Detection of urinary metabolites of arimistane in humans by gas chromatography coupled to high‐accuracy mass spectrometry for antidoping analyses

Abstract

Rationale The selection of the most appropriate metabolites of the substances included in the Prohibited List of the World Anti‐Doping Agency (WADA) is fundamental for setting up methods allowing the detection of their intake by mass spectrometric methods. The aim of this work is to investigate the metabolism of arimistane (an aromatase inhibitor included in the WADA list) in order to improve its detection capacity among the antidoping community. Methods Urinary samples collected after controlled single administration of arimistane in three healthy volunteers were analysed using the common routine sample preparation in antidoping laboratories to determine the steroid profile parameters considered in the steroid module of the Athlete Biological Passport by gas chromatography coupled to tandem mass spectrometry (GC/MS/MS). For the elucidation of the proposed metabolites, GC coupled to high‐accuracy MS (GC/qTOFMS) was used. Both mass spectrometers were operated in electron ionization mode. Non‐conjugated (free), glucuronated and sulfated fractions were analysed separately. Results No relevant effects on the steroid profile could be detected after a single oral dose (25 mg). Up to 15 metabolites, present only in the post‐administration samples, were detected and some structures were postulated. These metabolites are mainly excreted as glucuro‐conjugated into urine and only minor amounts of two metabolites are also excreted unconjugated or as sulfates. Conclusions Arimistane itself was not observed in the free or glucuronated fractions, but only in the sulfate fraction. The peaks showing mass spectra in agreement with hydroxylated metabolites did not match with those for 7‐keto‐DHEA, 7α‐ or 7β‐hydroxy‐DHEA. This suggests that the first hydroxylation did not occur on C3, but on C2. These newly described metabolites allow the specific detection of arimistane misuse in sports.

7‐keto‐DHEAmetabolism in humans. Pitfalls in interpreting the analytical results in the antidoping field

“…The presence of arimistane in concentrations higher than its 7β‐hydroxy metabolite could be an adequate marker of 7‐keto‐DHEA administration because after administration of arimistane, arimistane itself is almost undetectable in urine …”

“…The presence of arimistane in concentrations higher than its 7βhydroxy metabolite could be an adequate marker of 7-keto-DHEA administration because after administration of arimistane, arimistane itself is almost undetectable in urine. 43 To disclose whether arimistane is or is not an in vivo metabolite of 7-keto-DHEA requires the use of methodologies avoiding derivatization and/or the removal of the sulfate moiety as liquid chromatography coupled to mass spectrometry.…”

Detection of urinary arimistane metabolites in humans using liquid chromatography–mass spectrometry: Complementary results to gas chromatography mass spectrometric data and its application to antidoping analyses

“…Figure 1 shows the structures of previously proposed metabolites 4 . They correspond to reduced structures of Arim in positions C7 or C17, with molecular formula C 19 H 26 O 2 and monoisotopic mass m/z 286.1933 ([M + H] + m/z 287.2006).…”

“…This software predicts metabolic transformations related to cytochrome and flavin‐containing monooxygenase‐mediated reactions in phase I metabolism. It considers both enzyme‐substrate recognition and the chemical transformations induced by the enzyme 4,8 …”

Annual banned‐substance review: Analytical approaches in human sports drug testing 2019/2020

“…The formation of 4,5‐dihydrotestolactone and 1,2,4,5‐tetrahydrotestolactone as well as an increased concentration of T and androstenedione was determined by LC‐HRMS, suggesting that the animal model could be a valuable tool in studying steroid metabolic reactions for doping control purposes 141 . Biotransformation products of another steroidal aromatase inhibitor referred to as arimistane were investigated by Martinez Brito et al 142 Here, three study participants orally ingested 25 mg of arimistane, and urine samples were collected prior to and up to 95 hr post‐administration for subsequent GC‐QqQ‐ and GC‐Q/TOF‐MS analysis to probe for a potential influence of arimistane on the steroid profile as well as arimistane‐specific target analytes. While steroid profile analyses were conducted following standard protocols, a sequential purification of arimistane metabolites originating from different fractions (unconjugated, glucuronidated, and sulfated) was employed for in‐depth characterization of the drug's biotransformation.…”