Determination of S- and R-warfarin enantiomers by using modified HPLC method.

Warfarin is a commonly prescribed anticoagulant existing in two enantiomeric forms S- and R-warfarin. Many techniques have been used to analyze warfarin in plasma but less frequently for enantiomeric analysis. One of the HPLC method employed was further simplified and made economical. Method was validated according to ICH guidelines and was found to be sensitive, reliable and less time consuming. For both enantiomers, LLOQ was 12.5 ng/mL. The CV% and accuracy for method were in the range of 0.8-14.6% and 92-107% respectively. The recoveries for both enantiomers were in the range of 86-103.8%. Blood samples were collected from 170 stable patients taking warfarin and S- and R-warfarin levels were determined by this method. Majority of subjects were found to have S/R-warfarin ratio of about 1:2 as reported in previous studies due to rapid clearance of S-enantiomer than R-enantiomer. However individual subjects data was suggestive of presence of slow metabolizers of S-warfarin leading to altered S/R ratio. Previous studies have also pointed out CYP2C9 polymorphism being responsible for such inter-individual differences in S-warfarin metabolism. So plasma warfarin S/R ratio may serve as a useful phenotypic test for CYP2C9 polymorphism.