A Novel Method for the Quantitation of Warfarin and its Metabolites in Plasma

Abstract

A procedure for the rapid, quantitative recovery of warfarin and its metabolites (diastereoisomeric alcohol, 4′-, 6-, 7-, 8-benzylic-hydroxywarfarin and dehydrowarfarin) from plasma with Sep-Pak C₁₈ cartridges has been developed. A solution of warfarin and its metabolites in plasma was acidified with NH₄OAc buffer (pH 4.85), adsorbed on the Sep-Pak C₁₈ resin, washed free of polar constituents and eluted with methanol. Dilution of the eluate with buffer followed by gradient high performance liquid chromatography permitted accurate quantitation of the desired compounds when detected at 313 nm. The recovery of warfarin and each metabolite was greater than 95% over an investigated range of 0.5–10.0 μg/ml of plasma and the limit of quantitation by the assay was 0.1 μg/ml of plasma. For more rapid quantitation of warfarin, without simultaneous analysis of metabolites, the chromatographic parameters were modified so that elution of warfarin occurred within 13 minutes, and the sensitivity of the assay increased to 0.03 μg of warfarin/ml of plasma. The quantitative recovery of warfarin and its metabolites coupled with the chromatographic versatility of the method make it ideally suited for either detailed pharmacokinetic studies or routine plasma analysis of warfarin.