Detection and Identification of Pyrolysis Products from the Combustion of Synthetic Cannabinoids

1 Detection and identification of pyrolysis products from the combustion of synthetic cannabinoids Sofia Raquel Teixeira de Sousa Guedes Dissertation thesis for the Master Degree in Analytical, Clinical and Forensic Toxicology submitted to the Faculty of Pharmacy Dissertação de candidatura ao grau de Mestre em Toxicologia Analítica, Clínica e Forense apresentada à Faculdade de Farmácia Supervised by Doctor Maria Paula Amaral Alegria Guedes de Pinho and co-supervised by Professor Félix Dias Carvalho and Professor Maria de Lourdes Pinho de Almeida Souteiro Bastos Faculty of Pharmacy, University of Porto September 2014 iv AKNOWLEDGEMENTS Firstly I would like to thank Professor Doctor Maria Paula Amaral Alegria Guedes de Pinho for her efforts during this challenge, always available to clarify any doubts and for the essential contribution in the evolution and development of this dissertation such as the correspondent correction. I also thank to Professor Félix Dias Carvalho for his supervision, encouragement and enriching contribute during this research. I would to thank to Professor Maria de Lourdes Bastos, Coordinator of the Master Degree in Analytical, Clinical and Forensic Toxicology, for her support in the correction of this dissertation. ´ I have to thank to Professor Carlos Manuel Magalhães Afonso for his help and effort in clarify all the doubts presented. I want to express my gratitude to the Laboratory of Toxicology, Department of Biological Sciences, Faculty of Pharmacy of Porto University for providing all the equipment and financial support necessary to perform all the experimental work. I thank to the Department of Bromatologia, Faculty of Pharmacy of Porto University, for the provided equipment during the experiment. To my friends, thank for all the support. Special thanks to Johanna Domingos, Renato Martins, Filipa Dias and Sofia Borges for all the friendship, encouragement and care expressed. To my colleagues from the Laboratory of Toxicology for all the advice and support during this year. Lastly, I would like to thank to my family, specially my mum, dad, aunts and grandmother for the motivation, support, care and dedication transmitted throughout this journey. Without their support this work would never been possible. v ABSTRACT In the last years new psychoactive substances, initially termed as legal highs, appeared on the market, at breakneck speed, leading to a crescent concern due to its high consumption as well as to the serious adverse effects that have been reported. Among the new psychoactive substances emerged the synthetic cannabinoids category, being the class of drugs most commonly used in the large group of synthetic drugs. Initially, the first synthetic cannabinoids that emerged in the market had therapeutic purposes, being administered even in hospitals. However, they were later removed from clinical practice, due to the knowledge of its negative effects. Nevertheless, similar compounds have appeared in the market with recreational purposes as legal alternatives to illegal substances. The pharmacokinetics and pharmacodynamics of synthetic cannabinoids are similar to Δ9-THC, exerting similar effects on users. Nevertheless, synthetic cannabinoids affinity for cannabinoid receptors is generally much higher when compared with Δ9-THC, which may result in adverse reactions and toxic effects that are not experienced during the use of Cannabis, namely to heavier episodes of psychosis. Moreover, since the action of synthetic cannabinoids has been shown to be negative for the users, it is important to identify the compounds that are inhaled when the cigarette containing synthetic cannabinoids is burned, in order to assess the real danger concerning the consumption of such substances. Thus, several techniques were developed and tested trying to identify such compounds. The investigation samples, containing synthetic cannabinoids have been subjected to temperatures from 100 to 6000C (at intervals of 1000C) in a muffle furnace in order to simulate the combustion process. However, due to the experiment, volatiles were lost and the only conclusion obtained with this method was a decrease of synthetic cannabinoids concentration, as the temperature was increased, noting the complete volatilization of the majority, when subjected to 400°C. vi Next, a new method was developed, using an apparatus with a SPE column, enabling the simulation of the burning of cannabinoids and retention of new products formed within the column. The combustion products were analyzed by GC-MS. Using the mass spectrum of each compound and simulating the chemical breaks of the initial molecule of the synthetic cannabinoids, it was possible to identify new products, named combustion products. In short, it was concluded from this work that the consumption of synthetic cannabinoids not only becomes harmful because of the toxicity of cannabinoids themselves, but also because of new compounds formed after combustion. Some of these products may elicit toxic effects and excessive consumption of most of them can lead to serious consequences in the organism. vii RESUMO Nos últimos anos surgiram no mercado, a uma velocidade alucinante, uma série de novas substâncias psicoactivas, inicialmente denominadas como “drogas legais” cuja preocupação se tornou crescente devido ao elevado consumo que se começou a fazer sentir, bem como aos elevados riscos que foram sendo descobertos, à medida que novos casos de intoxicação iam surgindo. De entre as novas substâncias psicoactivas que surgiram, destacam-se os canabinóides sintéticos, constituindo a classe de novas substâncias psicoativas mais consumida. Inicialmente, os primeiros canabinóides sintéticos que surgiram no mercado, tinham finalidades terapêuticas, sendo administrados até mesmo em hospitais, acabando mais tarde por sair de uso, devido ao conhecimento dos seus efeitos negativos. Entretanto, começaram a surgir no mercado compostos semelhantes mas com fins recreativos, como alternativas das substâncias ilícitas. A farmacocinética e farmacodinâmica dos canabinóides sintéticos são similares às do ∆9-THC, exercendo efeitos similares nos consumidores. No entanto, a afinidade dos canabinóides sintéticos para os receptores canabinóides é muito maior quando comparada com o ∆9-THC, fazendo com que possam ocorrer reacções adversas e efeitos tóxicos que não são sentidos aquando do consumo de Cannabis, nomeadamente a episódios de psicose profunda. Tendo em consideração o elevado risco referido para os canabinóides sintéticos, tornou-se importante identificar os compostos que são inalados aquando da queima do cigarro contendo canabinóides sintéticos, por forma a aferir acerca do verdadeiro perigo do consumo de tais substâncias. Assim, foram testadas e desenvolvidas técnicas que possibilitassem a identificação de tais compostos. Inicialmente as amostras em estudo, contendo canabinóides sintéticos, foram submetidas a temperaturas de 100 a 6000C (em intervalos de 1000C) numa mufla, por forma a simular a combustão das mesmas. No entanto, devido à perda de compostos voláteis aquando da análise, a única conclusão possível de obter com este método foi o decréscimo da concentração dos canabinóides sintéticos, à medida que a viii temperatura aumentava, notando-se a total volatilização da maioria quando submetidos a 400ºC. Seguidamente foi desenvolvida uma nova metodologia, em que um equipamento contendo uma coluna de SPE, possibilitou a simulação da queima dos canabinóides e a retenção dos novos produtos formados no interior da coluna, que posteriormente foram analisados por GC-MS. A identificação dos fragmentos de massa (m/z) bem como a simulação teórica das quebras na molécula inicial dos canabinóides sintéticos permitiu a identificação de novos compostos, denominados produtos resultantes da combustão. Em suma, concluiu-se com este estudo que o consumo de canabinóides sintéticos não se torna prejudicial unicamente pela toxicidade dos próprios canabinóides, mas devido aos novos compostos formados após a sua inalação, sendo que alguns deles são potencialmente tóxicos, podendo o consumo excessivo da maioria deles levar a graves sequelas no organismo. ix INDEX AKNOWLEDGEMENTS ....................................................................................................... iv ABSTRACT .......................................................................................................................... v RESUMO ........................................................................................................................... vii INDEX ................................................................................................................................ ix ABREVIATION LIST ........................................................................................................... xii INDEX OF FIGURES .......................................................................................................... xiv INDEX OF TABLES .......................................................................................................... xviii PART I: GENERAL INTRODUCTION ................................................................................... 1 1.1 NEW PSYCHOACTIVE SUBSTANCES: AN OVERVIEW .......................................... 3 1.2 SYNTHETIC CANNABINOIDS ............................................................................... 6 1.2.1 General considerations ............................................................................... 6 1.2.2 Physico-chemical properties ...................................................................... 8 1.2.3 Pharmacokinetics and pharmacodynamics .............................................. 11 1.2.4 Physiology, pharmacology and toxicology ............................................... 15 1.2.5 Treatment ................................................................................................. 19 1.2.6 Analysis ..................................................................................................... 19 PART II: SCOPE AND OBJETIVES OF THE THESIS ............................................................. 25 PART III: EXPERIMENTAL ................................................................................................. 29 xvi Figure 25: SCORPION ULTRASTRONG herbal incense chromatograms analysis of the samples submitted to 100, 200, 300, 400, 500 and 600 degrees Celsius in a muffle furnace (focus on the main peaks related to the synthetic cannabinoids). .................. 59 Figure 26: Temperature ranging versus concentration of the synthetic cannabinoids found in the herbal incense SCORPION ULTRASTRONG. ............................................... 60 Figure 27: SHIVA ULTRASTRONG herbal incense chromatograms analysis of the samples submitted to 100, 200, 300, 400, 500 and 600 degrees Celsius in the muffle furnace. ........................................................................................................................... 