain The World Health Organisation reviews the world epidemiological situation annually and if necessary
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is held at a temperature of 53. If formaldehyde solution is used, the concentration does not exceed 0.2 g/l of formaldehyde at any time during inactivation; if betapropiolactone is used, the concentration does not exceed 0.1 per cent v/v at any time during inactivation. Before or after the inactivation procedure, the monovalent pooled harvest is concentrated and purified by high-speed centrifugation or other suitable method and the virus particles are disrupted into component subunits by the use of approved procedures. For each new strain, a validation test is carried out to show that the monovalent bulk consists predominantly of disrupted virus particles. Only a monovalent pooled harvest that complies with the following requirements may be used in the preparation of the final bulk vaccine. Haemagglutinin antigen. Determine the content of haemagglutinin antigen by an immunodiffusion test (2.2.14), by comparison with a haemagglutinin antigen reference preparation or with an antigen preparation calibrated against it. Carry out the test at 20 to 25. For some vaccines, the physical form of the haemagglutinin particles prevents quantitative determination by immunodiffusion after inactivation of the virus. For these vaccines, a determination of haemagglutinin antigen is made on the monovalent pooled harvest before inactivation. The production process is validated to demonstrate suitable conservation of haemagglutinin antigen and a suitable tracer is used for formulation, for example, protein content. Neuraminidase antigen. The presence and type of neuraminidase antigen are confirmed by suitable enzymatic or immunological methods (2.2.14) on the first three monovalent pooled harvests from each working seed lot. Sterility (2.2.11). Carry out the test for sterility using 10 ml for each medium. Viral inactivation. Carry out as described below under Tests. Chemicals used for disruption. Tests are carried out on the monovalent pooled harvest for the chemicals used for disruption, the limits being approved by the National Regulatory Authority. FINAL BULK VACCINE Appropriate quantities of the monovalent pooled harvests are blended to make the final bulk vaccine. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. 55
Sterility (2.2.11). Carry out the test for sterility using 10 ml for each sterility medium. FINAL LOT The final bulk vaccine is distributed aseptically into sterile, tamper-proof containers. The containers are closed so as to prevent contamination. Only a final lot that is satisfactory with respect to each of the requirements given below under Tests and Assay may be released for use. Provided that the test for viral inactivation has been performed with satisfactory results on each monovalent pooled harvest and that the tests for free formaldehyde, ovalbumin and total protein have been performed with satisfactory results on the final bulk vaccine, they may be omitted on the final lot. Description. The vaccine is a slightly opalescent liquid.
Identification
The assay serves to confirm the antigenic specificity of the vaccine.
Tests
Viral inactivation. Inoculate 0.2 ml of the vaccine into the allantoic cavity of each of ten fertilised eggs and incubate at 33 to 37 for 3 days. The test is not valid unless at least eight of the ten embryos survive. Harvest 0.5 ml of the allantoic fluid from each surviving embryo and pool the fluids. Inoculate 0.2 ml of the pooled fluid into a further ten fertilised eggs and incubate at 33 to 37 for 3 days. The test is not valid unless at least eight of the ten embryoes survive. Harvest about 0.1 ml of the allantoic fluid from each surviving embryo and examine each individual harvest for live virus by a haemagglutination test. If haemagglutination is found for any of the fluids, carry out for that fluid a further passage in eggs and test for haemagglutination; no haemagglutination occurs. Total protein (2.3.49). Not more than six times the total haemagglutinin content of the vaccine as determined in the assay, but in any case, not more than 100 g of protein per virus strain per human dose and not more than a total of 300 g of protein per human dose. Ovalbumin. Not more than 1 g of ovalbumin per human dose, determined by a suitable technique using a suitable reference preparation of ovalbumin. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Complies with the test for sterility. Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity.
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Bacterial endotoxins (2.2.3). Not more than 100 IU of endotoxin per human dose. Assay Determine the content of haemagglutinin antigen by an immunodiffusion test (2.2.14), by comparison with an appropriate haemagglutinin antigen reference preparation. Carry out the test at 20 to 25. The confidence interval (P = 0.95) of the assay is not greater than 80.0 per cent to 125.0 per cent of the estimated content. The lower confidence limit (P = 0.95) of the estimate of haemagglutinin antigen content is not less than 80.0 per cent of the amount stated on the label for each strain. For some vaccines, quantitative determination of haemagglutinin antigen with respect to available reference preparations is not possible. An immunological identification of the haemagglutinin antigen and a semi-quantitative determination are carried out instead by suitable methods. Labelling. The label complies with the requirements stated under Vaccines and also states (a) that the vaccine has been prepared on eggs; (b) the strain or strains of influenza virus used to prepare the vaccine; (c) the method of inactivation; (d) the haemagglutinin content in g per virus strain per dose; (e) the season during which the vaccine is intended to protect.
approved by the competent authority. Substrate for virus propagation Influenza virus seed to be used in the production of vaccine is propagated in fertilised eggs from chicken flocks free from specified pathogens or in suitable cell cultures, such as chickembryo fibroblasts or chick kidney cells obtained from chicken flocks free from specified pathogens. For production, the virus of each strain is grown in the allantoic cavity of eggs derived from SPF flocks. SEED LOT The production of vaccine is based on a seed-lot system. Working seed lots represent not more than fifteen passages from the approved reassorted virus or the approved virus isolate. The final vaccine represents one passage from the working seed lot. The haemagglutinin and neuraminidase antigens of each seed lot are identified as originating from the correct strain of influenza virus by suitable methods. Only a working virus seed lot that complies with the following requirements may be used in the preparation of the monovalent pooled harvest. Sterility (2.2.11). Carry out the test for sterility using 10 ml for each medium. Mycoplasmas (2.7.4). Carry out the test for mycoplasmas using 10 ml.
Inactivated Influenza Vaccine (Surface Antigen)

Influenza Vaccine (Surface Antigen, Inactivated) is a sterile, aqueous suspension of a strain or strains of influenza virus, type A or B, or a mixture of strains of the two types grown individually in eggs derived from specific pathogen free flocks or cell cultures, inactivated and treated so that the preparation consists predominantly of haemagglutinin and neuraminidase antigens, without diminishing the antigenic properties of these antigens. The stated amount of haemagglutinin antigen for each strain present in the vaccine is 15 g per dose, unless clinical evidence supports the use of a different amount.
PROPAGATION AND HARVEST An antimicrobial agent may be added to the inoculum. After incubation at a controlled temperature, the allantoic fluids are harvested and combined to form a monovalent pooled harvest. An antimicrobial agent may be added at the time of harvest. At no stage in the production penicillin or streptomycin is used. MONOVALENT POOLED HARVEST To limit the possibility of contamination, inactivation is initiated as soon as possible after preparation. The virus is inactivated by a method that has been demonstrated on three consecutive batches to be consistently effective for the manufacturer. The inactivation process shall have been shown to be capable of inactivating the influenza virus without destroying its antigenicity; the process should cause minimum alteration of the haemagglutinin and neuraminidase antigens. The inactivation process shall also have been shown to be capable of inactivating avian leucosis viruses and mycoplasmas. If the monovalent pooled harvest is stored after inactivation, it is held at a temperature of 53. If formaldehyde solution is used, the concentration does not exceed 0.2 g/l of formaldehyde at any time during inactivation; if betapropiolactone is used, the concentration does not exceed 56
Production
The production method is validated to demonstrate that the product, if tested, would comply with the test for safety and efficacy. Choice of vaccine strain The World Health Organisation reviews the world epidemiological situation annually and if necessary recommends new strains corresponding to prevailing epidemiological evidence. The origin and passage history of virus strains shall be
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INACTIVATED INFLUENZA VACCINE (SURFACE ANTIGEN)
0.1 per cent v/v at any time during inactivation. Before or after the inactivation process, the monovalent pooled harvest is concentrated and purified by high-speed centrifugation or other suitable method. Virus particles are disrupted into component subunits by approved procedures and further purified so that the monovalent bulk consists mainly of haemagglutinin and neuraminidase antigens. Only a monovalent pooled harvest that complies with the following requirements may be used in the preparation of the final bulk vaccine. Haemagglutinin antigen. Determine the content of haemagglutinin antigen by an immunodiffusion test (2.2.14), by comparison with a haemagglutinin antigen reference preparation or with an antigen preparation calibrated against it. Carry out the test at 20 to 25. Neuraminidase antigen. The presence and type of neuraminidase antigen are confirmed by suitable enzymatic or immunological methods (2.2.14) on the first three monovalent pooled harvests from each working seed lot. Sterility (2.2.11). Carry out the test for sterility, using 10 ml for each medium. Viral inactivation. Carry out the test described below under Tests. Purity. The purity of the monovalent pooled harvest is examined by polyacrylamide gel electrophoresis or by other approved techniques. Mainly haemagglutinin and neuraminidase antigens shall be present. Chemicals used for disruption and purification. Tests are carried out on the monovalent pooled harvest for the chemicals used for disruption and purification, the limits being approved by the competent authority. FINAL BULK VACCINE Appropriate quantities of the monovalent pooled harvests are blended to make the final bulk vaccine. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Carry out the test for sterility using 10 ml for each medium. FINAL LOT The final bulk vaccine is distributed aseptically into sterile, tamper-proof containers. The containers are closed so as to prevent contamination. 57
Only a final lot that is satisfactory with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided that the test for viral inactivation has been performed with satisfactory results on each monovalent pooled harvest and that the tests for free formaldehyde, ovalbumin and total protein have been performed with satisfactory results on the final bulk vaccine, they may be omitted on the final lot. Description. The vaccine is a clear liquid.
Identification
Tests
Viral inactivation. Inoculate 0.2 ml of the vaccine into the allantoic cavity of each of ten fertilised eggs and incubate at 33 to 37 for 3 days. The test is not valid unless at least eight of the ten embryos survive. Harvest 0.5 ml of the allantoic fluid from each surviving embryo and pool the fluids. Inoculate 0.2 ml of the pooled fluid into a further ten fertilised eggs and incubate at 33 to 37 for 3 days. The test is not valid unless at least eight of the ten embryos survive. Harvest about 0.1 ml of the allantoic fluid from each surviving embryo and examine each individual harvest for live virus by a haemagglutination test. If haemagglutination is found for any of the fluids, carry out for that fluid a further passage in eggs and test for haemagglutination; no haemagglutination occurs. Total protein (2.3.49). Not more than 40 g of protein other than haemagglutinin per virus strain per human dose and not more than a total of 120 g of protein other than haemagglutinin per human dose. Ovalbumin. Not more than 1 g of ovalbumin per human dose, determined by a suitable technique using a suitable reference preparation of ovalbumin. Free formaldehyde (2.3.20). Complies with the test for free formaldehyde as stated under General Requirements for Vaccines for Human Use. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity. Sterility (2.2.11). Complies with the test for sterility. Bacterial endotoxins (2.2.3). Not more than 100 IU of endotoxin per human dose. Assay Determine the content haemagglutinin antigen by an
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immunodiffusion test (2.2.14), by comparison with an appropriate haemagglutinin antigen reference preparation. Carry out the test at 20 to 25. The confidence interval (P = 0.95) of the assay is not greater than 80.0 per cent to 125.0 per cent of the estimated content. The lower confidence limit (P = 0.95) of the estimate of haemagglutinin antigen content is not less than 80.0 per cent of the amount stated on the label for each strain. Labelling. The label states (1) that the vaccine has been prepared on eggs; (2) the strain or strains of influenza virus used to prepare the vaccine; (3) the method of inactivation; (4) the haemagglutinin content in micrograms per virus strain per dose; (5) the season during which the vaccine is intended to protect.
SEED LOT The production of vaccine is based on a seed-lot system. Working seed lots represent not more than fifteen passages from the approved reassorted virus or the approved virus isolate. The final vaccine represents one passage from the working seed lot. The haemagglutinin and neuraminidase antigens of each seed lot are identified as originating from the correct strain of influenza virus by suitable methods. Only a working virus seed lot that complies with the following requirements may be used in the preparation of the monovalent pooled harvest. Sterility (2.2.11). Carry out the test for sterility using 10 ml for each medium. Mycoplasmas (2.7.4). Carry out the test for mycoplasmas using 10 ml.
Inactivated Influenza Vaccine (Whole Virion)

Influenza Vaccine (Whole Virion, Inactivated) is a sterile, aqueous suspension of a strain or strains of influenza virus, type A or B, or a mixture of strains of the two types grown individually in eggs derived from specific pathogen free flocks or cell culture and inactivated in such a manner that their antigenic properties are retained. The stated amount of haemagglutinin antigen for each strain present in the vaccine is 15 g per dose, unless clinical evidence supports the use of a different amount.
PROPAGATION AND HARVEST An antimicrobial agent may be added to the inoculum. After incubation at a controlled temperature, the allantoic fluids are harvested and combined to form a monovalent pooled harvest. An antimicrobial agent may be added at the time of harvest. At no stage in the production is penicillin or streptomycin used. MONOVALENT POOLED HARVEST To limit the possibility of contamination, inactivation is initiated as soon as possible after preparation. The virus is inactivated by a method that has been demonstrated on three consecutive batches to be consistently effective for the manufacturer. The inactivation process shall have been shown to be capable of inactivating the influenza virus without destroying its antigenicity; the process should cause minimum alteration of the haemagglutinin and neuraminidase antigens. The inactivation process shall also have been shown to be capable of inactivating avian leucosis viruses and mycoplasmas. If the monovalent pooled harvest is stored after inactivation, it is held at a temperature of 53. If formaldehyde solution is used, the concentration does not exceed 0.2 g/l of formaldehyde at any time during inactivation; if betapropiolactone is used, the concentration does not exceed 0.1 per cent v/v at any time during inactivation. Before or after the inactivation process, the monovalent pooled harvest is concentrated and purified by high-speed centrifugation or other suitable method. Only a monovalent pooled harvest that complies with the following requirements may be used in the preparation of the final bulk vaccine. Haemagglutinin antigen. Determine the content of haemagglutinin antigen by an immunodiffusion test (2.2.14), by comparison with a haemagglutinin antigen reference 58
Production
The production method is validated to demonstrate that the product, if tested, would comply with the test for safety and efficacy. Choice of vaccine strain The World Health Organisation reviews the world epidemiological situation annually and, if necessary, recommends new strains corresponding to prevailing epidemiological evidence. The origin and passage history of virus strains shall be approved by the competent authority. Substrate for virus propagation Influenza virus seed to be used in the production of vaccine is propagated in fertilised eggs from chicken flocks free from specified pathogens or in suitable cell cultures, such as chickembryo fibroblasts or chick kidney cells obtained from chicken flocks free from specified pathogens .For production, the virus of each strain is grown in the allantoic cavity of eggs derived from specific pathogen free flocks.
IP 2007
JAPANESE ENCEPHALITIS VACCINE (HUMAN)
preparation or with an antigen preparation calibrated against it. Carry out the test at 20 to 25. Neuraminidase antigen. The presence and type of neuraminidase antigen are confirmed by suitable enzymatic or immunological methods (2.2.14) on the first three monovalent pooled harvests from each working seed lot. Sterility (2.2.11). Carry out the test for sterility using 10 ml for each medium. Viral inactivation. Carry out the test described below under Tests. FINAL BULK VACCINE Appropriate quantities of the monovalent pooled harvests are blended to make the final bulk vaccine. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT The final bulk vaccine is distributed aseptically into sterile, tamper-proof containers. The containers are closed so as to prevent contamination. Only a final lot that is satisfactory with respect to each of the requirements given below under Tests and Assay may be released for use. Provided that the test for viral inactivation has been performed with satisfactory results on each monovalent pooled harvest and that the tests for free formaldehyde, ovalbumin and total protein have been performed with satisfactory results on the final bulk vaccine, they may be omitted on the final lot. Description. The vaccine is a slightly opalescent liquid.
the allantoic fluid from each surviving embryo and examine each individual harvest for live virus by a haemagglutination test. If haemagglutination is found for any of the fluids, carry out for that fluid a further passage in eggs and test for haemagglutination; no haemagglutination occurs. Total protein (2.3.49). Not more than six times the total haemagglutinin content of the vaccine as determined in the assay, but in any case, not more than 100 g of protein per virus strain per human dose and not more than a total of 300 g of protein per human dose. Ovalbumin. Not more than 1 g of ovalbumin per human dose, determined by a suitable technique using a suitable reference preparation of ovalbumin. Free formaldehyde (2.3.20). Maximum 0.2 g/l. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than the minimum amount shown to be effective and is not greater than 115.0 per cent of the quantity stated on the label. Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity. Sterility (2.2.11). Complies with the test for sterility. Bacterial endotoxins (2.2.3). Not more than 100 IU of endotoxin per human dose. Assay Determine the content of haemagglutinin antigen by an immunodiffusion test (2.2.14), by comparison with an appropriate haemagglutinin antigen reference preparation. Carry out the test at 20 to 25. The confidence interval (P = 0.95) of the assay is not greater than 80.0 per cent to 125.0 per cent of the estimated content. The lower confidence limit (P = 0.95) of the estimate of haemagglutinin antigen content is not less than 80.0 per cent of the amount stated on the label for each strain. Labelling. The label states (1) that the vaccine has been prepared on eggs; (2) the strain or strains of influenza virus used to prepare the vaccine; (3) the method of inactivation; (4) the haemagglutinin content in micrograms per virus strain per dose; (5) the season during which the vaccine is intended to protect.
Identification
Tests
Viral inactivation. Inoculate 0.2 ml of the vaccine into the allantoic cavity of each of ten fertilised eggs and incubate at 33 to 37 for 3 days. The test is not valid unless at least eight of the ten embryos survive. Harvest 0.5 ml of the allantoic fluid from each surviving embryo and pool the fluids. Inoculate 0.2 ml of the pooled fluid into a further ten fertilised eggs and incubate at 33 to 37 for 3 days. The test is not valid unless at least eight of the ten embryos survive. Harvest about 0.1 ml of 59
Japanese Encephalitis Vaccine (Human)

Japanese Encephalitis Vaccine for human use is a liquid or freeze dried preparation of Japanese encephalitis virus grown in approved substrate and inactivated by a validated method.
IP 2007
Production
General provisions The vaccine is produced on the basis of virus seed lot system The production method shall have been shown to yield consistently the vaccines that comply with the tests for immunogenicity, safety and stability. The production method is validated to demonstrate that the product, if tested, would comply with the test for safety and efficacy. Substrate for virus propagation The virus is propagated in an approved cell substrate like a Vero cell line. SEED LOT The strain of Japanese encephalitis virus used shall be identified by historical records that include information on the origin of the strain and its subsequent manipulation. The National Regulatory Authority shall determine the acceptable number of passages from the master virus seed lot to produce working virus seed lots. Only a working seed lot that complies with the following tests may be used for virus propagation.
if present are reduced to an acceptable level by suitable method of purification. Serum and trypsin used in the preparation of cell suspension and media are shown to be free from infectious extraneous agents. The cell culture media may contain a pH indicator such as phenol red. Not less than 500 ml of the cell cultures employed for vaccine production are set aside as uninfected cell cultures (control cells). The virus suspension is harvested on one or more occasions during incubation. Multiple harvests from the same production cell culture may be pooled and considered as a single harvest. Virus harvests that comply with the tests given under Identification and Virus concentration are pooled in the preparation of the inactivated viral harvest. Control cells. The control cells of the production cell culture from which the single harvest is derived should comply with the test for identification and with the tests for extraneous agents (2.7.3). Purification and inactivation The virus harvest may be concentrated and/or purified by suitable methods; the virus harvest is inactivated by a validated method at a fixed, well defined stage of the process which may be before, during or after any concentration or purification. The method shall have been shown to be capable of inactivating Japanese encephalitis virus without destruction of the immunogenic activity. If formalin is used, the concentration shall at no time exceed 1:2000. Only an inactivated viral suspension that complies with the following tests may be used in the preparation of the final bulk vaccine. Inactivation. Inactivation is confirmed by carrying out an amplification test for residual infectious Japanese encephalitis virus. Inoculate a quantity of inactivated viral suspension equivalent to not less than 25 doses into cell cultures of the same type as those used for production of the vaccine. Make a passage after 7 days. Maintain the cultures for a further period of 14 days and then examine the cell cultures for Japanese encephalitis virus using an immunofluorescence test. No Japanese encephalitis virus is detected. Alternatively, 5 ml of each culture fluid is pooled on day 14 and 21 and 0.03 ml is inoculated intracerebrally into each of the 10 mice weighing between 12 and 15 g. The mice are observed for 14 days for symptoms caused by Japanese encephalitis virus, and mice showing symptoms of Japanese encephalitis virus are sacrificed and virus presence is confirmed by immunofluorescence test. No Japanese encephalitis virus shall be detected. Residual host-cell DNA (2.2.15). The content of residual hostcell DNA, determined using a suitable method, should not be greater than 10 ng per single human dose if cells are used in the production. 60
Identification
Each working seed lot is identified as Japanese encephalitis virus using specific antibodies by an approved method. Virus concentration. The virus concentration of each working seed lot is determined by a cell culture method using immunofluoresence or any other approved method. Extraneous agents (2.7.3). The working seed lot complies with the tests for the virus seed lots. PROPAGATION AND HARVEST a) Mouse brain vaccine The vaccine is prepared by using a seed-lot system. An approved strain of virus is grown by inoculating intracerebrally into healthy mice. Virus harvests are pooled, concentrated and inactivated by addition of formalin or any other suitable inactivating agent. It may contain a suitable preservative. The vaccine may be issued in single or multidose containers. b) Cell culture vaccine All processing of the cell bank and subsequent cell cultures are done under aseptic conditions in an area where no other cells are handled. Approved animal (but not human) serum may be used in the media, but the final medium for maintaining cell growth during virus multiplication does not contain animal serum; the media may contain human albumin. Serum proteins,
IP 2007
FINAL BULK VACCINE The final bulk vaccine is prepared from one or more inactivated viral suspensions. An approved stabilizer may be added to maintain the activity of the product. Thiomersal can be used as preservative. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Formaldehyde (2.3.20). Not more than 0.01 per cent w/v. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT The final bulk vaccine is distributed aseptically into sterile containers. The containers are then sealed so as to prevent contamination. Only a final lot that complies with each of the tests given under Identification, Tests and Assay may be released for use. Provided that the test for inactivation has been carried out with satisfactory results on the inactivated virus suspension and the test for bovine serum albumin has been carried out with satisfactory results on the final bulk vaccine, these tests may be omitted on the final lot.
Antimicrobial preservative. Determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than the minimum amount shown to be effective and is not greater than 115.0 per cent of that stated on the label. Biological assay Potency of Japanese encephalitis virus vaccine is determined by titrating the neutralizing antibodies produced in the immunized mice by plaque reduction method or serum neutralization test (SNT) using appropriate cell culture. Standard preparation The Standard preparation is a freeze-dried Japanese encephalitis virus vaccine the potency of which has been determined in relation to the Japanese encephalitis reference vaccine obtained from the National Institute of Health, Tokyo, Japan. Suggested method Preparation of challenge virus suspension The approved challenge virus strain is stored in freeze-dried form or in liquid form stored below -70 . Prepare a working pool of the challenge virus strain by inoculating intracerebrally 0.03 ml of 100 fold dilution of the standard strain in Hanks balanced salt solution containing 5 per cent calf serum into a suitable number of 2-day old suckling mice. Sacrifice the animals after they show characteristic symptoms of encephalitis and become moribund. Harvest their brains aseptically, wash them in chilled sterile saline solution to remove blood clots. Homogenize the brains with Hanks balanced salt solution containing 5 per cent calf serum to make a 10 per cent emulsion. Centrifuge the emulsion at 2000 g for 30 minutes. Dilute the supernatant with Hanks balanced salt solution containing 5 per cent calf serum so as to contain about 200 Plaque-Forming Units (PFU) of the virus per 0.4 ml. Determination of potency Prepare appropriate dilutions of the vaccine under examination and of the Standard Preparation in a suitable medium. Inject intraperitoneally in two doses of 0.5 ml each at 7-day interval into at least 20 mice of 4 weeks of age. Bleed each mouse 7 days after the second injection, pool the separated serum from each group and inactivate the sera by heating at 56 for 30 minutes. The inactivated sera may be stored at -20, if necessary. Dilute the sera appropriately, e.g. 1:40. 1:160, 1:640 etc., mix with an equal volume of the challenge virus suspension and incubate at 37 for 90 minutes for neutralization. Inoculate the mixture into cell cultures and overlay the infected cells with 1 per cent agar. After incubation for an appropriate time (about 61
Identification
The vaccine is shown to contain Japanese encephalitis virus antigen by a suitable immunochemical method using specific antibodies, alternatively, the Assay also serves to identify the vaccine.
Tests
Complies with the test for Inactivation under final Purification and Inactivation. Sterility (2.2.11). Complies with the test for sterility. Bacterial endotoxins (2.2.3). Less than 25 IU per single human dose. Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity. Bovine serum albumin (for cell culture vaccine). Not more than 50 ng per single human dose, determined by a suitable immunochemical method (2.2.14). Residual host-cell DNA (for continuous cell line vaccines) (2.2.15). Not more than 10 ng per single human dose. Free formaldehyde (2.3.20). Not more than 0.01 per cent w/v.
MEASLES AND RUBELLA VACCINE (LIVE)
IP 2007
48 hours), stain the cells and count the number of plaques formed on the cultures to obtain the plaque reduction rates for the vaccine under examination and the Standard preparation. Calculate the neutralizing antibody titres for each group using standard statistical methods (5.7). The test is not valid unless (a) the mean number of plaques obtained with the Standard preparation is between 100 and 150 per dish and (b) the potency of the vaccine under examination is not less than that of the Standard preparation. Labelling. The label states (1) the biological origin of the cells used for the preparation of the vaccine; (2) the strain of virus used.
following test for thermal stability and with each of the tests given below under Identification and Tests and Assay may be released for use. Provided that the test for bovine serum albumin has been carried out with satisfactory results on the final bulk vaccine, it may be omitted on the final lot. Thermal stability. Maintain samples of the final lot of freezedried vaccine in the dry state at 37 for 7 days. Determine the virus concentration as described under Assay in parallel for the vaccine held at 37 for 7 days and for vaccine stored at 2 to 8. For each component, the virus concentration of the heated vaccine is not more than 1.0 log10 lower than that of the unheated vaccine.
Identification
Measles and Rubella Vaccine (Live)

Measles and Rubella Vaccine (Live) is a freeze-dried preparation of suitable attenuated strains of measles virus and rubella virus grown in suitable cell cultures. The vaccine is reconstituted immediately before use to give a clear liquid that may be coloured owing to the presence of a pH indicator.
When the vaccine reconstituted as stated on the label is mixed with antibodies specific for measles virus and rubella virus, it is no longer able to infect cell cultures susceptible to these viruses. When the vaccine reconstituted as stated on the label is mixed with quantities of specific antibodies sufficient to neutralize any one viral components, the second viral component infects susceptible cell cultures.
Tests
Water (2.3.43). Not more than 3.0 per cent, determined by Karl Fischer, semi-micro determination of water or by any suitable validated method. Sterility (2.2.11). The reconstituted vaccine complies with the test for sterility. Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity. Bovine serum albumin. Not more than 50 ng per single human dose, determined by a suitable immunochemical method (2.2.14). Assay A. Mix the vaccine with a sufficient quantity of antibodies specific for rubella virus.Titrate the vaccine for infective measles virus at least in triplicate, using at least eight cell cultures for each dilution 0.5 log10 step or by a method of equal precision. Use an appropriate virus reference preparation to validate each assay. The estimated measles virus concentration is not less than that stated on the label; the minimum measles virus concentration stated on the label is not less than 1x103 CCID50 per single human dose. The assay is not valid if the confidence limits (P = 0.95) of the logarithm of the virus concentration are greater than 0.3. Measles vaccine (Live) RS is suitable for use as a reference preparation. B. Titrate the vaccine for infective rubella virus at least in triplicate, using at least eight cell cultures for each 0.5 log10 62
Production
General provisions The two components are prepared as described in the monographs on Measles vaccine (live) and Rubella vaccine (live) and comply with the tests prescribed therein. The production method is validated to demonstrate that the product, if tested, would comply with the test for safety and efficacy. FINAL BULK VACCINE Virus harvests for each component are pooled and clarified to remove cells. A suitable stabilizer may be added and the pooled harvests diluted as appropriate. Suitable quantities of the pooled harvest for each component are mixed. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT For each component, a minimum virus concentration for release of the product is established such as to ensure, in the light of stability data, that the minimum concentration stated on the label will be present at the end of the period of validity. Only a final lot that complies with the tests for minimum virus concentration of each component for release, with the
IP 2007
MEASLES VACCINE (LIVE)
dilution step or by a method of equal precision. Use an appropriate virus reference preparation to validate each assay. The estimated rubella virus concentration is not less than that stated on the label; the minimum rubella virus concentration stated on the label are not less than 1x103 CCID50 per single human dose. The assay is not valid if the confidence limits (P = 0.95) of the logarithm of the virus concentration are greater than 0.3. Rubella vaccine (Live) RS is suitable for use as a reference preparation. Labelling. The label states (1) the strains of virus used in the preparation of the vaccine; (2) the type and origin of the cells used for the preparation of the vaccine; (3) the minimum virus concentration for each component of the vaccine; (4) the time within which the vaccine must be used after reconstitution; (5) that the vaccine must not be given to a pregnant woman and that a woman should not become pregnant within two months after having the vaccine.
information on the origin of the strain and its subsequent manipulation. Virus seed lots are prepared in large quantities and stored at temperatures below -20 if freeze-dried, or below -60 if not freeze-dried. Only a seed lot that complies with the following tests may be used for virus propagation.
Identification
The master and working seed lots are identified as measles virus by serum neutralization in cell culture, using specific antibodies. Virus concentration. The virus concentration of the master and working seed lots is determined to monitor consistency of production. Extraneous agents (2.7.3). The working seed lot complies with the tests for seed lots. Neurovirulence (2.7.5). The master/working seed lot complies with the test for neurovirulence of live virus vaccines. Macaca and Cercopithecus monkeys susceptible to measles virus are suitable for the test. PROPAGATION AND HARVEST All processing of the cell bank and subsequent cell cultures is done under aseptic conditions in an area where no other cells are handled. Suitable animal (but not human) serum may be used in the growth medium, but the final medium for maintaining cell growth during virus multiplication does not contain animal serum. Serum and trypsin used in the preparation of cell suspensions and culture media are shown to be free from extraneous agents. The cell culture medium may contain a pH indicator such as phenol red and suitable antibiotics at the lowest effective concentration. It is preferable to have a substrate free from antibiotics during production. Not less than 500 ml of the production cell culture is set aside as uninfected cell cultures (control cells). The viral suspensions are harvested at a time appropriate to the strain of virus being used. Only a single harvest that complies with the following tests may be used in the preparation of the final bulk vaccine.
Measles Vaccine (Live)

Measles Vaccine (Live) is a freeze-dried preparation of a suitable attenuated strain of measles virus. The vaccine is reconstituted immediately before use, as stated on the label, to give a clear liquid that may be coloured owing to the presence of a pH indicator.
Production
General provisions The production of vaccine is based on a virus seed-lot system and, if the virus is propagated in human diploid cells, a cellbank system. Unless otherwise justified and authorized, the virus in the final vaccine shall have undergone no more passages from the master seed lot than were used to prepare the vaccine shown in clinical studies to be satisfactory with respect to abnormal toxicity and efficacy; even with authorized exceptions, the number of passages beyond the level used for clinical studies shall not exceed five. The production method is validated to demonstrate that the product, if tested, would comply with the tests for abnormal toxicity and efficacy. Substrate for virus propagation The virus is propagated in human diploid cells or in cultures of chick embryo cells derived from a chicken flock free from specified pathogens. SEED LOT The strain of measles virus used in the production of measles vaccine shall be identified by historical records that include 63
Identification
The single harvest contains virus that is identified as measles virus by serum neutralisation in cell culture, using specific antibody. Virus concentration. The virus concentration in the single harvest is determined as prescribed under Assay to monitor consistency of production and to determine the dilution to be used for the final bulk vaccine. Extraneous agents (2.7.3). Complies with the test for extraneous agents.
MEASLES, MUMPS AND RUBELLA VACCINE (LIVE)
IP 2007
Control cells. If human diploid cells are used for production, the control cells comply with the test for identification and extraneous agents. FINAL BULK VACCINE Virus harvests that comply with the above tests are pooled and clarified to remove cells. A suitable stabilizer may be added and the pooled harvests diluted as appropriate. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Sterility (2.2.11). The final bulk vaccine complies with the test for sterility carried out using 10 ml for each medium. FINAL LOT A minimum virus concentration for release of the product is established so as to ensure, in the light of stability data, that the minimum concentration stated on the label will be present at the end of the period of validity. Only a final lot that complies with the tests for minimum virus concentration for release, with the following requirement for thermal stability and with each of the requirements given below under Identification, Tests and Assay may be released for use. Provided that the test for bovine serum albumin has been carried out with satisfactory results on the final bulk vaccine, it may be omitted on the final lot. Thermal stability. Maintain samples of the final lot of freezedried vaccine in the dry state at 37 for 7 days. Determine the virus concentration as described under Assay in parallel for the vaccine held at 37 for 7 days and for vaccine stored at 2 to 8. The virus concentration of the heated vaccine is not more than 1.0 log10 lower than that of the unheated vaccine.
Assay Titrate the vaccine for infective virus at least in triplicate, using at least five cell cultures for each 0.5 log10 dilution step or by a method of equal precision. Use an appropriate virus reference preparation to validate each assay. The estimated virus concentration is not less than that stated on the label; the minimum virus concentration stated on the label is not less than 1 103 CCID50 per human dose. The assay is not valid if the confidence limits (P = 0.95) of the logarithm of the virus concentration is greater than 0.3. Measles vaccine (Live) RS is suitable for use as a reference preparation. Labelling. The label states (1) the strain of virus used for the preparation of the vaccine; (2) the type and origin of the cells used for the preparation of the vaccine; (3) the minimum virus concentration; (4) the time within which the vaccine must be used after reconstitution.
Measles, Mumps and Rubella Vaccine (Live)

Measles, Mumps and Rubella Vaccine (Live) is a freeze-dried preparation of suitable attenuated strains of measles virus, mumps virus and rubella virus grown in suitable cell cultures. The vaccine is reconstituted immediately before use to give a clear liquid that may be coloured owing to the presence of a pH indicator.
Production
General provisions The three components are prepared as described in the monographs on Measles Vaccine (Live), Mumps Vaccine (Live) and Rubella Vaccine (Live) and comply with the tests prescribed therein. The production method is validated to demonstrate that the product, if tested, would comply with the test for safety and efficacy. FINAL BULK VACCINE Virus harvests for each component are pooled and clarified to remove cells. A suitable stabilizer may be added and the pooled harvests diluted as appropriate. Suitable quantities of the pooled harvest for each component are mixed. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Sterility (2.2.11). Carry out the test for sterility using 10 ml for each medium. 64
Identification
When the vaccine reconstituted as stated on the label is mixed with specific measles antibodies, it is no longer able to infect susceptible cell cultures.
Tests
Water (2.3.43). Not more than 3.0 per cent, determined by Karl Fischer, semi-micro determination of water or by any suitable validated method. Sterility ( 2.2.11). The reconstituted vaccine complies with the test for sterility. Abnormal toxicity ( 2.2.1). Complies with the test for abnormal toxicity. Bovine serum albumin. Not more than 50 ng per single human dose, determined by a suitable immunochemical method (2.2.14).
IP 2007
MENINGOCOCCAL POLYSACCHARIDE VACCINE
FINAL LOT For each component, a minimum virus concentration for release of the product is established such as to ensure, in the light of stability data, that the minimum concentration stated on the label will be present at the end of the period of validity. Only a final lot that complies with the tests for minimum virus concentration of each component for release, with the following requirement for thermal stability and with each of the requirements given below under Identification and Tests may be released for use. Provided that the test for bovine serum albumin has been carried out with satisfactory results on the final bulk vaccine, it may be omitted on the final lot. Thermal stability. Maintain samples of the final lot of freezedried vaccine in the dry state at 37 for 7 days. Determine the virus concentration as described under Assay in parallel for the vaccine held at 37 for 7 days and for vaccine stored at 2 to 8. The virus concentration of the heated vaccine is not more than 1.0 log10 lower than that of the unheated vaccine.
is not less than 1x103 CCID50 per single human dose. The assay is not valid if the confidence limits (P = 0.95) of the logarithm of the virus concentration are greater than 0.3. Measles vaccine (Live) RS is suitable for use as a reference preparation. B. Mix the vaccine with a sufficient quantity of antibodies specific for measles virus and rubella virus. Titrate the vaccine for infective mumps virus at least in triplicate, using at least eight cell cultures for each dilution 0.5 log10 step or by a method of equal precision. Use an appropriate virus reference preparation to validate each assay. The estimated mumps virus concentration is not less than that stated on the label; the minimum mumps virus concentration stated on the label is not less than 5 x 103 CCID50 per single human dose. The assay is not valid if the confidence limits (P = 0.95) of the logarithm of the virus concentration are greater than 0.3. Mumps vaccine (Live) RS is suitable for use as a reference preparation. C. Mix the vaccine with a sufficient quantity of antibodies specific for mumps virus. Titrate the vaccine for infective rubella virus at least in triplicate, using at least eight cell cultures for each dilution 0.5 log10 step or by a method of equal precision. Use an appropriate virus reference preparation to validate each assay. The estimated rubella virus concentration is not less than that stated on the label; the minimum rubella virus concentration stated on the label is not less than 1x103 CCID50 per single human dose. The assay is not valid if the confidence limits (P = 0.95) of the logarithm of the virus concentration are greater than 0.3. Rubella vaccine (Live) RS is suitable for use as a reference preparation. Labelling. The label states (1) the strains of virus used in the preparation of the vaccine; (2) the type and origin of the cells used for the preparation of the vaccine; (3) the minimum virus concentration for each component of the vaccine; (4) the time within which the vaccine must be used after reconstitution; (5) that the vaccine must not be given to a pregnant woman and that a woman should not become pregnant within two months after having the vaccine.
Identification
When the vaccine reconstituted as stated on the label is mixed with antibodies specific for measles virus, mumps virus and rubella virus, it is no longer able to infect cell cultures susceptible to these viruses. When the vaccine reconstituted as stated on the label is mixed with quantities of specific antibodies sufficient to neutralize any two viral components, the third viral component infects susceptible cell cultures.
Tests
Water (2.3.43). Not more than 3.0 per cent, determined by Karl Fischer, semi-micro determination of water or by any suitable validated method. Sterility (2.2.11). The reconstituted vaccine complies with the test for sterility. Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity. Bovine serum albumin. Not more than 50 ng per single human dose, determined by a suitable immunochemical method (2.2.14). Assay A. Mix the vaccine with a sufficient quantity of antibodies specific for mumps virus and rubella virus.Titrate the vaccine for infective measles virus at least in triplicate, using at least eight cell cultures for each dilution 0.5 log10 step or by a method of equal precision. Use an appropriate virus reference preparation to validate each assay. The estimated measles virus concentration is not less than that stated on the label; the minimum measles virus concentration stated on the label 65
Meningococcal Polysaccharide Vaccine

Meningococcal Polysaccharide Vaccine is a freeze-dried preparation of one or more purified capsular polysaccharides obtained from one or more suitable strains of Neisseria meningitidis group A, group C, group Y and group W135 that are capable of consistently producing polysaccharides known to be safe and effective in man.
IP 2007
N. meningitidis group A polysaccharide consists of partly O-acetylated repeating units of N-acetylmannosamine, linked with 16 phosphodiester bonds. N. meningitidis group C polysaccharide consists of partly O-acetylated repeating units of sialic acid, linked with 29 glycosidic bonds. N. meningitidis group Y polysaccharide consists of partly O-acetylated alternating units of sialic acid and D-glucose, linked with 26 and 14 glycosidic bonds. N. meningitidis group W135 polysaccharide consists of partly O-acetylated alternating units of sialic acid and D-galactose, linked with 26 and 14 glycosidic bonds.
medium. The liquid media used and the final medium are semisynthetic and free from substances precipitated by cetrimonium bromide (hexadecyltrimethylammonium bromide) and do not contain blood-group substances or highmolecular-mass polysaccharides. The bacterial purity of the culture is verified by microscopic examination of Gram-stained smears and by inoculation into appropriate media. The cultures are centrifuged and the polysaccharides precipitated from the supernatant by addition of cetrimonium bromide. The precipitate obtained is harvested and may be stored at or below -20 awaiting further purification. PURIFIED POLYSACCHARIDES The polysaccharides are purified, after dissociation of the complex of polysaccharide and cetrimonium bromide, using suitable procedures to remove successively nucleic acids, proteins and lipopolysaccharides. The purification step consists of ethanol precipitation of the polysaccharides or purification with chloroform and n-butanol or by cold phenol treatment, which are then dried and stored at or below -20. The loss on drying is determined by thermogravimetry, Karl Fischer or any other suitable method and the value is used to calculate the results of the other chemical tests with reference to the dried substance. Only purified polysaccharides that tested comply with the following requirements may be used in the preparation of the final bulk vaccine. Protein (2.7.1). Not more than 10 mg of protein per gram of purified polysaccharidefor group A and C organisms and less than 50 mg of protein per gram of polysaccharide for group Y and W135 calculated using bovine plasma albumin as a reference or other methods approved by National Regulatory Authority. Nucleic acids (2.7.1). Not more than 10 mg of nucleic acids per gram of purified polysaccharide, calculated with reference to the dried substance. O-Acetyl groups (2.7.1). Not less than 2 mmol of O-acetyl groups per gram of purified polysaccharide for group A, not less than 1.5 mmol per gram of polysaccharide for group C, not less than 0.3 mmol per gram of polysaccharide for groups Y and W135, all calculated with reference to the dried substance. Phosphorus (2.7.1). Not less than 80 mg of phosphorus per gram of group A purified polysaccharide, calculated with reference to the dried substance. Sialic acid (2.7.1). Not less than 800 mg of sialic acid per gram of group C polysaccharide and not less than 560 mg of sialic acid per gram of purified polysaccharide for groups Y and W135, all calculated with reference to the dried substance. Use the following reference solutions: 66
Production
General provisions Production of the meningococcal polysaccharides is based on a well defined seed-lot system. The method of production shall have been shown to yield consistently meningococcal polysaccharide vaccines of satisfactory immunogenicity and safety for man. The production method is validated to demonstrate that the product, if tested, would comply with the test of abnormal toxicity for antisera and vaccines. SEED LOT The strains of N. meningitidis used for the master seed lots shall be identified by historical records that include information on their origin and by their biochemical, serological, physicochemical or molecular characteristics. Cultures from the working seed lot shall have the same characteristics as the strain that was used to prepare the master seed lot. The strains have the following characteristics: a) Colonies obtained from a culture are round, uniform in shape and smooth with a mucous, opalescent, greyish appearance.
b) Gram staining reveals characteristic Gram-negative diplococci in coffee-bean arrangement. c) e) The oxidase test is positive. Suspensions of the culture agglutinate with specific antisera of known titre.
d) The culture utilizes glucose and maltose.
PROPAGATION AND HARVEST The working seed lots are cultured on solid media that do not contain blood-group substances or ingredients of mammalian origin. The inoculum may undergo one or more subcultures in liquid medium before being used for inoculating the final
IP 2007
Group C polysaccharide. A 150 mg/l solution of Nacetylneuraminic acid. Group Y polysaccharide. A solution containing 95 mg/l of Nacetylneuraminic acid and 55 mg/l of glucose. Group W135 polysaccharide. A solution containing 95 mg/l of N-acetylneuraminic acid and 55 mg/l of galactose. Calcium. If a calcium salt is used during purification, determination of calcium is carried out on the purified polysaccharide by a suitable method; the content is within the limits approved for the product. Molecular size. Examine by gel filtration or high performance size-exclusion chromatography (HPSEC) (2.4.16), using agarose for chromatography or cross-linked agarose for chromatography either alone or in combination with light scattering and refractive index detector (e.g. multiple angle LASER light scattering, MALLS) or any other suitable method. Use a column 0.9 m x 15 mm equilibrated with a solvent having an ionic strength of 0.2 mol/kg and a pH of 7.0 to 7.5. Apply to the column about 2.5 mg of polysaccharide in a volume of about 1.5 ml and elute at about 20 ml/h. Collect fractions of about 2.5 ml and determine the content of polysaccharide by a suitable method. At least 65.0 per cent of group A polysaccharide, 75.0 per cent of group C polysaccharide, 80.0 per cent of group Y polysaccharide and 80.0 per cent of group W135 polysaccharide is eluted before a distribution coefficient (K0) of 0.50 is reached. In addition, the percentages eluted before this distribution coefficient are within the limits approved for the particular product.
Only a final bulk vaccine that complies with the following requirement may be used in the preparation of the final lot. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT The final bulk vaccine is distributed aseptically into sterile containers. The containers are then closed so as to avoid contamination. Only a final lot that is satisfactory with respect to each of the requirements prescribed below under Identification, Tests and Assay may be released for use.
Identification
Carry out an identification test for each polysaccharide present in the vaccine by a suitable immunochemical method (2.2.14).
Tests
Sterility (2.2.11). Complies with the test for sterility. Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity. Pyrogens (2.2.8). Complies with the test for pyrogens. Inject each of the rabbit with 1ml per kg body weight of solution containing a) c) 0.025 g of polysaccharide for a monovalent vaccine, 0.075 g of polysaccharide for a trivalent vaccine,
b) 0.050 g of polysaccharide for a bivalent vaccine, d) 0.100 g of polysaccharide for a tetravalent vaccine. Water (2.3.43). Not more than 3.0 per cent, of moisture content by thermogravimetery, Karl Fischer or any other suitable method. Molecular size. Examine by gel filtration or size-exclusion chromatography (2.4.16). Use a column about 0.9 m long and 16 mm in internal diameter equilibrated with a solvent having an ionic strength of 0.2 mol/kg and a pH of 7.0 to 7.5. Apply to the column about 2.5 mg of each polysaccharide in a volume of about 1.5 ml and elute at about 20 ml/h. Collect fractions of about 2.5 ml and determine the content of polysaccharide by a suitable method. Use cross-linked agarose for chromatography and apply a suitable immunochemical method (2.2.14) to establish the elution pattern of the different polysaccharide(s). The vaccine complies with the test if: a) 65.0 per cent of Group A polysaccharide is eluted before K0 of 0.50,
Identification and serological specificity

The identity and serological specificity are determined by a suitable immunochemical method (2.2.14). Identity and purity of each polysaccharide shall be confirmed; it shall be shown that there is not more than 1.0 per cent m/m of groupheterologous N. meningitidis polysaccharide. Pyrogens (2.2.8). Complies with the test for pyrogens. Inject each of the rabbit with 1ml per kg body weight of solution containing a) c) 0.025 g of polysaccharide for a monovalent vaccine, 0.075 g of polysaccharide for a trivalent vaccine,
b) 0.050 g of polysaccharide for a bivalent vaccine, d) 0.100 g of polysaccharide for a tetravalent vaccine. FINAL BULK VACCINE One or more purified polysaccharides of one or more N. meningitidis groups are dissolved in a suitable solvent that may contain a stabilizer. 67
b) 75.0 per cent of Group C polysaccharide is eluted before K0 of 0.50, c) 80.0 per cent of Group Y & W135 polysaccharide is eluted before K0 of 0.50.
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For a tetravalent vaccine (group A + group C + group Y and group W135), use cross linked agarose for chromatography R1 and apply a suitable immunochemical method (2.2.14) to establish the elution pattern of the different polysaccharides. The vaccine complies with the test if K0 for the principal peak is a) not greater than 0.70 for group A and group C polysaccharide, b) not greater than 0.57 for group Y polysaccharide, c) not greater than 0.68 for group W135 polysaccharide. Assay Carry out an assay as stated under each polysaccharide present in the vaccine. For a divalent vaccine (group A + group C), use measurement of phosphorus (2.7.1) to determine the content of polysaccharide A and measurement of sialic acid (2.7.1) to determine the content of polysaccharide C. To determine sialic acid, use as reference solution a 150 mg/l solution of Nacetylneuraminic acid. For a tetravalent vaccine (group A + group C + group Y + group W135) a suitable immunochemical method (2.2.14) is used with a reference preparation of purified polysaccharide for each group. The vaccine contains not less than 70.0 per cent and not more than 130.0 per cent of the quantity of each polysaccharide stated on the label. Labelling. The label states (1) the group or groups of polysaccharides (A, C, Y or W135) present in the vaccine; (2) the number of g of polysaccharide per human dose.
shown in clinical studies to be satisfactory with respect to safety and efficacy even with authorized exceptions, the number of passages beyond the level used for clinical studies shall not exceed five. The production method is validated to demonstrate that the product, if tested, would comply with the tests for safety and efficacy. Substrate for virus propagation The virus is propagated in human diploid cells or in primary cultures of chick embryo cells derived from a chicken flock free from specified pathogens. SEED LOT The strain of mumps virus used shall be identified by historical records that include information on the origin of the strain and its subsequent manipulation. To avoid unnecessary use of monkeys in the test for neurovirulence, Virus seed lots are prepared in large quantities and stored at temperatures below -20 if freeze-dried, or below -60 if not freeze-dried. Only a seed lot that complies with the following tests may be used for virus propagation.
Identification
The master and working seed lots are identified as mumps virus by serum neutralisation in cell culture, using specific antibodies. Virus concentration. The virus concentration of the master and working seed lots is determined to ensure consistency of production. Extraneous agents (2.7.3). The working seed lot complies with the tests for seed lots. Neurovirulence (2.7.5). The master/working seed lot complies with the test for neurovirulence of live virus vaccines. Macaca and Cercopithecus monkeys are suitable for the test. PROPAGATION AND HARVEST All processing of the cell bank and subsequent cell cultures is done under aseptic conditions in an area where no other cells are handled. Suitable animal (but not human) serum may be used in the growth media. Serum and trypsin used in the preparation of cell suspensions and culture media are shown to be free from extraneous agents. The cell culture medium may contain a pH indicator such as phenol red and suitable antibiotics at the lowest effective concentration. It is preferable to have a substrate free from antibiotics during production. Not less than 500 ml of the production cell culture is set aside as uninfected cell culture (control cells). The viral suspensions are harvested at a time appropriate to the strain of virus being used. 68
Mumps Vaccine (Live)

Mumps Vaccine (Live) is a freeze-dried preparation of a suitable attenuated strain of mumps virus. The vaccine is reconstituted immediately before use to give a clear liquid that may be coloured owing to the presence of a pH indicator.
Production
General provisions The production of vaccine is based on a virus seed-lot system and, if the virus is propagated in human diploid cells, a cellbank system. The production method shall have been shown to yield consistently live mumps vaccines of adequate immunogenicity and safety in man. Unless otherwise justified and authorised, the virus in the final vaccine shall have undergone no more passages from the master seed lot than were used to prepare the vaccine
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PERTUSSIS VACCINE
Only a single harvest that complies with the following requirements may be used in the preparation of the final bulk vaccine.
Tests
Water (2.3.43). Not more than 3.0 per cent, determined by Karl Fischer, semi-micro determination of water or by any suitable validated method. Sterility ( 2.2.11). Complies with the test for sterility. Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity. Bovine serum albumin. Not more than 50 ng per single human dose, determined by a suitable immunochemical method (2.2.14). Assay Titrate the vaccine for infective virus at least in triplicate, using at least five cell cultures for each 0.5 log10 dilution step or by a method of equal precision. Use an appropriate virus reference preparation to validate each assay. The estimated virus concentration is not less than that stated on the label; the minimum virus concentration stated on the label is not less than 5 x 103 CCID50 per human dose. The assay is not valid if the confidence limits (P = 0.95) of the logarithm of the virus concentration is greater than 0.3. Mumps vaccine (Live) RS is suitable for use as a reference preparation. Labelling. The label states (1) the strain of virus used for the preparation of the vaccine; (2) the type and origin of the cells used for the preparation of the vaccine; (3) the minimum virus concentration and; (4) the time within which the vaccine must be used after reconstitution.
Identification
The single harvest contains virus that is identified as mumps virus by serum neutralization in cell culture, using specific antibodies. Virus concentration. The virus concentration in the single harvest is determined as prescribed under Assay to monitor consistency of production and to determine the dilution to be used for the final bulk vaccine. Sterility (2.2.11). Single harvest complies with sterility test should be processed further. Control cells. The control cells comply with a test for extraneous agents (2.7.3). FINAL BULK VACCINE Single harvests that comply with the above tests are pooled and clarified to remove cells. A suitable stabiliser may be added and the pooled harvests diluted as appropriate. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Sterility (2.2.11). The final bulk vaccine complies with the test for sterility, carried out using 10 ml for each medium. FINAL LOT A minimum virus concentration for release of the product is established such as to ensure, in the light of stability data, that the minimum concentration stated on the label will be present at the end of the period of validity. Only a final lot that complies with the tests for minimum virus concentration for release, with the following requirement for thermal stability and with each of the requirements given below under Identification and Tests and Assay may be released for use. Provided that the test for bovine serum albumin has been carried out with satisfactory results on the final bulk vaccine, it may be omitted on the final lot. Thermal stability. Maintain samples of the final lot of freezedried vaccine in the dry state at 37 for 7 days. Determine the virus concentration as described under Assay in parallel for the vaccine held at 37 for 7 days and for vaccine stored at 2 to 8. The virus concentration of the vaccine exposed to 37 for 7 days is not more than 1.0 log10 lower than that of the unheated vaccine.
Pertussis Vaccine
Pertussis Vaccine is a sterile saline suspension of inactivated whole cells of one or more strains of Bordetella pertussis.
Production
General provisions Inactivated B. pertussis suspension Production is based on a seed-lot system. One or more strains of B. pertussis with known origin and history are used. Strains, culture medium and cultivation method are chosen in such a way that agglutinogens 1, 2 and 3 are present in the final vaccine. Each strain is grown for 24 to 72 hours in a liquid medium or on a solid medium; the liquid medium used in the final cultivation stage does not contain blood or blood products. Human blood or blood products are not used in any culture media. The bacteria are harvested, washed to remove substances derived from the medium and suspended in a 0.9 per cent w/v solution of sodium chloride or other suitable 69
Identification
When the vaccine is reconstituted as stated on the label is mixed with specific mumps antibodies, it is no longer able to infect susceptible cell cultures.
PERTUSSIS VACCINE
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isotonic solution. The opacity of the suspension is determined not later than 2 weeks after harvest by comparison with the reference preparation of Opacity and used as the basis of calculation for subsequent stages in vaccine preparation. Single harvests are not used for the final bulk vaccine unless they have been shown to contain B. pertussis cells with the same characteristics with regard to growth and agglutinogens, as the parent strain and to be free from contaminating bacteria and fungi. The bacteria are killed and detoxified in controlled conditions by means of a suitable chemical agent or by heating or by a combination of these methods. Freedom from live B. pertussis is tested using a suitable culture medium. The suspension is maintained at 5 + 3 for a suitable period to diminish its toxicity. FINAL BULK VACCINE Suitable quantities of the inactivated single harvests are pooled to prepare the final bulk vaccine. Suitable antimicrobial preservatives may be added. The bacterial concentration of the final bulk vaccine does not exceed that corresponding to an opacity of 20 IU per single human dose. If 2 or more strains of B. pertussis are used, the composition of consecutive lots of the final bulk vaccine shall be consistent with respect to the proportion of each strain as measured in opacity units. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The amount is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT The final bulk vaccine is distributed aseptically into sterile, tamper-proof containers. The containers are closed so as to prevent contamination. Only a final lot that is satisfactory with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided the tests for specific toxicity, free formaldehyde and antimicrobial preservative and the assay have been carried out with satisfactory results on the final bulk vaccine, they may be omitted on the final lot.
to 16 g for the vaccine group and for the saline control. Use mice of the same sex or distribute males and females equally between the groups. Allow the animals access to food and water for at least 2 hours before injection and during the test. Inject each mouse of the vaccine group intraperitoneally with 0.5 ml, containing a quantity of the vaccine equivalent to not less than half the single human dose. Inject each mouse of the control group with 0.5 ml of a 0.9 per cent w/v sterile solution of sodium chloride, preferably containing the same amount of antimicrobial preservative as that injected with the vaccine. Weigh the groups of mice immediately before the injection, 72 hours and 7 days after the injection. The vaccine complies with the test if (a) at the end of 72 h the total mass of the group of vaccinated mice is not less than that preceding the injection; (b) at the end of 7 days the average increase in mass per vaccinated mouse is not less than 60.0 per cent of that per control mouse; and (c) not more than 5.0 per cent of the vaccinated mice die during the test. The test may be repeated and the results of the tests combined. Free formaldehyde (2.3.20). Maximum 0.2 g/l. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not more than 115.0 per cent of the intended amount. Sterility (2.2.11). Complies with the test for sterility. Assay Carry out the assay of Pertussis Vaccine as described below : Biological assay of pertussis vaccine The potency of PertussisVaccine is determined by comparison of the dose necessary to protect mice against the effects of a lethal dose of Bordetella pertussis challenge culture, administered intracerebrally, with the dose of a reference preparation, calibrated in International Units, required to give the same level of protection. For this comparison, the Standard preparation of Pertussis vaccine & a suitable strain of B. pertussis (e.g.18323, to be used as challenge strain), are required. Reference preparation The reference preparation is an International standard of Pertussis vaccine, consisting of a freeze dried vaccine or another suitable preparation, calibrated in comparison to International standard, from time to time. The International Unit is the activity contained in a stated amount of the International standard, which consists of a quantity of dried pertussis vaccine. The equivalence in International Units of the International Standard is stated by the World Health Organization. Use healthy mice of a suitable strain, weighing between 13 and 16 g from the same stock. Distribute the mice randomly, in 70
Identification
Identify pertussis vaccine by agglutination of the bacteria in the vaccine by antisera specific to B. pertussis.
Tests
Specific toxicity Use not less than 10 healthy mice each weighing between 14
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PNEUMOCOCCAL POLYSACCHARIDE VACCINE
six to eight groups of not less than 16 and not more than 24 and four groups of 10. The mice should all be of the same sex or the males and females should be distributed equally between the groups. Half of the groups of 16 to 24 should receive the reference preparation and the other half should receive the vaccine under examination. The four groups of 10 each should be used for the LD50 titration of challenge suspension. Use at least, three dilutions of the reference vaccine and similar dilutions of the vaccine under examination. In each case the dilutions are so selected that the dilution protecting 50 per cent of the mice (ED50) is as near as possible to middle of the dilution range. For example, suggested dilutions are (1/8, 1/40 and 1/200 of the human dose of the vaccine under examination) and (0.5 IU, 0.1 IU and 0.02 IU or any other suitable standardized dilutions, of the reference preparation), each dose being contained in a volume, not exceeding 0.5 ml. For each dilution use 16 to 24 mice and inject intraperitoneally, into each mouse one dose of the dilution. Select a suitable strain of B. pertussis (e.g. 18323), capable of causing the death of mice within 14 days of intracerebral injection. Make two subcultures after reviving the strain on a suitable medium (e.g. B.G. medium) and suspend the harvested growth in a solution containing 1 per cent w/v of casein hydrolysate (e.g. casamino acid) and 0.85 per cent w/v of sodium chloride and having a pH of 7.0 to 7.2 or in another suitable solution. Determine the opacity of the suspension by using 5th International reference preparation for opacity (10 OU) and/or spectophotometerically. Alternatively, aliquots of challenge suspension frozen in liquid nitrogen with a suitable preservative like 10 per cent DMSO may be used, to avoid heterogenicity. After 14 to 17 days of immunization, inject intracerebrally, a dose of 0.02 to 0.03 ml of the challenge dilution randomly, into each immunized mouse. The challenge should contain, approximately 1,00,000 organisms and 100 to 1000 LD50 per dose, in a volume of not more than 0.03 ml. In the same way, inject 4 groups of 10 control mice each, for LD50 titration of challenge preparation, prepared by a series of dilutions, from the dilution selected for challenge. The challenge should be completed within 2 to 2.5 hours of preparation. Exclude any mouse from consideration, that dies within 3 days of challenge. Count the number of mice surviving in each of the groups, after 14 days. On the basis of the numbers of animals surviving in each of the groups of 16 to 24 mice, calculate the potency of vaccine under examination, against the potency of reference preparation. Seed a suitable highest dilution of the challenge suspension, into each of two B.G medium plates, before and after challenge. Incubate the plates at 37 for 48 to 72 hours and calculate the number of colony forming units (CFUs). Calculate the potency of the vaccine by Probit analysis and LD 50 of the challenge suspension by Reed and Munch Method. 71
The test is not valid unless: a) for both the vaccine under examination and the reference preparation, the ED50 (protective dose), lies between the largest and the smallest doses given to the mice; b) the number of animals, which die in the four groups of 10 injected with the challenge suspension and its dilutions indicate that the challenge dose contains 100 to 1000 LD50 and 1 LD50 contains not more than 300 colony forming units; c) and the statistical analysis shows no deviation from linearity or parallelism, in terms of significance of the scope of dose response curve; d) the vaccine passes the requirements for potency, if the test results of a statistically valid assay show that the estimated potency of the vaccine is not less than 4.0 IU per single human dose and the lower fiducial limit (P = 0.95) of estimated potency is not less than 2.0 IU. The test may be repeated once, but when more than one test is performed the results of all valid tests must be combined, in the estimate of potency. Labelling. The label states (1) the minimum number of International Units per single human dose; (2) that the vaccine must be shaken before use; (3) that the vaccine is not to be frozen.
Pneumococcal Polysaccharide Vaccine

Pneumococcal Polysaccharide Vaccine consists of a mixture of equal parts of purified capsular polysaccharide of various serotype antigens prepared from suitable pathogenic strains of Streptococcus pneumoniae in different desired combinations whose capsules have been shown to be made up of polysaccharides that are capable of inducing satisfactory levels of specific antibodies in man. It contains upto 23 immunochemically different capsular polysaccharides listed in the Table 1.
Production
General provisions Production of the vaccine is based on a well defined seed-lot system for each type. The production method shall have been shown to yield consistently pneumococcal polysaccharide vaccines of acceptable immunogenicity and safety in man. The production method is validated to demonstrate that the product, if tested, would comply with the tests for abnormal toxicity of vaccines for human use, modified as follows for the tesonn guinea-pig; inject 10 human dose into each guineapig and observe for 12 days. Monovalent bulk polysaccharides The bacteria are grown in a suitable liquid medium that does not contain blood-group substances or high-molecular-mass polysaccharides. The bacterial purity of the culture is verified
PNEUMOCOCCAL POLYSACCHARIDE VACCINE
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Table 1- Specifications on monovalent bulk polysaccharides (per cent contents): Molecular Type* 1 2 3 4 5 6B 7F 8 9N 9V 10A 11A 12F 14 15B 17A or 17F 18C 19A 19F 20 22F 23F 33F Proteins Nucleic acids <2 <2 <5 <3 < 7.5 <2 <5 <2 <2 <2 <7 <3 <3 <5 <3 <2 <3 <2 <3 <2 <2 <2 < 2.5 <2 <2 <2 <2 <2 <2 <2 <2 <1 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 Total 3.5-6 0-1 0-1 4-6 2.5-6.0 0-2 1.5-4.0 0-1 2.2-4 0.5-3 0.5-3.5 0-2.5 3-5 1.5-4 1-3 0-1.5 0-1 0.6-3.5 1.4-3.5 0.5-2.5 0-2 0-1 0-2 Phosphorus Molecular size K0 Nitrogen CL-4B** CL-2B*** 0-1.5 0-1.0 0-1.0 0-1.5 <2 2.5-5.0 0-1.0 0-1.0 0-1.0 0-1.0 1.5-3.5 2.0-5.0 0-1.0 0-1.0 2.0-4.5 0-3.5 2.4-4.9 3.0-7.0 3.0-5.5 1.5-4.0 0-1.0 3.0-4.5 0-1.0 < 0.15 < 0.15 < 0.15 < 0.15 < 0.60 < 0.50 < 0.20 < 0.15 < 0.20 < 0.45 < 0.65 < 0.40 < 0.25 < 0.30 < 0.55 < 0.45 < 0.15 < 0.45 < 0.20 < 0.60 < 0.55 < 0.15 < 0.50 15 Uronic acids 45 15 40 12 40 20 15 13 25 20 15 28 13 12 9 25 20 15 20 14 20 20 25 37 HexoMethylsamines pentoses 38 O-acetyl Groups 1.8
12 12.5 12
* The different types are indicated using the Danish nomenclature ** Cross linked agarose for chromatography R *** Cross linked agarose for chromatography R1
and the culture is inactivated with phenol. Impurities are removed by such techniques as fractional precipitation, enzymatic digestion and ultrafiltration. The polysaccharide is obtained by fractional precipitation, washed, and dried in a vacuum to a residual moisture content shown to be favourable to the stability of the polysaccharide. The residual moisture content is determined by drying under reduced pressure over diphosphorus pentoxide or by thermogravimetric analysis and the value obtained is used to calculate the results of the tests shown below with reference to the dried substance. The monovalent bulk polysaccharide is stored at a suitable temperature in conditions that avoid the uptake of moisture. Only a monovalent bulk polysaccharide that complies with the following requirements may be used in the preparation of the final bulk vaccine. Percentage contents of components, 72
determined by the methods prescribed below, are shown in the Table 1. The purified polysaccharides comply with the following tests as applicable: Protein (2.7.1). Comply with the test for protein. Nucleic acids (2.7.1). Comply with the test for nucleic acids. Total nitrogen (2.3.30). Comply with the test for total nitrogen. Phosphorus (2.7.1). Comply with the test for phosphorus. Uronic acids (2.7.1). Comply with the test for uronic acids. Hexosamine (2.7.1). Comply with the test for hexosamine. Methylpentoses (2.7.1). Comply with the test for methylpentoses.
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POLYSACCHARIDE VACCINE (INACTIVATED)
O-Acetyl groups (2.7.1). Comply with the test for O-acetyl groups. Sterility (2.2.11). Comply with the test for sterility. Molecular size. Molecular size is determined by gel filtration or high performance size-exclusion chromatography (HPSEC) (2.4.16) using cross linked Agarose for chromatography R or chromatography Agarose for chromatograph R1, either alone or Multiple angle light laser scattering (MALLS) or any other suitable method.
on each monovalent bulk polysaccharide used in the preparation of the final lot.
Identification
The assay also serves to identify the vaccine.
Tests
Sterility (2.2.11). Complies with the test for sterility. Abnormal Toxicity (2.2.1). Complies with the test for abnormal toxicity with the following modifications. Inject 10 human doses each in two guinea pigs weighing between 250 and 350 g by intraperitoneal route and observe for 12 days, Pyrogens (2.2.8). Complies with the test for pyrogens. Inject each of the rabbit with 1 ml of a dilution of the vaccine containing 2.5 g/ml of each polysaccharide. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Phenol (2.3.36). Not more than 2.5 g/l. pH (2.4.24). 4.5 to 7.4. Assay Determine the content of each polysaccharide by a suitable biochemical, physicochemical or immunochemical method (2.2.14), using antisera specific for each polysaccharide contained in the vaccine, including factor sera for types within groups, and purified polysaccharides of each type as standards. The vaccine contains not less than 70.0 per cent and not more than 130.0 per cent of the quantity stated on the label for each polysaccharide. The confidence interval (P = 0.95) of the assay is not less than 80.0 and not more than 120.0 per cent of the estimated content. Labelling. The label states (1) the number of g of each polysaccharide per human dose; (2) the total amount of polysaccharide in the container.
Identification
Confirm the identity of the monovalent bulk polysaccharide by immunochemical method (2.2.14)(except for polysaccharides 7F, 14 and 33F). Specificity For establishing the specificity, no reaction should occur, when the antigens are tested against all the antisera specific for the other polysaccharides of the vaccine, including factor sera for distinguishing types within groups. The polysaccharides are tested at a concentration of 50 g/ml using a method capable of detecting 0.5 g/ml. FINAL BULK VACCINE The final bulk vaccine is obtained by aseptically mixing the different polysaccharide powders. The uniform mixture is aseptically dissolved in a suitable isotonic solution so that one human dose of 0.5 ml contains 25 g of each polysaccharide. An antimicrobial preservative may be added. The solution is sterilized by filtration through a bacteriaretentive filter. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT The final bulk vaccine is distributed and filled aseptically into sterile containers (vials or ampoules). Only a final lot that is satisfactory with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided that the tests for phenol and for antimicrobial preservative have been carried out with satisfactory results on the final bulk vaccine, they may be omitted on the final lot. When consistency of production has been established on a suitable number of consecutive batches, the assay may be replaced by a qualitative test that identifies each polysaccharide, provided that an assay has been performed 73
Poliomyelitis Vaccine (Inactivated)

Poliomyelitis Vaccine (Inactivated) is a liquid preparation of suitable strains of human polioviruses 1, 2 and 3 grown in suitable cell cultures and inactivated by a validated method.
Production
General provisions The production method should consistently yield vaccines of acceptable safety and immunogenicity in man.
IP 2007
Production of the vaccine is based on a virus seed-lot system. Cell lines are used according to a cell-bank system. If primary, secondary or tertiary monkey kidney cells are used, production complies with the requirements indicated below. Unless otherwise justified and authorised, the virus in the final vaccine shall not have undergone more passages from the master seed lot than was used to prepare the vaccine shown in clinical studies to be satisfactory with respect to safety and efficacy. The production method is validated to demonstrate that the product, if tested, would comply with the test for safety and efficacy. Substrate for virus propagation The virus is propagated in a human diploid cell line (2.7.2), in a continuous cell line (2.7.2) or in primary, secondary or tertiary monkey kidney cells. Primary, secondary or tertiary monkey kidney cells. The following special requirements for the substrate for virus propagation apply to primary, secondary or tertiary monkey kidney cells. Monkeys used in the preparation of kidney cell cultures for production and control of the vaccine. The animals used are of a species approved by the competent authority, in good health and, unless otherwise justified and authorised, have not been previously employed for experimental purposes. Kidney cells used for vaccine production and control are derived from monitored, closed colonies of monkeys bred in captivity, not from animals caught in the wild; a previously approved seed lot prepared using virus passaged in cells from wild monkeys may, subject to approval by the competent authority, be used for vaccine production if historical data on safety justify this. Monitored, closed colonies of monkeys. The monkeys are kept in groups in cages. Freedom from extraneous agents is achieved by the use of animals maintained in closed colonies that are subject to continuous and systematic veterinary and laboratory monitoring for the presence of infectious agents. The supplier of animals is certified by the competent authority. Each monkey is tested serologically at regular intervals during a quarantine period of not less than 6 weeks imposed before entering the colony and then during its stay in the colony. The monkeys used are shown to be tuberculin-negative and free from antibodies to simian virus 40 (SV40) and simian immunodeficiency virus. If Macaca spp. monkeys are used for production, the monkeys are also shown to be free from antibodies to herpesvirus B (Cercopithecine herpesvirus 1) infection. Human herpesvirus 1 has been used as an indicator for freedom from herpesvirus B antibodies on account of the danger of handling herpesvirus B 74
(Cercopithecine herpesvirus 1). Monkeys from which kidneys are to be removed are thoroughly examined, particularly for evidence of tuberculosis and herpesvirus B (Cercopithecine herpesvirus 1) infection. If a monkey shows any pathological lesion relevant to the use of its kidneys in the preparation of a seed lot or vaccine, it is not to be used nor are any of the remaining monkeys of the group concerned unless it is evident that their use will not impair the safety of the product. All the operations described in this section are conducted outside the area where the vaccine is produced. Monkey cell cultures for vaccine production. Kidneys that show no pathological signs are used for preparing cell cultures. Each group of cell cultures derived from a single monkey forms a separate production cell culture giving rise to a separate single harvest. The primary monkey kidney cell suspension complies with the test for mycobacteria ; disrupt the cells before carrying out the test. If secondary or tertiary cells are used, it shall be demonstrated by suitable validation tests that cell cultures beyond the passage level used for production are free from tumorigenicity. SEED LOT Each of the three strains of poliovirus used shall be identified by historical records that include information on the origin of the strain and its subsequent manipulation. Only a working seed lot that complies with the following requirements may be used for virus propagation.
Identification
Each working seed lot is identified as human poliovirus 1, 2 or 3 by virus neutralisation in cell culture using specific antibodies. Virus concentration. The virus concentration of each working seed lot is determined to define the quantity of virus to be used for inoculation of production cell cultures. Extraneous agents (2.7.3). The working seed lot complies with the requirements for seed lots for virus vaccines. In addition, if primary, secondary or tertiary monkey kidney cells have been used for isolation of the strain, measures are taken to ensure that the strain is not contaminated with simian viruses such as simian immunodeficiency virus, simian virus 40, filoviruses and herpesvirus B (Cercopithecine herpesvirus 1). A working seed lot produced in primary, secondary or tertiary monkey kidney cells complies with the requirements given below under Virus Propagation and Harvest for single harvests produced in such cells.
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PROPAGATION AND HARVEST All processing of the cell bank and subsequent cell cultures is done under aseptic conditions in an area where no other cells or viruses are being handled. Approved animal serum (but not human serum) may be used in the cell culture media. Serum and trypsin used in the preparation of cell suspensions and media are shown to be free from extraneous agents. The cell culture media may contain a pH indicator such as phenol red and approved antibiotics at the lowest effective concentration. Not less than 500 ml of the cell cultures employed for vaccine production is set aside as uninfected cell cultures (control cells); where continuous cell lines in a fermenter are used for production, 200 106 cells are set aside to prepare control cells; where primary, secondary or tertiary monkey kidney cells are used for production, a cell sample equivalent to at least 500 ml of the cell suspension, at the concentration employed for vaccine production, is taken to prepare control cell cultures. Only a single harvest that complies with the following requirements may be used in the preparation of the vaccine. The tests for Identification and Sterility may be carried out instead on the purified, pooled monovalent harvest. After demonstration of consistency of production at the stage of the single harvest, the test for virus concentration may be carried out instead on the purified, pooled monovalent harvest. Control cells. The control cells of the production cell culture comply with a test for Identification (if a cell-bank system is used for production) and with the requirements for extraneous agents, where primary, secondary or tertiary monkey kidney cells are used, the tests in cell cultures are carried out as shown below under Test in Rabbit Kidney Cell Cultures and Test in Cercopithecus Kidney Cell Cultures). Test in rabbit kidney cell cultures. Test a sample of at least 10 ml of the pooled supernatant fluid from the control cultures for the absence of herpesvirus B (Cercopithecine herpesvirus 1) and other viruses in rabbit kidney cell cultures. The dilution of supernatant in the nutrient medium is not greater than 1:4 and the area of the cell layer is at least 3 cm2 per ml of inoculum. Set aside one or more containers of each batch of cells with the same medium as non-inoculated control cells. Incubate the cultures at 37 and observe for at least 2 weeks. The test is not valid if more than 20 per cent of the control cells are discarded for non-specific, accidental reasons. Test in Cercopithecus kidney cell cultures. Test a sample of at least 10 ml of the pooled supernatant fluid from the control cultures for the absence of SV40 virus and other extraneous agents by inoculation onto cell cultures prepared from the kidneys of cercopithecus monkeys, or other cells shown to be at least as sensitive for SV40, by the method described under Test in Rabbit Kidney Cell Cultures. The test is not valid if more than 20 per cent of the control cell cultures are discarded 75
for non-specific, accidental reasons.
Identification
The single harvest is identified as containing human poliovirus 1, 2 or 3 by virus neutralisation in cell cultures using specific antibodies. Virus concentration. The virus concentration of each single harvest is determined by titration of infectious virus in cell cultures. Sterility (2.2.11). The single harvest complies with the test for sterility, carried out using 10 ml for each medium. Mycoplasmas (2.7.4). The single harvest complies with the test for mycoplasmas, carried out using 10 ml. Test in rabbit kidney cell cultures. Where primary, secondary or tertiary monkey kidney cells are used for production, test a sample of at least 10 ml of the single harvest for the absence of herpesvirus B (Cercopithecine herpesvirus 1) and other viruses in rabbit kidney cell cultures as described for the control cells. Test in Cercopithecus kidney cell cultures. Where primary, secondary or tertiary monkey kidney cells are used for production, test a sample of at least 10 ml of the single harvest for the absence of SV40 virus and other extraneous agents. Neutralise the sample by a high-titre antiserum against the specific type of poliovirus. Test the sample in primary cercopithecus kidney cell cultures or cells that have been demonstrated to be at least as susceptible for SV40. Incubate the cultures at 37 and observe for 14 days. At the end of this period, make at least one subculture of fluid in the same cell culture system and observe both primary cultures and subcultures for an additional 14 days. PURIFICATION AND PURIFIED MONOVALENT HARVEST Several single harvests of the same type may be pooled and may be concentrated. The monovalent harvest or pooled monovalent harvest is purified by validated methods. If continuous cell lines are used for production, the purification process shall have been shown to reduce consistently the content of substrate-cell DNA to not more than 500 pg per single human dose. Only a purified monovalent harvest that complies with the following requirements may be used for the preparation of the inactivated monovalent harvest.
Identification
The virus is identified by virus neutralisation in cell cultures using specific antibodies or by determination of D-antigen. Virus concentration. The virus concentration is determined by titration of infectious virus.
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Specific activity. The ratio of the virus concentration or the Dantigen content, determined by a suitable immunochemical method (2.2.14). to the total protein content (specific activity) of the purified monovalent harvest is within the limits approved for the particular product. INACTIVATION AND INACTIVATED MONOVALENT HARVEST Several purified monovalent harvests of the same type may be mixed before inactivation. To avoid failures in inactivation caused by the presence of virus aggregates, filtration is carried out before and during inactivation; inactivation is started within a suitable period, preferably not more than 24 h and in any case not more than 72 h, of the prior filtration. The virus suspension is inactivated by a validated method that has been shown to inactivate poliovirus without destruction of immunogenicity; during validation studies, an inactivation curve with at least four points (for example, time 0, 24, 48, and 96 h) is established showing the decrease in concentration of live virus with time. If formaldehyde is used for inactivation, the presence of an excess of formaldehyde at the end of the inactivation period is verified. Only an inactivated monovalent harvest that complies with the following requirements may be used in the preparation of a trivalent pool of inactivated monovalent harvests or a final bulk vaccine. Test for effective inactivation. After neutralisation of the formaldehyde with sodium bisulphite (where applicable), verify the absence of residual live poliovirus by inoculation on suitable cell cultures of two samples of each inactivated monovalent harvest, corresponding to at least 1500 human doses. Take one sample not later than three-quarters of the way through the inactivation period and the other at the end. Inoculate the samples in cell cultures such that the dilution of vaccine in the nutrient medium is not greater then and the area of the cell layer is at least 3 cm2 per ml of inoculum. Set aside one or more containers with the same medium as noninoculated control cells. Observe the cell cultures for at least 3 weeks. Make not fewer than two passages from each container, one at the end of the observation period and the other 1 week before; for the passages, use cell culture supernatant and inoculate as for the initial sample. Observe the subcultures for at least 2 weeks. No sign of poliovirus multiplication is present in the cell cultures. At the end of the observation period, test the susceptibility of the cell culture used by inoculation of live poliovirus of the same type as that present in the inactivated monovalent harvest. Sterility (2.2.11). The inactivated monovalent harvest complies with the test for sterility, carried out using 10 ml for each medium. D-antigen content. The content of D-antigen determined by a 76
suitable immunochemical method (2.2.14) is within the limits approved for the particular preparation. FINAL BULK VACCINE The final bulk vaccine is prepared directly from the inactivated monovalent harvests of human polioviruses 1, 2 and 3 or from a trivalent pool of inactivated monovalent harvests. If a trivalent pool of inactivated monovalent harvests is used, a test for effective inactivation is carried out on this pool instead of on the final bulk vaccine. A stabiliser and an antimicrobial preservative may be added. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. Inactivation. Before addition of any antimicrobial preservative, a sample of at least 1500 ml or, for a purified and concentrated vaccine, the equivalent of 1500 doses is tested for residual live poliovirus in cell cultures, as described for the inactivated monovalent harvest. If the final bulk vaccine is prepared from a trivalent pool of inactivated monovalent harvests, the test for inactivation is carried out on that pool rather than on the final bulk vaccine. FINAL LOT Only a final lot that complies with each of the requirements given below under Identification, Tests and Assay may be released for use. Provided that the tests for free formaldehyde and antimicrobial preservative and the in vivo assay have been performed with satisfactory results on the final bulk vaccine, they may be omitted on the final lot. Provided that the test for bovine serum albumin has been performed with satisfactory results on the trivalent pool of inactivated monovalent harvests or on the final bulk vaccine, it may be omitted on the final lot.
Identification
The vaccine is shown to contain human polioviruses 1, 2 and 3 by a suitable immunochemical method such as the determination of D-antigen by enzyme-linked immunosorbent assay (ELISA).
Tests
Free formaldehyde (2.3.20). Maximum 0.2 g/l. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical
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POLIOMYELITIS VACCINE, LIVE (ORAL)
method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Protein content (2.3.49). Not more than 10 g of protein nitrogen per human dose. Bovine serum albumin. Not more than 50 ng per single human dose, determined by a suitable immunochemical method (2.2.14). Sterility (2.2.11). Complies with the test for sterility. Bacterial endotoxins ( 2.2.3). Not more than 5 IU per human dose. Assay D-antigen content. As a measure of consistency of production, determine the D-antigen content for human polioviruses 1, 2 and 3 by a suitable immunochemical method (2.2.14) using an appropriate reference preparation calibrated in D-antigen units. For each type, the content, expressed with reference to the amount of D-antigen stated on the label, is within the limits approved for the particular product. In vivo test. The capacity of the vaccine to induce the formation of neutralizing antibodies is determined in-vivo by one of the following methods: Test in chicks or guinea-pigs. Prepare a suitable series of at least three dilutions of the vaccine under examination using a suitable buffered saline solution. Inject 0.5 ml of the dilutions intramuscularly into groups of ten 3-week-old chickens or groups of ten guinea-pigs, each weighing between 250 and 350 g, using a separate group for each dilution of vaccine. Bleed the animals on the fifth or sixth day after the injection and separate the sera. Examine the sera for the presence of neutralising antibody, at a dilution of 1 in 4, to each of the human polioviruses 1, 2 and 3. Mix 100 CCID50 of virus with the dilution of serum and incubate at 37 for 4 h 30 min to 6 h. Keep at 5 3 for 12 to 18 h. Inoculate the mixtures into cell cultures for the detection of unneutralised virus and read the results up to 7 days after inoculation. For each group of animals, note the number of sera which have neutralising antibody and calculate the dilution of the vaccine giving an antibody response in 50.0 per cent of the animals. Carry out in parallel a control test using a suitable reference preparation. The vaccine complies with the test if a dilution of 1 in 100 or more produces an antibody response for each of the three types of virus in 50.0 per cent of the animals. Test on rats. A suitable in vivo assay method consists of intramuscular injection into the hind limb(s) of not fewer than 3 dilutions of the vaccine under examination and a reference vaccine, using for each dilution a group of 10 specific pathogen-free rats of the suitable strain. Use of 4 dilutions is often necessary to obtain valid results for all 3 serotypes. The 77
number of animals per group must be sufficient to obtain results that meet the validity criteria; groups of 10 rats are usually sufficient although valid results may be obtained with fewer animals per group. The weight of individual animal must not vary by more than 10.0 per cent from the group mean. An inoculum of 0.5 ml per rat is used. The dose range is chosen such that a dose response to all 3 poliovirus types is obtained. Bleed the animals after 20 to 22 days. Neutralising titres against all 3 polivirus types are measured separately using 100 CCID50 of the Sabin strains as challenge viruses. Vero or Hep2 as indicator cells, and neutralization conditions of 3 h at 35 to 37 followed by 18 h at 2 to 8. Results are read following fixation and staining after 7 days of incubation at 35. For a valid antibody assay, the titre of each challenge virus must be shown to be within the range of 10 to 1000 CCID50 and the neutralizing antibody titre of a control serum must be within 2 twofold dilutions of the geometric mean titre of the serum. The potency is calculated by comparison of the preparation of responders for the vaccine under examination and the reference vaccine by the probit method or, after validation, using a parallel-line model. For the probit method it is necessary to establish a cut-off neutralising antibody titre for each poliovirus type to define a responder. Due to interlaboratory variation, it is not possible to define cut-off values that could be applied by all laboratories. Rather, the cut-off values are determined for each laboratory based on a minimum series of 3 tests with the reference vaccine. The midpoint on a log 2 scale of the minimum and maximum geometric mean titres of the series of 3 or more tests is used as the cutoff value. For each of the 3 poliovirus types, the potency of the vaccine is not significantly less than that of the reference preparation. The test is not valid unless (1) for both the test and reference vaccines the ED50 lies between the smallest and the largest doses given to the animals; (2) the statistical analysis shows no significant deviation from linearity or parallelism; (3) the fiducial limits of the estimated relative potency fall between 25.0 per cent and 400.0 per cent of the estimated potency. Labelling. The label states (1) the types of poliovirus contained in the vaccine; (2) the nominal amount of virus of each type (1, 2 and 3), expressed in units of D-antigen per single human dose; (3) the cell substrate used to prepare the vaccine.
Poliomyelitis Vaccine, Live (Oral)

Oral Poliomyelitis Vaccine is a preparation of approved strains of live attenuated poliovirus type 1, 2 or 3 grown in in vitro cultures of approved cells, containing any one type or any combination of the three types of Sabin strains, prepared in a form suitable for oral administration.
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Production
General provisions The vaccine strains and the production method should consistently yield vaccines that are both immunogenic and safe in man. The production of vaccine is based on a virus seed-lot system. Cell lines are used according to a cell-bank system. If primary monkey kidney cells are used, production complies with the requirements indicated below. Unless otherwise justified and authorised, the virus in the final vaccine shall not have undergone more than two passages from the master seed lot. Substrate for virus propagation The virus is propagated in human diploid cells (2.7.2) or in continuous cell lines (2.7.2) or in primary monkey kidney cells (including serially passaged cells from primary monkey kidney cells). Continuous cell lines are approved by the competent authority. Primary monkey cells. The following special requirements for the substrate for virus propagation apply to primary monkey cells. Monkeys used for preparation of kidney cell cultures and for testing of virus. If the vaccine is prepared in monkey kidney cell cultures, animals of a species approved by the competent authority, in good health, and not previously employed for experimental purposes shall be used. The monkeys shall be kept in well-constructed and adequately ventilated animal rooms in cages spaced as far apart as possible. Adequate precautions shall be taken to prevent cross-infection between cages. Not more than two monkeys shall be housed per cage and cage-mates shall not be interchanged. The monkeys shall be kept in the country of manufacture of the vaccine in quarantine groups for a period of not less than 6 weeks before use. A quarantine group is a colony of selected, healthy monkeys kept in one room, with separate feeding and cleaning facilities, and having no contact with other monkeys during the quarantine period. If at any time during the quarantine period the overall death rate of a shipment consisting of one or more groups reaches 5 per cent (excluding deaths from accidents or where the cause was specifically determined not to be an infectious disease), monkeys from that entire shipment shall continue in quarantine from that time for a minimum of 6 weeks. The groups shall be kept continuously in isolation, as in quarantine, even after completion of the quarantine period, until the monkeys are used. After the last monkey of a group has been taken, the room that housed the group shall be thoroughly cleaned and decontaminated before being used for a fresh group. If kidneys from near-term monkeys are used, the mother is quarantined for the term of pregnancy. 78
Monkeys from which kidneys are to be removed shall be anaesthetised and thoroughly examined, particularly for evidence of tuberculosis and cercopithecid herpesvirus 1 (B virus) infection. If a monkey shows any pathological lesion relevant to the use of its kidneys in the preparation of a seed lot or vaccine, it shall not be used, nor shall any of the remaining monkeys of the quarantine group concerned be used unless it is evident that their use will not impair the safety of the product. All the operations described in this section shall be conducted outside the areas where the vaccine is produced. The monkeys used shall be shown to be free from antibodies to simian virus 40 (SV40) and simian immunodeficiency virus. If Macaca spp. are used for production, the monkeys shall also be shown to be free from antibodies to cercopithecid herpesvirus 1 (B virus). Human herpesvirus has been used as an indicator for freedom from B virus antibodies on account of the danger of handling cercopithecid herpesvirus 1 (B virus). Monkey kidney cell cultures for vaccine production. Kidneys that show no pathological signs are used for preparing cell cultures. If the monkeys are from a colony maintained for vaccine production, serially passaged monkey kidney cell cultures from primary monkey kidney cells may be used for virus propagation, otherwise the monkey kidney cells are not propagated in series. Virus for the preparation of vaccine is grown by aseptic methods in such cultures. If animal serum is used in the propagation of the cells, the maintenance medium after virus inoculation shall contain no added serum. Each group of cell cultures derived from a single monkey or from fetuses from no more than ten near-term monkeys is prepared and tested as an individual group. SEED LOT The strains of poliovirus used shall be identified by historical records that include information on the origin and subsequent manipulation of the strains. Working seed lots are prepared by a single passage from a master seed lot and at an approved passage level from the original Sabin virus. Virus seed lots are prepared in large quantities and stored at a temperature below -60. Only a virus seed lot that complies with the following requirements may be used for virus propagation.
Identification
Each working seed lot is identified as poliovirus of the given type, using specific antibodies. Virus concentration. Determined by the method described below, the virus concentration is the basis for the quantity of virus used in the neurovirulence test.
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Extraneous agents (2.7.3). If the working seed lot is produced in human diploid cells (2.7.2) or in continuous cell lines (2.7.2) it complies with the requirements for seed lots for virus vaccines. If the working seed lot is produced in primary monkey cells, it complies with the requirements given below under Propagation and Harvest and Monovalent Pooled Harvest and with the tests in adult mice, suckling mice and guinea-pigs given under Tests for extraneous agents in viral vaccines for human use. Working seed lot shall be free from detectable DNA sequences from simian virus 40 (SV40) Neurovirulence (2.7.6). Each master and working seed lot complies with the test for neurovirulence of poliomyelitis vaccine (oral) in monkeys. Furthermore, the seed lot shall cease to be used in vaccine production if the frequency of failure of the monovalent pooled harvests produced from it is greater than predicted statistically. This statistical prediction is calculated after each test on the basis of all the monovalent pooled harvests tested; it is equal to the probability of false rejection on the occasion of a first test (i.e. 1 per cent), the probability of false rejection on retest being negligible. If the test is carried out only by the manufacturer, the test slides are provided to the control authority for assessment. Genetic markers. Each working seed lot is tested for its replicating properties at temperatures ranging from 36 to 40 as described under Monovalent Pooled Harvest. PROPAGATION AND HARVEST All processing of the cell-banks and subsequent cell-cultures is done under aseptic conditions in an area where no other cells are handled. Approved animal (but not human) serum may be used in the media, but the final medium for maintaining cell growth during virus multiplication does not contain animal serum. Serum and trypsin used in the preparation of cell suspensions and media are shown to be free from live extraneous agents. The cell-culture medium may contain a pH indicator such as phenol red and approved antibiotics at the lowest effective concentration. It is preferable to have a substrate free from antibiotics during production. Not less than 5 per cent and not more than 1000 ml of the cell cultures employed for vaccine production are set aside as uninfected cell cultures (control cells); special requirements, given below, apply to control cells when the vaccine is produced in primary monkey cells The virus suspension is harvested not later than 4 days after virus inoculation. After inoculation of the production cell culture with the virus working seed lot, inoculated cells are maintained at a fixed temperature, shown to be suitable, within the range 33 to 35; the temperature is maintained constant to 0.5; control cell cultures are maintained at 33 to 35 for the relevant incubation periods. Only a single virus harvest that complies with the following requirements may be used in the preparation of the monovalent 79
pooled harvest. Virus concentration. The virus concentration of virus harvests is determined as prescribed under Assay to monitor consistency of production and to determine the dilution to be used for the final bulk vaccine. Extraneous agents ( 2.7.3 ). Complies with tests for extraneous agents. Control cells. The control cells of the production cell culture from which the virus harvest is derived comply with a test for identity and with the requirements for extraneous agents or, where primary monkey cells are used, as shown below. Primary monkey cells. The following special requirements apply to virus propagation and harvest in primary monkey cells. Cell cultures. On the day of inoculation with virus seed, each cell culture is examined for degeneration caused by an infective agent. If, in this examination, evidence is found of the presence in a cell culture of any extraneous agent, the entire group of cultures concerned shall be rejected. On the day of inoculation with the virus working seed lot, a sample of at least 30 ml of the pooled fluid removed from the cell cultures of the kidneys of each single monkey or from fetuses from not more than ten near-term monkeys is divided into two equal portions. One portion of the pooled fluid is tested in monkey kidney cell cultures prepared from the same species, but not the same animal, as that used for vaccine production. The other portion of the pooled fluid is, where necessary, tested in monkey kidney cell cultures from another species so that tests on the pooled fluids are done in cell cultures from at least one species known to be sensitive to SV40. The pooled fluid is inoculated into bottles of these cell cultures in such a way that the dilution of the pooled fluid in the nutrient medium does not exceed 1 in 4. The area of the cell sheet is at least 3 cm2 per ml of pooled fluid. At least one bottle of each kind of cell culture remains uninoculated to serve as a control. If the monkey species used for vaccine production is known to be sensitive to SV40, a test in a second species is not required. Animal serum may be used in the propagation of the cells, provided that it does not contain SV40 antibody, but the maintenance medium after inoculation of test material contains no added serum except as described below. The cultures are incubated at a temperature of 35 to 37 and are observed for a total period of at least 4 weeks. During this observation period and after not less than 2 weeks incubation, at least one subculture of fluid is made from each of these cultures in the same cell culture system. The subcultures are also observed for at least 2 weeks. Serum may be added to the original culture at the time of subculturing, provided that the serum does not contain SV40 antibody.
IP 2007
Fluorescent-antibody techniques may be useful for detecting SV40 virus and other viruses in the cells. A further sample of at least 10 ml of the pooled fluid is tested for cercopithecid herpesvirus 1 (B virus) and other viruses in rabbit kidney cell cultures. Serum used in the nutrient medium of these cultures shall have been shown to be free from inhibitors of B virus. Human herpesvirus has been used as an indicator for freedom from B virus inhibitors on account of the danger of handling cercopithecid herpesvirus 1 (B virus). The sample is inoculated into bottles of these cell cultures in such a way that the dilution of the pooled fluid in the nutrient medium does not exceed 1 in 4. The area of the cell sheet is at least 3 cm2 per ml of pooled fluid. At least one bottle of the cell cultures remains uninoculated to serve as a control. The cultures are incubated at a temperature of 35 to 37 and observed for at least 2 weeks. A further sample of 10 ml of the pooled fluid removed from the cell cultures on the day of inoculation with the seed lot virus is tested for the presence of extraneous agents by inoculation into human cell cultures sensitive to measles virus. The tests are not valid if more than 20 per cent of the culture vessels have been discarded for non-specific accidental reasons by the end of the respective test periods. If, in these tests, evidence is found of the presence of an extraneous agent, the single harvest from the whole group of cell cultures concerned is rejected. If the presence of cercopithecid herpesvirus 1 (B virus) is demonstrated, the manufacture of oral poliomyelitis vaccine shall be discontinued and the competent authority shall be informed. Manufacturing shall not be resumed until a thorough investigation has been completed and precautions have been taken against any reappearance of the infection, and then only with the approval of the competent authority. If these tests are not done immediately, the samples of pooled cell-culture fluid shall be kept at a temperature of -60 or below, with the exception of the sample for the test for B virus, which may be held at 4, provided that the test is done not more than 7 days after it has been taken. Control cell cultures. On the day of inoculation with the virus working seed lot 25 per cent (but not more than 2500 ml) of the cell suspension obtained from the kidneys of each single monkey or from not more than ten near-term monkeys is taken to prepare uninoculated control cell cultures. These control cell cultures are incubated in the same conditions as the inoculated cultures for at least 2 weeks and are examined during this period for evidence of cytopathic changes. The tests are not valid if more than 20 per cent of the control cell cultures have been discarded for non-specific, accidental reasons. At the end of the observation period, the control cell cultures are 80
examined for degeneration caused by an infectious agent. If this examination or any of the tests required in this section shows evidence of the presence in a control culture of any extraneous agent, the poliovirus grown in the corresponding inoculated cultures from the same group shall be rejected. Tests for haemadsorbing viruses. At the time of harvest or within 4 days of inoculation of the production cultures with the virus working seed lot, a sample of 4 per cent of the control cell cultures is taken and tested for haemadsorbing viruses. At the end of the observation period, the remaining control cell cultures are similarly tested. The tests are made as described in (2.7.3), Tests for extraneous agents in viral vaccines for human use. Tests for other extraneous agents. At the time of harvest, or within 7 days of the day of inoculation of the production cultures with the working seed lot, a sample of at least 20 ml of the pooled fluid from each group of control cultures is taken and tested in two kinds of monkey kidney cell culture, as described above. At the end of the observation period for the original control cell cultures, similar samples of the pooled fluid are taken and the tests referred to in this section in the two kinds of monkey kidney cell culture and in the rabbit cell cultures are repeated, as described above under Cell cultures. If the presence of Cercopithecid herpesvirus 1 (B virus) is demonstrated, the production cell cultures shall not be used and the measures concerning vaccine production described above must be undertaken. The fluids collected from the control cell cultures at the time of virus harvest and at the end of the observation period may be pooled before testing for extraneous agents. A sample of 2 per cent of the pooled fluid is tested in each of the cell culture systems specified. Single harvests Tests for neutralised single harvests in monkey kidney cell cultures. A sample of at least 10 ml of each single harvest is neutralised by a type-specific poliomyelitis antiserum prepared in animals other than monkeys. In preparing antisera for this purpose, the immunising antigens used shall be prepared in non-simian cells. Half of the neutralised suspension (corresponding to at least 5 ml of single harvest) is tested in monkey kidney cell cultures prepared from the same species, but not the same animal, as that used for vaccine production. The other half of the neutralised suspension is tested, if necessary, in monkey kidney cell cultures from another species so that the tests on the neutralised suspension are done in cell cultures from at least one species known to be sensitive to SV40.
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The neutralised suspensions are inoculated into bottles of these cell cultures in such a way that the dilution of the suspension in the nutrient medium does not exceed 1 in 4. The area of the cell sheet is at least 3 cm2 per ml of neutralised suspension. At least one bottle of each type of cell culture remains uninoculated to serve as a control and is maintained by nutrient medium containing the same concentration of the specific antiserum used for neutralisation. Animal serum may be used in the propagation of the cells, provided that it does not contain SV40 antibody, but the maintenance medium, after the inoculation of the test material, contains no added serum other than the poliovirus neutralising antiserum, except as described below. The cultures are incubated at a temperature of 35 to 37 and observed for a total period of at least 4 weeks. During this observation period and after not less than 2 weeks incubation, at least one subculture of fluid is made from each of these cultures in the same cell-culture system. The subcultures are also observed for at least 2 weeks. Serum may be added to the original cultures at the time of subculturing, provided that the serum does not contain SV40 antibody. Additional tests are made for extraneous agents on a further sample of the neutralised single harvests by inoculation of 10 ml into human cell cultures sensitive to measles virus. Fluorescent-antibody techniques may be useful for detecting SV40 virus and other viruses in the cells. The tests are not valid if more than 20 per cent of the culture vessels have been discarded for non-specific accidental reasons by the end of the respective test periods. If any cytopathic changes occur in any of the cultures, the causes of these change are investigated. If the cytopathic changes are shown to be due to unneutralised poliovirus, the test is repeated. If there is evidence of the presence of SV40 or other extraneous agents attributable to the single harvest, that single harvest is rejected. MONOVALENT POOLED HARVEST Monovalent pooled harvests are prepared by pooling a number of satisfactory single harvests of the same virus type. Monovalent pooled harvests from continuous cell lines may be purified. Each monovalent pooled harvest is filtered through a bacteria-retentive filter. Only a monovalent pooled harvest that complies with the following requirements may be used in the preparation of the final bulk vaccine.
Virus concentration The virus concentration is determined by the method described below and serves as the basis for calculating the dilutions for preparation of the final bulk, for the quantity of virus used in the neurovirulence test and to establish and monitor production consistency. Genetic markers. A ratio of the replication capacities of the virus in the monovalent pooled harvest is obtained over a temperature range between 36 and 40 in comparison with the seed lot or a reference preparation for the marker tests and with appropriate rct/40- and rct/40+ strains of poliovirus of the same type. The incubation temperatures used in this test are controlled to within 0.1. The monovalent pooled harvest passes the test if, for both the virus in the harvest and the appropriate reference material, the titre determined at 36 is at least 5.0 log greater than that determined at 40. If growth at 40 is so low that a valid comparison cannot be established, a temperature in the region of 39.0 to 39.5 is used, at which temperature the reduction in titre of the reference material must be in the range 3.0 to 5.0 log of its value at 36; the acceptable minimum reduction is determined for each virus strain at a given temperature. If the titres obtained for one or more of the reference viruses are not concordant with the expected values, the test must be repeated. Neurovirulence (2.7.6). Each monovalent pooled harvest complies with the test for neurovirulence of poliomyelitis vaccine (oral). If the test is carried out only by the manufacturer, the test slides are provided to the competent authority for assessment. The TgPVR21 transgenic mouse model provides a suitable alternative to the monkey neurovirulence test for neurovirulence testing of types 1, 2 or 3 vaccines once a laboratory qualifies as being competent to perform the test and the experience gained is to the satisfaction of the competent authority. The test is carried out using a standard operating procedure approved by the competent authority. A suitable procedure (Neurovirulence test of type 1, 2 or 3 live poliomyelitis vaccines (oral) in transgenic mice susceptible to poliovirus) is available from WHO, Quality and Safety of Biologicals, Geneva. Primary monkey cells. The following special requirements apply to monovalent pooled harvests derived from primary monkey cells. Retroviruses. The monovalent pooled harvest is examined using a reverse transcriptase assay. No indication of the presence of retrovirus is found. Test on rabbits. A sample of the monovalent pooled harvest is tested for cercopithecid herpesvirus 1 (B virus) and other viruses by injection of not less than 100 ml into not fewer than 10 healthy rabbits each weighing between 1.5 and 2.5 kg. Each rabbit receives not less than 10 ml and not more than 20 ml, of 81
Identification
Each monovalent pooled harvest is identified as poliovirus of the given type, using specific antiserum.
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which 1 ml is given intradermally at multiple sites, and the remainder subcutaneously. The rabbits are observed for at least 3 weeks for death or signs of illness. All rabbits that die after the first 24 h of the test and those showing signs of illness are examined by autopsy, and the brain and organs removed for detailed examination to establish the cause of death. The test is not valid if more than 20.0 per cent of the inoculated rabbits show signs of intercurrent infection during the observation period. The monovalent pooled harvest passes the test if none of the rabbits shows evidence of infection with B virus or with other extraneous agents or lesions of any kind attributable to the bulk suspension. If the presence of B virus is demonstrated, the measures concerning vaccine production described above under Cell cultures are taken. Test on guinea-pigs. Administer to not less than five guineapigs, each weighing between 350 and 450 g, 0.1 ml of the monovalent pooled harvest by intracerebral injection and 0.5 ml by intraperitoneal injection. Measure the rectal temperature of each animal on each working day for 6 weeks. At the end of the observation period carry out autopsy on each animal. In addition, administer to not fewer than five guinea-pigs 0.5 ml by intraperitoneal injection and observe as described above for 2 to 3 weeks. At the end of the observation period, carry out a passage from these animals to not fewer than five guineapigs using blood and a suspension of liver or spleen tissue. Measure the rectal temperature of the latter guinea-pigs for 2 to 3 weeks. Examine by autopsy all animals that, after the first day of the test, die or are killed because they show disease or show for three consecutive days a body temperature higher than 39; carry out histological examination to detect infection with Marburg virus; in addition, inject a suspension of liver or spleen tissue or of blood intraperitoneally into not fewer than three guinea-pigs. If any signs of infection with Marburg virus are noted, confirmatory serological tests are carried out on the blood of the affected animals. The monovalent pooled harvest complies with the test if not fewer than 80.0 per cent of the guinea-pigs survive to the end of the observation period and remain in good health and no animal shows signs of infection with filoviruses virus. FINAL BULK VACCINE The final bulk vaccine is prepared from one or more satisfactory monovalent pooled harvests and may contain more than one virus type. Suitable flavouring substances and stabilisers may be added. Only a final bulk vaccine that complies with the following requirement may be used in the preparation of the final lot. Sterility (2.2.11). Complies with the test for sterility. 82
FINAL LOT Only a final lot that complies with the following requirement for thermal stability and is satisfactory with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Thermal stability. Expose samples of the final lot at 37 for 48 hours. Determine the total virus concentration as described under Assay in parallel for the heated vaccine and for unheated vaccine. The estimated difference between the total virus concentration of the unheated and heated vaccines is not greater than 0.5 log10 infectious virus units (CCID50) per single human dose.
Identification
The vaccine is shown to contain poliovirus of each type stated on the label, using specific antibodies.
Tests
Sterility (2.2.11). Complies with the test for sterility. Assay Titrate for infectious virus at least in triplicate using the method described below. Use an appropriate virus reference preparation to validate each assay. If the vaccine contains more than one poliovirus type, titrate each type separately, using appropriate type-specific antiserum (or preferably a monoclonal antibody) to neutralise each of the other types present. For a trivalent vaccine, the estimated mean virus titres must be: not less than 1 106.0 infectious virus units (CCID50) per single human dose for type 1; not less than 1 105.0 infectious virus units (CCID50) for type 2; and not less than 1 105.8 infectious virus units (CCID50) for type 3. For monovalent or divalent vaccine, the minimum virus titres are decided by the competent authority. Method. Groups of eight to twelve flat-bottomed wells in a microtitre plate are inoculated with 0.05 ml of each of the selected dilutions of virus followed by a suitable cell suspension of the Hep-2 (Cincinnati) line. The plates are incubated at a suitable temperature. Examine the cultures on days 7 to 9. The assay is not valid if (a) the confidence interval (P = 0.95) of the logarithm of the virus concentration is greater than 0.3; (b) the virus concentration of the reference preparation differs by more than 0.5 log CCID50 from the assigned value. Labelling. The label states (1) the types of poliovirus contained in the vaccine; (2) the minimum amount of virus of each type contained in one single human dose; (3) the cell substrate used for the preparation of the vaccine; (4) that the vaccine is not to be injected.
IP 2007
RABIES VACCINE, HUMAN
Rabies Vaccine, Human

Rabies Vaccine for Human use is a freeze-dried or liquid (adsorbed) preparation of a suitable approved, strain of fixed rabies virus grown in an approved cell culture/ embryos of duck/chicken and inactivated by a validated method. The freeze-dried vaccine is reconstituted immediately before use as stated on the label to give a clear or slightly opalescent solution/suspension. It may be coloured owing to the presence of a pH indicator. The vaccine complies with the General Requirements of Vaccines for Human Use.
immunofluoresence or any other approved method. Extraneous agents (2.7.3). The working seed lot complies with the requirements for the virus seed lots. PROPAGATION AND HARVEST All processing of the cell bank and subsequent cell cultures are done under aseptic conditions in an area where no other cells are handled. Approved animal (but not human) serum may be used in the media, but the final medium for maintaining cell growth during virus multiplication does not contain animal serum; the media may contain human albumin. Serum proteins, if present are reduced to an acceptable level by a suitable method of purification. Serum and trypsin used in the preparation of cell suspension and media are shown to be free from infectious extraneous agents. The cell culture media may contain a pH indicator such as phenol red and approved antibiotics at the lowest effective concentration. Not less than 500 ml of the cell cultures employed for vaccine production are set aside as uninfected cell cultures (control cells). The virus suspension is harvested on one or more occasions during incubation. Multiple harvests from the same production cell culture may be pooled and considered as a single harvest. When vaccine is prepared in embryonated eggs, the egg proteins are minimized by an appropriate method of purification. The eggs are inoculated with virus seed by the yolk sac route. The infected sterile living embryos are harvested, minced and emulsified in suitable diluent, and stabilizer with aseptic precautions. Emulsions are centrifuged, supernatants are collected and stored as raw virus harvest at a suitable temperature. Viral harvests that comply with the following requirements are pooled in the preparation of the inactivated viral harvest.
Production
General provisions The vaccine is produced on the basis of virus seed lot system and if a cell line is used for virus propagation, a cell-bank system shall be followed. The production method shall have been shown to yield consistently vaccines that comply with the requirements for immunogenicity, safety and stability. Unless otherwise justified and authorized, the virus in the final vaccine shall not have undergone more passages from the master seed lot than was used to prepare the vaccine shown in clinical studies to be satisfactory with respect to safety and efficacy. The production method is validated to demonstrate that the product, if tested, would comply with the test for safety and efficacy. Substrate for virus propagation The virus is propagated in any suitable approved cell substrate like a human diploid cell line (2.7.2), a continuous cell line, or in duck embroys or in cultures of chicken embryos derived from a flock certified as free from specified pathogens(2.7.7). SEED LOT The strain of rabies virus used shall be identified by historical records that include information on the origin of the strain and its subsequent manipulation. Working seed lots are prepared by not more than five passages from the master seed lot. Only a working seed lot that complies with the following requirements may be used for virus propagation.
Identification
The single harvest contains virus that is identified as rabies virus using specific antibodies by an approved method. Virus concentration. Titrate for infective virus in cell cultures or by any other approved method. The titre is used to monitor consistency of production. Control cells. The control cells of the production cell culture from which the single harvest is derived should comply with a test for identification and with the requirements for extraneous agents (2.7.3). Control eggs. Control eggs shall be tested for freedom from haemagglutinating agents, and other extraneous agents. PURIFICATION AND INACTIVATION The virus harvests may be concentrated and/or purified by suitable methods; the virus harvest is inactivated by a validated method at a fixed, well defined stage of the process which may 83
Identification
Each working seed lot is identified as rabies virus using specific antibodies by an approved method. Virus concentration. The virus concentration of each working seed lot is determined by a cell culture method using
IP 2007
be before, during or after any concentration or purification. The method shall have been shown to be capable of inactivating rabies virus without destruction of the immunogenic activity. If betapropiolactone is used, the concentration shall at no time exceed 1:3500. Cell culture vaccines Only an inactivated viral suspension that complies with the following requirements may be used in the preparation of the final bulk vaccine. Inactivation. Inactivation is confirmed by carrying out an amplification test for residual infectious rabies virus, not more than 4 days after inactivation or on the sample frozen after inactivation and stored at -70. Inoculate a quantity of inactivated viral suspension equivalent to not less than 25 vaccine doses into cell cultures of the same type as those used for production of the vaccine. Make a passage after 7 days. Maintain the cultures for a further period of 14 days and then examine the cell cultures for rabies virus using an immune fluorescence test. No rabies virus is detected. Alternatively, 5 ml of each culture fluid is pooled on days 14 and 21 and 0.03 ml is inoculated intracerebrally into each of the 10 mice weighing 12 to 15 g. The mice are observed for 14 days for symptoms caused by rabies virus, and mice showing symptoms of rabies are sacrificed and virus presence is confirmed by immunoflurescence test or tested for live virus in cell culture by immunofluorescence test or method of equal sensitivity. No rabies virus should be detected. Residual host-cell DNA (2.2.15). If a continuous cell line is used for virus propagation, the content of residual host-cell DNA, determined using a suitable method, should not be greater than 10 ng per single human dose. Embryonated egg vaccine Only concentrated viral suspensions that comply with the test for sterility, antigen content and endotoxin requirements may be used for preparation of bulk for inactivation. Inactivation is confirmed by carrying out Mice Inoculation Test for residual infectious rabies virus, not more than four days after inactivation or on the sample frozen 4 days after inactivation are stored at 70. Inoculate intracerebrally 0.03 ml into each of 20 mice weighing between 12 and 15 g. The mice are observed for 14 days for symptoms caused by rabies virus and mice showing symptoms of rabies are sacrificed and virus presence is confirmed by an immunoflurescence test or tested for live virus in cell culture by immunofluorescence test or method of equal sensitivity. No rabies virus should be detected. FINAL BULK VACCINE The final bulk vaccine is prepared from one or more inactivated viral suspensions. An approved stabilizer may be added to 84
maintain the activity of the product during and after freezedrying. Thiomersal can be used as preservative. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Antigen content. Determine the antigen content by a suitable approved in vitro or in vivo method. The content should be within the limits approved for the particular product. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT The final bulk vaccine is distributed aseptically into sterile containers and can be freeze-dried in case of lyophilized products. The containers are then sealed so as to prevent contamination and the introduction of moisture. Only a final lot that complies with each of the requirements given below under Identification, Tests and Assay may be released for use. Provided that the test for inactivation has been carried out with satisfactory results on the inactivated virus suspension and the test for bovine serum albumin has been carried out with satisfactory results on the final bulk vaccine, these tests may be omitted on the final lot.
Identification
The vaccine is shown to contain rabies virus antigen by a suitable immunochemical method using specific antibodies, alternatively, the Assay also serves to identify the vaccine.
Tests
Inactivation. Inoculate a quantity equivalent to not less than 25 human doses of vaccine into cell cultures of the same type as those used for production of the vaccine. Make a passage after 7 days. Maintain the culture for a further 14 days and then examine the cell culture for rabies virus using an immunofluorescence test. No rabies virus is detected. Alternatively, inject 0.03ml of the vaccine intracerebrally into each of the 10 mice weighing between 12 and 15g. Neither symptoms of disease in the central nervous system nor death occurs in any of the animal within 14 days. If the inactivation test is already performed on inactivated virus used for final lot, it may be omitted from the test on final lot. Sterility (2.2.11). Complies with the test for sterility. Bacterial endotoxins (2.2.3). Less than 25 IU per single human dose. Pyrogens (2.2.8). Complies with the test for pyrogens. Unless otherwise justified and authorized, inject into each rabbit a single human dose of the vaccine diluted to ten times its volume. Water (2.3.43). Not more than 3.0 per cent determined by an approved method.
IP 2007
Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity. Accelerated degradation. The potency determined by method described under Assay of a sample of the preparation under examination after storage at 37 for 4 weeks is not less than 2.5 units per single human dose. This may not be mandatory for lot release, once the consistency of the product is approved by National Regulatory Authority. Bovine serum albumin (for cell culture vaccine). Not more than 50 ng per single human dose, determined by a suitable immunochemical method (2.2.14). Aluminium content (for gel absorbed vaccine) (2.3.9). Not more than 1.25 mg per single human dose. Ovalbumin (for egg based vaccines). Not more than 1 g of ovalbumin per human dose, determined by a suitable technique using a suitable reference preparation of ovalbumin. Residual host-cell DNA (for continuous cell line vaccines) (2.2.15). Should not be greater than 10 ng per single human dose. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Assay The Standard preparation is the international standard or another suitable preparation, the potency of which has been determined in relation to the International standard. The potency of rabies vaccine is determined by comparing the dose necessary to protect mice against a lethal intracerebral dose of rabies vaccine necessary to provide the same protection. For this comparison, a reference preparation of rabies vaccine, calibrated in international units, and a suitable preparation of rabies virus for use as the challenge preparation are necessary. The international unit is the activity contained in a stated quantity of the international standard. The equivalence in international units of the international standard is stated by the World Health Organization. The test described below uses a parallel-line model with at least three points for the vaccine under examination and the reference preparation. Test animals. Use mice of a suitable strain, drawn from a uniform stock three to four weeks old, weighing between 11 and 15 g. Distribute the mice into six groups of at least 16 mice each and four groups of 10 mice each and must be of the same sex or the sexes must be equally distributed among the groups. Throughout the test all mice that die before the fifth day after challenge are excluded from the test and all mice that die with 85
signs of rabies between the fifth and fourteenth day after challenge are counted as failing to resist the challenge. The strain of mice suitable for the test is such that when 0.03 ml containing 5 to 50 LD50 of the challenge virus suspension is injected intracerebrally per mouse there is 100 per cent mortality. Standard challenge virus suspension. A working pool of the challenge virus strain is prepared by injecting intracerebrally 0.03ml of a 10 fold dilution of the CVS strain of rabies virus in 2 per cent v/v sterile inactivated normal horse serum in water for injection or another suitable diluent approved by the competent authority into a suitable number of test animals. The animals when moribund after showing characteristic signs of rabies are sacrificed and their brains harvested aseptically. They are then washed in chilled saline solution to remove blood clots. A 10 per cent suspension of the brains is prepared in a suitable diluent approved by the competent authority and thoroughly homogenised. After centrifuging lightly, the supernatant liquid is distributed into sterile vials and freeze dried. The sealed and freeze-dried supernatant liquid containing vials are stored at -20. When stored under prescribed conditions the virus titre of the freeze-dried preparation may be expected to be maintained for not less than 3 years. Alternatively, the washed brains are homogenised in a suitable diluent approved by the competent authority to give 10 per cent suspension. It is then centrifuged lightly, distributed into sterile ampoules or sterile plastic vials and sealed. The sealed ampoules or plastic vials can be stored at 60 or below. When stored under prescribed conditions, the virus titre may be expected to be maintained for not less than one year. Storage time needs to be validated by the manufacturer. Virus titre of the challenge virus. Prepare ten fold serial dilutions of the standard challenge virus suspension. Using the four groups of 10 mice each, inject 0.03 ml of the virus suspension intracerebrally into each mouse, using a different group for each suspension. Observe the mice for 14 days. Calculate the virus titre of the standard challenge virus suspension in LD50 per dose of 0.03 ml by standard statistical methods. Determination of potency of the vaccine. Reconstitute the standard preparation with a suitable diluent. Prepare at least three 5-fold serial dilutions of the solution of the standard preparation and three 5-fold serial dilutions of the vaccine under examination. For both, the standard preparation and the preparation under examination, the serial dilutions should be prepared in such a way that the lowest dilution protects more than 50 per cent of the injected mice. Allocate one dilution to each of the six groups of 16 mice each. Inject intraperitoneally each mouse in each group with dilutions of the vaccine and reference preparation and repeat the injections. After 7 days, prepare identical dilutions of the vaccine and
RUBELLA VACCINE (LIVE)
IP 2007
reference preparation and repeat the injections. After a further 7 days, inject each vaccinated mouse intracerebrally with 0.03 ml of the standard challenge virus suspension such that on the basis of preliminary titration, 0.03 ml contains between 5 to 50 LD50. Observe the mice for 14 days and record the number of mice surviving the challenge in each group. Calculate the potency of the preparation under examination by standard statistical methods. The vaccine complies with the test if the estimated potency is not less than 2.5 IU per single human dose. The test is not valid unless (a) for both the preparation under examination and the standard preparation, the 50 per cent protective dose lies between the largest and smallest doses given to the mice; (b) there is not deviation from linearity or parallelism of the dose response lines, the confidence limit (P = 0.95) are not less than 25.0 per cent and not more than 400 per cent of the estimated potency; (c) the titre of the challenge virus suspension lies between 5 to 50 LD50. Labelling. The label states the biological origin of the cells used for the preparation of the vaccine.
information on the origin of the strain and its subsequent manipulation. To avoid unnecessary use of monkeys in the test for neurovirulence virus seed lots are prepared in large quantities and stored at temperatures below -20 if freeze-dried, or below -60 if not freeze-dried. Only a seed lot that complies with the following tests may be used for virus propagation.
Identification
The master and working seed lots are identified as rubella virus by serum neutralisation in cell culture, using specific antibodies. Virus concentration. The virus concentration of the master and working seed lots is determined to ensure consistency of production. Extraneous agents (2.7.3). The working seed lot complies with the tests for seed lots. Neurovirulence (2.7.5). The master/working seed lot complies with the test for neurovirulence of live virus vaccines. Macaca and Cercopithecus monkeys are suitable for the test. PROPAGATION AND HARVEST All processing of the cell bank and subsequent cell cultures is done under aseptic conditions in an area where no other cells are handled. Suitable animal (but not human) serum may be used in the growth medium, but the final medium for maintaining cell growth during virus multiplication does not contain animal serum. Serum and trypsin used in the preparation of cell suspensions and culture media are shown to be free from extraneous agents. The cell culture medium may contain a pH indicator such as phenol red and suitable antibiotics at the lowest effective concentration. It is preferable to have a substrate free from antibiotics during production. Not less than 500 ml of the production cell culture is set aside as uninfected cell culture (control cells). The temperature of incubation is controlled during the growth of the virus. The virus suspension is harvested, on one or more occasions, within 28 days of inoculation. Multiple harvests from the same production cell culture may be pooled and considered as a single harvest. Only a single harvest that complies with the following requirements may be used in the preparation of the final bulk vaccine.
Rubella Vaccine (Live)

Rubella Vaccine (Live) is a freeze-dried preparation of a suitable attenuated strain of rubella virus. The vaccine is reconstituted immediately before use to give a clear liquid or may be coloured owing to the presence of a pH indicator.
Production
General provisions The production of vaccine is based on a virus seed-lot system and a cell-bank system. The production method shall have been shown to yield consistently live rubella vaccines of adequate immunogenicity and safety in man. Unless otherwise justified and authorised, the virus in the final vaccine shall have undergone no more passages from the master seed lot than were used to prepare the vaccine shown in clinical studies to be satisfactory with respect to safety and efficacy. The production method is validated to demonstrate that the product, if tested, would comply with the test for safety and efficacy. Substrate for virus propagation The virus is propagated in human diploid cells (2.7.2). SEED LOT The strain of rubella virus used in the production of rubella vaccine shall be identified by historical records that include 86
Identification
The single harvest contains virus that is identified as rubella virus by serum neutralization in cell culture, using specific antibodies. Virus concentration. The virus concentration in the single harvest is determined as prescribed under Assay to monitor
IP 2007
TETANUS VACCINE (ADSORBED)
consistency of production and to determine the dilution to be used for the final bulk vaccine. Extraneous agents (2.7.3). The single harvest complies with the tests for extraneous agents. Control cells. The control cells comply with a test for identification and with the tests for extraneous agents (2.7.3). FINAL BULK VACCINE Single harvests that comply with the above tests are pooled and clarified to remove cells. A suitable stabilizer may be added and the pooled harvests diluted as appropriate. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT A minimum virus concentration for release of the product is established such as to ensure, in the light of stability data, that the minimum concentration stated on the label will be present at the end of the period of validity. Only a final lot that complies with the tests for minimum virus concentration for release, with the following requirement for thermal stability and with each of the requirements given below under Identification and Tests may be released for use. Provided that the test for bovine serum albumin has been carried out with satisfactory results on the final bulk vaccine, it may be omitted on the final lot. Thermal stability. Maintain samples of the final lot of freezedried vaccine in the dry state at 37 for 7 days. Determine the virus concentration as described under Assay in parallel for the vaccine held at 37 for 7 days and for vaccine stored at 2 to 8. The virus concentration of the heated vaccine is not more than 1.0 log10 lower than that of the unheated vaccine.
Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity. Assay Titrate the vaccine for infective virus at least in triplicate, using at least five cell cultures for each 0.5 log10 dilution step or by a method of equal precision. Use an appropriate virus reference preparation to validate each assay. The estimated virus concentration is not less than that stated on the label; the minimum virus concentration stated on the label is not less than 1 103 CCID50 per human dose. The assay is not valid if the confidence limits (P=0.95) of the logarithm of the virus concentration is greater than 0.3. Rubella vaccine (Live) RS is suitable for use as a reference preparation. Labelling. The label states (1) the strain of virus used for the preparation of the vaccine; (2) the type and origin of the cells used for the preparation of the vaccine; (3) the minimum virus concentration; (4) the time within which the vaccine must be used after reconstitution; (5) that the vaccine must not be given to a pregnant woman and that a woman must not become pregnant within two months.
Tetanus Vaccine (Adsorbed)

