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Selective androgen receptor modulators (SARMs) have great therapeutic potential in various diseases including cancer cachexia, sarcopenia, and osteoporosis, and the number of drug candidates has been growing over the last decade. The SARM drug candidates S-22 and S-23 belong to one of the most advanced groups of androgen receptor modulators and are based on an arylpropionamide-derived core structure. Due to their anabolic effects, SARMs have been prohibited in elite sports and have been a subject of sports drug testing programmes since January 2008. Consequently, the structure of analytically useful urinary metabolites should be elucidated to provide targets for sensitive and retrospective analysis. In the present study, the phase-I and -II metabolism of S-22 and S-23 was simulated using hepatic human enzymes, and resulting metabolites were characterized by means of state-of-the-art mass spectrometric approaches employing high resolution/high accuracy Orbitrap mass spectrometry. Subsequently, the newly defined target compounds including the glucuronic acid conjugates of S-22 and S-23, their corresponding monohydroxylated and bishydroxylated analogs, as well as their B-ring depleted counterparts were implemented into an existing routine doping control procedure, which was examined for its specificity for the added substances. In order to obtain proof-of-concept data for authentic urine specimens, canine urine samples collected up to 72 h after oral administration of S-22 to dogs were analyzed using the established approach outlining the capability of the presented assay to detect the glucuronide of S-22 as well as the B-ring-depleted metabolite (M3) in all samples following therapeutic (31.4 µg/kg) dosing. Finally, M3 was chemically synthesized, characterized by nuclear magnetic resonance spectroscopy and high resolution/high accuracy mass spectrometry, and chosen as primary target for future doping control analyses., (Copyright © 2010 John Wiley & Sons, Ltd.)

Recent studies outlined the influence of exercise on the stability of the skeletal muscle calstabin1-ryanodine receptor1-complex, which represents a major Ca(2+) release channel. The progressive modification of the type-1 skeletal muscle ryanodine receptor (RyR1) combined with reduced levels of calstabin1 and phosphodiesterase PDE4D3 resulted in a Ca(2+) leak that has been a suggested cause of muscle damage and impaired exercise capacity. The use of 1,4-benzothiazepine derivatives such as the drug candidates JTV-519 and S-107 enhanced rebinding of calstabin1 to RyR1 and resulted in significantly improved skeletal muscle function and exercise performance in rodents. Due to the fact that the mechanism of RyR1 remodelling under exercise conditions were proven to be similar in mice and humans, a comparable effect of JTV-519 and S-107 on trained athletes is expected, making the compounds relevant for doping controls. After synthesis of JTV-519, S-107, and a putative desmethylated metabolite of S-107, target compounds were characterized using nuclear magnetic resonance spectroscopy and electrospray ionization (ESI)-high-resolution/high-accuracy Orbitrap mass spectrometry. Collision-induced dissociation pathways were suggested based on the determination of elemental compositions of product ions and H/D-exchange experiments. The most diagnostic product ion of JTV-519 was found at m/z 188 (representing the 4-benzyl-1-methyl piperidine residue), and S-107 as well as its desmethylated analog yielded characteristic fragments at m/z 153 and 138 (accounting for 1-methoxy-4-methylsulfanyl-benzene and 4-methoxy-benzenethiol residues, respectively). The analytes were implemented in existing doping control screening procedures based on liquid chromatography, multiple reaction monitoring and simultaneous precursor ion scanning modes using a triple quadrupole mass spectrometer. Validation items such as specificity, recovery (68-92%), lower limit of detection (0.1-0.2 ng/mL), intraday (5.2-18.5%) and interday (8.7-18.8%) precision as well as ion suppression/enhancement effects were determined., ((c) 2009 John Wiley & Sons, Ltd.)