61 Figure 28: SHIVA ULTRASTRONG herbal incense chromatograms analysis of the samples submitted to 100, 200, 300, 400, 500 and 600 degrees Celsius in a muffle furnace (focus on the main peaks related to the synthetic cannabinoids). .................. 62 Figure 29: Temperature ranging versus concentration of the synthetic cannabinoids found in the herbal incense SHIVA ULTRASTRONG. ....................................................... 63 Figure 30: Comparison between chromatograms resulting from the injection of CM21 incense before (1) and after (2) the combustion. .......................................................... 64 Figure 31: Chromatogram analysis of the injection of CM21 after the combustion and the main peaks found related to the combustion of the synthetic cannabinoids. ........ 65 Figure 32: Simulations of breaks in the molecule MAM2201 N-(4-fluoropentyl) isomer that could occur when the combustion process. ........................................................... 67 Figure 33: Simulations of breaks in the molecule JWH-122 N-(4-pentenyl) analog that could occur when the combustion process. ................................................................... 72 Figure 34: Simulations of breaks in the molecule AM2201 2’-naphthyl isomer that could occur when the combustion process. ................................................................... 76 xvii Figure 35: Comparison between chromatograms resulting of the injection of SCORPION ULTRASTRONG incense before (1) and after (2) the combustion. ................................. 80 Figure 36: Chromatogram analysis of the injection of CM21 after the combustion and the main peaks found related to the combustion of the synthetic cannabinoids. ........ 81 Figure 37: Simulations of breaks in the molecule JWH-122 7-methylnaphthyl isomer that could occur when the combustion process. ........................................................... 83 Figure 38: Comparison between chromatograms resulting from the injection of SHIVA ULTRASTRONG incense before (1) and after (2) the combustion. ................................. 87 Figure 39: Chromatogram analysis of the injection of CM21 after the combustion and the main peaks found related to the combustion of the synthetic cannabinoids. ........ 88 Figure 40: Simulations of breaks in the molecule AM2201 that could occur when the combustion process. ....................................................................................................... 90 Figure 41: Simulations of breaks in the molecule MAM2201 that could occur when the combustion process. ....................................................................................................... 94 Figure 42: Simulations of breaks in the molecule JWH-122 N-(4-pentenyl) analog that could occur when the combustion process. ................................................................... 98 Figure 43: Simulations of breaks in the molecule MAM2201 N-(5-chloropentyl) analog that could occur when the combustion process. ......................................................... 102 xviii INDEX OF TABLES Table 1: Summary of desired and toxicity effects and management (Aoun, Christopher et al. 2014). ....................................................................................................................... 5 Table 2: Classes of synthetic cannabinoids according to the chemical structure by UNODC (adapted by (De Brabanter 2013)). ..................................................................... 9 Table 3: Central effects of synthetic cannabinoids based in case reports (Font: (Seely, Lapoint et al. 2012)) ........................................................................................................ 17 Table 4: Cardiovascular effects of synthetic cannabinoids based in case reports (Font: (Seely, Lapoint et al. 2012)) ............................................................................................ 18 Table 5: Gastrointestinal effects of synthetic cannabinoids based in case reports (Font: (Seely, Lapoint et al. 2012)) ............................................................................................ 18 Table 6: Other effects of synthetic cannabinoids based in case reports (Font: (Seely, Lapoint et al. 2012)) ........................................................................................................ 18 Table 7: Analytical methodologies on the chromatographic/mass spectrometric for synthetic cannabinoids analysis in biological samples (Favretto et al. 2013). ............... 23 Table 8: CM21 herbal incense interesting peaks identification by the library swgdrug.44 Table 9: SCORPION ULTRASTRONG herbal incense interesting peaks identification by the library swgdrug. ....................................................................................................... 46 Table 10: SHIVA ULTRASTRONG herbal incense interesting peaks identification by the library swgdrug. .............................................................................................................. 48 Table 11: Peaks areas of the synthetic cannabinoids found in the herbal incense CM21 submitted to different temperatures in a muffle furnace. ............................................ 57 xix Table 12: Peaks areas of the synthetic cannabinoids found in the herbal incense SCORPION ULTRASTRONG at different temperatures in a muffle furnace. ................... 60 Table 13: Peaks areas of the synthetic cannabinoids found in the herbal incense SHIVA ULTRASTRONG at different temperatures in muffle furnace. ....................................... 63 Table 14: CM21 herbal incense analysis of the volatile contained in the SPE cartridge: main peaks founded and possible combustion products. ............................................. 66 Table 15: Resulting compounds of the simulation of breaks in the molecule MAM2201 N-(4-fluoropentyl) isomer that could occur when the combustion process. ................ 68 Table 16: Possible resulting compounds of the combustion of the synthetic cannabinoid MAM2201 N-(4-fluoropentyl) isomer present in CM21 blends. ............... 69 Table 17: Compounds resulting of the matching between the simulation of the breaks (theoretical approach) in the molecule of the synthetic cannabinoid MAM2201 N-(4fluoropentyl) isomer and the compounds found in the chromatograms resulting of the combustion assay of the compound CM21. ................................................................... 70 Table 18: Resulting compounds of the simulation of breaks in the molecule JWH-122 N- (4-pentenyl) analog that could occur when the combustion process. .......................... 73 Table 19: Possible resulting compounds of the combustion of the synthetic cannabinoid JWH-122 N-(4-pentenyl) analog present in CM21 blends. ........................ 74 Table 20: Compounds resulting of the matching between the simulation of the breaks (theoretical approach) in the molecule of the synthetic cannabinoid JWH-122 N-(4pentenyl) analog and the compounds found in the chromatograms resulting of the combustion assay of the compound CM21. ................................................................... 74 Table 21: Resulting compounds of the simulation of breaks in the molecule AM2201 2’- naphthyl isomer that could occur when the combustion process. ................................ 77 xx Table 22: Possible resulting compounds of the combustion of the synthetic cannabinoid AM2201 2’-naphthyl isomer present in CM21 blends. ............................. 78 Table 23: Compounds resulting of the matching between the simulation of the breaks (theoretical approach) in the molecule of the synthetic cannabinoid AM2201 2’- naphthyl isomer and the compounds found in the chromatograms resulting of the combustion assay of the compound CM21. ................................................................... 79 Table 24: SCORPION ULTRASTRONG herbal incense analysis of the volatile contained in the SPE cartridge: main peaks founded and possible combustion products. ................ 82 Table 25: Resulting compounds of the simulation of breaks in the molecule JWH-122 7methylnaphthyl isomer that could occur when the combustion process. .................... 84 Table 26: Possible resulting compounds of the combustion of the synthetic cannabinoid JWH-122 7-methylnaphthyl isomer present in SCORPION blends. ........... 85 Table 27: Compounds resulting of the matching between the simulation of the breaks (theoretical approach) in the molecule of the synthetic cannabinoid JWH-122 7methylnaphthyl isomer and the compounds found in the chromatograms resulting of the combustion assay of the compound SCORPION. ..................................................... 86 Table 28: SHIVA ULTRASTRONG herbal incense analysis of the volatile contained in the SPE cartridge: main peaks founded and possible combustion products. ...................... 89 Table 29: Resulting compounds of the simulation of breaks in the molecule AM2201 2'- naphthyl isomer that could occur when the combustion process. ................................ 91 Table 30: Possible resulting compounds of the combustion of the synthetic cannabinoid AM2201 2'-naphthyl isomer present in SHIVA blends. ............................. 92 Table 31: Compounds resulting of the matching between the simulation of the breaks (theoretical approach) in the molecule of the synthetic cannabinoid AM2201 2'- xxi naphthyl isomer and the compounds found in the chromatograms resulting of the combustion assay of the compound SHIVA. .................................................................. 93 Table 32: Resulting compounds of the simulation of breaks in the molecule MAM2201 N-(4-fluoropentyl) isomer that could occur when the combustion process. ................ 95 Table 33: Possible resulting compounds of the combustion of the synthetic cannabinoid MAM2201 N-(4-fluoropentyl) isomer present in SHIVA blends. ............... 96 Table 34: Compounds resulting of the matching between the simulation of the breaks (theoretical approach) in the molecule of the synthetic cannabinoid MAM2201 and the compounds found in the chromatograms resulting of the combustion assay of the compound SHIVA. ........................................................................................................... 97 Table 35: Resulting compounds of the simulation of breaks in the molecule JWH-122 N- (4-pentenyl) analog that could occur when the combustion process. .......................... 99 Table 36: Possible resulting compounds of the combustion of the synthetic cannabinoid JWH-122 N-(4-pentenyl) analog present in SHIVA blends. ..................... 100 Table 37: Compounds resulting of the matching between the simulation of the breaks (theoretical approach) in the molecule of the synthetic cannabinoid JWH-122 N-(4pentenyl) analog and the compounds found in the chromatograms resulting of the combustion assay of the compound SHIVA. ................................................................ 101 Table 38: Resulting compounds of the simulation of breaks in the molecule MAM2201 N-(-5-chloropentyl) analog that could occur when the combustion process. ............. 103 Table 39: Possible resulting compounds of the combustion of the synthetic cannabinoid MAM2201 N-(-5-chloropentyl) analog present in SHIVA blends. ........... 