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Wednesday, December 21, 2011

In-process and finished product QC tests for parentera-Free pharmacy books from

In-process and finished product QC tests for parenterals:-

Parenterals are defined as sterile dosage forms which are administered to the patient by a route other than oral.In other words,these dosage forms bypass GIT(gastrointestinal tract) and liver and enter directly into systemic circulation. They exhibit maximum bioavailability(100%).

Parenterals are classified into four types:

Based on route of administration Based on the volume Based on the formulation Based on the type of product BASED ON THE ROUTE OF ADMINISTRATION:

Intra venous Intra muscular Intra dermal Intra thecal Intra arterial Intra peritoneal Intra medullary BASED ON THE VOLUME:

Large volume Small volume BASED ON THE FORMULATION

Solutions Suspension Emulsions Dry powders which form solutions Dry powders which form suspensions Concentrated liquids which are diluted before injection. PURPOSE BASED

Injections Infusions Irrigations EVALUATION OF PARENTRALS:

In process quality control tests

Environmental control Finished product quality control tests

Leakage test (or) Sealing verification Sterility test Pyrogen test Uniformity of weight Uniformity of content Extractable volume Safety test Clarity test/ Particulate matter test. ENVIRONMENTAL CONTROL:

Environmental control is always needed during the preparation of Parenterals so it is necessary to design and implement in-process control and monitoring.

In-process monitoring and control may includes

Environmental Particulate


Filter Integrity Testing


Environmental Particulate monitoring should be carried out using appropriate air Particle Counting devices to check that the general environmental and work station air remain in conformity with specification.

This method is used to identify large pores, other openings in the membrane filter


As appropriate to the type of manufacturing process, consideration needs to be given to the following Microbiological Monitoring and Control.

Exposure of "Settle Plates" (Petri dishes of nutrient agar) at critical positions within the general Clean Room environment and at the controlled work station(s).

Use of Air Sampling devices to determine the number of viable organisms per cubic metre (or cubic foot) of air in the room, and within the work station(s).

Use of Contact Plates, or Swabs, to check the microbiological quality of surfaces.



As the containers are exposed to various environmental conditions, uneven expansion occurs and as a result the cracks occurs and it leads to the entry of microbes or particulate matter .

This test is done mainly for the ampoules which have been sealed by fusion method to ensure that no leakage is present in them.

There are two methods for the identification of leaks in parentrals they are

1)methylene blue dye test.

2)spark test.



Leakage test is done in a vacuum chamber.

The ampoules are dipped in 1% solution of methylene blue in vacuum chamber and vacuum is applied.

When this vacuum is released, the coloured solution will enter the ampoules with defective sealing and the Vaccum is applied for another 15-30min.

Result : The presence of dye in the ampoule, confirms the leakage and hence rejected.

NOTE: This test is not suitable for the vials and bottles because the methylene blue solution may enter through the caps or may diffuse through the rubber closure.


Electrical probe is introduced into the vial or the bottle and when the blue light appears the vial or bottle is said to have a hole.


The test is designed to identify the presence of viable forms of bacteria, fungi and yeasts in substances, preparations or articles which are required to be sterile. Principle:

when the suitable growth medium is provided and kept at favourable temperature condition, the organism grows and it is indicated by a turbidity in clear medium.

The culture media used are:

Fluid thioglycolate medium Soyabean- casein digest medium Special media for penicillin's and cephalosporin's Any other medium as directed by the manufacture. Fluid thioglycolate medium: It is primarly intended for the culture of anaerobic bacteria.

Special media for penicillin's and cephalosporin's:

The commonly used soya bean-casein digest medium and fluid thioglycollate is taken and the required amount of penicillinase required to inactivate the penicillin is calculated and added to the sample that is being tested.

The inactivation can be checked out by using Staphylococcus aureus as the challenge.

Typical microbial growth is to be observed as a confirmation that the penicillinase concentration is appropriate.

Steps involved in sterility testing

Selection of the sample size.

Selection of the quantity of product to be used.

Method of testing.

Observation and results.

