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 MS3 experiments of the intact conjugates and by comparison of resulting product ion mass spectra with reference material.
Highlights
Trenbolone (Tren) belongs to the class of synthetic anabolicandrogenic steroids (AAS) and is structurally characterized by a 4,9,11-triene-3-one structure composing a highly conjugated πelectron system (Figure 1)
The number of previously reported Tren metabolites is scarce, and for doping control purpose the analysis focuses at present on the main human urinary metabolites epitrenbolone (EpiTREN), epitrenbolone glucuronide (EpiTREN Glu), and trenbolone glucuronide (TREN Glu) (De Boer et al, 1991; Schänzer, 1996)
An approach utilizing stable isotope-labeled substrates facilitating the investigation of biotransformations by GC-TC-IRMS was employed
Summary
Trenbolone (Tren) belongs to the class of synthetic anabolicandrogenic steroids (AAS) and is structurally characterized by a 4,9,11-triene-3-one structure composing a highly conjugated πelectron system (Figure 1). The use of trenbolone has been prohibited by the World Anti-Doping Agency (WADA) at all times, categorized under S1 1. (anabolic androgenic steroids) in the Prohibited List (WADA, 2020). According to WADA’s annual statistics, anabolic agents are the most frequently misused substance group in sports with a total of 1,823 adverse analytical findings (AAFs) in 2018. Within this group, Tren occurrences account for 6% (WADA, 2019).
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