104 Table 40: Compounds resulting of the matching between the simulation of the breaks (theoretical approach) in the molecule of the synthetic cannabinoid MAM2201 and the xxii compounds found in the chromatograms resulting of the combustion assay of the compound SHIVA. ......................................................................................................... 105 Table 41: Effects and toxicity of the resulting compounds from the combustion of synthetic cannabinoids. ................................................................................................ 112 1 PART I: GENERAL INTRODUCTION 2 3 1.1 NEW PSYCHOACTIVE SUBSTANCES: AN OVERVIEW In the last years a growing number of novel psychoactive substances appeared in the legal market as recreational drugs. They are manufactured mainly in China, Pakistan and India and distributed all over the world (Aoun et al. 2014). Those substances are called legal highs or smart drugs and range from plant-based to synthetic compounds (Johnson et al. 2013). These novel substances comprise derivatives and analogues of cathinones or cannabis, falling into three categories: synthetic cathinones (bath salts), synthetic cannabinoids (spice or incense) and synthetic amphetamine-like drugs (Arnold 2013). Other authors divided these substances in three other groups: stimulants, depressants and hallucinogens; they comprise natural plants, synthetic substances and semi-synthetic substances originating from natural oils (Kapka-Skrzypczak 2011). These substances may differ in its chemical structures, potencies, half-lives, metabolism and severity of side effects (Zukiewicz-Sobczak et al. 2012). In the last years, the control of these drugs has been reinforced opening great challenges to analytical laboratories to provide rapid tests for detecting these new materials (AgilentTechnologies 2012). These products are usually available in various formats, as pills, roll-ups, incense sticks, herb mixtures, room fresheners, powders or even bath salts. They are mixtures of fragments of plants (herbs) added with extracts of other plants and synthetic chemical substances. The majority of these substances were not tested, neither in humans nor animals, before introducing in the market (Kapka-Skrzypczak 2011). Plant-products often contain more than ten herbal additives, which difficult the identification of each psychoactive constituent (Rosenbaum et al. 2012). Several research works performed on the botanical material showed that most of the plant species do not have psychoactive properties; instead, they are used to dilute the cannabimimetic compounds (De Brabanter 2013). 10 Eicosanoids Endocannabinoids and synthetic analogs Anandamide; Methanandamide Aminoalkylindoles Naphthoylindoles; Phenylacetylindoles; Naphthylmethylindoles; Benzoylindoles JWH-122 JWH-018; JWH-073 JWH-250; RCS-8; JWH-203 JWH-175; JWH-184; JWH-185 AM-630; AM-2233; RCS-4 Others Diarylpyrazoles, naphtoylpyrroles, etc. JWH-307; CRA-13 11 In the commercialized products containing synthetic cannabinoids, it can also be found: fatty acids and their esters (linoleic acid, palmitic acid), amide fatty acids (oleamide, palmitoylethanolamine), plant-derived substances (flavors compounds), preservatives (benzyl benzonate) and additives (alpha-tocopherol). They may also contain vitamin E and are frequently contaminated with β2-adrenergic agonist clenbuterol, producing the known sympathomimetic-like effects (Seely et al. 2012). 1.2.3 Pharmacokinetics and pharmacodynamics Although the pharmacokinetic and pharmacodynamic profiles of synthetic cannabinoids are not completely known, some studies reported oral and inhalation bioavailability, and no cases of parenteral or rectal routes of administration have been referred (Seely et al. 2012). Concerning to pharmacokinetic aspects, some studies were performed with JWH-018. The maximum concentrations were found five minutes after smoking, meaning that the absorption via lungs and the distribution over the other organs takes place quickly. The compound is detectable until 48 hours after the consumption. In general, these compounds are extensively metabolized in the human body (De Brabanter 2013). Some studies about the aminoalkylindole compounds described a lot of modifications occurring after the administration, as multiple hydroxylations, carboxylations, dehydrogenations, dealkylations and dehydrodiol formation. It was also showed that these metabolites are excreted as glucuronide and/or sulphate conjugates in urine. Monohydroxylated and carboxylated metabolites are also found in urine in high quantities. Some studies indicate that hydroxylation and carboxylation 12 are the preferred pathways of synthetic cannabinoids metabolism (Adamowicz et al. 2013). JWH-018 is an example of a synthetic cannabinoid (figure 2) having a short halflife in human blood following smoking (Teske et al. 2010). Nevertheless, specific metabolic pathways leading to detoxification and excretion remains to be determined (Gronewold and Skopp 2011). Specific cytochrome P450 enzymes responsible for the metabolism of the synthetic cannabinoids are not identified (Chimalakonda et al. 2011), but it has been reported that the major UDP-glucuronosyltransferases responsible for conjugation are UGT1A1, UGT1A3, UGT1A9, UGT1A10 and UGT2B7 (Seely et al. 2012). Figure 2: Summary representation about JWH-018 metabolism, excretion and potential downstream interactions with CB1 receptors and other physiological targets (Font: (Seely et al. 2012)). Legend: “+” indicates agonism; “-“ indicates antagonism; “X” indicates no binding affinity and “?” indicates not known) . 13 Based in user reports, some synthetic cannabinoids act like marijuana whose principal active is ∆9-THC. Hence, the hydrophobicity of a compound (as measured by its distribution coefficient, log D) affects strongly its pharmacokinetics and pharmacodynamics. The log D values indicate that synthetic cannabinoids are highly lipophilic and their absorption, distribution, metabolism and excretion are similar to ∆9-THC. This lipophilic characteristic favours the absorption of the cannabinoids across the lipid bilayer membranes, favoring the quick elimination from blood circulation. It can be expectable that after the consume of similar doses of synthetic cannabinoids and ∆9-THC, the levels of blood and urine can be comparable (Adamowicz et al. 2013). In conclusion to the pharmacokinetic aspects, it is possible to affirm that the diverse metabolite activity profile of aminoalkylindole metabolites may partially explain the mixed effects of this kind of drugs and their high potential safety concerns (Seely et al. 2012). In respect to pharmacodynamics, it is known the presence of two cannabinoid receptors in the human body, named CB1 and CB2 receptors. The CB1 receptor is located in the brain and in the central nervous system, expressed presynaptically, decreasing the release of GABA and increasing the extracellular glutamate and dopamine neurotransmitters levels (Seely et al. 2012), what makes the activation of this receptor responsible for the psychotropic effects. CB2 receptor is essentially present in immune cells and has an effect in the inflammatory process, the release of cytokines and cell migration (Rosenbaum et al. 2012). Both are G-protein coupled receptors responding to endogenous ligands anandamide and noladin ether and also to synthetic cannabinoids and ∆9-THC (Rosenbaum et al. 2012). The activation provokes presynaptic hyperpolarization through changes in calcium influx and potassium efflux resulting in neuronal hyperpolarization and a decrease in neurotransmitter release (Seely et al. 2012). Although chemical structure of synthetic cannabinoids and ∆9-THC structure are considerably different, both are agonists of the cannabinoid receptors and may exert activity on other receptor families. The affinity of the most cannabimimetic compounds is considerably higher, especially with the CB1 receptor, comparable to ∆9- 14 THC (Adamowicz et al. 2013), but also for the CB2 receptors, hence the synthetic cannabinoids, with affinity for this receptor can affect the immune system by modulating chemotaxis of T lymphocytes or inducing thymic atrophy and apoptosis (Seely et al. 2012). The prolonged use of synthetic cannabinoids compared to consume of marijuana is strongly related to persistent psychosis, because of its major affinity of the cannabinoid receptors and the high potency of compounds found in these products. In other hand, marijuana has in its composition cannabidiol, an antipsychotic compound that provokes a decrease of psychotic activity (Aoun et al. 2014). The knowledge of the pharmacological effects associated with cannabinoid receptors and their agonists led the chemists to find selective analogs of THC that separate the beneficial effects for the treatment of some diseases. The first analogs discovered, also classical cannabinoids, closely related structurally with THC, were the HU-210 and the Nabilone. This last one was approved by the FDA for the treatment of nausea and vomiting in chemotherapy-induced patients. The non-classical cannabinoids appear later, included the CP series developed by Pfizer and the aminoalkylindole series developed by Sterling Winthrop. Recently, the series JWH, above mentioned, were synthesized with a variety of core ring structures. These cannabinoid analogues were used with medicinal purposes (Kennedy). The synthetic cannabinoid receptor agonist may, in this way, inhibit tumor growth and metastasis of breast cancer and human tumor prostate PC-3 cell growth and interact with chemokine receptor agonists CXCR4 in modulating breast cancer growth and invasion. They can also affect basic neural cell processes like cell proliferation and survival, by the presence of CB2 receptors in neurons and glial cells in the brain (Seely et al. 2012). 