Urinary analysis of four testosterone metabolites and pregnanediol by gas chromatography-combustion-isotope ratio mass spectrometry after oral administrations of testosterone

J Anal Toxicol. 2004 Sep;28(6):426-31. doi: 10.1093/jat/28.6.426.


The most frequently used method to demonstrate testosterone abuse is the determination of the testosterone and epitestosterone concentration ratio (T/E ratio) in urine. Nevertheless, it is known that factors other than testosterone administration may increase the T/E ratio. In the last years, the determination of the carbon isotope ratio has proven to be the most promising method to help discriminate between naturally elevated T/E ratios and those reflecting T use. In this paper, an excretion study following oral administration of 40 mg testosterone undecanoate initially and 13 h later is presented. Four testosterone metabolites (androsterone, etiocholanolone, 5 alpha-androstanediol, and 5 beta-androstanediol) together with an endogenous reference (5 beta-pregnanediol) were extracted from the urines and the delta(13)C/(12)C ratio of each compound was analyzed by gas chromatography-combustion-isotope ratio mass spectrometry. The results show similar maximum delta(13)C-value variations (parts per thousand difference of delta(13)C/(12)C ratio from the isotope ratio standard) for the T metabolites and concomitant changes of the T/E ratios after administration of the first and the second dose of T. Whereas the T/E ratios as well as the androsterone, etiocholanolone and 5 alpha-androstanediol delta(13)C-values returned to the baseline 15 h after the second T administration, a decrease of the 5 beta-androstanediol delta-values could be detected for over 40 h. This suggests that measurements of 5 beta-androstanediol delta-values allow the detection of a testosterone ingestion over a longer post-administration period than other T metabolites delta(13)C-values or than the usual T/E ratio approach.

Publication types

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

MeSH terms

  • Administration, Oral
  • Adult
  • Algorithms
  • Androgens / urine
  • Biotransformation
  • Carbon Dioxide / metabolism
  • Gas Chromatography-Mass Spectrometry
  • Humans
  • Male
  • Mass Spectrometry
  • Pregnanediol / urine*
  • Solvents
  • Testosterone / administration & dosage
  • Testosterone / pharmacokinetics
  • Testosterone / urine*


  • Androgens
  • Solvents
  • Carbon Dioxide
  • Testosterone
  • Pregnanediol