Synthetic cannabinoids: analysis and metabolites.

LFS-13865; No of Pages 13 Life Sciences xxx (2014) xxx–xxx

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Synthetic cannabinoids: Analysis and metabolites ElSohly Laboratories, Inc., 5 Industrial Park Drive, Oxford, MS 38655, USA 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

a b s t r a c t

Article history: Received 21 October 2013 Accepted 24 December 2013 Available online xxxx

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Cannabimimetics (commonly referred to as synthetic cannabinoids), a group of compounds encompassing a wide range of chemical structures, have been developed by scientists with the hope of achieving selectivity 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 used the information generated by the scientific community to develop these compounds for illicit use as marijuana substitutes. This chapter reviews the different classes of these “synthetic cannabinoids” with particular emphasis on the methods used for their identification in the herbal products with which they are mixed and identification of their metabolites in biological specimens. © 2014 Published by Elsevier Inc.

Keywords: Synthetic cannabinoids Analysis Herbal products Metabolites

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Contents

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Naphthoylindoles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Liquid chromatography electrospray ionization tandem mass spectrometry (LC/MS/MS) . . Immunoassay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . GC/MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) Direct analysis in real time mass spectrometry (DART-MS) . . . . . . . . . . . . . . . Nano-liquid chromatography (nano-LC) . . . . . . . . . . . . . . . . . . . . . . . . Nuclear magnetic resonance (NMR) . . . . . . . . . . . . . . . . . . . . . . . . . . Phenylacetylindoles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . LC/MS/MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Immunoassay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MALDI-TOF-MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . GC/MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nano-LC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Benzoylindoles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . LC/MS/MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MALDI-TOF-MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . GC/MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nano-LC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Naphthoylpyrole . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cyclohexylphenols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adamantylindoles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adamantylindazoles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Miscellaneous . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . LC/MS/MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nano-LC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DART/MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Mahmoud A. ElSohly a,b,c, Waseem Gul a,b, Amira S. Wanas b,d, Mohamed M. Radwan b,e

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0024-3205/$ – see front matter © 2014 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.lfs.2013.12.212

Please cite this article as: ElSohly MA, et al, Synthetic cannabinoids: Analysis and metabolites, Life Sci (2014), http://dx.doi.org/10.1016/ j.lfs.2013.12.212

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Introduction

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122

Naphthoylindoles

123 124

Liquid chromatography electrospray ionization tandem mass spectrometry (LC/MS/MS)

125

Since JWH-018 itself cannot be detected in urine, Möller et al. (2010) developed a method based on enzymatic hydrolysis followed by liquid–

126

0 0 0 0

liquid extraction and LC/MS/MS analysis to detect its major metabolites in human urine for the purpose of doping control. After a successful confirmation of the JWH-018 phase-I metabolites, the method was then used in routine analysis of doping control samples. Hutter et al. (2012) analyzed and screened the urine samples of patients who had consumed synthetic cannabinoids using LC/MS/MS and HR/MS/MS, and reported metabolites of JWH-018, JWH-073, JWH-081, JWH-122, and JWH-210 by ion spectra and mass measurement. They found that the major metabolic pathways include monohydroxylation on the naphthoyl moiety, indole moiety or at the N-alkyl chain (Figs. 1–5). Carboxylation of the side chain was reported only for JWH-018 and JWH-073. LC–MS/MS and the software assisted library searching against reference spectra were applied to detect the urinary metabolites of JWH-018, JWH-073, JWH-081, JWH-122, JWH-200, JWH-210, and AM-2201 (Wohlfarth et al., 2013). The parent compounds for these synthetic cannabinoids were also identified, and the method was validated with a limit of detection ranging from 0.5 to 10 ng/mL. D5-JWH-200 and D9-JWH-081 were used as internal standards at concentrations of 800 and 100 ng/mL, respectively. JWH-018 was subjected to in vitro metabolism using human liver microsomal system. The compound was incubated with the microsomes for 30 min at 37 °C in the presence of NADP and G-6-PDH (ElSohly et al., 2011). This was followed by extraction of the reaction mixture and analysis by LC/MS/MS. LightSight® software program for metabolite identification was used to identify possible metabolites in the LC/MS/MS (Qtrap) run of the HLM preparation extract. A full scan mass spectrum was generated for each peak based on the ions trapped in the Qtrap. This allows for the generation of the total ion chromatogram for each group of metabolites sharing the same molecular ion. Two major monohydroxylated metabolites of JWH-018 (with m/z 358) were detected. A close examination of the fragmentation pattern of these metabolites showed that the hydroxy group of the early eluting metabolite is located on the terminal carbon of the side chain attached to the indole nitrogen. This is supported by the presence of ions at m/z 127 (unhydroxylated naphthalene nucleus), m/z 155 (the naphthalene nucleus with the carbonyl group), and m/z 284 (the molecular ion with loss of the terminal 4 carbon moiety of the side chain containing the hydroxy group). On the other hand, the spectrum of the second, later eluting, monohydroxy metabolite showed fragmentation consistent with hydroxylation on the indole moiety. This was proven to be the 6-hydroxy-metabolite by comparison with a reference sample made available by Cayman Chemical during the course of the work. A urine specimen received at ElSohly Laboratories, Inc. (ELI) (specimen CM504), from a subject who admitted the use of Spice, was analyzed following the same protocol used for the HLM metabolic study. Examination of the LightSight list of possible metabolites indicated the presence of several peaks with m/z 358 (monohydroxylated metabolites), m/z 372 (possible carboxy metabolite at the terminal carbon of the side chain), m/z 390 (possible trihydroxy metabolite), and m/z 374 (possible dihydroxy metabolite) (Fig. 1), these metabolites were also proposed by Sobolevskii et al. (2011). JWH-073, another naphthoylindole derivative analogous to JWH-018 with a C4 side chain 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 as previously described for JWH-018. Interestingly, only the 4-hydroxy metabolite of JWH-073 (Fig. 2) was confirmed by comparison with the standard made available in house at ELI. LC/MS/MS chromatograms of the HLM NADPH at 30 min showed peaks of four unidentified metabolites of JWH-073 with a

