Synthetic Cannabinoids: Analysis and Metabolites

Life Sciences 97 (2014) 78–90

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Minireview Synthetic cannabinoids: Analysis and metabolites

Mahmoud A. ElSohly a,b,c,⁎,WaseemGula,b, Amira S. Wanas b,d, Mohamed M. Radwan b,e a ElSohly Laboratories, Inc., 5 Industrial Park Drive, Oxford, MS 38655, USA b National Center for Natural Products Research, School of Pharmacy, The University of Mississippi, University, MS 38677, USA c Department of Pharmaceutics, School of Pharmacy, The University of Mississippi, University, MS 38677, USA d Department of Pharmacognosy, Faculty of Pharmacy, Minia University, Minia, Egypt e Department of Pharmacognosy, Faculty of Pharmacy, University of Alexandria, Alexandria, Egypt article info abstract

Article history: Cannabimimetics (commonly referred to as synthetic cannabinoids), a group of compounds encompassing a Received 21 October 2013 wide range of chemical structures, have been developed by scientists with the hope of achieving selectivity Accepted 24 December 2013 toward one or the other of the cannabinoid receptors CB1 and CB2. The goal was to have compounds that could possess high therapeutic activity without many side effects. However, underground laboratories have Keywords: used the information generated by the scientific community to develop these compounds for illicit use as Synthetic cannabinoids marijuana substitutes. This chapter reviews the different classes of these “synthetic cannabinoids” with particular Analysis fi Herbal products emphasis on the methods used for their identi cation in the herbal products with which they are mixed and Metabolites identification of their metabolites in biological specimens. © 2014 Published by Elsevier Inc.

Contents

Introduction...... 79 Naphthoylindoles...... 79 Liquidchromatographyelectrosprayionizationtandemmassspectrometry(LC/MS/MS)...... 79 Immunoassay...... 83 GC/MS...... 83 Matrix-assisted laser desorption ionization-time of ﬂightmassspectrometry(MALDI-TOF-MS)...... 84 Directanalysisinrealtimemassspectrometry(DART-MS)...... 84 Nano-liquidchromatography(nano-LC)...... 84 Nuclearmagneticresonance(NMR)...... 84 Phenylacetylindoles...... 85 LC/MS/MS...... 85 Immunoassay...... 86 MALDI-TOF-MS...... 86 GC/MS...... 87 Nano-LC...... 87 Benzoylindoles...... 87 LC/MS/MS...... 87 MALDI-TOF-MS...... 87 GC/MS...... 87 Nano-LC...... 88 Naphthoylpyrrol...... 88 Cyclohexylphenols...... 88 Adamantylindoles...... 89 Adamantylindazoles...... 89 Miscellaneous...... 89 LC/MS/MS...... 89 Nano-LC...... 89 DART/MS...... 89

⁎ Corresponding author. E-mail address: [email protected] (M.A. ElSohly).

Concludingremarks...... 89 Conﬂictofintereststatement...... 89 References...... 89

