Research Article

Received: 29 October 2010 Revised: 22 December 2010 Accepted: 11 January 2011

Drug Testing and Analysis

Published online in Wiley Online Library:

(www.drugtestinganalysis.com) DOI 10.1002/dta.268

The emergence and analysis of synthetic cannabinoids

Simon Hudsona and John Ramseyb
In late 2008, several synthetic cannabinoids were detected in herbal smoking mixtures. Typical of these products were Spice Gold, Spice Silver and Yucatan Fire, but many other products have since appeared. The analytes detected, such as JWH-018 and CP47,497 are experimental compounds, some of which were never designed for human use. Both scientic and anecdotal evidence suggest that these compounds are more potent than traditional cannabis and are being widely used. As a result, authorities around the world are now beginning to control them by either naming individual compounds or using generic legislation. This, however, is easier said than done as the synthetic cannabinoids detected are constantly changing in attempts by manufacturers to evade legislation. This paper includes background information in the style of a brief monograph, as an aid to rapidly understanding the pharmacological aspects of these compounds in the forensic context, and then presents a comprehensive set of data, obtained from analysis of purchased products by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Copyright c 2011 John Wiley & Sons, Ltd. Keywords: spice; cannabinoid; AM-694; JWH-018; HU-210; Orbitrap; Cannabicyclohexanol; accurate mass

Introduction
This paper includes background information in the style of a brief monograph, as an aid to rapidly understanding the pharmacological aspects of these compounds in the forensic context, and then presents a comprehensive set of data, obtained from analysis of purchased products by GC-MS and LC-MS/MS.

Synthetic cannabinoids, more correctly designated as cannabinoid receptor agonists that target the CB1 receptor, have been investigated and developed over the past 40 years as therapeutic agents, often for the treatment of pain. It has proved difcult, however, to separate the desired properties from unwanted psychoactive effects. Spice

Background
For centuries Cannabissativa and cannabis extracts have been used in natural medicine. In the 1970s, delta (9)-tetrahydrocannabinol (THC) was found to be the main active ingredient of cannabis, responsible for most of its therapeutic and psychotropic actions. In some countries, cannabis extracts (i.e. sativex) or synthetic drugs based on THC, such as nabilone and dronabinol, are used clinically for the treatment of conditions like chemotherapy-induced nausea and vomiting, multiple sclerosis, and glaucoma. The side effects of cannabis are well documented[1,2] and the abuse potential of these agents and their synthetic analogues represent a serious limitation in their medical use. In addition, diversion in the use of these active ingredients for recreational purpose is a concern due to their reported enhanced afnities for the cannabinoid receptors.[1,2] The rst evidence of a cannabinoid receptor was suggested by Howlett and Flemming in 1984.[3] In the early 1990s, two pharmacologically distinct cannabinoid receptors (CB1 CB2 ) were cloned and reported.[4,5] The CB1 receptor is expressed in the peripheral and central nervous system while CB2 receptors, which play a role in the regulation of the inammatory process, are located on different types of immune cells and immune-related organs. However, there is evidence that some of the effects of cannabis are not related to either receptor.[6]

In late 2008, several synthetic cannabinoids were detected in herbal smoking mixtures often marketed as incense or room odorizers. Typical of these were Spice Gold, Spice Silver, and Yucatan Fire, but many other products later appeared. These products, of which Spice is perhaps the best known example, have been available globally. Although declared as incense and not for human consumption, Spice-type products are smoked as an apparently legal alternative to cannabis to deliver a so-called herbal high. When analyzed, they have not been found to contain tobacco or cannabis but when smoked, produce effects similar to those of cannabis. The listed constituents of Spice products often include a long list of plant/herbal ingredients such as Baybean, Blue Lotus, Lions Tail, Lousewort, Indian Warrior, Dwarf Scullcap, Maconha Brava, Pink Lotus, Marshmallow, Red Clover, Rose, Siberian Motherwort, Vanilla, and Honey. The assumption initially was that it was a botanical ingredient that was responsible

Correspondence to: Simon Hudson, HFL Sport Science Ltd, Newmarket Road, Fordham, Cambridgeshire, CB7 5WW. E-mail: shudson@h.co.uk

a HFL Sport Science Ltd, Newmarket Road, Fordham, Cambridgeshire, CB7 5WW b TICTAC Communications Ltd, TICTAC Communications Ltd., St. Georges University of London, Cranmer Terrace, London, SW17 ORE

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Drug Testing and Analysis

To date these include:
H H O HO H H O HU-210 CP47,497 OH THC OH OH OH

S. Hudson and J. Ramsey

Figure 1. Structures of THC, HU-210-a classical cannabinoid and CP47,497, a non-classical cannabinoid.

for the cannabis-like effect. More recently, however, based on the reported effects of smoking these products, studies have focused on identifying any unlisted components that they may contain. Synthetic cannabinoids detected in Spice Several synthetic cannabinoid receptor agonist (cannabinomimetic) compounds have been identied in Spice-type products.
Naphthoylindoles R4

JWH-0187, 8, 9, personal nding JWH-07310, personal nding JWH-25011, personal nding JWH-39812 HU-21013 CP47,4978, 9, 14, personal nding CP47,497 C8 homologue (cannabicyclohexanol)8, 9, 14, personal nding Cannabicylohexanol + C2 variantpersonal nding AM694personal nding JWH-12212, EMCDDA communication, personal communication JWH-200personal nding JWH-08112, EMCDDA communication, personal nding JWH-253personal nding JWH-387personal nding JWH-210EMCDDA communication, personal communication JWH-019personal nding JWH-203EMCDDA communication (4-methoxyphenyl)(1-pentyl-1H-indol-3-yl)methanone
personal nding, EMCDDA communication

(4-hydroxymethylphenyl)(1-pentyl-1H-indol-3-yl)methanone
EMCDDA communication

Although often referred to simply as synthetic cannabinoids, many of the substances are not structurally related to the
Naphthylmethylindoles

O R3 N R1 R2

JWH-015 R1 = propyl R2 = methyl JWH-018 R1 = pentyl JWH-019 R1 = hexyl JWH-073 R1 = butyl JWH-081 R1 = pentyl R3 = methoxy JWH-122 R1 = pentyl R3 = methyl JWH-200 R1 = morpholinylethyl JWH-210 R1 = pentyl R3 = ethyl JWH-387 R1 = pentyl R3 = Br JWH-398 R1 = pentyl R3 = Cl

