Column-switching high-performance liquid chromatography for on-line simultaneous determination and resolution of enantiomers of verapamil and its metabolites.

An on-line simultaneous assay for the enantiomers of verapamil (VA) and its three metabolites in plasma was developed with column-switching HPLC. This system consists of an ovomucoid protein chiral stationary phase coupled to an achiral reversed-phase column via a dilution tube and a trapping column. The reversed-phase column was used to separate and quantitate VA, its metabolites, and internal standard, without interference from plasma components. Then each of the eluates containing VA and its metabolites was selectively switched into a sampling loop, and the samples were transferred successively to the trapping column after dilution with a new mobile phase. After concentration on the trapping column, each sample was passed to the ovomucoid column, where the enantiomers were resolved and quantitated. The mobile phases for both HPLC columns were independently optimized and the diffusion of the sample during column switching was minimized. This method was shown to be efficient and reliable.