Tetanus Vaccine (Adsorbed) is a preparation of tetanus formol toxoid adsorbed on mineral carrier. The formol toxoid is prepared from the toxin produced by the growth of Clostridium tetani.
Production
General provisions The maximum number of Lf per single human dose of tetanus vaccine is 25. The production method is validated to demonstrate that the product, if tested, would comply with the following test. BULK PURIFIED TOXOID For the production of tetanus toxin, from which toxoid is prepared, seed cultures are managed in a defined seed-lot system in which toxinogenicity is conserved and, where necessary, restored by deliberate reselection. A highly toxinogenic strain of Clostridium tetani with known origin and history is grown in a suitable liquid medium. At the end of cultivation, the purity of each culture is tested and contaminated cultures are discarded. Toxin-containing culture medium is collected aseptically. The toxin content (Lf per ml) is checked to monitor consistency of production. Single harvests may be pooled to prepare the bulk purified toxoid. 87
Identification
When the vaccine reconstituted as stated on the label is mixed with specific rubella antibodies, it is no longer able to infect susceptible cell cultures.
Tests
Sterility (2.2.11). The reconstituted vaccine complies with the test for sterility. Water (2.3.43). Not more than 3.0 per cent, determined by Karl Fischer, semi-micro determination of water or by any suitable validated method. Bovine serum albumin. Not more than 50 ng per single human dose, determined by a suitable immunochemical method (2.2.14).
IP 2007
The toxin is purified to remove components likely to cause adverse reactions in humans. The purified toxin is detoxified with formaldehyde by a method that avoids destruction of the immunogenic potency of the toxoid and reversion of toxoid to toxin, particularly on exposure to heat. Alternatively, purification may be carried out after detoxification. Only bulk purified toxoid that complies with the following requirements may be used in the preparation of the final bulk vaccine. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. Absence of tetanus toxin. Inject subcutaneously at least 500 Lf of purified toxoid in a volume of 1 ml into each of five healthy guinea-pigs, each weighing 250 to 350 g, that have not previously been treated with any material that will interfere with the test. If within 21 days of the injection any of the animals shows signs of or dies from tetanus, the toxoid does not comply with the test. If more than one animal dies from non-specific causes, repeat the test once; if more than one animal dies in the second test, the toxoid does not comply with the test. Irreversibility of toxoid. Using the buffer for the final vaccine, prepare a dilution of the bulk purified toxoid containing the same toxoid concentration as the final vaccine. Divide the dilution into two equal parts. Keep one of them at 2 to 8 and the other at 37 for 6 weeks. Test both dilutions by a suitable sensitive assay for active tetanus toxin, such as inoculation into mice or guinea-pigs. The toxoid complies with the test if neither sample produces any sign of a toxic reaction attributable to tetanus toxin. Antigenic purity. Not less than 1,000 Lf per mg of protein nitrogen. FINAL BULK VACCINE The final bulk vaccine is prepared by adsorption of a suitable quantity of bulk purified toxoid onto a mineral carrier such as hydrated aluminium phosphate or aluminium hydroxide; the resulting mixture is approximately isotonic with blood. Suitable antimicrobial preservatives may be added. Certain antimicrobial preservatives, particularly those of the phenolic type, adversely affect the antigenic activity and must not be used. Only final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Specific toxicity. Inject five times the dose stated on the label subcutaneously or intraperitoneally into each of five guineapigs. None of the guinea-pigs shows any symptoms of, or dies from, tetanus within 21 days. If more than one animal dies from non-specific causes within this period repeat the test. None of the second group of animals shows symptoms of
tetanus or dies from tetanus or any other cause within 21 days. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Free formaldehyde (2.3.20). Maximum 0.2 g/l. pH (2.4.24). 6.0 and 7.0 Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity for antisera and vaccine. FINAL LOT The final bulk vaccine is distributed aseptically into sterile, tamper-proof containers. The containers are closed so as to prevent contamination. Only a final lot that is satisfactory with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided the tests for antimicrobial preservative and the assay have been carried out with satisfactory results on the final bulk vaccine, they may be omitted on the final lot.
Identification
Tetanus toxoid is identified by a suitable immunochemical method (2.2.14). The following method, applicable to certain vaccines, is given as an example. Dissolve in the vaccine under examination sufficient sodium citrate to give a 10 per cent solution. Maintain at 37 for about 16 hours and centrifuge until a clear supernatant liquid is obtained. The clear supernatant liquid reacts with a suitable tetanus antitoxin, giving a precipitate.
Tests
Aluminium (2.3.9). Maximum 1.25 mg per single human dose, if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. Free formaldehyde (2.3.20). Maximum 0.2 g/l. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. Sterility (2.2.11). Complies with the test for sterility. Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity for antisera and vaccine.
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Potency of tetanus component Assay. Determine by either of the following methods. (1) Inject subcutaneously on each of two occasions separated by an interval of not more than 4 weeks, one-tenth of the stated human dose diluted to 1 ml with saline solution into each of 9 normal, healthy guinea-pigs weighing between 250 and 350 g. Not more than 2 weeks after the second injection, collect the serum from each animal and carry out the biological test for tetanus antitoxin, described under Tetanus antitoxin or any other method approved by National Regulatory Authority. Sera of at least 6 guinea-pigs out of 9 should contain not less than 0.5 Unit of tetanus antitoxin per ml. (2) Carry out the biological assay of adsorbed Tetanus Vaccine (Adsorbed) described below. If the lower limit of the 95.0 per cent confidence interval of estimated potency is less than 40 IU per single human dose then the limits of the 95.0 per cent confidence interval of the estimater of potency shall be within 50 to 200 per cent of the estimated potency unless the lower limit of the 95.0 per cent confidence interval of the estimated potency is greater than 40 IU per single human dose. Biological assay of adsorbed tetanus vaccine The potency of adsorbed tetanus vaccine is determined by comparing the dose of the vaccine required to protect guineapigs or mice from the paralytic effects of a subcutaneous injection of tetanus toxin with the dose of the Standard preparation needed to give the same protection. For this comparison, the Standard preparation of adsorbed tetanus toxoid and a suitable preparation of tetanus toxin, for use as a challenge toxin, are necessary. Standard preparation The Standard preparation is International standard for Tetanus toxoid, adsorbed or another suitable preparation, the potency of which has been determined in relation to the International standard. Suggested method (a) Test on guinea-pigs Test animals. Use healthy guinea-pigs from the same stock and weighing between 250 and 350 g. Distribute them into six groups of sixteen each. The guinea-pigs should all be of the same sex or the males and females should be distributed equally between the groups. If the challenge toxin to be used has not been shown to be stable or has not been adequately standardised, include four further groups of five guinea-pigs to serve as unvaccinated controls. Challenge toxin. Select a preparation of tetanus toxin containing not less than 50 times the 50 per cent paralytic 89
dose per ml. If the challenge toxin preparation has been shown to be stable, it is not necessary to verify the paralytic dose for every assay. Preparation of the challenge toxin solution. Immediately prior to use, prepare from the challenge toxin by dilution with phosphate buffered saline pH 7.4 a challenge toxin solution containing fifty times the 50 per cent paralytic dose per ml. If necessary, dilute portions of this challenge toxin solution 16-, 50- and 160-fold with the same buffer solution. Determination of potency. Prepare in saline solution three dilutions of the vaccine under examination and three dilutions of a solution of the Standard preparation such that, for each, the dilutions form a series differing by not more than 2.5-fold steps and in which the dilutions of intermediate concentration, when injected subcutaneously in 1-ml volumes into guineapigs, protect approximately 50 per cent of the animals from the paralytic effects of the subcutaneous injection of the quantity of tetanus toxin prescribed for this test. Allocate the six dilutions one to each of the six groups of sixteen guinea-pigs and inject subcutaneously 1.0 ml of each dilution into each guinea-pig in the group to which that dilution is allocated. After 28 days inject each animal subcutaneously with 1.0 ml of the challenge toxin solution containing fifty times the 50 per cent paralytic dose. If necessary, allocate the challenge toxin solution and the three dilutions made from it one to each of the four groups of five guinea-pigs and inject subcutaneously 1.0 ml of each toxin solution into each guineapig in the group to which that toxin solution is allocated. Examine the guinea-pigs twice daily, remove and kill all animals showing definite signs of tetanus paralysis. Count the number of guinea-pigs without paralysis 5 days after injection of the challenge toxin and calculate the potency of the vaccine under examination relative to the potency of the Standard preparation on the basis of the number of animals without paralysis in each of the six groups of sixteen, using standard statistical methods. The test is not valid unless (a) for both the vaccine under examination and the Standard preparation, the 50 per cent protective doses lie between the largest and smallest doses of the preparations given to the guinea-pigs; (b) if applicable, the number of paralysed animals among the four groups of five injected with the challenge toxin solution and its dilutions indicate that the challenge was approximately 50 times the 50 per cent paralytic dose; (c) the fiducial limits of assay lie between 50.0 per cent and 200.0 per cent of the estimated potency; (d) the statistical analysis shows no deviations from linearity or parallelism. The test may be repeated any number of times but when more than one test is performed the results of all valid tests must be combined in the estimate of potency. (b) Test on mice Test animals. Use healthy mice from the same stock, weighing
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between 14 and 20 g. Distribute them into six groups of sixteen each. If the challenge toxin to be used has not been shown to be stable or has not been adequately standardised, include four further groups of six mice to serve as unvaccinated controls. The mice should all be of the same sex or the males and females should be distributed equally among the groups. Challenge toxin. Select a preparation of tetanus toxin containing not less than 100 times the 50 per cent paralytic dose per ml. Preparation of the challenge toxin solutions. Immediately prior to use prepare from the challenge toxin by dilution with phosphate buffered saline pH 7.4 a challenge toxin solution containing fifty times the 50 per cent paralytic dose in each 0.5 ml. If necessary, dilute portions of this challenge toxin solution 16-, 50- and 160-fold with the same buffer solution. Determination of potency. Prepare in saline solution three dilutions of the vaccine under examination and three dilutions of a solution of the Standard preparation such that, for each, the dilutions form a series differing by not more than 2.5-fold steps and in which the dilutions of intermediate concentration, when injected subcutaneously in 0.5 ml volumes into mice, protect approximately 50 per cent of the animals from the paralytic effects of the subcutaneous injection of the quantity of tetanus toxin prescribed for this test. Allocate the six dilutions one to each of the six groups of sixteen mice and inject subcutaneously 0.5 ml of each dilution into each mouse in the group to which the dilution is allocated. After 28 days inject each animal subcutaneously with 0.5 ml of the challenge toxin solution containing fifty times the 50 per cent paralytic dose. If necessary, allocate the challenge toxin solution and the three dilutions made from it one to each of the four groups of six mice and inject subcutaneously 0.5 ml of each toxin solution into each mouse in the group to which that toxin solution is allocated. Count the number of mice without paralysis 4 days after injection of the challenge toxin and calculate the potency of the vaccine under examination relative to the potency of the Standard preparation on the basis of the numbers of animals without paralysis in each of the six groups of sixteen, using standard statistical methods. The test is not valid unless (a) for both the vaccine under examination and the Standard preparation, the 50 per cent protective doses lie between the largest and smallest doses of the preparations given to the mice; (b) if applicable, the number of paralysed animals among the four groups of six injected with the challenge toxin solution and its dilutions indicate that the challenge was approximately 50 times the 50 per cent paralytic dose; (c) the fiducial limits of the assay lie between 50.0 per cent and 200.0 per cent of the estimated potency; (d) the statistical analysis shows no deviation from linearity or parallelism. The test may be repeated any number of times but when more than one test is performed the results of all valid 90
tests must be combined in the estimate of potency. (c) Determination of antibodies in guinea-pigs Preparation of serum samples. For preparation of serum samples, the following technique has been found suitable. Invert the tubes containing blood samples 6 times and allow to stand at 37 for 2 h, then at 4 for 2 hours, centrifuge at room temperature at 800 g for 20 min. transfer the serum to sterile tubes and store at a temperature below -20. At least 40.0 per cent yield of serum is obtained by this procedure. Determination of antibody titre. The ELISA and ToBI tests shown below are given as examples of immunochemical methods that have been found suitable for the determination of antibody tire. Determination of antibody titre in guinea-pig serum by enzyme-linked immunosorbent assay (ELISA). Dilutions of test and reference sera are made on ELISA plates coated with tetanus toxoid. A positive guinea-pig serum control and a negative guinea-pig serum control are included on each plate to monitor the assay performance. Peroxidase-conjugated rabbit or goat antibody directed against guinea-pig-IgG is added followed by a peroxidase substrate. Optical density is measured and the relative antibody titre is calculated using the usual statistical methods. Reagents and equipment ELISA plates: 96 wells, columns 1-12, rows A-H. Clostridium tetani guinea-pig antiserum (for vaccines-human use) reference preparation (positive control serum). Peroxidase conjugate. Peroxidase-conjugated rabbit or goat antibody directed against guinea-pig IgG. Tetanus toxoid. Carbonate coating buffer pH 9.6. Dissolve 1.59 g of anhydrous sodium carbonate and 2.93 g of sodium hydrogen carbonate in 1000 ml of water. Distribute into 150 ml bottles and sterilise by autoclaving at 121 for 15 min. Phosphate buffered saline pH 7.4 (PBS). Dissolve with stirring 80.0 g of sodium chloride, 2.0 g of potassium dihydrogen phosphate, 14.3 g of disodium hydrogen phosphate dihydrate and 2.0 g of potassium chloride in 1000 ml of water. Store at room temperature to prevent crystallisation. Dilute to 10 times its volume with water before use. Citric acid solution. Dissolve 10.51 g of citric acid in 1000 ml of water and adjust the solution to pH 4.0 with a 400 g/l solution of sodium hydroxide. Washing buffer. PBS containing 0.5 g/l of polysorbate 20. Diluent block buffer. PBS containing 0.5 g/l of polysorbate 20 and 25 g/l of dried skimmed milk. Peroxidase substrate. Shortly before use, dissolve 10 mg of
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diammonium 2,2'-azinobis(3-ethylbenzothiazoline-6sulphonate) (ABTS) in 20 ml of citric acid solution. Immediately before use add 5 l of strong hydrogen peroxide solution. Method The description below is given as an example of a suitable plate lay-out but others may be used. Wells 1A-H are for negative control serum and wells 2A-H and 3A-H are for positive control serum for assay monitoring. Wells 4-12A-H are for test samples. Coat each well of the ELISA plates with 100 l of tetanus toxoid solution (0.5 Lf/ml in carbonate coating buffer). Allow to stand overnight at 4 in a humid atmosphere. To avoid interference from temperature gradient, do not stack more than 4 plates high. On the following day, wash the plates thoroughly with washing buffer. Block the plates by addition of 100 l of diluent block buffer to each well. Incubate in a humid atmosphere at 37 for 1 h. Wash the plates thoroughly with washing buffer. Place 100 I of diluent block buffer in each well of the plates, except those of row A. Prepare suitable dilutions of negative control serum, positive control serum (from about 0.01 IU/ml) and test sera. Allocate the negative control serum to column 1, positive control serum to columns 2 and 3 and test sera to columns 4-12 and add 100 I of each serum to the first 2 wells of the column to which it is allocated. Using a multi channel micropipette, make twofold serial dilutions from row B down the plate to row H by transferring 100 l to the following well. Discard 100 l from the last row so that all wells contain 100 I. Incubate at 37 for 2 h. Wash thoroughly with washing buffer. Prepare a suitable dilution (a 1 in 2000 dilution has been found suitable) of peroxidase conjugate in diluent block buffer and add 100 I to each well. Incubate at 37 in a humid atmosphere for 1 h. Wash the plates thoroughly with washing buffer. Add 100 I of peroxidase substrate to each well. Allow to stand at room temperature, protected from light, for 30 min. Read the plates at 405 nm in the same order as addition of substrate was made. Determination of antibody titre in guinea-pig serum by toxinor toxoid-binding inhibition (ToBI). Tetanus toxin or toxoid is added to serial dilutions of test and reference sera; the serum/ antigen mixtures are incubated overnight. To determine unbound toxin or toxoid, the mixtures are transferred to an ELISA plate coated with tetanus antitoxin. Peroxidaseconjugated equine anti-tetanus IgG is added followed by a peroxidase substrate. Optical density is measured and the antibody titre is calculated using the usual statistical methods. A positive control serum and a negative control serum are included on each plate to monitor assay performance. Reagents and equipment Round-bottomed, rigid polystyrene microtitre plates. 91
Flat-bottomed ELISA plates. Tetanus toxin or tetanus toxoid. Clostridium tetani guinea-pig antiserum (for vaccines-human use) reference preparation. Equine anti-tetanus IgG. Peroxidase-conjugated equine anti-tetanus IgG. Carbonate buffer pH 9.6. Dissolve 1.5 g of anhydrous sodium carbonate, 2.39 g of sodium hydrogen carbonate and 0.2 g of sodium azide in 1000 ml of water, adjust to pH 9.6 and autoclave at 121 for 20 min. Sodium acetate buffer pH 5.5. Dissolve 90.2 g of anhydrous sodium acetate in 900 ml of water, adjust to pH 5.5 using a saturated solution of citric acid monohydrate and dilute to 1000 ml with water. Phosphate buffered saline pH 7.2 (PBS). Dissolve 135.0 g of sodium chloride, 20.55 g of disodium hydrogen phosphate dihydrate and 4.80 g of sodium dihydrogen phosphate monohydrate in water and dilute to 15 litres with the same solvent. Autoclave at 100 for 60 min. Diluent buffer. PBS containing 5 g/l of bovine albumin and 0.5 g/l of polysorbate 80. Block buffer. PBS containing 5 g/l of bovine albumin. Tetramethylbenzidine solution. 6 g/l solution of tetramethylbenzidine in alcohol. The substance dissolves within 30-40 min at room temperature. Peroxidase substrate. Mix 90 ml of water, 10 ml of sodium acetate buffer pH 5.5, 1.67 ml of tetramethylbenzidine solution and 20 I of strong hydrogen peroxide solution. Washing solution. Tap water containing 0.5 g/l of polysorbate 80. Method Block the round-bottomed polystyrene microtitre plates by placing in each well 150 I of block buffer. Cover the plates with a lid or sealer. Incubate in a humid atmosphere at 37 for 1 h. Wash the plates thoroughly with washing solution. Place 100 I of PBS in each well. Place 100 l of reference guineapig tetanus antitoxin in the first well of a row. Place 100 l of undiluted test sera in the first well of the required number of rows. Using a multichannel micropipette, make it two fold serial dilutions across the plate (up to column 10), by transfer of 100 I to the following well. Discard 100 l from the last column so that all wells contain 100 l. Prepare 0.1 Lf/ml solution of tetanus toxin or toxoid using PBS a diluent. Add 40 l of this solution to all wells except those column 12. The wells of row 11 are a positive control. Add 40 l of PBS to the wells of column 12 (negative control). Shake the plates gently and cover them with lids. Coat the ELISA plates: immediately before use make
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a suitable dilution of equine anti-tetanus IgG in carbonate buffer pH 9.6 and add 100 l to all wells. Incubate the 2 series of plates overnight in a humid atmosphere at 37. To avoid temperature gradient effects, do not stack more than 4 plate high. Cover the plates with lids. On the following day, wash the ELISA plates thoroughly with washing solution. Block the plates by placing in each well 125 l of block buffer. Incubate at 37 in a humid atmosphere for 1 h. Wash the plates thoroughly with washing solution. Transfer 100 l of the preincubation mixture from the polystyrene plates to the corresponding wells of the ELISA plates, starting with column 12 and then from 1 to 11. Cover the plates with a lid. Incubate at 37 in a humid atmosphere for 2 h. Wash the ELISA plates thoroughly with washing solution. Make a suitable dilution (a 1 in 4000 dilution has been found suitable) of the peroxidaseconjugated equine anti-tetanus IgG in diluent buffer. Add 100 l of the dilution to each well and cover the plates with a lid. Incubate at 37 in a humid atmosphere for 1.5 h. Wash the ELISA plates thoroughly with washing solution. Add 100 l of peroxidase substrate to each well. A blue colour develops. Incubate the plates at room temperature. Stop the reaction at a given time (within 10 min) by the addition of 100 l of 2 M sulphuric acid to each well in the same order as the addition of substrate. The colour changes from blue to yellow. Measure the absorbance (2.4.7) at 450 nm immediately after addition of the sulphuric acid or maintain the plates in the dark until reading. (d) Any other validated serological assay in guinea-pigs/mice approved by National Regulatory Authority. Labelling. The label states (1) the human dose (ml); (2) the minimum units per single human dose or the minimum International Units per single human dose if potency test done by challenge method or both; (3) the name and the amount of the adsorbent and preservative; (4) that the vaccine must be shaken before use; (5) that the vaccine is not to be frozen.
consistently vaccines comparable with the vaccine of proven clinical efficacy and safety in man. The virus in the final vaccine shall not have undergone more passages from the master seed lot than the virus in the vaccine used in clinical trials. The production method is validated to demonstrate that the product, if tested, would comply with the test for abnormal toxicity and immunogenicity. Substrate for virus propagation The virus is propagated in chick embryo cells prepared from eggs derived from a chicken flock free from specified pathogens (2.7.7) or in other suitable cell cultures. SEED LOT The strain of virus used is identified by historical records that include information on the origin of the strain and its subsequent manipulation. Virus seed lots are stored at or below -60. Only a seed lot that complies with the following requirements may be used for virus propagation.
Identification
Each seed lot is identified as containing the vaccine strain of tick-borne encephalitis virus by a suitable immunochemical method, preferably using monoclonal antibodies. Virus concentration. The virus concentration of each seed lot is determined by titration in suitable cell cultures to monitor consistency of production. Extraneous agents (2.7.3). Each seed lot complies with the requirements for extraneous agents in viral vaccines for human use; the tests in cell cultures are carried out in human and simian cells only. PROPAGATION AND HARVEST All processing of the cell cultures if performed under aseptic conditions in an area where no other cells are being handled. Serum and trypsin used in the preparation of cell suspensions and media used must be shown to be free from extraneous agents. The cell culture media may contain a pH indicator such as phenol red and approved antibiotics at the lowest effective concentration. At least 500 ml of the cell cultures employed for vaccine production is set aside as uninfected cell cultures (control cells). Only a single harvest that complies with the following requirements may be used in the preparation of the inactivated harvest.
Tick-borne Encephalitis Vaccine (Inactivated)

Tick-borne Encephalitis Vaccine (Inactivated) is a liquid preparation of a suitable strain of tick-borne encephalitis virus grown in cultures of chick-embryo cells or other suitable cell cultures and inactivated by a suitable, validated method.
Production
General provisions The vaccine complies with the General Requirements of Vaccines for Human Use. Production of the vaccine is based on a virus seed lot system. The production method shall have been shown to yield 92
Identification
The single harvest is shown to contain tick-borne encephalitis virus by a suitable immunochemical method, preferably using
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TICK-BORNE ENCEPHALITIS VACCINE (INACTIVATED)
monoclonal antibodies, or by virus neutralization in cell cultures. Sterility (2.2.11). Complies with the test for sterility carried out using 10 ml for each medium. Mycoplasma (2.7.4). Complies with the test for mycoplasmas carried out using 1 ml for each medium. Control cells. The control cells comply with the tests for extraneous agents (2.7.3). If the vaccine is produced using a cell-bank system, the control cells comply with a test for identification. Virus concentration. Determine the virus concentration by titration in suitable cell culture to monitor consistency of production. Inactivation To avoid interference, viral aggregates are removed by filtration immediately before the inactivation process. The virus suspension is inactivated by a validated method; the method shall have been shown to be consistently capable of inactivating tick-borne encephalitis virus without destroying the antigenic and immunogenic activity; as part of the validation studies, an inactivation curve is plotted representing residual live virus concentration measured on not fewer than three occasions. If formaldehyde is used for inactivation, the presence of an excess of free formaldehyde is verified at the end of the inactivation process. Only an inactivated harvest that complies with the following requirements may be used in the preparation of the final bulk vaccine. Residual infective virus. Inoculate a quantity of the inactivated harvest equivalent to not less than ten human doses of vaccine in the final lot into primary chicken fibroblast cell cultures, or other cells shown to be at least as sensitive to tick-borne encephalitis virus with not less than 3 cm2 of cell sheet per ml of inoculum. Incubate at 37 1 for 14 days. No cytopathic effect is detected at the end of the incubation period. Collect the culture fluid and inoculate 0.03 ml intracerebrally into each of not fewer than ten mice about 4 weeks old. Observe the mice for 14 days. They show no evidence of tick-borne encephalitis virus infection. Purification Several inactivated single harvests may be pooled before concentration and purification by suitable methods, preferably by continuous-flow, sucrose density-gradient centrifugation. Only a purified, inactivated harvest that complies with the following requirements way be used in the preparation of final bulk vaccine. Sterility ( 2.2.11). Complies with the test for sterility, carried out using 10 ml for each medium. 93
Specific activity. Determine the antigen content of the purified, inactivated harvest by a suitable immunochemical method (2.2.14). Determine the total protein content by a suitable method. The specific activity, calculated as the antigen content per unit mass of protein, is within the limits approved for the specific product. FINAL BULK VACCINE The final bulk vaccine is prepared from one or more purified, inactivated harvests. Only a final bulk vaccine that complies with the following requirement may be used in the preparation of the final lot. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT Only a final lot that is satisfactory with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided that the tests for free formaldehyde, bovine serum albumin (where applicable) and pyrogens and the assay have been carried out satisfactory results on the final bulk vaccine, they may be omitted on the final lot.
Identification
The vaccine is shown to contain tick-borne encephalitis virus antigen by a suitable immunochemical method using specific antibodies or by the mouse immunogenicity test described under Assay.
Tests
Aluminium (2.3.9). Maximum 1.25 mg per single human dose, if aluminium hydroxide or hydrated aluminium phosphate is used as the adsorbent. Free formaldehyde (2.3.20). Maximum 0.1 g/l. Bovine serum albumin. If bovine serum albumin has been used during production, the vaccine contains not more than 50 ng per single human dose, determined by a suitable immunochemical method (2.2.14). Sterility (2.2.11). Complies with the test for sterility. Pyrogens (2.2.8). Complies with the test for pyrogens. Inject into each rabbit, per kg of body mass, one dose of vaccine. Assay The potency is determined by comparing the dose necessary to protect a given proportion of mice against the effects of a lethal dose of tick-borne encephalitis virus, administered intraperitoneally, with the quantity of a reference preparation
TUBERCULIN PURIFIED PROTEIN DERIVATIVE
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of tick-borne encephalitis vaccine necessary to provide the same protection. For this comparison an approved reference preparation and a suitable preparation of tick-borne encephalitis virus from an approved strain for use as the challenge preparation are necessary. The following is cited as an example of a method that has been found suitable for a given vaccine. Selection and distribution of test animals. Use healthy mice weighing between 11 and 17 g derived from the same stock. Distribute the mice into not less than six groups of a suitable size to meet the requirements for validity of the test; for titration of the challenge suspension, use not fewer than four groups of ten mice. Use mice of the same sex or distribute males and females equally between groups. Determination of potency of the vaccine. Prepare not less than three suitable dilutions of the vaccine under examination and of the reference preparation; in order to comply with validity criteria four to five dilutions will usually be necessary. Prepare dilutions such that the most concentrated suspension is expected to protect more than 50 per cent of the animals and the least concentrated suspension less than 50.0 per cent. Allocate each dilution to a different group of mice and inject subcutaneously into each mouse 0.2 ml of the dilution allocated to its group. Seven days later make a second injection using the same dilution scale. 14 days after the second injection prepare a suspension of the challenge virus containing not less than 100 LD50 in 0.2 ml. Inject 0.2 ml of this virus suspension intraperitoneally into each vaccinated mouse. To verify the challenge dose, prepare a series of not fewer than three dilutions of the challenge virus suspension at not greater than one-hundredfold intervals. Allocate the challenge suspension and the four dilutions, one to each of the five groups of ten mice, and inject intraperitoneally into each mouse 0.2 ml of the challenge suspension or the dilution allocated to its group. Observe the animals for 21 days after the challenge and record the number of mice that die in the period between 7 and 21 days after the challenge. Calculations. Calculate the results by the usual statistical methods for an assay with quantal responses (5.7). Validity criteria. The test is not valid unless (1) the concentration of the challenge virus is not less than 100 LD50; (2) for both the vaccine under examination and the reference preparation the 50.0 per cent protective dose (PD50) lies between the largest and smallest doses given to the mice; (3) the statistical analysis shows a significant slope and no significant deviation from linearity and parallelism of the doseresponse lines; (4) the fiducial limits (P = 0.95) are not less than 33.0 per cent and not more than 300.0 per cent of the estimated potency. Potency requirement. Include all valid tests to estimate the 94
mean potency and the fiducial limits (P = 0.95) for the mean potency; compute weighed means with the inverse of the squared standard error as weights. The vaccine complies with the test if the estimated potency is not less than that approved by the competent authority, based on data from clinical efficacy trials. Labelling. The label states (1) the strain of virus used in preparation; (2) the type of cells used for production of the vaccine.
Tuberculin Purified Protein Derivative

Tuberculin PPD; Tuberculin Purified Protein Derivative for Human Use Tuberculin Purified Protein Derivative is a preparation made from the heat-treated products of growth and lysis of one or more strains of Mycobacterium tuberculosis that reveal delayed hypersensitivity in animals sensitised by a microorganism of the same species.
Production
It is prepared from the water-soluble fraction obtained by heating in free-flowing steam or in an autoclave and subsequently filtering cultures of the mycobacteria grown in a suitable liquid medium. The active fraction in the filtrate, which is predominantly protein, is separated by precipitation, washed and redissolved. The preparation is free from mycobacteria. An antimicrobial preservative that does not give rise to false positive reactions, such as 0.5 per cent w/v of phenol, and a suitable stabiliser may be added. Phenol is not added to preparations that are to be freeze-dried. The final sterile product is distributed into sterile glass containers which are then sealed so as to prevent microbial contamination or alternatively it is freeze-dried and the containers subsequently sealed.*
* To ensure availability of a preparation of uniform potency, Tuberculin Purified Protein Derivative is produced and issued by the Statens Serum Institute ,Denmark as a powder to be reconstituted as stated on the label.
The preparation may be issued either as a sterile liquid or as a freeze-dried product. If issued as a liquid, it is in a ready-touse form and 0.1 ml constitutes one intradermal dose containing appropriate number of Units. If issued as a freezedried product, it should yield a ready-to-use preparation when reconstituted as per manufacturers instructions. Description. A colourless or pale, straw-coloured liquid, or dry cream-coloured powder, or pellet. The preparation, reconstituted if necessary as stated on the label, complies with the following requirements.
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TUBERCULIN PURIFIED PROTEIN DERIVATIVE
Identification
A. When progressively increasing doses are injected intradermally into specifically sensitised guinea-pigs, reactions occur at the points of injection, varying from erythema to necrosis. When similar injections are administered to nonsensitised guinea-pigs no such reactions occur. B. The potency test described below serves as a test for identity if it is performed on material from the final containers.
derivative with the dose of the appropriate Standard Preparation necessary to give the same effect. The estimated potency is not less 80 per cent and not more than 125 per cent of the stated potency. The fiducial limits of error are not less than 64 per cent and not more than 156 per cent of the stated potency. Standard Preparation The Standard Preparation is the 1st International Standard for Tuberculin, purified protein derivative (PPD), mammalian, established in 1951, consisting of PPD derived from cultures of M. tuberculosis (supplied in ampoules containing 5,00,000 Units) or another suitable preparation the potency of which has been determined in relation to the International Standard. Method Sensitise 12 guinea-pigs each weighing not less than 400 g by the intramuscular injection of a total of about 0.5 ml of a suspension in a suitable mineral oil with or without emulsifier and containing 0.1 mg per ml of heat-inactivated, dried mycobacteria of the same type as that used in the preparation of tuberculin. Use phosphate-buffered saline pH 7.4 containing 0.005 per cent w/v of polysorbate 80 to prepare the dilutions of the Standard preparation and of the preparation under examination and to reconstitute freeze-dried preparations containing no stabiliser. Not less than 1 month and not more than 6 months later carry out the following test : Shave the flanks to provide space for at least three reactions on each side, but not more than a total of 12 injection sites per animal. Use at least three doses of the Standard preparation and at least three doses of the preparation under examination, the highest dose being about 10 times as strong as the lowest. Dilute the preparations so that the lesions produced are 8 to 25 mm in diameter. Allocate the doses to the available sites in a random manner, in a Latin square design. Inject each dose intradermally in the same volume (0.1 or 0.2 ml) at the sites to which it has been allocated. Measure the diameters of the lesions after 24 to 48 hours and calculate the result of the test using standard statistical methods on the basis that the lesion diameters are directly proportional to the logarithm of the concentration of the tuberculin. Storage. Store in light-resistant containers at a temperature between 2 and 8. It should not be allowed to freeze. Labelling. The label states (1) the number of Units per dose of 0.1 ml or per ml or per mg; (2) the total volume in the container (for liquid preparation); (3) the nature and quantity of the reconstituting liquid (for the freeze-dried preparation); (4) the name and proportion of any added substances; (5) the species or strain used; (6) the storage conditions; (7) the date after which the contents are not intended to be used; (8) that care 95
Tests
pH (2.4.24). 6.5 to 7.5. Phenol (if present) (2.3.36). Not more than 0.5 per cent w/v. Sterility (2.2.11). Complies with the tests for sterility. Potency. Carry out the biological assay of tuberculin purified protein derivative described below. Tuberculin Purified Protein Derivative in freeze-dried form complies with the following additional requirements. Live mycobacteria A. Inject 5.0 ml intraperitoneally or subcutaneously into each of two guinea-pigs weighing between 300 and 400 g. Observe the animals for not less than 42 days. Kill the animals and carry out an autopsy. No guinea-pig shows signs of infection with mycobacteria. B. Carry out tests for live mycobacteria in the preparation under examination using suitable culture media. No growth of mycobacteria should occur. Sensitising effect. Inject intradermally into each of three guinea-pigs three times, at intervals of 5 days, a dose of the preparation under examination containing about 500 Units in a volume of 0.1 ml. Two to three weeks after the third injection inoculate the same dose intradermally into these animals and into a control group of three guinea-pigs of the same weight but that have not had any previous injections of tuberculin. The reactions of the two groups are not significantly different after 48 to 72 hours. Toxicity. Inject subcutaneously into 2 healthy guinea-pigs, weighing not less than 250 g and which have not previously been treated with any material which will interfere with the test, 0.5 ml of a solution containing 1,00,000 Units per ml. No harmful effects are produced within 7 days. Biological assay The potency of tuberculin purified protein derivative is determined by comparing the dose necessary to reveal delayed hypersensitivity in guinea-pigs or other animals hypersensitised with mycobacteria of the same type as that used in the preparation of the tuberculin purified protein
TYPHOID (STRAIN TY 21A) VACCINE, LIVE (ORAL)
IP 2007
should be taken to avoid inhaling the powder (for the freezedried preparation).
the growth conditions, strain Ty 21a does not agglutinate to Vi antiserum. Strain Ty 21a agglutinates to H:d flagellar antiserum. Biochemical markers Strian Ty 21a does not produce hydrogen sulphide on Kligler iron agar. This property serves to distinguish Ty 21a from other galactose-epimerase-negative S. typhi strains. Cell growth Strain Ty 21a cells lyse when grown in the presence of 1.0 per cent of galactose. PROPAGATION AND HARVEST The bacteria from the working seed lot are multiplied in a preculture, subcultured once and are then grown in a suitable medium containing 0.001 per cent of galactose at 30 for 13 to 15 hours. The bacteria are harvested. The harvest must be free from contaminating micro-organisms. Only a single harvest that complies with the following requirements may be used for the preparation of the freezedried harvest. pH (2.4.24). 6.8 to 7.5. Optical density The optical density of the culture, measured at 546 nm, is 6.5 to 11.0. Before carrying out the measurement, dilute the culture so that a reading in the range 0.1 to 0.5 is obtained and correct the reading to take account of the dilution.
Typhoid (Strain Ty 21a) Vaccine, Live (Oral)

Typhoid Vaccine (Live, Oral, strain Ty 21a) is a freeze-dried preparation of the live Salmonella typhi strain Ty 21a grown in a suitable medium. When presented in capsules, the vaccine complies with the tests stated under Capsules.
Production
Choice of vaccine strain The main characteristic of the strains is the defect of the enzyme uridine diphosphate-galactose-4 epimerase. The activities of galactopermease, galactokinase and galactose-1phosphate uridyl-transferase are reduced by 50 to 90 per cent. Whatever the growth conditions, the strain does not contain Vi antigen. The strain agglutinates to anti-O:9 antiserum only if grown in medium containing galactose. It contains the flagellar H:d antigen and does not produce hydrogen sulphide on Kligler iron agar. The strain is nonvirulent for mice. Cells of strain Ty 21a lyse if grown in the presence of 1.0 per cent of galactose. SEED LOT The vaccine is prepared using a seed-lot system. The working seed lots represent not more than one subculture from the master seed lot. The final vaccine represents not more than four subcultures from the original vaccine on which were made the laboratory and clinical tests showing the strain to be suitable. Only a master seed lot that complies with the following requirements may be used in the preparation of working seed lots. Galactose metabolism In a spectrophotometric assay, no activity of the enzyme uridine diphosphate-galactose-4-epimerase is found in the cytoplasm of strain Ty 21a compared to strain Ty 2. Biosynthesis of lipopolysaccharide Lipopolysaccharides are extracted by the hot-phenol method and examined by size-exclusion chromatography (2.4.16). Strain Ty 21a grown in medium free of galactose shows only the rough (R) type of lipopolysaccharide. Serological characteristics Strain Ty 21a grown in a synthetic medium without galactose does not agglutinate to specific anti-O:9 antiserum. Whatever 96
Identification
Culture bacteria on an agar medium containing 1.0 per cent of galactose and bromothymol blue. Light blue, concave colonies, transparent due to lysis of cells, should be found. No yellow colonies (galactose-fermenting) should be found. FREEZE-DRIED HARVEST The harvest is mixed with a suitable stabilizer and freeze-dried by a process that ensures the survival of at least 10.0 per cent of the bacteria and to a water content shown to be favourable to the stability of the vaccine. No antimicrobial preservative is added to the vaccine. Only a freeze-dried harvest that complies with the following tests may be used for the preparation of the final bulk.
Identification
Culture bacteria are examined on an agar medium containing 1.0 per cent of galactose and bromoethymol blue. Light blue, concave colonies, transparent due to lysis of cells, should be found. No yellow colonies (galactose-fermenting) should be found.
IP 2007
TYPHOID POLYSACCHARIDE VACCINE
Number of live bacteria Not less than 1 x 10 live S. typhi strain Ty 21a per gram. Water (2.4.43). 1.5 to 4.0 per cent, determined by the semimicro determination of water or any other validated method. FINAL BULK VACCINE The final bulk vaccine is prepared by aseptically mixing one or more freeze-dried harvests with a suitable sterile excipient. Only a final bulk that complies with the following requirement may be used in the preparation of the final lot. Number of live bacteria. Not less than 40 109 live S. typhi strain Ty 21a per gram. FINAL LOT The final bulk vaccine is distributed under aseptic conditions into capsules with a gastro-resistant shell or into suitable containers. Only a final lot that is satisfactory with respect to Identification, Tests and Number of live bacteria will be released for use, except that in the determination of number of live bacteria each dosage unit must contain not less than 4 x 109 live bacteria.
11
Labelling. The label states (1) the minimum number of live bacteria per dosage unit; (2) that the vaccine is for oral use only.
Typhoid Polysaccharide Vaccine

Typhoid Polysaccharide Vaccine is a preparation of purified Vi capsular polysaccharide obtained from Salmonella typhi Ty2 strain or some other suitable strain of known origin and history that has the capacity to produce Vi polysaccharide, and which have been characterized by suitable biochemical, physicochemical, serological or molecular methods. Capsular polysaccharide is a partly 3-O-acetylated repeated units of 2-acetylamino-2-deoxy-D-galactopyranuronic acid with -(1?4)-linkages.
Production
General provisions The production of Vi polysaccharide is based on a defined seed-lot system. The method of production shall have been shown to yield consistently Vi polysaccharide typhoid vaccines of adequate immunogenicity and safety in man. The production method is validated to demonstrate that the product, if tested, would comply with the tests for abnormal toxicity. SEED LOT The strain of S. typhi used for the master seed lot shall be identified by historical records that include information on its origin and by its biochemical and serological characteristics. Cultures from the working seed lot shall have the same characteristics as the strain that was used to prepare the master seed lot, shall be demonstrated along with adequate documentation. Only a strain that has the following characteristics may be used in the preparation of the vaccine: (a) stained smears from a culture are typical of S. typhi; (b) the culture utilises glucose without production of gas; (c) colonies on agar are oxidasenegative; (d) a suspension of the culture agglutinates specifically with an appropriate Vi antiserum or colonies form haloes on an agar plate containing a suitable Vi antiserum. PROPAGATION AND HARVEST The working seed lot is cultured on a solid medium, which may contain blood-group substances, or a liquid medium; the inoculum obtained is transferred to a liquid medium, which is used to inoculate the final medium. The liquid medium used and the final medium are semi-synthetic, free from substances that are precipitated by cetrimonium bromide and do not contain blood-group substances or high-molecular-mass 97
Identification
Culture bacteria from the vaccine under examination on an agar medium containing 1.0 per cent of galactose and bromothymol blue. Light blue, concave colonies transparent due to lysis of cells, should be found. No yellow colonies (galactose-fermenting) should be found.
Tests
Microbial contamination (2.2.9). Carry out the test using suitable selective media. Determine the total viable count using the plate-count method. The number of contaminating microorganisms per dosage unit is not greater than 102 bacteria and 20 fungi. No pathogenic bacterium, particularly Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and no Salmonella other than strain Ty 21a are found. Water (2.4.43). 1.5 to 4.0 per cent. Number of live bacteria Carry out the test using not less than five dosage units. Homogenise the contents of the dosage units in a 0.9 per cent w/v solution of sodium chloride at 4 using a mixer in a cold room with sufficient glass beads to emerge from the liquid. Immediately after homogenization prepare a suitable dilution of the suspension using cooled diluent and inoculate brain heart infusion agar, incubate at 36 1 for 20 to 36 hours. The vaccine contains not less than 2 x 109 live S. typhi Ty 21a bacteria per dosage unit.
TYPHOID POLYSACCHARIDE VACCINE
IP 2007
polysaccharides, unless it has been demonstrated that they are removed by the purification process. The bacterial purity of the culture is verified by microscopic examination of Gramstained smears and by inoculation into appropriate media. The culture is then inactivated at the beginning of the stationary phase by the addition of formaldehyde. Bacterial cells are eliminated by centrifugation; the polysaccharide is precipitated from the culture medium by addition of hexadecyltrimethylammonium bromide (cetrimonium bromide). The precipitate is harvested and may be stored at or below -20 before purification. Purified Vi Polysaccharide The polysaccharide is purified, after dissociation of the polysaccharide/cetrimonium bromide complex, using suitable procedures to eliminate successively nucleic acids, proteins and lipopolysaccharides. The polysaccharide is precipitated in its salt form and dried at 5+3; the powder obtained constitutes the purified Vi polysaccharide. The loss on drying is determined by thermogravimetry, Karl Fischer or any other suitable method and is used to calculate the results of the chemical tests shown below with reference to the dried substance. Only those pools of purified Vi polysaccharide that comply with the following requirements may be used in the preparation of the final bulk: Protein (2.7.1). Not more than 10 mg per gram of polysaccharide, calculated with reference to the dried substance. Nucleic acids (2.7.1). Not more than 20 mg per gram of polysaccharide, calculated with reference to the dried substance. O-Acetyl groups (2.7.1). Not less than 2 mmol per gram of polysaccharide, calculated with reference to the dried substance. Molecular size. Examine by gel filtration or size-exclusion chromatography (2.4.16) using cross-linked agarose for chromatography. Use a column 0.9 m long and 16 mm in internal diameter equilibrated with a solvent having an ionic strength of 0.2 mol per kg and a pH of 7.0 to 7.5. Apply about 5 mg of polysaccharide in a volume of 1 ml to the column and elute at about 20 ml/h. Collect fractions of about 2.5 ml. Determine the point corresponding to K0 = 0.25 and make two pools consisting of fractions eluted before and after this point. Determine O-acetyl groups on the two pools. Not less than 50 per cent of the polysaccharide is found in the pool containing fractions eluted before K0 = 0.25.
Bacterial endotoxins (2.2.3). Determine by a suitable method, the content should not be more than 150 IU per mg of polysaccharide. FINAL BULK VACCINE One or more batches of purified Vi polysaccharide are dissolved in a suitable solvent, which may contain an antimicrobial preservative, so that the volume corresponding to one dose contains 25 g of polysaccharide and the solution is isotonic with blood (250 mosm/kg to 350 mosm/kg). Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot; Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. If phenol has been used in the preparation, the content is not more than 2.5 g/l. FINAL LOT The final bulk vaccine is distributed and filled aseptically into sterile containers. The containers are closed so as to prevent contamination. Only a final lot that is satisfactory with respect to each of the requirements prescribed below under Identification, Tests and Assay and with the requirements for Bacterial endotoxins may be released for use. Provided the tests for free formaldehyde and antimicrobial preservative have been carried out on the final bulk vaccine, they may be omitted on the final lot. The vaccine contains minimum of 25 g purified ViPolysaccharide per dose of 0.5 ml.
Identification
Carry out an identification test using a suitable immunochemical method (2.2.14).
Tests
Sterility (2.2.11). Complies with the tests for sterility. Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity. pH (2.4.24). 6.5 to 7.5. O-Acetyl groups (2.7.1). 0.085 ( 25 per cent) mol per dose (25 g of polysaccharide). Test solution. Place 3 ml of the vaccine in each of three tubes (two reaction solutions and one correction solution). 98
Identification
Carry out an identification test using a suitable immunochemical method (2.2.14).
IP 2007
TYPHOID VACCINE (FREEZE DRIED)
Reference solutions. Dissolve 0.150 g of acetylcholine chloride in 10 ml of water (stock solution containing 15 g/l of acetylcholine chloride). Immediately before use, dilute 0.5 ml of the stock solution to 50 ml with water (working dilution containing 150 g/ml of acetylcholine chloride). In ten tubes, place in duplicate (reaction and correction solutions) 0.1 ml, 0.2 ml, 0.5 ml, 1.0 ml and 1.5 ml of the working dilution. Prepare a blank using 3 ml of purified water. Make up the volume in each tube to 3 ml with water. Add 0.5 ml of a mixture of 1 volume of water and 2 volumes of dilute hydrochloric acid to each of the correction tubes and to the blank. Add 1.0 ml of alkaline hydroxylamine solution to each tube. Allow the reaction to proceed for exactly 2 min and add 0.5 ml of a mixture of 1 volume of water and 2 volumes of dilute hydrochloric acid to each of the reaction tubes. Add 0.5 ml of a 20 per cent w/vl solution of ferric chloride in 0.2M hydrochloric acid to each tube, stopper the tubes and shake vigorously to remove bubbles. Measure the absorbance of each solution at 540 nm using the blank as the compensation liquid. For each reaction solution, subtract the absorbance of the corresponding correction solution. Draw a calibration curve from the corrected absorbance for the five reference solutions and the corresponding content of acetylcholine chloride and read from the curve the content of acetylcholine chloride in the test solution for each volume tested. Calculate the mean of the two values. 1 mol of acetylcholine chloride (181.7 g) is equivalent to 1 mol of O-acetyl (43.05 g). Free formaldehyde (2.3.20). Maximum 0.2 g/l. Antimicrobial preservative. Where applicable, determine the amount of antimicrobial preservative by a suitable chemical method. The content is not less than 85.0 per cent and not greater than 115.0 per cent of the intended amount. If phenol has been used in the preparation, the content is not more than 2.5 g/l. Assay Determine Vi polysaccharide content by a suitable immunochemical method (2.2.14) using a reference purified polysaccharide. The estimated amount of polysaccharide per dose is 80.0 per cent to 120.0 per cent of the content stated on the label. The fiducial limits of error (P = 0.95) of the estimated amount of polysaccharide are not less than 80.0 per cent and not more than 120.0 per cent. Labelling. The label states (1) the number of micrograms of polysaccharide per human dose (25 g); (2) the total quantity of polysaccharide in the container. 99
Typhoid Vaccine
Typhoid Vaccine is a sterile suspension of inactivated Salmonella typhi containing not less than 5 x 108 and not more than 1 x 109 bacteria per human dose. The human dose does not exceed 1.0 ml.
Production
The vaccine is prepared using a seed-lot system from a suitable strain of S. typhi such as, Ty 2. The final vaccine represents not more than 3 subcultures from the strain on which were made the laboratory and clinical tests that showed it to be suitable. The bacteria are inactivated by acetone, by formaldehyde, by phenol or by heating or by a combination of the last two methods. The production method is validated to demonstrate that the product, if tested, would comply with the test for abnormal toxicity (2.2.1) modified to the extent that 0.5 ml of the vaccine is injected subcutaneously or intramuscularly or intraperitoneally into each mouse and 1.0 ml into each guineapig.
Identification
It is identified by specific agglutination. Phenol (2.3.36). If phenol has been used in the preparation, the concentration is not more than 0.5 per cent w/v. Antigenic power. When injected into susceptible laboratory animals, it elicits anti-O, anti-H and, to a lesser extent, anti-Vi agglutinins. Sterility (2.2.11). Complies with the test for sterility. Labelling. The label states (1) the method used to inactivate the bacteria; (2) the number of bacteria per human dose.
Typhoid Vaccine (Freeze Dried)

Freeze Dried Typhoid Vaccine is a freeze-dried preparation of inactivated Salmonella typhi. The vaccine is reconstituted as stated on the label to give a uniform suspension containing not less than 5 x 108 and not more than 1 x 109 bacteria per human dose. The human dose does not exceed 1.0 ml of the reconstituted vaccine.
Production
The vaccine is prepared from a seed-lot system from a suitable strain of S. typhi, such as Ty 2. The final vaccine represents not more than 3 subcultures from the strain on which were made the laboratory and clinical tests that showed it to be suitable. The bacteria are inactivated either by acetone or by
VARICELLA VACCINE, LIVE
IP 2007
formaldehyde or by heat. Phenol is not used in the preparation. The vaccine is distributed into sterile containers and freezedried to a moisture content favourable to the stability of the vaccine. The production method is validated to demonstrate that the product, if tested, would comply with test for abnormal toxicity (2.2.1), modified to the extent that 0.5 ml of the vaccine is injected subcutaneously or intramuscularly or intraperitoneally into each mouse and 1.0 ml into each guineapig.
origin of the strain and its subsequent manipulation. The virus shall at no time have been passaged in continuous cell lines. Seed lots are prepared in the same kind of cells as those used for the production of the final vaccine. To avoid the unnecessary use of monkeys in the test for neurovirulence, virus seed lots are prepared in large quantities and stored at temperatures below -20, if freeze-dried, or below -60, if not freeze-dried. Only a virus seed lot that complies with the following requirements may be used for virus propagation.
Identification
The vaccine reconstituted as stated on the label is identified by specific agglutination. Antigenic power. When injected into susceptible laboratory animals, the reconstituted vaccine elicits anti-O, anti-H and, to a lesser extent, anti-Vi agglutinins. Sterility (2.2.11). The reconstituted vaccine complies with the tests for sterility. Water. Not more than 5.0 percent. Labelling. The label states (1) the method used to inactivate the bacteria; (2) the number of bacteria per human dose; (3) that the vaccine should be used within 8 hours of reconstitution.
Identification
The master and working seed lots are identified as varicella virus by serum neutralisation in cell culture, using specific antibodies. Virus concentration. The virus concentration of the master and working seed lots is determined as prescribed under Assay to monitor consistency of production. Extraneous agents (2.7.3). Complies with the requirements for seed lots for live virus vaccines; a sample of 50 ml is taken for the test in cell cultures. Neurovirulence (2.7.5). Complies with the test for neurovirulence of live virus vaccines. PROPAGATION AND HARVEST All processing of the cell bank and subsequent cell cultures is done under aseptic conditions in an area where no other cells are handled. Approved animal (but not human) serum may be used in the media. Serum and trypsin used in the preparation of cell suspensions and media are shown to be free from extraneous agents. The cell culture medium may contain a pH indicator such as phenol red and approved antibiotics at the lowest effective concentration. It is preferable to have a substrate free from antibiotics during production. 5.0 per cent, but not less than 50.0 ml, of the cell cultures employed for vaccine production is set aside as uninfected cell cultures (control cells). The infected cells constituting a single harvest are washed, released from the support surface and pooled. The cell suspension is disrupted by sonication. Only a virus harvest that complies with the following requirements may be used in the preparation of the final bulk vaccine.
Varicella Vaccine, Live

Varicella Vaccine (Live) is a freeze-dried preparation of a suitable attenuated strain of Herpesvirus varicellae.
Production
General provisions The production of vaccine is based on a virus seed-lot system and a cell-bank system. The production method shall have been shown to yield consistently live varicella vaccines of adequate immunogenicity and safety in man. The virus in the final vaccine shall not have been passaged in cell cultures beyond the 38th passage from the original isolated virus. The production method is validated to demonstrate that the product, if tested, would comply with the test for safety and efficacy. Substrate for virus propagation The virus is propagated in human diploid cells (2.7.2). SEED LOT The strain of varicella virus shall be identified as being suitable by historical records which shall include information on the
Identification
The virus harvest contains virus that is identified as varicella virus by serum neutralisation in cell culture, using specific antibodies. Virus concentration. The concentration of infective virus in virus harvests is determined as prescribed under assay to monitor consistency of production and to determine the
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IP 2007
VIPER VENOM
dilution to be used for the final bulk vaccine. Extraneous agents (2.7.3). Use 50 ml for the test in cell cultures. Control cells. The control cells of the production cell culture from which the single harvest is derived comply with a test for identity and with the requirements for extraneous agents (2.7.3). FINAL BULK VACCINE Virus harvests that comply with the above tests are pooled and clarified to remove cells. A suitable stabiliser may be added and the pooled harvests diluted as appropriate. Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. FINAL LOT The final bulk vaccine is distributed aseptically into sterile, tamper-proof containers and freeze-dried to a moisture content shown to be favourable to the stability of the vaccine. The containers are then closed so as to prevent contamination and the introduction of moisture. Only a final lot that is satisfactory with respect to each of the requirements given below under Identification, Tests and Assay may be released for use. Provided that the test for bovine serum albumin has been carried out with satisfactory results on the final bulk vaccine, it may be omitted on the final lot.
Labelling. The label states (1) the strain of virus used for the preparation of the vaccine; (2) the type and origin of the cells used for the preparation of the vaccine; (3) that contact with disinfectants is to be avoided; (4) the minimum virus concentration; (5) that the vaccine is not to be administered to pregnant women; (6) the time within which the vaccine must be used after reconstitution.
Viper Venom
Daboia Venom
Viper Venom is the dried secretion obtained from the poison glands of Viperae russelli and other species of Viperae (Fam. Viperidae). Viper Venom contains not less than 50 Mouse Units per mg.
Production
Immediately after extraction, the poisonous secretion is dried from the frozen state. The dried venom is pooled, mixed, dissolved in ice-cold water for injection and then filtered through a bacteria-proof filter to give a stock solution. Further dilutions of the stock solution are made with water for injection under aseptic conditions to give solutions with the required number of Mouse Units per ml. These solutions are then distributed in single dose sterile glass containers, dried from the frozen state and sealed at a pressure not exceeding 2.75 kPa. Description. An almost white or very light yellow, dry powder which when mixed with water yields a clear solution with some insoluble residue.
Identification
When the vaccine reconstituted as stated on the label is mixed with specific Herpesvirus varicellae antibodies, it is no longer able to infect susceptible cell cultures.
Identification
A. Produces almost immediate coagulation of blood and citrated human plasma. B. Mix the soluble fraction from at least 0.6 mg with 1 ml of polyvalent antisnake venom serum and incubate the mixture at 37o for 30 minutes. Inject 0.5 ml of the mixture intravenously into a group of mice weighing between 18 and 20 g. Observe the animals for 24 hours; no animal dies.
Tests
Sterility (2.2.11) Complies with the test for sterility. Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity. Bovine serum albumin. Not more than 0.5 g per human dose, determined by a suitable immunochemical method (2.2.14). Water (2.3.43). Not more than 3.0 per cent, determined by the semi-micro determination of water. Assay Titrate for infective virus, using at least ten cell cultures for each fourfold dilution or by a technique of equal precision. Use a suitable virus reference preparation to validate each assay. The virus concentration is not less than the minimum stated on the label.
Tests
Sterility (2.2.11). Complies with tests for sterility. Assay. Carry out the biological assay of snake venom described below: Biological assay of snake venom Dissolve a quantity of the freeze-dried venom equivalent to 50 Mouse Units in 25 ml of saline solution. Inject 0.5 ml
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YELLOW FEVER VACCINE (LIVE)
IP 2007
intravenously into each of 10 mice weighing between 18 and 20 g and observe the animals for 24 hours. Not less than 3 and not more than 8 of the mice die in 2 to 24 hours. If the number of deaths is not within this range, change the dilution of the venom suitably. Express the result in terms of number of Mouse Units per mg. NOTE The quantity in mg of the venom which will kill in 2 to 24 hours not less than 3 and not more than 8 mice represents one Mouse Unit. Storage. Store in single dose, light-resistant containers. Labelling. The label states (1) the number of Mouse Units per container; (2) the volume of water for injection to be used for reconstitution.
be only one passage from a master seed lot. A working seed lot shall be used without intervening passage as the inoculum for infecting the tissues used in the production of a vaccine lot, so that no vaccine virus is more than one passage from a seed lot that has passed all the safety tests. Only a virus seed lot that complies with the following requirements may be used for virus propagation.
Identification
The master and working seed lots are identified as containing yellow fever virus by serum neutralisation in cell culture, using specific antibodies. Extraneous agents (2.7.3). Each working seed lot complies with the test for extraneous agents. PROPAGATION AND HARVEST
Yellow Fever Vaccine (Live)

Yellow Fever Vaccine (Live) is a freeze-dried preparation of the 17D strain of yellow fever virus grown in fertilised hen eggs.
Production
General provisions The production of vaccine is based on a virus seed-lot system. The production method shall have been shown to yield consistently the yellow fever vaccine (live) of acceptable immunogenicity and safety for man. The production method is validated to demonstrate that the product, if tested, would comply with the test for safety and efficacy. Reference preparation. In the test for neurotropism, a suitable batch of vaccine known to have satisfactory properties in man is used as the reference preparation. Substrate for virus propagation Virus for the preparation of master and working seed lots and for all vaccine batches is grown in the tissues of chick embryos from a flock free from specified pathogens (2.7.7). SEED LOT The 17D strain shall be identified by historical records that include information on the origin of the strain and its subsequent manipulation. Virus seed lots are prepared in large quantities and stored at a temperature below -60. Master and working seed lots shall not contain any human protein or added serum. Unless otherwise justified and authorised, the virus in the final vaccine shall be between passage levels 204 and 239 from the original isolate of strain 17D. A working seed lot shall
All processing of the fertilised eggs is done under aseptic conditions in an area where no other infectious agents or cells are handled at the same time. Two per cent but not less than twenty and not more than fifty eggs are set aside as uninfected control eggs. After inoculation and incubation at a controlled temperature, only living and typical chick embryos are harvested. The age of the embryo at the time of virus harvest is reckoned from the initial introduction of the egg into the incubator and shall not be more than 12 days. After homogenisation and clarification by centrifugation, the extract of embryonic pulp is tested as described below and kept at -70 or colder until further processing. Virus harvests that comply with the prescribed tests may be pooled. No human protein is added to the virus suspension at any stage during production. If stabilisers are added, they shall have been shown to have no antigenic or sensitising properties for man. Only a single harvest that complies with the following tests may be used in the preparation of the final bulk vaccine.
Identification
The single harvest contains virus that is identified as yellow fever virus by serum neutralisation in cell culture, using specific antibodies. Extraneous agents (2.7.3). Complies with the tests for extraneous agents. Control eggs. Complies with the tests for extraneous agents (2.7.3). Virus concentration. In order to calculate the dilution for formulation of the final bulk, each single harvest is titrated as described under Assay. FINAL BULK VACCINE Single harvests that comply with the tests prescribed above are pooled and clarified again. A test for protein nitrogen
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content is carried out. A suitable stabiliser may be added and the pooled harvests diluted as appropriate. Only a final bulk vaccine that complies with the following tests may be used in the preparation of the final lot. Sterility (2.2.11). Carry out test for sterility using 10 ml of bulk for each sterility medium. Protein nitrogen content (2.3.30). The protein nitrogen content, before the addition of any stabiliser, is not more than 0.25 mg per human dose. FINAL LOT The final bulk vaccine is distributed aseptically into sterile, tamper-proof containers and freeze-dried to a moisture content shown to be favourable to the stability of the vaccine. The containers are then closed so as to prevent contamination and the introduction of moisture. Only a final lot that is satisfactory with respect to thermal stability and each of the tests given under Identification, Tests and Assay may be released for use. Provided that the test for ovalbumin has been performed with satisfactory results on the final bulk vaccine, it may be omitted on the final lot. Thermal stability. Maintain samples of the final lot of freezedried vaccine in the dry state at 37 for 14 days. Determine the virus concentration as described under Assay in parallel for the heated vaccine and for unheated vaccine. The difference in the virus concentration between unheated and heated vaccine does not exceed 1.0 log10, and the virus concentration of the heated vaccine is not less than the number of TCID50 or plaque-forming units (PFU) equivalent to 1 103 mouse LD50 per human dose.
The virus concentration is not less than the equivalent in TCID50 or PFU of 1 103 mouse LD50 per human dose. The relationship between mouse LD50 and TCID50 or PFU is established by each laboratory and approved by the competent authority. The method shown below, or another suitable technique, may be used to determine the mouse LD50. Mouse LD50. The statistically calculated quantity of virus suspension that is expected to produce fatal specific encephalitis in 50 per cent of mice of a highly susceptible strain, 4 to 6 weeks of age, after intracerebral inoculation. Appropriate serial dilutions of the reconstituted vaccine are made in diluent for yellow fever virus (0.75 per cent solution of bovine albumin in phosphate-buffered saline pH 7.4, or any other diluent that has been shown to be equivalent for maintaining the infectivity of the virus). Mice of a highly susceptible strain, 4 to 6 weeks of age, are injected intracerebrally under anaesthesia with 0.03 ml of the vaccine dilution. Groups of not less than eight mice are used for each dilution; the series of dilutions is chosen so as to cover the range 0 to 100.0 per cent mortality of the mice. Injection of the mice is performed immediately after the dilutions have been made. The mice are observed for 21 days and all deaths are recorded. Only survivors and deaths caused by typical yellow fever infections are counted in the computations. Mice paralysed on the twenty-first day of observation are counted as survivors. Tests in monkeys for Yellow Fever Vaccine Each master and working seed lot complies with the following tests in monkeys for viraemia (viscerotropism), immunogenicity and neurotropism. The monkeys shall be Macaca spp. susceptible to yellow fever virus and shall have been shown to be non-immune to yellow fever at the time of injecting the seed virus. They shall be healthy and shall not have received previously intracerebral or intraspinal inoculation. Furthermore, they shall not have been inoculated by other routes with neurotropic viruses or with antigens related to yellow fever virus. Not fewer than ten monkeys are used for each test. Use a test dose of 0.25 ml containing the equivalent of not less than 5000 mouse LD50 and not more than 50,000 mouse LD50, determined by a titration for infectious virus and using the established equivalence between virus concentration and mouse LD50 (see under Assay). Inject the test dose into one frontal lobe of each monkey under anaesthesia and observe the monkeys for not less than 30 days. Viraemia (Viscerotropism). Viscerotropism is indicated by the amount of virus present in serum. Take blood from each of the test monkeys on the second, fourth and sixth days after
Identification
When the vaccine reconstituted as stated on the label is mixed with specific yellow fever virus antibodies, there is a significant reduction in its ability to infect susceptible cell cultures.
Tests
Sterility (2.2.11). Complies with the test for sterility. Ovalbumin. Not more than 5 g of ovalbumin per human dose, determined by a suitable immunochemical method (2.2.14). Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity. Bacterial endotoxins (2.2.3). Not more than 5 IU of bacterial endotoxin per human dose. Water (2.3.43). Not more than 3.0 per cent, determined by the semi-micro determination of water. Assay Titrate for infective virus in cell cultures. Use an appropriate virus reference preparation to validate each assay.
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inoculation and prepare serum from each sample. Prepare 1:10, 1:100 and 1:1000 dilutions from each serum and inoculate each dilution into a group of at least six cell culture vessels used for the determination of the virus concentration. The seed lot complies with the test if none of the sera contains more than the equivalent of 500 mouse LD50 in 0.03 ml and at most one serum contains more than the equivalent of 100 mouse LD50 in 0.03 ml. Immunogenicity. Take blood from each monkey 30 days after the injection of the test dose and prepare serum from each sample. The seed lot complies with the test if at least 90.0 per cent of the test monkeys are shown to be immune, as determined by examining their sera in the test for neutralisation of yellow fever virus described below. It has been shown that a low dilution of serum (for example, 1:10) may contain non-specific inhibitors that influence this test; such serum shall be treated to remove inhibitors. Mix dilutions of at least 1: 10, 1:40 and 1: 160 of serum from each monkey with an equal volume of 17D vaccine virus at a dilution that will yield an optimum number of plaques with the titration method used. Incubate the serum-virus mixtures in a waterbath at 37 for 1 h and then cool in iced water; add 0.2 ml of each serum-virus mixture to each of four cell-culture plates and proceed as for the determination of virus concentration. Inoculate similarly ten plates with the same amount of virus plus an equal volume of a 1:10 dilution of monkey serum known to contain no neutralising antibodies to yellow fever virus. At the end of the observation period, compare the mean number of plaques in the plates receiving virus plus non-immune serum with the mean number of plaques in the plates receiving virus plus dilutions of each monkey serum. Not more than 10 per cent of the test monkeys have serum that fails to reduce the number of plaques by 50.0 per cent at the 1:10 dilution. Neurotropism. Neurotropism is assessed from clinical evidence of encephalitis, from incidence of clinical manifestations and by evaluation of histological lesions, in comparison with ten monkeys injected with the reference preparation. The seed lot is not acceptable if either the onset and duration of the febrile reaction or the clinical signs of encephalitis and pathological findings are such as to indicate a change in the properties of the virus. Clinical evaluation. The monkeys are examined daily for 30 days by personnel familiar with clinical signs of encephalitis in primates (if necessary, the monkeys are removed from their cage and examined for signs of motor weakness or spasticity). The seed lot is not acceptable if in the monkeys injected with it the incidence of severe signs of encephalitis, such as paralysis or inability to stand when stimulated, or mortality is greater than for the reference vaccine. These and other signs of encephalitis, such as paresis, in-coordination, lethargy, tremors or spasticity are assigned numerical values for the severity of symptoms by a grading method. Each day each
monkey in the test is given a score based on the scale: Grade 1 rough coat, not eating, high-pitched voice, inactive, slow moving, shaky, tremors, unco-ordinated, limb weakness, inability to stand, limb paralysis or death (a dead monkey receives a daily score of 4 from the day of death until day 30). Grade 2 Grade 3 Grade 4
A clinical score for a particular monkey is the average of its daily scores; the clinical score for the seed lot is the mean of the individual monkey scores. The seed lot is not acceptable if the mean of the clinical severity scores for the group of monkeys inoculated with it is significantly greater (P = 0.95) than the mean for the group of monkeys injected with the reference preparation. In addition, special consideration is given to any animal showing unusually severe signs when deciding on the acceptability of the seed lot. Histological evaluation. Five levels of the brain are examined including : Block 1 Block II the corpus striatum at the level of the optic chiasma, the thalamus at the level of the mamillary bodies,
Block III the mesencephalon at the level of the superior colliculi, Block IV the pons and cerebellum at the level of the superior olives, Block V the medulla oblongata and cerebellum at the level of the mid-inferior olivary nuclei.
Cervical and lumbar enlargements of the spinal cord are each divided equally into six blocks; 15 m sections are cut from the tissue blocks embedded in paraffin wax and stained with gallocyanin. Numerical scores are given to each hemisection of the cord and to structures in each hemisection of the brain as listed below. Lesions are scored as follows: Grade 1. Minimal: 1 to 3 small focal inflammatory infiltrates. Degeneration or loss of a few neurons. Grade 2. Moderate: 4 or more focal inflammatory infiltrates. Degeneration or loss of neurons affecting not more than one third of cells. Grade 3. Severe: moderate focal or diffuse inflammatory infiltration. Degeneration or loss of up to two third of the neurons. Grade 4. Overwhelming: variable but often severe inflammatory reaction. Degeneration or loss of more than 90.0 per cent of neurons. It has been found that inoculation of yellow fever vaccine into the monkey brain causes histological lesions in different
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anatomical formations of the central nervous system with varying frequency and severity (I. S. Levenbook et al., Journal of Biological Standardization, 1987, 15, 305-313). Based on these two indicators, the anatomical structures can be divided into target, spared and discriminator areas. Target areas are those which show more severe specific lesions in a majority of monkeys irrespective of the degree of neurovirulence of the seed lot. Spared areas are those which show only minimal specific lesions and in a minority of monkeys. Discriminator areas are those where there is a significant increase in the frequency of more severe specific lesions with seed lots having a higher degree of neurovirulence. Discriminator and target areas for Macaca cynomolgus and Macaca rhesus monkeys are shown in the table below: Table 1. The discriminator and target areas for monkey. Type of monkey Discriminator areas Target areas substantia nigra
Macaca cynomolgus globus pallidus putamen anterior/ median thalamic nucleus lateral thalamic nucleus Macaca rhesus
caudate nucleus substantia nigra globus pallidus cervical putamen anterior/ lumbar median thalamic enlargement nucleus lateral thalamic nucleus cervical enlargement lumbar enlargement enlargement
Scores for discriminator and target areas are used for the final evaluation of the seed lot. The individual monkey score is calculated from the sum of individual target area scores in each hemisection divided by the number of areas examined. A separate score is calculated similarly for the discriminator areas. Mean scores for the test group are calculated in two ways: (1) by dividing the sum of the individual monkey discriminator scores by the number of monkeys and (2) by dividing the sum of the individual monkey target and discriminator scores by the number of monkeys. These two mean scores are taken into account when deciding on the acceptability of the seed lot. The seed lot is not acceptable if either of the mean lesion scores is significantly greater (P = 0.95) than for the reference preparation. Labelling. The label states (1) the strain of virus used in preparation; (2) that the vaccine has been prepared in chick embryos; (3) the minimum virus concentration; (4) that contact
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MONOGRAPHS
HERBS AND HERBAL PRODUCTS

General Requirements ..................................................................................................... Monographs ..................................................................................................................... Acacia .................................................................................................................. Amalaki ................................................................................................................. Amra .................................................................................................................... Arachis Oil ............................................................................................................ Arjuna .................................................................................................................... Artemisia ............................................................................................................... Ashwagandha ........................................................................................................ Belladonna Dtry Extract ....................................................................................... Belladonna Leaves .................................................................................................. Bhibhitaki ................................................................................................................ Bhringraj ................................................................................................................ Bhuiamla ................................................................................................................ Brahmi ................................................................................................................. Castor Oil ............................................................................................................... Clove Oil ............................................................................................................... Coleus .................................................................................................................. Eucalyptus Oil ........................................................................................................ Garcinia ................................................................................................................. Gokhru ................................................................................................................. Guar Gum ............................................................................................................. Gudmar ................................................................................................................ Guduchi ............................................................................................................... ........................................................................................................ Guggul resin
Gugulipid ............................................................................................................... Gugulipid Tablets .................................................................................................... Haridra .................................................................................................................. Haritaki ................................................................................................................. Hydrogenated Castor Oil .......................................................................................
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Isapgol Husk ......................................................................................................... Kalmegh ................................................................................................................ Kunduru ................................................................................................................ Kutki ...................................................................................................................... Lausna ................................................................................................................... Malt Extract ............................................................................................................ Mendukaparni ........................................................................................................ Manjistha .............................................................................................................. Maricha ................................................................................................................ Mentha Oil ............................................................................................................. Opium ................................................................................................................... Opium Powder ....................................................................................................... Papain .................................................................................................................... Peppermint Oil ...................................................................................................... Pippali Large .......................................................................................................... Pippali Small ......................................................................................................... Punarnava Senna Pods Senna Leaf ......................................................................................................... ......................................................................................................... ......................................................................................................... Sarpagandha .........................................................................................................
Shatavari ................................................................................................................ Shati ...................................................................................................................... Shellac .................................................................................................................. Shunti ..................................................................................................................... Starch .................................................................................................................... Tolu Blasam ........................................................................................................... Tragacanth Tulasi Vasaka Yasti ......................................................................................................... ............................................................................................................... .................................................................................................................. ...................................................................................................................
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Herbs and Herbal Products

Herbs and products containing herb(s) have been in trade and commerce and are currently used for a variety of purposes. As a country, India has a rich history of use of herbs, processed herbs and formulations containing herbs both from traditional wisdom as well as cultural usage. Herbs and herbals products are also regulated by various laws. For the purposes of pharmacopoeial standards various considerations have been given. This monograph provides a general outline and policies towards the same.
Crude Herbs
This term means, unless specified otherwise, mainly whole, fragmented or cut, plants, parts of plants, algae, fungi, and lichen in a form which is not processed. Herbs are usually in dried form, but sometimes, when specified, may also be in a fresh form. In specific cases exudates which have not been processed further also are covered under the term herbs. Processing, does not include, normally expected value addition steps like grading, sizing, removal of weeds or parts of plants other than those specified herb and removal of adulterants. The term herbs, though botanically generally refer to plants of specified height and nature, for the purposes of pharmacopoeial reference, shall mean and include plants and parts of plants not necessarily from herbs and shrubs, but cover the entire range namely creepers, climbers, trees etc. Each monograph of a herb in the pharmacopoeia shall specify the botanical scientific name according the binomial system specifying the genus, species, variety and author. In cases where there are controversial botanical identity, as is seen with mainly herbs known in the Indian traditional system, the monograph shall specify the official name of the herb along with its botanical scientific name and guidance is taken from Ayurvedic Pharmacopoiea of India* to decide the same. In cases where, the same herb is available in different grades or sizes, if found appropriate and necessary, separate monographs may be introduced in the pharmacopoeia to cover each of them with appropriate standards. For example- Pippali (large) and Pippali (small). While deciding to introduce a monograph for a herb in the pharmacopeia, the criteria that would be kept in mind, but not limited to are herbs with specific name and a definitive botanical identity up to species, availability and usage in trade and commerce, regulatory interest, knowledge of and availability of a specific chemical compound of well characterised structure [either responsible for the biological activity of the herb (bio-marker) or a chemical compound known to be present in the herb even if not responsible for biological activity(chemical/analytical marker)], availability of a quantitative method for estimation of such a compound, knowledge of safety of the herb, and its sustainability. Herbs
which may figure in a regulated list under appropriate forest and other laws, may still be taken up for a monograph for inclusion in pharmacopoeia, if there is knowledge of efforts to cultivate or take care of sustainability issues and / or specific permission is available under law for use of the herb. As already specified under General Notices in the pharmacopeia, appearance of a monograph does not mean its approval as a drug under the law. Monograph of a herb in the pharmacopoeia is to provide qualitative and quantitative standards of quality for the herb for its use either as a food item or food ingredient or food supplement/ nutraceuticals, as a drug, and / or as an ingredient in cosmetics. Each such use would need to comply with applicable regulations. Each herb is regarded as one active substance, irrespective of the knowledge about the active constituents of the herb is available of not.
* Ayurvedic Pharmacopeia of India, Vol. 1-6, Ministry of Health and Family Welfare, Govt. of India.
Herbs may be exposed to low dose gamma radiation for purposes of reducing their microbial contamination. Herbs treated with low dose gamma radiation shall meet national laws related to such treatment and shall be labeled as per law.
Processed Herbs
Processed herbs means preparations obtained by subjecting herbs to treatment such as extraction, distillation, expression, fractionation, purification, concentration and partial or full fermentation. Processing may also be done by way of powdering herbs, preparing tincture, preparing extract, distilling to get essential oils, fatty oils (either expressed or solvent extracted or a blend of both) expressed juices, extracted exudates, gums and oleo resins, liquid extract where the solvent is evaporated to yield concentrated semi solid mass or dried mass. Extraction may be performed by means of appropriate technology such as infusion, maceration, soxhleting, boiling under ambient or higher pressure, with or without specified enzymes, with or without agitation and combination thereof. Drying of liquid extracts for removal of the solvent may be done by using various appropriate technologies like air drying, sun drying, drying under vacuum or with forced air circulation, drying at low temperature with air circulation, by way of lyophilization or freeze drying. Extracts of herbs may also be prepared by using carbon dioxide as a solvent-super critical fluid extraction. Extracts may be liquid extracts and tinctures, semi solid (soft extracts) or solid dry extracts of known consistency obtained from herbs. Standardized extract, a term commonly employed, would for pharmacopoeial purposes, mean an extract adjusted with in an acceptable tolerance to a given content of biomarker or chemical/ analytical marker. Standardization may be achieved by adjusting the extracts with approved inert material or by blending one or more batches of extracts. Wherever
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possible, extracts shall specify the defined range of the constituents (bio-marker or chemical/ analytical marker). Extracts not covered in the above description would be defined by the process of production of the herb to the extract, solvent used and technology applied. The difference between extracts and tinctures would be, in the type of solvent used for extracting a herb, and tincture would normally mean an extract where aqueous-ethanol is used as a solvent for extraction. Dry extracts usually have a loss on drying or water content not greater than 5 per cent w/w, unless specified otherwise in any monograph. It is normal to extrapolate safety aspects and history of use information for extracts as long as the process, solvents, extraction ratios are comparable to the processes used in documented traditional knowledge. All extracts should specify the extractive ratios for example 15: 1 meaning 15 parts of the herb provides one part of the extracts, either as w/w or w/v as the case may be. Additionally in cases of standardized extracts the inert excipients(s), if any used for standardization or adjustment of the content of constituents should also be declared on the label of such extracts. Extracts shall be free from solvent used for extraction and shall comply with a limit of not more than 10 ppm incases where the solvent is either acetone, iso-propyl alcohol, or methanol and not more than 100 ppm of hexane, if hexane is the solvent used, in the final extract. Harmful and carcinogenic solvents shall not be used for extraction purposes. Solvents and solvent systems may include use of propylene glycol, glycerin, sorbitol and such other polyhydroxy alcohols, as long as the content of such polyhydroxy alcohol are within safe limit in the final product. In cases where extraction and fractionation process leads to preparation of an extract, which consists of a single chemical compound of more than of at least 70 per cent purity, such extracts shall be treated as an active pharmaceutical ingredient or a food additive or a cosmetic ingredient and would be required to meet relevant laws. Extracts may also be offered as purified or enriched extracts. Such extract of a herb is processed in such a way to provide higher than normal proportion of the active constituent (s) of the herb as long as the active constituent (s) is/are are known. Such purified or enriched extracts may contain additional valuable components which may provide specific properties like enhanced efficacy or stability or solubility and availability of the active constituent (s). Purified and enriched extracts may also be prepared to reduce or remove other specific compound or group of compounds that is scientifically considered undesirable in the herb extracts. Pharmacological, toxicological, pharmaceutical considerations need to be applied while preparing such purified or enriched extracts. Mixed extracts may also be offered which would cover combination of more than one herb extract for purposes of providing simplification or economical way to manufacture herbal formulations.
Herbs may also be extracted using vegetable oils (approved by Food Law) for extraction purposes and such extracts shall specify the oil used for processing. Approved preservatives or preservatives system may be used during preparation of extracts. The names of such preservatives used which would remain in the final extract shall be listed on the label of such extract, and the proportion of preservatives used shall not exceed normally accepted safe limits of their usage as per relevant laws or pharmacopoeial standards. No artificial colours may be used in extracts of herbs. Extracts may be exposed to ethylene oxide fumigation or low dose gamma radiation for purposes of reducing their microbial contamination. In cases where they are fumigated, the final extracts exposed shall meet residual levels of ethylene oxide limits as applicable. Herbs treated with low dose gamma radiation shall meet national laws related to such treatment and shall be labelled as per law. Appearance of a monograph of an extract in the pharmacopeia does not mean its approval as a drug under the law. Monograph of an extract in the pharmacopoeia is to provide qualitative and quantitative standards of quality for the extract for its use either as a food item or food ingredient or food supplement/ nutraceuticals, as a drug and / or as an ingredient in cosmetics. Each such use would need to comply with applicable regulations. Each extract is regarded as one active substance irrespective of the knowledge about the active constituents of the herb is available or not.
Herbal Formulations
Herbal formulation shall mean a dosage form consisting of one or more herbs or processed herb(s) in specified quantities to provide specific nutritional, cosmetic benefits, and/or other benefits meant for use to diagnose treat, mitigate diseases of human beings or animals and/or to alter the structure or physiology of human beings or animals. Dosage forms commonly employed for food or cosmetic or pharmaceuticals may be employed to formulate one or more herb or processed herbs. Dosage forms known in traditional medicines may also be adopted for preparing herbal formulations, either for external use or for internal administration. Adequate consideration for uniform distribution of herb or processed herbs as well as stability of the same in the dosage form shall be provided during formulation development. Herbal formulation shall be labeled to comply with relevant labelling requirements under food or drug or cosmetics laws as applicable. Additionally, adequate information shall be provided on label of such formulations to include the name of the herb, parts used, nature and type of extract or processed herb used, extraction ratios, quantity per unit dose or per serving, name (s) of inert excipients used and any preservatives added shall be provided on the label.
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ACACIA
Appearance of a monograph of a herbal formulation in the pharmacopoeia does not mean its approval as a drug under the law. Monograph of a herbal formulation in the pharmacopoeia is to provide qualitative and quantitative standards of quality for the formulation for its use either as a food item or food ingredient or food supplement/ nutraceuticals, as a drug and / or as a cosmetic. Each such use would need to comply with applicable regulations.
addition of 0.5 ml of acetic acid, gives a white precipitate. Filter and add to the clear filtrate 50 ml of ammonium oxalate solution; the filtrate becomes cloudy. C. A 10 per cent w/v solution is either dextro-rotatory or slightly laevo-rotatory.
Tests
Sterculia gum and agar. To 50 mg of the powdered substance under examination add 0.2 ml of freshly prepared ruthenium red solution and examine microscopically; the particles do not acquire a red colour after irrigation with water. Agar and tragacanth. To 10 ml of a 10 per cent w/v solution add 0.2 ml of lead acetate solution; no precipitate is produced. Starch and dextrin. Boil 10 ml of a 10 per cent w/v solution and cool, add 0.1 ml of 0.05 M iodine; no blue or brown colour is produced. Tannins. To 10 ml of a 10 per cent w/v solution add 0.1 ml of ferric chloride test solution; a gelatinous precipitate is formed, but neither the precipitate nor the liquid shows a dark blue colour. Sucrose and fructose. To 1 ml of a 10 per cent w/v solution add 4 ml of water, 0.1 g of resorcinol and 2 ml of hydrochloric acid and heat on a water-bath; no yellow or pink colour develops. Water-insoluble matter. Dissolve 5 g, in fine powder, in 100 ml of water in a 250-ml flask, add 10 ml of dilute hydrochloric acid and boil gently for 15 minutes. Filter by suction while hot through a sintered-glass crucible, previously tared, wash thoroughly with hot water, dry at 105 and weigh; the residue does not exceed 50 mg. Microbial contamination (2.2.9). 1.0 g is free from Escherichia coli. Sulphated ash (2.3.18). Not more than 5.0 per cent. Acid-insoluble ash (2.3.19). Not more than 1.0 per cent, determined on 1.0 g by Method C. Loss on drying (2.4.19). Not more than 15.0 per cent, determined on 1.0 g by drying in an oven at 105. Storage. Store protected from heat, moisture and against attack by insects and rodents.
Acacia
Gum Acacia; Indian Gum
Acacia is the air-hardened, gummy exudation from the stem and branches of Acacia nilotica (Linn.) Del. subsp. indica (Benth.) Brenan (syn. A. arabica Willd. var. indica Benth.) (Fam. Leguminosae), or other species of Acacia. It is available as pieces (tears) or in the form of a powder. Description Tears Irregular and broken pieces of varying size, yellowishwhite, yellow or amber in colour, with numerous minute fissures; brittle fractured surface, glassy and occasionally iridescent; odourless. Powder A white or yellowish-white powder; odourless; on treatment with water it dissolves to give a mucilaginous liquid which is colourless or yellowish, dense, viscous, adhesive and translucent.
Identification
A. An aqueous solution is gelatinised by the addition of lead subacetate sol3either dextro-rotatory or slightly laevo-rotatory. B. To 5 ml of a 10 per cent w/v solution add gradually, while shaking, 10 ml of ethanol (95 per cent). The cloudy liquid, on 1381
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Amalaki
Emblic Myrobalan; Indian Gooseberry
and filter. Reflux the residue further for two times with 75 ml of methanol, cool and filter. Combine all the filtrates and concentrate under vacuum to 10 ml. Apply to the plate 10 l of each solution as bands 10 mm by 2 mm. Allow the mobile phase to rise 8 cm. Dry the plate in air, spray with anisaldehyde sulphuric acid reagent. Heat at 100 for 5-10 minutes and examine the plate in day light. The chromatographic profile of the test solution is similar to that of the reference solution.
Tests
Foreign organic matter (2.6.1). Not more than 3 per cent. Ethanol-soluble extractive (2.6.2). Not less than 30 per cent. Water-soluble extractive (2.6.3). Not less than 40 per cent by Method 1. Amalaki consists of the dried fruit pericarp of Emblica officinalis Gaertn. (Phyllanthus emblica Linn.) (Fam. Euphorbiaceae). Amalaki contains not less than 1.0 per cent w/w gallic acid calculated on the dried basis. Description. The dried fruit has a highly shriveled and wrinkled external surface. The taste is sour and astringent followed by delicately sweet tinge. Ash (2.3.19). Not more than 5.0 per cent. Acid-insoluble ash (2.3.19). Not more than 2.0 per cent. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B ( 20 ppm). Loss on drying (2.4.19). Not more than 12.0 per cent, determined on 5 g by drying in an oven at 105. Microbial contamination (2.2.9). Complies with the microbial contamination tests. Assay. Determine by liquid chromatography (2.4.14). Test solution. Weigh accurately about 0.5 g of coarsely powdered substance under examination, add 50 ml of water, sonicate for 3 minutes and heat on a boiling water bath for 15 minutes, cool and dilute to 100.0 ml with water and filter. Reference solution. A 0.01 per cent w/v solution of gallic acid RS in water. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a gradient mixtures of acetonitrile and a buffer solution prepared by dissolving 0.136 g of potassium di-hydrogen orthophosphate in 500 ml of water, add 0.5 ml of orthophosphoric acid and make upto 1000 ml with water, flow rate. 1.5 ml per minute, spectrophotometer set at 270 nm, a 20 l loop injector. Time Buffer solution Acetonitrile (min) (per cent v/v) (per cent v/v) 0 100 0 18 55 45 25 20 80 30 100 0
Identification
A. Macroscopic The dried fruit shows a broad, highly shriveled and wrinkled external convex surface, lateral surface transversely wrinkled, external surface exhibits few whitish specks, occasionally some pieces show a portion of stony testa. B. Microscopic The epicarpic cells are rectangular in shape and their walls are highly cuticularized. Anomocytic type of stomata is found rarely. Collateral fibrovascular bundles are scattered throughout the inner mesocarp. Pitted and helical tracheids with tapering ends are seen. At places in the phloem, large cavities filled with crystal mass are present. C. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel. Mobile phase. A mixture of 20 volumes of toluene, 45 volumes of ethyl acetate, 20 volumes of glacial acetic acid and 5 volumes of formic acid. Test solution. Reflux 2 g of the coarsely powdered substance under examination with 50-75 ml of methanol for 15 minutes, cool and filter. Reflux the residue further for two times with 75 ml of methanol, cool and filter. Combine all the filtrates and concentrate under vacuum to 50 ml. Reference solution. Reflux 0.4 g of the coarsely powdered amalaki RS with 50-75 ml of methanol for 15 minutes, cool
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AMRA
Inject the reference solution. The relative standard deviation for the replicate injections is not more than 2.0 per cent. Inject the test solution and reference solution. Calculate the content of gallic acid. Storage. Store protected from light, heat, moisture and against attack by insects and rodents.
Test solution. To 5 g of the coarsely powdered substance under examination, add 50 ml of methanol and reflux for 15 minutes, cool and filter. Reflux the residue further for three times with 50 ml of methanol, cool and filter. Combine all the filtrates and concentrate under vacuum to 10 ml. Reference solution. Weigh about 2 g of amra RS, add 50 ml of methanol and reflux for 15 minutes, cool and filter. Reflux the residue further three times with 50 ml of methanol, cool and filter. Combine all the filtrates and concentrate under vacuum to 4 ml. Apply to the plate 10 l of each solution as bands 10 mm by 2 mm. Allow the mobile phase to rise 8 cms. Dry the plate in air, spray with vanilin sulphuric acid reagent. Heat at 100 for 5-10 minutes and examine the plate in day light. The chromatographic profile of the test solution is similar to that of the reference solution.
Amra
Mango; Mangifera
Tests
Foreign organic matter (2.6.1). Not more than 2.0 per cent. Ethanol-soluble extractive (2.6.2). Not less than 10.0 per cent. Water-soluble extractive (2.6.3). Not less than 10.0 per cent by method I. Ash (2.3.19). Not more than 16.0 per cent. Acid-insoluble ash (2.3.19). Not more than 5.0 per cent. Amra consists of dried stem bark of Mangifera indica L.(Fam. Anacardiaceae). Amra contains not less than 1.5 per cent of mangiferin calculated on the dried basis. Description. The dried stem bark occurs in pieces of variable size and thickness, surface rough. Odour pleasant and taste astringent. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Loss on drying (2.4.19). Not more than 12.0 per cent, determined on 5 g by drying in an oven at 1050. Microbial contamination (2.2.9). Complies with the microbial contamination tests. Assay. Determine by liquid chromatography (2.4.14). Test solution. Weigh 2 g of coarsely powdered substance under examination, add 10 ml of dimethylformamide, sonicate for 5 minutes and add 50 ml of methanol and boil on a water bath for 10 minutes, cool and dilute to 100.0 ml with methanol and filter. Dilute 5.0 ml of this solution to 50.0 ml in methanol. Reference solution. Dissolve 10 mg of mangiferin RS in 10 ml of dimethylformamide and dilute to 100.0 ml with methanol. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5 m), mobile phase: filtered and degassed mixture of 15 volumes of acetonitrile and 85 volumes of buffer pH 2.8 prepared by dissolving 1.36 g of potassium dihydrogen orthophosphate in 950 ml of water, adjust the pH 2.8 with orthophosphoric acid and make upto 1000 ml, flow rate. 1 ml per minute,
Identification
A. Macroscopic The surface is rough due to longitudinal cracks, fissures and scattered, raised lenticels, greyish to dark brown externally and yellowish-white to reddish internally. B. Microscopic The mature bark shows a wide cork which has tangentially elongated cells a few outer layers are brown and inner lighter in colour, resin canals and yellow coloured tannin sacs are found in the phloem region, stone cells are thick walled and lignified, prismatic calcium oxalate crystals are present. C. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel. Mobile phase. A mixture of 100 volumes of ethyl acetate, 11 volumes of formic acid, 11 volumes of acetic acid and | 25 volumes of water.
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ARACHIS OIL
IP 2007
spectrophotometer set at 366 nm, a 20 l loop injector. Inject the reference solution. The relative standard deviation for the replicate injections is not more than 2.0 per cent. Inject the test solution and reference solution. Calculate the content of mangiferin. Storage. Store protected from moisture and against attack by insects and rodents.
Tests
Weight per ml (2.4.29). 0.908 g to 0.920 g. Refractive index (2.4.27). 1.467 to 1.470. Alkaline impurities. Mix 10 ml of recently distilled acetone and 0.3 ml of water in a test-tube, add 0.05 ml of a 0.04 per cent w/v solution of bromophenol blue in ethanol (95 per cent) and neutralise the solution, if necessary, with 0.01 M hydrochloric acid or 0.01 M sodium hydroxide. Add 10 ml of the substance under examination, shake and allow to stand. Not more than 0.1 ml of 0.01 M hydrochloric acid is required to change the colour of the upper layer to yellow. Semi-drying oils. Boil 1 g in a flask under a reflux condenser for 5 minutes with 5 ml of a mixture of 3 volumes of 2 M ethanolic potassium hydroxide and 1 volume of ethanol (95 per cent), add 1.5 ml of 6 M acetic acid and 50 ml of ethanol (70 per cent), warm until the solution is clear. Cool slowly with a thermometer in the liquid; the temperature at which the solution becomes turbid is not lower than 36. Peroxide value (2.3.35). Not more than 5.0. Acid value (2.3.23). Not more than 0.5. Iodine value (2.3.28). 85 to 105. Saponification value (2.3.37). 185 to 196. Rancidity. Shake 1 ml of a 10 per cent v/v solution in ether with 1 ml of hydrochloric acid, add 1 ml of a 0.1 per cent w/v solution of phloroglucinol in ether; no red or pink colour is produced. Unsaponifiable matter (2.3.39). Not more than 1.0 per cent. Cottonseed oil. In the Identification test, the chromatogram obtained with the test solution does not correspond to that obtained with reference solution (b) Sesame oil. In the Identification test, the chromatogram obtained with the test solution does not correspond to that obtained with reference solution (c). Heavy metals (2.3.13). 2.0 g complies with the limit test for heavy metals, Method B (10 ppm). Arachis Oil intended for use in the manufacture of parenteral preparations complies with the following additional requirement. Water (2.3.43). Not more than 0.3 per cent, determined on 3.0 g. Storage. Store protected from light and moisture, in a wellfilled container. Arachis Oil intended for use in the manufacture of parenteral preparations should be stored in a glass container. Labelling. The label states (1) whether the contents are suitable for use in the manufacture of parenteral preparations; (2) when the addition of antioxidants is authorised, the name and quantity of the added antioxidants.
Arachis Oil
Groundnut Oil; Peanut Oil
Arachis Oil is the refined fixed oil obtained from the seed kernels of one or more of the cultivated varieties of Arachis hypogaea Linn. (Fam. Leguminosae) and may contain suitable antioxidants as stabilisers. Description. A clear, colourless or pale yellow oily liquid; odour, faint and nutlike.
Identification
Determine by the thin layer chromatography (2.4.17), coating the plate with kieselguhr G. Mobile phase. Glacial acetic acid. Test solution. Dissolve 20 mg (one drop) of the substance under examination in 4 ml of chloroform. Reference solution (a). Dissolve 20 mg (one drop) of arachis oil in 4 ml of chloroform. Reference solution (b). Dissolve 20 mg (one drop) of cottonseed oil in 4 ml of chloroform. Reference solution (c). Dissolve 20 mg (one drop) of sesame oil in 4 ml of chloroform. Impregnate the plate by placing it to a depth of about 5 mm in a tank containing a shallow layer of a mixture of 95 volumes of light petroleum (60 to 80) and 5 volumes of liquid paraffin, allowing the solvent to rise at least 14 cm, removing the plate and allowing it to dry in air for 5 minutes; use with the flow of the mobile phase in the direction in which impregnation was carried out. Apply to the plate 2 l of each solution. Allow the mobile phase to rise 12 cm. Dry the plate at 110 for 10 minutes, allow to cool and expose it to iodine vapour in a saturated chamber. Remove the plate and allow it to stand for a few minutes until the brown background colour has disappeared. Spray the plate with starch solution; blue spots appear which become brown on drying and become blue again on spraying with water. The spots in the chromatogram obtained with the test solution correspond to those in the chromatogram obtained with reference solution (a).
1384
IP 2007
ARJUNA
Arjuna
Terminalia Bark
Apply to the plate 10 l of each solution as bands 10 mm by 2 mm. Allow the mobile phase to rise 8 cm. Dry the plate in air, spray with 10 per cent w/v solution of sulphuric acid in methanol and heat at 1100 for 5 minutes. The chromatographic profile of the test solution is similar to that of the reference solution.
Tests
Foreign organic matter (2.6.1). Not more than 2.0 per cent. Ethanol-soluble extractive (2.6.2). Not less than 20.0 per cent. Water-soluble extractive (2.6.3). Not less than 20 per cent by method I. Ash (2.3.19). Not more than 30.0 per cent. Acid-insoluble ash (2.3.19). Not more than 2 per cent. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Arjuna consists of dried stem bark of Terminalia arjuna (Roxb) Wight & Arn (Fam. Combretaceae) Arjuna contains not less than 0.02 per cent of arjungenin calculated on the dried basis. Description. A flat or minutely curved thick pieces of bark with reddish gray colour and astringent taste. Loss on drying (2.4.19). Not more than 10.0 per cent, determined on 5 g by drying in an oven at 1050. Microbial contamination (2.2.9). Complies with the microbial contamination tests. Assay. Determine by liquid chromatography (2.4.14). Test solution. Reflux 5 g of coarsely powdered substance under examination with 50 ml of chloroform for 15 minutes, cool and filter. Reflux the residue further with 50 ml of chloroform, cool and filter. Combine the filtrates and concentrate under vacuum to dryness, then extract dried residue with 10 ml of ethanol at 50 for 10 minutes and filter. Reference solution. A 0.1 per cent w/v solution of arjungenin RS in ethanol. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (10 m), mobile phase (A). acetonitrile (70 per cent) in water, (B). acetonitrile (30 per cent) in water, flow rate. 1.2 ml per minute, spectrophotometer set at 210 nm, a 20 l loop injector. Time Mobile phase A Mobile phase B (min) (per cent v/v) (per cent v/v) 0 30 70 10 50 50 30 70 30 50 30 70 Inject the reference solution. The test is not valid unless the relative standard deviation for the replicate injection for the analyte peak corresponding to arjungenin is not more than 2.0 per cent.
Identification
A. Macroscopic Stem bark pieces, flat or minutely curved, with reddish gray external surface and darker inner surface. Internal surface has longitudinal minute ridges. Fractures longitudinal. B. Microscopic Cork consisting of 6-10 layers of elongated cells, phloem broad, medullary rays uniseriate. Calcium oxalate clusters abundant. Few of the parenchyma cells contain colouring matter. C. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 5 volumes of toluene, 5 volumes of ethyl acetate and 0.5 volume of acetic acid. Test solution. Reflux 2 g of coarsely powdered substance under examination with 50 ml of chloroform for 15 minutes, cool and filter. Reflux the residue further with 50 ml of chloroform. Combine the filtrate and concentrate under vacuum to dryness. Dissolve the residue in 10 ml of ethanol at 50 for 10 minutes and filter. Reference solution. Reflux 1 g of arjuna RS with 50 ml of chloroform for 15 minutes, cool and filter. Reflux the residue further with 50 ml of chloroform. Combine the filtrate and concentrate under vacuum to dryness. Dissolve the residue in 5 ml of ethanol at 50 for 10 minutes and filter.
1385
ARTEMISIA
IP 2007
Inject the test solution and reference solution. Calculate the contents of arjungenin. Storage. Store protected from light, heat, moisture and against attack by insects and rodents.
because florets / achenes come out of it. Receptacle globular to oblong. Achenes minute; possess striated surface, yellowish brown in colour, 0.7-1.0 mm long, and oval to elliptic in shape. Stomatal index: 8-10-12. B. The powder of the herb is grayish green to greenish yellow. Examined under a microscope using chloral hydrate solution. The powder shows the following diagnostic characteristics: T-shaped and globular trichomes, epidermal cells with wavy walls, anomocytic to anisocytic stomata, minute druse crystals, tricolpate rounded pollen grains, stone cells, annular vessels, rod shaped palisade cells and stigma with small and club shaped papillae. C. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 1 volume of hexane and 1 volume of diethyl ether. Test solution. Boil 0.1 g of the coarsely powdered substance under examination with 10 ml of hexane for 10 minutes and filter. Evaporate the filterate to 1 ml. Reference solution. A 0.1 per cent w/v solution of artemisia RS in hexane.
Artemisia
Artemisia consists of dried leaves or the dried leaf and flowering tops of Artemisia annua L. (Fam. Asteraceae), known as Qinghao. Artemisia contains not less than 0.8 per cent of artemisinin, calculated on the dried basis. Description. Slightly camphoraceous odour and bitter taste.
Apply to the plate 5 l of each solution. Allow the mobile phase to rise 5 cm. Dry the plate in air and spray with a mixture of 50 volumes of glacial acetic acid, 1 volume of sulphuric acid and 0.5 volume of anisaldehyde. Heat the plate at 100 for 15 minutes. The chromatogram having pink colour spot obtained with the test solution corresponds to the reference solution.
Identification
A. The leaves grayish green, slightly darker upper surface, glabrous to sparsely hairy, break easily into small fragments, 3-pinnatipartite, petiolate and much variable in size (2.510.0 cm long). Leaf lobes narrow, oblong to elliptical with acuminate tip, about 1.0 mm wide. Petioles up to 1.0 cm long, base amplexicaul. Inflorescence panicled raceme. Dry capitula yellowish brown, pedicellate, heterogamous globose to subglobose or disc shaped, 2.0 mm in diameter, flower heads arranged in lax or drooping, involucral bracts 3- seriate, greenish yellow, glabrous and oblong in shape, measure 1.0-1.2 x 0.5 mm in size. Inner involucral bracts elliptic having a median greenish streak on its outer surface. Ray florets pistillate, 6-8 in number per capitulum and 1.0-1.2 mm long. Disc florets hermaphrodite, 20-36 florets per capitulum and 0.8-1.0 mm long. All florets possess capitate oil glands on the middle of the outer surface that are 54-83 m in diameter. Stamens 0.7 mm long attached to the corolla base, anther appendages lanceolate to triangular with acuminate tip. Anthers oblong and introrse. Pollen grains tricolpate, rounded 21-33 m in diameter. In dry condition, capitula become empty
Tests
Foreign organic matter (2.6.1). Not more than 2.0 per cent. Ash (2.3.19). Not more than 11.0 per cent. Acid-insoluble ash (2.3.19). Not more than 3.0 per cent. Assay. Determine by high performance thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 1 volume of hexane and 1 volume of diethyl ether. Test solution. To 0.1 g of the coarsely powdered substance under examination, add 10 ml of hexane and keep for 12 hours, filter. Repeat the process of extraction 3 times. Combine the extracts, evaporate and dissolve in 1.0 ml of hexane. Reference solution. A 0.1 per cent w/v solution of artemisinin RS in hexane. Apply to the plate 5 l of each solution as bands 10 mm by 2 mm. Allow the mobile phase to rise 8 cm. Dry the plate in air and spray with a mixture of 50 volumes of glacial acetic acid,
1386
IP 2007
ASHWAGANDHA
1 volume of sulphuric acid and 0.5 volume of anisaldehyde. Heat the plate at 100 for 15 minutes, scan the plate in absorbance mode at 540 nm. Record the chromatograms and measure the responses for the analyte peak. Calculate the content of artemisinin. Storage. Store protected from light and moisture and against attack by insects and rodents.
Reference solution. Reflux 0.6 g of coarsely powdered ashwagandha RS with 10 ml methanol for 15 minutes, cool and filter. Apply to the plate 10 l of each solution as bands 10 mm by 2 mm. Allow the mobile phase to rise 8 cms above the line of application. Dry the plate in air, spray with solution of anisaldehyde reagent. Heat at 100 for 5-10 minutes and examine the plate in day light. The chromatographic profile of the test solution is similar to that of the reference solution.
Ashwagandha
Indian Ginseng; Withania
Tests
Foreign organic matter (2.6.1). Not more than 2.0 per cent. Ethanol-soluble extractive (2.6.2). Not less than 10.0 per cent. Water-soluble extractive (2.6.3). Not less than 15 per cent by Method I. Ash (2.3.19). Not more than 7.0 per cent. Acid-insoluble ash (2.3.19). Not more than 1.2 per cent. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Loss on drying (2.4.19). Not more than 12.0 per cent, determined on 5 g by drying in an oven at 1050. Microbial contamination (2.2.9). Complies with the microbial contamination tests.
Ashwagandha consists of the dried mature roots of Withania somnifera Dunal (Fam. Solanaceae). Ashwagandha contains not less than 0.02 per cent of total withanolide A and withaferin A, calculated on the dried basis. Description. Buff to greyish-yellow roots. Taste, slightly mucilaginous, bitter and acrid.
Assay. Determine by liquid chromatography (2.4.14). Test solution. Reflux about 5 g of the coarsely powdered substance under examination with 50 ml of methanol on a water bath for 15 minutes, cool and filter. Reflux the residue further with methanol till the last extract turns colorless, cool and filter. Combine all the filtrates and concentrate to 50.0 ml. Reference solution (a). A solution containing 0.02 per cent w/v each of withanolide A RS and withaferin A RS in methanol, prepared by heating gently on a water bath. Reference solution (b). A 0.02 per cent w/v solution of withanolide A RS in methanol, prepared by heating gently on a water bath, for peak identification. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (10 m), mobile phase: a gradient mixtures of acetonitrile and a buffer solution prepared by dissolving 1.36 g of potassium dihydrogen phosphate in 900 ml water, adjust pH to 2.8 with dilute phosphoric acid and diluting it to 1000 ml with water, flow rate. 1.5 ml per minute, spectrophotometer set at 227nm, a 20 l loop injector.
Identification
A. Macroscopic Primary roots are straight, conical or finger like in shape, variable in thickness with the age. Secondary roots are thin and fibrous. Surface buff to greyish-yellow with longitudinal wrinkles. B. Microscopic Vessels with bordered pits and horizontal perforations. Fibres aseptate with pointed ends. Wood elements lignified. Starch grains abundant, simple, mostly spherical, reniform oval with central hilum. Microcrystals in parenchyma cells. Mobile phase. A mixture of 9 volumes of chloroform and 1 volume of methanol. Test solution. Reflux 3 g of coarsely powdered substance under examination with 50 ml methanol for 15 minutes, cool and filter.
1387
BELLADONNA DRY EXTRACT
IP 2007
Time Buffer solution Acetonitrile (min) (per cent v/v) ( per cent v/v) 0 90 10 18 55 45 25 20 80 27 90 10 37 90 10 Inject the reference solution (b) and determine the retention time of withanolide A. Inject the reference solution (a). Identify the retention time of withaferin A. The test is not valid unless the relative standard deviation for the replicate injections for both the analyte peaks corresponding to withanolide A and withaferin A is not more than 2.0 per cent and resolution is not less than 2.0. Inject the test solution. Calculate the contents of withanolide A and withaferin A. Storage. Store protected from heat, moisture and against attack by insects and rodents.
acid and discard the chloroform. Combine the acid solutions, neutralise with dilute ammonia solution, add 5 ml in excess and shake with successive quantities, each of 25 ml, of chloroform until complete extraction of the alkaloids is effected, washing each chloroform solution with the same 10 ml of water and filtering into a flask through a plug of cotton wool previously moistened with chloroform. Distil most of the chloroform from the combined extracts and transfer the remainder of the chloroform to a shallow open dish. Evaporate the remainder of the chloroform without the aid of a current of air, heat the residue in an oven at 100o for 15 minutes, dissolve in a little chloroform, evaporate to dryness without the aid of a current of air and again heat in an oven at 100o for 15 minutes. Dissolve the residue in 2 ml of chloroform, add 5.0 ml of 0.025 M sulphuric acid, warm to remove the chloroform, cool and titrate the excess of acid with 0.05 M sodium hydroxide using methyl red solution as indicator. 1 ml of 0.025 M sulphuric acid is equivalent to 0.01447 g of alkaloids, calculated as hyoscyamine. Storage. Store in small, wide-mouthed, tightly-closed containers in a cool place.
Belladonna Dry Extract