R3 N R1 R2

JWH-184 R1 = pentyl R3 = methyl

Naphthoylpyrroles

Naphthylmethylindenes

Phenylacetylindoles O R2

O N C5H11 R1

N R1

R2

N R1

R2

JWH-146 R1 = heptyl R2 = phenyl

JWH-176 R1 = pentyl

JWH-250 R2 = methoxyphenyl JWH-253 R1 = methyl R2 = methoxyphenyl

Cyclohexylphenols OH OH

Classical Cannabinoids (Dibenzopyranv) R2 OH

Benzoylindoles R2 O R3 N R1
AM-694 R1 = 5-fluoropentyl R2 = I RCS-4 R1= pentyl R3 = methoxy

R2

R1

R1
R1 = 1,1 dimethylheptyl R2 = hydroxymethyl R1 = 1,1 dimethylheptyl R2 = keto

CP47,497 R1 = 1,1 dimethylheptyl HU-210 Cannibicylohexanol R1 = 1,1 dimethyloctyl CP55,940 R1 = 1,1 dimethylheptyl Nabilone R2 = hydroxypropyl

Figure 2. Structures of the main synthetic cannabinoid groups with examples from the data or the ACMD.[13] (R=H unless specied).

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The emergence and analysis of synthetic cannabinoids

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Figure 3. High resolution accurate mass screening report for Spike 99 Ultra.

naturally occurring cannabinoids, i.e. compounds, like THC based on dibenzopyran. Consequently they may be divided into classical cannabinoids structurally similar to THC such as HU-210 and non-classical cannabinoids such as CP47,497, structurally unrelated to THC. Both series of compounds are characterized by a bicyclic structure and are reported in the scientic literature as having been tested successfully for cannabinoid action (Figure 1). The JWH compounds were developed as test compounds in the investigation of drug-receptor interactions in the study of the endocannabinoid system. The JWH prex is derived from the initials of John W. Huffman who developed a range of cannabinoid receptor agonists at Clemson University in the USA for researching receptor-drug interactions. The cannabinoid receptor agonists form a diverse group, but most are lipid soluble and non-polar, and consist of 22 to 26 carbon atoms; they would therefore be expected to volatilize readily when smoked. A common structural feature is a

side-chain, where optimal activity requires more than four and up to nine saturated carbon atoms. Most of these compounds can be classied in one of the following groups, although there are a few that fall outside this scheme. The generic structures together with examples from detected compounds are shown in Figure 2. 1. 2. 3. 4. 5. 6. 7. 8. Naphthoylindoles (e.g. JWH-018, JWH-073, JWH-398). Naphthylmethylindoles. Naphthoylpyrroles. Naphthylmethylindenes. Phenylacetylindoles (e.g. JWH-250, JWH-253). Cyclohexylphenols (e.g. CP 47,497, homologues of CP 47,497). Classical cannabinoids (e.g. HU-210, nabilone). Benzoylindoles (e.g AM-694, (4-methoxyphenyl)(1-pentyl-1Hindol-3-yl)methanone

The possible published variants of these compounds are listed in a document from the UK Advisory Council on the Misuse of Drugs (ACMD).[15]

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S. Hudson and J. Ramsey

+ve ESILCMS of 342 Accurate Mass 100 Relative Abundance 80 60 40 20 0 100 150 200 m/z +ve ESILCMS of 328 Accurate Mass 100 Relative Abundance 80 60 40 20 0 127.0539 100 150 200 m/z
2 2

m/z 155.0491 O

EI-GCMS 100 Relative Abundance 80 60 40 20 0 127 144 116 100 213 200 m/z 270 28 5 343 400 JWH-018 Mwt 341 214 284 324 N m/z 284 m/z 127 341 m/z 214 O

155.0491

214.1225 N 127.0541 250 300 JWH-018 Mwt 341 m/z 155.0491 m/z 214.1226

300

EI-GCMS m/z 200 Relative Abundance 100 80 60 40 20 0 127 116 100 144 241 200 m/z 200 284 270 328 N m/z 284 327 O m/z 127

155.0490 O

200.1070 N 270.2299 250 310.2614 300 JWH-073 Mwt 327 m/z 169.0648 m/z 200.1070

300

400

JWH-073 Mwt 327 m/z 214

+ve ESILCMS of 356 Accurate Mass 100 Relative Abundance 80 60 40 20 0 100 150
2

EI-GCMS 100 Relative Abundance 80 60 40 20 0 11 5 18 1 21 4 29 8 33 8 355

169.0646

O m/z 141 N 28 4 27 0 35 6 m/z 298

214.1226

N m/z 214.12 26

141.0697 200 m/z +ve ESILCMS of 376 AccurateMass 250 300 350 JWH-122 Mwt 355 m/z 189.0102

100 EI-GCMS 100 Cl Relative Abundance 80 60 40 20 0 144 126 102 116 100

200 m/z

300

400

JWH-122 Mwt 355 m/z 161

100 Relative Abundance 80 60 40 20 0 100

189.0102 O

375 O 214 318 161 269 358 320 377 378 200 m/z 300 400 JWH-398 Mwt 375 m/z 318 N m/z 214 Cl

N 214.1226 161.0152 150 200 250 m/z 300 350 JWH-398 Mwt 375 m/z 214.1226

Figure 4. EI GC-MS and accurate mass LC-MS2 data for JWH-018, JWH-073, JWH-122 and JWH-398.

Some of these compounds have been designated as controlled drugs in countries around the world. It is apparent, however, that as one compound or group of compounds is made illegal, more variants that fall outside current legislation take their place.[10,16]

Effect There is a considerable volume of anecdotal evidence on various internet sites regarding the efcacy of Spice products with different variants being said to deliver different effects to the user. There is also a documented case where a user reported that he had been smoking Spice Gold daily for eight months. He reported a tolerance to the product which led to increasing the amount he smoked each day. He also felt a conscious desire for the product. On treatment, the physical withdrawal symptoms were very similar to those seen with cannabis dependence.[17] The reported symptoms included profuse sweating leading to internal unrest, a strong desire for Spice, nightmares, nausea, tremor, and headaches. This led to a diagnosis of a dependency syndrome according to both the International Statistical Classication of Diseases and Related Health Problems 10th revision (ICD-10) and the Diagnostic and Statistical Manual of Mental Disorders 4th edition (DSM-IV). On admission to hospital, a urine sample was taken. This was subjected to the normal immunological tests performed for

