INTRODUCTION

Parenteral preparation are the sterile solution or suspension of drug in aqueous or oily vehicle meant for introduction into the body by means of an injectable needle under or through one or more layers of skin or mucous membrane.

Injection should be sterile, isotonic and free from foreign particles, such as dust, fibers etc.They should be introduce through the same route for which they are intended.

QUALITY CONTROL OF PARENTERAL PREPARATION

The finished parenteral products are subjected to the following tests, in order to maintain quality control,

1. Sterility test2. Pyrogen test

3. Clarity test

4. Leakage test

5. Assay

STERILITY TEST

The test for sterility are intended for detecting the presence of viable forms of micro-organisms in or on pharmacopoeial preparations. The test must be carried out under conditions designed to avoid accidental contamination of the product during the test. Precautions taken for this purpose should not adversely affect any micro-organisms which should be revealed in the test.

The working conditions in which the tests are performed should be monitored regularly by sampling the air and surfaces of the working area and by carrying out control tests. The tests are based upon the principle that if micro-organisms are placed in a medium which provides nutritive material and water, and kept at a favourable temperature, the organisms will grow and their presence can be indicated by a turbidity in the originally clear medium.

The probability of detecting viable micro-organisms the tests for sterility increases with the number present in a given amount of the preparation being examined and varies according to the species of micro – organisms present. Very low levels of contamination cannot be detected on the basis of random sampling of a batch. (A batch may be defined for the purposes of these tests as a homogeneous collection of sealed containers prepared in such a manner that the

risk of contamination is the same for each of the units in it). Moreover, if contamination is not uniform throughout the batch, random sampling cannot detect contamination with certainty. Compliance with the test for sterility alone cannot therefore constitute absolute assurance of freedom from microbial contamination. Greater assurance of sterility must come from reliable manufacturing practices.

 The tests for sterility are designed to reveal the presence of micro-organisms in the samples used in the tests; interpretation of results is based on the assumption that the contents of every container in the batch, had they been tested, would also have complied with the tests. Since every container cannot be tested, a sufficient number of containers should be examined to give a suitable degree of confidence in the results of the tests.

No sampling plan for applying the tests to a specified proportion of discrete units selected from a batch is capable of demonstrating that all of the untested units are in fact sterile. Therefore, in determining the number of units to be tested, the manufacturer should have regard to the environmental conditions of manufacture, the volume of preparation per container and other special considerations particular to the preparation being examined. Table 1 gives guidance on the minimum number of items recommended to be tested in relation to the number of items in the batch on the assumption that the preparation has been manufactured under conditions designed to exclude contamination.

CULTURE MEDIA

Fluid thioglycollate media

For use with clear fluid products. Mix the ingredients other than the thioglycollate and the resazurin, in the order given above in a mortar, with through grinding. Stir in some heated distilled water, transfer to a suitable container, add the remainder of the water and complete the solution by heating in a boiling water-bath. Add the Sodium thioglycollate, then 1M Sodium hydroxide, if necessary, so that (after sterilisation) the medium will have a pH of 7.1 + 0.2. Reheat the solution, but do not boil, filter (if necessary) through a moistened filter paper and add the resazurin solution.

Distribute into suitable vessel which provide a ration of surface to depth of medium such that not more than the upper half of the medium has undergone a colour change, indicative of oxygen uptake at the end of the incubating period. Sterilise in an autoclave at 121º for 20 minutes. Cool promptly to 25º and store at 20º to 30º, avoiding excess of light.Medium more than 3 weeks old should not be used. Use fluid thioglycollate medium by incubating it at 30º to 35º under aerobic conditions.

Heat the ingredients in a suitable container until solution is effected. Mix and adjust the reaction with 1M Sodium hydroxide, if necessary, so that the medium will have a pH of 7.1 + 0.2 after sterilisation. Filter if necessary, place in suitable vessels and sterilise in an autoclave at 121º for 20 minutes. The medium is freshly prepared or heated in a steam-bath and allowed to cool just prior to use. Do not reheat.