15 1.2.4 Physiology, pharmacology and toxicology Almost nothing about pharmacology, toxicology and safety profile of the synthetic cannabinoids is known. Some studies concluded that they are responsible for psychiatric complications and overdose symptoms may occur, depending on the quantity of cannabinoids added to the mixture (AgilentTechnologies 2012) Adverse reactions and toxic effects result principally from activation of central CB receptors and β2 adrenergic receptors (Aoun et al. 2014). The pharmacological effects are similar than those obtained with cannabis consumption, described as well-being and euphoria, sensory changes, visual, tactile and auditory perceptions, perceptual illusions, hallucinations and feeling of slowed time. Some studies showed that synthetic cannabinoids have serotonin-like or weak monoamine oxidase inhibitor properties (Rosenbaum et al. 2012). Their pharmacological activity is related with changes in the neurotransmitter system, intensifying the dopaminergic and catecholaminergic systems activity, inhibiting the reuptake of serotonin. It causes psychological and physical agitation, euphoria/dysphoria, impaired appetite and increased concentration (ZukiewiczSobczak et al. 2012). The most common administration Spice intake is the inhalation. The users described the effects similar to those obtained with marijuana consume. The adverse clinical effects are mainly anxiety, paranoia, headache, nausea, vomiting, convulsions and inducing psychosis. They can be classified into central, cardiovascular and gastrointestinal effects (Cottencin et al. 2014). The central effects (Table 3) include emotional alterations or, in some cases of vulnerable individuals, psychosis. There were also described seizure activity, cognitive impairment, sedation, confusion and impaired motor coordination difficulties. The adverse physic effects include anxiety, agitation and irritability. The cardiovascular effects (Table 4) are tachycardia, tachyarrhythmia, cardiotoxicity, myocardial infarction and chest pain. Gastrointestinal effects (Table 5) such as nausea associated with 16 agitation, tachycardia, drowsiness, vomiting and hallucinations were reported. Other effects (Table 6) described were dilated pupils, somnolence, brisk reflexes, emesis, increased pulse rates and blood pressure, hyperreflexia, flushed skin and appetite changes (Cottencin et al. 2014). There is no information about chronic effects of synthetic cannabinoids; however, some authors referred some possible effects of the prolonged use of these substances as significant alterations in emotional processing and cognitive functioning. Other authors speculate that the long-term effects could be as the same of the prolonged marijuana consume, as the reduction of brain volume, affecting amygdala and hippocampus, which are associated with memory and the pathophysiology of schizophrenia and emotional processing. Some case reports focus symptoms related to auditory and visual hallucinations, paranoid delusions, thought blocking, disorganized speech, anxiety and insomnia, stupor and suicidal ideas resulting in an increased risk of psychosis, an age-and-dose-dependent manner (Seely et al. 2012). In other hand, cannabinoids can modulate pre frontal cortex neural functioning by decreasing the release of GABA and increasing extracellular glutamate and dopamine levels what results in alterations in emotional processing and cognitive functioning (Seely et al. 2012). Some case reports described the symptoms of overdoses as loss of contact and consciousness, seizures, aphasia, tachycardia and hallucinations followed by drowsiness and impaired memory. Disorientation, insomnia, memory loss and lapses, jerky movements and problems with speech were present even after several days (Adamowicz et al. 2013). Vascular reactivity and dysfunctioning have been associated to intoxication symptoms, especially in patients with history of ischemic heart disease (Aoun et al. 2014). In the tables present below are shown the most common effects induced by the consumption of synthetic cannabinoids based in case reports 17 Table 3: Central effects of synthetic cannabinoids based in case reports (Font: (Seely, Lapoint et al. 2012)) Central effects Psychosis Acute exacerbation of cannabis-induced recurrent psychotic episodes (Müller et al., 2010) Enhanced risk of psychosis relapse in vulnerable individuals (Every-Palmer, 2010, 2011) New-onset psychosis in otherwise healthy young men (Hurst et al., 2011) Seizures Seizure activity approximately 1 h after smoking “SpicyXXX” (Simmons et al., 2011a,b) Generalized seizure in a healthy 48 years old man within 30 min after ingesting a spice mixture purchased from the Internet (Lapoint et al., 2011) Witnessed generalized convulsions (Schneir and Baumbacher, 2012) Anxiety Occurrence of anxiety shortly after smoking “Banana Cream Nuke” (Schneir et al., 2011) Frequent anxiety in 15–19 years old boys consuming synthetic cannabinoids (Castellanos et al., 2011) Self-reports of users experiencing anxiety after injection of “Aroma” (Every-Palmer, 2010, 2011) Agitation Reported to Texas poison centers by 20–23 years old patients following exposure to synthetic cannabinoids Forrester et al. (2011) Irritability Reported to South Miami Hospital Addiction Treatment Center by 15–19 years old individuals who smoked Spice or K2 (Castellanos et al., 2011) Memory changes Reported by all smokers evaluated at the South Miami Hospital Addiction Treatment Center (Castellanos et al., 2011) Sedation Detected in a 21 years old man an unknown time after smoking spice (Simmons et al., 2011a,b) Confusion Detected in a 27 years old man 1 h after smoking SpicyXXX (Simmons et al., 2011a,b) 18 Table 4: Cardiovascular effects of synthetic cannabinoids based in case reports (Font: (Seely, Lapoint et al. 2012)) Cardiovascular effects Tachycardia A common clinical feature in 21–27 years old patients smoking SpicyXXX and other spice products (Forrester et al., 2011; Simmons et al., 2011a,b) Tachyarrhytmia Detected in a healthy 48 years old man 30 min after ingesting an ethanol mixture containing a white powder he purchased from the Internet (Lapoint et al., 2011) Cardiotoxicity Chest pain, tachycardia, and bradycardia occurred in a 17 years old boy after smoking K9 (Young et al., 2011) Chest pain Three 16 years old boys complained of chest pain within days after using K2 (Mir et al., 2011) Table 5: Gastrointestinal effects of synthetic cannabinoids based in case reports (Font: (Seely, Lapoint et al. 2012)) Gastrointestinal effects Nausea Along with agitation, tachycardia, drowsiness, vomiting, and hallucinations, nausea was frequently reported after synthetic cannabinoid exposure (Forrester et al., 2011) Vomiting Described in a 19 years old healthy boy along with a generalized 1–2 min convulsion while smoking “Happy Tiger Incense” (Schneir and Baumbacher, 2012) Table 6: Other effects of synthetic cannabinoids based in case reports (Font: (Seely, Lapoint et al. 2012)) Other effects Somnolence; dilated pupils; brisk reflexes; emesis. All common clinical features in 21–27 years old patients smoking SpicyXXX (Simmons et al., 2011a,b) 19 Appetite changes Opposite changes have been reported, patients reporting both decreased and increased appetite (Castellanos et al., 2011) Tolerance; withdrawal and drug dependence Tolerance developed in a 20 years old boy after smoking “Spice Gold” daily for 8 months; during abstinence, he reported inner unrest, craving, nocturnal nightmares, copious sweating, headache, nausea, and tremor (Zimmermann et al., 2009) 1.2.5 Treatment There are no specific guidelines or recognized antidotes for the treatment of acute synthetic cannabinoids intoxication. It is generally supportive and dependent upon a patient specific presentation. They were related some interventions like intravenous benzodiazepines administration for agitation and anxiety (Aoun et al. 2014). However, there are some procedures that the patient should take when exposed to this kind of substances. Primarily, the consumer(s) should go immediately to health services or call to a poison control center. The professional must certificate that the vital signs, vomiting and psychiatric derangements are stabilized before leaving the patient (Rosenbaum et al. 2012). 1.2.6 Analysis To monitor the abuse of the clandestine compounds, the forensic toxicologists have adopted a strategy based on two analytical steps: a preliminary screening to identify all the “presumptive positives” followed by a confirmation test able to identify the “true positives” (Favretto et al. 2013), however, analytical laboratories had 26 27 Although consume of synthetic cannabinoids and ∆9-THC can be compared since their psychoactive effects are similar, synthetic cannabinoids consume seems to be more harmful because their constituents are based in chemical formulations. The study of synthetic cannabinoids has become more explored for the scientific community in the last years, however, the real danger of these drugs consume is not really elucidated. In this way, the knowledge of the combustion products resulting of synthetic cannabinoids present in commercial samples bought in on-line shops proved to be an interesting topic to be explored since few studies in this area were realized until now. In this way, the aims of the present work are the identification and characterization of synthetic cannabinoids and the identification of the new products formed during the combustion process, it is, mimicking what happens during their consume by inhalation. Those objectives allow the knowledge of the toxicity of synthetic cannabinoids when smoked, by the development of methodologies that simulate the referred process. Firstly, a muffle furnace apparatus was used to heat the samples until their combustion temperature, in order to obtain the resulting compounds of the pyrolysis of synthetic cannabinoids. Since no results were obtained unless the decrease of their concentration, a new method needed to be developed. For this purpose, an apparatus allowing the simulation of the combustion process of the synthetic cannabinoids enabled the identification of the intended compounds. Those results were obtained by GC-MS analysis. Although the synthetic cannabinoids themselves could not present a real danger, the combination of the new products formed during the combustion process can be pernicious, as will be show during the presentation of this experimental work. The developed combustion technique can be used likewise to identify combustion products of tobacco and other drugs. 28 29 PART III: EXPERIMENTAL 30 31 CHAPTER I: MATERIAL AND METHODS 32 33 1. MATERIAL 1.1 Commercial samples Three plant sample material with 1g each one, called CM21, SHIVA ULTRASTRONG and SCORPION ULTRASTRONG, were obtained in an online shop included in herbal incenses category with the description “potpourri”. The price of each package was 13 euros. The characterization of these products is described below. 1.1.1 HERBAL INCENSE: CM21 1.1.2 HERBAL INCENSE: SCORPION ULTRASTRONG CM 21 Herbal Incense is a blend of natural herbs and essential oils that refreshes the air with aromatic fragrance when burned. Creates an exotic and relaxing sensation. It is perfect to elevate your mood. Very nice aroma. Containing a mixture of herbal resins. Warning: This product is not for human consumption. The product should be kept out of reach of children. Swallowing should contact the health services responsible. Scorpion Ultra Strong Herbal Incense is a blend of natural herbs and essential oils that refreshes the air with aromatic fragrance when burned. Creates an exotic and relaxing sensation. It is perfect to elevate your mood. Very nice aroma. Containing a mixture of herbal resins. Warning: This product is not for human consumption. The product should be kept out of reach of children. Swallowing should contact the health services responsible. Figure 3: Herbal incense CM21 sold in online headshops Figure 4: Herbal incense SCORPION ULTRASTRONG sold in online headshops 34 1.1.3 HERBAL INCENSE: SHIVA ULTRASTRONG 1.2 Samples preparation Commercial samples were triturated with a mortar and a pestle. Firstly, five mg of each sample were dissolved in 1 mL of methanol. The mixture was homogenized in a vortex during 1 minute and centrifuged 10 minutes, filtered and analyzed in a GC-MS. Five mg of each samples were weighed and placed into an open ampoule that posteriorly was submitted to heating in a muffle furnace (section 2.2) in a way of being totally sealed, avoiding the escape of volatiles that would form upon combustion process. For combustion procedure, five mg of each sample were likewise weighted in triplicate and then put into the combustion tube according to the method described in the section 2.1 of this chapter. Shiva Ultra Strong Herbal Incense is a blend of natural herbs and essential oils that refreshes the air with aromatic fragrance when burned. Creates an exotic and relaxing sensation. It is perfect to elevate your mood. Very nice aroma. Containing a mixture of herbal resins. Warning: This product is not for human consumption. The product should be kept out of reach of children. Swallowing should contact the health services responsible. Figure 5: Herbal incense SHIVA ULTRASTRONG sold in online headshops. 