With respect to the continuous emergence of new synthetic cannabinoids on the market since 2008, evaluation of the metabolism of these compounds and the development of analytical methods for the detection of these drugs including their respective metabolites in biological fluids have become essential. Other than JWH-018 or JWH-073, AM-2201 is one of the frequently identified synthetic cannabinoids in Korea. Recently, in our laboratory, several JWH-018 metabolites have been detected in some urine samples obtained from subjects who were arrested for the possession of herbal mixtures containing only AM-2201 or from those who confessed AM-2201 abuse. In the present study, we identified major urinary metabolites of AM-2201 and several metabolites of JWH-018, i.e., N-5-hydroxylated and carboxylated metabolites from rats administered AM-2201 and found that the metabolic profile in rats was similar to those in human subjects in this study. Analytical results of the urine samples from suspects who had a considerable possibility of AM-2201 or JWH-018 intake were also compared to distinguish between AM-2201 and JWH-018 abuse. The presence of 6-indole hydroxylated metabolites of each drug and N-4-hydroxy metabolite of AM-2201 was found to contribute to the decisive differences in the metabolic patterns of the two drugs. In addition, the concentration ratio of the N-(5-hydroxypentyl) metabolite to the N-(4-hydroxypentyl) metabolite of JWH-018 may be used as a criterion to differentiate between AM-2201 and JWH-018 abuse.

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Despite efforts by legal authorities to control the abuse of synthetic cannabinoids, new derivatives have continually emerged on the market to circumvent regulations, and its abuse has become a threat to public health. Thus, development of analytical methods for confirming drug intake in biological fluids is essential to ensure effective drug control and to address further drug intoxication cases. Herein, a sensitive and reliable liquid chromatography-tandem mass spectrometry method was established and validated for the simultaneous determination of 37 synthetic cannabinoid metabolites, such as N-hydroxypentyl and carboxy metabolites, using 100 ll of urine. Urine specimens were treated by enzymatic hydrolysis and solid-phase extraction. Limits of detection for the evaluated drugs ranged from 0.1 to 1 ng/ml, and the linear range spanned from 0.25 or 1 to 100 ng/ml. Precision and accuracy bias were 1.4-12.1 % and -7.2-7.2 %, respectively. Matrix effects biases were in the range of 0.4 to 10.1 %, and extraction recoveries were 65-99 %. In addition, all analytes were stable under storage conditions of 4°C and -20°C for 14 days, and after three freeze-thaw cycles. The developed method was successfully applied to actual urine specimens obtained from synthetic cannabinoid users. The present method enabled simultaneous quantification of 37 synthetic cannabinoid metabolites, including their regioisomers, in urine in the field of clinical and forensic toxicology.

Recently, use of novel synthetic cannabinoids has increased greatly despite worldwide efforts to regulate these drugs. XLR-11 ((1-[5'-fluoropentyl]indol-3-yl)-(2,2,3,3-tetramethylcyclopropyl)methanone), a fluorinated synthetic cannabinoid with a tetramethylcyclopropyl moiety, has been frequently abused since 2012. XLR-11 produces a number of metabolites in common with its non-fluorinated parent analogue, UR-144 ((1-pentylindol-3-yl)-(2,2,3,3-tetramethylcyclopropyl)methanone). Therefore, it is essential to develop effective urinary markers to distinguish between these drugs. In this study, we investigated the metabolic profile of authentic human urine specimens from suspected users of XLR-11 using liquid chromatography-quadrupole time-of-flight mass spectrometry. Furthermore, we quantified four potential XLR-11 metabolites by using commercially available reference standards. In vitro metabolism of XLR-11 and UR-144 using human liver microsomes was also investigated to compare patterns of production of hydroxypentyl metabolites. Urine samples were prepared with and without enzymatic hydrolysis, and subjected to solid-phase extraction. We identified 19 metabolites generated by oxidative defluorination, hydroxylation, carboxylation, dehydrogenation, glucuronidation, and combinations of these reactions. Among the identified metabolites, 12 were generated from a cyclopropyl ring-opened XLR-11 degradation product formed during smoking. The XLR-11 metabolite with a hydroxylated 2,4-dimethylpent-1-ene moiety was detected in most specimens after hydrolysis and could be utilized as a specific marker for XLR-11 intake. Quantitative results showed that the concentration ratio of 5- and 4-hydroxypentyl metabolites should also be considered as a useful marker for differentiating between the abuse of XLR-11 and UR-144.