Belladonna Dry Extract is a dried and powdered ethanolic extract of Belladonna Herb. Belladonna Dry Extract contains not less than 0.95 per cent and not more than 1.05 per cent w/w of alkaloids, calculated as hyoscyamine.
Belladonna Leaf
Belladonna Leaf consists of the dried leaf and flowering tops of Atropa belladonna Linn. or of A. acuminata Royle ex Lindley (Fam. Solanaceae) or a mixture of both species. Belladonna Herb contains not less than 0.30 per cent of total alkaloids, calculated as hyoscyamine with reference to the material dried at 100o to 105o. Description. Green to greenish-brown leaves, slightly darker on the upper surface, often crumpled and rolled and partly matted together in the drug. When whole, the lamina is 5 to 25 cm long and 3 to 12 cm wide, elliptical to ovate; acuminate at the apex, narrowing at the base; margin entire. Petiole 0.5 to 4 cm in length. The young leaves are highly pubescent, the older leaves are slightly pubescent along the veins. In the flowering tops, the stems are hollow and flattened, with leaves in pairs of unequal size, in the axils of which are single flowers with campanulate corolla, about 2 cm long and 1.5 cm wide, purple or yellow-brown in colour, with five short, reflexed lobes, five epipetalous stamens and one bilocular ovary with numerous ovules.
Tests
Loss on drying (2.4.19). Not more than 5.0 per cent, determined on 0.5 g by drying in an oven at 105o. Assay. Weigh accurately about 3 g and wash into a separating funnel with 12 ml of a mixture of equal volumes of ethanol (95 per cent) and water, shake-well and frequently for 30 minutes, add 2 ml of dilute ammonia solution and 25 ml of chloroform. Shake well, allow to separate and filter the chloroform layer into a second separating funnel through a plug of absorbent cotton moistened with chloroform. Continue the extraction with further quantities, each of 25 ml, of chloroform until complete extraction of the alkaloids is effected (2.6.4), running each chloroform solution through the same plug of absorbent cotton. Extract the combined chloroform solutions with successive quantities of a mixture of 3 volumes of 0.1 M sulphuric acid and 1 volume of ethanol (95 per cent) until complete extraction of the alkaloids is effected, filtering each extract through a plug of absorbent cotton previously moistened with water. Wash the mixed acid solutions with 10, 5 and 5 ml of chloroform, extracting each chloroform solution with the same 20 ml of 0.05 M sulphuric
Identification
A. When examined under a microscope it shows epidermal cells with sinuate anticlinal walls and cuticle which is often striated and furrowed. Covering and glandular hairs infrequent, though more frequent in the young leaves and around the veins; covering hairs, multicellular, uniseriate, with thin smooth
1388
IP 2007
BELLADONNA LEAF
walls; glandular hairs; short clavate glands with multicellular heads and glands with a long uniseriate stalk and ovoid unicelluar head. Stomata, anisocytic, more frequent on the lower epidermis. The midrib is characterised by an open arc of vascular bundles with isolated groups of perimedullary phloem. Mesophyll dorsiventral with a single palisade layer. Throughout the parenchyma and particularly just below the palisade layer are cells containing microsphenoidal crystals of calcium oxalate or, very rarely, cluster crystals. The stems show pericyclic fibres and perimedullary bundles of phloem, few trichomes; the cortical parenchymatous cells and the pith cells contain microsphenoidal crystals of calcium oxalate. B. Powder 1 g and shake for 2 minutes with 10 ml of 0.05 M sulphuric acid. Filter and add to the filtrate 1 ml of strong ammonia solution and 5 ml of water. Extract with 15 ml of ether, taking care to prevent the formation of an emulsion. Dry the ether extract over anhydrous sodium sulphate and filter. Evaporate the filtrate to dryness, add 0.5 ml of fuming nitric acid and evaporate to dryness. Add 10 ml of acetone and, dropwise, a 3 per cent w/v solution of potassium hydroxide in ethanol (95 per cent); a deep violet colour develops. C. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G.. Mobile phase. A mixture of 90 volumes of acetone, 7 volumes of water and 3 volumes of strong ammonia solution. Test solution. Add 15 ml of 0.05 M sulphuric acid to 0.6 g of the material under examination, in fine powder, shake for 15 minutes, filter and wash the filter with 0.05 M sulphuric acid until 20 ml of filtrate is obtained; add 1 ml of strong ammonia solution to the filtrate, extract with two quantities, each of 10 ml, of peroxide-free ether, separate the ether layer, by centrifugation if necessary, dry the combined ether extracts over anhydrous sodium sulphate, filter, evaporate to dryness on a water-bath and dissolve the residue in 0.5 ml of methanol. Reference solution. Dissolve 50 mg of hyoscyamine sulphate in 9 ml of methanol (solution A) and dissolve 15 mg of hyoscine hydrobromide in 10 ml of methanol (solution B); mix 8 ml of solution A with 1.8 ml of solution B. Apply to the plate 10 l and 20 l of each solution as bands. After development, dry the plate at 105o for 15 minutes, allow to cool and spray with 10 ml of modified potassium iodobismuthate solution until the bands become visible as orange or brown on a yellow background. The bands in the chromatogram obtained with test solution have similar Rf values to those in the chromatograms obtained with reference solution (hyoscyamine in the lower third of the chromatogram; hyoscine in the upper third) and are similar in colour and atleast equal in size. Faint secondary bands may appear, particularly in the middle of the chromatogram obtained with 20 l of the test solution or near the line of application in the
chromatogram obtained with 10 l of test solution. Spray the plate with a freshly prepared 10 per cent w/v solution of sodium nitrite until transparent and examine after 15 minutes. The colours due to hyoscyamine in the chromatogram change from brown to reddish-brown but not to greyish-blue (atropine); any secondary bands are no longer visible. Foreign organic matter (2.6.1). Not more than 3 per cent. Acid-insoluble ash (2.3.19). Not more than 3 per cent. Assay. Powder 50 g and determine the loss on drying (2.4.19 ), by drying 2 g, accurately weighed, in an oven at 105o. From the remaining sample, weigh accurately about 10 g, moisten with a mixture of 5 ml of dilute ammonia solution, 10 ml of ethanol (95 per cent) and 30 ml of ether and mix thoroughly. Transfer the mixture to a percolator with the aid of an extracting solvent mixture consisting of 3 volumes of ether and 1 volume of chloroform. Allow to macerate for 4 hours and percolate with the solvent mixture until complete extraction of the alkaloids is effected, ( 2.6.4). Concentrate the percolate to about 50 ml by distilling off the solvent mixture on a water-bath, and transfer to a separator, previously rinsed with ether. Add a quantity of ether at least equal to 2.1 times the volume of the percolate and extract with three quantities, each of 20 ml, of 0.5 M sulphuric acid. Transfer each acid extract to another separating funnel. Combine the acid extracts, make the solution alkaline with dilute ammonia solution and extract with chloroform until complete extraction of the alkaloids has been effected. Wash the combined chloroform extracts with 10 ml water, discard the water, evaporate the chloroform layer to dryness and heat the residue for 15 minutes on a water-bath. Redissolve the residue in successive small quantities of chloroform, evaporating to dryness on a water-bath each time before adding the solvent. Heat for 15 minutes on a water-bath and dissolve the residue in 5 ml of chloroform. Add 20.0 ml of 0.01 M sulphuric acid, remove the chloroform by evaporation on a water-bath and titrate the excess of acid with 0.02 M sodium hydroxide using methyl red solution as indicator. 1 ml of 0.01 M sulphuric acid is equivalent to 0.005788 g of total alkaloids calculated as hyoscyamine. Calculate the content of total alkaloids with reference to the dried material. Storage. Store protected from light and moisture.
1389
BHIBHITAKI
IP 2007
Bhibhitaki
Belliric Myrobalan; Terminalia
75 ml of methanol, cool and filter. Combine all the filtrates and concentrate under vacuum to 50 ml. Reference solution. Reflux 0.4 g of the coarsely powdered bhibhitaki RS with 50-75 ml of methanol for 15 minutes, cool and filter. Reflux the residue further for two times with 75 ml of methanol, cool and filter. Combine all the filtrates and concentrate under vacuum to 10 ml. Apply to the plate 10 l of each solution as bands 10 mm by 2 mm. Allow the mobile phase to rise 8 cm. Dry the plate in air, spray with anisaldehyde sulphuric acid reagent. Heat at 100 for 5-10 minutes and examine the plate in day light. The chromatographic profile of the test solution is similar to that of the reference solution.
Tests
Foreign organic matter (2.6.1). Not more than 2 per cent. Ethanol-soluble extractive (2.6.2). Not less than 25 per cent. Bhibhitaki consists of the dried fruit pericarp of Terminalia belerica Roxb. (Fam. Combretaceae). Bhibhitaki contains not less than 0.3 per cent w/w of ellagic acid and 0.75 per cent w/w of gallic acid, calculated on the dried basis. Description. The dried pericarp appears as curved pieces of irregular shapes, the external surface is velvety, wrinkled grey to brown in colour and has astringent taste Water-soluble extractive (2.6.3). Not less than 35 per cent by Method 1. Ash (2.3.19). Not more than 8 per cent. Acid-insoluble ash (2.3.19). Not more than 2 per cent. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Loss on drying (2.4.19). Not more than 12.0 per cent, determined on 5 g by drying in an oven at 1050. Microbial contamination (2.2.9). Complies with the microbial contamination tests. Assay. Determine by liquid chromatography (2.4.14). Test solution. Weigh 0.5 g of coarsely powdered substance under examination, add 50 ml of water, sonicate for 3 minutes and heat on a boiling water bath for 15 minutes, cool and dilute to 100.0 ml with water and filter. Reference solution. A solution containing 0.01 per cent w/v each of gallic acid RS and ellagic RS in water. NOTE Use freshly prepared solution and protected from light. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a gradient mixtures of acetonitrile and a buffer solution prepared by dissolving 0.136 g of potassium di-hydrogen orthophosphate in 500 ml of water, add 0.5 ml of orthophosphoric acid and make upto 1000 ml with water, flow rate. 1.5 ml per minutes, spectrophotometer set at 270 nm, a 20 l loop injector.
Identification
A. Macroscopic The dried pericarp of the ripe fruit occurs as curved pieces of irregular shapes with convex external surface. The external surface appears velvety, slightly wrinkled grey to brown in colour. Internal surface is pale yellow. The cut surface is with occasional projecting threads, representing the vascular bundles. B. Microscopic The cells of the epidermis has a characteristic and a slightly bulged based with a hair like prolongation. Several vascular strands traverse the mesocarp in various directions. Peripheral layers of mesocarp have tangentially elongated cells, devoid of starch grains, containing rosettes of calcium oxalate crystals and few small stone cells. C. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel. Mobile phase. A mixture of 20 volumes of toulene, 45 volumes of ethyl acetate and 20 volumes of glacial acetic acid and 5 volumes of formic acid. Test solution. Reflux 2 g of the coarsely powdered substance under examination with 50-75 ml of methanol for 15 minutes, cool and filter. Reflux the residue further for two times with
1390
IP 2007
BHRINGRAJ
Time Buffer solution Acetonitrile (min) (per cent v/v) (per cent v/v) 0 95 5 18 65 35 25 45 55 30 95 5 Inject the reference solution. The relative standard deviation for the replicate injections is not more than 2.0 per cent. Inject the test solution and reference solution. Calculate the content of gallic acid and ellagic acid. Storage. Store protected from light, heat, moisture and against attack by insects and rodents.
Leaf. Opposite, sessile to sub sessile, usually oblong, lanceolate, sub-entire, sub -acute or acute, strigose with appressed hairs on both surfaces. Flower. Solitary or 2, together on unequal axillary peduncles, involucral bracts about 8, ovate, obtuse or acute, herbaceous, strigose with oppressed hairs; ray flowers ligulate, ligule small, spreading, scarcely as long as bracts, not toothed; white disc flowers tubular, corolla often 4 toothed; pappus absent, except occasionally very minute teeth on the top of achene; stamen 5, filaments epipetalous, free, anthers united into a tube with base obtuse; pistill bicarpellary; ovary inferior; unilocular with one basal ovule. Fruit. Achenial cypsella, one seeded, cuneate, with a narrow wing, covered with warty excrescences, brown. Seed. Dark brown, hairy and non endospermic. B. Microscopic Powder. Dark green; shows vessels in large groups or single broken pieces with pitted walls, numerous fibres entire or in pieces, trichomes entire or in pieces, warty, a few attached with epidermal and subsidiary cells, anomocytic and anisocytic stomata. Root. The cells of outer one or two rows of secondary cortex, elongated or rounded with air cavities, while cells of inner secondary cortex, elongated to irregular in shape. Stone cells scattered in secondary cortex. Phloem rays broader towards the periphery, cells rounded. Xylem rays distinct, run straight in tangential section, rarely uniseriate and biseriate, cells pitted. Stem. A few epidermal cells elongate to form characteristic non-glandular trichomes. Secondary cortex composed of large, rounded parenchymatous cells having wide air space. Vascular bundle in a ring, collateral, endarch, of varying size. Vessels barrel-shaped, some elongated with simple perforations, pitted with spiral thickening. A few xylem fibres bifurcate. Xylem rays uniseriate or biseriate.
Bhringraj
Eclipta
Bhringraj consists of the dried whole plant of Eclipta alba (L.) Hassk. (Fam. Asteraceae). Bhringraj contains not less than 0.1 per cent of wedelolcatone, calculated on the dried basis. Description. A green to greenish brown colour when completely dry.
Leaf. Anomocytic and anisocytic stomata and non-glandular hairs are present on both surface, more abundant on lower side. Vascular bundle, fine in mid rib, central one largest while four other small flanking either side of central bundle. C. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel F254. Mobile phase. A mixture of 9 volumes of toluene, 6 volumes of acetone and 1 volume of formic acid. Test solution. Reflux 1g of the coarsely powdered substance under examination with 25 ml of methanol for 30 minutes, cool and filter. Reflux the residue further with 3 25 ml of methanol, cool and filter. Combine all the filtrates and concentrate under vacuum to 10 ml. Reference solution. Reflux 0.5 g bhringraj RS with 25 ml of methanol for 30 minutes, cool and filter. Reflux the residue
Identification
A. Macroscopic Root. Well developed, a number of secondary branches arise from main root up to about 7 mm in dia, cylindrical, greyish. Stem. Herbaceous, branched occasionally rooting at nodes, cylindrical or flat, rough due to oppressed white hairs, node distinct, greenish, occasionally brownish.
1391
BHUIAMLA
IP 2007
further with 3 25 ml of methanol, cool and filter. Combine all the filtrates and concentrate under vacuum to 5 ml. Apply to the plate 10 l of each solution. Allow the mobile phase to rise 8 cm. Dry the plate in air and examine in ultra violet light at 254 nm. The chromatographic profile of the test solution is similar to that of the reference solution.
Bhuiamla
Phyllanthus
Tests
Foreign organic matter (2.6.1). Not more than 2 .0 per cent. Ethanol-soluble extractive (2.6.2). Not less than 5.0 per cent. Water- soluble extractive (2.6.3). Not less than 15.0 per cent by Method I. Ash (2.3.19). Not more than 22 per cent. Acid-insoluble ash (2.3.19). Not more than 11 per cent. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Loss on drying (2.4.19). Not more than 15.0 per cent, determined on 5 g by drying in an oven at 105. Microbial contamination (2.2.9). Complies with the microbial contamination tests. Assay. Determine by liquid chromatography (2.4.14). Test solution. Reflux 5 g of the coarsely powdered substance under examination with 30 ml of methanol on a water- bath for 30 minutes, cool and filter. Reflux the residue further with methanol till the last extract turns colourless, cool and filter. Combine all the filtrates and concentrate to 100 ml. Reference solution. 0.01 per cent w/v solution of wedelolactone RS in methanol. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5 m), mobile phase: 35 volumes of acetonitrile and 60 volumes of 0.1 per cent v/v phosphoric acid prepared by diluting 1ml of phosphoric acid to 1000 ml with water, flow rate. 1 ml per minute, spectrophotometer set at 249 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the relative standard deviation for the replicate injections is not more than 2.0 per cent. Inject the test solution and reference solution. Calculate the content of wedelolactone. Storage. Store protected from heat, moisture and against attack by insects and rodents. Bhuiamla consists of the dried aerial parts of Phyllanthus amarus Schum. & Thom. (Fam. Euphorbiaceae). Bhuiamla contains not less than 0.25 per cent of total phyllanthin and hypophyllanthin, calculated on the dried basis. Description. A green to greenish yellow in colour, taste, slightly bitter.
Identification
A. Macroscopic Stem teret, 1-4 mm in diameter. Leaves oblong 5 3 mm, short stalked, greenish brown in colour. B. Microscopic Stem, inner cortex chlorenchymatous; xylem rays 1-2-seriate. Leaf stomata mostly paracytic; epidermal cell wall markedly sinuous; rosette and prismatic crystals of calcium oxalate along the veins and midrib. C. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF 254. Mobile phase. A mixture of 6 volumes of toluene, 2 volumes of ethyl acetate, 1 volume of formic acid and 0.2 volume of methanol. Test solution. Reflux 2 g of coarsely powdered substance under examination with 50 ml methanol on a boiling water-bath for 30 minutes, cool and filter. Reflux the residue further with 2 50 ml of methanol, cool and filter. Combine all the filtrates and concentrate under vacuum to 10 ml. Reference solution. Reflux 1 g of bhuiamla RS with 50 ml methanol on a boiling water-bath for 30 minutes, cool and filter. Reflux the residue further with 2 50 ml of methanol, cool and filter. Combine all the filtrates and concentrate under vacuum to 5 ml.
1392
IP 2007
BRAHMI
Apply to the plate 10l of each solution as bands 10 mm by 2 mm. Allow the mobile phase to rise 8 cms above the line of application. Dry the plate in air and spray with methanolic sulphuric acid (10 per cent) Heat at 120 for 5-10 minutes and examine the plate in day light. The chromatographic profile of the test solution is similar to that of the reference solution.
Brahmi
Bacopa
Tests
Foreign organic matter (2.6.1). Not more than 2.0 per cent. Ethanol-soluble extractive (2.6.2). Not less than 6.0 per cent. Water-soluble extractive (2.6.3). Not less than 15.0 per cent by Method I. Ash (2.3.19). Not more than 8.0 per cent. Acid-insoluble ash (2.3.19). Not more than 5.0 per cent. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Loss on drying (2.4.19). Not more than 12.0 per cent, determined on 5 g by drying in an oven at 1050. Microbial contamination (2.2.9). Complies with the microbial contamination tests. Assay. Determine by liquid chromatography (2.4.14). Test solution. Reflux about 2 g of the coarsely powdered substance under examination with 50 ml of methanol on a water bath for 15 minutes, cool and filter. Reflux the residue further with methanol till the last extract turns colorless, cool and filter. Combine all the filtrates and concentrate to 10.0 ml. Reference solution (a). A 0.020 per cent w/v solution of phyllanthin RS in methanol. Reference solution (b). A 0.020 per cent w/v solution of hypophyllanthin RS in methanol. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5m), mobile phase: a mixture of 65 volumes of methanol and 35 volumes of water, flow rate. 1.5 ml per minute, spectrophotometer set at 230 nm, a 20 l loop injector. Inject the reference solution (a) and (b). The test is not valid unless the relative standard deviation for the replicate injections for both the analyte peaks corresponding to phyllanthin and hypophyllanthin is not more than 2.0 per cent. Inject the test solution, reference solutions (a) and (b). Calculate the contents of phyllanthin and hypophyllanthin. Storage. Store protected from heat, moisture and against attack by insects and rodents. Brahmi consists of the dried whole plant, preferably leaves and stem of Bacopa monnieri (Linn.) Pennell (Fam. Scrophulariaceae). Brahmi contains not less than 2.5 per cent of bacoside A, calculated on the dried basis. Description. A brown to reddish brown colour when completely dried or green colour when partially dried with slightly bitter taste.
Identification
A. Macroscopic Herbaceous comprising of stems, runner stems and leaves. Stems glabrous, leafless towards the base; internodes long. Leaves spatulate-obovate, sessile, and glabrous. B. Microscopic Cortex in stem composed of parenchyma cells enclosing large air spaces; xylem vessels radially arranged xylem rays uniseriate; pith parenchyma collapsed. In leaf, midrib indistinct, mesophyll isobilateral of spongy cells, a few prismatic crystals of calcium oxalate in mesophyll; stomata anomocytic on both the surfaces of leaf. C. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 70 volumes of chloroform and 30 volumes of methanol. Test solution. Reflux 2 g of coarsely powdered substance under examination with 25 ml methanol for 15 minutes, cool and filter. Reflux the residue further with 2 x 25 ml of methanol, cool and filter. Combine all the filtrates and concentrate under vacuum to 25 ml. Reference solution. Reflux 0.4 g of coarsely powdered brahmi RS with 5 ml methanol for 15 minutes, cool and filter.
1393
CASTOR OIL
IP 2007
Apply to the plate 10 l of each solution as bands 10 mm by 2 mm. Allow the mobile phase to rise 8 cms above the line of application. Dry the plate in air, spray with 20 per cent v/v sulphuric acid in methanol. Heat at 100 for 5-10 minutes and examine the plate in day light. The chromatographic profile of the test solution is similar to that of the reference solution. D. In the Assay, the chromatogram obtained with test solution corresponds to the chromatogram obtained with reference solution.
Inject the reference solution. The relative retention times are 1.00 for bacoside A3, about 1.04 for bacopaside II, 1.13 for jujubogenin isomer of bacopasaponine C and 1.19 for bacopasaponine C as bacoside A. The test is not valid unless the relative standard deviation for the replicate injections is not more than 2.0 per cent. Inject the test solution and reference solution. Calculate the content of bacoside A. Storage. Store protected from heat, moisture and against attack by insects and rodents.
Tests
Foreign organic matter (2.6.1). Not more than 2.0 per cent. Ethanol-soluble extractive (2.6.2). Not less than 6.0 per cent. Water-soluble extractive (2.6.3). Not less than 22 per cent by Method I. Ash (2.3.19). Not more than 18 per cent. Acid-insoluble ash (2.3.19). Not more than 6.0 per cent. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Loss on drying (2.4.19). Not more than 12.0 per cent, determined on 5 g by drying in an oven at 1050. Microbial contamination (2.2.9). Complies with the microbial contamination tests. Assay. Determine by liquid chromatography (2.4.14). Test solution. Reflux about 2 g of the coarsely powdered substance under examination with 50 ml of methanol on a water bath for 15 minutes, cool and filter. Reflux the residue further with methanol till the last extract turns colorless, cool and filter. Combine all the filtrates and concentrate to 100.0 ml. Reference solution. A 0.2 per cent w/v solution of bacoside A RS in methanol, prepared by heating gently on a water bath. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a gradient mixtures of acetonitrile and a buffer solution prepared by dissolving 0.5 g phosphoric acid in 800 ml of water, adjust pH to 2.8 with dilute phosphoric acid and dilute to 1000 ml with water, flow rate. 1.5 ml per minute, spectrophotometer set at 205 nm, a 20 l loop injector. Time Buffer (pH 2.8) Acetonitrile (min) ( per cent v/v) ( per cent v/v) 0 70 30 25 60 40 35 40 60 36 70 30 45 70 30
Castor Oil
Castor Oil is the fixed oil obtained by cold expression from the seeds of Ricinus communis Linn. (Fam. Euphorbiaceae). It may contain suitable antioxidants. Description. A pale yellowish or almost colourless, transparent, viscid liquid; odour, slight and characteristic.
Tests
Light absorption (2.4.7). Absorbance of a 1.0 per cent w/v solution in ethanol (95 per cent) at the maximum at about 269 nm, not more than 1.0. Weight per ml (2.4.29). 0.945 g to 0.965 g. Refractive index (2.4.27). 1.4758 to 1.4798. Optical rotation (2.4.22). +3.5 to +6.0. Peroxide value (2.3.35). Not more than 5.0. Acid value (2.3.23). Not more than 2.0. Acetyl value (2.3.22). Not less than 143. Hydroxyl value (2.3.27). Not less than 150. Saponification value (2.3.37). 176 to 187. Iodine value (2.3.28). 82 to 90. Foreign fatty substances. A mixture of 2 ml of the substance under examination and 8 ml of ethanol (95 per cent) is clear. B. Shake 10.0 ml with 20.0 ml of light petroleum (60 to 80) and allow to separate; the volume of the lower layer is not less than 16.0 ml. Storage. Store protected from light and moisture at a temperature not exceeding 15. Labelling. The label states (1) the name and quantity of any added antioxidant; (2) whether the contents are suitable for use in the manufacture of parenteral preparations.
1394
IP 2007
CLOVE OIL
Clove Oil
Clove oil is the oil distilled from the dried flower buds of Syzygium aromaticum (Linn.) Merrill and Perry [Eugenia caryophyllus (Spreng.) Bull. and Harr. Description. A clear, colourless or pale yellow liquid when freshly distilled, becoming darker and thicker by ageing or exposure to air; odour as of clove. Clove Oil contains not less than 85.0 per cent w/w and not more than 95.0 per cent w/w of phenolic substances, chiefly eugenol, C10H12O2.
Weight per ml (2.4.29). 1.038 g to 1.060 g. Refractive index (2.4.27). 1.527 to 1.535, determined at 20. Heavy metals (2.3.13). 0.5 g complies with the limit test for heavy metals, Method B (40 ppm). Phenol. Shake 1 ml with 20 ml of hot water; the mixture shows not more than a scarcely perceptible acid reaction with blue litmus paper. Cool the mixture, pass the aqueous layer through a wetted filter and treat the clear filtrate with 1 drop of ferric chloride test solution. The mixture has only a transient greyishgreen colour but not a blue or violet colour. Alkali-soluble matter. Place 80 ml of a 5 per cent w/v solution of potassium hydroxide in a 150-ml flask with a long neck which is graduated in tenths of a ml and is of such a diameter that not less than 15 cm in length has a capacity of 10 ml. Clean the flask with sulphuric acid and rinse well with water before use. Add 10 ml of the oil and shake thoroughly at 5 minute intervals for 30 minutes at ambient temperature. Raise the undissolved portion of the oil into the graduated part of the neck of the flask by the gradual addition of more of the potassium hydroxide solution; allow to stand for not less than 24 hours and read off the volume of the undissolved portion of the oil which measures between 1.0 and 1.5 ml. Assay. Determine by gas chromatography (2.4.14). Test solution (a). A 0.2 per cent w/v solution of the oil under examination in ethanol (95 per cent). Test solution (b). A 0.2 per cent w/v solution of the oil under examination and 0.15 w/v of 1-decanol (internal standard) in ethanol (95 per cent). Reference solution. A solution containing 0.2 per cent w/v solution of eugenol RS and 0.15 per cent w/v of the internal standard in ethanol (95 per cent). Chromatographic system a glass column 1.5 m x 4 mm, packed with 3 per cent w/w of dimethyl silicone fluid on acid-washed diatomaceous support (120 mesh), temperature: column.110 for 18 minutes, then increased to 170 at a rate of 12 per minute and maintained at this temperature for 2 minutes, inlet port. 220, detector. 300, flow rate 40 ml per minute of the carrier gas. Calculate the eugenol content in the oil under examination using the ratios of the area of the peak corresponding to eugenol to the area of the peak due to the internal standard in the chromatogram obtained with test solutions (b) and the reference solution. Storage. Store protected from light in well-filled containers at a temperature not exceeding 30. 1395
Identification
Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. Toluene Test solution. Dissolve 20 l of the substance under examination in 2 ml of toluene. Reference solution. Dissolve 20 l of eugenol RS in 2 ml of toluene. Apply to the plate 20 l of the test solution and 10 l of the reference solution as bands 20 mm by 3 mm. Use an unlined tank, develop the chromatogram immediately after pouring the mobile phase into the tank and allow the mobile phase to rise 10 cm. Dry the plate, allow to stand for 5 minutes and again allow the mobile phase to rise 10 cm under the same conditions. Following the second development, dry the plate in air, examine in ultraviolet light at 254 nm and mark the quenching bands. In the chromatogram obtained with the test solution there is a quenching band in the middle of the plate corresponding to the quenching band due to eugenol in the chromatogram obtained with the reference solution. A weak quenching band may also be seen in the chromatogram obtained with the test solution with an Rf value slightly lower than that of the band corresponding to eugenol (acetyleugenol). Spray the plate with about 10 ml of anisaldehyde solution, heat at 100 to 105 for 10 minutes and examine in daylight. In the chromatogram obtained with the test and reference solutions the bands corresponding to eugenol are strongly coloured brownish-violet and any band corresponding to acetyleugenol in the chromatogram obtained with the test solution is faintly violet-blue. Other coloured bands may be visible in the chromatogram obtained with the test solution, in particular a faint red band in the lower part of the chromatogram and a reddish-violet band in the upper part (caryophyllene).
Tests
Optical rotation (2.4.22). 0 to 1.50.
COLEUS
IP 2007
Coleus
10 minutes and examine the plate in day light. The chromatographic profile of the test solution is similar to that of the reference solution.
Tests
Foreign organic matter (2.6.1). Not more than 2.0 per cent. Ethanol-soluble extractive (2.6.2). Not less than 15.0 per cent. Water-soluble extractive (2.6.3). Not less than 18.0 per cent by method I. Ash (2.3.19). Not more than 15.0 per cent. Acid-insoluble ash (2.3.19). Not more than 5.0 per cent. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Coleus consists of the whole or cut dried roots of Coleus forskohlii Briq. (Fam. Lamiaceae). Coleus contains not less than 0.4 per cent w/w of forskolin, calculated on the dried basis. Description. The roots are light brown in color, generally long and radially spread. They have an aromatic characteristic odor and the taste is slightly pungent. Loss on drying (2.4.19). Not more than 12.0 per cent, determined on 5 g by drying in an oven at 1050. Microbial contamination (2.2.9). Complies with the microbial contamination tests. Assay. Determine by liquid chromatography (2.4.14). Test solution. Weigh 3 g of coarsely powdered substance under examination, add 50 ml of methanol and reflux on a water bath for 15 minutes, cool and filter. Reflux the residue two times with 75 ml of methanol, cool and filter, Concentrate the filterate to 100.0 ml. Reference solution. A 0.1 per cent w/v solution of forskolin RS in methanol. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5 m), mobile phase: filtered and degassed mixture of 45 volumes of acetonitrile and 55 volumes of water, flow rate. 1.8 ml per minute, spectrophotometer set at 220 nm, a 20 l loop injector. Inject the reference solution. The relative standard deviation for the replicate injections is not more than 2.0 per cent. Inject the test solution and reference solution. Calculate the content of forskolin. Storage. Store protected from moisture and against attack by insects and rodents.
Identification
A. Macroscopic Roots are brown, longitudinally wrinkled, fracture short, cut surface yellowish white. B. Microscopic The outermost layer consists of rectangular cork cells, cork cambium, rectangular parenchymatous region containing sclereids and calcium oxalate crystals. Vascular cambium is present in the form of a continuous ring. The tracheids and tracheidal fibres have bordered pits. C. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel. Mobile phase. A mixture of 75 volumes of benzene, and 25 volumes of ethyl acetate. Test solution. To 5 g of the coarsely powdered substance under examination, add 50 ml of methanol and reflux for 15 minutes, cool and filter. Reflux the residue further for two times with 50 ml of methanol, cool and filter. Combine all the filtrates and concentrate under vacuum to 100 ml. Reference solution. To 1 g of coleus RS add 50 ml of methanol and reflux for 15 minutes, cool and filter. Reflux the residue further for two times with 50 ml of methanol, cool and filter. Combine all the filtrates and concentrate under vacuum to 20 ml. Apply to the plate 20 l of each solution as bands 10 mm by 2 mm. Allow the mobile phase to rise 8 cms. Dry the plate in air, spray with vanilin sulphuric acid reagent. Heat at 100 for 5-
Eucalyptus Oil
Nilgiri Oil
Eucalyptus Oil is the essential oil obtained by steam distillation and rectification from the fresh leaves or the fresh terminal
1396
IP 2007
GARCINIA
branches of various species of eucalyptus like Eucalyptus globulus Labill., E. fruticetorum F. von Muell., and E. smithii (R. T. Baker) (Fam. Myrtaceae). Eucalyptus Oil contains not less than 60 per cent w/w of cineole, C10H18O. Description. A colourless or pale yellow liquid; odour, aromatic and camphoraceous; taste, pungent and camphoraceous, followed by a sensation of cold.
liquid of the first determination with the addition of 0.5 ml of 0.5 M potassium hydroxide in ethanol (60 per cent). Not more than 2.0 ml of 0.5 M potassium hydroxide in ethanol (60 per cent) is required in the second determination. Phellandrene. Mix 1 ml with 2 ml of glacial acetic acid and 5 ml of light petroleum (40 to 60), add 2 ml of a saturated solution of sodium nitrite and shake gently; no crystalline precipitate is produced in the upper layer within 1 hour. Assay (2.3.24) Determine the content of cineole in the oil.
Identification
Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 90 volumes of toluene and 10 volumes of ethyl acetate. Test solution. Dissolve 1 g of the substance under examination in 100 ml of toluene. Reference solution. A 1 per cent w/v solution of cineole RS in toluene. Apply to the plate 2 l of each solution. After development, dry the plate in air, spray with anisaldehyde solution, using about 10 ml for a 200 mm x 200 mm plate, heat at 105 for 10 minutes and examine in daylight and in ultraviolet light at 365 nm. In the chromatogram obtained with the reference solution a dark brown spot due to cineole is visible in daylight in the middle part; when examined in ultraviolet light at 365 nm, the spot shows a brown fluorescence. The principal spot in the chromatogram obtained with the test solution corresponds to that of cineole; no carmine-brown spot appears in daylight in the upper third of the chromatogram and when examined in ultraviolet light at 365 nm no spot showing a greenish brown fluorescence appears in the upper third (citronellal). Other spots may be visible in the upper and lower thirds of the chromatogram.
Storage. Store in well-filled, tightly-closed containers at a temperature not exceeding 30.
Garcinia
Vilayati Imli
Garcinia is the dried deseeded fruit of Garcinia cambogia Desr. (Fam. Guttiferae). Garcinia contains not less than 12.0 per cent of total hydroxycitric acid and hydroxycitric acid lactone, calculated on the dried basis. Description. Dark brown to blackish brown fruits. Taste, acidic.
Tests
Optical rotation (2.4.22). 0 to +10. Refractive index (2.4.27). 1.457 to 1.469. Weight per ml (2.4.29). 0.897 g to 0.924 g. Aldehydes. Place 10 ml in a glass-stoppered tube (150 mm x 25 mm) add 5 ml of toluene and 4 ml of ethanolic hydroxylamine solution, shake vigorously and titrate immediately with 0.5 M potassium hydroxide in ethanol (60 per cent) until the red colour changes to yellow. Continue the shaking and neutralising until the pure yellow colour of the indicator is permanent in the lower layer after shaking vigorously for 2 minutes and allowing separation to take place; the reaction is complete in about 15 minutes. Repeat the operation using a further 10 ml of the substance under examination, and as the standard for the end-point, the titrated
Identification
A. Macroscopic Dark brown to blackish brown fruits, ovoid, longitudinally grooved. B. Microscopic Mesocarp very wide composed of parenchymatous cells of various sizes and shapes. Compound starch grains and prismatic crystals of calcium oxalate traverse throughout the parenchymatous cells of the mesocarp. C. In the Assay, the principal peak in the chromatogram obtained with test solution has a retention time similar to that of the peak due to hydroxycitric acid in the chromatogram obtained with reference solution.
1397
GOKHRU
IP 2007
Tests
Citric Acid. Not more than 2.0 per cent. Determine by liquid chromatography (2.4.14). Test solution, reference solutions (a), (b) and chromatographic system as described under Assay. Inject test solution and reference solution (b). Calculate the content of citric acid. Foreign organic matter (2.6.1). Not more than 5.0 per cent. Water-soluble extractive (2.6.3). Not less than 40.0 per cent by Method I. Ash (2.3.19). Not more than 8.0 per cent. Acid-insoluble ash (2.3.19). Not more than 1.5 per cent. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Loss on drying (2.4.19). Not more than 12.0 per cent, determined on 5 g by drying in an oven at 1050. Microbial contamination (2.2.9). Complies with the microbial contamination tests. Assay. Determine by liquid chromatography (2.4.14). Test solution. Weigh about 5 g of the coarsely powdered substance under examination and transfer to a 250-ml beaker. Add 5 ml of dilute phosphoric acid and 100 ml of water, concentrate to half the volume by heating, cool and filter. Extract the residue further with water till the last extract turns colorless, cool and filter. Combine all the filtrates and concentrate under vacuum to 250.0 ml. Reference solution (a). A 0.8 per cent w/v solution of hydroxycitric acid calcium salt RS in dilute phosphoric acid. Reference solution (b). A 0.1 per cent w/v solution of citric acid in dilute phosphoric acid. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a buffer solution prepared by dissolving 1.36 g of potassium dihydrogen phosphate in 900 ml of water, adjusting the pH to 2.5 with dilute phosphoric acid and diluting to 1000.0 ml with water, flow rate. 1 ml per minute, spectrophotometer set at 215 nm, a 20 l loop injector. Inject the reference solution (a). The test is not valid unless the relative retention times are about 0.8 for hydroxycitric acid lactone, 1.0 for hydroxycitric acid and 2.0 for citric acid, the resolution factor between hydroxycitric acid lactone and hydroxycitric acid is not less than 1.8, the tailing factor is
not more than 1.5 and the relative standard deviation for the replicate injections is not more than 2.0 per cent. Inject the test solution and reference solution (a). Calculate the sum of the contents of hydroxycitric acid and hydroxycitric acid lactone. Storage. Store protected from heat, moisture and against attack by insects and rodents.
Gokhru
Tribulus
Gokhru consists of dried fruits of Tribulus terrestris L. Gokhru contains not less than 0.5 per cent of diosgenin, calculated on the dried basis. Description. Pedicellate and globose fruits, having wedgeshaped cocci, covered with short and stiff spines. Possesses faintly aromatic smell and acrid taste.
Identification
A. Macroscopic Fruit is pedicellate, having wedge-shaped cocci, covered with spines. Surface of schizocarp is rough. B. Microscopic Pericarp is differentiated into epicarp, mesocarp and endocarp. Epicarp is surrounded by nonglandular trichomes. C. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 8 volumes of toluene and 2 volumes of ethyl acetate. Test solution. Reflux 5 g of coarsely powdered substance under examination with 50 ml of methanol for 15 minutes, cool and filter. Reflux the residue further with 50 ml of methanol, cool and filter. Combine both the filtrates and concentrate under vacuum to dryness. Extract the dried residue with 10 ml of methanol at 50 for 10 minutes, filter the solution and use filtrates for analysis.
1398
IP 2007
GUAR GUM
Reference solution. Reflux 2.5 g of gokhru RS with 50 ml of methanol for 15 minutes, cool and filter. Reflux the residue further with 50 ml of methanol, cool and filter. Combine both the filtrates and concentrate under vacuum to dryness. Extract the dried residue with 5 ml of methanol at 50 for 10 minutes, filter the solution and use the filtrates for analysis. Apply to the plate 20 l of each solution as bands 10 mm by 2 mm. Allow the mobile phase to rise 8 cm. Dry the plate in air, spray with anisaldehyde sulphuric acid. The chromatographic profile of the test solution is similar to that of the reference solution.
Storage. Store protected from heat, moisture and against attack by insects and rodents.
Guar Gum
Guar Gum is a gum obtained from the ground endosperms of the seeds of Cyamopsis tetragonolobus (Linn.) Taub or other species of Cyamopsis (Fam. Leguminosae). It consists mainly of a high molecular weight hydrocolloidal polysaccharide, composed of galactan and mannan units combined through glycosidic linkages. Description. An almost white to pale yellowish white powder; odour, characteristic.
Tests
Foreign organic matter (2.6.1). Not more than 2 per cent. Ethanol-soluble extractive (2.6.2). Not less than 3 per cent. Water-soluble extractive (2.6.3). Not less than 15 per cent by method I. Ash (2.3.19). Not more than 11 per cent. Acid-insoluble ash (2.3.19). Not more than 1 per cent. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Loss on drying (2.4.19). Not more than 5 per cent, determined on 5 g by drying in an oven at 105. Microbial contamination (2.2.9). Complies with the microbial contamination tests. Assay. Determine by liquid chromatography (2.4.14). Test solution. Reflux 5.0 g of the substance under examination with 50 ml of sulphuric acid (10 per cent) for 4 hours. Cool and transfer to separating funnel. Extract with 50 ml of ethyl acetate. Repeat the extraction 3 times. Pass the ethyl acetate layer through sodium sulphate and evaporate. Dissolve the residue with 50 ml of methanol. Reference solution. A 0.1 per cent w/v solution of diosgenin RS in methanol. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5 m), mobile phase: 80 volumes of acetonitrile and 20 volumes of methanol, flow rate. 1 ml per minute, spectrophotometer set at 210 nm, a 20 l loop injector. Inject the reference solution. The relative standard deviation for the replicate injections is not more than 2.0 per cent. Inject the test solution. Calculate the content of diosgenin.
Identification
A. When mounted in lactophenol and examined under a microscope, irregular, angular particles of various sizes and shapes are seen. B. To 0.1 g add 1 ml of 0.2 M iodine; the mixture does not acquire an olive-green colour. C. Dissolve 0.1 g in 20 ml of water by shaking and add 0.5 ml of hydrogen peroxide solution (20 vol) and 0.5 ml of a 1 per cent w/v solution of benzidine in ethanol (90 per cent), shake and allow to stand; no blue colour is produced (distinction from acacia). D. Mount a small quantity in ruthenium red solution and examine under a microscope; the particles do not acquire a pink colour (distinction from sterculia gum and agar). E. To 2 ml of a 0.5 per cent w/v solution add 2 ml of a 20 per cent w/v solution of lead acetate; a flocculent precipitate is produced (distinction from acacia, ghatti gum and sterculia).
Tests
Acidity or alkalinity. A 0.5 per cent w/v solution is neutral to litmus paper. Tannin. To 5 ml of a 0.5 per cent w/v solution add 0.1 ml of ferric chloride test solution; no bluish black colour is produced. Arsenic (2.3.10). Mix 3.3 g with 3 g of anhydrous sodium carbonate, add 10 ml of bromine solution and mix thoroughly. Evaporate to dryness on a water-bath, gently ignite and dissolve the cooled residue in a mixture of 16 ml of brominated hydrochloric acid and 45 ml of water. Remove the excess of bromine with 2 ml of stannous chloride solution AsT. The resulting solution complies with the limit test for arsenic (3 ppm). Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm).
1399
GUDMAR
IP 2007
Protein. Not more than 5.0 per cent, determined by the following method. Carry out the determination of nitrogen (2.3.30), using about 3.5 g, accurately weighed, and multiplying the percentage of nitrogen determined by 6.25 to obtain the percentage of protein. Acid-insoluble matter. Not more than 3.0 per cent, determined by the following method. Weigh accurately about 1.5 g and disperse in 150 ml of water and 1.5 ml of sulphuric acid. Warm on a water-bath for 6 hours, replacing the water lost by evaporation. Add about 0.5 g of a suitable filter-aid, accurately weighed, and filter through a suitable ashless filter paper. Wash the residue several times with hot water, dry the filter and its contents at 105 for 3 hours. Cool in a desiccator, weigh and subtract the weight of the filter aid. Microbial contamination (2.2.9). Total bacterial count: Not more than 5000 per g. 1 g is free from Escherichia coli and 10 g is free from Salmonellae. Ash (2.3.19). Not more than 2.0 per cent, determined on 1.0 g. Loss on drying (2.4.19). Not more than 13.0 per cent, determined on 0.5 g by drying in an oven at 105.
Identification
A. Macroscopic Leaves, simple, petiolate about 2 to 6 cm long and 1 to 4 cm broad, yellowish brown on adaxial and dark green on abaxial side. B. Microscopic Upper and lower epidermis covered with cuticle having uni to tri cellular covering trichomes which are slightly curved at the bulbous base. Below the epidermis is single layer of palisade cells followed by 2-3 layered spongy parenchyma. Starch gains are simple and present in spongy parenchyma. Midrib region shows 2-7 layers of collenchymatous cells. Stomata are of paracytic type, mostly on lower the surface. There is a fan shaped vascular bundle in the centre. Each vascular bundle is collateral, closed and surrounded by parenchymatous sheath. Rosette crystal of calcium oxalate present in the spongy parenchyma. C. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 5 volumes of chloroforms, 1 volume of methanol and 1 volume of ethyl acetate. Test solution. Reflux 5 g of the coarsely powdered substance under examination with 50 ml of ethanol (50 per cent v/v) for 15 minutes, cool and filter. Reflux the residue further with 2 50 ml of ethanol (50 per cent v/v), cool and filter. Combine all the filtrates and concentrate under vacuum to 25 ml. Take 5 ml of resulting solution, add 5 ml ethanol and 2 ml of potassium hydroxide and reflux for 1 hour. Cool and add 1.8 ml of 12 M hydrochloric acid and heat on water bath. Cool and adjust the pH to 7.5-8.5 with 11 per cent potassium hydroxide. Dilute the solution with ethanol (50 per cent v/v) to 100 ml and filter. Reference Solution. Reflux 1 g of gudmar RS with 50 ml of ethanol (50 per cent v/v) for 15 minutes, cool and filter. Reflux the residue further with 2 50 ml of ethanol (50 per cent v/v), cool and filter. Combine all the filtrates and concentrate under vacuum to 10 ml. Take 5 ml of resulting solution, add 5 ml ethanol and 2 ml of potassium hydroxide and reflux for 1 hour. Cool and add 1.8 ml of 12 M hydrochloric acid and heat on water bath. Cool and adjust the pH to 7.5-8.5 with 11 per cent potassium hydroxide. Dilute the solution with ethanol (50 per cent v/v) to 50 ml and filter. Apply to the plate 10 l of each solution as bands 10 mm by 2 mm. Allow the mobile phase to rise 8 cm. Dry the plate in air, spray with anisaldehyde sulphuric acid solution. Heat at 100 for 10 minutes and examine the plate in day light. The chromatographic profile of the test solution is similar to that of the reference solution.
Gudmar
Gymnema
Gudmar consists of the dried mature leaves of Gymnema sylvestre R.Br. (Fam. Asclepiadaceae). Gudmar contains not less than 1.0 per cent of gymnemic acids (calculated as gymnemagenin), calculated on dried basis. Description. Greenish-yellow in colour, surface pubescent on both sides and characteristic odour with extremely bitter and acrid taste.
Tests
Foreign organic matter (2.6.1). Not more than 2.0 per cent. Ethanol-soluble extractive (2.6.2). Not less than 5.0 per cent.
1400
IP 2007
GUDUCHI
Water-soluble extractive (2.6.3). Not less than 20.0 per cent by method I. Ash (2.3.19). Not more than 15.0 per cent . Acid-insoluble ash (2.3.19). Not more than 6.0 per cent. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Loss on drying (2.4.19). Not more than 14.0 per cent, determined on 5 g by drying in an oven at 1050. Microbial contamination (2.2.9). Complies with the microbial contamination tests. Assay. Determine by liquid chromatography (2.4.14). Test solution. Reflux 5 g of the coarsely powdered substance under examination with 50 ml of ethanol (50 per cent v/v) for 15 minutes, cool and filter. Reflux the residue further with 2 50 ml of ethanol (50 per cent v/v), cool and filter. Combine all the filtrates and concentrate under vacuum to 25 ml. Take 5 ml of resulting solution, add 5 ml ethanol and 2 ml of potassium hydroxide and reflux for 1 hour. Cool and add 1.8 ml of 12 M hydrochloric acid and heat on water bath. Cool and adjust the pH to 7.5-8.5 with 11 per cent potassium hydroxide. Dilute the solution with ethanol (50 per cent v/v) to 100 ml and filter. Reference solution. A 0.01 per cent w/v solution of gymnemagenin RS in methanol. Chromatographic system a stainless steel column 25 cm 4.6 mm packed with octadecylsilane bonded to porous silica (5 m), mobile phase (A). 80 per cent v/v acetonitrile, (B). 0.1 per cent w/v dihydogen potassium phosphate, flow rate. 1.5 ml per minute, spectrophotometer set at 210 nm, a 20 l loop injector. Time Mobile phase A Mobile phase B (min) (per cent v/v) (per cent v/v) 0 25 75 20 50 50 30 25 75 Inject the reference solution. The test is not valid unless the relative standard deviation for the replicate injections is not more than 2.0 per cent. Inject the test solution and reference solution. Calculate the content of gymnemagenin. Storage. Store protected from heat, moisture and against attack by insects and rodents.
Guduchi
Tinospora, Giloe, Amrita
Guduchi consists of the dried, mature pieces of stem of Tinospora cordifolia (Willd.) Miers (Fam. Menispermaceae). Guduchi contains not less than 0.02 per cent w/w of cordifolioside A, calculated on the dried basis. Description. A greyish-black in colour, fibrous fracture and no distinct odour with bitter taste.
Identification
A. Macroscopic Stem-pieces glabrous, cylindrical, solid, lenticillate, 5-15 mm in diameter having light brown surface marked with warty protuberances due to circular lenticels. Transversely smoothened surface shows a radial structure with conspicuous medullary rays traversing porous tissues. B. Microscopic Transverse section of stem shows outermost layer of cork which is differentiated in to outer zone of thick walled, compressed cells and inner zone of thin walled, tangential cells. Cork broken at some places due to lenticels. Cortex consists of 3-5 rows of irregularly arranged tangential, chlorenchymatous cells with numerous intercellular spaces. Inner cortex filled with plenty of starch gains. Vascular zone consists of 10-12 wedge-shaped strips of xylem externally surrounded by semi-circular strips of phloem. Cambium composed of one to two layers of tangentially elongated cells. Primary phloem appears crushed and obliterated; secondary phloem groups are massive. Pith composed of large, thin walled cells mostly containing starch gains. C. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 85 volumes of chloroform and 15 volumes of methanol. Test solution. Reflux 2 g of the coarsely powdered substance
1401
GUGGUL RESIN
IP 2007
under examination with 25 ml of methanol for 15 minutes, cool and filter. Reflux the residue further with 2 25 ml of methanol, cool and filter. Combine all the filtrates and concentrate under vacuum to 5 ml. Reference solution. Reflux 2 g of the guduchi RS with 25 ml of methanol for 15 minutes, cool and filter. Reflux the residue further with 2 25 ml of methanol, cool and filter. Combine all the filtrates and concentrate under vacuum to 5 ml. Apply to the plate 10 l of each solution as bands 10 mm by 2 mm. Allow the mobile phase to rise 8 cm. Dry the plate in air, spray with a anisaldehyde solution. Heat at 110 for 10 minutes and examine the plate in day light. The chromatographic profile of the test solution is similar to that of the reference solution.
Time (min) 0 25 30
Water (per cent v/v) 80 20 80
Acetonitrile (per cent v/v) 20 80 20
Inject the reference solution. The test is not valid unless the relative standard deviation for the replicate injections is not more than 2.0 per cent. Inject the test solution and reference solution. Calculate the content of cordifolioside A. Storage. Store protected from heat, moisture and against attack by insects and rodents.
Tests
Foreign organic matter (2.6.1). Not more than 2.0 per cent. Ethanol-soluble extractive (2.6.2). Not less than 1.5 per cent. Water-soluble extractive (2.6.3). Not less than 9.0 per cent by method I. Ash (2.3.19). Not more than 10.0 per cent. Acid-insoluble ash (2.3.19). Not more than 3.0 per cent. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Loss on drying (2.4.19). Not more than 10.0 per cent, determined on 5 g by drying in an oven at 1050. Microbial contamination (2.2.9). Complies with the microbial contamination tests. Assay. Determine by liquid chromatography (2.4.14). Test solution. Reflux 2 g of the coarsely powdered substance under examination with 50 ml of methanol on a water-bath for 15 minutes, cool and filter. Reflux the residue further with methanol till the extract turns colourless, cool and filter. Combine all the filtrates and concentrate to a volume slightly less than 25 ml. Dilute with methanol to 25.0 ml. Reference solution. A 0.004 per cent w/v solution of cordifolioside A RS in methanol. Chromatographic system a stainless steel column 25 cm 4.6 mm packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a gradient mixtures of water and acetonitrile, flow rate. 1 ml per minute, spectrophotometer set at 210 nm, a 20 l loop injector.
Guggul Resin
Guggul, Commiphora
Guggul Resin is the oleoresin exudation from Commiphora wightii (Arnott) Bhandari (Commiphora mukul (Arn.) Bhandari, Balsamodendron mukul Hook. ex Stocks) (Fam. Burseraceae). Guggul Resin contains not less than 1.0 per cent and not more than 1.5 per cent of gugulsterones (Z and E). Description. Light to dark-brown conglomerates of tears, rounded or irregular, slightly sticky to touch; odour, faintly balsamic.
Identification
A. Prepare a 0.005 per cent w/v solution in ethanol (95 per cent) of the residue obtained in the test for Ethanol-soluble extractive. When examined in the range 230 nm to 360 nm (2.4.7), the resulting solution shows absorption maxima at about 245 nm and 327 nm.
1402
IP 2007
GUGULIPID
B. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 3 volumes of light petroleum (60 to 80) and 1 volume of ethyl acetate. Test solution. Dissolve 0.5 g of the residue obtained in the test for Ethanol-soluble extractive in 100 ml of ethanol (95 per cent). Reference solution. A 0.5 per cent w/v solution of the residue obtained similarly from guggul resin RS in ethanol (95 per cent). Apply to the plate 20 l of each solution. After development, dry the plate in air until the odour of the solvent is no longer detectable and spray with a 50 per cent w/v solution of sulphuric acid. The principal spots in the chromatogram obtained with the test solution correspond to the spots in the chromatogram obtained with the reference solution.
Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilyl silica gel (5 m), mobile phase: a filtered and degassed mixture of 45 volumes of acetonitrile and 55 volumes of water, flow rate. 2 ml per minute, spectrophotometer set at 242 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the relative retention times are about 0.69 for gugulsterone E and 1.0 for gugulsterone Z and the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject the test solution and the reference solution. Calculate the contents of gugulsterones (Z and E). Storage. Store protected from light at a temperature not exceeding 30.
Tests
Ethyl acetate-soluble extractive. Not less than 25.0 per cent, determined by the following method. Crush the substance under examination to a coarse powder. Shake 5.0 g of the powder with 25 ml of light petroleum (60 to 80) for 1 hour and separate the liquid by filtration. Repeat the extraction twice and dry the defatted material over phosphorus pentoxide at room temperature at a pressure not exceeding 2.75 kPa for 8 hours. Crush the dried material and extract with four quantities, each of 25 ml, of ethyl acetate by shaking each time for 1 hour followed by filtration through a sintered-glass funnel (porosity No. 3) and combining the filtrates. Evaporate the combined filtrates, dry over phosphorus pentoxide at room temperature at a pressure not exceeding 2.75 kPa for 12 hours and weigh. Ethanol-soluble extractive (2.3.46). Not less than 35.0 per cent, determined by the following method. Crush the substance under examination to a coarse powder. Macerate 5.0 g of the powder with 100 ml of ethanol (95 per cent) in a closed flask for 24 hours, shaking frequently during the first 6 hours and allowing to stand for 18 hours. Filter rapidly taking care to avoid loss of ethanol, evaporate 25 ml of the filtrate to dryness, dry at 105 and weigh. Sulphated ash (2.3.18). Not more than 10.0 per cent, determined on 0.5 g. Assay. Determine by liquid chromatography (2.3.14). Test solution. Weigh accurately about 3.0 g of the substance under examination, add 50 ml of acetonitrile, reflux on a waterbath for 30 minutes, cool and filter. Reflux the residue further with three portions, each of 30 ml, of acetonitrile, cool and filter. Combine the filtrates and concentrate to 100.0 ml. Reference solution. A 0.02 per cent w/v solution of gugulsterones (Z and E) RS in acetonitrile.
Gugulipid
Gugulipid is the ethyl acetate extractive of Guggul Resin. Gugulipid contains not less than 4.0 per cent and not more than 6.0 per cent of gugulsterones (Z and E). Description. A brown, viscous liquid.
Identification
A. When examined in the range 230 nm to 360 nm (2.4.7), a 0.005 per cent w/v solution in chloroform shows absorption maxima at about 245 nm and 327 nm; absorbance at about 245 nm, about 0.87 and at about 327 nm, about 0.52. B. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 3 volumes of light petroleum (60 to 80) and 1 volume of ethyl acetate. Test solution. Dissolve 0.25 g of the substance under examination in 100 ml of ethanol (95 per cent). Reference solution. A 0.25 per cent w/v solution of gugulipid RS in ethanol (95 per cent). Apply to the plate 5 l of each solution. After development, dry the plate in air until the odour of the solvent is no longer detectable and spray with a 50 per cent w/v solution of sulphuric acid. The principal spots in the chromatogram obtained with the test solution correspond to those in the chromatogram obtained with the reference solution.
Tests
Assay. Determine by liquid chromatography (2.4.14).
1403
GUGULIPID TABLETS
IP 2007
Test solution. Weigh accurately about 0.75 g of the substance under examination, add 50 ml of acetonitrile and warm on a boiling water-bath for 10 minutes. Cool and add sufficient acetonitrile to produce 100.0 ml. Reference solution. A solution containing 0.02 per cent w/v of gugulsterones (Z and E) in acetonitrile. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilyl silica gel (5 m), mobile phase: a filtered and degassed mixture of 45 volumes of acetonitrile and 55 volumes of water, flow rate. 2 ml per minute, spectrophotometer set at 242 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the relative retention times are about 0.69 for gugulsterone E and 1.0 for gugulsterone Z and the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject alternately the test solution and the reference solution. Calculate the contents of gugulsterones (Z and E). Storage. Store protected from light at a temperature not exceeding 30.
Apply to the plate 5 l of each solution. After development, dry the plate in air until the odour of the solvent is no longer detectable and spray with a 50 per cent w/v solution of sulphuric acid. The principal spots in the chromatogram obtained with the test solution correspond to those in the chromatogram obtained with the reference solution.
Tests
Disintegration (2.5.1). 60 minutes. Other tests. Comply with the tests stated under Tablets. Assay. Determine by liquid chromatography (2.4.13). Test solution. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing about 10 mg of gugulsterones (Z and E) and extract with five quantities, each of 20 ml, of acetonitrile, with the aid of heat. Combine the extracts and concentrate to 50.0 ml. Filter the solution through a membrane filter disc with an average pore diameter not greater than 1.0 m and use the filtrate. Reference solution. A solution containing 0.02 per cent w/v of gugulsterones (Z and E) in acetonitrile. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilyl silica gel (5 m), mobile phase: a filtered and degassed mixture of 45 volumes of acetonitrile and 55 volumes of water, flow rate. 2 ml per minute, spectrophotometer set at 242 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the relative retention times are about 0.69 for gugulsterone E and 1.0 for gugulsterone Z and the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject the test solution and the reference solution. Calculate the contents of gugulsterones (Z and E) in the tablets. Storage. Store protected from light at a temperature not exceeding 30. Labelling. The label states the strength in terms of the equivalent amount of gugulsterones (Z and E).
Gugulipid Tablets
Gugulipid Tablets contain not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of gugulsterones (Z and E). The tablets may be coated.
Identification
Extract a quantity of the powdered tablets containing about 20 mg of gugulsterones (Z and E) with two quantities, each of 15 ml, of ethyl acetate, combine the extracts, filter and evaporate to dryness. The residue complies with the following tests. A. When examined in the range 230 nm to 360 nm (2.4.7), a 0.005 per cent w/v solution in chloroform shows absorption maxima at about 245 nm and 327 nm; absorbance at about 245 nm, about 0.87 and at about 327 nm, about 0.52. B. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 3 volumes of light petroleum (60 to 80) and 1 volume of ethyl acetate. Test solution. A 0.25 per cent w/v solution of the substance under examination in ethanol (95 per cent). Reference solution. A 0.25 per cent w/v solution of gugulipid RS in ethanol (95 per cent).
1404
IP 2007
HARIDRA
Haridra
Haldi; Turmeric; Curcuma
Tests
Foreign organic matter (2.6.1). Not more than 2.0 per cent. Ethanol-soluble extractive (2.6.2). Not less than 6.0 per cent. Water-soluble extractive (2.6.3). Not less than 12 per cent by Method I. Ash (2.3.19). Not more than 10 per cent. Acid-insoluble ash (2.3.19). Not more than 2.0 per cent. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Water (2.3.43). Not more than 12.0 per cent, determined on 0.2 g. Microbial contamination (2.2.9). Complies with the microbial contamination tests. Assay. Determine by liquid chromatography (2.4.14). Test solution. Reflux about 1 g of the coarsely powdered substance under examination with 50 ml of methanol on a water bath for 15 minutes cool and filter. Reflux the residue further with 5 x 25 ml of methanol, cool and filter. Combine all the filtrates and concentrate to 100.0 ml. Reference solution. A 0.01 per cent w/v solution of curcumin RS in methanol. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with silicagel consisting of porous spherical particles with chemically bonded nitrile group, mobile phase: 35 volumes of tetrahydrofuran and 65 volumes of a buffer solution prepared by dissolving 10 g of citric acid in 1000 ml of water, adjusting the pH to 3.0 with dilute ammonia solution, flow rate. 1.2 ml per minute, spectrophotometer set at 430 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the relative standard deviation for the replicate injections is not more than 2.0 per cent. Inject the test solution and reference solution. Calculate the content of curcumin. Storage. Store protected from moisture.
Haridra consists of the dried rhizomes of Curcuma longa Linn. (Fam. Zingiberaceae). Haridra contains not less than 1.5 per cent of curcumin, calculated on the dried basis. Description. Externally yellowish to yellowish brown with root scars and annulations. Odour, aromatic; taste, warmly aromatic and bitter.
Identification
A. Macroscopic Rhizome oblong, conical or cylindrical to elongate, finger-like; internally orange yellow. Texture hard and heavy; fracture short. B. Microscopic Ground tissue of parenchyma cells; cells filled with gelatinized starch grains and yellow pigment. Fibrovascular bundles and oil cells scattered through out ground tissue. C. Determine by thin-layer chromatography (2.4.17), coating the1 plate with silica gel GF 254. Mobile phase. A mixture of 94 volumes of chloroform, 5 volumes of ethanol and 1 volume glacial acetic acid. Test solution. Extract 1 g of the coarsely powdered substance under examination with 5 ml methanol for 10 minutes with slight warming. Filter and use the filtrate. Reference solution. Reflux 1 g of coarsely powdered haridra RS with 5 ml methanol for 15 minutes, cool and filter. Apply to the plate 10 l of each solution as bands 10 mm by 2 mm. Allow the mobile phase to rise 8 cms above the line of application. Dry the plate in air and examine in daylight and ultra-violet light 365 nm. The chromatographic profile of the test solution is similar to that of the reference solution.
1405
HARITAKI
IP 2007
Haritaki
Harad; Chebulic myrobalan; Terminalia
Reference solution. To 0.1 g of the haritaki RS, add 50-75 ml of methanol and reflux for 15 minutes, cool and filter. Reflux the residue further for two times with 75 ml of methanol, cool and filter. Combine all the filtrates and concentrate under vacuum to 10 ml. Apply to the plate 10 l of each solution as bands 10 mm by 2 mm. Allow the mobile phase to rise 8 cms above the line of application. Dry the plate in air, spray with solution of 10 per cent w/v ferric chloride solution in water. Examine the plate in day light. The chromatographic profile of the test solution is similar to that of the reference solution. D. In the Assay, the chromatogram obtained with the test solution corresponds to the chromatogram obtained with reference solution (a).
Tests
Foreign organic matter (2.6.1). Not more than 2.0 per cent. Haritaki consists of pericarp of the dried fruit of Terminalia chebula Retz. (Fam. Combretaceae). Haritaki contains not less than 5 per cent and not more than 12.5 per cnet of chebulinic acid, calculated on the dried basis. Description. It has a shine on its external part and has longitudinal ridges. The colour varies from yellowish brown to light black. It has a astringent taste and is also slightly bitter Ethanol-soluble extractive (2.6.2). Not less than 35.0 per cent. Water-soluble extractive (2.6.3). Not less than 50 per cent by Method I. Ash (2.3.19). Not more than 6.0 per cent. Acid-insoluble ash (2.3.19). Not more than 3.0 per cent. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm) Loss on drying (2.4.19). Not more than 12 per cent, determined on 5 g by drying in an oven at 105. Microbial contamination (2.2.9). Complies with the microbial contamination tests. Assay. Determine by liquid chromatography (2.4.14). Test solution. Weigh 0.5 g of coarsely powdered sample, add 50 ml of water, sonicate for 3 minutes and heat on a boiling water bath for 15 minutes, cool and dilute to 100.0 ml with water and filter. Dilute 10.0 ml of the solution to 25.0 ml with water. Reference solution (a). Weigh 0.5 g of haritaki RS, add 50 ml of water, sonicate for 3 minutes and heat on a boiling water bath for 15 minutes, cool and dilute to 100.0 ml with water and filter. Dilute 10.0 ml of the solution to 25.0 ml with water. Reference solution (b). A 0.01 per cent w/v solution of chebulagic acid RS in water. Reference solution (c). A 0.01 per cent w/v solution of chebulinic acid RS in water. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5 m). mobile phase: filtered and degassed gradient mixtures
Identification
Test C may be omitted if tests A, B and D are carried out and test D may be omitted if tests A, B and C are carried out. A. Macroscopic The fruit is 2 to 3 cm in length and 1 to 2 cm in diameter with hard stony appearance. Externally it is shining and is adorned with longitudinal ridges. Color of the fruit rind varies from yellowish brown, uniform brown to light black. Internally the fruit is light yellow. B. Microscopic Epicarp has thick walls covered with cuticle. The mesocarp has many stone cells of various sizes and shapes which forms a reticulum. Large quantity of tannin is present in the mesocarp. Simple starch granules are present in plenty. C. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel 60. Mobile phase. A mixture of 35 volumes of toulene, 50 volumes of acetone, 15 volumes of glacial acetic acid and 5 volumes of formic acid. Test solution. To 1 g of the coarsely powdered substance being examined, add 50-75 ml of methanol and reflux for 15 minutes, cool and filter. Reflux the residue further for two times with 75 ml of methanol, cool and filter. Combined all the filtrates and concentrate under vacuum to 100 ml.
1406
IP 2007
ISPAGHULA HUSK
of acetonitrile and a buffer solution pH 2.5 prepared by dissolving 0.136 g of potassium di-hydrogen orthophosphate in 500 ml of water, add 0.5 ml of orthophosphoric acid and make upto 1000 ml with water, flow rate. 1.5 ml per min., spectrophotometer set at 270 nm, a 20 l loop injector. Time Buffer solution Acetonitrile (in min.) (per cent v/v) (per cent v/v) 0 95 5 18 65 35 25 45 55 28 45 55 35 95 5 Inject the reference solution (b) and (c). The relative standard deviation for the replicate injections is not more than 2.0 per cent. Inject the test solution and reference solution (a). Calculate the content of Chebulagic acid and Chebulinic acid. Storage. Store protected from heat, moisture and against attack by insects and rodents.
Acid value (2.3.23). Not more than 2.0. Hydroxyl value (2.3.27). 154 to 162. Iodine value (2.3.28). Not more than 5.0. Peroxide value (2.3.35). Not more than 5.0. Saponification value (2.3.37). 176 to 182. Storage. Store at a temperature not exceeding 30. Avoid exposure to excessive heat.
Ispaghula Husk
Isapgol Husk; Plantago
Hydrogenated Castor Oil

Castor Wax; Opalwax
Hydrogenated Castor Oil is refined, bleached, hydrogenated and deodorised castor oil. It consists mainly of the triglyceride of hydroxystearic acid. Description. A white to yellow powder of uniform consistency and texture. It may have a hard, waxy consistency. Ispaghula Husk consists of the epidermis and collapsed adjacent layers removed from the dried ripe seeds of Plantago ovata Forssk. Description. Pale buff, brittle flakes, more or less lanceolate, up to 2 mm long and 1 mm wide at the centre, much broken into smaller fragments; many of the flakes having a small, brownish, oval spot, about 0.8 to 1.0 mm long, in the centre; the material swells rapidly in water, forming a stiff mucilage.
Tests
Melting range (2.4.21). 85 to 88, determined by Method II. Free fatty acids. Weigh accurately about 20 g, melt on a waterbath, add 75 ml of hot ethanol (95 per cent), previously neutralised to phenolphthalein solution with 0.1 M sodium hydroxide, swirl, add 1 ml of phenolphthalein solution and titrate with 0.1 M sodium hydroxide, swirling vigorously until the solution remains faintly pink after being shaken for 60 seconds; not more than 11.0 ml of 0.1 M sodium hydroxide is required. Heavy metals (2.3.13). 2.0 g complies with the limit test for heavy metals, Method B (10 ppm). Acetyl value (2.3.22). Not less than 143.
Identification
When mounted in cresol and examined under a microscope, the particles are found to be transparent and angular, the edges straight or curved and sometimes rolled. They are composed of polygonal prismatic cells with four to six straight or slightly curved walls; the cells vary in size in different parts of the seed coat, from about 25 mm to 60 mm at the summit of the seed, that is, near and over the brown spot, to 25 mm to 100 mm for the remainder of the epidermis except at the edges of the seed, where the cells are smaller, about 45 mm to 70 mm. When mounted in ethanol (95 per cent) and irrigated with
1407
KALMEGH
IP 2007
water, the mucilage in the outer part of the epidermal cells swells rapidly and goes into solution, while the two inner layers of mucilage are more resistant and swell to form rounded papillae. When mounted in 0.005 M iodine, occasional simple and two- to four-compound starch granules, about 2 mm to 10 mm, can be seen in some of the cells. Occasional fragments of thick-walled, reddish brown endosperm, cells with pitted walls and elongated fragments of grey embryo may be present. Swelling power. Transfer 1 g to a 100-ml stoppered cylinder containing 90 ml of water, shake well for 30 seconds and allow to stand 24 hours, shaking gently on three occasions during this period. Add sufficient water to produce 100 ml, mix gently for 30 seconds, avoiding the entrapment of air, allow to stand for 5 hours and measure the volume of mucilage. Repeat the determination three times. The average of four determinations is not less than 40 ml. Ash (2.3.19). Not more than 4.5 per cent, determined on 1 g. Acid-insoluble ash (2.3.19). Not more than 0.45 per cent. Loss on drying (2.4.19). Not more than 12.0 per cent, determined on 0.5 g by drying in an oven at 105 for 5 hours. Storage. Store protected from moisture and from attack by insects and rodents.
Identification
A. Macroscopic Mixture of crisp, dark green-coloured broken leaves and quadrangular stems; leaves brittle. Stem fracture short, fibrous. B. Microscopic Stems quadrangular with collenchyma strands at angles and on side; small acicular crystals of calcium oxalate present in pith and cortex. Trichomes 1-3 celled, glandular hair disc-shaped and multicellular. C. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 7 volumes of chloroform and 1 volume of methanol. Test solution. Reflux 1 g of coarsely powdered substance under examination with 50 ml methanol for 15 minutes, cool and filter. Reflux the residue further with 2 50 ml of methanol, cool and filter. Combine all the filtrates and concentrate to 10 ml. Reference solution. Reflux 0.5 g of kalmegh RS with 50 ml methanol for 15 minutes, cool and filter. Reflux the residue further with 2 50 ml of methanol, cool and filter. Combine all the filtrates and concentrate to 5 ml. Apply to the plate 10 l of each solution as bands 10 mm by 2 mm. Allow the mobile phase to rise 8 cms. Dry the plate in air and spray with methanolic sulphuric acid (20 per cent). Heat at 120 for 5-10 minutes and examine the plate in day light. The chromatographic profile of the test solution is similar to that of the reference solution.
Kalmegh
Andrographis
Tests
Foreign organic matter (2.6.1). Not more than 2.0 per cent. Ethanol-soluble extractive (2.6.2). Not less than 3.0 per cent. Water-soluble extractive (2.6.3). Not less than 12.0 per cent by Method I. Ash (2.3.19). Not more than 15 per cent. Acid-insoluble ash (2.3.19). Not more than 3.0 per cent. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Loss on drying (2.4.19). Not more than 12.0 per cent, determined on 5 g by drying in an oven at 1050. Kalmegh consists of the dried aerial parts, mainly stems and leaves, of Andrographis paniculata Nees. (Fam. Acanthaceae). Kalmegh contains not less than 1.0 per cent of andrographolide, calculated on the dried basis. Description.Taste, intensely bitter. Microbial contamination (2.2.9). Complies with the microbial contamination tests. Assay. Determine by liquid chromatography (2.4.14). Test solution. Reflux about 2.5 g of the coarsely powdered substance under examination with 50 ml of methanol on a water bath for 15 minutes, cool and filter. Reflux the residue
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IP 2007
KUNDURU
further with methanol till the last extract turns colorless, cool and filter. Combine all the filtrates and concentrate to 50.0 ml. Reference solution. A 0.01 per cent w/v solution of andrographolide RS in methanol. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5 m), mobile phase: 65 volumes of methanol and 35 volumes of water, flow rate. 1 ml per minute, spectrophotometer set at 223 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the relative standard deviation for the replicate injections is not more than 2.0 per cent. Inject the test solution and reference solution. Calculate the content of andrographolide. Storage. Store protected from heat, moisture and against attack by insects and rodents.
aromatic. B. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF 254. Mobile phase. A mixture of 7 volumes of hexane and 3 volumes of ethyl acetate. Test solution. Reflux 1 g of coarsely powdered substance under examination with 50 ml methanol on a boiling water-bath for 30 minutes, cool and filter. Evaporate the filtrate to dryness and dissolve the residue in 5 ml of methanol. Reference solution. Reflux 1 g of coarsely powdered kunduru RS with 50 ml methanol on a boiling water-bath for 30 minutes, cool and filter. Evaporate the filtrate to dryness and dissolve the residue in 5 ml of methanol. Apply to the plate 10l of each solution as bands 10 mm by 2 mm. Allow the mobile phase to rise 8 cms above the line of application. Dry the plate in air and spray with methanolic sulphuric acid (10 per cent) and heat at 110 for 5-10 minutes and examine the plate in day light. The chromatographic profile of the test solution is similar to that of the reference solution.
Tests
Kunduru
Sallaki Gum; Gum of Boswellia Serrata
Foreign organic matter (2.6.1). Not more than 2.0 per cent. Ethanol-soluble extractive (2.6.2). Not less than 35 per cent. Ash (2.3.19). Not more than 10.0 per cent. Acid-insoluble ash (2.3.19). Not more than 2.0 per cent. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Water (2.3.43). Not more than 12.0 per cent, determined on 0.2 g. Microbial contamination (2.2.9). Complies with the microbial contamination tests. Assay. Determine by liquid chromatography (2.4.14). Test solution. Reflux about 2 g of the coarsely powdered substance under examination with 50 ml of methanol on a water bath for 15 minutes, cool and filter. Reflux the residue further with methanol till the last extract turns colorless, cool and filter. Combine all the filtrates and concentrate to 100.0 ml. Reference solution (a). A 0.01 per cent w/v solution of 11-keto--boswellic acid RS in methanol. Reference solution (b). A 0.05 per cent w/v solution of acetyl-11-keto--boswellic acid RS in methanol. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5 m), mobile phase: 90 volumes of methanol and 10 volumes of a mixture containing 5 ml of acetonitrile and 95 ml of
Kunduru is the gum-resin from Boswellia serrata Roxb. (Fam. Burseraceae). Kunduru contains not less than 1.0 per cent of total 11-keto-boswellic acid and acetyl-11-keto--boswellic acid, calculated on the dried basis. Description. Translucent, brittle, whitish yellow substance, in roundish, club-shaped, pear-shaped, or irregular tears.
Identification
A. Macroscopic Fracture dull. Slightly sticky to touch; odour, balsamic; taste slightly mucilaginous, bitter and
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KUTKI
IP 2007
water, adjusting the pH to 2.8 with dilute orthophosphoric acid, flow rate. 1.5 ml per minute, spectrophotometer set at 247 nm, a 20 l loop injector. Inject the reference solution (a) and (b). The test is not valid unless the relative retention times are about 0.65 for 11-keto-boswellic acid and 1.0 for acetyl-11-keto--boswellic acid and the relative standard deviation for the replicate injections is not more than 2.0 per cent. Inject the test solution. Calculate the sum of the contents of 11-keto--boswellic acid and acetyl-11-keto--boswellic acid. Storage. Store protected from heat, moisture and against attack by insects and rodents.
C. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 7.5 volumes of ethyl acetate, 2.2 volumes of methanol and 0.1 volume glacial acetic acid. Test solution. Reflux 5 g of coarsely powdered substance under examination with 50 ml of methanol for 15 minutes, cool and filter. Reflux the residue further with 50 ml of methanol, cool and filter. Combine both the filtrates and concentrate under vacuum to dryness. Extract the dried residue with 10 ml of methanol at 50 for 10 minutes, filter the solution and use the filtrate for analysis. Reference solution. Reflux 5 g of kutki RS with 50 ml of methanol for 15 minutes, cool and filter. Reflux the residue further with 50 ml of methanol, cool and filter. Combine both the filtrates and concentrate under vacuum to dryness. Extract the dried residue with 10 ml of methanol at 50 for 10 minutes, filter the solution and use the filtrate for analysis. Apply to the plate 20 l of each solution as bands 10 mm by 2 mm. Allow the mobile phase to rise 8 cm. Dry the plate in air, spray with anisaldehyde sulphuric acid. The chromatographic profile of the test solution is similar to that of the reference solution.
Kutki
Picrorhiza
Tests
Foreign organic matter (2.6.1). Not more than 2 per cent. Ethanol-soluble extractive (2.6.2). Not less than 15 per cent. Water-soluble extractive (2.6.3). Not less than 25 per cent by method I. Ash (2.3.19). Not more than 6 per cent. Acid-insoluble ash (2.3.19). Not more than 1 per cent. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Kutki consists of dried roots of Picrorhiza kurroa Royle ex Benth.(Fam. Scrophulariaceae) Kutki contains not less than 5 per cent of kutkin, calculated on the dried basis. Description. Rhizomes are sub cylindrical, straight or slightly curved, externally greyish with wrinkled surfaces, circular scars of roots and bud scales, with cork exposed at places. Loss on drying (2.4.19). Not more than 5 per cent, determined on 5 g by drying in an oven at 105. Microbial contamination (2.2.9). Complies with the microbial contamination tests. Assay. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 100 mg of the substance under examination in 25.0 ml of methanol, filter. Reference solution. A 0.1 per cent w/v solution of kutkin RS in methanol. Chromatographic system a stainless steel column 15 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5 m), mobile phase: 83 volumes of 1 per cent v/v orthophosphoric acid in water and 17 volumes of acetonitrile,
Identification
A. Macroscopic 3-6 cm long and about 1 cm thick, subcylindrical, straight, grayish brown, wrinkled roots. Odour is pleasant and tastes bitter. B. Microscopic There is well-developed periderm. About 7-10 layers of cork cells are seen. Cells of phelloderm loosely arranged.
1410
IP 2007
LASUNA
flow rate. 1 ml per minute, spectrophotometer set at 280 nm, a 20 l loop injector. Inject the reference solution. The relative standard deviation for the replicate injections is not more than 2.0 per cent. Inject the test solution and reference solution. Calculate the content of kutkin. Storage. Store protected from heat, moisture and against attack by insects and rodents.
B. Microscopic The protective leaf contains an epidermis enclosing a mesophyll free from chlorophyll. The outer epidermis consists of lignified sclereid cells of thick, pitted walls, elongated, covered with thin cuticle. The cortical cells are thick walled, non lignified, tending to collapse on maturity, isodiametric and contain purple pigments. The vascular bundles consist of lignified spiral and annular vessels. The storage leaves show an outer epidermis of thin, delicate cells of variable shape. Stomata are present on the outer epidermis only at extreme tip near the base of the foliage leaves.The mesophyll consists of swollen storage parenchyma cells filled with fine granular reserve material. C. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel F254. Mobile phase. A mixture of 3 volumes of butyl alcohol, 1 volume of n-propyl alcohol, 1 volume of glacial acetic acid and 1 volume of water. Test solution. Reflux 1 g of substance under examination with 20 ml of methanol (50 per cent) for 10 minutes, cool and filter. Reflux the residue with another 20 ml of methanol (50 per cent), cool and filter. Combine all the filtrates and concentrate under vacuum to 5 ml. Reference solution. Reflux 1 g of lasuna RS with 20 ml of methanol (50 per cent) for 10 minutes, cool and filter. Reflux the residue with another 20 ml of methanol (50 per cent), cool and filter. Combine all the filtrates and concentrate under vacuum to 5 ml.
Lasuna
Garlic, Allium sativum bulb
Lasuna consists of the fresh or dried compound bulbs of Allium sativum Linn. (Fam. Liliaceae). Lasuna contains not less than 0.2 per cent of alliin, calculated on the dried basis. Description. Bulbs made up of cloves and is wrapped in a white papery sheath with pungent taste and odour
Apply to the plate 10 l of each solution as bands 10 mm by 2 mm. Allow the mobile phase to rise 8 cm. Dry the plate in air, spray with 0.2 per cent w/v solution of ninhydrin in mixture of 95 volumes of butyl alcohol and 5 volumes of 2 M acetic acid. Heat at 100 to 105 for about 10 minutes and examine the plate in day light. The chromatographic profile of the test solution is similar to that of the reference solution. D. Transfer about 10 g of garlic bulbs that have been cut into small pieces to a beaker, add 10 ml of 1 M sodium hydroxide and 10 ml of water, heat the beaker in a boiling water for 10 minutes, cool and filter. To 2 ml of this filtrate add few drops of freshly prepared solution of sodium nitroprusside. Appearance of a red or orange red colour indicates the presence of sulphur containing compounds in the sample.
Identification
A. Macroscopic Each bulb has several cloves which are arranged in concentric rings and enclosed in a shining white or pinkish papery envelope. The cloves are attached to a flat, circular hard disc with numerous thin wiry roots from its underside and short, cylindrical out growth from the upper surface. Each clove is ovoid and further covered by papery sheath with a tail like structure at one and opposite to its attachment. The cloves in the outer ring are loose and white in colour where as cloves in the inner ring are adherent and pale pinkish in colour. Each clove covered with a white scale leaf and a pinkish white epidermis, easily separated from the solid portion. In the middle of the bulb a hollow, cylindrical, linear remnant of the scape is seen.
Tests
Foreign organic matter (2.6.1). Not more than 2.0 per cent. Ash (2.3.19). Not more than 5 per cent. Acid-insoluble ash (2.3.19). Not more than 1.0 per cent. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm).
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MALT EXTRACT
IP 2007
Loss on drying (2.4.19). Not more than 65.0 per cent for fresh bulbs, determined on 5 g by drying in an oven at 105. Microbial contamination (2.2.9). Complies with the microbial contamination tests. Assay. Determine by liquid chromatography (2.4.14). Test solution. Weigh accurately about 2 g of the freshly peeled substance under examination. Add 15 ml of hot water and grind in a porcelain mortor and filter. Grind the residue further with 2 15 ml of hot water and filter. Combine all the filtrates and make up to volume 50 ml with distilled water. Reference solution. A 0.004 per cent w/v solution of alliin RS in water. Chromatographic system a stainless steel column 25 cm 4.6 mm packed with octadecylsilane bonded to porous silica (5 m), mobile phase: 0.1 per cent v/v phosphoric acid prepared by diluting 1ml of phosphoric acid to 1000 ml with water, flow rate. 0.5 ml per minute, spectrophotometer set at 210 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the relative standard deviation for the replicate injections is not more than 2.0 per cent. Inject the test and reference solution. Calculate the content of alliin. Storage. Store protected from heat, moisture and against attack by insects and rodents.
Lipase. To 95 ml of water add 6.5 ml of triacetin and 0.2 ml of a 0.1 per cent w/v solution of bromocresol purple, neutralise with 0.5 M sodium hydroxide and add sufficient water to produce 110 ml. Place 50 ml of this solution in each of two large test-tubes (20 cm 3 cm) A and B kept in a water-bath at 30 1. Insert in each tube a rubber stopper having two holes, one for the tip of a burette and the other for a short glass tube through which passes a thread operating a glass stirring coil. Stir the contents of the tube until they attain the temperature of the water-bath. Prepare a solution of 5.0 g of the substance under examination in 10 ml of water. To tube A add 1 ml of this solution; to tube B add 1 ml of this solution after previous boiling. Adjust and maintain the pH of the two tubes to between 6.2 and 6.4 by the dropwise addition of 0.05 M sodium hydroxide, stirring frequently. After 6 hours, the difference between the amounts of 0.05 M sodium hydroxide added to the tubes is not more than 1.0 ml. Assay. Weigh accurately about 5.0 g into a 200-ml long-necked flask and carry out the determination of nitrogen, Method A (2.3.30), using 0.05 M sulphuric acid instead of 0.1 M sulphuric acid. 1 ml of 0.05 M sulphuric acid is equivalent to 0.001401 g of N and multiply the result by 6.25 to obtain the protein content. Storage. Store protected from moisture.
Mandukaparni
Centella, Gotu Kola
Malt Extract
Malt Extract is a product obtained by extracting malted grains of cereals (barley, cholam or wheat) with water at a suitable temperature and evaporation of the strained liquid until a viscous product is obtained. It may be mixed with 10 per cent by weight of Glycerin. Malt Extract contains nitrogen equivalent to not less than 4.0 per cent w/w of protein. Description. A sweet, viscous, light brown liquid; odour, pleasant and characteristic.
Tests
Refractive index (2.4.27). 1.489 to 1.498, determined at 20. Arsenic (2.3.10). Dissolve 10.0 g in 10 ml of water, add 10 ml of brominated hydrochloric acid, allow to stand for 5 minutes and remove the excess of bromine with a few drops of stannous chloride solution AsT. The resulting solution complies with the limit test for arsenic (1 ppm). Mandukaparni consists of the dried aerial parts of Centella asiatica (Linn.) Urban. (Fam. Umbelliferae). Mandukaparni contains not less than 0.5 per cent of asiaticoside, calculated on the dried basis. Description. A green to greenish yellow in colour, taste, slightly bitter and sweet.
1412
IP 2007
MANJISTHA
Identification
A. Macroscopic A slender trailing herb with rooted nodes and internodes. Leaves with elongated petioles and sheathing leaf bases; lamina reniform with crenate margin. B. Microscopic Animocytic stomata on both surfaces, more on lower surface; petiole epidermis has calcium oxalate prisms; vascular bundles seven arranged in U shape, parenchyma cells contain simple and compound starch granules. C. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G.. Mobile phase. A mixture of 60 volumes of chloroform, 32 volumes of glacial acetic acid, 12 volumes of methanol and 8 volumes of water. Test solution. Reflux 2 g of coarsely powdered substance under examination with 50 ml methanol for 15 minutes, cool and filter. Reflux the residue further with 2 50 ml of methanol, cool and filter. Combine all the filtrates and concentrate to 10 ml under reduced pressure. Reference solution. Reflux 1 g of mandukaparni RS with 50 ml methanol for 15 minutes, cool and filter. Reflux the residue further with 2 50 ml of methanol, cool and filter. Combine all the filtrates and concentrate to 5 ml. Apply to the plate 10 l of each solution as bands 10 mm by 2 mm. Allow the mobile phase to rise 8 cms above the line of application. Dry the plate in air, spray with anisaldehyde solution. Heat at 100 for 5-10 minutes and examine the plate in day light. The chromatographic profile of the test solution is similar to that of the reference solution.
water bath for 15 minutes, cool and filter. Reflux the residue further with methanol till the last extract turns colorless, cool and filter. Combine all the filtrates and concentrate to 100.0 ml. Reference solution. A 0.1 per cent w/v solution of asiaticoside RS in methanol. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with octylsilane bonded to porous silica (10 m), mobile phase: 25 volumes of acetonitrile and 75 volumes of water, flow rate. 1.5 ml per minute, spectrophotometer set at 210 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the relative standard deviation for the replicate injections is not more than 2.0 per cent. Inject the test solution and reference solution. Calculate the content of asiaticoside. Storage. Store protected from heat, moisture and against attack by insects and rodents.
Manjistha
Indian Madder; Rubia Cordifolia
Tests
Foreign organic matter (2.6.1). Not more than 2.0 per cent. Ethanol-soluble extractive (2.6.2). Not less than 6.0 per cent. Water-soluble extractive (2.6.3). Not less than 15.0 per cent by Method I. Ash (2.3.19). Not more than 24 per cent. Acid-insoluble ash (2.3.19). Not more than 5.0 per cent. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Loss on drying (2.4.19). Not more than 12.0 per cent, determined on 5 g by drying in an oven at 1050. Microbial contamination (2.2.9). Complies with the microbial contamination tests. Assay. Determine by liquid chromatography (2.4.14). Test solution. Reflux about 3 g of the coarsely powdered substance under examination with 50 ml of methanol on a Manjistha consists of dried stem of Rubia cordifolia Linn.sensu Hook.f. (Fam. Rubiaceae). Manjistha contains not less than 0.02 per cent of rubiadin, calculated on the dried basis. Description. A cylindrical, slightly flattened, wiry pieces of brown to purple coloured root with mild bitter taste.
Identification
A. Microscopic Exfoliating cork, consisting of 4-12 or more layered radially arranged, thin walled cells.
1413
MARICHA
IP 2007
B. Macroscopic Stem, slender, cylindrical, wiry and about 0.5 cm thick. It is brown to purple coloured and with longitudinal cracks. C. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G.. Mobile phase. A mixture of 7 volumes of toluene, 25 volumes of ethyl acetate and 0.5 volumes glacial acetic acid. Test solution. To 4 g of coarsely powdered substance under examination, add 100 ml of methanol, reflux for 15 minutes, cool and filter. Reflux the residue further for two times with 50 ml methanol, cool and filter. Combine all the filtrates and concentrate under vacuum to 100 ml. Reference solution. To 1 g of coarsely powdered manjistha, add 100 ml of methanol, reflux for 15 minutes, cool and filter. Reflux the residue further for two times with 50 ml methanol, cool and filter. Combine all the filtrates and concentrate under vacuum to 25 ml. Apply to the plate 20 l of each solution as bands 10 mm by 2 mm. Allow the mobile phase to rise 8 cm. Dry the plate in air and examine in the 254 nm, 366 nm and spray the plate with anisaldehyde sulphuric acid. The chromatographic profile of the test solution is similar to that of the reference solution.
octadecylsilane bonded to porous silica (5 m), mobile phase: filtered and degassed gradient mixtures of acetonitrile and a buffer solution pH 2.5 prepared by dissolving 0.136 g of potassium di-hydrogen orthophosphate in 900 ml of water, add 0.5 ml of orthophosphoric acid and dilute to 1000 ml with water, flow rate. 1.5 ml per minute, spectrophotometer set at 278 nm, a 20 l loop injector. Time Buffer solution Acetonitrile (in min.) (per cent v/v) ( per cent v/v) 0 65 35 5 50 50 15 20 80 30 20 80 35 50 50 40 65 35 Inject the reference solution. The relative standard deviation for the replicate injections is not more than 2.0 per cent. Inject the test solution and reference solution. Calculate the content of rubiadin. Storage. Store protected from heat, moisture and against attack by insects and rodents.
Tests
Foreign organic matter (2.6.1). Not more than 2 per cent. Ethanol-soluble extractive (2.6.2). Not less than 4 per cent. Water-soluble extractive (2.6.3). Not less than 20 per cent by method I. Ash (2.3.19). Not more than 10 per cent. Acid-insoluble ash (2.3.19). Not more than 0.5 per cent. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Loss on drying (2.4.19). Not more than 5 per cent, determined on 5 g by drying in an oven at 105. Microbial contamination (2.2.9). Complies with the microbial contamination tests. Assay. Determine by liquid chromatography (2.4.14). Test solution. Weigh 4 g of coarsely powdered substance under examination, add 100 ml of methanol, reflux on a waterbath 15 minutes, cool and filter. Reflux the residue 2 x 50 ml methanol, cool and filter. Combine all the filtrates and concentrate under vacuum to 100.0 ml. Reference solution. A 0.002 per cent w/v solution of rubiadin RS in methanol. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with
Maricha
Black pepper; pepper; Piper nigrum
Maricha consists of the unripe fruits of Piper nigrum Linn. (Fam. Piperaceae). Maricha contains not less than 2.5 per cent w/w of piperine, calculated on the dried basis. Description. Fruits are globular or oblong. They have a blackish brown cover, with raised reticulated wrinkles. The odor is aromatic and the taste is strong and pungent.
1414
IP 2007
MENTHA OIL
Identification
A. Macroscopic The fruits are globular or oblong, 4-6 mm in diameter. The outer cover is blackish brown, with raised reticulated wrinkles. One seeded, seeds are white and hollow. B. Microscopic The fruit has a well differentiated pericarp, testa and perisperm. Isolated, tangentially elongated oil cells are in the outer region of the mesocarp. Endocarp has beaker shaped stone cells and numerous polyhedral masses of starch grains. Testa has a single layer of yellow colored cells. C. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 60 volumes of benzene, 30 volumes of ethyl acetate and 10 volumes of diethyl ether. Test solution. To 2 g of the coarsely powdered substance under examination, add 50 ml of methanol and reflux for 15 minutes, cool and filter. Reflux the residue further for two times with 75 ml of methanol, cool and filter. Combine all the filtrates and concentrate under vacuum to 50 ml. Reference solution. To 2 g of the maricha RS, add 50 ml of methanol and reflux for 15 minutes, cool and filter. Reflux the residue further for two times with 75 ml of methanol, cool and filter. Combine all the filtrates and concentrate under vacuum to 50 ml. Apply to the plate 10 l of each solution as bands 10 mm by 2 mm. Allow the mobile phase to rise 8 cms. Dry the plate in air and examine in ultraviolet light at 254 nm. The chromatogram obtained with test solution corresponds to the band in the chromatogram obtained with reference solution. Spray with vanilin sulphuric acid reagent. Heat at 100 for 5-10 minutes and examine the plate in day light. The chromatographic profile of the test solution is similar to that of the reference solution.
Test solution. Weigh 2 g of coarsely powdered substance under examination, add 50 ml of methanol, sonicate for 3 minutes and heat on a boiling water bath for 15 minutes, cool and dilute to 100.0 ml with methanol and filter. Dilute further if necessary. Reference solution. A 0.01 per cent w/v solution of piperine RS in methanol. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5 m), mobile phase. filtered and degassed gradient mixtures of acetonitrile and a buffer solution pH 2.5 prepared by dissolving 0.136 g of potassium di-hydrogen orthophosphate in 500 ml of water, add 0.5 ml of orthophosphoric acid and dilute to 1000 ml with water, flow rate. 1.5 ml per minute, spectrophotometer set at 345 nm, a 20 l loop injector. Time Buffer solution. Acetonitrile (in min.) (per cent v/v) (per centv/v) 0 95 5 18 55 45 25 20 80 Inject the reference solution. The relative standard deviation for the replicate injections is not more than 2.0 per cent. Inject the test solution and reference solution. The relative retention time of piperine is 1. Calculate the content of piperine. Storage. Store protected from moisture and against attack by insects and rodents.
Tests
Foreign organic matter (2.6.1). Not more than 2.0 per cent. Ethanol-soluble extractive (2.6.2). Not less than 6.0 per cent. Water-soluble extractive (2.6.3). Not less than 6.0 per cent by method I. Ash (2.3.19). Not more than 7.0 per cent. Acid-insoluble ash (2.3.19). Not more than 2.0 per cent. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Loss on drying (2.4.19). Not more than 12.0 per cent, determined on 5 g by drying in an oven at 1050. Microbial contamination (2.2.9). Complies with the microbial contamination tests. Assay. Determine by liquid chromatography (2.4.14).
Mentha Oil
Mentha
Mentha Oil is the volatile oil distilled with steam from various species of Mentha (Fam. Labiatae) and rectified if necessary. Mentha Oil contains not less than 50.0 per cent w/w of total menthol, C10H20O. Description. A colourless or yellowish, clear liquid; odour, characteristic and pleasant.
Tests
Acidity or alkalinity. A solution of 1 ml in 3.5 ml of ethanol (70 per cent) is neutral to litmus. Weight per ml (2.4.29). 0.892 g to 0.910 g. Specific optical rotation (2.4.22). 18.0 to 33.0.
1415
OPIUM
IP 2007
Assay. Place about 10.0 g in an acetylation flask, add 10 ml of acetic anhydride and 1 g of anhydrous sodium acetate, attach a reflux condenser and boil for 2 hours. Cool, add 30 ml of water and warm on a water-bath for 15 minutes with occasional shaking. Transfer the contents of the flask to a separating funnel, reject the water layer and wash the remaining oil with water until the last washing no longer shows acid reaction. Dry the resulting oil by shaking with 2 g of anhydrous sodium sulphate, allow to stand for 30 minutes and filter through a dry filter paper. Weigh accurately about 1.5 g of the dry acetylated oil, add 3 ml of ethanol (95 per cent) and 0.1 ml of phenolphthalein solution and dropwise, 0.5 M ethanolic potassium hydroxide until the solution acquires a faint pink colour. Add a further 20.0 ml of the alkali, attach a reflux condenser and boil for 1 hour on a water-bath. Cool, add 1 ml of phenolphthalein solution and titrate the excess of alkali with 0.5 M hydrochloric acid. Repeat the operation with the same quantities of the same reagents in the same manner without the oil and calculate the amount of total menthol from the following expression.
Opium contains not less than 10.0 per cent of morphine, C17H 19NO3, and not less than 2.0 per cent of codeine, C18H21NO3, both calculated on the dried basis. Description. Masses of various sizes which tend to be soft and shiny and, after drying, hard and brittle; usually in somewhat irregularly shaped masses (natural opium) or moulded into masses of more uniform size and shape (manipulated opium); colour, blackish brown; odour, strong and characteristic.
Identification
Strip off any covering, cut the substance under examination into thin slices, if necessary, dry at about 60 for 48 hours and reduce to a powder. A. When examined under a microscope, a suspension in a 2 per cent w/v solution of potassium hydroxide appears as granules of latex agglomerated in irregular masses and light brown elongated filaments. Some fragments of vessels and rather elongated, refringent crystals are also visible, as well as a smaller number of round pollen grains and fragments of elongated fibres. Hairs of various lengths with sharp points and a few grains of starch introduced during the handling of the latex may be present. Fragments of epicarp consisting of polygonal cells with thick walls defining a stellate lumen may also be present. B. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A freshly prepared mixture of 20 volumes of acetone, 20 volumes of toluene, 3 volumes of ethanol (95 per cent) and 1 volume of strong ammonia solution. Test solution. Triturate 0.1 g of the powdered substance with 5 ml of ethanol (70 per cent), add 3 ml of ethanol (70 per cent), transfer to a 25-ml conical flask and heat in a water-bath at 50 to 60 for 30 minutes, with stirring. Cool, filter, wash the filter with ethanol (70 per cent) and dilute the filtrate to 10 ml with the same solvent. Reference solution. Dissolve 2 mg of papaverine hydrochloride RS, 12 mg of codeine phosphate RS, 12 mg of noscapine hydrochloride RS, and 25 mg of morphine hydrochloride RS in ethanol (70 per cent) and dilute to 25 ml with the same solvent. Apply to the plate 20 l of each solution as 20 mm bands. After development, dry the plate at 100 to 105 for 15 minutes, allow to cool and spray with potassium iodobismuthate solution and then with a 0.4 per cent w/v solution of sulphuric acid. The chromatogram obtained with the reference solution shows in the lower part an orange-red or red band (morphine), above it a similarly coloured band (codeine) and in the upper part an orange-red or red band (papaverine) and above it a similarly coloured band (noscapine). The chromatogram
where, S is the amount, in g, of the acetylated sample taken, a is the amount, in ml, of 0.5 M hydrochloric acid consumed in the blank test, and b is the amount, in ml, of 0.5 M hydrochloric acid consumed in saponification of the acetylated oil. Storage. Store protected from light and moisture.
Opium
Raw Opium; Papaver
Opium is the air-dried latex obtained by incision from the unripe capsules of Papaver somniferum Linn.
1416
IP 2007
OPIUM POWDER
obtained with the test solution shows orange-red or red bands corresponding to those in the chromatogram obtained with the reference solution. The chromatogram obtained with the test solution may also show a dark red band (thebaine) situated between those due to codeine and to papaverine. C. To 1 g of the powdered substance add 5 ml of water, shake for 5 minutes, filter and add to the filtrate 0.25 ml of ferric chloride solution; a red colour develops which does not disappear on the addition of 0.5 ml of 2 M hydrochloric acid.
octylsilane bonded to porous silica (5 m), fitted with a guard column 4 cm x 4.6 mm packed with octylsilane bonded to porous silica (5 m), mobile phase: 1.0 g of sodium heptanesulphonate in 420 ml of water, adjusting to pH 3.2 with phosphoric acid that has been diluted to contain 0.49 per cent w/v solution of H3PO4 (about 5 ml) and adding 180 ml of acetonitrile, flow rate. 1.5 ml per minute, spectrophotometer set at 280 nm. Inject suitable volumes of each solution. The assay is not valid unless the resolution between the peaks corresponding to morphine and codeine is at least 2.5. If necessary, adjust the volume of acetonitrile in the mobile phase. Inject the reference solution six times. The assay is not valid unless the relative standard deviation of the peak area for morphine is not more than 1.0 per cent. Inject the test solution and the reference solution. Calculate the percentage content of each alkaloid from the expression
Tests
Thebaine. Not more than 3 per cent, calculated on the dried basis. Determine by liquid chromatography (2.4.14). Test solution. Use the test solution prepared in the assay. Reference solution. Dissolve 25 mg of thebaine in sufficient mobile phase to produce 25.0 ml and dilute 10.0 ml of this solution to 100.0 ml with the mobile phase. Chromatographic system as described in the Assay. The test is not valid unless the capacity factor for thebaine is at least 3.0 and the number of theoretical plates is at least 3000. Calculate the percentage content of thebaine from the expression given in the assay. Ash (2.3.19). Not more than 6.0 per cent, determined on 0.5 g. w 1 A 2 625 100 Loss w 2 A1 5(100 h)on drying (2.4.19). Not more than 15.0 per cent, determined on 0.25 g cut into thin slices, by drying in an oven at 105 for 4 hours. Assay. Determine by liquid chromatography (2.4.14). Test solution. Suspend about 1.0 g, accurately weighed substance under examination, cut into thin slices, in 50 ml of ethanol (50 per cent), mix with the aid of ultrasound for 1 hour, allow to cool, dilute to 100.0 ml with the same solvent and allow to stand. To 10.0 ml of the supernatant liquid add 5 ml of ammonia buffer pH 9.5, dilute to 25.0 ml with water, mix, transfer 20.0 ml of the solution to a column (about 15 cm 30 mm) containing 15 g of kieselguhr for column chromatography and allow to stand for 15 minutes. Elute with two quantities, each of 40 ml, of a mixture of 85 volumes of dichloromethane and 15 volumes of 2-propanol, evaporate the eluate to dryness at 40 at a pressure of 2 kPa, transfer the residue to a 25-ml volumetric flask with the aid of the mobile phase and dilute to volume with the same solvent. Reference solution. Weigh accurately 0.1 g of morphine hydrochloride and 25 mg of codeine, dissolve in sufficient of the mobile phase to produce 25.0 ml and dilute 10.0 ml of this solution to 100.0 ml with the same solvent. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with
where, w1 = weight, in g, of the alkaloid used to prepare the reference solution, w2 = weight, in g, of the substance under examination used to prepare the test solution, A 1 = area of the peak corresponding to the alkaloid in the chromatogram obtained with the reference solution, A 2 = area of the peak corresponding to the alkaloid in the chromatogram obtained with the test solution, h = percentage loss on drying. For calculation, 1 mg of morphine hydrochloride may be taken as equivalent to 0.759 mg of morphine and 1 mg of codeine phosphate may be taken as equivalent to 0.943 mg of codeine. Storage. Store protected from light and moisture.
Opium Powder
Opium Powder is Opium dried at a temperature not exceeding 70, reduced to a fine or moderately fine powder and adjusted by the addition of Lactose, suitably coloured with Caramel, or other suitable diluent to contain about 10 per cent of morphine and 2 per cent of codeine. Opium Powder contains not less than 9.5 per cent and not more than 10.5 per cent of morphine, C17H19NO3, and not less
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OPIUM POWDER
IP 2007
than 1.9 per cent and not more than 2.1 per cent of codeine, C18H21NO3, both calculated on the dried basis. Description. A light brown powder consisting of yellowish brown or brownish red particles; odour, characteristic.
Identification
A. When examined under a microscope, the residue obtained after extraction with water, appears as granules of latex agglomerated in irregular masses and light brown elongated filaments. Some fragments of vessels and rather elongated, refringent crystals are also visible, as well as a smaller number of round pollen grains and fragments of elongated fibres. Hairs of various lengths with sharp points and a few grains of starch introduced during the handling of the latex may be present. Fragments of epicarp consisting of polygonal cells with thick walls defining a stellate lumen may also be present and if powdered cocoa husk is present, the following: brown colour of the fragments; narrow spiral vessels about 10 to 20 mm wide, in groups of from one to six, traversing a spongy parenchyma of thin-walled cells about 40 to 60 mm in either direction and united by arm-like projections enclosing almost circular intercellular spaces; fragments of the sclerenchymatous layer, consisting of thick-walled lignified brown, rectangular to polyhedral cells in a single layer, individual cells about 5 to 10 m wide and 10 to 30 m long; fragments of mucilage staining in ruthenium red solution. B. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G.. Mobile phase. A mixture of 20 volumes of acetone, 20 volumes of toluene, 3 volumes of ethanol (95 per cent) and 1 volume of strong ammonia solution. Test solution. Triturate 0.10 g of the powdered substance with 5 ml of ethanol (70 per cent), add 3 ml of ethanol (70 per cent), transfer to a 25-ml conical flask and heat in a water-bath at 50 to 60 for 30 minutes, with stirring. Cool, filter, wash the filter with ethanol (70 per cent) and dilute the filtrate to 10 ml with the same solvent. Reference solution. Dissolve 2 mg of papaverine hydrochloride RS, 12 mg of codeine phosphate RS, 12 mg of noscapine hydrochloride RS, and 25 mg of morphine hydrochloride RS in ethanol (70 per cent) and dilute to 25 ml with the same solvent. Apply to the plate 20 l of each solution as 20 mm bands. After development, dry the plate at 100 to 105 for 15 minutes, allow to cool and spray with potassium iodobismuthate solution and then with a 0.4 per cent w/v solution of sulphuric acid. The chromatogram obtained with the reference solution shows in the lower part an orange-red or red band (morphine), above it a similarly coloured band (codeine) and in the upper part an orange-red or red band (papaverine) and above it a
similarly coloured band (noscapine). The chromatogram obtained with the test solution shows orange-red or red bands corresponding to those in the chromatogram obtained with the reference solution. The chromatogram obtained with the test solution may also show a dark red band (thebaine) situated between those due to codeine and to papaverine. C. To 1 g of the powdered substance add 5 ml of water, shake for 5 minutes, filter and add to the filtrate 0.25 ml of ferric chloride solution; a red colour develops which does not disappear on the addition of 0.5 ml of 2 M hydrochloric acid.
Tests
Thebaine. Not more than 3 per cent, calculated on the dried basis. Determine by liquid chromatography (2.4.14). Test solution. Use the test solution prepared in the Assay. Reference solution. Dissolve 25 mg of thebaine in sufficient mobile phase to produce 25.0 ml and dilute 10.0 ml of this solution to 100.0 ml with the mobile phase. Chromatographic condition as described in the Assay. The test is not valid unless the capacity factor for thebaine is at least 3.0 and the number of theoretical plates is at least 3000. Calculate the percentage content of thebaine from the expression given in the Assay. Ash (2.3.19). Not more than 6.0 per cent, determined on 0.5 g. Loss on drying (2.4.19). Not more than 15.0 per cent, determined on 0.25 g by drying in an oven at 105 for 4 hours. Assay. Determine by liquid chromatography (2.4.14). Test solution. Suspend about 1.0 g, accurately weighed, of the substance under examination, cut into thin slices, in 50 ml of ethanol (50 per cent), mix with the aid of ultrasound for 1 hour, allow to cool, dilute to 100.0 ml with the same solvent and allow to stand. To 10.0 ml of the supernatant liquid add 5 ml of ammonia buffer pH 9.5, dilute to 25.0 ml with water, mix, transfer 20.0 ml of the solution to a column (about 15 cm 30 mm) containing 15 g of kieselguhr for column chromatography and allow to stand for 15 minutes. Elute with two quantities, each of 40 ml, of a mixture of 85 volumes of dichloromethane and 15 volumes of 2-propanol, evaporate the eluate to dryness at 40 at a pressure of 2 kPa, transfer the residue to a 25-ml volumetric flask with the aid of the mobile phase and dilute to volume with the same solvent. Reference solution. Weigh accurately 0.1 g of morphine hydrochloride RS and 25 mg of codeine phosphate RS, dissolve in sufficient of the mobile phase to produce 25.0 ml and dilute 10.0 ml of this solution to 100.0 ml with the same solvent.
1418
IP 2007
PEPPERMINT OIL
Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octylsilane bonded to porous silica (5 m), fitted with a guard column 4 cm x 4.6 mm packed with octylsilane bonded to porous silica (5 m), mobile phase: 1.0 g of sodium heptanesulphonate in 420 ml of water, adjusting to pH 3.2 with phosphoric acid that has been diluted to contain 0.49 per cent w/v solution of H3PO4 (about 5 ml) and adding 180 ml of acetonitrile, flow rate. 1.5 ml per minute, spectrophotometer set at 280 nm. Inject suitable volume of reference solution. The test is not valid unless the resolution between the peaks corresponding to morphine and codeine is at least 2.5. If necessary, adjust the volume of acetonitrile in the mobile phase. The relative standard deviation of the peak area for morphine is not more than 1.0 per cent. Inject the test solution and the reference solution. Calculate the percentage content of each alkaloid from the expression
w 1 A 2 625 100 w 2 A1 5(100 h)
Description. A white to light brown, amorphous or slightly granular powder; odour, characteristic.
Tests
Microbial contamination (2.2.9). 1.0 g is free from Escherichia coli and 10.0 g is free from salmonellae. Loss on drying (2.4.19). Not more than 7.0 per cent, determined on 1 g by drying in an oven at 60o at a pressure not exceeding 0.7 kPa for 4 hours. Assay. Weigh accurately about 0.5 g, triturate with 10 ml of cysteine hydrochloride solution and dilute to 100.0 ml with water. To 30 ml of water in each of two flasks add 15.0 ml of casein solution and maintain at 60o by heating on a waterbath. To the first flask add 5.0 ml of the solution of the substance under examination, and to the second flask add 5.0 ml of the same solution, previously boiled for 2 minutes and cooled. Maintain the solutions at 60o for 30 minutes, cool rapidly to room temperature and add to each flask 0.75 ml of phenolphthalein solution and 10 ml of formaldehyde solution, previously neutralised to phenolphthalein solution. Titrate both solutions with 0.1 M sodium hydroxide to the same definite pink colour; the difference between the two titrations is not less than 4.5 ml. Storage. Store protected from light and moisture. Labelling. The label states the name of any added substance.
where, w1 = weight, in g, of the alkaloid used to prepare the reference solution, w2 = weight, in g, of the substance under examination used to prepare the test solution, A 1 = area of the peak corresponding to the alkaloid in the chromatogram obtained with the reference solution, A 2 = area of the peak corresponding to the alkaloid in the chromatogram obtained with the test solution,h h = percentage loss on drying. For calculation, 1 mg of morphine hydrochloride may be taken as equivalent to 0.759 mg of morphine and 1 mg of codeine phosphate may be taken as equivalent to 0.943 mg of codeine. Storage. Store protected from light and moisture.
Peppermint Oil
Peppermint Oil is obtained by steam distillation from the aerial parts of the flowering plant of Mentha piperita L. and M. arvensis var. piperascens. Peppermint Oil contains not less than 4.5 per cent w/w and not more than 10.0 per cent w/w of esters, calculated as menthyl acetate, C12H22O2, not less than 44.0 per cent w/w of free alcohols, calculated as menthol, C10H20O, and not less than 15.0 per cent w/w and not more than 32.0 per cent w/w of ketones, calculated as menthone, C10H18O. Description. A colourless, pale yellow or pale greenish yellow liquid; odour, characteristic; taste, characteristic followed by sensation of cold.
Identification
Papain
Papain is an enzyme or a mixture of enzymes obtained from the juice of the unripe fruit of Carica papaya Linn. (Fam. Caricaceae). It may contain a suitable diluent such as Lactose. Papain contains not less than the minimum protease activity determined under the conditions of the Assay.
Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G F254. Mobile phase. A mixture of 95 volumes of toluene and 5 volumes of ethyl acetate. Test solution. Dissolve 1 g of the oil under examination in 100 ml of toluene.
1419
PEPPERMINT OIL
IP 2007
Reference solution. Dissolve 50 mg of ()-menthol RS, 20 l of cineole RS, 10 mg of thymol RS and 10 l of menthyl acetate RS in sufficient toluene to produce 10 ml. Apply to the plate 20 l of test solution and 10 l of reference solution as bands 20 mm by 3 mm. After development, dry the plate in air until the odour of solvent is no longer detectable and examine in ultraviolet light at 254 nm. In the chromatogram obtained with test solution there are no quenching bands at Rf values slightly lower than that of the faint band due to thymol in the chromatogram obtained with reference solution (carvone and pulegone). Spray the plate with anisaldehyde solution and examine in daylight after heating at 105o for 5 minutes. The chromatogram obtained with reference solution shows, in order of increasing Rf value, an intense blue to violet band (menthol) in the lower third, a violet-blue to brown band (cineole), a pink band (thymol) and a violet-blue band (menthyl acetate). The chromatogram obtained with test solution shows an intense band corresponding to menthol, a band corresponding to cineole, a violet-blue band corresponding to menthyl acetate in the middle of the chromatogram and at a slightly lower Rf value a greenish band (menthone). The chromatogram obtained with test solution does not show intense greyish green or faint bluish grey bands at Rf values between those of cineole and thymol in the chromatogram obtained with reference solution (carvone, pulegone, isomenthone). The chromatogram obtained with test solution shows an intense reddish violet band near the solvent front (hydrocarbons) and a brownish yellow band (menthofuran) at a slightly lower Rf value; other less intensely coloured bands may also be seen.
or a few pieces of porous pot and heat under a reflux condenser on a water-bath for 30 minutes. Add 1 ml of phenolphthalein solution and immediately titrate with 0.5 M hydrochloric acid. Repeat the operation without the substance under examination. The difference between the titrations represents the volume of alkali required to saponify the esters. 1 ml of 0.5 M ethanolic potassium hydroxide is equivalent to 0.09915 g of esters, calculated as menthyl acetate, C12H22O2 For free alcohols To 1 g in a dry, 150-ml acetylation flask, add 3 ml of a mixture of 3 volumes of pyridine and 1 volume of acetic anhydride. Determine the weight of the acetylation mixture to the nearest mg, keeping the flask closed while weighing. Boil under a reflux condenser in a water-bath for 3 hours, maintaining the water level 2 to 3 cm above the level of the liquid in the flask throughout. Remove the flask from the water-bath and add 50 ml of water through the condenser, remove the condenser and wash the walls of the flask with 10 ml of water. Allow to stand for 15 minutes and titrate with 0.5 M sodium hydroxide using 1 ml of phenolphthalein solution as indicator. Repeat the operation without the substance under examination. The difference between the titrations represents the volume of sodium hydroxide required. 1 ml of 0.5 M sodium hydroxide is equivalent to 0.07815 g of free alcohols, calculated as menthol, C10H20O. If the quantities of acetic anhydride in pyridine used in the two determinations differ by more than 5 mg, adjust the volume of alkali used in the second titration by multiplying with a/b where a is the weight, in g, of acetic anhydride in pyridine used in the first determination and b is the weight, in g, of acetic anhydride in pyridine used in the second test. For ketones To 2 g add 25 ml of a 5.0 per cent w/v solution of hydroxylamine hydrochloride in ethanol (95 per cent), heat on a water-bath for 1 hour, allow to cool, add about 1 mg of methyl orange and titrate with 0.5 M ethanolic potassium hydroxide until an orange-yellow colour is obtained. Repeat the heating for further periods of 1 hour until, after cooling, not more than 0.1 ml of 0.5 M ethanolic potassium hydroxide is required to neutralise the solution. 1 ml of 0.5 M ethanolic potassium hydroxide is equivalent to 0.07710 g of ketones, calculated as menthone, C10H18O. Storage. Store protected from light and moisture, in well-filled containers.
Tests
Acidity. To 2 g add 0.25 ml of phenolphthalein solution; not more than 0.1 ml of 0.5 M ethanolic potassium hydroxide is required to change the colour of the solution. Optical rotation (2.4.22). 10o to 30o. Refractive index (2.4.27). 1.460 to 1.467. Weight per ml (2.4.29). 0.900 g to 0.916 g. Dimethyl sulphide. Distil 25 ml, collect the first 1 ml of the distillate and carefully superimpose it on 5 ml of a 6.5 per cent w/v solution of mercuric chloride; no white film is produced within 1 minute at the interface of the two liquids. Fixed oils and resinified volatile oils. Allow 0.05 ml to fall on a filter paper. The oil evaporates completely within 24 hours without leaving a translucent or greasy mark. Assay. For esters To 2 g in a borosilicate glass flask add 2 ml of ethanol (90 per cent) and 0.25 ml of phenolphthalein solution, neutralise with 0.5 M ethanolic potassium hydroxide, add an additional 25.0 ml and a little pumice powder
1420
IP 2007
PIPPALI LARGE
Pippali, Large
Long pepper; Catkins (Big)
Reference solution. Reflux 0.4 g of the coarsely powdered pippali, Big RS with 50-75 ml of methanol for 15 minutes, cool and filter. Reflux the residue further for two times with 75 ml of methanol, cool and filter. Combine all the filtrates and concentrate under vacuum to 10 ml. Apply to the plate 10 l of each solution as bands 10 mm by 2 mm. Allow the mobile phase to rise 8 cm. Dry the plate in air and examine in ultra violet light at 254 nm. The chromatographic profile of the test solution corresponds to that in the chromatogram obtained with reference solution. Spray with solution of vanillin sulphuric acid reagent. Heat at 100 for 5-10 minutes and examine the plate in day light. The chromatographic profile of the test solution is similar to that of the reference solution.
Tests
Foreign organic matter (2.6.1). Not more than 2 per cent. Ethanol-soluble extractive (2.6.2). Not less than 8 per cent. Pippali, Large consists of the fruiting spikes of Piper longum Linn. (Syn. P. sarmentosum Wall., P. latifolium Hunter, Chavica roxburghii Miq., C. sarmentosa Miq.) (Fam. Piperaceae) Pippali, Large contains not less than 1 per cent w/w of piperine, calculated on the dried basis. Description. The spikes are blacking green to green in colour, cylindrical, erect and blunt. It has pungent taste and the odour is aromatic and characteristic. The spikes are 2 to 4 cm long and 0.4-0.7 cm in diameter. Water-soluble extractive (2.6.3). Not less than 10 per cent by Method I. Ash (2.3.19). Not more than 8 per cent. Acid-insoluble ash (2.3.19). Not more than 3 per cent. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Loss on drying (2.4.19). Not more than 12.0 per cent, determined on 5 g by drying in an oven at 1050. Microbial contamination (2.2.9). Complies with the microbial contamination tests. Assay. Determine by liquid chromatography (2.4.14). Test solution. Weigh 2 g of coarsely powdered substance under examination, add 50 ml of methanol, sonicate for 3 minutes and heat on a boiling water bath for 15 minutes, cool and dilute to 100.0 ml with methanol and filter. Dilute further if necessary. Reference solution. A 0.01 per cent w/v solution of piperine RS in methanol. Chromatographic system a stainless steel column 25 cm 4.6 mm packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a gradient mixture of acetonitrile and a buffer solution prepared by dissolving 0.136 g of potassium di-hydrogen orthophosphate in 900 ml of water, adjust the pH to 2.5 with orthophosphoric acid and dilute to 1000 ml with water, flow rate. 1.5 ml per minute, spectrophotometer set at 270 nm, a 20 l loop injector.
Identification
A. Macroscopic The spikes are blackish green to green in colour, surface rough. The spikes bear bracts and numerous small fruits sunk in solid spike. B. Microscopic Epidermis is a single layer of tangentially elongated cells. Cells are packed with abundant starch grains which are small. Crystals are present in some of the cells, they appear unequal in size. The epicarp is a single layer of thick walled cells having greenish content. Endocarp is wavy in outline. Mostly, endocarp and seed coat are fused together to form a deep zone with hyaline content in outer layer and orange red (brown) inner region. C Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 60 volumes of benzene, 30 volumes of ethyl acetate and 10 volumes of diethyl ether. Test solution. Reflux 2 g of the coarsely powdered substance under examination with 50-75 ml of methanol for 15 minutes, cool and filter. Reflux the residue further for two times with 75 ml of methanol, cool and filter. Combine all the filtrates and concentrate under vacuum to 50 ml.
1421
PIPPALI SMALL
IP 2007
Time (min) 0 18 25 30
Buffer solution (per cent v/v) 95 55 20 95
Acetonitrile (per cent v/v) 5 45 80 5
which are small. Crystals are present in some of the cells, they appear unequal in size. The epicarp is a single layer of thick walled cells having greenish content. Endocarp is wavy in outline. Mostly, endocarp and seed coat are fused together to form a deep zone with hyaline content in outer layer and orange red (brown) inner region. C Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 60 volumes of benzene, 30 volumes of ethyl acetate and 10 volumes of diethyl ether. Test solution. Reflux 2 g of the coarsely powdered substance under examination with 50-75 ml of methanol for 15 minutes, cool and filter. Reflux the residue further for two times with 75 ml of methanol, cool and filter. Combine all the filtrates and concentrate under vacuum to 50 ml. Reference solution. Reflux 0.4 g of the coarsely powdered pippali, small RS with 50-75 ml of methanol for 15 minutes, cool and filter. Reflux the residue further for two times with 75 ml of methanol, cool and filter. Combine all the filtrates and concentrate under vacuum to 10 ml. Apply to the plate 10 l of each solution as bands 10 mm by 2 mm. Allow the mobile phase to rise 8 cm. Dry the plate in air and examine in ultra violet light at 254 nm. The chromatographic profile of the test solution corresponds to that in the chromatogram obtained with reference solution. Spray with vanillin sulphuric acid reagent. Heat at 100 for 5-10 minutes and examine the plate in day light. The chromatographic profile of the test solution is similar to that of the reference solution.
Inject the reference solution. The relative standard deviation for the replicate injections is not more than 2.0 per cent. Inject the test solution and the reference solution. Calculate the content of piperine. Storage. Store protected from heat, moisture and against attach by insects and rodents.
Pippali, Small
Small pepper; Catkins (small)
Tests
Foreign organic matter (2.6.1). Not more than 2 per cent. Ethanol-soluble extractive (2.6.2). Not less than 8 per cent. Pippali, small consists of the fruiting spike of Piper longum Linn. (Fam. Piperaceae) Pippali, small contains not less than 0.4 per cent w/w of piperine, calculated on the dried basis. Description. The spikes are greenish black to black in colour, cylindrical, erect and blunt. It has pungent taste and the odour is aromatic and characteristic. The spikes are 1.0 to 1.9 cm long and 0.2 to 0.3 cm in diameter. Water-soluble extractive (2.6.3). Not less than 10 per cent by Method I. Ash (2.3.19). Not more than 8 per cent. Acid-insoluble ash (2.3.19). Not more than 3 per cent. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Loss on drying (2.4.19). Not more than 12.0 per cent, determined on 5 g by drying in an oven at 1050. Microbial contamination (2.2.9). Complies with the microbial contamination tests. Assay. Determine by liquid chromatography (2.4.14). Test solution. Weigh 2 g of coarsely powdered substance under examination, add 50 ml of methanol, sonicate for 3 minutes and heat on a boiling water bath for 15 minutes, cool and dilute to 100.0 ml with methanol and filter. Dilute further if
Identification
A. Macroscopic The spikes are greenish black to black in colour, surface rough. The spikes bear bracts and numerous small fruits sunk in solid spike. B. Microscopic Epidermis is a single layer of tangentially elongated cells. Cells are packed with abundant starch grains
1422
IP 2007
PUNARNAVA
necessary. Reference solution. A 0.01 per cent w/v solution of piperine RS in methanol. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a gradient mixtures of acetonitrile and a buffer solution prepared by dissolving 0.136 g of potassium di-hydrogen orthophosphate in 900 ml of water, adjust the pH to 2.5 with orthophosphoric acid and dilute to 1000 ml with water, flow rate. 1.5 ml per minute, spectrophotometer set at 270 nm, a 20 l loop injector. Time Buffer solution Acetonitrile (min) (per cent v/v) (per cent v/v) 0 18 25 30 95 55 20 95 5 45 80 5
Punarnava contains not less than 0.005 per cent of boeravinone B, calculated on the dried basis. Description. A greyish-brown in colour, short and fibrous fracture and no distinct odour with bitter taste.
Identification
A. Macroscopic Stout, tapering, somewhat knotty and twisted roots upto 30 cm or more long and 0.5-1.5 cm thick often crowned with stem bases, greyish-brown, surface is rough due to minute, irregular, longitudinal striations and root scars. B. Microscopic Transverse section of root shows outermost layer of cork which consists of thin walled tangentially elongated cells with brownish walls followed by thin walled 1-2 layered cork cambium. Cortex many layered and composed of thin walled cells. Secondary cortex 2-4 layered and parenchymtous. Concentric bands of xylem tissues alternating with parenchymatous tissues. Vessels are radial with reticulate thickening. Simple and compound starch grains with centric hilum and raphide crystals of calcium oxalate in cortex region. Fibres are aseptate and spindle shaped with pointed ends. C. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 35 volumes of chloroform, 6 volumes of methanol and 1 volume of glacial acetic acid. Test solution. Reflux 2 g of the coarsely powdered substance under examination with 25 ml of methanol for 15 minutes, cool and filter. Reflux the residue further with 2 25 ml of methanol, cool and filter. Combine all the filtrates and concentrate under vacuum to 5 ml. Reference solution. Reflux 2 g of the punarnava RS with 25 ml of methanol for 15 minutes, cool and filter. Reflux the residue further with 2 25 ml of methanol, cool and filter. Combine all the filtrates and concentrate under vacuum to 5 ml. Apply to the plate 10 l of each solution as bands 10 mm by 2 mm. Allow the mobile phase to rise 8 cm. Dry the plate in air, spray with a anisaldehyde solution. Heat at 110 for 10 minutes and examine the plate in day light. The chromatographic profile of the test solution is similar to that of the reference solution.
Inject the reference solution. The relative standard deviation for the replicate injections is not more than 2.0 per cent. Inject the test solution and the reference solution. The relative retention time of piperine is 1. Calculate the content of piperine. Storage. Store protected from heat, moisture and against attach by insects and rodents.
Punarnava
Boerhaavia; Hogweed
Tests
Foreign organic matter (2.6.1). Not more than 2.0 per cent. Ethanol-soluble extractive (2.6.2). Not less than 0.5 per cent. Water-soluble extractive (2.6.3). Not less than 9.0 per cent by method I. Punarnava consists of the dried root of Boerhaavia diffusa Ash (2.3.19). Not more than 10.0 per cent. Linn. (syn. B. reperis Linn) (Fam. Nyctaginaceae). Acid-insoluble ash (2.3.19). Not more than 3.0 per cent. 1423
SERPGANDHA
IP 2007
Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Loss on drying (2.4.19). Not more than 10.0 per cent, determined on 5 g by drying in an oven at 1050. Microbial contamination (2.2.9). Complies with the microbial contamination tests. Assay. Determine by liquid chromatography (2.4.14). Test solution. Reflux 2 g of the coarsely powdered substance under examination with 50 ml of methanol on a water-bath for 15 minutes, cool and filter. Reflux the residue further with methanol till the extract turns colourless, cool and filter. Combine all the filtrates and concentrate to a volume slightly less than 25 ml. Dilute to 25.0 ml with methanol. Reference solution. A 0.002 per cent w/v solution of boeravinone B RS in methanol. Chromatographic system a stainless steel column 25 cm 4.6 mm packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a gradient mixtures of water and acetonitrile, flow rate. 1 ml per minute, spectrophotometer set at 272 nm, a 20 l loop injector. Time Water Acetonitrile (min) (per cent v/v) ( per cent v/v) 0 90 10 25 10 90 35 90 10 Inject the reference solution. The test is not valid unless the relative standard deviation for the replicate injections is not more than 2.0 per cent. Inject the test solution and reference solution. Calculate the content of boeravinone B. Storage. Store protected from heat, moisture and against attack by insects and rodents.
Identification
A. Macroscopic Roots are sub cylindrical to tapering, tortuous or curved, rarely branched. Occurs as segments usually from 5 to 15 cms in length and 3 to 20 mm in diameter. Externally grayish yellow to brown, wood pale yellow. Roots tough with longitudinal marking & slightly wrinkled surface.
When scraped, bark separates readily from the wood. Fracture is short and irregular. B. Microscopic In transverse section, cork cells in 2 to 8 alternating bands of radically narrow and broader cells, thin, lignified up to 75 m in tangential width, broader cells up to about 90 m in radial length, phelloderm, tangentially elongated to isodimetric parenchyma cells containing starch and short latex cells with brown resinous matter; secondary cortex consists of parenchyma cells, heavily packed with starch grains secondary phloem contains phloem parenchyma and sieve elements, parenchyma contains starch and angular crystals of calcium oxalate 3 to 20 m in length. Xylem is about 4/5 of the diameter of the root, wood is transversed by medullay rays 1 to 5 cells in width. Xylem consists of vessels, tracheids, wood parenchyma & wood fibers. Xylem vessels are elongated up to 350 m in length and 50 m in width and contains simple or bordered pits; tracheids lignified, pitted; wood parenchyma with moderately thick, lignified and pitted walls containing starch; wood fibres highly thickened with pointed ends, stone cells absent. C. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G.. Mobile phase. A mixture of 70 volumes of chloroform and 30 volumes of acetone. Test solution. To 250 mg of the coarsely powdered substance under examination, add 5 ml methanol, shake for 10 minutes, and filter. Wash the residue with 5 ml of methanol and add the washing to the filtrate.
Serpgandha
Rauwolfia Root
Serpgandha consists of the dried roots of Rauwolfia Serpentina Bentham ex Kurz (Fam Apocynaceae). Serpgandha contains not less than 0.15 per cent of reserpine and ajmalicine, calculated on the dried basis. Description. Taste bitter, odour indistinct.
1424
IP 2007
SENNA PODS
Reference solution. To 250 mg of sepgandha RS, add 5 ml methanol, shake for 10 minutes, and filter. Wash the residue with 5 ml of methanol and add the washing to the filtrate. Apply to the plate 10 l of each solution as bands 10 mm by 2 mm. Allow the mobile phase to rise 12 cm. Dry the plate in air, spray with anisaldehyde solution and heat at 100 for 5 minutes and examine the plate in day light. The chromatogram obtained with test solution shows two pinkish-violet bands corresponding to the bands in the chromatogram obtained with reference solution. A dark brown band may also appear at the line of application in the chromatogram obtained with test solution.
a 10 l loop injector. Inject the reference solution (a). The test is not valid unless the relative standard deviation for the replicate injections is not more than 2.0 per cent. Inject reference solution (b) for peak identification. Inject the test solution and reference solution (a). Calculate the content of reserpine and ajmalicine. Storage. Store protected from heat, moisture and against attack by insects and rodents.
Tests
Foreign organic matter (2.6.1). Not more than 2.0 per cent Ethanol-soluble extractive (2.6.2). Not less than 2.0 per cent. Water-soluble extractive (2.6.3). Not less than 5.0 per cent. Ash (2.3.19). Not more than 8.0 per cent. Acid-insoluble ash (2.3.19): Not more than 2.0 per cent. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Loss on drying (2.4.19). Not more than 12.0 per cent, determined on 5 g by drying in an oven at 105. Microbial contamination limits (2.2.9). Complies with the microbial contamination tests. Assay. Determine by liquid chromatography (2.4.14). Test solution. Reflux about 2 g of the coarsely powdered substance under examination with 50 ml of ethanol (95 per cent), on a water bath for 15 minutes, cool and filter. Reflux the residue further with ethanol (95 per cent), till the last extract turns colorless, cool and filter. Combine all the filterates and concentrate to 100.0 ml. Reference solution (a). A solution containing a 0.002 per cent w/v reserpine RS and 0.002 per cent w/v ajmalicine RS in ethanol (95 per cent). Reference solution (b). A 0.002 per cent w/v reserpine RS in ethanol (95 per cent) for peak identification. Chromatographic system a stainless steel column 25 cm 4.6 mm packed with octadecylsilane bonded to porous silica (5m), mobile phase: a mixture of 35 volumes of acetonitrile and 65 volumes of a buffer solution prepared by dissolving 6.80 g of potassium dihydrogen phosphate in 1000 ml water, adjust the pH to 3.0 with dilute orthophosphoric acid, flow rate. 1 ml per minute, spectrophotometer set at 268 nm,
Senna Pods
Senna Fruit; Pods of cassia
Senna pods consist of the dried compound Pods of Cassia angustifolia or Cassia senna Vhal. (Family: Leguminosae). Senna Pods contains not less than 1 per cent of sennosides A and B, claculated on the dried basis. Description. Pale yellowish green coloured pods with slight odour. Leaflets with mucilaginous and faint odour.
Identification
A. Macroscopic Flattened reniform pods, brownish yellow at the edges, dark brown in the central area about 40 to 50 mm long and about at least 20 mm wide. At one end is a stylar point and at the other a short stalk. The pods contains 5 to 7 flattened and obovate seeds, green to pale brown, with a continuous network of prominent ridges on the tests and in complate wavy transverse ridges on the testa. Leaflets, 2.5 to 8 cm long and 5-15 mm wide at centre, pale yellowish green, elongated lanceolate, slightly asymmetric at base; margins entire, flat, apex acute with a sharp spine; both surface smooth with sparce trichomes; odour, faint but distinctive; taste, mucilaginous and disagreeable but not distinctly bitter.
1425
SENNA LEAF
IP 2007
B. Microscopic The pods present an epicarp with strongly cuticularised isodiametric cells, occasional anomocytic or paracytic stomata, and very few conical, unicellular and warty trichomes. Hypodermis with collenchymatous cells, mesocarp with parenchymatous tissue, a layer of prism s of calcium oxalate and containing vascular bundles incompletely surrounded by fibres with a crystals sheth of calcium oxalate prisms, endocarp consisting of thick-walled and inter lacing fibres. The seeds present a sub epidermal layers of palisade cells with thick outer walls, endosperm composed of polyhedral cells with mucilaginous wall. Reduce to moderately fine powder examine microscopically using chloral hydrate solution. The powder consists of epicarp with polygonal cells and a small number of warty trichomes and occasional anomocytic stomata, fibres in two crossed layers, accomparied by a crystal sheath of calcium oxalate prism, characteristic palisade cells in the seeds and stratified cells in the endosperm, cluster and prisms of calcium oxalate C. In the Assay, the chromatogram obtained with test solution corresponds to the chromatogram obtained with reference solution. D. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel F254. Mobile phase. A mixture of 40 volumes of n- propyl alcohol, 40 volumes of ethyl acetate, 29 volumes of water and 1 volume of glacial acetic acid. Test solution. Take 1 g of the dried leaves powder substance under examination. Add 25 ml of methanol, reflux for 10 minutes, cool and filter. Reflux the residue with another 20 ml of methanol, cool and filter. Combine all the filtrates and concentrate to 10 ml. Reference solution. A solution containing 0.01 per cent w/v of sennoside A and B RS in methanol. Apply to the plate 10 l of each solution as bands 10 mm by 6 mm. Allow the mobile phase to rise 8 cm. Dry the plate in air, spray with 20 per cent v/v of nitric acid solution. Heat at 1000 to 1050 for about 10 minutes and immediately examine the plate. The chromatographic profile of the test solution is similar to that of the reference solution.
Assay. Determine by liquid chromatography (2.4.14). Test solution. Weigh accurately 1.0 g of the coarsely powdered substance in a round bottom flask, add about 10 ml of 1 per cent v/v acetic acid and 25 ml of methanol and reflux on a water bath for about 30 minutes. Cool to room temperature; make up the volume up to 50 ml with methanol and filter. Reference solution. A 0.004 per cent w/v solution of sennosides RS in methanol. Chromatographic system: a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5 m), mobile phase. a mixtures of 82 volumes of 1 per cent v/v acetic acid in water and 18 volumes of acetonitrile. flow rate. 1 ml per minute, spectrophotometer set at 350 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the relative standard deviation for the replicate injections is not more than 2.0 per cent. Inject the test solution and reference solution. Calculate the content of sennoside A and B. Storage. Store protected from light and moisture.
Senna Leaf
Cassia leaf
Tests
Foreign organic matter (2.6.1). Not more than 1.0 per cent. Ash (2.3.19). Not more than 14.0 per cent. Acid-insoluble ash (2.3.19). Not more than 2.5 per cent. Loss on drying (2.4.19). Not more than 12.0 per cent, determined on 5 g by drying in an oven at 105. Microbial contamination (2.2.9). Complies with the microbial contamination tests. Senna leaf consists of the dried compound leaves of Cassia angustifolia or Cassia senna Vhal. (Family: Leguminosae). Description. Pale yellowish green coloured leaflets with mucilaginous and faint odour.
Identification
A. Macroscopic Leaflets,2.5 to 8 cm long and 5-15 mm wide at centre, pale yellowish green, elongated lanceolate,
1426
IP 2007
SHATAVARI
slightly asymmetric at base; margins entire, flat, apex acute with a sharp spine; both surface smooth with sparce trichomes; odour, faint but distinctive; taste, mucilaginous and disagreeable but not distinctly bitter. B. Microscopic Transverse section shows outer single layered mucilaginous epidermal cells. Unicellular hairs presents. Stomata paracytic, numerous on both surface. Mesophyll consists of upper and lower palisade layers with spongy layer in between , primatic crystals of calcium oxalate present on larger veins. C. In the Assay, the chromatogram obtained with test solution corresponds to the chromatogram obtained with reference solution. D. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel F254. Mobile phase. A mixture of 40 volumes of n- propyl alcohol, 40 volumes of ethyl acetate, 29 volumes of water and 1 volume of glacial acetic acid. Test solution. Take 1 g of the dried leaves powder substance under examination. Add 25 ml of methanol, reflux for 10 minutes, cool and filter. Reflux the residue with another 20 ml of methanol, cool and filter. Combine all the filtrates and concentrate to 10 ml. Reference solution. A 0.001 per cent w/v solution of Sennoside A and B in methanol. Apply to the plate 10 l of each solution as bands 10 mm by 6 mm. Allow the mobile phase to rise 8 cm. Dry the plate in air, spray with 20 per cent v/v of nitric acid solution. Heat at 1000 to 105 for about 10 minutes and immediately examine the plate. The chromatographic profile of the test solution is similar to that of the reference solution.
Reference solution. A 0.004 per cent w/v solution of sennosides RS in methanol. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixtures of 82 volumes of 1 per cent v/v acetic acid in water and 18 volumes of acetonitrile, flow rate. 1 ml per minute, spectrophotometer set at 350 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the relative standard deviation for the replicate injections is not more than 2.0 per cent. Inject the test solution and reference solution. Calculate the content of sennoside A and B. Storage. Store protected from light and moisture.
Shatavari
Asparagus racemosus root
Tests
Foreign organic matter (2.6.1). Not more than 1.0 per cent. Ash (2.3.19). Not more than 14.0 per cent. Acid-insoluble ash (2.3.19). Not more than 2.5 per cent. Loss on drying (2.4.19). Not more than 12.0 per cent, determined on 5 g by drying in an oven at 105. Microbial contamination (2.2.9). Complies with the microbial contamination tests. Assay. Determine by liquid chromatography (2.4.14). Test solution. Weigh accurately 1.0 g of the coarsely powdered substance in a round bottom flask, add about 10 ml of 1 per cent v/v acetic acid and 25 ml of methanol and reflux on a water bath for about 30 minutes. Cool to room temperature; make up the volume up to 50.0 ml with methanol and filter. Shatavari consists of the tuberous roots of Asparagus racemosus willd. (Fam .Liliaceae). Shatavari contains not less than 0.1 per cent of shatavarin IV, calculated on the dried basis. Description. The tuberous root bits are dirty white in color, longitudinally wrinkled with yellow hard central core. It is starchy and slightly bitter followed by sweet taste.
Identification
A. Macroscopic The tuberous roots are borne in a compact bunch and are fleshy, and spindle shaped. They are marketed in pieces 5-15 cm in length and 2 cm in thickness. They are silvery white or ash-colored externally and white internally,
1427
SHATAVARI
IP 2007
more or less smooth when fresh, developing longitudinal wrinkles when dry. B. Microscopic The inner parenchymatous zone of cortex is composed of 18-24 layers in upper and 42-47 layers in the middle tuberous portion of the roots. The cells are thin - walled cellulosic, with circular to oral outlines and distinct inter cellular spaces. In some roots 3-4 layers of cortex immediately adjacent to the endodermis are modified into a sheath of stone cells round the endodermis. The number of vascular bundles is 3035 in the upper levels and 35-45 in the middle tuberous portions of the roots. C. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel F254. Mobile phase. A mixture of 13 volumes of chloroform, 10 volumes of methanol and 2 volumes of water. Test solution. Reflux 1 g of the coarsely powdered substance under examination with 30 ml of methanol for 30 minutes, cool and filter. Reflux the residue further with 2 30 ml of methanol, cool and filter. Combine all the filtrates and concentrate under vacuum to 10.0 ml. Reference solution. Reflux 1 g of shatavari RS with 30 ml of methanol for 30 minutes, cool and filter. Reflux the residue further with 2 30 ml of methanol, cool and filter. Combine all the filtrates and concentrate under vacuum to 10.0 ml. Apply to the plate 5 l of each solution as bands 10 mm by 2 mm. Allow the mobile phase to rise 8 cm. Dry the plate in air, spray with vanillin sulphuric acid reagent prepared by mixing 1 per cent w/v vanillin in ethanol (95 per cent) and methanolic sulphuric acid (5 per cent) before spraying. Heat at 120 for 10 minutes and examine the plate in day light. The chromatographic profile of the test solution is similar to that of the reference solution.
Method I Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF 254. Mobile phase. A mixture of 6.5 volumes of chloroform and 3.5 volumes of methanol. Test solution. Reflux 4 g of the coarsely powdered substance under examination with 50 ml of methanol on a water-bath for 30 minutes, cool and filter. Reflux the residue further with methanol till the last extract turns colourless, cool and filter. Combine all the filtrates and concentrate to 50 ml. Reference solution. A 0.008 per cent w/v solution of shatavarin IV RS in methanol. Apply to the plate 5 l of each solution as bands 10 mm by 2 mm. Allow the mobile phase to rise 8 cm. Dry the plate in air and spray with 10 per cent v/v sulphuric acid in methanol. Heat the plate at 100 for 5 minutes, scan the plate in absorbance mode at 500 nm. Record the chromatograms and measure the responses for the analyte peak. Calculate the content of shatavarin IV. Method II Determine by liquid chromatography (2.4.14). Test solution. Reflux 5 g of the coarsely powdered substance under examination with 50 ml of methanol on a water-bath for 30 minutes, cool and filter. Reflux the residue further with methanol till the last extract turns colourless, cool and filter. Combine all the filtrates and concentrate to 50.0 ml. Reference solution. A 0.01 per cent w/v solution of shatavarin IV RS in methanol. Dilute suitably to prepare 0.0075- 0.075 per cent w/v solution. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 60 volumes of acetonitrile and 40 volumes of water, flow rate. 1 ml per minute, use evaporative light scattering detector, temperature evaporator 110, nebulizer 90, nebulizer gas nitrogen and gas flow 1 SLM, a 20 l loop injector. Inject the reference solution. The test is not valid unless the regression coefficient is not more than 0.9. Inject the test solution and reference solution. Calculate the content of shatavarin IV. Storage. Store protected from heat, moisture and against attack by insects and rodents.
Tests
Foreign organic matter (2.6.1). Not more than 2.0 per cent. Ethanol-soluble extractive (2.6.2). Not less than 15.0 per cent. Water- soluble extractive (2.6.3). Not less than 20.0 per cent by Method I. Ash (2.3.19). Not more than 15.0 per cent. Acid-insoluble ash (2.3.19). Not more than 3.0 per cent. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Loss on drying (2.4.19). Not more than 15.0 per cent, determined on 5 g by drying in an oven at 105. Microbial contamination (2.2.9). Complies with the microbial contamination tests. Assay. Perform the Assay by following method I or by method II. The result of the method II will be official in case of dispute.
1428
IP 2007
SHATI
Shati
Hedychium
and filter. Reflux the residue further with 2 25 ml of methanol, cool and filter. Combine all the filtrates and concentrate under vacuum to 25 ml. Reference solution. Reflux 0.5 g of the shati RS with 25 ml of methanol for 15 minutes, cool and filter. Reflux the residue further with 2 25 ml of methanol, cool and filter. Combine all the filtrates and concentrate under vacuum to 12.5 ml. Apply to the plate 10 l of each solution as bands 10 mm by 2 mm. Allow the mobile phase to rise 8 cm. Dry the plate in air, spray with a anisaldehyde solution. Heat at 110 for 10 minutes and examine the plate in day light. The chromatographic profile of the test solution is similar to that of the reference solution.
Tests
Foreign organic matter (2.6.1). Not more than 2.0 per cent. Ethanol-soluble extractive (2.6.2). Not less than 0.7 per cent. Shati consists of the dried rhizomes of Hedychium spicatum Buch.-Ham.ex Smith (Fam. Zingiberaceae). Shati contains not less than 0.80 per cent of p-methoxy cinnamic acid ethyl ester, calculated on the dried basis. Description. A reddish-brown outer surface and white in side, short and uneven fracture and camphoraceous odour with aromatic and pungent taste. Water-soluble extractive (2.6.3). Not less than 12 per cent by method I. Ash (2.3.19). Not more than 8 per cent. Acid-insoluble ash (2.3.19). Not more than 3.0 per cent. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Loss on drying (2.4.19). Not more than 14.0 per cent, determined on 5 g by drying in an oven at 1050. Microbial contamination (2.2.9). Complies with the microbial contamination tests. Assay. Determine by liquid chromatography (2.4.14). Test solution. Reflux 2 g of the coarsely powdered substance under examination with 50 ml of methanol on a water-bath for 15 minutes, cool and filter. Reflux the residue further with methanol till the extract turns colourless, cool and filter. Combine all the filtrates and concentrate to a volume slightly less than 100 ml. Dilute to 100.0 ml with methanol. Reference solution. A 0.01 per cent w/v solution of p-methoxy cinnamic acid ethyl ester RS in methanol. Chromatographic system a stainless steel column 25 cm 4.6 mm packed with octadecylsilane bonded to porous silica (5 m), mobile phase: 40 volumes of water and 60 volumes of acetonitrile, flow rate. 1 ml per minute, spectrophotometer set at 310 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the relative standard deviation for the replicate injections is not more than 2.0 per cent. Inject the test solution and reference solution.
Identification
A. Macroscopic Tuberous rhizome having reddish brown, rough outer surface with round root scars or rootlets which remain attached at some places. Transversely sliced pieces of dried rhizome are spherical, flat, 1 cm in thickness and 2-3 cm in diameter having white and starchy surface. B. Microscopic Transverse section of rhizome shows outermost layer of cork having 2-6 layers of isodiametic, nonlignified and suberised cells which are radially arranged. In old rhizome, the cork cells are exfoliated or crushed. Cortex is a broad zone with 20-25 layers of thin walled parenchymatous cells. Cortex region is filled with abundant starch grains and numerous oleo-resin cells. Vascular bundles are closed and collateral and scattered throughout the ground tissues. Cambium has 4-5 rows of tangentially elongated cells. It forms a complete ring between the xylem and phloem groups. Starch grains tissue are simple, circular or oval in shape, found in ground cells. C. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 80 volumes of n-haxane and 20 volumes of acetone. Test solution. Reflux 1 g of the coarsely powdered substance under examination with 25 ml of methanol for 15 minutes, cool
1429
SHELLAC
IP 2007
Calculate the content of p-methoxy cinnamic acid ethyl ester. Storage. Store protected from heat, moisture and against attack by insects and rodents.
Shellac
Lac
Shellac consists of a resinous substance prepared from a secretion that encrusts the bodies of a scale insect, Laccifer lacca Kerr (Fam. Ciccidae). Description. Lemon-yellow to brownish orange thin scales or hard, brittle masses; odourless or with a faint odour.
Arsenic ( 2.3.10). Heat gently 5.0 g with 2 ml of nitric acid and 0.5 ml of sulphuric acid in a long-necked flask, until the first reaction has subsided, cool, add carefully and in small portions, 15 ml of nitric acid and 6 ml of sulphuric acid taking care to avoid excessive foaming, and continue heating, adding further small portions of nitric acid, if necessary, until white fumes are evolved and the solution becomes colourless or almost colourless. Cool, add carefully 10 ml of water, evaporate until white fumes are evolved, and repeat the addition of water and evaporation until all the nitric acid has been removed, cool, dilute to 50 ml with water, and add 10 ml of stannated hydrochloric acid AsT. The resulting solution complies with the limit test for arsenic (2 ppm). Use 0.5 ml of arsenic standard solution (10 ppm As) for the standard stain. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Sulphated ash (2.3.18). Not more than 1.0 per cent, determined on 0.5 g. Storage. Store protected from moisture.
Identification
To 50 mg add a few drops of a mixture of 1 g of ammonium molybdate and 3 ml of sulphuric acid; a green colour is produced and it becomes lilac on standing for 5 minutes.
Tests
Acid value (2.3.23). 50 to 70, determined by the following method. Weigh accurately about 2.0 g and dissolve with the aid of gentle heat, in 50 ml of ethanol (95 per cent) previously neutralised to ethanolic thymol blue solution. Titrate with 0.1 M ethanolic potassium hydroxide using ethanolic thymol blue solution as an external indicator. Calculate the acid value from the expression 5.61 a /w where, a = number of ml of 0.1 M ethanolic potassium hydroxide and w = weight, in g, of the sample.
Starch
Starch consists of polysaccharide granules obtained from the caryopsis of maize or corn, Zea mays Linn., or of rice, Oryza sativa Linn., or of wheat, Triticum aestivum Linn., or from the tuber of potato, Solanum tuberosum Linn., or from the rhizomes of tapioca, Manihot utilissima Pohl. Description. A very fine, white or slightly yellowish powder or irregular white masses which are readily reducible to powder, creaks when pressed between the fingers; odourless and tasteless. The presence of granules showing cracks or edge irregularities is exceptional in starches other than wheat starch; wheat starch may contain granules with cracks on the edges.
Ethanol-insoluble matter. Not more than 2.0 per cent, determined by the following method. Weigh accurately about 5.0 g in an extraction thimble, cover with ethanol (95 per cent) and allow to stand for 16 hours. Place in an apparatus for the continuous extraction of drugs, extract with ethanol (95 per cent) for 4 hours, dry the residue at 100 for 3 hours and weigh. Colophony. Dissolve 2.0 g by shaking with 10 ml of ethanol, add slowly, with shaking 50 ml of light petroleum (40 to 60), wash with two successive portions, each of 50 ml, of water, filter the washed light petroleum solution, and evaporate to dryness; to the residue add 2 ml of a mixture of 1 volume of liquified phenol and 2 volumes of carbon tetrachloride and transfer to a cavity of a colour-reaction porcelain tile; fill an adjacent cavity with a mixture of 1 volume of bromine and 4 volumes of carbon tetrachloride, and cover both cavities with an inverted watch-glass; no purple or deep indigo-blue colour is produced in the liquid containing the residue.
Identification
A. Corn or maize starch Polyhedral granules, 2 to 23 m in size, or rounded granules, 25 to 32 m in diameter. The central hilum consists of a distinct cavity or two- to five-rayed cleft; no concentric striations. Viewed between crossed nicol prisms, a distinct black cross is seen intersecting at the hilum. Potato starch Single granules, either irregular, ovoid or pear-shaped, 30 to 100 m in size, or rounded, 10 to 35 m in size; compound granules consisting of groups of two to four elements are rare. Eccentric hilum; clearly visible concentric striations. Viewed between crossed nicol prisms, a distinct black cross is seen intersecting at the hilum. Rice starch Polyhedral granules, 2 to 5 m in size, either isolated or aggregated in ovoid masses, 10 to 20 m in size. Central hilum poorly visible; no concentric striations. Viewed
1430
IP 2007
SUNTHI
between crossed nicol prisms, a distinct black cross is seen intersecting at the hilum. Tapioca starch Principally simple granules, sub-spherical, muller-shaped or rounded polyhedral; smaller granules 5 to 10 m, larger granules 20 to 35 m in diameter; hilum, central, punctate, linear or triradiate; striations, faint, concentric; compound granules, few, of two to three unequal components. Wheat starch Large discoid or, more rarely, reniform granules, 10 to 45 m in size; profile, elliptical and fusiform, slit along the main axis. Small rounded or polyhedral granules, 2 to 10 m in size. Granules of intermediate size very rarely occur. Hilum and striations invisible or barely visible. Viewed between crossed nicol prisms, a distinct black cross is seen intersecting at the hilum. B. Heat to boiling for 1 minute a suspension of 1 g in 50 ml of water and cool; a thin and cloudy mucilage is produced with all starches except potato starch which gives a thick and more transparent mucilage. C. To 10 ml of the mucilage obtained in test B add 0.05 ml of 0.01 M iodine; a dark blue colour is produced, which disappears on heating and reappears on cooling.
Storage. Store protected from light and moisture. Labelling. The label states the type of starch.
Sunthi
Saunth; Ginger
Tests
Acidity. Add 10.0 g to 100 ml of ethanol (70 per cent) previously neutralised to phenolphthalein solution, shake for 1 hour, filter and titrate 50 ml of the filtrate with 0.1 M sodium hydroxide. Not more than 2.0 ml is required to change the colour of the solution. Iron (2.3.14). Dissolve the residue obtained in the test for sulphated ash in 4 ml of hydrochloric acid with the aid of gentle heat, dilute with water to 50 ml and mix; 25 ml of the resulting solution complies with the limit test for iron (40 ppm). Fluorescence. No fluorescence should be visible under screened ultra-violet light. Oxidising substances. To 5.0 g add 10 ml of water and 1 ml of acetic acid and stir until a homogeneous suspension is obtained. Add 0.5 ml of a freshly prepared saturated solution of potassium iodide, mix and allow to stand for 5 minutes; no distinct brown or blue colour is observed. Microbial Contamination (2.2.9). 1 g is free from Escherichia coli and salmonellae. Sulphated ash (2.3.18). Not more than 0.6 per cent (for all starches except rice starch) and not more than 0.8 per cent (for rice starch), determined on 2.0 g. Loss on drying (2.4.19). Not more than 15.0 per cent (for all starches except potato starch) and not more than 20.0 per cent (for potato starch), determined on 0.2 g by drying in an oven at 105.
Sunthi is the whole or cut scraped or unscraped, dried rhizomes of Zingiber officinale Rosc. (Fam. Zingiberaceae). Sunthi contains not less than 0.8 per cent of total gingerols, calculated on the dried basis. Description. Odour, agreeable and aromatic; taste, agreeable and pungent.
Identification
A. Macroscopic Rhizome laterally compressed, bearing short, flattened, oblique branches; outer surface buff-coloured, longitudinally striate; inner surface pale yellow, starchy and fibrous. Fracture short with projecting fibers. B. Microscopic Fibro-vascular bundles and oleoresin cells with yellow pigment scattered in ground tissue. Starch grains abundant in parenchyma cells, mostly simple, sack shaped, spherical; hilum eccentric. C. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G.. Mobile phase. A mixture of 30 volumes of hexane and 70 volumes of diethyl ether. Test solution. Reflux 1 g of the coarsely powdered substance under examination with 25 ml of methanol for 15 minutes, cool and filter. Wash the residue with 10 ml of methanol. Combine all the filtrates and concentrate to 10 ml. Reference solution. Reflux 0.5 g of coarsely powdered sunthi RS with 5 ml methanol for 15 minutes, cool and filter.
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Apply to the plate 10 l of each solution as bands 10 mm by 2 mm. Allow the mobile phase to rise 8 cms. Dry the plate in air, spray with 1 per cent w/v vanillin in ethanol (95 per cent) followed by 10 per cent w/v ethanolic sulphuric acid. Heat at 100 for 5-10 minutes and examine the plate in day light. The chromatographic profile of the test solution is similar to that of the reference solution.
Tolu Balsam
Balsam of Tolu
Tolu Balsam is a solid or semi-solid, balsam obtained by incision from the trunk of Myroxylon balsamum (Linn.) Harms (Fam. Leguminosae). Tolu Balsam contains not less than 35.0 per cent and not more than 50.0 per cent of total balsamic acids, calculated as cinnamic acid, C9H8O2, on the dry, ethanol-soluble matter. Description. A soft, tenacious, brownish yellow or brown mass, when first collected; subsequently, becoming harder and finally brittle. Transparent in thin films; odour, aromatic and vanilla-like. Warmed and pressed between pieces of glass and examined with a lens, it exhibits crystals of cinnamic acid.
Tests
Foreign organic matter (2.6.1). Not more than 2.0 per cent. Ethanol-soluble extractive (2.6.2). Not less than 2.0 per cent. Water-soluble extractive (2.6.3). Not less than 10.0 per cent by Method I. Ash (2.3.19). Not more than 8.0 per cent. Acid-insoluble ash (2.3.19). Not more than 1.5 per cent. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Water (2.3.43). Not more than 12.0 per cent, determined on 0.2 g. Microbial contamination (2.2.9). Complies with the microbial contamination tests. Assay. Determine by liquid chromatography (2.4.14). Test solution. Reflux about 3 g of the coarsely powdered substance under examination with 100 ml of methanol on a waterbath for 15 minutes cool and filter. Reflux the residue further with methanol till the last extract turns colorless, cool and filter. Combine all the filtrates and concentrate to 50.0 ml Reference solution. A 0.1 per cent w/v solution of 6-gingerol RS in methanol. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5 m), mobile phase: 55 volumes of acetonitrile and 45 volumes of water, flow rate. 1.3 ml per minute, spectrophotometer set at 278 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the relative standard deviation for the replicate injections is not more than 2.0 per cent. Inject the test solution and reference solution. Calculate the contents of total gingerols by summing the peak areas of 6-gingerol with all other peaks, which elute after 6gingerol and have a peak area of at least 5 per cent of the peak area of 6-gingerol. Storage. Store protected from heat, moisture and against attack by insects and rodents.
Identification
A. To a solution in ethanol (90 per cent) add ferric chloride test solution; a green colour is produced. B. To 1 g add to 5 ml of water, heat to boiling, filter, add 30 mg of potassium permanganate and continue heating; the odour of benzaldehyde is produced.
Tests
Acidity. A solution in ethanol (90 per cent) is acidic to litmus solution. Acid value (2.3.23). 97 to 160, calculated on the dry, ethanolsoluble matter, determined by the following method. Dissolve 5.0 g in 50 ml of boiling ethanol (90 per cent), add 3 ml of phenolphthalein solution and titrate the hot solution with 1 M ethanolic potassium hydroxide. When the colour becomes dark brown, attach to a reflux condenser, boil for a few minutes to break up the precipitate and complete the titration. Ethanol-insoluble matter. Not more than 5 per cent, determined by the following method. Digest 2.5 g with 50 ml of ethanol (90 per cent), filter through a sintered glass crucible, transfer the residue to the filter crucible with the aid of more ethanol (90 per cent), wash with hot ethanol (90 per cent) until all soluble matter is removed and dry to constant weight at 100. Colophony. Add 5 g to 25 ml of carbon disulphide, warm gently on a water-bath under a reflux condenser, filter, evaporate the solution to dryness, dissolve the residue in 6 ml of light petroleum (40 to 60) and shake with 10 ml of a 0.5 per cent w/v solution of cupric acetate; the light petroleum layer is not coloured green. Ester value (2.3.26). 47 to 95, calculated on the dry, ethanolsoluble basis. Saponification value. (2.3.37). 170 to 230, calculated on the dry, ethanol-soluble basis.
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TRAGACANTH
Loss on drying (2.4.19). Not more than 4 per cent, determined on 2.0 g by drying in a thin layer in an oven at 60 over phosphorus pentoxide at a pressure not exceeding 2.7 kPa. Assay. Weigh accurately about 2.0 g and boil with 25 ml of dilute ethanolic potassium hydroxide solution under a reflux condenser for 1 hour. Remove the ethanol and digest the residue with 50 ml of hot water until diffused. Cool the liquid, add 150 ml of water and 1.5 g of magnesium sulphate dissolved in 50 ml of water. Mix thoroughly and set aside for 10 minutes. Filter, wash the residue on the filter with 20 ml of water, acidify the combined filtrate and washings with hydrochloric acid and extract with successive quantities of 50, 40, 30, 30 and 30 ml of ether. Combine the ether extracts and discard the aqueous portion. Extract with successive quantities of 20, 20, 10, 10 and 10 ml of sodium bicarbonate solution, washing each aqueous extract with the same 20 ml of ether. Discard the ether layers, acidify the combined aqueous extracts with hydrochloric acid and extract with successive quantities of 30, 20, 20 and 10 ml of chloroform, filtering each chloroform extract through a plug of cotton wool on which a layer of anhydrous sodium sulphate is placed. Evaporate the chloroform on a water-bath until about 10 ml remains and remove the remainder in a current of air stopping immediately when the last trace of solvent is removed. Dissolve the residue by warming with 10 ml of ethanol (95 per cent), previously neutralised to phenol red solution, cool and titrate with 0.1 M sodium hydroxide using phenol red solution as indicator. 1 ml of 0.1 M sodium hydroxide is equivalent to 0.01482 g of total balsamic acids, calculated as cinnamic acid, C9H8O2. Storage. Store protected from light and moisture. Avoid exposure to excessive heat.
concentric transverse ridges; white, translucent, horny; fracture, short. May also be in the form of thicker, less brittle pieces, white to pale yellow and more opaque. B. Microscopic Reduce to powder. Examine under a microscope; the powder shows in the gummy mass numerous stratified cellular membranes which turn violet on the addition of iodinated zinc chloride solution. The mass includes starch granules, isolated or in small clusters, rounded or occasionally deformed, diameter 4 to 10 m, and up to 20 m, with a central hilum, visible in polarised light. C. Moisten 0.5 g of the powdered material with 1 ml of ethanol (95 per cent) and add gradually, while shaking, 50 ml of water until a homogeneous mucilage is obtained. To 5 ml of the mucilage add 5 ml of water and 2 ml of barium hydroxide solution. A slightly flocculent precipitate is formed which, when heated for 10 minutes on a water-bath, gives an intense yellow colour. D. Add 4 ml of a 0.5 per cent w/v dispersion in water to 0.5 ml of hydrochloric acid and heat on a water-bath for 30 minutes. To one half of the resulting liquid add 1.5 ml of sodium hydroxide solution and 3 ml of alkaline cupric-tartrate solution and heat on a water-bath; a reddish brown precipitate is formed. To the other half of the liquid, add a few drops of barium chloride solution; no precipitate is formed (freedom from agar).
Tests
Acacia and other soluble gums. To 20 ml of a 2.5 per cent w/v suspension of the powdered material prepared with freshly boiled water add 10 ml of lead acetate solution; a flocculent precipitate is formed. Filter and add to the filtrate 10 ml of lead subacetate solution; a slight cloudiness may appear, but there is no precipitate. Karaya gum. Boil 1 g with 20 ml of water until a mucilage is formed, add 5 ml of hydrochloric acid and again boil for 5 minutes; no pink or red colour develops. Sterculia. A. Shake 0.2 g of the powdered material with 10 ml of ethanol (60 per cent) in a 10-ml stoppered cylinder; any gel formed occupies not more than 1.5 ml. B. Shake 1 g of the powdered material with 100 ml of water and titrate with 0.01 M sodium hydroxide, using methyl red solution as indicator. Not more than 5.0 ml is required to change the colour of the solution. Foreign matter. Not more than 1.0 per cent, determined by the following method. To 2.0 g of the powdered material in a 250ml round-bottomed flask add 95 ml of methanol, swirl to moisten the powder and add 60 ml of 7 M hydrochloric acid. Add a few glass beads and heat under a reflux condenser in a water-bath for 3 hours, shaking occasionally. Remove the glass beads and filter the hot suspension under reduced pressure
Tragacanth
Tragacanth is the air-hardened gummy exudate, flowing naturally or obtained by incision, from the trunk and branches of Astragalus gummifer Labill. and certain other species of Astragalus. Description. Pale yellow, thin, flattened ribbons or brittle pieces; odourless and almost tasteless. On the addition of about 10 times its weight of water, it forms a mucilaginous gel. It has the macroscopic and microscopic characteristics described under Identification tests A and B.
Identification
A. Macroscopic Occurs as thin, flattened pieces, 30 mm long, 10 mm wide and up to 1 mm in thickness, more or less curved, marked on the surface by fine longitudinal striae and
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IP 2007
through a sintered-glass crucible (porosity No. 1). Rinse the flask with a small quantity of water, passing the rinsings through the filter. Wash the residue on the filter with about 40 ml of methanol and dry to constant weight at 110. Arsenic (2.3.10). Mix 3.3 g with 3 g of anhydrous sodium carbonate, add 10 ml of bromine solution and mix thoroughly. Evaporate to dryness on a water-bath, gently ignite, and dissolve the cooled residue in 16 ml of brominated hydrochloric acid AsT and 45 ml of water. Remove the excess of bromine with 2 ml of stannous chloride AsT. The resulting solution complies with the limit test for arsenic (3 ppm). Heavy metals (2.3.13). 0.5 g complies with the limit test for heavy metals, Method B (40 ppm). Ash (2.3.19). Not more than 4.0 per cent, determined on 1.0 g. Microbial contamination (2.2.9). 1 g is free from Escherichia coli and 10 g is free from salmonellae. Storage. Store protected from moisture.
5-7 mm in length. It has both male and female parts. Calyx: There are 5 sepals and it is greenish in colour. Corolla: There are 5 petals, bilabiate in shape and covered with scattered hairs. Petals whitish-purple. B. Microscopic Transverse section of leaf shows a pot shaped midrib. Upper epidermis consists of a layer of small, quadrangular transparent cells with thin walls and thin smooth cuticle. On tangential view, these cells are polygonal with straight or wavy walls. Lower epidermis consists of a layer of small, quadrangular transparent cells with thin walls and thin and thin smooth cuticle. Trichomes bent, consisting of 2-6 of 1 stalk cell and 2-4 cells with rounded heads. Palisade parenchyma consists of layer of long cylindrical cells containing chlorophyll; spongy parenchyma consists of polygonal cells with thin, straight or slightly wavy side walls. Vascular bundles collateral type. Stomata diacytic. C. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 97 volumes of toluene and 3 volumes of ethyl acetate. Test solution. Reflux 2 g of the coarsely powdered substance under examination with 25 ml of methanol for 15 minutes, cool and filter. Reflux the residue further with 2 25 ml of methanol, cool and filter. Combine all the filtrates and concentrate under vacuum to 10 ml. Reference solution. Reflux 1 g of tulasi RS with 25 ml of methanol for 15 minutes, cool and filter. Reflux the residue further with 2 25 ml of methanol, cool and filter. Combine all the filtrates and concentrate under vacuum to 5 ml. Apply to the plate 10 l of each solution as bands 10 mm by 2 mm. Allow the mobile phase to rise 8 cm. Dry the plate in air, spray with a anisaldehyde solution. Heat at 110 for 10 minutes and examine the plate in day light. The chromatographic profile of the test solution is similar to that of the reference solution.
Tulasi
Ocimum, Basil
Tests
Tulasi consists of leaves of Ocimum sanctum Linn (Fam. Lamiaceae). Tulasi contains not less than 0.40 per cent of eugenol, calculated on the dried basis. Description. Greyish-black in colour having characteristic odour with slightly pungent and aromatic taste. Foreign organic matter (2.6.1). Not more than 2.0 per cent. Ethanol-soluble extractive (2.6.2). Not less than 3.0 per cent. Water-soluble extractive (2.6.3). Not less than 10.0 per cent by method I. Ash (2.3.19). Not more than 15.0 per cent. Acid-insoluble ash (2.3.19). Not more than 5.0 per cent. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Loss on drying (2.4.19). Not more than 12.0 per cent, determined on 5 g by drying in an oven at 1050.
Identification
A. Macroscopic Leaves simple, elliptic, 2.7-7.5 cm long, 1-3 cm wide, with acute top, cuneate, obtuse to rounded base, margin entire, undulate or serrate, both surfaces thinly pubescent and dotted; petiole 0.2-3.0 cm long. Flowers are
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IP 2007
VASAKA
Microbial contamination (2.2.9). Complies with the microbial contamination tests. Assay. Determine by gas chromatography (2.4.13). Test solution. Reflux 0.5 g of the coarsely powdered substance under examination with 50 ml of methanol on a water-bath for 15 minutes, cool and filter. Reflux the residue further with methanol till the extract turns colourless, cool and filter. Combine all the filtrates and concentrate to a volume slightly less than 100 ml. Dilute to 100.0 ml with methanol. Reference solution. A 0.004 per cent w/v solution of eugenol RS in methanol. Chromatographic system a capillary column 30 m x 0.25 x 0.25 mm coated with100 per cent dimethylpolysiloxane temperature: oven 60 to 260 @10 per minute, (Initially and finally hold for 5 minutes respectively) Injector 240, detector 280, flow rate. 0.8 ml per minute, split flow 20 ml per minute. Inject 1 l of the reference solution. The test is not valid unless the relative standard deviation for the replicate injections is not more than 2.0 per cent. Inject 1 l of the test solution and reference solution. Calculate the content of eugenol. Storage. Store protected from heat, moisture and against attack by insects and rodents.
Vasaka contains not less than 0.6 per cent of vasicine, calculated on the dried basis. Description.Taste, bitter.
Identification
A. Macroscopic Leaf pieces membranous, brittle, greyishbrown, a few pieces green coloured. Floral bracts leaf-like. B. Microscopic Stomata diacytic, more on the lower epidermis; glandular and non-glandular hair on both surfaces of leaf; elongated cystoliths present in palisade cells. C. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G.. Mobile phase. A mixture of 8 volumes of ethyl acetate, 2 volumes of methanol and 0.2 volume of strong ammonia solution. Test solution. Reflux 1 g of coarsely powdered substance under examination with 50 ml methanol for 15 minutes, cool and filter. Reflux the residue further with 2 50 ml of methanol, cool and filter. Combine all the filtrates and concentrate to 10 ml. Reference solution. Reflux 0.5 g of vasaka RS with 50 ml methanol for 15 minutes, cool and filter. Reflux the residue further with 2 50 ml of methanol, cool and filter. Combine all the filtrates and concentrate to 5 ml. Apply to the plate 10 l of each solution as bands 10 mm by 2 mm. Allow the mobile phase to rise 8 cms. Dry the plate in air and spray with dragendorfs reagent. The chromatographic profile of the test solution is similar to that of the reference solution.
Vasaka
Adulasa; Adhatoda
Tests
Foreign organic matter (2.6.1). Not more than 2.0 per cent. Ethanol-soluble extractive (2.6.2). Not less than 3.0 per cent. Water-soluble extractive (2.6.3). Not less than 22 per cent by Method I. Ash (2.3.19). Not more than 21 per cent. Acid-insoluble ash (2.3.19). Not more than 2.0 per cent. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method B (20 ppm). Loss on drying (2.4.19). Not more than 12.0 per cent, determined on 5 g by drying in an oven at 1050. Microbial contamination (2.2.9). Complies with the microbial contamination tests. Assay. Determine by liquid chromatography (2.4.14).
Vasaka consists of the dried mature leaves of Adhatoda vasica Nees. (Fam. Acanthaceae).
Test solution. Reflux about 2 g of the coarsely powdered substance under examination with 50 ml of methanol on a
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YASTI
IP 2007
water bath for 15 minutes, cool and filter. Reflux the residue further with methanol till the last extract turns colorless, cool and filter. Combine all the filtrates and concentrate to 100.0 ml. Reference solution. Dissolve 25 mg of vasicine hydrochloride RS in 50 ml of methanol. Dilute 5.0 ml of this solution to 50.0 ml with methanol. Chromatographic system a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 3 volumes of the solution prepared by dissolving 1 g of sodium hexanesulphonate in 1000 ml of water, 1 volume of acetonitrile and 20 volumes of glacial acetic acid, flow rate. 1 ml per minute, spectrophotometer set at 300 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the relative standard deviation for the replicate injections is not more than 2.0 per cent. Inject the test solution and reference solution. Calculate the content of vasicine. Storage. Store protected from heat, moisture and against attack by insects and rodents.
Identification
A. Macroscopic Root with few branches, up to 1 m long and 0.5 to 3 cm in diameter. Bark, brownish-grey to brown with longitudinal striations, bearing traces of lateral roots. Stolons, cylindrical, 1 to 2 cm in diameter and up to several metres long, but may be cut into lengths of 10 to 15 cm; similar in external appearance to the root but with occasional small buds. Fracture of the root and stolon, granular and fibrous. Cork layer, thin; secondary phloem region, wide, light yellow with radial striations; xylem, compact, yellow, with radiate structure. The stolon has a central pith which is absent from the root. B. Microscopic Cork and phelloderm are narrow. Phloem consisting of bundles of thick-walled, yellow fibres with narrow lumina surrounded by cells each containing a calcium oxalate prism, alternating in the external layers with areas of strongly hyaline keratenchyma; functional sieve tissue near the cambium. Medullary rays parenchymatous, widening towards the exterior, 3 to 12 cells wide. Xylem composed of radial rows of tracheids and vessels alternating with bundles of lignified fibres with crystal sheaths similar to those of the secondary phloem; vessels 30 m to 150 m in diameter with thick walls (5 m to 10 m) having reticulate thickenings or numerous bordered pits with slit-shaped openings associated with lignified xylem parenchyma. Medullary rays, 2 to 5 cells wide. Parenchymatous cells throughout containing simple, round, oval or fusiform starch granules 2 m to 20 m, mostly 5 m to 12 m, in diameter; parenchymatous pith present solely in the stolon. C. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. The upper layer, even if turbid, of a mixture of 60 volumes of ethyl acetate, 27 volumes of 1 M ammonia and 13 volumes of ethanol, shaken together and allowed to stand for 5 minutes. Test solution. Shake 1 g, in No. 180 powder, with 20 ml of chloroform for 15 minutes, filter and reserve the extracted powder for the preparation of reference solution (a). Evaporate the filtrate to dryness and dissolve the residue in 2 ml of a mixture of equal volumes of chloroform and methanol. Reference solution (a). Add to the extracted powder 30 ml of 0.5 M sulphuric acid and heat under a reflux condenser for 1 hour, allow to cool and extract with two quantities, each of 20 ml, of chloroform; dry the combined chloroform extracts with anhydrous sodium sulphate, filter, evaporate to dryness and dissolve the residue in 2 ml of a mixture of equal volumes of chloroform and methanol. Reference solution (b). Dissolve 10 mg of glycyrrhetinic acid in 2 ml of a mixture of equal volumes of chloroform and methanol.
Yasti
Liquorice root; Mulethi; Glycyrrhiza
Yasti consists of the dried, unpeeled roots and stolons of Glycyrrhiza glabra Linn. (Fam. Leguminoseae). Yasti contains not less than 3.0 per cent of glycyrrhizinic acid. Description. Odour, characteristic and slightly aromatic; taste, very sweet and faintly astringent; the bark is not bitter.
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IP 2007
YASTI
Apply to the plate 10 l of test solution, reference solution (a) and 20 l of reference solution (b) as bands 20 mm by 3 mm. After development, dry the plate in air for 5 minutes and examine in ultraviolet light at 254 nm. The chromatogram obtained with reference solution (b) exhibits a band corresponding to b-glycyrrhetinic acid with an Rf value of about 0.1. The chromatogram obtained with reference solution (a) exhibits a corresponding band but this is not seen in the chromatogram obtained with the test solution. Spray the plate with anisaldehyde solution, using about 10 ml for a plate, 20 cm 20 cm in size, heat at 105 for 10 minutes and examine in daylight. The b-glycyrrhetinic acid bands become violetblue. One or two bands with an Rf value of about 0.6, visible in daylight before spraying, become orange-yellow and several other violet-blue bands appear in the chromatograms obtained with the test solution and reference solution (a). The band corresponding to b-glycyrrhetinic acid in the chromatogram obtained with reference solution (a) is at least equal in size to the band in the chromatogram obtained with reference solution (b). D. Mix a small quantity, in powder, with 0.05 ml of sulphuric acid; the powder particles become orange-yellow and some fragments change, more slowly, to pinkish red.
filtrate to dryness on a water-bath, dry the residue at 105 and weigh. Acid-insoluble ash (2.3.19). Not more than 2.0 per cent. Sulphated ash (2.3.18). Not more than 10.0 per cent. Assay. Determine by liquid chromatography (2.4.14). Test solution. Weigh accurately about 1.0 g of the coarsely powdered substance under examination in a 250-ml conical flask, add 100 ml of 0.1 M ammonia and mix with the aid of ultrasound for 30 minutes. Centrifuge a part of the supernatant liquid and dilute 1.0 ml to 5.0 ml with 0.1 M ammonia. Filter the solution through a membrane filter disc with an average pore diameter not greater than 1.0 m and use the filtrate. Reference solution. A 0.005 per cent w/v solution of glycyrrhizinic acid RS in 0.1 M ammonia. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 6 volumes of glacial acetic acid, 30 volumes of acetonitrile and 64 volumes of water, flow rate. 1.5 ml per minute, spectrophotometer set at 254 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the relative standard deviation for replicate injections is not more than 2.0 per cent. Inject the test solution and the reference solution and measure the responses for the analyte peak. Calculate the content of glycyrrhizinic acid. Storage. Store protected from light and moisture.
Tests
Curcuma. When examined under a microscope, none of the fragments of the powder in sulphuric acid (see Identification test D) should immediately take on a carmine-red colour. Water-soluble extractive (2.6.3). Not less than 20 per cent, determined by the following method. Mix 2.5 g of the finely powdered drug with 50 ml of water and allow to stand for 2 hours, shaking frequently. Filter, evaporate 10.0 g of the
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YASTI
IP 2007
1438
IP 2007
1439
MONOGRAPHS
BLOOD AND BLOOD-RELATED PRODUCTS

