1|Page

DRUG ANALYSIS
Drug Analysis is the set of techniques that allow the analysis and characterization of drugs. It
involves identifying novel drugs, evaluating their association and specificity, classifying their
molecular structures, analysis can be divided into two types.
1.Qualitative methods
2.Quantitative methods
Qualitative methods: These methods are commonly used to confirm the presence or detection
of component and\or impurities (predictable).
Quantitative methods: Determine the amount of known drugs in bulk form or formulation.
(a) Organic substances : A material is organic if it contains carbon bound to other atoms by
covalence. The other atoms most commonly contain hydrogen, oxygen, and/or nitrogen.
Example - Urea, sodium salicylate, diphenhydramine, emetine hydrochloride, meprobamate,
paramethadione, pyrazinamide etc., and
(b) Inorganic substances : Inorganic substances are a group of chemicals that contain no
carbon. Example - sodium bicarbonate, milk of magnesia, ammonium chloride, calcium
hydroxide, lithium carbonate, zinc oxide etc.

Direct Titration Method : It is an usual practice that when a solid substance is to be assayed, an
aliquot quantity of the same may be weighed accurately and dissolved in sufficient water so that
the resulting solution should have more or less the same equivalent concentration as that of the
acid used in the titration. Methyl orange (pH range = 3.0 to 4.4) is the indicator of choice for
obvious reasons, as phenolphthalein and most other indicators are instantly affected by the
carbonic acid (H2CO3) generated in the reaction which ultimately cause a change in color even
before the reaction attains completion.
2|Page

Residual / Indirect / back titration : Residual titration or back titration is normally employed in
the following two situations, namely :
Case I : when a chemical reaction proceeds rather slowly or sluggishly, and
Case II : when the substance under determination fails to give a sharp and distinctly visible end-
point with an indicator by direct titration. In usual practice, the residual titration is accomplished
by allowing to dissolve the substance under estimation in an accurately measured quantity of a
standard solution of known strength present in excess and subsequently titrating the excess of the
latter with another previously standardized solution. A good number of examples of this
particular method shall be discussed in subsequent exercises.

Acid-Base titration (Acidimetry and Alkalimetry): This includes the titration of a base with
standard acid (acidimetry) and titration of acids with standard base (alkalimetry). This reaction
involves the combination of hydrogen and hydroxide ions to form water.

Types of acid-base titration

1) Strong acid- strong base
2) Weak acid-strong base
3) Strong acid-weak base
4) Weak acid-weak base

Fig : Flow chart of acid base titration

3|Page

Redox Titration : The oxidation and reduction processes essentially take place simultaneously
in a reaction, thus one entity gets reduced in the process of oxidizing the second. ‘Redox’—is the
abbreviated form of reduction— oxidation systems. In the oxidation—reduction methods of
analysis a change in valence of the reacting products is a must which is contrary to precipitation
and neutralization methods of analysis where no change in valence occur. The major oxidizing
agents normally employed in volumetric titrations include, potassium permanganate, potassium
dichromate, and ceric sulphate. Example –
Sn2+ + 2Cl– + 2HgCl2 → Sn4+ + 4Cl– + Hg2Cl2
Iodimetry Titration : Iodimetry is a technique that involves titrating free iodine with a reducing
agent. In iodimetry, quantitative oxidation of reducing agents, such as arsenious acid (H2AsO3)
may be carried out by employing standard solutions of iodine as shown under :
H3AsO3 + H2O + I2 H3AsO4 + 2H+ + 2I–
This type of assay is known as ‘direct method of iodimetry’.
In another situation, a known excess quantity of standard iodine solution is added in the
substance (a reducing agent) to be assayed and then the excess iodine may be titrated with the
help of standard sodium thiosulphate solution, such as : the estimation of sodium bisulphite :
NaHSO3 + I2 + H2O → NaHSO4 + 2HI
I2 + 2Na2S2O3 → 2NaI + Na2S4O6

Iodometry Titration : Iodimetry is a technique that involves titrating free iodine with a reducing
agent. As a result, iodine decreases to iodide and oxidizes other species. Because a free iodine
solution is difficult to obtain, we must combine iodine with potassium iodide and KI solution to
get the needed solution.
2CuSO4 + 4 KI → 2CuI ↓ + I2 + 2K2SO4
I 2 + OH– → HI + IO–
3IO– → IO3 – + 2I
Principle of HPLC : The purification takes place in a separation column between a stationary
and a mobile phase.
** The stationary phase is a granular material with very small porous particles in a separation
column.
** The mobile phase, on the other hand, is a solvent or solvent mixture which is forced at high
pressure through the separation column.
** Via a valve with a connected sample loop, i.e. a small tube or a capillary made of stainless
steel, the sample is injected into the mobile phase flow from the pump to the separation column
using a syringe.
** Subsequently, the individual components of the sample migrate through the column at
different rates because they are retained to a varying degree by interactions with the stationary
phase.
** After leaving the column, the individual substances are detected by a suitable detector and
passed on as a signal to the HPLC software on the computer.
4|Page

** At the end of this operation/run, a chromatogram in the HPLC software on the computer is
obtained.
** The chromatogram allows the identification and quantification of the different substances.