35 1.3 Reagents and equipment Methanol (HPLC grade) was purchased from Fisher Chemical. Thymol was used as an internal standard (1mg/mL). SPE was performed using C18 Sep-Pack colum obtained from Waters (Milford, Massachusetts, USA) SPE Vacuum Pump from BUSHI LaborTechnik AG (Switzerland). The sample concentrator used to dry samples after the extraction from the SPE cartridge was from Barloworld Scientific Limited Stone (Staffordshire, UK). The vials used have the volume of 2mL and the sample containers for the combustion process 10mL. The syringes used to extract the volatile contained in the SPE cartridge were labelled BRAUN (Germany) Omnifix 1mL. The filters used to filter the standard samples have a 0,45µm of pore size and 25mm of diameter The muffle furnace Thermoline 48000 was from Barnstead/Thermoline (Kerper Boulevard, USA). 1.4 GC-MS conditions Figure 6: GC-MS equipment used in this experiment. 42 43 1. SAMPLES ANALYSIS 1.1 Herbal incense: CM21 Figure 10: CM21 chromatogram (5mg of the sample dissolved in 1mL of methanol and filtered) and the main synthetic cannabinoids peaks found. 3 1 2 4 44 (swgdrug) AM2201 2'-naphthyl isomer 388 408 428 448 468 488 508 25 56 87 O N F (Text File) 18.682 min, Scan: 1610 50 150 250 350 450 550 0 50 100 144 232 284 359 501 (swgdrug) JWH-122 N-(4-pentenyl) analog 50 60 70 80 90 100 110 28 61 94 N O (Text File) 18.895 min, Scan: 1630 50 110 170 230 290 350 0 50 100 89 115 141 169 212 254 298 353 (swgdrug) MAM2201 N-(4-fluoropentyl) isomer 305 315 325 335 345 355 365 375 51 72 93 N O F (swgdrug) MAM2201 N-(4-fluoropentyl) isomer 309 319 329 339 349 359 369 379 389 51 74 97 N O F (Text File) 20.456 min, Scan: 1777 50 110 170 230 290 350 0 50 100 51 89 115 141 169 232 298 374 (Text File) 24.442 min, Scan: 2152 50 120 190 260 330 400 0 50 100 89 115 169 248 298 372 Table 8: CM21 herbal incense interesting peaks identification by the library swgdrug. Peak number Retention time m/z Mass spectrum Library identification % of probability by swgdrug library Molecular structure 1 18,669 144, 232, 284, 359 AM2201 2'- naphthyl isomer 34,4% 2 18,897 115, 141, 169, 212, 298, 353 JWH-122 N-(4pentenyl) analog 57,4% 3 20,460 89, 115, 141, 139, 232, 298, 374 MAM2201 N-(4fluoropentyl) isomer 50,3% 4 24,450 89, 115, 169, 248, 298, 372 MAM2201 N-(4fluoropentyl) isomer 34,3% 45 1.2 Herbal incense: SCORPION ULTRASTRONG Figure 11: SCORPION ULTRASTRONG chromatogram (5mg of the sample dissolved in 1mL of methanol and filtered) and the main synthetic cannabinoids peaks found. 1 46 (Text File) 19.004 min, Scan: 1634 50 150 250 350 450 550 0 50 100 115 144 169 214 298340 545 (swgdrug) JWH-122 7-methylnaphthyl isomer 358 388 418 448 478 508 25 56 87 ON Table 9: SCORPION ULTRASTRONG herbal incense interesting peaks identification by the library swgdrug. Peak number Retention time m/z Mass spectrum Library identification % of probability by swgdrug library Molecular structure 1 19,004 115, 144, 169, 214, 298, 340 JWH-122 7methylnaphthyl isomer 18,5% 47 2 1.3 Herbal incense: SHIVA ULTRASTRONG Figure 12: SHIVA ULTRASTRONG chromatogram (5mg of the sample dissolved in 1mL of methanol and filtered) and the main synthetic cannabinoids peaks found. 3 1 4 48 (swgdrug) JWH-122 N-(4-pentenyl) analog 50 60 70 80 90 100 110 28 61 94 N O (Text File) 20.456 min, Scan: 1777 50 110 170 230 290 350 0 50 100 51 89 115 141 169 232 298 374 (swgdrug) AM2201 50 80 110 140 170 200 41 70 99 N O F (swgdrug) MAM2201 75 95 115 135 155 175 48 74 100 N O F (Text File) 19.101 min, Scan: 1575 50 130 210 290 370 450 0 50 100 144 232 284 359 401 477 (Text File) 19.348 min, Scan: 1598 50 150 250 350 450 550 0 50 100 115 141 169 212 298 353 441488 549 Table 10: SHIVA ULTRASTRONG herbal incense interesting peaks identification by the library swgdrug. Peak number Retention time (min.) m/z Mass spectrum Library identification % of probability by swgdrug library Molecular structure 1 19,101 144, 232, 284, 359 AM2201 68,0% 2 19,348 115, 141, 169, 212, 298, 353 JWH-122 N-(4pentenyl) analog 88,6% 3 21,044 115, 169, 232, 255, 284, 298, 313, 374 MAM2201 73,9% 49 (swgdrug) MAM2201 N-(5-chloropentyl) analog 440 470 500 530 560 590 30 59 88 N O Cl 4 25,146 115, 169, 248, 298, 389 MAM2201 N-(5chloropentyl) analog 96,9 % (Text File) 25.146 min, Scan: 2134 50 160 270 380 490 600 0 50 100 115 169 248 298 389 414463 535595 50 (swgdrug) MAM2201 N-(4-fluoropentyl) isomer 50 140 230 320 410 500 0 50 100 141 169 232 254 298 373 N O F (swgdrug) JWH-122 N-(4-pentenyl) analog 50 140 230 320 410 500 0 50 100 141 169 212 298 353 N O 2. SYNTHETIC CANNABINOIDS CHARACTERIZATION 2.1 Synthetic cannabinoids present in the CM21 sample MAM2201 N-(4-fluoropentyl) isomer (SYNTHETIC CANNABINOID 1) Figure 13: MAM2201 N-(4-fluoropentyl) isomer ion spectrum from the library swgdrug. JWH-122 N-(4-pentenyl) analog (SYNTHETIC CANNABINOID 2) Figure 14: JWH-122 N-(4-pentenyl) analog ion spectrum from the library swgdrug. Name: MAM2201 N-(4-fluoropentyl) isomer Formula: C25H24FNO MW: 373 ID#: 3299 DB: swgdrug Comment: Provided by www.caymanchem.com/app/template/landing%2CForensic.v m / Item #11782 Lot #0439334 10 largest peaks: 232 999 | 141 847 | 115 751 | 298 718 | 144 717 | 169 715 | 181 496 | 149 409 | 373 366 | 116 361 | Synonyms: 1.(1-(4-fluoropentyl)-1H-indol-3-yl)(4-methylnaphthalen-1yl)methanone Estimated Kovats RI: Value: 3011 iu Confidence interval (Diverse functional groups): 89(50%) 382(95%) iu Name: JWH-122 N-(4-pentenyl) analog Formula: C25H23NO MW: 353 ID#: 3141 DB: swgdrug Comment: Provided by www.caymanchem.com/app/template/landing%2CForensic.v m / Item #11611 Lot #0437478 10 largest peaks: 169 999 | 353 677 | 212 492 | 298 478 | 141 422 | 115 351 | 144 255 | 41 219 | 284 204 |352 202 | Synonyms: 1.MAM2201 N-(4-pentenyl) analog 2.JWH-022 4-methylnaphthyl analog 3.(4-methylnaphthalen-1-yl)(1-(pent-4-en-1-yl)-1H-indol-3yl)methanone Estimated Kovats RI: Value: 3092 iu Confidence interval (Diverse functional groups): 89(50%) 382(95%) iu 51 (swgdrug) AM2201 2'-naphthyl isomer 50 110 170 230 290 350 0 50 100 51 77 127 155 170 232 284 359 O N F (swgdrug) JWH-122 7-methylnaphthyl isomer 50 130 210 290 370 450 0 50 100 55 144181 214 298 355 ON AM2201 2'-naphthyl isomer (SYNTHETIC CANNABINOID 3) Figure 15: AM2201 2'-naphthyl isomer ion spectrum from the library swgdrug. 2.2 Synthetic cannabinoids present in the SCORPION ULTRASTRONG sample JWH-122 7-methylnaphthyl isomer (SYNTHETIC CANNABINOID 4) Figure 16: JWH-122 7-methylnaphthyl isomer ion spectrum from the library swgdrug. Name: AM2201 2'-naphthyl isomer Formula: C24H22FNO MW: 359 ID#: 3218 DB: swgdrug Comment: DEA SFL1 / Cayman lot 0430209-19 10 largest peaks: 359 999 | 127 986 | 232 869 | 155 597 | 284 563 | 144 480 | 128 305 | 360 259 | 41 238 | 116 199 Synonyms: 1.(1-(5-fluoropentyl)-1H-indol-3-yl)(naphthalen-2yl)methanone Estimated Kovats RI: Value: 2962 iu Confidence interval (Diverse functional groups): 89(50%) 382(95%) iu Name: JWH-122 7-methylnaphthyl isomer Formula: C25H25NO MW: 355 ID#: 2849 DB: swgdrug Comment: Provided by www.caymanchem.com/app/template/landing%2CForensic.v m / Item #9001036 Lot #0431265 10 largest peaks: 355 999 | 214 716 | 298 645 | 181 430 | 144 384 | 141 382 | 338 381 | 115 292 | 356 271 | 169 240 | Synonyms: 1.(7-methylnaphthalen-1-yl)(1-pentyl-1H-indol-3yl)methanone Estimated Kovats RI: Value: 3102 iu Confidence interval (Diverse functional groups): 89(50%) 382(95%) iu 58 3.2 Herbal incense: SCORPION ULTRASTRONG Figure 24: SCORPION ULTRASTRONG herbal incense chromatograms analysis of the samples submitted to 100, 200, 300, 400, 500 and 600 degrees Celsius in a muffle furnace. 59 Figure 25: SCORPION ULTRASTRONG herbal incense chromatograms analysis of the samples submitted to 100, 200, 300, 400, 500 and 600 degrees Celsius in a muffle furnace (focus on the main peaks related to the synthetic cannabinoids). In relation to the chromatograms presented above, there has only one main peak related to the synthetic cannabinoids already described in this experiment (Chapter 2 –Section 2): JWH-122 7-methylnaphthyl isomer (1). In the table is represented the respective peak areas of the named synthetic cannabinoids, in order to confirm the decrease of the concentration with the temperature increase. 1 60 0 20000000 40000000 60000000 80000000 100000000 120000000 140000000 Synthetic cannabinoids concentration SCORPION ULTRASTRONG JWH-122 7methylnaphthyl isomer Table 12: Peaks areas of the synthetic cannabinoids found in the herbal incense SCORPION ULTRASTRONG at different temperatures in a muffle furnace. Retention time 19,508 Compound name JWH-122 7-methylnaphthyl isomer Peak area (1000C) 1,177 x 108 Peak area (2000C) 1,111 x 108 Peak area (3000C) 1,022 x 108 Peak area (4000C) 2,137 x 107 Peak area (5000C) 1,092x 107 Peak area (6000C) 7,075 x 106 Figure 26: Temperature ranging versus concentration of the synthetic cannabinoids found in the herbal incense SCORPION ULTRASTRONG. 61 3.3 Herbal incense: SHIVA ULTRASTRONG Figure 27: SHIVA ULTRASTRONG herbal incense chromatograms analysis of the samples submitted to 100, 200, 300, 400, 500 and 600 degrees Celsius in the muffle furnace. 62 Figure 28: SHIVA ULTRASTRONG herbal incense chromatograms analysis of the samples submitted to 100, 200, 300, 400, 500 and 600 degrees Celsius in a muffle furnace (focus on the main peaks related to the synthetic cannabinoids). In relation to the chromatograms presented above, there are 4 main peaks related to the synthetic cannabinoids already described in this experiment (Chapter 2 – Section 2): AM2201 (1), JWH-122 N-(4-pentenyl) analog (2) and MAM2201 (3, 4). In the table are represented the respective peaks areas of the named synthetic cannabinoids, in order to confirm the decrease of the concentration with the temperature increase. 1 3 4 2 63 0 5000000 10000000 15000000 20000000 25000000 100ºC 200ºC 300ºC 400ºC 500ºC 600ºC Synthetic cannabinoids concentration SHIVA ULTRASTRONG AM2201 JWH-122 N-(4-pentenyl) analog MAM2201 MAM2201 Table 13: Peaks areas of the synthetic cannabinoids found in the herbal incense SHIVA ULTRASTRONG at different temperatures in muffle furnace. Retention time 18,642 18,864 20,459 25,258 Compound name AM2201 JWH-122 N-(4pentenyl) analog MAM2201 MAM2201 Peak area (1000C) 1,416x 106 1,881 x 106 1,993 x 107 7,857 x 106 Peak area (2000C) 1,403 x 106 1,016 x 106 1,873 x 107 6,111 x 106 Peak area (3000C) 523749 862040 9,882 x 106 829861 Peak area (4000C) ______ ______ ______ ______ Peak area (5000C) ______ ______ ______ ______ Peak area (6000C) ______ ______ ______ ______ Figure 29: Temperature ranging versus concentration of the synthetic cannabinoids found in the herbal incense SHIVA ULTRASTRONG. 