Abstract
Warfarin is the only oral anticoagulant used in the prevention of thromboembolism in Japan.The anticoagulant effect of warfarin is monitored by analysis of the prothrombin complex (International Normalised Ratio,INR),but in some cases,measurement of plasma warfarin concentrations is necessary.
We developed simple and sensitive LC/MS/MS methods for this purpose and validated it through assay of four warfarin monohydroxylated metabolites and warfarin enantiomers in human plasma.Preparation of samples from 0.05 mL of plasma involved simple protein precipitation using hydrochloric acid/acetonitrile.Analytes remained stable during sample processing and storage.Chromatographic separation was conducted using a chiral (CHIROBIOTIC V) column and an achiral (Symmetry Shield RP18)column.No significant interference from human plasma components was observed.Assay linearity was demonstrated for all analytes with the LC/MS/MS system over the calibration range 2 to 500 ng/mL.The intra-day and inter-day assay precisions expressed as coefficients of variation (C.V.) for the quality control samples (4,40 and 400 ng/mL) were not more than 9.0%.In conclusion,this system was considered to be reliable enough for the evaluation of pharmacokinetic profiles in planned clinical studies.