Anti-A Blood Grouping Serum Anti-D (Rho) Immunoglobulin ........................................................................................ .......................................................................................... ..........................................................
Anti-B Blood Grouping Serum .......................................................................................... Anti-D Immunoglobulin Human for Intravenous Use
Anti-Human Globulin Serum ................................................................................................ Blood Grouping Serums Anti-D, Anti-C, Anti-E, Anti-c, Anti-e ........................................... Concentrated Human Red Blood Cells ............................................................................... Cryoprecipitated Antihemophilic Factor .............................................................................. Dried Human Antihaemophilic Fraction ............................................................................... Fibrin Sealant Kit .............................................................................................................. Human Albumin ................................................................................................................ Human Coagulation Factor IX Human Coagulation Factor VII .......................................................................................... ........................................................................................
Human Coagulation Factor VIII (rDNA) ............................................................................ Human Normal Immunoglobulin ......................................................................................... Human Plasma Protein Fraction .......................................................................................... Human Prothrombin Complex ............................................................................................ Normal Immunoglobulin for Intravenous Use ...................................................................... Plasma for Fractionation ......................................................................................... Platelet Concentrate ........................................................................................................... Whole Human Blood ..........................................................................................................
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IP 2007
ANTI-A BLOOD GROUPING SERUM
Anti-A Blood Grouping Serum

Anti-A Blood Grouping Serum is a sterile, liquid or dried preparation containing the particular blood group antibodies derived from high-titered blood plasma or serum of human subjects, with or without stimulation by the injection of Blood Group Specific Substance A (or AB). It contains a suitable antimicrobial preservative. It is of two types polyclonal & monoclonal.
Storage. Store at a temperature between 2 and 8. Labelling. Label states that the source material was not reactive for hepatitis B surface antigen, but that no known test method offers assurance that products derived from human blood will not transmit hepatitis in case of polyclonal. Label also states that it is for in vitro diagnostic use. NOTEThe labeling is in black lettering imprinted on paper that is white or is coloured completely or in part to match the specified blue colour standard.
Production
The monoclonal antibody technique devised by Kohler and Milsten has proved useful in producing high titre and specific antibodies. Laboratory animals, usually mice are immunized for the production of monoclonal antibodies. After suitable immune response, mouse spleen cells containing antibody secreting lymphocytes are fused to neoplastic plasma cells of infinite reproductive capacity from a mouse (that is myeloma cells). The resulting hybridomas are screened for antibody with the required specificity and affinity. The antibody secreting clones may then be propagated in tissue culture or by inoculation into mice in which case the antibodies are collected as ascites. The clonal line produces a single antibody, there is no need for absorption or to remove heterospecific antibodies. All antibody molecules produced by a clone of hybridoma cells are identical in terms of antibody structure and antigen specificity. Once one antibody secreting clone of cells has been established, antibody with same specificity and reaction characteristics will be available indefinitely.
Anti-B Blood Grouping Serum

Anti-B Blood Grouping Serum is a sterile, liquid or dried preparation containing the particular blood group antibodies derived from high-titered blood plasma or serum of human subjects, with or without stimulation by the injection of Blood Group Specific Substance B (or AB). It contains a suitable antimicrobial preservative.
Production
The monoclonal antibody technique devised by Kohler and Milsten has proved useful in producing high titre and specific antibodies. Laboratory animals, usually mice are immunized for the production of monoclonal antibodies. After suitable immune response, mouse spleen cells containing antibody secreting lymphocytes are fused to neoplastic plasma cells of infinite reproductive capacity from a mouse (that is myeloma cells). The resulting hybridomas are screened for antibody with the required specificity and affinity. The antibody secreting clones may then be propagated in tissue culture or by inoculation into mice in which case the antibodies are collected as ascites. The clonal line produces a single antibody, there is no need for absorption or to remove heterospecific antibodies. All antibody molecules produced by a clone of hybridoma cells are identical in terms of antibody structure and antigen specificity. Once one antibody secreting clone of cells has been established, antibody with same specificity and reaction characteristics will be available indefinitely.
Identification
It agglutinates human red cells containing A-antigens, are blood groups A and AB (including subgroups A1, A2, A1B, and A2B but not necessarily weaker subgroups).
Tests
It meets the requirements to the test for potency, in parallel with, and not less than equivalent to, the Reference Blood Grouping Serum Anti-A, in agglutinating red blood cells from Group A1 and Group A2B donors. It complies with the tests for specificity with Group A1, A2B, B, and O cells and confirms the absence of contaminating antibodies reactive with Mg, Wra antigens as well as other antigens having an incidence of 1 per cent or greater in the general population (see under Blood Grouping Serums Anti-D, Anti-C, Anti-E, Anti-c, rAnti-e). It complies with the tests for avidity with Group A1 and A2B cells. All fresh or frozen red blood cell suspensions used for these tests are prepared under specified conditions and meet specified criteria. Anti-A Blood Grouping Serum may be artificially coloured blue. Expiration date. The expiration date for liquid serum is not later than 1 year, and for dried serum not later than 5 years after date of issue from manufacturers cold storage (5o, 1 year; or 00, 2 years), provided that the expiration date for dried serum is not later than 1 year after constitution.
Identification
It agglutinates human red cells containing B-antigens, i.e., blood groups B and AB (including subgroups A1B and A2B).
Tests
It complies with the test for potency, in parallel with, and not less than equivalent to, the Reference Blood Grouping Serum Anti-B, in agglutinating red blood cells from Group B donors. It complies with the tests for specificity with Group A1, B, and O cells and confirms the absence of contaminating antibodies reactive with Mg, Wra antigens as well as other antigens having an incidence of 1.0 per cent or greater in the general population (see under Blood Grouping Serums Anti-D, Anti-C, Anti-E, 1443
ANTI-D (Rho) IMMUNOGLOBULIN
IP 2007
Anti-c, Anti-e). It complies with the test for avidity with Group B cells. All fresh or frozen red blood cell suspensions used for these tests are prepared under specified conditions and meet specified criteria. Anti-B Blood Grouping Serum may be artificially coloured yellow. Expiration date. The expiration date for liquid serum is not later than 1 year and for dried serum not later than 5 years after date of issue from manufacturers cold storage (5, 1 year; or 0, 2 years), provided that the expiration date for dried serum is not later than 1 year after constitution. Storage. Store at a temperature between 2 and 8. Labelling. Label states that the source material was not reactive for hepatitis B surface antigen, but that no known test method offers assurance that products derived from human blood will not transmit hepatitis in case of polyclonal. Label also states that it is for in vitro diagnostic use. NOTEThe labelling is in black lettering imprinted on paper that is white or is coloured completely or in part to match the specified yellow color standard.
Immunisation Immunisation of the plasma donor is carried out under proper medical supervision. Recommendations concerning donor immunisation, including testing of erythrocyte donors, have been formulated by the World Health Organisation (Requirements for the collection, processing and quality control of blood, blood components and plasma derivatives, WHO Technical Report Series, No. 840, 1994 or subsequent revision). Pooled plasma To limit the potential B19 virus burden in plasma pools used for the manufacture of anti-D immunoglobulin, the plasma pool is tested for B19 virus using validated nucleic acid amplification techniques (2.8.1). B19 virus DNA. Maximum 104 IU per ml. A positive control with 104 IU of B19 virus DNA per ml and, to test for inhibitors, an internal control prepared by addition of a suitable marker to a sample of the plasma pool are included in the test. The test is invalid if the positive control is nonreactive or if the result obtained with the internal control indicates the presence of inhibitors. B19 virus DNA for NAT testing reference preparation is suitable for use as a positive control. If Human Normal Immunoglobulin is added to the preparation, the plasma pool from which it is derived complies with the above requirement for B19 virus DNA.
Anti-D (Rho) Immunoglobulin

Human Anti-D Immunoglobulin Human anti-D immunoglobulin is a liquid or freeze-dried preparation containing immunoglobulins, mainly immunoglobulin G. The preparation is intended for intramuscular administration. It contains specific antibodies against erythrocyte D-antigen and may also contain small quantities of other blood-group antibodies. Human normal immunoglobulin may be added. It complies with the monograph on Human Normal Immunoglobulin, except for the minimum number of donors and the minimum total protein content. For products prepared by a method that eliminates immunoglobulins with specificities other than anti-D, where authorised, the test for antibodies to hepatitis B surface antigen is not required.
Tests
Potency. Determine the assay of human anti-D immunoglobulin by Method A (2.8.2). The estimated potency is not less than 90.0 per cent of the stated potency. The confidence limits (P = 0.95) are not less than 80.0 per cent and not more than 120.0 per cent of the estimated potency. Method B or C (2.8.3) may be used for potency determination if a satisfactory correlation with the results obtained by Method A has been established for the particular product. Storage. For the liquid preparation, store protected from light, in a plastic container. For the freeze-dried preparation, store protected from light, in a plastic container. Labelling. The label states (1) for liquid preparations, the volume of the preparation in the container and the protein content expressed in grams per litre; (2) for freeze-dried preparations, the quantity of protein in the container; (3) the route of administration; (4) for freeze-dried preparations, the name or composition and the volume of the reconstituting liquid to be added; (5) where applicable, that the preparation is suitable for use in the prophylaxis of hepatitis A infection; (6) where applicable, the anti-hepatitis A virus activity in International Units per ml; (7) where applicable, the name and amount of antimicrobial preservative in the preparation. The label also states the number of International Units per container.
Production
Human anti-D immunoglobulin is preferably obtained from the plasma of donors with a sufficient titre of previously acquired anti-D antibodies. Where necessary, in order to ensure an adequate supply of human anti-D immunoglobulin, it is obtained from plasma derived from donors immunised with D-positive erythrocytes that are compatible in relevant blood group systems in order to avoid formation of undesirable antibodies. Erythrocyte donors Erythrocyte donors comply with the requirements for donors prescribed in the monograph on Human Plasma for Fractionation. 1444
IP 2007
ANTI-D IMMUNOGLOBULIN FOR INTRAVENOUS USE
Anti-D Immunoglobulin for Intravenous Use

Anti-D Immunoglobulin Human for Intravenous Use
Anti-D Immunoglobulin for intravenous administration is a liquid or freeze-dried preparation containing immunoglobulins, mainly immunoglobulin G. It contains specific antibodies against erythrocyte D-antigen and may also contain small quantities of other blood-group antibodies. Human normal immunoglobulin for intravenous use may be added. It complies with the monograph on Human Normal Immunoglobulin for Intravenous Administration, except for the minimum number of donors, the minimum total protein content, the limit for osmolality and the limit for prekallikrein activator. For products prepared by a method that eliminates immunoglobulins with specificities other than anti-D: where authorised, the test for antibodies to hepatitis B surface antigen is not required; a suitable test for Fc function is carried out.
immunisation, including testing of erythrocyte donors, have been formulated by the World Health Organisation (Requirements for the collection, processing and quality control of blood, blood components and plasma derivatives, WHO Technical Report Series, No. 840, 1994 or subsequent revision). Pooled plasma To limit the potential B19 virus burden in plasma pools used for the manufacture of anti-D immunoglobulin, the plasma pool is tested for B19 virus using validated nucleic acid amplification techniques (2.8.1).
Identification
Examine by a suitable immunoelectrophoresis technique. Using antiserum to normal human serum, compare normal human serum and sample under examination in a dilution of 10 g per litre of protein. The main component of the sample corresponds to the IgG component of normal human serum.
Production
Polyclonal (human) anti Rh (D) serum Human anti-D immunoglobulin is preferably obtained from the plasma of donors with a sufficient titre of previously acquired anti-D antibodies. Where necessary, in order to ensure an adequate supply of human anti-D immunoglobulin, it is obtained from plasma derived from donors immunised with D-positive erythrocytes that are compatible in relevant blood group systems in order to avoid formation of undesirable antibodies. Erythrocyte donors Laboratory tests are carried out for each donation to detect the following viral markers: 1. 2. 3. 4. Antibodies against human immunodeficiency virus 1 (anti-HIV-1), Antibodies against human immunodeficiency virus 2 (anti-HIV-2), Antibodies against hepatitis C virus (anti-HCV). Hepatitis B surface antigen (HBsAg),
Tests
B19 virus DNA . Maximum 104 IU per ml. A positive control with 104 IU of B19 virus DNA per ml and, to test for inhibitors, an internal control prepared by addition of a suitable marker to a sample of the plasma pool are included in the test. The test is invalid if the positive control is nonreactive or if the result obtained with the internal control indicates the presence of inhibitors. B19 virus DNA for NAT testing RP is suitable for use as a positive control. If Human normal immunoglobulin for intravenous administration is added to the preparation, the plasma pool from which it is derived complies with the above test for B19 virus DNA. Assay. Determine by method A of human anti-D immunoglobulin (2.8.2). The estimated potency is not less than 90.0 per cent of the stated potency. The confidence limits (P = 0.95) are not less than 80.0 per cent and not more than 120.0 per cent of the estimated potency. Method B or C (2.8.3) may be used for potency determination if a satisfactory correlation with the results obtained by Method A has been established for the particular product. Storage. For the liquid preparation, store protected from light, in a colourless glass container, at the temperature stated on the label. For the freeze-dried preparation, store protected from light, in colourless glass container, at a temperature not exceeding 25. Labelling. The label states (1) for liquid preparations, the volume of the preparation in the container and the protein content expressed in gram per litre; (2) for freeze-dried preparations, the quantity of protein in the container; (3) the amount of immunoglobulin in the container; (4) the route of 1445
Pending complete harmonisation of the laboratory tests to be carried out, the competent authority may require that a test for alanine aminotransferase (ALT) also be carried out. The test methods used are of suitable sensitivity and specificity and comply with the regulations in force. If a repeatreactive result is found in any of these tests, the donation is not accepted. Immunisation Immunisation of the plasma donor is carried out under proper medical supervision. Recommendations concerning donor
ANTI- HUMAN GLOBULIN SERUM
IP 2007
administration, (5) for freeze-dried preparations, the name or composition and the volume of the reconstituting liquid to be added; (6) the distribution of subclasses of immunoglobulin G present in the preparation; (7) where applicable, the amount of albumin added as a stabilizer; (8) the maximum content of immunoglobulin A. The label states the number of International Units per container.
also states the specific antibody activities present; the application for which the reagent is intended; a cautionary statement that it does not contain antibodies to immunoglobulins or that it does not contain antibodies to complement components, wherever and whichever is applicable; and states that it is for in vitro diagnostic use. NOTEThe lettering on the label of the general-purpose polyspecific reagent is black on a white background. The label of all other Anti-Human Globulin Serum containers is in white lettering on a black background.
Anti-Human Globulin Serum

Anti-Human Globulin Serum is a sterile, liquid preparation of serum produced by immunizing lower animals such as rabbits or goats with human serum or plasma, or with selected human plasma proteins. It is free from agglutinins and from hemolysins to nonsensitized human red cells of all blood groups. It contains a suitable antimicrobial preservative. Three varieties of Anti-Human Globulin Serum are recognized: (1) a general-purpose polyspecific reagent which, as a minimum, contains antibodies specific for immunoglobulin IgG, and at least the C3d component of human complement (for use in the direct antiglobulin test, it contains this Anti-C3d and Anti-IgG activity) and which may be artificially coloured green; (2) a reagent containing antibodies only against immunoglobulin IgG (not heavy chain specific) intended for use in the indirect antiglobulin test, and which may be artificially coloured green; and (3) reagents containing antibodies specific for individual or selected components of human complement, such as Anti-C3, and Anti-C3b-C3d-C4, or a single class of immunoglobulins, such as Anti-IgG (heavy chain specific), used only to identify plasma components coated on the surface of red blood cells. Anti-Human Globulin Serum containing Anti-IgG complies with the test for potency, in parallel with the Reference Anti-Human Globulin (AntiIgG) Serum (at a 1:4 dilution) when tested with red cells suspended in isotonic saline sensitized with decreasing amounts of nonagglutinating Anti-D (Anti-Rho) serum, and with cells sensitized in the same manner with an immunoglobulin IgG Anti-Fya serum of similar potency. AntiHuman Globulin Serum containing one or more Anticomplement components complies with the tests for potency in giving a 2+ agglutination reaction (i.e., agglutinated cells dislodged into many small clumps of equal size) by the lowionic sucrose or sucrosetrypsin procedures when tested as recommended in the labelling. Anti-Human Globulin Serum containing Anti-3Cd activity meets the requirements for stability, by potency testing of representative lots every 3 months during the dating period. Expiration date. Its expiration date is not later than 1 year after the date of issue from manufacturers cold storage (5, 1 year; or 0, 2 years). Storage. Store at a temperature between 2 and 8. Labelling. Label states the animal source of the product. Label 1446
Blood Grouping Serums Anti-D, AntiC, Anti-E, Anti-c, Anti-e

Anti-Rh Blood Grouping Serums Blood Grouping Serums Anti-D, Anti-C, Anti-E, Anti-c, Anti-e (Anti-Rh Group) are sterile, liquid or dried preparations derived from the blood plasma or serum of human subjects who have developed specific Rh antibodies. They are free from agglutinins for the A or B antigens and from alloantibodies other than those for which claims are made in the labelling. They contain a suitable antimicrobial preservative. Liquid serums are not artificially coloured. Two varieties of Anti-Rh Blood Grouping Serums are recognized, i.e., (1) saline agglutinating complete antiserums, which specifically agglutinate human red blood cells suspended in saline and (2) blocking or incomplete antiserums, which contain protein or other macromolecular substances, usually require the cells to be suspended in serum or plasma, and generally are for slide or rapid tube tests. The most commonly used of these blood grouping serums are listed in Table 1 each reacting with the antigen(s) designated by the corresponding letter(s) with the alternative nomenclature indicated parenthetically. Table - 1 ______________________________________________ Serum Antigen(s) Reacting ______________________________________________ Anti-D (Anti-Rho) Anti-C (Anti-rh) Anti-E (Anti-rh) Anti-CD (Anti-Rho) Anti-DE (Anti-Rho) Anti-CDE (Anti-Rho) Anti-c (Anti-hr) D (Rho) C (rh) E (rh) D (Rho) and C (rh) D (Rho) and E (rh) D (Rho), C (rh), and E (rh) c (hr)
Anti-e (Anti-hr) e (hr) ______________________________________________ Each serum meets the requirements of the test for potency in the case of serums for saline tube test in parallel with, and not less than equivalent to, the Reference Blood Grouping Serum
IP 2007
BLOOD GROUPING SERUM ANTI-D, ANTI-C, ANTI-E, ANTI-c, ANTI-e
for Anti-D, Anti-C, or Anti-E, whichever is applicable, or, in the case of Anti-c and Anti-e for saline tube test which have no reference preparations, the test for minimum agglutination reactivity at a specified dilution; and in the case of serums for slide or rapid tube test in parallel with, and not less than equivalent to, the Reference Blood Grouping Serum for AntiD, Anti-C, Anti-E, Anti-c, or Anti-e, whichever is applicable, in agglutinating as a minimum red blood cells from the donors indicated in Table 2 (which may be from Group A, B, AB, or O). Each serum for slide or rapid tube test complies with the tests for avidity with the cells as indicated under tests for potency above. Table - 2 ______________________________________________ Serum Phenotype of Cells ______________________________________________ Anti-D Anti-C Anti-E Anti-CD Anti-DE Anti-CDE Anti-c Anti-e cDe Ccde cdEe cDe, Ccde cDe, cdEe cdEe, cDe, Ccde CcDEe cdEe
Anti-DE Anti-CDE
cDe, Ccde, cdEe, A1 cde, B cde, O cde cDe, Ccde, cdEe, A1 cde, B cde, O cde, and where recommended for detection of the G antigen, rGr Ccde, A1 CDe, B CDe, O CDe, and CDEe or CDE or CdE cdEe, A1 cDE, B cDE, O cDE, and CcDE or CDE or CdE
Anti-c Anti-e
______________________________________________ All fresh or frozen red blood cell suspensions used for these tests are prepared under specified conditions and meet specified criteria. Monoclonal Rh (D) antibodies. Monoclonal antibodies are derived from hybridoma cell lines produced by fusing mouse antibody produced by B lymphocyte with mouse myeloma cells or are derived from a human B cell line through EpsteinBarr Virus (EBV) transformation. Each hybridoma cell line produces homogenous antibodies of only one immunoglobulin class, which are identical in their chemical structure and immunological activity. The type of monoclonal anti-D reagents are: 1. 2. 3. IgM anti-D monoclonal reagent, Blend of IgM and IgG monoclonal antibodies reagent, Blend of monoclonal IgM and polyclonal (human) IgG anti-D.
Each serum complies with the tests for specificity by the most sensitive method recommended by the manufacturer, in which not less than 4 positive and 4 negative phenotypes are included (see Table 3), and confirms the absence of contaminating antibodies reactive with Mg, Wra antigens as well as other antigens having an incidence of 1.0 per cent or greater in the general population, except where some of these confirmatory tests can not be done by the manufacturer, in which event such omissions are noted. Table 3 ______________________________________________ Serum Cells ______________________________________________ Anti-D CcDe, cDe, Ccde, cdEe, A1 cde, B cde, O cde, and where recommended for use by indirect antiglobulin technique, cde Bg(a+) cells from 3 different donors cDe, Ccde, cdEe, C + rhi neg. cells, A1 cde, B cde, O cde cDe, Ccde, cdEe, A1 cde, B cde, O cde cDe, Ccde, cdEe, A1 cde, B cde, O cde, and where recommended for detection of the G antigen, rGr
IgM anti-D monoclonal antibodies are highly specific and saline reacting equally well at room temperature and at 37. They are good for slide test or immediate spin tube tests as well as routine Rh(D) typing in tube. IgM anti D are unreliable for detection of weak D (Du) by AHG test. Blend of IgM and IgG (monoclonal) anti-D or blend of IgM (monoclonal) and polyclonal (human) IgG anti-D can be used for testing weak D (Du) antigen by AHG test. Mostly blended IgM and IgG (monoclonal) anti- Rh(D) or blended monoclonal IgM and polyclonal (human) IgG anti Rh (D) antibodies are used now in routine. Expiration date. The expiration date for liquid serums is not later than 1 year and for dried serums not later than 5 years after date of issue from manufacturers cold storage (5, 1 year; or 0, 2 years), provided that the expiration date for dried serums is not later than 1 year after constitution. Storage. Store at a temperature between 2 and 8. Labelling. Label each to state that the source material was not reactive for hepatitis B surface antigen, but that no known test method offers assurance that products derived from human blood will not transmit hepatitis. Label each to state that it is for in vitro diagnostic use.
Anti-C Anti-E Anti-CD
1447
CORPUSCLES
IP 2007
Concentrated Human Red Blood Corpuscles

Concentrate of Human Red Blood Cells; Packed Red Cells Concentrated Red Blood Cells (RBCs) are units of whole blood with most of the plasma removed. Additive red cell preservative systems consist of a primary collection bag containing an anticoagulant preservative with at least two satellite bags integrally attached, one is empty and one contains an additive solution (AS). Each 100 ml citrate phosphate dextrose (CPD) contains Citric acid (monohydrate) Sodium citrate (dihydrate) Sodium acid phosphate (dihydrate) Dextrose (monohydate) Water for injection 0.327 g 2.63 g 0.251 g 2.55 g 100 ml
Description. A dark red fluid when prepared; after standing, the red corpuscles may form a sediment, leaving a small supernatant layer of yellowish plasma.
Tests
Sterility (2.2.11). Complies with the tests for sterility, determined by Method B. Assay. Determine the haemoglobin content by photometric haemoglobinometry (2.8.12). Storage. Store in containers which are made of colourless and transparent glass, or of a suitable plastic material, are sterile and sealed so as to exclude micro-organisms. Store at a temperature between 2o and 8o. Labelling. The label states (1) the reference number of the Whole Human Blood from which the preparation was made; (2) the ABO and Rh groups of the Whole Human Blood; (3) the date of collection of the Whole Human Blood from which the preparation was made; (4) the storage conditions; (5) the date after which the preparation is not suitable for transfusion; (6) that the preparation should not be used if there is any visible evidence of haemolysis or other deterioration.
AS contains sodium chloride, dextrose, adenine and other substances that support red cell survival and function up to 42 days. The volume of AS in 350 ml is 49 ml and in 450 ml is 63 ml. AS is added to the red cells remaining in the primary bag after most of the plasma has been removed. This allows blood centres to use or recover a maximum amount of plasma, yet still prepare a red cell component with a final haematocrit between 55.0 per cent and 65.0 per cent, a level that facilitates excellent flow rates and allows easy administration.
Cryoprecipitated Antihaemophilic Factor

Cryoprecipitated Antihaemophilic Factor is a sterile, frozen concentrate of human antihaemophilic factor prepared from the Factor VIII-rich cryoprotein fraction of human venous plasma obtained from suitable whole-blood donors from a single unit of plasma derived from whole blood or by plasmapheresis, collected and processed in a closed system. It contains no preservative. It complies with the test for potency by comparison with the Standard Antihaemophilic Factor (Factor VIII) or with a working reference that has been calibrated with it, in having an average potency of not less than 80 Antihaemophilic Factor Units per container, made at intervals of not more than 1 month during the dating period. Expiration date. The expiration date is not later than 1 year from the date of collection of source material. Storage. Store in hermetic containers at a temperature of - 18 or lower.
o
Production
It may be prepared by centrifugation or undisturbed sedimentation for the separation of plasma and anticoagulant solution equivalent to not less than 40.0 per cent of the total volume of Whole Human Blood. A portion of the plasma, sufficient to ensure optimal cell preservation, shall be left. All surfaces that come in contact with the red cells and plasma shall be sterile and pyrogen-free and the entire processing of the blood shall be conducted in a sterile system or in a closed system by use of satellite bags. The final containers used for the Concentrated Human Red Blood Corpuscles shall be the original blood containers unless the method of processing requires a different container. Immediately after processing, the containers are stored at a temperature between 2o and 8o and not opened until immediately before transfusion. Human Red Blood Corpuscles may be stored for a period not longer than that for which the Whole Human Blood from which it is prepared. However, if the hermetic seal is broken during processing, the product must be used within 24 hours. From the tube of the blood bag sample is taken for compatibility and infections markers testing. Concentrated Human Red Blood Corpuscles should be administered only with suitable equipment meant for the transfusion of blood and blood components. Concentrated Human Red Blood Corpuscles contains not less than 15.5 per cent w/v of haemoglobin. 1448
Labelling. Label it to indicate (1) the ABO blood group designation and the identification number of the donor from whom the source material was obtained; (2) with the type and result of a serologic test for syphilis, or to indicate that it was non-reactive in such test; with the type and result of a test for hepatitis B surface antigen, or to indicate that it was nonreactive in such test; with a warning not to use it if there is evidence of breakage or thawing; with instructions to thaw it before use to a temperature between 20 and 37, after which it is to be stored at room temperature and used as soon as possible but within 6 hours after thawing; (3) to state that it is
IP 2007
DRIED HUMAN ANTIHAEMOPHILIC FRACTION
to be used within 4 hours after the container is entered; (4) to state that it is for intravenous administration; (5) that a filter is to be used in the administration equipment.
Dried Human Antihaemophilic Fraction

Dried Factor VIII Fraction; Freeze-dried Human Coagulation Factor VIII Dried Human Antihaemophilic Fraction is a preparation of antihaemophilic factor which is obtained from human plasma. It is rich in clotting factor VIII. When the contents of a sealed container of Dried Human Antihaemophilic Fraction are dissolved in a volume of water equal to the volume of water for injection stated on the label, the resulting solution contains not less than 3.0 Units per ml, not less than 0.1 Unit per mg of protein, not more than 80.0 per cent of which is fibrinogen, and not more than 200 millimoles of sodium ions per litre.
containers and dried from the frozen state. The air is removed or replaced by oxygen-free nitrogen and the containers are sealed so as to exclude micro-organisms. No antimicrobial preservative is added but an antiviral agent may be added provided that it can be demonstrated to have no deleterious effect on the final product in the amount present and to cause no adverse reaction in man. Heparin may be used. For the following tests, where it is directed that a solution is to be used, dissolve the contents of a sealed container in a volume of the appropriate solvent equal to the volume of water for injection stated on the label. Description. A white or pale yellow powder or friable solid.
Identification
A. Precipitation tests with a suitable range of species specific antisera give positive results for the presence of plasma proteins of human origin and negative results with antisera specific to plasma proteins of the other species. B. A freshly prepared solution in water causes a reduction in clotting time when treated as directed under Assay.
Production
The plasma to be used for preparing Dried Human Antihaemophilic Fraction is obtained from blood of healthy human donors who are, as far as can be ascertained after clinical examination, laboratory tests on their blood and consideration of their medical history, free from detectable agents of infection transmissible by blood transfusion. The examinations and tests to be carried out are decided by the National Regulatory Authority. In particular, the blood must be tested with negative results for (a) evidence of syphilitic infection; (b) hepatitis B surface antigen and (c) HIV antibodies by suitably sensitive methods. The haemoglobin value of the donors blood is not less than 12.5 per cent w/v. The blood is withdrawn aseptically through a closed system of sterile tubing into a sterile container in which a suitable anticoagulant solution has been placed before sterilisation. During the withdrawal there is no interruption in the flow from the donor, and the container is gently agitated. Immediately after the withdrawal is completed, the blood is cooled to 4o; if the plasma is to be stored frozen it is separated from the cellular components by centrifugation and frozen to 30o or below, preferably within 12 hours of collection; if the plasma is not to be frozen it is separated from the cellular components by centrifugation as soon as possible and not later than 18 hours after collection, and fractionation begun without delay. Dried Human Antihaemophilic Fraction may be prepared from human plasma so obtained by precipitation under controlled conditions of pH, ionic strength and temperature with organic solvents, or by freezing and thawing. The precipitate may be washed by extraction with suitable solvents, dissolved in a solution of sodium citrate adjusted to a pH of 6.8 to 7.2, which may also contain sodium chloride. The solution is sterilised by filtration through a membrane filter, distributed in sterile
Tests
pH (2.4.24). 6.8 to 7.4, determined on the reconstituted solution. Loss on drying (2.4.19). Not more than 2.0 per cent, determined by drying 0.5 g over phosphorus pentoxide at a pressure not exceeding 3 kPa for 24 hours. Haemagglutinins, anti-A and anti-B. Dissolve in water. Dilute the solution with saline solution to produce a solution containing 3 Units per ml. Carry out the test for haemagglutinins anti-A and anti-B using a suitable indirect method such as that described below. Prepare in duplicate serial dilutions of the preparation under examination in saline solution. To each dilution of one series add an equal volume of a 5.0 per cent v/v suspension of group A1 red blood cells previously washed three times with saline solution. To each dilution of the other series add an equal volume of a 5.0 per cent v/v suspension of group B red blood cells previously washed three times with saline solution. Incubate the suspensions at 37o for 30 minutes and then wash the cells three times with saline solution. Leave the cells in contact with a polyvalent anti-human globulin reagent for 30 minutes. Without centrifuging, examine each suspension for agglutination under a microscope. The 1 in 64 dilutions do not show agglutination. Hepatitis B surface antigen. Dissolve in water. Examine the solution by a suitably sensitive method such as radioimmunoassay. Hepatitis B surface antigen is not detected. Abnormal toxicity (2.2.1). When dissolved in water for injection, complies with the test for abnormal toxicity, Method B, injecting into each mouse a volume containing 1.5 Units and into each guinea-pig a volume containing 15 Units. Pyrogens (2.2.8). When dissolved in water for injection, complies with the test for pyrogens, using a volume containing 1449
IP 2007
10 Units per kg of the rabbits weight in rabbits that have not previously received blood products. Sterility (2.2.11). Complies with the tests for sterility. Assay For potency Carry out the biological assay of human antihaemophilic fraction described below. The estimated potency is not less than 80.0 per cent and not more than 125.0 per cent of the stated potency. The fiducial limits of error are not less than 64.0 per cent and not more than 156.0 per cent of the stated potency. Biological assay The potency of human antihaemophilic fraction is determined by comparing the amount necessary to reduce the clotting time of a test mixture containing substances that cause clotting of blood with the amount of the Standard Preparation necessary to produce the same effect under the conditions of the following method of assay. Standard preparation The Standard preparation is the 4th International Standard for Blood coagulation factor VIII:C, concentrate, human, established in 1989, consisting of an intermediate purity concentrate of human blood clotting factor VIII (supplied in ampoules containing 6.3 Units of clotting factor VIII), or another suitable preparation the potency of which has been determined in relation to the International Standard The Unit is the specific antihaemophilic factor contained in such an amount of the Standard Preparation as the Ministry of Health & Family Welfare, Govt. of India may from time to time indicate as the quantity exactly equivalent to the Unit accepted for international use. Special reagents Normal serum reagent. Collect normal human blood in a dry, sterile, glass bottle, shake continuously until coagulation is complete, incubate at 37o for 3 hours, maintain at 4o overnight, remove the serum, store at 20o, dry from the frozen state and keep in a vacuum desiccator over phosphorus pentoxide. Dissolve a quantity of the dried serum calculated to have been obtained from 1 ml of the serum in sufficient imidazole buffer pH 7.4 to produce 10 ml and allow to stand at 4o for 16 to 24 hours. Phospholipid. Wash a quantity of normal human or bovine brain freed from meninges and blood vessels and macerate in a suitable blender. Weigh 1,000 to 1,300 g of the macerate and measure its volume (V). Extract with three quantities, each of 4V ml, of acetone, filter by suction and dry the precipitate at 37o for 18 hours. Extract the dried precipitate with two quantities, each of 2V ml, of a mixture of two volumes of light petroleum (boiling range 30o to 40o) and 3 volumes of light petroleum (boiling range 40o to 60o), filtering each extract through a filter paper previously washed with the light 1450
petroleum mixture. Combine the extracts and evaporate to dryness at 45o at a pressure not exceeding 0.7 kPa. Dissolve the residue in 0.2V ml of ether and allow to stand at 4o until a deposit forms. Centrifuge and evaporate the clear supernatant liquid under reduced pressure until the volume is about 100 ml per kg of the original macerate. Allow to stand at 4o until a precipitate forms (12 to 24 hours) and centrifuge. To the clear supernatant liquid add 5 times its volume of acetone, centrifuge, discard the supernatant liquid, dry the precipitate and store protected from light in a vacuum desiccator. Phospholipid reagent. Suspend 0.125 g of phospholipid in 5 ml of water, shake and stir until a uniform suspension is obtained. Prepare a dilution with saline solution that will give minimum clotting times consistent with the largest clotting time differences between consecutive dilutions of the Standard preparation and the preparation under examination. The concentration usually lies between 50 and 250 g per ml. The diluted suspension may be kept at 20 for 6 weeks. Clotting factor V solution. Prepare from fresh oxalated bovine plasma by fractionation at 4o with a saturated solution of ammonium sulphate prepared at 4 o . Use the fraction precipitating between 38 per cent and 50 per cent saturation (which contains clotting factor V not significantly contaminated with clotting factor VIII), dialysed to remove ammonium sulphate and diluted with saline solution to give a solution containing between 10 per cent and 20 per cent of the amount of clotting factor V present in fresh normal human plasma. Determine the clotting factor V content of the solution as follows. Prepare two dilutions in imidazole buffer pH 7.4 to contain 1 volume of the solution under examination in 10 volumes and 20 volumes respectively. Test each dilution as follows. Mix 0.1 ml each of substrate plasma deficient in clotting factor V, the dilution under test, thrombokinase extract and 0.025M calcium chloride. Record as the clotting time the interval between the addition of the calcium chloride solution and the first indication of fibrin formation, which may be observed visually or by mechanical means. Similarly determine the clotting times, in duplicate, for four dilutions of pooled normal human plasma in imidazole buffer pH 7.4 containing 1 volume in 10 volumes (equivalent to 100 per cent of clotting factor V), in 50 volumes (20 per cent), in 100 volumes (10 per cent), and in 1,000 volumes (1 per cent), respectively. To calculate the result, plot the mean of the clotting times for each dilution of human plasma on double cycle log/log paper against the equivalent percentage of clotting factor V and read the percentage of clotting factor V for the two dilutions of clotting factor V solution by interpolation from the curve. The mean of the two results is taken as the percentage of clotting factor V in the solution. Substrate plasma. Separate the plasma from 9 volumes of human or bovine blood collected in 1 volume of a 3.8 per cent w/v solution of sodium citrate or from 3.5 volumes of human or bovine blood collected in 1 volume of a solution containing 2.0 per cent w/v of sodium acid citrate and 2.5 per cent w/v of
IP 2007
dextrose. In the former case, prepare the substrate plasma on the day of collection of the blood. In the latter case, the substrate plasma may be prepared up to 2 days after collection of the blood. Store at 20o. Substrate plasma deficient in clotting factor V. Preferably use congenitally deficient plasma or, alternatively, prepare as follows. Separate the plasma from human blood collected in one-tenth its volume of a 1.34 per cent w/v solution of sodium oxalate and incubate at 37o for 24 to 36 hours. This plasma should have a clotting time, when tested by the assay method given under clotting factor V solution, of 70 to 100 seconds; if the clotting time is less than 70 seconds, incubate the plasma for a further 12 to 24 hours. Storage. Store in small amounts, at -20o or below. Suggested method Dissolve the contents of the sealed container of the substance under examination in the volume of the liquid stated on the label and use immediately. Reconstitute the entire contents of one ampoule of the Standard preparation as stated on the label and use immediately. To the reconstituted Standard preparation and the preparation under examination, add sufficient imidazole buffer pH 7.4 to produce solutions containing between 0.5 and 2 Units per ml; these solutions are stable for 15 minutes at 20o. Using a mixture of 1 volume of a 3.8 per cent w/v solution of sodium citrate and 5 volumes of saline solution as the diluent, make from the solutions three successive 2-fold dilutions in the range 1 in 16 to 1 in 256 so that all the clotting times are between 17 and 35 seconds; the dilutions must be accurately made and used immediately. Introduce into each of six glass incubation tubes (75 mm 100 mm) 0.1 ml each of clotting factor V solution, phospholipid reagent and normal serum reagent. To the first tube add 0.1 ml of the highest dilution of the Standard Preparation, place the tube in a water-bath at 37o, add 0.1 ml of 0.05M calcium chloride and start a stop-watch. During the next minute add 0.1 ml of the second highest dilution of the standard to a second tube, place it in the water-bath, and add 0.1 ml of 0.05M calcium chloride at exactly 1 minute by the stop-watch. Repeat the procedure with the lowest dilution of the standard and the highest to lowest dilutions of the preparation under examination so that the calcium chloride solution is added at 2, 3, 4 and 5 minutes by the stop-watch, respectively. Place in a water-bath at 37o twelve glass tubes each containing 0.2 ml of 0.025M calcium chloride and a further tube containing about 3 ml of substrate plasma. At 14 minutes, 40 seconds by the stop-watch, transfer 0.1 ml of the mixture from the first incubation tube to one of the tubes containing 0.2 ml of 0.025M calcium chloride solution and mix. At 15 minutes add 0.2 ml of the warmed substrate plasma and, using a second stop-watch, record as the clotting time the interval between the addition of the substrate plasma and the first indication of fibrin formation, which may be observed visually or by mechanical means. Repeat the procedure with the other
incubation tubes at 1-minute intervals and carry out a second series of determinations at 21 to 26 minutes. The period of incubation should, if necessary, be adjusted so that the clotting times recorded in the corresponding tests in the two series of determinations do not differ by more than 5.0 per cent, showing that a stable plateau of prothrombin activator formation has been reached. Carry out a blank determination using in place of the preparation under examination, an equal quantity of a mixture of 1 volume of a 3.8 per cent w/v solution of sodium citrate and 5 volumes of saline solution. The result of the assay is not valid unless the clotting time in the blank determination is more than 40 seconds. Calculate the result of the assay by standard statistical methods. For total protein. Dilute 1.0 ml to 10.0 ml with saline solution. Determine the assay described under Human Plasma using 5.0 ml of the dilution and beginning at the words add 0.2 ml of a 7.5 per cent w/v solution..... For fibrinogen. Dilute 1.0 ml of the solution to 10.0 ml with a phosphate-saline buffer pH 6.5 and ionic strength 0.15. Clot 5.0 ml of the dilution with the minimum amount of thrombin, collect the clot and add three drops of a 30 per cent w/v solution of copper sulphate and 1 ml of nitrogen-free sulphuric acid and boil gently for 10 minutes; cool, add 1 g of anhydrous sodium sulphate and 10 mg of selenium, boil gently for 1 hour and cool. Transfer to an ammonia distillation apparatus, add 6 ml of a saturated solution of sodium hydroxide and pass steam through the flask; distil for seven minutes, collecting the distillate in a mixture of five ml of a saturated solution of boric acid, 5 ml of water, and 1 drop of a saturated solution of methyl red in alcohol containing 0.1 per cent of methylene blue, and titrate with 0.02 M hydrochloric acid. 1 ml of 0.02M hydrochloric acid is equivalent to 0.00175 g of fibrinogen. For sodium ions. To 10.0 ml of the solution add sufficient water to produce 100 ml, dilute 10.0 ml to 500 ml with water and determine the content of sodium ions by Method B for flame photometry (2.4.4), measuring at about 589 nm and using sodium solution FP suitably diluted with water as the standard solution. Storage. Store protected from light, in an atmosphere of nitrogen at a temperature below 8o. The containers are sterile and sealed so as to exclude micro-organisms. Labelling. The label states (1) the ABO blood group designation of the source of blood; (2) the number of Units in the container; (3) that 1 Unit is approximately equivalent to the antihaemophilic activity of 1 ml of average normal plasma; (4) the concentration of protein in g per litre and of sodium ions in millimoles per litre of the solution constituted as directed; (5) the maximum fibrinogen content; (6) where applicable, the number of Units of heparin in the container; (7) the name and amount of any other added substance contained in it; (8) the volume of Water for Injections necessary to constitute the solution; (9) the instructions for constitution 1451
FIBRIN SEALANT KIT
IP 2007
and that reconstitution may take upto 30 minutes; (10) that if the solution is not complete or if a gel forms on constitution, the preparation should not be used; (11) that the solution should be used as soon as possible and in any case within 3 hours of constitution and any unused solution should be discarded; (12) that the solution should be administered only with equipment that includes a filter; (13) the storage conditions.
and water. Component 1 (Fibrinogen Concentrate)
Identification
A. Complies with the limits of the assay of fibrinogen. B. Complies with the limits of the assay of factor XIII (where applicable).
Fibrin Sealant Kit

Fibrin Sealant Kit is essentially composed of two components, namely fibrinogen concentrate (component 1), a protein fraction containing human fibrinogen and a preparation containing human thrombin (component 2). A fibrin clot is rapidly formed when the two thawed or reconstituted components are mixed. Other ingredients (for example, human coagulation factor XIII, a fibrinolysis inhibitor or calcium ions) and stabilisers (for example, Human albumin solution may be added. No antimicrobial preservative is added. Human constituents are obtained from plasma that complies with the requirements of the monograph on Human Plasma for Fractionation. No antibiotic is added to the plasma used. When thawed or reconstituted as stated on the label, component 1 contains not less than 4.0 per cent of clottable protein; the thrombin activity of component 2 varies over a wide range (approximately 4-1000 IU per ml).
Tests
pH (2.4.24). 6.5 to 8.0. Stability of solution. No gel formation appears at room temperature during 120 min following thawing or reconstitution. Water. Determine by semi-microdetermination (2.3.43), loss on drying (2.4.19) or near infrared spectrophotometry (2.4.6), the water content is within the limits approved by the competent authority. Sterility (2.2.11). Complies with the test for sterility. Assay Fibrinogen (Clottable Protein) The estimated content in mg of clottable protein is not less than 70.0 per cent and not more than 130.0 per cent of the content stated on the label. Mix 0.2 ml of the reconstituted preparation with 2 ml of a suitable buffer solution pH 6.6 to 7.4 containing sufficient human thrombin (approximately 3 IU per ml) and calcium (0.05mol per l). Maintain at 37 for 20 minutes, separate the precipitate by centrifugation (5000 g, 20 minutes), wash thoroughly with a 0.9 per cent solution of sodium chloride and determine the protein as nitrogen by sulphuric acid digestion (2.3.30). Calculate the protein content by multiplying the result by 6.0. If for a particular preparation this method cannot be applied, use another validated method for determination of fibrinogen. Factor XIII Where the label indicates that the human coagulation factor XIII activity is greater than 10 Units per ml, the estimated activity is not less than 80.0 per cent and not more than 120.0 per cent of the activity stated on the label. Make at least 3 suitable dilutions of thawed or reconstituted component 1 and of human normal plasma (reference preparation) using as diluent coagulation factor XIII deficient plasma or another suitable diluent. Add to each dilution suitable amounts of the following reagents: a. activator reagent, containing bovine or human thrombin, a suitable buffer, calcium chloride and a suitable inhibitor such as Gly-Pro-Arg-Pro-Ala-NH2 which inhibits clotting of the sample but does not prevent coagulation factor XIII
Production
The method of preparation includes a step or steps that have been shown to remove or to inactivate known agents of infection; if substances are used for inactivation of viruses during production, the subsequent purification procedure must be validated to demonstrate that the concentration of these substances is reduced to a suitable level and any residues are such as not to compromise the safety of the preparation for patients. Constituents or mixtures of constituents are passed through a bacteria-retentive filter and distributed aseptically into sterile containers. Containers of freeze-dried constituents are closed under vacuum or filled with oxygen-free nitrogen or other suitable inert gas before being closed. In either case, they are closed so as to exclude micro-organisms. If the human coagulation factor XIII content in component 1 is greater than 10 Units per ml, the assay of coagulation factor XIII is carried out. Description. Freeze-dried constituents are hygroscopic, white or pale yellow powders or friable solids. Frozen constituents are colourless or pale yellow, opaque solids. Liquid constituents are colourless or pale yellow. For the freeze-dried or frozen constituents, reconstitute or thaw as stated on the label immediately before carrying out the Identification and the Tests, except those for solubility 1452
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HUMAN ALBUMIN
activation by thrombin, b. detection reagent, containing a suitable factor XIIIa-specific peptide substrate, such as Leu-Gly-Pro-Gly-Glu-Ser-Lys-ValIle-Gly-NH2 and glycine ethyl ester as 2nd substrate in a suitable buffer solution, c. NADH reagent, containing glutamate dehydrogenase, aketoglutarate and NADH in a suitable buffer solution. After mixing, the absorbance changes (A per min) are measured at a wavelength of 340 nm, after the linear phase of the reaction is reached. 1 Unit of factor XIII is equal to the activity of 1 ml of human normal plasma. Calculate the activity of the test preparation by the usual statistical methods. The confidence limits (P = 0.95) are not less than 80.0 per cent and not more than 125.0 per cent of the estimated activity. Component 2 (Thrombin Preparation)
of clottable protein), thrombin (International Units) per container, and coagulation factor XIII, if this is greater than 10 Units per ml; (2) where applicable, the name and volume of solvent to be used to reconstitute the components.
Human Albumin
Human Normal Albumin; Human Albumin Solution
Human Albumin is a sterile non-pyrogenic solution of the albumin component obtained from pooled human blood or from normal placentae frozen immediately after collection. It is obtained by fractionating source material such as blood, plasma, serum or placentae from healthy human donors and tested individually for the absence of hepatitis B surface antigen, HCV antibodies and HIV antibodies and complies with other tests and requirements prescribed by the appropriate national control authority. Source material obtained from donors who do not meet all the requirements stated may be used provided that it has been demonstrated to the national control authority that the process of fractionation will remove any known agent capable of adversely affecting the health of subjects treated with the preparation. It may be prepared from pooled source materials by precipitation with organic solvents under controlled conditions of pH, ionic strength and temperature or by chromatography or by any other method which does not affect the integrity of the product and has been shown to yield consistently a product containing not less than 95.0 per cent w/v of the total protein as albumin which is safe for intravenous injection. Residual organic solvent, if present, is removed by freeze-drying or other suitable treatment. The product is dissolved in sufficient water to obtain a suitable concentration, and a suitable stabilising agent is added to stabilise it to heat. It is prepared as a solution containing 15.0 to 25.0 per cent w/v of total protein or as an isotonic solution containing 4.0 to 5.0 per cent w/v of total protein. No antimicrobial agent is added at any stage during preparation and all processing steps are conducted in a manner to minimise risk of contamination from either micro-organisms or other deleterious matter. The solution is sterilised by filtration and distributed aseptically into containers which are then sealed so as to exclude micro-organisms. The solution is then heated to and maintained at 60o 0.5o for 10 hours so as to prevent the transmission of agents of infection transmissible by transfusion of blood or blood derivatives. Finally, the containers are stored for not less than 14 days at 30o to 32o or for not less than 4 weeks at 20o and examined visually. Those showing abnormalities such as abnormal colour, turbidity, microbial contamination, or presence of atypical particles must be discarded. Albumin Solution should be tested in accordance with the requirements decided by the National Regulatory Authority; in particular, tests for the absence of hepatitis B surface antigen and HIV antibodies are carried out by suitably sensitive methods.
Identification
Complies with the limits of the assay of thrombin.
Tests
pH (2.4.24). 5.0 to 8.0. Water. Determine by semi-microdetermination of water (2.3.43), loss on drying (2.4.19) or near infrared spectrophotometry (2.4.6), the water content is within the limits approved by the competent authority. Sterility (2.2.11). Complies with the test for sterility. Assay Thrombin The estimated activity is not less than 80.0 per cent and not more than 125.0 per cent of the activity stated on the label. If necessary, dilute the reconstituted preparation under examination to approximately 2-20 IU of thrombin per ml using as diluent a suitable buffer pH 7.3 to 7.5, such as imidazole buffer solution pH 7.3 containing 1.0 per cent of human albumin or bovine albumin. To a suitable volume of the dilution, add a suitable volume of fibrinogen solution (0.1 per cent of clottable protein) warmed to 37 and start measurement of the clotting time immediately. Repeat the procedure with each of at least 3 dilutions, in the range stated above, of a reference preparation of thrombin, calibrated in International Units. Calculate the activity of the test preparation by the usual statistical methods. The confidence limits (P = 0.95) are not less than 80.0 per cent and not more than 125.0 per cent of the estimated activity. Storage. Store protected from light. Labelling. The label states (1) the amount of fibrinogen (mg
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HUMAN ALBUMIN
IP 2007
Human Albumin contains not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of protein. It contains not less than 95.0 per cent and not more than 105.0 per cent of the contents of Na and K stated on the label which are, in any case, not more than 160 millimoles of Na per litre and 2 millimoles of K per litre. Description. A clear, slightly viscous liquid, ranging in colour from almost colourless to greenish-yellow or amber depending on protein concentration and the method of fractionation used.
the volume, V, of the eluate from the entry of the sample into the column to the apex of the first peak. Dilute the substance under examination with the salinephosphate solution to contain about 5.0 per cent w/v of protein, apply 2.5 ml to the column and elute under the above conditions, collecting the eluate in 5-ml portions. Three peaks may appear in similar positions to those in the chromatogram obtained from the normal human serum but the relative peak heights may be different. To the fraction eluted between volume 0.85 V and 1.15 V, add for each 10 ml, 0.4 ml of a 7.5 per cent w/v solution of sodium molybdate and 0.4 ml of a mixture of 1 part of nitrogen-free sulphuric acid and 30 parts of water, shake, centrifuge for 5 minutes and complete the Assay described under Human Plasma beginning at the words decant the supernatant.... The weight of protein in the fraction of the eluate is not more than 3.5 per cent of the weight of protein in the volume of the substance under examination applied to the column. Heat the substance under examination for 50 hours at 56.5o to 57.5o and repeat the chromatographic separation and the determination of the weight of protein in the fraction eluted between 0.85 V and 1.15 V. When expressed as a percentage of the weight of protein in the volume of the substance under examination applied to the column, it exceeds the percentage obtained before heating by not more than 1.5 per cent w/v. Protein composition. Not less than 95.0 per cent w/v of the total protein as albumin, when determined by the following method. Carry out Method II for cellulose acetate electrophoresis (2.4.12), using one strip of cellulose for each solution. Test solution. Dilute the substance under examination with saline solution to contain 2.0 per cent w/v of total protein. Reference solution. Dilute human albumin for electrophoresis RS with saline solution to obtain a solution containing 2.0 per cent w/v of total protein. Not more than 5.0 per cent of total protein is contained in bands other than the principal band in the strip obtained with test solution. The test is not valid if the proportion of the protein in the principal band is not within the limits stated in the leaflet supplied with human albumin for electrophoresis RS. Stability. The contents of the final container remain unchanged, as determined by visual inspection, after heating at 57o for 50 hours, when compared to its control consisting of a sample from the same lot which has not undergone this heating. Pyrogens (2.2.8). Complies with the test for pyrogens, using 3 ml per kg of the rabbits weight, irrespective of the protein content, in rabbits that have not previously received blood products. Sterility (2.2.11). Complies with the tests for sterility. Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity, using Method B and 0.5 ml of the solution for each mouse and 5 ml for each guinea-pig irrespective of the protein
Identification
A. It contains plasma proteins of human origin only as determined by precipitation tests with specific antisera. B. Ditermine the cellulose acetate by electrophoresis (2.4.12), using barbitone buffer pH 8.6, ionic strength 0.1 and human albumin for electrophoresis RS; 96.0 per cent of the protein has the mobility of human albumin.
Tests
Acidity or alkalinity (2.4.24). Dilute with sufficient saline solution to produce a solution containing 1.0 per cent w/v of protein; pH of the resulting solution, 6.7 to 7.3. Alkaline phosphatase. Not more than 0.1 Unit per g of protein, determined by the following method. Transfer a mixture of 0.5 ml of the substance under examination and 0.5 ml of diethanolamine buffer pH 10.0 to a spectrophotometer cell maintained at a temperature of 37o 0.2o and add 0.1 ml of nitrophenyl phosphate solution. Using a continuously recording spectrophotometer record the absorbance of the solution at about 405 nm (2.4.7), over a period of at least 30 seconds from the time of addition of the nitrophenyl phosphate solution. Calculate the alkaline phosphatase activity at 37o in Units per g of protein from the expression 118.3x/P, where x is the rate of increase of absorbance per minute and P is the content of total protein in g per litre, as determined in the Assay. Haem content. Dilute with sufficient saline solution to produce a solution containing 1.0 per cent w/v of protein; absorbance of the resulting solution at about 403 nm, not more than 0.15 (2.4.7). Denatured protein. Equilibrate a column (60 to 75 cm x 2.5 to 3.0 cm) of a gel of a cross-linked dextran suitable for fractionation of proteins in the range of molecular weight from 5,000 to 3,50,000 with a lower molecular weight for complete exclusion of globulin proteins of molecular weight between 4,00,000 and 5,00,000 (Sephadex G 150 is suitable) at 20o to 25o with a saline-phosphate solution prepared by mixing 2 volumes of saline solution and 1 volume of mixed phosphate buffer pH 7.0 with azide. Apply to the column 2.5 ml of normal human serum, previously clarified by centrifuging, and elute with the saline-phosphate solution at a rate of 20 ml per hour. Prepare a chromatogram by recording the absorbance (2.4.7), of the eluate at about 280 nm in relation to its volume. The chromatogram exhibits three well-defined peaks. Determine 1454
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HUMAN COAGULATION FACTOR IX
content. Assay For protein. Dilute to about 0.75 per cent w/v of total protein with saline solution. Take 2 ml of this solution in a roundbottomed centrifuge tube, add 2 ml of a 7.5 per cent w/v solution of sodium molybdate and 2 ml of a mixture of 30 volumes of water and 1 volume of nitrogen-free sulphuric acid. Shake, centrifuge for 5 minutes, decant the supernatant liquid and let the inverted tube stand on a filter paper to drain the fluid. Carry out Method E for determination of nitrogen (2.3.30), on the residue thus obtained and multiply the result by 6.25 to obtain the protein content. For sodium. Dilute to 0.01 per cent w/v of protein with water and determine by Method A for atomic absorption spectrophotometry (2.4.2), or by Method B for flame photometry (2.4.4), measuring at about 589 nm and using sodium solution FP suitably diluted with water as the standard solution. For potassium. Dilute to 0.25 per cent w/v of protein with water and determine by Method A for atomic absorption spectrophotometry (2.4.2), or by Method B for flame photometry (2.4.4), measuring at about 767 nm and using potassium solution FP suitably diluted with water as the standard solution. Human Albumin intended for administration to patients undergoing dialysis or to premature infants complies with the following additional test. Aluminium (2.3.8). Not more than 200 g of Al per litre. Determine by atomic absorption spectrophotometry (2.4.2), with a furnace as atomic generator and measuring at 309.3 nm and using as standard solutions a suitable range of dilutions in water of aluminium standard solution (10 ppm Al) further diluted, as necessary, with a solution containing 0.17 per cent w/v of magnesium nitrate and 0.05 per cent w/v of octoxinol 10 in a solution of nitric acid containing 1 per cent w/v of nitric acid. Prepare suitable dilutions of the preparation under examination and human albumin for aluminium validation RS with water. Dilute the solutions, as necessary, with the magnesium nitrate-octoxinol 10-nitric acid solution used for dilution of the standard solution. The test is valid only if the aluminium content determined for human albumin for aluminium validation RS is within 20 per cent of the stated value. NOTE Wash all equipments with a solution containing 20.0 per cent w/v of nitric acid before use and use plastic containers only to prepare all solutions. Storage. Store protected from light, at a temperature between 2o and 25o. Human Albumin stored at 2o to 8o may be expected to continue to meet the requirements of the monograph for 5 years from the date on which it was heated at 60o for 10 hours. Human Albumin stored at a temperature not exceeding 25o may be expected to continue to meet the requirements of the monograph for 3 years from the date on which it was heated at
60o for 10 hours. Labelling. The label states (1) the volume in the container; (2) the total amount of protein in the container expressed in g per litre or as percentage; (3) the concentration of sodium and potassium ions expressed in millimoles per litre; (4) the names and concentrations of any stabilising agents and any other additives in the final solution; (5) the type of source material used to manufacture the product; (6) the words Do not use if turbid; (7) that the contents must not be used more than 4 hours after the container has been penetrated and any remnant portion must be discarded; (8) the storage conditions; (9) the date after which the solution is not intended to be used; (10) either that the preparation is suitable for administration to patients undergoing dialysis and to premature infants or that it is not intended for such purpose.
Human Coagulation Factor IX

Human Coagulation Factor IX is a plasma protein fraction containing coagulation factor IX, prepared by a method that effectively separates factor IX from other prothrombin complex factors (factors II, VII and X). It is obtained from human plasma that complies with the monograph on Human Plasma for Fractionation. The potency of the preparation, reconstituted as stated on the label, is not less than 20 IU of factor IX per ml.
Production
The method of preparation is designed to maintain functional integrity of factor IX, to minimise activation of any coagulation factor (to minimise potential thrombogenicity) and includes a step or steps that have been shown to remove or to inactivate known agents of infection; if substances are used for inactivation of viruses during production, the subsequent purification procedure must be validated to demonstrate that the concentration of these substances is reduced to a suitable level and that any residues are such as not to compromise the safety of the preparation for patients. The specific activity is not less than 50 IU of factor IX per mg of total protein, before the addition of any protein stabiliser. The factor IX fraction is dissolved in a suitable liquid. Heparin, antithrombin and other auxiliary substances such as a stabiliser may be included. No antimicrobial preservative is added. The solution is passed through a bacteria-retentive filter, distributed aseptically into the final containers and immediately frozen. It is subsequently freeze-dried and the containers are closed under vacuum or under an inert gas. Consistency of the method The consistency of the method of production is evaluated by suitable analytical procedures that are determined during process development and which normally include (1) assay of factor IX; (2) determination of activated coagulation factors;
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HUMAN COAGULATION FACTOR VII
IP 2007
(3) determination of activities of factors II, VII and X which shall be shown to be not more than 5.0 per cent of the activity of factor IX. Description. A white or pale yellow, hygroscopic powder or friable solid. Reconstitute the preparation under examination as stated on the label, immediately before carrying out the Identification, Tests (except those for solubility and water ) and Assay.
IX (2.8.8). The estimated potency is not less than 80.0 per cent and not more than 125.0 per cent of the stated potency. The confidence limits (P = 0.95) are not less than 80.0 per cent and not more than 125.0 per cent of the estimated potency. Storage. Store protected from light. Labelling. The label states (1) the number of International Units of factor IX per container; (2) the amount of protein per container; (3) the name and quantity of any added substances including, where applicable, heparin; (4) the name and volume of the liquid to be used for reconstitution; (5) that the transmission of infectious agents cannot be totally excluded when medicinal products prepared from human blood or plasma are administered.
Identification
It complies with the limits of the Assay.
Tests
pH (2.4.24). 6.5 to 7.5. Osmolality (2.4.23). Minimum 240 mosmol per kg. Total protein. If necessary, dilute an accurately measured volume of the preparation under examination with a 0.9 per cent solution of sodium chloride, to obtain a solution which may be expected to contain about 15 mg of protein in 2 ml. To 2.0 ml of that solution, in a round-bottomed centrifuge tube, add 2 ml of a 7.5 per cent solution of sodium molybdate and 2 ml of a mixture of 1 volume of nitrogen-free sulphuric acid and 30 volumes of water. Shake, centrifuge for 5 minutes decant the supernatant liquid and allow the inverted tube to drain on filter paper. Determine the nitrogen in the residue by the method of sulphuric acid digestion (2.3.30) and calculate the amount of protein by multiplying the result by 6.25. For some products, especially those without a protein stabiliser such as albumin, this method may not be applicable. Another validated method for protein determination must therefore be performed. Activated coagulation factors (2.8.4). If necessary, dilute the preparation under examination to contain 20 IU of factor IX per ml. For each of the dilutions the coagulation time is not less than 150 seconds. Heparin. If heparin has been added during preparation, determine the amount by the assay of heparin in coagulation factor concentrates (2.8.10). The preparation under examination contains not more than the amount of heparin stated on the label and in any case not more than 0.5 IU of heparin per International Unit of factor IX. Water. Determine by semi-micro determination of water (2.3.43), loss on drying (2.4.19) or near infrared spectrophotometry (2.4.6), the water content is within the limits approved by the competent authority. Sterility (2.2.11). Complies with the test for sterility. Pyrogens (2.2.8). Complies with the test for pyrogens. Inject per kg of the rabbits mass a volume equivalent to not less than 50 IU of factor IX. Assay. Determine the assay of human blood coagulation factor 1456
Human Coagulation Factor VII

Human Coagulation Factor VII is a plasma protein fraction that contains the single-chain glycoprotein factor VII and may also contain small amounts of the activated form, the twochain derivative factor VIIa. It may also contain coagulation factors II, IX and X and protein C and protein seconds. It is obtained from human plasma that complies with the monograph on Human Plasma for Fractionation. The potency of the preparation, reconstituted as stated on the label, is not less than 15 IU of factor VII per ml.
Production
The method of preparation is designed to minimise activation of any coagulation factor (to minimise potential thrombogenicity) and includes a step or steps that have been shown to remove or to inactivate known agents of infection; if substances are used for inactivation of viruses during production, the subsequent purification procedure must be validated to demonstrate that the concentration of these substances is reduced to a suitable level and that any residues are such as not to compromise the safety of the preparation for patients. The specific activity is not less than 2 IU of factor VII per mg of total protein, before the addition of any protein stabiliser. The factor VII fraction is dissolved in a suitable liquid. Heparin, antithrombin and other auxiliary substances such as a stabiliser may be added. No antimicrobial preservative is added. The solution is passed through a bacteria-retentive filter, distributed aseptically into the final containers and immediately frozen. It is subsequently freeze-dried and the containers are closed under vacuum or under an inert gas. Consistency of the method The consistency of the method of production with respect to
IP 2007
HUMAN COAGULATION FACTOR VII
the activities of factors II, IX and X of the preparation, expressed in International Units relative to the activity of factor VII, shall be demonstrated. The consistency of the method of production with respect to the activity of factor VIIa of the preparation shall be demonstrated. The activity of factor VIIa may be determined, for example, using a recombinant soluble tissue factor that does not activate factor VII but possesses a cofactor function specific for factor VIIa; after incubation of a mixture of the recombinant soluble tissue factor with phospholipids reagent and the dilution of the test sample in factor VII-deficient plasma, calcium chloride is added and the clotting time determined; the clotting time is inversely related to the factor VIIa activity of the test sample. Description. A hygroscopic powder or friable solid that may be white, pale yellow, green or blue. Reconstitute the preparation under examination as stated on the label immediately before carrying out the Identification, Tests (except those for solubility and water ) and Assay.
1 IU of heparin). In each of 2 test-tubes, mix equal volumes of the reconstituted preparation and a 0.3 per cent solution of fibrinogen. Keep one of the tubes at 37 for 6 hours and the other at room temperature for 24 hours. In a third tube, mix a volume of the fibrinogen solution with an equal volume of a solution of human thrombin (1 IU per ml) and place the tube in a water-bath at 37. No coagulation occurs in the tubes containing the preparation under examination. Coagulation occurs within 30 seconds in the tube containing thrombin. Factor II Determine the assay of human coagulation factor II (2.8.5). The estimated content is not more than 125.0 per cent of the stated content. The confidence limits (P = 0.95) are not less than 90.0 per cent and not more than 111.0 per cent of the estimated potency. Factor IX Determine the assay of human coagulation factor IX (2.8.8). The estimated content is not more than 125.0 per cent of the stated content. The confidence limits (P = 0.95) are not less than 80.0 per cent and not more than 125.0 per cent of the estimated potency. Factor X Determine the assay of human coagulation factor X (2.8.9). The estimated content is not more than 125.0 per cent of the stated content. The confidence limits (P = 0.95) are not less than 90.0 per cent and not more than 111.0 per cent of the estimated potency. Water. Determine by semi-micro determination of water (2.3.43), loss on drying (2.4.19) or near-infrared spectrophotometry (2.4.6), the water content is within the limits approved by the competent authority. Sterility (2.2.11). Complies with the test for sterility. Pyrogens (2.2.8). Complies with the test for pyrogens. Inject per kg of the rabbits mass a volume equivalent to not less than 30 IU of factor VII. Assay Determine the assay of human coagulation factor VII (2.8.6). The estimated potency is not less than 80.0 per cent and not more than 125.0 per cent of the stated potency. The confidence limits (P = 0.95) are not less than 80.0 per cent and not more than 125.0 per cent of the estimated potency. Storage. Store protected from light. Labelling. The label states (1) the number of International Units of factor VII per container; (2) the maximum content of International Units of factor II, factor IX and factor X per
Identification
It complies with the limits of the assay.
Tests
pH (2.4.24). 6.5 to 7.5. Osmolality (2.4.23). Minimum 240 mosmol per kg. Total protein. If necessary, dilute an accurately measured volume of the reconstituted preparation with a 0.9 per cent solution of sodium chloride to obtain a solution expected to contain about 15 mg of protein in 2 ml. To 2.0 ml of the solution in a round-bottomed centrifuge tube add 2 ml of a 7.5 per cent solution of sodium molybdate and 2 ml of a mixture of 1 volume of nitrogen-free sulphuric acid and 30 volumes of water. Shake, centrifuge for 5 minutes, decant the supernatant liquid and allow the inverted tube to drain on filter paper. Determine the nitrogen in the residue by the method of sulphuric acid digestion (2.3.30) and calculate the amount of protein by multiplying the result by 6.25. Activated coagulation factors (2.8.4). For each of the dilutions, the coagulation time is not less than 150 seconds. Heparin. If heparin has been added during preparation, determine the amount present by the assay of heparin in coagulation factor concentrates (2.8.10). The preparation under examination contains not more than the amount of heparin stated on the label and in any case not more than 0.5 IU of heparin per International Unit of factor VII. Thrombin. If the preparation under examination contains heparin, determine the amount present as described in the test for heparin and neutralise the heparin by addition of protamine sulphate (10 g of protamine sulphate neutralises
1457
HUMAN COAGULATION FACTOR VIII (rDNA)
IP 2007
container; (3) the amount of protein per container; (4) the name and quantity of any added substances, including where applicable, heparin; (5) the name and volume of the liquid to be used for reconstitution; (6) that the transmission of infectious agents cannot be totally excluded when medicinal products prepared from human blood or plasma are administered.
Human Coagulation Factor VIII (rDNA)

Human Coagulation Factor VIII (rDNA) is a freeze-dried preparation of glycoproteins having the same activity as coagulation factor VIII in human plasma. It acts as a cofactor of the activation of factor X in the presence of factor IXa, phospholipids and calcium ions. It circulates in plasma mainly as a two-chain glycosylated protein with 1 heavy (relative molecular mass of about 2,00,000) and 1 light (relative molecular mass 80,000) chain held together by divalent metal ions. Human coagulation factor VIII (rDNA) is prepared as full-length factor VIII (octocog alfa), or as a shortened twochain structure (relative molecular mass 90,000 and 80,000), in which the B-domain has been deleted from the heavy chain (moroctocog alfa). Full-length human rDNA coagulation factor VIII contains 25 potential N-glycosylation sites, 19 in the B domain of the heavy chain, 3 in the remaining part of the heavy chain (relative molecular mass 90,000) and 3 in the light chain (relative molecular mass 80,000). The different products are characterised by their molecular size and post-translational modification and/or other modifications.
production, and for control of bulk and final preparation. They are derived from representative batches of purified bulk factor VIII (rDNA) that are extensively characterised by tests including those described below and whose procoagulant and other relevant functional properties have been ascertained and compared, wherever possible, with the International Standard for factor VIII concentrate. The reference preparations are suitably characterised for their intended purpose and are stored in suitably sized aliquots under conditions ensuring their stability. Purified bulk factor VIII (rDNA) The purified bulk complies with a suitable combination of the following tests for characterisation of integrity of the factor VIII (rDNA). Where any substance added during preparation of the purified bulk interferes with a test, the test is carried out before addition of that substance. Where applicable, the characterisation tests may alternatively be carried out on the finished product. Specific biological activity or ratio of factor VIII activity to factor VIII antigen Determine the assay of human coagulation factor VIII (2.8.7). The protein content, or where a protein stabiliser is present, the factor VIII antigen content, is determined by a suitable method and the specific biological activity or the ratio of factor VIII activity to factor VIII antigen is calculated. Protein composition The protein composition is determined by a selection of appropriate characterisation techniques which may include peptide mapping, Western blots, HPLC, gel electrophoresis, capillary electrophoresis, mass spectrometry or other techniques to monitor integrity and purity. The protein composition is comparable to that of the reference preparation. Molecular size. Using size-exclusion chromatography (2.4.16), the molecular size distribution is comparable to that of the reference preparation. Peptide mapping (2.3.47). There is no significant difference between the test protein and the reference preparation. Carbohydrates/sialic acid. To monitor batch-to-batch consistency, the monosaccharide content and the degree of sialylation or the oligosaccharide profile are monitored and correspond to those of the reference preparation. Final lot It complies with the tests under Identification, Tests and Assay. Excipients. 80.0 per cent to 120.0 per cent of the stated content, determined by a suitable method, where applicable. Description. A white or slightly yellow powder or friable mass.
Production
Human coagulation factor VIII (rDNA) is produced by recombinant DNA technology in mammalian cell culture. It is produced under conditions designed to minimise microbial contamination. Purified bulk factor VIII (rDNA) may contain added human albumin and/or other stabilising agents, as well as other auxiliary substances to provide, for example, correct pH and osmolality. The specific activity is not less than 2,000 IU of factor VIII:C per mg of total protein before the addition of any protein stabiliser, and varies depending on purity and the type of modification of molecular structure of factor VIII. The quality of the bulk preparation is controlled using reference preparations. Reference preparations During development, reference preparations are established for subsequent verification of batch consistency during
Identification
1458
IP 2007
HUMAN NORMAL IMMUNOGLOBULIN
A. It complies with the limits of the Assay. B. The distribution of characteristic peptide bands corresponds with that of the reference preparation (SDSPAGE or Western blot).
Tests
Reconstitute the preparation as stated on the label immediately before carrying out the Tests (except those for solubility and water ) and Assay. pH (2.4.24). 6.5 to 7.5. Osmolality (2.4.23). Minimum 240 mosmol per kg. Water. Determine by semi-micro determination of water (2.3.43), loss on drying (2.4.19) or near infrared spectrophotometry (2.4.6), the water content is within the limits approved by the competent authority. Sterility (2.2.11). Complies with the test for sterility. Bacterial endotoxins (2.2.3). Less than 3 IU in the volume that contains 100 IU of factor VIII activity. Assay Determine the assay of human coagulation factor VIII (2.8.7). The estimated potency is not less than 80.0 per cent and not more than 125.0 per cent of the stated potency. The confidence limits (P = 0.95) are not less than 80.0 per cent and not more than 120.0 per cent of the estimated potency. Storage. Store protected from light. Labelling. The label states (1) the factor VIII content in International Units; (2) the name and amount of any excipient; (3) the composition and volume of the liquid to be used for reconstitution.
Regulatory Authority; in particular, tests for hepatitis B surface antigen, HCV antibodies and for HIV antibodies must be carried out by suitable sensitive methods and must show negative results in both cases. Plasma, serum or placentae obtained from donors who do not meet all the requirements stated above may be used as source material provided that it has been demonstrated to the national authority that process of fractionation and production removes any known agent capable of adversely affecting the health of subjects treated with the preparation. No antibiotic is added to the source materials used for the preparation of immunoglobulin. It is prepared from pooled material of a minimum volume of 25 litres by a method which has been shown (a) to be capable of concentrating tenfold from source material at least two different antibodies, one viral and one bacterial, for which an International Standard or Reference Preparation is available; (b) not to affect the integrity of the globulins; (c) to consistently yield a product which is safe for intramuscular injection and; (d) to yield a product that does not transmit viral hepatitis or any other infection. The liquid preparation is prepared as a stabilised solution in saline solution or a 2.25 per cent w/v solution of glycine or other suitable agent and is sterilised by filtration and distributed into previously sterilised containers which are then sealed so as to exclude micro-organisms. An antimicrobial preservative may be added except when the preparation is to be freeze-dried. Any antimicrobial preservative or stabilising agent added must be such that neither deleterious effect on the final product in the amounts present nor capability to cause untoward reactions in human beings is demonstrated. An accelerated degradation test is carried out on the final liquid or freeze-dried preparation by heating at 37o for 4 weeks. The difference between the percentages of protein eluted in the fractions following the main peak as determined by sizeexclusion chromatography (2.4.16), before and after exposure at 37o does not exceed 5.0 per cent. Human Normal Immunoglobulin contains not less than 90.0 per cent and not more than 110.0 per cent of the quantity of protein stated on the label and in any case, not less than 10.0 per cent w/v and not more than 18.0 per cent w/v of protein. Description. The liquid preparation is clear pale yellow or brownish in colour; on storage it may show turbidity or a small amount of particulate matter. The freeze-dried preparation is a white to slightly yellow powder or solid, friable mass.
Human Normal Immunoglobulin

Normal Immunoglobulin; Immune Human Serum Globulin; Human Gamma Globulin Human Normal Immunoglobulin is a sterile solution or freezedried preparation containing immunoglobulins, mainly immunoglobulin G (IgG), together with smaller amounts of other plasma proteins.
Production
It is obtained from source materials such as the blood, plasma, serum or placentae frozen immediately after collection from healthy donors who must as far as can be ascertained after clinical examination, laboratory tests on their blood and a study of their medical history, be free from disease transmissible by transfusion of blood or blood products. The examinations and tests to be carried out are decided by the National
Identification
A. Precipitation tests with a suitable range of species-specific antisera which give positive results for the presence of proteins of human origin and negative results with antisera specific to plasma proteins of the other species. B. Examine by electrophoresis (2.4.12), using the moving boundary technique and a 1.0 per cent w/v solution in barbitone buffer solution pH 8.6 of ionic strength 0.1. At 1459
HUMAN NORMAL IMMUNOGLOBULIN
IP 2007
least 90.0 per cent w/v of the protein has a mobility not greater than 2.8 x 10-5 cm2V-1S-1.
cent of the total area of the chromatogram represents proteins eluted after IgG monomer and albumin (area C).
Tests
pH (2.4.24). 6.4 to 7.2, determined in a solution prepared by dilution of a quantity with saline solution so as to contain 1.0 per cent w/v of protein. Protein composition. Determine by cellulose acetate electrophoresis (2.4.12), but applying an electric field such that the albumin band of normal human serum applied in a control strip migrates at least 30 mm and using one strip of cellulose acetate for each of the following solutions: Test solution. Dilute the preparation under examination with saline solution to produce a solution containing 5 per cent w/ v of protein. Reference solution. Reconstitute human immunoglobulin for electrophoresis RS with saline solution to produce a solution containing 5 per cent w/v of protein. Calculate the result as the mean of three measurements of the absorbance of each strip. In the electrophoretogram obtained with test solution not more than 10.0 per cent of the protein is contained in bands other than the principal band. The test is not valid unless the proportion of protein in the principal band in the electrophoretogram obtained with reference solution is within the limits stated in the leaflet supplied with human immunoglobulin for electrophoresis RS. Molecular size. Determine by size-exclusion chromatography (2.4.16), applying 2 ml of the preparation under examination diluted with mixed phosphate buffer pH 7.0 with azide to produce a solution containing 4.0 to 5.0 per cent w/v of protein. Chromatographic system a column 1 m x 25 mm packed with agarose trapped within a cross-linked polyacrylamide network and having a linear fraction range suitable for fractionation of globular proteins in the range of molecular weights from 20,000 to 350,000, mobile phase: mixed phosphate buffer pH 7.0 with azide, flow rate 20 ml per hour (4 ml per cm2 of column cross-sectional area), spectrophotometer set at 280 nm. Collect the eluate in fractions of about 4 ml. Examine the chromatogram in comparison with that in Fig.1. The sum of the areas of the peaks containing IgG monomer, dimer, albumin and other proteins of similar molecular size (area B) is not less than 85.0 per cent of the total area of the chromatogram. Not more than 10.0 per cent of the total area of the chromatogram represents proteins eluted ahead of IgG dimer (area A); if area A can be subdivided into two distinct areas, the area corresponding to larger proteins does not exceed 5.0 per cent of the total area of the chromatogram. Not more than 5.0 per 1460 Fig.1 Typical Chromatogram for Human Normal Immunoglobulin
Stability. Heat approximately 2 ml at 57o for 4 hours in a stoppered glass tube (75 mm x 12 mm); no gelation or flocculation occurs. Pyrogens (2.2.8). Complies with the test for pyrogens, using 1 ml of the preparation under examination per kg of the rabbits weight. Sterility (2.2.11). Complies with the tests for sterility. Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity, Method A, injecting 0.5 ml into each mouse and 5 ml into each guinea-pig. Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. Dilute a suitable volume with water to produce a solution containing 1.0 per cent w/v of protein. Take 1.5 ml of the dilution in a round-bottomed centrifuge tube. Add 5 ml of water, mix, add 0.2 ml of 7.5 per cent w/v solution of sodium molybdate and 2 ml of a mixture consisting of 1 volume nitrogen free sulphuric acid and 30 volumes of water. Shake, centrifuge for five minutes, decant the supernatant liquid and allow the inverted tube to drain on filter paper. To the residue in the tube add three drops of a 30 per cent w/v solution of copper sulphate and 1 ml of nitrogen-free sulphuric acid and boil gently for 10 minutes; cool, add 1 g of anhydrous sodium sulphate and 10 mg of selenium, boil gently for 1 hour and cool. Transfer to an ammonia distillation apparatus, add 6 ml of a saturated solution of sodium hydroxide and pass steam through the flask; distil for seven minutes, collecting the distillate in a mixture of 5 ml of a saturated solution of boric acid, 5 ml of water, and 1 drop saturated solution of
IP 2007
HUMAN PLASMA PROTEIN FRACTION
methyl red in alcohol containing 0.1 per cent of methylene blue, and titrate with 0.02 M hydrochloric acid. 1ml of 0.02 M hydrochloric acid is equivalent to 0.00175 g of protein. Freeze-dried Human Normal Immunoglobulin complies with the following additional requirements. Solubility rate. Add the volume of the liquid stated on the label and allow it to stand for 15 minutes at a temperature of 20o to 25o; it dissolves completely. Loss on drying (2.4.19). Not more than 2.0 per cent, determined on 0.5 g, by drying over phosphorus pentoxide at a pressure not exceeding 3 Pa for 24 hours. Human Normal Immunoglobulin intended for use in the prevention of infective hepatitis (hepatitis A) complies with the following additional requirement. Anti-hepatitis A activity. Determine the anti-hepatitis A activity by comparison with the activity of the Standard preparation, using an immunoassay of suitable sensitivity and specificity. The stated potency is not less than 100 Units per ml. The estimated potency is not less than the stated potency. The fiducial limits of error are not less than 80.0 per cent and not more than 125.0 per cent. Standard Preparation. The Standard Preparation is the 1st International Reference Preparation for Hepatitis A immunoglobulin, established in 1981, consisting of freeze-dried material derived from fractionated plasma (supplied in ampoules containing 100 Units), or another suitable preparation the antigen binding of which has been determined in relation to the International Reference Preparation. Storage. Store protected from light, the liquid preparation in sealed, colourless, glass containers, at a temperature between 2o and 8o. Store the freeze-dried preparation under vacuum or under an inert gas. Labelling. The label states (1) the volume and the protein concentration expressed in g per litre or, for freeze-dried preparations, the total amount of protein in the container; (2) the type of source material; (3) the name and quantity of any added preservative or stabilising agent; (4) the recommended human dose; (5) that it is meant for intramuscular injection only; (6) the storage conditions.
Production
The plasma or serum is obtained from healthy human donors who must, after clinical examination, laboratory tests on their blood and a study of their medical history, be free from detectable agents of infection transmissible by transfusion of blood or blood derivatives. The examinations and tests to be carried out are decided by the National Regulatory Authority; in particular tests for hepatitis B surface antigen and for HIV antibodies are carried out by suitable sensitive methods and must give negative results in both cases. Other diseasecausative agents that are not destroyed or removed by the processing method must not be present. Plasma or serum obtained from donors who do not meet all the stated requirements may be used as source material provided that it has been demonstrated to the national authority that the process of fractionation will remove any known agent capable of adversely affecting the health of recipients of the Human Plasma Protein Fraction. The separation of the protein may be done by precipitation with suitable organic solvents under controlled conditions, particularly of pH, ionic strength and temperature, so that in the final product not less than 85.0 per cent of the total protein is albumin. Residual solvent, if present, may be removed by freeze-drying or other suitable treatment. Alternative methods of preparation which shall not affect the integrity of the product and shall have been shown to yield consistently a product which is safe for intravenous injection may be adopted. The product is dissolved in water and sufficient quantities of a suitable stabiliser against the effect of heat, like sodium caprylate in a suitable concentration, and sufficient sodium chloride to adjust the sodium ions to between 130 and 160 millimoles per litre may be added but no antibiotic or antimicrobial preservative is added at any stage during preparation. The solution is sterilised by filtration through a bacteria-retentive filter and distributed aseptically into sterile containers which are then closed so as to prevent microbial contamination. The solution in its final container is heated at 60o 0.5o and maintained at this temperature for 10 hours. The containers are then incubated at 30o to 32o for not less than 14 days or at 20o to 25o for not less than 4 weeks and examined visually for evidence of microbial contamination. Those showing abnormalities such as abnormal colour, turbidity, presence of atypical particles or microbial contamination are discarded. Human Plasma Protein Fraction contains not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of total protein. Description. A clear, almost colourless or pale yellow liquid; almost odourless. On storage a dust-like precipitate may develop but it disappears on shaking.
Human Plasma Protein Fraction

Plasma Protein Solution; Human Albumin Fraction (Saline); Plasma Protein Fraction; PPF Human Plasma Protein Fraction is a sterile isotonic aqueous solution of proteins of plasma or serum containing albumin and globulins. It is prepared as an isotonic solution containing 4.0 to 5.0 per cent w/v of total protein. It contains no fibrinogen or antibodies.
Identification
A. Precipitation tests with specific antisera show that the preparation consists only of plasma proteins of human origin only and gives negative results with antisera specific to plasma 1461
HUMAN PLASMA PROTEIN FRACTION
IP 2007
proteins of other species. B. Examine by a suitable immunoelectrophoresis technique. Using antiserum to normal human serum, compare normal human serum and the preparation under examination, both diluted to contain 1.0 per cent w/v of protein. The main component of the preparation under examination corresponds to the main component of the normal human serum. The solution may show the presence of small quantities of other plasma proteins.
electrophoresis RS. Sodium. Not less than 95.0 per cent and not more than 105.0 per cent of the stated amount and, in any case not more than 160 millimoles of Na per litre, determined by Method A for atomic absorption spectrophotometry (2.4.2), measuring at 589 nm and using sodium solution AAS suitably diluted with water to prepare the standard solutions. Potassium. Not more than 50 mol of K per g of protein, determined by atomic absorption spectrophotometry (2.4.2), measuring at 766 nm and using potassium solution AAS, suitably diluted with water to prepare the standard solutions. Alkaline phosphatase. Not more than 0.1 Unit per g of protein, determined by the following method. Transfer a mixture of 0.5 ml of the substance under examination and 0.5 ml of diethanolamine buffer pH 10.0 to a spectrophotometer cell maintained at a temperature of 37o 0.2o and add 0.1 ml of nitrophenyl phosphate solution. Record the absorbance of the solution at about 405 nm (2.4.7), over a period of at least 30 seconds from the time of addition of the nitrophenyl phosphate solution. Calculate the alkaline phosphatase activity at 37o in Units per g of protein from the expression 118.3x/P, where x is the rate of increase of absorbance per minute and P is the content of total protein in g per litre, as determined in the Assay. Haem content. Dilute with sufficient saline solution to produce a solution containing 1.0 per cent w/v of protein; absorbance of the resulting solution at about 403 nm, not more than 0.15 (2.4.7). Sterility (2.2.11). Complies with the tests for sterility. Abnormal toxicity (2.2.1). Complies with the test for abnormal toxicity, using Method B and 0.5 ml of the solution for each mouse and 5 ml for each guinea-pig irrespectiveof the protein content. Pyrogens (2.2.8). Complies with the test for pyrogens, using 3 ml per kg of the rabbits weight in rabbits that have not previously received blood products. Assay For protein. Dilute to about 0.75 per cent w/v of total protein with saline solution. Take 2 ml of this solution in a roundbottomed centrifuge tube, add 2 ml of a 7.5 per cent w/v solution of sodium molybdate and 2 ml of a mixture of 30 volumes of water and 1 volume of nitrogen-free sulphuric acid. Shake, centrifuge for 5 minutes, decant the supernatant liquid and let the inverted tube stand on a filter paper to drain the fluid. Carry out Method E for determination of nitrogen (2.3.30), on the residue thus obtained and multiply the result by 6.25 to obtain the protein content. Storage. Store protected from light at a temperature between 2o and 25o. Labelling. The label states (1) the volume in the container; (2) the total amount of protein in the container expressed as a percentage or in grams per litre; (3) the concentration of sodium
Tests
pH (2.4.24). 6.7 to 7.3, determined in a solution prepared by diluting with sufficient of saline solution to produce a solution containing 1.0 per cent w/v of protein. Polymers and aggregates. Determine by size-exclusion chromatography (2.4.16), applying 2 ml of the preparation under examination. Chromatographic system a column 1 m x 25 mm packed with a cross-linked dextran suitable for fractionation of globular proteins in the range of molecular weights from 5,000 to 3,50,000 (such as Sephadex G-150), mobile phase. mixed phosphate buffer pH 7.0 with azide, flow rate 20 ml per hour (4 ml per square centimeter of column cross-sectional area), spectrophotometer set at 280 nm. Collect the eluate in fractions of about 4 ml and combine the fractions corresponding to each peak. For each combined fraction, determine by Method E for determination of nitrogen (2.3.30). 1 ml of 0.02M hydrochloric acid is equivalent to 0.00028 g of nitrogen. Not more than 10.0 per cent of the total nitrogen is present in the combined fraction associated with non-retained proteins. Protein composition. Carry out by cellulose acetate electrophoresis (2.4.12), Method II using one strip for each solution. Test solution. Dilute the preparation under examination with saline solution to obtain a solution containing 2.0 per cent w/v of protein. Reference solution. Dilute human plasma protein fraction for electrophoresis RS with saline solution to obtain a solution containing 2.0 per cent w/v of protein. Calculate the result as the mean of three measurements of the absorbance of each of the 10 strips. In the electrophoretogram obtained with test solution not more than 15.0 per cent of the protein is contained in bands other than the principal band. The test is not valid unless the proportion of protein in the principal band in the electrophoretogram obtained with reference solution is within the limits stated in the leaflet supplied with human plasma protein fraction for 1462
IP 2007
HUMAN PROTHROMBIN COMPLEX
ions in millimoles per litre; (4) the names and concentrations of stabilising agents and of any other added substances present in the final solution; (5) that the solution should not be used if the solution is cloudy or shows a deposit which does not disappear on shaking; (6) that, once the container has been penetrated, the contents must be used within 4 hours and any unused solution discarded; (7) the storage conditions.
Tests
pH (2.4.24). 6.5 to 7.5. Osmolality (2.4.23). Minimum 240 mosmol per kg. Total protein. If necessary, dilute an accurately measured volume of the reconstituted preparation with a 0.9 per cent w/v solution of sodium chloride to obtain a solution expected to contain about 15 mg of protein in 2 ml. To 2.0 ml of the solution in a round-bottomed centrifuge tube add 2 ml of a 7.5 per cent w/v solution of sodium molybdate and 2 ml of a mixture of 1 volume of nitrogen-free sulphuric acid and 30 volumes of water. Shake, centrifuge for 5 minutes, decant the supernatant liquid and allow the inverted tube to drain on filter paper. Determine the nitrogen in the residue by the method of sulphuric acid digestion (2.3.30) and calculate the amount of protein by multiplying the result by 6.25. Activated coagulation factors (2.8.4). If necessary, dilute the preparation under examination to contain 20 IU of factor IX per ml. For each of the dilutions, the coagulation time is not less than 150 seconds. Heparin. If heparin has been added during preparation, determine the amount present by the assay of heparin in coagulation factor concentrates (2.8.10). The preparation under examination contains not more than the amount of heparin stated on the label and in any case not more than 0.5 IU of heparin per International Unit of factor IX. Thrombin. If the preparation under examination contains heparin, determine the amount present as described in the test for heparin and neutralise it by addition of protamine sulphate (10 g of protamine sulphate neutralises 1 IU of heparin). In each of 2 test-tubes, mix equal volumes of the reconstituted preparation and a 0.3 per cent w/v solution of fibrinogen. Keep one of the tubes at 37 for 6 hours and the other at room temperature for 24 hours. In a third tube, mix a volume of the fibrinogen solution with an equal volume of a solution of human thrombin (1 IU/ml) and place the tube in a water-bath at 37. No coagulation occurs in the tubes containing the preparation under examination. Coagulation occurs within 30 seconds in the tube containing thrombin. Water. Determine by semi-micro determination of water (2.3.43), loss on drying (2.4.19) or near-infrared spectrometry (2.4.6), the water content is within the limits approved by the competent authority. Sterility (2.2.11). Complies with the test for sterility. Pyrogens (2.2.8). Complies with the test for pyrogens. Inject per kg of the rabbits mass a volume of the reconstituted preparation equivalent to not less than 30 IU of factor IX. Assay Factor IX Determine the assay of human coagulation factor IX (2.8.8). The estimated potency is not less than 80.0 per cent and not 1463
Human Prothrombin Complex

Human Prothrombin Complex is a plasma protein fraction containing blood coagulation factor IX together with variable amounts of coagulation factors II, VII and X; the presence and proportion of these additional factors depends on the method of fractionation. It is obtained from human plasma that complies with the monograph on Human Plasma for Fractionation. The potency of the preparation, reconstituted as stated on the label, is not less than 20 IU of factor IX per ml.
Production
The method of preparation is designed to minimise activation of any coagulation factor (to minimise potential thrombogenicity) and includes a step or steps that have been shown to remove or to inactivate known agents of infection; if substances are used for inactivation of viruses during production, the subsequent purification procedure must be validated to demonstrate that the concentration of these substances is reduced to a suitable level and that any residues are such as not to compromise the safety of the preparation for patients. The specific activity is not less than 0.6 IU of factor IX per mg of total protein, before the addition of any protein stabiliser. The prothrombin complex fraction is dissolved in a suitable liquid. Heparin, antithrombin and other auxiliary substances such as a stabiliser may be added. No antimicrobial preservative is added. The solution is passed through a bacteria-retentive filter, distributed aseptically into the final containers and immediately frozen. It is subsequently freezedried and the containers are closed under vacuum or under an inert gas. Description. A white or slightly coloured powder or friable solid, very hygroscopic. Reconstitute the preparation under examination as stated on the label immediately before carrying out the Identification, Tests (except those for solubility and water ) and Assay.
Identification
It complies with the limits of the assay for coagulation factor IX activity and, where applicable, those for factors II, VII and X.
NORMAL IMMUNOGLOBULINFOR INTRAVENOUS USE
IP 2007
more than 125.0 per cent of the stated potency. The confidence interval (P = 0.95) of the estimated potency is not greater than 80.0 per cent to 125.0 per cent. Factor II Determine the assay of human coagulation factor II (2.8.5). The estimated potency is not less than 80.0 per cent and not more than 125.0 per cent of the stated potency. The confidence interval (P = 0.95) of the estimated potency is not greater than 90.0 per cent to 111.0 per cent. Factor VII If the label states that the preparation contains factor VII, Determine the assay of human coagulation factor VII (2.8.6). The estimated potency is not less than 80.0 per cent and not more than 125.0 per cent of the stated potency. The confidence interval (P = 0.95) of the estimated potency is not greater than 80.0 per cent to 125.0 per cent. Factor X Determine the assay of human coagulation factor X (2.8.9). The estimated potency is not less than 80.0 per cent and not more than 125.0 per cent of the stated potency. The confidence interval (P = 0.95) of the estimated potency is not greater than 90.0 per cent to 111.0 per cent. Storage. Store protected from light. Labelling. The label states (1) the number of International Units of factor IX, factor II and factor X per container; (2) where applicable, the number of International Units of factor VII per container; (3) where applicable, that the preparation contains protein C and/or protein S; (4) the amount of protein per container; (5) the name and quantity of any added substances, including where applicable, heparin; (6) the name and quantity of the liquid to be used for reconstitution; (7) that the transmission of infectious agents cannot be totally excluded when medicinal products prepared from human blood or plasma are administered.
Human normal immunoglobulin for intravenous administration is obtained from plasma that complies with the requirements of the monograph on Human plasma for fractionation. No antibiotic is added to the plasma used.
Production
The method of preparation includes a step or steps that have been shown to remove or to inactivate known agents of infection; if substances are used for inactivation of viruses, it shall have been shown that any residues present in the final product have no adverse effects on the patients treated with the immunoglobulin. The product shall have been shown, by suitable tests in animals and evaluation during clinical trials, to be well tolerated when administered intravenously. Human normal immunoglobulin for intravenous administration is prepared from pooled material from not fewer than 1,000 donors by a method that has been shown to yield a product that (a) does not transmit infection; (b) at an immunoglobulin concentration of 50 g per litre, contains antibodies for at least 2 of which (one viral and one bacterial) an International Standard or Reference Preparation is available, the concentration of such antibodies being at least 3 times that in the initial pooled material; (c) has a defined distribution of immunoglobulin G subclasses; (d) complies with the test for Fc function of immunoglobulin. Test for Fc function of immunoglobulin Stabilised human blood. Collect group O human red blood into ACD anticoagulant solution. Store the stabilised blood at 4 for not more than 3 weeks. Phosphate buffered saline pH 7.2. Dissolve 1.022 g of anhydrous disodium hydrogen phosphate, 0.336 g of anhydrous sodium dihydrogen phosphate and 8.766 g of sodium chloride in 800 ml of water and dilute to 1,000 ml with the same solvent. Magnesium and calcium stock solution. Dissolve 1.103 g of calcium chloride and 5.083 g of magnesium chloride in water and dilute to 25 ml with the same solvent. Barbital buffer stock solution. Dissolve 207.5 g of sodium chloride and 25.48 g of barbital sodium in 4,000 ml of water and adjust to pH 7.3 using 1 M hydrochloric acid. Add 12.5 ml of magnesium and calcium stock solution and dilute to 5,000 ml with water. Filter through a membrane filter (pore size 0.22 m). Store at 4 in glass containers. Albumin barbital buffer solution. Dissolve 0.150 g of bovine albumin in 20 ml of barbital buffer stock solution and dilute to 100 ml with water. Tannic acid solution. Dissolve 10 mg of tannic acid in 100 ml of phosphate-buffered saline pH 7.2. Prepare immediately before use. Guinea-pig complement. Prepare a pool of serum from the blood of not fewer than 10 guinea-pigs. Separate the serum
Normal Immunoglobulin for Intravenous Use

Human Normal Immunoglobulin for Intravenous Administration Human Normal Immunoglobulin for Intravenous Administration is a liquid or freeze-dried preparation containing immunoglobulins, mainly immunoglobulin G (IgG). Other proteins may be present. Human normal immunoglobulin for intravenous administration contains the IgG antibodies of normal subjects. This monograph does not apply to products intentionally prepared to contain fragments or chemically modified IgG. 1464
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from the clotted blood by centrifugation at about 4. Store the serum in small amounts below -70. Immediately before starting complement-initiated haemolysis, dilute to 125 to 200 CH50 per ml with albumin barbital buffer solution and store in an icebath during the test. Rubella antigen. Suitable rubella antigen for haemagglutination-inhibition titre (HIT). Titre > 256 HA units. Preparation of tanned human red blood cells. Separate human red blood cells by centrifuging an appropriate volume of stabilised human blood and wash the cells at least 3 times with phosphate-buffered saline pH 7.2 and suspend at 2 per cent v/v in phosphate-buffered saline pH 7.2. Dilute 0.1 ml of tannic acid solution to 7.5 ml with phosphate-buffered saline pH 7.2 (final concentration 1.3 mg per litre). Mix 1 volume of the freshly prepared dilution with 1 volume of human red blood cell suspension and incubate at 37 for 10 minutes. Collect the cells by centrifugation (400 to 800 g for 10 minutes), discard the supernatant and wash the cells once with phosphate-buffered saline pH 7.2. Resuspend the tanned cells at 1.0 per cent v/v in phosphate-buffered saline pH 7.2. Antigen coating of tanned human red blood cells. Take a suitable volume (Vs) of tanned cells, add 0.2 ml of rubella antigen per 1.0 ml of tanned cells and incubate at 37 for 30 minutes Collect the cells by centrifugation (400 to 800 g for 10 minutes) and discard the supernatant, leaving a volume of 200 l. Add a volume of albumin barbital buffer solution equivalent to the discarded supernatant, resuspend and collect the cells as described and repeat the washing procedure. Make up the remaining 200 l to three-quarters of Vs, thereby obtaining the initial volume (Vi). Mix 900 l of albumin barbital buffer solution with 100 l of Vi, which is thereby reduced to the residual volume (Vr), and determine the initial absorbance at 541 nm (A). Dilute Vr by a factor equal to A using albumin barbital buffer solution, thereby obtaining the final adjusted volume Vf = Vr A of sensitised human red blood cells and adjusting A to 1.0 0.1 for a tenfold dilution. Antibody binding of antigen-coated tanned human red blood cells. Prepare the following solutions in succession and in duplicate, using for each solution a separate half-micro cuvette (for example, disposable type) or test-tube: Test solutions. If necessary, adjust the immunoglobulin under examination to pH 7, for example by addition of 1 M sodium hydroxide. Dilute volumes of the preparation under examination containing 30 mg and 40 mg of immunoglobulin with albumin barbital buffer solution and adjust the volume to 900 l. Reference solutions. Prepare as for the test solutions using human immunoglobulin RS. Complement control. 900 l of albumin barbital buffer solution. Add to each cuvette/test-tube 100 l of sensitised human red blood cells and mix well. Incubate at room temperature for 15 minutes, add 1,000 l of albumin barbital buffer solution, collect the cells by
centrifugation (1,000 g for 10 min) of the cuvette/test-tube and remove 1,900 l of the supernatant. Replace the 1,900 l with albumin barbital buffer solution and repeat the whole of the washing procedure, finally leaving a volume of 200 l. Test samples may be stored in sealed cuvette/test-tubes at 4 for 24 hours. Complement-initiated haemolysis. To measure haemolysis, add 600 l of albumin barbital buffer solution warmed to 37 to the test sample, re-suspend the cells carefully by repeated pipetting (not fewer than 5 times) and place the cuvette in the thermostatted cuvette holder of a spectrophotometer. After 2 minutes, add 200 l of diluted guinea-pig complement (125 to 200 CH50/ml), mix thoroughly by pipetting twice and start immediately after the second pipetting the time-dependent recording of absorbance at 541 nm, using albumin barbital buffer solution as the compensation liquid. Stop the measurement if absorbance as a function of time has clearly passed the inflexion point. Evaluation. Determine the slope (S) of the haemolysis curve at the approximate inflexion point by segmenting the steepest section in suitable time intervals t (for example, t = 1 minute) and calculate S between adjacent intersection points, expressed as A per minute. The largest value for S serves as (Sexp). In addition, determine the absorbance at the start of measurement (As) by extrapolating the curve, which is almost linear and parallel to the time axis within the first few minutes. Correct (Sexp) using the expression:
S =
'
S exp As
Calculate the arithmetic mean of the values of S for each preparation. Calculate the index of Fc function (IFc) from the expression:
I Fc = 100 S ' S ' c S' s S'c
_ S = arithmetic mean of the corrected slope for the preparation under examination, _ Ss = arithmetic mean of the corrected slope for the reference preparation, _ Sc = arithmetic mean of the corrected slope for the complement control. Calculate the index of Fc function for the preparation under examination; the value is not less than that stated in the leaflet accompanying the reference preparation. Human normal immunoglobulin for intravenous administration is prepared as a stabilised solution or as a freeze-dried 1465
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preparation. A stabiliser may be added. In both cases the preparation is passed through a bacteria-retentive filter. The preparation may subsequently be freeze-dried and the containers closed under vacuum or under an inert gas. No antimicrobial preservative is added either during fractionation or at the stage of the final bulk solution. The stability of the preparation is demonstrated by suitable tests carried out during development studies. Description. The liquid preparation is clear or slightly opalescent and colourless or pale yellow. The freeze-dried preparation is a hygroscopic, white or slightly yellow powder or solid friable mass. For the freeze-dried preparation, reconstitute as stated on the label immediately before carrying out the identification and the tests, except those for solubility and water.
0.9 per cent w/v solution of sodium chloride to an immunoglobulin concentration of 3.0 per cent w/v. Reference solution. Reconstitute human immunoglobulin for electrophoresis reference preparation and dilute with a 0.9 per cent w/v solution of sodium chloride to a protein concentration of 3.0 per cent w/v. To a strip apply 4 l of the test solution as a 10 mm band or apply 0.4 l per mm if a narrower strip is used. To another strip apply in the same manner the same volume of the reference solution. Apply a suitable electric field such that the albumin band of normal human serum applied on a control strip migrates at least 30 mm. Stain the strips with amido black 10B solution for 5 minutes. Decolourise with a mixture of 10 volumes of glacial acetic acid and 90 volumes of methanol so that the background is just free of colour. Develop the transparency of the strips with a mixture of 19 volumes of glacial acetic acid and 81 volumes of methanol. Measure the absorbance of the bands at 600 nm in an instrument having a linear response over the range of measurement. Calculate the result as the mean of 3 measurements of each strip. System suitability. In the electropherogram obtained with the reference preparation, the proportion of protein in the principal band is within the limits stated in the leaflet accompanying the reference preparation. Results. In the electropherogram obtained with the test solution, not more than 5.0 per cent of protein has a mobility different from that of the principal band. This limit is not applicable if albumin has been added to the preparation as a stabiliser; for such preparations, a test for protein composition is carried out during manufacture before addition of the stabiliser. Molecular size. Determine by liquid chromatography (2.4.14). Test solution. Dilute the preparation under examination with a 0.9 per cent w/v solution of sodium chloride to obtain a concentration in the range of 0.4 to1.2 per cent w/v and injection of 50 to 600 g of protein are usually suitable. Reference solution. Dilute human immunoglobulin RS with a 0.9 per cent w/v solution of sodium chloride to the same protein concentration as the test solution. Chromatographic system a stainless steel column 60 cm x 7.5 mm or 30 cm x 7.8 mm packed with hydrophilic silica gel, mobile phase. dissolve 4.873 g of disodium hydrogen phosphate dihydrate, 1.741 g of sodium dihydrogen phosphate monohydrate, 11.688 g of sodium chloride and 50 mg of sodium azide in 1000 ml of water. flow rate 0.5 ml per minute, spectrophotometer set at 280 nm. In the chromatogram obtained with the reference solution, the principal peak corresponds to IgG monomer and there is a peak corresponding to dimer with a relative retention to the principal peak of about 0.85. Identify the peaks in the chromatogram obtained with the test solution by comparison
Identification
Examine by a suitable immunoelectrophoresis technique. Using antiserum to normal human serum, compare normal human serum and the preparation under examination, both diluted to contain 1.0 per cent w/v of protein. The main component of the preparation under examination corresponds to the IgG component of normal human serum. The preparation under examination may show the presence of small quantities of other plasma proteins; if human albumin has been added as a stabiliser, it may be seen as a major component.
Tests
pH (2.4.24). 4.0 to 7.4. Dilute the preparation under examination with a 0.9 per cent solution of sodium chloride to obtain a solution containing 1.0 per cent of protein. Osmolality (2.4.23). Minimum 240 mosmol per kg. Total protein. Minimum 3.0 per cent w/v and between 90.0 to 110.0 per cent of the quantity of protein stated on the label. Dilute the preparation under examination with a 0.9 per cent solution of sodium chloride to obtain a solution containing about 15 mg of protein in 2 ml. To 2.0 ml of this solution in a round-bottomed centrifuge tube add 2 ml of a 7.5 per cent w/ v solution of sodium molybdate and 2 ml of a mixture of 1 volume of nitrogen-free sulphuric acid and 30 volumes of water. Shake, centrifuge for 5 minutes, decant the supernatant liquid and allow the inverted tube to drain on filter paper. Determine the nitrogen in the centrifugation residue by the method of sulphuric acid digestion (2.3.30) and calculate the content of protein by multiplying the result by 6.25. Protein composition. Determine by zone electrophoresis (2.4.12). Use strips of suitable cellulose acetate gel as the supporting medium and barbital buffer solution pH 8.6 as the electrolyte solution. Test solution. Dilute the preparation under examination with a 1466
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with the chromatogram obtained with the reference solution; any peak with a retention time shorter than that of dimer corresponds to polymers and aggregates. Results. In the chromatogram obtained with the test solution: a. relative retention: for monomer and dimer, the relative retention to the corresponding peak in the chromatogram obtained with the reference solution is 1 02; b. peak area: the sum of the peak areas of monomer and dimer represent not less than 90.0 per cent of the total area of the chromatogram and the sum of the peak area of polymers and aggregates represents not more than 3.0 per cent of the total area of the chromatogram. This requirement does not apply to products where albumin has been added as a stabiliser; for products stabilised with albumin, a test for distribution of molecular size is carried out during manufacture before addition of the stabiliser. Anticomplementary activity. The consumption of complement is not greater than 50.0 per cent (1 CH50/mg of immunoglobulin). Test for anticomplementary activity of immunoglobulin For the measurement of anticomplementary activity (ACA) of immunoglobulin, a defined amount of test material (10 mg of immunoglobulin) is incubated with a defined amount of guinea-pig complement (20 CH 50 ) and the remaining complement is titrated; the anticomplementary activity is expressed as the percentage consumption of complement relative to the complement control considered as 100.0 per cent. The haemolytic unit of complement activity (CH50) is the amount of complement that, in the given reaction conditions, will produce the lysis of 2.5 108 out of a total of 5 108 optimally sensitised red blood cells. Magnesium and calcium stock solution. Prepare as stated earlier. Barbital buffer stock solution. Prepare as stated earlier. Gelatin solution. Dissolve 12.5 g of gelatin in about 800 ml of water and heat to boiling in a water-bath. Cool to 20 and dilute to 10 litres with water. Filter through a membrane filter (pore size: 0.22 m). Store at 4. Use clear solutions only. Citrate solution. Dissolve 8.0 g of sodium citrate, 4.2 g of sodium chloride and 20.5 g of glucose in 750 ml of water. Adjust to pH 6.1 using a 10.0 per cent w/v solution of citric acid and dilute to 1,000 ml with water. Gelatin barbital buffer solution. Add 4 volumes of gelatin solution to 1 volume of barbital buffer stock solution and mix. Adjust to pH 7.3, if necessary, using 1 M sodium hydroxide or 1 M hydrochloric acid. Maintain at 4. Prepare fresh solutions daily. Stabilised sheep blood. Collect one volume of sheep blood into one volume of citrate solution and mix. Store at 4 for not less than 7 days and not more than 28 days. (Stabilised sheep blood and sheep red blood cells are available from a number
of commercial sources.) Haemolysin. Antiserum against sheep red blood cells prepared in rabbits. Guinea-pig complement. Prepare a pool of serum from the blood of not fewer than ten guinea-pigs. Separate the serum from the clotted blood by centrifugation at about 4. Store the serum in small amounts below -70. Method Preparation of standardised 5 per cent sheep red blood cell suspension. Separate sheep red blood cells by centrifuging an appropriate volume of stabilised sheep blood and wash the cells at least three times with gelatin barbital buffer solution and prepare as a 5.0 per cent v/v suspension in the same solution. Measure the cell density of the suspension as follows: add 0.2 to 2.8 ml of water and centrifuge the lysed solution for 5 minutes at 1,000 g; the cell density is suitable if the absorbance (2.4.7) of the supernatant liquid at 541 nm is 0.62 0.01. Correct the cell density by adding gelatin barbital buffer solution according to the formula: Vix A Vf = 0.62 Vf = final adjusted volume, Vi = initial volume, A = absorbance of the original suspension at 541 nm. The adjusted suspension contains about 1 109 cells per ml. Haemolysin titration Prepare haemolysin dilutions as shown in Table 1. Add 1.0 ml of 5.0 per cent sheep red blood cell suspension to each tube of the haemolysin dilution series, starting at the 1:75 dilution, and mix. Incubate at 37 for 30 minutes. Transfer 0.2 ml of each of these incubated mixtures to new tubes and add 1.10 ml of gelatin barbital buffer solution and 0.2 ml of diluted guinea-pig complement (for example, 1:150). Perform this in duplicate. As the unhaemolysed cell control, prepare three tubes with 1.4 ml of gelatin barbital buffer solution and 0.1 ml of 5.0 per cent sheep red blood cell suspension. As the fully haemolysed control, prepare three tubes with 1.4 ml of water and 0.1 ml of 5.0 per cent sheep red cell suspension. Incubate all tubes at 37 for 60 minutes and centrifuge at 1,000 g for 5 minutes. Measure the absorbance (2.4.7) of the supernatants at 541 nm and calculate the percentage degree of haemolysis in each tube using the expression: Aa A1 x 100 Ab A1 Aa = absorbance of tubes with haemolysin dilution, 1467
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Ab = mean absorbance of the three tubes with full haemolysis, A1 = mean absorbance of the three tubes with no haemolysis. Plot the percentage degree of haemolysis as the ordinate against the corresponding reciprocal value of the haemolysin dilution as the abscissa on linear graph paper. Determine the optimal dilution of the haemolysin from the graph by inspection. Select a dilution such that further increase in the amount of haemolysin does not cause appreciable change in the degree of haemolysis. This dilution is defined as one minimal haemolytic unit (1 MHU) in 1.0 ml. The optimal haemolytic haemolysin dilution for preparation of sensitised sheep red blood cells contains 2 MHU per ml. The haemolysin titration is not valid unless the maximum degree of haemolysis is 50.0 per cent to 70.0 per cent. If the maximum degree of haemolysis is not in this range, repeat the titration with more or less diluted complement solution. Table - 1 ________________________________________________ Required Prepared using dilution of haemolysin Gelatin barbital buffer solution
containing 2 MHU/ml and an equal volume of standardised 5.0 per cent sheep red blood cell suspension. Add the haemolysin dilution to the standardised cell suspension and mix. Incubate at 37 for 15 minutes, store at 2 to 8 and use within 6 hours. Titration of complement. Prepare an appropriate dilution of complement (for example, 1:250) with gelatin barbital buffer solution and perform the titration in duplicate as shown in Table 2. Add 0.2 ml of sensitised sheep red blood cells to each tube, mix well and incubate at 37 for 60 minutes. Cool the tubes in an ice-bath and centrifuge at 1,000 g for 5 minutes. Measure the absorbance of the supernatant liquid at 541 nm and calculate the degree of haemolysis (Y) using the expression: Ac A1 Ab A1 Ac = absorbance of tubes 1 to 12,
Haemolysin
Ab = mean absorbance of tubes with 100 per cent haemolysis, A1 = mean absorbance of cell controls with 0 per cent haemolysis. Plot Y/(1 - Y) as the abscissa against the amount of diluted complement in ml as the ordinate on loglog graph paper. Fit the best line to the points and determine the ordinate for the 50.0 per cent haemolytic complement dose where Y/(1 - Y) = 1.0. Calculate the activity in haemolytic units (CH50/ml) from the expression: Cd Ca x 5 Cd = reciprocal value of the complement dilution, Ca = volume of diluted complement in ml resulting in 50.0 per cent haemolysis, 5 = scaling factor to take account of the number of red blood cells. The test is not valid unless the plot is a straight line between 15.0 per cent and 85.0 per cent haemolysis and the slope is 0.15 to 0.40, and preferably 0.18 to 0.30.
Volume Dilution Volume ml (1 : ) ml _________________________________________________ 7.5 10 75 100 150 200 300 400 600 800 1200 1600 2400 3200* 0.65 0.90 1.80 1.80 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 undiluted undiluted 7.5 10 75 100 150 200 300 400 600 800 1200 1600 0.1 0.1 0.2 0.2 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
4800* 1.00 2400 1.0 _______________________________________________ * discard 1.0 ml of the mixture Preparation of optimised sensitised sheep red blood cells (haemolytic system) Prepare an appropriate volume of diluted haemolysin 1468
Table - 2 _______________________________________________ Tube Number Volume of diluted Volume of gelatin
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complement in ml barbital buffer (for example 1:250) solution in ml _______________________________________________ 1 2 3 4 5 6 7 8 9 10 11 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.1 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2
and of gelatin barbital buffer solution are added if the immunoglobulin concentration varies from 50 mg per ml; for example, 0.47 ml of gelatin barbital buffer solution is added to 0.33 ml of immunoglobulin containing 30 mg per ml to give 0.8 ml. Close the tubes and incubate at 37 for 60 minutes. Add 0.2 ml of each incubation mixture to 9.8 ml of gelatin barbital buffer solution to dilute the complement. Perform complement titrations as described above on each tube to determine the remaining complement activity (Table 2). Calculate the anticomplementary activity of the preparation under examination relative to the complement control considered as 100.0 per cent, from the expression: a-b
x 100
a a = mean complement activity (CH50/ml) of complement control, b = complement activity (CH50/ml) of tested sample. The test is not valid unless: a. the anticomplementary activities found for ACA negative control and ACA positive control are within the limits stated in the leaflet accompanying the reference preparation, b. the complement activity of the complement control (a) is in the range 80 to 120 CH50 per ml. Prekallikrein activator. Maximum 35 IU per ml, calculated with reference to a dilution of the preparation under examination containing 3.0 per cent w/v of immunoglobulin. Test for prekallikrein activator Prekallikrein activator (PKA) activates prekallikrein to kallikrein and may be assayed by its ability to cleave a chromophore from a synthetic peptide substrate so that the rate of cleavage can be measured spectrophotometrically and the concentration of PKA calculated by comparison with a reference preparation calibrated in International Units. The International Unit is the activity of a stated amount of the International Standard which consists of freeze-dried prekallikrein activator. The equivalence in International Units of the International Standard is stated by the World Health Organisation. Reagents. Prekallikrein activator in albumin RS is calibrated in International Units by comparison with the International Standard. Buffer A. Dissolve 6.055 g of tris(hydroxymethyl)aminomethane, 1.17 g of sodium chloride, 50 mg of hexadimethrine bromide and 0.100 g of sodium azide in water. Adjust to pH 8.0 with 2 M hydrochloric acid and dilute to 1000 ml with water. Buffer B. Dissolve 6.055 g of tris(hydroxymethyl)aminomethane and 8.77 g of sodium chloride in water. Adjust to pH 8.0 with 2 M hydrochloric acid and dilute to 1,000 ml 1469
12 1.2 0.1 _______________________________________________ Three tube as cell 1.3 control at 0 per cent haemolysis _______________________________________________ Three tube at 100 1.3 ml of water per cent haemolysis _______________________________________________ Test for anticomplementary activity Prepare a complement dilution having 100 CH50 per ml by diluting titrated guinea-pig complement with gelatin barbital buffer solution. If necessary, adjust the immunoglobulin under examination to pH 7. Prepare incubation mixtures as follows for an immunoglobulin containing 50 mg per ml: Table - 3 ______________________________________________ Immunoglobulin Complement under examination control (in duplicate) ______________________________________________ Immunoglobulin (50 mg per ml) 0.2 ml Gelatin barbital buffer 0.6 ml 0.8 ml Complement 0.2 ml 0..2 ml ______________________________________________ Carry out the test on the immunoglobulin under examination and prepare ACA negative and positive controls using human immunoglobulin RS, as indicated in the leaflet accompanying the reference preparation. Higher or lower volumes of sample
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with water. Preparation of prekallikrein substrate To avoid coagulation activation, blood or plasma used for the preparation of prekallikrein must come into contact only with plastics or silicone-treated glass surfaces. Draw 9 volumes of human blood into 1 volume of anticoagulant solution (ACD, CPD or 3.8 per cent w/v solution of sodium citrate) to which 1 mg per ml of hexadimethrine bromide has been added. Centrifuge the mixture at 3,600 g for 5 minutes. Separate the plasma and centrifuge again at 6,000 g for 20 minutes to sediment platelets. Separate the platelet-poor plasma and dialyse against 10 volumes of buffer A for 20 hours. Apply the dialysed plasma to a chromatography column containing agarose-DEAE for ion exchange chromatography which has been equilibrated in buffer A and is equal to twice the volume of the plasma. Elute from the column with buffer A at 20 ml per cm2 per hours. Collect the eluate in fractions and record the absorbance (2.4.7) at 280 nm. Pool the fractions containing the first protein peak so that the volume of the pool is about 1.2 times the volume of the platelet-poor plasma. Test the substrate pool for absence of kallikrein activity by mixing 1 part with 20 parts of the pre-warmed chromogenic substrate solution to be used in the assay and incubate at 37 for 2 minutes. The substrate is suitable if the increase in absorbance is less than 0.001 per minute. Add to the pooled solution 0.7 per cent w/v of sodium chloride and filter using a membrane filter (porosity 0.45 m). Freeze the filtrate in portions and store at -25; the substrate may be freeze-dried before storage. Carry out all procedures from the beginning of the chromatography to freezing in portions during a single working day. Method. The assay may be carried out using an automated enzyme analyser or a suitable microtitre plate system allowing kinetic measurements, with appropriate software for calculation of results. Standards, samples and prekallikrein substrate may be diluted as necessary using buffer B. Incubate diluted standards or samples with prekallikrein substrate for 10 minutes such that the volume of the undiluted sample does not exceed 1/10 of the total volume of the incubation mixture to avoid errors caused by variation in ionic strength and pH in the incubation mixture. Incubate the mixture or a part thereof with at least an equal volume of a solution of a suitable synthetic chromogenic substrate, known to be specific for kallikrein (for example, N-benzoyl-L-prolylL-phenylalanyl-L-arginine 4-nitroanilide acetate or Dprolyl-L-phenylalanyl-L-arginine-4-nitroanilidedihydrochloride), dissolved in buffer B. Record the rate of change in absorbance per minute for 2 to 10 minutes at the wavelength specific for the substrate used. Prepare a blank for each mixture of sample or standard using buffer B instead of prekallikrein substrate.
Depending on the method used, A per minutes has to be corrected by subtracting the value obtained for the corresponding blank without the prekallikrein substrate. The results may be calculated using a standard curve, a parallelline or a slope ratio assay or any other suitable statistical method. Plot a calibration curve using the values thus obtained for the reference preparation and the respective concentrations; use the curve to determine the PKA activity of the preparation under examiantion. Anti-A and anti-B haemagglutinins. Carry out the tests for anti-A and anti-B haemagglutinins as stated under Dried human haemophilic fraction If the preparation under examination contains more than 3.0 per cent of immunoglobulin, dilute to this concentration before preparing the dilutions to be used in the test. The 1 to 64 dilutions do not show agglutination. Anti-D antibodies. It complies with the test for anti-D antibodies in human immunoglobulin for intravenous administration. Test for anti-D antibodies in human immunoglobulin for intravenous administration Materials Phosphate-buffered saline (PBS). Dissolve 8.0 g of sodium chloride, 0.76 g of anhydrous disodium hydrogen phosphate, 0.2 g of potassium chloride, 0.2 g of potassium dihydrogen phosphate and 0.2 g of sodium azide in water and dilute to 1,000 ml with the same solvent. Papain solution. Use serological grade papain from a commercial source, the activity of which has been validated. Red blood cells. Use pooled red blood cells from not fewer than 3 donors of group OR2R2 and 3 donors of group Orr respectively. Wash the cells 4 times with PBS or until the supernatant is clear. Centrifuge the cells at 1, 800 g for 5 minutes to pack. Treat the packed red cells with papain solution according to the manufacturers instructions. Store in Alsevers solution for not more than 1 week. Microtitre plates. Use V-bottomed rigid micro-titre plates. Reference standards. Immunoglobulin (anti-D antibodies test) reference preparation and Immunoglobulin (anti-D antibodies test negative control) reference preparation are suitable for use as the reference preparation and negative control, respectively. Method Reference preparation and negative control solutions. Reconstitute the reference preparation and the negative control according to instructions. Dilute the reconstituted preparations with an equal volume of PBS containing 0.2 per cent w/v of bovine albumin and then prepare a further 7 serial two-fold dilutions using PBS containing 0.2 per cent w/v of bovine albumin to give a total dilution range from 1/2 to 1/ 256. Make 2 independent sets of dilutions for each preparation. Add 20 l of each dilution to the microtitre plate.
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PLASMA FOR FRACTIONATION
Test solutions. Initially dilute the test samples to give a starting immunoglobulin G (IgG) concentration of 2.5 per cent w/v using PBS containing 0.2 per cent w/v of bovine albumin and then prepare a further 7 serial two-fold dilutions using PBS containing 0.2 per cent w/v of bovine albumin to give a total dilution range from 1/2 to 1/256. Make 2 independent sets of dilutions for each test sample. Add 20 l of each dilution to the microtitre plate. Prepare 3.0 per cent v/v suspensions of papain-treated Dpositive (OR2R2) and D-negative (Orr) red cells in PBS containing 0.2 per cent w/v of bovine albumin. Add 20 l of D-positive cells to one dilution series of each of the test sample, the reference preparation and the negative control, and 20 l of D-negative cells to the other dilution series of each of the test samples, the reference preparation and the negative control. Mix by shaking the plate on a shaker for 10 seconds. Centrifuge the plate at 80 g for 1 minutes to pellet the cells. Place the plate at an angle of approximately 70. Read after 4 to 5 minutes (or until the cells have streamed in the wells containing the negative control and the wells where the Dnegative cells have been added). A cell button at the bottom of the well indicates a positive result. A stream of cells represents a negative result. Record the endpoint titre as the reciprocal of the highest dilution that gives rise to a positive result. The titre of the preparation under examination is not greater than the titre of the reference preparation. Water. Determine by semi-micro determination of water (2.3.43), loss on drying (2.4.19) or near infrared spectrophotometry (2.4.6), the water content is within the limits approved by the competent authority. Sterility (2.2.11). Complies with the test for sterility. Pyrogens (2.2.8). Complies with the test for pyrogens. Inject per kg of the rabbits mass a volume equivalent to 0.5 g of immunoglobulin but not more than 10 ml per kg of body mass. Antibody to hepatitis B surface antigen Minimum 0.5 IU per g of immunoglobulin, determined by a suitable immunochemical method. Storage. For the liquid preparation, store in a colourless glass container, protected from light, at the temperature stated on the label. For the freeze-dried preparation, store in an airtight colourless glass container, protected from light, at a temperature not exceeding 25. Labelling. The label states (1) for liquid preparations, the volume of the preparation in the container and the protein content expressed in grams per litre; (2) for freeze-dried preparations, the quantity of protein in the container; (3) the amount of immunoglobulin in the container; (4) the route of administration; (5) for freeze-dried preparations, the name or composition and the volume of the reconstituting liquid to be added; (6) the distribution of subclasses of immunoglobulin
G present in the preparation; (7) where applicable, the amount of albumin added as a stabilizer; (8) the maximum content of immunoglobulin A.
Plasma for Fractionation

