Biochemical applications of a quantitative high-pressure liquid chromatographic assay of warfarin and its metabolites.

A quantitative high-pressure liquid chromatographic assay of warfarin and its diastereoisomeric alcohols and 4'-, 6-, 7-, 8- and benzylic hydroxylated metabolites was accomplished using a reversed-phase, microparticle column (muBondapak/C18) employing 1.5% acetic acid (pH 4.7)-acetonitrile (69:31) as solvent. Concentration of these compounds on the column prior to their elution permitted the loading of 0.01-0.5 ml solutions of warfarin and metabolites without consequent alterations in peak heights and resolution. Details of the use of this technique in the study of R and S warfarin metabolism by hepatic mixed function oxidases and in the determination of warfarin and its metabolites in blood are presented.