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 times are 6.9, 8.0, 7.0, and 7.9 min for S-WAR, R-WAR, S-IS and R-IS, respectively. The detection was by 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, and <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.