Human Plasma for Fractionation Human Plasma for Fractionation is the liquid part of human blood remaining after separation of the cellular elements from blood collected in a receptacle containing an anticoagulant, or separated by continuous filtration or centrifugation of anticoagulated blood in an apheresis procedure; it is intended for the manufacture of plasma-derived products.
Production
Donors Only a carefully selected, healthy donor who, as far as can be ascertained after medical examination, laboratory blood tests and a study of the donors medical history, is free from detectable agents of infection transmissible by plasma-derived products may be used. Immunisation of donors Immunisation of donors to obtain immunoglobulins with specific activities may be carried out when sufficient supplies of material of suitable quality can not be obtained from naturally immunised donors. Recommendations for such immunisations are formulated by the World Health Organisation (Requirements for the collection, processing and quality control of blood, blood components and plasma derivatives, WHO Technical Report Series, No. 840, 1994 or subsequent revision). Records Records of donors and donations made are kept in such a way that, while maintaining the required degree of confidentiality concerning the donors identity, the origin of each donation in a plasma pool and the results of the corresponding acceptance procedures and laboratory tests can be traced. Laboratory tests Laboratory tests are carried out for each donation to detect the following viral markers: 1. Antibodies against human immunodeficiency virus 1 (antiHIV-1), 2. Antibodies against human immunodeficiency virus 2 (antiHIV-2), 3. Antibodies against hepatitis C virus (anti-HCV), 4. Hepatitis B surface antigen (HBsAg). Pending complete harmonisation of the laboratory tests to be carried out, the competent authority may require that a test for 1471
PLASMA FOR FRACTIONATION
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alanine aminotransferase (ALT) also be carried out. The test methods used are of suitable sensitivity and specificity and comply with the regulations in force. If a repeatreactive result is found in any of these tests, the donation is not accepted. Individual plasma units The plasma is prepared by a method that removes cells and cell debris as completely as possible. Whether prepared from whole blood or by plasmapheresis, the plasma is separated from the cells by a method designed to prevent the introduction of micro-organisms. No antibacterial or antifungal agent is added to the plasma. The containers comply with the requirements for plastic containers for blood and blood components (6.2). The containers are closed so as to prevent any possibility of contamination. If 2 or more units are pooled prior to freezing, the operations are carried out using sterile connecting devices or under aseptic conditions and using containers that have not previously been used. When obtained by plasmapheresis, plasma intended for the recovery of proteins that are labile in plasma is frozen by cooling rapidly in a chamber at - 30 or below as soon as possible and at the latest within 24 hours of collection. When obtained from whole blood, plasma intended for the recovery of proteins that are labile in plasma is separated from cellular elements and is frozen by cooling rapidly in a chamber at -30 or below as soon as possible and at the latest within 24 hours of collection. When obtained from whole blood, plasma intended solely for the recovery of proteins that are not labile in plasma is separated from cellular elements and frozen in a chamber at 20 or below as soon as possible and at the latest within 72 hours of collection. It is not intended that the determination of total protein and factor VIII shown below be carried out on each unit of plasma. They are rather given as guidelines for good manufacturing practice, the test for factor VIII being relevant for plasma intended for use in the preparation of concentrates of labile proteins. The total protein content of a unit of plasma depends on the serum protein content of the donor and the degree of dilution inherent in the donation procedure. When plasma is obtained from a suitable donor and using the intended proportion of anticoagulant solution, a total protein content complying with the limit of 5 per cent is obtained. If a volume of blood or plasma smaller than intended is collected into the anticoagulant solution, the resulting plasma is not necessarily unsuitable for pooling for fractionation. The aim of good manufacturing practice must be to achieve the prescribed limit for all normal donations. Preservation of factor VIII in the donation depends on the collection procedure and the subsequent handling of the blood and plasma. With good practice, 0.7 IU/ml can usually 1472
be achieved, but units of plasma with a lower activity may still be suitable for use in the production of coagulation factor concentrates. The aim of good manufacturing practice is to conserve labile proteins as much as possible. Total protein. Carry out the test using a pool of not less than 10 units. Dilute the pool with a 0.9 per cent solution of sodium chloride to obtain a solution containing about 15 mg of protein in 2 ml. To 2.0 ml of this solution in a round-bottomed centrifuge tube add 2 ml of a 7.5 per cent solution of sodium molybdate and 2 ml of a mixture of 1 volume of nitrogen-free sulphuric acid and 30 volumes of water. Shake, centrifuge for 5 minutes, decant the supernatant liquid and allow the inverted tube to drain on filter paper. Determine the nitrogen in the residue by the method of sulphuric acid digestion (2.3.30) and calculate the protein content by multiplying the quantity of nitrogen by 6.25. The total protein content is not less than 5.0 per cent. Factor VIII Carry out the test using a pool of not less than 10 units. Thaw the samples under examination, if necessary, at 37. Determine the assay of factor VIII (2.8.7), using a reference plasma calibrated against the International Standard for human coagulation factor VIII in plasma. The activity is not less than 0.7 IU per ml. Pooled plasma During the manufacture of plasma products, the first homogeneous pool of plasma (for example, after removal of cryoprecipitate) is tested for HBsAg, HCV antibodies and for HIV antibodies using test methods of suitable sensitivity and specificity; the pool must give negative results in these tests. The plasma pool is also tested for hepatitis C virus RNA using a validated nucleic acid amplification technique (2.8.1). A positive control with 100 IU per ml of hepatitis C virus RNA and, to test for inhibitors, an internal control prepared by addition of a suitable marker to a sample of the plasma pool are included in the test. The test is invalid if the positive control is non-reactive or if the result obtained with the internal control indicates the presence of inhibitors. The plasma pool complies with the test if it is found non-reactive for hepatitis C virus RNA. Description. Before freezing, a clear to slightly turbid liquid without visible signs of haemolysis; it may vary in colour from light yellow to green. Storage. Frozen plasma is stored in conditions designed to maintain the temperature at or below -20; for accidental reasons, the storage temperature may rise above -20 on one or more occasions during storage but the plasma is nevertheless considered suitable for fractionation if all the following conditions are fulfilled (1) the total period of time during which the temperature exceeds -20 does not exceed 72 hours; (2) the temperature does not exceed -15 on more than one occasion; (3) the temperature at no time exceeds -5. Labelling. The label enables each individual unit to be traced to a specific donor.
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PLATELET CONCENTRATE
Platelet Concentrate
Platelets separated from whole blood within 4 to 6 hours of collection and suspended in 40 to 50 ml of plasma are designated as platelet concentrate.
Production
Platelet Concentrate is prepared from units of whole blood that have not been allowed to cool below 20. Platelet rich plasma (PRP) is separated within 4-6 hours after completion of the phlebotomy. The final component should contain resuspended platelets in an amount of plasma adequate to maintain an acceptable pH - generally 40 to 70 ml is used.
22, label it also to state that a continuous gentle agitation shall be maintained, or where labelled for storage at 1 to 6, to state that such agitation is optional. Label it also with the type and result of a serologic test for syphilis, or to indicate that it was nonreactive in such test; with the type and result of a test for hepatitis B surface antigen, or to indicate that it was nonreactive in such test; with a warning that it is to be used as soon as possible but not more than 6 hours after entering the container; to state that a filter is to be used in the administration equipment; and to state that the instruction circular provided is to be consulted for directions for use.
Tests
Four PC per month should be assayed for pH and for platelet, RBC and leucocyte counts. PRP Counts Volume Total Count PRC Counts 8 - 10 x 105 per l 350 ml bag (approximately) 10 - 12 x 105 per l 450 ml bag (approximately) Volume 40 - 50 ml per bag Platelet counts > 5.5 x 1010 per bag in 75 per cent of bags ( if prepared from 450 ml bags ) > 4.2 x 1010 per bag in 75 per cent of bags ( if prepared from 350 ml bags ) Leucocyte count < 0.12 x 109 per bag. RBC counts < 1.2 x 109 per bag. pH < 6.3 at the end of 5 days storage. Expiration time. The expiration time is not more than 72 hours from the time of collection of the source material. Storage. Store at 20 to 22 in polyvinylchloride plastic bags. Preserve at the temperature relevant to the volume of resuspension plasma, either between 20 and 22 or between 1 and 6, the latter except during shipment, when the temperature may be between 1 and 10. Labelling. In addition to the labelling requirements of Whole Blood applicable to this product, label it to state the volume of original plasma present, the kind and volume of anticoagulant solution present in the original plasma, the blood group designation of the source blood, and the hour of expiration on the stated expiration date. Where labelled for storage at 20 to 3.0 - 4.5 x 105 per l (approximately) 170 - 250 ml (approximately) 9 x 1010 per bag (approximately)
Whole Human Blood

Whole Blood (Human) Whole Human Blood is blood drawn aseptically from selected human donors and mixed with a suitable anticoagulant. Whole Human Blood is the final mixture of blood and anticoagulant solution contains not less than 9.7 per cent w/v of haemoglobin, calculated from the haemoglobin content of the donors blood and the dilution due to the anticoagulant solution. It is obtained from healthy donors who must: (a) be in the age range of 18 to 60 years and be in good health as indicated in part by normal temperature and blood pressure within normal limits; (b) not be pregnant, if females; (c) not have undergone major surgery within 6 months of donation; (d) as far as can be ascertained after clinical and laboratory examination and the study of medical history of the donor be free from disease transmissible by blood transfusion; (e) have blood containing not less than 12.5 per cent w/v of haemoglobin; (f) be free from acute respiratory diseases; (g) be free from any infectious skin disease at the site of phlebotomy; (h) have no history of malarial fever within 12 months of donation; (i) have no history of viral hepatitis or of close contact with an individual having viral hepatitis within 12 months of donation and have blood that has given negative results in tests for the presence of hepatitis-B antigen; (j) have blood that has been tested with negative results for evidence of syphilitic infection, HCV antibodies, HIV antibodies and malarial parasites. The examinations and tests to be carried out are decided by the National Regulatory Authority. The frequency of donations of whole blood shall not exceed once every 3 months with a maximum volume of 1.5 litres in any consecutive 12 month period. 1473
WHOLE HUMAN BLOOD
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The blood is drawn aseptically through a closed system into a suitable sterile container containing a specific amount of Anticoagulant Citrate Dextrose Solution (ACD Solution) or Anticoagulant Citrate Phosphate Dextrose Solution (CPD Solution) which is placed before the container is sterilised. The quantity of anticoagulant solution should not exceed 22.0 per cent v/v of the final volume of the mixture. No antimicrobial preservative is added. During the withdrawal of blood the container is gently agitated to ensure thorough mixing. When withdrawal is complete the container is immediately sealed and cooled to 2o to 8o. It is not opened until immediately before transfusion. With every container of blood, a separate sample mixed with the appropriate quantity of anticoagulant solution, is collected for compatibility and other tests; this small container is firmly attached to the main container. Whole Human Blood in containers from which samples have been removed for tests should not be used for transfusion. Consequently, it is not intended that the Tests and the Assay should be carried out on the contents of the container. The Blood Bank or the service collecting the blood is responsible for ensuring that the conditions in which the blood is collected and stored are such that, if and when tested, the blood will comply with the requirements of the monograph. Blood group. Determine the blood group (in the sample accompanying each donation) under the ABO system (2.8.11), by examination of both corpuscles and serum, and under the Rh system (2.8.11), by examination of the corpuscles. Description. Deep red fluid which, on standing separates into a lower layer of sedimented-red cells and a yellowish, almost clear upper layer of plasma, free from visible signs of
haemolysis, with a greyish layer between the two consisting of leucocytes and thrombocytes. A layer containing emulsified fat may form on the surface.
Tests
Sterility (2.2.11). Complies with the tests for sterility, determined by Method B. Assay. Determine the haemoglobin content by photometric haemoglobinometry (2.8.12). Storage. Store in colourless, transparent and sterile containers into which it was originally drawn. The containers should be provided with a hermetic, contamination-proof closure. Store at a temperature between 2o to 8o. Labelling. The label states (1) the distinctive code number by reference to which the details of the donor are available; (2) the ABO group with the approved colour scheme for different groups as specified by the National Regulatory Authority; (3) the Rh group; (4) the total volume of fluid, the proportion of blood, and the nature and volume of anticoagulant solution; (5) the date on which the blood was withdrawn; (6) the expiry date which should not exceed 21 days from the date of withdrawal of blood; (7) the storage conditions; (8) that the blood must not be used for transfusion if there is any visible evidence of haemolysis or other deterioration; (9) for blood of group O whether haemolysins are present or not and if they are, that the blood must be administered only to recipients of blood group O; (10) that the blood has given negative results in the tests for the presence of malarial parasites, hepatitis-B antigen, syphilis and HIV antibodies and any other tests prescribed by the National Regulatory Authority.
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MONOGRAPHS
BIOTECHNOLOGY PRODUCTS
General Monographs r-DNA Biotechnology Products of Therapeutic Value Monographs Erythropoietin concentrated solution Granulocyte Colony Stimulating Factor, Human ( Filgrastim ) Interferon alfa-2 concentrated solution Sereptokinase .... .... .... .... ....
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r-DNA BIOTECHNOLOGY PRODUCTS OF THERAPEUTIC VALUE
r-DNA Biotechnology Products of Therapeutic Value

This monograph states the general requirements for the manufacture of products of recombinant DNA technology that are produced by genetic modification in which the DNA coding for the required product is introduced into a suitable cell line or microorganism by means of a plasmid or viral vector. The DNA is expressed and translated into protein of interest in the genetically modified organism. Therapeutic proteins are derived from several processes. One of them is the recombinant DNA technology, the products of which are often referred to as r-DNA products. The process involves isolation of a specific active gene and inserting into a host cell. The host cell expresses the protein encoded in the transferred gene. The host cell could be a microorganism or an animal cell. Large-scale propagation yields large quantities of crude protein. Purification of the crude protein using multiple techniques derives a safe, pure and biologically active product. The product being a protein may cause immunological sensitization in the recipients and therefore needs a high degree of characterization. Testing of the product for safety, identity, strength, quality and purity are similar to those applied to other pharmaceutical products, although the tests themselves may require some modification due to the pretentious nature of the active compound. Recombinant DNA technology products are produced by genetic manipulation of a cell line or microorganism usually through a plasmid transfer or viral vector. The recovery of the desired product expressed by such genetically modified organisms is by multistage purification. Monoclonal antibodies too fall under this category. The cell or microorganism used for expressing the protein is referred to as the host cell and the transformed cell after the gene insertion is referred to as the host-vector system. The host cells could be Prokaryotic or Eukaryotic cells. The differences in their metabolic pathway result in differences in the type proteins expressed by them. Prokaryotic cells do not have the ability to do glycosylation. Therefore, proteins expressed by the Prokaryotic cells may require post translation modification to make them biologically active. Monoclonal antibodies are derived from a single clone of B lymphocyte .They differs from polyclonal antibodies that are a mixture of antibodies of several antigenic epitopes. Blymphocytes have a short life span and hence need to be immortalized if they are to be used for production for a long period. Fusion with myeloma cells make them immortal. Such fused hybridoma cells inherit the property of producing the desired antibody from the B lymphocyte and immortal property from the Myeloma cells. Large scale culturing of these 1
hybridoma cells using various techniques yield monoclonal antibodies. r-DNA derived products suffer from a danger of microheterogeneity in the expressed protein and therefore require extensive process validation. In addition, the concern of adventitious viruses , presence of host cell DNA, presence of oncogenic genes, residual host proteins, endotoxins, residual media proteins etc calls for stringent methods of testing to ensure their removal to acceptable levels. r-DNA derived products are not significantly different from other therapeutic proteins derived from natural sources after the final purification step and therefore basic requirements of validation of the process, environmental conditions, and aseptic techniques during manufacturing, quality assurance and quality control do not differ in approach from conventional pharmaceutical products. Handling of genetically modified organisms in the production process require adherence to the rules governing environmental safety. r-DNA products are produced in Prokaryotic or Eukaryotic systems. The choice of the system primarily depends upon the size of the protein and the extent of glycosylation required to make them biologically active. The choice of bacteria in the Prokaryotic system is E.Coli due to the extensive information available of this bacterium. The disadvantages in use of the organism are: 1) The proteins are produced in the reduced state. 2) Need to remove the N formyl methionine sequence. 3) Product degradation during the production process due to the presence of protease enzyme. 4) Need for endotoxin removal step. Eukaryotic cells including yeasts on the other hand have the ability to produce proteins that are properly folded due to its glycosylation ability. Primary cell lines have a short life span. Cloning, selection, amplification, master cell bank creation, scaling up requires several passages. Primary Cell lines do not last these many passages. Use of immortal cell lines has helped overcome the problem but brought in new challenges in the form of oncogenes, potential viral and retroviral contamination, and presence of animal derived growth promoters etc., which are required to be removed by various purification steps. Exhaustive analysis, validation of the process and safety tests are done on the Master Banks are done to rule out presence of adventitious agents. Cells are used only after this for largescale production. Yeast offers an advantage against the complexities of using animal cell lines but have the disadvantage that they maintain plasmids extra chromosally creating a danger of losing the inserted gene. However, their ability to produce glycosylated protein makes them a better choice than Escherichia coli.
r-DNA BIOTECHNOLOGY PRODUCTS OF THERAPEUTIC VALUE
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Monoclonal antibodies are produced in two ways depending on the source of the lymphocyte murine or Human. Antibodies of Murine origin are produced by selecting the lymphocytes from the spleen of previously inoculated mice or rats and fusing them with an immortal cell line like myeloma cells. Such hybridoma cells are cloned, propagated in large scale and used for antibody production. Antibody produced is as per the chromosomal information acquired during the hybridoma formation process. Large-scale propagation of bacteria and Yeast are often referred to as Fermentation. During the process the growth rate, purity of the culture and yields are monitored. Nucleotide sequence analysis or DNA restriction mapping checks the genetic stability of the plasmid. Peptide mapping of the expressed protein is carried out for confirmation. Proteolytic degradation of the expressed protein is a concern and therefore optimization of the fermentation process is essential to minimize it. Recovery of the protein often requires lysis of the cells. Proteases released during the process of cell lysis calls for rapid processing to minimize the damage caused by it. Large-scale cell culture is quite well established but poses it own challenges like genetic stability, protein folding and culture conditions. Genetic stability cannot be rapidly checked as in the case of Prokaryotic as the gene is incorporated in the cell genome. Peptide mapping of the expressed protein is the only check but lacks sensitivity to detect minor genetic mutations. Manufacturers are required to demonstrate the purity of the cultures against contaminating organisms including mycoplasma and adventitious viruses, at the end of purification. The advantage of cell culture is that the protein is directly secreted into the medium and therefore cell separation is sufficient to achieve a significant level of purification.
iii. The method of transfer of the expression gene into the host cell. iv. Methods employed in the clone selection. v. Amplification role of various components like promoter, enhancers, antibiotic resistance gene etc. vi. A complete annotated sequence of the plasmid indicating those regions that have been sequenced. Characterization of the cell bank system The master cell bank is a collection of vials containing homogeneous suspension of the original cells already transformed by the expression vector containing the desired gene. One or more of the Master Cell Bank vials may be amplified for creating a Working Cell Bank. The cell banks should be characterized for relevant phenotypic and genotypic markers, by expressing the recombinant protein and confirming the nucleotide sequence. The number of passages from the Master Bank level or the cell doubling permitted during the production process will be determined from the data derived from long tem cultivation. Based on the analysis of the molecular integrity of the proteinexpressing gene, the phenotypic and the genotypic characteristics of the host cell, passage or cell doubling limits are established. Analysis of the cell bank systems for copy number, insertions and deletions and the number of integration sites is done. When the expression system is extra chromosomal, the percentage of the host cells retaining the expression system should be determined. The nucleic acid sequence should be identical and should correspond to that expected for the protein sequence. Characterization of the host-vector system and the final purified protein are done to ensure consistent production of the r-DNA protein. Validation of the cell banks Validation of the cell banks shall include: i. Stability by measuring viability and the retention of the vector. ii. Identity of the cells by phenotypic features. iii. Establishing that the cell banks are free from potentially oncogenic or infective adventitious agents (viral, bacterial, fungal or mycoplasma). iv. For mammalian cells, details of the tumorogenic potential of the cell bank shall be obtained. Validation of the purification process The ability of the different steps of the extraction and purification process to remove and/or inactivate contaminating substances derived from the host cell or culture medium, 2
Production
Production is based on a validated stable host vector combination using a seed-lot system. The seed-lot system uses a master cell bank and a working cell bank derived from the master seed lot of the host-vector combination. The validation to determine the suitability shall include the following. Characterization of the Host-Vector System Characterization of the host-vector system is used for establishing its suitability for production. Characterization of the host-vector system involves several tests that are performed to establish the purity. Characterization includes documentation of: i. ii. The origin of the nucleotide sequence coding for the protein. The methods used to prepare the DNA coding.
IP 2007
ERYTHROPOIETIN CONCENTRATED SOLUTION
including, virus particles, proteins, nucleic acids and added substances, must be validated. Validation studies should demonstrate that the production process routinely meets the following criteria: i. Removal of extraneous agents from the product. ii. Removal of vector, host-cell, culture medium and reagentderived contaminants below the acceptable levels. iii. Capability to reduce the DNA below acceptable levels. iv. Adequate stability of any partially purified product if stored during the process. v. Minimum yields in the process. Characterization of the substance The identity, purity, potency and stability of the final bulk product are established initially by carrying out a wide range of chemical, physical, immunochemical and biological tests. Prior to release, each batch of the product is tested by the manufacturer for identity and purity and an appropriate assay is carried out. Production Consistency Suitable tests for demonstrating the consistency of the production and purification are performed. The tests include, especially characterization tests, in-process controls and finalproduct tests, for example: Amino-acid composition Partial amino acid sequence analysis test for confirmation of the correct N-terminal processing and detect loss of the Cterminal amino acids. Peptide mapping Peptide mapping using chemical and/or enzymatic cleavage of the protein product must be done that no significant difference between the test protein and reference preparation is found. Two-dimensional gel electrophoresis, capillary electrophoresis or liquid chromatography may be used for analysis. Peptide mapping can also be used to demonstrate correct disulphide bonding. Determination of molecular mass Cloned-gene Retention The relevant authority approves the minimum amount in percentage of the cells containing the vector or the cloned gene after cultivation. Total Protein. The yield of protein is determined. Chemical Purity The purity of the protein product is analyzed in comparison with a reference preparation by a suitable method such as 3
liquid chromatography, capillary electrophoresis or sodium dodecyl sulphate polyacrylamide gel electrophoresis. Host-cell-derived proteins Host-cell-derived proteins are detected by immunochemical methods, using, polyclonal antisera rose against protein components of the host-vector system. Liquid-phase displacement assays (radioimmunoassay), liquid-phase directbinding assays and direct-binding assays using antigens immobilized on nitrocellulose (or similar) membranes (for example, dot-immunoblot assays, Western blots) may be used for the assay. The Antisera are raised against a preparation of antigens derived from the host organism, into which has been inserted the vector used in the manufacturing process that lacks the specific gene coding for the product. This host cell is cultured, and proteins are extracted, using conditions identical to those used for culture and extraction in the manufacturing process. Partly purified preparations of antigens, using some of the purification steps in the manufacturing process, may also be used for the preparation of antisera. Host-cell- and vector-derived DNA Residual DNA is detected by hybridization analysis, using suitably sensitive, sequence-independent analytical techniques or other suitably sensitive analytical techniques. However, the method used must be validated to ensure parallelism with the DNA standard used, linearity of response and non-interference of either the drug substance or excipients of the formulation at the dilutions used in the assay. The limits for the presence of these are as currently prescribed by W.H.O. or competent authorities.
Erythropoietin Concentrated Solution

APPRLICDSR EHCSLNENIT VEVWQGLALL LQLHVDKAVS PPDAASAAPL GKLKLYTGEA VLERYLLEAK VPDTKVNFYA SEAVLRGQAL GLRSLTTLLR RTITADTFRK CRTGD EAENITTGCA WKRMEVGQQA LVNSSQPWEP ALGAQKEAIS LFRVYSNFLR
Mol. Wt. 30,600 (approx) Erythropoietin Concentrated Solution contains a family of closely-related glycoproteins which are not different from the
IP 2007
naturally occurring human erythropoietin (urinary erythropoietin) in terms of the amino acid sequence (165 amino acids) and average glycosylation pattern, at a concentration of 0.5 mg per ml to 10 mg per ml. It has a potency of not less than 100000 IU per mg of active substance.
Pour into a suitable gel cassette, with approximate dimensions of 15 cm x 15 cm x 0.05 cm. Insert a suitable sample well comb and allow to polymerize. Use as the anode solution the anolyte for isoelectric focusing pH 3 to 5 and as the cathode solution the catholyte for isoelectric focusing pH 3 to 5. Allow prefocusing to take place for 1 hour at a constant power of 10 W, with maximum voltage and current settings of 2000 V and 100 mA respectively. Dilute the test solution to 0.5 mg per ml with water. Apply to the gel 10 l of each solution. Carry out focusing for a further 30 minutes at the same power supply settings. Take the gel out of the focusing chamber, and transfer the proteins onto a membrane suitable for immobilization of proteins (such as Polyvinylidene Fluoride), using commercially available electrotransfer equipment and following the manufacturers instructions. After electrotransfer, incubate the membrane in a neutral isotonic buffer containing a suitable blocking agent (for example, 50 g per litre of dried milk), for 1 hour, followed by incubation in the same blocking solution with a suitable dilution of a polyclonal anti-erythropoietin antibody. Detect the erythropoietin-bound antibody using a suitable enzyme- or radiolabelled antibody (for example, an alkaline phosphataseconjugated second antibody). The precise details of blocking details of blocking agents, concentrations and incubation times should be optimized using the principles set out in Immunochemical methods. The test is not valid unless the distribution of bands in the electrophoretogram obtained with the reference solution contains at least 6 well separated bands. If necessary, the voltage settings and duration may be altered to optimize the separation of the isoforms. In the electrophoretogram obtained with the test solution, the pH range of the bands observed corresponds to that of the electrophoretogram obtained with the reference solution. The predominant bands correspond to isoforms 4, 5, 6 and 7. Additional, fainter bands corresponding to isoforms 1, 2, 3 and 8 may also be present. Other bands are present in no more than trace amounts. C. Determine by capillary electrophoresis (capillary zone electrophoresis) (2.4.32). All the solutions should be filtered through a 0.45 m membrane filter before use. Test solution. Dilute the substance under examination with water or concentrate it to obtain a concentration of 1 mg per ml. Desalt 0.25 ml of the solution by passage through a microconcentrator cartridge provided with a membrane with a molecular mass cut-off of not more than 10000. Add 0.2 ml of water to the sample and desalt again. Repeat the desalting procedure once more. Dilute the sample with water, determine its protein concentration as described under Tests and adjust 4
Production
Erythropoietin is produced in rodent cells in vitro by a method based on recombinant DNA technology. All batches are tested as described below, prior to release. Host cell-derived proteins The limit is as prescribed by WHO. Host cell-derived proteins (HCP). This must be routinely monitored using a scientifically accepted method that demonstrates that: 1. the assay is sensitive (a useful target for the limit of detection is 1 to 100 ppm) 2. the assay is specific for the HCP (defined by proprietary detection reagents such as antibodies elicited against process-specific contaminating HCP) consistently found in the product from a specific manufacturing / purification process. Acceptable limits should be set empirically based on the above by the manufacturer. Host cell- and vector-derived DNA The limit is as prescribed by the regulatory authority. Description. A clear and colourless solution.
Identification
A. It gives the appropriate response when examined using the conditions described under Assay. B. Determine by capillary electrophoresis (capillary isoelectric focusing) (2.4.32). Test solution. Dilute if necessary the preparation in water and desalt the preparation. Using a membrane filtration system suitable for desalting proteins, desalt the sample. Make up the volume to the original volume with water. Reference solution. Dissolve erythropoietin RS in water to produce a solution containing 1mg per ml and desalt as above. The isoelectric focusing procedure may be carried out using a 0.5mm thick polyacrylamide slab gel containing ampholytes covering the pH of range 3 to 10, prepared as follows. Mix 9 g of urea, 6.0 ml of 30 per cent acrylamide / bisacrylamide solution, 1.05 ml of pH 3 to 5 ampholyte, 0.45 ml pH 3 to 10 ampholyte and 13.5 ml of water. Degas. Add 15 l of tetramethylethylene diamine and 0.3 ml of a 100 g per litre freshly prepared solution of ammonium persulphate.
IP 2007
to a concentration of approximately 1 mg per ml with water. Reference solution. Dissolve erythropoietin RS in water to produce a solution containing 1 mg per ml. Desalt the sample as described for the test solution. Capillary system material. uncoated fused silica, size. effective length = about 100 cm, = 50 m, temperature. 35, spectrophotometer set at 214 nm, injection. under pressure or vacuum. CZE buffer concentrate (0.1 M sodium chloride, 0.1 M tricine, 0.1 M sodium acetate). Dissolve 0.584 g of sodium chloride, 1.792 g of tricine and 0.820 g of anhydrous sodium acetate in water and dilute to 100.0 ml with the same solvent. 1 M putrescine solution. Dissolve 0.882 g of putrescine in 10 ml of water. Distribute in 0.5 ml aliquots. CZE buffer. (0.01 M tricine, 0.01 M sodium chloride, 0.01 M sodium acetate, 7 M urea, 2.5 mM putrescine). Dissolve 21.0 g of urea in 25 ml of water by warming in a water-bath at 30. Add 5.0 ml of CZE buffer concentrate and 125 ml of 1 M putrescine solution. Dilute to 50.0 ml with water. Using dilute acetic acid, adjust the pH to 5.55 at room temperature and filter through a 0.45 m membrane filter. Set the auto sampler to store the samples at 4 during analysis.
Preconditioning of the capillary. Rinse the capillary for 60 minutes with 0.1 M sodium hydroxide filtered through a 0.45 m membrane filter and for 60 minutes with CZE buffer. Apply voltage for 12 hours (20 kV). Between-run rinsing. Rinse the capillary for 10 minutes with water, for 5 minutes with 0.1 M sodium hydroxide filtered through a 0.45 m membrane filter and for 10 minutes with CZE buffer. Migration. Apply a field strength of 143 V/cm (15.4 kV for capillaries of 107 cm total length) for 80 min, using CZE buffer as the electrolyte in both buffer reservoirs. System suitability. In the electropherogram obtained with the reference solution, a pattern of well- separated peaks corresponding to the peaks in the reference electropherogram of erythropoietin RS is seen, and the largest peak is at least 50 times greater than the baseline noise. If necessary, adjust the sample load to give peaks of sufficient height. Identify the peaks corresponding to isoforms 1 to 8. The peak corresponding to isoform 1 is detected; the resolution between the peaks corresponding to isoforms 5 and 6 is not less than 1. Repeat the separation at least 3 times. The baseline is stable, showing little drift, and the distribution of peaks is qualitatively and quantitatively similar to the distribution of peaks in the reference electropherogram of erythropoietin RS. The relative standard deviation of the migration time of the peak corresponding to isoform 2 is less than 2 per cent.
Reference electropherogram of Erythropoietin 5
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Identify the peaks corresponding to isoforms 1 to 8 in the electropherogram obtained with the test solution by comparison with the electropherogram obtained with the reference solution. Calculate the percentage content of each isoform from the corresponding peak area. The percentages are within the following ranges: Isoform Number 1 2 3 4 5 6 7 8 Content (per cent) 0 15 0 15 0 20 10 35 15 40 10 35 0 20 0 15
Casting the Stacking gel: 4 per cent Decant the water-saturated butanol and rinse the separating gel with water. (If the gel has not polymerized and flows out, discard and prepare fresh). Place the comb in position above the separating gel. Prepare the stacking mix by adding the reagents in the same sequence as given in the table below: Solutions Water Acrylamide (30 per cent) 0.5 M Tris Cl pH. 6.8 20 per cent SDS Ammonium persulphate (10 per cent w/v) (APS) TEMED For 5 ml (1 gel) 1.8 ml 0.6 ml 2.5 ml 25 l 50 l 5 l For 10 ml (2 gels) 3.6 ml 1.2 ml 5.0 ml 50 l 100 l 10 l
D. Immunoblotting. Determine by electrophoresis (sodium dodecyl sulphate polyacrylamide gel electrophoresis) (SDSPAGE)(2.4.12). Use a 1-mm thick spacer with six-well comb, a well capacity of 45 l and plate size of 100 x 120 mm Assemble the gel casting as recommended by the manufacturer. Use the composition of gel given below: Separating gel. 12 per cent Add the reagents into a clean glass/plastic container in the same sequence as given in the table below: Solutions Water Acrylamide (30per cent) 1.5 M Tris-Cl pH.8.8 20 per cent SDS Ammonium persulphate (10 per cent w/v) TEMED For 7 ml (for 1gel) 2.16 ml 2.8 ml 1.75 ml 35 ml 70 ml 3 ml For 14 ml (for 2 gels) 4.32 ml 5.6 ml 3.4 ml 70 ml 140 ml 6 ml
Mix the above ingredients by swirling gently and pour over the separating gel. Avoid bubbles. Keep aside for at least 45 minutes for polymerization at room temperature. Preparation of samples and loading Samples include: 1. 2. 3. Test sample Reference standard RS Prestained Marker 1.0 g 1.0 g 20 l
Test solution. Take X l of the test sample and add an equal volume of 2X non-reducing sample buffer where, X (l) = 1g per (concentration of the test sample in mg per ml) Reference solution. Dilute a part of 1mg per ml reference standard RS stock 10 times to get a final concentration of 0.1 mg per ml with water as follows: 10 l of 1 mg per ml reference standard RS + 90 ml water. To 10 l of 0.1 mg per ml reference standard RS solution add 10 ml of 2X non-reducing sample buffer. Prestained marker: Take 20 ml of prestained marker, reconstituted as recommended by the manufacturer. Loading of samples Boil the samples for two minutes, centrifuge, bring to room temperature and load the entire volume on the gel. Sample No. 1 2 3 6 Samples Test Sample Reference Marker Amount of protein Total Vol. to be loaded/well (g) loaded. (l) 1 1 20 20
Mix the above contents by swirling gently (do not mix vigorously) in the container and pour into the casting unit using a 1-ml pipette to 1 to 1.5 cm from the top edge of the plate. After addition of ammonium persulphate and TEMED, mix the solution and pour quickly (in less than 1minute) or it will begin to polymerize. Pour 200 1000 l of water-saturated butanol on the top of separation gel. Keep aside for at least 45 minutes for polymerization at room temperature.
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Running the gel 1. 2. 3. 4. Fix the gel apparatus in the running unit according to the manufacturers instructions. Chill the 1X running buffer to 4 for at least 1 hour. Pour 1X running buffer in the upper as well as lower chambers of the running unit. Set parameters on the Power pack as follows: Constant Voltage: 130 V Max Current: 200 mA. Run until dye front just goes out at the gel bottom (usually about 1.5 hrs)
5.
Transfer, blotting and development of membrane: 1. 2. 3. 4. 5. 6. Disassemble the running unit according tohe manufacturers instructions. Cut Nitrocellulose Membrane (NCM) having the same dimensions as of the gel. Equilibrate the membrane in chilled transfer buffer for 15 20 minutes. Equilibrate the gel in chilled transfer buffer for 15 20 minutes. Soak the nylon pads in transfer buffer and place on the pad on the cathode. Take 5 sheets of a suitable filter paper, cut to the size of the gel and soak in chilled transfer buffer. Place them on the nylon pad placed on cathode plate. Carefully place the equilibrated gel on the filter papers. Place the equilibrated membrane onto the gel.
17. Incubate for 1hour with gentle agitation at room temperature. 18. Wash the membrane with 1 X transfer buffer saline with change of buffer in-between at room temperature with gentle agitation. (3 times X 5 minutes) 19. Discard the transfer buffer saline. Add 25 ml of goat antirabbit ALP conjugated antibody (secondary antibody) solution. Incubate for 1 hour with gentle shaking at room temperature. 20. Wash the membrane with 1 X transfer buffer saline (3 times X 5 minutes) 21. Develop the color by adding 5 ml of the substrate solution. (Usually it takes 15 20 minutes). 22. Stop the reaction by pouring off the substrate solution and washing it with water before the color of the background gets dark. 23. Scan the blot while it is wet. 24. Air dry the blot and preserve it. The molecular mass markers are resolved on the membrane into discrete bands with a linear relationship between distance migrated and logarithm10 of the molecular mass. To evaluate the linear relationship between the distance migrated and logarithm10 of the molecular mass, calculate a) log molecular weights corresponding to marker bands b) migration distance of protein band c) plot (a) vs (b) and perform linear regression analysis The graph should be linear The single broad band of the test solution and of reference standard RS match in position and intensity. E. Peptide mapping (2.3.47). Test solution. Dilute the substance under examination in trisacetate buffer solution pH 8.5 to a concentration of 1.0 mg per ml. Equilibrate the solution in tris-acetate buffer solution pH 8.5 using a suitable procedure (such as dialysis against tris-acetate buffer solution pH 8.5, or membrane filtration using the procedure described under Identification C, but reconstituting the desalted sample with tris-acetate buffer solution pH 8.5). Transfer the dialyzed solution to a polypropylene centrifuge tube. Freshly prepare a solution of trypsin for peptide mapping at a concentration of 1 mg per ml in water, and add 5 ml to 0.25 ml of the dialysed solution. Cap the tube and place in a water-bath at 37 for 18 hours. Remove the sample from the water-bath and stop the reaction immediately by freezing. Reference solution. Dissolve the contents of a vial of erythropoietin RS in 0.25 ml of water. Prepare as for the test solution, ensuring that all procedures are carried out simultaneously, and under identical conditions. 7
7. 8. 9.
Roll a clean glass rod dipped in transfer buffer on top of the membrane to get rid of any air bubbles trapped between the gel and the membrane. 10. Place 5 sheets of a suitable filter paper soaked in chilled transfer buffer over the membrane. Once again roll the glass rod to remove any air bubbles. 11. Close the cassette. 12. Place the cassette in the running unit. 13. Connect the electrodes with the Power pack and set following parameters for transfer: Current: 200 mA Voltage: 100 V Time: 1 hour. 14. After the transfer is over, disassemble the cassette. 15. Transfer the membrane to the blocking solution. Incubate the membrane in the blocking buffer for 1 hour with gentle shaking at room temperature. 16. Discard the blocking buffer. Add 25 ml of rabbit anti r-Hu EPO antibody (Primary antibody) solution.
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Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with butylsilyl silica gel (5-10 m), mobile phase: A. 0.06 per cent v/v solution of trifluoroacetic acid, B. to 100 ml of water add 0.6 ml of trifluoroacetic acid and dilute to 1000 ml with acetonitrile, a linear gradient programme using the conditions given below, spectrophotometer set at 214 nm, a 50 l loop injector. Time (min) 0 10 10 125 125 135 135 145 145 150 Flow rate (ml/min) 0.75 0.75 1.25 1.25 1.25 Mobile phase A Mobile phase B (per cent v/v) (per cent v/v) 100 0 100 39 0 61 39 17 17 0 100 6183 83 100 0
cartridge using the protocol provided by the manufacturer. Run 15 sequencing cycles, using the reaction conditions for proline when running the second and third cycles. Identify the phenylthiohydantoin (PTH)-amino acids released at each sequencing cycle by reverse-phase liquid chromatography. The procedure may be carried out using the column and reagents recommended by the manufacturer of the sequencing equipment for the separation of PTH-aminoacids. The separation procedure is calibrated using: the mixture of PTH-amino acids provided by the manufacturer of the sequencer, with the gradient conditions adjusted as indicated to achieve optimum resolution of all amino acids, a sample obtained from a blank sequencing cycle obtained as recommended by the equipment manufacturer. If a sample does not comply with the limits set for the n-1 and n-2 sequences repeat the analysis.
Equilibrate at initial conditions for at least 15 minutes. Carry out a blank run using the above-mentioned gradient. The chromatogram obtained with each solution is qualitatively similar to the reference chromatogram of erythropoietin RS digest. The profile of the chromatogram obtained with the test solution corresponds to that of the chromatogram obtained with the reference solution. F. Determine by N-terminal sequence analysis The first 15 amino acids are: Alanine - Proline - Proline Arginine - Leucine - Isoleucine - (no recovered peak) - Aspartic acid - Serine - Arginine - Valine - Leucine - Glutamic acid Arginine - Tyrosine. Perform the Edman degradation using an automated solidphase sequencer, operated in accordance with the manufacturers instructions. Desalt the equivalent of 50 g of erythropoietin. For example, dilute a volume of the substance under examination containing 50 g of the active substance in 1 ml of a 0.1 per cent v/v solution of trifluoroacetic acid. Pre-wash a C18 reverse-phase sample preparation cartridge according to the instructions supplied by the manufacturer and equilibrate the cartridge in a 0.1 per cent v/v solution of trifluoroacetic acid. Apply the sample to the cartridge, and wash successively with a 0.1 per cent v/v solution of trifluoroacetic acid containing 0 per cent, 10 per cent and 50 per cent v/v of acetonitrile according to the manufacturers instructions. Lyophilise the 50 per cent v/ v acetonitrile eluate. Redissolve the desalted sample in 50 l of a 0.1 per cent v/v solution of trifluoroacetic acid and couple to a sequencing 8
Tests
Protein. 80 per cent to 120 per cent of the stated amount. Test solution. Dilute the substance under examination in a 0.4 per cent w/v solution of ammonium hydrogen carbonate or the appropriate blank solution. When examined in the range 250 nm to 400 nm (2.4.7), the solution shows an absorption maximum between 276 nm and 282 nm. Calculate the content of erythropoietin taking the specific absorbance to be 7.43. Dimers and related substances of higher molecular mass A. Determine by size-exclusion chromatography (2.4.16). Test solution. Dilute the substance under examination in the mobile phase to obtain a concentration of 0.2 mg per ml. Reference solution. To 0.02 ml of the test solution add 0.98 ml of the mobile phase (2 per cent). Chromatographic system a stainless steel column 6 cm x 7.5 mm, packed with hydrophilic silica gel, of a grade suitable for fractionation of globular proteins in the molecular mass range of 20 000 to 200 000, mobile phase: Dissolve 1.15 g of anhydrous disodium hydrogen phosphate, 0.2 g of potassium dihydrogen phosphate and 23.4 g of sodium chloride in 1 litre of water (1.5 mM potassium dihydrogen phosphate, 8.1 mM disodium hydrogen phosphate, 0.4M sodium chloride, pH 7.4); adjust the pH to 7.4 if necessary, flow rate. 0.5 ml per minute, spectrophotometer at 214 nm,
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a 100 l loop injector. Inject the test solution and the reference solution. Continue the chromatography for 1 hour. The area of the principal peak in the chromatogram obtained with the reference solution is 1.5 to 2.5 per cent of the area of the principal peak in the chromatogram obtained with the test solution. The total area of any peaks eluted before the principal peak is not more than the area of the principal peak in the chromatogram obtained with the reference solution (2 per cent). B. Determine by electrophoresis (sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) (2.4.12). Casting the Gels Use a 1-mm thick spacer with eight-well comb. The well capacity is 65 l. Assemble the gel - casting unit according to the manufacturers instructions. Use the assembly of plate size, 160 x 160 mm. a) Casting the separating gel: 12 per cent Add the reagents into a clean glass/plastic container in the same sequence as given in the table below: Solutions Water Acrylamide (30 per cent) 1.5 M Tris Cl pH. 8.8 20 per cent SDS Ammonium persulphate (10 per cent w/v) (APS) TEMED For 20 ml (1 gel) 6.17 ml 8.0 ml 5.0 ml 100 l 200 l 8 l For 40 ml (2 gels) 12.34 ml 16.0 ml 10.0 ml 200 l 400 l 16 l
Place the comb in position above the separating gel. Prepare the stacking mix by adding the reagents in the same sequence as given in the table below: Solutions Water Acrylamide (30 per cent) 0.5 M Tris Cl pH. 6.8 20 per cent SDS Ammonium persulphate (10 per cent w/v) (APS) TEMED For 5 ml (1 gel) 1.8 ml 0.6 ml 2.5 ml 25 l 50 l 5 l For 10 ml (2 gels) 3.6 ml 1.2 ml 5.0 ml 50 l 100 l 10 l
Mix the above contents by swirling gently and pour over the separating gel. Avoid bubbles. Keep aside for at least 45 minutes for polymerization at room temperature. Preparation of samples Samples include: 1. Test sample 2. 3. Reference standard RS Marker for non-reducing gel 10 g 10 g 20 l
Test solution. To a volume containing 10 g of protein add an equal volume of 2X non-reducing sample buffer. Reference solution. Take 10 l of the reference standard RS from 1mg per ml stock in a micro-centrifuge tube and add 10 l of 2X non-reducing sample buffer. Molecular weight marker. Take 20 l of low molecular weight markers for SDS-PAGE, which is reconstituted according to the manufacturers instruction: Sample loading Keep all the samples in a boiling water-bath for 2 minutes. Centrifuge, bring to room temperature and load the entire volume on to the gel. Sample No. 1 2 3 Samples Test Sample Reference Marker Amount of protein Total Vol. to be loaded/well (g) loaded. (l) 1 1 20 20
Mix the above ingredients by swirling gently and pou Mix the above contents by swirling gently (do not mix vigorously) in the container and pour into the casting unit using a 1-ml pipette till 1 - 1.5 cm from the top edge of the plate. After addition of ammonium persulphate and TEMED, the solution should be mixed and poured quickly (less than 1minute) or it will begin to polymerize. Pour 200 1000 l of water-saturated butanol on the top of the separation gel. Keep aside for at least 45 minutes for polymerization at room temperature (RT). b) Casting the Stacking gel: 4 per cent Decant the water-saturated butanol and rinse the separating gel with water. (If the gel has not polymerized and flows out, discard and prepare fresh) 9
Running the gel Fix the gel apparatus in the running unit as given in the instruction manual of the manufacturer.
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Chill the 1X running buffer to 4 for at least 1 hour. Pour 1X running buffer in the upper as well as lower chambers of the running unit. Set parameters on the Power pack as follows: Constant Voltage: 130 V Max Current: 200 mA. Run until the dye front reaches the bottom of the gel (usually about 5.0 hrs. The dye front should have run at least 80 per cent of the gel). Staining of Gel Stop the run. Disassemble the casting unit and transfer the gel carefully into a staining tray. Do not touch the gel with naked hands; wear gloves while handling the gel. Detect proteins in the gel by silver staining. Scanning Scan and save the image of the stained gel. Gel drying The stained gel is placed in between two cellophane sheets and clamped with the gel dryer frames (taking care that no air bubbles are present in between the two cellophane sheets). The set up is placed in the gel dryer apparatus and left at least 2 hrs for drying. The dye front is run for at least 80 per cent of the total gel length. Molecular weight markers are resolved on the gel into discrete bands, with a linear relationship between distance migrated and logarithm10 of the molecular mass. To evaluate the linear relationship between distance migrated and logarithm10 of the molecular mass, calculate a) c) log molecular weights corresponding to marker bands plot (a) vs (b) and perform linear regression analysis. b) migration distance of protein bands The graph should be linear. The single diffuse band of the test solution and of the reference solution match in position and intensity. Sialic acids. Not less than 10 mol of Sialic acids (calculated as N-acetylneuraminic acid) per mole of erythropoietin, determined in the following manner. Test solution (a). Dilute the preparation under examination in the mobile phase used in the test for dimers and related substances of higher molecular mass to obtain a concentration of 0.3 mg/ml. Test solution (b). To 0.5 ml of test solution (a) add 0.5 ml of the mobile phase used in the test for dimers and related substances of higher molecular mass. 10
Reference solution (a). Dissolve a suitable amount of Nacetylneuraminic acid in water to produce a solution containing 0.1 mg per ml. Reference solution (b). To 0.8 ml of reference solution (a) add 0.2 ml of water. Reference solution (c). To 0.6 ml of reference solution (a) add 0.4 ml of water. Reference solution (d). To 0.4 ml of reference solution (a) add 0.6 ml of water. Reference solution (e). To 0.2 ml of reference solution (a) add 0.8 ml of water. Reference solution (f). Use water Carry out the test in triplicate. Transfer 100 ml each of the test and reference solutions to 10-ml glass test tubes. To each tube add 1.0 ml of resorcinol reagent. Stopper the tubes and incubate at 100 for 30 minutes. Cool on ice. To each tube, add 2.0 ml of a mixture of 12 volumes of butanol and 48 volumes of butyl acetate. Mix vigorously, and allow the 2 phases to separate. Ensuring that the upper phase is completely clear, remove the upper phase, taking care to exclude completely any of the lower phases. Measure the absorbance. of all samples at 580 nm (2.4.7). Using the calibration curve generated by the reference solutions, determine the content of sialic acids in each of the two test solutions and calculate the mean. Calculate the number of moles of sialic acids per mole of erythropoietin assuming that the relative molecular mass of erythropoietin is 30 600 and that the relative molecular mass of Nacetylneuraminic acid is 309. System suitability. The individual replicates agree to within 10 per cent of each other; the value obtained from reference solution (a) is between 1.5 and 2.5 times that obtained with test solution (a). Bacterial endotoxins (2.2.3). Not more than 20 Endotoxin Units in the volume that contains 100 000 IU of erythropoietin. Assay. The activity of the preparation is compared with that of erythropoietin RS and expressed in International Units (IU). The estimated potency is not less than 80 per cent and not more than 125 per cent of the stated potency. The fiducial limits of error of the estimated potency (P = 0.95) are not less than 64 per cent and not more than 156 per cent of the stated potency. Determine by Method A or Method B. A. In polycythaemic mice The activity of the preparation is estimated by examining, under given conditions, its effect in stimulating the
IP 2007
incorporation of 59Fe into circulating red blood cells of mice made polycythaemic by exposure to reduced atmospheric pressure. The following schedule, using treatment in a hypobaric chamber, has been found to be suitable. Induce polycythaemia in female mice of the same strain, weighing 16 g to 18 g. Place the mice in a hypoxic chamber and reduce the pressure to 0.6 atmospheres. After 3 days at 0.6 atmospheres, further reduce the pressure to 0.4-0.5 atmospheres and maintain the animals at this pressure for a further 11 days (the partial vacuum is interrupted daily for a maximum of 1 h at about 11:00 a.m., in order to clean the cages and feed the animals). At the end of the specified period, return the mice to normal atmospheric conditions. Randomly distribute the mice into cages, each containing 6 animals, and mark them. Test solution (a). Dilute the substance under examination in phosphate-albumin buffered saline pH 7.2 to obtain a concentration of 0.2 IU per ml. Test solution (b). Mix equal volumes of test solution (a) and phosphate-albumin buffered saline pH 7.2. Test solution (c). Mix equal volumes of test solution (b) and phosphate-albumin buffered saline pH 7.2. Reference solution (a). Dissolve erythropoietin RS in phosphate-albumin buffered saline pH 7.2 to obtain a concentration of 0.2 IU per ml. Reference solution (b).Mix equal volumes of reference solution (a) and phosphate-albumin buffered saline pH 7.2. Reference solution (c). Mix equal volumes of reference solution (b) and phosphate-albumin buffered saline pH 7.2. Radiolabelled ferric [59Fe] chloride solution, concentrated. Use a commercially available solution of [59Fe] ferric chloride (approximate specific activity: 100-1000 MBq per mg of Fe). Radiolabelled [59Fe] ferric chloride solution. Dilute the concentrated radiolabelled [59Fe] ferric chloride solution in sodium citrate buffer solution pH 7.8 to obtain a solution with an activity of 3.7 104 Bq per ml. The concentrations of the test solutions and reference solutions may need to be modified, based on the response range of the animals used. 3 days after returning the animals to atmospheric pressure, inject each animal subcutaneously with 0.2 ml of one of the solutions. The 6 animals in each cage must each receive one of the 6 different treatments (3 test solutions and 3 reference solutions), and the order of injection must be separately randomised for each cage. A minimum of 8 cages is recommended. 2 days after injection of the test or reference solution, inject each animal intraperitoneally with 0.2 ml of 11
radiolabelled [59Fe] ferric chloride solution. The order of the injections must be the same as that of the erythropoietin injections, and the time interval between administration of the erythropoietin and the radiolabelled ferric chloride solution must be the same for each animal. After a further 48 h, anaesthetise each animal by injection of a suitable anaesthetic, record body weights and withdraw blood samples (0.65 ml) into haematocrit capillaries from the bifurcation of the aorta. After determining the packed cell volume for each sample, measure the radioactivity. Calculate the response (percentage of iron-59 in total circulating blood) for each mouse using the expression:
A s M 7.5 A t Vs
Where, As At 7.5 M Vs
= = = = =
radioactivity in the sample, total radioactivity injected, total blood volume as per cent body weight, body weight, in grams, sample volume.
Calculate the potency by the usual statistical methods for a parallel line assay. Eliminate from the calculation any animal where the packed cell volume is less than 54 per cent, or where the body weight is more than 24 g. B. In normocythaemic mice The assay is based on the measurement of stimulation of reticulocyte production in normocythaemic mice. Test solution (a). Dilute the substance under examination in phosphate-albumin buffered saline pH 7.2 to obtain a concentration of 80 IU per ml. Test solution (b). Mix equal volumes of test solution (a) and phosphate-albumin buffered saline pH 7.2. Test solution (c). Mix equal volumes of test solution (b) and phosphate-albumin buffered saline pH 7.2. Reference solution (a). Dissolve erythropoietin RS in phosphate-albumin buffered saline pH 7.2 to produce a solution containing 80 IU per ml. Reference solution (b). Mix equal volumes of reference solution (a) and phosphate-albumin buffered saline pH 7.2. Reference solution (c). Mix equal volumes of reference solution (b) and phosphate-albumin buffered saline pH 7.2. The exact concentrations of the test solutions and reference solutions may need to be modified, based on the response range of the animals used. At the beginning of the assay procedure, randomly distribute mice of a suitable age and strain (8-week old B6D2F1 mice are
FILGRASTIM CONCENTRATED SOLUTION
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suitable. Other strains like Swiss Albino, Balb/C of suitable age can also be used) into 6 cages. A minimum of 8 mice per cage is recommended. Inject each animal subcutaneously with 0.5 ml of the appropriate treatment (one solution per cage) and put the animal in a new cage. Combine the mice in such a way that each cage housing the treated mice contains one mouse out of the 6 different treatments (3 test solutions and 3 reference solutions, 6 mice per cage). 4 days after the injections, collect blood samples from the animals and determine the number of reticulocytes using the following procedure. The volume of blood, dilution procedure and fluorescent reagent may need to be modified to ensure maximum development and stability of fluorescence. Colorant solution, concentrated. Use a solution of thiazole orange suitable for the determination of reticulocytes. Prepare at a concentration twice that necessary for the analysis. Proceed with the following dilution steps. Dilute whole blood 500-fold in the buffer used to prepare the colorant solution. Dilute this solution 2-fold in the concentrated colorant solution. After staining for 3-10 min, determine the reticulocyte count microfluorometrically in a flow cytometer. The percentage of reticulocytes is determined using a biparametric histogram: number of cells/red fluorescence (620 nm). Calculate the potency by the usual statistical methods for a parallel line assay. Storage. Store in an airtight container at a temperature below -20. Avoid repeated freezing and thawing. Labelling. The label states (1) the erythropoietin content in mg per ml; (2) the activity in International Units per ml; (3) the name and the concentration of any other excipients.
Filgrastim Concentrated Solution contains not less than 1.5 mg of protein per ml, and not less than 1.0 x 108 IU of filgrastim per mg of protein. Prior to release, the following tests are carried out on each batch of the final bulk product, unless the regulatory authority has granted exemption. Description. A clear, colourless to slightly yellowish liquid.
Identification
A. It shows the biological activity as decribed under Assay. B. Determine by capillary electrophoresis (capillary isoelectric focusing) (2.4.32). In the test for impurities with charges different from that of filgrastim the principal band in the electropherogram obtained with the test solution is similar in position to the principal band in the electropherogram obtained with the reference solution. C. In the test for impurities of molecular masses higher than that of filgrastim, the retention time, of the principal peak obtained with the test solution is similar to that of the principal peak obtained with the reference solution. D. In the test for impurities with molecular masses differing from that of rG-CSF under both reducing and non-reducing conditions, the principal band in the electropherogram obtained with test solution (a) is similar in position to the principle band in the electropherogram obtained with reference solution (a). E. Determine by peptide mapping (2.3.47). Test solution. Introduce 50 l of a 0.05 M sodium phosphate buffer pH 8.0 into a polypropylene tube. Add a volume of the substance under examination corresponding to 25 g of protein, and 25 l of a 0.1 mg per ml solution of Glu-C2 protease; dilute to 1 ml with water, stopper the tube and incubate at about 37 for 18 hours. Add 125 l of a 7.64 per cent w/v solution of guanidine hydrochloride and mix well. Add 10 l of a 1.542 per cent w/v solution of dithiothreitol and mix well. Place the capped tube in boiling water for 1 minute. Allow to cool to room temperature. Reference solution. Prepare at the same time and in the same manner as for the test solution but use G-CSF RS instead of the test preparation under examination. Chromatographic system a stainless steel column 10 cm x 2 mm, packed with octadecylsilyl silica gel (5 m) with a pore size of 20 nm, mobile phase: A. Dilute 0.5 ml of trifluoroacetic acid to 950 ml with water add 50 ml of acetonitrile and mix, B. Dilute 0.5 ml of trifluoroacetic acid to 50 ml with water; add 950 ml of acetonitrile, flow rate. 0.2 ml per minute, 12
Filgrastim Concentrated Solution

Granulocyte Colony Stimulating Factor Solution
MTPLGPASSL PQSFLLKCLE QVRKIQGDGA ALQEKLCATY KLCHPEELVL LGHSLGIPWA PLSSCPSQAL QLAGCLSQLH SGLFLYQGLL QALEGISPEL GPTLDTLQLD VADFATTIWQ QMEELGMAPA LQPTQGAMPA FASAFQRRAG GVLVASHLQS FLEVSYRVLR HLAQP
C845H1343N223O243S9
Mol. Wt. 18799
Filgrastim Concentrated Solution is a solution of a protein having the structure of the granulocyte colony-stimulating factor (G-CSF) produced and secreted by various human blood cell types. The protein stimulates the differentiation and proliferation of leucocyte stem cells into mature granulocytes. It is produced by a method based on rDNA technology, using bacteria as host cells.
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A linear gradient programme using the conditions given below, spectrophotometer set at 215 nm, a 10 l loop injector. Time (min) 0 8.0 8 25 25 40 40 45 45 46 46 65 Mobile phase A (per cent v/v) 36 6 34 34 90 90 90 3 3 Mobile phase B (per cent v/v) 97 94 94 66 66 100 10 10 97 97
sulphate polyacrylamide gel electrophoresis) (2.4.12) under both reducing and non-reducing conditions. Gel dimensions. 1.0 mm thick. 160 X 160 mm Resolving gel. 13 per cent Acrylamide Sample buffer A. Sample buffer B (reducing conditions). Test solution (a). Dilute the preparation under examination with water to produce a solution containing 0.1 mg per ml. To 50 volumes of this solution, add 13 volumes of concentrated SDS-PAGE sample buffer. Test solution (b). Prepare in the same manner as test solution (a) but using concentrated SDS-PAGE sample buffer for reducing conditions. Reference solution (a). Dilute the preparation under examination with water to obtain a concentration of 0.1 mg per ml. To 50 volumes of this solution, add 13 volumes of concentrated SDS-PAGE sample buffer. Reference solution (b.) Prepare as for reference solution (a), but using concentrated SDS-PAGE sample buffer for reducing conditions. Reference solution (c). Use a solution of molecular mass marker suitable for calibrating SDS-PAGE gels in the range of 14 400 to 94 000. Dissolve in sample buffer of sample buffer (reducing conditions), as appropriate. Reference solution (a) (b) (c) (d) (e) (f) (g) Protein amount (g) 100 50 20 10 5 2 1 Sample Volume (l) 20 20 20 20 20 20 20 SDS-PAGE Sample Buffer 20 20 20 20 20 20 20
Equilibrate the column at the initial conditions for at least 30 minutes. Inject the test solution and the reference solution. The chromatograms obtained with the reference solution and the test solutions are qualitatively similar. The profile of chromatogram obtained with the reference solution and the test solution corresponds to that of the chromatogram obtained with the reference solution. F. Determine by N-Terminal Sequencing. Perform the Edman degradation using an automated solidphase sequencer, operated in accordance with the manufacturers instructions. Load about 1ml of the test preparation to a sequencing cartridge using the protocol provided by the manufacturer. Run 16 sequencing cycles, noting, if appropriate the presence of praline at positions 3, 6 and 111. Identify PTH-amino acids released at each sequencing cycle by RPHPLC. The procedure may be carried out using the column and reagents recommenced by the manufacturer of the sequencing equipment for the separation of PTH-amino acids. The separation procedure is calibrated using: a) the mixture of PTH-amino acids provided by the manufacturer of the sequencer, with the gradient conditions adjusted as indicated to achieve optimum resolution of all amino acids; b) a sample obtained from a blank sequencing cycle obtained as recommended by the equipment manufacturer. The first 16 amino acids should match the sequence given in the beginning starting with Met.
Sample treatment: boil for 5 minutes. Apply 20 l of each reduced and non-reduced solutions to separate gels. Detection: Silver staining as described below. Immerse the gel for 30 minutes in a mixture of 10 volumes of acetic acid, 40 volumes of water and 50 volumes of methanol. Transfer the gel to 5 per cent methanol and shake for 5 minutes. Repeat this washing step thrice. Replace the 5 per cent v/v solution of methanol with 0.2 g per litre sodium thiosulphate. Wash the gel thrice in water for 30 seconds each. Transfer the gel to a 0.2 per cent w/v solution of silver nitrate. 13
Tests
Impurities with molecular masses differing from that of Filgrastim. Determine by electrophoresis (sodium dodecyl
IP 2007
This solution is prepared immediately before use. Place the gel on shaker for 25 minutes. Wash the gel for 1 minute. Repeat this washing step thrice. Transfer the gel into a mixture containing 30 g per litre solution of sodium carbonate, 0.05 per cent v/v solution of formaldehyde and 0.2 g per litre solution of sodium thiosulphate in water. Protein bands become visible during this step. Keep the gel in the solution until sufficiently stained and then stop the staining by soaking the gel in a 14 g per litre solution of disodium edetate. The test is not valid unless the proteins of the molecular weight marker are distributed along 80 per cent of the gel and over the required separation range (the range covering the product and its dimer or the product and its related impurities). In the electropherogram obtained with test solution (a) no band is more intense that the principal band in the electropherogram obtained with reference solution (f). Impurities with charges differing from that of Filgrastim. Determine by capillary electrophoresis (capillary isoelectric focusing) (2.4.32). Test solution. Dilute the preparation under examination to produce a solution containing 0.3 mg per ml. Reference solution (a). A solution of filgrastim RS containing 0.3 mg per ml. Reference solution (b). A solution of filgrastim RS containing 0.06 mg per ml. Reference solution (c). Use an isoelectric point (pI) calibration solution, in the pI range of 2.5-6.5, prepared according to manufacturers instructions. Focusing: pH gradient. 4.5 - 8.0, catholyte. 1 M sodium hydroxide, anolyte: 0.04 M glutamic acid in a 0.0025 per cent v/v solution of phosphoric acid, Application 20 l. Detection. Proceed as described in Isoelecric Focusing (2.4.33). Detect the product and its dimer or the product and its related impurities. In the electropherogram obtained with reference solution (c), relevant isoelectric point markers are distributed along the entire length of the gel. In the electropherogram obtained with reference solution (a), the pI of the principal band is 5.7-6.3. No band is more intense that the principal band in the electropherogram obtained with reference solution (b). Related Proteins. Determine by liquid chromatography (2.4.14). 14
Test solution. Dilute the preparation under examination with mobile phase A to obtain a concentration of 0.3 mg per ml. Reference solution (a). Dilute G-CSFRS with the same mobile phase to obtain a concentration of 0.3 mg per ml. Reference solution (b). To 570 l of reference solution (a), add 6.8 l of a 0.45 per cent v/v solution of hydrogen peroxide; mix and incubate at 25 for 1hour, then add 2.5 mg of methionine RS. Chromatographic system a stainless steel column 15 cm x 4.6 mm, packed with octadecylsilyl silica gel (5 m) with a pore size of 20 nm and a guard column 7.5 cm x 7.5mm packed with octadecylsilyl silica gel, column temperature. 65, mobile phase: A. dilute 1 ml of trifluoroacetic acid to 500 ml with water and add 499 ml of acetonitrile, B. dilute 1 ml of trifluoroacetic acid to 950 ml with acetonitrile and add 49 ml of water, flow rate. 1 ml per minute, a linear gradient programme using the conditions given below, spectrophotometer set at 215 nm, a 50 l loop injector. Time (min) 04 4 19 19 19.1 19.1 21 21 21.1 21.1 25 Mobile phase A (per cent v/v) 92 92 72 72 0 0 0 92 92 Mobile phase B (per cent v/v) 8 8 28 28 100 100 100 8 8
Inject the test solution and reference solutions (a) and (b). Relative retention with reference to filgrastim (retention time=about 12 minutes.): oxidized filgrastim 2= about 0.95 The profile of the chromatogram obtained with reference solution (b) is similar to that of the chromatogram of oxidized filgrastim supplied with filgrastim RS. Two peaks corresponding respectively to oxidized filgrastim 2 elute before the principal peak, the second peak not being completely separated form the principal peak. In the chromatogram obtained with the test solution, the area of any peak other than the principal peak is not greater than 2.0 per cent of the total area of all the peaks. The sum of the areas of any peaks other than the principal peak is not greater than 3.5 per cent of the total area of all of the peaks. Dimers and Related Substance of Higher Molecular Mass. Determine by size exclusion chromatography (2.4.14).
IP 2007
INTERFERON ALFA-2 CONCENTRATED SOLUTION
Solution A. Dissolve 4.1 g of sodium acetate in 400 ml of water, adjust to pH 4.0 with acetic acid and dilute to 500 ml with water. Test solution. Dilute the preparation under examination with solution A to obtain a concentration of 0.4 mg per ml. Reference solution. Dilute filgrastim RS with solution A to obtain a concentration of 0.4 mg per ml. Resolution solution. Mix a sample of the reference solution for about 30 seconds using a vortex mixer. Chromatographic system a stainless steel column 30 cm x 7.8 mm, packed with hydrophilic silica gel column temperature. 30, mobile phase: dissolve 7.9 g of ammonium hydrogen carbonate in 1000 ml of water and adjust to pH 7.0 with phosphoric acid; dilute to 2000 ml with water, flow rate. 0.5 ml per minute, spectrophotometer set at 215 nm, a 200 l loop injector. Relative retention times with reference to filgrastim monomer (retention time=about 19 minutes): aggregates = about 0.60; filgrastim oligomer 1= about 0.75; filgrastim oligomer 2= about 0.80; filgrastim dimer=about 0.85. Inject the resolution solution. The retention time of filgrastim monomer is 17 minutes to 20 minutes. The resolution between the peaks due to filgrastim dimer and filgrastim monomer is not less than 4.0. Calculate the content of dimer, oligomers and aggregates. The sum of the peaks with retention times less that that of the principal peak is not more than 2.5 per cent. Bacterial Endotoxins. Not more than 2 Endotoxin Unit per mg of protein. Assay. A. Protein - Determine by liquid chromatography (2.4.14) as described under the test for Related proteins. Inject the test solution and reference solution (a). Calculate the content of filgrastim. B. Potency- Determination of the biological activity of rGCSF concentrated solution is based on the stimulation of NFS60 cells (murine myeloblastic cell line) by rG-CSF. The following method uses the conversion of tetrazolium bromide (MTS) as a staining method. Alternative methods of quantifying cell proliferation, such as measurement of intracellular ATP by luciferase bioluminescence have also been found suitable, and may be used as the assay readout, subject to appropriate validation. NFS-60 cells are incubated with varying dilution of test and reference preparations of rG-CSF. They are then incubated with a solution of MTT. This cytochemical stain is converted by cellular dehydrogenases to a purple formazan product. 15
The formazan is then measured spectrophotometrically. The potency of the test preparation is determined by comparison of the dilutions of the test preparation with the dilutions of the appropriate International Standard of rG-CSF or with a reference preparation calibrated in International Units, which yield the same response (50 per cent maximal stimulation). The International Unit is the activity contained in a stated amount of the appropriate International Standard. The equivalence in International Units of the International standard is stated by the World Health Organization. Add 50 l of the dilution medium to all wells of a 96 well microtitre plate. Add an additional 50 l of this solution to the wells designed for blanks. Add 50 l of each solution to be tested in triplicate (test preparation and reference preparation at a concentration of about 800 IU per ml, plus a series of 10 twofold dilutions to obtain a standard curve). Prepare a suspension of NFS-60 cells containing 7x105 cells per ml. immediately before use, add 2-mercaptoethanol to a final concentration of 0.1 mM, and add 50 l of the prepared cell suspension to each well, maintaining the cells in a uniform suspension during addition. Incubate the plate at 36.0- 38.0 for a minimum of 24 hours in a humidified incubator using 6 1 per cent CO2. Add 20 ml of a 5.0 g per litre sterile solution of tetrazolium bromide to each well and re-incubate 4 hours. Estimate the quantity of formazan produced using a microtitre well plate reader at 490 nm. Analyze the data by fitting a sigmoidal dose-response curve to the data obtained and by using a suitable stastical method, for example the 4-parameter or parallel line models. The estimated potency is not less than 80 per cent and not more than 125 per cent of the stated potency. The confidence limits (P=0.95) of the estimated potency are not less than 74 per cent and not more than 136 per cent of the stated potency. Storage. Store protected from light in a refrigerator (2 to 8). Labeling. The label states the content, in mg of protein per ml; the potency, in IU per mg of protein.
Interferon Alfa-2 Concentrated Solution

CDLPQTHSLG EEFGNQFQKA LDKFYTELYQ KYFQRITLYL LRSKE SRRTLMLLAQ ETIPVLHEMI QLNDLEACVI KEKKYSPCAW MRX1ISLFSCL QQIFNLFSTK QGVGVTETPL EVVRAEIMRS KDRHDFGFPQ DSSAAWDETL MKEDSILAVR FSLTSNLQES
alfa-2a: C860H1353N227O255S9 alfa-2b: C860H1353N229O255S9
Mol. Wt.19,241 Mol. Wt.19,269
IP 2007
Interferon alfa-2 concentrated solution is a solution of an rDNA derived therapeutic protein which exhibits non-specific antiviral activity, at least in homologous cells. Interferon alfa2 concentrated solution also exerts antiproliferative and immunomodulator activity. Two different types of alfa-2 interferon, varying in the amino acid residue at position 23, are found and are named as alfa-2a and alfa-2b. Designation alfa-2a alfa-2b Residue at position 23 (X1) Lys Arg
Horizontal Electrophoresis Select and use a suitable horizontal isoelectric focusing apparatus with facility for connecting a circulating bath chiller capable of maintaining 10. Select gels for isoelectric focusing with a pH gradient from 3.5 to 9.5 Use phosphoric acid as anode solution (98 g per litre phosphoric acid) and 1 M sodium hydroxide as the cathode solution. Using filter paper apply 15 l of the test solution and the reference solution to the gel close to the cathode. Start the isoelectric focusing at 1500 V and 50 mA. Turn off the power after 30 minutes. Remove the application filters and reconnect the power supply for 1 hour. Keep the power constant during the focusing process. Immerse the gel in a solution containing 115 g per litre of trichloroacetic acid and 34.5 g per litre of sulphosalicylic acid in water and agitate the container gently for 60 minutes. Prepare a mixture of 32 volumes of glacial acetic acid, 100 volumes of ethanol and 268 volumes of water. Transfer the gel to the mixture and soak for 5 minutes. Immerse the gel for 10 minutes in a staining solution prewarmed to 60.The staining solution is prepared by adding 1.2 g per litre of acid blue 83 to the mixture of glacial acetic acid, ethanol and water. Wash the gel several times to destain with the mixture of glacial acetic acid, ethanol and water and keep the gel in this mixture for about 12-24 hours until the background is clear. Add glycerol 10 per cent v/v to the mixture of glacial acetic acid, ethanol and water. Soak the gel for 1 hour in the solution. The principal bands of the electropherogram obtained with the test solution correspond in position to the principal bands of the electropherogram obtained with the reference solution. Plot the migration distances of the isoelectric point markers versus their isoelectric points and determine the isoelectric points of the principal components of the test solution and the reference solution. They do not differ by more than 0.2 pI unit. The test is not valid unless the isoelectric point markers are distributed along the entire length of the gel and the isoelectric points of the principal bands in the electropherogram obtained with the reference solution are between 5.8 and 6.3. C. Examine the electropherograms obtained under reducing conditions in the test for impurities of molecular masses differing from that of interferon alfa-2. The principal band in the electropherogram obtained with test solution (a) corresponds in position to the principal band in the electropherogram obtained with reference solution (a). D. Determine by peptide mapping (2.3.47). Test solution. Dilute the preparation under examination with water to produce a solution containing 0.5 mg protein per ml. 16
This monograph applies to interferon alfa-2a and 2b concentrated solutions. Interferon alfa-2 concentrated solution contains not less than 1.4 108 IU per mg of protein and not less than 0.5 mg of interferon alfa-2 per ml.
Production
Interferon alfa-2 concentrated solution is produced by a method based on recombinant DNA (rDNA) technology. It is produced under controlled conditions designed to ensure sterility of the product. Interferon alfa-2 concentrated solution complies with the following additional requirements. Host-cell-derived proteins The limit is approved by WHO guidelines. Host-cell- or vector-derived DNA The limit is approved by WHO guidelines. Description. A clear,colourless or slightly yellowish liquid.
Identification
A. It shows the expected biological activity as described under Assay for potency. B. Determine by isoelectric focusing. Test solution. Dilute the preparation under examination with water to obtain a solution containing 0.5 mg protein per ml. Reference solution. A 0.5 mg per ml solution of interferon alfa-2 RS in water. Isoelectric point calibration solution pI range 3.0 to 10.0. Prepare and use according to the manufacturers instructions. Isoelectric focusing is carried out using either horizontal electrophoresis system or by vertical electrophoresis system as per the procedure described below or by any appropriate validated method.
IP 2007
Transfer 25 l to a microfuge tube of 1.5 ml capacity. Add 1.6 l of 1 M phosphate buffer solution pH 8.0, 2.8 l of a freshly prepared 1.0 mg per ml solution of trypsin in water (suitable for peptide mapping) and 3.6 l of water and mix vigorously. Cap the tube and place it in a water-bath at 37 for 18 hours. Add 100 l of a 573 g per litre solution of guanidine hydrochloride and mix well. Add 7 l of 154.2 g per litre solution of dithiothreitol and mix well. Place the capped tube in boiling water for 1 minute and cool to room temperature. Reference solution. A 0.5 mg per ml solution of the appropriate interferon alfa-2 RS prepared at the same time and in the same manner as the test solution. Chromatographic system a stainless steel column 100 cm x 4.6 mm, packed with octadecylsilyl silica gel (5 m) with a pore size of 30 nm, mobile phase A.1 ml of trifluoroacetic acid dilute to 1000 ml with water, B. Add 1 ml of trifluroacetic acid to 100 ml of water and dilute to 1000 ml with acetonitrile, flow rate. 1 ml per minute, a linear gradient programme using the conditions given below, spectrophotometer set at 214 nm, a 100 l loop injector. Time Mobile Mobile Comment phase A phase B (min) (per cent v/v) (per cent v/v) 0.8 8.68 68.72 72.75 75.08 100 100 04 40 40 100 100 0 0 60 60 60 0 0 isocratic linear gradient isocratic linear gradien re-equilibration
Sample buffer (reducing conditions). Mix equal volumes of water and concentrated SDS PAGE sample buffer for reducing conditions containing 2-mercaptoethanol as the reducing agent. Test solution (a).Dilute the preparation under examination in the sample buffer to obtain a solution containing a concentration of 0.5 mg protein per ml. Test solution (b). Dilute 0.2 ml of test solution (a) to 1 ml with the sample buffer. Reference solution (a). Prepare a 0.625 mg per ml solution of the appropriate interferon alfa-2 RS in the sample buffer. Reference solution (b). Dilute 0.2 ml of reference solution (a) to 1 ml with the sample buffer. Reference solution (c). Dilute 0.2 ml of reference solution (b) to 1 ml with the sample buffer. Reference solution (d). Dilute 0.2 ml of reference solution (c) to 1 ml with the sample buffer. Reference solution (e). Dilute 0.2 ml of reference solution (d) to 1 ml with the sample buffer. Reference solution (f). Use a solution of molecular mass standards suitable for calibrating SDS-PAGE gels in the range 15 kDa to 67 kDa. Place the test and reference solutions, contained in covered test-tubes, on a water-bath for 2 minutes. Apply 10 l of reference solution (f) and 50 l of each of the other solutions to the stacking gel wells. Perform the electrophoresis under the conditions recommended by the manufacturer of the equipment. Detect proteins in the gel by silver staining. The test is not valid unless (1) the validation criteria are met; (2) a band is seen in the electropherogram obtained with reference solution (e); (3) a gradation of intensity of staining is seen in the electropherograms obtained, respectively, with test solution (a) and test solution (b) and with reference solutions (a) to (e). The electropherogram obtained with test solution (a) under reducing conditions may show, additional bands but no such band should be more intense than the band obtained with reference solution (d). Further, not more than 3 such bands should be more intense than the principal band obtained with reference solution (e). The electropherogram obtained with test solution (a) under non-reducing conditions may show, in addition to the principal band, less intense bands with molecular masses higher than the principal band. No such band is more intense than the principal band in the electropherogram obtained with reference solution (d) and not more than three such bands are more 17
Equilibrate the column with mobile phase A for at least 15 minutes maintaining the temperature of the column at 30. Inject the test solution and the reference solution. The chromatogram obtained with each solution should be qualitatively similar to the chromatogram of interferon alfa-2. The profile of the chromatogram obtained with the test solution should also correspond to that of the chromatogram obtained with the reference solution.
Tests
Impurities of molecular masses differing from that of interferon alfa-2. Determine by electrophoresis (sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE) (2.4.12). The test is performed under both reducing and non-reducing conditions, using resolving gels of 14 per cent acrylamide and silver staining as the detection method. Sample buffer (non-reducing conditions). Mix equal volumes of water and concentrated SDS PAGE sample buffer.
IP 2007
intense than the principal band in the electropherogram obtained with reference solution (e). Related proteins. Determine by liquid chromatography (2.4.14). 0.25 per cent w/w hydrogen peroxide solution. Dilute hydrogen peroxide solution with water to obtain 0.25 per cent w/w solution. Test solution. Dilute the preparation under examination with water to obtain a solution containing 0.5 mg protein per ml. Reference solution. To a volume of the test solution, add a suitable volume of the 0.25 per cent hydrogen peroxide solution to give a final hydrogen peroxide concentration of 0.005 per cent, and allow to stand at room temperature for 1 hour, to generate about 5 per cent oxidised interferon. Add 12.5 mg of L-methionine per ml of the solution. Allow to stand at room temperature for 1 hour. Store the solutions for not longer than 24 hours at a temperature of 2 to 8. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilyl silica gel (5 m) with a pore size of 30 nm, mobile phase: A. To 700 ml of water add 2 ml of trifluoroacetic acid and 300 ml of acetonitrile, B. To 200 ml of water add 2 ml of trifluoroacetic acid and 800 ml of acetonitrile, flow rate. 1 ml per minute, a linear gradient programme using the conditions given below, spectrophotometer set at 210 nm, a 100 l loop injector Time (min) 0.1 1.5 5 20 20 30 30 40 40 42 42 50 50 60 Mobile Mobile phase A phase B (per cent v/v) (per cent v/v) 72 280 72 67 28 33 67 63 33 37 63 57 37 43 57 40 43 60 40 60 40 72 60 28 72 28 Comment
1.0. Consider only the peaks whose retention time is 0.7 to 1.4 relative to that of the principal peak. In the chromatogram obtained with the test solution, the area of any peak, apart from the principal peak, is not greater than 3.0 per cent of the total area of all of the peaks. The sum of the areas of any peaks other than the principal peak is not greater than 5.0 per cent of the total area of all of the peaks. Bacterial endotoxins (2.2.3). Not more than 100 Endotoxin Units per mg of protein. Assay Protein Test solution. Dilute the preparation under examination with water to obtain a concentration of about 0.5 mg of interferon alfa-2 per ml. Reference solutions. Prepare a stock solution of 0.5 mg per ml of bovine albumin. Prepare eight dilutions of the stock solution containing between 3 g per ml and 30 g per ml of bovine albumin. Prepare 30-fold and 50-fold dilutions of the test solution. Prepare a mixture of 2.0 ml of a 2.0 per cent w/v solution of copper sulphate in water, 2.0 ml of a 4.0 per cent w/v solution of sodium tartrate in water and 96.0 ml of a 4.0 per cent w/v solution of sodium carbonate in 0.2 M sodium hydroxide. Add 1.25 ml of the above mixture to the test-tube containing 1.5 ml of water to prepare the blank, 1.25 ml to the test tube containing different dilutions of the sample and 1.25 ml to the test tube with the reference solution. Mix after each addition and after approximately 10 minutes, add to each test-tube 0.25 ml of a mixture of equal volumes of water and phosphomolybdotungstic reagent. Mix after each addition. After 30 minutes, measure the absorbance of each solution at 750 nm (2.4.7) using the blank as the compensation liquid. Draw a calibration curve from the absorbances of the eight reference solutions and the corresponding protein contents and read from the curve the content of protein in the test solution. Potency The potency of interferon alfa-2 is estimated based on its ability to protect cells against a viral cytopathic effect compared to the protection accorded by an appropriate International Standard of human recombinant interferon alfa-2 or of a reference preparation calibrated in International Units. The International Unit is the activity contained in a stated amount of the appropriate International Standard. The equivalence in International Units of the International Standard is stated by the World Health Organisation. 18
isocratic linear gradient linear gradient linear gradient linear gradient isocratic linear gradien re-equilibration
Equilibrate the column with the mobile phases in the initial gradient ratio for at least 15 minutes. Inject alternatively the test solution and the reference solution. Interferon alfa-2 elutes at a retention time of about 20 minutes in the chromatogram. With the reference solution a peak related to oxidized interferon appears at a retention time of about 0.9 relative to the principal peak. The test is not valid unless the resolution between the peaks corresponding to oxidised interferon and interferon is at least
IP 2007
STREPTOKINASE BULK SOLUTION
Carry out the assay by a suitable method, based on the following design. Use, an established cell line sensitive to the cytopathic effect of a suitable virus, responsive to interferon. The following cell cultures and virus have shown to be suitable: MDBK cells (ATCC No. CCL22), or Mouse L cells (NCTC clone 929; ATCC No.CCL 1) as the cell culture and vesicular stomatitis virus VSV, Indiana strain (ATCC No. VR-158) as the infective agent; or human diploid fibroblast FS-71 cells responsive to interferon as the cell culture, and encephalomyocarditis virus (ATCC No.VR-129B) as the infective agent. Incubate in at least three groups, cells with three or more different concentrations of the preparation under examination and one with the reference preparation in a microtitre plate. Include appropriate controls of untreated cells in each group. Choose the concentrations of the preparations such that the lowest concentration produces some protection and the largest concentration produces less than maximal protection against the viral cytopathic effect. Add the cytopathic virus after the cells have established to all wells except the control wells. Determine the cytopathic effect of virus quantitatively and calculate the potency of the preparation to be examined by the usual statistical methods for a parallel line assay. The estimated potency is not less than 80 per cent and not more than 125 per cent of the stated potency. The confidence limits of the estimated potency (P= 0.95) are not less than 64 per cent and not more than 156 per cent of the stated potency. Storage. Store protected from light, at or below 20. Labelling. The label states (1) The type of interferon (alfa-2a or alfa-2b); (2) the type of production.
demonstrate that the product, if tested, would comply with the following test. Abnormal toxicity Inject into each mouse a quantity of the preparation under examination (diluted, if necessary, with water for injections) containing 50,000 IU of streptokinase activity in 0.5 ml, the injection lasting 15-20 seconds. Description. A clear, colourless liquid.
Identification
A. Place 0.5 ml of citrated human plasma in a haemolysis tube maintained in a water-bath at 37. Add 0.1 ml of a dilution of the preparation under examination containing 10,000 IU of streptokinase activity per ml in phosphate buffer pH 7.2 and 0.1 ml of a solution of human thrombin RS containing 20 IU per ml in phosphate buffer pH 7.2. Mix immediately. A clot forms and lyses within 30 minutes. Repeat the procedure using citrated bovine plasma. The clot does not lyse within 60 minutes. B. Dissolve 0.6 g of agar in 50.0 ml of barbitone buffer pH 8.6, heating until a clear solution is obtained. Use glass plates 50 mm square (transparency mounts) free from traces of grease. Using a pipette, apply to each plate 4 ml of the agar solution. Maintain the plates horizontal. Allow to cool. Pierce a cavity 6 mm in diameter in the centre of the agar and an appropriate number of cavities (not exceeding 6) at distances of 11 mm from the central cavity. Remove the residual agar from the cavities using a cannula connected to a vacuum pump. Using pipettes graduated in microlitres, place in the central cavity about 80 l of goat or rabbit antistreptokinase serum containing 10,000 units of antistreptokinase activity per ml; place in each of the surrounding cavities about 80 l of a dilution of the preparation under examination containing 125,000 IU of streptokinase activity per ml. Allow the plates to stand in a humidified tank for 24 hours. Only one precipitation arc appears and it is well defined and localised between the application point of the serum and each cavity containing the solution of the preparation under examination.
Streptokinase Bulk Solution

Streptokinase Bulk Solution is a fibrinolytic enzyme present in certain strains of haemolytic Streptococcus group C. It has the property of combining with human plasminogen to form plasminogen activator. Streptokinase is also produced by a method based on recombinant DNA technology using bacteria or suitable genetically engineered host cells. Streptokinase Bulk Solution has a potency of not less than 96,000 IU per mg of protein.
Tests
pH (2.4.24). 6.8 to 7.5, determined in a solution prepared by diluting the preparation under examination in carbon dioxidefree water to produce a solution containing 5000 IU of streptokinase activity per ml. Streptodornase. Not more than 10 IU of streptodornase activity per 100,000 IU of streptokinase activity. Test solution. Dilute the preparation under examination in imidazole buffer pH 6.5 to obtain a solution containing 150,000 IU of streptokinase activity per ml. 19
Production
If intended for use in the manufacture of parenteral preparations, the method of manufacture is validated to
IP 2007
Reference solution. Dissolve in imidazole buffer pH 6.5 a reference preparation of streptodornase, calibrated in International Units against the International Standard of streptodornase, to obtain a solution containing 20 IU of streptodornase activity per ml. The equivalence in International Units of the International Standard is stated by the regulatory authority. To each of 8 numbered centrifuge tubes, add 0.5 ml of a 0.1 per cent solution of sodium deoxyribonucleate in imidazole buffer pH 6.5. To tube number 1 and tube number 2 add 0.25 ml of imidazole buffer pH 6.5, 0.25 ml of the test solution and, immediately, 3.0 ml of 2.5 per cent w/v of perchloric acid. Mix, centrifuge at about 3000 rpm for 5 minutes and measure the absorbances of the supernatant liquids at 260 nm (2.4.7), using as the compensation liquid a mixture of 1.0 ml of imidazole buffer pH 6.5 and 3.0 ml of 2.5 per cent w/v solution of perchloric acid (absorbances A1 and A2). To the other 6 tubes (numbers 3 to 8) add 0.25 ml, 0.25 ml, 0.125 ml, 0.125 ml, 0 ml and 0 ml respectively of imidazole buffer solution pH 6.5; add to each tube 0.25 ml of the test solution and 0 ml, 0 ml, 0.125 ml, 0.125 ml, 0.25 ml and 0.25 ml respectively of the reference solution. Mix the contents of each tube and heat at 37 for 15 minutes. To each tube add 3.0 ml of 2.5 per cent w/v of perchloric acid, mix and centrifuge. Measure the absorbances of the supernatant liquids at 260 nm (2.4.7), using the compensation liquid described above (absorbances A3 to A8). The absorbances comply with the following test.
Potency The potency of streptokinase is determined by comparing its capacity to activate plasminogen to form plasmin with the same capacity of a reference preparation of streptokinase calibrated in International Units; the formation of plasmin is determined using a suitable chromogenic substrate. The International Unit is the activity of a stated amount of the International Standard for streptokinase. The equivalence in International Units of the International Standard is stated by the regulatory authority. Reference and test solutions. Prepare 2 independent series of 4 dilutions of each of the substance under examination and of the reference preparation of streptokinase in tris (hydroxymethyl) aminomethane sodium chloride buffer pH 7.4, in the range of 0.5-4.0 IU per ml. Prepare and maintain all solutions at 37. Substrate solution. Mix 1.0 ml of tris (hydroxymethyl) aminomethane buffer pH 7.4 with 1.0 ml of chromophore substrate. Add 5 l of a 10 per cent w/v solution of polysorbate 20. Keep at 37 in a water-bath. Immediately before commencing the activation assay, add 45 l of a 1 mg per ml solution of human plasminogen. Analyse each streptokinase dilution, maintained at 37, in duplicate. Initiate the activation reaction by adding 60 l of each dilution to 40 l of substrate solution. For blank wells, use 60 l of tris (hydroxymethyl) aminomethane sodium chloride buffer solution pH 7.4 instead of the reference and test solutions. Allow the reaction to proceed at 37 for 20 minutes and read the absorbance at 405 nm (2.4.7). If a suitable thermostated plate reader is available, this may be used to monitor the reaction. Alternatively, it may be necessary to stop the reaction after 20 minutes using 50 l of a 50 per cent v/v solution of glacial acetic acid. Best results are obtained when the absorbance for the highest streptokinase concentration is between 0.1 and 0.2 (after blank subtraction). If necessary, adjust the time of incubation in order to reach this range of absorbances. Calculate the regression of the absorbance on log concentrations of the solutions of the substance under examination and of the reference preparation of streptokinase and calculate the potency of the substance under examination using the usual statistical methods for parallel-line assays. The estimated potency is not less than 90 per cent and not more than 111 per cent of the stated potency. The confidence limits (P = 0.95) of the estimated potency are not less than 80 per cent and not more than 125 per cent of the stated potency. Streptokinase Bulk Solution intended for use in the manufacture of parenteral preparations without a further appropriate procedure for the removal of bacterial 20
(A3 + A4) (A1 + A2) <

Streptolysin
(A5 + A6 + A7 + A8) (A3 + A4) 2
In a haemolysis tube, use a quantity of the preparation under examination containing 500,000 IU of streptokinase activity and dilute to 0.5 ml with a mixture of 1 volume of phosphate buffer pH 7.2 and 9 volumes of a 0.9 per cent w/v solution of sodium chloride. Add 0.4 ml of a 2.3 per cent w/v solution of sodium thioglycollate. Heat in a water-bath at 37 for 10 minutes. Add 0.1 ml of a solution of a reference preparation of human antistreptolysin O containing 5 IU per ml. Heat at 37 for 5 minutes. Add 1 ml of rabbit erythrocyte suspension. Heat at 37 for 30 minutes. Centrifuge at about 1000 rpm. In the same manner, prepare a haemolysis tube in which the solution of the preparation under examination has been replaced by 0.5 ml of a mixture of 1 volume of phosphate buffer pH 7.2 and 9 volumes of a 0.9 per cent w/v solution of sodium chloride. Measure the absorbances of the supernatant liquids at 550 nm (2.4.7). The absorbance of the test solution is not more than 50 per cent than that of the reference solution. Protein. Determine the nitrogen content (2.3.30). 1 mg of N is equivalent to 6.25 mg of protein.
IP 2007
endotoxins complies with the following additional requirement. Bacterial endotoxins (2.2.3). Less than 0.02 Endotoxin Unit per 100 IU of streptokinase activity. Storage. Store protected from light and at a temperature of about - 20. If it is intended for the manufacture of parenteral preparations, the container should be sterile and sealed so as to exclude micro-organisms.
Labelling. The label states (1) the number of International Units of streptokinase activity per mg, calculated on the dried basis; (2) the name and quantity of any added substance; (3) where applicable, that the substance is free from bacterial endotoxins; (4) where applicable, that the substance is suitable for use in the manufacture of parenteral preparations.
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GENERAL MONOGRAPHS
VETERINARY PRODUCTS
General Monographs Dip Concentrates .................................................................................................. Intramammary Infusions ......................................................................................... Premixes ................................................................................................................ Veterinary Aerosols ................................................................................................ Veterinary Diagnostics ............................................................................................ Veterinary Oral Liquids .......................................................................................... Veterinary Oral Powders ....................................................................................... Veterinary Parenteral Preparations .......................................................................... Veterinary Tablets .................................................................................................. Monographs Non Biological ....................................................................................................... Biological ............................................................................................................... Veterinary Vaccines ..............................................................................................
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GENERAL MONOGRAPHS
Veterinary Preparations
General Requirements
The general requirements relating to a specific type of dosage form of an active pharmaceutical ingredient or ingredients, that have been given in the chapter on General Monographs on Dosage Forms of Active Pharmaceutical Ingredients apply to all veterinary dosage forms or preparations of the type defined. However, a valid interpretation of the appropriateness of a test or requirement should be done in the context of the monograph as a whole and of the relevant General Notices. The requirement for compliance with the tests given under each dosage form or preparation is indicated in each monograph of a drug product or preparation under the heading Other tests. These tests are mandatory and are additional to the tests given in the individual monograph.
suitable vehicle. They may contain stabilizing, emulsifying, suspending and thickening agents. If a sediment is formed in a suspension, it is readily dispersible on shaking. In emulsions, phase separation may occur but this is readily miscible on shaking. There are two main types of Intramammary Infusions. One is intended for administration to lactating animals as qualified by the term Lactating Cow/Buffalo and the other, qualified as Non-lactating or Dry Cow/Buffalo, is intended for administration to animals at the end of lactation or during the non-lactating period for the prevention or treatment of infection during the dry period. Intramammary Infusions are prepared by dissolving or suspending the sterile medicaments in the sterilized vehicle using aseptic precautions, unless a process of terminal sterilisation is employed. Containers. Intramammary Infusions are usually supplied in single dose containers for administration into a single teat canal of an animal. If supplied in multiple dose containers, aqueous preparations contain an antimicrobial preservative in adequate concentration except when the preparation itself has antimicrobial properties. The containers are made as far as possible from materials that meet the requirements for Parenteral Preparations intended for use in human beings. The containers are sealed so as to exclude micro-organisms and each container is fitted with a smooth, tapered nozzle to facilitate the introduction of the infusion into the teat canal. The containers are sterilised and filled aseptically unless the preparation is subjected to a process of terminal sterilisation.
Dip Concentrates
Dip concentrates are preparations for the prevention and treatment of ectoparasitic infestations of animals. They contain one or more medicaments, usually in the form of wettable powders, pastes or solutions from which diluted suspensions or emulsions are prepared by appropriate dilution with the recommended liquid. The diluted preparations are applied by complete immersion of the animal or by spraying, as appropriate. They contain suitable antimicrobial preservatives. Labelling. The label states (1) the name(s) and proportion(s) of medicament(s); (2) the name and proportion of any added antimicrobial preservative; (3) the name and quantity of the diluent and the manner of preparing the diluted dip solution or spray; (4) any special precautions to be taken for use of the preparation; (6) the storage conditions; (6) the date after which the preparation is not intended to be used. If the preparation contains an organophosphorus compound the label also states (1) that the preparation contains an organophosphorus compound; (2) and special precautions on the use of the preparation.
Tests
Sterility. Intramammary Infusions comply with the test for sterility (2.2.11), using Method A or B, as appropriate, using the contents of 10 containers mixed thoroughly before use in the test. Use for each medium 0.5 to 1.0 g or 0.5 to 1.0 ml, as appropriate, of the mixed sample. Storage. Store in sterile, single dose or multiple dose, tamperevident containers. Labelling. The label states (1) the strength in terms of the weight or the number of Units of activity of the active ingredient(s) or that may be expressed from the container using normal techniques; (2) whether the preparation is intended for use in lactating cow/buffalo or in dry or non-lactating cow/buffalo; (3) for Intramammary Infusions (Non-lactating or Dry Cow/Buffalo), that the preparation is not intended for use in lactating animals; (4) in the case of infusions in multiple dose containers, the name of any added antimicrobial preservative.
Intramammary Infusions
Intramammary Infusions for Veterinary Use; Intramammary Injections.
Intramammary Infusions are sterile products intended for injection into the mammary gland through the teat canal. They are solutions, emulsions or suspensions or semi-solid preparations containing one or more active ingredients in a
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Premixes
Premixes are mixtures of one or more active ingredients with suitable bases intended for mixing with feedstuffs before administration to the animals. They are used to dilute medicament(s) with the feed and are usually issued as pellets, granules or powders. If issued as granules, these are freeflowing and free from aggregates. Suitable precautions are taken during manufacture for ensuring that the premix is homogeneous. Unless otherwise stated in the individual monograph, the concentration of the premix in medicated feedstuffs is not less than 0.5 per cent.
Labelling. The label states (1) that the aerosol is intended for external use only; (2) the instructions for use; (3) any special precautions in the use of the preparation.
Veterinary Diagnostics
Veterinary Diagnostics are antigenic materials of bacterial or viral origin employed for various tests. These will also include polyclonal or monoclonal antibodies. The preparations are examined for their purity at various critical stages of production. The diagnostic kits may be prepared using bacterial or viral antigens and antisera.
Tests
Loss on drying (2.4.19). Not more than 15.0 per cent, determined on 3 g by drying in an oven at 105 for 2 hours. Labelling. The label states (1) the strength in terms of the amount of active ingredient(s) as a percentage; (2) the category of animal for which the premix is intended to be used; (3) the directions for the preparation of the medicated feed; (4) where applicable, the minimum interval between the stoppage of feeding of the diluted premix and the slaughter of the animal for human consumption; (5) any special precautions to be taken for use of the premix; (6) the storage conditions; (7) the date after which the preparation is not intended to be used.
Tests
Veterinary Diagnostics, reconstituted where necessary, comply with the following tests unless otherwise stated in the individual monograph.
Identification
Unless otherwise stated in the individual monograph, Veterinary Diagnostics give specific reaction when injected into the skin of a healthy white guinea-pig or rabbit that has not been previously treated with any material that will interfere with the test but fails to produce this reaction when mixed with a sufficient quantity of the specific antitoxin or antiserum. Sterility. Unless otherwise stated, Veterinary Diagnostics comply with the test for sterility (2.2.11), except that in the case of preparations containing living bacteria there may be growth of the organism from which the diagnostic was prepared. Use suitable solid media for streaking the preparation under examination and incubate at 32 to 37 for 72 hours for detecting bacteria and at 20 to 25 for 72 hours for detecting fungi. The media selected will depend upon the nature of the product to be tested. The contents of each randomly selected sealed container of the preparation under examination or portions or dilutions thereof, as appropriate, are used for the test. Other tests to determine the nature and identity of contaminating microorganisms, if any, detected during the test include examination for mobility of the organisms, fermentation reactions, thermo-agglutination tests and dye inhibitor tests (in the case of Brucella cultures). Unless otherwise stated in the monograph, the preparation passes the test if no growth of microorganisms, other than those from which the veterinary diagnostic was prepared, is observed in any of the media during the incubation period. Repeat the tests if growth of organisms, other than those from which the veterinary diagnostic was prepared, is observed. The vaccine passes the test if no growth of microorganisms,
Veterinary Aerosols
Veterinary Sprays
Veterinary Aerosols are solutions, suspensions or emulsions of one or more active ingredients intended for use by external application. They may contain auxiliary substances such as solvents, solubilising agents, emulsifying agents and suspending agents. They are delivered in the form of an aerosol by the actuation of an appropriate valve or by means of a suitable atomizing device that is either an integral part of the container or is supplied separately. They may be presented in special containers under pressure of a gas and contain propellants or mixtures of propellants. The medicaments are released from the container in the form of an aerosol upon actuation of an appropriate valve. Veterinary Aerosols supplied in special pressurized containers comply with the appropriate requirements for Inhalation Preparations. The following requirements also apply for any veterinary aerosol that is the subject of an individual monograph. Containers. A suitable atomizing device may form part of the container or is supplied separately.
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other than those from which the diagnostic was prepared, is observed in any of the media. The preparation fails the test if growth of a microorganism that was seen after the first test, other than those from which the veterinary diagnostic was prepared, is observed. If growth of a different microorganism is observed, the test may be repeated a second time. The preparation passes the test if no growth of a microorganism, other than those from which the veterinary diagnostic was prepared, is observed in any of the media. The number of containers recommended to be drawn by the manufacturer for performing the test for sterility depends on the environmental conditions of manufacture, the volume of preparation per container and any other special considerations applicable to the preparation concerned. For preparations intended for veterinary use, 1 per cent of the containers in a batch, with a minimum of three and a maximum of ten, is considered a suitable number assuming that the preparation has been manufactured under appropriately validated conditions designed to exclude contamination. Storage. Store protected from light in a refrigerator (2 to 8) unless otherwise stated in the individual monograph. Labelling. The label states (1) the name and quantity of any antibacterial substance added; (2) for a dried preparation, the nature and quantity of the liquid to be used for reconstitution.
given in the chapter on General Monographs on Dosage Forms of Active Pharmaceutical Ingredients.
Veterinary Tablets
Veterinary tablets are usually solid, circular cylinders the end surfaces of which are flat or biconvex and the edges of which are bevelled except that those weighing 5 g of more may be elongated or biconical.
Tests
Disintegration (2.5.1). The test may have to be suitably modified in the case of large tablets; the discs may have to be omitted because they would otherwise be dislodged from the disintegration tubes. It may also be necessary to adjust the volume of the disintegration medium so that the tablet does not break the surface of the medium at the top of the upstroke, care being taken to apply the minimum practical volume of liquid for this purpose. For certain tablets where the diameter of the tablet may not permit adequate movement of the disintegration medium, the apparatus and the method should be suitably modified.
Veterinary Oral Liquids

Veterinary oral liquids intended for administration in large animals may also be called Drenches.
Veterinary Vaccines
Vaccines for Veterinary Use
Vaccines are a heterogeneous class of medicinal products containing immunogenic substances capable of inducing specific, active and protective host immunity against infectious diseases. They may be prepared from bacteria, viruses, parasites or other organisms or their toxins. Vaccines may contain live attenuated or avirulent microorganisms or these may consist of killed or inactivated microorganisms. Some vaccines consist of antigenic fractions or substances produced by the same pathogenic organisms but rendered harmless whilst retaining their immunogenecity. Vaccines may be prepared from one species or from two or more species of microorganisms. Vaccines may be prepared by the method described in the individual monograph or by any other appropriate method provided the identity of the antigens is maintained and the preparations are free from microbial contamination and extraneous agents. Suitable adjuvants may be added during the preparation of the vaccines. The addition of antibiotics during the manufacturing process is normally restricted to cell culture fluids and other media, egg inocula and material harvested from skin or other tissues. A suitable bactericide may be added to sterile and inactivated vaccines. The final products are distributed aseptically into sterile containers that
Veterinary Oral Powders

Veterinary Oral Powders are intended for oral administration, usually after dilution in drinking water or the feed. They may be in the form of soluble or wettable powders. Labelling. The label states (1) for single dose containers, the name and quantity of active medicament(s) per container; (2) for multiple dose containers, the name and quantity of active medicament(s) by weight; (3) the name of any added antimicrobial preservative(s); (4) the directions for use of the preparation.
Veterinary Parenteral Preparations

Veterinary Parenteral Preparations prepared with oily vehicles are not meant for intravenous administration but are suitable for intramuscular or subcutaneous use. Veterinary Parenteral Preparations comply with the appropriate requirements for Parenteral Preparations (Injections) that are
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are then sealed to exclude extraneous microorganisms. Unless otherwise indicated in the monograph, the final vaccine may be filled into single dose or multiple dose containers; however, inactivated vaccines in multiple dose containers must invariably contain a bactericide. Bacterial vaccines. Bacterial vaccines are either suspensions of live or killed bacteria or sterile antigenic extracts or derivatives of bacteria pathogenic to animals. They may be simple vaccines prepared from one species or may be combined or polyvalent vaccines prepared by blending two or more simple vaccines from different species or strains. Bacterial vaccines may be prepared from cultures grown on suitable solid or liquid media. The identity, antigenic potency and purity of each bacterial culture must be carefully controlled. Bacterial vaccines are suspensions of varying degrees of opacity in colourless or slightly coloured liquids or they may be freeze-dried so that the water content is not more than 3.0 per cent w/w unless otherwise stated in the individual monograph. They may be standardised in terms of international opacity units or, where appropriate, by numbers of live or killed bacteria determined by viable count or by direct cell count. Live bacterial vaccines. Live bacterial vaccines are prepared from avirulent or attenuated strains of the specific bacteria that are capable of stimulating immune response against pathogenic strains of the same or of antigenically related species of bacteria. Inactivated bacterial vaccines. Inactivated bacterial vaccines are either prepared from bacteria or their immunogenic components that have been inactivated in a suitable way that they retain adequate immunogenicity. Bacterial toxoids. Bacterial toxoids are prepared from toxins by diminishing their toxicity to a very low level or by completely eliminating it by physical or chemical means whilst retaining adequate immunising potency. The toxins are obtained from selected strains of specific microorganisms, grown in suitable media devoid of agents capable of inducing undesirable immunological reactions in animals. Bacterial toxoids may be liquid or may be prepared by adsorbing on suitable agents such as aluminium phosphate, aluminium hydroxide or any other suitable adsorbents. Bacterial toxoids are clear or slightly opalescent liquids, colourless or slightly yellow. Adsorbed toxoids may be white or greyish-white suspensions or paleyellow liquids with sediment at the bottom of the container. Freeze-dried preparations are greyish-white or yellowish-white powders or pellets. Viral vaccines. Viral vaccines are suspensions of viruses or preparations obtained from tissues or blood of animals artificially infected with viruses pathogenic to animals or from cultures in fertile eggs, or from cell or tissue cultures, they may be live, inactivated / killed and may be freeze-dried.
Live viral vaccines. Live viral vaccines are prepared using avirulent or attenuated strains of the specific viruses that are capable of stimulating immunogenic response against pathogenic strains of the same or of antigenically related viruses. Inactivated viral vaccines. Inactivated viral vaccines contain viruses that have been inactivated by suitable chemical or physical means in such a way that the preparations retain adequate immunogenicity or they are suspensions of immunogenic components of such viruses. Combined vaccines. Combined vaccines consist of two or more antigens, combined by the manufacturer at the final formulation stage or mixed immediately before administration. Such vaccines are intended to protect against either more than one disease, or against one disease caused by different strains or serotypes of the same organism. Stability. Stability is the ability of a vaccine to retain its chemical, physical, microbiological and biological properties within specified limits throughout its shelf life. Adjuvants. Substances that are intended to enhance relevant immune response and subsequent clinical efficacy of the vaccine.
Tests
Vaccines comply with the tests prescribed in the individual monographs including, where applicable, the following: Aluminium (2.3.9). Where an aluminium adsorbent has been used in the vaccine, not more than 1.25 mg of aluminium (Al) per single dose, unless otherwise stated. Calcium (2.3.11). Where a calcium adsorbent has been used in the vaccine, not more than 1.3 mg of calcium (Ca) per single dose, unless otherwise stated. Formaldehyde (2.3.20). Where formaldehyde has been used in the preparation of the vaccine, not more than 0.2 g/l of free formaldehyde is present in the final product, unless otherwise stated. Phenol (2.3.36). Where phenol has been used in the preparation of the vaccine, not more than 2.5 g/l is present in the final product, unless otherwise stated. Water (2.3.43). For freeze-dried vaccines, not more than 3.0 per cent, unless otherwise stated. Thiomersal (2.3.12) (2.3.48). Where thiomersal has been used in the preparation of the vaccine, not more than 0.02 per cent w/v. Extraneous pathogens. Unless otherwise stated in the individual monograph, live viral vaccines other than those intended for poultry comply with the following test. The mixture obtained after neutralisation of the vaccine with specific
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antiserum does not cause cytopathic effects in cell cultures known to be sensitive to agents pathogenic for the species in which the vaccine is intended to be used. Sterility (2.2.11). Unless otherwise stated in the individual monograph, use method A. Incubate the media for not less than 14 days at 30 to 37 in the test for detecting bacteria and at 20 to 25 in the test for detecting fungi. However, for live bacterial vaccines growth of the organism from which the vaccine was prepared is permitted. The number of containers to be drawn for the test should be 1 per cent of the containers in a batch, with a minimum of 3 and a maximum of 10, assuming that the preparation has been manufactured under appropriately validated conditions designed to exclude contamination. Safety Test. Unless otherwise stated in the individual monograph, vaccines other than live viral vaccines intended for poultry comply with the following test. Inject at least 2 healthy, susceptible animals of one of the species in which the vaccine is intended to be used by the route recommended by the manufacturer for field use. The quantity to be injected in each animal is twice the appropriate vaccinating dose. Observe the animals for not less than 7 days. No animal exhibits an abnormal reaction. Abnormal toxicity. Where stated in the individual monograph vaccines comply with the following test. Inject 0.5 ml subcutaneously into each of five mice and 2 ml intraperitoneally into each of two guinea pigs. If the vaccine
being examined contains an adjuvant, inject 2 ml of the vaccine subcutaneously into each guinea pig. Observe the animals for 7 days. None of the animals shows significant local or systemic reaction. If one animal dies or shows signs of ill health during the observation period repeat the test. None of the animals of the second group dies or shows signs of ill health. This test may be omitted if a safety test is carried out on animals of the species for which the vaccine is intended. Potency. Determine the potency of the vaccine using the method described in the individual monograph. The vaccine complies with the level of immune response specified in the monograph. A combined vaccine complies with the level specified in the respective monographs for each individual component. If the immunogenicity test (potency test) has been performed with satisfactory results on representative batch of live vaccine from the same seed lot, it may be omitted as a routine control test during production of other batches of the vaccine prepared from the same seed lot. Storage. Store protected from light in a refrigerator (2 to 8), unless otherwise directed. Do not freeze. Store freeze-dried vaccines at a temperature not exceeding 20. Labelling. The label states (1) the potency of the preparation; (2) the route of administration and dose; (3) the date up to which the product is expected to remain within specifications; (4) the storage conditions.
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ACEPROMAZINE MALEATE
Acepromazine Maleate
CH3 N N S
H3 C
O CH3 ,
H H
COOH COOH
C19H22N2OS, C4H4O4 Acepromazine Maleate is 2-acetyl-10-(3dimethylaminopropyl)phenothiazine maleate. Description. A yellow, crystalline powder.
Mol. Wt. 442.5
liquid and allow the impregnating solvent to ascend almost to the top. Use the plate immediately after removing it from the tank. Apply to the plate 1 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution corresponds to the spot in the chromatogram obtained with the reference solution. A secondary spot due to maleic acid is also observed in both chromatograms. Spray the plate with ethanolic sulphuric acid (10 per cent v/v). The spot in the chromatogram obtained with the test solution corresponds to the spot in the chromatogram obtained with the reference solution. D. Dissolve 5 mg in 2 ml of sulphuric acid; a yellow colour is produced which changes to deep orange on warming for 2 minutes. E. Dissolve 0.2 g in a mixture of 3 ml of water and 2 ml of 5 M sodium hydroxide and shake with three quantities, each of 3 ml, of ether. Discard the ether extracts. Add 2 ml of bromine solution to the aqueous solution, warm in a water-bath for 10 minutes, heat to boiling, cool and add 0.25 ml to a solution of 10 mg of resorcinol in 3 ml of sulphuric acid; a bluish-black colour is produced on heating for 15 minutes in a water-bath. F. Melting range ( 2.4.21). 136 to 139.
Acepromazine Maleate contains not less than 98.5 per cent and not more than 101.0 per cent of C19H22N2OS,C4H4O4, calculated on the dried basis.
Identification
Tests B and D may be omitted if tests A, C, E and F are carried out. Tests A and E may be omitted if tests B, C, D and F are carried out. NOTE Carry out the tests in subdued light. A. Dissolve 20 mg in 2 ml of water, add 3 ml of 2 M sodium hydroxide, extract with 5 ml of cyclohexane and remove the solvent under reduced pressure. The residue complies with the following test. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with acepromazine RS or with the reference spectrum of acepromazine. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.002 per cent w/v solution in 0.1 M hydrochloric acid, exhibits maxima at about 244 nm and 280 nm; absorbance at about 244 nm, about 1.1 and at about 280 nm, about 0.82. C. Determine by thin-layer chromatography (2.4.17), coating the plate with kieselguhr G. Mobile phase. A mixture of 100 volumes of light petroleum ( 40 to 60 ), 2 volumes of diethylamine and 6 to 8 volumes of 2-phenoxyethanol. Shake and use the supernatant liquid. Test solution. Dissolve 0.2 g of the substance under examination in 100 ml of dichloromethane. Reference solution. A 0.2 per cent w/v solution of acepromazine maleate RS in dichloromethane. Impregnate the dry plate by placing it in a tank containing a shallow layer of a mixture of 85 volumes of acetone, 10 volumes of 2-phenoxyethanol and 5 volumes of polyethyleneglycol 300 so that the plate dips about 5 mm below the surface of the
Tests
pH (2.4.24). 4.0 to 4.5, determined in a 1.0 per cent w/v solution. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Solvent mixture. A mixture of 95 volumes of methanol and 5 volumes of diethylamine. Mobile phase. A mixture of 75 volumes of hexane, 17 volumes of 2-butanol and 8 volumes of diethylamine. Test solution. Dissolve 0.2 g of the substance under examination in 10 ml of the solvent mixture. Reference solution. A 0.010 per cent w/v solution of the substance under examination in the solvent mixture. Apply to the plate 10 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Sulphated ash (2.3.18) Not more than 0.2 per cent. Loss on drying (2.4.19). Not more than 0.1 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.4 g, dissolve in 50 ml of acetic anhydride. Titrate with 0.1 M perchloric acid, using crystal violet solution as indicator. Carry out a blank titration.
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1 ml of 0.1 M perchloric acid is equivalent to 0.04425 g of C19H22N20S,C4H4O4. Storage. Store protected from moisture.
chromatogram obtained with the reference solution. A secondary spot due to maleic acid is also observed in both chromatograms. Spray the plate with ethanolic sulphuric acid (10 per cent v/v). The spot in the chromatogram obtained with the test solution corresponds to the spot in the chromatogram obtained with the reference solution. D. To a volume containing 25 mg of acepromazine add 2 ml of 5 M sodium hydroxide and shake with three quantities, each of 3 ml, of ether. Discard the ether extracts. Add 2 ml of bromine solution to the aqueous solution, warm in a water-bath for 10 minutes, heat to boiling, cool and add 0.25 ml to a solution of 10 mg of resorcinol in 3 ml of sulphuric acid; a bluish-black colour is produced on heating for 15 minutes in a water-bath.
Acepromazine Injection
Acepromazine Maleate Injection
Acepromazine Injection is a sterile solution of Acepromazine Maleate in Water for Injections. Acepromazine Injection contains not less than 92.5 per cent and not more than 107.5 per cent of the stated amount of acepromazine, C19H22N2OS.
Tests
pH (2.4.24). 4.5 to 5.5. Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. To an accurately measured volume containing 40 mg of acepromazine add 5 ml of 1 M sodium hydroxide and extract with three or more quantities, each of 50 ml, of dichloromethane until the dichloromethane extract is colourless. Wash the extracts with the same 10 ml of water and filter through a plug of absorbent cotton previously moistened with dichloromethane. Evaporate the combined extracts to dryness, dissolve the residue in 15 ml of acetic anhydride. Titrate with 0.02 M perchloric acid, using crystal violet solution as indicator. Carry out a blank titration. 1 ml of 0.02 M perchloric acid is equivalent to 0.006529 g of C19H22N2OS. Storage. Store protected from light. Labelling. The label states the strength in terms of the equivalent amount of acepromazine.
Identification
NOTE Carry out the tests in subdued light. A. To a volume containing 20 mg of acepromazine, add 2 ml of water and 3 ml of 2 M sodium hydroxide, extract with two quantities, each of 5 ml, of cyclohexane and remove the solvent under reduced pressure. The residue complies with the following test. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with acepromazine RS or with the reference spectrum of acepromazine. B. To 5 mg of the residue obtained in test A, add 2 ml of sulphuric acid; a yellow colour is produced which changes to deep orange on warming for 2 minutes. C. Determine by thin-layer chromatography (2.4.17), coating the plate with kieselguhr G. Mobile phase. A mixture of 100 volumes of light petroleum (40 to 60), 2 volumes of diethylamine and 6 to 8 volumes of 2-phenoxyethanol. Shake and use the supernatant liquid. Test solution. Extract a volume containing 20 mg of acepromazine with two quantities, each of 5 ml, of dichloromethane and use the combined extracts. Reference solution. A 0.2 per cent w/v solution of acepromazine maleate RS in dichloromethane. Impregnate the dry plate by placing it in a tank containing a shallow layer of a mixture of 85 volumes of acetone, 10 volumes of 2-phenoxyethanol and 5 volumes of polyethyleneglycol 300 so that the plate dips about 5 mm below the surface of the liquid and allow the impregnating solvent to ascend almost to the top. Use the plate immediately after removing it from the tank. Apply to the plate 1 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution corresponds to the spot in the
Acepromazine Tablets
Acepromazine Maleate Tablets
Acepromazine Tablets contain not less than 92.5 per cent and not more than 107.5 per cent of the stated amount of acepromazine, C19H22N2OS.
Identification
NOTE Carry out the tests in subdued light. A. To a quantity of the powdered tablets containing 20 mg of acepromazine add 2 ml of water and 3 ml of 2 M sodium hydroxide. Extract with two quantities, each of 5 ml, of cyclohexane and remove the solvent under reduced pressure. The residue complies with the following test.
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AMITRAZ
Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with acepromazine RS or with the reference spectrum of acepromazine. B. To 5 mg of the residue obtained in test A add 2 ml of sulphuric acid; a yellow colour is produced which changes to deep orange on warming for 2 minutes. C. Determine by thin-layer chromatography (2.4.17), coating the plate with kieselguhr G. Mobile phase. A mixture of 100 volumes of light petroleum (40 to 60), 2 volumes of diethylamine and 6 to 8 volumes of 2-phenoxyethanol. Shake and use the supernatent liquid. Test solution. Extract a quantity of powdered tablets containing 20 mg of acepromazine with two quantities, each of 5 ml, of dichloromethane and use the combined extracts. Reference solution. A 0.2 per cent w/v solution of acepromazine maleate RS in dichloromethane. Impregnate the dry plate by placing it in a tank containing a shallow layer of a mixture of 85 volumes of acetone, 10 volumes of 2-phenoxyethanol and 5 volumes of polyethyleneglycol 300 so that the plate dips about 5 mm below the surface of the liquid and allow the impregnating solvent to ascend almost to the top. Use the plate immediately after removing it from the tank. Apply to the plate 1 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution corresponds to the spot in the chromatogram obtained with the reference solution. A secondary spot due to maleic acid is also observed in both chromatograms. Spray the plate with ethanolic sulphuric acid (10 per cent v/v). The spot in the chromatogram obtained with the test solution corresponds to the spot in the chromatogram obtained with the reference solution. D. Dissolve a quantity of the powdered tablets containing 25 mg of acepromazine as completely as possible in a mixture of 3 ml of water and 2 ml of 5 M sodium hydroxide and shake with three quantities, each of 3 ml, of ether. Discard the ether extracts. Add 2 ml of bromine solution to the aqueous solution, warm in a water-bath for 10 minutes, heat to boiling, cool and add 0.25 ml of a solution of 10 mg of resorcinol in 3 ml of sulphuric acid; a bluish-black colour is produced on heating for 15 minutes in a water-bath.
Test solution. Shake a quantity of the powdered tablets containing 50 mg of acepromazine with 10 ml of dichloromethane, filter, evaporate to dryness and dissolve the residue in 5 ml of methanol containing 0.5 per cent v/v of strong ammonia solution. Reference solution. Dilute 1 ml of the test solution to 100 ml with methanol containing 0.5 per cent v/v of strong ammonia solution. Apply to the plate 10 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Other tests. Comply with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing 60 mg of acepromazine, add 5 ml of water and extract with three or more quantities, each of 50 ml, of dichloromethane until the dichloromethane extract is colourless. Wash the extracts with the same 10 ml of water and filter through a plug of absorbent cotton previously moistened with dichloromethane. Evaporate the combined extracts to dryness, dissolve the residue in 15 ml of acetic anhydride. Titrate with 0.02 M perchloric acid, using crystal violet solution as indicator. Carry out a blank titration. 1 ml of 0.02 M perchloric acid is equivalent to 0.006529 g of C19H22N2OS. Labelling. The label states the strength in terms of the equivalent amount of acepromazine.
Amitraz
CH3 H3C
C19H23N3 Description. A white to buff powder. Amitraz contains not less than 95.0 per cent and not more than 101.5 per cent of C19H23N3, calculated on the anhydrous basis.
H3C CH3 N C N C N H H
CH3
Mol. Wt. 293.41
Amitraz is N,N-di-(2,4-xylyliminomethyl)methylamine.
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Solvent mixture. A mixture of 95 volumes of methanol and 5 volumes of diethylamine. Mobile phase. A mixture of 75 volumes of hexane, 17 volumes of 2-butanol and 8 volumes of diethylamine.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with amitraz RS or with the reference spectrum of amitraz.
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IP 2007
B. In the test for Related substances, the principal spot in the chromatogram obtained with test solution (b) corresponds to that in the chromatogram obtained with reference solution (a). C. In the Assay, the principal peak in the chromatogram obtained with the test solution (b) corresponds to the peak in the chromatogram obtained with the reference solution.
Test solution (a). Dissolve 0.8 g of the substance under examination in 100 ml of methyl acetate. Test solution (b). Dissolve 0.8 g of the substance under examination in 100 ml of the internal standard solution. Reference solution. A 0.8 per cent w/v solution of amitraz RS in the internal standard solution. Chromatographic system a glass column 1.5 m x 4 mm, packed with acid-washed, silanised diatomaceous support (80 to 100 mesh) coated with 3 per cent w/w methyl silicone gum or fluid (such as OV-1 or OV-101), temperature: column. 250, inlet port and detector. 280, flow rate. 30 ml per minute of the carrier gas. Calculate the content of C19H23N3. Storage. Store in containers which may contain paraformaldehyde packed in separate sachets as stabiliser.
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel HF254. Mobile phase. A mixture of 50 volumes of cyclohexane, 30 volumes of ethyl acetate and 20 volumes of triethylamine. Test solution (a). Dissolve 1 g of the substance under examination in 10 ml of toluene. Test solution (b). Dissolve 20 mg of the substance under examination in 10 ml of toluene. Reference solution (a). A 0.20 per cent w/v solution of amitraz RS in toluene. Reference solution (b). A 0.030 per cent w/v of 2,4-dimethylaniline in toluene. Impregnate the plate to a depth of about 3.5 cm with a solution prepared by dissolving 35 g of acetamide in 100 ml of methanol, adding 100 ml of triethylamine and diluting to 250 ml with methanol, before standing it in a stream of cold air for about 30 seconds. Immediately apply to the plate, at a level 1 cm below the top of the impregnated zone, 2 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with test solution (a) is not more intense than the spot in the chromatogram obtained with reference solution (a). Expose the plate to the vapours of hydrochloric acid until the plate smells strongly of acid. Expose to the vapours of nitrogen dioxide (prepared by the action of nitric acid on granulated zinc) for 10 minutes, remove the excess of nitrogen dioxide with air and spray with a 0.5 per cent w/v solution of N-(1-naphthyl)ethylenediamine dihydrochloride in a 50 per cent v/v solution of methanol. Any secondary spot corresponding to 2,4-dimethylaniline in the chromatogram obtained with test solution (a) is not more intense than the corresponding spot in the chromatogram obtained with reference solution (b). Water (2.3.43). Not more than 0.1 per cent , determined on 5 g and using anhydrous pyridine in place of anhydrous methanol. Sulphated ash (2.3.18). Not more than 0.2 per cent. Assay. Determine by gas chromatography (2.4.13). Internal Standard Solution. A 1.0 per cent v/v solution of squalane in methyl acetate.
Liquid Amitraz Dip Concentrate