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Synthetic cannabinoids, better referred to as cannabimimetic 73 compounds, are compounds prepared by scientists around the world 74 targeting an interaction with the endocannabinoid system, namely CB1 75 and CB2 receptors. Although many of these compounds have been de76 scribed in the literature for many years, their illicit use appeared only in 77 the last few years. They appeared in the United States market in late 78 2008. They were mixed with herbal products and sold as incense or pot79 pourri on the internet, gas stations and tobacco shops under many brand 80 names (Spice, Spice gold, Aroma, K2, Spike 99, etc.) with labels stating 81 “not for human consumption”. These products are claimed to contain 82 only natural non-illegal compounds and consequently have no limitations 83 in their commercial distribution (Carroll et al., 2012). The consumption of 84 these products has become a popular alternative to marijuana, as they are 85 of high-potency and high efficacy as cannabinoid receptor full agonists. 86 The number of patients presented to the emergency department with 87 problems associated with these drugs has dramatically increased. In 88 March, 2011 the US Drug Enforcement Administration (DEA) scheduled 89 five synthetic cannabinoids (JWH-018, JWH-073, JWH-200, CP-47,497, 90 and CP-47,497 C8 homologue) as schedule 1 controlled substances. 91 Many of these products especially Spice and K2 have been banned in 92 many European countries (ElSohly et al., 2011; Wells and Ott, 2011) 93 and in May 2013, three synthetic cannabinoids (UR-144, XLR-11 and 94 AKB-48) were also placed in schedule 1. 95 Moreover, compared to THC, some synthetic cannabinoids possess a 96 4–5 times improved binding affinity to the cannabinoid CB1 receptor 97 and many toxicity symptoms were reported including anxiety, para98 noia, tachycardia, irritability, hallucination, numbness, seizures, high 99 blood pressure, drowsiness, and slurred speech (Seely et al., 2012). 100 Over the past few years a great effort has been exerted to identify and 101 quantify synthetic cannabinoids in herbal products, and detect their 102 metabolites in body fluids (urine, serum, and saliva) and also in hair 103 specimens. These methods include liquid chromatography tandem 104 mass spectroscopy (LC–MS/MS) (Teske et al., 2010), high mass resolu105 tion techniques like matrix-assisted laser desorption/ionization time 106 of flight mass spectroscopy (MALDI-TOF) (Gottardo et al., 2012), direct 107 analysis in real time mass spectrometry (DART-MS) (Musah et al., 108 2012), nuclear magnetic resonance (NMR) (Rollins et al., 2013), 109 gas chromatography/mass spectrometry (GC/MS) (Sobolevskii et al., 110 Q10 2011), and immunoassays (Arntson et al., 2013). 111 Recently, many reviews on the chemistry, toxicity, and pharmacolo112 Q11 gy of synthetic cannabinoids have been published (Carroll et al., 2012; Q12113 Q13 Favretto et al., 2013; Seely et al., 2012; Spaderna et al., 2013; Wells 114 and Ott, 2011). In this chapter, the focus will be on the analysis of the 115 different classes of synthetic cannabinoids in herbal mixtures and the 116 identification/analysis of their metabolites in biological fluids. 117 Synthetic cannabinoids can be chemically classified into 118 naphthoylindoles, benzoylindoles, phenylacetylindoles, adam119 antylindoles, cyclophenols and a miscellaneous group. Different analyt120 ical techniques have been applied to the detection and quantitation of 121 different members of each of these classes. Details are outlined below.

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Concluding remarks . . . . . . . Conflict of interest statement . . . Uncited reference . . . . . . . . . References . . . . . . . . . . . .

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O

N

3

COOH

(a)

O

OH

N

OH

(b)

N O

N

(f)

JWH-018

O

HO O

(c)

OH

P

N

N

R O

OH

HO

O

OH

F

O

O

D

N

E

(e)

OH

T

(d)

C

R

R

E

Q2

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. (2011) and Lovett et al. (2013).

N C O

O

N

U

JWH-073 MW = 326.2

O

O

O

N

(a) MW = 358.3

O

OH

(b)

OH

MW = 344.3

(c)

MW = 344.3

O

O

N

N

OH

N

O

N

N

4

3

O

(d)

HO

MW = 344.4

Q5

(e)

HO

MW = 344.4

(f)

OH

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. (2011) and Lovett et al. (2013).


