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.

Conjugation of Synthetic Cannabinoids JWH-018 and JWH-073, Metabolites by Human UDP-Glucuronosyltransferases

“…Analysis of human urine from individuals known to have ingested either JWH-018 or JWH-073 have identified several products of Phase I and II metabolism (Moller et al, 2010;Sobolevsky et al, 2010;Beuck et al, 2011;Grigoryev et al, 2011;Moran et al, 2011;Schneir et al, 2011). Many of these hydroxylated metabolites have been shown to be monohydroxy derivatives of the naphthalene, indole, or side-chain moieties (Sobolevsky et al, 2010) that are excreted in urine primarily as glucuronic acid conjugates (Sobolevsky et al, 2010;Wintermeyer et al, 2010;Moran et al, 2011), thus implicating the involvement of UGTs in this process.…”

“…These products are typically vegetative material laced with these potent CB-receptor agonists, and recent clinical data provide evidence of development of anxiety (Schneir et al, 2011), tolerance (Zimmermann et al, 2009), and enhancement of psychoses (Every-Palmer, 2010) in individuals using these products. There is also an evolving body of information available on the metabolism of synthetic cannabinoids, JWH-018 and JWH-073, in body fluids (Moller et al, 2010;Sobolevsky et al, 2010;Beuck et al, 2011;Grigoryev et al, 2011;Moran et al, 2011) and in vitro . Continued characterization of these metabolic pathways is important because recent studies have shown that oxidized products of JWH-018 retain significant in vitro and in vivo activity (L. K. Brents, E. E. Reichard, S. M. Zimmerman, J. H. Moran, W. E. Fantegrossi, and P. L. Prather, unpublished data).…”

Atypical Diabetic Ketoacidosis: Case Report

“…Affordability and easy accessibility via the internet contribute to the popularity of these drugs (2). Synthetic cannabinoids induce metabolic effects such as nausea and vomiting, hyperglycaemia, hypokalaemia and acidosis (3); however, they are undetectable in routine blood and urine drug screening methods (4,5). Ingestion usually occurs via smoking and the consumption of synthetic cannabinoids, and is associated with severe adverse effects and life-threatening conditions, including tachycardia, agitation, high blood pressure, excess sedation, loss of Background: Diabetic ketoacidosis (DKA) is a potentially life-threatening complication of diabetes mellitus and can lead to death if untreated.…”

Bioanalytical methods for the determination of synthetic cannabinoids and metabolites in biological specimens

“…The metabolism studies of SCs are based on in vivo experiments [40,[43][44][45][46][47][48][49][50][51][52][53] or on in vitro experiments with human or rat liver microsomes [24,41,43,44,[53][54][55][56][57][58][59][60][61][62][63]. Due to the speed of both the emergence and spread of SCs on the world market, the pharmacokinetics properties of some SCs have not been satisfactorily investigated.…”

“…In SCs extraction, LLE is usually used because of their high hydrophobicity. It has been used for the extraction of SCs in the following matrices: blood [35,[66][67][68][69][70][71][72], serum [39,[73][74][75][76], plasma [77], urine [40,41,47,49,51,59,72,[78][79][80][81][82][83], oral fluid [84,85] and hair [86][87][88][89][90]. It involves adding an immiscible organic solvent such as tert-butyl methyl ether [41,82], chloroform [50] and diethyl ether [40,52,79] to the sample, and mixing, removing the organic solvent.…”