Liquid Amitraz Dip Concentrate contains Amitraz in a suitable emulsifiable vehicle. It may contain a suitable stabilising agent. Liquid Amitraz Dip Concentrate contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of amitraz, C19H23N3.
Identification
A. In the test for Related substances, the principal spot in the chromatogram obtained with test solution (b) corresponds to that in the chromatogram obtained with reference solution (a). B. In the Assay, the principal peak in the chromatogram obtained with the test solution (b) corresponds to the peak in the chromatogram obtained with reference solution (b).
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel HF254. Mobile phase. A mixture of 50 volumes of cyclohexane, 30 volumes of ethyl acetate and 20 volumes of triethylamine. Test solution (a). Dilute the dip concentrate with toluene to obtain 5.0 per cent w/v of Amitraz. Test solution (b). Dilute the dip concentrate with toluene to obtain 0.2 per cent w/v of Amitraz. Reference solution (a). A 0.20 per cent w/v solution of amitraz RS in toluene.
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IP 2007
AMITRAZ DIP CONCENTRATE POWDER
Reference solution (b). A 0.030 per cent w/v of 2,4-dimethylaniline in toluene. Impregnate the plate to a depth of about 3.5 cm in a solution prepared by dissolving 35 g of acetamide in 100 ml of methanol, adding 100 ml of triethylamine and diluting to 250 ml with methanol, before standing it in a stream of cold air for about 30 seconds. Immediately apply to the plate, at a level 1 cm below the top of the impregnated zone, 2 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution (a) is not more intense than the spot in the chromatogram obtained with the reference solution (a). Expose the plate to the vapours of hydrochloric acid until the plate smells strongly of acid. Expose to the vapours of nitrogen dioxide (prepared by the action of nitric acid on granulated zinc) for 10 minutes, remove the excess of nitrogen dioxide with air and spray with a 0.5 per cent w/v solution of N-(1-naphthyl)ethylenediamine dihydrochloride in a 50 per cent v/v solution of methanol. Any secondary spot corresponding to 2,4-dimethylaniline in the chromatogram obtained with test solution (a) is not more intense than the corresponding spot in the chromatogram obtained with reference solution (b). Water (2.3.43). Not more than 0.15 per cent w/v, determined in 5 ml of the dip concentrate and using anhydrous pyridine in place of anhydrous methanol. Other tests. Complies with the tests stated under Dip Concentrates. Assay. Determine by gas chromatography (2.4.13). Internal Standard Solution. A 1.0 per cent v/v solution of squalane in methyl acetate. Test solution (a). Dissolve an accurately measured volume of the dip concentrate containing 80 mg of Amitraz in 10 ml of methyl acetate. Test solution (b). Dissolve an accurately measured volume of the dip concentrate containing 80 mg of Amitraz in 10 ml of the internal standard solution. Reference solution. A 0.8 per cent w/v solution of amitraz RS in the internal standard solution. Chromatographic system a glass column 1.5 m x 4 mm, packed with acid-washed, silanised diatomaceous support (80 to 100 mesh) coated with 3 per cent w/w methyl silicone gum or fluid (such as OV-1 or OV-101), temperature: column. 250, inlet port and detector. 280, flow rate. 30 ml per minute of the carrier gas. Calculate the content of C19H23N3.
Amitraz Dip Cconcentrate Powder

Amitraz Dip Concentrate Powder consists of Amitraz mixed with suitable wetting, dispersing and suspending agents. It may contain a suitable stabilising agent. Amitraz Dip Concentrate Powder contains not less than 92.0 per cent and not more than 108.0 per cent of the stated amount of amitraz, C19H23N3.
Identification
A. Shake a quantity of the powder containing 0.1 g of Amitraz with 10 ml of acetone for 5 minutes, filter and evaporate the filtrate to dryness. The residue complies with the following test. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with amitraz RS or with the reference spectrum of amitraz. B. In the test for Related substances, the principal spot in the chromatogram obtained with test solution (b) corresponds to that in the chromatogram obtained with reference solution (a). C. In the Assay, the principal peak in the chromatogram obtained with the test solution (b) corresponds to the peak in the chromatogram obtained with the reference solution.
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel HF254. Mobile phase. A mixture of 50 volumes of cyclohexane, 30 volumes of ethyl acetate and 20 volumes of triethylamine. Test solution (a). The supernatant liquid obtained by shaking a quantity of the powder containing 0.5 g of Amitraz with 10 ml of toluene for 5 minutes and centrifuging the suspension. Test solution (b). The supernatant liquid obtained by shaking a quantity of the powder containing 20 mg of Amitraz with 10 ml of toluene for 5 minutes and centrifuging the suspension. Reference solution (a). A 0.20 per cent w/v solution of amitraz RS in toluene. Reference solution (b). A 0.030 per cent w/v of 2,4-dimethylaniline in toluene. Impregnate the plate to a depth of about 3.5 cm in a solution prepared by dissolving 35 g of acetamide in 100 ml of methanol, adding 100 ml of triethylamine and diluting to 250 ml with methanol, before standing it in a stream of cold air for about 30 seconds. Immediately apply to the plate, at a level 1 cm below the top of the impregnated zone, 2 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution (a) is not more
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AMPICILLIN AND CLOXACILLIN INTRAMAMMARY INFUSION (LACTATING COW/BUFFALO)
IP 2007
intense than the spot in the chromatogram obtained with the reference solution (a). Expose the plate to the vapours of hydrochloric acid until the plate smells strongly of acid. Expose to the vapours of nitrogen dioxide (prepared by the action of nitric acid on granulated zinc) for 10 minutes, remove the excess of nitrogen dioxide with air and spray with a 0.5 per cent w/v solution of N-(1-naphthyl)ethylenediamine dihydrochloride in a 50 per cent v/v solution of methanol. Any secondary spot corresponding to 2,4-dimethylaniline in the chromatogram obtained with test solution (a) is not more intense than the corresponding spot in the chromatogram obtained with reference solution (b). Other tests. Complies with the tests stated under Dip Concentrates. Assay. Determine by gas chromatography (2.4.13). Internal Standard Solution. A 1.0 per cent v/v solution of squalane in methyl acetate. Test solution (a). Shake a quantity of the powder containing 80 mg of Amitraz with 10 ml of methyl acetate, centrifuge and use the supernatant liquid. Test solution (b). Shake a quantity of the powder containing 80 mg of Amitraz with 10 ml of the internal standard solution, centrifuge and use the supernatant liquid. Reference solution. A 0.8 per cent w/v solution of amitraz RS in the internal standard solution. Chromatographic system a glass column 1.5 m x 4 mm, packed with acid-washed, silanised diatomaceous support (80 to 100 mesh) coated with 3 per cent w/w methyl silicone gum or fluid (such as OV-1 or OV-101), temperature: column. 250, inlet port and detector. 280, flow rate. 30 ml per minute of the carrier gas. Calculate the content of C19H23N3.
Ampicillin and Cloxacillin Intramammary Infusion (Lactating Cow/Buffalo) contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of each of ampicillin, C16H19N3O4S, and cloxacillin, C19H18ClN3O5S.
Identification
A. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 10 volumes of butyl acetate, 6 volumes of glacial acetic acid, 1 volume of 1-butanol and 2 volumes of solution A (see below). Test solution. Extract a quantity of the infusion containing 50 mg of ampicillin with three successive quantities, each of 15 ml, of light petroleum ( 120 to 160). Discard the extracts, wash the residue with 10 ml of ether and dry in a current of air. Dissolve the residue in 50 ml of phosphate buffer pH 7.0, shake well, filter and use the filtrate. Reference solution. A 0.12 per cent w/v solution of ampicillin trihydrate RS in phosphate buffer pH 7.0. Impregnate the plate by spraying it with a 0.1 per cent w/v solution of disodium edetate in a 5 per cent w/v solution of sodium dihydrogen phosphate (solution A), allow the plate to dry in air and heat it at 105 for 1 hour. Apply to the plate 1 l of each solution. After development, dry the plate in air and heat at 150 for 10 to 15 minutes and spray with a mixture of 100 volumes of starch mucilage, 6 volumes of glacial acetic acid and 2 volumes of a 1 per cent w/v solution of iodine in a 4 per cent w/v solution of potassium iodide. The principal spot in the chromatogram obtained with the test solution corresponds to the spot in the chromatogram obtained with the reference solution. B. Determine by thin-layer chromatography (2.4.17), coating the plate with silanised silica gel GF254. Mobile phase. A mixture of 70 volumes of 0.05 M potassium hydrogen phthalate, 30 volumes of acetone and 1 volume of formic acid that has been adjusted first to pH 6.0 with 5 M sodium hydroxide and then to pH 9.0 with 0.1 M sodium hydroxide. Test solution. Extract a quantity of the infusion containing 130 mg of cloxacillin with three successive quantities, each of 15 ml, of light petroleum ( 120 to 160). Discard the extracts, wash the residue with 10 ml of ether and dry in a current of air. Dissolve the residue in 50 ml of phosphate buffer pH 7.0, shake well, filter and use the filtrate. Reference solution. A 0.28 per cent w/v solution of cloxacillin sodium RS in phosphate buffer pH 7.0. Apply to the plate 1 l of each solution. After development, dry the plate in air and heat at 150 for 10 to 15 minutes and spray with a mixture of 100 volumes of starch mucilage, 6
Ampicillin and Cloxacillin Intramammary Infusion (Lactating Cow/Buffalo)

Ampicillin Sodium and Cloxacillin Sodium Intramammary Infusion (LC/B)
Ampicillin and Cloxacillin Intramammary Infusion (Lactating Cow/Buffalo) is a sterile suspension of Ampicillin Sodium and Cloxacillin Sodium in a suitable vehicle containing suitable suspending agents.
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AMPICILLIN AND CLOXACILLIN BENZATHINE INTRAMAMMARY INFUSION (DRY COW/BUFFALO)
volumes of glacial acetic acid and 2 volumes of a 1 per cent w/v solution of iodine in a 4 per cent w/v solution of potassium iodide. The principal spot in the chromatogram obtained with the test solution corresponds to the spot in the chromatogram obtained with the reference solution. C. Extract a quantity containing 50 mg of ampicillin with three successive quantities, each of 15 ml, of light petroleum ( 120 to 160). Discard the extracts, wash the residue with 10 ml of ether and dry the residue at 55. The residue produces an intense, persistent yellowish orange colour when introduced into a non-luminous flame on a platinum wire moistened with hydrochloric acid.
out the procedure simultaneously using 2.0 ml of a solution prepared by dissolving 0.14 g of cloxacillin sodium RS in 100.0 ml of water. Labelling. The label states the quantity of Ampicillin Sodium in terms of the equivalent amount of ampicillin and the quantity of Cloxacillin Sodium in terms of the equivalent amount of cloxacillin.
Tests
Water (2.3.43). Not more than 1.0 per cent , determined on 1.5 g using a mixture of 70 volumes of dichloromethane and 30 volumes of anhydrous methanol as the solvent. Other tests. Complies with the tests stated under Intramammary Infusions. Assay. Weigh and mix the contents of 10 containers. Weigh accurately a quantity of the mixed contents containing 50 mg of ampicillin and extract with three successive quantities, each of 15 ml, of light petroleum ( 120 to 160) previously saturated with ampicillin sodium and cloxacillin sodium. Discard the extracts, wash the residue with ether previously saturated with ampicillin sodium and cloxacillin sodium, dry in a current of air, dissolve in water and dilute to 100.0 ml with water. Centrifuge and use the clear supernatant liquid (solution B). For ampicillin Dilute 2.0 ml of solution B to 50.0 ml with buffered cupric sulphate solution pH 5.2, transfer 10.0 ml of the resulting solution to a stoppered test-tube and heat in a water-bath at 75 for 30 minutes. Cool to room temperature rapidly, dilute to 20.0 ml with buffered cupric sulphate solution pH 5.2 and measure the absorbance of the resulting solution at the maximum at about 320 nm (2.4.7), using as the blank a solution prepared by diluting 2.0 ml of solution B to 100.0 ml with buffered cupric sulphate solution pH 5.2. Calculate the content of C16H19N3O4S in a container of average content from the absorbance obtained by carrying out the procedure simultaneously using 2.0 ml of a solution prepared by dissolving 60 mg of ampicillin trihydrate RS in 100.0 ml of water, diluting to 50.0 ml with buffered cupric sulphate solution pH 5.2 and beginning at the words transfer 10.0 ml..... . For cloxacillin Dilute 2.0 ml of solution B to 100.0 ml with 1 M hydrochloric acid. Measure the absorbance of the resulting solution at 20 after exactly 12 minutes at the maximum at about 350 nm (2.4.7), using 1 M hydrochloric acid as the blank. Calculate the content of C19H18ClN3O5S in a container of average content from the absorbance obtained by carrying
Ampicillin and Cloxacillin Benzathine Intramammary Infusion (Dry Cow/ Buffalo)

Ampicillin and Cloxacillin Benzathine Intramammary Infusion (Dry Cow/Buffalo) is a sterile suspension of Ampicillin Trihydrate and Cloxacillin Benzathine in a suitable vehicle containing suitable suspending agents. Ampicillin and Cloxacillin Benzathine Intramammary Infusion (Dry Cow/Buffalo) contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amounts of ampicillin, C16H19N3O4S, and cloxacillin, C19H18ClN3O5S.
Identification
A. Extract a quantity containing 250 mg of ampicillin with three quantities, each of 15 ml, of light petroleum ( 120 to 160). Discard the extracts, wash the residue with 10 ml of ether and dry in a current of air. Shake with 10 ml of dichloromethane and filter. Keep both the residue and the filtrate. Wash the residue with two quantities, each of 5 ml, of dichloromethane and dry in a vaccum desiccator. On the residue determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with ampicillin trihydrate RS or with the reference spectrum of ampicillin trihydrate. B. Wash the filtrate with two quantities, each of 5 ml, of water, dry the dichloromethane layer with anhydrous sodium sulphate, filter and dilute the filtrate to 20 ml with dichloromethane. On the filtrate determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with cloxacillin benzathine RS or with the reference spectrum of cloxacillin benzathine.
Tests
Water (2.3.43). Not more than 3.0 per cent, determined on 1.5 g using a mixture of 70 volumes of dichloromethane and 30 volumes of anhydrous methanol as the solvent.
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AMPICILLIN VETERINORY ORAL POWDER
IP 2007
Other tests. Complies with the tests stated under Intramammary Infusions. Assay. Weigh and mix the contents of 10 containers. Weigh accurately a quantity of the mixed contents containing 60 mg of ampicillin and extract with three quantities, each of 15 ml, of light petroleum ( 120 to 160) previously saturated with ampicillin trihydrate and cloxacillin benzathine. Discard the extracts, wash the residue with ether previously saturated with ampicillin trihydrate and cloxacillin benzathine, dry in a current of air, dissolve in 50 ml of methanol and dilute to 100.0 ml with water. Centrifuge and use the clear supernatant liquid (solution A). For ampicillin Dilute 2.0 ml of solution A to 50.0 ml with buffered cupric sulphate solution pH 5.2, transfer 10.0 ml to a stoppered test-tube and heat in a water-bath at 75 for 30 minutes. Cool to room temperature rapidly, dilute to 20.0 ml with buffered cupric sulphate solution pH 5.2 and measure the absorbance of the resulting solution at the maximum at about 320 nm (2.4.7), using as the blank the unheated buffered solution of the infusion. Calculate the content of C16H19N3O4S in a container of average content from the absorbance obtained by carrying out the procedure simultaneously using 2.0 ml of a solution prepared by dissolving 70 mg of ampicillin trihydrate RS in 100.0 ml of a 50 per cent v/v solution of methanol, diluting to 50.0 ml with buffered cupric sulphate solution pH 5.2, and beginning at the words transfer 10.0 ml..... . For cloxacillin Dilute 2.0 ml of solution A to 100.0 ml with 1 M hydrochloric acid and measure the absorbance of the resulting solution at 20 after exactly 12 minutes at the maximum at about 350 nm, (2.4.7), using 1 M hydrochloric acid as the blank. Calculate the content of C19H18ClN3O5S in a container of average content from the absorbance obtained by carrying out the procedure simultaneously using 2.0 ml of a solution prepared by dissolving 0.165 g of cloxacillin benzathine RS in 100.0 ml of a 50 per cent v/v solution of methanol. Labelling. The label states the strength of Ampicillin Trihydrate in terms of the equivalent amount of ampicillin and that of Cloxacillin Benzathine in terms of the equivalent amount of cloxacillin.
Description. A fine granular powder.
Identification
A. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. NOTE Prepare the solutions immediately before use. Mobile phase. A mixture of 10 volumes of butyl acetate, 6 volumes of glacial acetic acid, 2 volumes of a 0.1 per cent w/ v solution of disodium edetate in mixed phosphate buffer pH 4.0 and 1 volume of 1-butanol. Test solution. Shake a quantity of the powder containing 0.1 g of ampicillin with 50 ml of phosphate buffer pH 7.0 for 15 minutes, filter and use the filtrate. Reference solution. A 0.2 per cent w/v solution of ampicillin trihydrate RS in phosphate buffer pH 7.0. Impregnate the dry plate by placing it in a tank containing a shallow layer of a 0.1 per cent w/v solution of disodium edetate in mixed phosphate buffer pH 4.0, allowing the solvent to ascend to the top, removing the plate from the tank and allowing the solvent to evaporate. Use the plate with the flow of the mobile phase in the direction in which impregnation was carried out. Before use heat the plate at 100 for 1 hour and allow to cool. Apply to the plate 1 l of each solution. After development, dry the plate in air and spray with a mixture of 100 volumes of a 1 per cent w/v solution of starch, 6 volumes of glacial acetic acid and 2 volumes of a 1 per cent w/v solution of iodine in a 4 per cent w/v solution of potassium iodide. The principal spot in the chromatogram obtained with the test solution corresponds to the spot in the chromatogram obtained with the reference solution. B. To a quantity of the powder containing 10 mg of ampicillin add sufficient water to produce 10 ml, shake for 15 minutes and filter. Place 0.1 ml of a 0.1 per cent w/v solution of ninhydrin on a filter paper, dry at 105, superimpose 0.1 ml of the solution of the preparation under examination, heat for 5 minutes at 105 and allow to cool; a mauve colour is produced. C. Suspend a quantity of the powder containing 10 mg of ampicillin in 1 ml of water and add 2 ml of a mixture of 2 ml of potassium cupri-tartrate solution and 6 ml of water; a magenta-violet colour is immediately produced.
Ampicillin Veterinary Oral Powder

Ampicillin TrihydrateVeterinary Oral Powder
Ampicillin Veterinary Oral Powder is a mixture of Ampicillin Trihydrate and Lactose or other suitable diluent. Ampicillin Veterinary Oral Powder contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of ampicillin, C16H19N3O4S.
Tests
Uniformity of weight. When supplied in containers intended for use on one occasion, complies with the test for Uniformity of weight described under Parenteral Preparations (Powders for Injection). Other tests. Complies with the tests stated under Veterinary Oral Powders.
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IP 2007
AMPROLIUM HYDROCHLORIDE AND ETHOPABATE PREMIX
Assay. Weigh accurately a quantity of the powder containing 0.1 g of ampicillin, add sufficient water to produce 100.0 ml, shake for 15 minutes and filter. Dilute 2.0 ml to 100.0 ml with buffered cupric sulphate solution pH 5.2, transfer 10.0 ml of the resulting solution to a stoppered test-tube and heat in a water-bath at 75 for 30 minutes. Cool to room temperature rapidly, adjust the volume if necessary to 10.0 ml with water and measure the absorbance at the maximum at about 320 nm (2.4.7), using as the blank the unheated buffered solution of the powder under examination. Calculate the content of C16H19N3O4S from the absorbance obtained by carrying out the procedure simultaneously using 0.12 g of ampicillin trihydrate RS. When the powder is supplied in containers intended for use on more than one occasion, take the quantity of powder without previous mixing. Storage. Store protected from moisture, at a temperature not exceeding 30. Labelling. The label states the strength in terms of the equivalent concentration of ampicillin.
C. To 1 mg add 5 ml of naphthalenediol reagent; a deep violet colour is produced. D. Gives the reactions of chlorides (2.3.1).
Tests
Picoline. Dissolve 1.5 g in 30 ml of water in a distillation flask, add 20 ml of a saturated solution of potassium carbonate sesquihydrate, connect the flask to a ground-glass aerator extending to the bottom of a l00-ml graduated cylinder containing 50 ml of 0.05 M hydrochloric acid and pass air, which has previously been passed through sulphuric acid and glass wool, through the system for 60 minutes. To 5 ml of the hydrochloric acid solution add sufficient 0.05 M hydrochloric acid to produce 200 ml. Absorbance of the resulting solution at about 262 nm (2.4.7), not more than 0.52. Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 1.0 per cent, determined on 1.0 g by drying to constant weight at 100 at a pressure not exceeding 0.7 kPa. Assay. Weigh accurately about 0.3 g, dissolve in 20 ml of anhydrous glacial acetic acid, add 10 ml of mercuric acetate solution. Titrate with 0.1 M perchloric acid, using 1-naphtholbenzein solution as indicator. Carry out a blank titration.
Amprolium Hydrochloride
N N H3C N NH2 CH3 Cl , HCl
1 ml of 0.1 M perchloric acid is equivalent to 0.01577 g of C14H19ClN4,HCl.
C14H19ClN4,HCl
Mol. Wt. 315.2
Amprolium Hydrochloride and Ethopabate Premix

Amprolium Hydrochloride and Ethopabate Premix contains Amprolium Hydrochloride and Ethopabate. Amprolium Hydrochloride and Ethopabate Premix contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amounts of amprolium hydrochloride, C14H19ClN4,HCl and of ethopabate, C12H15NO4.
Amprolium Hydrochloride is hydrochloride salt of 1-[(4amino-2-propyl-5-pyrimidinyl)methyl]-2-methylpyridinium chloride. Amprolium Hydrochloride contains not less than 97.5 per cent and not more than 101.0 per cent of C14H19ClN4,HCl, calculated on the dried basis. Description. A white or almost white powder.
Identification
A. Shake a quantity containing 20 mg of Amprolium Hydrochloride with 90 ml of methanol and filter. Add 5 ml of the filtrate to 5 ml of naphthalenediol reagent; a deep violet colour is produced. B. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. NOTE Prepare the solution immediately before use. Mobile phase. A mixture of 90 volumes of dichloromethane and 10 volumes of methanol.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with amprolium hydrochloride RS or with the reference spectrum of amprolium hydrochloride. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.002 per cent w/v solution in 0.1 M hydrochloric acid exhibits maxima at about 246 nm and 262 nm; absorbance at about 246 nm, about 0.84 and at about 262 nm, about 0.80.
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AMPROLIUM, ETHOPABATE AND SULPHAQUINOXALINE PREMIX
IP 2007
Test solution. Shake continuously for 10 minutes a quantity containing 10 mg of Ethopabate with 25 ml of acetone that has been warmed to 50, filter and use the filtrate. Reference solution. A 0.04 per cent w/v solution of ethopabate RS in acetone. Apply to the plate 2 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution corresponds to the spot in the chromatogram obtained with the reference solution.
at the words add 2.5 ml of 2 M hydrochloric acid....... Calculate the content of C12H15NO4 from the absorbance obtained by carrying out the procedure simultaneously, using 10.0 ml of a 0.006 per cent w/v solution of ethopabate RS in methanol and beginning at the words add 10 ml of 1 M sodium hydroxide and evaporate to dryness......
Amprolium, Ethopabate and Sulphaquinoxaline Premix

Amprolium Hydrochloride, Sulphaquinoxaline Premix Ethopabate and
Tests
pH (2.4.24). 2.5 to 4.0, determined in a 25 per cent w/v slurry in carbon dioxide-free water. Other tests. Complies with the tests stated under Premixes. Assay. For amprolium hydrochloride Weigh accurately a quantity containing 50 mg of Amprolium Hydrochloride, shake continuously for 20 minutes with 100.0 ml of a mixture of 2 volumes of methanol and 1 volume of water and filter. Dilute 5.0 ml of the filtrate to 100.0 ml with the methanol-water mixture. To 4.0 ml of the resulting solution add 10.0 ml of naphthalenediol reagent, allow to stand for 20 minutes and measure the absorbance of the resulting solution at the maximum at about 520 nm (2.4.7), using as the blank a solution obtained by mixing 4.0 ml of a mixture of 2 volumes of methanol and 1 volume of water with 10.0 ml of naphthalenediol reagent and allowing to stand for 20 minutes. Calculate the content of C14H19ClN4,HCl from the absorbance obtained by carrying out the procedure simultaneously, using 4.0 ml of a 0.0025 per cent w/v solution of amprolium hydrochloride RS in a mixture of 2 volumes of methanol and 1 volume of water and beginning at the words, To 4.0 ml of the resulting solution add 10.0 ml of..... . For ethopabate Weigh accurately a quantity containing 6 mg of Ethopabate, add 75 ml of methanol, shake continuously for 20 minutes, dilute to 100.0 ml with methanol and filter. To 10.0 ml of the filtrate add 10 ml of 1 M sodium hydroxide and evaporate to dryness. Dissolve the residue in 10.0 ml of water, heat on a water-bath for 15 minutes, add 10 ml of 2 M hydrochloric acid, dilute to 100.0 ml with water and filter. To 25.0 ml of the filtrate, add 2.5 ml of 2 M hydrochloric acid and 5 ml of a 0.1 per cent w/v solution of sodium nitrite prepared immediately before use. Allow to stand for 3 minutes and add 2.0 ml of a freshly prepared 0.5 per cent w/v solution of ammonium sulphamate. Allow to stand for 2 minutes, add 5.0 ml of a freshly prepared 0.1 per cent w/v solution of N-(1-naphthyl)ethylenediamine dihydrochloride, allow to stand for 10 minutes and dilute to 50.0 ml with water. Measure the absorbance of the resulting solution at the maximum at about 540 nm (2.4.7), using as the blank a solution obtained by repeating the procedure with 25 ml of water and beginning
Amprolium, Ethopabate and Sulphaquinoxaline Premix contains Amprolium Hydrochloride, Ethopabate and Sulphaquinoxaline. Amprolium, Ethopabate and Sulphaquinoxaline Premix contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amounts of amprolium hydrochloride, C 14 H 19 ClN 4 ,HCl, of ethopabate, C 12 H 15 NO 4 , and of sulphaquinoxaline, C14H12N4O2S.
Identification
A. Shake a quantity containing 20 mg of Amprolium Hydrochloride with 90 ml of methanol and filter. Add 5 ml of the filtrate to 5 ml of naphthalenediol reagent; a deep violet colour is produced. B. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. NOTE Prepare the solution immediately before use. Mobile phase. A mixture of 90 volumes of dichloromethane and 10 volumes of methanol. Test solution. Shake continuously for 10 minutes a quantity containing 10 mg of Ethopabate with 25 ml of acetone that has been warmed to 50, filter and use the filtrate. Reference solution (a). A 0.04 per cent w/v solution of ethopabate RS in acetone. Reference solution (b). A 0.4 per cent w/v solution of sulphaquinoxaline RS in acetone. Apply to the plate 2 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution corresponds to the spot in the chromatogram obtained with reference solutions (a) and (b).
Tests
pH (2.4.24). 2.5 to 4.0, determined in a 25 per cent w/v slurry in carbon dioxide-free water.
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IP 2007
CALCIUM BOROGLUCONATE INJECTION
Other tests. Complies with the tests stated under Premixes. Assay. For amprolium hydrochloride Weigh accurately a quantity containing 50 mg of Amprolium Hydrochloride, shake continuously for 20 minutes with 100.0 ml of a mixture of 2 volumes of methanol and 1 volume of water and filter. Dilute 5.0 ml of the filtrate to 100.0 ml with the methanol-water mixture. To 4.0 ml of the resulting solution add 10.0 ml of naphthalenediol reagent, allow to stand for 20 minutes and measure the absorbance of the resulting solution at the maximum at about 520 nm (2.4.7), using as the blank a solution obtained by mixing 4.0 ml of a mixture of 2 volumes of methanol and 1 volume of water with 10.0 ml of naphthalenediol reagent and allowing to stand for 20 minutes. Calculate the content of C14H19ClN4,HCl from the absorbance obtained by carrying out the procedure simultaneously, using 4.0 ml of a 0.0025 per cent w/v solution of amprolium hydrochloride RS in a mixture of 2 volumes of methanol and 1 volume of water and beginning at the words, To 4.0 ml of the resulting solution add 10.0 ml of..... . For ethopabate Weigh accurately a quantity containing 6 mg of Ethopabate, add 75 ml of methanol, shake continuously for 20 minutes, dilute to 100.0 ml with methanol and filter. To 10.0 ml of the filtrate add 10 ml of 1 M sodium hydroxide and evaporate to dryness. Dissolve the residue in 10.0 ml of water, heat on a water-bath for 15 minutes, add 10 ml of 2 M hydrochloric acid, dilute to 100.0 ml with water and filter. To 25.0 ml of the filtrate, add 2.5 ml of 2 M hydrochloric acid and 5 ml of a 0.1 per cent w/v solution of sodium nitrite prepared immediately before use. Allow to stand for 3 minutes and add 2.0 ml of a freshly prepared 0.5 per cent w/v solution of ammonium sulphamate. Allow to stand for 2 minutes, add 5.0 ml of a freshly prepared 0.1 per cent w/v solution of N-(1-naphthyl)ethylenediamine dihydrochloride, allow to stand for 10 minutes and dilute to 50.0 ml with water. Measure the absorbance of the resulting solution at the maximum at about 540 nm (2.4.7), using as the blank a solution obtained by repeating the procedure with 25 ml of water and beginning at the words add 2.5 ml of 2 M hydrochloric acid....... Calculate the content of C12H15NO4 from the absorbance obtained by carrying out the procedure simultaneously, using 10.0 ml of a 0.006 per cent w/v solution of ethopabate RS in methanol and beginning at the words add 10 ml of 1 M sodium hydroxide and evaporate to dryness...... For sulphaquinoxaline Weigh accurately a quantity containing 40 mg of Sulphaquinoxaline, shake continuously for 10 minutes with a mixture of 75 ml of water and 4 ml of 2 M sodium hydroxide, dilute to 250.0 ml with water and centrifuge. To 10.0 ml of the supernatant liquid add 5 ml of 2 M hydrochloric acid and dilute to 200.0 ml with water. To 10.0 ml of the diluted solution add 2.5 ml of 2 M hydrochloric acid and 5 ml of a freshly prepared 0.1 per cent w/v solution of sodium nitrite and allow to stand for 3 minutes. Add 5.0 ml of
a freshly prepared 0.5 per cent w/v solution of ammonium sulphamate and allow to stand for 2 minutes. Add 5.0 ml of a freshly prepared 0.1 per cent w/v solution of N-(1-naphthyl)ethylenediamine dihydrochloride, allow to stand for 10 minutes and dilute to 50.0 ml with water. Measure the absorbance of the resulting solution at the maximum at about 540 nm (2.4.7), using as the blank the solution obtained by repeating the procedure with 10 ml of water and beginning at the words add 2.5 ml of 2 M hydrochloric acid....... Calculate the content of C14H12N4O2S from the absorbance obtained by carrying out the procedure simultaneously, using 10.0 ml of a 0.0008 per cent w/v solution of sulphaquinoxaline RS in 0.001 M sodium hydroxide and beginning at the words To 10.0 ml of the diluted solution add 2.5 ml of 2 M hydrochloric acid......
Calcium Borogluconate Injection

Calcium Borogluconate Injection is a sterile solution of Calcium Gluconate and Boric Acid in Water for Injections. The solution may contain up to 0.2 per cent w/v of Chlorocresol. Calcium Borogluconate Injection contains not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of calcium, Ca, and boric acid, H3BO3 equiivalent to not more than 2.3 times the stated content of calcium.
Identification
A. Dilute 1 ml with sufficient water to produce a solution containing about 0.75 per cent w/v of calcium and add 0.05 ml of ferric chloride test solution; an intense yellow or yellowish green colour is produced. B. Gives the reactions of calcium salts (2.3.1). C. To 1 ml add 0.15 ml of sulphuric acid and 5 ml of methanol and ignite; the mixture burns with a flame tinged with green.
Tests
pH (2.4.24). 3.0 to 4.0, determined in a solution diluted if necessary with carbon dioxide-free water to produce a solution containing 1.5 per cent w/v of calcium. Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. For calcium Dilute an accurately measured volume containing 45 mg of calcium to about 50 ml with water. Titrate with 0.05 M disodium edetate to within a few ml of the expected end-point, add 4 ml of a 40 per cent w/v solution of sodium hydroxide and 10 mg of calcon mixture and continue the titration until the colour changes from pink to blue. 1 ml of 0.05 M disodium edetate is equivalent to 0.002004 g of Ca.
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CALCIUM MAGNESIUM BOROGLUCONATE INJECTION
IP 2007
For boric acid Dilute an accurately measured volume containing 0.1 g of Boric Acid to 50 ml with water, add 3 g of mannitol and titrate with 0.1 M sodium hydroxide using phenolphthalein solution as indicator. 1 ml of 0.1 M sodium hydroxide is equivalent to 0.006183 g of H3BO3. Storage. Store protected from light, at a temperature not exceeding 30. Labelling. The label states (1) the strength in terms of the equivalent amount of calcium in a suitable dose-volume; (2) the proportion of boric acid present; (3) the proportion of chlorocresol, if present.
Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Bacterial endotoxins (2.2.3). Not more than 0.5 Endotoxin Unit per ml. Sterility (2.2.11). Complies with the test for sterility. Assay. For calcium Dilute a volume containing 45 mg of calcium to about 50 ml with water. Add 1 ml of 1 M sodium hydroxide solution. Titrate with 0.05 M disodium edetate to within a few ml of the expected end-point, add 5 ml of strong ammonia-ammonium chloride solution and 10 mg of calcon mixture as indicator and continue the titration until the colour changes from pink to blue. Calculate the volume of 0.05 M disodium edetate consumed by substracting the volume of 0.05 M disodium edetate consumed in the assay for magnesium. 1 ml of 0.05 M disodium edetate is equivalent to 0.002004 g of Ca. For magnesium Dilute a volume containing 10 mg of magnesium to about 50 ml with water. Add 1 g of ammonium chloride and 1 g of ammonium oxalate. Neutralise to litmus paper with dilute ammonia solution and add 5 ml in excess. Boil for 5 minutes and allow to stand for 1 hour. Filter and wash the residue with hot water. Collect the filtrate and washings and add 5 ml of strong ammonia-ammonium chloride solution. Titrate with 0.05 M disodium edetate using eriochrome black T mixture as indicator. 1 ml of disodium edetate is equivalent to 0.001216 g of magnesium or 0.0107894 g of magnesium hypophosphite, Mg(H2PO2)2,6H2O. For boric acid Dilute a volume containing 0.1 g of boric acid to 50 ml with water, add 3 g of mannitol and titrate with 0.1 M sodium hydroxide using phenolphthalein solution as indicator. 1 ml of 0.1 M sodium hydroxide is equivalent to 0.006183 g of H3BO3. For dextrose Dilute a volume containing 200 mg of dextrose to 50 ml with water; add 30 ml of 0.1 M iodine solution and 10 ml of a 5 per cent w/v solution of sodium carbonate and allow to stand for 20 minutes. Add 15 ml of 1 M hydrochloric acid and titrate the excess of iodine with 0.1 M sodium thiosulphate solution using starch solution as indicator. Carry out a blank titration. 1 ml of 0.1 M iodine solution is equivalent to 0.009008 g of dextrose, C6H12O6. Storage. Store protected from light. Labelling. The label states (1) the strength in terms of the equivalent amount of calcium and magnesium in a suitable dose-volume; (2) the proportion of boric acid to calcium; (3) the percentage of any added stabilising agent.
Calcium Magnesium Borogluconate Injection

Calcium Magnesium Borogluconate Injection is a sterile solution of Calcium Gluconate, Boric Acid, Magnesium Hypophosphite and Dextrose in Water for Injections. It may contain upto 0.2 per cent w/v of Chlorocresol. Calcium Magnesium Borogluconate Injection contains not less than 95.0 per cent and not more than 105.0 per cent of the stated amounts of calcium, Ca, of magnesium, calculated as magnesium hypophosphite, Mg(H2 PO2 )2 ,6H2O, and of dextrose, C6H12O6, and the content of boric acid, H3BO3, is not more than 2.3 times the stated content of calcium.
Identification
A. Dilute 1 ml with sufficient water to produce a solution containing about 0.75 per cent w/v of calcium and add 0.05 ml of ferric chloride test solution; an intense yellow or yellowish green colour is produced. B. Gives the reactions of calcium salts (2.3.1). C. Gives the reactions of magnesium salts (2.3.1). D. To 1 ml add 5 ml of water, neutralise to pH 7.0 with dilute ammonia solution and add 5 ml of silver nitrate solution. A yellow precipitate is produced which does not change colour on boiling but dissolves on addition of dilute ammonia solution. E. To 1 ml add 0.15 ml of sulphuric acid and 5 ml of methanol and ignite; the mixture burns with a flame tinged with green. F. To 1 ml add 2 ml of 2 M sodium hydroxide solution and 0.05 ml of copper sulphate solution. The solution is blue and clear. Heat to boiling. A copious red precipitate is produced.
Tests
pH (2.4.24). 3.0 to 4.0, determined in a solution diluted, if necessary, with carbon dioxide-free water so as to contain 1.5 per cent w/v of calcium.
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IP 2007
CHLORAMPHENICOL INJECTION
Chloral Hydrate
HO HO
C2H3Cl3O2
Chloramphenicol Injection
Cl Cl Cl
Mol. Wt. 165.4 Chloramphenicol Injection is a sterile suspension of Chloramphenicol in Water for Injections containing suitable suspending and stabilising agents. Chloramphenicol Injection contains not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of chloramphenicol, C11H12Cl2N2O5.
Chloral Hydrate is 2,2,2-trichloro-1,1-ethane-diol. Description. A colourless, transparent crystals; odour, pungent. Chloral Hydrate contains not less than 98.5 per cent and not more than 101.0 per cent of the stated amount of chloral hydrate, C2H3Cl302.
Identification
Centrifuge a volume containing 0.15 g of Chloramphenicol; wash the residue with water and dry over self-indicating silica gel and then for 1 hour at 105. The dried residue complies with the following tests. A. Wash 75 mg of the residue with two quantities, each of 10 ml, of light petroleum ( 60 to 80) and allow to dry. The residue complies with the following test. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with chloramphenicol RS or with the reference spectrum of chloramphenicol. B. In the test for Related substances, the principal spot in the chromatogram obtained with 1 l of test solution corresponds to that in the chromatogram obtained with reference solution (a). C. To 5 ml of a 0.1 per cent w/v solution add a few drops of silver nitrate solution; no precipitate is produced. Heat about 50 mg with 3 ml of ethanolic potassium hydroxide solution on a water-bath for 15 minutes, add 15 mg of decolorising charcoal, shake and filter. The filtrate gives the reactions of chlorides (2.3.1).
Identification
A. To 10 ml of a 10 per cent w/v solution in carbon dioxide-free water (solution A) add 2 ml of 2 M sodium hydroxide. The mixture becomes cloudy and when heated gives an odour of chloroform. B. To 1 ml of solution A add 2 ml of sodium sulphide solution; a yellow colour develops which quickly turns reddish brown and may yield a red precipitate on standing.
Tests
pH (2.4.24). 3.5 to 5.5, determined in solution A. Appearance of solution. Solution A is clear (2.4.1) and colourless (2.4.1). Heavy metals (2.3.13). 12 ml of a 5 per cent w/v solution in water, complies with the limit test for heavy metals, Method D (20 ppm). Chlorides (2.3.12). Dissolve 2.5 g in 15 ml with water; the resulting solution complies with the limit test for chlorides (100 ppm). Chloral alcoholate. Warm 1 g with 10 ml of 2 M sodium hydroxide, filter the upper layer and add 0.05 M iodine dropwise until a yellow colour is produced. Set aside for 1 hour; no yellow precipitate is produced and no smell of iodoform is perceptible. Assay. Weigh accurately about 4 g, dissolve in 10 ml of water and add 40 ml of 1 M sodium hydroxide. Allow the mixture to stand for exactly 2 minutes and titrate the residual alkali immediately with 0.5 M sulphuric acid using phenolphthalein solution as indicator. 1 ml of 1 M sodium hydroxide is equivalent to 0.1654 g of C2H3Cl3O2. Storage. Store protected from moisture.
Tests
pH (2.4.24). 3.5 to 6.5. Consistence. Chloramphenicol Injection containing 150 mg per ml passes readily through a 23G hypodermic needle. 2-Amino-1-(4-nitrophenyl)propane-1,3-diol Determine by liquid chromatography (2.4.14). Test solution. Dilute the injection with sufficient of the mobile phase to produce a solution containing 0.030 per cent w/v of Chloramphenicol. Reference solution. A 0.00225 per cent w/v of 2-amino-1-(4-nitrophenyl) propane-1,3-diol RS in the mobile phase. Chromatographic system a stainless steel column 10 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a filtered and degassed mixture of 85 volumes of 0.012 M sodium pentanesulphonate, 15 volumes of acetonitrile and 1 volume of glacial acetic acid,
1519
CHLORTETRACYCLINE HYDROCHLORIDE
IP 2007
flow rate. 2 ml per minute, spectrophotometer set at 272 nm, a 20 l loop injector. Inject alternatively the test solution and the reference solution. In the chromatogram obtained with test solution the area of any peak corresponding to 2-amino-1-(4- nitrophenyl)propane-1,3-diol is not more than the area of the peak obtained with the reference solution. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 90 volumes of dichloromethane, 10 volumes of methanol and 1 volume of water. Test solution. A 1.0 per cent w/v solution of the dried residue obtained in the test for Identification in acetone. Reference solution (a). A 1.0 per cent w/v solution of chloramphenicol RS in acetone. Reference solution (b). Dilute 1 ml of reference solution (a) to 200 ml with acetone. Apply to the plate 1 l and 20 l of the test solution, 1 l of reference solution (a) and 20 l of reference solution (b). After development, dry the plate in air and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (b). Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. To an accurately measured volume containing 0.75 g of Chloramphenicol add sufficient water to produce 1000.0 ml and shake until a clear solution is obtained. Dilute 5.0 ml of this solution to 200.0 ml with water and measure the absorbance of the resulting solution at the maximum at about 278 nm (2.4.7). Calculate the content of C11H12Cl2N2O5 in the injection taking 297 as the specific absorbance at 278 nm. Storage. Store protected from light. Do not freeze. Labelling. The label states (1) the name of any added suspending agent; (2) that the injection is for intramuscular injection only; (3) the date after which the contents are not intended to be used.
Chlortetracycline Hydrochloride is [4S(4,4a,5a,6,12a)]-7-chloro-4- dimethylamino1,4,4a,5,5a,6,11,12a-octahydro-3,6,10,12,12a-pentahydroxy-6methyl-1,11-dioxo-2-naphthacenecarboxamide hydrochloride. Chlortetracycline Hydrochloride contains not less than 89.5 per cent of chlortetracycline hydrochloride and the sum of the contents of chlortetracycline hydrochloride and tetracycline hydrochloride is not less than 94.5 per cent and not more than 100.5 per cent, calculated on the anhydrous basis. Description. Yellow powder.
Identification
A. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel H. NOTE Use freshly prepared solution. Mobile phase. A mixture of 59 volumes of dichloromethane, 35 volumes of methanol and 6 volumes of water. Test solution. Dissolve 50 mg of the substance under examination in 100 ml of methanol. Reference solution (a). A 0.05 per cent w/v of chlortetracycline hydrochloride RS in methanol. Reference solution (b). A solution containing 0.05 per cent w/ v each of chlortetracycline hydrochloride RS, tetracycline hydrochloride RS and metacycline hydrochloride RS in methanol. Adjust the pH of a 10 per cent w/v solution of disodium edetate to 8.0 with 10 M sodium hydroxide and spray this solution evenly on the plate (about 10 ml for a plate of 100 mm x 200 mm size). Allow the plate to dry in a horizontal position for at least 1 hour. Dry the plate in an oven at 100 for 1 hour before use. Apply to the plate 1 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows three clearly separated spots. B. To about 2 mg add 5 ml of sulphuric acid; a deep blue colour develops which becomes bluish green. Add the solution to 2.5 ml of water; the colour changes to brownish. C. Gives reaction A of chlorides (2.3.1).
Chlortetracyline Hydrochloride
OH O OH O HO O NH2 H Cl H3C OH
C22H23ClN2O8,HCl
, HCl
Tests
pH (2.4.24). 2.3 to 3.3, determined in a 1 per cent w/v solution in carbon dioxide-free water prepared by slight heating, if necessary.
OH H N(CH3)2
Mol. Wt. 515.3
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IP 2007
CHLORTETRACYCLINE HYDROCHLORIDE
Specific optical rotation (2.4.22). 235 to 250, determined at 20 in a 0.25 per cent w/v solution in water, calculated on the anhydrous basis. Light absorption. When examined in the range 430 nm to 460 nm of a 0.5 per cent w/v solution in water is not greater than 0.40. Related substances. Carry out the method described under Assay injecting test solution, reference solutions (e) and (f). The test is not valid unless the peak in the chromatogram obtained with reference solution (f) is properly integrated. In the chromatogram obtained with test solution the area of the peak corresponding to 4-epichlortetracycline is not more than the area of the peak corresponding to 4-epichlortetracycline in the chromatogram obtained with reference solution (e) (4 per cent) and the total area of any secondary peaks, other than the peaks due to tetracycline and 4-epichlortetracycline, is not more than 25 per cent of the area of the peak corresponding to 4-epichlortetracycline in the chromatogram obtained with reference solution (e) (1 per cent). Ignore any peak with an area smaller than that of the principal peak in the chromatogram obtained with reference solution (f) (0.1 per cent). Tetracycline hydrochloride. Not more than 8.0 per cent, calculated on the anhydrous basis and determined as described under the Assay, injecting separately test solution and reference solution (e). Heavy metals (2.3.13). 0.5 g complies with the limit test for heavy metals, Method D (50 ppm), using 2.5 ml of lead standard solution (10 ppm Pb) as the standard. Sulphated ash (2.3.18). Not more than 0.5 per cent. Water (2.3.43). Not more than 2.0 per cent , determined on 0.3 g. Assay. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 100 mg of the substance under examination in 0.01 M hydrochloric acid. Reference solution (a). A 0.1 per cent w/v solution of chlortetracycline hydrochloride RS in 0.01 M hydrochloric acid. Reference solution (b). A 0.04 per cent w/v of 4-epichlortetracycline hydrochloride RS in 0.01 M hydrochloric acid. Reference solution (c). A 0.08 per cent w/v of tetracycline hydrochloride RS in 0.01 M hydrochloric acid. Reference solution (d). Mix 5 ml of reference solution (a) and 10 ml of reference solution (b) and dilute to 25 ml with 0.01 M hydrochloric acid.
Reference solution (e). Mix 5 ml of reference solution (b) and 5 ml of reference solution (c) and dilute to 50 ml with 0.01 M hydrochloric acid. Reference solution (F). Dilute 1 ml of reference solution (c) to 20 ml with 0.01 M hydrochloric acid and dilute 2.5 ml of this solution to 100 ml with 0.01 M hydrochloric acid. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octylsilyl groups (5 m), column temperature 35, mobile phase: a filtered and degassed mixture of 450 ml of dimethyl sulphoxide, 50 ml of 1 M perchloric acid and 500 ml of water, flow rate. 1 ml per minute, spectrophotometer set at 280 nm, a 20 l loop injector. Inject reference solution (d) and adjust the instrument so that the peak heights correspond to at least 50 per cent of the full scale deflection of the recorder. If necessary, adjust the dimethyl sulphoxide content in the mobile phase. The test is not valid unless the resolution factor between the first peak (4-epichlortetracycline) and the second (chlortetracycline) is not less than 2.0 and the symmetry factor for the second peak is not more than 1.3. Inject reference solution (a). The test is not valid unless the relative standard deviation of the peak area for chlortetracycline hydrochloride is not more than 1.0 per cent. If necessary, adjust the integrator parameters. Inject alternately the test solution and reference solution (a). Calculate the content of C22H23ClN2O8,HCl. Chlortetracycline Hydrochloride intended for use in the manufacture of Parenteral Preparations without a further appropriate procedure for removal of bacterial endotoxins complies with the following additional requirement. Bacterial endotoxins (2.2.3). Not more than 1.1 EU per mg. Chlortetracycline Hydrochloride intended for use in the manufacture of Parenteral Preparations without a further appropriate sterilisation procedure complies with the following additional requirement. Sterility (2.2.11). Complies with the test for sterility. Storage. Store protected from light. If it is intended for use in the manufacture of Parenteral Preparations, the container should be sterile, tamper-evident and sealed so as to exclude micro-organisms. Labelling. The label states (1) the date after which the material is not intended to be used; (2) the storage conditions; (3) where applicable, that the material is sterile and free from Bacterial endotoxins.
1521
CHLORTETRACYCLINE VETERINORY ORAL POWDER
IP 2007
Chlortetracycline Veterinary Oral Powder

Chlortetracycline Hydrochloride Veterinary Oral Powder; Chlortetracycline Soluble Powder Chlortetracycline Veterinary Oral Powder is a mixture of Chlortetracycline Hydrochloride and Lactose or other suitable diluent. Chlortetracycline Veterinary Oral Powder contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of chlortetracycline hydrochloride, C22H23ClN2O8,HCl
Tests
Other tests. Complies with the tests stated under Veterinary Oral Powders. Assay. Weigh accurately a quantity containing 0.25 g of Chlortetracycline Hydrochloride, add 500 ml of water, mix thoroughly and carry out the microbiological assay of antibiotics (2.2.10). Calculate the content of chlortetracycline hydrochloride taking each 1000 Units found to be equivalent to 1 mg of chlortetracycline hydrochloride. Storage. Store protected from moisture, at a temperature not exceeding 30. Labelling. The label states the strength in terms of the concentration of Chlortetracycline Hydrochloride.
Identification
A. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel H. NOTE Use freshly prepared solutions. Mobile phase. A mixture of 59 volumes of dichloromethane, 35 volumes of methanol and 6 volumes of water. Test solution. The supernatant liquid obtained by extracting a quantity containing 5 mg of Chlortetracycline Hydrochloride with 10 ml of methanol and centrifuging. Reference solution (a). A 0.05 per cent w/v of chlortetracycline hydrochloride RS in methanol. Reference solution (b). A solution containing 0.05 per cent w/ v each of chlortetracycline hydrochloride RS, tetracycline hydrochloride RS and metacycline hydrochloride RS in methanol. Adjust the pH of a 10 per cent w/v solution of disodium edetate to 8.0 with 10 M sodium hydroxide and spray this solution evenly on the plate (about 10 ml for a plate of 100 mm x 200 mm size). Allow the plate to dry in a horizontal position for at least 1 hour. Dry the plate in an oven at 100 for 1 hour before use. Apply to the plate 1 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows three clearly separated spots. B. To a quantity containing 10 mg of Chlortetracycline Hydrochloride, add 20 ml of warm ethanol (95 per cent), allow to stand for 20 minutes, filter and evaporate to dryness on a water-bath. Dissolve the residue in sufficient phosphate buffer pH 7.6 to produce a 0.1 per cent w/v solution and heat at 100 for 1 minute; it exhibits a strong blue fluorescence in ultra-violet light.
Cloxacillin Benzathine
O Cl N O O N N S H H H COOH CH3 CH3
2
N H
H N
C19H18ClN3O5S)2,C16H20N2
Mol. Wt. 1112.1
Cloxacillin Benzathine is N,N-dibenzylethylenediammonium bis-[(6R)-6-{3-(2-chlorophenyl)-5-methylisoxazole-4-carboxamido}penicillanate]. Cloxacillin Benzathine contains not less than 92.0 per cent of (C19H18ClN3O5S)2,C16H20N2 and not less than 20.0 per cent and not more than 22.0 per cent of benzathine, C16H20N2, both calculated on the anhydrous basis. Description. A white or almost white powder.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with cloxacillin benzathine RS or with the reference spectrum of cloxacillin benzathine. B. Shake 0.1 g with 1 ml of 1 M sodium hydroxide for 2 minutes, add 2 ml of ether, shake for 1 minute and allow to separate. Evaporate 1 ml of the ether layer to dryness, dissolve the residue in 2 ml of glacial acetic acid and add 1 ml of dilute potassium dichromate solution; a golden yellow precipitate is produced. C. Shake 50 mg with 10 ml of water and filter. To 5 ml of the filtrate add a few drops of silver nitrate solution; no precipitate is produced. Heat 50 mg with 2 ml of ethanolic potassium
1522
IP 2007
CLOXACILLIN BENZATHINE INTRAMAMMARY INFUSION (DRY COW/BUFFALO)
hydroxide solution on a water-bath for 15 minutes, add 15 mg of decolorising charcoal, shake and filter. Acidify the filtrate with 2 M nitric acid; the solution gives reaction A of chlorides (2.3.1).
Cloxacillin Benzathine Intramammary Infusion (Dry Cow/ Buffalo)

Cloxacillin Benzathine Intramammary Injection; Cloxacillin Intramammary Infusion (Dry Cow/Buffalo); Cloxacillin Intramammary Infusion (DC/B) Cloxacillin Benzathine Intramammary Infusion (Dry Cow/ Buffalo) is a sterile suspension of Cloxacillin Benzathine in a suitable non-aqueous vehicle containing suitable suspending agents. Cloxacillin Benzathine Intramammary Infusion (Dry Cow/ Buffalo) contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of cloxacillin, C19H18ClN3O5S.
Tests
Water (2.3.43). Not more than 5.0 per cent w/w, determined on 0.5 g. Assay. For cloxacillin benzathine Weigh accurately about 60 mg, add 40 ml of methanol, shake to dissolve, add 25 ml of 1 M sodium hydroxide and allow to stand for 30 minutes. Add 27.5 ml of 1 M hydrochloric acid and sufficient water to produce 100.0 ml, mix, transfer 20.0 ml of the solution to a stoppered conical flask, add 30.0 ml of 0.01 M iodine, close the flask with a wet stopper and allow to stand for 15 minutes protected from light. Titrate the excess of iodine with 0.02 M sodium thiosulphate, using starch mucilage, added towards the end of the titration, as indicator. Add a further 12 mg of the substance under examination to 10 ml of water, swirl to disperse, add 30 ml of 0.01 M iodine and titrate immediately with 0.02 M sodium thiosulphate, using starch mucilage, added towards the end of the titration, as indicator. The difference between the titrations represents the volume of 0.01 M iodine equivalent to the total penicillins present. Calculate the content of (C19H18ClN3O5S)2,C16H20N2 from the difference obtained by carrying out the procedure simultaneously using cloxacillin benzathine RS. For benzathine Weigh accurately about 1 g, add 30 ml of a saturated solution of sodium chloride and 10 ml of 5 M sodium hydroxide, shake well and extract with four quantities, each of 50 ml, of ether. Wash the combined extracts with three quantities, each of 10 ml, of water, extract the combined washings with 25 ml of ether and add the extract to the main ether solution. Evaporate the ether solution to low volume, add 2 ml of ethanol and evaporate to dryness. To the residue add 50 ml of anhydrous glacial acetic acid. Titrate with 0.1 M perchloric acid, using 0.1 ml of 1-naphtholbenzein solution as indicator. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.01202 g of C16H20N2. Cloxacillin Benzathine intended for use in the manufacture of either parenteral preparations or intramammary infusions without a further appropriate sterilisation procedure complies with the following additional requirement. Sterility (2.2.11). Complies with the test for sterility. Storage. Store protected from moisture. If it is intended for use in the manufacture of parenteral preparations or intramammary infusions, the container should be sterile, tamper-evident and sealed so as to exclude micro-organisms.
Identification
Extract a quantity containing 75 mg of cloxacillin with three quantities, each of 15 ml, of light petroleum ( 120 to 160). Discard the extracts, wash the residue with 10 ml of ether and dry in a current of air. The residue complies with the following tests. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with cloxacillin benzathine RS or with the reference spectrum of cloxacillin benzathine. B. Shake 50 mg with 1 ml of 1 M sodium hydroxide for 2 minutes, add 2 ml of ether, shake for 1 minute and allow to separate. Evaporate 1 ml of the ether layer to dryness, dissolve the residue in 2 ml of glacial acetic acid and add 1 ml of dilute potassium dichromate solution; a golden yellow precipitate is produced.
Tests
Water (2.3.43). Not more than 2.0 per cent , determined on 3 g and using a mixture of 70 volumes of dichloromethane and 30 volumes of anhydrous methanol as the solvent. Other tests. Complies with the tests stated under Intramammary Infusions. Assay. Weigh and mix the contents of 10 containers. Weigh accurately a quantity of the mixed contents containing 80 mg of cloxacillin and extract with three quantities, each of 15 ml, of light petroleum ( 120 to 160) previously saturated with cloxacillin benzathine. Discard the extracts, wash the residue with ether previously saturated with cloxacillin benzathine. Dry in a current of air, dissolve in 25 ml of methanol and dilute to 50.0 ml with water. Dilute 2.0 ml to 100.0 ml with buffered cupric sulphate solution pH 2.0, transfer 10.0 ml to a stoppered test-tube and heat in a water-bath at 70 for 20 minutes. Cool to room temperature rapidly, dilute to 20.0 ml with ethanol and measure the absorbance of the resulting solution at the
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CLOXACILLIN SODIUM INTRAMAMMARY INFUSION (LACTATING COW/BUFFALO)
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maximum at about 338 nm (2.4.7), using as the blank 10.0 ml of the unheated buffered solution of the substance under examination after dilution to 20.0 ml with ethanol. Calculate the content of C19H18ClN3O5S in a container of average weight from the absorbance obtained by carrying out the procedure simultaneously using 2.0 ml of a solution prepared by dissolving 105 mg of cloxacillin benzathine RS in 50.0 ml of a mixture of equal volumes of methanol and water. Labelling. The label states the strength in terms of the equivalent amount of cloxacillin in the sealed container.
cloxacillin sodium. Discard the extracts, wash the residue with ether previously saturated with cloxacillin sodium. Dry in a current of air, dissolve in water and dilute to 50.0 ml with the same solvent. Dilute 2.0 ml to 100.0 ml with buffered cupric sulphate solution pH 2.0, transfer 10.0 ml to a stoppered test-tube and heat in a water-bath at 70 for 20 minutes. Cool to room temperature rapidly, dilute to 20.0 ml with ethanol and measure the absorbance of the resulting solution at the maximum at about 338 nm (2.4.7), using as the blank 10.0 ml of the unheated buffered solution of the substance under examination after dilution to 20.0 ml with ethanol. Calculate the content of C19H18ClN3O5S in a container of average content from the absorbance obtained by carrying out the procedure simultaneously using 2.0 ml of a solution prepared by dissolving 85 mg of cloxacillin sodium RS in 50.0 ml of water, diluting to 100 ml with buffered cupric sulphate solution pH 2.0, and beginning at the words transfer 10.0 ml...... Labelling. The label states the strength in terms of the equivalent amount of cloxacillin.
Cloxacillin Sodium Intramammary Infusion (Lactating Cow/Buffalo)

Cloxacillin Intramammary Injection; Cloxacillin Intramammary Infusion (Lactating Cow/Buffalo); Cloxacillin Intramammary Infusion (LC/B)
Cloxacillin Sodium Intramammary Infusion (Lactating Cow/ Buffalo) is a sterile suspension of Cloxacillin Sodium in a suitable non-aqueous vehicle containing suitable suspending and dispersing agents. Cloxacillin Sodium Intramammary Infusion (Lactating Cow/ Buffalo) contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of cloxacillin, C19H18ClN3O5S.
Dichlofenthion
H3 C H3 C S O P O O Cl Cl
Identification
Extract a quantity containing 75 mg of cloxacillin with three quantities, each of 15 ml, of light petroleum ( 120 to 160). Discard the extracts, wash the residue with 10 ml of ether and dry in a current of air. The residue complies with the following tests. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with cloxacillin sodium RS or with the reference spectrum of cloxacillin sodium. B. Gives reaction A of sodium salts (2.3.1).
C10H13Cl2O3
Mol. Wt. 315.2
Dichlofenthion is O-2,4-dichlorophenyl-O,O-diethyl phosphorothioate. Dichlofenthion contains not less than 95.0 per cent and not more than 100.5 per cent of C10H13Cl2O3PS. Description. A colourless or pale yellow, oily substance.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with dichlofenthion RS or with the reference spectrum of dichlofenthion. B. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 95 volumes of hexane and 5 volumes of 2-butanone. Test solution. Dissolve 0.5 g of the substance under examination in 100 ml of methanol. Reference solution. A 0.5 per cent w/v solution of dichlofenthion RS in methanol.
Tests
Water (2.3.43). Not more than 1.0 per cent, determined on 3 g using a mixture of 70 volumes of dichloromethane and 30 volumes of anhydrous methanol as the solvent. Other tests. Complies with the tests stated under Intramammary Infusions. Assay. Weigh and mix the contents of 10 containers. Weigh accurately a quantity of the mixed contents containing 80 mg of cloxacillin and extract with three quantities, each of 15 ml, of light petroleum ( 120 to 160) previously saturated with
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DICHLOROPHEN
Apply to the plate 2 l of each solution. After development, dry the plate in air and spray with a 2 per cent w/v solution of 4-(4-nitrobenzyl)pyridine in ethyl acetate. Heat the plate at 130 for 10 minutes, allow to cool and spray with a 2 per cent w/v solution of lithium hydroxide in a mixture of 8 volumes of methanol, 1 volume of diethylene glycol and 1 volume of water. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution. C. Burn 50 mg by the oxygen-flask method (2.3.34), using 20 ml of 1 M sodium hydroxide as the absorbing liquid. The solution obtained, after acidification with 2 M nitric acid gives reaction A of chlorides and reaction C of phosphates (2.3.1).
Dichlorophen contains not less than 97.0 per cent and not more than 101.0 per cent of C13H10Cl2O2, calculated on the dried basis. Description. A white or almost white powder; odour, slightly phenolic.
Identification
A. When examined in the range 220 nm to 360 nm (2.4.7), a 0.002 per cent w/v solution in 0.1 M sodium hydroxide shows absorption maxima at about 245 nm and 304 nm. The absorbances of the solution after further dilution with an equal volume of 0.1 M sodium hydroxide at these maxima are about 0.65 and 0.27 respectively. B. Dissolve 0.2 g in 10 ml of 2.5 M sodium hydroxide, cool in ice and add a solution prepared by mixing 1 ml of sodium nitrite solution with a cold solution containing 0.15 ml of aniline in a mixture of 4 ml of water and 1 ml of hydrochloric acid; a reddish-brown precipitate is produced. C. Fuse 0.5 g with 2 g of anhydrous sodium carbonate, cool, extract the residue with water and filter. The filtrate gives reaction A of chlorides (2.3.1). D. Melting point (2.4.21). about 175.
Tests
Refractive index (2.4.27). 1.530 to 1.533. Weight per ml (2.4.29). 1.296 to 1.316 g. Assay. Determine by gas chromatography (2.4.13). Test solution (a). Dissolve 0.3 g of the substance under examination in 100 ml of dichloromethane. Test solution (b). A solution containing 0.3 per cent w/v of the substance under examination and 0.2 per cent w/v of methyl stearate (internal standard) in dichloromethane. Reference solution. A solution containing 0.3 per cent w/w of dichlofenthion RS and 0.2 per cent w/v of methyl stearate (internal standard) in dichloromethane. Chromatographic system a glass column 1.5 m x 4 mm, packed with 3 per cent w/w of phenyl methyl silicone fluid (50 per cent phenyl) on acid-washed, silanised diatomaceous support (80 to 100 mesh) (such as OV-17), temperature: column 190, inlet port and detector. 280, flow rate. 30 ml per minute of the carrier gas. Calculate the content of C10H13Cl2O3PS.
Tests
Chlorides (2.3.12). Shake 3.0 g with 6 ml of ethanol (95 per cent), dilute with water to 100 ml, allow to stand for 5 minutes and filter. 25 ml of the filtrate complies with the limit test for chlorides (330 ppm). Sulphates (2.3.17). Shake 1.0 g with 20 ml of water for 2 minutes and filter. 5 ml of the filtrate complies with the limit test for sulphates (600 ppm). Related substances. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 100 mg of the substance under examination in 10 ml of the mobile phase. Reference solution (a). A 1.0 per cent w/v solution of dichlorophen impurity standard RS in the mobile phase. Reference solution (b). A 0.0010 per cent w/v solution of 4-chlorophenol in the mobile phase.
Dichlorophen
OH OH
Cl
Cl
C13H10Cl2O2 Mol. Wt. 269.1 Dichlorophen is 2,2-methylenebis(4-chlorophenol).
Chromatographic system a stainless steel column 20 cm x 5 mm, packed with octadecylsilane bonded to porous silica (10 m) (such as Spherisorb ODS 1), mobile phase: a filtered and degassed mixture of 75 volumes of methanol, 25 volumes of water and 1 volume of glacial acetic acid , flow rate. 1.5 ml per minute, spectrophotometer set at 280 nm,
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DICHLOROPHEN VETERINARY AEROSOL
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a 20 l loop injector. Inject the test solution and reference solution (b). In the chromatogram obtained with the test solution the area of the peak corresponding to 4-dichlorophenol is not more than the area of the principal peak in the chromatogram obtained with reeference solution (b). The content of 4,4-dichloro-2,2(2-hydroxy-4-chloro-m-xylene-a,a-diyl) diphenol in the substance under examination does not exceed 8.0 per cent w/ w and the sum of the contents of any other impurities, excluding 4-chlorophenol, is not more than 2.0 per cent w/w. Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 3 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.5 g, dissolve in 20 ml of 2-propanol. Titrate with 0.1 M tetrabutylammonium hydroxide, determining the end-point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M tetrabutylammonium hydroxide is equivalent to 0.02691 g of C13H10Cl2O2. Labelling. The label states that the substance is intended for animal treatment only.
cent w/v of potassium ferricyanide. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution.
Tests
Other tests. Complies with the tests stated under Veterinary Aerosols. Assay. Weigh the intact container. Place the container in an ice-bath for 15 minutes. Make a small hole about 1 cm from the top of the body of the container and let the propellant escape. When the flow of propellant stops, enlarge the hole. Transfer the contents of the container to a tared vessel capable of being fitted with a reflux condenser. Remove the top of the container carefully retaining all fragments. Wash the container with suitable solvents and add the washings to the tared vessel. Dry the container and reweigh to obtain the net weight of the contents. Heat the contents of the tared vessel under reflux for 30 minutes, cool and weigh (solution A). Dilute an accurately measured volume of the resulting solution containing about 0.25 g of Dichlorophen to 100.0 ml with acetone. Dilute 2.0 ml of this solution to 200.0 ml with ammonia buffer pH 10.9 and mix. To 10.0 ml of the resulting solution add 20 ml of ammonia buffer pH 10.9 and 2 ml of a freshly prepared 2 per cent w/v solution of 4-aminophenazone, mix, and add 2 ml of a freshly prepared 8 per cent w/v solution of potassium ferricyanide. Dilute to 50.0 ml with ammonia buffer pH 10.9 and allow to stand for 15 minutes. Measure the absorbance of the resulting solution at the maximum at about 510 nm (2.4.7), using as the blank a solution obtained in a similar manner by carrying out the procedure simultaneously, beginning at the words To 10.0 ml of the resulting solution..... but omitting the 4-aminophenazone solution. Calculate the weight of C13H10Cl2O2 in the container from the absorbance obtained by repeating the operation using a 0.25 per cent w/v solution of dichlorophen in acetone beginning at the words Dilute 2.0 ml...... Labelling. The label states (1) the weight of Dichlorophen present in the container; (2) the total weight of contents; (3) the name and proportion of any added dye.
Dichlorophen Veterinary Aerosol

Dichlorophen Veterinary Aerosol Spray; Dichlorophen Veterinary Spray
Dichlorophen Veterinary Aerosol is a solution of Dichlorophen in a suitable solvent to which suitable propellants have been added. It may contain a suitable dye as a marker. Dichlorophen Veterinary Aerosol contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of dichlorophen, C13H10Cl2O2.
Identification
Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 60 volumes of hexane and 40 volumes of acetone. Test solution. Solution A obtained in the Assay diluted with methanol to contain the equivalent of 1 per cent w/v of Dichlorophen. Reference solution. A 1 per cent w/v solution of dichlorophen RS in methanol. Apply to the plate 5 l of each solution. After development, dry the plate in air and spray with a freshly prepared solution containing 3.5 per cent w/v of ferric chloride and 0.25 per
Dichlorophen Tablets
Dichlorophen Tablets contain not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of dichlorophen, C13H10Cl2O2.
Identification
A. Shake a quantity of the powdered tablets containing 0.1 g of Dichlorophen with 50 ml of 0.1 M sodium hydroxide for 15
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DIETHYLCARBAMAZINE INJECTION
minutes, add sufficient 0.1 M sodium hydroxide to produce 100 ml, centrifuge and dilute a suitable volume of the supernatant liquid with 0.1 M sodium hydroxide to produce a solution containing 0.002 per cent w/v of Dichlorophen. When examined in the range 220 to 360 nm (2.4.7), the resulting solution shows absorption maxima at about 245 nm and 304 nm; absorbances at about 245 nm and 304 nm, about 1.3 and 0.54 respectively. B. Shake a quantity of the powdered tablets containing 0.2 g of Dichlorophen with a mixture of 5 ml of water and 5 ml of 5 M sodium hydroxide, filter, cool in ice and add a solution prepared by mixing 1 ml of sodium nitrite solution with a cold solution containing 0.15 ml of aniline in a mixture of 4 ml of water and 1 ml of hydrochloric acid; a reddish-brown precipitate is produced. C. Fuse a quantity of the powdered tablets containing 0.5 g of Dichlorophen with 2 g of anhydrous sodium carbonate, cool, extract the residue with water and filter. The filtrate gives reaction A of chlorides (2.3.1).
Other tests. Comply with the tests stated under Tablets. Assay. Weigh and powder 20 tablets. Weigh accurately a quantity of the powder containing 0.1 g of Dichlorophen, shake with 50 ml of 0.1 M sodium hydroxide for 15 minutes and add sufficient 0.1 M sodium hydroxide to produce 100.0 ml. Centrifuge and dilute 10.0 ml of the clear supernatant liquid to 100.0 ml with 0.1 M sodium hydroxide. Dilute 20.0 ml of this solution to 100.0 ml with 0.1 M sodium hydroxide and measure the absorbance of the resulting solution at the maximum at about 304 nm (2.4.7). Calculate the content of C13H10Cl2O2 taking 275 as the specific absorbance at 304 nm.
Diethylcarbamazine Injection
Diethylcarbamazine Citrate Injection.
Diethylcarbamazine Injection is a sterile solution of Diethylcarbamazine Citrate in Water for Injections. Diethylcarbamazine Injection contains not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of diethylcarbamazine citrate, C10H21N3O,C6H8O7.
Tests
Related substances. Determine by liquid chromatography (2.4.14). Test solution. Shake a quantity of the powdered tablets containing 0.50 g of Dichlorophen with 20 ml of methanol for 10 minutes, filter, add 7 ml of water and dilute to 50 ml with the mobile phase. Reference solution (a). A 1.0 per cent w/v solution of dichlorophen impurity standard RS in the mobile phase. Reference solution (b). A 0.0010 per cent w/v solution of 4-chlorophenol in the mobile phase. Chromatographic system a stainless steel column 20 cm x 5 mm, packed with octadecylsilane bonded to porous silica (10 m) (such as Spherisorb ODS 1), mobile phase: a filtered and degassed mixture of 75 volumes of methanol, 25 volumes of water and 1 volume of glacial acetic acid , flow rate. 1.5 ml per minute, spectrophotometer set at 280 nm, a 20 l loop injector. Inject the test solution and reference solution (b). In the chromatogram obtained with the test solution the area of the peak corresponding to 4-dichlorophenol is not more than the area of the principal peak in the chromatogram obtained with reeference solution (b). The content of 4,4-dichloro-2,2-(2-hydroxy-4-chloro-m-xylene-a,a-diyl) diphenol in the substance under examination does not exceed 8.0 per cent w/w and the sum of the contents of any other impurities, excluding 4-chlorophenol, is not more than 2.0 per cent w/w.
Identification
A. To a volume containing 0.5 g of Diethylcarbamazine Citrate add 2 ml of water and make alkaline with 5 M sodium hydroxide. Extract with four quantities, each of 5 ml, of dichloromethane, reserve the aqueous solution for test B, wash the combined dichloromethane extracts with water and remove the dichloromethane by evaporation. Add 0.5 ml of iodoethane to the residue and heat gently under a reflux condenser for 5 minutes. Remove the excess iodoethane with a current of air, dissolve the viscous yellow oil in 2 ml of ethanol (95 per cent) and add, with continuous stirring, sufficient ether to precipitate the quaternary ammonium salt. Decant off the ether, dissolve the residue in 2 ml of ethanol (95 per cent), reprecipitate with ether and dry at 105; the residue melts at about 152 (2.4.21). B. Neutralise the aqueous solution obtained in test A with 1 M sulphuric acid, add an excess of mercuric sulphate solution, boil and add a few drops of potassium permanganate solution; a white precipitate is produced.
Tests
pH (2.4.24). 6.0 to 7.0. N,N-Dimethylpiperazine and N-methylpiperazine. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 65 volumes of methanol, 30 volumes of 2-butanone and 5 volumes of strong ammonia solution.
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Test solution. Dilute a volume of the injection with sufficient methanol to produce a solution containing the equivalent of 5.0 per cent w/v of Diethylcarbamazine Citrate. Reference solution (a). A 5.0 per cent w/v solution of diethylcarbamazine citrate RS in methanol. Reference solution (b). A 0.010 per cent w/v solution of N,N-dimethylpiperazine in methanol. Reference solution (c). A 0.010 per cent w/v solution N-methylpiperazine in methanol. Apply to the plate 10 l of each solution. Allow the mobile phase to rise 12 cm. Dry the plate at 105 and expose it to iodine vapours for 30 minutes. Any spots corresponding to N,N-dimethylpiperazine and N- methylpiperazine in the chromatogram obtained with the test solution are not more intense than the spots in the chromatograms obtained with reference solutions (b) and (c) respectively. Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. To an accurately measured volume containing 8 g of Diethylcarbamazine Citrate add sufficient water to produce 100.0 ml. To 10.0 ml of this solution add 2 ml of 5 M sodium hydroxide and extract with four quantities, each of 25 ml, of dichloromethane. Wash each extract with the same two quantities, each of 20 ml, of water and with a third quantity if the second becomes alkaline to phenolphthalein solution. Extract the combined dichloromethane extracts in succession with 25.0 ml of 0.05 M sulphuric acid and 15 ml and 10 ml of water. Combine the acid and water extracts, remove the dichloromethane, by warming, cool and titrate the excess of acid with 0.1 M sodium hydroxide using bromocresol green solution as indicator. 1 ml of 0.05 M sulphuric acid is equivalent to 0.03914 g of C10H21N3O,C6H8O7. Storage. Store protected from light.
Dihydrostreptomycin Sulphate
NH NH H2N NH OH HN OH OH O O R' R OH OH O R' RHN O R = CH3 R = CH2OH , 3H2SO4 NH2
OH
C21H41N7O12)2,3H2SO4
Mol. Wt. 1461.4
Dihydrostreptomycin sulphate is O-2-deoxy-2-methylaminod-L-lyxofuranosyl-(14)-N1,N3-diamidino-D-streptamine sulphate. Dihydrostreptomycin Sulphate has a potency equivalent to not less than 730 Units per mg, calculated on the dried basis. Description. A white or almost white powder; may be hygroscopic.
Identification
A. Determine by thin-layer chromatography (2.4.17), coating the plate in the following manner. Mix 0.3 g of carbomer with 240 ml of water, allow to stand with moderate stirring for 1 hour, adjust to pH 7.0 by the gradual addition with constant shaking of 2 M sodium hydroxide and add 30 g of silica gel H. Spread a uniform layer of the resulting suspension 0.75 mm thick. Heat the plate at 110 for 1 hour, allow to cool and use immediately. Mobile phase. A 7 per cent w/v solution of potassium dihydrogen phosphate. Test solution. Dissolve 100 mg of the substance under examination in 100 ml of water. Reference solution (a). A 0.10 per cent w/v of dihydrostreptomycin sulphate RS in water. Reference solution (b). A solution containing 0.10 per cent w/v of dihydrostreptomycin sulphate RS, 0.10 per cent w/v of neomycin sulphate RS and 0.10 per cent w/v of kanamycin sulphate RS in water.
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DIHYDROSTREPTOMYCIN SULPHATE
Apply to the plate 10 l of each solution. Allow the mobile phase to rise 12 cm. Dry the plate in a current of warm air, spray it with a mixture of equal volumes of a 0.2 per cent w/v solution of naphthalene-1,3-diol in ethanol (95 per cent) and a 46 per cent w/v solution of sulphuric acid and heat at 150 for 5 to 10 minutes. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows three clearly separated spots. B. Dissolve 0.1 g in 2 ml of water and add 1 ml of dilute 1-naphthol solution and 2 ml of a mixture of equal volumes of sodium hypochlorite solution (3 per cent Cl) and water; a red colour is produced. C. Dissolve 10 mg in 5 ml of water and add 1 ml of 1 M hydrochloric acid. Heat in a water-bath for 2 minutes. Add 2 ml of a 0.5 per cent w/v solution of 1-naphthol in 1 M sodium hydroxide and heat in a water-bath for 1 minute; a violet-pink colour is produced (distinction from streptomycin). D. Gives the reactions of sulphates (2.3.1).
20 minutes after the addition of the ferric ammonium sulphate solution, measure the absorbance of a 2-cm layer at the maximum at about 525 nm (2.4.7), using as the blank a solution prepared in the same manner, omitting the substance under examination. The absorbance is not more than that obtained by carrying out the procedure simultaneously using 5.0 ml of a solution prepared by dissolving 10 mg, accurately weighed, of streptomycin sulphate RS in sufficient water to produce 50 ml and beginning at the words Add 5.0 ml...., both absorbances being calculated on the dried basis. Methanol. Determine by gas chromatography (2.4.13). Test solution. Dissolve 4.0 g of the substance under examination in 100 ml of water. Reference solution. A 0.008 per cent w/v of methanol. Chromatographic system a glass column 1.5 to 2.0 m x 2 to 4 mm, packed with ethylvinylbenzene-divinylbenzene copolymer (150 to 180 mm) porous polymer beads (such as Porapak Q), temperature: column 50, inlet port and detector. 280, flow rate. 30 to 40 ml per minute of the carrier gas. The area of any peak corresponding to methanol in the chromatogram obtained with test solution is not more than that of the peak in the chromatogram obtained with reference solution (0.2 per cent). Sulphated ash (2.3.18). Not more than 1.0 per cent. Loss on drying (2.4.19). Not more than 5 per cent, determined on 1 g by drying over phosphorus pentoxide at 60 at a pressure not exceeding 0.1 kPa for 4 hours. Assay. Carry out the microbiological assay of antibiotics, Method A or B (2.2.10), and express the result in Units of dihydrostreptomycin per mg. Dihydrostreptomycin Sulphate intended for use in the manufacture of parenteral preparations without a further appropriate procedure for the removal of bacterial endotoxins complies with the following additional requirement. Bacterial endotoxins (2.2.3). Not more than 0.5 Endotoxin Unit per mg of dihydrostreptomycin sulphate. Dihydrostreptomycin Sulphate intended for use in the manufacture of parenteral preparations without a further appropriate sterilization procedure complies with the following additional requirement. Sterility (2.2.11). Complies with the test for sterility. Storage. Store protected from light, at a temperature not exceeding 30. If it is intended for use in the manufacture of parenteral preparations or intramammary infusions, the
Tests
Appearance of solution. A 25 per cent w/v solution in carbon dioxide-free water is not more intensely coloured than degree 4 of the appropriate range of reference solutions (2.4.1). The solution, after standing protected from light at a temperature of about 20 for 24 hours, is not more opalescent than opalescence standard OS2 (2.4.1). pH (2.4.24). 5.0 to 7.0, determined in a 25 per cent w/v solution. Specific optical rotation (2.4.22). 83 to 91, calculated on the dried basis, determined in a 2 per cent w/v solution in water. Sulphate. 18.0 per cent to 21.5 per cent, calculated on the dried basis. Dissolve 0.25 g in 100 ml of water, adjust the pH to 11 with strong ammonia solution and add 10.0 ml of 0.1 M barium chloride and 0.5 mg of metalphthalein. Titrate the excess of barium chloride with 0.1 M disodium edetate, adding 50 ml of ethanol (95 per cent) when the colour of the solution begins to change and continuing the titration until the violet-blue colour disappears. 1 ml of 0.1 M barium chloride is equivalent to 0.009606 g of sulphate, SO4. Streptomycin. Weigh accurately about 0.10 g and dissolve in sufficient water to produce 5.0 ml. Add 5.0 ml of 0.2 M sodium hydroxide and heat for exactly 10 minutes in a water-bath. Cool in ice for exactly 5 minutes, add 3 ml of a 1.5 per cent w/ v solution of ferric ammonium sulphate in 0.25 M sulphuric acid and sufficient water to produce 25.0 ml, and mix. Exactly
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IP 2007
container should be sterile and sealed so as to exclude micro-organisms. Labelling. The label states (1) the number of Units per mg; (2) the name and quantity of any added stabiliser; (3) whether or not the contents are intended for use in the manufacture of Parenteral Preparations or intramammary infusions; (4) that the substance is meant for veterinary use only; (5) the storage conditions; (6) the date after which the contents are not intended to be used.
neomycin sulphate RS and 0.10 per cent w/v of kanamycin sulphate RS in water. Apply to the plate 10 l of each solution. Allow the mobile phase to rise 12 cm. Dry the plate in a current of warm air, spray it with a mixture of equal volumes of a 0.2 per cent w/v solution of naphthalene-1,3-diol in ethanol (95 per cent) and a 46 per cent w/v solution of sulphuric acid and heat at 150 for 5 to 10 minutes. The principal spot in the chromatogram obtained with test solution corresponds to that in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows three clearly separated spots. B. Dissolve 0.1 g in 2 ml of water and add 1 ml of dilute 1-naphthol solution and 2 ml of a mixture of equal volumes of sodium hypochlorite solution (3 per cent Cl) and water; a red colour is produced. C. Dissolve 10 mg in 5 ml of water and add 1 ml of 1 M hydrochloric acid. Heat in a water-bath for 2 minutes. Add 2 ml of a 0.5 per cent w/v solution of 1-naphthol in 1 M sodium hydroxide and heat in a water-bath for 1 minute; a violet-pink colour is produced (distinction from streptomycin). D. Gives the reactions of sulphates (2.3.1).
Dihydrostreptomycin Injection
Dihydrostreptomycin Sulphate Injection.
Dihydrostreptomycin Injection is a sterile solution of Dihydrostreptomycin Sulphate in Water for Injections. It is prepared by dissolving the contents of a sealed container in the requisite amount of Water for Injections immediately before use. Dihydrostreptomycin Injection contains not less than 90.0 per cent and not more than 115.0 per cent of the stated amount of dihydrostreptomycin, C21H41N7O12, calculated on the dried basis. Description. A white or almost white powder which yields a clear, colourless or faintly yellow solution when dissolved in water. The injection complies with the tests stated under Parenteral Preparations (Powders for Injection). The contents of the sealed container comply with the following requirements.
Tests
Appearance of solution. A 25 per cent w/v solution in carbon dioxide-free water is not more intensely coloured than degree 4 of the appropriate range of reference solutions (2.4.1). The solution, after standing protected from light at a temperature of about 20 for 24 hours, is not more opalescent than opalescence standard OS2 (2.4.1). pH (2.4.24). 5.0 to 7.0, determined in a 25 per cent w/v solution. Specific optical rotation (2.4.22). 83 to 91, calculated on the dried basis, determined in a 2 per cent w/v solution in water. Sulphate. 18.0 per cent to 21.5 per cent, calculated on the dried basis. Dissolve 0.25 g in 100 ml of water, adjust the pH to 11 with strong ammonia solution and add 10.0 ml of 0.1 M barium chloride and 0.5 mg of metalphthalein. Titrate the excess of barium chloride with 0.1 M disodium edetate, adding 50 ml of ethanol (95 per cent) when the colour of the solution begins to change and continuing the titration until the violet-blue colour disappears. 1 ml of 0.1 M barium chloride is equivalent to 0.009606 g of sulphate, SO4. Streptomycin. Weigh accurately about 0.10 g and dissolve in sufficient water to produce 5.0 ml. Add 5.0 ml of 0.2 M sodium hydroxide and heat for exactly 10 minutes in a water-bath.
Identification
A. Determine by thin-layer chromatography (2.4.17), prepared by mixing 0.3 g of carbomer with 240 ml of water, allow to stand with moderate stirring for 1 hour, adjust to pH 7.0 by the gradual addition with constant shaking of 2 M sodium hydroxide and add 30 g of silica gel H. Spread a uniform layer of the resulting suspension 0.75 mm thick. Heat the plate at 110 for 1 hour, allow to cool and use immediately. Mobile phase. A 7 per cent w/v solution of potassium dihydrogen phosphate. Test solution. Dissolve 100 mg of the sunbstance under examination in 100 ml of water. Reference solution (a). A 0.10 per cent w/v of dihydrostreptomycin sulphate RS in water. Reference solution (b). A solution containing 0.10 per cent w/ v of dihydrostreptomycin sulphate RS, 0.10 per cent w/v of
1530
IP 2007
DIMETRIDAZOLE
Cool in ice for exactly 5 minutes, add 3 ml of a 1.5 per cent w/ v solution of ferric ammonium sulphate in 0.25 M sulphuric acid and sufficient water to produce 25.0 ml, and mix. Exactly 20 minutes after the addition of the ferric ammonium sulphate solution, measure the absorbance of a 2-cm layer at the maximum at about 525 nm (2.4.7), using as the blank a solution prepared in the same manner, omitting the substance under examination. The absorbance is not more than that obtained by carrying out the procedure simultaneously using 5.0 ml of a solution prepared by dissolving 10 mg, accurately weighed, of streptomycin sulphate RS in sufficient water to produce 50 ml and beginning at the words Add 5.0 ml...., both absorbances being calculated on the dried basis. Methanol. Determine by gas chromatography (2.4.13). Test solution. Dissolve 4.0 g of the substance under examination in 100 ml of water. Reference solution. A 0.008 per cent w/v of methanol. Chromatographic system a glass column 1.5 to 2.0 m x 2 to 4 mm, packed with ethylvinylbenzene-divinylbenzene copolymer (150 to 180 mm) porous polymer beads (such as Porapak Q), temperature: column 50, inlet port and detector. 280, flow rate. 30 to 40 ml per minute of the carrier gas. The area of any peak corresponding to methanol in the chromatogram obtained with test solution is not more than that of the peak in the chromatogram obtained with reference solution (0.2 per cent). Sulphated ash (2.3.18). Not more than 1.0 per cent. Loss on drying (2.4.19). Not more than 5 per cent, determined on 1 g by drying over phosphorus pentoxide at 60 at a pressure not exceeding 0.1 kPa for 4 hours. Assay. On the mixed contents of ten containers carry out the microbiological assay, Method A or B (2.2.10), and express the result in Units of dihydrostreptomycin per mg. Dihydrostreptomycin Sulphate intended for use in the manufacture of Parenteral Preparations without a further appropriate procedure for the removal of bacterial endotoxins complies with the following additional requirement. Bacterial endotoxins (2.2.3). Not more than 0.5 Endotoxin Unit per mg of dihydrostreptomycin sulphate. Dihydrostreptomycin Sulphate intended for use in the manufacture of Parenteral Preparations without a further appropriate sterilization procedure complies with the following additional requirement. Sterility (2.2.11). Complies with the test for sterility.
Storage. Store protected from light. Use the injection within 7 days of the preparation of the solution when stored in a cool place or within 1 month when stored in a cold place. Labelling. The label states (1) the strength in terms of the equivalent amount of dihydrostreptomycin in a suitable dose-volume; (2) that the contents are meant for veterinary use only; (3) the storage conditions; (4) the date after which the contents are not intended to be used.
Dimetridazole
CH3 N N
C5H7N3O2 Mol. Wt. 141.1
O2N
CH3
Dimetridazole is 1,2-dimethyl-5-nitro-1H-imidazole. Dimetridazole contains not less than 98.0 per cent and not more than 101.0 per cent of the stated amount of dimetridazole, C5H7N3O2, calculated on the anhydrous basis. Description. An almost white to brownish yellow powder; darkens on exposure to light, odourless or almost odourless.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with dimetridazole RS or with the reference spectrum of dimetridazole. B. When examined in the range of 230 to 360 nm (2.4.7), a 0.002 per cent w/v solution in methanol shows a well-defined absorption maximum only at about 309 nm; absorbance at about 309 nm, about 1.3. C. Dissolve 0.1 g in 20 ml of ether, add 10 ml of a 1 per cent w/ v solution of picric acid in ether, induce crystallisation by scratching the sides of the vessel and allow to stand. Wash the precipitate obtained with ether and dry at 105; the residue melts at about 160 (2.4.21).
Tests
2-Methyl-5-nitroimidazole. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 90 volumes of dichloromethane and 10 volumes of 2-propanol. Test solution. Dissolve 2 g of the substance under examination in 100 ml of dichloromethane.
1531
DIMETRIDAZOLE PREMIX
IP 2007
Reference solution. A 0.010 per cent w/v of 2-methyl5-nitroimidazole RS in dichloromethane. Apply to the plate 5 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. Any spot corresponding to 2-methyl-5-nitroimidazole in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Sulphated ash (2.3.18). Not more than 0.1 per cent. Water (2.3.43). Not more than 1.0 per cent, determined on 1 g. Assay. Weigh accurately about 0.3 g, dissolve in 30 ml of anhydrous glacial acetic acid. Titrate with 0.1 M perchloric acid, using crystal violet solution as indicator. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.01411 g of C5H7N3O2. Storage. Store protected from light.
Dimetridazole Veterinary Oral Powder

Dimetridazole Veterinary Oral Powder is a mixture of Dimetridazole and a suitable water-soluble diluent. Dimetridazole Veterinary Oral Powder contains not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of dimetridazole, C5H7N3O2.
Identification
Mix a quantity containing 0.1 g of Dimetridazole with 20 ml of ether, shake and filter. To the filtrate add 10 ml of a 1 per cent w/v solution of picric acid in ether, stir to induce crystallisation and allow to stand. Wash the precipitate obtained with ether and dry at 105; the residue melts at about 160 (2.4.21).
Tests
Other tests. Complies with the tests stated under Veterinary Oral Powders. Assay. Weigh accurately a quantity containing 0.4 g of Dimetridazole, transfer to a sintered glass funnel (porosity No. 4), add 10 ml of dichloromethane, stir for 1 minute, and apply gentle suction. Repeat the extraction with four further quantities, each of 10 ml, of dichloromethane. To the combined dichloromethane extracts add 50 ml of anhydrous glacial acetic acid previously neutralised to crystal violet solution by the dropwise addition of 0.1 M perchloric acid. Titrate with 0.1 M perchloric acid, using crystal violet solution as indicator. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.01411 g of C5H7N3O2. Storage. Store protected from light.
Dimetridazole Premix
Dimetridazole Premix contains not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of dimetridazole, C5H7N3O2.
Identification
Mix a quantity containing 0.1 g of Dimetridazole with 20 ml of ether, shake and filter. To the filtrate add 10 ml of a 1 per cent w/v solution of picric acid in ether, stir to induce crystallisation and allow to stand. Wash the precipitate obtained with ether and dry at 105; the residue melts at about 160 (2.4.21).
Tests
Other tests. Complies with the tests stated under Premixes. Assay. Weigh accurately a quantity containing 0.45 g of Dimetridazole, transfer to a sintered glass funnel (porosity No. 4), add 10 ml of dichloromethane, stir for 1 minute, and apply gentle suction. Repeat the extraction with four further quantities, each of 10 ml, of dichloromethane. To the combined dichloromethane extracts add 50 ml of anhydrous glacial acetic acid previously neutralised to crystal violet solution by the dropwise addition of 0.1 M perchloric acid. Titrate with 0.1 M perchloric acid, using crystal violet solution as indicator. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.01411 g of C5H7N3O2. Storage. Store protected from light.
Dinitolmide
O NH2 CH3 O2N NO2
C8H7N3O5 Dinitolmide is 3,5-dinitro-2-methylbenzamide.
Mol. Wt. 225.2
Dinitolmide contains not less than 98.0 per cent and not more than 100.5 per cent of C8H7N3O5, calculated on the dried basis. Description. A cream to light tan powder.
1532
IP 2007
ETHOPABATE
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with dinitolmide RS or with the reference spectrum of dinitolmide. B. Heat 1 g with 20 ml of 9 M sulphuric acid under a reflux condenser for 1 hour, cool, add 50 ml of water and filter. The residue after washing with water and drying at 105 melts at about 205 (2.4.21).
1 ml of 0.1 M titanium trichloride is equivalent to 0.001876 g of C8H7N3O5.
Ethopabate
O O H3C
C12H15NO4
OCH3 N H O CH3
Mol. Wt. 237.3
Tests
Acid value (2.3.23). Not more than 5.0, determined on 0.5 g and using 50 ml of ethanol (95 per cent) as the solvent. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 85 volumes of dichloromethane, 10 volumes of methanol and 5 volumes of glacial acetic acid. Test solution. Dissolve 2.5 g of the substance under examination in 100 ml of acetone. Reference solution (a). A 0.0125 per cent w/v of the substance under examination in acetone. Reference solution (b). A 0.0125 per cent w/v of o-toluic acid in acetone. Apply to the plate 10 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. Spray with titanium trichloride solution, diluted 5 times with water, heat at 100 for 5 minutes and spray with ethanolic dimethylaminobenzaldehyde solution. When viewed under ultraviolet light at 354 nm the spot in the chromatogram obtained with reference solution (b) is more intense than any corresponding spot in the chromatogram obtained with the test solution. By both methods of visualisation any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (a). Loss on drying (2.4.19). Not more than 1.0 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.15 g, dissolve in acetone and dilute to 50.0 ml. To 10.0 ml of the solution add 10 ml of glacial acetic acid and 15 ml of a 40 per cent w/v solution of sodium acetate. Maintain a stream of carbon dioxide through the flask throughout the determination. Add 25.0 ml of 0.1 M titanium trichloride and allow to stand for 5 minutes. Add 10 ml of hydrochloric acid, 10 ml of water and 1 ml of potassium thiocyanate solution. Titrate with 0.1 M ferric ammonium sulphate until the solution becomes first colourless and then orange. Repeat the operation without the substance under examination. The difference between the titrations represents the amount of titanium trichloride required.
Ethopabate is methyl 4-acetamide-2-ethoxybenzoate. Ethopabate contains not less than 96.0 per cent and not more than 104.0 per cent of ethopabate, C12H15NO4, calculated on the dried basis. Description. A white or pinkish white powder, odourless or almost odourless.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with ethopabate RS or with the reference spectrum of ethopabate. B. When examined in the range 230 to 360 nm (2.4.7), a 0.0016 per cent w/v solution in methanol shows absorption maxima at about 268 nm and at about 299 nm; absorbance at about 268 nm, about 1.3 and at about 299 nm, about 0.58. C. Melts at about 148 (2.4.21).
Tests
Diazotisable substances. Dissolve 0.2 g in 10 ml of dichloromethane and extract in succession with 100 ml and 90 ml of 0.1 M hydrochloric acid, combine the acid extracts, wash with 5 ml of dichloromethane, dilute to 200 ml with 0.1 M hydrochloric acid and filter. To 5 ml, add 6 ml of 1 M hydrochloric acid and 1 ml of a 0.1 per cent w/v solution of sodium nitrite, mix, and allow to stand for 4 minutes. Add 1 ml of a 0.5 per cent w/v solution of ammonium sulphamate, mix and allow to stand for 3 minutes. Add 1.0 ml of a 0.1 per cent w/v solution of N-(1-naphthyl)ethylenediamine dihydrochloride, mix, and allow to stand for 30 minutes. Absorbance of the resulting solution at about 545 nm (2.4.7), not more than 0.70. Phenolic substances. Dissolve 0.25 g in 15 ml of methanol and add sufficient methanol to produce 25 ml. To 5 ml add 5 ml of a 3 per cent w/v solution of anhydrous ferric chloride, mix and allow to stand for 10 minutes. Absorbance of the resulting solution at about 525 nm (2.4.7), not more than 0.70,
1533
FURAZOLIDONE VETERINARY ORAL SUSPENSION
IP 2007
using as the blank a solution prepared by adding 5 ml of a 3 per cent w/v solution of anhydrous ferric chloride to 5 ml of methanol. Sulphated ash (2.3.18). Not more than 0.5 per cent. Loss on drying (2.4.19). Not more than 1.0 per cent, determined on 1.0 g, by drying in an oven at 105 at a pressure not exceeding 0.7 kPa. Assay. Weigh accurately about 75 mg and dissolve in sufficient methanol to produce 250.0 ml. Dilute 10.0 ml to 100.0 ml with water, transfer 10.0 ml of this solution to an evaporating dish, add 10 ml of 1 M sodium hydroxide and evaporate to dryness on a water-bath. Moisten the dish with 10 ml of water, evaporate to dryness, again moisten with 10 ml of water and heat on a water-bath for 15 minutes. Add 20 ml of 1 M hydrochloric acid, transfer to a flask and add sufficient water to produce 100.0 ml. To 25.0 ml add 5.0 ml of 1 M hydrochloric acid and 5.0 ml of a 0.1 per cent w/v solution of sodium nitrite, mix and allow to stand for 2 minutes. Add 5.0 ml of 0.5 per cent w/v solution of ammonium sulphamate, mix and allow to stand for 2 minutes. Add 5.0 ml of a 0.1 per cent w/v solution of N-(1-naphthyl)ethylenediamine dihydrochloride, mix and allow to stand for 10 minutes. Add sufficient water to produce 50.0 ml and measure the absorbance of the resulting solution at the maximum at about 545 nm (2.4.7). Calculate the content of C12H15NO4 from the absorbance obtained by carrying out the procedure simultaneously, using ethopabate RS in place of the substance under examination.
Test solution. Shake a quantity of the suspension containing 5 mg of Furazolidone with 1 ml of acetone, allow to stand, and use the supernatant liquid. Reference solution. A 0.5 per cent w/v solution of furazolidone RS in acetone. Apply to the plate 10 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution.
Tests
Other tests. Complies with the tests stated under Veterinary Oral Liquids. Assay. Protect the solutions from light throughout the assay. Weigh accurately a quantity of the well-shaken suspension containing 35 mg of Furazolidone, add slowly and with stirring, 50 ml of dimethylformamide. Warm on a water-bath, with occasional stirring, until most of the solid is dissolved. Decant the supernatant liquid and extract the residue further with two quantities, each of 50 ml, of dimethylformamide, decanting the supernatant solution. No yellow colour should be visible in the third extract. Cool the combined dimethylformamide extracts, add sufficient water to produce 500.0 ml and filter. To 10.0 ml of the filtrate add sufficient water to produce 100.0 ml and measure the absorbance of the resulting solution at the maximum at about 367 nm (2.4.7). Calculate the content of C8H7N3O5 taking 754 as the specific absorbance at 367 nm. Determine the weight per ml of the suspension (2.4.29), and calculate the content of furazolidone, weight in volume. Storage. Store protected from light. Labelling. The label states that the oral suspension should be administered undiluted.
Furazolidone Veterinary Oral Suspension

Furazolidone Veterinary Mixture; Furazolidone Mixture; Furazolidone Drench
Furazolidone Veterinary Oral Suspension is an aqueous suspension of Furazolidone. Furazolidone Veterinary Oral Suspension contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of furazolidone, C8H7N3O5.
Furazolidone Premix
Furazolidone Premix contains not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of furazolidone, C8H7N3O5.
Identification
A. Add 0.2 ml to a mixture of 15 ml of dimethylformamide and 1 ml of 0.5 M ethanolic potassium hydroxide; a blue colour is produced. B. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 50 volumes of dichloromethane and 40 volumes of nitromethane and 10 volumes of methanol.
Identification
A. To a mixture of 15 ml of dimethylformamide and 1 ml of 0.5 M ethanolic potassium hydroxide add 5 mg of the premix; a blue colour is produced. B. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G.
1534
IP 2007
HALOXON
Mobile phase. A mixture of 50 volumes of dichloromethane and 40 volumes of nitromethane and 10 volumes of methanol. Test solution. The supernatant liquid obtained by shaking a quantity of the premix containing 5 mg of furazolidone with 1 ml of acetone. Reference solution. A 0.5 per cent w/v solution of furazolidone RS in acetone. Apply to the plate 10 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution.
When examined in the range 230 to 360 nm (2.4.7), the resulting solution exhibits a maximum at about 290 nm and a less well defined maximum at about 312 nm. Ratio of the absorbance at about 312 nm to that at about 290 nm, about 1.08. B. Dissolve 0.1 g in 5 ml of 5 M sodium hydroxide with the aid of warming, cool, acidify 1 ml of the solution by the addition of 2 M nitiric acid and add 1 ml of silver nitrate solution, a white precipitate is formed. The precipitate is soluble in 5 M ammonia giving a brown solution which exhibits a green fluorescence when viewed under screened ultraviolet light. C. Melting range (2.4.21). 88 to 93.
Tests
Acidity. Dissolve 0.1 g in 10 ml of ethanol (95 per cent) previously neutralised to methyl red solution; the solution requires for neutralisation not more than 0.1 ml of 0.1 M sodium hydroxide. 3-Chloro-4-methylumbelliferone. Not more than 2.0 per cent. NOTE Prepare the solutions immediately before use and protected from light. Dissolve 0.20 g in 50 ml of 0.01 M methanolic hydrochloric acid and dilute 5 ml to 100 ml with 0.01 M methanolic hydrochloric acid. Measure the fluorescence of the resulting solution (2.4.5), using an excitation wavelength of about 345 nm and an emission wavelength of about 400 nm and setting the spectrofluorimeter to zero with 0.01 M methanolic hydrochloric acid and to 100 with a standard solution prepared by dissolving 25 mg of 3-chloro-4-methylumbelliferone RS in sufficient 0.01 M methanolic hydrochloric acid to produce 250 ml (solution A) and diluting 5 ml to 100 ml with 0.01 M methanolic hydrochloric acid. Calculate the content of 3-chloro-4-methylumbelliferone from a calibration curve prepared by measuring the fluorescence of suitable dilutions of solution A. Loss on drying (2.4.19). Not more than 1.0 per cent, determined on 1.0 g by drying in an oven at 80 at a pressure not exceeding 0.7 kPa. Assay. Weigh accurately about 0.25 g and dissolve in sufficient acetonitrile to produce 10 ml and record the infrared absorption of a 0.2 mm layer of the solution at the maximum at about 1155 cm 1(2.4.6). Construct a base line between the minima at about 1125 cm 1 and 1180 cm 1. Calculate the content of C14H14Cl3O6P from the absorption obtained by repeating the procedure using haloxon RS in place of the substance under examination. Storage. Avoid contact with metals.
Tests
Other tests. Complies with the tests stated under Premixes. Assay. Protect the solutions from light throughout the assay. Weigh accurately a quantity of the premix containing 35 mg of Furazolidone, add 50 ml of dimethylformamide and shake for 20 minutes. Add sufficient water to produce 500.0 ml and filter. To 10.0 ml of the filtrate add sufficient water to produce 100.0 ml and measure the absorbance of the resulting solution at the maximum at about 367 nm (2.4.7). Calculate the content of C8H7N3O5 taking 754 as the specific absorbance at 367 nm. Determine the weight per ml, (2.4.29), and calculate the content of furazolidone, weight in volume. Storage. Store protected from light.
Haloxon
Cl Cl O P O O O O O Cl CH3
C14H14Cl3O6P
Mol. Wt. 415.6
Haloxone is phosphoric acid bis(2-chloroethyl) 3-chloro-4methyl-2-oxo-2H-1-benzopyran-7-yl ester. Haloxon contains not less than 95.0 per cent and not more than 100.5 per cent of C14H14Cl3O6P, calculated on the dried basis. Description. A white or almost white powder.
Identification
A. Dissolve about 20 mg in 10 ml of dioxan, add 0.5 ml of 0.1 M hydrochloric acid and dilute to 25 ml with methanol. Dilute 1 ml to 25 ml with methanol.
1535
IVERMECTION
IP 2007
Ivermectin
OCH3 HO O H3C O H H3 C O O H3C O OH Component B1a R = CH2CH3 Component B1b R = CH3 O H OH O OCH3 CH3 H CH3 O O H CH3 R
Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 15 volumes of water, 34 volumes of methanol and 51 volumes of acetonitrile, flow rate. 1 ml per minute, spectrophotometer set at 254 nm, a 20 l loop injector. Inject reference solution (a). This test is not valid unless resolution between the component H2B1b (first peak) and component H2B1b (second peak) is not less than 3.0. Inject the test solution and reference solution (b). In the chromatogram obtained with the test solution the impurity with a relative retention of 1.3 to 1.5 with reference to the principal peak is not more than 2.5 times the area of the principal peak in the chromatogram obtained with reference solution (b) (2.5 per cent). The area of any other peak is not more than the area of the principal peak in the chromatogram obtained with reference solution (b) (1.0 per cent) and the sum of all the secondary peaks is not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (b) (5.0 per cent). Ethanol and formamide. Determine by gas chromatography (2.4.13). Internal standard solution. Dilute 0.5 ml of propanol to 100 ml with water. Test solution. Dissolve 0.120 g of the substance under examination in 2.0 ml of m-xylene by heating on a water-bath at 40 to 50, add 2.0 ml of water, mix thoroughly and centrifuge. Remove the upper layer and extract it with 2.0 ml of water. Discard the upper layer and combine the aqueous layers. Add 1.0 ml of the internal standard solution. Centrifuge and discard any remaining m-xylene. Reference solution (a). Dilute 3.0 g of ethanol to 100 ml with water. Reference solution (b). Dilute 1.0 g of formamide to 100 ml with water. Reference solution (c). Dilute 5.0 ml of reference solution (a) and 5 ml of reference solution (b) to 50.0 ml with water. Transfer 2.0 ml of this solution to a centrifuge tube, add 2 ml of mxylene, mix thoroughly and centrifuge. Remove the upper layer and extract it with 2.0 ml of water. Discard the upper layer and combine the aqueous layers. Add 1.0 ml of the internal standard solution. Centrifuge and discard any remaining m-xylene. Reference solution (d). Dilute 10.0 ml of reference solution (a) and 10.0 ml of reference solution (b) to 50.0 ml with water. Transfer 2.0 ml of this solution to a centrifuge tube, add 2 ml of m-xylene, mix thoroughly and centrifuge. Remove the upper layer and extract it with 2.0 ml of water. Discard the upper layer and combine the aqueous layers. Add 1.0 ml of the internal
CH3
C48 H74O14, H2B1a C47 H72O14, H2B1b
Mol.Wt. 875.0 Mol.Wt. 861.0
Ivermectin contains not less than 95.0 per cent and not more than 102.0 per cent of H2B1a + H2B1b, calculated on the dried basis. The ratio H 2B1a/(H 2B1a + H2B1b), determined by liquid chromatography is not less than 90.0 per cent. Description. A white crystalline powder, slightly hygroscopic.
Identification
A. Determine by infrared absorption spectrophotometery (2.4.6). Compare the spectrum with that obtained with ivermectin RS. B. In the Assay, the principal peak in the chromatogram obtained with the test solution corresponds to the peak in the chromatogram obtained with reference solution (a).
Tests
Appearance of solution. A 2.0 per cent w/v solution in toluene is clear (2.4.1) and not more intensely colored than reference solution BY57 (2.4.1). Specific optical rotation (2.4.22) -17.0 to -20.0, determined on a 2.5 per cent w/v solution in methanol. Related substances. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 40 mg of the substance under examination in 50 ml of methanol. Reference solution (a). A 0.08 per cent w/v solution of ivermectin RS in methanol. Reference solution (b). Dilute 1.0 ml of reference solution (a) to 100 ml with methanol. Reference solution (c). Dilute 5 ml of reference solution (b) to 100 ml with methanol.
1536
IP 2007
KAOLIN VETERINARY ORAL SUSPENSION
standard solution. Centrifuge and discard any remaining mxylene. Chromatographic system a glass column 30 m x 0.53 mm, packed with fused silica with macrogol 20,000 with film thickness 1 mm, temperature column 80 increase @ 60 per minute to 240, injection port 220 and detector 280, flow rate. 7.5 ml per minute of helium as carrier gas. Inject 1 l of the test solution and reference solutions (c) and (d). Calculate the content of ethanol is not more than 5 per cent and formamide not more than 1 per cent. Heavy metals (2.3.13). 1 g complies with the limit test for heavy metals, Method C (20 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Water (2.3.43). Not more 0.1 per cent, determined on 0.5 gm. Assay. Determine by liquid chromatography (2.4.14), as described under Related substances. Inject the test solution and reference solution (a). Calculate the percentage contents of ivermectin (H2B1a + H2B1b) and the ratio H2B1a/(H2B1a + H2B1b). Storage. Store protected from light.
Assay. Determine by liquid chromatography (2.4.14). Test solution. Dilute accurately a volume of the injection containing 5 mg of Ivermectin to 100 ml with water. Reference solution. A 0.005 per cent w/v solution of ivermectin RS in water. Chromatographic system a stainless steel column 25 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m), mobile phase: a mixture of 9 volumes of methanol and 1 volume of water, flow rate. 1 ml per minute, spectrophotometer set at 245 nm, a 20 l loop injector. Inject the reference solution. The test is not valid unless the tailing factor is not less than 2.0 Inject alternatively the test solution and the reference solution. Calculate the content of ivermectin in the injection. Storage. Store protected from light. Labelling. The label states (1) the strength in mg of Ivermectin per ml; (2) that the contents are to be used for subcutaneous use only; (3) the names of any preservatives used.
Kaolin Veterinary Oral Suspension

Kaolin Veterinary Mixture; Kaolin Mixture
Light Kaolin Light Magnesium Carbonate Sodium Bicarbonate Water to produce 200 g 50 g 50 g 1000 ml
Ivermectin Injection
Ivermectin Injection is a sterile solution of Ivermectin with or with out one or more anaesthetics, preservatives and solvents. Ivermectin Injection contains not less than 90 per cent and not more than 110 per cent of H2B1a, and not more than 5 per cent of H2B1b. The content of H2B1a + H2B1b is not less than 95 per cent and not more than 110 per cent of the stated amount of Ivermectin. Description. A clear, colourless to yellow colour solution.
Kaolin Veterinary Oral Suspension should be freshly prepared, unless the Light Kaolin has been sterilised. Kaolin Veterinary Oral Suspension contains not less than 1.04 per cent w/w and not more than 1.25 per cent w/w of the stated amount of magnesium, Mg and not less than 4.05 per cent w/ w and not more than 4.65 per cent w/w of the stated amount of sodium bicarbonate, NaHCO3.
Identification
When examined in the range 220 nm to 360 nm (2.4.7), a 0.001 per cent w/v solution in methanol shows an absorption maximum at about 245 nm.
Tests
Acid-insoluble matter. 13.8 to 18.4 per cent w/w, determined by the following method. Weigh accurately about 3 g, add 15 ml of water and make acid to litmus paper by the cautious addition of 2 M hydrochloric acid; boil for 5 minutes, replacing water lost by evaporation, cool and decant the supernatant layer through a filter. Boil the residue with 20 ml of water and 10 ml of 2 M hydrochloric acid, cool, filter through the same filter, and wash the residue with water until the washings are
Tests
pH (2.4.24). 5.5 to 7.0. Pyrogens (2.2.8). Complies with the test for pyrogens, by injecting 0.2 mg of Ivermectin per kg body weight of rabbit. Other tests. Complies with the tests stated under Parenteral Preparations (Injections).
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LEVAMISOLE INJECTION
IP 2007
free from chloride, reserving the filtrate and washings for the Assay for magnesium. Dry and ignite the residue to constant weight at red heat. Other tests. Complies with the tests stated under Veterinary Oral Liquids. Assay. For magnesium Dilute the combined filtrate and washings reserved in the determination of acid-insoluble matter to 100.0 ml with water. To 20.0 ml add 0.1 g of ascorbic acid, make slightly alkaline to litmus paper with 5 M ammonia and add 10 ml of triethanolamine, 10 ml of ammonia buffer pH 10.9 and 1 ml of potassium cyanide solution. Titrate with 0.05 M disodium edetate using eriochrome black T solution as indicator. 1 ml of 0.05 M disodium edetate is equivalent to 0.001215 g of Mg. For sodium bicarbonate Weigh accurately about 10 g, boil with 100 ml of water for 5 minutes and filter. Boil the residue with 100 ml of water for 5 minutes and filter. Cool the combined filtrates and titrate with 0.5 M hydrochloric acid using methyl orange-xylene cyanol FF solution as indicator. Add 10 ml of ammonia buffer pH 10.9 and titrate with 0.05 M disodium edetate using eriochrome black T solution as indicator. 1 ml of 0.5 M hydrochloric acid after subtracting one fifth of the volume of 0.05 M disodium edetate is equivalent to 0.0420 g of NaHCO3.
Apply to the plate 1 l of each solution. After development, dry the plate in air and spray with potassium iodoplatinate solution. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution. B. Dilute a volume of the injection containing 0.75 g of Levamisole Hydrochloride to 20 ml with water and add 6 ml of 1 M sodium hydroxide. Extract with 20 ml of dichloromethane, discard the aqueous layer and wash the dichloromethane layer with 10 ml of water. Dry by shaking with anhydrous sodium sulphate, filter and evaporate the solvent at room temperature. The residue, after drying over phosphorus pentoxide at a pressure of 1.5 to 2.5 kPa at a temperature not exceeding 40, melts at about 59 (2.4.21). C. The injection is laevorotatory. D. Gives reaction B of chlorides (2.3.1).
Tests
pH (2.4.24). 3.3 to 3.7. 2,3-Dihydro-6-phenylimidazo[2,1-b]thiazole hydrochloride. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 45 volumes of toluene, 8 volumes of methanol and 4 volumes of anhydrous glacial acetic acid. Test solution. Dilute a volume of the injection with methanol to produce a solution containing 5.0 per cent w/v of Levamisole Hydrochloride. Reference solution. A 0.025 per cent w/v of 2,3-dihydro-6phenylimidazo[2,1-b]thiazole hydrochloride RS in methanol. Apply to the plate 10 l of each solution. After development, dry the plate in air and spray with potassium iodoplatinate solution. Any spot corresponding to 2,3-dihydro-6phenylimidazo[2,1-b]thiazole hydrochloride in the chromatogram obtained with test solution is not more intense than the spot in the chromatogram obtained with reference solution. Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. To an accurately measured volume containing 0.75 g of Levamisole Hydrochloride add 50 ml of water and 15 ml of 2 M sodium hydroxide, extract with three quantities, each of 25 ml, 20 ml and 15 ml of dichloromethane, wash the combined extracts with two quantities, each of 10 ml, of water and discard the washings. To the clear dichloromethane solution, after drying with anhydrous sodium sulphate, add 50 ml of anhydrous glacial acetic acid. Titrate with 0.1 M perchloric acid, using 1-naphtholbenzein solution as indicator. Carry out a blank titration.
Levamisole Injection
Levamisole Hydrochloride Injection
Levamisole Injection is a sterile solution of Levamisole Hydrochloride in Water for Injections. It may contain suitable colouring agents. Levamisole Injection contains not less than 92.5 per cent and not more than 107.5 per cent of the stated amount of levamisole hydrochloride, C11H12N2S,HCl.
Identification
A. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 100 volumes of ethyl acetate, 10 volumes of methanol and 1 volume of strong ammonia solution. Test solution. Dilute a volume of the injection to produce a solution containing 1.0 per cent w/v of Levamisole Hydrochloride in methanol. Reference solution. A 1.0 per cent w/v of levamisole hydrochloride RS in methanol.
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IP 2007
LINCOMYCIN PREMIX
1 ml of 0.1 M perchloric acid is equivalent to 0.02408 g of C11H12N2S,HCl. Storage. Store protected from light.
Mobile phase. A mixture of 45 volumes of toluene, 8 volumes of methanol and 4 volumes of anhydrous glacial acetic acid. Test solution. Dilute a volume of the preparation under examination with methanol to produce a solution containing 1.0 per cent w/v of Levamisole Hydrochloride. Reference solution. A 0.025 per cent w/v of 2,3-dihydro-6phenylimidazo[2,1-b]thiazole hydrochloride RS in methanol. Apply to the plate 50 l of the test solution and 10 l of the reference solution. After development, dry the plate in air and spray with potassium iodoplatinate solution. Any spot corresponding to 2,3-dihydro-6-phenylimidazo[2,1-b]thiazole hydrochloride in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Other tests. Complies with the tests stated under Veterinary Oral Liquids. Assay. Weigh accurately a quantity containing 0.75 g of Levamisole Hydrochloride add 15 ml of 2 M sodium hydroxide, extract with three quantities each of 25 ml, 20 ml and 15 ml of dichloromethane, wash the combined extracts with two quantities, each of 10 ml, of water and discard the washings. To the clear dichloromethane solution, after drying with anhydrous sodium sulphate, add 50 ml of anhydrous glacial acetic acid. Titrate with 0.1 M perchloric acid, using 1-naphtholbenzein solution as indicator. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.02408 g of C11H12N2S,HCl.
Levamisole Hydrochloride Veterinary Oral Solution

Levamisole Hydrochloride Veterinary Mixture; Levamisole Veterinary Oral Solution; Levamisole Veterinary Mixture
Levamisole Hydrochloride Veterinary Oral Solution is an aqueous solution of Levamisole Hydrochloride containing suitable stabilising agents. Levamisole Hydrochloride Veterinary Oral Solution contains not less than 92.5 per cent and not more than 107.5 per cent of the stated amount of levamisole hydrochloride, C11H12N2S,HCl.
Identification
A. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 100 volumes of ethyl acetate, 10 volumes of methanol and 1 volume of strong ammonia solution. Test solution. Dilute a volume of the preparation under examination with methanol to produce a solution containing 1.0 per cent w/v of Levamisole Hydrochloride. Reference solution. A 1.0 per cent w/v of levamisole hydrochloride RS in methanol. Apply to the plate 1 l of each solution. After development, dry the plate in air and spray with potassium iodoplatinate solution. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution. B. To a quantity containing 0.3 g of Levamisole Hydrochloride add 10 ml of water and 6 ml of 1 M sodium hydroxide. Extract with 20 ml of dichloromethane, discard the aqueous layer and wash the dichloromethane layer with 10 ml of water. Dry by shaking with anhydrous sodium sulphate, filter and allow the dichloromethane to evaporate at room temperature. The residue, after drying over phosphorus pentoxide at a pressure of 1.5 to 2.5 kPa at a temperature not exceeding 40, melts at about 59 (2.4.21). C. The solution is laevorotatory.
Lincomycin Premix
Lincomycin Hydrochloride Premix.
Lincomycin Premix contains Lincomycin Hydrochloride. Lincomycin Premix contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of lincomycin, C18H34N2O6S.
Identification
In the Assay, the chromatogram obtained with the test solution (b) corresponds to the chromatogram obtained with the reference solution.
Tests
Lincomycin B. Examine test solution (a) as described in the Assay but increasing the sensitivity by 8 to 10 times while recording the peak due to the trimethylsilyl derivative of lincomycin B, which is eluted immediately before the
Tests
2,3-Dihydro-6-phenylimidazo[2,1-b]thiazole hydrochloride. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G.
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LITHIUM ANTIMONY THIOMALATE
IP 2007
trimethylsilyl derivative of lincomycin. The area of the peak due to the trimethylsilyl derivative of lincomycin B, after correction for the sensitivity factor, is not more than 5 per cent of the area of the peak due to the trimethylsilyl derivative of lincomycin. Other tests. Complies with the tests stated under Premixes. Assay. Determine by gas chromatography (2.4.13). Solution A. Weigh accurately a quantity of the premix containing about 90 mg of lincomycin, shake with 10.0 ml of dimethylformamide and filter. Test solution (a). Add 1 ml of a 1 per cent w/v solution of tetraphenylcyclopentadienone (internal standard) in dimethylformamide and 0.4 ml of a mixture of 9 volumes of N,O-bis(trimethylsilyl)acetamide and 1 volume of trimethylchlorosilane to 1 ml of solution A, mix and allow to stand for 15 minutes. Test solution (b). Add 1 ml of dimethylformamide and 0.4 ml of a mixture of 9 volumes of N,O-bis(trimethylsilyl)acetamide and 1 volume of trimethylchlorosilane to 1 ml of solution A, mix and allow to stand for 15 minutes. Reference solution. Add 1 ml of a 1 per cent w/v solution of tetraphenylcyclopentadienone (internal standard) in dimethylformamide and 0.4 ml of a mixture of 9 volumes of N,O-bis(trimethylsilyl)acetamide and 1 volume of trimethylchlorosilane to 1 ml of a 1 per cent w/v solution of lincomycin hydrochloride RS in dimethylformamide, mix and allow to stand for 15 minutes. Chromatographic system a glass column 1.5 m x 3 mm, packed with silanised diatomaceous support (100 to 120 mesh) coated with 3 per cent w/w of methyl silicone gum (such as SE 30), temperature: column 260, inlet port and detector. 260 to 290, flow rate. 30 ml per minute of the carrier gas. Calculate the content of C18H34N2O6S. Labelling. The label states the strength in terms of the equivalent amount of lincomycin.
5.1 per cent and not more than 5.7 per cent of Li, calculated on the dried, solvent-free basis. Description. A pinkish white or creamy powder; hygroscopic.
Identification
A. To 0.2 g dissolved in 5 ml of water add 2 ml of hydrochloric acid and 5 ml of sodium sulphide solution; a yellowish- orange precipitate is produced which does not dissolve on addition of dilute ammonia solution. B. When moistened with hydrochloric acid and introduced on a platinum wire it imparts a red colour to a non-luminous flame.
Tests
Appearance of solution. A 6 per cent w/v solution in carbon dioxide-free water is clear (2.4.1), and not more intensely coloured than reference solution RS3 (2.4.1). pH (2.4.24). 9.0 to 10.5, determined in a 6 per cent w/v solution in carbon dioxide-free water. Assay. For antimony Weigh accurately about 0.50 g , add 35 ml of water and swirl to dissolve. Add 5 g of ammonium persulphate, 10 ml of sodium hydroxide solution and 3 or 4 glass beads (approximately 0.5 cm diameter). Place a small funnel in the neck of the flask and boil gently for 20 minutes at such a rate that the volume is not reduced appreciably. Cool, add through the funnel 0.25 ml of phenolphthalein solution and sufficient 0.1 M hydrochloric acid until the last trace of pink colour disappears. Add 25 ml of a 10 per cent w/v solution of oxalic acid through the funnel and boil vigorously for 3 minutes. Rinse the funnel, with a small quantity of water, remove it and add 5 ml of hydrochloric acid and 2 g of potassium iodide. Allow to stand for 10 minutes and boil until the solution becomes yellow and shows no further decrease in colour, but taking care to see that the volume is not reduced to less than about 30 ml. Cool and remove a small drop of the solution with a sealed capillary melting point tube and add to starch iodide paper. If a bluish colour is produced, add 1 drop of 0.1 M sodium thiosulphate while swirling and again test with starch iodide paper. Repeat if necessary until a bluish colour is no longer produced. Add 5 g of sodium potassium tartrate, cool to about 15 to 20 and cautiously add small portions of sodium bicarbonate until no further effervescence is produced. Add 2 to 4 g more of sodium bicarbonate and titrate with 0.1 M iodine until the first permanent light yellow colour is produced. 1 ml of 0.1 M iodine is equivalent to 0.006088 g of Sb. For lithium Weigh accurately about 0.2 g, dissolve in 50 ml of glacial acetic acid . Titrate with 0.1 M perchloric acid, using 1 ml of crystal violet solution as indicator. Carry out a blank titration.
Lithium Antimony Thiomalate

LiOOC C CHCOOLi H2 S Sb
C12H9Li6O12S3Sb,9H2O
3
, 9H2O
Mol. Wt. 766.9
Lithium Antimony Thiomalate contains not less than 15.5 per cent and not more than 16.5 per cent of Sb and not less than
1540
IP 2007
MAGNESIUM HYPOPHOSPHITE
1 ml of 0.1 M perchloric acid is equivalent to 0.000694 g of Li. Storage. Store protected from light and moisture.
loss by spurting due to effervescence) till alkaline to litmus paper and titrate with 0.05 M iodine using 1 ml of starch solution, added towards the end of the titration, as indicator. 1 ml of 0.05 M iodine is equivalent to 0.03834 g of C12H9Li6O12S3Sb,9H2O. Storage. Store protected from light.
Lithium Antimony Thiomalate Injection