cannabinoids, benzodiazepines, amphetamines, cocaine, opiates, and methadone. The results were all negative. It should be noted that the Spice Gold in this report was not tested for cannabinomimetic compounds or drugs of abuse. In another study, a self experiment was conducted by two authors.[7] One cigarette containing 0.3g of Spice Diamond was smoked. After approximately 10 min, the rst noticeable and cannabis-like effects occurred. These included considerably reddened conjunctivae, a signicant increase in pulse rates, xerostomia, and an alteration in mood and perception. In psychomotor tests, no abnormalities were detected, although the subjects had the impression of being moderately impaired. These effects slowly subsided over a period of 6 h. The following day, some minor after effects were reported. Further analytical work on the administered Spice Diamond identied the presence of JWH-018 and the C8 homologue of CP47,497. A review of clinical reports was recently published in which addiction and withdrawal symptoms similar to those seen with cannabis abuse were linked to the use of Spice.[18] A huge amount of anecdotal evidence surrounding the effects of Spice and often individual Spice synthetic cannabinoids is available on the internet. For example, the website http://www.bluelight.ru/vb/home.php contains personal reports of use and differences in effects between compounds. It is apparent that different Spice variants have different active ingredients.[12,14] It is also apparent that the active ingredients

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Table 1. Summary of compounds, monoisotopic masses, and data locations Analyte JWH-018 JWH-073 JWH-122 JWH-398 JWH-015 JWH-019 WIN 55212-2 JWH-387 JWH-200 JWH-210 JWH-081 JWH-250 AM-694 (4-methoxyphenyl)(1-pentyl-1H-indol-3yl)methanone (RCS-4) CRA-13 RCS-4 methylated A RCS-4 methylated B CP47,497 Cannabicyclohexanol Cannabicyclohexanol + C2 variant CP 55490 HU-308 HU-210 Nabilone JWH-210 ethyl derivative JWH-253 Methylated JWH-073 JWH-200 piperidine variant (4-hydroxymethylphenyl)(1-pentyl-1Hindol-3-yl)methanone JWH-203 Formula C24 H23 NO C23 H21 NO C25 H25 NO C24 H22 ClNO C23 H21 NO C25 H25 NO C27 H26 N2 O3 C25 H22 BrNO C25 H24 N2 O2 C26 H27 NO C25 H25 NO2 C22 H25 NO2 C20 H19 FINO C21 H23 NO2 C26 H24 O2 C22 H25 NO2 C22 H25 NO2 C21 H34 O2 C22 H36 O2 C24 H40 O2 C25 H40 O3 C27 H42 O3 C25 H38 O3 C24 H36 O3 C28 H31 NO C23 H27 NO2 C24 H23 NO C26 H26 N2 O C21 H23 NO2 C21 H22 ClNO Monoisotopic mass 341.1774 327.1618 355.1931 375.1384 327.1618 355.1931 429.1938 419.0879 384.1832 369.2087 371.1880 335.1880 435.0490 321.1723 368.1771 335.1880 335.1880 318.2553 332.2710 360.2022 388.2972 414.3128 386.2815 372.2658 397.2400 349.2036 341.1774 382.2040 321.1723 339.1384 Source Spice products and customs seizure Spice products and reference material Star of Fire spice product Numerous spice products Reference material Spice product and reference material Reference material Spice products Customs seizure Reference material Spice product and reference material Numerous spice products and reference material Spice products and reference material Jah Rush spice product Reference material Jah Rush spice product (minor component) Jah Rush spice product (minor component) Numerous spice products and reference material Numerous spice products Numerous spice products Reference material Reference material Reference material Reference material Minor component of JWH-210 reference material Spice products Personal communication Personal communication EMCDDA communication EMCDDA communication Figure No. 4 4 4 4 5 5 5 5 6 6 6 6 7 7 7 7 8 8 8 8 9 9 9 9 10 10 10 10 10 10

may differ in relative amounts from one variant to another and also from batch to batch of the same variant.[12,14] Legislation Legislation has been put in place in many countries based on either controlling individual named substances or as in the UK, through the introduction of a generic legislation based on modications to a core structure as in the recommendation to the UK home ofce from the ACMD.[15] The position of the World Anti-Doping Agency (WADA) at the time of writing is that synthetic cannabinoids related structurally to traditional cannabinoids only, are prohibited. Availability Spice products are readily available on the Internet and in head shops on the high street. Although most now claim to be legal (i.e. free of controlled substances), this is generally not the case, as recent studies in the UK have shown.[16] There have also been several seizures of relatively large amounts (100g or more) of the active substances. It is not clear if these were destined to be added to herbal products for sale or whether they were intended for distribution to the end users as white powders.

Metabolism Most of the work so far has been performed on the raw ingredient with very little consideration of the detection in mammalian body uids. A recent publication has shown that both JWH018 and CP 47,497 can be detected as parent compounds and metabolites after administrations to a rat.[19] Hydroxylation and n-dealkylation occurred with the most abundant analytes being n-dealkylated hydroxylated metabolites. Hydroxylation occurred in both aromatic systems and in the aliphatic side chain. This has more recently also been shown in the human[20,21] and in vitro.[22] The formation of similar metabolites in vitro for another naphthoylindole compound JWH-015, had been reported previously.[23] The metabolism of CP 47,497 in the rat was reported to have been hydroxylation in both aromatic and aliphatic portions of the molecule. Detection The synthetic cannabinoids are readily resolved using gas chromatography (GC) and liquid chromatography (LC), but their identication and quantitative analysis is limited by the availability of pure reference materials. Conventional cannabinoid tests utilizing immunoassay are ineffective in detecting these

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S. Hudson and J. Ramsey

+ve ESILCMS2 of 328 Accurate Mass 100 Relative Abundance 80 60 40 20 200.1062 155.0484

m/z 155.0491 O

EI-GCMS 100 Relative Abundance 80 60 40 20 0 77 115 100 127 155 159 200 m/z 300 200 270 254 310 326

327

m/z 200 O m/z 127 N m/z 270 JWH-015 Mwt 327

N m/z 200.1070

298

0 100 200 m/z 300 JWH-015 Mwt 327 m/z 155.0491

100 Relative Abundance 80 60 40 20

+ve ESILCMS2 of 356 Accurate Mass 155.0484

EI-GCMS 100 Relative Abundance 355

m/z 228 O

O 228.1374 N m/z 228.1377 0 100 200 m/z +ve ESILCMS2 of 427 Accurate Mass 155.0486 300 JWH-019 Mwt 355 m/z 155.0491

