Development and validation of a sensitive and robust LC-tandem MS method for the analysis of warfarin enantiomers in human plasma

A liquid chromatography-tandem mass spectrometry method (LC-MS-MS) was developed and validated for measuring warfarin (WAR) enantiomers (R-wAR and S-WAR) in human EDTA plasma. Liquid-liquid extraction using ethyl ether was used to extract the analytes from the plasma. Baseline resolution of S- and R-WAR as well as the internal standard enantiomers (S- and R-p-ClWAR. S-IS and R-IS) was achieved on a beta -cyclodextrin column with a mobile phase of acetonitrile-acetic acid-triethylamine (1000:3:2.5, v/v/v). The retention rimes are 6.9. 8.0, 7.0. and 7.9 min for S-WAR. R-WAR. S-IS and R-IS. respectively. The detection M as bq monitoring S- and R-WAR at m/z 307 --> 161 and S- and R-IS at m/z 341 --> 161 using (-) ESI. The standard curve range was 1-100 ng ml(-1) for both S- and R-WAR. The inter-day precision and accuracy of the quality control (QC) samples were <7.3% relative standard deviation (RSD) and <7.3% bias for S-WAR, acid <6.5% RSD and <5.8% bias for R-WAR, respectively. Analyte stability during sample processing and storage were established. Method ruggedness was demonstrated by the reproducible performance from analysis of clinical samples. (C) 2001 Elsevier Science B.V. All rights reserved.