Determination of 13C/12C ratios of endogenous urinary steroids: method validation, reference population and application to doping control purposes
Article first published online: 6 JUN 2008
Copyright © 2008 John Wiley & Sons, Ltd.
Rapid Communications in Mass Spectrometry
Volume 22, Issue 14, pages 2161–2175, 30 July 2008
How to Cite
Piper, T., Mareck, U., Geyer, H., Flenker, U., Thevis, M., Platen, P. and Schänzer, W. (2008), Determination of 13C/12C ratios of endogenous urinary steroids: method validation, reference population and application to doping control purposes. Rapid Commun. Mass Spectrom., 22: 2161–2175. doi: 10.1002/rcm.3601
- Issue published online: 6 JUN 2008
- Article first published online: 6 JUN 2008
- Manuscript Accepted: 13 MAY 2008
- Manuscript Revised: 9 MAY 2008
- Manuscript Received: 7 MAR 2008
- Federal Ministry of the Interior of the Federal Republic of Germany, the Manfred Donike Institute (MDI), Cologne and the World Antidoping Agency (WADA)
The application of a comprehensive gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS)-based method for stable carbon isotopes of endogenous urinary steroids is presented. The key element in sample preparation is the consecutive cleanup with high-performance liquid chromatography (HPLC) of underivatized and acetylated steroids, which allows the isolation of ten analytes (11β-hydroxyandrosterone, 5α-androst-16-en-3β-ol, pregnanediol, androsterone, etiocholanolone, testosterone, epitestosterone, 5α-androstane-3α,17β-diol, 5β-androstane-3α,17β-diol and dehydroepiandrosterone) from a single urine specimen. These steroids are of particular importance to doping controls as they enable the sensitive and retrospective detection of steroid abuse by athletes.
Depending on the biological background, the determination limit for all steroids ranges from 5 to 10 ng/mL for a 10 mL specimen. The method is validated by means of linear mixing models for each steroid, which covers repeatability and reproducibility. Specificity was further demonstrated by gas chromatography/mass spectrometry (GC/MS) for each analyte, and no influence of the sample preparation or the quantity of analyte on carbon isotope ratios was observed. In order to determine naturally occurring 13C/12C ratios of all implemented steroids, a reference population of n = 61 subjects was measured to enable the calculation of reference limits for all relevant steroidal Δ values. Copyright © 2008 John Wiley & Sons, Ltd.