Analysis of acenocoumarin and its amino and acetamido metabolites in body fluids by high-performance liquid chromatography.

Acenocoumarin and its acetamido metabolite, after extraction at pH 4.4, were analysed by isocratic reversed-phase high-performance liquid chromatography using aqueous acetonitrile (pH 4.90) as eluent. Warfarin was used as internal standard. The amino metabolite, after back-extraction into 0.5 N HCl, was derivatized by diazotization and heat treatment. The resulting product was analysed by the same reversed-phase system. The sensitivity of the method for acenocoumarin and its amino metabolite was in the range of 20 ng/ml. To achieve this sensitivity for the analysis of the acetamido compound, the acetonitrile content of the eluent had to be lowered. The assay was applied to the analysis of plasma samples of patients under acenocoumarin therapy. The disposition of the amino compound in rats was investigated.