Development of a liquid chromatographic method for the determination of sildenafil in seminal plasma

A sensitive high performance liquid chromatographic (HPLC) method with ultraviolet absorption detection (230 nm) was developed and validated for the determination of a phosphodiesterase V inhibitor, Sildenafil, in seminal plasma. A single step liquid-liquid extraction procedure using ethyl acetate was performed to recover sildenafil from 1.0 mL of seminal plasma combined with 200 muL of NaOH 0.1 M. A symmetry C-18 column (150 x 4.6 mm I.D. 5 mum) was used as a stationary phase and the mobile phase consisted of 32% acetonitrile and 68% phosphoric acid (0.016 M; pH 5.3) at a flow rate of 1.0 mL/min. The quantitation limit was 5 ng/mL. Intra- and inter-day relative standard deviation (RSD) did not exceed 6.6%. This HPLC method has been successfully used in medical laboratories to assay seminal plasma samples for studies on the treatment with sildenafil.