Analytical strategies based on mass spectrometric techniques for the study of steroid metabolism

doi:10.1016/j.trac.2013.08.010

TrAC Trends in Analytical Chemistry

Volume 53, January 2014, Pages 106-116

https://doi.org/10.1016/j.trac.2013.08.010 Get rights and content

Highlights

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The use of mass spectrometry for the study of steroid metabolism is reviewed.
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The evaluation of metabolism of both phase I and phase II is discussed.
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Both direct and indirect (after hydrolysis) detection of conjugates is considered.
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An integrated approach to the broad detection of steroid metabolism is suggested.

Abstract

Anabolic androgenic steroids (AASs) are synthetic substances derived from testosterone, whose use in sports is prohibited. This review underlines the advantages and the drawbacks of different coupled mass spectrometry-based approaches to the identification of AAS metabolites. We discuss diverse aspects of the applications in the doping-control field, including sample preparation and instrumental analysis, with special emphasis on the potential scope of each strategy.

Introduction

Anabolic androgenic steroids (AAS) are included in the list of forbidden substances in sports due to their performance-enhancing and adverse health effects. They are the most prominent group of forbidden substances detected in doping controls, reflecting the wide misuse of these compounds as performance-enhancing drugs by athletes [1]. The general structure and some examples of AAS structures are depicted in Fig. 1A.

Antidoping laboratories have to develop analytical tools to detect the use of AASs in sports. AASs suffer extensive metabolism, so their misuse is normally monitored through the analysis of the metabolites excreted in urine [2], [3]. It has to be kept in mind that the best marker is not always the most abundant metabolite but the metabolite excreted for longer times after the administration of the drug. For this reason, antidoping laboratories need to perform comprehensive and in-depth metabolic studies in order to identify as many metabolites as possible and to select the best markers. These studies have to include:

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detection of potential metabolites;
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identification of potential metabolites based on the structural information provided by mass spectrometry (MS) data;
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the excretion profile of each metabolite to evaluate the excretion time; and,
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confirmation of the postulated structure of each metabolite by synthesis of the authentic material [4].

Metabolic studies of AASs have been traditionally performed using gas chromatography coupled to MS (GC-MS), and the detection of the misuse of AASs by antidoping laboratories has relied for many years on the detection of the metabolites identified in the 1980s by GC-MS methods [2]. GC-MS has some limitations for the execution of metabolic studies, among which the most significant are:

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the need for derivatization of polar compounds; and,
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the impossibility of detecting conjugated metabolites directly.

In recent years, liquid chromatography coupled to tandem MS (LC-MS/MS) has shown several novel possibilities for the detection and the identification of new phase I and phase II metabolites. In particular, LC-MS/MS has the capacity to detect conjugated metabolites directly and the possibility to apply open methods [neutral loss (NL) and precursor ion (PI) scan methods] for the detection of metabolites having a common chemical structure [5].

The detection of AASs and their metabolites based on MS techniques has been reviewed [6], [7], [8], [9]. These reviews deal mainly with the potential of MS for the target detection of several doping agents (including AASs and their known metabolites) in different biological matrices. However, they are not focused on the analytical strategies for the detection and the elucidation of these metabolites.

In this article, we review the potential of MS for the study of AAS metabolism in human urine samples. First, we briefly summarize the metabolism for AASs. Second, we present different approaches to the detection and the characterization of phase I metabolites. Then, we discuss the study of phase II metabolism, considering two approaches: indirect detection of conjugates after hydrolysis, and direct detection of the phase II metabolites. Finally, we suggest an integrated approach based on different methods for the comprehensive study of AAS metabolism.

Section snippets

Metabolism of anabolic androgenic steroids

Consideration of general metabolic pathways is fundamental for the detection and the characterization of the main metabolites of new steroids, but also for the discovery of previously unreported metabolites of known steroids.

