SARM-S4 and metabolites detection in sports drug testing: A case report

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Abstract

Recently, pharmaceutical industry developed a new class of therapeutics called Selective Androgen Receptor Modulator (SARM) to substitute the synthetic anabolic drugs used in medical treatments. Since the beginning of the anti-doping testing in sports in the 1970s, steroids have been the most frequently detected drugs mainly used for their anabolic properties. The major advantage of SARMs is the reduced androgenic activities which are the main source of side effects following anabolic agents’ administration.

In 2010, the Swiss laboratory for doping analyses reported the first case of SARMs abuse during in-competition testing. The analytical steps leading to this finding are described in this paper. Screening and confirmation results were obtained based on liquid chromatography tandem mass spectrometry (LC–MS/MS) analyses. Additional information regarding the SARM S-4 metabolism was investigated by ultra high-pressure liquid chromatography coupled to quadrupole time-of-flight mass spectrometer (UHPLC–QTOF-MS).

Introduction

Arylpropionamide-derived substances are analogs of androgen receptor (AR) agonists like bicalutamide. Since the discovery of these compounds has been reported by Dalton et al. in 1998 [1], these nonsteroidal androgens have been developed and investigated by many authors, especially for their anabolic therapeutics properties [2], [3]. Because of their high affinity binding capacity to the androgen receptor, various synthetic drugs have been named Selective Androgen Receptor Modulator or SARMs. At this time four categories of nonsteroidal androgen receptor agonists have been identified as arylpropionamides, bicyclic hydantoins, quinolines and tetrahydroquinoline analogues [4] but none of them are actually available as approved pharmaceutical compound.

The main advantages of SARMs compared to classical steroid replacement therapies have been shown through recent animal models studies which demonstrated that SARMs have a high tissue selectivity in muscle and bone whereas the steroid side effects linked to the androgens tissues are significantly reduced [5], [6]. This is mainly due to the AR activation by the SARMs which are not substrates for 5α-reductase and aromatases, thus excluding the amplified androgenic and estrogenic functions in tissues like prostate and seminal vesicles, observed after administration of testosterone and other anabolic androgenic steroids [7], [8], [9].

The high anabolic potency of SARMs coupled to a limited or even an absence of androgenic effects are properties which should attract athletes looking for power and strength improvement without undergoing deleterious physiological side effects. Consequently, the SARMs have been prohibited by the World Anti-Doping Agency (WADA) since January 2008 [10] and are included in the S1 class of anabolic agents.

A recent review paper described analytical methods to put forth a doping with SARMs in urine matrix with LC–MS/MS or GC–MS depending on the structure and physicochemical properties of the analyzed product [11]. Also, each of the four above-cited SARMs categories has been investigated regarding the detection of parent compounds and/or metabolites either in vitro or in vivo [12], [13], [14], [15]. Considering these published papers, WADA accredited laboratories are currently able to screen for SARMs and their metabolites in doping control urine samples.

In 2008, Thevis et al. identified a significant amount of Andarine (also called SARM-S4 or S-3-(4-acetylamino-phenoxy)-2-hydroxy-2-methyl-N-(4-nitro-3-trifluoromethyl-phenyl)-propionamide) in a freely available internet product [16]. This finding demonstrated the easy accessibility of SARMs products. Thus, athletes could obtain these forbidden substances and use them to improve their physical skills straightforwardly.

Since the introduction of SARMs on the WADA prohibited list, no adverse analytical finding (AAF) was declared by any of the world accredited laboratory. We report here the first AAF in regards to the presence of Andarine and its metabolites identified by LC–MS/MS and UHPLC–QTOF-MS in an in-competition female athlete urine sample.

Section snippets

Chemicals and reagents

All solvents and reagents were of analytical grade purity. Potassium carbonate anhydrous was obtained from Acros Organics (Geel, Belgium). Ethyl acetate was purchased from Panreac (Barcelona, Spain). Sodium hydrogen carbonate and potassium dihydrogen phosphate were supplied by Merck (Darmstadt, Germany). Ultrapure water was produced by a Milli-Q Gradient A10 water purification system with a Q-Gard® 2 and a Quantum™ EX Ultrapure organex cartridge purchased by Millipore Corp. (Billerica, MA,

Results and discussion

The recent developments of the Selective Androgen Receptor Modulator as therapeutics as well as the increasing internet availability have led the WADA to introduce these synthetic compounds on the prohibited list. Since the metabolic pathway description of the arylpropionamide-derived molecule named as SARM S-4 [17], we report here the first adverse analytical finding in the fight against doping.

After reception to the laboratory, the athlete's urine sample followed the usual screening

Conclusions

This case report presents the first adverse analytical finding regarding a Selective Androgen Receptor Modulator, SARM S-4 also named Andarine, classified in the S1 class of the WADA prohibited list. Established screening procedures revealed the presence of the parent compound and the M5 metabolite. State-of-the-art mass spectrometric techniques allowed the confirmation and the identification of SARM S-4 and several metabolites previously described following in vivo and in vitro studies.

Acknowledgements

The authors acknowledge the Institute of Biochemistry, Center for Preventive Doping Research of the German Sport University in Cologne (Germany), especially Prof. Mario Thevis, for his scientific advices and his help in the analytical procedures. The authors are also grateful to Stop-Doping Foundation and the Société de la Loterie de la Suisse Romande for their financial support.

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    Several in vitro and in vivo studies have been conducted to elucidate the metabolic pathways of these compounds in humans (see Table 1 for the summary of the data reported in the literature about the in vitro and in vivo studies). Different LC–MS applications have been reported, most with respect to the analysis of a specific SARM or of a small group of SARMs characterized by a similar chemical structure [6,7,9–26]. Recently an LC–MS/MS method that allows the simultaneous detection of 15 SARMs in urine, targeting the parent drugs, has been developed [27].

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This paper is part of the special issue entitled: Fight Against Doping in 2011, Guest-edited by Neil Robinson (Managing Guest Ediyor), Martial Saugy, Patrice Mangin, Jean-Luc Veuthey, Serge Rudaz and Jiri Dvorak.

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