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.
Zuo
Zhong
Z
School of Pharmacy, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, SAR. joanzuo@cuhk.edu.hk
Wo
Siu Kwan
SK
Lo
Cindy M Y
CM
Zhou
Limin
L
Cheng
Gregory
G
You
Joyce H S
JH
D000925:Anticoagulants / Q000097:blood* / Q000378:metabolism; D002138:Calibration; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods*; D004355:Drug Stability; D005615:Freezing; D006801:Humans; D015394:Molecular Structure; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D052616:Solid Phase Extraction; D013237:Stereoisomerism; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D013696:Temperature; D013997:Time Factors; D014859:Warfarin / Q000031:analogs & derivatives* / Q000097:blood* / Q000378:metabolism
D000925:Anticoagulants; D014859:Warfarin