QC Of Parenterals- Anitha Sri


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QUALITY CONTROL TESTS FOR PARENTERALS M. Anitha Sri, Y11MPH448, I/II M. Pharmacy, Industrial Pharmacy, Chalapathi Institute Of Pharmaceutical Sciences.


Introduction Parenterals are the sterile dosage forms, administered by routes other than the oral route. These preparations bypass the gastrointestinal tract and liver and directly enter the systemic circulation. The finished products are forwarded to the quality control department where the various quality control tests are performed. 2

Quality Control Tests Of Parenterals:

Quality Control Tests Of Parenterals Leakage Test Clarity Test Pyrogen Test Sterility Test 3

Leakage Test:

Leakage Test Ampoules are subjected to this test. Leakage test not done for vials and bottles. 1. using methylene blue solution 2. spark test 4

Leakage Test (with methylene blue solution):

Leakage Test (with methylene blue solution) 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. 5

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6 Leakage Test machine Autoclave

Spark Test:

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. All products with a measured voltage higher than a defined maximum value are separated from the good products. 7

High Voltage Leak Detection:

High Voltage Leak Detection 8

Advantages Of HVLD Test:

Advantages Of HVLD Test Vials and ampoules also can be tested. High accuracy of inspection. High speed of processing. 9

Clarity Test:

Clarity Test Test to detect the particulate matter. 1. Visual method 2. Microscopic count method 3. Light obstruction method 4. Coulter counter method 10

Visual method:

Visual method 11 The containers are examined against strong illuminated screen. Black background is used for the detection of light colored particles and white background for dark colored particles. Permissible limits: Particle size ( ≥ µm) Max. no. of particles per mL 10 50 25 5 50 Nil

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12 Visual Inspection machine

Microscopic count method:

Microscopic count method Membrane filters and microscopes are used. 13

Light obstruction method:

Light obstruction method This method uses an electronic counter that produces a light beam of high intensity. The solution is allowed to pass under this bright light. A shadow is formed if a particle is present. The particles are counted by the no. of shadows. 14 Particle size SVP LVP ≥ 10µm 6000/container 25 / ml ≥ 20µm 600/container 3 / ml Limits for subvisible particulate matter as prescribed in USP

Coulter Counter method:

Coulter Counter method 15 The sample solution is added to an electrolyte solution which is drawn through a small orifice. As particle passes through the orifice it displaces its own volume of electrolyte. Particle detected by the increase in electrical resistance. Voltage pulses are proportional to the particle size. Particles below 0.2 µm can also be detected.

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16 Particle size SVP LVP ≥ 10µm 3000/container 12/ml ≥ 25µm 300/container 2/ml Limits for detection of subvisible Particulate matter as prescribed in USP

Pyrogen Test:

Pyrogen Test Pyrogens are the metabolic products of gram negative bacteria. Endotoxin - complex of pyrogenic lipopolysaccharide , a protein and inert lipid; lipid part of the lipopolysaccharide is the main pyrogenic agent; polysaccharide part increases solubility 17

Sources of pyrogen contamination:

18 Sources of pyrogen contamination solvent - possibly the most important source the medicament the apparatus the method of storage between preparation and sterilization

Animals and equipment:

Animals and equipment 19 selection of animals (healthy, adult, not less than 1.5 kg) housing of animals equipment and material used in test (glassware, syringes, needles) retaining boxes (comfortable for rabbits as possible) thermometers (standardized position in rectum, precision of 0.1°C)

Housing of Rabbits:

Housing of Rabbits 20

Preliminary Test:

Preliminary Test 21 intravenous injection of sterile pyrogen-free saline solution to exclude any animal showing an unusual response to the trauma (shock) of injection any animal showing a temperature variation greater than 0.6  C is not used in the main test

Main Test:

Main Test 22 group of 3 rabbits preparation and injection of the product: warming the product dissolving or dilution duration of injection: not more than 4 min the injected volume: not less than 0.5 ml per 1 kg and not more than 10 ml per kg of body mass determination of the initial and maximum temperature all rabbits should have initial T: from 38.0 to 39.8  C the differences in initial T should not differ from one another by more than 1  C

