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Updated: Sep 6, 2025

Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures
Published on: March 28, 2017
Investigations on the in vivo metabolism of 5α-androst-2-en-17-one
Thomas Piper1, Gregor Fusshöller1, Wilhelm Schänzer1
1German Sport University Cologne, Center for Preventive Doping Research - Institute of Biochemistry, Köln, Germany.
Rationale:
The anabolic steroid 5α-androst-2-en-17-one (2EN) is sold as a prohormone and has been investigated regarding its potential as a steroidal aromatase inhibitor. The administration of 2EN was detected in a doping control sample in 2015, and investigations into its metabolism allowed for the identification and characterization of three urinary metabolites. Unfortunately, the utility of the main metabolite 2β,3α-dihydroxy-5α-androstan-17-one for doping control purposes was hampered under routine doping control conditions due to chromatographic issues, thus warranting further studies on the metabolism of the prohibited substance.
Methods:
The metabolism of 2EN was reinvestigated after oral administration of twofold-deuterated 2EN employing hydrogen isotope ratio mass spectrometry (IRMS) in combination with high-accuracy/high-resolution mass spectrometry. After a single dose of 50 mg of doubly labeled 2EN, urine samples were collected for 9 days. All samples were processed using routine doping control methods for IRMS analysis, and all detected metabolites were further characterized by mass spectrometry-based investigations.
Results:
More than 15 different metabolites still containing the deuterium label were detected after administration. The presence of steroids exhibiting a 5β-configuration was unexpected as the administered 2EN features a 5α-configured pharmacophore. Further investigations corroborated a significant impact of the administered 2EN on etiocholanolone and 5β-androstanediol. Seven metabolites of 2EN not present as endogenous compounds were identified as potential candidates for routine doping controls and could be detected for up to 9 days after administration.
Conclusions:
The new metabolites identified in this study enable the detection of the misuse of 2EN for up to 9 days. The conversion of a 5α-steroid to urinary metabolites with 5β-configuration has not been reported so far and should be further investigated.
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