Detection of the Selective Androgen Receptor Modulator Andarine (S-4) in a Routine Equine Blood Doping Control Sample Adam T
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Drug Testing Correspondence case report and Analysis Received: 25 June 2015 Revised: 5 August 2015 Accepted: 16 August 2015 Published online in Wiley Online Library: 12 October 2015 (www.drugtestinganalysis.com) DOI 10.1002/dta.1867 Detection of the selective androgen receptor modulator andarine (S-4) in a routine equine blood doping control sample Adam T. Cawley,* Corrine Smart, Candace Greer, Marcus Liu Lau and John Keledjian Introduction (Pointe-Claire, Quebec, Canada). Mefruside was purchased from Bayer AG (Pymble, NSW, Australia). All general laboratory reagents Andarine (S-4) is the arylpropionamide-derived compound [S-3-(4- and solvents used were of HPLC grade and were purchased from acetylamino-phenoxy)-2-hydroxy-2-methyl-N-(4-nitro-3-trifluoro- Thermo Fisher Scientific (Fair Lawn, NJ, USA). Water was obtained methyl-phenyl)-propionamide] first described by Yin et al.[1] to be a using a Millipore filtration system (Bedford, MA, USA). selective androgen receptor modulator (SARM). Androgen receptor mediated transcription was reported to be 93% that of dihydrotes- [1] tosterone, the most potent endogenous steroid. This work built Sample preparation on the discovery of nonsteroidal androgens by Dalton et al.[2] follow- ing investigations of electrophilic ligands binding to the androgen An equine blood doping control sample consisting of 6x6 mL lith- receptor. Andarine (S-4) was shown by in vitro and in vivo studies to ium heparin tubes (BD Vacutainer®LH 102 IU ref 367885) sealed in be an effective anabolic agonist, without side-effects often associ- a plastic integrity pack divided into 2x3 tubes representing the A- ated with anabolic-androgenic steroids,[1] relating principally to sub- and B-portions was submitted to the Australian Racing Forensic strate affinity for 5α-reductase to produce DHT in the prostate.[3,4] Laboratory (ARFL) by an Australian principal racing authority. Fol- Unfortunately the benefits that SARMs such as andarine (S-4) lowing sample receipt, inspection and chain of custody documen- may provide to clinical medicine have the potential for misuse in tation, 3 tubes (A-portion) were centrifuged at 3000 rpm for sports where athletes and/or their handlers may seek to gain unfair 10 min to obtain plasma for routine analysis. Blood tubes were advantage with the assumption that these compounds are unde- stored at 4 °C until analyzed. tectable by anti-doping laboratories. The pharmacological activity and oral bioavailability of andarine (S-4) at relevant doses in rats Screening analysis (≤10 mg/kg)[5] highlights the potential threat to the integrity of an- imal and human sports. SARMs are therefore prohibited for use by The plasma sample (2 mL) was fortified with mefruside and d3-caf- the International Federation of Horseracing Authorities[6] and the feine (2 μg/mL, 50 μL) as surrogates before addition of trichloroace- World Anti-Doping Agency (WADA).[7] tic acid (10% v/v, 400 μL) with mixing and pH adjustment to 3.0–3.5. Analytical methods utilizing the sensitivity and specificity of liquid A quality control sample fortified with andarine (S-4; 1 μg/mL, chromatography-highresolutionaccurate mass(LC-HRAM)spectrom- 20 μL) was concurrently analyzed. The acid-neutral fraction contain- etry to detect andarine (S-4) have been reported since 2006.[8,9] A ing arylpropionamide SARM compounds was isolated by solid- black-market product was subsequently found by the Cologne anti- phase extraction (SPE) using a mixed-mode C8/strong cation doping laboratory to contain andarine (S-4) in 2009.[10] For the benefit exchange XTRACKT® column (200 mg, 3 cc, UCT, Bristol, PA, USA) of urine analysis in equine[11–13] and human[13,14] anti-doping, in vitro with the aid of a UCT positive pressure manifold. The cartridge and in vivo metabolic studies of andarine (S-4) have been performed. was conditioned with methanol (2 mL) and water (2 mL) before This assisted the reporting of two adverse analytical findings in human loading supernatant plasma and washing with acetic acid (0.1 M, doping control urine samples analysed by the Lausanne and Los 2 mL). The cartridge was dried using N2 gas under positive pressure Angeles anti-doping laboratories in 2011 and 2013, respectively.