title: Structure characterisation of urinary metabolites of the cannabimimetic JWH-018 using chemically synthesised reference material for the support of LC-MS/MS-based drug testing.
abstract: 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.
authors: Institute of Biochemistry-Center for Preventive Doping Research, German Sport University Cologne, Cologne, Germany.
authors: Simon
authors: S
authors: Beuck
authors: Ines
authors: I
authors: Möller
authors: Andreas
authors: A
authors: Thomas
authors: Annika
authors: A
authors: Klose
authors: Nils
authors: N
authors: Schlörer
authors: Wilhelm
authors: W
authors: Schänzer
authors: Mario
authors: M
authors: Thevis
mesh_terms: D002853:Chromatography, Liquid; D004300:Doping in Sports; D006801:Humans; D007211:Indoles / Q000737:chemistry* / Q000378:metabolism / Q000652:urine*; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D009281:Naphthalenes / Q000737:chemistry* / Q000378:metabolism / Q000652:urine*; D012680:Sensitivity and Specificity; D015813:Substance Abuse Detection / Q000379:methods*; D053719:Tandem Mass Spectrometry
chemical_list: D007211:Indoles; D009281:Naphthalenes; C552597:1-pentyl-3-(1-naphthoyl)indole