Bioassay of Heparin

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  • please enhance the calculation of the potency also, with out that its a waste slide show. after that its useful to real time application.
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Bioassay of Heparin

  1. 1. Bioassay of Heparin Dr. S. Parasuraman Senior Lecturer, Faculty of Pharmacy AIMST University, Malaysia
  2. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 12. Bioassay of Heparin • • • • Three independent assay Limit of errors: P=0.99 90-110 with three determination 92-108 with four determination
  13. 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.

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