64 4. GC/MS RESULTS OBTAINED FROM THE APPARATUS TO COLLECT VOLATILES 4.1 Herbal incense: CM21 Figure 30: Comparison between chromatograms resulting from the injection of CM21 incense before (1) and after (2) the combustion. The first chromatogram presented in figure 30 corresponds to the injection of 2 µL of the solution prepared - 5mg of the sample CM21 dissolved in 1 mL of methanol. This chromatogram serves as a reference to compare with the chromatograms relative to the simulation of the combustion of these samples, giving rise to the appearance of combustion products of synthetic cannabinoids present in the herbal incense. Thus, the second chromatogram correspond to the injection of 2 µL of the solution obtained after the extraction of the SPE cartridge, shown in the apparatus presented in figure 9, with 2 mL of methanol and subsequent drying in a nitrogen flow, and resuspended in 100uL of methanol. Thus, it is possible to observe that after being subjected to heating, the sample undergoes several processes of transformation, leading to the formation of new products, as we will verify later, on a closer examination of the chromatogram in question. 65 4.1.1 Volatile contained in the SPE cartridge – analysis of the possible combustion products Figure 31: Chromatogram analysis of the injection of CM21 after the combustion and the main peaks found related to the combustion of the synthetic cannabinoids. In the chromatogram present in figure 31 is possible to verify that, after the heating, there are a lot of new substances formed. The main peaks found in this chromatogram are: peak 1, with the retention time 5,832 minutes; peak 2, whose retention time is 6,873 minutes; peak 3, with retention time 7,588 minutes; The peak 4 has a retention time of 11,340 minutes; peak 5, that appears at 11,457 minutes; peak 6 whose retention time is 17,561 minutes; peak 7 appearing at 18,665 minutes; peak 8, at 18,905 minutes; peak 9 appears at 20,469 minutes and finally the peak 10 at 24,451 minutes. Some of these peaks have an especial interest because of the coincidence of their m/z ratio with the m/z ratio of the molecules formed when theoretical breaks in the synthetic cannabinoid are simulated. In table 14, the main peaks are referred along with the synthetic cannabinoids precursors. The m/z ratio of the new founded compound is taken into account to be compared with the m/z ratio resultant of the breaks of the synthetic cannabinoid(s) 4 2 1 7 6 5 8 9 10 66 (figure 32). Moreover, it is represented in the table the identification of the compounds by the swgdrug library. Table 14: CM21 herbal incense analysis of the volatile contained in the SPE cartridge: main peaks founded and possible combustion products. Peak number Retention time m/z of the unknown compound Synthetic cannabinoid(s) that possibly originate this compound 1 5,832 89, 117, 145, 169 MAM2201 N-(4-fluoropentyl) isomer or AM2201 2’-naphtyl isomer 2 6,873 55, 69, 83, 101, 111, 129, 143, 215 MAM2201 N-(4-fluoropentyl) isomer or JWH122 N-(4-fluoropentyl) isomer 3 7,588 55, 83, 99, 155,173, 217 AM2201 2’-naphthyl isomer 4 11,340 89, 113, 157, 255, 326 MAM2201 N-(4-fluoropentyl) isomer or AM2201 2’-naphtyl isomer 5 11,457 75, 89, 113 MAM2201 N-(4-fluoropentyl) isomer or AM2201 2’-naphtyl isomer 6 17,561 63, 115, 144, 169, 268, 355 MAM2201 N-(4-fluoropentyl) isomer or AM2201 2’-naphtyl isomer 7 18,665 127, 144, 232, 256, 284, 342 MAM2201 N-(4-fluoropentyl) isomer; AM2201 2’-naphtyl isomer or JWH-122 N-(4fluoropentyl) isomer 8 18,905 89, 115, 141, 169, 212, 254, 298, 353 MAM2201 N-(4-fluoropentyl) isomer or JWH122 N-(4-fluoropentyl) isomer 9 20,469 115, 141, 169, 232, 254, 298, 374 MAM2201 N-(4-fluoropentyl) isomer 10 24,451 115, 144, 248, 298, 372 AM2201 2’-naphtyl isomer 67 4.1.1.1 MAM2201 N-(4-fluoropentyl) isomer (SYNTHETIC CANNABINOID 1) Simulations of breaks in the molecule (theoretical approach) – Identification of combustion products Figure 32: Simulations of breaks in the molecule MAM2201 N-(4-fluoropentyl) isomer that could occur when the combustion process. Each number refers to a simulation of a break in the initial molecule of the synthetic cannabinoid MAM2201 N-(4-fluoropentyl) isomer (figure 32). In table 15 it is represented the molecular weight as a result of the referred break and the correspondent molecular formulation. 8 2 1 3 4 7 9 6 5 74 m/z (284.1) that could be a result of the break 5 of the synthetic cannabinoid. The chromatographic peaks 8, 9 and 10 correspond to a compound with a m/z ratio of 169.1, meaning that it could result from the break 6 of the synthetic cannabinoid. The peak 8 corresponds to a molecule with a m/z ratio of 141.1 that could be result of the break 7. Table 19: Possible resulting compounds of the combustion of the synthetic cannabinoid JWH122 N-(4-pentenyl) analog present in CM21 blends. Combustion products After an extensive analysis of the results obtained, molecules that could possibly be combustion products of the synthetic cannabinoid JWH-122 N-(4-pentenyl) analog are presented in the table 20. This result was obtained comparing the molecular weight of the resultant molecules obtained by the simulation of the breaks in the synthetic cannabinoid with the molecular weight of the identified compound in the resultant chromatogram of the combustion experiment. Table 20: Compounds resulting of the matching between the simulation of the breaks (theoretical approach) in the molecule of the synthetic cannabinoid JWH-122 N-(4-pentenyl) Peak number Retention time (minutes) Interesting peak Break (number) 2 6,873 55,2 4 4 11,032 55,2 4 6 17,561 284,1 5 7 18,665 284,1 5 8 18,905 169,1 6 8 18,905 141,1 7 9 20,448 169,1 6 10 24,451 169,1 6 75 analog and the compounds found in the chromatograms resulting of the combustion assay of the compound CM21. C20H14NO 3-(1-(4-methylnaphthalen-1yl)vinyl)-1-(pent-4-en-1yl)indoline C4H8 but-1-ene C12H10O 4-methyl-1-naphtaldehyde 76 4.1.1.3 AM2201 2’-naphthyl isomer (SYNTHETIC CANNABINOID 3) Simulations of breaks in the molecule (theoretical approach) – Identification of combustion products Figure 34: Simulations of breaks in the molecule AM2201 2’-naphthyl isomer that could occur when the combustion process. Each number refers to a simulation of a break in the initial molecule of the synthetic cannabinoid AM2201 2’-naphthyl isomer. In table 21 it is represented the 8 7 6 4 3 2 1 5 77 molecular weight as a result of the referred break and the correspondent molecular formulation. Table 21: Resulting compounds of the simulation of breaks in the molecule AM2201 2’- naphthyl isomer that could occur when the combustion process. Break number Resulting molecule (1) Molecular weight (1) Resulting molecule (2) Molecular weight (2) 1 C24H22NO● 340,17 F 19,00 2 C23H20NO● 326,15 CH2F● 33,01 3 C22H18NO● 312,14 C2H4 F● 47,03 4 C21H16NO● 299,13 C4H8F● 75,06 5 C3H6F● 61,05 C20H14NO● 284,11 6 C19H12NO● 270,09 C5H10F● 89,08 7 C13H15FN● 204,12 C11H7O● 155,05 8 C14H15FNO● 232,11 C10H7● 127,05 In each assay it was obtained a chromatogram with the peaks that allows the study of the combustion products of the synthetic cannabinoids presents in the drugs used in this experiment. The peaks represented are those corresponding to compounds whose compounds m/z ratio is coincident to the m/z ratio of some molecule resultant of the break of the initial molecule. In this way, the resultant molecule could possibly be a combustion product of the initial molecule, the synthetic cannabinoid. Thus, the peaks 1, 3, 4 and 5 of the chromatogram presented in the Figure 31 are referent to 78 compounds with a common m/z ratio (89.1) that can probably be a result of the break 6 of the synthetic cannabinoid (Figure 34). Also the peak 3 is correspondent to molecules having in their mass spectrum an interesting m/z (155.1) making that this compound could be a result of the break 7 of the synthetic cannabinoid. The peak 7 corresponds to a molecule that has a m/z ratio of 75.1, meaning that it could be a resulting compound of the breaks 5 of the synthetic cannabinoid. The peak 6 is referent to a molecule that has also a special interest because of their m/z ratio of 284.1 that could be a result of the breaks 5. The peaks 8 and 9 correspond to compounds that have m/z ratios 232.1 and 232.2 that might result of the break 8 of the initial molecule. Table 22: Possible resulting compounds of the combustion of the synthetic cannabinoid AM2201 2’-naphthyl isomer present in CM21 blends. Peak number Retention time (minutes) Interesting peak Break (number) 1 5,382 89,1 6 3 7,588 155,1 7 4 11,340 89,1 6 5 11,457 75,1 5 5 11,457 89,1 6 6 17,550 89,1 6 6 17,550 284,1 5 7 18,675 232,2 8 9 20,480 232,1 8 79 Combustion products After an extensive analysis of the results obtained molecules that could possibly be combustion products of the synthetic cannabinoid AM2201 2’-naphthyl isomer are presented in the table 23. This result was obtained comparing the molecular weight of the resultant molecules obtained by the simulation of the breaks in the synthetic cannabinoid with the molecular weight of the identified compound in the resultant chromatogram of the combustion experiment. Table 23: Compounds resulting of the matching between the simulation of the breaks (theoretical approach) in the molecule of the synthetic cannabinoid AM2201 2’-naphthyl isomer and the compounds found in the chromatograms resulting of the combustion assay of the compound CM21. C5H11F 1-fluoropentane C20H15NO (1-methyl-1H-indol-3yl)(naphthalene-2-yl)methanone C4H9F 1-fluorobutane C11H8O 2-naphthaldehyde C14H16FNO 1-(5-fluoropentyl)-1H-indole-3carbaldehyde 80 4.2 Herbal incense: SCORPION ULTRASTRONG Figure 35: Comparison between chromatograms resulting of the injection of SCORPION ULTRASTRONG incense before (1) and after (2) the combustion. The first chromatogram presented in figure 35 is related to the injection of 2 µL of the solution prepared - 5mg of the sample SCORPION ULTRASTRONG dissolved in 1 mL of methanol. This chromatogram serves as a reference for comparison with the chromatograms relative to the simulation of the combustion of these samples, giving rise to the appearance of combustion products of synthetic cannabinoids present in the herbal incense. Thus, the second chromatogram is related to the injection of 2 µL of the solution obtained after the extraction of the SPE cartridge with 2 mL of methanol and subsequent drying in a nitrogen flow, and resuspended in 100uL of methanol. Thus, it is possible to observe that after being subjected to heating, the sample undergoes several processes of transformation, leading to the formation of new products, as we will verify later, on a closer examination of the chromatogram in question. 