Determination of phenprocoumon, warfarin and their monohydroxylated metabolites in human plasma and urine by liquid chromatography–mass spectrometry after solid-phase extraction

A high-performance liquid chromatography–mass spectrometry (HPLC–MS) method for the quantification of phenprocoumon, warfarin, and their known monohydroxylated metabolites in human plasma and urine was developed using a simple, selective solid-phase extraction scheme. Chromatographic separation was achieved on a reversed-phase Luna C18 column and step gradient elution resulted in a total run time of about 13 min. Limits of quantification (LOQ) were ≤40 nM for the parent compounds and ≤25 nM for the metabolites and the limit of detection (LOD) was ≤2.5 nM for all analytes. Average recovery was 84% (±3.7) and 74% (±13.2) in plasma and urine, respectively. Intra- and inter-day coefficients of variation were ≤8.6 and ≤10.6% in plasma and urine, respectively. The method was successfully applied to the analysis of phenprocoumon samples from four healthy volunteers and should prove useful for future comparative studies of warfarin and phenprocoumon pharmacokinetics.