Application of HPLC in Pharmaceutical:

** To control the drug stability
** Quantity of drug determination from pharmaceutical dosages form
** Quantity of drug determination from biological fluids
** Therapeutic drug monitoring
** Purity checking
** Toxicology: e.g. Paracetamol poisoning
Principle of IR spectroscopy : IR spectroscopy works on the principle that molecules absorb
specific frequencies that are characteristic of their structure. At temperatures above absolute
zero, all the atoms in molecules are in continuous vibration with respect to each other. The IR
spectrum of a sample is recorded by passing a beam of IR radiation through the sample.
When the frequency of a specific vibration is equal to the frequency of the IR radiation directed
on the molecule, the molecule absorbs the radiation. The examination of the transmitted light
reveals how much energy was absorbed at each frequency (or wavelength). Using various
sampling accessories, IR spectrometers can accept a wide range of sample types such as gases,
liquids, and solids.

Application of IR in Pharmaceutical:
5|Page

Principle Of UV-Visible spectroscopy : The Principle of UV-Visible Spectroscopy is based

on the absorption of ultraviolet light or visible light by chemical compounds, which results in the
production of distinct spectra. Spectroscopy is based on the interaction between light and matter.
When the matter absorbs the light, it undergoes excitation and de-excitation, resulting in the
production of a spectrum.
When matter absorbs ultraviolet radiation, the electrons present in it undergo excitation. This
causes them to jump from a ground state (an energy state with a relatively small amount of
energy associated with it) to an excited state (an energy state with a relatively large amount of
energy associated with it). It is important to note that the difference in the energies of the ground
state and the excited state of the electron is always equal to the amount of ultraviolet radiation or
visible radiation absorbed by it.

Application of UV-Visible spectroscopy in Pharmaceuticals :

Figure 5: application of UV in pharmaceutical

6|Page

METHODS OF STERILIZATION
Sterilization : Sterilization is the process of killing or removing bacteria and all other form of
living microorganisms and their spores from preparations or articles . A product is said to be
sterile when it is free from all living microorganisms and passes the sterility test .
Methods of Sterilization : Methods of sterilizations is divided into following categories
1. Physical methods
a) Dry heat sterilization
b) Moist heat sterilization
c) Sterilization by radiations

2. Chemical methods

a) Gaseous Sterilization
b) Sterilization by disinfectants
3. Mechanical methods :
In this methods of sterilization the solution to be sterilized is passed through a mechanical device
known as bacteria proof filter which include Millipore filter , Setiz filter ,sintered glass filter ,
berkefeld filter .
1) Physical Methods – (Dry Heat Sterilization )
Substance which get destroyed by moist air or due to their physical characteristics can not be
sterilized by moist heat must be sterilized by dry heat in ovens .
Flaming: This is a method of passing the article over a Bunsen flame, but not heating it to
redness. Articles such as scalpels, mouth of test tubes, flasks, glass slides and cover slips are
passed through the flame a few times. Even though most vegetative cells are killed, there is no
guarantee that spores too would die on such short exposure. This method too is limited to those
articles that can be exposed to flame. Cracking of the glassware may occur.
Hot air oven: This method was introduced by Louis Pasteur. Articles to be sterilized are
exposed to high temperature (160o C) for duration of one hour in an electrically heated oven.
Since air is poor conductor of heat, even distribution of heat throughout the chamber is achieved
by a fan. The heat is transferred to the article by radiation, conduction and convection. The oven
should be fitted with a thermostat control, temperature indicator, meshed shelves and must have
adequate insulation.
Advantage of Dry Heat Sterilization :
1) It is most suitable method of sterilization for substances which are destroyed by moisture
e.g. oily substances and dry powders
7|Page

