QUALITY TEST OF PARENTERALS, SUSPENSIONS ND STERILTY

OF STERILE PRODUCTS
Quality is suitability of drugs for their intended use determined by their efficiency
weighed against safety, according to label claim, or as promoted or publicized
their conformity to specifications regarding identity, purity and other
characteristics.

The International Standard of Organization (ISO) definition states that quality

control is “the operational techniques and activities that are used to fulfill
requirements for quality”

QC OF PARENTERALS:
Paenterals:

Injectable Preparations are also called as Parenteral preparations. Injectable

preparations are sterile preparations and are administered by injection, infusion
or implantation.

How to use parenterals:

An injection is an infusion method of putting fluid into the body, usually with a
syringe and a hollow needle which is pierced through the skin to a sufficient depth
for the material to be administered into the body.

Route to administer parenterals:

An injection follows a Parenteral route of administration; that is, administration

via a route other than through the digestive tract. There are several methods of
injection or infusion used in humans, including intradermal

 Subcutaneous
 Intramuscular
 Intravenous
 Intraosseous
 Intraperitoneal
  Intrathecal
 Epidural
 Intracardiac
 Intraarticular
 Intracavernous
 Intravitreal.

Injections are among the most common health care procedures, with at least 16
billion administered in developing and transitional countries each year.

95% of injections are administered in curative care, 3% are for immunization, and
the rest for other purposes, such as blood transfusions.

Approximately 40% of injections worldwide are administered with unsterilized,

reused syringes and needles, and in some countries this proportion is 70%,
exposing millions of people to infections.

Quality Control Testing of Injectable Preparations:

The quality of Injectable is the sum of all parameters that contribute to safety and
therapeutic efficacy of the drug. The USP compendial requirements has
recommended the following tests for Injectable products.

1. Content uniformity
2. Extractable volume
3. Particulate matter in injections
4. Bacterial endotoxin test
5. Pyrogen test
6. Sterility test
7. Membrane filteration process
8. Direct inoculation method

1) Content uniformity & weight

Determine the content of the active ingredient of each of 10 containers taken

at random. The preparation under examination complies with the test if the
 individual values thus obtained are all between 85 and 115 percent of the
average value. The preparation under the examination fails to comply with the
test if more than one individual value is outside the limits 85 to 115 percent of
the average value or if any one individual value is outside the limits 75 to 125
percent of the average value. If one individual value is outside the limits 85 to
115 percent but within the limits 75 to 125 percent of the average value,
repeat the determination using another 20 containers taken at random. The
preparation under examination complies with the test if in the total sample of
30 containers not more than one individual value is outside the limits 85 to
115 percent and none is outside the limits 75 to 125 percent of the average
value.

2) Extractable volume
a) Single dose containers

Method I: Where the nominal volume does not exceed 5ml.

Use 6 containers, 5 for the tests and 1 for rinsing the syringe used. Using a
syringe with appropriate capacity, rinse the syringe and withdraw as much as
possible the contents of one of the containers reserved for the test and
transfer, without emptying the needle, to a dry graduated cylinder of such
capacity that the total combined volume to be measured occupies not less
than 40% of the nominal volume of the cylinder. Repeat the procedure until
the contents of the 5 containers have been transferred and measure the
volume. The average content of the 5 containers is not less than the nominal
volume and not more than 115% of the nominal volume. Alternatively the
volume of contents in milliliter can be calculated as mass in grams divided by
the density.
 Method II: Where the nominal volume is more than 5ml

Transfer the contents of not less than 3 containers separately to dry graduated
cylinders such that the volume to be measured occupies not less than 40% of
the nominal volume of the cylinder and measure the volume transferred. The
contents of each container are not less than the nominal volume and not more
than 110% of the nominal volume.

3) Particulate matter in injections:

The preparations intended for parenteral use should be free from particulate
matter and should be clear when inspected visually. Two methods are
described by USP according to the filled volume of the product to be tested.

For large volume parenterals (LVP's), a filtration followed by microscopical

examination procedure is used.

