Determination of verapamil and norverapamil in human plasma by liquid chromatography: Comparison between a liquid—liquid extraction procedure and an automated liquid—solid extraction method for sample preparation

Abstract

A conventional liquid—liquid extraction (LLE) procedure with high-performance liquid chromatography (HPLC) has been developed for the determination of verapamil and its main metabolite, norverapamil, in plasma. After addition of the internal standard, plasma samples were basified with phosphate buffer (pH 9.0) and extracted with a mixture of cyclohexane—dichloromethane. After centrifugation, the organic layer was separated and the analytes were extracted back into a 0.1 N sulphuric acid solution containing 2-aminoheptane. An aliquot of this aqueous phase was then injected directly onto the HPLC column. This LLE procedure has been compared with an automated liquid—solid extraction (LSE) method that has been developed in parallel. Good linearity was obtained using both extraction methods. The absolute recoveries for the two analytes were ca 95% with the automated LSE procedure and slightly lower ( ca 84%) for the LLE method. The automated method gives better results with respect to detectability and precision, but the LLE procedure is simpler to develop, requires much less expensive equipment, and remains a useful alternative when the number of samples to be analysed is limited.