Screening urine for exogenous testosterone by isotope ratio mass spectrometric analysis of one pregnanediol and two androstanediols

• Published in: Journal of chromatography. B, Biomedical sciences and applications Vol. 727; no. 1; pp. 95 - 105
• Main Authors: Aguilera, Rodrigo, Catlin, Don H., Becchi, Michel, Phillips, Andy, Wang, Cristina, Swerdloff, Ronald S., Pope, Harrison G., Hatton, Caroline K.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 30.04.1999; Elsevier Science; Elsevier
• Subjects: Adult; Analytical, structural and metabolic biochemistry; Androstane-3,17-diol - analogs & derivatives; Androstane-3,17-diol - urine; Biochemistry, Molecular Biology; Biological and medical sciences; Biomarkers; Doping in Sports; Fundamental and applied biological sciences. Psychology; Humans; Isotopes; Life Sciences; Male; Mass Spectrometry - methods; Middle Aged; Other biological molecules; Pregnanediol - urine; Reference Standards; Reference Values; Terpenes, steroids. Hormones; Testosterone - urine; Testosterone; Steroids; Human; Urine; Androgen; Doping; Isotopic composition; Solid liquid separation; Sport; Analytical method; Testicular hormone; Sex steroid hormone; Anabolic agent; Mass spectrometry
• ISSN: 0378-4347; 1387-2273
• DOI: 10.1016/S0378-4347(99)00066-3

We propose a new screening method for testosterone (T) doping in sport. The current method for detecting T administration is based on finding a T to epitestosterone ratio (T/E) in urine that exceeds six. The difficulties with T/E are that T administration does not always result in a T/E>6 and that a rare individual will have T/E>6 in the absence of T administration. Our previous studies reveal that carbon isotope ratio helps to determine the origin of the urinary T because the values for T and its metabolites decrease after the administration of exogenous T. In this study, we present a rapid and efficient screening sample preparation method based on three successive liquid–solid extractions, deconjugation with E. coli β-glucuronidase after the first extraction, acetylation after the second extraction, and a final extraction of the acetates. The 13C/ 12C of two T metabolites (5β-androstane-3α,17β-diol and 5α-androstane-3α,17β-diol) and one pregnanediol as endogenous reference (5β-pregnane-3α,20α-diol) was measured by gas chromatography–combustion–isotope ratio mass spectrometry (GC–C–IRMS) on 10 ml of urine collected from 10 healthy men before and after T administration. Following T administration, the 13C/ 12C of 5β-androstane-3α,17β-diol diacetate and 5α-androstane-3α,17β-diol diacetate declined significantly from −26.2‰ to −30.8‰ and from −25.2‰ to −29.9‰, respectively and the 13C/ 12C of 5β-pregnane-3α,20α-diol diacetate was unchanged. In addition, the ratio of androstanediols to pregnanediol increased in the post-T urines.