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.
Department of Bioanalytical Chemistry, Covance Laboratories, Inc., 3301 Kinsman Boulevard, Madison, WI 53704, USA. naidong.weng@covance.com
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D000925:Anticoagulants / Q000097:blood*; D002614:Chelating Agents; D002853:Chromatography, Liquid; D004492:Edetic Acid; D006801:Humans; D007202:Indicators and Reagents; D013058:Mass Spectrometry; D011786:Quality Control; D015203:Reproducibility of Results; D013237:Stereoisomerism; D014859:Warfarin / Q000031:analogs & derivatives / Q000097:blood*
D000925:Anticoagulants; D002614:Chelating Agents; D007202:Indicators and Reagents; D014859:Warfarin; D004492:Edetic Acid