For small volume parenterals (SVP's) a light obscuration based sensor

containing electronic liquidborne particle counter system is used.

The USP standards are met if the LVP's under test contain NMT 50 particles per
ml of 10μ m, and NMT 5 particles per ml of 25μm in an effective linear
dimensional fashion. The USP standards are met if the SVP's under test contain
NMT 10,000 particles per container of 10 μm, and NMT 1000 particles per
container of 25μm in an effective spherical diameter.
4) Bacterial endotoxin test:

LAL (Limulus Amebocyte Lysate) test is used to characterize the bacterial

endotoxin that may be present. The USP reference standard contains 10,000
USP endotoxins per vial. The LAL reagent is used for gel-clot formation. The
test is performed using stated amounts of volumes of products, standard,
positive control, negative control of endotoxin.

The tubes are incubated at 37±1ºC FOR 60 ±2 minutes. When the tubes are
inverted at 180ºC angle, formation of firm gel confirms positive reaction. While
formation of a viscous gel that doesn't maintain its integrity or absence of a
firm gel confirms negative reaction. The test is invalid if the standard
endotoxin or positive product control doesn't show end point within ± 1.

Two fold dilution from label claim sensitivity of LAL reagent or if the negative
control shows gel-clot end point.

The following methods can be used to monitor the endotoxin concentration:

Method A - Gel- clot limit test method

Method B -Semi quantitative gel clot method

Method C - Kinetic turbidimetric method

Method D - Kinetic chromogenic method

Method E - End point chromogenic method

5) Pyrogen test

It is performed by using rabbits as test animals. Initially 10 ml/kg body weight

of an animal is injected through rat vein at 37±2ºC within ten minutes from
start of administration. The temperatures are recorded at 1, 2 and 3 hours
after injection.

6) Clarity of Solution
 Clarity is performed to ensure that parenteral product is free from foreign
particles Constitute the injection as directed on the label.

a) The solid dissolves completely, leaving no visible residue as undissolved

matter.

b) The constituted injection is not significantly less clear than an equal volume
of diluents for water for injections contained in a similar container and
examined in the same manner.

7) Leak Test

Performed only for ampoules which have been sealed by fusion to ensure that
their should not be any leakage in them.

a) Vacuum Chamber Test

b) Dye Bath Test

A) Dye Bath Test :

The test container is immersed in a dye bath. Vacuum and pressure is applied
for some time. The container is removed from the dye bath and washed. The
container is then inspected for the presence of dye either visually or by means
of UV spectroscopy. The dye used may be of blue, green, yellowish-green
color. The dye test can be optimized by use of a surfactant and or a low
viscosity fluid in the dye solution to increase the capillary migration through
the pores. The dye test is widely accepted in industry and is approved in drug
use. The test is inexpensive and is requires no special equipment required for
visual dye detection. However, the test is qualitative, destructive and slow. The
test is used for ampoules and vials.

8) Sterility test
Growth promotion medium and incubation conditions are selected based on
the test microorganism. The sterility test is done using direct transfer and
membrane filtration techniques. Membrane filtration technique is suitable for
liquids, soluble powders with bacteriostatic or fungi static properties, oils,
creams and ointments. Sterility test by direct transfer is performed by aseptic
transfer of specified volume from test container to culture medium and
incubated for 14 days and visual observation of medium is done on 3rd, 4th,
5th, 7th, 8th and 14th day. A membrane filter with porosity of 0.45μm with
diameter of 47mm with flow rate of 55-75 ml of water per minute at a
pressure of 70 cm of mercury should be used. The test meets the required
second stage and generally second stage is repeated with double the number
of specimens tested in first stage when the test was found to be conducted
under faulty or inadequate aseptic techniques.

 Membrane Filtration Method

Use membrane filters having a nominal pore size not greater than 0.4μm
whose effectiveness to retain microorganisms has been established.

Cellulose nitrate filters, for example, are used for aqueous, oily, and weakly
alcoholic solutions; and cellulose acetate filters, for example, are used for
strongly alcoholic solutions. Specially adapted filters may be needed for certain
products (e.g., for antibiotics).

