Simultaneous determination of warfarin and 7-hydroxywarfarin enantiomers by high-performance liquid chromatography with ultraviolet detection.

Warfarin is administered clinically as a racemic mixture of two enantiomers, (R)-warfarin and (S)-warfarin. (S)-Warfarin has more potent anticoagulant activity than (R)-warfarin and is metabolized mainly to (S)-7-hydroxywarfarin by CYP2C9. A simple, rapid, and sensitive high-performance liquid chromatography method with ultraviolet detection was developed for the simultaneous quantitative determination of the (R)- and (S)-enantiomers of warfarin and 7-hydroxywarfarin in human plasma. Analytes and the internal standard (p-chlorowarfarin) were separated using a mobile phase of 0.5% KH2PO4 (pH 3.5)-methanol (41:59, v/v) on a Chiral CD-Ph column at a flow rate of 0.5 mL/min and were detected at a ultraviolet absorbance of 305 nm. Analysis required 200 μL of plasma and involved a simple and rapid solid-phase extraction with an Oasis HLB cartridge, which gave high recovery (greater than 91.8%) and good selectivity for all analytes. The lower limit of quantification for the (R)- and (S)-enantiomers of warfarin and 7-hydroxywarfarin was 2.5 ng/mL for each analyte. Inter- and intraday coefficients of variation for all analytes were less than 14.2% and accuracies were within 6.6% over the linear range. Our results indicate that this method is applicable to the simultaneous monitoring of the enantiomers of warfarin and 7-hydroxywarfarin in human plasma. The S/R-enantiomer ratio of warfarin and the (S)-warfarin/(S)-7-hydroxywarfarin ratio 3 hours after administration in 67 CYP2C9*1/*1 patients ranged from 0.24 to 0.75 and from 1.83 to 19.02, respectively, whereas these ratios in a CYP2C9*3/*3 patient were 1.12 and 17.02, respectively.