Introduction liquid extraction and LC/MS/MS analysis to detect its major metabolites in human urine for the purpose of doping control. After a successful Synthetic cannabinoids, better referred to as cannabimimetic confirmation of the JWH-018 phase-I metabolites, the method was compounds, are compounds prepared by scientists around the world then used in routine analysis of doping control samples. targeting an interaction with the endocannabinoid system, namely CB1 Hutter et al. (2012) analyzed and screened the urine samples of pa- and CB2 receptors. Although many of these compounds have been de- tients who had consumed synthetic cannabinoids using LC/MS/MS and scribed in the literature for many years, their illicit use appeared only in HR/MS/MS, and reported metabolites of JWH-018, JWH-073, JWH-081, the last few years. They appeared in the United States market in late JWH-122, and JWH-210 by ion spectra and mass measurement. They 2008. They were mixed with herbal products and sold as incense or pot- found that the major metabolic pathways include monohydroxylation pourri on the internet, gas stations and tobacco shops under many brand on the naphthoyl moiety, indole moiety or at the N-alkyl chain names (Spice, Spice gold, Aroma, K2, Spike 99, etc.) with labels stating (Figs. 1–5). Carboxylation of the side chain was reported only for “not for human consumption”. These products are claimed to contain JWH-018 and JWH-073. only natural non-illegal compounds and consequently have no limitations LC–MS/MS and the software assisted library searching against refer- in their commercial distribution (Carroll et al., 2012). The consumption of ence spectra were applied to detect the urinary metabolites of JWH-018, these products has become a popular alternative to marijuana, as they are JWH-073, JWH-081, JWH-122, JWH-200, JWH-210, and AM-2201 of high-potency and high efficacy as cannabinoid receptor full agonists. (Wohlfarth et al., 2013). The parent compounds for these synthetic can- The number of patients presented to the emergency department with nabinoids were also identified, and the method was validated with problems associated with these drugs has dramatically increased. In a limit of detection ranging from 0.5 to 10 ng/mL. D5-JWH-200 and March, 2011 the US Drug Enforcement Administration (DEA) scheduled D9-JWH-081 were used as internal standards at concentrations of 800 five synthetic cannabinoids (JWH-018, JWH-073, JWH-200, CP-47,497, and 100 ng/mL, respectively. and CP-47,497 C8 homologue) as schedule 1 controlled substances. JWH-018 was subjected to in vitro metabolism using human liver Many of these products especially Spice and K2 have been banned in microsomal system. The compound was incubated with the micro- many European countries (ElSohly et al., 2011; Wells and Ott, 2011) somes for 30 min at 37 °C in the presence of NADP and G-6-PDH and in May 2013, three synthetic cannabinoids (UR-144, XLR-11 and (ElSohly et al., 2011). This was followed by extraction of the reaction AKB-48) were also placed in schedule 1. mixture and analysis by LC/MS/MS. LightSight® software program for Moreover, compared to THC, some synthetic cannabinoids possess a metabolite identification was used to identify possible metabolites in 4–5 times improved binding affinity to the cannabinoid CB1 receptor the LC/MS/MS (Qtrap) run of the HLM preparation extract. A full scan and many toxicity symptoms were reported including anxiety, para- mass spectrum was generated for each peak based on the ions trapped noia, tachycardia, irritability, hallucination, numbness, seizures, high in the Qtrap. This allows for the generation of the total ion chromato- blood pressure, drowsiness, and slurred speech (Seely et al., 2012). gram for each group of metabolites sharing the same molecular ion. Over the past few years a great effort has been exerted to identify and Two major monohydroxylated metabolites of JWH-018 (with m/z quantify synthetic cannabinoids in herbal products, and detect their 358) were detected. A close examination of the fragmentation pattern metabolites in body fluids (urine, serum, and saliva) and also in hair of these metabolites showed that the hydroxy group of the early eluting specimens. These methods include liquid chromatography tandem metabolite is located on the terminal carbon of the side chain attached mass spectroscopy (LC–MS/MS) (Teske et al., 2010), high mass resolu- to the indole nitrogen. This is supported by the presence of ions at m/z tion techniques like matrix-assisted laser desorption/ionization time 127 (unhydroxylated naphthalene nucleus), m/z 155 (the naphthalene of flight mass spectroscopy (MALDI-TOF) (Gottardo et al., 2012), direct nucleus with the carbonyl group), and m/z 284 (the molecular ion analysis in real time mass spectrometry (DART-MS) (Musah et al., with loss of the terminal 4 carbon moiety of the side chain containing 2012), nuclear magnetic resonance (NMR) (Rollins et al., 2013), the hydroxy group). On the other hand, the spectrum of the second, gas chromatography/mass spectrometry (GC/MS) (Sobolevskii et al., later eluting, monohydroxy metabolite showed fragmentation consis- 2011), and immunoassays (Arntson et al., 2013). tent with hydroxylation on the indole moiety. This was proven to be Recently, many reviews on the chemistry, toxicity, and pharmacolo- the 6-hydroxy-metabolite by comparison with a reference sample gy of synthetic cannabinoids have been published (Carroll et al., 2012; made available by Cayman Chemical during the course of the work. A Favretto et al., 2013; Seely et al., 2012; Spaderna et al., 2013; Wells urine specimen received at ElSohly Laboratories, Inc. (ELI) (specimen and Ott, 2011). In this chapter, the focus will be on the analysis of the CM504), from a subject who admitted the use of Spice, was analyzed different classes of synthetic cannabinoids in herbal mixtures and the following the same protocol used for the HLM metabolic study. Exami- identification/analysis of their metabolites in biological fluids. nation of the LightSight list of possible metabolites indicated the Synthetic cannabinoids can be chemically classified into presence of several peaks with m/z 358 (monohydroxylated metabo- naphthoylindoles, benzoylindoles, phenylacetylindoles, adam- lites), m/z 372 (possible carboxy metabolite at the terminal carbon of antylindoles, cyclophenols and a miscellaneous group. Different analyt- the side chain), m/z 390 (possible trihydroxy metabolite), and m/z 374 ical techniques have been applied to the detection and quantitation of (possible dihydroxy metabolite) (Fig. 1), these metabolites were different members of each of these classes. Details are outlined below. also proposed by Sobolevskii et al. (2011). JWH-073, another naphthoylindole derivative analogous to JWH-018 with a C4 side chain Naphthoylindoles instead of the C5, has also been detected in some K2 (Spice) samples. The HLM metabolism of JWH-073 was carried out in a similar manner Liquid chromatography electrospray ionization tandem mass spectrometry as previously described for JWH-018. (LC/MS/MS) Interestingly, only the 4-hydroxy metabolite of JWH-073 (Fig. 2) was confirmed by comparison with the standard made available in Since JWH-018 itself cannot be detected in urine, Möller et al. (2010) house at ELI. LC/MS/MS chromatograms of the HLM NADPH at 30 min developed a method based on enzymatic hydrolysis followed by liquid– showed peaks of four unidentified metabolites of JWH-073 with a 80 M.A. ElSohly et al. / Life Sciences 97 (2014) 78–90

O N COOH

OH (a) O N

OH O N (b) O N OH

(f) JWH-018 O N

HO OH

HO (c) OH O N

O N (e)

OH (d)

Fig. 1. Chemical structures of JWH-018 metabolites. Carboxylation at the N-alkyl chain (a), monohydroxylation at the N-alkyl chain (b), monohydroxylation at the indole moiety (c). trihydroxylation (d), dihydroxylation (e) and monohydroxylation at N-alkyl chain (f). ElSohly et al. (2011), Hutter et al. (2012) and Lovett et al. (2013).

