Determination of warfarin enantiomers and hydroxylated metabolites in human blood plasma by liquid chromatography with achiral and chiral separation

An assay comprising two simple, selective and isocratic HPLC methods with UV detection was developed and validated for measuring warfarin enantiomers and all five warfarin monohydroxylated metabolites in patient blood plasma. Following liquid/liquid extraction from 1 ml of blood plasma a baseline separation of analytes was achieved on chiral (α1 acid glycoprotein – AGP) and achiral (C18) column. Both methods were consistent (R.S.D. < 6.9% for warfarin enantiomers and < 8.9% for monohydroxylated metabolites) and linear (r > 0.998). The limits of detection were 25 ng/ml for warfarin enantiomers, 25 ng/ml for 4′-, 10-, 6- and 7-hydroxywarfarin, 35 ng/ml for 8-hydroxywarfarin and 50 ng/ml for racemic warfarin. In a clinical study in 204 patients, it was confirmed that the assay is appropriate for evaluation of influences of genetic polymorphisms, demographic factors and concomitant drug treatment on warfarin metabolism.