The result of pyrogen test: :

No. Of Rabbits Individual Temp. rise( 0 C) Rise in group( 0 C) Inference 3 0.6 1.4 Pass 3 + 5 = 8 0.6 3.7 Pass 23 The result of pyrogen test:

Limulus Amoebocyte LysateTest:

Limulus Amoebocyte LysateTest To detect or quantify endotoxins of gram-negative bacterial origin reagent: amoebocyte lysate from horseshoe crab ( Limulus polyphemus or Tachypleus tridentatus ). Bacterial Endotoxin test (BET) Monocyte Activation Test (MAT) 24

The endotoxin characteristics:

25 The endotoxin characteristics thermostable water-soluble unaffected by the common bactericides non-volatile These are the reasons why pyrogens are difficult to destroy once produced in a product

Horse shoe crab:

Horse shoe crab 26

Mechanism Of LAL Test:

Mechanism Of LAL Test 27 the test is based on the primitive blood-clotting mechanism of the horseshoe crab enzymes located within the crab's amoebocyte blood cells + endotoxins initiation of an enzymatic coagulation cascade proteinaceous gel

Commercially derived LAL reagent:

Commercially derived LAL reagent 28 bleeding adult crabs into an anticlotting solution washing and centrifuging to collect the amoebocyte lysing in 3% NaCl lysate is washed and lyophilized for storage


Procedure Test: equal Volume of LAL reagent and test solution (usually 0.1 ml of each) are mixed in a depyrogenated test-tube incubation at 37°C, 1 hour remove the tube - invert in one smooth motion (180°) Observe the result 29

Different Techniques:

Different Techniques Three different techniques: the gel-clot technique - gel formation the turbidimetric technique - the development of turbidity after cleavage of an endogenous substrate the chromogenic technique - the development of color after cleavage of a synthetic peptide - chromogen complex 30

Gel Clot Technique:

Gel Clot Technique A solid gel is formed in the presence of endotoxins. This technique requires positive and negative controls. Positive control – A known concentration of endotoxin added to the lysate solution Negative control – Water, free from endotoxins, added to the lysate solution. 31

Turbidimetric Technique:

Turbidimetric Technique This test is based on the measurement of opacity change due to the formation of insoluble coagulin. Opacity is directly proportional to the endotoxin concentration. This technique is used for water systems and simple pharmaceutical produts . 32

Chromogenic Technique:

Chromogenic Technique This is based on the measurement of colour change which is caused by the release of the chromogenic chemical p – nitroanilide. The quantity of the p-nitroanilide produced is directly proportional to the endotoxin concentration. 33

Advantages Of LAL Test:

Advantages Of LAL Test 34 Fast Greater Sensitivity Less Variability Much Less Expensive Alternative to Animal Model more accurate than other is performed in the pharmaceutical laboratory specific for endotoxins of gram-negative origin particularly useful for: Radiopharmaceuticals and cytotoxic agents Products with marked pharmacological or toxicological activity in the rabbit (e.g. insulin) Blood products which sometime give misleading results in the rabbit Water for injection where LAL test is potentially more stringent and readily applied

Sterility Test:

Sterility Test Sterility testing attempts to reveal the presence or absence of viable micro-organisms in a sample number of containers taken from batch of product. Based on results obtained from testing the sample a decision is made as to the sterility of the batch. Sterility testing is made after the product exposition to the one of the possible sterilization procedures 35


Principle Sterility test is based on the principle that when microorganisms are supplied with nutrient medium and water, and incubated at favorable temperatures, they multiply. The presence of micro organisms can be identified by turbidity in the clear medium. 36

Culture Media:

Culture Media The culture media used for sterility test must be capable of promoting the growth of a wide range of microorganisms. Types of media- 1. Fluid thioglycollate medium (for anaerobic bacteria). 2. Soyabean-casein digest medium (for aerobic bacteria and fungi). Incubation of media: 14 days at 30-35 0 C 37

Fluid Thioglycolate Medium:

Fluid Thioglycolate Medium Ingredients Quantity for 1000 ml L- cysteine 0.5 g Sodium chloride 2.5 g Dextrose 5.5 g Agar 0.75 g Yeast extract 5.0 g Pancreatic digest of casein 15.0 g Sodium thioglycollate 0.5 g Resazurin(0.1 % fresh solution) 1.0 ml Distilled water Upto 1000 ml 38

Soyabean - Casein Digest Medium:

Soyabean - Casein Digest Medium Ingredients Quantity for 1000 ml Pancreatic digest of casein 17 g Peptic digest of soyabean meal 3 g Sodium chloride 5 g Dibasic potassium phosphate 2.5 g Dextrose 2.5 g Distilled water Upto 1000 ml 39

Minimum sample size related to batch size:

Minimum sample size related to batch size No. of items in the batch Min. no. of item recommended to be tested Injectable preparations a) upto 100 containers 10% or 4 containers whichever is greater b) 101-500 containers 10 containers c) > 500 containers 2% or 20 containers which ever is less Ophthalmic and other non - Injectable preparations a) upto 20 containers 5% or 2 containers whichever is greater b) > 200 containers 10 containers 40

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No of items in the batch Min no. of items recommended to be tested Bulk Solids a) <4 containers All containers b) 4 – 50 containers 20% or 4 containers whichever is greater c) > 50 containers 2% or 20 containers whichever is greater Surgical Dressings a)< 100 packages 10% or 4 packages whichever is greater b) > 100 but < 500 packages 10 packages c) > 500 packages 2% or 20 packages whichever is less 41

For injectable preparations:

For injectable preparations Quantity of each container Min Quantity to be used for each culture medium For liquids a) < 1 ml Total contents of a container b) 1 ml – 4 ml Half the contents of a container c) 4 ml – 20 ml 2 ml d) 20 – 100 ml 10% of contents of a container e) > 100 ml NLT half of the containers For solids a) < 50 mg Total contents of a container b) 50 mg – 200 mg Half the contents of a container c) > 200 mg 100 mg 42

Test methods:

Test methods The test methods for the sterility of the products are: Membrane filtration method Direct inoculation of the culture medium 43

Membrane Filtration Method:

Membrane Filtration Method Appropriate for : (advantage) filterable aqueous preparations alcoholic preparations oily preparations preparations miscible with or soluble in aqueous or oily (solvents with no antimicrobial effect) solutions to be examined must be introduced and filtered under aseptic conditions 44

Selection of the filters:

Selection of the filters pore size of 0.45  m effectiveness established in the retention of micro-organisms appropriate composition the size of filter discs is about 50 mm in diameter 45


Procedure sterilization of filtration system and membrane filtration of examined solution under aseptic conditions (suitable volume, dissolution of solid particles with suitable solvents, dilution if necessary) The membrane is removed, aseptically transferred to container of appropriate culture medium 46

Scheme for sterility test by membrane filtration:

Scheme for sterility test by membrane filtration 47

Direct inoculation of the culture medium:

Direct inoculation of the culture medium suitable quantity of the preparation to be examined is transferred directly into the appropriate culture medium volume of the product is not more than 10% of the volume of the medium suitable method for aqueous solutions, oily liquids, ointments an creams 48

Scheme for sterility test by direct inoculation method:

Scheme for sterility test by direct inoculation method 49

Interpretation of the results:

Interpretation of the results The culture media is examined during and after the incubation period to detect the possible microbial growth. a) the sample passes the test if microbial growth is not found. b) If microbial growth is present, a retest is performed. If growth absent. Then sample passes the test. If microbial growth is present in the retest also, identify the organisms. If same organisms are found as in the first test, then the sample fails the test. If different organisms are found, retest is performed using twice the number of samples. Passes if microbial growth is not found. 50


References USP, Appendix 788, 56. IP page no: 659 to 660 Remington, The science and Practice of Pharmacy, 21 st ed. Page no. 1367 to 1374 Michael J. Akers and Daniel S. Larrimore, Parenteral Quality Control. 51

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