4


OH O O

(a)

197 198 199 200 201 202 203 204

F

O

et al., 2011). The method was fully validated on 101 serum samples which underwent liquid–liquid extraction. Detection of the synthetic cannabinoids JWH-018, JWH-073, JWH-200 in hair samples was carried out using a fully validated method by Salomone et al. (2012). Liquid–liquid extraction was performed followed by an analysis of the extract on an ultra-high performance liquid chromatography system (UPLC system) coupled to a triple quadrupole mass spectrometer (UHPLC–MS/MS) operated in the selected reaction monitoring mode. Out of 179 hair samples, 14 were positive for at least one synthetic cannabinoid. All the synthetic cannabinoids showed 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). Liquid–liquid extraction was used to detect the presence of synthetic cannabinoids in 100 μL aliquots of blood samples (Ammann et al., 2012). An LC/MS/MS method was used to separate and detect 25 synthetic cannabinoids of which seven belong to the naphthoylindole class (JWH-015, JWH-073, JWH-018, JWH-081, JWH-007, JWH-398,

R O

mass of m/z 344 (Fig. 2), presumably monohydroxylated and possibly one of them is the three carbon-N-carboxylic acid metabolite shown in Fig. 2. The only metabolite identified in the LC/MS/MS chromatogram was the terminal hydroxylated metabolite which showed a mass of m/z 344 with Rt and fragmentation identical with a standard synthesized in-house. A new metabolite, JWH-72 N-propanoic acid (Fig. 2, d) was detected in urine specimens at the Air Force Drug Testing Laboratory using LC/MS/MS. Thirty urine samples were collected and subjected to enzymatic hydrolysis followed by analysis using UPLC/TQD for detection of parent and daughter metabolites. The chemical structure of the metabolite was confirmed by total synthesis followed by 1H NMR, 13C NMR, IR and HRMS analyses (Lovett et al., 2013). JWH-018 metabolite f (Fig. 1) and JWH-73 metabolite e (Fig. 2) were also identified by the same authors using LC/MS/MS. The LC/MS/MS method was developed to quantitate JWH-015, JWH-018, JWH-073, JWH-081, and JWH-200 and detect JWH-019 and JWH-020 (Fig. 6) due to the increasing demand for detection and quantification of synthetic cannabinoids in biological samples (Dresen

R

E

205

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. (2011).

P

195 196

MW = 388.2

D

193 194

(c)

E

191 192

(b)

T

189 190

N

MW = 388.5

OH

MW = 388.3

C

187 188

O N

OH

N

JWH-081 MW = 372.3

O

O

O

N

Q6

O

O

N

C

O

R

OH

O

OH

N

N JWH-122 MW = 356.2

Q7

N N

(a)

(b)

MW = 372.2

MW = 372.3

U

O

O

O

OH

(c)

MW = 372.2

O

O

N

N

(d)

(e)

MW = 354.2

MW = 372.2

OH

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. (2011) and Uchiyama et al. (2013).


206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224


5

OH

O

O

JWH-210

(a)

(b)

MW = 370.3

MW = 386.3

MW = 386.2

F

JWH-210). The method was validated according to three nationally

241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261

262

Table 1 Chemical structures of naphthoylindoles.

t1:1 t1:2

P

D

E

T

C

239 240

E

238

R

236 237

In vivo metabolism of JWH-200 was conducted in mice where the parent compound was orally administered and urine samples were collected and purified by Solid Phase Extraction (SPE). Human liver microsomes were also used for in vitro metabolism of JWH-200, followed by SPE extraction using methanol and water mixture. The parent compound and 11 metabolites (Fig. 7) from the in vivo and in vitro studies were analyzed by LC/MS (Brabanter et al., 2013). A validated LC/ESI/MS/MS method was applied to quantitate 28 synthetic cannabinoids in oral fluids. The oral fluid samples were treated with ice-cold acetonitrile to allow protein precipitation, the supernatant was purified by HPLC using water with 0.2% formic acid and 2 mmol/L ammonium formate as solvent A and methanol as solvent B, the mobile phase consisted of A/B (1:1). The analysis of the oral fluid of a volunteer who ingested AM-2201 (5 mg) revealed that the synthetic cannabinoid diffused from the blood stream into the oral fluids, but at a very low rate (Kneisel et al., 2013). JWH-018, JWH-019, JWH-073, JWH-081, JWH-200, JWH-210, JWH250, RCS-8, AM-2201 were detected and identified in a variety of incense products using Thin Layer Chromatography (TLC), GC/MS, HPLC and liquid chromatography time of flight mass spectrometry (LC/TOF)

