Simultaneous direct determination of the enantiomers of verapamil and norverapamil in plasma using a derivatized amylose high-performance liquid chromatographic chiral stationary phase.

Chiralpak AD is a commercially available high-performance liquid chromatographic column containing a chiral stationary phase composed of 3,5-dimethylphenylcarbamate-derivatized amylose coated on silica. This column was applied to the assay for the plasma concentrations of the enantiomers of verapamil and its major metabolite norverapamil. After the extraction from plasma, the analytes were separated on a diol silica column (LiChrocart DIOL) and Chiralpak AD column which were connected in series in this order and detected using a fluorescence detector (excitation at 272 nm, emission at 317 nm). The enantiomers of verapamil and norverapamil were separated from each other and other metabolites using a mobile phase of hexane-isopropanol-ethanol (85:7.5:7.5, v/v/v) containing 1.0% triethylamine. The calibration curves were linear (R greater than 0.9989) in the plasma concentration range 2.5-100 ng/ml for verapamil enantiomers and 5.0-100 ng/ml for norverapamil enantiomers. The intra-day and inter-day reproducibility tests showed good reproducibilities; coefficients of variation of each enantiomer were less than 14.9% at the lowest concentration and less than 2.0% at the highest concentration. The effect of organic modifier content and column temperature upon the retention and the enantioseparation were also discussed.