SIMULTANEOUS HPLC ANALYSIS OF S- AND R-VERAPAMIL AND METABOLITES, S- AND R-NORVERAPAMIL IN HUMAN PLASMA

Abstract

This study aimed to develop a simple, sensitive, and accurate HPLC method for the simultaneous determination of verapamil and norverapamil enantiomers in human plasma. An internal standard (diphenhydramine) was used and separations of verapamil and norverapamil enantiomers were done at room temperature. Chromatographic separation was achieved using a Chiralcel^® OD-RH column (5 μm, 4.6 mm i.d. × 15 cm, Daicel Chemical Industries Ltd., Tokyo, Japan) and a mobile phase consisting of 30 mM hexafluorophosphate and acetonitrile (66 : 34, v/v, pH 4.6). Detection of S- and R-verapamil and the metabolites, S- and R-norverapamil was accomplished with a fluorescence detector. The wavelengths of excitation and emission were set at 280 nm and 315 nm respectively.

The inter-day coefficients of variation (CV) of S-verapamil, R-verapamil, S-norverapamil, and R-norverapamil from extracted human plasma samples (at a high concentration, 250 ng/mL) were 9.8%, 5.3%, 4.3%, and 5.8%, respectively, while at a low concentration of 10 ng/mL, the CV were 15.9%, 17.5%, 15.6%, and 14.9%, respectively. The recovery was 117%, 117%, 110%, and 96.3% for S-verapamil, R-verapamil, S-norverapamil, and R-norverapamil respectively at low concentration (10 ng/mL) and was 95%, 108%, 124%, and 107%, at high concentration (250 ng/mL), respectively. The plasma samples were stable for at least three months after the samples were collected and stored at 20°C. The lower detection limit for each of S- and R-verapamil and the metabolites, S- and R-norverapamil in human plasma was 10 ng/mL. The present method is simple because it does not require a column-switching system or organic solvent as a component in the mobile phase, and the sample preparation is more economic as solid-phase extraction is not required. Thus, the assay described in this report is suitable for studying the pharmacokinetics and metabolism of verapamil.

Acknowledgments