263 264 265 266 267 Q16 268 269 270 271 272 273 274 275 276 277 278 279 280 281

R

234 235

N C O

232 233

A method was developed to analyze 15 naphthoylindoles (JWH-007, JWH-015, JWH-019, JWH-020, JWH-073, JWH-081, JWH-122 5 fluoropentyl derivative, JWH-200, JWH-210, JWH-387, JWH-398, JWH-412, AM-1220, and AM-2201) in human serum by liquid–liquid extraction and LC/MS/MS (Kneisel and Auwärter, 2012) (Table 1). All but three of the synthetic cannabinoids were quantitated using this method which was validated according to the guidelines of the German Society of Toxicological and Forensic Chemistry (Peters et al., 2009). More than 800 serum samples were successfully analyzed by this method during routine analysis. A high-resolution mass spectrometry with mass defect filtering method was applied to detect synthetic cannabinoids that are closely related to JWH-018 (Grabenauer et al., 2012). The use of a mass defect filter and precursor ion searching or mass defect filtering of fragment ions encompassed a broad range of JWH-018-related compounds. An herbal mixture named “Spice 99 GI Joe” was analyzed, in which a filter at 0.051 with a window of ±20 mDa produced a chromatogram with 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 135.0459 which are similar to those of JWH-018. Not only is mass defect 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-(5hydroxypentyl)-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 spectrometry (LC/QTOF/MS) was applied to study the diffusion of synthetic cannabinoids in hair and 435 samples were screened, of which eight were positive for the presence of JWH-018, JWH-073, JWH-081 and JWH-122 in concentrations ranging from 0.010 to 1.28 ng/mg (Gottardo et al., 2013).

U

230 231

(c)

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. (2011).

226 Q15 accepted guidelines (Peters and Maurer, 2002; Peters et al., 2007; U.S. 227 Department of Health, Human Services, 2001). 228 229

OH

O

225

N

N

R O

Q8

OH

N

N

O

O

O

N R1

R3 R2

JWH-015: R1 = propyl, R2 = methyl, R3 = H JWH-018: R1 = pentyl, R2 = H, R3 = H JWH-019: R1 = hexyl, R2 = H, R3 = H JWH-020: R1 = heptyl, R2 = H, R3 = H JWH-073: R1 = butyl, R2 = H, R3 = H JWH-081: R1 = pentyl, R2 = H, R3 = methoxy JWH-200: R1 = 4-ethylmorpholino, R2 = H, R3 = H

Fig. 6. Chemical structures of synthetic cannabinoids detected in serum by Dresen et al. (2011).

t1:3 t1:4 Compound name

R

R1

R2

t1:5

JWH-018 JWH-073 JWH-081 JWH-122 JWH-210 JWH-015 JWH-200 JWH-019 JWH-020 JWH-007 JWH-398 JWH-387 JWH-412 AM-1220 AM-2201 MAM-2201 JWH-213 N-(5-hydroxypentyl)-JWH-122

H H OCH3 CH3 CH2CH3 H H H H H Cl Br F H H CH3 CH3CH2 CH3

C5H11 C4H9 C5H11 C5H11 C5H11 C3H7 4-Ethylmorpholino C6H13 C7H15 C5H11 C5H11 C5H11 C5H11 1-Methylpiperidin-2-yl-methyl C5H10F C5H10F C5H11 5-Hydroxypentyl (C5H11O)

H H H H H CH3 H H H CH3 H H H H H H H H

t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14 t1:15 t1:16 t1:17 t1:18 t1:19 t1:20 t1:21 t1:22 t1:23


6


O O

M11 O

M10

N N NH

N

N

M1

O O OH

OH

N

M9

O

N

HO

F

HO

O

O

R O

O N N N

M2

JWH-200

OH

OH

M3

O O

O

E

N N

R

M4

N

M5

Immunoassay

286

An Enzyme Linked Immunosorbent Assay (ELISA) was used to detect JWH-018 and its metabolites in urine specimens. The assay was calibrated at 5 ng/mL with the 5-OH metabolite of JWH-018, validated with 114 urine samples and confirmed by using LC/MS (Arntson et al., 2013). Four commercially available herbal incense products (Spike Max, California Spice, K2 and Blueberry Haze) were analyzed for the detection of synthetic cannabinoids on both the kinetic interaction of microparticles in a solution (KIMS) immunoassay and an enzyme multiplied immunoassay technique (EMIT) (Penn et al., 2011). A tea and methanolic extract were prepared from each herbal incense product which was analyzed by GC/MS. Only JWH-018 and JWH-073 were detected in three of the herbal incense products (there were no synthetic cannabinoids detected in Blueberry Haze). There was no JWH-073 detected in Spike Max from the tea or methanolic

293 294 295 296 297 298 299

U

N

C

285

291 292

OH M6

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 analyzed products ranged from 5 to 20 mg/g. Many products contained more than one drug.

289 290

OH

N

M7 OH

N

283 284

287 288

O

N

O

R

O

282

O

T

N

OH

OH

C

N

OH

O

OH

E

OH

M8 N

OH

D

O

O

O

P

N O

OH

extract. Since the concentration of JWH-018 and JWH-073 was not 300 quantified, the extract may not have had enough drug to give a positive 301 result in either the KIMS or EMIT immunoassay screen. 302 GC/MS

303

Two main hydroxylated metabolites of JWH-018 were identified in a urine sample using GC and HPLC coupled with tandem mass spectrometry (Sobolevskii et al., 2011). First, the composition of a smoking mixture was determined by extracting the plant material with methanol and analyzing on GC/MS. Then, a urine sample was extracted and analyzed on both GC/MS/MS and HPLC/MS/MS which ionization gave mass spectra of a and b (Fig. 8). At 50 pg/mL, JWH-018 was not detected in urine 12 h after administration. Grigoryev et al. (2011a) studied the metabolism of JWH-018 and JWH-073 in 26 herbal smoking mixtures in human and rat urine samples using GC/MS. Human urine samples were extracted using liquid–liquid and Solid Phase Extraction and the rat urine was collected from rats that have been injected intraperitoneally with a tarry residue resulted from the extraction of smoking mixture then suspended in 2%

304


305 306 307 308 309 310 311 312 313 314 315 316 317


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 method to analyze synthetic cannabinoids in dried leaves, bulk powders 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 GC/MS analysis.

