Drug Abuse Patterns in Coimbra Recreational Nightlife

Drug abuse patterns in Coimbra recreational nightlife Renata Isabel Braga Fernandes Rocha Lima Dissertação de Mestrado em Medicina Legal 2013 III Renata Isabel Braga Fernandes Rocha Lima Drug abuse patterns in Coimbra recreational nightlife Dissertação de Candidatura ao grau de Mestre em Medicina Legal submetida ao Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto. Orientador – Doutor Félix Dias Carvalho Categoria – Professor catedrático Afiliação – REQUIMTE, Laboratório de Toxicologia, Departamento de Ciências Biológicas, Faculdade de Farmácia da Universidade do Porto. Co-orientador – Doutor Fernando Mendes Categoria – Psicólogo AfiliaçãoIREFREA, Portugal IV V ACKNOWLEDGMENTS Firstly I would like to express my deepest gratitude to my supervisor, Professor Doctor Félix Dias Carvalho for accepting to orient this thesis. Thanks also for the availability, readiness and for the contribution to my training and the provision of all necessary means to carry out this work. I also express my gratitude and appreciation to Doctor Fernando Mendes for all the support and collaboration given during the elaboration of this thesis. I also thank to Professor Doctor Paula Guedes for the availability, sympathy and for the transmitted knowledge through the Master Degree. I would like to thank to Professor Doctor Irma Brito whose support was relentless, especially during data collection. I would also like to thank for the revision of all work. I thank to Professor Doctor Maria José Pinto da Costa, Coordinator of the Master Degree of Legal Medicine. I thank to all students of Laboratory of Toxicology, Department of Biological Sciences of Faculty of Pharmacy of Porto, specially Master Márcia Monteiro for all the support during the realization of this work, encouragement, friendship and care. A special thanks to my family, mainly my parents who participated in the study, for the extraordinary kindness, patience and dedication. VI VII ABSTRACT Background: The use of psychoactive substances among young people, in recreational spaces, has been a subject of growing concern. The uncontrolled consumption of these substances, associated to risk behaviors, morbility and morbidity, corresponds to a heavy burden for the modern society. Objective: The main objective of this study was to analyze the drug abuse patterns in Coimbra recreational nightlife. Methods: The study was applied in the form of the questionnaire and the collection of biological samples from volunteers gathered at nighttime recreational spaces in Coimbra. Each blood and oral fluid sample was qualitatively and quantitatively analyzed according to each psychoactive substance reported to be used by the interviewed volunteer. Ethanol was also evaluated in exhaled air and oral fluid. A GC-MS methodology was developed for the measurement of Δ9-tetrahydrocannabinol (THC), and standard methods were used for the other psychoactive substances analyzed. The data were gathered by a nonrandomized process of accidental sampling, and quantitative analyzes using the statistics tests of Mann-Whitney, Kruskal-Wallis, Pearson Chi-Square, Odd’s Ratio. Results: 78 young adults, between 18 to 30 years, participated in the study. 26,92% were females and 73,08% males. The majority of them were students (75,64%). Alcohol was the psychoactive substance more used (87,2%), followed by tobacco (64,1%), cannabis (19,2%), cocaine and amphetamines (2,6%) and finally LSD and smartshop substances (1,3%). Males used more psychoactive substances than females, both in quantity and in diversity. In relation to risk behaviors or health problems, the cocaine users had the highest risk of both types of problems, following cannabis, alcohol and finally tobacco. Relatively to biological samples, the ethanol and Δ9-THC were the compounds quantitatively analyzed in this study due to their high frequency. Ethanol was the substance that had more discordance, as compared to the surveys. The results of Δ9THC had more concordance with answers of the surveys. Conclusion: Alcohol, tobacco and cannabis were the psychoactive substances more used in recreational spaces. Alcohol is the major substance used among young people, usually in an excessive way. Exhaled air reveal to be the best method for analyzing ethanol. The Δ9-THC method validated in the present study, showed to be selective, linear, efficient, and precise. Keywords: psychoactive substances, alcohol, THC, recreational nightlife. VIII RESUMO Introdução: O consumo de substâncias psicoativas entre jovens, em espaços de lazer, tem sido um tema de crescente preocupação. O consumo incontrolado destas substâncias, associadas a comportamentos de risco, morbilidade e morbidade constituem uma tarefa árdua para a sociedade moderna. Objetivo: O objetivo principal deste estudo foi analisar os padrões de consumo recreativo, de substâncias psicoativas, na cidade de Coimbra. Métodos: O estudo foi efetuado sob a forma de um questionário acompanhado de uma amostra biológica, colhida a jovens voluntários que se encontravam em espaços recreativos noturnos, na cidade de Coimbra. Cada amostra de sangue e fluido oral foi qualitativamente e quantitativamente analisada de acordo com o tipo de substância psicoativa consumida, reportada pelo participante. O etanol foi também colhido sob a forma de ar exalado e fluido oral. Para analisar Δ9-tetrahydrocannabinol desenvolveu-se uma metodologia analítica, enquanto que as restantes substâncias foram analisadas segundo metodologias padrão. A recolha de dados foi efetuada segundo um processo não aleatório de amostra acidental, e a sua análise foi realizada quantitativamente recorrendo a testes estatísticos de Mann-Whitney, Kruskal-Wallis, Pearson Chi-Square e Odd’s Ratio. Resultados: 78 jovens adultos, entre 18 e 30 anos, participaram neste estudo. 