HPLC-MS for the determination of sildenafil citrate (Viagra®) in biological fluids. Application to the salivary excretion of sildenafil after oral intake

Abstract

An original high-performance liquid chromatography-mass spectrometry (HPLC-MS) procedure was developed for the determination of sildenafil in biological fluids. Liquid-liquid extraction was performed by chloroform/2-propanol/n-heptane (25:10:65, v/v) at pH 9.5 with 300 ng of buprenorphine-d4 as the internal standard (IS). After agitation (10 min) and centrifugation (3500 × g, 10 min), the organic phase was evaporated and the dry extract resuspended in 25 μL methanol, from which 2 μL was injected onto a NovaPak C18 (Waters) HPLC column. Separation was carried out by a gradient of (acetonitrile + 10 μg/mL trimethylamine) in 2mM NH4COOH pH 3.0 buffer (35-70% in 9 min). Detection was done by a PerkinElmer Sciex API-100 single-quadrupole mass analyzer with an ionspray interface operated in positive-ion mode. MS data were collected as either TIC or SIM at m/z {475 + 534} or {475 + 283} for sildenafil, depending on the potential applied at the ion sampling orifice (0 V or + 100 V). The retention times of sildenafil and the IS were 4.20 and 5.07 min, respectively. Extraction recoveries were always > 87%. LOD and LOQ were 0.2 and 0.5 ng/mL whatever the biological fluid tested. The method appears specific, extremely sensitive, and relatively simple in both equipment and sample preparation. As an example, we present the results of a preliminary study on the salivary excretion of sildenafil following the oral intake (T0) of 25 mg Viagra in a 38-year-old volunteer. Sildenafil was detectable in oral fluid at T0 + 0.5 h (1.2 ng/mL) and peaked at T0 + 1.5 h (8.3 ng/mL), whereas at the same time its plasma concentration was 72.4 ng/mL. Salivary concentrations then rapidly decreased, and the last detectable value (0.9 ng/mL) was at T0 + 5.5 h. It is suggested that the salivary excretion pattern of sildenafil resembles that of benzodiazepines (high plasma protein binding, low saliva-to-plasma ratio).