Simultaneous measurement of S-warfarin, R-warfarin, S-7-hydroxywarfarin and R-7-hydroxywarfarin in human plasma by liquid chromatography-tandem mass spectrometry.

Clinically used anticoagulant warfarin is usually available as a racemic mixture of S- and R-warfarin that are both metabolized mainly by cytochrome P450 isoenzymes. Determination of warfarin enantiomers and their enantiomeric 7-hydroxywarfarin (7-OH-warfarin) metabolites in the human plasma sample allows probing of cytochrome P450 isoenzyme activities and detection of ingestion of warfarin-containing products for the investigation of unexplained bleeding. The present study aims to develop a sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous detection of S-warfarin, R-warfarin, S-7-OH-warfarin and R-7-OH-warfarin in human plasma. Plasma samples were extracted with mixed-mode cation-exchange (MCX) cartridge with recoveries of greater than 87.0% for all four analytes. The selectivity of 7-OH-warfarin from other monohydroxylated warfarin metabolites such as 4'-, 6-, 8- and 10-hydroxywarfarins using a Chirobiotic V chiral column and multiple reaction monitoring (MRM) was addressed. The developed LC-MS/MS method is simple, specific and has been fully validated with satisfactory accuracy and adequate reproducibility with limit of quantification (LOQ) of 5 ng/ml for all four analytes. The method was successfully applied to analyze the steady state plasma concentrations of the four analytes in 30 patients. Copyright (c) 2010 Elsevier B.V. All rights reserved.