26,92% eram do género feminino e 73,08% do género masculino. A maioria dos participantes eram estudantes (75,64%). O álcool foi a substância psicoativa mais consumida (87,2%), seguindo o tabaco (64,1%), canábis (19,2%), cocaína e anfetaminas (2,6%) e finalmente LSD e substâncias vendidas em smartshop (1,3%). Os participantes do género masculino consomem mais substâncias psicoativas do que os do género feminino, tanto em quantidade como em diversidade. Em relação aos riscos comportamentais ou de saúde, os consumidores de cocaína apresentavam maior risco de possuírem ambos os problemas, seguindo os consumidores de cannabis, álcool e por último tabaco. Relativamente às amostras biológicas, o etanol e o Δ9-THC foram as substâncias analisadas quantitativamente neste estudo, devido á sua maior frequência. O etanol foi a substância que teve maior discordância, quando comparado com as respostas dos questionário, enquanto que os resultados do Δ9-THC mostraram-se mais concordantes. Conclusão: Álcool, tabaco e cannabis são as substâncias mais consumidas em espaços recreativos noturnos. O álcool é a substancia mais consumida pelos jovens de uma forma exacerbada. A análise ao álcool por ar exalado foi aquele que obteve resultados mais IX fidedignos. O método de Δ9-THC validado neste estudo mostrou ser seletivo, linear, eficiente e preciso. Palavras-chave: Substâncias psicoativas, álcool, THC, ambiente recreativo noturno XVI XVII INDEX ACKNOWLEDGMENTS ................................................................................................... V ABSTRACT .................................................................................................................... VII RESUMO…………………………………………………………………………………………VIII ABBREVIATION LIST ...................................................................................................... XI INDEX OF FIGURES ..................................................................................................... XIII INDEX OF TABLES ........................................................................................................ XV INDEX .......................................................................................................................... XVII Part I: Introduction ............................................................................................................ 1 1. Drug use among young people..................................................................................... 3 2. Why do some people use, abuse, and become dependent on drugs? .......................... 5 2.1 The nature and the special properties of psychoactive substances .......................... 6 2.2 The outcomes of drug use ....................................................................................... 6 2.3 The dynamics of use behavior ................................................................................. 7 2.4 The individual differences in vulnerabilities .............................................................. 7 3. Predisposing factors for drug use ................................................................................. 8 3.1 Curiosity .................................................................................................................. 8 3.2 Peer group influence ................................................................................................ 8 3.3 Ordalic behaviour ..................................................................................................... 9 3.4 Hedonism ................................................................................................................ 9 4. Brain Reward System ..................................................................................................10 4.1 Neuroanatomy of brain reward system ...................................................................10 4.2 Molecular physiology of brain reward system ..........................................................11 4.3 Action of drug abuse in brain reward system ..........................................................13 4.3.1 Alcohol .............................................................................................................13 4.3.2 Opiates ............................................................................................................13 4.3.3 Cannabis .........................................................................................................14 4.3.4 Cocaine ...........................................................................................................15 XVIII 5. From pleasure to drug addiction ..................................................................................16 6. Tolerance and sensitization in drug of