HO N

O

N

O

(a)

7

OH

(b)

Fig. 8. Metabolites of JWH-018 identified by GC/MS/MS in urine. Sobolevskii et al. (2011).

F

1-[(5-fluoropentyl)-1H-indol-3yl](4-methylnaphthalen-1-yl)methanone

325 326 327 328 329 330

Q3

N

JWH-250

(a)

MW = 336.3

MW = 352.2

349

O

Nano-liquid chromatography (nano-LC)

359

Merola et al. (2012) applied nano-LC to separate the synthetic cannabinoids, JWH-018, JWH-019, JWH-073, JWH-081, JWH-122, JWH-200, JWH-210 and AM-2201 in herbal blends (Table 1). An LCQ™ ion trap electrospray mass spectrometer was used to identify and characterize each analyte. The analytes were separated on the nano-LC in less than 30 min in one run using an isocratic elution mode at 93% (v/v) ACN.

360

Nuclear magnetic resonance (NMR)

366

352 353 354 355 356 357 358

361 362 363 364 365

Rollins et al. (2013) applied a fluorine specific NMR spectroscopy for 367 the qualitative and quantitative analysis of AM-2201. 368

O O

N

347 348

351

O O

345 346

DART-MS was applied to detect synthetic cannabinoids from solid herbal matrices to bypass sample preparation and extraction (Musah et al., 2012). This study rapidly identified JWH-015 which was added to dried plant material. JWH-018 was identified from an extract of ‘Spice’ using the same technique (Dunham et al., 2012). ‘Spice’ was extracted using an automated accelerated solvent extraction (ASE) instrument which is known to provide clean extracts.

OH

O O

350

P

T

C

E

U

331 332

R

323 324

R

321 322

Tween-80. Native JWH-018 and JWH-073 were not detected in this study which was consistent with previous reports (Sobolevsky et al., 2010) but contradicts that of Kraemer et al. (2009). The conflict of detecting the parent compounds in urine may be attributed to the difference in detection limits among laboratories. Because of the lack of commercial standards of synthetic cannabinoids used in herbal mixtures, Moosmann et al. (2012a) extracted products purchased on the internet and purified the detected compounds which were then used as standards for analysis. AM-2201 and JWH-200 (Table 1) were prepared following this process and identified by GC/MS. Other synthetic cannabinoids identified as 1-[(5-fluoropentyl)-1Hindol-3yl]-(4-methylnaphthalen-1-yl)-methanone isolated from the herbal mixture, ‘Xoxo’ and JWH-412 (provided by German authorities as a microcrystalline powder) were identified by Moosmann et al.

N C O

319 320

340 341

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

E

JWH-412

Fig. 9. Chemical structures of the two synthetic cannabinoids identified by Moosmann et al. (2012b).

318

339

344

D

F

F

337 338

The 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 in which 21 contained at least one of the synthetic cannabinoids JWH-018, JWH-073, JWH-081, JWH-210, and JWH-019.

R O

F

N

N

335 336

Matrix-assisted laser desorption ionization-time of flight mass spectrometry 342 (MALDI-TOF-MS) 343

O

O

333 334

N

OH

O O

N

(b)

(c)

MW = 352.2

MW = 352.4

OH

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. (2011).


8


O

O

O OH

O

O

N

JWH-250

O

N M4

OH

O

O

HO

O

HO

N M1 - M3

2OH O

O

N

O M10, M11 OH

N

M6 - M9

O

HO

R O

O

HO

O

F

M5

N

2OH

O

N

M14 - M18

O

O

O

HO

O

O OH

O

NH

M19

P

M12, M13

N

D

O

O

NH

NH

M22

E

M20, M21

C

T

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

Phenylacetylindoles

370

LC/MS/MS

371

To analyze and screen urine samples of patients who had consumed synthetic cannabinoids, LC/MS/MS and HR/MS/MS were used (Hutter et al., 2012). JWH-250 and its metabolites were identified by ion spectra and mass measurements (Fig. 10). Twenty-two metabolites of the synthetic cannabinoid JWH-250 (Fig. 11) in human urine and serum samples as well as in rat urine were identified by Grigoryev et al. (2011b). The consumption of JWH-250 can be established by detection of these metabolites in urine collected within a day of consumption. The detection of the

R

N

378 379

R

376 377

O

374 375

C

372 373

E

369

U

O

O

O

R N

monohydroxylated metabolite (M1) was the most convenient for diagnosis of drug intoxication. Nineteen JWH-250 metabolites (products of mono- and polyhydroxylation, trihydroxylation with dehydration of the N-alkyl chain and N-dealkylation with monohydroxylation) could be detected in human urine, and five mono- and dihydroxylated products (M1, M5, M7, M8 and M9) could be detected in smoker's serum (Grigoryev et al., 2011b). The primary urinary metabolites detected in human included the monohydroxylated components excreted as conjugates with urinary acids. At least eleven metabolites could be detected in rat urine: N-dealkylated, dihydroxylated and N-dealkylated combined with monohydroxylation components (high concentration). Native JWH-250 was not detected. This seems contrary to a previous study (Dresen et al., 2011) that used LC linked to tandem mass

O

O N

N

N R = -Cl JWH-203 R = -O-CH3 JWH-250 R = -CH3 JWH-251 Q4

RCS-8

Cannabipipreidiethanone

Fig. 12. Chemical structures of synthetic cannabinoids isolated from herbal mixtures. Kneisel and Auwarter (2012) and Moosmann et al. (2012a, b).