81 4.2.1 Volatile contained in the SPE cartridge Figure 36: Chromatogram analysis of the injection of CM21 after the combustion and the main peaks found related to the combustion of the synthetic cannabinoids. In the chromatogram present in figure 36 it is possible to verify that, after the heating, there are a lot of new substances formed. The main peaks found in this chromatogram are: peak 1, with the retention time 6,873 minutes; peak 2, whose retention time is 7,469 minutes; peak 3, with retention time 18,910 minutes; peak 4, that appears at 20,512 minutes. Some of these peaks have an especial interest because of the coincidence of their m/z ratio with the m/z ratio of the molecules formed when the breaks in the synthetic cannabinoid are simulated. In the table 24, the main peaks are referred along with the synthetic cannabinoids precursors. The m/z ratios of the new found compounds were taken into account and were compared with the m/z ratio resultant of the breaks of the synthetic cannabinoid(s) (figure 37). Furthermore, it is also represented in the table the identification of the compounds by the swgdrug library. 1 2 3 4 82 Table 24: SCORPION ULTRASTRONG herbal incense analysis of the volatile contained in the SPE cartridge: main peaks founded and possible combustion products. Peak number Retention time m/z of the unknown compound Synthetic cannabinoid(s) that possibly originate this compound 1 6,873 55, 111, 129, 143, 184, 215, 239, 323 JWH-122 7-methylnaphthyl isomer 2 8,469 57, 91, 219, 341 JWH-122 7-methylnaphthyl isomer 3 18,910 115, 141, 169, 212, 241, 298 JWH-122 7-methylnaphthyl isomer 4 20,512 115, 169, 232, 254, 298, 374 JWH-122 7-methylnaphthyl isomer 83 4.2.1.1 JWH-122 7-methylnaphthyl isomer (SYNTHETIC CANNABINOID 4) Simulations of breaks in the molecule – Identification of combustion products – theoretical approach Figure 37: Simulations of breaks in the molecule JWH-122 7-methylnaphthyl isomer that could occur when the combustion process. Each number refers to a simulation of a break in the initial molecule of the synthetic cannabinoid JWH-122 7-methylnaphthyl isomer (figure 37). In the table 25 is 5 2 1 3 4 6 7 8 90 4.3.1.1 AM2201 (SYNTHETIC CANNABINOID 5) Simulations of breaks in the molecule (theoretical approach) – Identification of combustion products Figure 40: Simulations of breaks in the molecule AM2201 that could occur when the combustion process. Each number refers to a simulation of a break in the initial molecule of the synthetic cannabinoid AM2201. In the table 29 it is represented the molecular weight as a result of the referred break and the correspondent molecular formulation. 1 6 5 4 3 2 8 7 91 Table 29: Resulting compounds of the simulation of breaks in the molecule AM2201 2'- naphthyl isomer that could occur when the combustion process. In each assay it was obtained a chromatogram with the peaks that allows the study of the combustion products of the synthetic cannabinoids presents in the drugs used in this study. The peaks represented are those corresponding to compounds whose m/z ratio is coincident to the m/z ratio of some molecule resultant of the break of the initial molecule. In this way, the resultant molecule could possibly be a combustion product of the initial molecule, the synthetic cannabinoid. Thus, the peak 3 of the chromatogram presented in the Figure 39 is correspondent to a compound with a m/z ratio (155.1) that can probably be a result of the break 7 of the synthetic cannabinoid (Figure 40). In turn, the peak 6, 7 and 8 are correspondent to molecules having in their mass spectrum an interesting m/z (89.1) making that this compound could be a result of the break 6 of the synthetic cannabinoid. On the other hand, the peak 7 Break number Resulting molecule (1) Molecular weight (1) Resulting molecule (2) Molecular weight (2) 1 C24H22NO● 340,17 F 19,00 2 C23H20NO● 326,15 CH2F● 33,01 3 C22H18NO● 312,14 C2H4 F● 47,03 4 C21H16NO● 299,13 C3H6F● 61,05 5 C20H14NO● 284,11 C4H8F● 75,06 6 C19H12NO● 270,09 C5H10F● 89,08 7 C13H15FN● 204,12 C11H7O● 155,05 8 C14H15FNO● 232,11 C10H7● 127,05 92 corresponds to a compound that has a m/z ratio of 75.1, meaning that it could be a resulting compound of the break 5 of the synthetic cannabinoid. The peaks 9 and 11 are correspondent to molecules that also have a special interest because of their m/z ratio (232.1) that could be a result of the breaks 5. Table 30: Possible resulting compounds of the combustion of the synthetic cannabinoid AM2201 2'-naphthyl isomer present in SHIVA blends. Combustion products After an extensive analysis of the results obtained, molecules that could possibly be combustion products of the synthetic cannabinoid AM2201 are presented in the table 31. This result was obtained comparing the molecular weight of the resultant molecules obtained by the simulation of the breaks in the synthetic cannabinoid with the molecular weight of the identified compound in the resultant chromatogram of the combustion experiment. Peak number Retention time (minutes) Molecular weight Break (number) 3 7,591 155,1 7 6 11,338 89,1 6 7 11,455 89,1 6 7 11,455 75,1 5 8 17,555 89,1 6 9 18,670 232,1 8 11 20,463 232,1 8 93 Table 31: Compounds resulting of the matching between the simulation of the breaks (theoretical approach) in the molecule of the synthetic cannabinoid AM2201 2'-naphthyl isomer and the compounds found in the chromatograms resulting of the combustion assay of the compound SHIVA. C4H9F 1-fluorobutane C5H11F 1-fluoropentane C14H16FNO 1-(5-fluoropentyl)-1H-indole-3carbaldehyde C11H8O 2-naphthaldeyde C10H8 Naphthalene 94 4.3.1.2 MAM2201 (SYNTHETIC CANNABINOID 6) Simulations of breaks in the molecule (theoretical approach) – Identification of combustion products Figure 41: Simulations of breaks in the molecule MAM2201 that could occur when the combustion process. Each number refers to a simulation of a break in the initial molecule of the synthetic cannabinoid MAM2201 (figure 41). In the table 32 is represented the molecular weight as a result of the referred break and the correspondent molecular formulation. 8 2 1 3 4 7 9 6 5 95 Table 32: Resulting compounds of the simulation of breaks in the molecule MAM2201 N-(4fluoropentyl) isomer that could occur when the combustion process. Break number Resulting molecule (1) Molecular weight (1) Resulting molecule (2) Molecular weight (2) 1 C24H21FNO● 358,16 CH3 15,02 2 C25H24NO● 354,19 F● 19,00 3 C23H20NO● 326,15 C2H4F● 47,03 4 C22H18NO● 312,14 C3H6F● 61,05 5 C21H16NO● 298,12 C4H8F● 75,06 6 C20H14NO● 284,11 C5H10F● 89,08 7 C13H15FN● 204,12 C12H9O● 169,07 8 C12H15FN3● 192,12 C13H10O● 169,07 9 C24H21FNO● 358,16 CH3 15,02 In each assay it was obtained a chromatogram with the peaks that allows the study the combustion products of the synthetic cannabinoids presents in the drugs used in this study. The peaks represented are those corresponding to compounds whose m/z ratio is coincident to the m/z ratio of some molecule resultant of the break of the initial molecule. In this way, the resultant molecule could possibly be a combustion product of the initial molecule, the synthetic cannabinoid. Thus, the peaks 6, 7 and 8 of the chromatogram presented in the Figure 39 have a chromatographic peak corresponding to compounds having a m/z ratio (89.1) that could probably be a result of the break 6 of the synthetic cannabinoid (Figure 41). In turn, the peak 7 correspond to a molecule having in their mass spectrum a m/z of 75.1, making that this compound could be a result of the break 5 of the synthetic cannabinoid. Finally, the peaks 10, 11 96 and 12 are correspondent to a compound with a common m/z ration of 169.1 meaning that could be a result of the break 8. Table 33: Possible resulting compounds of the combustion of the synthetic cannabinoid MAM2201 N-(4-fluoropentyl) isomer present in SHIVA blends. Combustion products After an extensive analysis of the results obtained, molecules that could possibly be combustion products of the synthetic cannabinoid MAM2201 are presented in the table 34. This result was obtained comparing the molecular weight of the resultant molecules obtained by the simulation of the breaks in the synthetic cannabinoid with the molecular weight of the identified compound in the resultant chromatogram of the combustion experiment. Peak number Retention time (minutes) Molecular weight Break (number) 6 11,338 89,1 6 7 11,455 75,1 5 7 11,455 89,1 6 8 17,555 89,1 6 10 18,892 169,1 8 11 20,463 169,1 8 12 24,445 169,1 8 97 Table 34: Compounds resulting of the matching between the simulation of the breaks (theoretical approach) in the molecule of the synthetic cannabinoid MAM2201 and the compounds found in the chromatograms resulting of the combustion assay of the compound SHIVA. C4H9F 2-fluorobutane C5H11F 2-fluoropentane 98 4.3.1.3 JWH-122 N-(4-pentenyl) analog (SYNTHETIC CANNABINOID 2) Simulations of breaks in the molecule (theoretical approach) – Identification of combustion products Figure 42: Simulations of breaks in the molecule JWH-122 N-(4-pentenyl) analog that could occur when the combustion process. Each number refers to a simulation of a break in the initial molecule of the synthetic cannabinoid JWH-122 N-(4-pentenyl) analog. In the table 35 is represented 3 6 7 4 1 1 1 1 1 5 8 1 2 99 the molecular weight as a result of the referred break and the correspondent molecular formulation. Table 35: Resulting compounds of the simulation of breaks in the molecule JWH-122 N-(4pentenyl) analog that could occur when the combustion process. Break number Resulting molecule (1) Molecular weight (1) Resulting molecule (2) Molecular weight (2) 1 C24H23NO● 341,18 CH2 14,02 2 C23H20NO● 326,15 C2H3● 27,02 3 C22H18NO● 312,14 C3H5● 41,04 4 C21H16NO● 298,12 C4H7● 55,05 5 C20H14NO● 284,11 C5H9● 69,07 6 C13H14N● 184,11 C12H9O● 169,07 7 C14H14NO● 212,11 C11H9● 141,07 8 C24H20NO● 338,15 CH3 15,02 In each assay it was obtained a chromatogram with the peaks that allows the study of the combustion products of the synthetic cannabinoids presents in the drugs used in this experiment. The peaks represented are those corresponding to compounds whose m/z ratio is coincident to the m/z ratio of some molecule resultant of the break of the initial molecule. In this way, the resultant molecule could possibly be a combustion product of the initial molecule, the synthetic cannabinoid. Thus, the chromatographic peaks 1, 2 and 3 of the chromatogram presented in the Figure 39 are correspondent to a molecule with a m/z ratio (55.1) that can probably be a result of the break 4 of the 106 107 CHAPTER III: DISCUSSION 108 109 In order to mimic what happens when consumers smoke these drugs, and the effects of high temperatures, several techniques were tested aiming the identification of compounds produced during the combustion process of the synthetic cannabinoids. Initially, vials containing the investigation samples were put inside a muffle furnace, however, they could not be screwed because they have a plastic lid, which melted during the heating. In this way, the new volatile compounds formed were lost during its formation. As result, only ashes obtained were analyzed showing a decrease of the synthetic cannabinoids concentration dependent on the temperature. Later, due to the fail of the experiment described to get volatiles, another method (in a muffle apparatus – Chapter 1 – section 2.1) was tested using the glass screwed ampoules. This method enables the retention