O O O O

N N N N OH O OH JWH-073 (a) OH (b) (c) MW = 326.2 MW = 358.3 MW = 344.3 MW = 344.3

O O O

N N N 4 3 OH O (e)HO (f) (d) HO MW = 344.4 MW = 344.4 MW = 344.4

Fig. 2. Chemical structures of JWH-073 metabolites. Carboxylated JWH-073 at the N-alkyl chain (a), monohydroxylated JWH-073 at the N-alkyl chain (b), monohydroxylated JWH-073 at the indole moiety (c), demethylation and carboxylation at the N-alkyl chain (d) and monohydroxylated at the N-alkyl chain (e and f)chain. ElSohly et al. (2011), Hutter et al. (2012) and Lovett et al. (2013). M.A. ElSohly et al. / Life Sciences 97 (2014) 78–90 81

O O O O O O O O

H N N N O N

JWH-081 (a) OH (b) (c) MW = 372.3 MW = 388.3 MW = 388.5 MW = 388.2

Fig. 3. Chemical structures of JWH-081 metabolites. Monohydroxylated JWH-081 at the N-alkyl chain (a), monohydroxylated JWH-081 at the indole moiety (b), and the metabolite monohydroxylated at the naphthalene moiety (c). Hutter et al. (2012). mass of m/z 344 (Fig. 2), presumably monohydroxylated and possibly et al., 2011). The method was fully validated on 101 serum samples one of them is the three carbon-N-carboxylic acid metabolite shown which underwent liquid–liquid extraction. in Fig. 2. The only metabolite identified in the LC/MS/MS chromatogram Detection of the synthetic cannabinoids JWH-018, JWH-073, was the terminal hydroxylated metabolite which showed a mass of JWH-200 in hair samples was carried out using a fully validated method m/z 344 with Rt and fragmentation identical with a standard synthe- by Salomone et al. (2012). Liquid–liquid extraction was performed sized in-house. followed by an analysis of the extract on an ultra-high performance liq- A new metabolite, JWH-72 N-propanoic acid (Fig. 2, d)wasde- uid chromatography system (UPLC system) coupled to a triple quadru- tected in urine specimens at the Air Force Drug Testing Laboratory pole mass spectrometer (UHPLC–MS/MS) operated in the selected using LC/MS/MS. Thirty urine samples were collected and subjected reaction monitoring mode. Out of 179 hair samples, 14 were positive to enzymatic hydrolysis followed by analysis using UPLC/TQD for detec- for at least one synthetic cannabinoid. All the synthetic cannabinoids tion of parent and daughter metabolites. The chemical structure of the showed a lower limit of detection (LOD; 0.02–0.18 pg/mg) and limit metabolite was confirmed by total synthesis followed by 1H NMR, 13C of quantitation (LOQ; 0.07–0.59 pg/mg) than that of cannabidiol NMR, IR and HRMS analyses (Lovett et al., 2013). JWH-018 metabolite (CBD), cannabinol (CBN), and Δ9-THC (LOD; 1.2–5.4 pg/mg and LOQ; f (Fig. 1) and JWH-73 metabolite e (Fig. 2) were also identified by the 3.9–18 pg/mg). same authors using LC/MS/MS. Liquid–liquid extraction was used to detect the presence of synthetic The LC/MS/MS method was developed to quantitate JWH-015, cannabinoids in 100 μLaliquotsofbloodsamples(Ammann et al., JWH-018, JWH-073, JWH-081, and JWH-200 and detect JWH-019 2012). An LC/MS/MS method was used to separate and detect 25 syn- and JWH-020 (Fig. 6) due to the increasing demand for detection and thetic cannabinoids of which seven belong to the naphthoylindole quantification of synthetic cannabinoids in biological samples (Dresen class (JWH-015, JWH-073, JWH-018, JWH-081, JWH-007, JWH-398,

O O O

N N OH N

(a) (b)OH (c) MW = 372.2 MW = 372.2 O MW = 372.3

N O O

JWH-122 MW = 356.2 N N

(d) (e) OH MW = 354.2 MW = 372.2

Fig. 4. Chemical structures of JWH-122 metabolites. Monohydroxylated at the N-alkyl chain (a), monohydroxylated at the naphthalene moiety (b), monohydroxylated at the indole moiety (c), dehydrogenated at the N-alkyl chain (d), and hydroxylated at the N-alkyl chain, C-5 (e). Hutter el al. (2012) and Uchiyama et al. (2013). 82 M.A. ElSohly et al. / Life Sciences 97 (2014) 78–90

O O O O

N N OH N N

JWH-210 (a) (b) OH (c) MW = 370.3 MW = 386.3 MW = 386.2 MW = 386.3

Fig. 5. Chemical structures of JWH-210 metabolites. monohydroxylated JWH-210 at the N-alkyl chain (a), the metabolite monohydroxylated at the naphthalene moiety (b), and monohydroxylated JWH-210 at the indole moiety (c). Hutter et al. (2012).

