Development of a mass spectrometric hydroxyl- position determination method for the hydroxyindole metabolites of JWH-018 by GC-MS/MS Maiko Kusano, a Kei Zaitsu, a,b * Mayumi Yamanaka, a Kazuaki Hisatsune, a,c Tomomi Asano, a Kentaro Taki, a Yumi Hayashi, b,d Hitoshi Tsuchihashi a and Akira Ishii a One of the many issues of designer drugs of abuse like synthetic cannabinoids (SCs) such as JWH-018 is that details on their metabolism has yet to be fully elucidated and that multiple metabolites exist. The presence of isomeric compounds poses further challenges in their identification. Our group has previously shown the effectiveness of gas chromatography-electron ionization- tandem mass spectrometry (GC-EI-MS/MS) in the mass spectrometric differentiation of the positional isomers of the naphthoylindole-type SC JWH-081, and speculated that the same approach could be used for the metabolite isomers. Using JWH-018 as a model SC, the aim of this study was to differentiate the positional isomers of its hydroxyindole metabolites by GC-MS/MS. Standard compounds of JWH-018 and its hydroxyindole metabolite positional isomers were first analyzed by GC-EI- MS in full scan mode, which was only able to differentiate the 4-hydroxyindole isomer. Further GC-MS/MS analysis was performed by selecting m/z 302 as the precursor ion. All four isomers produced characteristic product ions that enabled the differentiation between them. Using these ions, MRM analysis was performed on the urine of JWH-018 administered mice and determined the hydroxyl positions to be at the 6-position on the indole ring. GC-EI-MS/MS allowed for the regioisomeric differentiation of the hydroxyindole metabolite isomers of JWH-018. Furthermore, analysis of the fragmentation patterns suggests that the present method has high potential to be extended to hydroxyindole metabolites of other naphthoylindole type SCs in identifying the po- sition of the hydroxyl group on the indole ring. Copyright © 2016 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: synthetic cannabinoids; JWH-018 metabolites; positional isomers; GC-MS/MS Introduction Since their first detection in herbal blends marketed as ‘Spice’ or ‘K2’ in 2008, [1] synthetic cannabinoids (SCs) quickly emerged and continued to rapidly grow in varying structural analog forms as popular alternatives to Δ 9 -tetrahydrocannabinol (THC). One of the many issues of designer drugs of abuse like SCs is related to their metabolism or elimination mechanisms, leading to the absence or only trace amounts of the parent compound in the biological spec- imens of those who have consumed the drug causing challenges to forensic drug testing. Because the parent compounds are usually not detected in common specimen samples like urine, it is of critical importance to test for their metabolites; however, here in itself lies another major problem that multiple metabolites exist and refer- ence standards are not always commercially available for identity confirmation. Moreover, positional isomers may also exist within the aforementioned multiple metabolites. It is thus of forensic im- portance to develop a method capable of identifying these metab- olites as well as their isomers. In recent years, gas chromatography-mass spectrometry (GC-MS) has been proven to be useful for the discrimination of isomers of drugs. [2–9] Particularly, there is a recent interest in the use of GC- tandem mass spectrometry (GC-MS/MS) for the isomer differentiation of newer designer drugs taking advantage of the spectral differ- ences from characteristic product ion fragmentation patterns as well as their high reproducibility of the produced fragment ions and their ion intensity ratios in the GC-MS/MS spectra. [10–16] Our group has also previously reported the effectiveness of GC-MS/ MS in the mass spectrometric differentiation of the methoxy posi- tions on the naphthoylindole ring of the synthetic cannabinoid JWH-081. [12] We speculated that the same approach could be ap- plied to positional isomers of the metabolites of similar synthetic cannabinoids. * Correspondence to: Kei Zaitsu, Department of Legal Medicine and Bioethics, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi 466-8550, Japan. E-mail: kzaitsu@med.nagoya-u.ac.jp a Department of Legal Medicine and Bioethics, Nagoya University, Nagoya, Japan b Institute for Advanced Research, Nagoya University, Nagoya, Japan c Forensic Science Laboratory, Aichi Prefectural Police Headquarters, Aichi, Japan d Department of Radiological and Medical Laboratory Sciences, Nagoya University Graduate School of Medicine, Nagoya, Japan J. Mass Spectrom. (2016), 51, 350–357 Copyright © 2016 John Wiley & Sons, Ltd. Research article Journal of MASS SPECTROMETRY Received: 22 January 2016 Revised: 3 March 2016 Accepted: 11 March 2016 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/jms.3761 350