HPLC Determination of Verapamil and Norverapamil in Plasma Using Automated Solid Phase Extraction for Sample Preparation and Fluorometric Detection

Abstract

A sensitive and fully automated method for the simultaneous determination of verapamil and its main metabolite norverapamil in plasma is described, which involves the solid phase extraction (SPE) of the analytes from plasma on disposable extraction cartridges (DECs) and reversed phase HPLC with fluorescence detection. The DEC filled with endcapped cyanopropyl silica (50 mg) was first conditioned with methanol and phosphate buffer of pH 7.4. A 1.0-mL volume of plasma sample containing the internal standard was then applied on the DEC. The washing step was performed with the same buffer. The analytes were eluted with 0.24 mL of methanol containing 0.2 % of 2-aminoheptane. A 0.41-mL volume of acetate buffer of pH 3.0 was then passed through the DEC and 0.25 mL of the resultant extract was directly introduced into the HPLC system. The absolute recoveries of the drugs were around 95 % and the limit of detection of verapamil was 1.0 ng/mL. The within-day and between-day reproducibilities at a plasma concentration of 100 ng/mL were 1.4 % and 1.9 %, respectively.