JWH-210). The method was validated according to three nationally In vivo metabolism of JWH-200 was conducted in mice where the accepted guidelines (Peters and Maurer, 2002; Peters et al., 2007; U.S. parent compound was orally administered and urine samples were Department of Health, Human Services, 2001). collected and purified by Solid Phase Extraction (SPE). Human liver mi- A method was developed to analyze 15 naphthoylindoles (JWH-007, crosomes were also used for in vitro metabolism of JWH-200, followed JWH-015, JWH-019, JWH-020, JWH-073, JWH-081, JWH-122 5 by SPE extraction using methanol and water mixture. The parent com- fluoropentyl derivative, JWH-200, JWH-210, JWH-387, JWH-398, pound and 11 metabolites (Fig. 7)fromthein vivo and in vitro studies JWH-412, AM-1220, and AM-2201) in human serum by liquid–liquid were analyzed by LC/MS (Brabanter et al., 2013). extraction and LC/MS/MS (Kneisel and Auwärter, 2012)(Table 2). All A validated LC/ESI/MS/MS method was applied to quantitate 28 syn- but three of the synthetic cannabinoids were quantitated using this thetic cannabinoids in oral fluids. The oral fluid samples were treated method which was validated according to the guidelines of the German with ice-cold acetonitrile to allow protein precipitation, the supernatant Society of Toxicological and Forensic Chemistry (Peters et al., 2009). was purified by HPLC using water with 0.2% formic acid and 2 mmol/L More than 800 serum samples were successfully analyzed by this ammonium formate as solvent A and methanol as solvent B, the mobile method during routine analysis. phase consisted of A/B (1:1). The analysis of the oral fluid of a volunteer A high-resolution mass spectrometry with mass defect filtering who ingested AM-2201 (5 mg) revealed that the synthetic cannabinoid method was applied to detect synthetic cannabinoids that are closely diffused from the blood stream into the oral fluids, but at a very low rate related to JWH-018 (Grabenauer et al., 2012). The use of a mass defect (Kneisel et al., 2013). filter and precursor ion searching or mass defect filtering of fragment JWH-018, JWH-019, JWH-073, JWH-081, JWH-200, JWH-210, JWH- ions encompassed a broad range of JWH-018-related compounds. An 250, RCS-8, AM-2201 were detected and identified in a variety of in- herbal mixture named “Spice 99 GI Joe” was analyzed, in which a filter cense products using Thin Layer Chromatography (TLC), GC/MS, HPLC at 0.051 with a window of ±20 mDa produced a chromatogram with and liquid chromatography time of flight mass spectrometry (LC/TOF) a single peak at 2.31 min (no background peaks). The mass spectrum of this peak contained two fragment ions at m/z 107.0504 and Table 1 135.0459 which are similar to those of JWH-018. Not only is mass defect Chemical structures of naphthoylindoles. great for metabolite identification (Zhang et al., 2003), but it also can be used to remove interferences from complex matrices (Zhu et al., 2006; Bateman et al., 2007). Recently, Uchiyama et al. (2013) identified the metabolites, N- (4-pentenyl)-JWH-122, JWH-213, AM-2232, AM-2201 and N-(5- hydroxypentyl)-JWH-122 (Table 1) in a herbal-type product sold in Japan. The product was extracted with methanol and analyzed by UPLC/ESI/MS. A liquid chromatography–quadrupole-time of flight mass spectrom- Compound name RR1 R2 etry (LC/QTOF/MS) was applied to study the diffusion of synthetic JWH-018 HC5H11 H cannabinoids in hair and 435 samples were screened, of which eight JWH-073 H C4H9 H JWH-081 OCH C H H were positive for the presence of JWH-018, JWH-073, JWH-081 3 5 11 JWH-122 CH3 C5H11 H and JWH-122 in concentrations ranging from 0.010 to 1.28 ng/mg JWH-210 CH2CH3 C5H11 H

(Gottardo et al., 2013). JWH-015 H C3H7 CH3 JWH-200 H 4-Ethylmorpholino H

JWH-019 HC6H13 H

JWH-020 H C7H15 H JWH-015: R1 = propyl, R2 =methyl, R3 = H JWH-007 H C5H11 CH3 JWH-018: R1 = pentyl, R2 = H, R3 = H O JWH-398 Cl C5H11 H R JWH-019: R = hexyl R = H R = H 3 1 , 2 , 3 JWH-387 Br C5H11 H - : = = = JWH 020 R1 heptyl, R2 H, R3 H JWH-412 F C5H11 H JWH-073: R1 = butyl, R2 = H, R3 = H AM-1220 H 1-Methylpiperidin-2-yl-methyl H R2 N JWH-081: R1 = pentyl, R2 = H, R3 = methoxy AM-2201 H C5H10FH WH-2 : R = 4-ethylmorpholino R = H R = H MAM-2201 CH3 C5H10FH R1 J 00 1 , 2 , 3 JWH-213 CH3CH2 C5H11 H N-(5-hydroxypentyl)-JWH-122 CH 5-Hydroxypentyl H Fig. 6. Chemical structures of synthetic cannabinoids detected in serum by Dresen et al. 3 (C H O) (2011). 5 11 M.A. ElSohly et al. / Life Sciences 97 (2014) 78–90 83

O M11 O O M10 N N NH N N O OH O N OH M9 M1 HO O HO N OH

O O O N N M8 N N O M2 N O OH JWH-200 O

O OH OH OH OH OH OH OH N O N O O O M3 O N M7 N N N OH N OH M4 N OH M5 M6 O O

Fig. 7. Chemical structures of JWH-200 metabolites. Brabanter et al. (2013).

