Development and validation of an ultra performance liquid chromatography tandem mass method for sildenafil and N-desmethyl sildenafil plasma determination and quantification

•Only fully FDA validated method for sildenafil and N-desmethyl sildenafil quantification.•The only method using UPLC–MS/MS technology for adult patients.•Easy to use method in clinical routine.

Sildenafil is a selective inhibitor of cGMP-specific type 5 phosphodiesterase (PDE5) used for the treatment of masculine erectile dysfunction and Pulmonary Arterial Hypertension (PAH). Sildenafil causes vasodilatation; relax of the smooth muscle and reduction of pulmonary arterial pressure. In the liver cytocrome P450 metabolizes sildenafil into its active metabolite, N-desmethyl sildenafil. The determination of plasma levels of sildenafil and N-desmethyl sildenafil could be useful for therapy optimization and pharmacokinetic studies. We have developed and validated a new method for the quantification of sildenafil and its metabolite in human plasma by rapid protein precipitation extraction, using an UPLC system, coupled with a tandem mass spectrometric detector (UPLC–MS/MS). The calibration range was fitted at least square model (r2 ≥ 0.999), with an accuracy and an intra- and inter-day RSD% (Relative Standard Deviation), both for sildenafil and N-desmethyl sildenafil, lower than 15%, as required by the FDA guidelines; LLOQ, LLOD, ULOQ were 3.9 ng/mL, 1.95 ng/mL and 1000 ng/mL, respectively, for both analytes. Matrix effect, expressed as mean percent deviation of peak areas, was in the range between 2.6% and 5.8%, lower than 15% as required by guidelines. The mean recovery was 83.2 % for sildenafil and 84.5% for N-desmethyl sildenafil. This method has successfully been applied to a clinical pharmacokinetic study of sildenafil and N-desmethyl sildenafil in patients with PAH undergoing cardiac surgery.