Lithium Antimony Thiomalate Injection is a sterile solution of Lithium Antimony Thiomalate in Water for Injection containing a suitable antimicrobial preservative. Lithium Antimony Thiomalate Injection contains not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of lithium antimony thiomalate, C12H9Li6O12S3Sb,9H2O.
Magnesium Hypophosphite
HO P Mg P OH OH OH , 6H2O
Identification
A. Dilute a volume containing 0.2 g of Lithium Antimony Thiomalate to 5 ml with water. Add 2 ml of hydrochloric acid and 5 ml of sodium sulphide solution; a yellowish orange precipitate is produced which does not dissolve on addition of dilute ammonia solution. B. Dilute 0.2 ml of the injection under examination to 10 ml with a 5 per cent w/v solution of sodium potassium tartrate. To 2 ml of the solution add sodium sulphide solution dropwise; a reddish orange precipitate is produced. The precipitate dissolves on adding dilute sodium hydroxide solution.
Mg(H2PO2)2,6H2O
Mol. Wt. 262.4
Magnesium Hypophosphite contains not less than 98.5 per cent and not more than 101.0 per cent of Mg(H2PO2)2,6H2O. Description. Colourless crystals or white crystalline powder.
Identification
A. Gives the reactions of magnesium salts (2.3.1). B. Dissolve about 50 mg in 5 ml of water and add 0.5 ml of mercuric chloride solution; a white precipitate is produced. C. Dissolve about 50 mg in 5 ml of water and acidify with sulphuric acid. Add 0.5 ml of cupric sulphate solution and warm; a red precipitate is produced.
Tests
Appearance of solution. The solution is clear (2.4.1), and not more intensely coloured than reference solution RS3 (2.4.1). pH (2.4.24). 9.0 to 10.5. Pyrogens. Complies with the test for pyrogens (2.2.8), using per 1.5 kg of the rabbits weight, a volume containing 0.012 g of Lithium Antimony Thiomalate. Sterility (2.2.11). Complies with the test for sterility. Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. Dilute 10.0 ml with 25 ml of water, add 7.5 g of ammonium persulphate and 16 ml of sodium hydroxide solution, boil gently for 20 minutes, cool and add 0.5 ml of phenolphthalein solution. Neutralise the solution with dilute hydrochloric acid and boil for 3 minutes. Add 50 ml of a 10 per cent w/v solution of oxalic acid, 7.5 ml of hydrochloric acid and sufficient water to make up the volume, if necessary. Add 2 g of potassium iodide to the hot solution, allow to stand for 10 minutes and boil until it acquires a pale yellow colour (about 10 minutes). Cool and remove the colour by adding 0.1 M sodium thiosulphate using starch iodide solution as an external indicator. Add 7.5 g of sodium potassium tartrate and dilute to 200 ml. Add sodium bicarbonate carefully (avoiding
Tests
Appearance of solution. A 5 per cent w/v solution is clear (2.4.1) and colourless (2.4.1). Heavy metals (2.3.13). Dissolve 1.0 g in 20 ml of water, add 2 ml of dilute hydrochloric acid and sufficient water to produce 25 ml. The resulting solution complies with the limit test for heavy metals, Method A (20 ppm). Chlorides (2.3.12). To 1 g add 200 ml of water and filter. 10 ml of the filtrate complies with the limit test for chlorides (0.1 per cent). Sulphates (2.3.17). 1 g complies with the limit test for sulphates (0.015 per cent). Assay. Weigh accurately about 0.2 g, dissolve in 50 ml of water, add 5 ml of strong ammonia-ammonium chloride solution and titrate with 0.05 M disodium edetate using 0.1 g of mordant black II mixture as indicator, until a blue colour is obtained. 1 ml of 0.05 M disodium edetate is equivalent to 0.01312 g of Mg(H2PO2)2,6H2O. Storage. Store protected from moisture.
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MECLEOFENAMIC ACID
IP 2007
Meclofenamic Acid
C14H11Cl2NO2
Mol. Wt. 296.2
Meclofenamic acid is N-(2,6-dichloro-3-methylphenyl) anthranilic acid. Meclofenamic Acid contains not less than 98.5 per cent and not more than 100.5 per cent of the stated amount of C14H11Cl2NO2, calculated on the dried basis. Description. A white or almost white, crystalline powder.
Chromatographic system a stainless steel column 20 cm x 4 mm, packed with particles of silica gel (10 m) the surface of which has been modified with chemically-bonded octadecasilyl groups (such as Spherisorb ODS), mobile phase: a mixture of 75 volumes of methanol, 25 volumes of water and 1 volume of glacial acetic acid, flow rate. 2 ml per minute, spectrophotometer set at 254 nm, a 20 l loop injector. Inject reference solution (a). The test is not valid unless the column efficiency is not less than 4500 theoretical plates. Inject reference solution (b). The area of the peak immediately preceding the peak due to meclofenamic acid is not more than one-seventh of the area of the peak due to the internal standard. The area of any other peak is not more than the area of the peak due to the internal standard. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method D (20 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.6 g, dissolve in 100 ml of warm ethanol previously neutralised to phenol red solution and titrate with 0.1 M sodium hydroxide, using phenol red solution as indicator. 1 ml of 0.1 M sodium hydroxide is equivalent to 0.02962 g of C14H11Cl2NO2. Storage. Store protected from moisture.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with meclofenamic acid RS or with the reference spectrum of meclofenamic acid. B. When examined in the range 220 nm to 360 nm (2.4.7), a 0.002 per cent w/v solution in 0.1 M sodium hydroxide shows absorption maxima at about 279 nm, and 371 nm; absorbance at about 279 nm, about 0.45, and at about 317 nm, about 0.33. C. Dissolve 25 mg in 15 ml of dichloromethane; the solution exhibits a strong blue fluorescene when examined under ultraviolet light. D. Dissolve 1 mg in 2 ml of sulphuric acid and add 0.05 ml of 0.02 M potassium dichromate; an intense purple colour is produced, which rapidly fades to purple brown.
Tests
Appearance of solution. A 5.0 per cent w/v solution in 1 M sodium hydroxide is not more opalescent than reference suspension OS2 (2.4.1) and is not more intensely coloured than reference solution BYS5 (2.4.1). Light absorption (2.4.7). Absorbance of a 0.002 per cent w/v solution in 0.01 M methanolic hydrochloric acid at the maximum at about 279 nm, not less than 0.400 and not more than 0.445, and at the maximum at about 335 nm, not less than 0.440 and not more than 0.490. Related substances. Determine by liquid chromatography (2.4.14). Test solution. Dissolve 1.0 g of the substance under examination in 100 ml of ethanol. Reference solution (a). A 0.0035 per cent w/v solution of ethyl meclofenamate RS (internal standard). in ethanol. Reference solution (b). A solution containing 1.0 per cent w/ v of the substance under examination and 0.0035 per cent w/v of ethyl meclofenamate RS (internal standard) in ethanol.
Mepyramine Injection
Mepyramine Maleate Injection; Pyrilamine Maleate Injection; Pyrilamine Injection
Mepyramine Injection is a sterile solution of Mepyramine Maleate in Water for Injections. Mepyramine Injection contains not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of mepyramine maleate, C17H23N3O,C4H4O4. Description. Colourless or almost colourless solution.
Identification
A. To a volume containing 0.1 g of Mepyramine Maleate add 2 ml of 5 M sodium hydroxide and shake with three quantities, each of 3 ml, of ether. Warm the aqueous layer in a water-bath for 10 minutes with 2 ml of bromine solution, heat to boiling, cool, and add 0.2 ml to a solution of 10 mg of resorcinol in 3 ml
1542
IP 2007
MONOSULFIRAM SOAP
of sulphuric acid; a blue-black colour develops on heating for 15 minutes in a water-bath. B. Dilute a volume containing 20 mg of mepyramine maleate to 2 ml with water, add 1 ml of cyanogen bromide solution and 5 ml of a 2 per cent w/v solution of potassium hydrogen phthalate, mix, allow to stand for 15 minutes and add 1 ml of a 4 per cent solution of aniline in ethanol (95 per cent); a yellow colour is produced.
C. Boil 0.1 g with 2 M hydrochloric acid; hydrogen sulphide is evolved which has a characteristic odour and turns filter paper treated with lead acetate solution, black.
Tests
Freezing point (2.4.11). 28.5 to 32.0. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. NOTE Carry out the test in subdued light. Mobile phase. A mixture of 70 volumes of n-hexane and 30 volumes of butyl acetate. Test solution. Dissolve 2.5 g of the substance under examination in 100 ml of ethyl acetate. Reference solution (a). A 0.125 per cent w/v solution of disulfiram RS in ethyl acetate. Reference solution (b). A 0.050 per cent w/v solution of the substance under examination in ethyl acetate. Apply to the plate 5 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. In the chromatogram obtained with the test solution any secondary spot is not more intense than the spot in the chromatogram obtained with reference solution (a) and any other spot, apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (b). Water (2.3.43). Not more than 1.0 per cent, determined on 1 g. Sulphated ash (2.3.18). Not more than 0.1 per cent. Assay. Weigh accurately about 0.35 g, dissolve in 8 ml of nitrogen-free sulphuric acid and carry out the method for the determination of nitrogen ( 2.3.30). 1 ml of 0.05 M sulphuric acid is equivalent to 0.01322 g of C10H20N2S3. Storage. Store protected from light.
Tests
pH (2.4.24). 5.5 to 6.5. Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. To an accurately measured volume containing about 25 mg of mepyramine maleate add sufficient 0.01 M hydrochloric acid to produce 100.0 ml. Dilute 10.0 ml of this solution to 100.0 ml with 0.01 M hydrochloric acid and measure the absorbance of the resulting solution at the maximum at about 316 nm (2.4.7). Calculate the content of C17H23N3O,C4H4O4, taking 206 as the specific absorbance at 316 nm.
Monosulfiram
Sulfiram
H3 C H3 C N S S S N CH3 CH3
C10H20N2S3
Mol. Wt. 264.5
Monosulfiram is bis(diethylthiocarbamoyl)sulphide. Monosulfiram contains not less than 98.0 per cent and not more than 101.0 per cent of C10H20N2S3, calculated on the anhydrous basis. Description. A yellow or yellowish-brown soft solid; odour, sulphurous.
Monosulfiram Soap
Monosulfiram Soap contains not less than 5 per cent w/w of monosulfiram in a toilet soap basis which may be perfumed. Monosulfiram Soap contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of monosulfiram, C10H20N2S3.
Identification
A. When examined in the range 230 nm to 360 nm (2.4.7), a 2-cm layer of a 0.001 per cent w/v solution in methanol shows a well-defined absorption maximum only at about 281 nm; absorbance at about 281 nm, about 1.3. B. Dissolve 0.1 g in a mixture of 0.15 ml of a 1 per cent w/v solution of cupric sulphate and 5 ml of ethanol (95 per cent), evaporate on a water-bath and dissolve the residue in dichloromethane; a deep yellowish brown colour is produced.
Identification
In the test for Related substances, the principal spot in the chromatogram obtained with test solution (b) corresponds to that in the chromatogram obtained with reference solution (c).
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MONOSULFIRAM SOLUTION
IP 2007
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. NOTE Carry out the test in subdued light. Mobile phase. A mixture of 70 volumes of n-hexane and 30 volumes of butyl acetate. Test solution (a). Shake a quantity of the finely shredded soap containing 20 mg of Monosulfiram with 10 ml of dichloromethane, filter and wash the filtrate with dichloromethane. Evaporate the combined filtrate and washings just to dryness at room temperature in a current of nitrogen and dissolve the residue in 1 ml of ethanol (95 per cent). Test solution (b). Dilute 0.5 ml of test solution (a) to 10 ml with ethanol (95 per cent). Reference solution (a). A 0.10 per cent w/v solution of disulfiram RS in ethanol (95 per cent). Reference solution (b). A 0.040 per cent w/v solution of monosulfiram RS in ethanol (95 per cent). Reference solution (c). A 0.10 per cent w/v solution of monosulfiram RS in ethanol (95 per cent). Apply to the plate 5 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. In the chromatogram obtained with test solution any spot running ahead of the principal spot and corresponding in position to disulfiram is not more intense than the spot in the chromatogram obtained with reference solution (a) and any spot running behind the principal spot is not more intense than the spot in the chromatogram obtained with reference solution (b). Ignore any subsidiary spots due to the soap basis which may also be observed ahead of the principal spot in the chromatogram obtained with test solution (a). Assay. Protect the solutions from light throughout the assay. Determine by gas chromatography (2.4.13). Test solution (a). Weigh accurately a quantity of the finely shredded soap containing about 0.25 g of Monosulfiram, shake for 10 minutes with 50 ml of dimethylformamide, centrifuge and use the supernatant liquid. Test solution (b). Weigh accurately a quantity of the finely shredded soap containing about 0.25 g of Monosulfiram, shake for 10 minutes with 50 ml of dimethylformamide containing 0.125 g of N-phenylcarbazole (internal standard), centrifuge and use the supernatant liquid. Reference solution. A solution containing 0.5 per cent w/v of monosulfiram RS and 0.25 per cent w/v of N-phenylcarbazole (internal standard) in dimethylformamide.
Chromatographic system a glass column 1.5 m x 4 mm, packed with 2 per cent w/w of methyl silicone gum on acid-washed, silanised diatomaceous support (80 to 100 mesh) (such as SE 30), temperature: column 180, inlet port 180 and detector 280, flow rate. 30 ml per minute of the carrier gas. Calculate the content of C10H20N2S3. Labelling. The label states (1) the proportion of Monosulfiram in the preparation; (2) the method of use of the preparation.
Monosulfiram Solution
Monosulfiram Solution is a solution of Monosulfiram in Ethanol (95 per cent) containing a suitable dispersing agent. In making Monosulfiram Solution the ethanol (95 per cent) may be replaced by Industrial Methylated Spirit provided that the statutory requirements governing the use of Industrial Methylated Spirit are observed. Monosulfiram Solution contains not less than 94.0 per cent and not more than 106.0 per cent of the stated amount of monosulfiram, C10H20N2S3. Description. Clear, bright, deep reddish-brown liquid; crystals from which may deposit slowly at low temperatures but dissolve on warming. Yields a pale yellow dispersion on dilution with water.
Identification
In the test for Related substances, the principal spot in the chromatogram obtained with test solution (b) corresponds to that in the chromatogram obtained with reference solution (c).
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. NOTE Carry out the test in subdued light. Mobile phase. A mixture of 70 volumes of n-hexane and 30 volumes of butyl acetate. Test solution (a). Dilute a quantity of the solution under examination with ethanol (95 per cent) so as to contain of 2.0 per cent w/v of Monosulfiram. Test solution (b). Dilute 0.5 ml of test solution (a) to 10 ml with ethanol (95 per cent). Reference solution (a). A 0.10 per cent w/v solution of disulfiram RS in ethanol (95 per cent). Reference solution (b). A 0.040 per cent w/v solution of monosulfiram RS in ethanol (95 per cent).
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NANDROLONE LAURATE
Reference solution (c). A 0.10 per cent w/v solution of monosulfiram RS in ethanol (95 per cent). Apply to the plate 5 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. In the chromatogram obtained with the test solution any spot running ahead of the principal spot and corresponding in position to disulfiram is not more intense than the spot in the chromatogram obtained with reference solution (a) and any spot running behind the principal spot is not more intense than the spot in the chromatogram obtained with reference solution (b). Assay. Protect the solutions from light throughout the assay. Determine by gas chromatography (2.4.13). Test solution (a). Dilute the solution under examination in dimethylformamide containing the equivalent of 0.5 per cent w/v of Monosulfiram. Test solution (b). Weigh accurately a quantity of the finely shredded soap containing about 0.25 g of Monosulfiram, shake for 10 minutes with 50 ml of dimethylformamide containing 0.125 g of N-phenylcarbazole (internal standard), centrifuge and use the supernatant liquid. Reference solution. A solution containing 0.5 per cent w/v of monosulfiram RS and 0.25 per cent w/v of N-phenylcarbazole (internal standard) in dimethylformamide. Chromatographic system a glass column 1.5 m x 4 mm, packed with 2 per cent w/w of methyl silicone gum on acid-washed, silanised diatomaceous support (80 to 100 mesh) (such as SE 30), temperature: column 180, inlet port 180 and detector 280, flow rate. 30 ml per minute of the carrier gas. Calculate the content of C10H20N2S3. Labelling. The label states (1) the percentage w/w of monosulfiram; (2) the method of use of the preparation.
Nandrolone Laurate is 3-oxoestr-4-en-17-yl-dodecanoate. Nandrolone Laurate contains not less than 97.0 per cent and not more than 103.0 per cent of C30H48O3, calculated on the dried basis. Description. A white to creamy white, crystalline powder; odour, faint and characteristic.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with nandrolone laurate RS or with the reference spectrum of nandrolone laurate. B. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254, surface of which has been modified by chemically bonded octadecylsilyl groups (such as Whatman KC 18F plates). Mobile phase. A mixture of 60 volumes of 2-propanol, 40 volumes of acetonitrile and 20 volumes of water. Test solution. Dissolve 0.5 g of the substance under examination in dichloromethane. Reference solution (a). A 0.5 per cent w/v of nandrolone laurate RS in dichloromethane. Reference solution (b). A mixture of equal volumes of the test solution and reference solution (a) Apply to the plate 5 l of each solution. After development, dry the plate in air until the odour of the solvent is no longer detectable and heat at 100 for 10 minutes. Allow to cool and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution (a). The test is not valid unless the principal spot in the chromatogram obtained with reference solution (b) appears as a single, compact spot. C. Melts at about 47 (2.4.21).
Tests
Nandrolone Laurate
O H3C O H H O H H CH2(CH2)9CH3
Specific optical rotation (2.4.22). +31.0 to +35.0, determined in a freshly prepared 2 per cent w/v solution in dioxan. Nandrolone. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 70 volumes of n-heptane and 30 volumes of acetone. Test solution. Dissolve 1.5 g of the substance under examination in dichloromethane. Mol. Wt. 456.7 Reference solution. A 0.030 per cent w/v of nandrolone RS in dichloromethane.
C30H48O3
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NANDROLONE LAURATE INJECTION
IP 2007
Apply to the plate 1 l of each solution. After development, dry the plate in air until the odour of the solvent is no longer detectable, spray with a 10 per cent v/v solution of sulphuric acid in ethanol (95 per cent), heat at 105 for 30 minutes and examine in ultraviolet light at 365 nm. Any spot in the chromatogram obtained with the test solution corresponding to nandrolone is not more intense than the spot in the chromatogram obtained with the reference solution. Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying over phosphorus pentoxide at a pressure not exceeding 0.7 kPa for 24 hours. Assay. Weigh accurately about 0.1 g, dissolve in sufficient ethanol to produce 100.0 ml and dilute 10.0 ml to 100.0 ml with ethanol. Dilute 10.0 ml of this solution to 100.0 ml with ethanol and measure the absorbance of the resulting solution at the maximum at about 240 nm (2.4.7). Calculate the content of C30H48O3 taking 380 as the specific absorbance at 240 nm. Storage. Store protected from light.
chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution (a). The test is not valid unless the principal spot in the chromatogram obtained with reference solution (b) appears as a single, compact spot.
Tests
Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. To an accurately measured volume containing about 0.1 g of Nandrolone Laurate add sufficient dichloromethane to produce 100.0 ml. Dilute 3.0 ml of the resulting solution to 50.0 ml with dichloromethane. To 5.0 ml of this solution add 10 ml of isoniazid solution and sufficient methanol to produce 20.0 ml. Allow to stand for 45 minutes and measure the absorbance of the resulting solution at the maximum at about 380 nm (2.4.7), using as the blank 5 ml of dichloromethane treated in a similar manner. Calculate the content of C30H48O3 from the absorbance obtained by repeating the procedure using a suitable quantity of nandrolone RS. 1 mg of C18H26O2 is equivalent to 0.001664 g of C30H48O3.
Nandrolone Laurate Injection

Nandrolone Laurate Injection is a sterile solution of Nandrolone Laurate in Ethyl Oleate or other suitable ester, in a suitable fixed oil, or in any mixture of these. Nandrolone Laurate Injection contains not less than 92.5 per cent and not more than 107.5 per cent of the stated amount of nandrolone laurate, C30H48O3.
Niclosamide Veterinary Oral Powder

Niclosamide Dispersible Powder for Veterinary Use Niclosamide Veterinary Oral Powder contains Niclosamide with suitable auxiliary substances. Niclosamide Veterinary Oral Powder contains not less than 97.0 per cent and not more than 103.0 per cent of the stated amount of niclosamide, C13H8Cl2N2O4.
Identification
Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254, surface of which has been modified by chemically bonded octadecylsilyl groups (such as Whatman KC 18F plates). Mobile phase. A mixture of 60 volumes of 2-propanol, 40 volumes of acetonitrile and 20 volumes of water. Test solution. Dilute a suitable volume with dichloromethane to produce a solution containing 0.5 per cent w/v of Nandrolone Laurate . Reference solution (a). A 0.5 per cent w/v of nandrolone laurate RS in dichloromethane. Reference solution (b). A mixture of equal volumes of the test solution and reference solution (a) Apply to the plate 5 l of each solution. After development, dry the plate in air until the odour of the solvent is no longer detectable and heat at 100 for 10 minutes. Allow to cool and examine in ultraviolet light at 254 nm. The principal spot in the
Identification
Heat 50 mg with 5 ml of 1 M hydrochloric acid and 0.1 g of zinc powder in a water-bath for 10 minutes, cool and filter. To the filtrate add 0.5 ml of a 1 per cent w/v solution of sodium nitrite and allow to stand for 10 minutes. Add 2 ml of a 2 per cent w/v solution of ammonium sulphamate, shake, allow to stand for 10 minutes and add 2 ml of a 0.5 per cent w/v solution of N-(1-naphthyl)ethylenediamine dihydrochloride; a deep red colour is produced.
Tests
2-Chloro-4-nitroaniline. Boil a quantity containing 0.10 g of Niclosamide with 20 ml of methanol for 2 minutes, cool, add sufficient 1 M hydrochloric acid to produce 50 ml and filter. To 10 ml of the filtrate add 0.5 ml of a 0.5 per cent w/v solution of sodium nitrite and allow to stand for 10 minutes. Add 1 ml
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NITROXYNIL INJECTION
of a 2 per cent w/v solution of ammonium sulphamate, shake, allow to stand for 10 minutes and add 1 ml of a 0.5 per cent w/v solution of N-(1-naphthyl)ethylenediamine dihydrochloride. The colour produced is not more than that produced by simultaneously treating 10 mg of 2-chloro-4-nitroaniline in the same manner. 5-Chlorosalicylic acid. Boil a quantity containing 0.50 g of Niclosamide with 10 ml of water for 2 minutes, cool and filter. To the filtrate add a few drops of ferric chloride solution; no red or violet colour is produced. Loss on drying (2.4.19). Not more than 1.0 per cent, determined on 1 g by drying in an oven at 105 for 4 hours. Other tests. Complies with the tests stated under Veterinary Oral Powders. Assay. Weigh accurately a quantity containing about 0.3 g of Niclosamide, dissolve in 60 ml of dimethylformamide with the aid of gentle heat, cool. Titrate with 0.1 M tetrabutylammonium hydroxide, maintaining a stream of nitrogen through the solution throughout the titration, and determining the end-point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M tetrabutylammonium hydroxide is equivalent to 0.03271 g of C13H8Cl2N2O4.
C. When heated with sulphuric acid, iodine vapours are evolved. D. Melting range (2.4.21). 136 to 139.
Tests
Inorganic iodide. To 0.40 g add 0.35 g of N-methylglucamine and 10 ml of water. Shake to dissolve and add sufficient water to produce 50 ml. To 10 ml of the resulting solution add 4 ml of 1 M sulphuric acid and extract with three quantities, each of 10 ml, of dichloromethane. Add to the aqueous extract 1 ml of hydrogen peroxide solution (100 vol) and 1 ml of dichloromethane, shake for 2 minutes and allow to separate. Any purple colour in the dichloromethane layer is not more intense than that obtained by adding 2 ml of a 0.0026 per cent w/v solution of potassium iodide to a mixture of 4 ml of 1 M sulphuric acid and 8 ml of water, adding 10 ml of dichloromethane, shaking for 2 minutes, adding to the aqueous layer 1 ml of hydrogen peroxide solution (100 vol) and 1 ml of dichloromethane, shaking for 2 minutes and allowing to separate (500 ppm). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 1.0 per cent, determined on 1 g by drying in an oven at 105 for 4 hours. Assay. Carry out the oxygen flask method for iodine (2.3.34), using 25 mg.
Nitroxynil
OH I NO2
1 ml of 0.02 M sodium thiosulphate is equivalent to 0.0009667 g of C7H3IN2O3. Storage. Store protected from light.
Nitroxynil Injection
CN
C7H3IN2O3
Mol. Wt. 290.0
Nitroxynil Injection is a sterile solution of the N-ethylglucamine salt of Nitroxynil in Water for Injections. Nitroxynil Injection contains not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of nitroxynil, C7H3IN2O3.
Nitroxynil is 4-hydroxy-3-iodo-5-nitrobenzonitrile. Nitroxynil contains not less than 98.0 per cent and not more than 101.0 per cent of C7H3IN2O3, calculated on the dried basis. Description. A yellow to yellowish brown powder.
Identification
A. When examined in the range 230 nm to 360 nm (2.4.7), of the final solution obtained in the Assay exhibits a maximum at about 271 nm. B. Heat 0.5 ml with 3 ml of sulphuric acid; iodine vapours are evolved.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with nitroxynil RS or with the reference spectrum of nandrolone nitroxynil. B. When examined in the range 220 nm to 360 nm (2.4.7), a 0.002 per cent w/v solution in 0.01 M sodium hydroxide exhibits maxima at about 225 nm and at about 271 nm; absorbance at about 271 nm, about 1.3.
Tests
pH (2.4.24). 5.0 to 7.0, determined by using a 20 per cent w/v solution of N-ethylglucamine hydrochloride instead of a
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saturated solution of potassium chloride as the liquid junction solution. Inorganic iodide. To a volume containing 0.4 g of Nitroxynil add 0.35 g of N-methylglucamine and dilute to 100 ml with water. To 10 ml of the diluted solution add 4 ml of 1 M sulphuric acid and extract with three quantities, each of 10 ml, of dichloromethane. Add to the aqueous extract 1 ml of hydrogen peroxide solution (100 vol) and 1 ml of dichloromethane, shake for 2 minutes and allow to separate. Any purple colour in the dichloromethane layer is not more intense than that obtained by adding 2 ml of a 0.0026 per cent w/v solution of potassium iodide to a mixture of 4 ml of 1 M sulphuric acid and 8 ml of water, adding 10 ml of dichloromethane, shaking for 2 minutes, adding to the aqueous layer 1 ml of hydrogen peroxide solution (100 vol) and 1 ml of dichloromethane, shaking for 2 minutes and allowing to separate (500 ppm) (0.1 per cent w/v of iodide). Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. To an accurately measured volume containing about 1.7 g of Nitroxynil add sufficient 0.01 M sodium hydroxide to produce 500.0 ml. Dilute 20.0 ml of this solution to 500.0 ml with 0.01 M sodium hydroxide. To 5.0 ml of this solution add sufficient 0.01 M sodium hydroxide to produce 100.0 ml and measure the absorbance of the resulting solution at the maximum at about 271 nm (2.4.7). Calculate the content of C7H3IN2O3 taking 660 as the specific absorbance at 271 nm. Storage. Store protected from light.
and dry at 105 at a pressure not exceeding 2.7 kPa. The residue complies with the following test. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with oxfendazole RS or with the reference spectrum of oxfendazole. B. When examined in the range 220 nm to 360 nm (2.4.7), a 0.001 per cent w/v solution in methanol exhibits two maxima at about 228 nm and about 297 nm; absorbances at about 228 nm, about 1.4 and at about 297 nm, about 0.55.
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Solvent mixture. 40 volumes of ethyl acetate and 10 volumes of glacial acetic acid. Mobile phase. A mixture of 95 volumes of ethyl acetate and 5 volumes of glacial acetic acid. Test solution. Dissolve 0.5 g of the substance under examination in 100 ml of solvent mixture. Reference solution (a). A 0.010 per cent w/v solution of the substance under examination in solvent mixture. Reference solution (b). A 0.0050 per cent w/v solution of methyl 5-phenylthio-1H-benzimidazol-2-yl carbamate RSin solvent mixture.) Apply to the plate 20 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. Any spot corresponding to methyl 5-phenylthio-1Hbenzimidazol-2-yl carbamate in the chromatogram obtained with test solution is not more intense than the spot in the chromatogram obtained with reference solution (b). Any other secondary spot in the chromatogram obtained with test solution is not more intense than the spot in the chromatogram obtained with reference solution (a). Sulphated ash (2.3.18). Not more than 0.2 per cent. Loss on drying (2.4.19). Not more than 1.0 per cent, determined on 1 g by drying in an oven at 105 for 2 hours at a pressure not exceeding 0.7 kPa. Assay. Weigh accurately about 0.3 g, dissolve in 20 ml of glacial acetic acid, add 3 g of potassium iodide and 1 ml of acetyl chloride and stir for 10 minutes. Add 50 ml of 1 M hydrochloric acid and 10 ml of dichloromethane and titrate immediately with 0.1 M sodium thiosulphate, shaking after each addition, until the dichloromethane layer is colourless. Repeat the operation omitting the substance under examination; the difference between the titrations represents the amount of sodium thiosulphate required. 1 ml of 0.1 M sodium thiosulphate is equivalent to 0.01577 g of C15H13N3O3S.
Oxfendazole
C15H13N3O3S Oxfendazole is methyl 5-(phenylsulphinyl)-2benzimidazolecarbamate.
Mol. Wt. 315.4
Oxfendazole contains not less than 97.0 per cent and not more than 100.5 per cent of C15H13N3O3S, calculated on the dried basis. Description. A white or almost white powder; odour, slight and characteristic.
Identification
A. Dissolve 0.1 g in 50 ml of methanol, evaporate to a volume of about 2 ml, cool, filter, wash the residue with 2 ml of water
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IP 2007
OXYCLOZANIDE
Oxfendazole Veterinary Oral Suspension

Oxfendazole Veterinary Mixture; Oxfendazole Mixture; Oxfendazole Oral Suspension
Oxfendazole Veterinary Oral Suspension is an aqueous suspension of Oxfendazole containing suitable suspending or dispersing agents. Oxfendazole Veterinary Oral Suspension contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of oxfendazole, C15H13N3O3S.
benzimidazol-2-yl carbamate in the chromatogram obtained with test solution is not more intense than the spot in the chromatogram obtained with reference solution (b). Any other secondary spot in the chromatogram obtained with test solution is not more intense than the spot in the chromatogram obtained with reference solution (a). Other tests. Complies with the tests stated under Veterinary Oral Liquids. Assay. Weigh accurately a quantity of the well-mixed suspension containing about 0.1 g of Oxfendazole and disperse in 15 ml of water. Add 200 ml of methanol and mix in an ultrasonic bath for 15 minutes, cool, add sufficient methanol to produce 500.0 ml and filter. Dilute 4.0 ml of the filtrate to 100.0 ml with methanol and measure the absorbance of the resulting solution at the maximum at about 296 nm (2.4.7). Calculate the content of C15H13N3O3S taking 550 as the specific absorbance at 296 nm. Determine the weight per ml of the suspension (2.4.29), and calculate the content of oxfendazole, weight in volume.
Identification
Shake a quantity containing 0.1 g of Oxfendazole with 50 ml of methanol for 15 minutes, centrifuge, evaporate the supernatant liquid to a volume of about 2 ml, cool, filter and wash the residue with 2 ml of water and dry at 105 for 1 hour at a pressure not exceeding 2.7 kPa. The residue complies with the following tests. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with oxfendazole RS or with the reference spectrum of oxfendazole. B. When examined in the range 220 nm to 360 nm (2.4.7), a 0.001 per cent w/v solution in 1 M hydrochloric acid exhibits three maxima, at about 226, 284 and 291 nm.
Oxyclozanide
Cl OH O Cl Cl Cl N H Cl OH
Tests
pH (2.4.24). 4.3 to 5.3. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Solvent mixture. 40 volumes of ethyl acetate and 10 volumes of glacial acetic acid. Mobile phase. A mixture of 95 volumes of ethyl acetate and 5 volumes of glacial acetic acid. Test solution. Shake a quantity containing 0.1 g of Oxfendazole with 20 ml of solvent mixture and filter. Reference solution (a). Dilute 1 volume of test solution to 50 volumes with the solvent mixture. Reference solution (b). A 0.0050 per cent w/v solution of methyl 5-phenylthio-1H-benzimidazol-2-yl carbamate RSin solvent mixture.) Apply to the plate 20 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. Any spot corresponding to methyl 5-phenylthio-1HC13H6Cl5NO3
Mol. Wt. 401.5
Oxyclozanide is 2,3,5-trichloro-N-(3,5-dichloro-2hydroxyphenyl)-6-hydroxybenzamide. Oxyclozanide contains not less than 98.0 per cent and not more than 101.0 per cent of C13H6Cl5NO3, calculated on the dried basis. Description. A pale cream to cream-coloured powder.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with oxyclozanide RS or with the reference spectrum of oxyclozanide. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.003 per cent w/v solution in 1 M methanolic hydrochloric acid exhibits a maximum only at about 300 nm; absorbance at about 300 nm, about 0.76. C. Melting range (2.4.21). 208 to 211.
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OXYCLOZANIDE VETERINARY ORAL SUSPENSION
IP 2007
Tests
Ionisable chlorine. Dissolve 2 g in 100 ml of methanol, add 10 ml of 1.5 M nitric acid and titrate with 0.1 M silver nitrate, determining the end-point potentiometrically (2.4.25). Not more than 1.4 ml is required (0.25 per cent). Related substances. Determine by liquid chromatography (2.4.14). Test solution. A 0.1 per cent w/v solution of the substance under examination prepared by dissolving it in a suitable volume of methanol and slowly diluting with water containing 0.1 per cent v/v of phosphoric acid to give a solution containing about the same proportion of methanol to water as in the mobile phase. Reference solution. Dilute 1 ml of test solution to 100 ml with the mobile phase. Chromatographic system a stainless steel column 20 cm x 5 mm, packed with octadecylsilane bonded to porous silica (5 m) (such as Hypersil ODS), mobile phase: a filtered and degassed mixture of 62 volumes of methanol and 38 volumes of water containing 0.1 per cent v/v of phosphoric acid, flow rate. 2 ml per minute, spectrophotometer set at 300 nm, a 20 l loop injector. Inject alternatively test solution and the reference solution. In the chromatogram obtained with test solution the area of any secondary peak with a retention time less than that of the principal peak is not more than one-third of the area of the principal peak in the chromatogram obtained with reference solution and the area of any secondary peak with a retention time greater than that of the principal peak is not more than the area of the principal peak in the chromatogram obtained with reference solution. Sulphated ash (2.3.18). Not more than 0.2 per cent. Loss on drying (2.4.19). Not more than 1.0 per cent, determined on 1 g by drying in an oven at 60 at a pressure not exceeding 0.7 kPa. Assay. Weigh accurately about 0.25 g, dissolve in 75 ml of anhydrous pyridine and pass a stream of nitrogen through the solution for 5 minutes. Titrate with 0.1 M tetrabutylammonium hydroxide, maintaining a stream of nitrogen through the solution throughout the titration, determining the end-point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M tetrabutylammonium hydroxide is equivalent to 0.02007 g of C13H6Cl5NO3.
Oxyclozanide Veterinary Oral Suspension

Oxyclozanide Oral Suspension; Oxyclozanide Suspension; Oxyclozanide Mixture; Oxyclozanide Drench
Oxyclozanide Veterinary Oral Suspension is an aqueous suspension of Oxyclozanide containing suitable suspending or dispersing agents. Oxyclozanide Veterinary Oral Suspension contains not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of oxyclozanide, C13H6Cl5NO3.
Identification
In test A for Related substances, the principal spot in the chromatogram obtained with 10 ml of test solution corresponds to that in the chromatogram obtained with reference solution (b).
Tests
Related substances. A. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 60 volumes of light petroleum ( 60 to 80), 20 volumes of acetone and 5 volumes of glacial acetic acid. Test solution. Dilute a quantity with acetone to contain 1.0 per cent w/v of Oxyclozanide, centrifuge and use the supernatant liquid. Reference solution (a). A 0.050 per cent w/v solution of 3,5,6-trichloro- 2-hydroxybenzoic acid RS in acetone. Reference solution (b). A 1.0 per cent w/v solution of oxyclozanide RS in acetone. Apply to the plate 40 l and 10 l of test solution, 4 l of reference solution (a) and 10 l of reference solution (b). After development, dry the plate in air and spray with a 3 per cent w/ v solution of ferric chloride in methanol. In the chromatogram obtained with 40 l of test solution any spot corresponding to 3,5,6-trichloro-2-hydroxybenzoic acid RS is not more intense than that in the chromatogram obtained with reference solution (a). B. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 100 volumes of ethyl acetate, 10 volumes of methanol and 1 volume of strong ammonia solution. Test solution. Dilute a quantity with acetone to contain 1.0 per cent w/v of Oxyclozanide, centrifuge and use the supernatant liquid.
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OXYCLOZANIDE PREMIX
Reference solution. A 0.040 per cent w/v of 2-amino4,6-dichlorophenol RS in acetone. Apply to the plate 40 l of test solution and 4 l of reference solution. After development, dry the plate in air and spray with lithium and sodium molybdotungstophosphate solution. In the chromatogram obtained with test solution any spot corresponding to 2-amino-4,6-dichlorophenol is not more intense than that in the chromatogram obtained with reference solution. Other tests. Complies with the tests stated under Veterinary Oral Liquids. Assay. Protect the solutions from light throughout the procedure. Weigh accurately a quantity containing about 60 mg of Oxyclozanide, add 60 ml of acidified methanol and boil gently on a water-bath. Shake continuously for 20 minutes, cool to 2 and dilute to 100.0 ml with acidified methanol. Filter, dilute 5.0 ml of the filtrate to 100.0 ml with acidified methanol and measure the absorbance of the resulting solution at the maximum at about 300 nm (2.4.7). Calculate the content of C13H6Cl5NO3 taking 254 as the specific absorbance at 300 nm. Determine the weight per ml of the suspension (2.4.29), and calculate the content of oxyclozanide, weight in volume.
Reference solution (a). A 0.050 per cent w/v solution of 3,5,6-trichloro- 2-hydroxybenzoic acid RS in acetone. Reference solution (b). A 1.0 per cent w/v solution of oxyclozanide RS in acetone. Apply to the plate 40 l and 10 l of test solution, 4 l of reference solution (a) and 10 l of reference solution (b). After development, dry the plate in air and spray with a 3 per cent w/ v solution of ferric chloride in methanol. In the chromatogram obtained with 40 l of test solution any spot corresponding to 3,5,6-trichloro-2-hydroxybenzoic acid RS is not more intense than that in the chromatogram obtained with reference solution (a). B. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 100 volumes of ethyl acetate, 10 volumes of methanol and 1 volume of strong ammonia solution. Test solution. Extract the finely powdered preparation under examination with sufficient acetone to produce a mixture containing 1.0 per cent w/v of Oxyclozanide, centrifuge and use the supernatant liquid. Reference solution. A 0.040 per cent w/v of 2-amino-4,6-dichlorophenol RS in acetone. Apply to the plate 40 l of test solution and 4 l of reference solution. After development, dry the plate in air and spray with lithium and sodium molybdotungstophosphate solution. In the chromatogram obtained with test solution any spot corresponding to 2-amino-4,6-dichlorophenol is not more intense than that in the chromatogram obtained with reference solution. Other tests. Complies with the tests stated under Premixes. Assay. Protect the solutions from light throughout the procedure. Weigh accurately a quantity of the finely powdered preparation under examination containing 60 mg of Oxyclozanide, add 60 ml of acidified methanol and boil gently on a water-bath. Shake continuously for 20 minutes, cool to 2 and dilute to 100.0 ml with acidified methanol. Filter, dilute 5.0 ml of the filtrate to 100.0 ml with acidified methanol and measure the absorbance of the resulting solution at the maximum at about 300 nm (2.4.7). Calculate the content of C13H6Cl5NO3 taking 254 as the specific absorbance at 300 nm. Labelling. The label states (1) the proportion of oxyclozanide in the premix and (2) the method of use of the preparation.
Oxyclozanide Premix
Oxyclozanide Granules
Oxyclozanide Premix contains Oxyclozanide. Oxyclozanide Premix contains not less than 92.5 per cent and not more than 107.5 per cent of the stated amount of oxyclozanide, C13H6Cl5NO3.
Identification
In test A for Related substances, the principal spot in the chromatogram obtained with 10 ml of test solution corresponds to that in the chromatogram obtained with reference solution (b).
Tests
Related substances. A. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 60 volumes of light petroleum ( 60 to 80), 20 volumes of acetone and 5 volumes of glacial acetic acid. Test solution. Extract the finely powdered preparation under examination with sufficient acetone to produce a mixture containing 1.0 per cent w/v of Oxyclozanide, centrifuge and use the supernatant liquid.
1551
OXYTETRACYCLINE VETERINARY ORAL POWDER
IP 2007
Oxytetracycline Veterinary Oral Powder

Oxytetracycline Hydrochloride Veterinary Oral Powder; Oxytetracycline Hydrochloride Soluble Powder; Oxytetracycline Soluble Powder
Oxytetracycline Veterinary Oral Powder is a mixture of Oxytetracycline Hydrochloride and Lactose or other suitable diluent. Oxytetracycline Veterinary Oral Powder contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of oxytetracycline hydrochloride, C22H24N2O9,HCl.
it and filter again. The filtrate gives the reactions of chlorides (2.3.1).
Tests
Assay. To a quantity of the powder containing 0.25 g of Oxytetracylcine Hydrochloride, add 250.0 ml of water, shake, filter and carry out the microbiological assay (2.2.10), Method A or B. Storage. Store at a temperature not exceeding 15.
Pentobarbitone Injection
Pentobarbitone Sodium Injection; Pentobarbital Sodium Injection
Pentobarbitone Injection is a sterile solution of Pentobarbitone Sodium in a suitable vehicle. Solutions containing 20 per cent w/v of Pentobarbitone Sodium in 100-ml and 500-ml containers are also available for use other than for injection. Such solutions may be coloured and need not be sterile but must comply with all other requirements of this monograph. Pentobarbitone Injection contains not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of pentobarbitone sodium, C11H17N2NaO3. Description. A clear, colourless or almost colourless solution.
Identification
A. Determine by thin-layer chromatography (2.4.17), coating the plate with a substance prepared by mixing 25 g of silica gel G with 50 ml of a mixture of 2.5 ml of glycerin and 47.5 ml of 0.1 M disodium edetate previously adjusted to pH 7.0 with dilute ammonia solution. After spreading the plate, allow it to stand at room temperature till it is dry (70 to 90 minutes). Mobile phase. The lower layer formed after shaking 200 ml of a mixture of 2 volumes of ethyl acetate, 2 volumes of dichloromethane and 1 volume of acetone with 25 ml of 0.1 M disodium edetate previously adjusted to pH 7.0 with dilute ammonia solution. Test solution. Extract a quantity of the oral powder containing 10 mg of Oxytetracycline Hydrochloride with 20 ml of methanol, centrifuge and use the supernatant liquid. Reference solution (a). A 0.05 per cent w/v solution of oxytetracycline hydrochloride RS in methanol. Reference solution (b). A solution containing 0.05 per cent w/v each of demethylchlortetracycline hydrochloride RS, oxytetracycline hydrochloride RS and tetracycline hydrochloride RS in methanol. Apply to the plate 1 l of each solution. After development, dry the plate in air, expose to the vapours of ammonia and examine in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with reference solution (a). The test is not valid unless the chromatogram obtained with reference solution (b) shows three clearly separated spots. B. To a quantity of the powder containing 0.4 mg of Oxytetracycline Hydrochloride add 5 ml of a 1 per cent w/v solution of sodium carbonate, shake and add 2 ml of diazotised sulphanilic acid solution; a light brown colour is produced. C. Shake a quantity of the powder containing 100 mg of Oxytetracyline Hydrochloride with 10 ml of 2 M nitric acid and filter. To the filtrate add activated charcoal to decolorise
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with pentobarbitone RS or with the reference spectrum of pentobarbitone. B. The residue obtained in the Assay melts at about 128 (2.4.21). C. When introduced on a platinum wire into a Bunsen burner flame, a golden yellow colour is imparted to the flame.
Tests
pH (2.4.24). 10.0 to 11.5. Isomer. To a volume of the injection containing 0.3 g of Pentobarbitone Sodium diluted, if necessary, to 5 ml with water add 0.3 g of 4-nitrobenzyl bromide dissolved in 10 ml of ethanol (95 per cent). Heat under a reflux condenser for 30 minutes, cool to 25, scratch the sides of the vessel with a glass rod if necessary to induce crystallisation, filter and wash the residue with five quantities, each of 5 ml, of water. Transfer the residue as completely as possible to a small flask, add 25 ml of ethanol (95 per cent) and heat under a reflux condenser for 10 minutes. Filter the hot solution, cool to 25 and scratch the sides of the vessel with a glass rod to induce
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IP 2007
PROGESTERONE INJECTION
crystallisation. Filter and wash the residue with two quantities, each of 5 ml, of water and dry at 105 for 30 minutes. The dried residue melts completely between 136 and 148 (2.4.21). Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. To an accurately measured volume containing about 0.5 g of Pentobarbitone Sodium diluted to 15 ml with water add 5 ml of 2 M hydrochloric acid, extract with 50 ml of ether and then with successive quantities, each of 25 ml, of ether until complete extraction is effected. Wash the combined extracts with two quantities, each of 5 ml, of water and wash the combined aqueous extracts with 10 ml of ether. Add the ether washings to the main ethereal extract, filter and wash the filter with ether. Evaporate the solvent and dry the residue to constant weight at 105. 1 g of the residue is equivalent to 1.097 g of C11H17N2NaO3.
Reference solution. A 0.1 per cent w/v of progesterone RS in a mixturance at 241 nm. Storage. Store protected from light.
Progesterone Injection
Progesterone Injection is a sterile solution of Progesterone in Ethyl Oleate or other suitable ester, in a suitable fixed oil or in any mixture of these. It may contain suitable alcohols. Progesterone Injection contains not less than 92.5 per cent and not more than 107.5 per cent of the stated amount of progesterone, C21H30O2.
Identification
Dissolve a volume containing 50 mg of Progesterone in 8 ml of light petroleum ( 40 to 60) and extract with three quantities, each of 8 ml, of a mixture of 7 volumes of glacial acetic acid and 3 volumes of water until the solution becomes turbid, allow to stand in ice for 2 hours and filter. The precipitate, after washing with water and drying at 105, complies with the following tests. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with progesterone RS or with the reference spectrum of progesterone. If the spectra are not concordant, prepare spectra using 5 per cent w/v solutions in chloroform IR.
Progesterone
O H3 C H3 C H O H H CH3
C21H30O2 Progesterone is pregn-4-en-3,20-dione.
Mol. Wt. 314.5
B. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Solvent mixture. A mixture of 90 volumes of acetone and 10 volumes of 1,2-propanediol. Mobile phase. A mixture of equal volumes of cyclohexane and light petroleum (40 to 60 ). Test solution. Dissolve 25 mg of the substance under examination in 10 ml of the solvent mixture. Reference solution (a). A 0.25 per cent w/v solution of progesterone RS in the solvent mixture. Reference solution (b). Mix equal volumes of the test solution and reference solution (a). Place the dry plate in a tank containing a shallow layer of the solvent mixture, allow the solvent mixture to ascend to the top, remove the plate from the tank and allow the solvent to evaporate. Use within 2 hours, with the flow of the mobile phase in the direction in which the aforementioned treatment was done. Apply to the plate 2 l of each solution. Allow the mobile phase to rise 12 cm. Dry the plate in a current of warm air, allow the solvent to evaporate, heat at 120 for 15 minutes and spray
Progesterone contains not less than 97.0 per cent and not more than 103.0 per cent of C21H30O2, calculated on the dried basis. Description. Colourless crystals or a white or almost white crystalline powder.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with progesterone RS or with the reference spectrum of progesterone. If the spectra are not concordant, prepare spectra using 5 per cent w/v solutions in chloroform IR. B. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 66 volumes of dichloromethane and 33 volumes of ethyl acetate. Test solution. Dissolve 0.1 g of the substance under examination in 100 ml of a mixture of 9 volumes of dichloromethane and 1 volume of methanol.
1553
PROMAZINE HYDROCHLORIDE
IP 2007
the hot plate with ethanolic sulphuric acid (20 per cent v/v). Heat at 120 for a further 10 minutes, allow to cool and examine in daylight and in ultraviolet light at 365 nm. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with reference solution (a). The principal spot in the chromatogram obtained with reference solution (b) appears as a single, compact spot.
Identification
Test A may be omitted if tests B, C, D and E are carried out. Tests B, C and D may be omitted if tests A and E are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with promazine hydrochloride RS or with the reference spectrum of promazine hydrochloride. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.001 per cent w/v solution in 0.01 M hydrochloric acid shows an absorption maximum at about 252 nm and a less well-defined maximum at about 302 nm; absorbance at about 252 nm, about 0.93. C. Dissolve 5 mg in 2 ml of sulphuric acid and allow to stand for 5 minutes; an orange colour is produced. D. Melting range (2.4.21). 177 to 181. E. Gives reaction A of chlorides (2.3.1).
Tests
Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. To an accurately measured volume containing about 50 mg of Progesterone add sufficient dichloromethane to produce 100.0 ml. Dilute 3.0 ml to 50.0 ml with dichloromethane. To 5.0 ml of the solution add 10 ml of isoniazid solution and sufficient methanol to produce 20.0 ml. Allow to stand for 45 minutes and measure the absorbance of the resulting solution at the maximum at about 380 nm (2.4.7), using as the blank 5 ml of dichloromethane treated in the same manner. Calculate the content of C 21H30 O2 from the absorbance obtained by repeating the procedure using a 0.003 per cent w/v solution of progesterone RS in dichloromethane and beginning at the words To 5.0 ml of the solution...... Storage. Store protected from light. If solid matter separates on standing, it should be redissolved by heating before use. Labelling. The label states (1) the composition of the solvent; (2) that the preparation is intended for veterinary use by subcutaneous or intramuscular injection only.
Tests
pH (2.4.24). 4.2 to 5.4, determined in a 5 per cent w/v solution. Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.6 g, dissolve in 100 ml of acetone. Titrate with 0.1 M perchloric acid, using 3 ml of a saturated solution of methyl orange in acetone as indicator. Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.03209 g of C17H20N2S,HCl. Storage. Store protected from light.
Promazine Hydrochloride
CH3 N CH3 N S , HCl
Promazine Injection
Promazine Hydrochloride Injection
Promazine Injection is a sterile solution of Promazine Hydrochloride in Water for Injections free from dissolved air and containing suitable buffering and stabilising agents. The solution is distributed in containers, the air in which is replaced by nitrogen or other suitable gas. Promazine Injection contains not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of promazine hydrochloride, C17H20N2S,HCl. Description. A colourless or almost colourless liquid.
C17H20N2S,HCl
Mol. Wt. 320.9
Promazine Hydrochloride is 10-(3-dimethylaminopropyl) phenothiazine hydrochloride. Promazine Hydrochloride contains not less than 99.0 per cent and not more than 101.0 per cent of C17H20N2S,HCl, calculated on the dried basis. Description. A white or almost white, crystalline powder; slightly hygroscopic.
Identification
A. To a volume containing 0.1 g of Promazine Hydrochloride add 20 ml of water and 2 ml of 10 M sodium hydroxide. Shake
1554
IP 2007
RAFOXANIDE
and extract the mixture with 25 ml of ether. Wash the ether extract with two quantities, each of 5 ml, of water, dry with anhydrous sodium sulphate and evaporate the ether. A 10 per cent w/v solution of the oily residue in chloroform complies with the following test. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with promazine hydrochloride RS, treated in the same manner. B. To a volume containing 5 mg of Promazine Hydrochloride add carefully 2 ml of sulphuric acid and allow to stand for 5 minutes; an orange colour is produced. C. To a volume containing 0.2 g of Promazine Hydrochloride add 1 ml of 1 M sodium hydroxide and extract with four quantities, each of 10 ml, of ether. Wash the combined extracts with 10 ml of water, remove the ether and dissolve the residue in 4 ml of methanol. Heat on a water-bath almost to boiling, immediately add 2 ml of a boiling 3.5 per cent w/v solution of picric acid in methanol and boil for 2 minutes. Cool in ice, filter, wash the crystals thrice with methanol, dissolve in 10 ml of hot methanol and repeat the crystallisation and washing. The rust-red crystals so obtained, after drying at 105 for 1 hour, melt at about 144 (2.4.21).
of 0.1 M hydrochloric acid, dilute to 100.0 ml with water and measure the absorbance of the resulting solution at the maximum at about 251 nm (2.4.7). Calculate the content of C17H20N2S,HCl taking 935 as the specific absorbance at 251 nm. Storage. Store protected from light.
Rafoxanide
O I N H OH I O Cl Cl
C19H11Cl2I2NO3
Mol. Wt. 626.0
Rafoxanide is N-[3-chloro-4-(4-chlorophenoxy)phenyl]-2hydroxy-3,5-diiodobenzamide. Rafoxanide contains not less than 98.0 per cent and not more than 101.0 per cent of C19H11Cl2I2NO3, calculated on the dried basis. Description. A greyish-white to brown powder.
Tests
pH (2.4.24). 4.4 to 5.2. Related substances. Carry out the test for identification of related substances in phenothiazines (2.3.5), using mobile phase A and applying separately to the plate 10 l of each of the following freshly-prepared solutions. Test solution. Dilute a volume of the injection with sufficient methanol to produce a solution containing the equivalent of 1.0 per cent w/v of Promazine Hydrochloride. Reference wsoslution (a). Dilute 1 volume of the test solution to 40 volumes with methanol. Reference wsoslution (b). Dilute 1 volume of the test solution to 200 volumes with methanol. Any secondary spot in the chromatogram obtained with the test solution is more intense than the spot in the chromatogram obtained with reference solution (a) and not more than one such spot is more intense than the spot in the chromatogram obtained with reference solution (b). Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. Protect the solutions from light throughout the procedure. To an accurately measured volume containing about 50 mg of Promazine Hydrochloride, add 5 ml of 2 M hydrochloric acid and sufficient water to produce 1000.0 ml. To 10.0 ml add 10 ml
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with rafoxanide RS or with the reference spectrum of rafoxanide. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.004 per cent w/v solution in 0.1 M methanolic hydrochloric acid shows absorption maxima at about 280 nm and at 335 nm; absorbance at about 280 nm, about 0.97 and at about 335 nm, about 0.59. C. Burn 20 mg by the oxygen-flask method (2.3.34), using 5 ml of 2 M sodium hydroxide as the absorbing liquid, and dilute to 25 ml with water. To 5 ml add 1 ml of silver nitrate solution; a yellow precipitate is produced; add 5 ml of 5 M ammonia, shake, filter, and acidify the filtrate with nitric acid; a white precipitate is produced. D. Shake 10 mg with 10 ml of ethanol (80 per cent) and add 0.1 ml of ferric chloride test solution; a violet colour is produced. E. Melts at about 175 (2.4.21).
Tests
Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254.
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RAFOXANIDE VETERINARY ORAL SUSPENSION
IP 2007
NOTE Carry out the test in subdued light and use freshly prepared solution5. Mobile phase. A mixture of 170 volumes of dichloromethane, 30 volumes of methanol and 2 volumes of strong ammonia solution. Test solution. Dissolve 2 g of the substance under examination in 100 ml in dichloromethane. Reference solution. A 0.010 per cent w/v of rafoxanide RS in dichloromethane. Apply to the plate 5 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution. Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 0.5 per cent, determined on 1.0 g by drying in an oven at 90 at a pressure not exceeding 0.7 kPa for 2 hours. Assay. To 50 ml of dioxan add 1 ml of phenolphthalein solution, replace the air in the flask with nitrogen and titrate with 0.1 M sodium hydroxide. Weigh accurately about 1.25 g, dissolve it in the mixture and again titrate with 0.1 M sodium hydroxide. The difference between the titrations represents the amount of 0.1 M sodium hydroxide required. 1 ml of 0.1 M sodium hydroxide is equivalent to 0.06260 g of C19H11Cl2I2NO3. Storage. Store protected from light.
B. In addition to the absorbance at about 335 nm, measure the absorbance at about 280 nm (2.4.7), of the final solution obtained in the Assay. The ratio of the absorbance at about 280 nm to that at about 335 nm is 1.59 to 1.69.
Tests
Other tests. Complies with requirements stated under Veterinary Oral Liquids. Assay. Weigh accurately a quantity of the well-mixed suspension containing about 0.12 g of Rafoxanide in a stoppered 50-ml test tube and add 15 ml of 0.1 M sodium hydroxide and 15 ml of ether. Shake for 5 minutes and centrifuge. Remove the ether layer and repeat the extraction with three further quantities, each of 15 ml, of ether. Dilute the combined ether solutions to 250.0 ml with ether and mix. Dilute 5.0 ml of this solution to 100.0 ml with 0.1 M methanolic hydrochloric acid, mix and measure the absorbance of the resulting solution at about 335 nm (2.4.7). Calculate the content of C19H11Cl2I2NO3 taking 149 as the specific absorbance at 335 nm. Determine the weight per ml of the suspension (2.4.29), and calculate the content of rafoxanide, weight in volume.
Ronidazole
O2N CH3 N O N
C6H8N4O4 Mol. Wt. 200.2
O NH2
Rafoxanide Veterinary Oral Suspension

Rafoxanide Suspension; Rafoxanide Veterinary Mixture; Rafoxanide Mixture
Rafoxanide Veterinary Oral Suspension is an aqueous suspension of Rafoxanide containing suitable suspending and dispersing agents and antimicrobial preservatives. Rafoxanide Veterinary Oral Suspension contains not less than 90.0 per cent and not more than 110.0 per cent of rafoxanide, C19H11Cl2I2NO3.
Ronidazole is 1-methyl-2-[(carbamoyloxy)methyl]-5nitroimidazole. Ronidazole contains not less than 98.5 per cent and not more than 101.0 per cent of C6H8N4O4, calculated on the anhydrous basis. Description. A white to yellowish-brown powder; odourless or almost odourless.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with ronidazole RS or with the reference spectrum of ronidazole. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.002 per cent w/v solution in 0.1 M methanolic hydrochloric acid shows an absorption maximum only at about 270 nm; absorbance at about 270 nm, about 0.64. C. Melts at about 167 (2.4.21).
Identification
A. Evaporate a volume containing 0.2 g of Rafoxanide to dryness on a water-bath and heat the residue over a Bunsen burner flame; the vapours turn moistened starch-iodide paper blue.
1556
IP 2007
SERUM GONADOTROPHIN FOR VETERINARY USE
Tests
Appearance of solution. A 0.5 per cent w/v solution in methanol is not more intensely coloured than reference solution YS6 (2.4.1). (1-Methyl-5-nitroimidazol-2-yl)methano .Determine by thinlayer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 80 volumes of toluene, 5 volumes of methanol and 5 volumes of glacial acetic acid. Test solution. Dissolve 1 g of the substance under examination in 100 ml in acetone. Reference solution. A 0.0050 per cent w/v of (1-methyl-5-nitroimidazol-2-yl)methanol RS in acetone. Apply to the plate 20 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution corresponding to (1-methyl-5-nitroimidazol-2 -yl)methanol RS is not more intense than the spot in the chromatogram obtained with the reference solution. Sulphated ash (2.3.18). Not more than 0.1 per cent. Water (2.3.43). Not more than 0.5 per cent , determined on 5 g. Assay. Weigh accurately about 0.3 g, dissolve in 50 ml of anhydrous glacial acetic acid. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.4.25). Carry out a blank titration. 1 ml of 0.1 M perchloric acid is equivalent to 0.02002 g of C6H8N4O4. Storage. Store proted from light.
B. When examined in the range 230 nm to 360 nm (2.4.7), the final solution obtained in the Assay shows an absorption maximum only at about 281 nm.
Tests
(1-Methyl-5-nitroimidazol-2-yl)methanol .Determine by thinlayer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 80 volumes of toluene, 5 volumes of methanol and 5 volumes of glacial acetic acid. Test solution. Shake a quantity of the powder containing 0.1 g of Ronidazole with 10 ml of acetone for 15 minutes and filter. Reference solution. A 0.0050 per cent w/v of (1-methyl-5-nitroimidazol-2-yl)methanol RS in acetone. Apply to the plate 20 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. Any secondary spot in the chromatogram obtained with the test solution corresponding to (1-methyl-5-nitroimidazol-2-yl)methanol RS is not more intense than the spot in the chromatogram obtained with the reference solution. Other tests. Complies with the tests stated under Veterinary Oral Powders. Assay. Weigh accurately a quantity of powder containing 2 g of Ronidazole, dissolve in 450 ml of water and add sufficient water to produce 500.0 ml. Dilute 5.0 ml of this solution to 100.0 ml with 0.1 M hydrochloric acid and measure the absorbance of the resulting solution at the maximum at about 281 nm (2.4.7). Calculate the content of C6H8N4O4 taking 279 as the specific absorbance at 281 nm. Storage. Store proted from light.
Ronidazole Veterinary Oral Powder

Ronidazole Veterinary Oral Powder is a mixture of Ronidazole with suitable diluents. Ronidazole Veterinary Oral Powder contains not less than 92.5 per cent and not more than 107.5 per cent of the stated amount of ronidazole, C6H8N4O4.
Serum Gonadotrophin for Veterinary Use

Equine Serum Gonadotrophin for Veterinary Use
Serum Gonadotrophin for Veterinary Use is a dry preparation of a glycoprotein fraction, obtained from the serum or plasma of pregnant mares in their 60th to 75th day of pregnancy, which stimulates the formation of follicles and induces leutinising activity. Serum Gonadotrophin for Veterinary Use contains not less than 1000 Units per mg, calculated on the anhydrous basis. Description. A white or pale grey, amorphous powder.
Identification
A. Shake a quantity of the powder containing 0.1 g of Ronidazole with 10 ml of acetone for 15 minutes, filter and evaporate the filtrate to dryness. The residue complies with the following test. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with ronidazole RS or with the reference spectrum of ronidazole.
Identification
Causes enlargement of the ovaries of immature female rats when administered as directed in the Assay.
1557
SERUM GONADOTROPHIN INJECTION FOR VETERINARY USE
IP 2007
Tests
Water (2.3.43). Not more than 10.0 per cent, determined on 80 mg. Assay. Carry out the biological assay of serum gonadotrophin described below. The potency of serum gonadotrophin for veterinary use is determined by comparing its effect in increasing the weight of the ovaries of immature rats with that of the Standard Preparation of serum gonadotrophin under the conditions of the following method of assay. Standard Preparation The Standard Preparation is the 2nd International Standard for serum gonadotrophin, equine, for bioassay, established in 1966, consisting of the freeze-dried active principle from the serum of pregnant mares, with lactose (supplied in ampoules containing 1600 Units), or other suitable preparation the potency of which has been determined in relation to the International Standard. Method Test animals. Use immature female rats of the same strain, 21 to 28 days old, differing in age by not more than 3 days and of approximately equal weights such that the difference between the heaviest and the lightest rat is not more than 10 g. Assign the rats at random to six equal groups of not less than five animals. If sets of six litter-mates are available, allot one litter-mate from each set at random to each group and mark according to the litter. Procedure. Choose three doses of the Standard Preparation and three doses of the preparation under examination such that the smallest dose is sufficient to produce a positive response in some of the rats and the largest dose does not produce a maximal response in all of the rats. Use doses in geometric progression. As an initial approximation total doses of 8, 12 and 18 Units may be tried although the dose will depend on the sensitivity of the animals used, which may vary widely. Dissolve separately the total quantities of the preparation under examination and of the Standard Preparation corresponding to the doses to be used in sufficient of a sterile saline solution containing 1 mg of bovine albumin per ml such that each single dose may be administered by the injection of 6 equally-divided portions, in the same volume of about 0.2 ml. Store the solutions at a temperature 2 to 8. Inject subcutaneously into each rat the dose allocated to its group. Repeat the injections 18, 21, 24, 42 and 48 hours after the first injection. Kill the rats between 40 hours and 72 hours after the last injection and remove the ovaries. Remove any extraneous fluid and tissue and immediately weigh the two ovaries from each rat.
Calculate the result of the assay by standard statistical methods using the combined weight of the two ovaries of each animal as the response. Limits of error - The estimated potency is not less than 80 per cent and not more than 125 per cent of the stated potency. The fiducial limits of error (P = 0.95) of the estimated potency are not less than 64 per cent and not more than 156 per cent of the stated potency. Serum Gonadotrophin for Veterinary Use intended for use in the manufacture of parenteral preparations without a further appropriate procedure for the removal of pyrogens complies with the following additional requirement. Pyrogens. Complies with the test for pyrogens (2.2.8), using per kg of the rabbits weight 1 ml of a solution in sodium chloride injection containing 500 Units per ml. Serum Gonadotrophin for Veterinary Use intended for use in the manufacture of parenteral preparations without a further appropriate sterilisation procedure complies with the following additional requirement. Sterility (2.2.11). Complies with the test for sterility. Storage. Store protected from moisture and light in a refrigerator (2 to 8). If the contents are sterile, the containers should be sterile, tamper-evident and sealed so as to exclude micro-organisms. Labelling. The label states (1) the number of Units per mg; (2) the total number of Units in the container; (3) the date after which the material is not intended to be used; (4) the storage conditions; (5) whether or not it is intended for use in the manufacture of parenteral preparations.
Serum Gonadotrophin Injection for Veterinary Use

Serum Gonadotrophin Injection for Veterinary Use is a sterile material consisting of Serum Gonadotrophin for Veterinary Use with or without buffering agents and other excipients. It is filled in a sealed container. The injection is constituted by dissolving the contents of the sealed container in the requisite amount of sterile Water for Injections, immediately before use. The constituted solution complies with the requirements for Clarity of solution and Particulate matter stated under Parenteral Preparations (Injections). Storage. The constituted solution should be used immediately after preparation but, in any case, within the period recommended by the manufacturer.
1558
IP 2007
SPECTINOMYCIN HYDROCHLORIDE
Serum Gonadotrophin Injection for veterinary Use contains not less than 80.0 per cent and not more than 125.0 per cent of the stated potency. The contents of the sealed container comply with the requirements stated under Parenteral Preparations (Powders for Injection) and with the following requirements.
Identification
Causes enlargement of the ovaries of immature female rats when administered as directed in the Assay.
Tests
Appearance of solution. A solution containing 5000 Units per ml (solution A) is clear (2.4.1), and colourless (2.4.1). pH (2.4.24). 6.0 to 8.0, determined on solution A. Water (2.3.43). Not more than 10.0 per cent, determined on 80 mg. Assay. Carry out the biological assay of serum gonadotrophin described below. The potency of serum gonadotrophin for veterinary use is determined by comparing its effect in increasing the weight of the ovaries of immature rats with that of the Standard Preparation of serum gonadotrophin under the conditions of the following method of assay. Standard Preparation The Standard Preparation is the 2nd International Standard for serum gonadotrophin, equine, for bioassay, established in 1966, consisting of the freeze-dried active principle from the serum of pregnant mares, with lactose (supplied in ampoules containing 1600 Units), or other suitable preparation the potency of which has been determined in relation to the International Standard. Test animals. Use immature female rats of the same strain, 21 to 28 days old, differing in age by not more than 3 days and of approximately equal weights such that the difference between the heaviest and the lightest rat is not more than 10 g. Assign the rats at random to six equal groups of not less than five animals. If sets of six litter-mates are available, allot one litter-mate from each set at random to each group and mark according to the litter. Procedure. Choose three doses of the Standard Preparation and three doses of the preparation under examination such that the smallest dose is sufficient to produce a positive response in some of the rats and the largest dose does not produce a maximal response in all of the rats. Use doses in geometric progression. As an initial approximation total doses of 8, 12 and 18 Units may be tried although the dose will depend on the sensitivity of the animals used, which may
vary widely. Dissolve separately the total quantities of the preparation under examination and of the Standard Preparation corresponding to the doses to be used in sufficient of a sterile saline solution containing 1 mg of bovine albumin per ml such that each single dose may be administered by the injection of 6 equally-divided portions, in the same volume of about 0.2 ml. Store the solutions at a temperature 2 to 8. Inject subcutaneously into each rat the dose allocated to its group. Repeat the injections 18, 21, 24, 42 and 48 hours after the first injection. Kill the rats between 40 hours and 72 hours after the last injection and remove the ovaries. Remove any extraneous fluid and tissue and immediately weigh the two ovaries from each rat. Calculate the result of the assay by standard statistical methods using the combined weight of the two ovaries of each animal as the response. Limits of error - The estimated potency is not less than 80 per cent and not more than 125 per cent of the stated potency. The fiducial limits of error (P = 0.95) of the estimated potency are not less than 64 per cent and not more than 156 per cent of the stated potency. Pyrogens. Complies with the test for pyrogens (2.2.8), using per kg of the rabbits weight 1 ml of a solution in sodium chloride injection containing 500 Units per ml. Storage. Store protected from light in a refrigerator (2 to 8) . Labelling. The label states the number of Units contained in the sealed container.
Spectinomycin Hydrochloride
H HO H H N O O H O HO NH O
H3C
CH3 , 2HCl, 5H2O
HO H3 C
C14H24N2O7,2HCl,5H2O
Mol. Wt. 495.4
Spectinomycin Hydrochloride is [2R(2,4a,5a,6,7,8,9,9a,10a)]-decahydro-4a,7,9trihydroxy-2-methyl-6,8-bis(methylamino)-4H-pyrano[2,3b][1,4]benzodioxin-4-one dihydrochloride pentahydrate. Spectinomycin Hydrochloride contains not less than 95.0 per cent and not more than 100.5 per cent of C14H24N2O7,2HCl, calculated on the anhydrous basis. Description. A white or almost white, crystalline powder.
1559
SPECTINOMYCIN INJECTION
IP 2007
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with spectinomycin hydrochloride RS or with the reference spectrum of spectinomycin hydrochloride. B. Gives reaction A of chlorides (2.3.1).
ml of a solution containing 0.15 per cent w/v of phenazone (internal standard) in dimethylformamide and 2.0 ml of hexamethyldisilazane, shake intermittently for 1 hour and dilute to 20.0 ml with dimethylformamide. Chromatographic system a glass column 1.5 m x 4 mm, packed with acid-washed, silanised diatomaceous support (100 to 120 mesh) coated with 3 per cent w/w of phenylmethylsilicone fluid (50 per cent phenyl), temperature: column 200, inlet port 200 and detector 230, flow rate. 45 ml per minute of the carrier gas. Inject the chosen volumes of test solutions (a) and (b). The test is not valid unless the resolution factor between the peak due to the internal standard and the principal peak in the chromatogram obtained with test solution (a) is not less than 8.0. Inject alternately test solution (b) and the reference solution. Calculate the content of C14H24N2O7,2HCl. Spectinomycin Hydrochloride intended for use in the manufacture of parenteral preparations without a further appropriate procedure for the removal of bacterial endotoxins complies with the following additional requirement. Bacterial endotoxins (2.2.3). Not more than 0.09 Endotoxin Unit per mg determined in a 0.42 per cent w/v solution of sodium bicarbonate. Spectinomycin Hydrochloride intended for use in the manufacture of parenteral preparations without a further appropriate sterilisation procedure complies with the following additional requirement. Sterility (2.2.11). Complies with the test for sterility. Storage. Store protected from moisture, at a temperature not exceeding 30. If the substance is sterile, the container should be sterile, tamper-evident and sealed so as to exclude micro-organisms. Labelling. The label states (1) the date after which the material is not intended to be used; (2) the storage conditions; (3) whether or not it is intended to be used for manufacture of parenteral preparations.
Tests
Appearance of solution. A 10 per cent w/v solution is clear (2.4.1), and colourless (2.4.1). pH (2.4.24). 3.8 to 5.6, determined in a 10 per cent w/v solution. Specific optical rotation (2.4.22). +15.0 to +21.0, determined in a 10 per cent w/v solution within 20 minutes of preparation, on the anhydrous basis. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 50 volumes of 1-propanol, 40 volumes of water, 5 volumes of glacial acetic acid and 5 volumes of pyridine. Test solution. Dissolve 2 g of the substance under examination in 100 ml water. Reference solution. A 0.020 per cent w/v solution of the substance under examination in water. Apply to the plate 10 l of each solution. After development, dry the plate in air and spray with a 5 per cent w/v solution of potassium permanganate. Allow the plate to stand for 2 to 3 minutes. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with the reference solution (1 per cent). Sulphated ash (2.3.18). Not more than 1.0 per cent w/v. Water (2.3.43). 16.0 to 20.0 per cent , determined on 0.2 g. Assay. Determine by gas chromatography (2.4.13). NOTE Use the solutions within 1 hour after preparation. Test solution (a). Take 60 mg of the substance under examination in a glass-stoppered conical flask, add 10.0 ml of dimethylformamide and 2.0 ml of hexamethyldisilazane, shake intermittently for 1 hour and dilute to 20.0 ml with dimethylformamide. Test solution (b). Take 60 mg of the substance under examination in a glass-stoppered conical flask, add 10.0 ml of a solution containing 0.15 per cent w/v of phenazone (internal standard) in dimethylformamide and 2.0 ml of hexamethyldisilazane, shake intermittently for 1 hour and dilute to 20.0 ml with dimethylformamide. Reference solution. Take 60 mg of the spectinomycin hydrochloride RS in a glass-stoppered conical flask, add 10.0
Spectinomycin Injection
Spectinomycin Hydrochloride Injection
Spectinomycin Injection is a sterile material consisting of Spectinomycin Hydrochloride with or without auxiliary substances. It is filled in a sealed container.
1560
IP 2007
SPECTINOMYCIN INJECTION
The injection is constituted by suspending the contents of the sealed container in the requisite amount of sterile Water for Injections, immediately before use. Storage. The constituted suspension should be used immediately after preparation but, in any case, within the period recommended by the manufacturer. Spectinomycin Injection contains not less than 90.0 per cent and not more than 110.0 per cent the stated amount of spectinomycin, C14H24N2O7. The contents of the sealed container comply with the requirements stated under Parenteral Preparations (Powders for Injection) and with the following requirements.
Assay. Determine by gas chromatography (2.4.13). NOTE Use the solutions within 1 hour after preparation. Test solution (a). Weigh and mix the contents of the 10 containers. To an accurately weighed quantity containing about 60 mg of Spectinomycin Hydrochloride in a glass-stoppered conical flask, add 10.0 ml of dimethylformamide and 2.0 ml of hexamethyldisilazane, shake intermittently for 1 hour and dilute to 20.0 ml with dimethylformamide. Test solution (b). To an accurately weighed quantity containing about 60 mg of Spectinomycin Hydrochloride in a glass-stoppered conical flask, add 10.0 ml of a solution containing 0.15 per cent w/v of phenazone (internal standard) in dimethylformamide and 2.0 ml of hexamethyldisilazane, shake intermittently for 1 hour and dilute to 20.0 ml with dimethylformamide. Reference solution. T0 about 60 mg, accurately weighed, of spectinomycin hydrochloride RS in a glass-stoppered conical flask, add 10.0 ml of a solution containing 0.15 per cent w/v of phenazone (internal standard) in dimethylformamide and 2.0 ml of hexamethyldisilazane, shake intermittently for 1 hour and dilute to 20.0 ml with dimethylformamide. Chromatographic system a glass column 1.5 m x 4 mm, packed with acid-washed, silanised diatomaceous support (100 to 120 mesh) coated with 3 per cent w/w of phenylmethylsilicone fluid (50 per cent phenyl), temperature: column 200, inlet port 200 and detector 230, flow rate. 45 ml per minute of the carrier gas. Inject the chosen volumes of test solutions (a) and (b). The test is not valid unless the resolution factor between the peak due to the internal standard and the principal peak in the chromatogram obtained with test solution (a) is not less than 8.0. Inject alternately test solution (b) and the reference solution. Calculate the content of C14H24N2O7,2HCl. Storage. Use the injection immediately after preparation but, in any case, within the period recommended by the manufacturer provided it is prepared and stored in accordance with the manufacturers instructions. Labelling. The label states the strength in terms of the equivalent amount of spectinomycin.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with spectinomycin hydrochloride RS or with the reference spectrum of spectinomycin hydrochloride. B. Gives reaction A of chlorides (2.3.1).
Tests
pH (2.4.24). 4.0 to 7.0, determined in a suspension of the contents of a sealed container in the volume of the liquid stated on the label. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 50 volumes of 1-propanol, 40 volumes of water, 5 volumes of glacial acetic acid and 5 volumes of pyridine. Test solution. Prepare a solution containing the equivalent of 1.4 per cent w/v of spectinomycin in water. Reference solution. Prepare a solution containing the equivalent of 0.014 per cent w/v of spectinomycin in water. Apply to the plate 10 l of each solution. After development, dry the plate in air and spray with a 5 per cent w/v solution of potassium permanganate. Allow the plate to stand for 2 to 3 minutes. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution. Water (2.3.43). Not more than 20.0 per cent, determined on 0.2 g. Bacterial endotoxins (2.2.3). Not more than 0.09 Endotoxin Unit per ml, determined on a solution prepared by dissolving the contents in a solution containing 0.05 M sodium bicarbonate in water BET to give a solution containing the equivalent of 1 mg of spectinomycin per ml (solution A), and using the maximum valid dilution of solution A calculated from the declared sensitivity of the lysate used in the test.
1561
SPIRAMYCIN
IP 2007
Spiramycin
O CH3 CHO HO O OR O CH3 N CH3 CH3 O O OH CH3 OH CH3 CH3 N CH CH3 3 Spiramycin I R = H Spiramycin II R = COCH3 Spiramycin III R = COCH2CH3
Mobile phase. A mixture of 45 volumes of ethyl acetate, 40 volumes of a 15 per cent w/v of ammonium acetate previously adjusted to pH 9.6 with 10 M sodium hydroxide and 20 volumes of 2-propanol. Use the upper layer. Test solution. Dissolve 0.4 g of the substance under examination in 100 ml of methanol. Reference solution (a). A 0.40 per cent w/v solution of spiramycin RS in methanol. Mol. Wt. 843.1 Reference solution (b). A 0.040 per cent w/v solution of spiramycin RS in methanol. Reference solution (c). A 0.020 per cent w/v solution of spiramycin RS in methanol. Reference solution (d). A 0.0080 per cent w/v solution of spiramycin RS in methanol. Reference solution (e). A 0.40 per cent w/v of erythromycin RS in methanol. Apply to the plate 5 l of each solution. After development, dry the plate in air and spray with ethanolic anisaldehyde solution and heat at 110 for 5 minutes. In the chromatogram obtained with reference solution (a) there are two spots with Rf values slightly higher than that of the principal spot; the spot nearer the principal spot corresponds to the monoacetate ester and the one farther from the principal spot corresponds to the monopropionate ester. In the chromatogram obtained with the test solution any spot corresponding to the monoacetate is not more intense than the spot in the chromatogram obtained with reference solution (c), any spot corresponding to the monopropionate is not more intense than the spot in the chromatogram obtained with reference solution (b) and any other secondary spot is not more intense than the spot in the chromatogram obtained with reference solution (d). Sulphated ash (2.3.18). Not more than 0.1 per cent w/v. Loss on drying (2.4.19). Not more than 3.5 per cent, determined on 0.5 g by drying over phosphorus pentoxide at 80 at a pressure not exceeding 0.7 kPa for 6 hours. Assay. Carry out the microbiological assay of antibiotics (2.2.10), Method A. Storage. Store protected from moisture.
H3CO O H3CO O
C43H74N2O14
Spiramycin is a mixture comprised primarily of spiramycin I produced by Streptomyces ambofacien from soil of northern France. Spiramycin contains not less than 3900 Units per mg, calculated on the dried basis. Description. A white or slightly yellowish powder; odour, slight; slightly hygroscopic.
Identification
A. When examined in the range 220 nm to 360 nm (2.4.7), a 0.001 per cent w/v solution in methanol shows an absorption maximum only at about 232 nm; absorbance at about 232 nm, about 0.34. B. In the test for Related substances the principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with reference solution (a). If in the chromatogram obtained with the test solution one or two additional spots appear with Rf values slightly higher than that of the principal spot, these spots correspond to the secondary spots in the chromatogram obtained with reference solution (a) but differ from the spots in the chromatogram obtained with reference solution (e). C. Dissolve 0.5 g in a mixture of 10 ml of 0.05 M sulphuric acid and 25 ml of water. Adjust the pH to about 8 by addition of 0.1 M sodium hydroxide and dilute to 50 ml with water. To 5 ml of the resulting solution add 2 ml of a mixture of 1 volume of water and 2 volumes of sulphuric acid; a brown colour is produced.
Tests
pH (2.4.24). 8.5 to 10.5, determined in a solution prepared by dissolving 0.5 g in 5 ml of methanol and diluting to 100 ml with carbon dioxide-free water. Specific optical rotation (2.4.22). 80.0 to 85.0, determined in a 2 per cent w/v solution in 0.2 M acetic acid. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals, Method A (20 ppm). Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G.
Sulphadiazine and Trimethoprim Injection

Trimethoprim and Sulphadiazine Injection; Co-trimazine Injection
Sulphadiazine and Trimethoprim Injection is a sterile suspension in Water for Injections containing Sulphadiazine and Trimethoprim in the proportion of five parts to one part respectively.
1562
IP 2007
SULPHADIAZINE AND TRIMETHOPRIM VETERINARY ORAL POWDER
Sulphadiazine and Trimethoprim Injection contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amounts of sulphadiazine, C 10H 10N 4O 2 S and of trimethoprim, C14H18N4O3.
Identification
A. When examined in the range 230 nm to 360 nm (2.4.7), the solution obtained in the Assay for trimethoprim shows an absorption maximum only at about 271 nm. B. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 75 volumes of ethyl acetate, 15 volumes of dimethylformamide and 5 volumes of water. Test solution. Add 4 ml of hydrochloric acid to 2.5 ml of the well-mixed contents of the container and dilute to 50 ml with 1.4 M methanolic ammonia. Reference solution (a). A 2.0 per cent w/v of sulphadiazine RS in 1.4 M methanolic ammonia. Reference solution (b). A 0.4 per cent w/v of trimethoprim RS in 1.4 M methanolic ammonia. Apply to the plate 1 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. One of the principal spots in the chromatogram obtained with the test solution corresponds to the principal spot in the chromatogram obtained with reference solution (a) and the other corresponds to the principal spot in the chromatogram obtained with reference solution (b). C. To 5 ml of the filtrate obtained in the Assay for sulphadiazine add 10 ml of water and 5 ml of thiobarbituric acid-citrate buffer. Mix and heat on a water-bath for 30 minutes; a pink colour is produced.
200.0 ml with water. Dilute 10.0 ml of this solution to 100.0 ml with water. To 3.0 ml of the resulting solution add 1 ml of 2 M hydrochloric acid and 1 ml of a 0.1 per cent w/v solution of sodium nitrite and allow to stand for 2 minutes. Add 1 ml of a 0.5 per cent w/v solution of ammonium sulphamate and allow to stand for 3 minutes. Add 1 ml of a 0.1 per cent w/v solution of N-(1-naphthyl)ethylenediamine dihydrochloride, allow to stand for 10 minutes, add sufficient water to produce 25.0 ml and measure the absorbance of the resulting solution at the maximum at about 538 nm (2.4.7). Calculate the content of C10H10N4O2S from the absorbance obtained by carrying out the procedure simultaneously, using 3.0 ml of a solution prepared by dissolving 200 mg of sulphadiazine RS in 50 ml of 0.1 M sodium hydroxide, adding sufficient water to produce 200.0 ml, diluting 5.0 ml to 250.0 ml with water and beginning at the words add 1 ml of 2 M hydrochloric acid......... For trimethoprim Extract the dichloromethane solution reserved in the Assay for sulphadiazine with three quantities, each of 100 ml, 50 ml and 50 ml, of 1 M acetic acid and dilute the combined extracts to 500.0 ml with 1 M acetic acid. To 5.0 ml add 35 ml of 1 M acetic acid and sufficient water to produce 200.0 ml and measure the absorbance of the resulting solution at the maximum at about 271 nm (2.4.7). Calculate the content of C14H18N4O3 taking 204 as the specific absorbance at 271 nm. Labelling. The label states the content of Sulphadiazine and Trimethoprim in a suitable dose-volume.
Sulphadiazine and Trimethoprim Veterinary Oral Powder

Trimethoprim and Sulphadiazine Veterinary Oral Powder; Sulphadiazine and Trimethoprim Dispersible Powder; Co-trimazine Veterinary Oral Powder
Sulphadiazine and Trimethoprim Veterinary Oral Powder consists of Sulphadiazine and Trimethoprim in the proportion of five parts to one part respectively, mixed with suitable wetting, dispersing and suspending agents. Sulphadiazine and Trimethoprim Veterinary Oral Powder contains not less than 92.5 per cent and not more than 107.5 per cent of the stated amounts of sulphadiazine, C10H10N4O2S, and of trimethoprim, C14H18N4O3.
Tests
pH (2.4.24). 10.0 to 10.5. Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. For sulphadiazine Disperse the trimethoprim evenly throughout the injection solution by gently inverting the container several times without foam formation. Transfer an accurately measured quantity of the injection containing 2 g of Sulphadiazine to a separating funnel containing 50 ml of 0.1 M sodium hydroxide and extract with two quantities, each of 100 ml and 50 ml of dichloromethane, washing the extract with the same 25-ml quantity of 0.1 M sodium hydroxide. Reserve the combined dichloromethane extracts for the assay for trimethoprim. Dilute the combined aqueous solutions and washings to 250.0 ml with water and filter, and dilute 5.0 ml of the filtrate to
Identification
Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 75 volumes of ethyl acetate, 15 volumes of dimethylformamide and 5 volumes of water. Test solution (a). The supernatant liquid obtained by shaking a quantity of the powder containing 0.2 g of Sulphadiazine
1563
SULPHADIAZINE AND TRIMETHOPRIM VETERINARY ORAL SUSPENSION
IP 2007
with sufficient 1.4 M methanolic ammonia to produce 100 ml and centrifuging. Test solution (b). The supernatant liquid obtained by shaking a quantity of the powder containing 0.2 g of Trimethoprim with sufficient 1.4 M methanolic ammonia to produce 100 ml and centrifuging. Reference solution (a). A 0.2 per cent w/v solution of sulphadiazine RS in 1.4 M methanolic ammonia. Reference solution (b). A 0.2 per cent w/v solution of trimethoprim RS in 1.4 M methanolic ammonia. Apply to the plate 5 l of each solution. After development, dry the plate in air and spray with a 0.1 per cent w/v solution of 4-dimethylaminobenzaldehyde in a mixture of 1 ml of hydrochloric acid and 100 ml of ethanol (95 per cent), allow to dry and spray with dilute potassium iodobismuthate solution. The spot in the chromatogram obtained with test solution (a) having Rf value of about 0.7 corresponds to the principal spot in the chromatogram obtained with reference solution (a). The spot in the chromatogram obtained with test solution (b) having Rf value of about 0.3 corresponds to the principal spot in the chromatogram obtained with reference solution (b).
2.0 ml of a 0.0025 per cent w/v solution of sulphadiazine RS in 0.0005 M sodium hydroxide and beginning at the words add 0.5 ml of 4 M hydrochloric acid........ For trimethoprim - Extract the combined dichloromethane extracts from the Assay for sulphadiazine with four quantities, each of 50 ml, of a 5 per cent v/v solution of 6 M acetic acid; wash the combined aqueous extracts with 5 ml of dichloromethane, discard the dichloromethane layer and dilute to 250.0 ml with a 5 per cent v/v solution of 6 M acetic acid. Dilute 20.0 ml to 100.0 ml with water and determine the absorbance of the resulting solution at the maximum at about 271 nm (2.4.7). Calculate the content of C14H18N4O3 taking 204 as the specific absorbance at 271 nm.
Sulphadiazine and Trimethoprim Veterinary Oral Suspension

Sulphadiazine and Trimethoprim Mixture; Trimethoprim and Sulphadiazine Veterinary Oral Suspension; Co-trimazine Oral Suspension; Co-trimazine Mixture
Sulphadiazine and Trimethoprim Veterinary Oral Suspension is a suspension of Sulphadiazine and Trimethoprim in the proportion of five parts to one part respectively, containing suitable suspending and dispersing agents. It may contain suitable antimicrobial preservatives. Sulphadiazine and Trimethoprim Veterinary Oral Suspension contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amounts of sulphadiazine, C10H10N4O2S, and of trimethoprim, C14H18N4O3.
Tests
Other tests. Complies with the tests stated under Veterinary Oral Powders. Assay. For sulphadiazine - Weigh accurately a quantity of the powder containing about 0.125 g of Sulphadiazine, transfer into a separator containing 20 ml of 0.1 M sodium hydroxide and extract with four quantities, each of 50 ml, of dichloromethane. Wash each dichloromethane extract with the same two quantities, each of 10 ml, of 0.1 M sodium hydroxide. Combine the aqueous washings and the aqueous layer from the separator and reserve the combined dichloromethane extracts for the Assay for trimethoprim. Dilute the combined aqueous solutions to 250.0 ml with water, filter and dilute 10.0 ml of the filtrate to 200.0 ml with water. To 2.0 ml of the resulting solution add 0.5 ml of 4 M hydrochloric acid and 1 ml of a 0.1 per cent w/v solution of sodium nitrite and allow to stand for 2 minutes. Add 1 ml of a 0.5 per cent w/ v solution of ammonium sulphamate and allow to stand for 3 minutes. Add 1 ml of a 0.1 per cent w/v solution of N-(1-naphthyl)ethylenediamine dihydrochloride, allow to stand for 10 minutes. Dilute the solution to 25.0 ml with water and measure the absorbance of the resulting solution at the maximum at about 538 nm (2.4.7), using as the blank a solution prepared in the same manner using 2 ml of water and beginning at the words add 0.5 ml of 4 M hydrochloric acid......... Calculate the content of C10H10N4O2S from the absorbance obtained by carrying out the procedure simultaneously, with
Identification
Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 75 volumes of ethyl acetate, 15 volumes of dimethylformamide and 5 volumes of water. Test solution (a). A dilution of the oral suspension in 1.4 M methanolic ammonia containing the equivalent of 0.2 per cent w/v of Sulphadiazine. Test solution (b). A dilution of the oral suspension in 1.4 M methanolic ammonia containing the equivalent of 0.2 per cent w/v of Trimethoprim. Reference solution (a). A 0.2 per cent w/v solution of sulphadiazine RS in 1.4 M methanolic ammonia. Reference solution (b). A 0.2 per cent w/v solution of trimethoprim RS in 1.4 M methanolic ammonia. Apply to the plate 5 l of each solution. After development, dry the plate in air and spray with a 0.1 per cent w/v solution
1564
IP 2007
SULPHAQUINOXALINE
of 4-dimethylaminobenzaldehyde in a mixture of 1 ml of hydrochloric acid and 100 ml of ethanol (95 per cent), allow to dry and spray with dilute potassium iodobismuthate solution. The spot in the chromatogram obtained with test solution (a) having Rf value of about 0.7 corresponds to the principal spot in the chromatogram obtained with reference solution (a). The spot in the chromatogram obtained with test solution (b) having Rf value of about 0.3 corresponds to the principal spot in the chromatogram obtained with reference solution (b).
Determine the weight per ml of the suspension (2.4.29), and calculate the contents of sulphadiazine and trimethoprim, weight in volume. Labelling. The label states the strength in terms of the amounts of Sulphadiazine and Trimethoprim.
Sulphaquinoxaline
N N H N NH2
Tests
Other tests. Complies with the tests stated under Veterinary Oral Liquids. Assay. For sulphadiazine - Transfer an accurately weighed quantity of the oral suspension containing about 0.125 g of Sulphadiazine, into a separator containing 20 ml of 0.1 M sodium hydroxide and extract with four quantities, each of 50 ml, of dichloromethane. Wash each dichloromethane extract with the same two quantities, each of 10 ml, of 0.1 M sodium hydroxide. Combine the aqueous washings and the aqueous layer from the separator and reserve the combined dichloromethane extracts for the Assay for trimethoprim. Dilute the combined aqueous solutions to 250.0 ml with water, filter and dilute 10.0 ml of the filtrate to 200.0 ml with water. To 2.0 ml of the resulting solution add 0.5 ml of 4 M hydrochloric acid and 1 ml of a 0.1 per cent w/v solution of sodium nitrite and allow to stand for 2 minutes. Add 1 ml of a 0.5 per cent w/ v solution of ammonium sulphamate and allow to stand for 3 minutes. Add 1 ml of a 0.1 per cent w/v solution of N-(1-naphthyl)ethylenediamine dihydrochloride, allow to stand for 10 minutes. Dilute the solution to 25.0 ml with water and measure the absorbance of the resulting solution at the maximum at about 538 nm (2.4.7), using as the blank a solution prepared in the same manner using 2 ml of water and beginning at the words add 0.5 ml of 4 M hydrochloric acid......... Calculate the content of C10H10N4O2S from the absorbance obtained by carrying out the procedure simultaneously, with 2.0 ml of a 0.0025 per cent w/v solution of sulphadiazine RS in 0.0005 M sodium hydroxide and beginning at the words add 0.5 ml of 4 M hydrochloric acid........ For trimethoprim - Extract the combined dichloromethane extracts from the Assay for sulphadiazine with four quantities, each of 50 ml, of a 5 per cent v/v solution of 6 M acetic acid; wash the combined aqueous extracts with 5 ml of dichloromethane, discard the dichloromethane layer and dilute to 250.0 ml with a 5 per cent v/v solution of 6 M acetic acid. Dilute 20.0 ml to 100.0 ml with water and determine the absorbance of the resulting solution at the maximum at about 271 nm (2.4.7). Calculate the content of C14H18N4O3 taking 204 as the specific absorbance at 271 nm. C14H12N4O2S
S O O
Mol. Wt. 300.3
Sulphaquinoxaline is 4-amino-N-2-quinoxalinylbenzenesulphonamide. Sulphaquinoxaline contains not less than 98.0 per cent and not more than 101.0 per cent of C14H12N4O2S, calculated on the dried basis. Description. A yellow colour powder.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with sulphaquinoxaline RS or with the reference spectrum of sulphaquinoxaline. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.001 per cent w/v solution in 0.01 M sodium hydroxide shows an absorption maximum only at about 252 nm; absorbance at about 252 nm, about 1.1. C. Dissolve 4 mg in 2 ml of warm 2 M hydrochloric acid. The solution gives the reaction of primary aromatic amines (2.3.1).
Tests
Acidity. To 2 g add 100 ml of water, heat at 70 for 5 minutes, cool to 20, and filter. Titrate 50 ml of the filtrate to pH 7.0 with 0.1 M sodium hydroxide; not more than 0.2 ml of 0.1 M sodium hydroxide is required. Heavy metals. Dissolve the residue obtained in the test for Sulphated ash in 1 ml of 2 M hydrochloric acid and dilute to 14 ml with water. 12 ml of the solution complies with limit test for heavy metals, Method D (2.3.13) (20 ppm). Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 60 volumes of dichloromethane, 40 volumes of methanol and 20 volumes of strong ammonia solution.
1565
SULPHAQUINOXALINE SODIUM SOLUTION
IP 2007
Test solution. Dissolve 0.20 g of the substance under examination in 2 ml of 1 M sodium hydroxide and add sufficient methanol to produce 50 ml. Reference solution (a). A 0.012 per cent w/v solution of N1,N2-diquinoxalin- 2-ylsulphanilamide RS in methanol. Reference solution (b). A 0.0040 per cent w/v solution of sulphanilamide RS in methanol. Apply to the plate 5 l of each solution. After development, dry the plate in air until the odour of the solvent is no longer detectable and examine in ultraviolet light at 254 nm. Any spot corresponding to N1,N2-diquinoxalin-2-ylsulphanilamide in the chromatogram obtained with the test solution not more intense than that of the spot in the chromatogram obtained with reference solution (a). Any other secondary spot in the chromatogram obtained with the test solution is not more intense than that in the chromatogram obtained by reference solution (b). Sulphated ash (2.3.18). Not more than 0.1 per cent. Loss on drying (2.4.19). Not more than 1.0 per cent, determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.65 g and dissolve in 10 ml of a mixture of equal volumes of 1 M sodium hydroxide and water. Add 20 ml of glycerin, 20 ml of 9 M sulphuric acid and 5 g of potassium bromide, cool in ice and carry out the nitrite titration (2.3.31). 1 ml of 0.1 M sodium nitrite is equivalent to 0.03003 g of C14H12N4O2S. Storage. Store protected from light.
sulphaquinoxaline RS or with the reference spectrum of sulphaquinoxaline. B. Dissolve 4 mg of the residue obtained in test A in 2 ml of warm 2 M hydrochloric acid. The solution gives the reaction of primary aromatic amines (2.3.1). C. Acidify with 6 M acetic acid, filter and evaporate the filtrate to dryness. The incinerated residue, when moistened with hydrochloric acid and introduced on a platinum wire into a Bunsen burner flame, gives a yellow colour to the flame.
Tests
pH (2.4.24). 12.2 to 12.8, determined in a 9.6 per cent w/v solution in carbon dioxide-free water. Related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 60 volumes of dichloromethane, 40 volumes of methanol and 20 volumes of strong ammonia solution. Test solution. Dilute a solution containing 0.20 g of Sulphaquinoxaline to 50 ml with methanol. Reference solution (a). A 0.012 per cent w/v solution of N1,N2-diquinoxalin- 2-ylsulphanilamide RS in methanol. Reference solution (b). A 0.0040 per cent w/v solution of sulphanilamide RS in methanol. Apply to the plate 5 l of each solution. After development, dry the plate in air until the odour of the solvent is no longer detectable and examine in ultraviolet light at 254 nm. Any spot corresponding to N1,N2-diquinoxalin-2-ylsulphanilamide in the chromatogram obtained with the test solution not more intense than that in the chromatogram obtained with reference solution (a). Any other secondary spot in the chromatogram obtained with the test solution is not more intense than that of the spot in the chromatogram obtained with reference solution (b). Other tests. Complies with the tests stated under Veterinary Oral Liquids. Assay. To an accurately measured volume containing about 0.48 g of Sulphaquinoxaline add 30 ml water, 20 ml of glycerin, 20 ml of 9 M sulphuric acid and 5 g of potassium bromide, cool in ice and carry out the nitrite titration (2.3.31). 1 ml of 0.1 M sodium nitrite is equivalent to 0.03003 g of C14H12N4O2S. Storage. Store protected from light. Labelling. The label states the strength in terms of the equivalent amount of Sulphaquinoxaline in a suitable dose-volume.
Sulphaquinoxaline Sodium Solution

Sulphaquinoxaline Sodium Solution is an aqueous solution of sulphaquinoxaline sodium prepared by the interaction of Sulphaquinoxaline and Sodium Hydroxide. Sulphaquinoxaline Sodium Solution contains not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of sulphaquinoxaline, C14H12N4O2S. Description. A clear, yellow to brown solution.
Identification
A. To a volume containing 1 g of Sulphaquinoxaline add 10 ml of water and 3 ml of 2 M hydrochloric acid, filter, wash the precipitate with water and dry for 2 hours at 105. The residue complies with the following test. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with
1566
IP 2007
THIABENDAZOLE VETERINARY ORAL SUSPENSION
Sulphathiazole Sodium
O O S S N N H2N
C9H8N3NaO2S2,H2O C9H8N3NaO2S2,5H2O Mol. Wt. 304.3 Mol. Wt. 367.4
1 ml of 0.1 M sodium nitrite is equivalent to 0.02773 g of C9H8N3NaO2S2. Storage. Store protected from light.
Na
, 5H2O
Labelling. The label states whether the substance is the sesquihydrate or the pentahydrate.
Sulphathiazole Sodium is sodium salt of 4-amino-N-2thiazolylbenzenesulphonamide with five or one and half molecules of water. Sulphathiazole Sodium contains not less than 99.0 per cent and not more than 101.0 per cent of C9H8N3NaO2S2, calculated on the dried basis. Description. A white or yellowish white, crystalline powder or granules.
Thiabendazole Veterinary Oral Suspension

Thiabendazole Oral Suspension; Thiabendazole Mixture; Thiabendazole Drench
Thiabendazole Veterinary Oral Suspension is an aqueous suspension of Thiabendazole containing suitable suspending agents and antimicrobial preservatives. Thiabendazole Veterinary Oral Suspension contains not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of thiabendazole, C10H7N3S.
Identification
A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with sulphathiazole sodium RS or with the reference spectrum of sulphathiazole sodium. B. Dissolve 1 g in 25 ml of water and add 2 ml of 6 M acetic acid. Wash the precipitate formed with water and dry for 4 hours at 105. The residue melts at about 201 (2.4.21). C. The precipitate obtained in test B gives the reaction of primary aromatic amines (2.3.1).
Identification
Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 50 volumes of toluene, 20 volumes of glacial acetic acid, 8 volumes of acetone and 2 volumes of water. Test solution. Add 50 ml of ethyl acetate and 2 ml of glacial acetic acid to a volume of the well-mixed oral suspension containing about 0.25 g of Thiabendazole. Shake for 5 minutes, heat to boiling, cool, shake for a further 15 minutes and filter. Reference solution. Dissolve 0.25 g of thiabendazole RS in 50 ml of ethyl acetate and add 2 ml of glacial acetic acid. Apply to the plate 10 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with reference solution.
Tests
pH (2.4.24). 9.0 to 10.0, determined in a 1 per cent w/v solution. Heavy metals. Dissolve 2.5 g of the substance under examination in 10 ml of water, add 15 ml of 2 M acetic acid, shake for 30 minutes and filter. 12 ml of this solution complies with the limit test for heavy metals, Method D (2.3.13) (20 ppm). Related substances. Complies with test A for related substances in sulphonamides (2.3.7). Loss on drying (2.4.19). Not less than 6.0 per cent and not more than 10.0 per cent (sesquihydrate) or not less than 22.0 per cent and not more than 27.0 per cent (pentahydrate), determined on 1.0 g by drying in an oven at 105. Assay. Weigh accurately about 0.5 g, dissolve in a mixture of 75 ml of water and 10 ml of hydrochloric acid, add 3 g of potassium bromide, cool in ice and carry out the nitrite titration (2.3.31).
Tests
Other tests. Complies with the tests stated under Veterinary Oral Liquids. Assay. Weigh accurately a quantity of the well-mixed oral suspension containing about 1 g of Thiabendazole, add to 700 ml of 0.1 M hydrochloric acid, shake for 30 minutes, add sufficient 0.1 M hydrochloric acid to produce 1000.0 ml, mix and filter. Dilute 10.0 ml of the filtrate to 100.0 ml with 0.1 M hydrochloric acid. Dilute 5.0 ml to 100.0 ml with 0.1 M hydrochloric acid and measure the absorbance of the resulting
1567
THIABENDAZOLE AND RAFOXANIDE VETERINARY ORAL SUSPENSION
IP 2007
solution at the maximum at about 302 nm (2.4.7). Calculate the content of C10H7N3S taking 1230 as the specific absorbance at 302 nm. Determine the weight per ml of the suspension (2.4.29), and calculate the content of thiabendazole, weight in volume. Labelling. The label states that the suspension should be administered undiluted.
For rafoxanide - Protect the solutions from light throughout the determination. Weigh accurately a volume of the well-mixed suspension containing about 0.1 g of Rafoxanide in a 500-ml stoppered flask and add sufficient water to produce 100 ml. Swirl to disperse, add 20 ml of 1 M hydrochloric acid, mix well and add 300 ml of ethyl acetate. Shake the mixture for 1 hour, set aside for separation of the immiscible layers and centrifuge a portion of the ethyl acetate layer. Transfer 15.0 ml of the clear solution to a 50-ml centrifuge tube, add 20 ml of 0.1 M hydrochloric acid, stopper the tube, shake for 15 minutes, and centrifuge. Remove and discard the aqueous layer. Repeat the washing with two quantities, each of 20 ml, of 0.1 M hydrochloric acid. Evaporate the ethyl acetate solution almost to dryness in a warm water-bath, passing a stream of nitrogen over the surface of the liquid. Add 10 ml of water, warm on a water-bath for 10 minutes, add 5 ml of 1 M sodium hydroxide and mix. Add 15 ml of ether, shake for 15 minutes, centrifuge, and remove the ether layer. Repeat the extraction with two quantities, each of 15 ml, of ether. Evaporate the combined ether extracts almost to dryness on a warm water-bath, passing a stream of nitrogen over the surface of the liquid. Dissolve the residue in sufficient 0.1 M methanolic hydrochloric acid to produce 200.0 ml and measure the absorbance of the resulting solution at the maximum at about 335 nm (2.4.7). Calculate the content of C19H11Cl2I2NO3 from the absorbance obtained by carrying out the procedure simultaneously, using 0.1 g of rafoxanide RS and beginning at the words, add sufficient water to produce 100 ml...... Determine the weight per ml of the suspension (2.4.29), and calculate the content of thiabendazole and rafoxanide, weight in volume.
Thiabendazole and Rafoxanide Veterinary Oral Suspension

Thiabendazole and Rafoxanide Suspension; Thiabendazole and Rafoxanide Mixture
Thiabendazole and Rafoxanide Veterinary Oral Suspension is an aqueous suspension of Thiabendazole and Rafoxanide containing suitable suspending and dispersing agents. Thiabendazole and Rafoxanide Veterinary Oral Suspension contains not less than 92.5 per cent and not more than 107.5 per cent of the stated amount of thiabendazole, C10H7N3S, and not less than 90.0 per cent and not more than 110.0 per cent of the stated amount of rafoxanide, C19H11Cl2I2NO3.
Identification
A. Mix a volume containing 20 mg of Thiabendazole with 5 ml of 0.1 M hydrochloric acid, add 3 mg of 4-phenylenediamine dihydrochloride, mix, add 0.1 g of zinc powder and allow to stand for 2 minutes. Add 10 ml of ferric ammonium sulphate solution; a deep blue or blue violet colour is produced. B. In addition to the absorbance at about 335 nm, measure the absorbance at about 280 nm (2.4.7), of the final solution obtained in the Assay. The ratio of the absorbance at about 280 nm to that at about 335 nm is 1.59 to 1.69.
Thiabendazole Premix
Thiabendazole Premix contains not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of thiabendazole, C10H7N3S.
Tests
Other tests. Complies with the tests stated under Veterinary Oral Liquids. Assay. For thiabendazole - Weigh accurately a volume of the well-mixed suspension containing about 85 mg of Thiabendazole, add 20 ml of water and 9 ml of 0.1 M hydrochloric acid and warm on a water-bath for 30 minutes with occasional stirring. Transfer the suspension to a flask, rinse the vessel with water and add the washings to the flask. Cool, add sufficient water to produce 1000.0 ml and filter. Dilute 5.0 ml of the filtrate to 100.0 ml with 0.1 M hydrochloric acid and measure the absorbance of the resulting solution at the maximum at about 302 nm (2.4.7). Calculate the content of C10H7N3S taking 1230 as the specific absorbance at 302 nm.
Identification
Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 50 volumes of toluene, 20 volumes of glacial acetic acid, 8 volumes of acetone and 2 volumes of water. Test solution. To a quantity of the premix containing 0.25 g of Thiabendazole, finely powdered if necessary, add 50 ml of ethyl acetate and 2 ml of glacial acetic acid, shake for 5 minutes, heat to boiling, cool, shake for a further 15 minutes and filter.
1568
IP 2007
TYLOSIN
Reference solution. Dissolve 0.25 g of thiabendazole RS in 50 ml of ethyl acetate and add 2 ml of glacial acetic acid. Apply to the plate 10 l of each solution. After development, dry the plate in air and examine in ultraviolet light at 254 nm. The principal spot in the chromatogram obtained with the test solution corresponds to that in the chromatogram obtained with the reference solution.
(solution A) shows an absorption maximum only at about 290 nm; absorbance at about 290 nm, about 0.94. C. To 10 ml of solution A add 1 ml of 2 M sodium hydroxide, heat on a water-bath for 20 minutes and cool. When examined in the range 250 nm to 430 nm (2.4.7), of the resulting solution shows an absorption maximum only at about 332 nm. D. In the test for Tylosin A and other tylosins, the retention time and size of the principal peak in the chromatogram obtained with the test solution are approximately the same as those of the principal peak in the chromatogram obtained with reference solution (a). E. Dissolve about 30 mg in a mixture of 0.15 ml of water, 2.5 ml of acetic anhydride and 7.5 ml of pyridine. Allow to stand for 10 minutes; no green colour develops.
Tests
Other tests. Complies with the tests stated under Premixes. Assay. Weigh accurately a quantity containing about 0.1 g of Thiabendazole, add 700 ml of 0.1 M hydrochloric acid, shake for 30 minutes, dilute to 1000.0 ml with 0.1 M hydrochloric acid and filter. Dilute 5.0 ml of the filtrate to 100.0 ml with 0.1 M hydrochloric acid and measure the absorbance of the resulting solution at the maximum at about 302 nm (2.4.7). Calculate the content of C10H7N3S, taking 1230 as the specific absorbance at 302 nm.
Tests
pH (2.4.24). 8.5 to 10.5, determined in a 2.5 per cent w/v suspension in carbon dioxide-free water. Heavy metals. To the residue obtained in the test for Sulphated ash add 2 ml of hydrochloric acid and evaporate slowly to dryness on a water-bath. Moisten the residue with 0.05 ml of hydrochloric acid, add 10 ml of boiling water and heat for 10 minutes on a water-bath. Cool and dilute to 25 ml with water. 12 ml of the solution complies with the limit test for heavy metals, Method D (2.3.13) (20 ppm).
Tylosin
O CH3 H3C H3C HO H3CO O O O CH3 H3C O OH CHO HO O O CH3 N CH3 CH3 O O OH CH3 OH CH3
OCH3
C46H77NO17
Mol. Wt. 916.1
Tylosin is a macrolide antibiotic isolatede from a strain of Stryptomycetes fradiae found in soil from Thailand. Tylosin has a potency of not less than 900 Units per mg, calculated on the dried basis. The content of tylosin A is not less than 80.0 per cent and the sum of the contents of tylosin A, tylosin B, tylosin C and tylosin D is not less than 95.0 per cent. Description. Almost white or slightly yellow powder.
Tyramine. Dissolve 50 mg in 5 ml of 0.03 M phosphoric acid in a 25-ml volumetric flask, add 1 ml of pyridine and 2 ml of a saturated solution of ninhydrin in water (approximately 4 per cent w/v). Close the flask by covering with a piece of aluminium foil and heat in a water-bath at 85 for at least 20 minutes. Cool rapidly and add sufficient water to produce 25 ml. Mix and measure without delay the absorbance of the solution at about 570 nm (2.4.7), using as the blank a solution prepared in a similar manner but omitting the substance under examination. The absorbance is not more than that obtained by carrying out the procedure simultaneously, using 5 ml of a solution in 0.03 M phosphoric acid containing 35 mg of tyramine per ml and beginning at the words add 1 ml of pyridine...... (0.35 per cent). Sulphated ash (2.3.18). Not more than 3.0 per cent. Loss on drying (2.4.19). Not more than 5.0 per cent, determined on 1.0 g by drying at 60 at a pressure not exceeding 0.7 kPa for 3 hours. Tylosin A and other tylosins. Determine by liquid chromatography (2.4.14). NOTE- Use freshly prepared solutions. Test solution. Dissolve 20 mg of the substance under examination in 100 ml of a mixture of equal volumes of acetonitrile and water.
Identification
Tests B and C may be omitted if tests A, D and E are carried out. Tests D and E may be omitted if tests A, B and C are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with tylosin RS or with the reference spectrum of tylosin. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.004 per cent w/v solution in 0.1 M hydrochloric acid
1569
TYLOSIN INJECTION
IP 2007
Reference solution (a). A 0.02 per cent w/v solution of tylosin RS in a mixture of equal volumes of acetonitrile and water. Reference solution (b). A solution containing 0.02 per cent w/ v each of tylosin A RS and tylosin D RS in a mixture of equal volumes of acetonitrile and water. Chromatographic system a stainless steel column 20 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m) (such as Nucleosil C18), column temperature 35, mobile phase: a filtered and degassed mixture of 60 volumes of 0.85 M sodium perchlorate and 40 volumes of acetonitrile adjusted to pH 2.5 with 1 M hydrochloric acid, flow rate. 1 ml per minute, spectrophotometer set at 290 nm, a 20 l loop injector. Inject reference solution (b). If necessary, adjust the molarity of the sodium perchlorate or increase the temperature of the column to a maximum of 50 so as to obtain a retention time of about 12 minutes for tylosin A. The test is not valid unless the resolution between the peaks due to tylosin A and tylosin D is at least 2.0. Inject reference solution (a). The column efficiency, determined using the peak due to tylosin A, should be not less than 22,000 theoretical plates per metre. Inject alternatively the test solution and reference solution (a). The order of elution of the major components of the substance under examination is desmycinosyltylosin, tylosin C, tylosin B, tylosin D, tylosin A aldol and tylosin A. Calculate the percentage content of components from the areas of the peaks in the chromatogram obtained with the test solution by normalisation. Assay. Carry out the microbiological assay of antibiotics (2.2.10). Tylosin intended for use in the manufacture of Parenteral Preparations without a further appropriate sterilisation procedure complies with the following additional requirement. Sterility (2.2.11). Complies with the test for sterility. Storage. Store protected from light. If it is intended for use in the manufacture of Parenteral Preparations, the container should be sterile, tamper-evident and sealed so as to exclude micro-organisms. Labelling. The label states (1) the number of Units per mg; (2) the date after which the material is not intended to be used; (3) the storage conditions; (4) where applicable, that it is suitable for use in the manufacture of Parenteral Preparations; (5) that the preparation is intended for veterinary use.
Tylosin Injection
Tylosin Injection is a sterile solution of Tylosin in a mixture of equal volumes of Propylene Glycol and Water for Injections. Tylosin Injection contains not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of tylosin. The content of tylosin A is not less than 80.0 per cent and the sum of the contents of tylosin A, tylosin B, tylosin C and tylosin D is not less than 90.0 per cent. Description. A pale yellow to amber-coloured solution.
Identification
A. To a volume containing 0.1 g of Tylosin add sufficient water to obtain a solution containing 0.02 per cent w/v of Tylosin. To 5 ml of this solution add 10 ml of 0.1 M sodium hydroxide and extract with 10 ml of dichloromethane. Separate the dichloromethane layer and extract it with 25 ml of 0.1 M hydrochloric acid. Discard the dichloromethane layer, wash the aqueous layer with 3 ml of dichloromethane, discard the washings and filter. When examined in the range 230 nm to 360 nm (2.4.7), of the resulting solution exhibits a maximum only at about 290 nm; absorbance at about 290 nm, about 0.94. B. To 10 ml of the filtrate obtained in test A add 1 ml of 2 M sodium hydroxide, heat in a water-bath for 20 minutes and cool. When examined in the range 250 nm to 430 nm (2.4.7), exhibits a maximum only at about 332 nm.
Tests
Tyramine. Dilute a volume containing 100 mg of Tylosin with 5 ml of 0.03 M phosphoric acid in a 25-ml volumetric flask, add 1 ml of pyridine and 2 ml of a saturated solution of ninhydrin in water (approximately 4 per cent w/v). Close the flask by covering with a piece of aluminium foil and heat in a water-bath at 85 for at least 20 minutes. Cool rapidly and add sufficient water to produce 25 ml. Mix and measure without delay the absorbance of the resulting solution at about 570 nm (2.4.7), using as the blank a solution prepared in a similar manner but omitting the preparation under examination. The absorbance is not more than that obtained by carrying out the procedure simultaneously, using 5 ml of a solution in 0.03 M phosphoric acid containing 30 mg of tyramine per ml and beginning at the words add 1 ml of pyridine...... (0.15 per cent). Tylosin A and other tylosins. Determine by liquid chromatography (2.4.14). NOTE - Use freshly prepared solutions. Test solution. Dilute the injection with sufficient of a mixture of equal volumes of acetonitrile and water to produce a solution containing 0.02 per cent w/v of Tylosin.
1570
IP 2007
TYLOSIN TABLETS
Reference solution (a). A 0.02 per cent w/v solution of tylosin RS in a mixture of equal volumes of acetonitrile and water. Reference solution (b). A solution containing 0.02 per cent w/ v each of tylosin A RS and tylosin D RS in a mixture of equal volumes of acetonitrile and water. Chromatographic system a stainless steel column 20 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m) (such as Nucleosil C18), column temperature 35, mobile phase: a filtered and degassed mixture of 60 volumes of 0.85 M sodium perchlorate and 40 volumes of acetonitrile adjusted to pH 2.5 with 1 M hydrochloric acid, flow rate. 1 ml per minute, spectrophotometer set at 290 nm, a 20 l loop injector. Inject reference solution (b). If necessary, adjust the molarity of the sodium perchlorate or increase the temperature of the column to a maximum of 50 so as to obtain a retention time of about 12 minutes for tylosin A. The test is not valid unless the resolution between the peaks due to tylosin A and tylosin D is at least 2.0. Inject reference solution (a). The column efficiency, determined using the peak due to tylosin A, should be not less than 22,000 theoretical plates per metre. Inject alternatively test solution and reference solution (a). The order of elution of the major components of the substance under examination is desmycinosyltylosin, tylosin C, tylosin B, tylosin D, tylosin A aldol and tylosin A. Calculate the percentage content of components from the areas of the peaks in the chromatogram obtained with the test solution. Other tests. Complies with the tests stated under Parenteral Preparations (Injections). Assay. Carry out the microbiological assay of antibiotics (2.2.10). Calculate the content of tylosin in the injection, taking each 1000 Units found to be equivalent to 1 mg of tylosin. Storage. Store protected from moisture. Labelling. The label states that the preparation is intended for veterinary use by intramuscular injection only.
Identification
A. Triturate a quantity of the powdered tablets containing 0.2 g of Tylosin with 20 ml of dichloromethane and filter. Dry the dichloromethane extract by shaking with anhydrous sodium sulphate, filter and evaporate the filtrate to dryness. Dry the residue over phosphorus pentoxide at a pressure not exceeding 0.7 kPa for 1 hour. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with tylosin RS or with the reference spectrum of tylosin. B. Triturate a quantity of the powdered tablets containing 0.2 g of Tylosin with two quantities, each of 10 ml, of 0.1 M hydrochloric acid, filter and dilute the filtrate to 100 ml with 0.1 M hydrochloric acid. Dilute 10 ml of the resulting solution to 50 ml with the same solvent. Dilute 5 ml of this solution further to 50 ml with the same solvent. When examined in the range 230 nm to 360 nm (2.4.7), the resulting solution shows an absorption maximum only at about 290 nm; absorbance at about 290 nm, about 0.94. C. To 10 ml of the final solution obtained in test B add 1 ml of 2 M sodium hydroxide, heat in a water-bath for 20 minutes and cool. When examined in the range 250 nm to 430 nm (2.4.7), exhibits a maximum only at about 332 nm.
Tests
Tyramine. Shake a quantity of the powdered tablets containing 50 mg of Tylosin with 5 ml of 0.03 M phosphoric acid. Filter into a 25-ml volumetric flask, add 1 ml of pyridine and 2 ml of a saturated solution of ninhydrin in water (approximately 4 per cent w/v). Close the flask by covering with a piece of aluminium foil and heat in a water-bath at 85 for at least 20 minutes. Cool rapidly and add sufficient water to produce 25 ml. Mix and measure without delay the absorbance of the solution at about 570 nm (2.4.7), using as the blank a solution prepared in a similar manner but omitting the substance under examination. The absorbance is not more than that obtained by carrying out the procedure simultaneously, using 5 ml of a solution in 0.03 M phosphoric acid containing 35 mg of tyramine per ml and beginning at the words add 1 ml of pyridine...... (0.35 per cent). Tylosin A and other tylosins. Determine by liquid chromatography (2.4.14). NOTE Use freshly prepared solutions. Test solution. Shake a quantity of the powdered tablets containing 0.2 g of Tylosin with 50 ml of methanol, filter and dilute 5 ml of the filtrate to 100 ml with a mixture of equal volumes of acetonitrile and water. Reference solution (a). A 0.02 per cent w/v solution of tylosin RS in a mixture of equal volumes of acetonitrile and water.
Tylosin Tablets
Tylosin Tablets contain not less than 95.0 per cent and not more than 105.0 per cent of the stated amount of tylosin. The content of tylosin A is not less than 80.0 per cent and the sum of the contents of tylosin A, tylosin B, tylosin C and tylosin D is not less than 90.0 per cent.
1571
TYLOSIN TARTRATE
IP 2007
Reference solution (b). A solution containing 0.02 per cent w/ v each of tylosin A RS and tylosin D RS in a mixture of equal volumes of acetonitrile and water. Chromatographic system a stainless steel column 20 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m) (such as Nucleosil C18), column temperature 35, mobile phase: a filtered and degassed mixture of 60 volumes of 0.85 M sodium perchlorate and 40 volumes of acetonitrile adjusted to pH 2.5 with 1 M hydrochloric acid, flow rate. 1 ml per minute, spectrophotometer set at 290 nm, a 20 l loop injector. Inject reference solution (b). If necessary, adjust the molarity of the sodium perchlorate or increase the temperature of the column to a maximum of 50 so as to obtain a retention time of about 12 minutes for tylosin A. The test is not valid unless the resolution between the peaks due to tylosin A and tylosin D is at least 2.0. Inject reference solution (a). The column efficiency, determined using the peak due to tylosin A, should be not less than 22,000 theoretical plates per metre. Inject alternatively the test solution and reference solution (a). The order of elution of the major components of the substance under examination is desmycinosyltylosin, tylosin C, tylosin B, tylosin D, tylosin A aldol and tylosin A. Calculate the percentage content of components from the areas of the peaks in the chromatogram obtained with test solution. Other tests. Comply with the tests stated under Tablets. Assay. Carry out the microbiological assay of antibiotics (2.2.10). Calculate the content of tylosin in the tablets, taking each 1000 Units found to be equivalent to 1 mg of tylosin.
strains of Streptomyces fradiae or by any other means. It consists largely of tylosin A tartrate but tartrates of tylosin B (desmycosin), tylosin C (macrocin) and tylosin D (relomycin) may also be present. Tylosin Tartrate contains not less than 800 Units per mg, calculated on the dried basis. The content of tylosin A is not less than 80.0 per cent and the sum of the contents of tylosin A, tylosin B, tylosin C and tylosin D is not less than 95.0 per cent. Description. An almost white or slightly yellow, hygroscopic powder.
Identification
Tests B and C may be omitted if tests A, D and E are carried out. Tests D and E may be omitted if tests A, B and C are carried out. A. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with tylosin tartrate RS or with the reference spectrum of tylosin tartrate. B. When examined in the range 230 nm to 360 nm (2.4.7), a 0.004 per cent w/v solution in 0.1 M hydrochloric acid (solution A) shows an absorption maximum only at about 290 nm; absorbance at about 290 nm, about 0.88. C. To 10 ml of solution A add 1 ml of 2 M sodium hydroxide, heat on a water-bath for 20 minutes and cool. When examined in the range 250 nm to 430 nm (2.4.7), the resulting solution shows an absorption maximum only at about 332 nm. D. In the test for Tylosin A and other tylosins, the retention time and size of the principal peak in the chromatogram obtained with the test solution are approximately the same as those of the principal peak in the chromatogram obtained with the reference solution. E. Dissolve about 30 mg in a mixture of 0.15 ml of water, 2.5 ml of acetic anhydride and 7.5 ml of pyridine. Allow to stand for 10 minutes; a green colour develops.
Tylosin Tartrate
O CH3 H3C H3C HO H3CO O O O CH3 H3C O OH CHO HO O O CH3 N CH3 CH3 O O OH CH3 OH CH3
2
Tests
pH (2.4.24). 5.0 to 7.2, determined in a 2.5 per cent w/v solution in carbon dioxide-free water.
H OH HOOC HO COOH H
OCH3
(C46H77NO17)2, C4H6O6
Mol. Wt. 1983.3
Tylosin Tartrate is the tartrate of Tylosin, which is a mixture of antimicrobial macrolides produced by the growth of certain
Tyramine. Dissolve 50 mg in 5 ml of 0.03 M phosphoric acid in a 25-ml volumetric flask, add 1 ml of pyridine and 2 ml of a saturated solution of ninhydrin in water (approximately 4 per cent w/v). Close the flask by covering with a piece of aluminium foil and heat in a water-bath at 85 for at least 20 minutes. Cool rapidly and add sufficient water to produce 25 ml. Mix and measure without delay the absorbance of the solution at about 570 nm (2.4.7), using as the blank a solution prepared in a
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IP 2007
TYLOSIN TARTRATE AND SULPHATHIAZOLE SODIUM VETERINARY ORAL POWDER
similar manner but omitting the substance under examination. The absorbance is not more than that obtained by carrying out the procedure simultaneously, using 5 ml of a solution in 0.03 M phosphoric acid containing 35 mg of tyramine per ml and beginning at the words add 1 ml of pyridine...... (0.35 per cent). Sulphated ash (2.3.18). Not more than 2.5 per cent. Loss on drying (2.4.19). Not more than 4.5 per cent, determined on 1.0 g by drying at 60 at a pressure not exceeding 0.7 kPa for 3 hours. Tylosin A and other tylosins. Determine by liquid chromatography (2.4.14). NOTE - Use freshly prepared solutions. Test solution. Dissolve a quantity containing 20 mg of tylosin in 100 ml of a mixture of equal volumes of acetonitrile and water.. Reference solution (a). A 0.02 per cent w/v solution of tylosin RS in a mixture of equal volumes of acetonitrile and water. Reference solution (b). A solution containing 0.02 per cent w/ v each of tylosin A RS and tylosin D RS in a mixture of equal volumes of acetonitrile and water. Chromatographic system a stainless steel column 20 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m) (such as Nucleosil C18), column temperature 35, mobile phase: a filtered and degassed mixture of 60 volumes of 0.85 M sodium perchlorate and 40 volumes of acetonitrile adjusted to pH 2.5 with 1 M hydrochloric acid, flow rate. 1 ml per minute, spectrophotometer set at 290 nm, a 20 l loop injector. Inject reference solution (b). If necessary, adjust the molarity of the sodium perchlorate or increase the temperature of the column to a maximum of 50 so as to obtain a retention time of about 12 minutes for tylosin A. The test is not valid unless the resolution between the peaks due to tylosin A and tylosin D is at least 2.0. Inject reference solution (a). The column efficiency, determined using the peak due to tylosin A, should be not less than 22,000 theoretical plates per metre. Inject alternatively the test solution and the reference solution (a). The order of elution of the major components of the substance under examination is desmycinosyltylosin, tylosin C, tylosin B, tylosin D, tylosin A aldol and tylosin A. Calculate the percentage content of components from the areas of the peaks in the chromatogram obtained with test solution.
Assay. Carry out the microbiological assay of antibiotics (2.2.10). Tylosin Tartrate intended for use in the manufacture of parenteral preparations complies with the above requirements with the following modification. Tyramine. Carry out the procedure described in the test for Tyramine but using 100 mg in 5 ml of 0.03 M phosphoric acid. Measure the absorbance of the solution under the conditions described in the test. The absorbance is not more than that obtained by simultaneously carrying out the procedure using 5 ml of a solution in 0.03 M phosphoric acid containing 30 mg of tyramine per ml and beginning at the words add 1 ml of pyridine...... (0.15 per cent). Tylosin Tartrate intended for use in the manufacture of parenteral preparations without a further appropriate sterilisation procedure complies with the following additional requirement. Sterility (2.2.11). Complies with the test for sterility. Storage. Store protected from light. If it is intended to be used in the manufacture of parenteral preparations, the container should be sterile, tamper-evident and sealed so as to exclude micro-organisms. Labelling. The label states (1) the number of Units per mg; (2) the quantity of Tylosin Tartrate in terms of equivalent amount of tylosin; (3) the date after which the material is not intended to be used; (4) the storage conditions; (5) where applicable, that it is suitable for use in the manufacture of parenteral preparations; (6) that the preparation is intended for veterinary use.
Tylosin Tartrate and Sulphathiazole Sodium Veterinary Oral Powder

Tylosin Tartrate and Sulphathiazole Sodium Veterinary Oral Powder is a mixture of Tylosin Tartrate and Sulphathiazole Sodium. It contains 3 parts of Sulphathiazole Sodium for 1 part, by weight, of Tylosin. Tylosin Tartrate and Sulphathiazole Sodium Veterinary Oral Powder contains not less than 90.0 per cent and not more than 110.0 per cent of the stated amounts of tylosin and sulphathiazole sodium sesquihydrate, C9H8NaO2S2,11/2H2O.
Identification
A. Triturate a quantity of the powder containing 0.25 g of Tylosin with two quantities, each of 25 ml, of dichloromethane and filter. Reserve the dichloromethane-insoluble matter for test B. Wash the combined filtrates by shaking for 1 minute with 20 ml of 0.1 M sodium hydroxide and dry the dichloromethane layer by the addition of anhydrous sodium
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IP 2007
sulphate. Evaporate the filtrate to dryness and dry the residue over phosphorus pentoxide at a pressure not exceeding 0.7 kPa for 1 hour. The residue complies with the following test. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with tylosin RS or with the reference spectrum of tylosin. B. Dry the dichloromethane-insoluble matter reserved in test A at 105 for 1 hour. The residue complies with the following test. Determine by infrared absorption spectrophotometry (2.4.6). Compare the spectrum with that obtained with sulphathiazole sodium RS or with the reference spectrum of sulphathiazole sodium.
Nucleosil C18), column temperature 35, mobile phase: a filtered and degassed mixture of 60 volumes of 0.85 M sodium perchlorate and 40 volumes of acetonitrile adjusted to pH 2.5 with 1 M hydrochloric acid, flow rate. 1 ml per minute, spectrophotometer set at 290 nm, a 20 l loop injector. Inject reference solution (b). If necessary, adjust the molarity of the sodium perchlorate or increase the temperature of the column to a maximum of 50 so as to obtain a retention time of about 12 minutes for tylosin A. The test is not valid unless the resolution between the peaks due to tylosin A and tylosin D is at least 2.0. Inject reference solution (a). The column efficiency, determined using the peak due to tylosin A, should be not less than 22,000 theoretical plates per metre. Inject alternatively the test solution and the reference solution (a). The order of elution of the major components of the substance under examination is desmycinosyltylosin, tylosin C, tylosin B, tylosin D, tylosin A aldol and tylosin A. Calculate the percentage content of components from the areas of the peaks in the chromatogram obtained with the test solution by normalisation. In the chromatogram obtained with the test solution the content of tylosin A is not less than 80.0 per cent and the sum of the contents of tylosin A, tylosin B, tylosin C and tylosin D is not less than 95.0 per cent. Other tests. Complies with the tests stated under Veterinary Oral Powders. Assay. For tylosin activity - Weigh accurately a quantity of the powder containing about 0.2 g of Tylosin, transfer to a 100-ml volumetric flask with three quantities, each of 10 ml, of methanol, swirl to dissolve and add sufficient sterile phosphate buffer pH 7.0 to produce 100.0 ml. Filter and dilute 5.0 ml of the filtrate to 100.0 ml with sterile phosphate buffer pH 7.0. Carry out the microbiological assay of antibiotics (2.2.10). Calculate the content of tylosin taking each 1000 Units found to be equivalent to 1 mg of tylosin. For sulphathiazole sodium - Weigh accurately a quantity of the powder containing about 0.4 g of sulphathiazole sodium sesquihydrate, dissolve in a mixture of 75 ml of water and 10 ml of hydrochloric acid, add 3 g of potassium bromide, cool in ice and titrate slowly with 0.1 M sodium nitrite, stirring constantly and determine the end-point potentiometrically (2.4.25). 1 ml of 0.1 M sodium nitrite is equivalent to 0.03043 g of C9H8N3NaO2S2,11/2H2O. Storage. Store protected from moisture.
Tests
Sulphonamide-related substances. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel H. Solvent mixture. A mixture of 9 volumes of ethanol (95 per cent) and 1 volume of strong ammonia solution. Mobile phase. A mixture of 90 volumes of 1-butanol and 18 volumes of 10 M ammonia. Test solution. Shake a quantity of the powder containing 0.1 g of sulphathiazole sodium sesquihydrate with 10 ml of the solvent mixture. Reference solution. A 0.0050 per cent w/v solution of sulphanilamide in the solvent mixture. Apply to the plate 10 l of each solution. After development, dry the plate by heating it at 105 for 10 minutes and spray with a 0.1 per cent w/v solution of 4-dimethylaminobenzaldehyde in a mixture of 99 volumes of ethanol (95 per cent) and 1 volume of hydrochloric acid. Any secondary spot in the chromatogram obtained with the test solution is not more intense than the spot in the chromatogram obtained with reference solution (0.5 per cent). Tylosin A and other tylosins. Determine by liquid chromatography (2.4.14). NOTE - Use freshly prepared solutions. Test solution. Dissolve a quantity containing 20 mg of tylosin in 100 ml of a mixture of equal volumes of acetonitrile and water. Reference solution (a). A 0.02 per cent w/v solution of tylosin RS in a mixture of equal volumes of acetonitrile and water. Reference solution (b). A solution containing 0.02 per cent w/ v each of tylosin A RS and tylosin D RS in a mixture of equal volumes of acetonitrile and water. Chromatographic system a stainless steel column 20 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 m) (such as
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IP 2007
Labelling. The label states the strength of Tylosin Tartrate in terms of the equivalent amount of tylosin and that of
Sulphathiazole Sodium in terms of the equivalent amount of sulphathiazole sodium sesquihydrate.
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IP 2007
AVIAN INFECTIOUS BRONCHITIS VACCINE, INACTIVATED
Anthrax Spore Vaccine, Live

Anthrax Spore Vaccine, Live consists of a suspension of live spores of an attenuated, non-capsulated strain of Bacillus anthracis.
stated on the label for sheep and observe the animal for 21 days. None of the sheep shows any untoward reaction. If more than 2 animals die from non-specific causes, repeat the test. Inject subcutaneously into each vaccinated guinea pig or rabbit or sheep at least 100 MLD and to each of these control animals 10 MLD of a strain of B.anthracis pathogenic for the species of animal used in the test. Observe all the animals for 10 days, all vaccinated animals survive and all the controls die from anthrax during the observation period. If a vaccinated animal dies after the challenge, repeat the test. If in the second test, a vaccinated animal dies, the vaccine fails the test. Labelling. The label states (1) the strain used for the preparation of the vaccine; (2) the number of viable spores per ml.
Production
The strain used may either be not lethal to guinea pigs or the mouse or lethal to guinea pigs but not to the rabbit or lethal to some rabbits. At the end of growth the spores are suspended in 50 per cent glycerin saline and counted. The vaccine may contain an adjuvant.
Identification
B. anthracis present in the vaccine is identified by means of morphological, serological test, culture and biochemical test.
Tests
Safety. Carry out the test on one of the species for which the vaccine is intended. If the vaccine is intended for several species including the goat, carry out the test on sheep and goats. Administer subcutaneously or intramuscularly to each of two animals having no antibodies against B. anthracis, twice the dose (example, 2 million spores) stated on the label for the species used and observe the animals for 10 days. No abnormal systemic reaction is produced but a mild local reaction may occur at the site of injection. The severity of the local reaction may vary according to the strain of the spores and the adjuvants used in the preparation, but necrosis does not occur. Sterility (2.2.11). Complies with the test for sterility. Spore count. Determine the number of viable spores by plate count. The number of live spores is not less than 80 per cent of that stated on the label. Potency. For a strain of B. anthracis which is not lethal to the guinea pig or the mouse, the test may be carried out in the guinea pigs. For a strain which is lethal to the guinea pig but not to the rabbit, the test may be carried out in rabbits. For a strain which is lethal to some rabbits, carry out the test in sheep. If the test is carried out in guinea pigs or rabbits. Inject into animals (each guinea pig weighing not less than 500 g and each rabbit weighing not less than 4.5 kg) subcutaneously or intramuscularly, 1/10th of the smallest dose of the vaccine stated on the label for sheep. Observe the animals for 21 days. If more than two animals die from non-specific causes, repeat the test. If the test is carried out in sheep use 5 healthy animals. Inject subcutaneously or intramuscularly into each sheep weighing not less than 20 kg, 1/10th of the smallest dose of the vaccine
Avian Infectious Bronchitis Vaccine, Inactivated

Avian Infectious Bronchitis Vaccine, Inactivated consists of an emulsion or a suspension of one or more serotypes of avian infectious bronchitis virus which have been inactivated in such a manner that the immunogenic activity is retained. The vaccine may contain one or more suitable adjuvants. These vaccines intended to protect against a drop in egg production or quality; for vaccines also intended to protect against respiratory symptoms, a demonstration of efficacy (2.7.12) additional to that described under Potency is required.
Production
The virus is propagated in fertilised hen eggs (2.7.7) from SPF flock or in suitable cell cultures. The master seed lot complies with the tests for extreneous agents in seed lot (2.7.10). Inactivation An amplification test for residual live avian infectious bronchitis virus is carried out on each batch of antigen immediately after inactivation and on the final bulk vaccine or, if the vaccine contains an adjuvant, on the bulk antigen or mixture of bulk antigens immediately before the addition of adjuvant; the test is carried out in fertilised hen eggs from flocks free from specified pathogens (SPF) or in suitable cell cultures and the quantity of inactivated virus used is equivalent to not less than 10 doses of vaccine. No live virus is detected.
Identification
In susceptible birds, the vaccine stimulates the production of specific antibodies against each of the virus serotypes in the vaccine, detectable by virus neutralisation and protects chicken against infectious bronchitis.
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AVIAN INFECTIOUS BRONCHITIS VACCINE, LIVE
IP 2007
Tests
Sterility (2.2.11). Complies with the test for sterility. Safety. Inject intramuscularly a quantity equivalent to 2 doses into each of twenty healthy chickens, 2 to 4 weeks old, free from specific antibodies (2.7.7). Observe the chickens for 14 days. No abnormal systemic or local reaction is seen. Inactivation A. For vaccine prepared with embryo-adapted strains of virus, inject 2/5 of a dose into the allantoic cavity of ten 9 to 11-dayold fertilised hen eggs from an SPF flock and incubate. Observe for 5 to 6 days and pool separately the allantoic liquid from eggs containing live embryos and that from eggs containing dead embryos, excluding those that die within the first 24 hours after injection. Examine for abnormalities all embryos which die after 24 hours of injection or which survive 5 to 6 days. No death or abnormality attributable to the vaccine virus occurs. Inject into the allantoic cavity of each of ten 9 to 11-day-old fertilised hen eggs from an SPF flock, 0.2 ml of the pooled allantoic liquid from the live embryos and into each of 10 similar eggs 0.2 ml of the pooled liquid from the dead embryos and incubate for 5 to 6 days. Examine for abnormalities all embryos which die after 24 hours of injection or which survive 5 to 6 days. No death or abnormality attributable to the vaccine virus occurs. If more than 20 per cent of the embryos die at either stage repeat the test from that stage. The vaccine complies with the test if there is no death or abnormality attributable to the vaccine virus. B. For vaccine prepared with cell-culture-adapted strains of virus, inoculate 10 doses of the vaccine into suitable cell cultures. If the vaccine contains an oil adjuvant, eliminate it by suitable means. Incubate at 37 1 for 7 days. Make a passage on another set of cell cultures and incubate at 37 1 for 7 days. None of the cultures shows signs of infection. Extraneous agents. Use the chickens from the test for safety. 21 days after injection of the double dose of vaccine, inject 1 dose by the same route into each chicken. Collect serum samples from each chicken 2 weeks later and carry out tests for antibodies to the following agents: avian encephalomyelitis virus, avian leucosis viruses, haemagglutinating avian adenovirus, infectious bursal disease virus, infectious laryngotracheitis virus, influenza A virus, Mareks disease virus, Newcastle disease virus. The vaccine does not stimulate the formation of antibodies against these agents. Potency. Inject one dose by the route stated on the label into each of twenty chickens, 3 to 4 weeks old, complying with the requirements stated under Test on chickens flocks free from pathogens for the production and quality control of vaccines
(2.7.7 ). Use ten similar chickens as controls and house them together with the vaccinated chickens. After 28 days, collect serum samples from each of the vaccinated and control chickens and perform haemagglutination inhibition (HI) test on each serum using 8 haemagglutinating (HA) units of antigen and chicken erythrocytes, testing all serum samples at the same time. The vaccine passes the test if the mean antibody titre of the vaccinated group is not less than 1:64 and no specific antibody is detected in the control chickens. Alternatively, serum neutralization test may be carried out in SPF eggs. Serum neutralization titer should not be less than 102 neutralization units. If the immunogenicity test (potency test) has been performed with satisfactory results on representative batch of the vaccine from the same seed lot, it may be omitted as a routine control test during production of other batches of the vaccine prepared from the same seed lot. Labelling. The label states (1) the strain of virus used in preparing the vaccine; (2) the name of any added adjuvant.
Avian Infectious Bronchitis Vaccine, Live