(Logan et al., 2012). The average concentration of these drugs in the an- extract. Since the concentration of JWH-018 and JWH-073 was not alyzed products ranged from 5 to 20 mg/g. Many products contained quantiﬁed, the extract may not have had enough drug to give a positive more than one drug. result in either the KIMS or EMIT immunoassay screen.

Immunoassay GC/MS

An Enzyme Linked Immunosorbent Assay (ELISA) was used to detect Two main hydroxylated metabolites of JWH-018 were identiﬁed in a JWH-018 and its metabolites in urine specimens. The assay was calibrat- urine sample using GC and HPLC coupled with tandem mass spectrom- ed at 5 ng/mL with the 5-OH metabolite of JWH-018, validated with 114 etry (Sobolevskii et al., 2011). First, the composition of a smoking mix- urine samples and conﬁrmed by using LC/MS (Arntson et al., 2013). ture was determined by extracting the plant material with methanol Four commercially available herbal incense products (Spike Max, and analyzing on GC/MS. Then, a urine sample was extracted and ana- California Spice, K2 and Blueberry Haze) were analyzed for the detec- lyzed on both GC/MS/MS and HPLC/MS/MS which ionization gave tion of synthetic cannabinoids on both the kinetic interaction of micro- mass spectra of a and b (Fig. 8). At 50 pg/mL, JWH-018 was not detected particles in a solution (KIMS) immunoassay and an enzyme multiplied in urine 12 h after administration. immunoassay technique (EMIT) (Penn et al., 2011). Grigoryev et al. (2011a) studied the metabolism of JWH-018 and A tea and methanolic extract were prepared from each herbal JWH-073 in 26 herbal smoking mixtures in human and rat urine incense product which was analyzed by GC/MS. Only JWH-018 and samples using GC/MS. Human urine samples were extracted using liq- JWH-073 were detected in three of the herbal incense products (there uid–liquid and Solid Phase Extraction and the rat urine was collected were no synthetic cannabinoids detected in Blueberry Haze). There from rats that have been injected intraperitoneally with a tarry residue was no JWH-073 detected in Spike Max from the tea or methanolic resulted from the extraction of smoking mixture then suspended in 2% 84 M.A. ElSohly et al. / Life Sciences 97 (2014) 78–90

HO HO (2012b) (Fig. 9). Both compounds were analyzed by GC/MS and NMR spectroscopy. Choi et al. (2013) developed a validated rapid and simple GC/MS N N O O OH method to analyze synthetic cannabinoids in dried leaves, bulk pow- ders and tablets seized in Korea during drug trafﬁcking. JWH-018 and JWH-073 were the most frequently detected compounds. The ground powdered leaves (10 mg) were extracted with methanol (10 mL) and sonicated for 10 min, ﬁltered and then subjected to (a) (b) GC/MS analysis.

Fig. 8. Metabolites of JWH-018 identiﬁed by GC/MS/MS in urine. Sobolevskii et al. (2011). Matrix-assisted laser desorption ionization-time of ﬂight mass spectrometry (MALDI-TOF-MS)

The MALDI-TOF-MS method was developed for direct and rapid screening of herbal blends for synthetic cannabinoids (Gottardo et al., O O 2012). Each herbal blend was grounded and loaded onto a MALDI F plate. The method successfully analyzed 31 herbal blends in which 21 contained at least one of the synthetic cannabinoids JWH-018, JWH-073, JWH-081, JWH-210, and JWH-019. N N

Direct analysis in real time mass spectrometry (DART-MS)

DART-MS was applied to detect synthetic cannabinoids from solid F F herbal matrices to bypass sample preparation and extraction (Musah et al., 2012). This study rapidly identified JWH-015 which was added 1-[(5-fluoropentyl)-1H-indol-3yl]- to dried plant material. JWH-412 (4-methylnaphthalen-1-yl)methanone JWH-018 was identified from an extract of ‘Spice’ using the same technique (Dunham et al., 2012). ‘Spice’ was extracted using an Fig. 9. Chemical structures of the two synthetic cannabinoids identified by Moosmann et automated accelerated solvent extraction (ASE) instrument which is al. (2012b). known to provide clean extracts.

