METHOD DEVELOPMENT AND VALIDATON FOR DETERMINATION OF ANTIFACTOR IIA POTENCY OF HEPARIN SODIUM BY CHROMOGENIC METHOD

Abstract

A novel, precise, rapid, economical, and Accurate Chromogenic method for determination of Antifactor IIA potency of Heparin sodium.Tris-buffer preparation(PH 8.4) and 6%w/v solution of citric acid are the reagents used. Anti IIA reagents (AntithrombinIIIreagent (R1), Bovine thrombin IIa reagent (R2) ,Spectrozyme TH substrate(R3)) are reconstituted. Switch on the versamax instrument and wait for 15min for the temperature to attain 37ºc±0.2ºc. Use new microplates for every test and perform pre-read of empty plate.Heparin reference standard solution and test solution (API) are prepared. sample and standard preparations are incubated with different reagents at 37ºc. Incubation time depends on the potency of different lots of reagents(anti IIa) used.On the receipt of the reagents the preliminary assays are carried out over different incubation time to determine the suitable time combination to get a linear standard curve for USP heparin standard and use the corrected incubation time in sub seeding steps.