In general, synthetic AASs follow the metabolic pathways observed for testosterone and have been comprehensively reviewed [2], [3]. Metabolic reactions are grouped into two types – phase I and phase II – and they convert the steroid into more polar compounds in order

Evaluation of phase I metabolism

Most of the metabolic studies published for AASs focus on the detection of phase I metabolites. Several parameters in sample treatment and instrumental analysis have to be considered, since the results dramatically depend on the suitability of these parameters.

Evaluation of phase II metabolism

Phase II metabolic reactions have normally been studied using indirect methods consisting of a selective hydrolysis of the steroid conjugates to release the corresponding phase I metabolites, which were subsequently analyzed by MS. In addition to the factors discussed in Section 3 (above), the selection of the hydrolysis conditions is one of the key steps in these studies, so we treat it separately in this section. LC-MS(/MS) permits the direct detection of phase II metabolites, skipping the

Comprehensive approach for the study of AAS metabolism

The complexity of the analytical methods to study the different metabolic urinary fractions means that it is not feasible to have a comprehensive approach starting from a single urine aliquot. A possible strategy for a comprehensive metabolic study would start with five urine aliquots (see Fig. 3):

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Aliquot 1 is devoted to the study of the free and glucuronide fractions. The free fraction is extracted from the urine using organic solvents (free fraction extract). The remaining aqueous phase is

Conclusions

MS plays a crucial role in AAS metabolic studies due to its high sensitivity, specificity and versatility up to the point that AAS metabolic studies cannot be understood without MS. The identification of new AAS metabolites goes hand in hand with the evolution of MS, so we expect that several previously unreported metabolites will be detected by the use of the new generation of MS coupled to both LC and GC. The development of more potent chromatographic approaches might help also to

Acknowledgements

This work was supported by Grant from Instituto de Salud Carlos III FEDER (CP10/00576). Financial support of WADA (09A20RV, 11A9RV, 11A22OP), Ministerio de Economía y Competitividad (Spanish Government) (DEP2009-11454, DEP2012-35612) and Grant by the Generalitat de Catalunya (2009GR00492 to the research team) are also acknowledged.

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ReviewAnalytical strategies based on mass spectrometric techniques for the study of steroid metabolism

Highlights

Abstract

Introduction

Section snippets

Metabolism of anabolic androgenic steroids

Evaluation of phase I metabolism

Evaluation of phase II metabolism

Comprehensive approach for the study of AAS metabolism

Conclusions

Acknowledgements

J. Steroid Biochem. Mol. Biol.

Anal. Chim. Acta

Trends Anal. Chem.

J. Chromatogr. B

Steroids

Steroids

Steroids

J. Steroid Biochem. Mol. Biol.

J. Chromatogr. Biomed. Appl.

J. Pharm. Biomed. Anal.

J. Chromatogr.

Anal. Chim. Acta

J. Chromatogr.

Trends Anal. Chem.

J. Chromatogr. A

J. Steroid. Biochem. Mol. Biol.

Steroids

J. Steroid Biochem. Mol. Biol.

J. Chromatogr. A

Steroids

Steroids

Anal. Chim. Acta

J. Pharm. Biomed. Anal.

J. Chromatogr. B. Biomed. Sci. Appl.

J. Chromatogr. B

J. Chromatogr. B

Steroids

J. Steroid Biochem. Mol. Biol.

J. Chromatogr. B

Drug Metab. Pharmacokinet.

J. Chromatogr. B: Anal. Technol. Biomed. Life Sci.

Talanta

Forensic Sci. Int.

Metabolism of anabolic androgenic steroids

Clin. Chem.

Recent developments in MS for small molecules: application to human doping control analysis

Bioanalysis

Current use of high-resolution mass spectrometry in drug screening relevant to clinical and forensic toxicology and doping control

Anal. Bioanal. Chem.

Review
Analytical strategies based on mass spectrometric techniques for the study of steroid metabolism