DETERMINATION OF UNBOUND WARFARIN ENANTIOMERS IN HUMAN PLASMA AND 7-HYDROXYWARFARIN IN HUMAN URINE BY CHIRAL STATIONARY-PHASE LIQUID-CHROMATOGRAPHY WITH ULTRAVIOLET OR FLUORESCENCE AND ONLINE CIRCULAR-DICHROISM DETECTION

Enantiomers of warfarin and 7-hydroxywarfarin in human plasma and urin e, respectively, were determined by high-performance liquid chromatogr aphy using a cellulose-derivative column with UV or fluorescent detect ion, and their absolute configuration was determined simultaneously by a circular dichroism spectropolarimeter connected in series. Enantiom ers of warfarin and its major metabolites [i.e., (R)-6-hydroxywarfarin , (S)-7-hydroxywarfarin and (RS)-warfarin alcohol] were well resolved. The method was precise and sensitive: within-and between-day coeffici ents of variation were <9.6% for warfarin enantiomers in plasma and <7 .1% for 7-hydroxywarfarin enantiomers in urine, respectively, and the lower detection limits were 20 ng/ml for (R)-warfarin, 40 ng/ml for (S )-warfarin, 2.5 ng/ml for (R)-7-hydroxywarfarin and 4.5 ng/ml for (S)- 7-hydroxywarfarin in 0.5 ml of both plasma and urine. The ultrafiltrat ion technique was used for determining unbound concentrations of warfa rin enantiomers in plasma using [C-14]warfarin enantiomers resolved by the present HPLC system. Clinical applicability of the method was eva luated by determining unbound concentrations of warfarin enantiomers i n five consecutive plasma samples obtained from a patient exhibiting a n unstable anticoagulant response to warfarin (4 mg/day, p.o.). Result s indicated that the present method would be useful in clarifying fact ors responsible for a large intra-and inter-patient variability in war farin effects with regard to unbound plasma enantiomer pharmacokinetic s. (C) 1997 Elsevier Science B.V.