Development and application of analytical procedures for the GC-MS/MS analysis of the sulfates metabolites of anabolic androgenic steroids: The pivotal role of chemical hydrolysis

Development and application of analytical procedures for the GC-MS/MS analysis of the sulfates metabolites of anabolic androgenic steroids : The pivotal role of chemical hydrolysis / Michele Iannone, Francesco Botrè, Dayamin Martinez-Brito, Roberta Matteucci, Xavier de la Torre. - (Journal of Chromatography B 1155 (2020) 122280 (15 October))

  • PMID: 32763466
  • DOI: 10.1016/j.jchromb.2020.122280


In this work, we present a gas-chromatography tandem mass spectrometry (GC-MS/MS) method for the identification of the sulfo-conjugate metabolites of pseudo-endogenous steroids (endogenous steroids when administered exogenously). We have preliminarily evaluated the performances of different preparations of sulfatases from Pseudomonas aeruginosa and Helix pomatia, characterized by various origins and catalytic activities, and compared the efficacy of the enzymatic hydrolysis with chemical hydrolysis, performed with a mixture of ethyl acetate, methanol, and sulphuric acid. A procedure for the selective isolation of steroid conjugates from the urine matrix has been designed and optimized, based on the "sequential" extraction of the glucuro-conjugated and of the sulfo-conjugated fractions, performed by two different direct methods, i.e. by ion paired extraction or solid-phase extraction. More specifically, the former method is based on the use of N,N-dimethylephedrinium bromide as the ion paired extraction reagent, while the latter on the use of WAX® (weak anion exchange) cartridges. The performance of the newly developed procedure has been assessed by the analysis of real urine excretion samples collected after the oral intake of a single dose of dehydroepiandrosterone (DHEA) or androstenedione (AED), measuring the concentration of epiandrosterone (EpiA) sulfate. Our results have shown the following: (i) although the yields of chemical hydrolysis and enzymatic hydrolysis are in some cases quite similar, the former is generally preferable since it results in the quantitative cleavage of sulfate moiety; (ii) ion paired extraction has been selected as the most reliable method for direct isolation of sulfate steroids from urine matrices; (iii) EpiA sulfate allows to prolong the detectability of DHEA and AED when compared to routinely used steroidal target compounds.


27 July 2020
Botrè, Francesco
de la Torre, Xavier
Iannone, Michele
Martinez-Brito, Dayamin
Matteucci, Roberta
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Sapienza Università di Roma - Sapienza University of Rome
Roma, Italia: Laboratorio Antidoping FMSI
Analytical aspects
Mass spectrometry analysis
Testing method development
Doping classes
S1. Anabolic Agents
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Date generated
2 September 2020
Date of last modification
13 September 2020
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