80 127 60 40 43 20 0 100 200 m/z 101 11 6 155 167 270 254 228

28 4 338 m/z 284 28 5 33 6 300 JWH-019 Mwt 355 m/z 155 N m/z 127

EI-GCMS 100 Relative Abundance 100

100 Relative Abundance 80 60

80 60 40 O 20 0 200 m/z 400 WIN 55212-2 Mwt 426 O 56 127 155 426

N 40 20 0 200 m/z +ve ESILCMS2 of 420 Accurate Mass 232.9591 m/z 232.9596 271.1434 300 400 WIN 55212-2 Mwt 426 O O N m/z 271.1441

m/z 100

100 Relative Abundance 80 60 40 20 0

Relative Abundance

O Br

100 80 60 40 20 0

+ve ESILCMS2 of 421 AccurateMass with wide isolation window (79Br) 232.9592 234.9570 (81Br)

m/z 232.9596

O Br

N 214.1222 200 m/z 300 400 JWH-387 Mwt 419 m/z 214.1226

N 214 .1221 20 4.9644 200 m/z 300 400

JWH-387 Mwt 419

m/z 214.1226

Figure 5. EI GC-MS and accurate mass LC-MS2 data for JWH-015, JWH-019 and WIN 55212-2. Accurate mass LC-MS2 for JWH-387.

compounds. Studies on Spice products have generally been performed by GC linked to mass spectrometry (GC-MS)[7,8,9,10,14] or by LC linked to mass spectrometry (LC-MS)[7,8,9,14] but with these technologies, covering the range of over 100 possible compounds without reference materials is difcult. Previous work in our laboratory[12] focused on the use of full-scan high-resolution LC-MS to detect these compounds based on their elemental compositions. Any suspect analytes were then re-analyzed using multistage tandem LC-MS (LC-MSn ) measuring products ions with accurate mass detection. The accurate mass product ions produced helped in the tentative identication of several new compounds. Monitoring In a regulatory framework, knowing what is being found in other countries and thereby appreciating which compounds are likely to be seen is very important in maintaining a viable detection service. The European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) operates an early warning system (EWS) that provides a fast-track mechanism for 30 countries throughout Europe to report new drugs. Any new ndings are then communicated back to the relevant organisations in the member countries. In the rapidly evolving world of designer drugs, this resource is invaluable. We present data from the investigation and analysis of the contents of test-purchased Spice products and seizures by customs and borders agencies. Where available, reference materials have

been analyzed and the data included. Additional information was collected and added when not available in-house. The information presented will act as a reference point for laboratories aiming to detect these compounds in products and body uids.

Experimental
High performance liquid chromatography (HPLC) grade methanol, toluene, and acetic acid were obtained from ThermoFisher (Loughborough, UK). N-Methyl-N-trimethylsilyltriuoroacetamide (MSTFA), ephedrine, and uracil were obtained from Sigma (Poole, UK). Herbal products were obtained from a variety of sources including police seizures, Internet purchases, and amnesty bins. This was coordinated by TICTAC Communications Ltd (London, UK). Reference materials for JWH-015, JWH-019, WIN55212-2, CP55490, and HU-308 were obtained from Cambridge Bioscience Ltd (Cambridge, UK). HU-210 was obtained from Tocris Bioscience Ltd (Bristol, UK). Individual compounds were kindly supplied by other workers in this eld who are acknowledged accordingly. Samples of herbal product were prepared for analysis using a simple extraction into methanol. The materials tested did not need any treatment prior to extraction.

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m/z 155.0491 +ve ESILCMS2 of 385 Accurate Mass 100 Relative Abundance 80 60 40 20 0 150 127.0541 229.1336 200 250 m/z 298.1230 300 350 JWH-200 Mwt 384 N m/z 298.1226 N O m/z 183.0803 155.0491 Relative Abundance O EI-GCMS 100 80 60 40 m/z 284 20 0 100 200 m/z EI-GCMS 100 Relative Abundance 80 60 40 20 102 0 100 m/z 185.0596 EI-GCMS Relative Abundance O 100 80 60 40 20 0 100 m/z 121.0648 100 Relative Abundance 80 60 40 20 132.0812 0 100 150 200 m/z 250 300 JWH-250 Mwt 335 200.1436 308.2015 N +ve ESILCMS2 of 336 AccurateMass 121.0649 188.1437 200 m/z EI-GCMS 100 Relative Abundance O O 80 60 40 20 0 100 200 m/z 300 144 91 116 335 JWH-250 Mwt 335 N 214 O 300 102 214 144 185 114 157 314 354 285 300 315 N m/z 314 373 JWH-081 Mwt 371 371 O 200 m/z 300 JWH-210 Mwt 369 144 128 183 214 312 352 m/z 312 254 270 340 370 371 N 369 O 300 400 JWH-200 Mwt 384 O 56 127 155 254 284 384 N N 100 O

m/z 127

m/z 100

+ve ESILCMS2 of 370 Accurate Mass 100 Relative Abundance 80 60 40 20 0 100 150 200 250 m/z +ve ESILCMS2 of 372 Accurate Mass 185.0596 300 350 153.0697 JWH-210 Mwt 369 214.1225 183.0803

m/z 183

m/z 214.1226

m/z 214

m/z 185

100 Relative Abundance 80 60 40 20 0 100

214.1225

157.0646 150 200 250 m/z 300 350 JWH-081 Mwt 371

m/z 214.1226

m/z 214

m/z 188.1434

m/z 214

Figure 6. EI GC-MS and accurate mass LC-MS2 data for JWH-200, JWH-210, JWH-081 and JWH-250.

Approximately 50 mg of the herbal product was placed in a 20ml glass vial and 2 ml of methanol was added. This was sonicated for 10 min then left for 10 min to settle; 1 ml of the methanol extract was then transferred to a 1-ml glass vial. For all LC-MS analyses, 100 l of the methanol extract was transferred to a glass autosampler vial followed by the addition of 900 l of ultrapure water. For GC-MS analysis, 100 l of the methanol extract was transferred to a GC vial and evaporated to dryness by leaving overnight at room temperature. The sample was reconstituted in 30 l toluene followed by 20 l of MSTFA and then submitted for analysis. No heating was performed prior to injection. Instrumentation LC-MS The samples were analyzed on an Accela UPLC system interfaced to an LTQ Orbitrap Discovery both from ThermoFisher (Hemel Hempstead, UK). The chromatographic separation was performed using a Pursuit Diphenyl 3.0 m 2 mm 100 mm HPLC column from Varian (Oxford, UK) linked to a Waters (Elstree, UK) Acquity in-line lter operating in gradient mode at 40 C. The mobile phases were 0.1% acetic acid with 300 g/l uracil and 0.1% acetic acid in methanol with 300 g/l uracil. The uracil was added to the mobile phase as a lock mass for the Orbitrap. The gradient was formed from starting conditions of 80% aqueous mobile phase. Organic mobile phase was increased