of the new compounds formed inside the ampoule. However, due to the fact that the glass is highly sensitive to high temperatures, in the majority of the cases, the ampoule melted (above 400ºC), and volatiles were lost. This technique could work if the glass material were more resistant to high temperatures. Anyway, results were analyzed showing also a decrease of the synthetic cannabinoids parallel with the increase of the temperature. Both techniques described below were tested in a similar way, heating the samples until 600ºC. Based in internet reports, the synthetic cannabinoids combustion temperature varies from 450ºC to 550ºC. Since no studies in this topic are available in scientific literature, this experiment was based in the mentioned information. After that, a new technique was developed aiming to get volatiles, as described in the Chapter 1 – section 2.2. For this purpose an apparatus was developed in order to retain in a SPE column volatile compounds formed during the combustion process. The work was divided in two steps, first the identification of the cannabinoids present in each sample, second, after submitted by two different methods samples to high temperatures, samples were dissolved in methanol and analyzed by GC-MS. Chromatographic peaks obtained were identified by using mass spectra libraries for synthetic cannabinoids (swgdrug library). For combustion products each 110 mass spectrum was compared to m/z obtained by theoretical fragmentation of the initial cannabinoid. According to the results previously presented we are able to identify seven different synthetic cannabinoids in the three commercial products bought in an on-line shop: - In the herbal incense CM21 were found the follow synthetic cannabinoids: MAM2201 N-(-4-fluoropentyl) isomer, JWH-122 N-(4-pentenyl) analog and AM2201 2’-naphthyl isomer; - In the sample SCORPION ULTRASTRONG the synthetic cannabinoid found was JWH-122 7-methylnaphthyl; - In the herbal incense SHIVA ULTRASTRONG were founded the next synthetic cannabinoids: JWH-122 N-(4-pentenyl) analog (also present in the sample CM21), AM2201, MAM2201 and MAM2201 N-(-5-chloropentyl) analog. To the accomplishment of the objective of this experiment, identify the combustion products of the synthetic cannabinoids present, theoretical breaks were simulated in each cannabinoids. Subsequently, based on the chromatograms obtained from the injection of the samples obtained from the extraction of the SPE column after the combustion methodology, the peaks of interest were selected and the ions (m/z) of these chromatographic peaks were analyzed. Thereafter, it was performed a comparison between the peaks referred and the ratio mass/charge obtained by the simulation of the breaks in the initial compound leading to the conclusion that these molecules could possibly be the result from the heating of the starting compound, namely the products of combustion thereof. Thus, fulfilled the described above, according to the results obtained, concerning to the blend CM21, the molecules C4H9F (2-fluorobutane), C5H11F (2fluoropentane), C20H15NO (1h-inden-2-yl)(4-methylnaphthalen-1-yl)methano, C12H10O (4-methyl-1-naphtaldehyde), C14H16FNO (1-(4-fluoropentyl)-3-methyl-1H-indole) and 111 C11H10 (1-methylnaphthalene) can be combustion products of the synthetic cannabinoid MAM2201 N-(4-fluoropentyl) isomer, resulting of the breaks 5, 6, 6, 7, 8 and 8, respectively, in the original molecule. In respect to the cannabinoid JWH-122 N-(4-pentenyl) analog, also concerning to the blend CM21, the molecules C20H14NO (3-(1-(4-methylnaphthalen-1-yl)vinyl)-1- (pent-4-en-1-yl)indoline), C4H8 (but-1-ene) and C12H10O (4-methyl-1-naphtaldehyde), can also be combustion products, resultant of the breaks 5, 4 ad 6. Still referring to CM21, the cannabinoid AM2201 2’naphthyl isomer can have as combustion products C5H11F, C20H15NO, C4H9F, C11H8O, C14H16FNO and C19H20FNO, ensuing of the breaks 6, 5, 4, 7 and 8. In relation to herbal incense SCORPION, the molecules C4H10 (Butane), C12H10O (1-pentyl-1H-indole) and C11H10 (2-methylnaphtalene), can be combustion products of the synthetic cannabinoid JWH-122 7-methylnaphtyl isomer, resultant of the breaks 2, 6 and 7, respectively. Relating to the blend SHIVA, the molecules C4H9F (2-fluorobutane) and C5H11F (2-fluoropentane) may be combustion products of the synthetic cannabinoid MAM2201 N-(4-fluoropentyl) isomer, resulting of the breaks 5 and 6, correspondingly. Concerning to the cannabinoid JWH-122 N-(4-pentenyl) analog, the molecules C12H10O (4-methyl-1-naphtalenecarboxaldehyde), C4H8 (1-butane) and C14H15NO (1-(pent-4-en1-yl)-1H-indole-3-carbaldehyde) can also be combustion products, resultant of the breaks 6, 4 and 7. Still referring to SHIVA, the cannabinoid AM2201 can have as combustion products the C4H9F (2-fluorobutane), C5H11F (2-fluoropentane), C14H16FNO (1-(5-fluoropentyl)-1H-indole-3-carbaldehyde), C11H7O (2-naphthaldehyde) and C10H8 (naphthalene) resultant of the breaks 5, 6, 8, 7 and 8. Finally, the synthetic cannabinoid MAM2201 N-(-5-chloropentyl) analog has as combustion products C4H9Cl (1-chlorobutane), C12H10O (4-methyl-1-naphtalenecarboxaldehyde) and C11H10 2methylnaphtalene) resulting of the breaks 5, 7 and 8 of the initial molecule. Most of the molecules characterized as combustion products of the synthetic cannabinoids are not well-known or little studied. In this way, the effects of each one 112 in the human organism is difficult to predict. Nevertheless, some toxicological effects were related in relation to some known molecules as shown in the table 41. Table 41: Effects and toxicity of the resulting compounds from the combustion of synthetic cannabinoids. Compound Effects Toxicity 2-chlorobutane Hazardous in case of skin or eye contact, ingestion and inhalation. No references to any toxicological development. 2-fluoropentane Specific therapeutic activities of chemical compounds and medical preparations. No references. 4-methyl-1naphtaldehyde Skin and mucous membranes irritant and has an irritant effect to the eyes. No references. 1-methylnaphtalene May cause skin and eye irritation, photosensitization Inhalation of the vapors may produce airway irritation, headache, nausea, vomiting, extensive sweating, weakness and collapse. Dysuria, hematuria and the acute hemolytic reaction were also related. Ingestion may lead to systemic poisoning involving the gastrointestinal tract, kidneys and blood-forming tissue. But-1-ene may It is central nervous system depressant in high concentration. May cause asphyxia. On direct eye and skin contact can cause burns and frostbite. It has a low acute toxicity. Butane In low concentrations has not been reported to cause adverse effects in humans. It is anesthetic to both humans and experimental animals. Unexpected death or critical effects may occur when inhaled at high concentrations. Effects on the central nervous system in chronically exposed individuals were related. 1-pentyl-1h-indole Is used for specific therapeutic activity of chemical compounds or medicinal preparations as anti-infective, i.e. antibiotic, antiseptic and chemotherapeutic. No references. 113 2-methylnaphtalene Inhalation exposure can cause cough. It is skin, eyes, mucous membranes and upper respiratory tract irritating. May cause headaches, nausea, vomiting, diarrhea, anemia, jaundice, euphoria, dermatitis and visual disturbances. Convulsions and comatose may occur. 2-fluorobutane May be harmful if inhaled causing respiratory tract irritation and if swallowed. Causes skin and eye irritation. No references. 2-naphthaldehyde May cause eye irritation and may be harmful if absorbed through skin. Is also harmful if inhaled, causing lungs and respiratory tract irritation, and if swallowed. Symptoms of overexposure may be headache, dizziness, tiredness, nausea and vomiting. Higher exposure causes unconsciousness. Severe overexposure can result in injury or death. 1-chlorobutane Causes irritation to the respiratory tract. May cause coughing, shortness of breath. High concentrations have a narcotic effect. Also causes irritation to the gastrointestinal tract, may include symptoms as nausea, vomiting and diarrhea. Causes irritation to skin and eye. May causes systemic poisoning. Similar compounds demonstrate toxic effects after repeated exposures too low to result in acute effects. Investigated as carcinogenic and mutagenic. Naphtalene Exposure by inhalation, ingestion, and dermal contact is associated with hemolytic anemia, damage to the liver and lungs, and neurological damage. Symptoms of acute exposure include headache, nausea, vomiting, diarrhea, malaise, confusion, anemia, jaundice, convulsions, and coma. May cause laryngeal carcinomas or neoplasms of the pylorus and cecum. An increased number of alveolar/bronchiolar adenomas and carcinomas were reported by inhalation exposure. 114 The toxicological properties of the majority of the products referred have not been fully determined. Most of them do not present mutagenic, carcinogenic or teratogenic effects, reproductive toxicity or developmental toxicity, only presenting irritation when exposed, as the case of 2-chlorobutane and 4-methyl-1naphthaldehyde. However, a few of them, as the case of naphthalene and 1chlorobutane seem to be highly prejudicial to human health due to their carcinogenic and mutagenic properties. In turn, compounds as 2-fluoropentane and 1-pentyl-1Hindole are used for medicinal purposes, may provide a good sensation to their users, for example, functioning as analgesics. Compounds such as 2-fluorobutane, 1methylnaphtalene, butane, 2-methylnaphthalene and 2-naphthaldehyde with known toxicity, consumption in high doses can lead to the death or several problems as injury and affects the central nervous system. The compounds C20H14NO (3-(1-(4-methylnaphthalen-1-yl)vinyl)-1-(pent-4-en-1yl)indoline), C20H15NO (1h-inden-2-yl)(4-methylnaphthalen-1-yl)methano, C14H16FNO (1- (4-fluoropentyl)-3-methyl-1H-indole), C14H15NO (1-(pent-4-en-1-yl)-1H-indole-3carbaldehyde), C14H16FNO (1-(5-fluoropentyl)-1H-indole-3-carbaldehyde), C12H10O (4methyl-1-naphtalenecarboxaldehyde), also identified as combustion products, are not described in literature, therefore toxicological information cannot be present. Although the knowledge of toxicological effects of some of the compounds described, the real danger or toxicity cannot be proved because the lethal quantities are not known and a quantitative analysis was not realized, thus, the concentration of new substances formed during the combustion process is not elucidated. For the accomplishment of this procedure, patterns should be used. In this experiment, the identification of the compounds is based in a theoretical simulation. For better results, the detected substances should be compared with patterns instead of mass spectra. However, this analysis was not possible because patterns of the synthetic cannabinoids in study were not available. 115 CHAPTER IV: CONCLUSIONS AND FUTURE PERSPECTIVES 122 Kapka-Skrzypczak, L., et al. (2011). "Legal highs - legal aspects and legislative solutions." Ann Agric Environ Med 18(2): 304-309. Kennedy, P. C. W. 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