abuse...............................................................17 6.1 Alcohol ....................................................................................................................19 6.2 Opiates ...................................................................................................................19 6.3 Cannabis ................................................................................................................19 6.4 Cocaine ..................................................................................................................19 7. Addiction and physical dependence in drug of abuse ..................................................20 7.1 Alcohol ....................................................................................................................20 7.2 Opiates ...................................................................................................................21 7.3 Cannabis ................................................................................................................22 7.4 Cocaine ..................................................................................................................22 8. Withdrawal syndrome in drug of abuse........................................................................22 8.1 Alcohol ....................................................................................................................23 8.2 Opiates ...................................................................................................................24 8.3 Cannabis ................................................................................................................25 8.4 Cocaine ..................................................................................................................25 9. Toxic reactions and overdose in drug of abuse ...........................................................26 9.1 Alcohol ....................................................................................................................27 9.2 Opiates ...................................................................................................................27 9.3 Cannabis ................................................................................................................28 9.4 Cocaine ..................................................................................................................29 Part II: General and specific objectives of the thesis ........................................................31 1. Objectives of the thesis ...............................................................................................33 Part III: Experimental Part ................................................................................................35 Chapter I: Materials and Methods Surveys ......................................................................37 1. Surveys .......................................................................................................................39 1.1 Characterization of the study .................................................................................39 1.2 Study population .....................................................................................................39 1.3 Study sample………………………………………………………………………………40 XIX 1.4 Legal and administrative procedures ......................................................................39 1.5 Study variables .......................................................................................................39 1.6 Collection of surveys ...............................................................................................40 1.7 Statistic treatment ...................................................................................................40 Chapter I I: Materials and Methods Identification and quantification of psychoactive drugs in biological samples ........................................................................................................43 1. Identification and quantification of psychoactive drugs in biological samples ...............45 1.1 Ethics statement .....................................................................................................45 1.2 Reagents and standards .........................................................................................45 1.3 Biological specimens ..............................................................................................45 1.4 Analysis of ethanol in blood and oral fluid samples .................................................46 1.4.1 Preparation of stock solution ............................................................................46 1.4.2 Preparation of calibration standard solution .....................................................46 1.4.3 Preparation of internal standard .......................................................................46 1.4.4 Sample preparation for gas-chromatography flame ionization detector ............46 1.4.5 Gas-chromatography flame ionization detector conditions ...............................47 1.5 Analysis of 9-THC in blood and oral fluid samples .................................................48 1.5.1 Preparation of stock solution ............................................................................48 