Soyabean- casein digest medium

Dissolve the solids in distilled water, warming slightly to effect solution. Cool to room temperature and add, if necessary, sufficient 0.1M sodium hydroxide to give a final pH of 7.1 + 0.2 after sterilisation. Filter, if necessary, distribute into suitable containers and sterilise in an autoclave at 121º for 20 minutes.

NOTE – Where fluid thioglycollate medium and soyabean- casein digest medium are to be used in Method B for samples of the penicillin or cephalosporin class of antibiotic, eseptically transfer to each tube of medium a quantity of penicillinase sufficient to inactivate the amount of antibiotic in the sample under test. Determine the appropriate quantity of penicillinase to be used for this purpose by using a penicillinase preparation that has been assayed previously for its penicillin or cephalosporin-inactivating power. Alternatively, confirm that the appropriate quantity of penicillinase has been transferred to a tube of fluid thioglycollate medium by adding to it an amount of penicillin or cephalosporin antibiotic equivalent to the amount of antibiotic in the sample being examined, inoculating the medium with 1 ml of a 1:1000 dilution of an 18- to 24 hour culture of Staphylococcus aureus (ATCC 29737) in fluid thioglycollate mediaum and incubating it for 24 hour at 30º to 35º at this time typical microbial growth must be observed. Perform this confirmatory test in an area completely separate from that used for sterility testing.

Precautions : The tests for sterility should be carried out under conditions designed to avoid accidental contamination of the product during the test using, for example, a laminar sterile airflow cabinet. The precautions taken to avoid contamination must be such that they do not affect any micro-organisms that should be revealed in the test.

The working conditions in which the test is performed should be monitored

regularly by sampling the air and surfaces of the working area and by carrying

out control tests.

Observation and Interpretation of Results

At intervals during the incubation period, and at its conclusion, examine the media for macroscopic evidence of microbial growth. If no evidence of growth is found, the preparation being examined passes the test for sterility. If evidence of

microbial growth is found, reserve the containers showing this and, unless it is demonstrated by any other means that their presence is due to causes unrelated to the preparation being examined and hence that the tests for sterility are invalid and may therefore be recommenced, perform a retest using the same number of samples, volumes to be tested and the media as in the original test. If no evidence of microbial growth is then found, the preparation being examined passes the tests for sterility. If evidence of microbial growth is found, isolate and identify the organisms. If they are readily distinguishable from those growing in the containers reserved in the first test, perform a second retest using twice the number of samples. If no evidence of microbial growth is found in the second retest, the preparation being examined passes the tests for sterility. If evidence of growth of any micro-organisms is found in the second retest, the preparation being examined fails the tests for sterility

PYROGEN TEST The test involves measurement of the rise in body temperature of rabbits following the intravenous injection of a sterile solution of the substance being examined. It is designed for products that can be tolerated by the test rabbit in a dose not exceeding 10ml per kg injected intravenously within a period of not more than 10 minutes. Test Animals Use healthy, adult rabbits of either sex, preferably of the same variety, weighing not less than 1.5 kg, fed on a complete and balanced diet and not showing loss of body weight during the week preceding the test. House the animals individually in an area of uniform temperature (± 2° ), preferably with uniform humidity, and free from disturbances likely to excite them. Do not use animals for pyrogen tests more frequently than once every 48 hours. After a pyrogen test in the course of which a rabbits temperature has risen by 0.6° or more, or after a rabbit has been given a test substance that was adjudged pyrogenic, at least 2 weeks must be allowed to elapse the animals is used again.

Materials All glassware, syringes and needles must be thoroughly washed with water for injection and heated in a hot air oven at 250° for 30 minutes or at 200° for 1 hour. Treat all diluents and solutions for washing and rinsing of devices in a manner that will assure that will assure that they are sterile and pyrogen-free. Recording of Temperature Use an accurate temperature-sensing device such as a clinical thermometer or thermistor or other suitable 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 thermometer or temperature sensing probe into the rectum of the test rabbit to a depth of about 5-cm. The depth of insertion is constant for any one rabbit in any one test. If an electrical device is used, it should be inserted in the rectum of the rabbit 90 minutes before the

injection of the solution being examined and left in position throughout the test. After a period of time not less than that previously determined as sufficient, record the rabbit's body temperature.