Avian Infectious Bronchitis Vaccine, live is a preparation of one or more suitable strains of different types of avian infectious bronchitis virus.
Production
The vaccine virus is grown in embryonated hens eggs or in cell cultures. Substrate for virus propagation Embryonated hens eggs. If the vaccine virus is grown in embryonated hens eggs, they are obtained from SPF (2.7.7).
Identification
Carry out either the test A or B. A. Inoculate 0.2 ml undiluted vaccine in the allantoic sac of SPF embryonated eggs and incubate at 37 for 7 days. Lesions typical of infectious bronchitis are observed in the embryos and the allantoic fluid does not agglutinate chicken erythrocytes. B. Specific antiserum against the strain or each of the strains of the avian infectious bronchitis virus used in the vaccine are neutralised by the vaccine.
Tests
Water (2.3.43). Not more than 3.0 per cent.
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IP 2007
BLACKQUARTER VACCINE
Mycoplasmas (2.7.9). Complies with the test for mycoplasmas. Safety. Inject 10 times the dose by the route stated on the label into each of 10 healthy chickens free from specific antibodies (2.7.7) to 10 days old. Use five healthy chickens from the same source and stock as controls. Observe the birds for 21 days. Not more than one of the vaccinated chickens shows symptoms of or dies from infectious bronchitis. The test is not valid unless all the control chickens are free of clinical symptoms of infectious bronchitis. If during the period of observation more than 2 of the vaccinated chickens die from causes not attributable to the vaccine, repeat the test. Sterility (2.2.11). Complies with the test for sterility. Virus titre. Titrate the vaccine in cell cultures derived from SPF embryos or by inoculating into the allantoic sac of SPF embryonated eggs, 9 to 11 days old. One dose of the vaccine contains not less than 103.5 TCID50/EID50. Immunogenicity. Carry out a test for each route of administration recommended on the label and for each serotype against which protection is claimed. Administer to each of 20 healthy chickens, free from specific antibodies (2.7.7) 3 to 4 weeks old, for each of the stated routes a volume of reconstituted vaccine containing a quantity of virus equivalent to the minimum titre stated on the label. Ten additional healthy chickens of same flock for each serotype against which protection is claimed are used as unvaccinated controls. Three to four weeks later, administer by eye drop a virulent strain of bronchitis virus with a titre of at least 103.5 EID50 per ml to all the vaccinated and control birds. Between the fourth and seventh day after the challenge, take tracheal swabs from each of the vaccinated and control birds. Place each swab in a sterile test tube containing 3 ml of tryptose phosphate broth and antibiotics. Swirl thoroughly the tubes and swabs and store at 70 pending inoculation into eggs. For each chicken swab, inoculate at least 5 chicken embryos, 9 to 11 days old, with 0.2 ml of the broth from each tube into the allantoic cavity. All the embryos surviving on the third day after inoculation are used in the evaluation. A tracheal swab is considered positive for recovery of the virus if any of the embryos shows typical infectious bronchitis lesions such as stunting, curling, kidney urates, clubbing down or death between the fourth and seventh day after inoculation. The vaccine complies with the test if not less than 80 per cent of the controls and not more than 20 per cent of the vaccinated chickens are positive for virus recovery. If less than 80 per cent of the vaccinated chickens are negative for virus recovery the stock seed is unsatisfactory. The stock seed virus may be tested for immunogenicity once in 5 years provided it is maintained under standard conditions of storage of the bronchitis virus. Labelling. The label states (1) the minimum virus titre per dose; (2) the recommended age of the birds in which the vaccine is to be used.
Avian Spirochaetosis Vaccine

Avian Spirochaetosis Vaccine is a suspension prepared from viscera and membranes of developing chicken embryos of SPF eggs infected with antigenic strains of Borrelia anserina which has been inactivated in such a manner that its immunogenic activity is retained.
Production
The organism is grown in embryonated eggs derived from SPF flocks (2.7.7).
Identification
Protects chickens against infection with B. anserina.
Tests
Safety. Complies with the requiremented stated under Veterinary Vaccine. Sterility (2.2.11). Complies with the test for sterility. Potency. Inject at least twelve healthy chicken, free from antibodies, 8 to 12 weeks old, with the minimum dose of the vaccine by the route stated on the label. Use five chickens of the same stock as controls. Ten days later challenge all the chickens intraperitoneally with an adequate dose of a virulent culture of B. anserina used to prepare the vaccine or with a suspension of liver or kidney tissue obtained from infected chicken. Observe the chickens for 10 days. The vaccinated chickens do not show any symptoms of the disease. The test is not valid unless the control chickens show typical symptoms of spirochaetosis. If the potency test has been performed with satisfactory results on representative batch of the vaccine from the same seed lot, it may be omitted as a routine control test during production of other batches of the vaccine prepared from the same seed lot. Labelling. The label states (1) strain of virus used; (2) recommended age for vaccination.
Blackquarter Vaccine
Blackquarter Vaccine is a culture of suitable strain or strains of Clostridium chauvoei grown in a suitable anaerobic fluid medium and rendered sterile and non-toxic by addition of solution of formaldehyde in such a manner that it retains its immunizing properties. The vaccine may contain a suitable adjuvant.
Identification
Protects susceptible animals against infection with Cl. chauvoei.
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CANINE CONTAGIOUS HEPATITIS VACCINE, INACTIVATED
IP 2007
Tests
Safety. Use two healthy susceptible animals of one of the species for which the vaccine is intended. Inject into each animal by the recommended route twice the maximum dose stated on the label. Observe the animals for 7 days. No significant local or systemic reaction is produced. Sterility (2.2.11). Complies with the test for sterility. Potency. Inject ten healthy adult guinea pigs weighing between 350 and 450 g with a quantity of the product not greater than the minimum dose and route as stated on the label. Repeat the inoculation with the same dose of the vaccine after 28 days. None of the vaccinated guinea pigs shows any systemic reaction. However a minimal local reaction may be observed in the animal. Fourteen days after the second vaccination, inoculate intramuscularly into each of 10 vaccinated and into each of 5 control guinea pigs with a suitable quantity of virulent culture or spore suspension of Cl. Chauvoei, activated if necessary, with the solution of calcium chloride. The vaccine complies with the test, if not more than 10 per cent of the vaccinated guinea pigs die from Cl. chauvoei infection within 5 days and all the control animals die from Cl. chauvoei infection within 48 hours of challenge if virulent culture was used or within 72 hours if a spore suspension was used for the challenge. Repeat the test if 20 per cent of the vaccinated animals die. On repetition, the vaccine complies with the test if not more than 10 per cent of the vaccinated animals die within 5 days and all of the control animals die within 48 hours of challenge if virulent culture was used or within 72 hours if a spore suspension was used for challenge. Labelling. The label states (a) the method of preparation; (b) the strains of bacteria used to prepare the vaccine.
Identification
When injected into a susceptible dog, the animal develops specific neutralizing antibodies.
Tests
Water (2.3.43). Not more than 3.0 per cent (for freeze dried vaccine only). Safety. Inject each of two healthy dogs, between 8 and 14 weeks old, that have been previously tested and shown to be free from canine adenovirus neutralizing antibodies, with twice the dose and by the route stated on the label. Observe the animals for 14 days. No abnormal systemic or local reaction occurs. Sterility (2.2.11). Complies with the test for sterility. Potency. Inject each of two healthy susceptible dogs, between 8 and 14 weeks old, that have been previously tested and shown to be free from canine adenovirus neutralizing antibodies, with the minimum dose and by the route stated on the label. After14 days inject a second dose. Between 14 and 21 days after the second injection collect serum from each dog separately and examine each sample as described below. Inactivate the serum by heating at 56 for 30 min and prepare serial dilutions in a suitable medium. Add to each dilution an equal volume of serum-virus suspension containing approximately 102 TCID50. Incubate the mixtures for 1 hour at 37. Add suitable cell culture with minimum of four replicates for each dilution and incubate at 37 for 4 to 8 days. Examine each culture for evidence of specific cytopathic effect. Calculate the antibody titre. The serum from each dog contains not less than 80 SN50 per ml. Labelling. The label states (1) the strain used for the preparation; (2) the name of any added adjuvant.
Canine Contagious Hepatitis Vaccine, Inactivated

Canine Contagious Hepatitis Vaccine, Inactivated is a preparation containing canine contagious hepatitis virus inactivated in such a manner that its immunogenic activity is retained. It may be a freeze-dried preparation or a liquid preparation containing a suitable adjuvant.
Canine Contagious Hepatitis Vaccine, Live

Canine Contagious Hepatitis/Canine Adenovirus Vaccine is a freeze dried preparation containing one or more attenuated strains of canine adenovirus.
Production
The virus is propagated in a suitable cell cultures, harvested, titrated and may be mixed with a suitable stabilizing solution. The stock seed virus should be tested for immunogenicity at least once in 5 years, if maintained under suitable conditions of storage.
Production
The virus is propagated in suitable cell culture systems, the viral suspension is harvested, titrated and may contain a suitable stabilizing solution.
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CANINE CORONA VIRUS VACCINE, INACTIVATED
Identification
The vaccine, mixed with one or more specific antisera of canine adenovirus(s), does not produce specific cytopathic effects in susceptible cell cultures.
liquid or as a freeze-dried preparation to be reconstituted with a suitable liquid immediately before use. The liquid vaccine may contain a suitable adjuvant.
Production
The virus is grown in suitable cell cultures systems. The vaccine may contain a suitable adjuvant. Only an inactivated viral suspension that complies with the requirements mentioned under final bulk vaccine of each batch is used in the preparation of the final vaccine.
Tests
Water (2.3.43). Not more than 3.0 per cent. Virus titre. Not less than103 TCID 50 virus per dose, determining the titre of the vaccine in a suitable cell culture. Safety. Inject each of two susceptible dogs, between 8 and 14 weeks old, that have been previously tested and shown to be free from canine adenovirus neutralising antibodies, with twice the dose and by the route stated on the label. Observe the animals for 21 days. No abnormal systemic or local reaction occurs. Sterility (2.2.11). Complies with the test for sterility. Potency. Inject five healthy susceptible dogs, between 8 and 14 weeks old, that have been previously tested and shown to be free from canine adenovirus neutralizing antibodies, with a quantity of the vaccine equivalent to the minimum titre and by the route stated on the label. Observe the animals for 21 days. Inject intravenously each of the five vaccinated animals and each of two control animals of the same stock and weight range with a quantity of a virulent strain of canine contagious hepatitis virus sufficient to cause death or typical signs of the disease in a susceptible dog. Observe the animals for a further period of 21 days. The vaccinated animals remain in normal health and the controls die from hepatitis or show typical signs of serious infection. If one of the controls does not show any signs of the disease, repeat the test. The vaccinated animals of the second group remain in normal health and the control animals die from hepatitis or show typical signs of serious infection. If the potency test has been performed with satisfactory results on a representative batch of the vaccine from the seed lot, it may be omitted as a routine control test during production on other batches of the vaccine prepared from the same seed lot. Labelling. The label states (1) the minimum virus titre; (2) the species of the animals in which use of the vaccine is recommended.
Identification
When inoculated into dogs, the vaccine stimulates the production of specific neutralizing antibodies against canine corona virus as determined by suitable serological tests.
Tests
Water (2.3.43). Not more than 3.0 per cent (for freeze dried vaccine only). Safety. Inject each of two healthy susceptible dogs, between 8 and 14 weeks old, free from canine corona virus antibodies with a quantity equivalent to 2 doses by the route stated on the label. Observe the animals for 14 days. No abnormal systemic or local reaction occurs. Sterility (2.2.11). Complies with the test for sterility. Potency. Either of the test A or B may be carried out. A. Inject each of five healthy susceptible guinea pigs, each weighing between 350 and 400 g, with half the minimum dose and by the route stated on the label. Repeat the injection after 14 days. Fourteen days after the second injection collect blood samples and obtain the serum from each guinea pig separately. Inactivate each serum by heating at 56 for 30 min. Examine the serum samples for antibodies by the following method. Prepare 2-fold serial dilutions of serum in a medium suitable for canine cells. Add to each dilution an equal volume of a virus suspension containing approximately 102 TCID50 and incubate the mixtures at 37 for 1 hour. Inoculate a suitable volume of canine cells into at least 4 replicates of serum virus mixtures and incubate at 37 for 4 days. Examine for evidence of specific cytopathic effects and calculate the antibody titre. The vaccine complies with the test if the mean antibody titre is not less than 32 SN50 per 0.05 ml of serum. B. Inject each of two healthy susceptible dogs, between 8 and 14 weeks old, having antibody titre less than 4 SN50 per 0.05 ml of serum, with the dose and by the route recommended on the label. Fourteen days later collect the serum samples from each dog separately. Inactivate each serum by heating at 56
Canine Corona Virus Vaccine, Inactivated

Canine Corona Virus Vaccine, Inactivated is a preparation containing canine corona virus inactivated in such a manner that its immunogenic activity is retained. It may be issued as a
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IP 2007
for 30 min. Examine the serum samples individually for neutralizing antibodies by the method described in test A. If one dog fails to respond, i.e., the antibody titre is less than 4 SN50 per 0.05 ml of serum, repeat the test with two more dogs and calculate the mean titres of the three dogs that have responded. The vaccine complies with the test if the median antibody titre of the sera is not less than 32 SN50 per 0.05 ml. Labelling. The label states (1) the recommended routes of administration; (2) that the preparation should be shaken well before use; (3) that the liquid preparation should not be allowed to freeze; (4) that the vaccine should be used immediately after reconstitution.
tenths of the dose of the vaccine is injected. Inject each mouse intracerebrally with 0.03 m1 of the vaccine. Observe for 21 days. Not more than two mice die within the first 48 hours. If more than two mice die within the first 48 hours, repeat the test. From the third day to 21 days after the injection, the mice do not show any abnormalities attributable to the vaccine. Mycoplasmas (2.7.8). Complies with the test for freedom from mycoplasmas. Safety. Reconstitute the vaccine as recommended on the label and carry out the following tests. A. For chicken embryo-adapted vaccine only. Inject 0.3 m1 intracerebrally into each of a group of eight mice, between 3 and 4 weeks old, and 0.5 m1 intraperitoneally into each of another eight mice of the same age group. Observe both the groups for 7 days. Not more than one mouse in either group shows any abnormal local or systemic reaction attributable to the vaccine. B. Inject each of two susceptible dogs, between 8 and 14 weeks old which do not have antibodies against canine distemper virus with twice the dose and by the route stated on the label. Observe the animals for 21 days. None of the dogs shows any abnormal local or systemic reaction. Sterility (2.2.11). Complies with the test for sterility. Potency. Inject each of five susceptible dogs, between 8 and 14 weeks old, that have been previously tested and shown to be free from distemper virus neutralizing antibodies with a volume of the reconstituted vaccine containing a quantity of the virus equivalent to the minimum titre and by the route stated on the label. Use another two dogs of the same stock and age group as unvaccinated controls. Observe the animals for 21 days. Inject intravenously each of the seven animals with a quantity of virulent strain of canine distemper virus sufficient to cause death or produce typical signs of the disease in a susceptible dog. Observe the animals for a further 21 days. The vaccinated animals survive and show no clinical signs of canine distemper. The test is not valid unless the control dogs die or show symptoms typical of canine distemper. If one of the control animals does not show any sign of distemper, repeat the test. The vaccinated animals of the second group remain in normal health and the control animals die from distemper or show symptoms typical of canine distemper. If the potency test has been performed with satisfactory results on a represntative batch of the vaccine from the seed lot, it may be omitted as a routine control test during production on other batches of the vaccine prepared from the same seed lot. Labelling. The label states (1) the strain used for the preparation; (2) virus titre per dose.
Canine Distemper Vaccine, Live

Canine Distemper Vaccine, Live is a freeze-dried preparation of a strain of canine distemper virus that has been attenuated for dogs and is grown either in SPF embryonated eggs or in suitable cell cultures.
Production
The virus is propagated in suitable cell culture systems, the viral suspension is harvested, titrated and may contain a suitable stabilizing solution. The stock seed virus should be tested for potency at least once in 5 years, if maintained under suitable conditions of storage.
Identification
A. The vaccine infects the chorio-allantoic membranes of SPF embryonated eggs or suitable cell cultures. This effect is neutralized by canine distemper antiserum. B. An injection into susceptible ferrets or dogs does not cause distemper but immunizes them against the disease. The vaccine strain is satisfactory with respect to absence of increase in virulence.
Tests
Water (2.3.43). Not more than 3.0 per cent. Virus titre. Not less than 103 EID50/TCID50 of the virus per dose, determining the titre of the vaccine in a suitable cell culture or SPF eggs (2.7.7). Extraneous pathogens A. Mix the vaccine under examination with a mono specific distemper antiserum. It no longer provokes cytopathic effects in susceptible cell cultures and shows no evidence of haemagglutinating or haemadsorbing agents. B. Use a sufficient number of mice, not less than ten, each weighing between 11 and 15 g, such that a total of three-
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CANINE PARVOVIRUS VACCINE, INACTIVATED
Canine Parainfluenza Virus Vaccine, Live

Canine Parainfluenza Virus Vaccine, Live is a freeze-dried preparation of tissue culture fluid containing the cell cultureadapted attenuated canine parainfluenza virus of stock seed which has been established as pure, safe and immunogenic.
If the potency test has been performed with satisfactory results on a representative batch of the vaccine it may be omitted as a routine test during production on the other batches of vaccine prepared from the same seed lot. Labelling. The label states (1) the strain used for the preparation; (2) the virus titre per dose.
Production
The virus is propagated in suitable cell culture systems, the viral suspension is harvested, titrated and may contain a suitable stabilizing solution.
Canine Parvovirus Vaccine, Inactivated

Canine Parvovirus Vaccine, Inactivated is a liquid or freeze dried preparation of canine parvovirus inactivated by a suitable method so that its immunogenic activity is retained.
Identification
When inoculated into dogs, the vaccine stimulates the production of specific neutralizing antibodies against canine parainfluenza virus as determined by suitable serological tests.
Production
The virus is grown in suitable cell culture systems. The virus may be cloned, purified and concentrated. The vaccine may contain a suitable adjuvant.
Tests
Water (2.3.43). Not more than 3.0 per cent. Virus titre. Not less than 103 TCID50 per dose, determining the titre of the vaccine in a suitable cell culture. Safety. Inject each of two susceptible dogs, between 8 and 14 weeks old, free from canine parainfluenza virus haemaggulatinating antibodies with a dose of the vaccine reconstituted with the sterile diluent equivalent to 10 times the dose and by the route stated on the label. Observe the animals for 14 days. None of the dogs shows any systemic or local reactions. Sterility (2.2.11). Complies with the test for sterility. Potency. Use two healthy susceptible dogs, between 8 and 14 weeks old, having haemagglutination inhibition antibody titres less than 1:4 per 0.1 ml of serum. Keep equal number of dogs as unvaccinated controls. Vaccinate each test group dog with the vaccine as per schedule stated on the label. After 21 days, collect the serum from each dog separately and examine each sample as described below. Heat the serum of each animal at 56 for 30 minutes and prepare serial dilutions with saline solution. To 0.025 ml of each dilution add 0.025 ml of a canine parainfluenza virus suspension containing 4 haemagglutinating units. Allow the mixture to stand at room temperature for 30 minutes and add 0.05 ml of a suspension of chicken erythrocytes containing 2 x 107 erythrocytes per ml. Allow to stand for 1 hour and note the last dilution of serum that completely inhibits haemugglutination. Calculate the median antibody litre of the sera which should not be less than 1:32 per 0.1 ml of the serum. If one dog fails to respond, repeat the test using two more dogs and calculate the result as the mean of titres obtained from all of three dogs that have responded which is not less than 1 :32 per 0.1 ml of the serum.
Identification
When inoculated into dogs, the development of specific neutralizing antibodies against canine parvovirus can be demonstrated by suitable serological tests.
Tests
Safety. Inject into each of two healthy susceptible dogs, between 8 and 14 weeks old, preferably sero negative ones with a quantity equivalent to 2 doses by the route stated on the label. Observe the animals for 14 days. No abnormal systemic or local reaction occurs. Sterility (2.2.11). Complies with the test for sterility. Potency. Either of the test A or B may be carried out. A. Inject subcutaneously each of five healthy susceptible guinea-pigs, each weighing between 350 and 400g, with half of the dose stated on the label. After 14 days, inject again half of the dose stated on the label. Fourteen days after the second injection collect blood samples and obtain the serum from each guinea pig separately. Inactivate each serum by heating at 56 for 30 min. To 1 volume of each serum add 9 volumes of a 20 per cent suspension of light kaolin in phosphate buffered saline pH 7.4. Shake the mixture for 20 minutes and centrifuge at 2,000 rpm for 10 minutes. Collect the supernatant liquid and mix with 1 volume of a concentrated suspension of Rhesus monkey/pig erythrocytes. Allow the mixture to stand at 4 for 60 minutes and centrifuge at 2,000 rpm for 10 minutes and collect the supernatant serum obtained in 10-fold dilution. Using each serum, prepare a series of 2-fold dilutions. To 0.025 ml of each of the latter dilutions add 0.025 ml of a
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suspension of canine parvovirus antigen containing approximately 8 haemagglutinating (HA) units and allow to stand at 37 for 30 minutes. Add 0.05 ml or other suitable quantity of a 1 per cent suspension of Rhesus monkey/pig erythrocytes containing 30 x 106 cells per ml to at least four replicates of each dilution. Allow to stand at 4 for 90 minutes and note the last dilution of the serum that completely inhibits haemagglutination. The vaccine complies with the test if the median antibody titre of the sera is not less than 1:80. B. Inject each of two healthy susceptible dogs, between 8 and 14 weeks old, having antibody titres less than 4 ND50 (50 percent neutralizing dose) per 0.1 ml of serum, with the dose and by the route recommended on the label. Fourteen days later collect the blood/serum samples from each dog separately. Inactivate each serum by heating at 56 for 30 min. Examine the serum samples individually for neutralizing antibodies as follows: Prepare 2-fold serial dilutions of serum in a medium suitable for canine cells. Add to each dilution an equal volume of virus suspension containing approximately 104 TCID50 and incubate the mixtures at 37 for one hour. Inoculate a suitable volume of canine cells into at least four replicates of serum virus mixtures and incubate at 37 for 7 days. Examine for evidence of specific cytopathic effects and calculate the antibody titre. The vaccine complies with the test if the mean antibody titre is not less than 32 ND50 per 0.1 ml of serum. If one dog fails to respond repeat the test using two more dogs and calculate the mean titres of the three dogs that have responded. Labelling. The label states (1) the method of preparation; (2) the types and strains of virus used to prepare the vaccine.
Virus titre. Not less than 103 TCID50 per dose, determining the titre of the vaccine in a suitable cell culture. Safety. Inject each of two susceptible dogs, between 8 and 14 weeks old, free from canine parvovirus haemaggulatinating antibodies, a quantity of the vaccine reconstituted with the sterile diluent equivalent to 10 times the dose and by the route stated on the label. Observe the animals for 21 days. No abnormal systemic or local reaction occurs. Sterility (2.2.11). Complies with the test for sterility. Potency. Inject each of seven dogs, between 8 and 14 weeks old, free from canine parvovirus haemagglutinating antibodies, subcutaneously with the minimum dose stated on the label. Use two dogs of the same stock, weight and age range as controls. Not less than 21 days after injection of the vaccine, challenge the vaccinated and control animals through the oronasal route with a virulent strain of infectious canine parvovirus. Observe the animals for 14 days. Not less than five of the vaccinated dogs survive. The test is not valid unless the control dogs die or show clinical signs of canine parvovirus infection. If the potency test has been performed with satisfactory results on a representative batch of the vaccine it may be omitted as a routine test during production of the other batches of vaccine prepared from the same seed lot. Labelling. The label states (1) the strain used for the preparation; (2) virus titre per dose.
Canine Parvovirus Vaccine, Live

Canine Parvovirus Vaccine, Live is a freeze-dried preparation of a strain of canine parvovirus that is attenuated for the target species of dogs.
Clostridium Multicomponent Vaccine, Inactivated

Clostridium Multicomponent Vaccine, Inactivated consists of five highly antigenic components containing the toxoids of C. perfringens type B, C. perfringens type C, C. perfringens type D, C. oedematiens and C. septicum which are prepared in double strength and then combined in such a proportion that would invoke adequate anti-toxin response in the vaccinated sheep against each antigen incorporated in the vaccine.
Production
The attenuated virus is grown in suitable cell culture systems. The viral harvest is titrated and mixed with a suitable stabilizing solution. The stock seed virus should be tested for immunogenicity at least once in 5 years, if maintained under suitable conditions of storage.
Identification
When injected into susceptible animals, it stimulates the production of epsilon and beta antitoxin against C. perfringens types B, C and D and also antitoxins against C. septicum and toxin of C. oedematiens.
Identification
When inoculated into dogs, the development of specific neutralizing antibodies against canine parvovirus can be demonstrated by suitable serological tests.
Tests
Safety. Four sheep each are inoculated with two times the dose of vaccine subcutaneously and are observed for 7 days
Tests
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CLOSTRIDIUM NOVYI (TYPE B) VACCINE FOR VETERINARY USE
during which period the animals do not show any local or systemic reaction. Sterility (2.2.11). Complies with the test for sterility. Potency. Eight sheep each are inoculated with 2 doses of vaccine subcutaneously at an interval of 21 to 28 days and bled on 10th day after second inoculation for collection of serum for assessing the antitoxin titre against each antigen incorporated in the vaccine. The post-inoculation serum contains not less than 2 IU of epsilon antitoxin and 10 units of beta antitoxins of C. perfringens types B and C and 2.5 IU of C. septicum antitoxin and 3.5 IU of C. oedematiens antitoxin. Labelling. The label states (1) the types of Clostridia contained in the vaccine; (2) the preparation should be shaken before use.
Tests
Sterility (2.2.11). Complies with the test for sterility. Potency. Inject subcutaneously into each of not less than 10 healthy rabbits, 3 to 6 months old, a quantity of vaccine not exceeding the minimum dose stated on the label as the first dose. After 21 to 28 days, inject into the same animals a quantity of the vaccine not exceeding the minimum dose stated on the label as the second dose. 10 to 14 days after the second injection, bleed the rabbits and pool the sera. The alpha antitoxin titre of the pooled sera is not less than 3.5 IU per ml. The International Unit is the specific neutralising activity for C. novyi alpha toxin contained in a stated amount of the International standard, which consists of a quantity of dried immune horse serum. The equivalence in International Units of the International standard is stated by the World Health Organisation. The potency of the pooled sera obtained from the rabbits is determined by comparing the quantity necessary to protect mice or other suitable animals against the toxic effects of a fixed dose of C. novyi alpha toxin with the quantity of a reference preparation of Clostridium novyi alpha antitoxin, calibrated in International Units, necessary to give the same protection. For this comparison, a suitable preparation of C. novyi alpha toxin for use as a test toxin is required. The dose of the test toxin is determined in relation to the reference preparation; the potency of the serum under examination is determined in relation to the reference preparation using the test toxin. Preparation of test toxin. Prepare the test toxin from a sterile filtrate of an approximately 3 to 5 day culture in liquid medium of C. novyi type B and dry by a suitable method. Select the test dose of the toxin in mice by determining the L+ 10 dose and the LD50 the observation period being 72 hours. A suitable alpha toxin contains not less than one L+/10 dose in 0.05 mg and not less than 10 LD50 in each L+/10 dose. Determination of test dose of toxin. Prepare a solution of the reference preparation in a suitable liquid so that it contains 1 IU per ml. Prepare a solution of the test toxin in a suitable liquid so that 1 ml contains a precisely known amount such as 1 mg. Prepare mixtures of the solution of the reference preparation and the solution of the test toxin such that each mixture contains 1.0 ml of the solution of the reference preparation (1 IU), one of a series of graded volumes of the solution of the test toxin and sufficient of a suitable liquid to bring the total volume to 2.0 ml. Allow the mixtures to stand at room temperature for 60 min. Using not less than 2 mice, each weighing between 17 and 22 g, for each mixture, inject a dose of 0.2 ml subcutaneously into each mouse. Observe the mice for 72 hours. If all the mice die, the amount of toxin present in
Clostridium novyi (Type B) Vaccine for Veterinary Use

Clostridium novyi (Type B) Vaccine for Veterinary Use is prepared from a liquid culture of a suitable strain of Clostridium novyi type B.
Production
The whole culture or its filtrate or a mixture of the two is inactivated in such a manner that toxicity is eliminated and immunogenic activity is retained. Toxoids and/or inactivated cultures may be treated with a suitable adjuvant, after concentration if necessary. Choice of vaccine composition. The vaccine is shown to be satisfactory with respect to safety and efficacy (2.7.12). For the latter, it shall be demonstrated that for each target species the vaccine, when administered according to the recommended schedule, stimulates an immune response (for example, induction of antibodies) consistent with the claims made for the product. Batch testing Safety. Administer by a recommended route, to each of 2 sheep that have not been vaccinated against C. novyi type B twice the maximum dose of the vaccine stated on the label. Observe the animals for not less than 14 days. No abnormal local or systemic reaction occurs. Residual toxicity. Inject 0.5 ml of the vaccine subcutaneously into each of 5 mice, each weighing between 17 and 22 g. Observe the animals for 7 days. No abnormal local or systemic reaction occurs.
Identification
The vaccine stimulates the formation of C. novyi type B alpha antitoxin when injected into animals.
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0.2 ml of the mixture is in excess of the test dose. If none of the mice dies, the amount of toxin present in 0.2 ml of the mixture is less than the test dose. Prepare fresh mixtures such that 2.0 ml of each mixture contains 1.0 ml of the solution of the reference preparation (1 IU) and one of a series of graded volumes of the solution of the test toxin separated from each other by steps of not more than 20 per cent and covering the expected end-point. Allow the mixtures to stand at room temperature for 60 min. Using not less than two mice for each mixture, inject a dose of 0.2 ml subcutaneously into each mouse. Observe the mice for 72 hours. Repeat the determination at least once and combine the results of the separate tests that have been made with mixtures of the same composition so that a series of totals is obtained, each total representing the mortality due to a mixture of a given composition. The test dose of toxin is the amount present in 0.2 ml of that mixture which causes the death of one half of the total number of mice injected with it. Determination of the potency of the serum obtained from rabbits Preliminary test. Dissolve a quantity of the test toxin in a suitable liquid so that 1 ml contains 10 times the test dose (solution of the test toxin). Prepare a series of mixtures of the solution of the test toxin and of the serum under examination such that each mixture contains 1.0 ml of the solution of the test toxin, one of a series of graded volumes of the serum under examination and sufficient of a suitable liquid to bring the final volume to 2.0 ml. Allow the mixtures to stand at room temperature for 60 min. Using not less than 2 mice for each mixture, inject a dose of 0.2 ml subcutaneously into each mouse. Observe the mice for 72 h. If none of the mice dies, 0.2 ml of the mixture contains more than 0.1 IU. If all the mice die, 0.2 ml of the mixture contains less than 0.1 IU. Final test. Prepare a series of mixtures of the solution of the test toxin and of the serum under examination such that 2.0 ml of each mixture contains 1.0 ml of the solution of the test toxin and one of a series of graded volumes of the serum under examination, separated from each other by steps of not more than 20 per cent and covering the expected end-point as determined by the preliminary test. Prepare further mixtures of the solution of the test toxin and of the solution of the reference preparation such that 2.0 ml of each mixture contains 1.0 ml of the solution of the test toxin and one of a series of graded volumes of the solution of the reference preparation, in order to confirm the test dose of the toxin. Allow the mixtures to stand at room temperature for 60 min. Using not less than 2 mice for each mixture, proceed as described in the preliminary test. The test mixture which contains 0.1 IU in 0.2 ml is that mixture which kills the same or almost the same number of mice as the reference mixture containing 0.1 IU in 0.2 ml. Repeat the determination at least once and calculate the average of all
valid estimates. The test is valid only if the reference preparation gives a result within 20 per cent of the expected value. The confidence limits (P = 0.95) have been estimated to be (a) 85 per cent and 114 per cent when 2 animals per dose are used; (b) 91.5 per cent and 109 per cent when 4 animals per dose are used; (c) 93 per cent and 108 per cent when 6 animals per dose are used. Labelling. The label states (1) whether the product is a toxoid, a vaccine prepared from a whole inactivated culture or a mixture of the two; (2) that the preparation is to be shaken before use; (3) for each target species, the immunising effect produced (for example, antibody production, protection against signs of disease or infection).
Clostridium Perfringens Vaccine, Inactivated

The vaccines contains cultures strains of C. perfringens type B (Lamb Dysentery Vaccine), type C (Struck Vaccine) or type D (Enterotoxaemia Vaccine; Pulpy Kidney Vaccine) or any combination of these types.
Production
The organisms grown in an anaerobic medium, the whole culture or their filtrates or a mixture of the two are inactivated in such a manner that toxicity is eliminated and immunogenic activity is retained. Toxoid and or inactivated cultures may contain a suitable adjuvant. Batch tesing Safety. Inject subcutaneously two healthy susceptible sheep weighing about 18 kg each or two healthy susceptible rabbits weighing between 1.5 and 2.0 kg each with twice the dose stated on the label and observe for 7 days. No systemic or local reaction is observed. Observe the animals for 14 days. No abnormal local or systemic reaction occurs. Residual toxicity. Inject 0.5 ml of the vaccine subcutaneously into each of 5 mice, each weighing 17 to 22 g. Observe the animals for 7 days. No abnormal local or systemic reaction occurs. Sterility (2.2.11). Complies with the test for sterility. Potency C. perfringens Type B Vaccine. Inject subcutaneously into each of six healthy susceptible sheep weighing about 18 kg or ten healthy susceptible rabbits weighing between 1.5 and 2.0
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kg with the minimum dose of the vaccine stated on the label. Repeat the dose after an interval of 21 to 28 days. Bleed the animals 10 to 14 days after the second dose of the vaccine and determine beta antitoxin titres in the pooled serum sample by testing in mice as per the method described for C. perfringens Type D Vaccine. Product passes the test if the post-inoculation pooled serum contains not less than 10 IU of beta antitoxins, and not less than 5 IU of episilon toxin per milliliter. C. perfringens Type C Vaccine. Carry out the test for potency as described for C. perfringens Type B Vaccine. 1 ml of serum contains not less than 10 IU of beta antitoxin per ml. C. perfringens Type D (Enterotoxaemia) Vaccine. Carry out the test as described below. 1 ml of serum contains not less than 5 IU of C. perfringens epsilon toxin per ml.
The test dose of each toxin is established in relation to the appropriate Standard preparation of antitoxin and the potency of antitoxin under examination is then determined in relation to the appropriate Standard preparation using the appropriate test toxin. International standard for the standard preparations The International units of the antitoxin is the specific neutralizing activity of C. perfringens epsilon toxin contained in the stated amount in relation to International standards in the dried Horse serum. The International units of the antitoxins is the specific neutralizing activity for the C. perfringens beta toxin contained in the stated amount in relation to International standards in the dried Horse serum. Test animals Use healthy mice having body weights such that the difference between the lightest and heaviest is not more than 5 g. Suggested method for preparation of test toxin. Prepare C. perfringens toxins from sterile supernatants/filtrates of early cultures of C. perfringens type B, type C or type D. The supernatants may be purified by precipitation with ammonium sulphate and the resulting precipitate collected. This may then be dried over phosphorus pentoxide at a pressure of 1.5 to 2.5 kPa, powdered and kept dry or re-dissolved and freezedried. Selection of test toxin. Select toxin for use as the test toxin by determining the following quantities. L+ and L+/10 doses. These are the smallest quantities of toxin that when mixed respectively with 1 Unit of antitoxin and with 0.1 Unit of antitoxin and injected intravenously into mice cause the death of the animals within 72 hours. LD50. This is the quantity of toxin that when injected intravenously into mice causes the death of one-half of the mice injected within 72 hours. A suitable C. perfringens beta toxin is one that has an L+ dose in 0.2 mg or less and contains not less than 25 LD50 in an L+ dose. A suitable Clostridium perfringens epsilon toxin is one that has an L+/10 dose in 0.005 mg or less and contains not less than 20 LD50 in an L+/10 dose. Determination of test dose of C. perfringens beta toxin. Dissolve a quantity of dried toxin in a suitable liquid such that 1.0 ml contains a precise amount such as 10 mg. Reconstitute the Standard preparation of C. perfringens beta antitoxin with a suitable liquid to give a solution containing 5 Units of C. perfringens beta antitoxin in 1 ml.
Identification
A. When injected into susceptible animals, the C. perfringens Type B Vaccine stimulates the production of C. perfringens beta and epsilon antitoxins. B. When injected into susceptible animals, the C. perfringens Type C Vaccine stimulates the production of C. perfringens beta antitoxin. C. When injected into susceptible animals, the C. perfringens Type D Vaccine stimulates the production of C. perfringens epsilon antitoxin.
Tests
Potency test. Inject subcutaneously into each of at least six healthy susceptible sheep weighing about 18 kg or ten healthy susceptible rabbits weighing between 2.0 and 2.5 kg with the minimum dose of the vaccine stated on the label. Repeat the dose in each sheep/rabbit after an interval of 21 to 28 days. The rabbits are inoculated with the same dose after one month of the first inoculation. Bleed the animals 10 to 14 days after the second inoculation. The sera of sheep or rabbits are pooled separately and estimated for the antitoxin levels. Biological assay of C. perfringens antitoxins The potency of C. perfringens beta and epsilon antitoxins is determined by comparing the dose of antitoxin necessary to protect mice or other suitable animals against the toxic effects of C. perfringens beta toxin or epsilon toxin with the dose of a standard preparation of the respective antitoxin necessary to give the same protection. For this comparison, the Standard preparations of C. perfringens beta antitoxin and C. perfringens epsilon antitoxin and suitable preparations of C. perfringens beta and epsilon toxins are required.
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IP 2007
Prepare mixtures such that 5.0 ml of each mixture contains 2.0 ml of the solution of the Standard preparation (10 Units) and one of a series of graded volumes of the solution of the toxin. Dilute each mixture to the same final volume with a suitable liquid. Allow the mixtures to stand at room temperature, protected from light, for 30 minutes and then inject a dose of 0.5 ml of each mixture intravenously into each of not less than two mice. Observe the mice for 72 hours. If all the mice die, the amount of toxin present in 0.5 ml of the mixture is in excess of the test dose. If none of the mice dies, the amount of toxin present in 0.5 ml of the mixture is less than the test dose. Prepare similar fresh mixtures such that 5.0 ml of each mixture contains 2.0 ml of the solution of the Standard preparation (10 Units) and one of a series of graded volumes of the solution of the toxin, separated from each other by steps of not more than 20 per cent and covering the expected end-point. Allow the mixtures to stand at room temperature, protected from light, for 30 minutes, Inject a dose of 0.5 ml of each mixture intravenously into each of not less than two mice. Observe the mice for 72 hours. Repeat the determinations at least once and add together the results of the separate tests that have been made with mixtures of the same composition such that a series of totals is obtained, each total representing the mortality due to a mixture of a given composition. The test dose of toxin is the amount present in 0.5 ml of that mixture that causes the death of one-half of the total number of mice injected with it within 72 hours. Determination of test dose of C. perfringens epsilon toxin. Carry out the method described for the determination of test dose of C. perfringens beta toxin with the following modification. Dissolve a quantity of dried toxin in a suitable liquid such that 1.0 ml contains a precise amount such as 1 mg. Reconstitute the Standard preparation of C. perfringens epsilon antitoxin with a suitable liquid to give a solution containing 0.5 Unit in 1 ml (the prepared mixtures will therefore contain 1 Unit of the Standard preparation in 5 ml). The test dose of toxin is the amount present in 0.5 ml of that mixture that causes the death of one-half of the total number of mice injected with it within 72 hours. Determination of potency of C. perfringens beta antitoxin Preliminary test. Dilute the test toxin with a suitable liquid such that 2.0 ml contains 10 times the test dose. Prepare mixtures such that 5.0 ml of each mixture contains 2.0 ml of the solution of the toxin and one of a series of graded volumes of the preparation under examination. Adjust each mixture to the same final volume with a suitable liquid. Allow the mixtures to stand at room temperature, protected from light, for 30 minutes. Inject a dose of 0.5 ml of each mixture intravenously into each of not less than two mice and observe the mice for 72 hours. If
all the mice die, 0.5 ml of the mixture contains less than I Unit of antitoxin. If none of the mice dies 0.5 ml of the mixture contains more than 1 Unit of antitoxin. Final test. Prepare similar fresh mixtures such that 5.0 ml of each mixture contains 2.0 ml of the solution of the toxin and one of a series of graded volumes of the preparation under examination, separated from each other by steps of not more than 20 per cent and covering the expected end-point. Prepare further mixtures of 5.0 ml containing 2.0 ml of the solution of the toxin and graded volumes of the Standard preparation of C. perfringens beta antitoxin to confirm the test dose of the toxin. Allow the mixtures to stand at room temperature, protected from light, for 30 minutes. Inject a dose of 0.5 ml of each mixture intravenously into each of not less than two mice and observe the mice for 72 hours. The mixture of the antitoxin under examination which contains 1 Unit of C. perfringens beta antitoxin in 0.5 ml is that mixture which causes the death of the same or almost the same number of mice as the mixture containing 1 Unit of the Standard preparation of C. perfringens beta antitoxin in 0.5 ml. Repeat the determinations at least once and calculate the average of all valid estimates. Estimates are not valid unless the Standard preparation gives a result within 20 per cent of the expected value. Determination of potency of C. perfringens epsilon antitoxin. Carry out the preliminary test and final test as described for the determination of potency of C. perfringens beta antitoxin with the following amendments. Dilute a quantity of the test toxin in a suitable liquid such that 2.0 ml contains 10 times the test dose. The Standard preparation used in these tests is that of C. perfringens epsilon antitoxin. The mixture of the antitoxin under examination which contains 0.1 Unit of C. perfringens epsilon antitoxin in 0.5 ml is that mixture which causes the death of the same or almost the same number of mice as the mixture containing 0.1 Unit of the Standard preparation of C. perfringens epsilon antitoxin in 0.5 ml. Limits of error. For the suggested method, the limits of error (P = 0.95) have been estimated to be 85 to 114 per cent when two mice are used per dose, 91.5 to 109 per cent when four mice are used per dose, and 93 to 108 per cent when six mice are used per dose. Labelling. The label states (a) the type or types of C. perfringens from which the vaccine has been prepared; (b) whether the preparation is a toxoid or a vaccine prepared from a whole inactivated culture or a mixture of the two; (c) that the preparation is to be shaken before use; (d) for each target species, the immunising effect produced (for example, antibody production, protection against signs of disease or infection).
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IP 2007
CLOSTRIDIUM SEPTICUM VACCINE
Clostridium Septicum Vaccine

Clostridium Septicum Vaccine is a suspension of a culture of a highly toxigenic strain of C. septicum grown in an anaerobic medium, or a filtrate from such a culture.
serum (supplied in ampoules containing 500 Units) or another suitable preparation the potency of which has been determined in relation to the International standard. Safety. Inject subcutaneously each of two healthy susceptible sheep, between 8 and 12 months old, with twice the dose stated on the label. Observe the animals for 7 days; none of the animals shows any systemic or local reaction. Observe the animals for 14 days. Test animals. Use healthy mice having body weights such that the difference between the lightest and heaviest is not more than 5 g. Preparation of test toxin. Prepare C. septicum toxin by growing C. septicum in a liquid culture medium, filtering the supernatant aseptically and precipitating with ammonium sulphate. The resulting precipitate, which contains the toxin, is collected, dried over phosphorus pentoxide at a pressure of 1.5 to 2.5 kPa, powdered and kept dry. Selection of test toxin. Select toxin for use as the test toxin by determining the following quantities. L+/5 dose. This is the smallest quantity of the toxin which when mixed with 0.2 Unit of antitoxin and injected intravenously into mice causes the death of the animals within 72 hours. LD50. This is the quantity of toxin which when injected intravenously into mice causes the death of one-half of the animals within 72 hours. A suitable C. septicum toxin is one that has an L+/5 dose in 1 mg or less and contains not less than 10 LD50 in an L+/5 dose. Determination of test dose of toxin. Weigh accurately a quantity of the dried toxin and dissolve it in a suitable liquid so that 1.0 ml contains a precise known amount, such as 4 mg. Prepare a solution of the Standard preparation in a suitable liquid such that 1.0 ml contains 1 Unit. Prepare mixtures such that 5.0 ml of each mixture contains 2.0 ml of the solution of the Standard preparation (2 Units) and one of a series of graded volumes of the solution of the toxin. Dilute each mixture with a suitable liquid to the same final volume (5.0 ml). Allow the mixtures to stand at room temperature, protected from light, for 60 minutes and then inject a dose of 0.5 ml of each mixture intravenously into each of not less than 2 mice. Observe the mice for 72 hours. If all the mice die the amount of toxin present in 0.5 ml of the mixture is in excess of the test dose. If none of the mice dies, the amount of toxin present in 0.5 ml of the mixture is less than the test dose. Prepare similar fresh mixtures such that 5.0 ml of each mixture contains 2.0 ml of the solution of the Standard preparation (2 Units) and one of a series of graded volumes of the solution of the toxin separated from each other by steps of not more than 20 per cent and covering the expected endpoint.
Production
The whole culture or its filtrate or a mixture of the two is inactivated in such a manner that toxicity is eliminated and immunogenic activity is retained. Toxoid and/or inactivated cultures may be treated with a suitable adjuvant. Batch testing Residual toxicity. Inject 0.5 ml of the vaccine subcutaneously into each of 5 mice, each weighing between 17 and 22 g. Observe the animals for 7 days. No abnormal local or systemic reaction occurs. Sterility (2.2.11). Complies with the test for sterility. Potency. Inject subcutaneously each of eight healthy susceptible sheep, between 8 and 12 months old, or ten rabbits, between 3 and 6 months old, with the minimum dose of the vaccine stated on the label. Repeat the dose after an interval of 21 to 28 days. Ten days after the second inoculation, bleed the animals. Pool the sera samples from individual animals and determine the antitoxin titre by the biological assay of C. septicum antitoxin described below. 1 ml of serum contains not less than 2.5 Units of C. septicum antitoxin by biological assay of C. septicum antitoxin. The potency of Clostridium septicum antitoxin is determined by comparing the dose of antitoxin necessary to protect mice or other suitable animals against the lethal effects of Clostridium septicum toxin with the dose of a Standard preparation of Clostridium septicum antitoxin necessary to give the same protection. For this purpose, the Standard preparation of C. septicum antitoxin and a suitable preparation of C. septicum toxin for use as a test toxin are required.
Identification
When injected into healthy susceptible animals, it stimulates the production of antitoxins to C. septicum.
Tests
The test dose of the toxin is determined in relation to the Standard preparation of antitoxin and the potency of antitoxin under examination is then determined in relation to the Standard preparation using the test toxin. Assay Standard preparation The Standard preparation is the 3rd International standard, established in 1957, consisting of dried hyperimmune horse
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DUCK PASTEURELLA VACCINE, INACTIVATED
IP 2007
Allow the mixtures to stand at room temperature, protected from light, for 60 minutes. Inject a dose of 0.5 ml of each mixture intravenously into each of not less than two mice. Observe the mice for 72 hours. Repeat the determinations at least once and add together the results of the separate tests that have been made with mixtures of the same composition such that a series of totals is obtained, each total representing the mortality due to a mixture of a given composition. The test dose of toxin is the amount present in 0.5 ml of that mixture that causes the death of one-half of the total number of mice injected within 72 hours. Determination of potency of the antitoxin Preliminary test. Dilute the test toxin with a suitable liquid such that 2.0 ml contains 10 times the test dose. Prepare mixtures such that 5.0 ml of each mixture contains 2.0 ml of the solution of the toxin and one of a series of graded volumes of the preparation under examination. Adjust each mixture to the same final volume with a suitable liquid. Allow the mixtures to stand at room temperature, protected from light, for 60 minutes. Inject a dose of 0.5 ml of each mixture intravenously into each of not less than two mice and observe the mice for 72 hours. If all the mice die, 0.5 ml of the mixture contains less than 0.2 Unit of antitoxin. If none of the mice dies, 0.5 ml of the mixture contains more than 0.2 Unit of antitoxin. Final test. Prepare similar fresh mixtures such that 5.0 ml of each mixture contains 2.0 ml of the solution of the toxin and one of a series of graded volumes of the preparation under examination, separated from each other by steps of not more than 20 per cent and covering the expected end-point. Prepare further mixtures of 5.0 ml containing 2.0 ml of the solution of the toxin and graded volumes of the Standard preparation to confirm the test dose of the toxin. Allow the mixtures to stand at room temperature, protected from light, for 60 minutes. Inject a dose of 0.5 ml of each mixture intravenously into each of not less than two mice and observe the mice for 72 hours. The mixture of the antitoxin under examination which contains 0.2 Unit in 0.5 ml is that mixture which causes the death of the same or almost the same number of mice as the mixture containing 0.2 Unit of the Standard preparation in 0.5 ml. Repeat the determinations at least once and calculate the average of all valid estimates. Estimates are not valid unless the Standard preparation gives a result within 20 per cent of the expected value. Limits of error. For the suggested method, the limits of error (P = 0.95) have been estimated to be (a) 85 per cent and 114 per cent when 2 animals per dose are used; (b) 91.5 per cent and 109 per cent when 4 animals per dose are used; (c) 93 per cent and 108 per cent when 6 animals per dose are used.
The vaccine passes the test if the pooled serum contains 2.5 IU of C. septicum antitoxins. Labelling. The label states (1) whether the preparation is a toxoid or a vaccine prepared from a whole inactivated culture, or a mixture of the two; (2) that the preparation is to be shaken before use; (3) for each target species, the immunising effect produced (for example, antibody production, protection against signs of disease or infection).
Duck Pasteurella Vaccine, Inactivated

Duck Pasteurella Vaccine, Inactivated consists of an emulsion or suspension of a virulent strain of Pasteurella multocida which has been inactivated in such a manner that the toxicity is eliminated and the immunogenic activity is retained.
Identification
Protects susceptible ducks against infection with P. multocida.
Tests
Safety. Either test A or test B may be carried out. A. Inject 5 ml subcutaneously into each of four healthy rabbits, weighing between 1.0 and 1.5 kg. Observe the animals for 7 days. No untoward reaction except slight and transient local swelling occurs. B. Inject 5 ml subcutaneously into each of two healthy rabbits, each weighing between 1.0 and 1.5 kg, and 0.5 ml subcutaneously into each of six mice, each weighing between 25 and 30 g. Observe the animals for 7 days. No untoward reaction except slight and transient local swelling occurs in both species of animals. Sterility (2.2.11). Complies with the test for sterility. Potency. Either test A or test B may be carried out. A. Inject subcutaneously with the minimum dose of the vaccine stated on the label three healthy susceptible ducks, between 4 and 6 weeks old. Use another two ducks of the same stock and age as unvaccinated controls. Three weeks later, challenge each of the vaccinated and control ducks, subcutaneously with 102 mouse LD50 viable organisms in 0.2 ml of a suitably diluted 18-hour broth culture of the homologous virulent strain of P. multocida. Observe the ducks for 7 days. Not less than two of the vaccinated ducks remain in normal health and both the controls die of pasteurellosis. B. Inject subcutaneously each of six mice, each weighing between 25 and 30 g, with 0.2 ml of the vaccine under examination. Use another six mice of the same stock and weight range as unvaccinated controls. Three weeks later, challenge each of the vaccinated and control mice subcutaneously with
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IP 2007
EGG DROP SYNDROME 76 (ADENOVIRUS) VACCINE, INACTIVATED
0.2 ml of a suitably diluted 18-hour broth culture of the homologous virulent strain of P. multocida containing 50 mouse LD5o viable organisms. Observe the animals for 7 days. All the vaccinated mice survive. The test is not valid unless all the control mice die of pasteurellosis during the observation period. Labelling. The label states (1) type of strain; (2) the recommended age for vaccination.
clinical symptoms of plague. The test is not valid unless the control ducks die from duck plague or show typical signs of serious infection during the observation period. If potancy test has been performed with satisfactoery results on a representative batch of the vaccine, it may be omitted as a vaccine test during production on the other batches of vaccine prepared from the same seed lot. Labelling. The label states (1) the minimum virus titre per dose; (2) the recommended age of the birds in which the vaccine is to be used.
Duck Plague Vaccine, Live

Duck Plague Vaccine, Live is a preparation of attenuated strain of duck plague virus. This monograph applies to vaccines intended for administration to duck for active immunisation against duck plague disease.
Egg Drop Syndrome 76 (Adenovirus) Vaccine, Inactivated

Egg Drop Syndrome 76 (Adenovirus) Vaccine, Inactivated consists of an emulsion or a suspension of a suitable strain of egg drop syndrome 76 virus (haemagglutinating avian adenovirus) which has been inactivated in such a manner that immunogenic activity is retained.
Production
The vaccine virus is grown in SPF eggs (2.7.7) or in cell cultures. The master seed lot complies with the tests for extreneous agents in seed lot (2.7.10). Substrate for virus propagation The vaccine virus is grown in embryonated hens eggs or in cell cultures obtained from flocks free from specified pathogens SPF (2.7.7). If the vaccine virus is grown in cell cultures, they comply with the requirements for cell cultures for production of veterinary vaccines. The vaccine virus is filled with suitable stabilizing agent and freeze dried.
Production
The vaccine strain is propagated in fertilized SPF hen or duck eggs from healthy flocks (2.7.7) or in suitable cell cultures. Test for inactivation. The test for inactivation is carried out in fertilized duck eggs from a flock free from egg drop syndrome 76 virus infection or hen eggs from a flock free from specified pathogens, or in suitable cell cultures, whichever is the most sensitive for the vaccine strain; the quantity of virus used in the test is equivalent to not less than ten doses of the vaccine. No live virus is detected. The vaccine may contain adjuvant
Identification
Protects ducks against duck plague.
Tests
Water (2.3.43). Not more than 3.0 per cent. Safety: Inject subcutaneously each of four healthy susceptible ducks, between 8 and 12 weeks old and each weighing not less than 600 g, with 1 ml of a 1 : 10 dilution of the reconstituted vaccine. Observe the ducks for 14 days. None of the ducks shows any untoward reaction. Sterility (2.2.11). Complies with the test for sterility. Potency. Inject subcutaneously each of four healthy susceptible ducks, between 8 and 12 weeks old and each weighing not less than 600 g, with a volume of the reconstituted vaccine containing a quantity of the virus equivalent to the minimum dose stated on the label. Fourteen days later, challenge each of the vaccinated ducks and each of two control ducks of the same stock and weight range, subcutaneously with 10 2 ID50 of virulent duck plague virus. Observe the ducks for 14 days. None of the vaccinated ducks dies or shows any
Identification
When inoculated into chicken, the development of specific neutralizing antibodies against egg drop syndrome 76 (adenovirus) can be demonstrated by suitable serological tests.
Tests
Safety. Inject each of ten chickens between 2 and 4 weeks old, with two doses and by the route stated on the label. Observe the chicken for 14 days. None of the chicken shows any abnormal local or systemic reaction. Sterility (2.2.11). Complies with the test for sterility. Potency. Inject each of twenty healthy chickens free of antibodies (2.7.7) between 3 to 4 weeks old, with the dose and by the route stated on the label. After 21 days, collect serum
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FOOT-AND-MOUTH DISEASE VACCINE, INACTIVATED
IP 2007
samples from each of the birds as well as ten-control chickens of the same stock and perform haemagglutination inhibition test on each serum using 4 haemagglutinating units of antigen and chicken erythrocytes. The vaccine passes the potency test if the mean antibody titre of the vaccinated group is greater than 1:128. The test is valid only if no specific antibody is found in the control chicken. If the potency has been performed with satisfactory results on representative batch of the vaccine from the same seed lot, it may be omitted as a routine control test during production of other batches of the vaccine prepared from the same seed lot. Labelling. The label states (1) the strain used for the preparation; (2) the name of any added adjuvant; (3) the route of administration.
Only a final bulk vaccine that complies with the following requirements may be used in the preparation of the final lot.
Tests
Inactivation. A proportion of each batch of bulk inactivated antigen representing at least 200 doses are tested for freedom from infectious virus by inoculation into sensitive cell culture. A sample of inactivated antigen is concentrated to volumes adequate for inoculation into cell cultures and it must show that the concentrated antigen does not interfere with the sensitivity or reading of the assay. The sample is passaged 3 times at an interval of 24 to 48 hours and inoculated cell cultures are examined for the presence of foot-and-mouth disease virus by suitable tests. No cytopathic changes attributable to foot-and-mouth disease virus replication should be detected. If infectious foot-and-mouth disease virus is detected, the bulk antigen is rejected. FINAL LOT The final bulk vaccine is distributed aseptically into sterile containers. The containers are closed so as to avoid contamination. Only a final lot that complies with each of the requirements given below under Identification, Tests and Assay may be released for use
Foot-and-Mouth Disease Vaccine, Inactivated

Foot-and-Mouth Disease Vaccine, Inactivated is a liquid preparation containing one or more types of foot-and-mouth disease virus that have been inactivated in such a manner that its immunogenic activity is retained. Depending on the number of types of virus incorporated, the vaccine is described as monovalent, bivalent, trivalent or polyvalent.
Identification
The serum of a foot-and-mouth disease susceptible animal that has been immunized with the vaccine neutralizes the types of virus used to prepare the vaccine, when tested by a suitable method.
Production
The virus is grown in suitable cell cultures. The virus is separated from cellular material by filtration or other suitable procedures and the virus is inactivated using binary ethyleneimine (BEI) in suitable conditions. The antigen may be concentrated and purified. The antigen is used for the preparation of vaccine. The vaccine contains a suitable adjuvant. Only an inactivated antigen suspension that complies with the requirements mentioned under final bulk vaccine may be used in the preparation of the final lot. FINAL BULK VACCINE The final bulk vaccine is prepared from one or more inactivated antigen suspensions. During inactivation of the virus, samples should be taken at regular intervals for the purpose of monitoring the rate and linearity of the inactivation process. Virus titres in the samples are determined by inoculation into sensitive cell culture. The infectivities of the timed samples are plotted against time, and the inactivation procedure is not considered to be satisfactory unless the extrapolation indicates that there would be less than one infectious particle per 104 litres of liquid preparation at the end of the inactivation period.
Tests
Safety. Use two non-vaccinated cattle not less than 6 months old free from foot-and-mouth disease antibodies. Inoculate each animal with whole vaccine in case of gel adjuvant vaccine and the final bulk in case of oil adjuvant vaccine intradermally into the tongue at not less than twenty sites with 0.1 ml per site. Observe the animals for 4 days. No lesions or signs of foot-and-mouth disease infection should occur. At the end of the observation period, inject into the same animals three times the dose by the prescribed route as stated on the label. Observe the animals for a further period of 6 days. No lesions or clinical signs due to foot-and-mouth disease infection should occur on the feet or tongue and no evidence of toxicity shall be noticed. Sterility (2.2.11). Complies with the test for sterility. Assay. Use three groups of not less than five cattle per group, not less than 6 months old, which have never been vaccinated and are free from antibodies neutralizing the different types of foot-and-mouth disease virus in the vaccine. Vaccinate the 3
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IP 2007
FOWL POX VACCINE, LIVE
groups by the route stated on the label. Use different doses of the vaccine for each group without diluting the vaccine. For example, if 3 ml is one dose, a 1/3 dose of vaccine would be obtained by injecting 1 ml, and a 1/10 dose would be obtained by injecting 0.3 ml. Three weeks later, challenge all the vaccinated animals and a control group of two, susceptible to foot-and-mouth disease with a suspension of virus that is fully virulent and of the same type as that used for preparation of the vaccine by inoculating 10,000 ID50 (50 per cent bovine infectious dose) intradermally into two sites into the tongue (0.1ml per site). The challenge of oil adjuvanted vaccines is effected 28 days post vaccination. Observe the animals for 8 days and then sacrifice them. Unprotected animals show lesions at sites other than the tongue. Protected animals may display lingual lesions. The test is not valid unless control animals show lesions on at least three feet. From the number of animals protected in each group, calculate the PD50 content of the vaccine. The potency of the vaccine is expressed as the number of 50 per cent cattle protective doses (PD50) contained in the dose stated on the label. The vaccine must contain at least 3 PD50 per dose for cattle. Indirect tests, including post vaccination measurement of virus neutralizing antibodies in cell culture, or ELISA, may be used to assess the potency of a vaccine provided that a statistical evaluation has established a satisfactory correlation between the results obtained by the test on the relevant vaccine serotype and the potency test in cattle. The description applies to the testing of a monovalent vaccine. Polyvalent vaccines may be potency tested by challenging each valency as described above. Labelling. The label states (1) the method of preparation; (2) the types and strains of virus used to prepare the vaccine.
Tests
Safety. Administer double dose of vaccine subcutaneously into each of ten healthy chickens, free of antibodies (2.7.7) of 4 to 6 weeks age. Observe the chickens for 7 days; none of the chicken shows untoward reaction other than slight transient local swelling. Sterility (2.2.11). Complies with the test for sterility. Potency. Inject subcutaneously each of ten healthy chickens free from antibodies (2.7.7) between 4 to 6 weeks old, with the minimum dose stated on the label. Use five healthy chickens of the same age group and from the same stock as controls. Three weeks later challenge the vaccinated and control chickens by injecting subcutaneously with 0.2 ml of an 18- hour broth culture of the homologous virulent strain of P. multocida diluted suitably so as to contain 102 mouse LD50 or 200 to 300 CFU in the injected dose in each chicken. Observe the chickens for 14 days; not less than eight out of ten of the vaccinated chickens survive. The test is not valid unless 100 per cent of the control chickens die of pasteurellosis within the observation period. Labelling. The label states (1) the serovar(s) used to prepare the vaccine, the serovar(s) against which protection is claimed; (2) the method of preparation.
Fowl Pox Vaccine, Live

Fowl Pox Vaccine, Live is a preparation of a suitable strain of avian pox virus. This monograph applies to vaccines intended for administration to chickens for active immunization.
Production
The vaccine virus is grown in embryonated hens eggs or in cell cultures. Substrate for virus propagation The vaccine virus is grown either in embryonated hens eggs or in avian cell cultures obtained from flocks free from specified pathogens SPF (2.7.7). The master seed lot complies with the test for extreneous agents in seed lot (2.7.10).
Fowl Cholera Vaccine, Inactivated

Fowl Cholera Vaccine, Inactivated is a preparation of 1 or more suitable strains of 1 or more serovars of Pasteurella multocida. This monograph applies to vaccines intended for the active immunisation of chickens, turkeys, ducks and geese against acute fowl cholera.
Production
The seed material is inoculated in a suitable medium. If the vaccine contains more than 1 strain of bacterium, the different strains are grown and harvested separately. The bacterial harvests are inactivated with suitable agent. The vaccine may contain suitable adjuvant.
Identification
The vaccine protects susceptible chicken against fowl pox. Carry out an immunostaining test in cell cultures to demonstrate the presence of the vaccine virus. For egg adapted strains, inoculate the vaccine into eggs and notice the characteristic lesions.
Identification
Protects susceptible chicken against infection with P. multocida.
Tests
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GOAT POX VACCINE, LIVE
IP 2007
Mycoplasmas (2.7.4). The vaccine complies with the test for mycoplasmas. Extraneous agents (2.7.11). The vaccine complies with the tests for extraneous agents in batches of finished product. Safety. Administer 10 doses of the vaccine to each of ten chicken 6 to 8 weeks old complying with the requirements of test B of the test for freedom from specified pathogens and antibodies (2.7.7), SPF chicks and by the route stated on the label. Observe the birds for 21 days. No chicken dies from causes attributable to the vaccine or shows signs of toxicity other than mild, transient, local reactions. If during the observation period more than two chickens die from causes not attributable to the vaccine, repeat the test. Virus titre. Not less than 102 EID50/TCID50 of the virus per dose, determining the titre by inoculation into the chorioallantoic membrane of SPF embryonated eggs, between 9 and 11 days old, or in a cell culture derived from SPF embryos (2.7.7). Sterility (2.2.11). Complies with the test for sterility. Potency. Carry out a separate potency test for each of the routes of administration stated on the label. Use not less than ten susceptible chickens, 6 to 8 weeks old, complying with the requirements of test B of the test for freedom from specific pathogens and antibodies (2.7.7), and of the minimum age for vaccination stated on the label. Use ten birds from the same flock and weight range as controls. Administer to each chicken a volume of the reconstituted vaccine containing a quantity of the virus equivalent to the minimum titre stated on the label. After 21 days, challenge each chicken by intrafollicular administration or by scarification with a virulent strain of fowl poxvirus. Observe the birds for 14 days. The vaccinated chickens survive and show no signs of disease except transient local reactions of fowl pox within 6 days following the challenge. All control chickens show lesions of fowl pox. If the potency test has been performed with satisfactory results on a representative batch of the vaccine it may be omitted as a routine test during production of the other batches of the vaccine prepared from the same seed lot. Labelling. The label states (1) the minimum virus titre; (2) the age of vaccination; (3) the types and strains of virus used to prepare the vaccine.
Production
The virus is propagated in suitable cell culture. The viral suspension is harvested, titrated and may be mixed with a suitable stabilizing agents. The vaccine is then freeze-dried.
Identification
The vaccine protects susceptible animals against goat pox.
Tests
Water (2.3.43). Not more than 3.0 per cent. Safety A. Inoculate 10 doses of the reconstituted vaccine in each animal of the three species mentioned below by the route stated against each. Administer 0.2 ml intraperitoneally to each of six mice and 0.5 ml and 1.0 ml subcutaneously to each of three guinea pigs and three rabbits respectively. Observe the animals for 10 days. None of the animals shows an abnormal reaction. B. Inject 100 doses of the vaccine contained in 1 ml of the reconstituted vaccine subcutaneously into each of two susceptible goats, 6 to 8 months old. Observe the goats for 10 days. None of the animals shows abnormalities other than local erythema of not more than 3 cm in diameter around the site of injection. Virus titre. Not less than 103 TCID50 of the virus per dose, determining the titre in a suitable cell culture or by inoculation into the chorio-allantoic membrane of SPF embryonated eggs (2.7.7), 9 to 11 days old. Sterility (2.2.11). Complies with the test for sterility. Potency. Use nine susceptible goats, 8 to 10 months old. Divide them into three groups of three goats each. Inject subcutaneously 1/20th of the dose of the vaccine stated on the label into each goat of one group. Administer a quantity equivalent to the dose of the vaccine stated on the label into each goat of the second group. Use the third group as unvaccinated controls which should be kept along with the inoculated goats. Observe the animals for 14 days and record the rectal temperature daily of each goat during the observation period. None of the vaccinated goats shows any thermal reaction or local or generalised lesion. After 21 days, challenge the vaccinated and control animals with sufficient quantity of a virulent goat pox virus by intradermal injection. Observe the animals for 14 days and record the rectal temperature daily of each goat during the observation period. None of the vaccinated goats shows any thermal reaction or local or generalised lesion. The test is valid only if the control animals develop high fever or show local or generalised lesions. If the test for potency has been carried out with satisfactory results on a representative batch of vaccine, this test may be omitted
Goat Pox Vaccine, Live

Goat Pox Vaccine, Live is a freeze-dried preparation of an attenuated strain of goat poxvirus propagated in a suitable cell culture. It is reconstituted immediately before use by a suitable diluent.
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IP 2007
INCLUSION BODY HEPATITIS (IBH) VACCINE, INACTIVATED
as a routine control on other batches of vaccine prepared from the same seed lot, subject-to agreement by the competent authority. Labelling. The label states (1) the strain used for the preparation; (2) virus titer; (3) dose and age of vaccination.
weight and of the same stock as controls. After 21 days, challenge each of the vaccinated rabbits as well as the control rabbits with an 18 hour old culture of P. multocida containing not less than 10 LD50 of virulent organisms. Observe the animals for 7 days; none of the vaccinated animals dies of pasteurellosis. The test is not valid unless both the control rabbits die of pasteurellosis. (c) Test on calves. Inject each of not less than 3 healthy susceptible calves, weighing not less than 140 kg each with 2 ml of vaccine. Three weeks later, these animals along with two healthy animals of the same type are challenged subcutaneously with 18-hours old broth culture of P. multocida equivalent to at least 50 million mouse minimum infective. Observe the animals for 7 days. Both the controls should die of pasteurellosis and at least two out of three vaccinated animals should survive. Potency is conducted on each seed lot or for every fifth batch produced from the seed lot. Labelling. The label states (1) the type and strains of bacteria used to prepare the vaccine; (2) adjuvant used.
Haemorrhagic Septicaemia Vaccine

Haemorrhagic Septicaemia Vaccine (Inactivated) is a preparation of Pasteurella multocida. The whole culture is inactivated which may be heated with a suitable adjuvant.
Identification
The vaccine protects susceptible animals against infection with P. multocida.
Tests
Safety. Inject intraperitoneally or intramuscularly into each of six mice, weighing not less than 18 g, with 0.2 m1 of the vaccine under examination. Observe the animals for 5 days; no abnormal systemic reaction occurs. Inject two seronegative cattle with twice the maximum dose stated on the label and observe for 10 days for adverse effects. Sterility (2.2.11). Complies with the test for sterility. Potency. Carry out any of the following three tests. (a) Test on mice. Inject intramuscularly each of fifty mice, weighing not less than 18 g, with 0.2 ml of the preparation under examination. Repeat the dose 14 days later. After 7 days, divide the mice into ten groups of five each. Use another fifty mice of the same weight and from the same stock as controls. Divide the controls also into ten groups of five each. Challenge the vaccinated and the control mice with an 8 to 12-hour old broth culture of a virulent strain of P. multocida in the range of 10-1 to 10-10. Observe the mice for 5 days and record the number of vaccinated and control mice found dead in each group. Calculate the median lethal dose (LD50) for the vaccinated and control mice by standard methods. The protection provided by the vaccine is calculated as number of protection units using following formula: Number of protection units=LD50 in control animals - LD50 in vaccinated animals. The vaccine passes the test if it provides minimum protection of 104 units (b) Test on rabbits. Inject intramuscularly each of not less than six rabbits, each weighing not less than 2.0 kg, with 2 m1 of the vaccine under examination. Use two rabbits of the same
Inclusion Body Hepatitis (IBH) Vaccine Inactivated

Inclusion Body Hepatitis (IBH)/Hydropericardium Syndrome (HPS) Vaccine (Inactivated) consists of an emulsion or a suspension of avian adenovirus type 4 virus which have been inactivated in such a manner that the immunogenic activity is retained. The vaccine may contain one or more suitable adjuvants.
Production
Substrate for virus propagation Vaccine virus is grown in SPF chicks (2.7.7). Inactivation An amplification test for residual live IBH/HPS disease virus is carried out on each batch of antigen immediately after inactivation and on the final bulk vaccine or, if the vaccine contains an adjuvant, on the bulk antigen or the mixture of bulk antigens immediately before the addition of any adjuvant, to confirm inactivation; the test is carried out on chickens from a flock free from specified pathogens. The quantity of inactivated virus used in the test is equivalent to not less than ten doses of the vaccine. No live virus is detected.
Identification
Protects chickens against infection of IBH/HPS.
1595
INFECTIOUS AVIAN ENCEPHALOMYELITIS VACCINE, LIVE
IP 2007
Tests
Safety. Inject subcutaneously a quantity equivalent to 2 doses into each of 20 healthy chickens free from specific antibodies, of the recommended age at which vaccine is to be used. Observe the chickens for 14 days, no abnormal systemic or local reaction is seen. Sterility (2.2.11). Complies with the test for sterility. Potency. Inject one dose by the route stated on the label into each of 20 healthy chickens free from specific antibodies at the minimum recommended age at which vaccine is to be used. Use 10 similar chickens from same source as controls. Challenge chickens after 21 days by intraperitoneal route with 104 Chick Infective Dose (CID50) of virulent strain of IBH/ HPS. Observe the chickens of both groups for 14 days. Vaccine complies with the test if not more than 2 of vaccinated chickens die or show signs of disease. At the end of observation period sacrifice all the chickens. The test is valid only if at least 80 per cent of control chicks either die or show symptoms or show post-mortem lesions on sacrifice at the end of observation period post challenge. If the potency test has been performed with satisfactory results on representative batch of the vaccine from the same seed lot, it may be omitted as a routine control test during production of other batches of the vaccine prepared from the same seed lot. Labelling. The label states (1) strain used for vaccine production; (2) recommended age for vaccination.
Tests
Water (2.3.43). Not more than 3.0 per cent. Safety. Administer twenty five healthy chickens of 6 to 10 weeks old free from specific antibodies (2.7.7) ten doses of the vaccine by the recommended route. Observe the chickens for 21 days. No chicken develops signs of the disease or dies from causes attributable to the vaccine. Repeat the test if more than two chickens die from causes not attributable to the vaccine during the observation period. Virus titre. Not less than 102.5 TCID50/EID50 of the virus per dose, determining the titre of the virus in cell cultures derived from SPF embryos or by inoculation into the yolk sac of SPF embryonated hen eggs (2.7.7), between 5 and 6 days old. Sterility (2.2.11). Complies with the test for sterility. Potency. Carry out a separate potency test for each of the routes of administration stated on the label. For each of the stated routes, use not less than ten susceptible SPF chickens, 3 weeks old. Administer to each chicken a volume of the reconstituted vaccine containing a quantity of the virus equivalent to the minimum virus titre stated on the label. Use ten chickens of the same flock and age as controls. After 21 days, challenge each chicken in the vaccinated and control groups with intracerebral injection of a suitable quantity of a virulent avian encephalomyelitis virus. Observe the chickens for another 21 days. Not less than 80 per cent of the vaccinated chickens survive or show no signs of disease and not less than 70 per cent of the controls die or develop signs or lesions of avian encephalomyelitis. If the immunogenicity test (potency test) has been performed with satisfactory results on representative batch of the vaccine from the same seed lot, it may be omitted as a routine control test during production of other batches of the vaccine prepared from the same seed lot.
Infectious Avian Encephalomyelitis Vaccine, Live

Infectious Avian Encephalomyelitis Vaccine, Live is a freezedried preparation of an attenuated strain of infectious avian encephalomyelitis virus.
Production
The virus is grown in SPF embryonated eggs (2.7.7) or in suitable cell cultures. The master seed lot complies with the test for extraneous agents in seed lot (2.7.10).
Infectious Bursal Vaccine, Inactivated

InInfectious Avian Bursal Disease Vaccine, Inactivated consists of an emulsion or a suspension of a suitable strain of infectious avian bursal disease virus type 1 which has been inactivated in such a manner that immunogenic activity is retained. The vaccine may contain one or more suitable adjuvant.
Identification
Inoculate 0.1 ml of the undiluted reconstituted vaccine into the yolk sac of SPF embryonated eggs, between 5 and 6 days old. Keep them in an incubator and transfer to the brooder for hatching. Observe the hatched chickens for 7 days. Not less than 50 per cent of the chickens show the typical symptoms characteristic of infectious avian encephalomyelitis-like weakness or paralysis of legs and tremors.
Production
The virus is propagated in fertilized eggs from SPF flocks, in suitable cell cultures or in chickens from a flock free from specified pathogens (2.7.7).
1596
IP 2007
INFECTIOUS CORYZA VACCINE
Inactivation An amplification test for residual live infectious avian bursal disease virus is carried out on each batch of antigen immediately after inactivation and on the final bulk vaccine or, if the vaccine contains an adjuvant, on the bulk antigen or the mixture of bulk antigens immediately before the addition of any adjuvant, to confirm inactivation; the test is carried out in fertilized hen eggs or in suitable cell culture or, where chickens have been used for production of the vaccine, in chickens from a flock free from specified pathogens the quantity of inactivated virus used in the test is equivalent to not less than ten doses of the vaccine. No live virus is detected.
the bursa of each chicken from the first group and the second group. No evidence of infectious bursal disease is seen and no abnormal local reaction develops. Safety. Inject each of twenty healthy chickens free from specific antibodies (2.7.7) between 14 and 28 days old with twice the minimum vaccinating dose and by one of the routes stated on the label. Observe the chickens for 14 days. No abnormal local or systemic reaction is seen. Sterility (2.2.11). Complies with the test for sterility. Potency. Inject each of twenty healthy chickens free from specific antibodies (2.7.7) between 3 and 4 weeks old, with the minimum dose and by the route stated on the label. Use ten chickens of the same flock and age as controls. After 21 days, collect serum samples from each bird including the tencontrol chickens and perform quantitative agar gel precipitation test on each serum sample. The mean antibody titre of sera in vaccinated group shall be 8.0 and there are no specific antibodies in the sera of control chicken. If the potency test has carried out been with satisfactory results on representative batch of the vaccine from the same seed lot, it may be omitted as a routine control test during production of other batches of the vaccine prepared from the same seed lot. Labelling. The label states (1) the type of strain; (2) the recommended age for vaccination.
Identification
Protects susceptible chickens against infectious bursal disease by producing specific antibodies on inoculation.
Tests
Inactivation For vaccine prepared with embryo-adapted strains of the virus. Inject two-fifths of a dose into the allantoic cavity or onto the chorio-allantoic membrane of the SPF embryonated hen eggs, between 9 and 10 days old, and incubate at 37. Observe for 6 days and pool separately the allantoic fluid from eggs containing live embryos, and that from eggs containing dead embryos, excluding those dying from nonspecific causes within the first 24 hours after inoculation. Inject into the allantoic cavity of each of the SPF embryonated hen eggs, between 9 and 10 days old, 0.2 ml of the pooled allantoic fluid from the live embryos or membrane from the dead embryos and incubate at 37 for 6 days. Examine each embryo for lesions of infectious bursal disease. The vaccine complies with the test if there is no evidence of lesions of infectious bursal disease. The test is valid only if not more than 20 per cent of the embryos die at either stage of the test. If more than 20 per cent of the embryos die at either one of the stages of the test, repeat that stage. In any repeat test, not more than 20 per cent of the embryos die from non-specific causes. Antibiotics may be used to control extraneous bacterial infection. For vaccine prepared with strains of virus not adapted to embryos. Inject two doses intramuscularly into each of twenty chickens, between 14 and 28 days old, complying with the requirements stated under Test on chicken flocks free from pathogens for the production and quality control vaccines (2.7.7). Four day later, kill ten of the chickens and remove bursa of fabricius from each chicken, pool the bursa and homogenise in an equal volume of a suitable liquid. Inject 1 ml of the homogenate into each of a further ten chickens of the same flock and age. After 21 days, examine microscopically
Infectious Coryza Vaccine

Infectious Coryza Vaccine for chickens is a suspension of inactivated culture of one or more virulent strain of Haemophilus paragallinarium. A suitable adjuvant may be added.
Identification
Protects susceptible chicken against infection with H. paragallinarium organism.
Tests
Safety. Inject 1.0 ml subcutaneously into each of 10 healthy chickens free from antibodies (2.7.7) at the minimum age group at which vaccine is intended. Observe these birds for 7 days; no bird shows untoward reactions other than slight transient local swelling. Sterility (2.2.11). Complies with the test for sterility. Potency. Inject subcutaneously each of 10 healthy chickens free from antibodies (2.7.7) of the minimum age group at which vaccine is used, with minimum dose stated on the label. Repeat the vaccination after 2 weeks. Use 10 healthy chickens of
1597
LARYNGOTRACHEITIS VACCINE, LIVE
IP 2007
same age group and of same stock as controls. Three weeks later, challenge vaccinated and control chickens by instillation with 0.2 ml of 18-hour broth culture of homologous virulent strain of H. paragallinarium diluted suitably so as to contain lxl06 Chick ID50 by infra-orbital sinus inoculation. Observe the chickens for 7 days for unilateral eye swelling, nasal discharge, isolation of the virulent organisms from infra-orbital sinus in a suitable medium. Not less than 7 vaccinated chickens show prevention from lesions and isolation of homologous virulent organisms. The test is not valid unless 70 per cent of control chickens exhibit typical symptoms of eye swelling and nasal discharge typical of infectious coryza. Labelling. The label states (1) strains used for preparation; (2) route of administration.
more than 20 per cent of the chickens show abnormal clinical signs or die from causes not attributable to the vaccine. The vaccine complies with the test if no chicken shows notable clinical signs of disease or dies from causes attributable to the vaccine. Sterility (2.2.11). Complies with the test for sterility. Potency. A test is carried out for each route and method of administration to be recommended using in each case chickens not older than the youngest age to be recommended for vaccination. The quantity of the vaccine virus administered to each of 20 chickens is not greater than the minimum virus titre to be stated on the label and the virus is at the most attenuated passage level that will be present in a batch of the vaccine. Vaccinate by a recommended route not less than 20 chickens. Maintain not less than 10 chickens as controls. Challenge each chicken after 21 days by the intratracheal route with a sufficient quantity of virulent infectious laryngotracheitis virus. Observe the chickens at least daily for 7 days after challenge. Record the deaths and the number of surviving chickens that show clinical signs of disease. At the end of the observation period kill all the surviving chickens and carry out examination for macroscopic lesions: mucoid, hemorrhagic and pseudomembraneous inflammation of the trachea and orbital sinuses. The test is not valid if during the observation period after challenge less than 90 per cent of the control chickens die or show severe clinical signs of avian infectious laryngotracheitis or notable macroscopic lesions of the trachea and orbital sinuses, or if during the period between the vaccination and challenge more than 10 per cent of the vaccinated or control chickens show notable clinical signs of disease or die from causes not attributable to the vaccine. The vaccine virus complies with the test if during the observation period after challenge not less than 90 per cent of the vaccinated chickens survive and show no notable clinical signs of disease and/or macroscopically lesions of the trachea and orbital sinuses. If the potency test has been performed with satisfactory results on a representative batch of the vaccine from the seed lot, it may be omitted as a routine control test during production on other batches of the vaccine prepared from the same seed lot. Labelling. The label states (1) strain of virus used; (2) recommended age for vaccination.
Laryngotracheitis Vaccine, Live

Laryngotracheitis Vaccine, Live is a preparation of a suitable strain of Avian infectious laryngotracheitis virus (gallid herpes virus 1). This monograph applies to vaccines intended for administration to chickens for active immunization against laryngotracheitis disease in chickens.
Production
The vaccine virus is grown in embryonated hens eggs or in cell cultures. If the vaccine virus is grown in embryonated hens eggs, they are obtained from flocks free from specified pathogens (SPF) (2.7.7). If the vaccine virus is grown in cell cultures, they comply with the requirements for cell cultures for production of veterinary vaccines . The vaccine virus is filled with suitable stabilizing agent and freeze dried. The master seed lot complies with the tests for extraneous agents in seed lot (2.7.10).
Identification
When mixed with mono specific laryngotracheitis disease virus antiserum the vaccine no longer infects susceptible cell cultures or embryonated hen eggs, 9 to 11 days old.
Tests
Water (2.3.43). Not more than 3.0 per cent. Virus titre. Titrate the vaccine virus by inoculation into embryonated hens eggs from an SPF flock or into suitable cell cultures. The vaccine complies with the test if 1 dose contains not less than the minimum titre stated on the label. Safety. Use not less than 10 chickens from a healthy flock and of the youngest age recommended for vaccination. Administer by eye-drop to each chicken 10 doses of the vaccine. Observe the chickens at least daily for 21 days. The test is not valid if
Leptospira Veterinary Vaccine, Inactivated

Canine Leptospirosis Vaccine (Inactivated) is a suspension of inactivated whole organisms and/or antigenic extract(s) of one or more suitable strains of one or more of Leptospira
1598
IP 2007
PESTE DES PETITIS RUMINANTS VACCINE, LIVE
interrogans serovar canicola, serovar icterohaemorrhagiae or any other epidemiologically appropriate serovar, inactivated and prepared in such a way that adequate immunogenicity is maintained.
Not less than four of the control animals die showing typical leptospira infection. Not less than four of the vaccinated animals remain in good health for not less than 14 days after the death of the four control animals. Labelling. The label states (1) the strain used for the preparation; (2) the name of any added adjuvant.
Production
The seed material is cultured in a suitable medium; each strain is cultivated separately. During production, various parameters such as growth rate are monitored by suitable methods; the values are within the limits approved for the particular product. Purity and identity are verified on the harvest using suitable methods. After cultivation, the bacterial harvests are collected separately and inactivated by a suitable method. The antigen may be concentrated. The vaccine may contain an adjuvant. Inactivation Carry out a test for inactivation by inoculation on to a specific medium. Inoculate 1 ml of the vaccine into100 ml of the medium. Incubate at 30 for 14 days, subculture into a further quantity of the medium and incubate both media at 30 for 14 days: no growth occurs in either medium. At the same time, carry out a control test by inoculating a further quantity of the medium with the vaccine together with a quantity of a culture containing approximately 100 leptospirae and incubating at 30 Growth of leptospirae occurs within 14 days.
Peste Des Petitis Ruminants Vaccine, Live

Peste Des Petitis Ruminants Vaccine, Live is a preparation of a suitable strain of PPR virus that is attenuated for sheep and goats.
Production
The vaccine strain is grown in suitable cell cultures. The viral suspension is harvested, mixed with a suitable stabilizing liquid and freeze-dried. Batch testing If the test for potency has been carried out with satisfactory results on the representative batch of vaccine, this test may be omitted as a routine control on other batches of vaccine prepared from the same seed lot, subject to agreement by a National Regulatory Authority.
Identification
When administered to experimental animals causes the appearance of agglutinating antibodies against the serotype or serotypes used to prepare the vaccine.
Identification
When injected into the target animals, the vaccine stimulates the production specific neutralizing antibodies.
Tests
Safety. Use 2 dogs of the minimum age recommended for vaccination and which do not have antibodies to the leptospira serovar(s) present in the vaccine. Administer 2 doses of the vaccine to each dog by a recommended route. Observe the animals for 14 days. The animals remain in good health and no abnormal local or systemic reaction occurs. Sterility (2.2.11). Complies with the test for sterility. Potency. Carry out a separate potency test for each serotype if the vaccine is prepared with different serotypes. Inject each of five hamsters not more than 3 months old, the animals being drawn from the same stock, subcutaneously with 1/40 of the dose of the vaccine stated on the label for dogs. Use an equal number of animals of the species used for the test as controls. After 15 to 20 days inject intraperitoneally into each of the vaccinated and control animals an adequate dose of a virulent culture of leptospirae of the serotype used to prepare the vaccine or a suspension of liver or kidney tissue obtained from animals infected with the serotype used to prepare the vaccine. Observe the animals for 14 days after the injection.
Tests
Safety. Administer 0.5 ml of vaccine equivalent to 5 doses intramuscularly into each of two healthy guinea pigs weighing between 200 and 250 g and 0.5 ml each into two healthy guinea pigs weighing between 200 and 250 g intraperitoneally and 0.1 ml of vaccine equivalent to one dose intraperitoneally in each of six healthy mice weighing between 17 and 22 g. Keep two guinea pigs and two mice as uninoculated controls. Observe the animals for 3 weeks. At the end of 3 weeks of observation, all animals are killed for post-mortem examination. The vaccine is considered safe if during the first or second test at least 80 per cent of animals remain in good health during the period of observation, and no significant post-mortem lesion is found. Inject two susceptible goats of one year old free from antibodies to rinderpest or PPR by subcutaneous route with a 100 times the dose of vaccine stated on the label. Observe the animals for 21 days. No sign of illness attributable to PPR is noticed
1599
RABIES VETERINARY VACCINE, INACTIVATED (CELL CULTURE)
IP 2007
Water (2.3.43). Not more than 3 per cent. Virus titer. Virus titer not less than 10 TCID50 per dose. Extraneous viruses. The reconstituted vaccine when mixed with specific anti-PPR serum should not produce cytopathic effects in susceptible cell cultures and the cells should show no evidence of the presence of haemadsorbing agents. Sterility (2.2.11). Complies with the test for sterility. Potency. Use not less than six healthy goats and six healthy sheep of 1 year old free from antibodies to rinderpest or PPR. Collect sera from the animals before the time of vaccination and 3 weeks after vaccination and just before challenge. Vaccinate two goats and two sheep subcutaneously with 100 doses per ml; vaccinate two goats and two sheep subcutaneously with 1 dose per ml. Keep the remaining animals as the in-contact controls. Monitor each animal for clinical signs, in particular respiratory symptoms and record temperature measurements daily for three weeks. Three weeks after vaccination challenge the vaccinated animals and incontact controls group with a suspension of virus containing 103 LD50 pathogenic PPRV by subcutaneous route. The animals are observed for clinical signs and the body temperatures are recorded daily for two weeks. The vaccine passes the test if all vaccinated animals resist challenge infection and all the incontact controls develop signs of PPR. The serum neutralization test must be positive for PPR antibody in vaccinated animal only, in samples taken three weeks after vaccination. If the potency test has been performed with satisfactory results on a representative batch of the vaccine from the seed lot, it may be omitted as a routine control test during production on other batches of the vaccine prepared from the same seed lot. Labelling. The label states (1) cell line used for vaccine manufacture; (2) virus titer per dose; (3) recommended age for vaccination.
5
virus suspension is harvested on one or more occasions within 28 days of inoculation. Multiple harvests from a single production cell culture may be pooled and considered as a single harvest. The rabies virus is inactivated by a suitable method. The vaccine may contain one or more adjuvants. Inactivation A. The test for residual live rabies virus is carried out by inoculation of the inactivated virus into the same type of cell culture as that used in the production of the vaccine or a cell culture shown to be at least as sensitive. The quantity of inactivated virus used in the test is equivalent to not less than 25 doses of the vaccine. After incubation for 4 days, a subculture is made using trypsinised cells; after incubation for a further 4 days, the cultures are examined for residual live rabies virus by an immunofluorescence test. No live virus is detected. B. Inject each of twenty suckling mice, each weighing between 12 and 16 g, intracerebrally with not less than 0.03 ml of the vaccine or antigen under examination. Observe the animals for 21 days. None of the mice dies or shows any abnormalities attributable to the vaccine. If more than two mice die within 48 hours, repeat the test.
Identification
When injected into animals, the vaccine stimulates production of specific neutralising antibodies.
Tests
Water (2.3.43). Not more than 3.0 per cent (for freeze dried vaccine only). Safety. Inject each of twenty mice, each weighing between 12 and 16 g, intracerebrally with not less than 0.03 ml of the vaccine under examination. Observe the animals for 21 days. None of the mice dies or shows any abnormalities attributable to the vaccine. If more than two mice die within 48 hours repeat the test. If the vaccine is intended for more than one species including one belonging to the order of Carnivore, carry out the test in dogs. Otherwise use one of the species for which the vaccine is intended. Administer, by a recommended route, a double dose of vaccine to each of 2 animals having no antibodies against rabies virus. Observe the animals for 14 days. No abnormal local or systemic reaction occurs. Sterility (2.2.11). Complies with the test for sterility. Potency. The potency of rabies vaccine is determined by comparing the dose necessary to protect mice against the clinical effects of the dose of rabies virus defined below, administered intracerebrally, with the quantity of a reference preparation, calibrated in International Units, necessary to provide the same protection.
Rabies Veterinary Vaccine, Inactivated (Cell Culture)

Rabies Vaccine for Veterinary Use is a preparation of rabies fixed virus adapted to and propagated in cell culture and inactivated by a suitable method. It may be issued as a liquid containing a suitable adjuvant or as a freeze-dried preparation to be reconstituted with a suitable liquid immediately before use.
Production
The vaccine is prepared from virus grown either in suitable cell lines or in primary cell cultures from healthy animals. The
1600
IP 2007
RANIKHET DISEASE VACCINE, INACTIVATED
Preparation of the challenge suspension. Inoculate a group of mice intracerebrally with the CVS strain of rabies virus and when the mice show signs of rabies, but before they die, kill the mice and remove the brains and prepare a homogenate of the brain tissue in a suitable diluent. Separate gross particulate matter by centrifugation and use the supernatant liquid as challenge suspension. Distribute the suspension in small volumes in ampoules, seal and store at a temperature below -60. Thaw one ampoule of the suspension and make serial dilutions in a suitable diluent. Allocate each dilution to a group of 10 mice and inject intracerebrally into each mouse 0.03 ml of the dilution allocated to its group. Observe the animals for 14 days and record the number in each group that, between the fifth and the fourteenth day, develop signs of rabies. Calculate the ID50 of the undiluted suspension. Determination of potency of the vaccine Use in the test healthy mice about 4 weeks old and from the same stock. Distribute the mice into at least 10 groups of not less than 10 mice. Prepare at least three serial dilutions of the vaccine under examination and three similar dilutions of the reference preparation. Prepare the dilutions such that those containing the largest quantity of vaccine may be expected to protect more than 50 per cent of the animals into which they are injected and those containing the smallest quantities of vaccine may be expected to protect less than 50 per cent of the animals into which they are injected. Allocate each dilution to a different group of mice and inject intraperitoneally into each mouse 0.5 ml of the dilution allocated to its group. Fourteen days after the injection prepare a suspension of the challenge virus such that, on the basis of the preliminary titration, it contains about 50 ID50 in each 0.03 ml. Inject intracerebrally into each vaccinated mouse 0.03 ml of this suspension. Prepare 3 suitable serial dilutions of the challenge suspension. Allocate the challenge suspension and the 3 dilutions one to each of 4 groups of 10 unvaccinated mice and inject intracerebrally into each mouse 0.03 ml of the suspension or one of the dilutions allocated to its group. Observe the animals in each group for 14 days. The test is not valid if more than 2 mice of any group die within the first 4 days after challenge. Record the numbers in each group that show signs of rabies in the period 5 days to 14 days after challenge. The test is not valid unless (a) for both the vaccine under examination and the reference preparation the 50 per cent protective dose lies between the smallest and the largest dose given to the mice; (b) the titration of the challenge suspension shows that 0.03 ml of the suspension contained at least 10 ID50 and not more than 50 ID50; (c) the confidence limits (P = 0.95) are not less than 25 per cent and not more than 400 per cent of the estimated potency; (d) the statistical analysis shows a significant slope and no significant deviations from linearity or parallelism of the dose-response lines.
The vaccine complies with the test if the estimated potency is not less than 1 IU in the smallest prescribed dose. Labelling. The label states (1) the strain used for the preparation; (2) the name of any added adjuvant.
Ranikhet Disease Vaccine, Inactivated

Newcastle Disease Vaccine, Inactivated
Ranikhet Disease Vaccine, Inactivated consists of an emulsion or a suspension of a suitable strain of Newcastle disease virus (avian paramyxovirus 1) that has been inactivated in such a manner that immunogenic activity is retained.
Production
Preparation of the vaccine The vaccine virus is grown either in embryonated hens eggs or in avian cell cultures obtained from flocks free from specified pathogens (SPF) (2.7.7). Inactivation. Inject 2/5 of a dose into the allantoic cavity of each of 10 embryonated hen eggs that are 9 to 11 days old SPF eggs, and incubate. Observe for 6 days and pool separately the allantoic fluid from eggs containing live embryos and that from eggs containing dead embryos, excluding those dying within 24 hours of the injection. Examine embryos that die within 24 hours of injection for the presence of Newcastle disease virus: the vaccine does not comply with the test if Newcastle disease virus is found. Inject into the allantoic cavity of each of 10 SPF eggs, 9 to 11 days old, 0.2 ml of the pooled allantoic fluid from the live embryos and, into each of 10 similar eggs, 0.2 ml of the pooled fluid from the dead embryos and incubate for 5 to 6 days. Test the allantoic fluid from each egg for the presence of haemagglutinins using chicken erythrocytes. The vaccine complies with the test if there is no evidence of haemagglutinating activity and if not more than 20 per cent of the embryos die at either stage. If more than 20 per cent of the embryos die at one of the stages, repeat that stage; the vaccine complies with the test if there is no evidence of haemagglutinating activity and not more than 20 per cent of the embryos die at that stage. Antibiotics may be used in the test to control extraneous bacterial infection.
Identification
When injected into susceptible healthy chicken, free of antibodies (2.7.7) the vaccine stimulates the production of specific antibodies against Newcastle disease virus.
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RANIKHET DISEASE VACCINE, LIVE (LENTOGENIC STRAIN)
IP 2007
Tests
Safety. Inject ten healthy chickens, free of antibodies (2.7.7) between 2 and 4 weeks old, with twice the dose and by the route stated on the label. Observe the birds for 21 days. No abnormal local or systemic reaction is observed. Sterility (2.2.11). Complies with the test for sterility. Potency. Either test A or test B may be carried out. A. Inject intramuscularly each of ten healthy chickens, free from antibodies (2.7.7) between 3 and 4 weeks old, with a volume of the vaccine equivalent to one-fiftieth of a dose. Use ten chickens of the same stock and age group as controls. After 21 days, collect serum samples from each of the vaccinated and unvaccinated chicken. Perform haemagglutination inhibition test using the method described below. Use the positive control serum calibrated against a Standard preparation of anti-Newcastle disease serum. The vaccine passes the test if a mean HI titre of the vaccinated group is equal to or greater than 1:16 and that of the unvaccinated controls is equal to or less than 1:4. Standard preparation The Standard preparation is the 1st International reference preparation, established in 1966, consisting of freeze-dried chicken serum (supplied in ampoules containing 320 Units), or another suitable preparation, the potency of which has been determined in relation to the International reference preparation. Suggested method of haemagglutination inhibition test. Inactivate the serum samples by heating at 56 for 30 minutes. Add 0.05 ml of saline solution to all the wells in a microtitre plate and 0.05 ml of the test sera to the first row of wells. Prepare two-fold dilutions of the serum samples across the plate. Add 0.05 ml ofa suspension of Newcastle disease virus containing 4 haemagglutinating units of inactivated Newcastle disease virus. Incubate the plate at 4 for one hour. Add 0.05 ml of a 1 per cent suspension of erythrocytes collected from chicken, between 3 and 4 weeks old, susceptible to Newcastle disease. Incubate the plate at 4 for one hour. It must be ensured that negative and positive control sera are included in the test. The positive control serum must show a titre of 300 to 400 Units determined by calibration against the Standard reference Preparation. B. Inject intramuscularly each of three groups of twenty healthy chicken, free from antibodies (2.7.7) between 3 and 4 weeks old, with five fold dilution of vaccine. Use minimum three dilutions. Allocate a different volume to each vaccination group. Vaccinate each chicken by the intramuscular route with the volume of vaccine allocated to its group. Maintain not
less than 10 chickens as controls. Challenge each chicken after 21 days by the intramuscular route with 6 log10 chick LD50 of the virulent strain of avian paramyxovirus 1. Observe the chickens at least daily for 7 days after challenge. At the end of the observation period, calculate the PD50 by standard statistical methods from the number of chickens that survive in each vaccinated group without showing any signs of Newcastle disease during the 7 days. The vaccine complies with the test if the smallest dose stated on the label corresponds to not less than 50 PD50 and the lower confidence limit is not less than 35 PD50 per dose. If the lower confidence limit is less than 35 PD50 per dose, repeat the test; the vaccine must be shown to contain not less than 50 PD50 in the repeat test. The test is not valid unless all the control birds die within 6 days of challenge. Labelling. The label states (1) strain of virus used; (2) recommended age for vaccination of vaccines for veterinary use.
Ranikhet Disease Vaccine, Live (Lentogenic Strain)

Newcastle Disease Vaccine, Live (Lentogenic strain)
Ranikhet Disease Vaccine Live (Lentogenic Strain) is a preparation of a suitable strain of Newcastle disease/Ranikhet disease virus (avian paramyxovirus 1). This monograph applies to vaccines intended for administration to chickens and/or other avian species for active immunization.
Production
Preparation of the vaccine The vaccine virus is grown in embryonated hens eggs or in cell cultures derived from SPF flocks (2.7.7). The master seed lot complies with the tests for extraneous agents in seed lot (2.7.10).
Identification
The vaccine, diluted if necessary and mixed with a monospecific Newcastle disease virus antiserum, no longer provokes haemagglutination of chicken red blood cells or infects embryonated hens eggs from an SPF flock or susceptible cell cultures into which it is inoculated.
Tests
Water (2.3.43). Not more than 3.0 per cent. For vaccines recommended for use in healthy chickens, free of antibodies (2.7.7) use not less than 10 chickens from an SPF flock and of the youngest age recommended for vaccination.
1602
IP 2007
RANIKHET DISEASE VACCINE, LIVE (MESOGENIC STRAIN)
For vaccines recommended for use only in avian species other than the chicken, use not fewer than 10 birds of the species likely to be most sensitive to Newcastle disease, that do not have antibodies against Newcastle disease virus and of the minimum age recommended for vaccination. Administer to each bird by eye-drop, or parenterally if only parenteral administration is recommended, 10 doses of the vaccine in a volume suitable for the test. Observe the birds at least daily for 21 days. The test is not valid if more than 20 per cent of the birds show abnormal clinical signs or die from causes not attributable to the vaccine. The vaccine complies with the test if no bird shows notable clinical signs of disease or dies from causes attributable to the vaccine. Virus titre. Not less than 106 TCID50/EID50 of the virus per dose, determining the titre in suitable cell culture or by inoculation into the allantoic cavity of SPF embryonated eggs, 9 to 11 days old. Sterility (2.2.11). Complies with the test for sterility. Potency. Carry out a potency test for each of the routes of administration stated on the label. For each of the stated routes, use at least twenty susceptible chickens and of the minimum age recommended for vaccination. Administer each chicken with a volume of the reconstituted vaccine containing a quantity of the virus equivalent to the minimum titre stated on the label. Use eight chicken of the same flock and age as controls. After 14 to 21 days, challenge each chicken by intramuscular injection with 105 LD50, of a virulent strain of Newcastle disease virus. Observe the animals for 14 days. The vaccine complies with the test if not more than two of the vaccinated chickens die or show signs of disease. The test is valid only if all the control animals die within 6 days of inoculation of the virulent challenge strain. If the potency test has been performed with satisfactory results on a representative batch of the vaccine from the seed lot, it may be omitted as a routine control test during production on other batches of the vaccine prepared from the same seed lot. Labelling. The label states (1) strain of virus used; (2) recommended age for vaccination of vaccines for veterinary use.
applies to vaccines intended for administration to chickens for active immunization.
Identification
The vaccine, diluted if necessary and mixed with a monospecific Newcastle disease virus antiserum, no longer provokes haemagglutination of chicken red blood cells or infects embryonated hens eggs from an SPF flock (2.7.7) or susceptible cell cultures into which it is inoculated. The master seed lot complies with the tests for extraneous agents in seed lot (2.7.10).
Tests
Water (2.3.43). Not more than 3.0 per cent. Safety. Administer fifteen healthy chickens free from antibodies (2.7.7), 8 to 9 weeks old, with the minimum ten dose and by the route stated on the label. Observe the chickens for 21 days. None of them shows abnormal clinical signs or dies due to causes attributable to the vaccine. If more than two chickens die during the period of observation due to causes other than those attributable to the vaccine, repeat the test. Virus titre. Not less than 105 TCID50/EID50 of the virus per dose, determining the titre in suitable cell culture or by inoculation into the allantoic cavity of SPF embryonated eggs, (2.7.7) between 9 and 11 days old. Sterility (2.2.11). Complies with the test for sterility. Potency. Carry out a potency test for each of the routes of administration stated on the label. For each of the stated routes, use not less than twenty susceptible healthy chickens free of antibodies (2.7.7) and of the minimum age recommended for vaccination. Administer each chicken with a volume of the reconstituted vaccine containing a quantity of the virus equivalent to the minimum titre stated on the label. Use eight chickens of the same flock and age as controls. After 14 to 21 days, challenge each chicken by intramuscular injection with 105 LD50 of a virulent strain of Newcastle disease virus. Observe the animals for 14 days. The vaccine complies with the test if not more than two of the vaccinated chickens die or show signs of disease. The test is valid only if all the control chickens die within 6 days of inoculation of the virulent challenge strain. If the potency test has been performed with satisfactory results on a representative batch of the vaccine from the seed lot, it may be omitted as a routine control test during production on other batches of the vaccine prepared from the same seed lot. Labelling. The label states (1) strain of virus used; (2) recommended age for vaccination.
Ranikhet Disease Vaccine, Live (Mesogenic Strain)

Newcastle Disease Vaccine, Live (Mesogenic strain)
Ranikhet Disease Vaccine, Live (Mesogenic Strain) is a preparation of a suitable strain of Newcastle disease virus (naturally modified avian paramyxovirus 1). This monograph
1603
RINDERPEST VACCINE, LIVE
IP 2007
Rinderpest Vaccine, Live

Rinderpest Vaccine, Live is a freeze-dried preparation of a live attenuated strain of rinderpest virus that has been modified by adaptation to and propagation in suitable cell cultures in such a manner that it remains avirulent but retains its immunogenicity in cattle. It is reconstituted immediately before use with a suitable diluent.
containing not less than 100 times the minimum dose stated on the label, using pooled reconstituted contents of not less than ten containers taken at random. Observe the animals for 21 days. No sign of disease attributable to the vaccine other than mild transient pyrexia is seen. Virus titer. Not less than 103 TCID50 per dose of cell culture vaccine determining the virus content of the reconstituted vaccine in a suitable cell culture system. Sterility (2.2.11). Complies with the test for sterility. Potency. Inject subcutaneously each of two susceptible cattle, free from rinderpest specific antibodies, with a field dose and 1/l0th of the minimum dose respectively stated on the label, considering 103 TCID50 of cell culture vaccine. Use two animals of the same stock and age as controls. Observe the animals for 21 days. Challenge intramuscularly each animal with a dose of not less than 104 ID50 of virulent rinderpest virus. Observe the animals for 14 days. None of the vaccinated animals shows any clinical signs suggestive of rinderpest. The test is not valid unless both the control animals develop signs of rinderpest. If the potency test has been performed with satisfactory results on a representative batch of the vaccine from the seed lot, it may be omitted as a routine control test during production on other batches of the vaccine prepared from the same seed lot, provided the National Regulatory Authority permits. Labelling. The label states (1) the strain of the virus used; (2) the number of doses in the container; (3) that the vaccine should be used immediately after reconstitution.
Production
SEED LOT The seed lots should be validated for the following tests: a) Purity. It should be free from contaminations with viruses, bacteria, fungi and mycoplasmas; b) Should not induce any abnormal clinical reaction on inoculation into rinderpest susceptible cattle; c) Efficacy. It should induce an immunity to rinderpest in the susceptible cattle. CELLS. The primary cells/subcultured cells/continuous cell lines when used should be free from BVD and other contaminating viruses.
Identification
A. The vaccine protects cattle against virulent rinderpest virus. B. The seed and the vaccine must be titrated in a suitable cell culture system capable of supporting the multiplication of the rinderpest virus. C. When neutralised with a specific rinderpest antiserum, the vaccine is no longer capable of protecting cattle against rinderpest infection.
Tests
Water (2.3.43). Not more than 3.0 per cent. Mycoplasmas (2.7.4). Complies with the test for absence of mycoplasmas. Extraneous pathogens. Complies with the requirements stated under Veterinary Vaccines. Safety A. Use four healthy guinea-pigs, each weighing not less than 400 g. Inject two of them intramuscularly and two intraperitoneally with 0.5 ml of the vaccine under examination. In addition, inject intraperitoneally each of six mice, each weighing between 18 and 25 g, with 0.1 ml of the vaccine. Observe the animals for 21 days. All the animals remain healthy during the observation period. At the end of the observation period sacrifice the animals and perform autopsy on each. None of the animals shows any unusual changes. B. Inject subcutaneously each of two susceptible cattle, free from specific antibodies, with a quantity of the vaccine
Salmonella Abortus Equi Vaccine

Salmonella Abortus Equi Vaccine is a suspension of killed mixture of equal parts of pure formalized cultures of smooth laboratory strains of Salmonella abortus equi.
Production
The whole culture or its filtrate or a mixture is inactivated in such a manner that pathogenecity is eliminated and immunogenic activity is retained. The inactivated cultures may be treated with a suitable adjuvant.
Identification
It protects susceptible animals against infection with Salmonella abortus equi.
Tests
Safety. Inject 0.5 ml of the vaccine intraperitoneally to each of six mice, each weighing not less than 18 g. Observe the mice for 96 hours, none of the mice dies of salmonellosis.
1604
IP 2007
SWINE FEVER VACCINE, LIVE
Sterility (2.2.11). Complies with the test for sterility. Potency. Inject each of twelve mice, each weighing not less than 18 g, subcutaneously with 0.5 ml of the preparation under examination. Use another twelve mice of the same weight range and from the same stock as controls. Three weeks later, challenge the mice from both groups by injecting intraperitoneally each animal with 0.5 ml of a suspension of an 18-hour old culture containing 10 LD50 virulent organisms of S. abortus equi. Observe the mice for 7 days. The vaccine passes the test if not less than nine mice of the vaccinated group survive. The test is not valid unless not less than nine of the control mice succumb to the challenge. Labelling. The label states (1) the method of preparation; (2) the strains of bacteria used to prepare the vaccine.
the dose of the vaccine and by the route stated on the label. Use two sheep of the same stock and age as un-vaccinated controls. Shave the animals closely on the flank from the shoulder to the proctodal area. Challenge each animal after 21 days post-vaccination by inoculating intradermally with 0.1 ml of a suspension six ten fold dilution of the sheep pox challenge virus. Make five separate inoculations in a vertical line for each serial dilution from the anterior to the posterior of the animals. The titer of the challenge virus is calculated using a standard statistical method for the vaccinated and control sheep by the number of pox lesions observed in each dilution. The titer of the challenge virus is calculated for the vaccinated and control animals. The vaccine passes the test if there is a difference of log titer of more than log10 2.5. Labelling. The label states (1) the strain of virus used in preparing the vaccine; (2) the virus titre; (3) the minimum dose and the routes of administration; (4) the volume of the liquid to be used for reconstitution of the vaccine.
Sheep Pox Vaccine, Live Attenuated

Sheep Pox Vaccine, Live Attenuated is a freeze dried preparation obtained by producing attenuated sheep poxvirus in a suitable cell culture and mixed with a suitable stabilizer and freeze dried. The freeze dried vial is reconstituted with a suitable diluent and used immediately.
Swine Fever Vaccine, Live

Swine Fever Vaccine, Live is a preparation of a modified strain of classical swine fever virus, which is devoid of pathogenicity for the pig by adaptation either to cell cultures or to the rabbit. It is prepared immediately before use by reconstitution from the dried vaccine with a suitable diulent.
Production
The vaccine reconstituted with a suitable liquid and diluted if necessary to provide a concentration appropriate to the particular test, complies with the requirements stated under Veterinary Vaccines with the following modifications. The seed lots used for vaccine preparation must be free from extraneous pathogens.
Production
The virus is propagated in suitable cell culture. The viral suspension is harvested, titrated and may be mixed with a suitable stabilizing agents. The vaccine is then freeze-dried
Identification
The vaccine specifically protects sheep against sheep pox.
Identification
LAPINISED VACCINE. Administer 0.5 ml intravenously into one or more non-immunised rabbits , immunized either with an identical dose of a vaccine of the same type injected by the same route between 10 and 60 days before hand or with a sufficient dose of antiserum administered a few hours before the injection of the vaccine. Twenty-four hours after the injection, start recording the temperature of the rabbits in the mornings and the evenings until the fifth day after the injection. The immunised rabbits do not exhibit a rise in temperature of more than 1.5. The test is not valid unless the nonimmunised rabbits exhibit a rise in temperature of not less than 1.5. CELL CULTURE VACCINE. For non-lapinised vaccines prepared in cell cultures, on administration to pigs immunised with the vaccine specific neutralizing antibodies develop.
Tests
Water (2.3.43). Not more than 3.0 per cent. Safety. Inoculate not less than 2 sheep of 8 to 12 months old, free from neutralizing antibodies against sheep pox virus, with ten times the field dose of the vaccine contained in 1 ml by subcutaneous route. Observe the animals for 14 days. The vaccine complies the test if none of the vaccinated animals show deep necrotic lesion and generalization. Virus titre. Not less than 102.5TCID50 of the virus per dose as determined by the titre of the vaccine in a suitable cell culture system. Sterility (2.2.11). Complies with the test for sterility. Potency. Administer each of three sheep, between 8 and 12 months old, free from sheep pox neutralizing antibodies, with
Tests
Test for extraneous pathogens. The vaccine mixed with a mono specific antiserum does not cause cytopathic effects in
1605
TETANUS VETERINARY VACCINE
IP 2007
susceptible cell cultures. The cells also show no evidence of the presence of haemadsorbing agents and the cell-culture fluids are free of haemagglutinating agents when tested with chicken erythrocytes. Water (2.3.43) Not more than 3.0 per cent. Safety. Inject intramuscularly 10 times the minimum dose stated on the label into each of three healthy piglets, between 6 and 7 weeks old, free from swine fever virus antibodies. Observe the animals for 21 days. Temperature curve should be normal and animals remain in apparent good health and display normal growth. Inject intracerebrally 0.03 ml of the vaccine, reconstituted in a manner that 1.0 ml contains 1 ml dose, into each of ten mice, weighing between 11g and 15g. Observe the mice for 21 days. If more than two mice die within the first 48 hours repeat the test. The mice show no abnormalities attributable to the vaccine within the third and twenty-first day after the injection. Virus titre. Not less than minimum virus titre per dose stated on the label, determining the titre in a suitable cell culture. Sterility (2.2.11). Complies with the test for sterility. Potency. All the animals are healthy and must have had no contact with swine fever virus and serologically must be free from CSF and BVDV antibodies. Use four healthy piglets, between 6 and 7 weeks old, for each of the 1:50, 1:200 and 1:400 dilutions of the vaccine prepared in a suitable diluent or buffer. Inject intramuscularly 1 ml of these dilutions into each of the piglets in respective groups. Use two healthy susceptible piglets of the same stock and age as control animal group. After 21 days, inoculate intramuscularly with a sufficient quantity of the challenge virus in each vaccinated piglet and in each of the two unvaccinated control animals so that at least one of the two unvaccinated control animals die within 7 to 14 days. Observe the vaccinated animals for 14 days. Calculate the number of PD50 contained in the vaccine by standard statistical methods from the number of animals, which survive without showing any signs of swine fever. The vaccine contains not less than 100 PD50 per dose. The test is not valid unless the control animals die within 7 to 14 days after inoculation. PD50 correlation studies with virus titres can replace the potency test on routine basis. If the test for potency has been carried out with satisfactory results on a representative batch of vaccine, this test may be omitted as a routine control on other batches of vaccine prepared from the same seed lot, subject-to agreement by the competent authority. Labelling. The label states (1) the minimum dose; (2) the recommended routes of administration; (3) the name of any added adjuvant.
Tetanus Veterinary Vaccine

Tetanus Vaccine for Veterinary Use is a preparation of the neurotoxin of Clostridium tetani treated in a manner that eliminates toxicity while maintaining adequate immunogenic properties.
Production
The C. tetani strain used for production is cultured in a suitable medium. The toxin is purified and then detoxified or it may be detoxified before purification. The antigenic purity is determined in Lf units of tetanus toxoid per milligram of protein and shown to be not less than the value approved for the particular product. Choice of vaccine composition The C. tetani strain used in the preparation of the vaccine is shown to be satisfactory with respect to the production of the neurotoxin. The vaccine is shown to be satisfactory with respect to safety and immunogenicity for each species of animal for which it is intended. As part of the studies to demonstrate these characteristics, the tests described below may be used. Production of antigens. The production of the neurotoxin of C. tetani is verified by a suitable immunochemical method carried out on the neurotoxin obtained from the vaccine strain under the conditions used for the production of the vaccine. Safety. Carry out the test for each recommended route of administration and species of animal for which the vaccine is intended; use animals of the minimum age recommended for vaccination and of the most sensitive category for the species. Use not less than 15 animals, free from antitoxic antibodies for each test. Administer a double dose of vaccine to each animal. Administer a single dose of vaccine to each animal after the interval stated on the label. Observe the animals until 14 days after the last administration. The vaccine complies with the test if no animal shows abnormal local or systemic signs of disease or dies from causes attributable to the vaccine. DETOXIFIED HARVEST Absence of toxin and irreversibility of toxoid. Carry out a test for reversion to toxicity on the detoxified harvest using 2 groups of 5 guinea-pigs, each weighing between 350 to 450 g; if the vaccine is adsorbed, carry out the test with the shortest practical time interval before adsorption. Prepare a dilution of the detoxified harvest so that the guinea-pigs each receive 10 times the amount of toxoid (measured in Lf units) that will be present in a dose of vaccine. Divide the dilution into 2 equal parts. Keep one part at 5 3 and the other at 37 for 6 weeks. Attribute each dilution to a separate group of guinea-pigs and inject into each guinea-pig the dilution attributed to its
1606
IP 2007
TETANUS VETERINARY VACCINE
group. Observe the animals for 21 days. The toxoid complies with the test if no guinea-pig shows clinical signs of disease or dies from causes attributable to the neurotoxin of C. tetani. FINAL LOT The final bulk vaccine is distributed aseptically into sterile containers. The containers are closed so as to avoid contamination.
normally uses rabbits, the potency test described above may be carried out using ten healthy rabbits, between 3 and 6 months old. 1 m1 of serum contains not less than 2.5 Units. Biological assay of Cl. tetani antitoxin The potency of Cl. tetani antitoxin is determined by comparing the dose necessary to protect mice or other suitable animals against the toxic effects of a fixed dose of Cl. tetani toxin with the quantity of a Standard preparation of Cl. tetani antitoxin necessary to give the same protection. For this purpose, the Standard preparation of Cl. tetani antitoxin and a suitable preparation of Cl. tetani toxin are required. The test dose of the toxin is determined in relation to the Standard preparation of antitoxin and the potency of the preparation under examination is then determined in relation to the Standard preparation using the test toxin. Standard preparation The Standard preparation is the 2nd International standard, established in 1969, consisting of freeze-dried hyperimmune horse serum (supplied in ampoules containing 1400 Units) or another suitable preparation, the potency of which has been determined in relation to the International standard. Suggested method NOTE - The severity of tetanic paralysis to be regarded as the end-point is such that the paralysis is readily recognised but not sufficiently extensive to cause significant suffering. For humane reasons the animals should be examined at least twice a day and should be killed as soon as the end-point is reached. In practice, when using high levels of toxin to determine the test dose, or when using low levels of antitoxin in the preliminary and final tests, the development of paralysis is so rapid that the defined end-point is usually synchronous with death. Where death occurs, the combined totals of animals dying or reaching the paralytic end-point are used in the calculations. Preparation of test toxin. Prepare Cl. tetani toxin by growing Cl. tetani in liquid culture for 8 to 10 days and then adding 1 volume of a sterile filtrate of the culture to 1 or 2 volumes of glycerine. Store at 0 or at temperatures slightly below it. The toxin may be dried by a suitable method. Selection of test toxin. Select toxin for use as the test toxin by determining the following quantities: LP/10 dose (Limes paralyticum). This is the smallest quantity of toxin that when mixed with 0.1 Unit of antitoxin and injected subcutaneously into mice (or guinea-pigs) causes tetanic paralysis in the animals on or by the fourth day after injection.
Identification
Carry out test A if permitted by the nature of the adjuvant. Otherwise carry out test B. A. Separate the toxoid from the adjuvant. For vaccines adsorbed on aluminium hydroxide, the following treatment is suitable. Dissolve sufficient sodium citrate in the vaccine under examination to give a 10 per cent w/v concentration. Maintain at 37 for about 16 hours and centrifuge. The clear supernatant liquid reacts with a suitable tetanus antitoxin and yields a precipitate. B. When inoculated into healthy susceptible animals, the vaccine stimulates the formation of antitoxin to the neurotoxin of Clostridium tetani or protects the animals against the paralytic effects of the toxin.
Tests
Safety. Inject 5 ml of the vaccine subcutaneously as two equally divided doses at separate sites into each of five guinea pigs, each weighing between 350 and 450g. Observe the guinea pigs for 21 days. None of the animals shows any symptoms of tetanus or dies from tetanus. If more than one animal dies of non-specific causes, repeat the test. No animal dies in the second test. Sterility (2.2.11). Complies with the test for sterility. Potency. Test A may be omitted if test B is carried out. Test B may be ommited if test A is carried out. A. Inject subcutaneously each of ten guinea pigs, each weighing between 350 and 450 g, with a quantity of the vaccine not more than the minimum dose stated on the label as the primary dose, and 28 days later with a quantity of the vaccine not more than the minimum dose stated on the label as the secondary dose. Fourteen days after the second dose, collect the blood from each guinea pig, pool the sera and determine the antitoxin titre by the biological assay of Cl. tetani antitoxin described below. 1 ml of serum contains not less than 7.5 IU per ml or, for vaccine intended for use in equine, not less than 30 IU per ml. When Cl. tetani vaccine is presented as a component of a mixed vaccine intended for use in animals other than equine and the potency test of the other component or components
1607
THEILERIOSIS VACCINE, LIVE
IP 2007
Paralytic dose 50. This is the quantity of toxin that when injected subcutaneously into mice (or guinea-pigs) causes tetanic paralysis in one-half of the animals injected on or by the fourth day after injection. A suitable toxin is one that contains not less than 1000 paralytic dose 50 in an LP/10 dose. Determination of test dose of toxin. Measure or weigh a quantity of the test toxin and dilute with or dissolve in a suitable liquid. Reconstitute or dilute the Standard preparation with a suitable liquid to give a solution containing 0.5 Unit in 1 ml. Prepare mixtures of the solution of the Standard preparation and the solution of the test toxin such that each mixture contains 0.1 Unit of antitoxin in the volume selected for injection and one of a series of graded volumes of the solution of the toxin, separated from each other by steps of not more than 20 per cent and covering the expected end-point. Adjust each mixture to the same final volume (0.4 to 0.6 ml if mice are used or 4.0 ml if guinea-pigs are used) with a suitable liquid. Allow the mixtures to stand at room temperature, protected from light, for 60 minutes and then inject a dose of the selected volume of each mixture subcutaneously into each of not less than 2 animals of the group to which each mixture has been allocated. Observe the animals for 4 days and record daily the degree of tetanus developing in each group of animals. Repeat the determination at least once, add together the results of the separate tests that have been made with mixtures of the same composition such that a series of totals is obtained and determine the mean values. The test dose of the toxin is the amount present in that mixture that causes tetanic paralysis in one-half of the total number of animals injected with it. When the test dose of the test toxin has been determined, a concentrated solution of the test toxin may be prepared in a mixture consisting of 1 volume of saline solution and 1 or 2 volumes of glycerine. This concentrated solution may be stored frozen and diluted as required. The specific activity of such a solution must be determined at frequent intervals. Determination of potency of the antitoxin. Preliminary test. Measure or weigh a quantity of the test toxin and dilute with or dissolve in a suitable liquid such that the solution contains 5 test doses per ml. Prepare mixtures of the solution of the test toxin and the preparation under examination such that for each mixture the volume selected for injection contains the test dose of toxin and one of a series of graded volumes of the preparation under examination. Adjust each mixture to the same final volume with a suitable liquid. Allow the mixtures to stand at room temperature, protected from light, for 60 minutes. Inject a dose of the selected volumes of each mixture subcutaneously into each of not less than two animals of the group to which each mixture has been allocated. Observe the animals for 4 days and record daily the
degree of tetanus developing in each group of animals. From the results select suitable mixtures for the final test. Final test. Prepare similar fresh mixtures of the test toxin and the preparation under examination such that for each mixture the volume selected for injection contains the test dose of toxin and one of a series of graded volumes of the preparation under examination, separated from each other by steps of not more than 20 per cent and covering the expected end-point as determined in the preliminary test. Prepare further mixtures with the same amount of test toxin and graded volumes of the Standard preparation, centered on 0.1 Unit in the volume selected for injection to confirm the test dose of the toxin. Adjust each mixture to the same final volume with a suitable liquid. Allow the mixture to stand at room temperature, protected from light, for 6 minutes. Inject a dose of the selected volume of each mixture subcutaneously into each of not less than two animals of the group to which each mixture has been allocated. Observe the animals for 4 days and record daily the degree of tetanus developing in each group of animals. The mixture of antitoxin under examination that contains 0.1 Unit in the volume injected is that mixture which causes tetanic paralysis in the same, or almost the same number of animals as the mixture containing 0.1 Unit of the Standard preparation in the volume injected. Repeat the determination at least once and calculate the average of all valid estimates. Estimates are not valid unless the Standard preparation gives a result within 20 per cent of the expected value. Limits of error. For the suggested method, the limits of error (P = 0.95) have been estimated to be 85 to 114 per cent when two animals are used per dose, 91.5 to 109 per cent when three animals are used per dose, and 93 to 108 per cent when six animals are used per dose. B. Carry out the biologycal assay of adsorbed tetanus vaccine as stated under Tetanus Vaccine (Adsorbed). This method may only be used for those preparations for which it has been shown to be suitable and in particular may not be suitable for vaccine with an oil adjuvant. Where this alternative method is used the estimated potency is not less than 150 units in the smallest dose stated on the label. Labelling. The label states (1) the name of the adjuvant used; (2) the preparation should be shaken before use.
Theileriosis Vaccine, Live

Theileriosis Vaccine, Live is a lymphoblast cell culture containing Theileria annulata macroschizonts attenuated by passage in such a manner that it remains avirulent while it retains its immunogenicity. The concentrate of the vaccine may be diluted with a suitable diluent after thawing.
1608
IP 2007
THEILERIOSIS VACCINE, LIVE
Production
Production is based on approved seed lot system. The working seeds are prepared from the master seeds. The production seed may be prepared by propagating a large number of cells either in suspension/monolayer cultures.
Sterility (2.2.11). Complies with the test for sterility. Potency. Inject each of three susceptible cattle not less than 9 months old with the minimum dose by the route stated on the label. Use two cattle of the same stock and age as controls. After 30 days, challenge each of the vaccinated as well as the control animals with a preparation of gut homogenate of ticks containing suitable quantity of sporozoites to infect adult cattle. Observe the animals for 30 days; none of the vaccinated animals shows any abnormal signs. The test is not valid unless both the control animals show typical signs of theileriosis. If these tests have been performed with satisfactory results on a representative batch of the vaccine from the seed lot, they may be omitted by the manufacturer as a routine control on other batches of the vaccine prepared from the same seed lot. Labelling. The label states (1) the number of doses in the container; (2) the recommended dose; (3) the method of thawing and reconstitution; (4) that the reconstituted vaccine should be used within 3 hours after thawing and reconstitution.
Identification
Protects susceptible cattle against theileriosis.
Tests
Safety. Inject each of two healthy susceptible cattle not less than 9 months old with twice the dose as recommended on the label. Observe the animals for 30 days. None of the animals shows systemic reactions other than mild pyrexia and mild swelling of superficial lymph nodes. No schizonts/piroplasms should be seen in the blood smears/lymph node smear. Cell count. Contains not less than 2 million live lymphoblast cells in each dose.
1609

Indian Pharmacopoeia Vol-3

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INDIAN PHARMACOPOEIA 2007

THE INDIAN PHARMACOPOEIA COMMISSION GHAZIABAD

INDIAN PHARMACOPOEIA 2007

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INDIAN PHARMACOPOEIA 2007

INDIAN PHARMACOPOEIA 2007

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INDIAN PHARMACOPOEIA 2007

DRUG SUBSTANCES, DOSAGE FORMS AND PHARMACEUTICAL AIDS

INDIAN PHARMACOPOEIA 2007

INDIAN PHARMACOPOEIA 2007 2007

NALIDIXIC ACID TABLETS

Nalidixic Acid Tablets

Mol. Wt. 347.8

Nalorphine Hydrochloride is 17-allyl-7,8-didehydro-4,5epoxymorphinan-3,6-diol hydrochloride.

Storage. Store protected from light and moisture.

Mol. Wt. 428.7

Nandrolone Decanoate is 3-oxo-4-estren-17-yl decanoate.

NANDROLONE DECANOATE INJECTION

Nandrolone Decanoate Injection

NANDROLONE PHENYLPROPIONATE INJECTION

Nandrolone Phenylpropionate Injection

Mol. Wt. 273.3

CH3 CH3 CH3 H

Mol. Wt. 663.9

Nelfinavir Mesylate is (3S,4aS,8aS)-N-(tert-butyldecahydro2-[(2R,3R)-3-(3-hydroxy-o-toluamido)-hydroxy-4(phenylthio)butyl]isoquinoline-3-carboxamide methyl sulphonate.

NELFINAVIR MESTYLATE ORAL POWDER

Nelfinavir Mesylate Oral Powder

NEOMYCIN EYE DROPS

Neomycin Eye Drops

NEOMYCIN EYE OINTMENT

Neomycin Eye Ointment

NEOMYCIN EYE OINTMENT

Mol. Wt. 303.2

spectrophotometer set at 215 nm, a 10 l loop injector.

Mol. Wt. 266.3

NEVIRAPINE ORAL SUSPENSION

Nevirapine Oral Suspension

Mol. Wt. 327.1

C6H6N2O Nicotinamide is pyridine-3-carboxamide.

C6H5NO2 Nicotinic Acid is pyridine-3-carboxylic acid.

Mol. Wt. 353.3

Mol. Wt. 346.3

NIFEDIPINE SUSTAINED-RELEASE TABLETS

Nifedipine Sustained-release Tablets

Mol. Wt. 178.2

Mol. Wt. 238.2 (anhydrous) Mol. Wt. 256.2 (hydrous)

Mol. Wt. 198.1

H OH , HOOC COOH , H2O H OH

NORADRENALINE BITARTRATE INJECTION

Noradrenaline Bitartrate Injection

Description. A white or yellowish-white, crystalline powder; odourless.

Mol. Wt. 319.3

NORFLOXACIN EYE DROPS

Storage. Store protected from light and moisture.

Norfloxacin Eye Drops

NORGESTREL AND ETHINYLOESTRADIOL TABLETS

Mol. Wt. 312.5

Norgestrel and Ethinyloestradiol Tablets

Mol. Wt. 299.8