1.5.2 Preparation of working solutions ......................................................................48 1.5.3 Preparation of calibration standard solution .....................................................48 1.5.4 Preparation of internal standard .......................................................................48 1.5.5 Sample preparation for gas-chromatography mass spectrometry ....................48 1.5.6 Gas-chromatography mass spectrometry conditions ........................................49 1.6 Method validation of 9-THC ...................................................................................50 1.6.1 Selectivity ........................................................................................................50 1.6.2 Linearity ...........................................................................................................50 1.6.3 Limit of detection and lower limit of quantification ............................................50 1.6.4 Precision ..........................................................................................................51 1.6.5 Accuracy ..........................................................................................................51 XX 1.6.6 Recovery .........................................................................................................51 1.6.7 Reproducibility .................................................................................................51 1.7 Analysis of amphetamines ......................................................................................52 1.7.1 Sample preparation for gas-chromatography mass spectrometry .......................52 1.8 Analysis of cocaine .................................................................................................52 1.8.1 Sample preparation ..........................................................................................52 Chapter III Results Surveys .............................................................................................53 1. Surveys .......................................................................................................................55 1.1 Socio-demographic characteristics .........................................................................55 1.2 Recreational nightlife habits ....................................................................................59 1.3 Health information ...................................................................................................60 1.4 Problems due to use of psychoactive substances ...................................................60 1.5 Drug abuse patterns ...............................................................................................62 1.6 Drug abuse patterns and socio-demographic characteristics ..................................64 1.7 Drug abuse patterns and recreational nightlife habits .............................................68 1.8 Drug abuse patterns and health information ...........................................................69 1.9 Drug abuse patterns and problems due to use of psychoactive substances ...........70 Chapter IV: Implementation of methodologies for the identification and quantification of psychoactive substances in biological samples ................................................................73 1. Implementation of methodologies for the identification and quantification of psychoactive substances in biological samples ................................................................75 1.1 Analysis of ethanol ..................................................................................................75 1.1.1 Pre-treatment of samples .................................................................................75 1.1.2 Detection by gas-chromatography flame ionization detector ............................75 1.1.3 Selectivity of the method ..................................................................................76 1.1.4 Calibration curve ..............................................................................................77 2.1 Analysis of 9-THC .................................................................................................78 2.1.1 Sample preparation for gas-chromatography mass spectrometry ....................78 2.2 Method Validation of 9-THC ..................................................................................80 XXI 2.2.1 Detection by gas-chromatography mass spectrometry .....................................80 2.2.2 Selectivity ........................................................................................................81 2.2.3 Linearity ...........................................................................................................82 2.2.4 Limit of detection and lower limit of quantification ............................................83 2.2.5 Precision ..........................................................................................................83 2.2.6 Accuracy ..........................................................................................................83 2.2.7 Recovery .........................................................................................................83 2.2.8 