Analytical and Bioanalytical Chemistry

, Volume 405, Issue 16, pp 5455–5466 | Cite as

Combination of carbon isotope ratio with hydrogen isotope ratio determinations in sports drug testing

  • Thomas PiperEmail author
  • Caroline Emery
  • Andreas Thomas
  • Martial Saugy
  • Mario Thevis
Research Paper


Carbon isotope ratio (CIR) analysis has been routinely and successfully applied to doping control analysis for many years to uncover the misuse of endogenous steroids such as testosterone. Over the years, several challenges and limitations of this approach became apparent, e.g., the influence of inadequate chromatographic separation on CIR values or the emergence of steroid preparations comprising identical CIRs as endogenous steroids. While the latter has been addressed recently by the implementation of hydrogen isotope ratios (HIR), an improved sample preparation for CIR avoiding co-eluting compounds is presented herein together with newly established reference values of those endogenous steroids being relevant for doping controls. From the fraction of glucuronidated steroids 5β-pregnane-3α,20α-diol, 5α-androst-16-en-3α-ol, 3α-Hydroxy-5β-androstane-11,17-dione, 3α-hydroxy-5α-androstan-17-one (ANDRO), 3α-hydroxy-5β-androstan-17-one (ETIO), 3β-hydroxy-androst-5-en-17-one (DHEA), 5α- and 5β-androstane-3α,17β-diol (5aDIOL and 5bDIOL), 17β-hydroxy-androst-4-en-3-one and 17α-hydroxy-androst-4-en-3-one were included. In addition, sulfate conjugates of ANDRO, ETIO, DHEA, 3β-hydroxy-5α-androstan-17-one plus 17α- and androst-5-ene-3β,17β-diol were considered and analyzed after acidic solvolysis. The results obtained for the reference population encompassing n = 67 males and females confirmed earlier findings regarding factors influencing endogenous CIR. Variations in sample preparation influenced CIR measurements especially for 5aDIOL and 5bDIOL, the most valuable steroidal analytes for the detection of testosterone misuse. Earlier investigations on the HIR of the same reference population enabled the evaluation of combined measurements of CIR and HIR and its usefulness regarding both steroid metabolism studies and doping control analysis. The combination of both stable isotopes would allow for lower reference limits providing the same statistical power and certainty to distinguish between the endo- or exogenous origin of a urinary steroid.


Reference population Carbon isotope ratio Hydrogen isotope ratio Doping control Steroid metabolism 



This project was partly funded by the Federal Ministry of the Interior of the Federal Republic of Germany and the Manfred Donike Institute for Doping Analysis, Cologne.

Supplementary material

216_2013_6949_MOESM1_ESM.pdf (166 kb)
ESM 1 (PDF 165 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Thomas Piper
    • 1
    • 2
    Email author
  • Caroline Emery
    • 2
  • Andreas Thomas
    • 1
  • Martial Saugy
    • 2
  • Mario Thevis
    • 1
  1. 1.Center for Preventive Doping Research—Institute of BiochemistryGerman Sport University CologneKölnGermany
  2. 2.Swiss Laboratory for Doping AnalysisUniversity Center of Legal Medicine, Geneva and Lausanne, Centre Hospitalier Universitaire Vaudois and University LausanneEpalingesSwitzerland

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