Evaluation test of Parenterals
The following tests are performed on the finished parenteral products to maintain quality control:
- Leakage test
- Clarity test
- Pyrogen test
- Sterility test
- Ampoules are subjected to leakage test because ampoules are sealed by fusion.
- Therefore, there is a chance of incomplete sealing or for micropores to exist, allowing the contents to leak or micro-organisms and other contaminants to enter the ampoules.
- This test is performed by immersing the ampoules in a vacuum chamber consisting of a dye such as 1% methylene blue solution.
- A negative pressure (or vacuum) of about 27 inch Hg or more is created for about 15 to 30 minutes.
- This negative pressure causes the methylene blue solution to enter the ampoules with defective sealing.
- The vacuum is released and ampoules are washed externally and observed for the presence of dye in the ampoules.
- The colored (blue) solution because of the dye in the ampoules confirm the leakage and hence those ampoules are discarded.
Detection of leakage is more effective when the ampoules are subjected to autoclaving in the presence of a dye. This method has the advantage that both; the detection of leakage and sterilization can be done in a single operation. Bottles and vials are not subjected to vacuum test because of the flexibility of the rubber closures.
- High-frequency spark test which detects pin holes in the ampoules is another means of detecting leakage.
- This has been developed by the Nikka Densok Company of Japan.
- The advantages of this test are:
- High accuracy of inspection
- The high speed of processing
- This test is done to detect the presence of particulate matter.
- The parenteral products must be free from particulate matter such as glass, fiber, floaters etc.
- If the particle size is more than RBC than it can cause an obstruction of the blood vessels leading to severe consequences like emboli in the vital organs of humans and animals.
“Particulate matter is defined as the presence of unintentional and undesirable extraneous, mobile and undissolved substances (other than gas bubbles) within the parenteral preparations.”
Table: Permissible limits (Maximum limits) for particulate matter according to IP
- Injectable solutions in containers with 100 ml or more of a single dose large volume injection intended for administration by intravenous (i.v) infusion comply with the above limits of particulate matter
- The following are the methods used to detect the particulate matter:
- Visual method
- Microscopic count method (or membrane filtration method)
- Light obscuration (obstruction) method
- Coulter counter method
- The filled containers are examined by holding the neck of the containers against the strong illuminated screen.
- The containers are slowly inverted and rotated and the contents are examined for the presence of any foreign particles.
- The black surface is used for the detection of light-colored particles and white surface for dark colored particles.
- If any foreign particle is visible, then that container is discarded.
Microscopic count method (or membrane filtration method)
- A measured sample solution is filtered through a membrane filter.
- The collected particles on the surface of the filter are then counted with the help of a microscope at 100X magnification.
- The whole method is carried out under aseptic conditions.
- It’s also a time-consuming process.
Light obscuration (obstruction) method
- This method uses an electronic instrument that produces a light beam of high intensity.
- The solution is allowed to pass under this bright light.
- If the particle present in the solution passes through this light, a shadow cast is produced due to the obstruction of light by the particle.
- The particles are measured and counted automatically by the device by means of shadow castings.
Table: Limits for subvisible particulate matter in injectable preparations as prescribed in USP
Significance of particulate matter detection:
- The presence of foreign particles in intravenous solutions may cause obstruction of small blood vessels leading to severe consequences like emboli, infusion phlebitis, etc.
- The presence of particulate matter implies that the product is of inferior quality.
- Pyrogens are fever producing metabolic products of micro-organisms.
- The most potent pyrogens are the high molecular weight endotoxins found in the outer membrane of gram-negative bacteria.
- Pyrogens are lipopolysaccharides that may be soluble, insoluble or colloidal in nature.
- Pyrogens are thermostable and can pass through bacteria-proof filters.
- Even minute amount of pyrogens can cause fever, platelet aggregation, alterations in blood coagulation, etc.
- In large amounts, pyrogens can also produce shock and eventually death.
- So, the detection of pyrogens and their elimination from parenteral preparations is very essential.
- Test for pyrogens can be carried out by 2 methods:
- Rabbit test (In vivo)
- LAL (Limulus Amoebocyte Lysate) test (In vitro)
Rabbit test (In vivo)
- The test involves the measurement of a 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 1.5 kg, fed on a balanced diet are used for the test.