Reproducibility .................................................................................................83 2.3 Analysis of amphetamines ......................................................................................84 2.4 Analysis of cocaine .................................................................................................84 Chapter V: Application of the GC-MS and GC-FID methods to blood and oral fluid samples collected from the volunteers participating in this study .....................................85 1. Application of GC-FID for detect ethanol in biological samples ....................................87 1.1 Differences between blood, oral fluid and exhaled air samples ...............................89 1.2 Levels of ethanol in biological samples and its effects ............................................90 2. Application of GC-MS for detecting 9-THC in biological samples ..............................91 2.1 9-THC levels in blood samples and its effects ......................................................93 Chapter VI: Comparison of participants answers with the identification of psychoactive substances in collected biological samples ......................................................................95 1. Alcohol analyzed in biological samples vs answers about use of alcohol ...................97 2. THC analyzed in biological samples vs answers about use of cannabis ................... 100 3. Amphetamines analyzed in biological samples vs answers about use of amphetamines…………………………………………………………………………………..101 4. Cocaine analyzed in biological samples vs answers about use of cocaine ................ 101 Part IV: Discussion ........................................................................................................ 103 1. Discussion ................................................................................................................. 105 Part V: Conclusion and future perspectives ................................................................... 109 1. Conclusion ................................................................................................................. 111 2. Future Perspectives .................................................................................................. 113 Part VI: References ....................................................................................................... 115 XXII PART I: Introduction 2 3 1. Drug use among young people In the last few years, most of the consumption of alcohol and illicit drugs among young people in Europe, occurs in night-time recreational activities, typically with friends, either in formal recreational settings such as pubs, bars and nightclubs, or in informal settings such as on the street and at home. Much of party-goers young people’s drug use occurs on weekends and during holiday periods (Calafat et al., 2012). A wide range of diverse studies have shown that the levels of drug use among young people are higher in adolescents frequenting night clubs than among young people in the general population (Figure 1) (EMCDDA, 2010).The annual European Report (EMCDDA) shows that high levels of cocaine use occurs in regular attendees in clubs and other recreational settings. In 2010, 24% of visitors to pubs in Amsterdam were cocaine users. In Czech Republic, more than 1000 respondents to a online questionnaire, 29% reported having used cocaine in the last 12 months (Figure 1) (EMCDDA, 2012). Cannabis is another drug very common among adolescents. An estimated 15.4 million young Europeans between 15 to 34 years (11.7% of this age group) used cannabis in the last year, together with alcohol, in nightlife (EMCDDA, 2013). Figure 1. Proportion of 16 to 24 year olds reporting use of the most prevalent drugs in the last year, by frequency of nightclub visits (adapted from EMCDDA, 2012) 10 happiness, especially the use of hard drugs. People who have never tried drugs are half a point happier than their compatriots who have used them (Figure 3) (Veenhoven, 2003). Figure 3. Experience with drugs and happiness among young people (adapted from Veenhoven, 2003). 4. Brain Reward System 4.1 Neuroanatomy of brain reward system In 1953, the scientists Olds and Milner discovered that rats learned to return to the portions of their environment where they had been given direct electrical stimulation of the septal area of the brain. This stimulation was rewarding and the scientists confirmed that they could train rats to lever-press, by making short pulse trains of septal brain stimulation contingent upon this arbitrary response. Following studies have shown that brain stimulation reward establishes and maintains response habits in patterns very analogous to those established and maintained by natural rewards such as water, food and sexual activity (Wise, 1996). The reason for studying the laboratory reward of brain stimulation is to understand the mechanisms of natural rewards and drug rewards. Such as natural rewards, the drugs of abuse can also influence the brain stimulation and activate directly the reward circuits. This fact helps us to understand better the anatomy and neurochemistry of endogenous reward substrates (Wise, 1980). The core structure of the brain reward pathway is constituted be two dopamine pathways particularly