1. A seminar
on
Bioassay of official
drugs
By :PARTH
M.pharma-1(Q.A)
APMC Pharmacy College
1

2. Content
1. Definition
2. Principle of bioassay
3. Importance of bioassay
4. Types of bioassay
5. Methods of bioassay
6. Limitation of bioassay
7. Bioassay of official drugs
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3. Definition:
Estimation of the conc. or potency of a
substance by measuring its biological response
in living systems.
Observation of pharmacological effects on
[1] living tissues, or cells
[2] microorganisms
[3] animals
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4. Principle of bioassay:
Compare the biological effect produced by the
test substance with that of standard preparation
and find out how much test substance is
required to produce same biological effect as
produced by the standard.
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5. Importance of bioassay:
 Active principle of drug is unknown
 Active principle cannot be isolated, e.g posterior
pituitary extract etc.
 Chemical method is either
◦ not available
◦ if available, too complex,
◦ insensitive to low doses e.g. Histamine can be
bioassay in microgram conc.
 Unknown Chemical composition, e.g. long acting
thyroid stimulator.
 Chemical composition of drug is different but has same
pharmacological action e.g. cardiac glycosides isolated
from diff sources, catecholamines etc.
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6. To ascertain the potency of a drug.
Stability studies are also conducted by
bioassays.
It also measure toxicity.
Bacterial products like toxins, antitoxins,
vaccines, are assayed by only bioassay.
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7. Limitation of bioassay:

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8. Bioassay of official drugs:
1)HEPARIN SODIUM:(IP’96)
by comparing the conc. necessary to prevent the clotting
of sheep, goat or human plasma with the conc. of the
std. preparation.
 Standard preparation:
The freeze-dried sodium salt of the purified active
principle from bovine intestinal mucous membranes.
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9. Special reagent:
Prepared plasma:
collect the blood in to vessel containing 8%w/v sol. Of sodium citrate
&blood(1:19)
mix &centrifuge to pool out plasma
To 1 ml of plasma add 0.2 ml of 1% w/v of calcium chloride sol.
mix it
The plasma is suitable if clot forms within 5 mins.
Solution of standard preparation:
The minimum quantity of std. preparation of heparin sodium
which, when added in 0.8 ml of saline solution, maintain fluidity in 1
ml of prepared plasma for 1 hour after the addition of 0.2 ml of 1%
w/v calcium chloride.
On the day of assay prepare a solution of std. preparation such that
it contains in each 0.8ml of saline solution the above determined qty.
of the std. preparation.
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10. Test solution:
Weight accurately about 25mg of the test sample
dissolved in sufficient saline solution to give the conc. of
1mg/ml
dilute to concentration corresponds to that of standard
Method:
To very clean test tubes add graded amt. of std. preparation,
the largest dose doesn’t exceed 0.8 ml.
add sufficient saline solution to make volume 0.8ml & add
1.0 ml of prepared plasma to each test tube.
add 0.2 ml of 1% of calcium chloride, note the time
mix properly so that entire inner surface of test tubes is wet
In the same manner set up a series of test preparation
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11. completing the entire process within 20 minute after addition of prepared
plasma
after 1 hour the addition of calcium chloride solution, determined the
extent of clotting in each test tubes,
recognize three grades between zero and full clotting.
Dilution of test preparation which contain same concentration as that of
standard show same degree of clotting
If the degree of clotting in dilution of the std. preparation lies between that
observed in 2 of the dilution of test preparation, the potency of later is
estimated.
If there is no correspondence between the degree of clotting by standard &
test, new dilution prepared & assay is repeated.
Calculate the estimated potency of the test preparation by combining the
result of assay with standard statical methods.
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12. 2)OXYTOIN:(IP’07)
The potency of oxytocin is determined by comparing its activity with
that of the Standard Preparation of oxytocin under the conditions of a suitable
method of assay.
Standard Preparation:
consisting of freeze-dried synthetic oxytocin peptide with human albumin and
citric acid (supplied in ampoules containing 12.5 Units).
Method:
By contraction of the rat uterus:
Inject 100 mg of oestradiol benzoate intramuscularly into a female rat
weighing 120 to 200 g 18 to 24 hours before the assay.
Kill the rat and suspend one horn of the uterus in a bath containing a
solution of the following composition.
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13. Composition (% w/v)
Sodium chloride 0.662
Potassium chloride 0.045
Calcium chloride 0.007
Sodium bicarbonate 0.256
Disodium hydrogen phosphate 0.029
Sodium dihydrogen phosphate 0.003
Magnesium chloride 0.010
Dextrose 0.050
Maintain the bath at 32o C at which spontaneous contractions of
the uterus are abolished and the preparation maintains its sensitivity.
Oxygenate the solution with a mixture of 95% of oxygen and 5% of
carbon dioxide
record the contractions of the muscle using a suitable instrument
giving a linear response
Record the contractions produced by the addition of two doses of
the Standard Preparation suitably diluted with the above solution. 13

14. The doses should be such as to produce clearly discriminated
contractions
The required doses normally lie between 10 and 50 micro Units per ml of
bath liquid.
The doses should be added at regular intervals of 3 to 5 minutes
depending upon the rate of recovery of the muscle.
Dilute test preparation so as to produce same response as that of
standard
The ratio between the two doses of the preparation being examined
should be the same as that of the Standard Preparation and this ratio
should be kept constant throughout the assay.
The two doses of Standard Preparation and the preparation being
examined should be given according to a randomized block or a Latin
square design and at least six responses to each should be recorded.
calculate the result of the assay by standard statistical methods. 14