through the run to give 60% organic at 1.0 min and 99% at 2.5 min. The 99% organic step was held for 3.5 min before reverting to starting conditions at 6.01 min. The ow rate throughout was 400 l/min. The LC-MS interface was an Ion Max API source tted with an electrospray probe. A sheath gas ow rate and auxiliary gas ow rate of 30 and 10 arbitrary units, respectively, were used together with a source voltage of 4.5 KV and a heated capillary transfer temperature of 200 C. To optimize the remaining source parameters, a 10 g/ml solution of ephedrine was introduced at a rate of 3 l/min into a solvent ow of 400 l/min, consisting of 50% organic mobile phase and 50% aqueous mobile phase. This was achieved using the autotune functionality within the Xcalibur operating software using m/z 166 from ephedrine. The Orbitrap was calibrated for positive ion analysis using the standard ThermoFisher Orbitrap calibration solutions and procedures. The instrument was operated in positive ion mode with a resolution setting of 30 000 with an internal lock mass of 113.03455 derived from uracil in the mobile phase. Data were acquired in full scan mode over a mass range of 50650 Da. Further investigation of suspicious peaks Components identied by accurate mass as possible synthetic cannabinoids based on information from the UK Advisory Council on the Misuse of Drugs[15] and from information from Internet searches were subjected to further investigation by LC-MSn

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S. Hudson and J. Ramsey

100 Relative Abundance 80 60 40 20 0

+ve ESILCMS2 of 436 Accurate Mass 230.9301 436.0567 309.1523 N 234.0910 300 m/z 400 AM-694 Mwt 435 O

m/z230.9301 EI-GCMS I Relative Abundance 100 80 60 40 20 0 F m/z 135.0441 O O Relative Abundance EI-GCMS 100 80 264 60 40 20 0 77 50 EI-GCMS 100 Relative Abundance O 80 60 40 20 0 100 m/z 149.0597 O O CH3 Relative Abundance EI-GCMS 100 80 60 149 40 20 0 50 100 150 200 m/z 250 300 125 144 77 91 191 214 204 219 279 297 264 RCS-4 Methylated A Mwt 335 278 m/z 278 N m/z 214 335 200 m/z 300 CRA13 Mwt 368 m/z 149 O O CH3 127 115 128 75 171 239 299 226 351 370 O 281 298 368 m/z 171 O 144 119 100 152 150 186 200 m/z 135 214 m/z 264 222 250 265 306 322 320 300 m/z 214 323 RCS-4 Mwt 321 N 100 200 m/z 321 O 300 76 144 130 204 233 25 6 308 220 360 415 414 400 m/z 360 437 AM-694 Mwt 435 F m/z 135O 232 435 m/z 232 O I

m/z 309.1523

202.9351 200

100 Relative Abundance 80 60 40 20 0 100

+ve ESILCMS2 of 322 Accurate Mass 135.0439

N 214.1226 150 200 m/z 250 300 RCS-4 Mwt 321 m/z155.0491 m/z 214.1226

100 Relative Abundance 80 60 40 20 0 100

+ve ESILCMS2 of 369 Accurate Mass 299.1067

155.0491 171.0438 241.1224 m/z 299.1066 O m/z 241.1223 150 200 250 m/z +ve ESILCMS2 of 336 Accurate Mass 149.0595 300 350 CRA13 Mwt 368

m/z 298

100 Relative Abundance 80 60 40 20 0 100

N 214.1225 135.0439 150 200 m/z 250 300 m/z 214.1226 RCS-4 Methylated A Mwt 335

Figure 7. EI GC-MS and accurate mass LC-MS2 data for AM-694, (4-methoxyphenyl)(1-pentyl-1H-indol-3-yl)methanone (RCS-4), CRA-13 and methylated RCS-4.

analyses in either positive or negative ion mode, depending on which gave the more successful or diagnostic fragmentation. For product ions generated in positive ion mode, the Orbitrap was used to detect the fragments, thereby aiding in structural assignment. Negative ion LC-MSn was performed using the LTQ using nominal mass measurement. A collision energy of 35 units was used for all positive ion work and 30 units was used for all negative ion work. GC-MS The samples were analyzed on an Agilent 6890 GC interfaced to an Agilent 5973 MSD. The chromatographic separation was performed on a 25 metre 0.25 mm i.d. 0.25 m lm BPX5 capillary column from SGE. The chromatographic conditions were as follows: Carrier gas: Helium @15 psi constant pressure Injection: Splitless for 1.0 minutes at 280 C. Oven: Initial temp 150 C for 2 min Ramp 25 C/min to 230 C Ramp 5 C/min to 330 C Hold for 10 min The mass spectrometer was tuned and calibrated using peruorotributylamine and data were acquired in full-scan mode over

a mass range of 50 to 700Da acquiring spectra at a rate of 4 scans/second. 1 l of sample was injected on the system Data processing of full scan accurate mass LC-MS data Acquired full scan accurate mass LC-MS data were then subjected to analysis using ToxID, a program supplied by ThermoFisher. This utilizes a simple accurate mass database containing compound information including protonated monoisotopic mass. Narrow range mass lters, typically 2 ppm, are then employed to selectively lter the data for reporting. In this application, where few retention times are available for the compounds in the database, an expected retention of 4 min was used with a retention window of 3 min as the instrument run time was 7 min. The accurate mass database was created for compounds listed in a UK Advisory Council on the Misuse of Drugs communication[15] containing cannabinoid receptor agonists considered for control under the Misuse of Drugs Act 1971 and from other compounds identied on the Internet as being synthetic cannabinoids. The information in the communication was compiled by reviewing the literature on synthetic cannabinoids over the past 20 years.[24 33] Information on the major substances was extracted. These compounds, in experiments, had all acted as agonists at the CB1 (cannabinoid) receptor as measured by afnity constants (Ki ). Other compounds that have been developed by drug companies

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+ve ESILCMS2 of 336 Accurate Mass Relative Abundance 100 135.0439

m/z 135.0441 O O

m/z 135 EI-GCMS Relative Abundance 100 80 60 40 20 0 100 200 m/ z 300 RCS-4 Methylated Mwt 335 Si O Si 80 60 40 20 0 75 147 100 205 289 233 287 290 200 300 m/z 378 462 m/z 419 380 400 464 500 m/z 377 CP47,497 Mwt 318 Si O 476 60 40 20 0 23 3 14 7 205 200 EI-GCMS of TMS derivative 287 24 5 289 290 31 9 300 m/z 100 Relative Abundance 80 60 40 20 0 200 300 m/z 233 109 207 234 317 31 5 318 407 399 433 400 48 9 505 m/z 405 507 Cannabicyclohexanol + C2 variant Mwt 360 C2H5 m/z 433 405 504 400 379 433 478 500 m/z 377 Cannabicyclohexanol Mwt 332 Si O Si m/z 433 Si 73 O B 74 78 135 228 186 19 3 230 281 m/z 278 341 m/z 228 N 278 335 O O