380 381 382 383 384 385 386 387 388 389 390 391 392


HO O

O

O

O

O

N HO

(a)

(b) MW = 336.5

MW = 336.5

MW = 322.1

t3:3 t3:4

N

410 411 412 413

C

408 409

E

406 407

R

404 405

R

402 403

N C O

401

t2:3 t2:4

U

399 400

t2:5

Analyte

t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13 t2:14 t2:15 t2:16 t2:17 t2:18 t2:19 t2:20

JWH-007 JWH-015 JWH-018 JWH-019 JWH-020 JWH-073 JWH-081 JWH-122 5-fluoropentyl derivative JWH-200 JWH-210 JWH-387 JWH-398 JWH-412 AM-1220 AM-2201

X

Y

Z

H H H H H H OCH3 CH3 H C2H5 Br Cl F H H

C5H11 C3H7 C5H11 C6H13 C7H15 C4H9 C5H11 C5H10F 2-Morpholin-4-yl-ethyl C5H11 C5H11 C5H11 C5H11 1-Methylpiperidin-2-yl-methyl C5H10F

CH3 CH3 H H H H H H H H H H H H H

Z

R

t3:5

C5H10F C5H11 C4H9 C5H11 C5H11

H H H H H

H H H H OCH3

t3:6 t3:7 t3:8 t3:9 t3:10

OCH3 OH

H H

2-Morpholin-4-yl-ethyl 1-Methylpiperidin-2-yl-methyl

CH3 H

H H

t3:11 t3:12

liquid extraction and LC/MS/MS (Fig. 12) (Kneisel and Auwärter, 2012). These analytes were quantitated using this method which was validated according to the guidelines of the German Society of Toxicological and Forensic Chemistry (Peters et al., 2009). More than 800 serum samples were successfully analyzed by this method during routine analysis. The parent compounds JWH 250 and RCS-4 and their metabolites were identified in human urine using LC/MS/MS and library search (Wohlfarth et al., 2013). A liquid–liquid extraction was used to detect the presence of synthetic cannabinoids in 100 μL aliquots of blood samples (Ammann et al., 2012). A LC/MS/MS method was developed for this extraction which separated and detected 25 synthetic cannabinoids including those in Fig. 12 The method was validated according to three nationally accepted guidelines (Peters and Maurer, 2002; Peters et al., 2007; U.S. Department of Health, Human Services, 2001).

416

Immunoassay

431

P

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

397 398

Y

I H H OCH3 H

D

t2:1 t2:2

395 396

T

414 415

spectrometry to quantify the native JWH-250 in serum. Such disagreement 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 of naphthoylindoles, has resulted in the conclusion that the detection of metabolites in urine is preferred over the native compounds themselves. Overall, both GC/MS (after derivatization of samples by TMS or AC) and LC/MS/MS (MRM mode) can be used to establish JWH-250 consumption. A method for the detection of the synthetic cannabinoid JWH-250 along with Δ9-THC, CBD, and CBN in hair samples was developed and fully validated (Salomone et al., 2012). A liquid–liquid extraction was performed followed by the analysis of the extract on an ultra-high performance liquid chromatography system (UPLC system) coupled to a triple quadrupole mass spectrometer (UHPLC–MS/MS) operated in the selected reaction monitoring mode. Out of 179 hair samples, 14 were positive for at least one synthetic cannabinoid. JWH-250 showed 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). A method was developed to analyze 4 synthetic phenylacetylindoles (JWH-203, JWH-250, JWH-251, and RCS-8) in human serum by liquid–

X

H OCH3 OCH3 H H

E

393 394

W

AM-694 RCS-4 RCS-4-C4 RCS-4 ortho isomer RCS-4-3-methoxyisomer WIN 48,098 AM-1241

R O

Fig. 13. Chemical structures of RCS-4 and its monohydroxylated metabolites at the indole moiety (a), and at the phenyl moiety (b). Hutter et al. (2012).

Analyte

F

N RCS-4

t3:1 t3:2

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

O

O

9

417 418 419 420 421 422 423 424 425 426 427 428 429 Q17 430

Arntson et al. (2013) used enzyme linked immunoassay to detect 432 Q18 JWH 250 and its 4-OH metabolite in 84 urine samples. The method 433 showed 98% accuracy and high sensitivity. 434

MALDI-TOF-MS

435

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.

436 437

O O N N

JWH-030

F JWH-307

Fig. 14. Chemical structures of the synthetic cannabinoids JWH-030 and JWH-307. Uchiyama et al. (2013).


438 439 440 Q19


GC/MS

442

447

Moosmann et al. (2012a) isolated synthetic cannabinoids from herbal mixtures by a flash chromatography system, which was a faster approach for obtaining reference standards from new drugs that appear on the market, than waiting for the standards to become commercially available. They isolated and identified JWH-203, JWH-250, JWH-251 and cannabipiperidiethanone by GC/MS (Fig. 12).