Preliminary Test (Sham Test) If animals are used for the first time in a pyrogen test or have not been used during the 2 previous weeks, condition them 1 to 3 days before testing the substance being examined by injecting intravenously into them 10 ml per kg of body weight of a pyrogen-free saline solution warned to about 38.5°. Record the temperatures of the animals, beginning at least 90 minutes before injection and continuing for 3 hours after injection of the solution being examined. Any animal showing a temperature variation of 0.6° or more must not be used in the main test.

Main Test Carry out the test using a group of three rabbits.

Preparation of the sample: Dissolve the substance being examined in, or dilute with, pyrogen-free saline solution or other solution prescribed in the monograph. Warm the liquid being examined to approximately 38.5° before injection.

Procedure: Record the temperature of each animal at intervals of not more than 30 minutes, beginning at least 90 minutes before the injection of the solution being examined and continuing for 3 hours after the injection. Not more than 40 minutes immediately preceding the injection of the test dose, record the "initial temperature " of each rabbit, which is the mean of two temperatures recorded for that rabbit at an interval of 30 minutes in the 40 minute period. Rabbits showing a temperature variation greater than 0.2° between two successive readings in the determination of "initial temperature " should not be used for the test. In any one group of test animals, use only those animals whose " initial temperatures" do not vary by more than 10 from each other ,and do not use any rabbit having a temperature higher than 39.8° and lower than 38° Inject the solution being examined slowly into the marginal vein of the ear of each rabbit over a period not exceeding 4 minutes, unless otherwise prescribed in the monograph. The amount of sample to be injected varies according to the preparation being examined and is prescribed in the individual monograph. The volume of injection is not less than 0.5 ml per kg and not more than 10 ml per kg of body weight. Records the temperature of each animal at half-hourly intervals for 3 hours after the injection. The difference between the "initial temperature " and the "maximum temperature" which is the highest temperature recorded for a rabbit is taken to be its response. When this difference is negative, the results is counted as a zero response.Interpretation of results: if the sum of the responses of the group of three rabbits is less than 0.6° , the preparation being examined passes the test. If the response of any rabbit is 0.6° or more, or if the sum of the response of the

three rabbits exceeds 1.4° , continue the test using five other rabbits. If not more than three of the eight rabbits show individual responses of 0.6° or more, and if the sum of responses of the group of eight rabbits does not exceed 3.7° , the preparation being examined passes the test.

CLARITY TEST

Injectable solutions including solutions constituted from sterile solids must essentially be free from particles of approximately 50 µm or more that can be observed by inspection with the unaided eye. Injectable solutions in containers that are labelled as containing 100 ml or more of a single dose large volume injection intended for administration by intravenous infusion comply with limits of particulate matter prescribed in this test.

For the purpose of this test particulate matter is defined as extraneous mobile, undissolved substances, other than gas bubbles, unintentionally present in injections. The test need not be done where particles in an injectable solution can be observed on visual inspection. The limits do not apply to multiple dose injections, to single dose small volume parenterals and to injectable solutions constituted from sterile solids.

The results obtained in the following test by examining a discrete unit or group of units for particulate matter cannot be extrapolated with certainty in other units that have not been sampled or tested from a lot. It is essential to have statistically sound sampling plans based upon a known set of given operational factors if valid inferences are to be drawn from observed data in order to determine the level of particulate matter in a large group of units (such as a production batch).

This limit test is suitable for revealing the presence of particles whose longest axis, or effective linear dimension, is 10 µm or more. Alternative methods such as the automatic measurement of particle size operating on the electrical zone sensing principle or on the basis of light blockage may be employed provided the results obtained are of equivalent reliability. However, where a difference appears, or in the event of dispute, only the result obtained by the procedure given in this Pharmacopoeia is conclusive.