50 228.1382 0 100 150 200 m/z 250 300

N m/z 228.1377 RCS-4 Methylated Mwt 335 OH OH B

-ve ESI LCMS3 of 317 and 299 100 Relative Abundance 80 60 40 20 0 100 150 200 m/z 250 300 245

EI-GCMS of TMS derivative 100 Relative Abundance 377

m/z 299 271 299 CP47,497 Mwt 318 OH OH m/z271

m/z 245

-ve ESI LCMS3 of 331 and 313 100 Relative Abundance 80 60 40 20 0 100 135 160 186 213 233 150 200 m/z 250 260 285 313 300 259

EI-GCMS of TM S derivative 100 Relative Abundance 80 377 O

m/z 313 m/z285

m/z 259

Cannabicyclohexanol Mwt 332 OH

100 Relative Abundance 80 60 40 20 0 100

-ve ESI LCMS3 of 359 and 341

341

OH C2H5

285 259 241 150 200 m/z 250 300 m/z 341 m/z 285 323 350

m/z 259 Cannabicyclohexanol + C2 variant Mwt 360

500

Figure 8. EI GC-MS and accurate mass LC-MS2 data for methylated RCS-4. EI GC-MS and LC-MS3 data for CP 47,497, cannabicyclohexanol and cannabicyclohexanol+C2 variant.

as potential synthetic cannabinoids, were also added. A total of 142 compounds were initially included in the database with accurate masses derived from elemental compositions. Not all, however, had high receptor afnities (i.e. low values of Ki ); less than half would probably have misuse potential. It may be noted that of the various synthetic cannabinoids already reported, all, like 9 -THC itself, have low values of Ki .

Results
The rst pass analysis of Spice products was performed using full scan accurate mass LC-MS with data analysis using ToxID and the above database. An example of a report is shown in Figure 3. The data displayed are from an analysis of Spike 99 Ultra and shows the presence of six different spice compounds. In Figure 3, there is a result for a component at 4.63 min for JWH-007, JWH-019 and JWH-047. These have the same elemental composition and are indistinguishable by accurate mass alone. Follow-up LC-MSn analyses generating MS2 and MS3 data was used to characterize and identify all the analytes. In this case the compound was determined to be JWH-019. Where sufcient analyte existed, full-scan, electronic ionization (EI) GC-MS data were acquired. The data acquired to date from investigations into Spice products and related compounds are presented in Figures 4, 5, 6, 7, 8, 9 and 10 and are summarized in Table 1.

In addition, a number of reference materials have been analyzed where available, even if not yet detected in Spice products. The data are included as these are not readily available from the literature. The data includes a reference to cannabicyclohexanol + C2 variant (Figure 8). This was detected in a product called Ice Bud and is the rst report of this compound. This was identied with a combination of accurate mass LC-MS, LC-MSn and full-scan GC-MS of the derivatized and underivatized sample. All compounds listed are covered by current UK legislation with the exception of AM-694, CRA13, (4-hydroxymethylphenyl)(1-pentyl-1H-indol-3-yl)methanone and (4-methoxyphenyl)(1-pentyl-1H-indol-3-yl)methanone (and its methylated derivatives).

Discussion
It should rst of all be noted that most of the compounds reported here have been identied in Spice-type herbal products. These data have been generated through the analysis of acquired materials and in two instances from data circulated by EMCDDA. A few compounds have been acquired as seized powders and purchased reference materials and their analytical data have been included for future reference. When reviewing accurate mass by only screening data for many of the JWH series of compounds, it is important to remember

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S. Hudson and J. Ramsey

OH -ve ESI LCMS3 of 375 and 357 100 Relative Abundance 80 60 40 20 0 111 133 100 150
2

327

OH

EI-GCMS of TMS derivative Relative Abundance 100 73 305 327 219 50 75 85 0 100 200 300 m/z Si 400 500 600 147 205 233 420 328 412 454 419 592 577 594

Si O Si

m/z 299 339 219 245 200 250 m/z 299 300 m/z 327 355 350 CP 55,940 Mwt 376 HO

OH

O CP 55,940 Mwt 376 O

Si

100 Relative Abundance 80 60 40 20 0

+ve ESILCMS of 415 Accurate Mass 215.1059 271.1684 O 151.0748 289.1789 m/z 271.1693

EI-GCMS of TMS derivative Relative Abundance 100 73 277 318 50 103 157 0 100 200 300 m/z 400 500 239 443 486 487 396 353

m/z289.1798 200 m/z +ve ESILCMS2 of 387 Accurate Mass 243.1379 300 400 HU-308 Mwt 414 HO

HU-308 Mwt 414 Si

100 Relative Abundance 80 60 40 20

m/z 243.1379 OH Relative Abundance

EI-GCMS of TMS derivative 100 80 60 40 20 0 133 100 216 273 200 73 446 530

O O Si

261. 1487 369. 2790 201.090 9 161.0595 313.2166 200 250 m/z 300 350 HU-210 Mwt 386 O OH O m/z 261.1485

75 359 356

531 O 445 448 533 HU-210 Mwt 386 Si O Si m/z 446

0 150

300 m/z

400

500

100 Relative Abundance 80 60 40 20 0 100

+ve ESILCMS2 of 373 Accurate Mass 247.1330 229.1224

EI-GCMS of TMS derivative 73 100 Relative Abundance 80 60 40 20 0 100 200 300 m/z 400 500 Nabilone Mwt 372 74 259 290 359 432 431 516 501

O m/z 247.1331 189.0910 173.0597 150 200 250 m/z 315.2322 300 350 Nabilone Mwt 372

O m/z 432

Figure 9. EI GC-MS and LC-MS3 data for CP55490. EI GC-MS and accurate mass LC-MS2 data for HU-308, HU-210 and nabilone.