important for the reward system: the mesocortical and the 11 mesolimbic. The mesocortical dopamine pathway projects to multiple cortical areas and it is important for many aspects of reward-processing, including hedonic evaluation, comparative valuation, and option-assessment. This pathway projects primarily to prefrontal, cingulate, and entorhinal cortices in rodents, but to the entire cortical mantle in primates (Taber et al., 2012). The mesolimbic dopamine pathway projects primarily from ventral tegmental area (VTA -via the medial forebrain bundle) to nucleus accumbens (NA) and ventral striatum, secondly projects to other limbic areas such as amygdala, olfactory tubercle, septum. This pathway is important for the positive reinforcing effects of both natural rewards and drugs of abuse. It is responsible to modulate the activity of the ventral striatum, a brain region thought to be involved in converting emotion into motivated action and movement (Figure 4) (Koob, 1992). Figure 4. Neuroanatomy of the Brain Reward System (Kibiuk, 2012). 4.2 Molecular physiology of brain reward system Neuronal communications in the brain occurs through an electrochemical process, with electrical impulses in a neuron modulating the release of neurotransmitters, such as dopamine, serotonin, endogenous opiates, N -methyl-Daspartate (NMDA), gammaaminobutyric acid (GABA), and acetylcholine. These neurotransmitters diffusing across small spaces (called synapse) between adjacent neurons, and binding with proteins (called receptors) on the membranes of the adjacent neurons to modulate electrical signals and other activities. Then, neurotransmitters are deactivated through metabolism or reabsorbed by neurons for reuse (Figure 5) (Kelly et al., 2009). 12 Figure 5. Neurotransmitters mediate communication between adjacent neurons (Foundation for Alternative and Integrative Medicine, 2013). Psychoactive drugs capitalize on this system, modulating action at the receptor level and altering the manner in which neurons regulate neurotransmitters. When drugs act on this system effects the hormonal action and then other capacities stay affected such as memory, mood, reward, learning and behavior (Kelly et al., 2009). Although drugs of abuse act through separate mechanisms and on various locations in the brain reward system, all of them increase dopamine levels. Dopamine is a main neurotransmitter of the reward pathway, however there are more neurotransmitters such as: serotonin, endogenous opiates and GABA that are closely involved in the brain reward pathway and also modulate dopamine levels (Esch and Stefano, 2004). Psychoactive drugs could act in the central nervous system as agonists or as antagonists at the receptors for endogenous chemical messengers. In general, most drugs of abuse act as agonist and increase dopamine neurotransmitter levels in the reward pathway. For example, cocaine and amphetamines blocks reuptake of dopamine and stimulate of release (Wise, 1998). Serotonin neurotransmitter is also affected with the use of drugs. This neurotransmitter is mainly involved in the modulation of motivational factors and it appears to regulate dopamine release at the nucleus accumbens. The consumption of alcohol increases the synaptic availability of serotonin with precursor loading, blockade of serotonin reuptake, or blockade of certain serotonin receptor subtypes (Koob et al., 1999). GABA is an inhibitory neurotransmitter and has long been implicated in the modulation of dopaminergic reward systems, playing a role in the mediation of effects of many drugs of abuse such as: alcohol, benzodiazepines and barbiturates. These drugs inhibit the release of GABA and release more dopamine in the reward system (Wallner et al., 2006). Further neurotransmitters are also affected with the 13 presence of drugs such as: opiates act at receptors for endogenous opioid neurotransmitters; nicotine acts at a subclass nicotinic of acetylcholine receptors; cannabis acts at receptors an endogenous cannabanoid; phencyclidine acts at the Nmethyl-D-aspartate subtype of glutamate receptor and caffeine acts at adenosine receptors. (Koob et al.,1999). 4.3 Action of drug abuse in brain reward system 4.3.1 Alcohol Alcohol is a drug of use/abuse most widely used in our civilization, however it affecting the neurochemical system in diverse areas of the CNS. The severity of its effects depends on the dose, genetic susceptibilities and the type of administration (acute or chronic). Alcohol causes changes in the brain neurochemical dopamine and norepinephrine, especially in VTA (ventral tegmental area). The use of alcohol increases the release of dopamine and norepinephrine. The acute administration of alcohol increases the release of serotonin within the brain, but chronic administration of this drug tends to decrease the amount of serotonin stored in the CNS (Marc and Schuckit, 2006). Another neurochemical mechanism that has a major impact on the effects of alcohol is GABA. It is the main inhibitory neurotransmitter in the brain and short-term alcohol exposure increases the inhibitory effect of GABAA receptors. Alcohol has been shown to increase the function of glycine receptors in laboratory and also increase inhibitory neurotransmission by increasing the activity of inhibitory neuromodulators such as adenosine (Valenzuela, 1997). Alcohol induces sedative effects by reducing the major excitatory neurotransmitters in the brain, called amino acids aspartate and glutamate, which act in NMDA receptors. Acute doses of alcohol inhibit both NMDA and non-NMDA receptor activity, potentially resulting in sedation (Valenzuela, 1997). 