Selection of the sample size:

Number of items in the batch

Min.No.of items to be tested

NMT 100 containers

10% or 4 containers which ever is greater

NMT 100 but NMT 500 containers

10 containers

NMT 500 containers

2% or 20 containers which ever is less

2. Selection of the quantity of product to be used.

Quantity of each container of injectable preparation

Minimum quantity to be used for each culture medium

Less than 1ml

Total contents of a container

1ml/ more but less than 40ml

Half the contents of a container

40ml/ more but less than 100ml


100ml or more

10% of the contents of a container but not less than 20ml

Antibiotic liquid


Other preparations soluble in water(or) in isopropyl myristate

The whole contents of each container to provide not less than 200mg.

Insoluble preparations ie creams and ointments to be suspended / emulsified

The whole contents of each container to provide not less than 200mg.

The two methods for the sterility testing are:

Membrane filteration

Direct inoculation


The method is preferred in the following cases

An oil or oily preparation Liquid product where the volume in a container is 100ml or more. Method:

Filtration of the sample through a membrane filter having the nominal size of 0.45ยต and a diameter of 47mm.

After filteration the membrane is removed aseptically from the metallic holder and divided into two halves.

The first half is transferred into 100 ml of culture media meant for fungi and incubated at 20˚ to 25 ˚c for not less than seven days.

The other half is transferred into 100ml of fluid thioglycolate medium and incubated at 30 to 35 ˚c for not less than 7 days.

Observe the growth in the media.

Fluid –A: it has 1g peptic digest dissolved in 1 liter of water

Fluid – B: it has 1ml of polysorbate 80 added to the fluid A

The various types of filters used are :

Cellulose nitrate filters for aqueous , oily solutions , weakly alcoholic

Cellulose acetate filters are mainly used for strongly alcoholic


The medium is prepared and then it is sterilized.

Then the sample to be tested is poured into the medium plates as per the prescribed volumes.

Then it is incubated and the growth of the microbes is examined for not less than 14 days.

The visual testing is done by comparing the test results with the positive and negative controls.


At the end of the incubation period the following observations are possible:

No evidence of growth; hence the preparation being examined passes the test for sterility. If there is evidence of growth, retesting is performed 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 test for sterility. If There is again evidence of the microbial growth then isolate and identify the orgainism. If they are not readily distinguishable from those growing in the containers of the first test then the preparation being examined fails the test for sterility. If they are distinguishable from the organisms of the first test then again do the test using twice the number of samples. The prepration being examined passes the test for sterility in case there is no evidence of microbial growth. In case there is evidence of growth of any micro organisms in second re –test, the preparation being examined fails the tests for sterility. PYROGEN TEST:

Pyrogens are fever producing metabolic products of micro organisms.

The presence of pyrogens in parenteral preperations is evaluated by a qualitative fever response test in rabbits.

Test for pyrogens can be carried out by in-vitro and in-vivo methods.

A) Rabbit test (in-vivo)

B) LAL test (Limulus amoebocyte lysate) (in-vitro)



The test involves measurement of rise in body temperature of the rabbits following the intravenous injection of a sterile solution of the substance to be tested.

The body temperature of the rabbits increases if pyrogens are present in the injected test solution.


Healthy , adult rabbits of either sex, each weighing not less than 1.5kg.

Do not use any rabbit for main test if:

1) It shows a temperature variation greater than 0.20c between two successive readings noted during the determination of initial temperature .

2) And it’s temperature is higher than 39.80c or lower than 380 c.


All glass ware, syringes, needles and thermometer must be thoroughly washed with water for injection and heated in a hot air oven at 2500c for 30 minutes or at 2000 c for 1 hr.

Retaining boxes for rabbits.


The test is carried out on a group of three rabbits.


1.Preparation of the sample:

Dissolve the test substance with a pyrogen-free saline solution. Warm the solution to about 38.50c before the injection.

2)Determination of initial temperature of rabbits:

Insert a clinical thermometer into the rectum of each rabbit and normal readings of body temperature are taken prior to the injection of test solution.

Two such readings are taken at an interval of 30 minutes and the mean is calculated.

This mean reading is taken as the initial temperature of the rabbits.

3)Determination of the response of rabbits:

The test solution is injected into the ear vein of each rabbit.

The volume of injection is not less than 0.5 ml/kg of the body weight.

This volume varies according to the test substance and is prescribed in the individual monograph.

Record the temperature of each rabbit at an interval of 30 minutes for 3 hours after the injection.