2) Glass wares like flasks , test tubes , pipettes , all glass syringes etc. can be easily and
thoroughly sterilized which may not be possible in moist heat sterilization .
3) It is less damaging to glass and metal equipment than moist heat .
Disadvantage of Dry Heat Sterilization:
1) It requires very long heating up times , high temperature and long exposure time.
2) Most medicaments , rubber and plastic articles which are thermolabile get destroyed by
this method .
3) Preparations containing water , alcohol or other volatile substances cannot be sterilized
by this method because the liquid may evaporate at high temperature .
b) Moist heat:
Moist heat sterilization is also known as steam sterilization .It is done in autoclave but on small
scale pressure cooker can be used . It is the most reliable method in sterilization because in the
presence of moisture bacteria are destroyed at a considerably lower temperature rather then when
moisture is absent . Moist heat acts by coagulation and denaturation of proteins.
At temperature below 100o C:
Pasteurization: There are two methods of pasteurization, the holder method (heated at 63o C for
30 minutes) and flash method (heated at 72o C for 15 seconds) followed by quickly cooling to
13o C. Other pasteurization methods include Ultra-High Temperature (UHT), 140o C for 15 sec
and 149o C for 0.5 sec. This method is suitable to destroy most milk borne pathogens like
Salmonella, Mycobacteria, Streptococci, Staphylococci and Brucella, however Coxiella may
survive pasteurization.
At temperature 100o C:
Boiling: Boiling water (100o C) kills most vegetative bacteria and viruses immediately. Certain
bacterial toxins such as Staphylococcal enterotoxin are also heat resistant. Some bacterial spores
are resistant to boiling and survive; hence this is not a substitute for sterilization. The killing
activity can be enhanced by addition of 2% sodium bicarbonate. When absolute sterility is not
required, certain metal articles and glass wares can be disinfected by placing them in boiling
water for 10-20 minutes.
At temperature above 100o C:
Autoclave: Sterilization can be effectively achieved at a temperature above 100oC using an
autoclave. Water boils at 100oC at atmospheric pressure, but if pressure is raised, the temperature
at which the water boils also increases. In an autoclave the water is boiled in a closed chamber.
As the pressure rises, the boiling point of water also raises. At a pressure of 15 lbs inside the
autoclave, the temperature is said to be 121o C. Exposure of articles to this temperature for 15
minutes sterilizes them. To destroy the infective agents associated with spongiform
8|Page

encephalopathies (prions), higher temperatures or longer times are used; 135o C or 121o C for at
least one hour are recommended.
Advantage of Moist Heat Sterilization :
1) Because of high penetration power of stem under pressure , microorganisms are killed
more efficiently and in lesser time at low temperatures than dry heat .
2) In large size autoclaves large quantities of materials can be sterilized in one batch .
3) Solutions packed in sealed containers as ampoules , are readily sterilized by this methods.
Disadvantage of Moist Heat Sterilization :
1) This method is unsuitable for materials which can not withstand the heating at 1150 C or
more
2) This method is not useful for , oils , fats , ointments , powders , oily injections and other
preparations through which cannot steam penetrate .
Chemical method : (Gaseous Sterilization )
Gaseous sterilization is a special type of chemical sterilization in which the chemicals used are
gaseous or vapours and not the liquid or solid .
1) Formaldehyde
Mode of action: Acts through alkylation of amino-, carboxyl- or hydroxyl group, and probably
damages nucleic acids. It kills all microorganisms, including spores.
Application: 40% Formaldehyde (formalin) is used for surface disinfection and fumigation of
rooms, chambers, operation theatres, biological safety cabinets, wards, sick rooms etc.
Fumigation is achieved by boiling formalin, heating paraformaldehyde or treating formalin with
potassium permanganate. It also sterilizes bedding, furniture and books. 10% formalin with 0.5%
tetraborate sterilizes clean metal instruments. 2% gluteraldehyde is used to sterilize
thermometers, cystoscopes, bronchoscopes, centrifuges, anasethetic equipments etc. An exposure
of at least 3 hours at alkaline pH is required for action by gluteraldehyde. 2% formaldehyde at
40o C for 20 minutes is used to disinfect wool and 0.25% at 60o C for six hours to disinfect
animal hair and bristles.
Disadvantages: Vapors are irritating (must be neutralized by ammonia), has poor penetration,
leaves non-volatile residue, activity is reduced in the presence of protein. Gluteraldehyde
requires alkaline pH and only those articles that are wettable can be sterilized.
2) Ethylene Oxide :
Mode of action: It is an alkylating agent. It acts by alkylating sulfydryl-, amino-, carboxyl- and
hydroxyl- groups. Properties: It is a cyclic molecule, which is a colorless liquid at room
temperature. It has a sweet ethereal odor, readily polymerizes and is flammable.
9|Page

