SIMULTANEOUS ANALYSIS OF VERAPAMIL AND NORVERAPAMIL ENANTIOMERS IN HUMAN PLASMA BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

An improved HPLC method for the simultaneous determination of the enan tiomers of verapamil (V) and its major metabolite norverapamil (NV) in human plasma samples is presented. NV is acetylated immediately to N- acetylnorverapamil (ANV) in the extraction solvent (2% butanol in hexa ne). Acetylation is so rapid that it does not delay sample processing. ANV and V enantiomers are then separated on an alpha(1)-acid glycopro tein chiral column with a mobile phase of phosphate buffer (0.01 M, pH 6.65) and acetonitrile. The fluorescence detector wavelengths are set at 227 nm for excitation and 308 nm for emission. Introduction of the internal standard (I.S.) (+)-glaucine improves accuracy, precision an d robustness of the method. The assay is sensitive and specific. Basel ine separation is achieved for both V and ANV. Limits of quantitation are 3 ng/ml for V and 2 ng/ml for NV (single enantiomer) with precisio n and accuracy better than 15% at those levels. Detector response is l inear in the range tested (3-200 ng/ml for V and 2-100 ng/ml for NV, s ingle enantiomer). This assay has been applied to a clinical study of the pharmacodynamics of V involving six healthy volunteers.