This is the maximum temperature recorded for that rabbit.

The difference between maximum temperature and initial temperature is taken as its response.

If this difference is negative, it is taken as a zero response.

Interpretation of results:

If the response of any individual rabbit is less than 0.60c and if the some of the responses of the 3 rabbits is more than 1.40 c, the preparation being examined passes the test.

If the response of any rabbit is 0.60c or more or if the sum of the responses of the 3 rabbits is more than 1.40 c, then the same test is repeated on another 5 rabbits.

If not more than 3 of the 8 rabbits show individual responses of 0.60c or more and if the some of the responses of the 8 rabbits is not more than 3.70c,the preparation being examined passes the test.


It is used to identify the presence of a wide range of pyrogens.


It is a time - consuming method.


This is a sensitive test used to detect endotoxins from gram-negative bacteria .therefore it is also called as bacterial endotoxin test.

The endotoxins upon reaction with lysate form an insoluble gel clot.

The lysate is obtained by the lysis of amoebocyte (blood cells) of the horseshoe crab,LIMULUS POLYPHEMUS.

The sensitivity of lysate is expressed in terms of endotoxin units(EU).


The test is based on the formation of a gel in the presence of bacterial endotoxins and the lysate solution .

The lysate consists of a proclotting enzyme and coagulogen which are required for the reaction to occur.


1) The gel clot test

2) The turbidimetric test

3) The kinetic chromogenic test

1) The Gel clot test:

It is based on the formation of a solid gel clot.


The lysate solution is mixed with an equal volume of the test solution in a pyrogen-free test tube.

The test tube is allowed to stand for about 60 minutes.

Now, the tube is inverted and observed for the formation of gel clot.

The formation of solid gel confirms the presence of endotoxin.

If the solid gel is not formed,it indicates the absence of endotoxins and the test solution passes the test.

2) The turbidimetric test:

This test is based on the measurement of opacity change in the LAL test due to the formation of gel clot.

Opacity is directly proportional to the endotoxin concentration.

This test is used for water systems and simple pharmaceutical products.

3)The kinetic chromogenic test:

The test is based on the measurement of colour change which is caused by the release of chromogenic chemical, para-nitroanilide.

This p-nitroanilide is a by product of the clotting reaction during the LAL test.

The quantity of para-nitroanilide produced is directly proportional to the endotoxin concentration.

Advantages of LAL test over rabbit test:

It is easy to perform

It is a rapid method

It is an economical method

It is more sensitive test than rabbit test. Even the minute amounts of endotoxin can be detected by LAL test.

Disadvantage of LAL test:

It is used to identify the presence of endotoxins only.


Unless otherwise stated in the individual monograph, suspensions for injection that are presented in single dose containers and that contain less than 10mg or less than 10 per cent of active ingredient comply with the following test.

For suspensions for injection containing more than one active ingredient carry out the test for each active ingredient that corresponds to above conditions.

The test for uniformity of contents should be carried out only after the content of active ingredient in a pooled sample of the preparation has been shown to be within accepted limits of the stated content.

Determine the content of active ingredient of each of 10 containers taken at random, using the suitable analytical method of equivalent accuracy and precision.

The preparation under examination complies with the test if the individual values thus obtained are all between 85 and 115 % of the average value.

The preparation under examination fails to comply with the test if more than one individual value is outside the limits 85 to 115% of the average value or if any one individual value is outside the limits 75 to 125% of the average value.

If one individual value is outside the limits 85 to 115% but with in the limits 75 to 125% of 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% and none is out side the limits 75 to 125% of the average value.


For powders for injection that are required to comply with the test for uniformity of content of all active ingredients ,the test for uniformity of weight is not required.

Remove any adherent labels from a container and immediately weigh the container and its contents.

Empty the container as completely as possible by gentle tapping, rinse if necessary with water and then with Ethanol (95%) and dry at 1000 c to 1050 c for 1hr.

If the nature of the container precludes such treatment, dry at a lower temperature to constant weight.

Allow to cool in a desiccator and weigh.

The difference between the weights represents the weight of the contents.

Repeat the procedure with a further 19 containers and determine the average weight.

Not more than two of the individual weights deviate from the average weight by more than 10% and none deviates by more than 20%.


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