The technique described below assumes that membranes about 50 mm in

diameter will be used. If filters of a different diameter are used, the volumes of
the dilutions and the washings should be adjusted accordingly. The filtration
apparatus and membrane are sterilized by appropriate means. The apparatus
is designed so that the solution to be examined can be introduced and filtered
under aseptic conditions: it permits the aseptic removal of the membrane for
transfer to the medium, or it is suitable for carrying out the incubation after
adding the medium to the apparatus itself.
 After filtration the preparation membrane is cut into two halves. One halve is
transferred in to 100ml of culture medium meant for the growth of the
bacteria and incubated at 30 to 35°C for not less than 7 days.

The another halve is transferred to 100 ml of culture medium meant for fungi
and incubated at 20 - 25 o C for not less than 7 days.

Direct Inoculation
Method

Although international pharmacopoeias recommend using standard

membrane filtration for sterility testing, there are certain products that are not
filterable or deformable. These products are normally tested using direct
inoculation. In this method, the test sample is added directly into the required
media, ensuring that the amount of sample is below 10%. To comply with your
different direct inoculation method requirements, we offer sterility test media
in various volumes, from 9mL tubes up to 75 mL bottles. In this method an
aliquot quantity of the material being tested is drawn aseptically from the
container and transferred to a vessel containing a measured quantity of a
suitable culture medium. The culture is incubated at appropriate temperature
for not less than 14 days. The culture medium is observed at periodic intervals
during the incubation period and at the end to detect presence of any
microbial growth.
Rabbit pyrogen test:

The pyrogen test is designed to limit to an acceptable level the risks of febrile
reaction in the patient to the administration, by injection, of the product
concerned. The test involves measuring the rise in temperature of rabbits
following the intravenous injection of a test solution and is designed for products
that can be tolerated by the test rabbit in a dose not to exceed 10 mL per kg
injected intravenously within a period of not more than 10 minutes. For products
that require preliminary preparation or are subject to special conditions of
administration, follow the additional directions given in the individual monograph
or, in the case of antibiotics or biologics, the additional directions given in the
federal regulations.

APPARATUS AND DILUENTS

Render the syringes, needles, and glassware free from pyrogens by heating at 250
 for not less than 30 minutes or by any other suitable method. Treat all diluents
and solutions for washing and rinsing of devices or parenteral injection assemblies
in a manner that will assure that they are sterile and pyrogen-free. Periodically
perform control pyrogen tests on representative portions of the diluents and
solutions for washing or rinsing of the apparatus. Where Sodium Chloride
Injection is specified as a diluent, use Injection containing 0.9 percent of NaCl.

TEMPERATURE RECORDING
Use an accurate temperature-sensing device such as a clinical thermometer, or
thermistor probes or similar probes that have been calibrated to assure an
accuracy of ±0.1  and have been tested to determine that a maximum reading is
reached in less than 5 minutes. Insert the temperature-sensing probe into the
rectum of the test rabbit to a depth of not less than 7.5 cm, and, after a period of
time not less than that previously determined as sufficient, record the rabbit's
body temperature.

TEST ANIMALS
Use healthy, mature rabbits. House the rabbits individually in an area of uniform
temperature between 20  and 23  and free from disturbances likely to excite
them. The temperature varies not more than ±3  from the selected temperature.
Before using a rabbit for the first time in a pyrogen test, condition it not more
than seven days before use by a sham test that includes all of the steps as
directed for Procedure except injection. Do not use a rabbit for pyrogen testing
more frequently than once every 48 hours, nor prior to 2 weeks following a
maximum rise of its temperature of 0.6  or more while being subjected to the
pyrogen test, or following its having been given a test specimen that was
adjudged pyrogenic.

