Internally standardized simultaneous assay of verapamil and N-demethyl-verapamil enantiomers in human plasma by means of high performance liquid chromatography.

A high performance liquid chromatographic method with internal analog standardization for the simultaneous assay of the verapamil and N-demethyl-verapamil enantiomers in human plasma after administration of racemic verapamil is described. The method comprises extraction from alkalinized plasma into an n-hexane/2-propanol phase (95:5), separation of the optical antipodes on a chiral phase (Daicel OJ) and quantification by means of fluorescence detection. A detection limit of about 6 pmol was determined for all four enantiomers. Under routine conditions the limit of quantification (variation coefficient < 10%) was about 8 pmol for the (R)- and (S)-enantiomers. The determination method for verapamil and N-demethyl-verapamil enantiomers is not impaired by their known metabolites. The suitability of the method for clinical pharmacology studies is demonstrated with samples obtained from healthy subjects. First results of comparative kinetics studies in humans and various animal species show marked species-dependency with prevailing (R)-enantiomer levels in humans and dogs, and the opposite in rats.