Validated high-performance liquid chromatography method for the determination of a phosphodiesterase inhibitor SK5474 in rat plasma and its application to pharmacokinetic study of SK5474

Abstract In this study, a method for the quantitation of SK5474 in rat plasma was developed and validated using high performance liquid chromatography (HPLC). The plasma samples were prepared by deproteinization, and sildenafil was used as an internal standard. Chromatographic separation was achieved using a reversed-phase (C18) column. The mobile phase, 0.02 M ammonium acetate buffer:acetonitrile (48:52, v/v), was run at a flow rate of 1.0 mL/min, and the column eluent was monitored using an ultraviolet detector at 254 nm at room temperature. The retention times of sildenafil and SK5474 were approximately 5.8 and 7.2 min, respectively. The quantitation limit of SK5474 in rat plasma was 0.03 μg/mL. Pharmacokinetic parameters of SK5474 was evaluated after intravenous (i.v.; at doses of 15 mg/kg) and oral (p.o.; at doses of 30 mg/kg) administration of SK5474 in rats. After p.o. administration (30 mg/kg) of SK5474, F (fraction absorbed) value was approximately 46.0%.