Analytical methods for anti-doping control in sport: anabolic steroids with 4,9,11-triene structure in urine

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Abstract

Doping control in sport is mandatory to detect and to control the use of prohibited substances. Due to the growing number of targets, the analysis of doping compounds and their metabolites is carried out using established screening methods. However, detection of anabolic steroids with 4,9,11-triene structure in urine is problematic, so it is necessary to improve the methods.

We review the state of the art in doping-control analysis of 4,9,11-trien-3-one steroids, providing an overview of the screening and confirmatory methods developed for these analytes in human urine. First, we review chromatographic techniques. We discuss difficulties in the derivatization of those compounds prior to gas chromatography analyses. In recent years, liquid chromatography has been the preferred technique in drug testing in sport, due to the reduced sample pre-treatment, improved limits of detection and comprehensiveness. We also report on advances and limitations of immunochemical techniques for the analysis of this group of substances.

Introduction

Analysis of athletes’ urine is performed during training and competition periods for doping control [1]. The analyses are carried out at two levels:

  • (1)

    screening, applied to all the samples; and,

  • (2)

    confirmatory analyses of suspicious screening test samples.

The requirements of maximum speed with the minimum cost make this approach appropriate for the fight against doping in sport, promoted, coordinated and monitored by the International Olympic Committee (IOC) [2] and the World Anti-Doping Agency (WADA) [3].

The WADA’s prohibited list is an International Standard identifier of substances and practices banned in competition, out of competition and in particular sports. It is updated and modified annually based on most recent developments and scientific data. The 2010 WADA list [4] included nine classes of substances and three prohibited methods.

The number of substances used by athletes grows constantly [5], so testing for the above list of compounds is technically challenging, expensive and performed by only 34 WADA-accredited laboratories worldwide. Moreover, the inclusion of new compounds in the prohibited list makes the laboratories perform studies about its metabolism and excretion in urine [6], and develop methods to detect the intake through analysis of urine samples.

Doping controls during major competitions impose a significant analytical challenge in the laboratory. For example, in The Games of the XXIX Olympiad, Beijing 2008, requests were to report negative results in 24 h and positive ones in 48 h after sample collection [7]. Collected urine is divided by the athlete in two samples called A and B [8]. These are sent to a WADA-accredited laboratory, which inspects the samples upon their arrival to ensure there is no evidence of tampering. The laboratory ensures that the custody chain is maintained at all times, according to the international standard for laboratories [9]. The A sample is analyzed for substances on the prohibited list. The B sample is securely stored at the laboratory.

Standard methods are currently not available for doping-control analysis. The laboratory needs to develop, to validate and to document methods for the detection of substances present in the prohibited list and for associated metabolites, markers or related substances [9]. In order to minimize the complexity of analysis and reduce the time needed to produce results, the detection of the targets and/or their metabolites is carried out using screening methods for multi-classes of substances, based mainly on mass spectrometry (MS) coupled to chromatography [10]. However, in some cases, due to the physico-chemical characteristics of the compounds and metabolites, and their concentrations in urine, it is necessary to develop specific methods, which also increase the volume requirements for urine samples. WADA has established the minimum required performance limit (MRPL), the concentration that a doping-control laboratory is expected to detect and to confirm reliably in its routine work [11].

Anabolic androgenic steroids, commonly referred to as anabolic steroids, have been the most frequently detected prohibited compounds in doping-control analysis for many years [6], which is regarded as a major concern in enhancing detection methods for these substances [12]. Because of the large number of compounds involved, the structural similarity of exogenous and endogenous steroids, the complexity of the urine matrix, the low steroid concentrations in urine and WADA’s sensitivity requirements (MRPLs of 2–10 ng/mL), the analysis of anabolic steroids is challenging [13]. Most methods used routinely for detection of these compounds and their metabolites in human urine, comprising both screening and confirmatory analysis, had been based on gas chromatography-MS (GC-MS) techniques [14], [15]. However, among synthetic steroids, the detection of analytes bearing a 4,9,11-triene nucleus (Fig. 1), has proved to be a complex task using conventional GC-MS, due to its reduced thermal stability [16]. Liquid chromatography-(tandem) MS (LC-MS(/MS)) methods [17], [18], [19] have complemented most of the doping-testing strategies for several reasons: reduced sample pretreatment; superior limits of detection (LODs) and the possibility of detecting thermolabile compounds. Nowadays, the detection of conjugated double-bond anabolic agents using this technique is crucial, due to the poor yield of the classical derivatization protocol for these steroids.

This article comprises an overview of the reported analytical methods for determining 4,9,11-trien-3-one steroids, focusing on human-doping control. First, we discuss derivatization strategies associated with GC for this sub-group of anabolic steroids, and the assessment of HPLC-MS, evaluating its importance for a sensitive and comprehensive multi-residue analysis in doping control. Second, we discuss the increasing number of immunochemical methods for the analysis of these substances and their limitations for human-doping control.