Application: It is a highly effective chemisterilant, capable of killing spores rapidly. Since it is

highly flammable, it is usually combined with CO2 (10% CO2+ 90% EO) or
dichlorodifluoromethane. It requires presence of humidity. It has good penetration and is well
absorbed by porous material. It is used to sterilize heat labile articles such as bedding, textiles,
rubber, plastics, syringes, disposable petri dishes, complex apparatus like heart-lung machine,
respiratory and dental equipments. Efficiency testing is done using Bacillus subtilis var niger.
Disadvantages: It is highly toxic, irritating to eyes, skin, highly flammable, mutagenic and
carcinogenic.
Microbiological assay
A microbiological assay defined as qualitative or quantitative determination of chemical
compound from a simple or even complex material with the use of microorganisms.
Principles of microbiological assay:
The microbiological assay is the technique in which the potency or concentration of a compound
is assessed by determines its effect on the microorganism. It is a legal quality control
requirement for the assay of several antibiotics in both the British Pharmacopoeia and United
States Pharmacopoeia. Many therapeutic agents that are either inhibit the growth of
microorganisms (antibiotics) or essential for their growth (vitamins and amino acids) are
standardized by microbiological assays. The microbiological assay of an antibiotic is based upon
a comparison of the inhibition of growth of microorganisms by measured concentrations of the
antibiotics under examination with that produced by the known concentration of a standard
preparation of the antibiotic having a known activity. Microbiological assay of vitamins is a type
of biological assay performed with the help of microorganisms.
Sterility and Pyrogen
Sterility can be defined as the freedom from the presence of viable microorganisms. However,
the conditions that guarantee absolute sterility are usually too harsh for active ingredients, and
the definition of sterility for a medicinal product must be defined in functional terms.
Methods for sterility test are:
a) Membrane filtration
b) Direct transfer
Membrane filtration: The Membrane Filtration Sterility Test is the method of choice for
pharmaceutical products. An appropriate use of this test is for devices that contain a preservative
and are bacteriostatic and fungistatic under the direct transfer method. With membrane filtration,
the concept is that the microorganisms will collect onto the surface of a sub-micron pore size
filter. This filter is segmented and transferred to appropriate media. The test media are fluid
thioglycollate medium (FTM) and soybean casein digest medium (SCDM). FTM is selected
based upon its ability to support the growth of anaerobic and aerobic microorganisms. SCDM is
10 | P a g e

selected based upon its ability to support a wide range of aerobic bacteria and fungi (i.e., yeasts
and molds). Incubation time is 14 days.
Direct Transfer This method is the method of choice for medical devices because the device is
in direct contact with test media throughout the incubation period. Viable microorganisms that
may remain in or on a product after sterilization have an ideal environment within which to grow
and proliferate. This is especially true with damaged microorganisms where the damage is due to
a sub-lethal sterilization process. All microorganisms have biological repair mechanisms that can
take advantage of environmental conditions conducive to growth. The direct transfer method
benefits these damaged microorganisms. The entire product should be immersed in test fluid.
With large devices, patient contact areas should be immersed. Nova understands that appropriate
modifications are required due to the size and shape of test samples. The method requires that the
product be transferred to separate containers of both FTM and SCDM. The product is aseptically
cut, or transferred whole, into the media containers. After being transferred, the samples are
incubated for 14 days.
A Pyrogen is a substance i.e. products of the growth of micro-organisms or may be parts of dead
cells or metabolic products which cause febrile reactions like fever, chills, back pain etc.
Methods for pyrogen test are:
a) Sham Test (Rabbit test)
b) LAL test (Limulus Amoebecyte Lysate Test )

LAL test (Limulus Amoebecyte Lysate Test ) : In Vitro assay used to detect the presence and
concentration of bacterial endotoxins in drugs and biological products. Limulus Amoebocyte
Lysate (LAL) is an aqueous extract of blood cells (amoebocytes) from the horseshoe crab,
Limulus polyphemus. LAL reacts with bacterial endotoxin or lipopolysaccharide (LPS), which is
a membrane component of Gram-negative bacteria and forms gel which is then used for the
detection and quantification of bacterial endotoxins.
Limitations of LAL Test:
1. Disturbed by endotoxin binding components like lipids, blood components etc.
2. Difficult to correlate with rabbit test.
3. False positive for cellulose and many herbal preparations.
Rabbit pyrogen testing: The test involves the measurement of rise in body temperature of
rabbits following IV injection of sterile solution of substance being examined.
Animals used: Select same variety of healthy mature rabbits weighing less than 1.5Kg and
should maintain balanced diet. They should not show any loss of body weight during the
preceding week of test.