Liquid chromatography/tandem mass spectrometry method for the simultaneous determination of vardenafil and its major metabolite, N-desethylvardenafil, in human plasma: Application to a pharmacokinetic study

A rapid and sensitive LC-MS/MS method for the determination of vardenafil and its major metabolite, N-desethylvardenafil, in human plasma using sildenafil as an internal standard was developed and validated. The analytes were extracted from 0.25-mL aliquots of human plasma by liquid-liquid extraction, using 1 mL of ethyl acetate. Chromatographic separation was carried on a Luna C₁₈ column (50 mm × 2.0 mm, 3 μm) at 40 °C, with an isocratic mobile phase consisting of 10 mM ammonium acetate (pH 5.0) and acetonitrile (10:90, v/v), a flow rate of 0.2 mL/min, and a total run time of 2 min. Detection and quantification were performed using a mass spectrometer in the selected reaction-monitoring mode with positive electrospray ionization at m/z 489.1 → 151.2 for vardenafil, m/z 460.9 → 151.2 for N-desethylvardenafil, and m/z 475.3 → 100.1 for the internal standard (IS), respectively. This assay was linear over a concentration range of 0.5-200 ng/mL with a lower limit of quantification of 0.5 ng/mL for both vardenafil and N-desethylvardenafil. The coefficient of variation for the assay precision was <13.6%, and the accuracy was >93.1%. This method was successfully applied to a pharmacokinetic study after oral administration of vardenafil 20 mg tablet in Korean healthy male volunteers.