Quantification of total and free concentrations of R- and S-warfarin in human plasma by ultrafiltration and LC-MS/MS

A sensitive LC-MS/MS assay for quantification of total and free concentrations of R-and S-warfarin in plasma was required to support clinical studies on warfarin enantiomers. Several ultrafiltration devices were evaluated for separation of free warfarin from plasma proteins. The highest precision and lowest non-specific binding was obtained for Centrifree ultrafiltration devices. R- and S-warfarin were extracted from plasma (total) and ultrafiltrate (free) by liquid-liquid extraction with methyl tert-butyl ether using d(6)-warfarin as internal standard. Mean extraction recovery was 68 +/- 4%. The enantiomers were separated on a Chirobiotic V column with isocratic elution using 40% methanol and 0.03% acetic acid in water. Negative mode electrospray ionisation was used for MS/MS detection, monitoring the ion transition m/z 307/161. Calibration curves (quadratic, weighted 1/x) were fitted over the range of 20-2,000 ng/ml (r(2)>= 0.995) in plasma and 0.5-20 ng/ml (r(2)>= 0.998) in ultrafiltrate. The lower limit of quantification for R- and S-warfarin was 0.5 ng/ml in ultrafiltrate. Intra- and interday precision (% RSD) and bias were within 10% in all cases, and matrix effects were negligible. The assay was applied successfully to analysis of samples from clinical studies.