+ve ESILCMS2 of 398 Accurate Mass 100 Relative Abundance 80 60 40 20 0 155.0857 150 EI-GCMS 100 200 250 m/z 300 350 214.1226 211.1120

m/z 211.1117 100 O C2H5 Relative Abundance 80 60 40 20 0 100 EI-GCMS 100 O 98 146 121

+ve ESILCMS2 of 350 202

m/z 121

O O N JWH-253 Mwt 349 m/z 202 m/z 155

188 200

350 236 250 m/z 294 300 350

m/z 214.1226

JWH-210 ethylated Mwt 397 m/z 169

150

341

O m/z 127

50 115 144 89 0 80 EI-GCMS 100 135 140

200 241 226 200

298 m/z 298 284 270 260 320

m/z 141 N

50 42 70 127 155 254 324 382 360

N N m/z 98 Methyl piperidinyl analogue of JWH-200 Mwt 382

18 1

JWH-73 - methylated Mwt 341 m/z 135 O 321 304 N OH

0 40 EI-GCMS 100 214 120 200 280

O Cl

144

264 50 43 77 118 105 92 0 40 70 100 130 160 190 220 250 280 310 200 165 187

m/z 264

50 m/z 214 144 89 116 110 140 170 200 230 260 290

N m/z 214 339 JWH-203 Mwt 339 80 320 350

(4-hydroxymethylphenyl) (1-pentyl-1H-indol-3-yl) methanone Mwt 321

Figure 10. Accurate mass LC-MS2 data for ethylated JWH-210. LC-MS2 data for JWH-253. EI GC-MS data for methylated JWH-073, the methylpiperidinyl analogue of JWH-200, (4-hydroxymethylphenyl) (1-pentyl-1H-indol-3-yl) methanone and JWH-203.

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+ve ESILCMS3 of 328 and 200 Accurate Mass 100 Relative Abundance 80 60 40 20 0 100 150 200 m/z 250 300 m/z 144.0444 O 200.1069 JWH-015 N 158.0599 O m/z 158.0600

+ve ESILCMS3 of 328 and 200 Accurate Mass 100 Relative Abundance 80 60 40 20 0 100 150 200 m/z 25 0 300 158.0598 200.1067 JWH-073 144.0441

Figure 11. Accurate mass LC-MS3 data for JWH-015 and JWH-073 showing spectral differences due to positioning of additional methyl group.

that there are several isobars for some elemental compositions. Whereas this is not an issue for a screening analysis as performed here using accurate mass, the differentiation of one isobar from another is very important in a regulatory investigation. Once a compound has been detected, a follow-up analysis by full-scan EI GC-MS gives more structural information and a greater chance of structural assignment. Much of the early work in this eld, by Auw arters group[7] [8,9,14] and Uchiyamas group proposed the origin of many of the ions seen in both GC-MS and LC-MS analyses. This information is utilized and built upon in the identication of the synthetic cannabinoids presented here. As can be seen from both the GC-MS and LC-MSn data, the naphthoylindole compounds generally fragment in the same manner. The predominant fragment in LC-MS2 for all these compounds is from the naphthoyl fragment. Any substitution in the naphthoyl portion is seen in a modication to the m/z 155 fragment. Exceptions to this include JWH-200 which, due to the morpholinylethyl side chain, fragments differently with predominantly the generation of a morpholinylmethyl fragment of m/z 100 in EI GC-MS. The recent report of a piperidinyl version of JWH-200 (Figure 10) follows this pattern with a base peak in the EI GC-MS spectrum of m/z 98. In the case of the isobars JWH-018 and methylated JWH-073, which share the same elemental composition, full-scan highresolution LC-MS without retention time information would not be able to determine one from the other. EI GC-MS can, however, be used to differentiate these compounds. The m/z 284 ion seen in JWH-073 is modied by the addition of the methyl group to the indole portion of the molecule rather than the alkyl side chain, to give an ion of m/z 298. The modication of JWH-073, however, to give JWH-018 through the addition of a methyl group to the alkyl side chain would still yield an ion of m/z 284. The use of LC-MSn can aid in some identications. In Figure 11, the indole fragments generated by the MS2 experiment have been subjected to further fragmentation to yield MS3 spectra. The methyl group in the 2 position adjacent to the alkyl side

chain (JWH-015) gives a different MS3 spectrum to that from an addition of the methyl group to the side chain itself (JWH-073). This knowledge can be used to investigate such structural isomers. There are some compounds where only one type of data is presented. This is usually where the level is too low for satisfactory analysis by GC-MS. In many of these cases the LC-MS2 data were acquired on the LTQ rather than the Orbitrap to gain sensitivity. The negative ion LC-MS3 work on the CP 47,497 compounds was performed on the LTQ element of the LTQ Orbitrap and nominal mass data were obtained. New compounds are regularly being reported through EMCDDA as new Spice products come onto the market in attempts to evade detection and legislation. A recent example of this was the detection of AM-694 in a seizure of a Spice product from the Glastonbury music festival in the UK. At the time of writing, this is not covered by UK legislation or apparently any European legislation but is a potent and selective agonist for the cannabinoid receptors based on the limited data available which report a high binding afnity (Ki = 0.08 nM) for the CB1 receptor and an 18fold selectivity over the CB2 receptor.[34] Further recent seizures of Spice-type products in the UK contained AM-694 but also contained low levels of JWH-018 and JWH-073. The responses for these two materials were less than 1% of the response of AM-694. When the additional synthetic cannabinoids are at such low levels, they are still readily detectable by both accurate mass LC-MS and LC-MS2 . Detection by GC-MS however is less straightforward as the levels present are not readily detected. This is due in part to the relatively non-volatile nature of the analytes being tested. Recently, a new naphthoylindole, similar to JWH-200 but with a piperidine ring rather than the morpholine ring was identied (pers. comm.). This compound is not covered by current UK legislation and does not feature in the literature. Many of the samples submitted for analysis in the authors laboratory appeared to contain small amounts of the naturally occurring cannabinoid agonist oleamide. This may originate in the plant material that forms the bulk of the Spice product. One sample, however, had a level of oleamide that was between 1000

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and 10 000 times greater than that seen as probable background. Crudely, based on the response for 10 mg/gram for JWH-018, the level was in excess of 100 mg/gram of product. Whether or not this product had been fortied with oleamide is a point of debate From an anti-doping in sport and workplace drug testing perspective, details around the metabolism of these compounds in the human are beginning to emerge. The recent paper on JWH018[20] details how this compound is metabolized and further work in vitro has supported this.[22] This information can be used to determine the possible metabolites of many of the related compounds presented here. The metabolism of CP47,497 has also been reported and once again this information gives a starting point for possible detection by laboratories. Very recently, there was a report of ndings of JWH-018 metabolites in doping control samples.[21] These drugs are not currently covered by the WADA 2010 prohibited list[35] except for those based on the traditional cannabinoid structure, such as nabilone and HU-210. The new prohibited list for 2011 does include other compounds, specically JWH-018 and JWH-073.[35] The generic term cannabimimetic could be used effectively to cover variants without having to put in place complex legislation such as the December 2009 modication to the Misuse of Drugs Act in the UK. Acknowledgements The authors would like to thank Volker Auw arter and Stefan Kneisel from the Forensic Toxicology Department of the University of Freiburg in Germany for JWH-210 and JWH-081 as reference materials and Star of Fire containing JWH-122; and Istvan Ujvary who synthesized CRA-13.

