SIMULTANEOUS DETERMINATION OF THE ENANTIOMERS OF PIMOBENDAN AND ITS MAIN METABOLITE IN RAT PLASMA BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

A high-performance liquid chromatographic method for the simultaneous analysis of the enantiomers of pimobendan and its main metabolite, wit h an n-hexane-ethanol-acetic acid solvent system, has been developed. After solid-phase extraction from plasma, the enantiomers were separat ed from each other using a Sumichiral OA-4400 column, which is commerc ially available and contains a chiral stationary phase composed of (S) -proline and (S)-1-(alpha-naphthyl)ethylamine coated on silica. The en antiomers were detected with a fluorescence detector (excitation at 33 0 nm, emission at 415 nm). The intra- and inter-day precision studies showed good reproducibilities: the coefficients of variation were less than 10.3% for pimobendan enantiomers and 13.0% for metabolite enanti omers. The calibration curves were linear (r2 > 0.996) in the concentr ation range 1.25 200 ng/ml. The minimum measurable level was 125 pg pe r 100 mul of plasma. The method was used in a preliminary pharmacokine tic study in three male rats after intravenous administration of racem ic pimobendan (2 mg/kg).