Analysis of warfarin and its metabolites by reversed-phase ion-pair liquid chromatography with fluorescence detection.

A high-performance liquid chromatographic method was developed for the determination of warfarin and its metabolites (diastereomeric warfarin alcohols and 6-, 7-, 8-, 4'- and 3'-hydroxywarfarin) in microbial cultures. Ion-pair chromatography with tetrabutylammonium ion as the counter ion allowed for the complete resolution of all compounds at pH 7.5 on a reversed-phase (C18) column, thus permitting direct fluorescence detection without the use of post-column pH switching techniques. Analysis of cell suspension cultures of the fungus Cunninghamella elegans (ATCC 36112) indicated that this organism metabolizes warfarin to all known mammalian metabolites, plus the previously unreported 3'-hydroxywarfarin. Detection limits for all compounds were in the low nanogram range.