Authors: Tracqui A.; Ludes B.

Source: Journal of Analytical Toxicology, Volume 27, Number 2, March 2003 , pp. 88-94(7)

Publisher: Preston Publications

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).

Document Type: Research article

Affiliations: 1: Institut de Médecine Légale, Faculté de Médecine de Strasbourg, 11 rue Humann, 67085 Strasbourg, France

Publication date: 2003-03-01

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• The Journal of Analytical Toxicology (JAT), established in 1977 and published 9 times a year, is the international source covering a broad range of clinical, forensic, and industrial laboratory topics regarding the isolation, identification, and quantitation of potentially toxic substances.

  With an emphasis on practical application, JAT articles provide improved and novel techniques for use in clinical, forensic, workplace, sports testing (doping), and other toxicology laboratories. Articles describe newly developed methods in immunoassay testing, gas chromatography, liquid chromatography, mass spectrometry, atomic absorption spectrometry, solid and liquid phase extraction techniques, and other analytical approaches. Worldwide readership includes toxicologists, pathologists, chemists, clinicians, researchers, and educators working in medical examiner and law enforcement laboratories, hospitals, university and independent analytical laboratories, as well as the drug manufacturing industry.

  Each year in October, we publish a special issue from the Society of Forensic Toxicologists.

  JAT, as determined by ISI Citation Index, is one of the two most referenced international journals in forensic science.

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