Direct determination of verapamil in rat plasma by coupled column microbore-HPLC method

This report describes an automated coupled column microbore-high-performance liquid chromatography (HPLC) with fluorescence detection for direct determination of verapamil in small volume of rat plasma. We used HPLC system consisting of three columns such as precolumn, intermediate and analytical column and six-port switching valve and injected small volume of rat plasma to the system without sample preparation. An aliquot of sample was directly injected into Capcell Pak MF Ph precolumn for clean-up and enrichment, 35 mm Capcell Pak C18, intermediate column for concentration of compounds and 250 mm Capcell Pak C18 analytical column for separation of compounds and two mobile phases are used as mobile phase A (50 mM ammonium phosphate, pH 4.5) and B (50 mM ammonium phosphate:acetonitrile = 70:30 v/v). Analysis of verapamil and internal standard, propranolol was performed with direct injection of 10 μl of rat plasma to the system and were eluted at 22 and 12 min, respectively, at a mobile phase flow rate of 0.5 (mobile phase A) and 0.15 ml/min (mobile phase B). The peaks of verapamil and internal standard were good shapes and well separated from any interfering endogenous peaks during a total run time of 25 min. The calibration curve for verapamil showed good linearity (r2 = 0.9997) over the concentration range of 0.01-2.50 μg/ml. The mean RSD (%) values of intra-day (n = 5) and inter-day (n = 5) variability of verapamil ranged from 1.96 to 9.06 and 0.62 to 3.08%, respectively. The LOD and LOQ were 0.01 and 0.025 μg/ml, respectively, for verapamil using 10 μl of rat plasma. An automated coupled column microbore-HPLC method was successfully applied to a pharmacokinetic study after intravenous injection of 3 mg/kg of verapamil to the normal and dimethylnitrosamine (DMN)-induced hepatofibrotic rats.