Universität zu Köln

Putz, Marlen, Piper, Thomas and Thevis, Mario (2020). Identification of Trenbolone Metabolites Using Hydrogen Isotope Ratio Mass Spectrometry and Liquid Chromatography/High Accuracy/High Resolution Mass Spectrometry for Doping Control Analysis. Front. Chem., 8. LAUSANNE: FRONTIERS MEDIA SA. ISSN 2296-2646

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Abstract

Trenbolone is a synthetic anabolic-androgenic steroid, which has been misused for performance enhancement in sports. The detection of trenbolone doping in routine sports drug testing programs is complex as methods utilizing gas chromatography/mass spectrometry are complicated by unspecific derivatization products and artifacts, and liquid chromatography/mass spectrometry-based assays have shown to allow for comparably high limits-of-detection only. The number of previously reported metabolites in human urine is limited, and most analytical methods rely on targeting epitrenbolone, trenbolone glucuronide, and epitrenbolone glucuronide. In order to probe for the presence of additional trenbolone metabolites and to re-investigate the metabolism, an elimination study was conducted. One single dose of 10 mg of 5-fold deuterated trenbolone was administered to a healthy male volunteer and urine samples were collected for 30 days. For sample processing, published protocols were combined considering unconjugated, glucuronic acid-, sulfo- and alkaline-labile conjugated steroid metabolites. The sample preparation strategy consisted of solid-phase extractions, liquid-liquid extractions, metabolite de-conjugation, HPLC fractionation, and derivatization. Analytical methods included gas chromatography/thermal conversion/hydrogen isotope ratio mass spectrometry combined with single quadrupole mass spectrometry as well as liquid chromatography/high accuracy/high resolution mass spectrometry of the hydrolyzed and non-hydrolyzed samples. Twenty deuterium-labeled metabolites were identified including glucuronic acid-, sulfo- and potential cysteine-conjugates, and characterized by parallel reaction monitoring experiments yielding corresponding product ion mass spectra. Main metabolites were attributed to trenbolone-diol and potential trenbolone-diketone derivatives excreted as glucuronic acid and sulfo-conjugated analytes with detection windows of 5, respectively 6 days. Further characterization was conducted with pseudo MS(3)experiments of the intact conjugates and by comparison of resulting product ion mass spectra with reference material.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Putz, Marlen UNSPECIFIED UNSPECIFIED UNSPECIFIED Piper, Thomas UNSPECIFIED UNSPECIFIED UNSPECIFIED Thevis, Mario UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-333337
DOI:	10.3389/fchem.2020.00435
Journal or Publication Title:	Front. Chem.
Volume:	8
Date:	2020
Publisher:	FRONTIERS MEDIA SA
Place of Publication:	LAUSANNE
ISSN:	2296-2646
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language ENDOGENOUS URINARY STEROIDS; CONJUGATED ANDROSTENEDIONE; C-13/C-12 RATIOS; ANABOLIC AGENTS; TESTOSTERONE; TETRAHYDROGESTRINONE; DISCOVERY; ACETATE Multiple languages Chemistry, Multidisciplinary Multiple languages
URI:	http://kups.ub.uni-koeln.de/id/eprint/33333