Description

Trenbolone is a synthetic androgenic anabolic steroid that has been abused to improve athletic performance. Detection of trenbolone doping in routine sports doping screening programs is complex because gas chromatography / mass spectrometry methods complicate non-specific derivatization products and liquid chromatography-based artifacts and tests. Mass spectrometry has been shown to only allow detection limits.

The number of metabolites previously reported in urine is limited and most analytical methods are based on epitrenbolone, trenbolone glucuronide and epitrenbolone glucuronide.

An elimination study was performed to detect the presence of other metabolites of trenbolone and to reexamine the metabolism. A single 10 mg dose of deuterated trenbolone was administered 5 times to a healthy male volunteer and urine samples were taken for 30 days. For sample processing, published protocols have been published with respect to the unconjugated, glucuronic, sulfo- and alkaline-labile conjugated steroid metabolites. The sample preparation strategy consisted of solid phase extractions, liquid-liquid extractions, metabolite deconjugation, HPLC fractionation and derivatization.

Analytical methods included gas chromatography / thermal conversion / isotopic ratio of hydrogen mass spectrometry combined with simple four-pole mass spectrometry and high precision / high resolution liquid chromatography / mass spectrometry of hydrolyzed samples. and not hydrolyzed.

Twenty deuterium-labeled metabolites, including glucuronic acid, sulfo, and potential conjugates of cysteine, were identified and characterized by experiments to monitor side reactions that produced the corresponding mass spectra of the outputs. The major metabolites were attributed to trenbolone diol and a potential derivative of trenbolone diketone secreted as glucuronic acid and by a sulfoconjugated analyte with a detection window of 5 and 6 days, respectively.

Further characterization was performed by pseudo MS3 experiments of intact conjugates and by comparing the ion mass spectra of the final product with a reference material.