Published:Journal of Chromatographic Science,
ISSN 0021-9665Volume
41, Number 5, May/June 2003, pp 261-266
Development and Method Validation for Testosterone and Epitestosterone
in Human Urine Samples by Liquid Chromatography Applications
R. Gonzalo-Lumbreras, D. Pimentel-Trapero, and R. Izquierdo-Hornillos*
Department of Analytical Chemistry, Faculty of Chemistry, Universidad Complutense,
28040 Madrid, Spain
An isocratic high-performance liquid chromatographic method
for the determination of testosterone (T) and epitestosterone (ET) in human
urine using liquid–liquid or solid-phase extraction (SPE) is developed
and validated. The optimum separation is achieved using a Hypersil C18 column,
water–acetonitrile (57:43, v/v) as the mobile phase and UV-absorbance
detection at 245 nm. The recoveries obtained for T and ET in liquid–liquid
and SPE demonstrate that these procedures are interchangeable. Quantitation
limits for T and ET are 8.6 and 5.4 ng/mL using solvent extraction and 7.3
and 5.7 ng/mL using SPE, respectively. The proposed method is used to evaluate
the urinary T, ET, and the T/ET ratio for a healthy male population using liquid–liquid
extraction, and the T and ET excretion profile for nine healthy men using SPE.
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