1. PARENTERALS QUALITY CONTROL TESTS
DEPARTMENT OF PHARMACEUTICS
CHALAPATHI INSTITUTE OF PHARMACEUTICAL SCIENCES

2. Introduction The term parenteral is derived from The Greek word
Para (outside)
Enteron(intestine)
It denotes the route of administration other than oral route.
Because this route of administration bypasses the normal body defense mechanisms it is essential that these products are prepared with a higher degree of care and skills than utilized in preparing conventional oral or topical products.
The finished product must be sterile, non-pyrogenic and free from extraneous insoluble materials.

3. Quality Control tests for Parenterals
Quality control is a concept which strives to produce a perfect product by a series of measures designed to prevent and eliminate errors at different stages of production.
There are mainly four Quality control tests performed. They are
leakage test
Pyrogen test
Sterility test
Particulate evaluation (clarity test)

4. Leakage test
This test is performed only for ampoules which have been sealed by fusion to ensure that there should not be any leakage in them.
Vials & bottles not subjected to this test because of flexibility of rubber.
There are two methods for the identification of leaks in parenterals they are
Methylene blue dye test
spark test

5. Methylene blue dye test
The ampoules are immersed in vacuum chamber consisting of 1% methylene blue solution
A vacuum of about 27 inch Hg is created for about 15 to 30 min.
This causes the solution to enter the ampoules with defective sealing.
The vacuum is released and ampoules are observed.
If a leakage is present, the solution in the ampoules appear blue color.

6. Spark test
The machine uses high precision electrodes to inspect the full circumference of the containers, including the closure zone.
All containers are presented individually to the electrodes.
Any moisture that has penetrated through capillary forces in a crack, pinhole or just weak glass is registered as a change in resistance.
Appearance of blue color confirms the presence of leakage.

7. Sterilty test
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.
The primary official test is performed by means of filteration , but direct transfer is used for membrane filteration when unsuitable.
Methods of testing:
Membrane filtration method
Direct inoculation method
media used
Fluid thioglycollate medium
Soya bean casein digest medium

8. Membrane filtration method
Parenteral preparation
membrane filter ( having a nominal size of 0.45 µ and diameter of 47 mm )
Cut aseptically the filter paper in to two halves
Transfer one of the parts to each type of culture media
Then incubate under prescribed conditions
Note: Cellulose acetate filters for aqueous , oily solutions , weakly alcoholic Cellulose nitrate filters are mainly used for strongly alcoholic

9. Direct inoculation method
Parenteral preparation
Inoculate to culture media
Incubate at prescribed conditions for 14 days
suitable method for aqueous solutions, oily liquids, ointments and creams
No growth then the test is positive

10. Pyrogen test Pyrogens are Produced mostly by gram-negative bacteria .
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)

11. Rabbit test
PRINCIPLE:  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.
SELECTION OF TEST ANIMALS: 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.

12. EQUIPMENTS REQUIRED FOR THE TEST: 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. TEST: The test is carried out on a group of three rabbits.

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

14. Interpretation of results
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 less 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.

15. LAL ( BET) test
Bacterial endotoxin test(BET) is an invitro test based on the formation of gel or the development of color in the presence of the amoebocytes of horseshoe crab ( Limulus polyphemus).
Principle:
The addition of a test solution containing of endotoxin (if present) to a solution of lysate produce turbidity or precipitation or gel formation of mixture. The rate of reaction depends on the concentration of the endotoxin .
Types :
1) The gel clot test 2) The turbidimetric test  3) The kinetic chromogenic test 

16. Types
The Gel clot test: It is based on the formation of a solid gel clot.  Procedure:
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.

17. 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.

18. 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.

19. Procedure Interpretation of results
Into each test tubes dispense a volume appropriate to chosen receptacle of positive control, negative control and test solution
Add to each test tube equal volume of the appropriately constituted lysate.
Place and incubate at 37± 10c for 1 hr.
The preparation or substance being examined complies with pyrogen test if result of the positive product control is positive and negative product control is negative.
LAL test is cheaper, quicker and more accurate than other tests.
Interpretation of results

20. Clarity test
Particulate matter can be detected in parenteral product by two methods, including visual inspection and electronic particulate counting.
Visual methods
Visual inspection by naked eye
Automated visual inspection
Particle count method
Optical microscopic method
Automated particle counter

21. Visual methods Visual inspection by naked eye:
In visual inspection, each injectable is inspected visually against white and black backgrounds. The white background aids in detection of dark colored particles. The light or reflective particles will appear against the black back ground.
Automated visual inspection:
The automatic systems evaluates the particles in injectables automatically. However, this method requires destruction of the ampoule/container for the particle examination.

22. Particle count method i. Microscopic count method:
Membrane filters and microscopes are used.
ii. Automated particle counter s:
The automated particle counters are based on the light obscuration, light scattering method and the electrical resistance methods. Coulter Counter counts the particles in a sample based on the change in the electrical resistance. Particle size detection limit in this instrument is from 0.1 to 1000 micrometer.

23. References REFERENCES United States Pharmacopoeia , Appendix 788, 56.
Indian Pharmacopoeia page no: 659 to 660
Remington, The science and Practice of Pharmacy, 21st ed. Page no to 1374
Michael J. Akers and Daniel S. Larrimore, Parenteral Quality Control.
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