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

Abstract

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.

• Authors: Zhong Zuo, Siu Kwan Wo, Cindy M Y Lo, Limin Zhou, Gregory Cheng, Joyce H S You
• Journal: Journal of pharmaceutical and biomedical analysis (J Pharm Biomed Anal) Vol. 52 Issue 2 Pg. 305-10 (Jun 05 2010) ISSN: 1873-264X [Electronic] England
• PMID: 20133101 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright (c) 2010 Elsevier B.V. All rights reserved.

Chemical References

• Anticoagulants
• Warfarin

Topics

• Anticoagulants (blood, metabolism)
• Calibration
• Chromatography, Liquid (instrumentation, methods)
• Drug Stability
• Freezing
• Humans
• Molecular Structure
• Reference Standards
• Reproducibility of Results
• Sensitivity and Specificity
• Solid Phase Extraction
• Stereoisomerism
• Tandem Mass Spectrometry (instrumentation, methods)
• Temperature
• Time Factors
• Warfarin (analogs & derivatives, blood, metabolism)