Tween-80. Native JWH-018 and JWH-073 were not detected in this study which was consistent with previous reports (Sobolevsky et al., Nano-liquid chromatography (nano-LC) 2010) but contradicts that of Kraemer et al. (2009).Theconflict of detecting the parent compounds in urine may be attributed to the Merola et al. (2012) applied nano-LC to separate the synthetic difference in detection limits among laboratories. cannabinoids, JWH-018, JWH-019, JWH-073, JWH-081, JWH-122, Because of the lack of commercial standards of synthetic cannabi- JWH-200, JWH-210 and AM-2201 in herbal blends (Table 1). An LCQ™ noids used in herbal mixtures, Moosmann et al. (2012a) extracted ion trap electrospray mass spectrometer was used to identify and charac- products purchased on the internet and purified the detected com- terize each analyte. The analytes were separated on the nano-LC in less pounds which were then used as standards for analysis. AM-2201 than 30 min in one run using an isocratic elution mode at 93% (v/v) ACN. and JWH-200 (Table 1) were prepared following this process and identified by GC/MS. Other synthetic cannabinoids identified as 1-[(5-fluoropentyl)-1H- Nuclear magnetic resonance (NMR) indol-3yl]-(4-methylnaphthalen-1-yl)-methanone isolated from the herbal mixture, ‘Xoxo’ and JWH-412 (provided by German authorities Rollins et al. (2013) applied a fluorine specific NMR spectroscopy for as a microcrystalline powder) were identified by Moosmann et al. the qualitative and quantitative analysis of AM-2201.

O O O O O O O O

N N OH N N

JWH-250 (a) OH (b) (c) MW = 336.3 MW = 352.2 MW = 352.2 MW = 352.4

Fig. 10. Chemical structures of JWH-250 and its monohydroxylated metabolite at the N-alkyl chain (a), the monohydroxylated metabolite at the indole moiety (b), and the monohydroxylated metabolite at the phenyl moiety (c). Hutter et al. (2012). M.A. ElSohly et al. / Life Sciences 97 (2014) 78–90 85

O O O OH O O N N O N M1 - M3 M4 JWH-250 OH

O O O HO HO 2OH O N O N O N M6 - M9 M10, M11 M5 OH

O O HO HO 2OH O N O N M12, M13 M14 - M18 O

O O O HO OH

O O O NH NH NH M22 M19 M20, M21

Fig. 11. Proposed structures of JWH-250 urinary metabolites. Grigoryev et al. (2011a).

Phenylacetylindoles monohydroxylated metabolite (M1) was the most convenient for diag- nosis of drug intoxication. Nineteen JWH-250 metabolites (products of LC/MS/MS mono- and polyhydroxylation, trihydroxylation with dehydration of the N-alkyl chain and N-dealkylation with monohydroxylation) could To analyze and screen urine samples of patients who had consumed be detected in human urine, and five mono- and dihydroxylated prod- synthetic cannabinoids, LC/MS/MS and HR/MS/MS were used (Hutter ucts (M1, M5, M7, M8 and M9) could be detected in smoker's serum et al., 2012). JWH-250 and its metabolites were identified by ion spectra (Grigoryev et al., 2011b). The primary urinary metabolites detected in and mass measurements (Fig. 10). human included the monohydroxylated components excreted as conju- Twenty-two metabolites of the synthetic cannabinoid JWH-250 gates with urinary acids. At least eleven metabolites could be detected (Fig. 11)inhumanurineandserumsamplesaswellasinraturine in rat urine: N-dealkylated, dihydroxylated and N-dealkylated com- were identified by Grigoryev et al. (2011b). The consumption of bined with monohydroxylation components (high concentration). JWH-250 can be established by detection of these metabolites in Native JWH-250 was not detected. This seems contrary to a previous urine collected within a day of consumption. The detection of the study (Dresen et al., 2011) that used LC linked to tandem mass

O O O R O O

N N N

R =-Cl JWH-203 R - Cannabipipreidiethanone R =-O-CH3 JWH-250 CS 8 R =-CH3 JWH-251

Fig. 12. Chemical structures of synthetic cannabinoids isolated from herbal mixtures. Kneisel and Auwarter (2012) and Moosmann et al. (2012a, b). 86 M.A. ElSohly et al. / Life Sciences 97 (2014) 78–90

HO Table 3 Structures of 7 benzoylindoles covered by the LC/ESI/MS/MS method. O O O O O O