448

Nano-LC

449 450

Nano-LC was used to separate 2 synthetic phenylacetylindoles (JWH-203 and JWH-250) and Δ9-THC in herbal blends (Merola et al., 2012). An LCQ™ ion trap electrospray mass spectrometer was used to identify and characterize each analyte. The analytes were separated on the nano-LC in less than 30 min in one run using an isocratic elution mode at 93% (v/v) ACN.

457 458

MALDI-TOF-MS

R

OH OH

472

RCS-4 and AM-694 were detected and identified by Thin Layer Chromatography (TLC), GC/MS, and HPLC LCTOF in a variety of incense products (Logan et al., 2012). The average concentration of these synthetic cannabinoids in the materials ranged from 5 to 20 mg/g and many products contained more than one drug.

OH

OH OH OH OH

OH M6 m/z 333

O OH OH

OH

CP-47,497 m/z 317

M3/M5 m/z 333

OH O

OH

464 465 Q20

GC/MS

OH

M1/M4 m/z 331

462 463

467

R

E

M7/M8 m/z 333

460 461

466

C

OH

459

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 AM-694 (Table 3).

T

OH

O

453 454

C

451 452

N

445 446

U

443 444

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 and mass measurement (Fig. 13). Ammann et al., 2012 developed and validated an LC/MS/MS method to detect the presence of 7 synthetic benzoylindoles (WIN 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 (Table 3). (See Table 2.)

E

441

456

F

Fig. 15. Chemical structures of two cyclohexylphenols isolated from herbal mixtures. Moosmann et al. (2012a).

LC/MS/MS

O

trans-CP-47,497-C8

cis-CP-47,497-C8

455

R O

OH

OH

Benzoylindoles

P

OH

OH

D

10

O

M2 m/z 347 Fig. 16. Chemical structures of CP47-497 metabolites proposed by Jin et al. (2013).


468 469 470 471

473 474 475 476 477


O

11

O

O N H

N

N

AB-001

N H

N

F

APICA

STS-135

O

F

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

Nano-LC

Cyclohexylphenols

479

Nano-liquid chromatography (nano-LC) was used to separate AM-694 (Table 3) in herbal blends as previously described (Merola et al., 2012).

The synthetic cannabinoids cis-CP-47, 497-C8 and trans-CP-47, 497-C8 (Fig. 15) were isolated from herbal mixtures by Moosmann 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 blends using different chromatographic techniques. A validated LC/MS/MS method was used to detect 2 synthetic cyclophenols (CP 47,497 and CP 47,497 C-8 homolog) in blood samples (Ammann et al., 2012). The metabolism of CP47,497 in human liver microsomes was studied by Jin et al. (2013), where eight metabolites were identified

480 481

E O

b, c, d

R

a

N

N C O

O N H

N

N

N

(OH)2

N H

N O

h e, f, g O

O OH

N

(OH)2

OH

O N H

N

N H

N

R

OH

C

N

N

E

O

N H

O

O

OH

N H

N

T

O HO

N

D

Two new synthetic cannabinoids namely JWH-030 and JWH307 (Fig. 14) were identified by Uchiyama et al. (2013) using UPLC/ESIMS in a methanolic extract of an illegal Japanese herbal product.

N

N H

N

(OH)2 N

N

(OH)3

N H

m, n i

j, k, l

APINACA (AKB-48)

U

485 486

P

482 Q21 Naphthoylpyrole 483 484

487

R O

478

O

N

N H

N

OH

O

O (OH)2

N

N H

N

O HO HO

O OH

N

N

N H

O

OH

HO HO

O o

p

O OH OH

q

Fig. 18. AKB-48 and its metabolites (s) by human hepatocytes. Gandhi et al. (2013).


488 489 490 491 492 493 494 495 496 497 498

12


N

O

N

OH

O

O

F

Methanadamide

O URB-754

5-Fluoropentyl-3-pyridinoylindole

H N

O N

O

O

N

N

O

Cl

I

N

O

N N

AM-251

WIN55, 212-2

XLR-11

by LC/MS/MS analysis, which included mono-oxygenated, monohydroxylated and di-oxygenated derivatives (Fig. 16).

D

507

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

508

Adamantylindazoles

509 510

513

Gandhi et al. (2013) used human hepatocytes and TripleTOF mass spectrometry to identify 17 novel phase I and II metabolites of the adamantylindazole AKB-48 (APINACA) (Fig. 18, a–q). The metabolism involved monohydroxylation, dihydroxylation, trihydroxylation, oxidation and glucuronidation of the hydroxy metabolites of AKB-48.

514

Miscellaneous

515

LC/MS/MS

C

E R

R

O

C N

516

E

502

DART/MS

536

T

Adamantylindoles

511 512

532

Merola et al. (2012) used nano-liquid chromatography (nano-LC) to 533 separate WIN-55,212-2 in herbal blends. The compound was identified 534 by ESI/MS. 535

501

505 506

Nano-LC

UR-144

P

Fig. 19. Miscellaneous synthetic cannabinoids.