[15,16] before elution with ethyl acetate/hexane (3:2 v/v, 3 mL). The extract Here we describe, to the best of the authors’ knowledge, the first de- was evaporated to dryness under nitrogen at 60 °C before being tection of andarine (S-4) in a routine equine blood sample. reconstituted in 0.1% formic acid/water (50 μL) and 0.1% formic acid/methanol (50 μL) for LC-HRAM spectrometry analysis. Materials and methods Reference materials, chemicals, and reagents Andarine (S-4) [S-3-(4-acetylamino-phenoxy)-2-hydroxy-2-methyl- * Correspondence to: Adam Cawley, Australian Racing Forensic Laboratory, Racing NSW, Royal Randwick Racecourse, NSW 1465, Australia. E-mail: N-(4-nitro-3-trifluoromethyl-phenyl)-propionamide] was purchased [email protected] from the Institute of Biochemistry at the German Sports University 257 (Cologne, Germany). d3-Caffeine was purchased from CDN Isotopes Australian Racing Forensic Laboratory, Racing NSW, Randwick, NSW, Australia Drug Test. Analysis 2016,8, 257–261 Copyright © 2015 John Wiley & Sons, Ltd. Drug Testing and Analysis A. T. Cawley et al. LC-HRAM analysis Results and discussion LC-HRAM spectrometry analysis was undertaken using an Ultimate Screening 3000 HPLC coupled to an Exactive Plus benchtop orbitrap mass spec- trometer (Thermo Fisher Scientific, Bremen, Germany) equipped with On review of LC-HRAM data from the Exactive Plus instrument a a SunFire® C18 column (100 x 2.1 mm, 3.5 um; Waters, Milford, peak was found at a retention time (RT) of 12.29 min (relative reten- MA, USA) operating at 40 °C. Injection volume was 10 μL. The mo- tion (RR) 1.23 to mefruside) for both m/z 440.1020-440.1108 and bile phase consisted of A: 0.1% formic acid/water and B: 0.1% m/z 442.1176–442.1264 intervals. Specificity of these m/z intervals formic acid/methanol. Gradient elution was performed with a flow has been demonstrated with no response in bona fide blank equine rate of 0.4 mL/min according to the following programme: 5% Β for plasma used by the ARFL nor any of the more than 4000 routine 1 min, increased to 95% B at 15.85 min, before returning to 5% B at equine plasma samples analyzed since the inclusion of the parent 16.35 min and held until 20.85 min. The presence of andarine (S-4) substance andarine (S-4) as a target. Additional confidence was at- was monitored using deprotonated and protonated heated tributed to the screening abnormality for andarine (S-4) in the sam- electrospray ionization (HESI) in full-scan mode at a resolution of ple by having dual deprotonated and protonated ion responses. 70 000 (FWHM) and deconvolution of all-ion fragmentation using the m/z 440.1020–440.1108 and m/z 442.1176–442.1264 intervals, respectively, between 11.80 min and 12.80 min. The total scan range Confirmation was m/z 50 to m/z 850. Mass calibration was performed prior to Targeted MS2 experiments enabled confirmation of andarine (S-4) analysis using Pierce® ESI negative (P/N 88324) and positive (P/N in the equine plasma sample with comparison to the reference 88323) calibration solutions (Thermo Fisher Scientific, Bremen, standard using the identification criteria for LC-MS techniques pre- Germany) but no lock mass was used. Source temperature, spray scribed by the Association of Official Racing Chemists (Table 2).[17] voltage, sheath gas (high purity N2) and auxiliary gas (ultra-high Accurate mass assignments for deprotonated and protonated À purity N2) were set at 350 °C, +/ 4000 V, 63.74 and 10.30 arbitrary spectra are summarized in Tables 3 and 4, respectively, with good units, respectively. Instrument control and processing were per- agreement to the characterization of andarine (S-4) reported by formed using Xcalibur software (version 2.2 SP1). Thevis et al.[8–10] The deprotonated product ion spectrum (Figure 1) shows elimination of N-(4-hydroxyphenyl)-acetamide (À151 Da) Confirmation analysis yielding m/z 289.0447, followed by carbon monoxide to give m/z 261.0497. 4-Nitro-3-trifluouromethyl-aniline provides m/z A separate plasma aliquot (2 mL) from a previously unopened blood 205.0229, while the base ion m/z 150.0556 represents the tube was used for confirmation. Isolation of the acidic-neutral fraction by SPE was performed as described above for the screening analysis. Table 2. Confirmation criteria for andarine (S-4) in the equine plasma LC-HRAM spectrometry was undertaken using an Ultimate 3000 HPLC sample coupled to a QExactive benchtop orbitrap mass spectrometer Criterion Sample Standard % difference Requirement* (Thermo Fisher Scientific, Bremen, Germany). LC separation was per- formed as described for the screening analysis. The presence of Retention 12.33 12.34 0.08 <2% andarine (S-4) was confirmed using deprotonated and protonated time (RT) 2 À HESI with polarity switching in a single analysis. Targeted MS experi- [M-H] %rel. ments were performed using parameters in Table 1, together with cal- abundance ibration and source conditions described for the screening analysis. 150.0556 100 100 0 The following sequence order was followed; solvent blank (0.1% 205.0229 16.50 16.01 0.49 ±20% (abs.) formic acid/water (50 μL) and 0.1% formic acid/methanol (50 μL)), 261.0497 19.30 20.07 0.77 ±20% (abs.) equine plasma blank, reagent (MilliQ water extracted concurrently 289.0447 5.16 5.59 0.43 ±20% (abs.) with the sample) blank, test sample, solvent blank, equine plasma [M + H]+ %rel.