N N N

HO Analyte WXY ZR RCS-4 (a) (b) AM-694 HIC H FHH MW = 322.1 MW = 336.5 MW = 336.5 5 10 RCS-4 OCH3 HC5H11 HH

RCS-4-C4 OCH3 HC4H9 HH Fig. 13. Chemical structures of RCS-4 and its monohydroxylated metabolites at the indole RCS-4 ortho isomer H OCH3 C5H11 HH moiety (a), and at the phenyl moiety (b). RCS-4-3-methoxy- HHC5H11 HOCH3 Hutter et al. (2012). isomer WIN 48,098 OCH3 H 2-Morpholin-4-yl-ethyl CH3 H AM-1241 OH H 1-Methylpiperidin-2-yl-methyl H H spectrometry to quantify the native JWH-250 in serum. Such dis- agreement can be explained by the difference in sensitivity of the applied analytical methods. This assumption, combined with ﬁndings (Grigoryev et al., 2010; Teske et al., 2010) on the rapid metabolism liquid extraction and LC/MS/MS (Fig. 12)(Kneisel and Auwärter, 2012). of naphthoylindoles, has resulted in the conclusion that the detection These analytes were quantitated using this method which was validated of metabolites in urine is preferred over the native compounds according to the guidelines of the German Society of Toxicological and themselves. Forensic Chemistry (Peters et al., 2009). More than 800 serum samples Overall, both GC/MS (after derivatization of samples by TMS or AC) were successfully analyzed by this method during routine analysis. and LC/MS/MS (MRM mode) can be used to establish JWH-250 The parent compounds JWH 250 and RCS-4 and their metabolites consumption. were identiﬁed in human urine using LC/MS/MS and library search A method for the detection of the synthetic cannabinoid JWH-250 (Wohlfarth et al., 2013). along with Δ9-THC, CBD, and CBN in hair samples was developed and A liquid–liquid extraction was used to detect the presence of syn- fully validated (Salomone et al., 2012). A liquid–liquid extraction was thetic cannabinoids in 100 μL aliquots of blood samples (Ammann performed followed by the analysis of the extract on an ultra-high et al., 2012). A LC/MS/MS method was developed for this extraction performance liquid chromatography system (UPLC system) coupled to which separated and detected 25 synthetic cannabinoids including a triple quadrupole mass spectrometer (UHPLC–MS/MS) operated in those in Fig. 12 The method was validated according to three nationally the selected reaction monitoring mode. Out of 179 hair samples, 14 accepted guidelines (Peters and Maurer, 2002; Peters et al., 2007; U.S. were positive for at least one synthetic cannabinoid. JWH-250 showed Department of Health, Human Services, 2001). a lower limit of detection (LOD; 0.02–0.18 pg/mg) and limit of quantitation (LOQ; 0.07–0.59 pg/mg) than that of cannabidiol (CBD), cannabinol (CBN), and Δ9-THC (LOD; 1.2–5.4 pg/mg and LOQ; 3.9–18 pg/mg). Immunoassay A method was developed to analyze 4 synthetic phenylacetylindoles (JWH-203, JWH-250, JWH-251, and RCS-8) in human serum by liquid– Arntson et al. (2013) used enzyme linked immunoassay to detect JWH 250 and its 4-OH metabolite in 84 urine samples. The method showed 98% accuracy and high sensitivity.

Table 2 Structures of the 15 naphthoylindoles covered by the LC/ESI/MS/MS. MALDI-TOF-MS

A MALDI-TOF-MS method was developed for direct and rapid screening of herbal blends for synthetic cannabinoids (Gottardo et al., 2012). Each herbal blend was grounded and loaded onto a MALDI plate. The method successfully analyzed 31 herbal blends of which 21 were positive for the synthetic cannabinoid JWH-250.

Analyte XY Z

JWH-007 HC5H11 CH3

JWH-015 HC3H7 CH3

JWH-018 HC5H11 H O

JWH-019 H C6H13 H JWH-020 H C7H15 H O JWH-073 H C4H9 H

JWH-081 OCH3 C5H11 H N

JWH-122 5-ﬂuoropentyl derivative CH3 C5H10FH JWH-200 H 2-Morpholin-4-yl-ethyl H N F JWH-210 C2H5 C5H11 H

JWH-387 Br C5H11 H JWH-398 Cl C5H11 H JWH-030 JWH-307 JWH-412 F C5H11 H AM-1220 H 1-Methylpiperidin-2-yl-methyl H Fig. 14. Chemical structures of the synthetic cannabinoids JWH-030 and JWH-307. AM-2201 H C H FH 5 10 Uchiyama et al. (2013). M.A. ElSohly et al. / Life Sciences 97 (2014) 78–90 87

OH OH Benzoylindoles OH OH LC/MS/MS

To analyze and screen the urine samples of patients who had consumed synthetic cannabinoids, HR/MS/MS was used (Hutter et al., 2012) and RCS-4 and its metabolites were identiﬁed by ion spectra cis-CP-47,497-C8 trans-CP-47,497-C8 and mass measurement (Fig. 13). Ammann et al., 2012 developed and validated an LC/MS/MS Fig. 15. Chemical structures of two cyclohexylphenols isolated from herbal mixtures. method to detect the presence of 7 synthetic benzoylindoles (WIN Moosmann et al. (2012a). 48,098, AM-1241, AM-694, RCS-4 C-4 homolog, RCS-4 2-methoxy homolog, RCS-4, and RCS-4 3-methoxy homolog) in blood samples GC/MS (Table 3).