- Do not use the rabbit for the main test if any or both of the following happens:
- It shows temperature variation greater than 0.2°C between 2 successive readings noted during the determination of initial temperature and/or
- If the temperature of the rabbit is higher than 39.8 °C or lower than 38 °C.
- Requirements for the test
- All the glassware, syringes, needles, and thermometer must be thoroughly washed for injection and heated in a hot air oven at 250 °C for 30 minutes or at 200 °C for 1 hour.
- Retaining boxes for rabbits.
- Preliminary test
- This test is performed to identify and exclude any rabbit with an unusual response to the trauma of the injection.
- The test is performed by taking pyrogen-free saline solution (10 ml/kg of the body weight), which is warmed to 38.5°C and injected intravenously to the rabbits.
- The temperatures of the rabbits are recorded 90 minutes before the injection and continued for 3 hours after the injection.
- Any animal showing a temperature variation of 0.6°C or more must not be used for the main test.
- Main test
- This test is carried on a group of 3 rabbits.
- The complete procedure is as mentioned below:
- Step 1: Preparation of the sample
- Dissolve the test substance in a pyrogen-free saline solution.
- This can be further diluted with the same pyrogen-free saline if needed.
- Warm the solution to about 38.5°C before the injection.
- Step 2: Determination of initial temperature of rabbits
- Insert a clinical thermometer into the rectum of each rabbit and normal readings of the body temperature (rectal temperature) are taken prior to the injection of test solution.
- 2 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 rabbits.
- Step 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 and not more than 10 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.
- Preliminary test
Interpretation of results
- If the response of any individual rabbit is less than 0.6°C and if the sum of the responses of the 3 rabbits is not more than 1.4°C, the preparation being examined passes the test.
- If the response of any rabbit is 0.6°C or more or if the sum of the responses of the 3 rabbits is more 1.4°C, then the same test is repeated on another 5 rabbits.
- If not more than 3 of the 8 rabbits show individual responses of 0.6°C or more and if the sum of the responses of the 8 rabbits is not more than 3.7°C, the preparation being examined passes the test.
Advantage of rabbit test
- It is used to identify the presence of a wide range of pyrogens.
Disadvantage of the rabbit test
- It is a time-consuming method.
- It requires permissions from the animal ethical committee which may consume more time on certain occasions.
- Requires precaution regarding the handling of animals.
LAL (Limulus Amoebocyte Lysate) test (In vitro)
- This is a sensitive test used to detect endotoxins from gram-negative bacteria.
- Therefore, it is also known as the bacterial-endotoxin test.
- The endotoxins upon reaction with lysate form an insoluble gel clot.
- The lysate is obtained by the lysis of ameobocytes (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 (or the development of color) in the presence of bacterial endotoxins and the lysate solution.
- The lysate consists of a proclotting enzyme coagulogen (clottable protein) which are required for the reaction to occur.
Diagram: Mechanism of Lysate clotting
Types of LAL test
- There are 3 types of LAL tests, which are:
- The gel-clot test
- The turbidimetric test
- The kinetic chromogenic test
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 a solid gel confirms the presence of endotoxins.
- If the solid gel is not formed, it indicates the absence of endotoxins and the test solution passes the test.
- The test requires positive and negative controls.
- Positive control: A known concentration of endotoxin added to the lysate solution is used as a positive control.
- Negative control: Water (free from endotoxins) added to the lysate solution can be used as a negative control.
The turbidimetric test
- This test is based on the measurement of opacity change in the LAL test due to the formation of gel clot (insoluble coagulin).
- Opacity is directly proportional to the endotoxin concentration.
- This test is used for water systems and simple pharmaceutical products.
The kinetic chromogenic test
- The test is based on the measurement of color change which is caused by the release of chromogenic chemical containing para nitroaniline.
- This compounds containing p-nitroaniline is a byproduct of the clotting reaction during the LAL test.
- The quantity of the p-nitroaniline 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 than the rabbit test. Even the minute amounts of endotoxin can be detected by the LAL test.
Disadvantage of the LAL test
- It is used to identify the presence of endotoxins only (pyrogens of gram-negative bacteria).