Identification of Trenbolone Metabolites Using Hydrogen Isotope Ratio Mass Spectrometry and Liquid Chromatography/High Accuracy/High Resolution Mass Spectrometry for Doping Control Analysis

Identification of Trenbolone Metabolites Using Hydrogen Isotope Ratio Mass Spectrometry and Liquid Chromatography/High Accuracy/High Resolution Mass Spectrometry for Doping Control Analysis / Marlen Putz, Thomas Piper, Mario Thevis. - (Frontiers in Chemistry (2020) 435 (20 May); p. 1-15)

  • PMID: 32509736
  • PMCID: PMC7251174
  • DOI: 10.3389/fchem.2020.00435

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.

Original document

Parameters

Science
Study
Date
20 May 2020
People
Piper, Thomas
Putz, Marlen
Thevis, Mario
Country
Germany
Language
English
Other organisations
Deutsche Sporthochschule Köln (DSHS) - German Sport University Cologne
Analytical aspects
Mass spectrometry analysis
Testing method development
Doping classes
S1. Anabolic Agents
Substances
Trenbolone (17β-hydroxyestr-4,9,11-trien-3-one)
Document category
Scientific article
Document type
Pdf file
Date generated
2 December 2020
Date of last modification
5 December 2020
Category
  • Legal Source
  • Education
  • Science
  • Statistics
  • History
Country & language
  • Country
  • Language
Other filters
  • ADRV
  • Legal Terms
  • Sport/IFs
  • Other organisations
  • Laboratories
  • Analytical aspects
  • Doping classes
  • Substances
  • Medical terms
  • Various
  • Version
  • Document category
  • Document type
Publication period
Origin