A novel, rapid and simple UHPLC-MS/MS method for quantification of warfarin in dried blood spots

Warfarin is a common first line anticoagulant with a narrow therapeutic window. Because of the large blood volume needed, previous warfarin determination methods were not applicable to small animals, such as mice. To reduce the number of small animals used needed, we developed and validated a sensitive rapid assay for the simultaneous detection of warfarin enantiomers in mouse dried blood spot (DBS) samples. Analytes were extracted by tert-butyl methyl ether and then separated by a chiral Cellulose-1 column with a mobile phase of 75% acetonitrile (containing 0.1% formic acid). The total chromatographic run time was 3 min. Negative mode electrospray ionization was used for MS/MS detection, where the monitored ion transitions were m/z 307.1 → 161.0 and 341.1 → 284.0 for warfarin and coumachlor (internal standard) respectively. The calibration curves were linear with a correlation coefficient of ≥0.994 for both enantiomers over a concentration range of 10-1000 ng/mL. The satisfactory accuracy and adequate reproducibility of both warfarin enantiomers were validated in terms of intra- and interday precision with mouse DBS cards. The samples were stable at room temperature for at least 14 days. The validated method was applied to a pharmacokinetic study in mice.