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Published:
Journal of Analytical Toxicology,
ISSN 0146-4760,
Volume 27, Number 2, March 2003,
pp. 88-94
HPLC–MS for the Determination of Sildenafil Citrate
(Viagra®) in Biological Fluids. Application to the Salivary Excretion
of Sildenafil after Oral Intake
A. Tracqui* and B. Ludes
Institut de Médecine Légale, Faculté de Médecine
de Strasbourg, 11 rue Humann, 67085 Strasbourg, France
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). Reproduction
of editorial content of this journal is prohibited without publisher’s
permission.
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