503 504

N

F

Cl

500

R O

O

499

F

O

H N

N

H N

U

A new designer drug found in illegal herbal products (sold in Japan) 517 known as 6-methyl-2-[(4-methylphenyl)amino]-1-benzoxazin-4-one) 518 (URB-754) was identified along with 5-fluoropentyl-3-pyridinoylindole, 519 Q22 UR-144 and XLR-11 by UPLC/MS (Uchiyama 2013). 520 Nico et al. (2013) analyzed eight herbal smoking blends and could 521 identify UR-144 and XLR-11 along with 7 other synthetic cannabinoids 522 by using EI/MS, and ESI/MS/MS. The compounds were isolated by col523 umn chromatography and their chemical structures were confirmed 524 by ID and 2D NMR spectroscopy. 525 A LC/MS/MS method was also developed to quantitate 526 methanandamide due to the increasing demand for detection and 527 quantification of synthetic cannabinoids in biological samples (Dresen 528 et al., 2011). The method (which was fully validated) was tested on 529 101 serum samples which underwent liquid–liquid extraction. 530 The chemical structures of the miscellaneous synthetic cannabinoids 531 are shown in Fig. 19.

DART/MS was used by Musah et al. (2012) to identify AM-251 in 537 herbal preparations. 538 Concluding remarks

539

It is clear that the list of drugs with activity on the cannabinoid receptors is large and expanding every day. Many of these synthetic cannabinoids are finding their way into illicit markets as marijuana substitutes without any safety studies. The scientific community is continuing to generate new compounds and new chemical classes in their search for CB1 and CB2 antagonists for medicinal applications. It is anticipated that many of these compounds will find their way to the illicit market, thus the race will continue!

540 541

Conflict of interest statement

548

We, all four authors, have no conflict of interest.

Uncited reference Showalter et al., 1996

542 543 544 545 546 547

549 550 Q23 551

References

552

Ammann J, McLaren JM, Gerostamoulos D, Beyer J. Detection and quantification of new designer drugs in human blood: part 1 — synthetic cannabinoids. J Anal Toxicol 2012;36:372–80. Arntson A, Ofsa B, Lancaster D, Simon R, McMullin M, Logan B. Validation of a novel immunoassay for the detection of synthetic cannabinoids and metabolites in urine specimens. J Anal Toxicol 2013;37(5):284–90. Bateman KP, Castro-Perez J, Wrona M, Shockcor JP, Yu K, Oballa R, et al. MSE with mass defect filtering for in vitro and in vivo metabolite identification. Rapid Commun Mass Spectrom 2007;21(9):1485–96. Brabanter N, Esposito S, Tudela E, Lootens L, Meuleman P, Leroux-Roels G, et al. In vivo and in vitro metabolism of the synthetic cannabinoid JWH-200. Rapid Commun Mass Spectrom 2013;27(18):2115–26. Carroll F, Lewin A, Mascarella W, Seltzman H, Reddy P. Designer drugs: a medicinal chemistry perspective. Ann N Y Acad Sci 2012;1248:18–38.

553 554 555 556 557 558 559 560 561 562 563 564 565 566



700

N C O

R

R

E

C

D

P

R O

O

F

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Analysis of Parent Synthetic Cannabinoids in Blood and Urinary Metabolites by Liquid Chromatography Tandem Mass Spectrometry.

Analysis of Cannabinoids and Their Metabolites in Human Urine.

New Nightmare "Synthetic Cannabinoids".

Deaths linked to synthetic cannabinoids.

Synthetic cannabinoids: epidemiology, pharmacodynamics, and clinical implications.

Synthetic cannabinoids and acute kidney injury.

Synthetic cannabinoids: undesirable alternatives to natural marijuana.

Determination and identification of synthetic cannabinoids and their metabolites in different matrices by modern analytical techniques - a review.

Detection Times of Carboxylic Acid Metabolites of the Synthetic Cannabinoids JWH-018 and JWH-073 in Human Urine.

Neurotoxicity of Synthetic Cannabinoids JWH-081 and JWH-210.

Simultaneous quantification of 20 synthetic cannabinoids and 21 metabolites, and semi-quantification of 12 alkyl hydroxy metabolites in human urine by liquid chromatography-tandem mass spectrometry.

Qualitative analysis of seized synthetic cannabinoids and synthetic cathinones by gas chromatography triple quadrupole tandem mass spectrometry.

The Wide and Unpredictable Scope of Synthetic Cannabinoids Toxicity.

Synthetic cannabinoids pharmacokinetics and detection methods in biological matrices.

Endogenous and Synthetic Cannabinoids as Therapeutics in Retinal Disease.

A rapid quantitative method for the analysis of synthetic cannabinoids by liquid chromatography-tandem mass spectrometry.

Assessing the experience of using synthetic cannabinoids by means of interpretative phenomenological analysis.

DART-MS as a preliminary screening method for "herbal incense": chemical analysis of synthetic cannabinoids.

Effect of synthetic enantiomeric cannabinoids on platelet aggregation.

Analysis of 62 synthetic cannabinoids by gas chromatography-mass spectrometry with photoionization.

Rapid analysis of synthetic cannabinoids using a miniature mass spectrometer with ambient ionization capability.

Bilateral renal cortical necrosis associated with smoking synthetic cannabinoids.

New challenges of the pediatric emergency department: synthetic cannabinoids.

Synthetic cannabinoids as a cause for black carbonaceous bronchoalveolar lavage.