Moosmann et al. (2012a) isolated synthetic cannabinoids from herb- MALDI-TOF-MS al mixtures by a flash chromatography system, which was a faster approach for obtaining reference standards from new drugs that appear A MALDI-TOF-MS method was developed for direct and rapid on the market, than waiting for the standards to become commercially screening of herbal blends for synthetic cannabinoids (Gottardo et al., available. They isolated and identified JWH-203, JWH-250, JWH-251 2012). Each herbal blend was grounded and loaded onto a MALDI and cannabipiperidiethanone by GC/MS (Fig. 12). plate. The method successfully analyzed 31 herbal blends of which 21 were positive for AM-694 (Table 3). Nano-LC GC/MS Nano-LC was used to separate 2 synthetic phenylacetylindoles (JWH-203 and JWH-250) and Δ9-THC in herbal blends (Merola et al., RCS-4 and AM-694 were detected and identified by Thin Layer 2012). An LCQ™ ion trap electrospray mass spectrometer was used to Chromatography (TLC), GC/MS, and HPLC LCTOF in a variety of incense identify and characterize each analyte. The analytes were separated on products (Logan et al., 2012). The average concentration of these the nano-LC in less than 30 min in one run using an isocratic elution synthetic cannabinoids in the materials ranged from 5 to 20 mg/g and mode at 93% (v/v) ACN. many products contained more than one drug.

OH OH

M7/M8 m/z 333 OH OH

OH OH OH OH OH M6 m/z 333 O OH

OH OH M1/M4 m/z 331 CP-47,497 m/z 317

M3/M5 m/z 333 OH

OH O

M2 m/z 347

Fig. 16. Chemical structures of CP47-497 metabolites proposed by Jin et al. (2013). 88 M.A. ElSohly et al. / Life Sciences 97 (2014) 78–90

O O O

N N H H N N N

F AB-001 APICA STS-135

Fig. 17. Chemical structures of adamantylindoles identiﬁed in herbal mixtures. Nico et al. (2013) and Uchiyama et al. (2013).

Nano-LC Cyclohexylphenols

Nano-liquid chromatography (nano-LC) was used to separate The synthetic cannabinoids cis-CP-47, 497-C8 and trans-CP-47, AM-694 (Table 3) in herbal blends as previously described (Merola 497-C8 (Fig. 15) were isolated from herbal mixtures by Moosmann et al., 2012). et al. (2012a) using flash chromatography technique and their chemical identity was determined by GC/MS. Logan et al. (2012) identified CP47,497-C8 in a variety of incense Naphthoylpyrrol blends using different chromatographic techniques. A validated LC/MS/MS method was used to detect 2 synthetic Two new synthetic cannabinoids namely JWH-030 and JWH- cyclophenols (CP 47,497 and CP 47,497 C-8 homolog) in blood samples 307 (Fig. 14)wereidentified by Uchiyama et al. (2013) using (Ammann et al., 2012). UPLC/ESIMS in a methanolic extract of an illegal Japanese herbal The metabolism of CP47,497 in human liver microsomes was product. studied by Jin et al. (2013), where eight metabolites were identified

O HO O O N OH O H N (OH)2 N (OH)2 N O N H N N N H N H N N N OH O OH h a b, c, d e, f, g

O O O O (OH)3 (OH)2 N OH N N H N H N H N N N N N H N N m, n i j, k, l

APINACA (AKB-48)

O OH O O N O O N H (OH)2 N N N H O O OH N N H HO N N HO OH HO OH O HO OH p o q

Fig. 18. AKB-48 and its metabolites (s) by human hepatocytes. Gandhi et al. (2013). M.A. ElSohly et al. / Life Sciences 97 (2014) 78–90 89

O N

H H N N N N OH O O F O Methanadamide 5-Fluoropentyl-3-pyridinoylindole URB-754

O H O N N O O O N N N Cl I N N N O

Cl F AM-251 WIN55, 212-2 XLR-11 UR-144

Fig. 19. Miscellaneous synthetic cannabinoids. by LC/MS/MS analysis, which included mono-oxygenated, mono- Nano-LC hydroxylated and di-oxygenated derivatives (Fig. 16). Merola et al. (2012) used nano-liquid chromatography (nano-LC) to separate WIN-55,212-2 in herbal blends. The compound was identiﬁed Adamantylindoles by ESI/MS.

Two new adamantylindoles (AB-001 and APICA) were identiﬁed in DART/MS Japanese illegal herbal products (Fig. 17). The synthetic cannabinoids were extracted from the powdered herbal product with methanol and DART/MS was used by Musah et al. (2012) to identify AM-251 in the samples were analyzed by UPLC–ESI-MS (Uchiyama et al., 2013). herbal preparations. STS-135 (Fig. 17) was detected in Spice like herbal mixtures by using EI-MS and ESI/MS/MS (Nico et al., 2013). Concluding remarks

Adamantylindazoles It is clear that the list of drugs with activity on the cannabinoid receptors is large and expanding every day. Many of these synthetic Gandhi et al. (2013) used human hepatocytes and TripleTOF mass cannabinoids are finding their way into illicit markets as marijuana spectrometry to identify 17 novel phase I and II metabolites of the substitutes without any safety studies. The scientific community is adamantylindazole AKB-48 (APINACA) (Fig. 18, a–q). The metabolism continuing to generate new compounds and new chemical classes in involved monohydroxylation, dihydroxylation, trihydroxylation, oxida- their search for CB1 and CB2 antagonists for medicinal applications. It tion and glucuronidation of the hydroxy metabolites of AKB-48. is anticipated that many of these compounds will find their way to the illicit market, thus the race will continue!

Miscellaneous Conﬂict of interest statement

LC/MS/MS We, all four authors, have no conﬂict of interest.

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