Development of a high performance liquid chromatography method for quantification of PAC-1 in rat plasma

A sensitive and specific high performance liquid chromatography method with UV detection was developed and validated for the determination of PAC-1 in rat plasma. After extraction with ethyl acetate, the chromatographic separation was carried out on a Diamonsil C₁₈ column (150 mm × 4.6 mm i.d., 5 μm particle size, Zhonghuida) protected by a ODS guard column (10 mm × 4.6 mm i.d., 5 μm particle size), using acetonitrile–methanol–phosphate buffer (pH 3.0, 30 mM) (31:3:66, v/v/v) as mobile phase at a flow rate of 1.0 mL/min, and wavelength of the UV detector was set at 281 nm. No interference from any endogenous substances was observed during the elution of PAC-1 and internal standard (IS, indapamide). The calibration curve was linear over the range of 0.05–20 μg/mL (r > 0.99). The lower limit of quantification was evaluated to be 50 ng/mL. The method was successfully applied to the pharmacokinetic study of PAC-1 after intravenous and oral administration in rats, respectively.