Precautions during testing:

It is important to avoid the introduction of extraneous particulate contamination into the preparations being examined during the testing procedure. All the manipulative procedures should be carried out in a laminar air-flow cabinet or hood (of the horizontal type), equipped with HEPA filters and preferably located in a separate room

Method:

Using flat-ended forceps, carefully remove a colour contrast grid membrane filter (pore size less than or equal to 5 µm and of diameter 25 mm) from its container. Wash both sides of the membrane with a stream of water that has been further purified by filtration through a suitable membrane to remove particulate matter; start washing at the top of the non-gridded (unprinted) side, sweeping the stream back and forth across the surface, working slowly from top to bottom so as to wash any loose particles from the surface; repeat the process on the gridded side. Place the membrane (grid side up) on the filter holder base and install the filtering funnel on the base without sliding the funnel over the membrane filter. Invert the assembled unit and wash the inside of the funnel for about 10 seconds with a jet of filtered water. Allow the water to drain and place the unit on the filter flask.

Invert the container of the preparation being examined 20 times in order to mix the contents. Wash the outer surface of the container with a jet of water and remove the closure carefully, avoiding contamination of the contents. Transfer 25 ml of the solution to the funnel, allow to stand for 1 minute, and apply the vacuum and filter. Release the vacuum gently and wash the inner walls of the funnel with a jet of 25 ml of the filtered water. Direct the jet of water in such a manner as to wash the walls of the funnel free from any particles that may have become lodged on the walls but avoid directing the stream onto the filter surface. Filter under suction the rinsing. Carefully remove the upper section of the filter assemble while maintaining vacuum. Release the vacuum and remove the membrane filter with flat-ended forceps. Place the filter in a plastic Petri slide to which a very thin film of silicone grease may be applied to hold the filter flat and in place. Ensure that the filter is placed with gridded surface up. Partially cover the Petri slide with the lid and allow the filter to dry. Cover the slide carefully on the micrometer stage of the microscope and count the particles on the filter as described below.

Calibrate the eyepiece graticule of a suitable microscope under 100x magnification (or any other suitable magnification) using a standard stage micrometer grid. Examine the entire membrane filter under 100x magnification with the incident light at an angle of 10° to 20° with the horizontal. Count the number of particles having effective linear dimensions equal to or larger than 10 µm, equal to or larger than 25 µm and equal to or larger than 50 µm .

Perform a blank determination, using a membrane filter and assembly as directed above and beginning words ‘wash the linear walls of the funnel with a jet of 25 ml….’. Subtract the total counts obtained in the blank determination from the uncorrected total counts for the test solution.

If the blank determination yields more than 5 particles having effective linear dimension of 25µm or larger, the operational environment should be considered unsatisfactory and the test is invalid.

The preparation meets the requirements of the test if it contains particles within the maximum limits shown in Table 1.

TABLE 1 – Permitted limits of particulate matter

Particle size in µm

(Equal to or larger than)

Maximum number of

particles per ml

10 50

25 5

50 Nil

LEAKAGE TEST

DefinitionLeakage occurs when a discontinuity exists in the wall of a package that

can allow the passage of gas under the action of a pressure or concentration differential existing across the wall. Leakage is mathematically defined as the rate at which a unit of gas mass (or volume) .goes into or out of the leak underspecific conditions of temperature and pressure. A method of testing the integrity of ampules containing a liquid therein for leakage comprising the steps of: (a) Immersing the ampules in an aqueous solution of a water soluble salt of a dye, (b) Applying negative pressure or a vacuum to the top of the solution containing the dye to generate a pressure differential between said solution and the liquid within the ampules at least once, and releasing said pressure or vacuum, (c) Removing the ampules from the solution or draining the solution from the container, (d) Decontaminating the ampules by removing any solution containing dye adhering to the external surface thereof.(e) Color testing the ampules to detect faulty ampules into which dye has leaked, (f) Rejecting said faulty ampules, if any, and subsequently.

ASSAY Assay is performed according to the method given in the monograph of

that parenteral preparation in the pharmacopoeia. Assay is done to check the quantity of medicament present in the parenteral preparation

REFERENCE:

1. Leon Lachman, H. A. Lieberman, The theory and practical of industrial pharmacy, second edition 1991, Varghese publication house, page no- 673 to 684.

2. R. M. Mehta, text book of pharmaceutics –II, second edition 2007, vallabh prakashan, page no- 231 to 246.