PROCEDURE
Perform the test in a separate area designated solely for pyrogen testing and
under environmental conditions similar to those under which the animals are
housed and free from disturbances likely to excite them. Withhold all food from
the rabbits used during the period of the test. Access to water is allowed at all
times, but may be restricted during the test. If rectal temperature-measuring
probes remain inserted throughout the testing period, restrain the rabbits with
light-fitting neck stocks that allow the rabbits to assume a natural resting posture.
Not more than 30 minutes prior to the injection of the test dose, determine the
“control temperature” of each rabbit: this is the base for the determination of any
temperature increase resulting from the injection of a test solution. In any one
group of test rabbits, use only those rabbits whose control temperatures do not
vary by more than 1  from each other, and do not use any rabbit having a
temperature exceeding 39.8 .
Unless otherwise specified in the individual monograph, inject into an ear vein of
each of three rabbits 10 mL of the test solution per kg of body weight, completing
each injection within 10 minutes after start of administration. The test solution
is either the product, constituted if necessary as directed in the labeling, or the
material under test treated as directed in the individual monograph and injected
in the dose specified therein. For pyrogen testing of devices or injection
assemblies, use washings or rinsings of the surfaces that come in contact with the
parenterally administered material or with the injection site or internal tissues of
the patient. Assure that all test solutions are protected from contamination.
Perform the injection after warming the test solution to a temperature of 37 ± 2 .
Record the temperature at 30-minute intervals between 1 and 3 hours
subsequent to the injection.

TEST INTERPRETATION AND CONTINUATION

Consider any temperature decreases as zero rise. If no rabbit shows an individual
rise in temperature of 0.5  or more above its respective control temperature, the
product meets the requirements for the absence of pyrogens. If any rabbit shows
an individual temperature rise of 0.5  or more, continue the test using five other
rabbits. If not more than three of the eight rabbits show individual rises in
temperature of 0.5  or more and if the sum of the eight individual maximum
temperature rises does not exceed 3.3 , the material under examination meets
the requirements for the absence of pyrogens.

RADIOACTIVE PHARMACEUTICALS
Test Dose for Preformulated, Ready-to-Use Products Labeled with Radioactivity

AGGREGATED ALBUMIN AND OTHER PARTICLE-CONTAINING PRODUCTS

For the rabbit pyrogen test, dilute the product with Sodium Chloride Injection to
not less than 100 µCi per mL, and inject a dose of 3 mL per kg of body weight into
each rabbit.
OTHER PRODUCTS:

Where Physical Half-life of Radionuclide Is Greater Than 1 Day— Calculate the

maximum volume of the product that might be injected into a human subject.
This calculation takes into account the maximum recommended radioactive dose
of the product, in µCi, and the radioactive assay, in µCi per mL, of the product at
its expiration date or time. Using this information, calculate the maximum volume
dose per kg to a 70-kg human subject.
For the rabbit pyrogen test, inject a minimum of 10 times this dose per kg of body
weight into each rabbit. If necessary, dilute with Sodium Chloride Injection. The
total injected volume per rabbit is not less than 1 mL and not more than 10 mL of
solution.
Where Physical Half-life of Radionuclide is Less Than 1 Day— For products labeled
with radionuclides having a half-life of less than 1 day, the dosage calculations are
identical to those described in the first paragraph under Other Products. These
products may be released for distribution prior to completion of the rabbit
pyrogen test, but such test shall be initiated at not more than 36 hours after
release.
TEST DOSE FOR PHARMACEUTICAL CONSTITUENTS OR REAGENTS TO BE
LABELED:
The following test dose requirements pertain to reagents that are to be labeled or
constituted prior to use by the direct addition of radioactive solutions such as
Sodium Pertechnetate Tc 99m Injection, i.e., “cold kits”.
Assume that the entire contents of the vial of nonradioactive reagent will be
injected into a 70-kg human subject, or that 1/70 of the total contents per kg will
be injected. If the contents are dry, constitute with a measured volume of Sodium
Chloride Injection.
For the rabbit pyrogen test, inject (1/7) of the vial contents per kg of body weight
into each rabbit. The maximum dose per rabbit is the entire contents of a single
vial. The total injected volume per rabbit is not less than 1 mL and not more than
10 mL of solution.