This document summarizes the bioassay method used to test the potency of heparin preparations. The method involves comparing the clotting time of plasma incubated with the test heparin sample to the clotting time of plasma incubated with a reference standard heparin preparation. Several dilutions of both the standard and test sample are tested to identify the dilution for each that results in equivalent clotting time. The potency of the test sample can then be estimated based on its equivalent dilution relative to the standard. The bioassay follows specific procedures for obtaining and preparing plasma, making standard and test solutions, conducting the clotting time assays, and calculating potency results.

1. Bioassay of
Heparin
Dr. S. Parasuraman
Senior Lecturer, Faculty of Pharmacy
AIMST University, Malaysia

2. Bioassay of Heparin
• Heparin: anticoagulant
• Preparation: Injection
• Appearance: White or almost white, hygroscopic
nature
• Solubility: Freely soluble in water
• Identification:
• delays the clotting of recalcified citrated sheep plasma
• Nuclear magnetic resonance spectrometry
• Assay potency: 90-111% of stable potency
• Unit: I.U., (One mg of heparin = 130 I.U.)

3. Bioassay of Heparin
Principle:
•The potency of heparin sodium is determined in
vitro by comparing the concentration necessary to
prevent the clotting of sheep or goat or human
plasma with the concentration of the standard
preparation of heparin sodium.
•Its is necessary to give the same effect under the
same conditions of the method of assay.

4. Bioassay of Heparin (flow chart)
Principle: Potency of heparin (test)
determine by comparing the
clotting time with that of standard
Requirements:
1.Sheep/ goat/ human plasma
2.Standard
3.Test
4.Calcium chloride
sodium citrate
Check the Quality
0.2 ml of 1% w/v
of CaCl2  Clot
within 5 min

5. Bioassay of Heparin (flow chart)
1 ml Plasma +
Minimum quantity of STD
(total vol. 0.8 ml in normal Saline)
Serial dilution of
three conc. Both
STD and Test
1 hr
If fluidity maintains add 0.2 ml of
1% w/v of CaCl2
25 mg of sample
(make volume of 1 mg/ml)  make
test concentration equal to STD
1 ml Plasma +
Minimum quantity of STD
(total vol. 0.8 ml in normal Saline)
1 hr
If fluidity maintains add 0.2 ml of
1% w/v of CaCl2
monitored by clotting assays such as the activated
partial thromboplastin time (APTT).

6. Bioassay of Heparin
Requirements:
•Standard heparin (for assay)
•Plasma (form sheep or goats, human)
•Solution of standard solution
•Test drug/ heparin
•Standard heparin:
Purified freeze-dried heparin sodium salt from bovine/
pork intestinal mucous. The potency of standard
heparin has been determined in relation to the
International Standard stated by the World Health
Organization.

7. Bioassay of Heparin
• Prepared plasma:
Collect the blood from sheep or goat or human in vessel
containing 8% w/v of sodium citrate (The ratio of
sodium citrate and blood is 1:19)
Mix gently and centrifuge to pool out plasma
Clean test tube  In one ml of pooled plasma, add 0.2
ml of 1% w/v of calcium chloride solution and mix it
The plasma is suitable if clot form within 5 min

8. Bioassay of Heparin
• Solution of standard preparation:
The minimum quantity of standard preparation of
heparin sodium which, when added in 0.8 ml of saline
solution, maintain fluidity in 1 ml of prepared plasma
for 1 h after addition of 0.2 ml of 1% w/v calcium
chloride.
• Test solution:
Weigh accurately about 25 mg of the test sample
Dissolve in sufficient saline to give the concentration of
1 mg/ml
Dilute the test solution corresponds to that of standard

9. Bioassay of Heparin
Method:
•In cline test tube, add graded amount of the solution of
standard preparation (the largest dose not exceed 0.8
ml)
•Add sufficient volume of saline to make total volume of
0.8 ml and add 1 ml of prepared plasma to each test tube
•Add 0.2 ml of 1 % w/v solution of calcium chloride, note
the time
•Mix the content properly so entire inner surface of the
tube is wet

10. Bioassay of Heparin
Method Cont.,
•In same manner setup a series of test preparation
(complete the entire process within 20 min after addition
of prepared plasma)
•After 1 hr the addition of calcium chloride solution,
determine the extent of clotting in each test tube
•Recognize three grades between zero and full clotting
•Dilution of the test solution which contains same
concentration as that of standard shows same
degree of clotting

11. Bioassay of Heparin
Method Cont.,
•If the degree of clotting in dilution of the standard
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 and test, new dilution prepared
and assay is repeated
•Calculate the estimated potency of the preparation by
combining the result of assay with standard statistical
method

12. Bioassay of Heparin
•
•
•
•
Three independent assay
Limit of errors: P=0.99
90-110 with three determination
92-108 with four determination

13. Further reading
• Shah GA, Dhall TZ, Ferguson I, Davis MR, Graham DT,
Pomeroy AR, Dhall DP. An evaluation of the British
Pharmacopoeial assay of heparin: a comparison with
other methods. J Clin Pathol. 1980;33:562-5.
• Indian Pharmacopeia 1996.
• British
Pharmacopeia
2012,
online:
http://bp2012.infostar.com.cn/Bp2012.aspx?tab=browse&a=displa
(freely accessible)
• Swoap OF, Kuizenga MH. The sheep plasma method for
the bioassay of heparin preparations. J Am Pharm Assoc
Am Pharm Assoc. 1949;38:563-5.