15. 3)STERPTOKINASE:(IP’96)
Bioassay by comparing its ability to activate human plasminogen to form
plasmin with that of the Standard Preparation. The plasmin generated is
determined by measurement of the time taken to lyse a fibrin clot under
the conditions of a suitable method of assay.
Standard Preparation:
The Standard Preparation is consisting of freeze-dried streptokinase
(supplied in ampoules containing 700 Units of streptokinase activity).
Suggested Method:
Use citro-phosphate buffer pH 7.2 containing 3% w/v of bovine
serum albumin for the preparation of solutions and dilutions.
Prepare the Standard Preparation to contain 1000 Units of
streptokinase activity per ml and prepare a solution of the preparation
being examined of the same concentration;
keep the solutions in ice and use within 6 hours.
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16. Prepare three serial dilutions of the Standard Preparation so
longest clot-lysis time is less than 20 minutes.
prepare three similar dilutions of the solution of the preparation
being examined.
Keep the solutions in ice and use within 1 hour
take 24 tubes(8 mm), three for the dilutions of the Standard
Preparation and three for the dilutions of the Standard Preparation
being examined, allocating four tubes to each dilution
Add 0.2ml of dilution, 0.2 ml of citro-phosphate buffer pH 7.2
containing 3% w/v of bovine serum albumin and 0.1 ml of a solution
containing 20 Units of thrombin per ml,
Place the tubes in a water-bath at 37o and allow to stand for 2 minutes
to attain temperature equilibrium.
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17. Add 0.5 ml of a 1% w/v solution of human euglobulins in each tubes at
interval of 5-sec.
measure the time in seconds that elapses between the addition of the
euglobulin and the lysis of the clot.
calculate the result of the assay by standard statistical methods.
4)Vitamin D: (USP 23 NF 18)
 It is bioassayed by measuring the ability of vitamin D to stimulate
calcification of the rachitic metaphysis in rats.
 Assay uses young rats (not less than 55 days old) that have developed
rickets on a rachitogenic diet.
 These rats are divided in to groups and fed the rachitogenic diet
with either USP cholecalciferol reference standard, unknown or no
supplementation (control).
One half the dose of vitamin D as cholecalciferol standard or unknown
given to rats on day 1 to 3 of assay period.
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18. At the end of fixed period (7to10 days) they are weighted& scarifies.
Any rats whose wt. decrease has removed from further analysis.
The leg bones of remaining rats are dissected out & assayed for amt.of
recalcification of bones.
The activity of vitamin D may be determined by amt. of recalcification
in relation with reference standard.
5) Plague vaccine:
The potency of plague vaccine is estimated by determining the dose
necessary to protect mice against a lethal dose of a virulent strain of
Yersinia pestis.
Test Animals:
Use white mice, 6 to 7 weeks old, each weighing between 20 and 28 g
and of a strain susceptible to plague infection.
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19. •The animals should be healthy and free from intercurrent infection
with organisms such as Salmonella.
Suggested Method:
Selection of suitable virulent strain:
•A freeze-dried virulent culture of Y. pestis is revived by subculturing
0.5 ml in 9.5 ml of nutrient broth in test-tube and incubating at 28o
for exactly 48 hours.
•Such a culture should contain 300 to 600 million organisms per ml.
•Make 10-fold dilutions in nutrient broth and test for virulence.
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20. Standard challenge dose:
Freshly reconstitute the freeze-dried culture and dilute with nutrient
broth to strength such that 0.2 ml contains 60 to 120 organisms.
Measurement of protective power:
Prepare a series of five graded doses of the preparation being
examined arranged in such a manner that the 50% protective dose
(ED50) lies about the middle of the selected series.
16 mice are used for each dose.
Inject subcutaneously the selected dose in two equal parts with an
interval of 7 days between them.
Inject subcutaneously the standard challenge dose in each group of
mice 7 day after second half of dose.
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21. Observe the animals for 15 days and record the number of deaths in
each group.
Carry out a post-mortem, look for signs of plague .
If plague organisms are not seen, such deaths are excluded from the
calculation.
After observation kill all the surviving animals and examine for
signs of plague.
Calculate the median effective immunising dose, ED 50, by standard
statistical methods.
The vaccine passes the test if it has an ED50 of 0.004 ml or less per
mouse.
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22. 6) RABIES ANTISERUM:
The potency of rabies antiserum is determined by comparing
the dose necessary to protect mice against a lethal
intracerebral dose of rabies virus with the dose of the
Standard Preparation of rabies antiserum necessary to give the
same protection.
Standard Preparation:
The standard preparation is a dried serum the potency of
which has been determined in relation to the International
Standard.
Suggested Method:
Test animals:
Use healthy mice of either sex weighing between 10 and 14
gm.
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23. Test virus:
Any suitable strain of rabies virus of known potency such as the
CVS strain may be used.
Determination of potency of the rabies antiserum:
Prepare a series of 2-fold dilutions of the Standard Preparation and of
the preparation being examined with water containing 2% v/v of heat
inactivated normal horse serum
Add a quantity of a suspension of the test virus containing the test
dose.
keep the mixtures at 37o for 1 hour.
Inject intracerebrally 0.03 ml of each mixture into 10 mice.
Observe the mice for 14 days after the injection.
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24. Mice dying before the fifth day after inoculation with the virus
are eliminated from the test.
All the mice dying between the fifth and fourteenth days after showing
signs of rabies are considered to have died of rabies.
Mice living up to the fourteenth day but showing signs of rabies are also
counted as having died from rabies.
Calculate the result of the test by standard statistical methods.
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25. References :
Indian pharmacopoeia 1996,VOL.I, page no.
361,550.
Indian pharmacopoeia 1996, VOL.II, page no.
602,656.
United state pharmacopoeia 23,National
Formulary 18,asian edition.
Elements of pharmacology ,15th edition by Dr.
R.K. Goyal ,page no. 578-582.
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