Section snippets

Anabolic steroids with 4,9,11-trien-3-one structure

Trenbolone (17β-hydroxy-estra-4,9,11-trien-3-one, TRE), gestrinone (17α-ethynyl-17β-hydroxyestra-4,9,11-trien-3-one, GST) and related compounds (Fig. 1) are a group of anabolic steroids with a common 4,9,11-trien-3-one structure. TRE and its derivatives such as methyltrienolone (methylTRE) represent a class of highly potent anabolic androgenic steroids, which have been used for years as growth promoters for cattle. The misuse of TRE in elite sportsmen was reported in 42 cases by drug-testing

Sample preparation

The determination of anabolic steroids in human urine by chromatographic methods involves a sample-pre-treatment step. Direct analysis of urine is the ideal situation, but it is not feasible in the majority of cases due to matrix interferences, the effect of aqueous media in GC columns and the LODs required for these compounds. The large number of metabolic products carried in urine makes it necessary to extract the target compounds from the sample matrix. Fig. 3 shows the steps required for

LC-MS2 analysis of 4,9,11-trien-3-one steroids

Steroids with a 4,9,11-triene nucleus present difficulties in derivatization, and reduced thermal stability in GC analysis, so they need other methods. Moreover, the discovery of synthetic steroids {e.g., THG [25]}, undetectable by common GC-MS methods, increased the need to develop alternative ways to determine this type of substances [39]. Due to the good proton affinities resulting from a large, conjugated π-electron system, liquid chromatography-tandem MS (LC-MS2) has been the preferred

Immunochemical methods for the analysis of 4,9,11-trien-3-one steroids

The application of immunoassays to doping control started in the 1970s, radioimmunoassay being a new technique that allowed for the first time the measurement of hormonal steroids in serum or urine with high sensitivity [54]. However, because of its reduced specificity due to cross-reactivity with other compounds and the disadvantages of utilizing radioisotopes, they were abandoned in the 1980s.

Concerning 3-keto-4,9,11-triene steroids, few immunoassays have been developed for the detection of

Remarks and future trends

Chemical analyses of steroids in human urine have developed rapidly within the past few years. GC-MS-based methods, which usually require a derivatization step, have been replaced by LC-MS techniques. In particular, the detection of steroids with estra-4,9,11-triene core structures was a complex task for sports drug testing laboratories using conventional GC-MS approaches. Designer anabolic steroids bearing that nucleus were synthesized and used for several years without being detected.

Acknowledgments

This work is financed by Project DEP2006-56177-C03-03 (Spanish Ministerio de Ciencia e Innovación). We thank Jorge Segura, Head of the Doping Control Laboratory Barcelona (Bioanalysis Research Group, IMIM-Hospital del Mar, Barcelona, Spain) for his valuable assistance in correcting the manuscript. We also thank the financial support of the Prometeo program from the Generalitat Valenciana.

References (60)

  • R.K. Müller
  • M.K. Parr et al.

    J. Steroid Biochem. Mol. Biol.

    (2010)
  • O.J. Pozo et al.

    Trends Anal. Chem.

    (2008)
  • K. Saito et al.

    J. Pharm. Biomed. Anal.

    (2010)
  • M. Thevis et al.

    Steroids

    (2009)
  • A. La Marca et al.

    Fertil. Steril.

    (2004)
  • X. Gao et al.

    Contraception

    (2007)
  • P. Van Eenoo et al.

    J. Steroid Biochem. Mol. Biol.

    (2006)
  • B. Spranger et al.

    J. Chromatogr.

    (1991)
  • M.A. Marques et al.

    J. Chromatogr., A.

    (2007)
  • K. Fang et al.

    Chin. J. Anal. Chem.

    (2010)
  • M.H. Spyridaki et al.

    Anal. Chim. Acta

    (2006)
  • E.M. Brun et al.

    Talanta

    (2010)
  • N. Tort et al.

    Trends Anal. Chem.

    (2009)
  • The International Olympic Committee...
  • World Anti-Doping Agency...
  • The 2010 Prohibited List International Standard. World Anti-Doping Agency: Montreal, Canada, 2009...
  • M. Thevis et al.

    Eur. J. Mass Spectrom.

    (2010)
  • The International Olympic Committee Anti-doping Rules Applicable to the Games of the XXIX Olympiad, Beijing 2008...
  • Guidelines for urine sample collection, 2010...
  • International Standard for laboratories, 2009...
  • M. Thevis
  • Minimum required performance levels for detection of prohibited substances, 2010...
  • M. Thevis et al.

    Drug Test. Anal.

    (2009)
  • A. Vonaparti et al.

    Rapid Commun. Mass Spectrom.

    (2010)
  • M. Galesio et al.

    Rapid Commun. Mass Spectrom.

    (2010)
  • M. Thevis et al.

    Drug Test. Anal.

    (2010)
  • M. Thevis et al.

    J. Mass Spectrom.

    (2005)
  • M. Kolmonen et al.

    Drug Test. Anal.

    (2009)
  • World Anti-Doping Agency. Laboratory statistics, 2010...
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