S. Hudson and J. Ramsey

[13] DEA (US Drugs Enforcement Administration). Spice Plant material(s) laced with synthetic cannabinoids or cannabinoid mimicking compouinds. Microgram Bulletin 2009, 42(3), 23. [14] N. Uchiyama, R. Kikura-Hanajiri, J. Ogata, Y. Goda. Chemical analysis of synthetic cannabinoids as designer drugs in herbal products. Forensic Sci. Int. 2010, 198, 31. [15] Advisory Council on the Misuse of Drugs, Consideration of the major cannabinoid agonists. Home Ofce, London, 2009, Available at: http://drugs.homeofce.gov.uk/publicationsearch/acmd/acmd-report-agonists?view=Binary Accessed 2 June 2010. [16] P. I. Dargan, S. Hudson, J. Ramsey, D. M. Wood. The impact of changes in UK classication of the synthetic cannabinoid receptor agonists in Spice. Int. J. Drug Policy 2010, Accepted for publication December 2010. [17] U. S. Zimmermann, P. R. Winkelmann, M. Pilhatsch, J. A. Nees, R. Spanagel, K. Schulz. Withdrawal phenomena and dependence syndrome after the consumption of Spice Gold. Dtsch Arztebl Int. 2009, 106, 464. [18] I. Vardakou, C. Pistos, Ch. Spiliopoulou. Spice drugs as a new trend: mode of action, identication and legislation. Toxicol. Lett. 2010, 197, 157. [19] T. Kraemer, K. Y. Rust, M. R. Meyer, D. K. Wissenbach, D. Bregel, M. Hopf, H. H. Maurer, J. Wilske. Studies on the metabolism of JWH-018 and of a homologue of CP 47,497, pharmacologically active ingredients of different misused incense (Spice) using GC-MS and LCMSn techniques. Ann. Toxicol. Anal. 2009, 21(S1), O43. [20] T. Sobolevsky, I. Prasolov, G. Rodchenkov. Detection of JWH-018 metabolites in smoking mixture post-administration urine. Forensic Sci. Int. 2010, 200, 141. [21] I. Moller, A. Wintermeyer, K. Bender, M. Jubner, A. Thomas, O. Krug, W. Sch anzer, M. Thevis. Screening for the synthetic cannabinoid JWH-018 and its major metabolites in human doping controls. Drug Testing Anal. 2010, DOI:10.1002/dta.158. [22] A. Wintermeyer, I. Moller, M. Thevis, M. Jubner, J. Beike, M. A. Rothschild, K. Bender. In vitro phase I metabolism of the synthetic cannabimimetic JWH-018. Anal. Bioanal. Chem. 2010, 398, 214. [23] Q. Zhang, P. Ma, R. Cole, G. Wang. Identication of in vitro metabolites of JWH-015, an aminoalkylindole agonist for the peripheral cannabinoid receptor (CB2 ) by HPLC-MS/MS. Anal. Bioanal. Chem. 2006, 386, 1345. [24] J. W. Huffman, R. Mabon, M. J. Lu, R. Hurt, D. P. Hurst, P. H. Regio, J. L. Wiley, B. R. Martin. 3-Indolyl-1-naphthylmethanes: new cannabimimetic indoles provide evidence for aromatic stacking interactions with the CB1 cannabinoid receptor. Bioorg. Med. Chem. 2003, 11, 539. [25] A. C. Howlett, F. Barth, T. I. Bonner, G. Cabral, P. Casellas, W. A. Devane, C. C. Felder, M. Herkenham, K. Mackie, B. R. Martin, R. Mechoulam, R. G. Pertwee. International Union of Pharmacology. XXVII. Classication of cannabinoid receptors. Pharmacol. Rev. 2002, 54, 161. [26] M. M. Aung, G. Grifn, J. W. Huffman, M. J. Wu, C. Keel, B. Yang, V. M. Showalter, M. E. Abood, B. R. Martin. Inuence of the N-1 alkyl chain length of cannabimimetic indoles upon CB1 and CB2 receptor binding. Drug Alcohol Depen. 2000, 60, 133. [27] J. W. Huffman, G. Zengin, M. J. Wu, J. Lu, G. Hynd, K. Bushell, A. L. Thompson, S. Bushell, C. Tartal, D. P. Hurst, P. H. Reggio, D. E. Selley, M. P. Cassidy, J. L. Wiley, B. R. Martin. Structureactivity relationships for 1-alkyl-3-(1-naphthoyl)indoles at the cannabinoid CB1 and CB2 receptors: steric and electronic effects of naphthoyl substituents. New highly selective CB2 receptor agonists Bioorg. Med. Chem. 2005, 13, 89. [28] J. L. Wiley, D. R. Compton, D. Dai, J. A. H. Lainton, M. Phillips, J. W. Huffman, B. R. Martin. Structure-activity relationships of indole-and pyrrole-derived cannabinoids. J. Pharmacol. Exp. Ther. 1998, 285, 995. [29] J. W. Huffman. Cannabimimetic indoles, pyrroles, and indenes: structure-activity relationships and receptor interactions, in The Cannabinoid Receptors, (Ed: P. H. Reggio), Humana Press: Totowa, New Jersey, 2009, pp. 4994. [30] R. G. Pertwee. Pharmacological actions of cannabinoids, in Cannabinoids, (Ed. R. Pertwee), Springer Press: Heidelberg, 2005, pp. 151.

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relationships: Correlation of receptor binding and in vivo activities. J. Pharmacol. Exp. Ther. 1993, 265, 218. [34] A. Makriyannis, H. Deng. US Patent Application Cannabimimetic indole derivatives. US Patent No. 208/0090871, 2008. [35] WADA prohibited list 2010 and 2011. Available at: http:// www.wada-ama.org/en/World-Anti-Doping-Program/Sports-andAnti-Doping-Organizations/International-Standards/ProhibitedList Accessed 5 December 2010.

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