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

J Chromatogr B Biomed Sci Appl. 1999 Apr 30;727(1-2):95-105. 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 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.

Publication types

  • Clinical Trial
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Androstane-3,17-diol / analogs & derivatives
  • Androstane-3,17-diol / urine*
  • Biomarkers
  • Doping in Sports*
  • Humans
  • Isotopes
  • Male
  • Mass Spectrometry / methods*
  • Middle Aged
  • Pregnanediol / urine*
  • Reference Standards
  • Reference Values
  • Testosterone / urine*


  • Biomarkers
  • Isotopes
  • Androstane-3,17-diol
  • Testosterone
  • Pregnanediol