Epiandrosterone sulfate prolongs the detectability of testosterone, 4-androstenedione, and dihydrotestosterone misuse by means of carbon isotope ratio mass spectrometry Thomas Piper, a * Marlen Putz, a Wilhelm Schänzer, a Valentin Pop, b Malcolm D. McLeod, c Dimanthi R. Uduwela, c Bradley J. Stevenson c and Mario Thevis a,d In the course of investigations into the metabolism of testosterone (T) by means of deuterated T and hydrogen isotope ratio mass spectrometry, a pronounced influence of the oral administration of T on sulfoconjugated steroid metabolites was observed. Espe- cially in case of epiandrosterone sulfate (EPIA_S), the contribution of exogenous T to the urinary metabolite was traceable up to 8 days after a single oral dose of 40 mg of T. These findings initiated follow-up studies on the capability of EPIA_S to extend the detection of T and T analogue misuse by carbon isotope ratio (CIR) mass spectrometry in sports drug testing. Excretion study urine samples obtained after transdermal application of T and after oral administration of 4-androstenedione, dihydrotestosterone, and EPIA were investigated regarding urinary concentrations and CIR. With each administered steroid, EPIA_S was significantly depleted and prolonged the detectability when compared to routinely used steroidal target compounds by a factor of 2 to 5. In order to simplify the sample preparation procedure for sulfoconjugated compounds, enzymatic cleavage by Pseudomonas aeruginosa arylsulfatase was tested and implemented into CIR measurements for the first time. Further simplification was achieved by employing multidimensional gas chromatography to ensure the required peak purity for CIR determinations, instead of sample purification strategies using liquid chromatographic fractionation. Taking into account these results that demonstrate the unique and broad applicability of EPIA_S for the detection of illicit ad- ministrations of T or T-related steroids, careful consideration of how this steroid can be implemented into routine doping control analysis appears warranted. Copyright © 2017 John Wiley & Sons, Ltd. Keywords: testosterone; carbon isotope ratio; epiandrosterone; doping; long-term detectability; sulfoconjugated steroids Introduction Detecting the misuse of testosterone (17β-hydroxyandrost-4-en-3- one, T) is still of major concern for doping control analysis and remains challenging due to the ubiquitous presence of T in human urine. Urinary concentration thresholds or diagnostic ratios such as T / epitestosterone (17α-hydroxyandrost-4-en-3-one, E) may suffice to identify suspicious samples in initial testing procedures but it cannot unambiguously prove the administration of an illicit compound. Confounding factors like ethanol intake or microbial degradation together with intra- and inter-individual variation diminish the probative force of these markers. [1] Carbon isotope ratios (CIR) allow for discrimination between an endogenous or exogenous source of excreted urinary T and T- metabolites. [2,3] Endogenous steroids reflect the isotopic composi- tion of the individual´s diet, whereas exogenous steroids commonly exhibit more depleted CIR, i.e. pharmaceutical preparations contain a lower 13 C content. CIR are expressed as δ 13 C-values against the international standard Vienna Pee Dee Belemnite (VPDB) following equation (1) and given in ‰ or mUr: [4] * Correspondence to: Thomas Piper, German Sport University Cologne, Center for Preventive Doping Research, Am Sportpark Müngersdorf 6, 50933 Cologne, Germany. E-mail: t.piper@biochem.dshs-koeln.de a German Sport University Cologne, Center for Preventive Doping Research, Am Sportpark Müngersdorf 6, 50933 Cologne, Germany b Romanian Doping Control Laboratory, National Anti-Doping Agency, Bvd. Basarabia, nr. 37–39, Bucharest, Romania c Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia d European Monitoring Center for Emerging Doping Agents (EuMoCEDA), Cologne/ Bonn, Germany Drug Test. Analysis 2017, 9, 1695–1703 Copyright © 2017 John Wiley & Sons, Ltd. Research article Drug Testing and Analysis Received: 14 July 2017 Revised: 10 August 2017 Accepted: 17 August 2017 Published online in Wiley Online Library: 27 October 2017 (www.drugtestinganalysis.com) DOI 10.1002/dta.2291 1695