Simple method for determination of the active metabolite of the inotropic drug pimobendan in rat liver microsomes

A rapid and sensitive high-performance liquid chromatographic method for quantitation of the O-demethylated active metabolite formed in a liver microsomal assay system has been developed. The metabolite was separated on an Inertsil ODS-2 column and quantitated by fluorescence detection (excitation at 338 nm, emission at 405 nm). The retention times for pimobendan and its metabolite were 5.6 and 2.8 min, respectively. The intra- and inter-assay relative standard deviations in the measurement of pimobendan O-demethylase activity at the substrate concentrations of 1 μM and 500 μM were 2.0%, 6.8%, 2.1% and 5.6%, respectively, and the limit of detection was 0.1 ng for the demethylated metabolite.