Stereospecific fluorescence high-performance liquid chromatographic analysis of warfarin and its metabolites in plasma and urine.

A stereospecific assay for the simultaneous determination of the enantiomers of warfarin and its major metabolites, 6- and 7-hydroxywarfarin and warfarin alcohols, in plasma and urine was developed. Involved in this determination was the formation of diastereoisomeric esters with carbobenzyloxy-L-proline, separation by normal-phase high-performance liquid chromatography, and detection by fluorescence after postcolumn aminolysis with n-butylamine. The determination limit for any enantiomer is in the order of 50-100 ng. The method was applied to the analysis of the enantiomers of warfarin and metabolites in plasma and urine of human subjects receiving racemic drug. The results for warfarin enantiomers are comparable with those obtained by an MS method, involving administration of a synthetic pseudoracemate [12C(R), 13C(S)]warfarin. In addition to all known metabolites, the detection of 7-R-hydroxywarfarin indicates that 7-hydroxylation is stereoselective rather than stereospecific.