Screening urine for exogenous testosterone by isotope ratio mass spectrometric analysis of one pregnanediol and two androstanediols.
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 beta-glucuronidase after the first extraction, acetylation after the second extraction, and a final extraction of the acetates. The 13C/12C of two T metabolites (5beta-androstane-3alpha,17beta-diol and 5alpha-androstane-3alpha,17beta-diol) and one pregnanediol as endogenous reference (5beta-pregnane-3alpha,20alpha-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 13 C/12C of 5beta-androstane-3alpha,17beta-diol diacetate and 5alpha-androstane-3alpha,17beta-diol diacetate declined significantly from -26.2 per thousand to -30.8 per thousand and from -25.2 per thousand to -29.9 per thousand, respectively and the 13C/12C of 5beta-pregnane-3alpha,20alpha-diol diacetate was unchanged. In addition, the ratio of androstanediols to pregnanediol increased in the post-T urines.
Aguilera
R
R
Department of Molecular and Medical Pharmacology, University of California at Los Angeles, 90025-6106, USA. rodrigoa@ucla.edu
Catlin
D H
DH
Becchi
M
M
Phillips
A
A
Wang
C
C
Swerdloff
R S
RS
Pope
H G
HG
Hatton
C K
CK
D000328:Adult; D015113:Androstane-3,17-diol / Q000031:analogs & derivatives / Q000652:urine*; D015415:Biomarkers; D004300:Doping in Sports; D006801:Humans; D007554:Isotopes; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D008875:Middle Aged; D011276:Pregnanediol / Q000652:urine*; D012015:Reference Standards; D012016:Reference Values; D013739:Testosterone / Q000652:urine*
D015415:Biomarkers; D007554:Isotopes; D015113:Androstane-3,17-diol; D013739:Testosterone; D011276:Pregnanediol