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As recently reported, the synthetic cannabinoid JWH-018 is the subject of extensive phase I and II metabolic reactions in vivo. Since these studies were based on LC-MS/MS and/or GC-MS identification and characterisation of analytes, the explicit structural assignment of the metabolites was only of preliminary nature, if possible at all. Here, we report the chemical synthesis of five potential in vivo metabolites of JWH-018 derivatives featuring an alkylcarboxy (M1), a terminal alkylhydroxy (M2), a 5-indolehydroxy (M3), an N-dealkylated 5-indolehydroxy (M4) and a 2'-naphthylhydroxy (5) analogue, respectively, and their characterisation by nuclear magnetic resonance spectroscopy. The collision-induced dissociation (CID) patterns of the protonated compounds were studied by high-resolution/high-accuracy tandem mass spectrometry (MS( n )) applying an LTQ Orbitrap with direct infusion and electrospray ionisation of target analytes. An unusual dissociation behaviour including a reversible ion-molecule reaction between a naphthalene cation (m/z 127) and water in the gas phase of the MS was shown to be responsible for nominal neutral losses of 10 u in the course of the CID pathway. LC-MS/MS-supported comparison of synthesised reference standards with an authentic urine sample using an API 4000 QTrap mass spectrometer identified the synthetic JWH-018 analogues M1-M4 as true in vivo metabolites, presuming a chromatographic separation of potentially present regioisomeric analogues. Existing doping control methods were expanded and validated according to international guidelines in order to allow for the detection of the carboxy and the alkylhydroxy metabolites, respectively, as urinary markers for the illegal intake of the synthetic cannabinoid JWH-018. Both metabolites were quantified in authentic doping control urine samples that had been suspicious of JWH-018 abuse after routine screening procedures, and a stable isotope-labelled (13)C(8)-(15)N-carboxy metabolite was synthesised for future analytical applications.

Benfluorex [1-(m-trifluoromethylphenyl)-2-(β-benzoyloxyethyl)aminopropane] has been widely used for the treatment of atherogenic metabolic disorders and impaired carbohydrate metabolism (particularly in obese type-II diabetic patients) as well as an anorectic drug. Due to its potentially performance-enhancing properties, benfluorex has been added to the list of prohibited compounds and methods of doping by the World Anti-Doping Agency (WADA) in 2010, necessitating the implementation of the drug as well as its major metabolites into routine doping control procedures. In the present study, human urinary metabolites of benfluorex were characterized by gas chromatography-electron ionization-mass spectrometry (GC-EI-MS) as well as liquid chromatography-electrospray ionization-high resolution/high accuracy tandem mass spectrometry (LC-ESI-MS/MS). Commonly employed sports drug testing approaches consisting of liquid-liquid extraction followed by GC-MS or urine dilution and immediate LC-MS/MS analysis were expanded and validated with regard to specificity, recovery (48-54%, GC-MS only), intra- and interday precision (<25%), limits of detection (5-8 ng/mL for LC-MS/MS and 80 ng/mL for GC-MS), and ion suppression (for LC-ESI-MS/MS only) to allow the detection of benfluorex metabolites 1-(m-trifluoromethylphenyl)-2-(2-hydroxyethyl)aminopropane (M1), 1-(m-trifluoromethylphenyl)-2-(2-carboxymethyl)aminopropane (M2), and 1-(m-trifluoromethylphenyl)-2-aminopropane (M3) as well as the glucuronic acid conjugate of M1.

Selective androgen receptor modulators (SARMs) represent a novel class of drugs with tissue-specific agonistic and antagonistic properties, which are prohibited in sports from January 2008 according to the World Anti-Doping Agency. Preventive approaches to restrict the use of SARMs include early implementation of target analytes into doping control screening assays. Five model SARMs were synthesized, four of which are analogs to prostate-specific androgen receptor antagonists with a 5,6-dichloro-benzimidazole nucleus. The fifth SARM is a muscle-tissue specific agonist with a bicyclic hydantoin structure (BMS-564929). Dissociation pathways after negative electrospray ionization were studied using an LTQ-Orbitrap mass analyzer, and diagnostic product ions and common fragmentation patterns were employed to establish a screening procedure that target the intact SARMs as well as putative metabolic products. Sample preparation based on solid-phase extraction and subsequent LC-MS/MS measurement allowed for detection limits of 1-20 ng/mL, intra- and interday precisions of between 2.4 and 13.2% and between 6.5 and 24.2%, respectively. Recoveries varied from 89 to 106%, and tests for ion suppression or enhancement effects were negative for all analytes. [figure: see text]