pyrogen testing
TRANSCRIPT
PYROGEN TESTING
Present by :- Guided by:-Mr.N.P.Sawadadkar Prof.Tushar Thakur Sir
Pyrogens
Pyrogens - fever inducing organic substances Responsible for many febrile reaction These are Endotoxin.
Having nature Endogenous (inside body)Exogenous (outside body)
Exogenous pyrogens – mainly lipopolysaccharides bacterial origin, but not necessary
Endotoxin characteristic
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
Test for pyrogens = Rabbit test
the development of the test for pyrogens reach in 1920 a pyrogen test was introduced into the USP XII (1942) The test consists of measuring the rise in body
temperature in healthy rabbits by the intravenous injection of a sterile solution of the substance under the test.
Why the Rabbit?
Reproducible pyrogenic response Other species not predictable Similar threshold pyrogenic response to
humans Rabbit chosen for economic purposes
Rabbit Pyrogen Test
Rabbits must be healthy and mature New Zealand or Belgian Whites used mostly Either sex may be used Must be individually housed between 20 and
23°C Not varies more than ± 3º c. Free from disturbances likely to excite them.
equipment and material used in test (glassware, syringes, needles etc)
Must be free from Pyrogens by heating at 250º c for not less then 30 minutes or any other method
retaining boxes (comfortable for rabbits as possible)
Thermometers or thermistor probe (standardized position in rectum, precision of ± 0.1°C)
Rabbit pyrogen test
Preliminary test (Sham Test) intravenous injection of sterile pyrogen-free
saline solution Warm the pyrogen free solution up to 38.5ºc to exclude any animal showing an unusual
response to the trauma (shock) of injection any animal showing a temperature variation
greater than 0.6C is not used in the main test
Rabbit pyrogen test - main test:
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 Temperature: from 38.0 to 39.8C
the differences in initial Temperature should not differ from one another by more than 1C
Interpretation of the results: the test is carried out on the first group of 3 rabbits; if
necessary on further groups of 3 rabbits to a total of 4 groups, depending on the results obtained
intervals of passing or failing of products are on the basis of summed temperature response
The result of pyrogen test:
No.of Rabbits Individual Tempt. rise (°c)
Tempt. Rise in group (°c)
Test
3 rabbits 0.6 1.4 PassesIf above not
passes 3+5 = 8 rabbits
0.6 3.7 Passes
If above test not passes the sample is said to pyrogenic.
LAL Test
Limulus amebocyte lysate test. to measure the concentration of endotoxins of
gram-negative bacterial origin reagent: amoebocyte lysate from horseshoe
crab, Limulus polyphemus
Limulus polyphemus = horseshoe crab
Principle The addition of solution containing endotoxin
to a solution of lysate produce turbidity. The rate of reaction depends upon
concentration of endotoxin , the pH and the temperature.
The endotoxin reference standard is the freeze dried.
The test is based on the primitive blood-clotting mechanism of the horseshoe crab
Commercially derived LAL reagents
bleeding adult crabs into an anticlotting solution washing and centrifuging to collect the amebocyte lysing in 3% NaCl lysate is washed and lyophilized for storage activity varies on a seasonal basis and
standardization is necessary.
Test performance (short)
avoid endotoxin contamination Before the test:
interfering factors should not be present equipment should be depyrogenated the sensitivity of the lysate should be known
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°) - read
(observe) the result
Endotoxin concentration monitoring
Following method are used to monitor the endotoxin concentration Method A: El-clot method: limit test Method B: semi-quantitative gel-clot method Method C: kinetic turbidimetric method Method D: kinetic chromogenic method Method E: end-point chromomeric method
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
REFERENCES
U.S.PHARMACOPEIA Pharmaceutical Formulations by
M.E.Aulton, H.C. Ansel.page no 195-196.