4.3.2 Opiates Actually, there are three families of endogenous opioid peptides known: the endorphins, enkephalins, and dynorphins, and three major receptors: mu, kappa, and delta. All opioids have major impact on the receptor mu, more precisely on mu1 and mu2. The activation of these receptors causes analgesia and a feeling reinforcement, having an important impact on the neurotransmitter dopamine, in VTA. Opioids can also mediate acute effects on NMDA, cholinergic, GABA, cannabinoid, and serotonin systems (Marc 14 and Schuckit, 2006). The use of heroin, the most common opioid of drug abuse, modifies the action of dopamine. Once crossing the blood-brain barrier, heroin is converted to morphine, which acts as a powerful agonist at the mu opioid receptors subtype. This binding inhibits the release of GABA from the nerve terminal, reducing the inhibitory effect of GABA on dopaminergic neurons. The increased action of dopaminergic neurons and the release of dopamine into the synaptic cause a sustained activation of post-synaptic membrane. This continued action leads to the feelings of euphoria associated with heroin use (Figure 6). Figure 6. The mechanism of action of heroin (adapted from www.cnsforum.com). 4.3.3 Cannabis Cannabis has two neuronal cannabinoid receptors: CB1 and CB2 were recently discovered in human cells. Both of these receptor types are coupled through G-proteins, negatively to adenylate cyclase and positively to mitogen-activated protein kinase. CB1 receptors are also coupled to ion channels through G-proteins, negatively to N-type and P/Q-type calcium channels and positively to A-type and inwardly rectifying potassium channels. Under certain conditions, CB1 receptors may also activate adenylate cyclase through G-proteins (Pertwee, 1999). The activation of CB1 receptors produces a cascade of effects in the second messenger system within the cells, with an impact on dopaminerich areas (located in nucleus accumbens) as well as opioid, gamma aminobutyric acid (GABA) and glutamate systems (Marc and Schuckit, 2006). The majority of THC effects are mediated through agonistic actions at cannabinoid receptors of the human body. In general, when ∆9-THC binds CB1 on pre-synaptic nerve terminals in the brain, activates 15 Gproteins. G-protein activation also activates inwardly, rectifying potassium channels and the MAP kinase signalling pathway. This effect on these pathways causes euphoric feelings associated with cannabis use (Figure 7) (Grotenhermen, 2006). Figure 7. The mechanism of action of cannabis (adapted from www.cnsforum.com). 4.3.4 Cocaine Cocaine is a strong stimulant drug of abuse derived from leafs of Erythroxylon coca. However the same drug has also local anesthetic properties that may be applied in ophthalmology (Dackis and O’Brien, 2001). During the early years of the last century, it became evident that cocaine was addicting and producing serious medical complications, especially with the availability of cocaine powder for intranasal or intravenous use (Dackis and O’Brien, 2001). Cocaine affects three major dopaminergic systems: the mesolimbic (ventral tegmental area to nucleus accumbens), the mesocortical (VTA to medial prefrontal cortex and orbitofrontal cortex), and the nigrostriatal. The main synaptic action of cocaine in the reward pathway is to block reuptake of dopamine, norepinephrine, and serotonin, acting through cocaine-binding sites on bioamine uptake transporters (Figure 8) (Brust, 2004). Recent studies have confirmed an increase of 20% or more in dopamine activity in the mesolimbic and mesocortical brain areas in humans that use relevant doses of cocaine. There is also evidence that the level of euphoria experienced with cocaine correlates with the degree of dopamine change in the corpus striatum (Marc and Schuckit, 2006). 16 Figure 8. The mechanism of action of cocaine (adapted from Moussa et al., 2006). 5. From pleasure to drug addiction One of the main reasons for the consumption of drugs is the pleasure that it causes. According to folk psychology, humans tend to repeat behaviors that bring pleasure and relieve suffering. Most drugs of abuse act on ancient and remarkably conserved neural mechanisms, associated with positive emotions that evolved to mediate incentive behavior. Drugs stimulate positive emotions and block bad ones such as: anxiety, low mood, emotional suffering and others. However this pleasure tends to disappear with the continued use of drugs and bad effects in other systems such as memory, mood, reward, learning and behavior begins to appear (Nesse and Berridge, 1997). A study realized with three different groups of ecstasy users: the first is a group of regular ecstasy users who had taken MDMA (3,4-methylenedioxymethamphetamine) on ten or more occasions; the second is a group of novice ecstasy users who had taken MDMA on fewer than ten previous occasions; and the third is a control group who had never taken MDMA, shows the disappearance of good feelings (good mood and pleasure) with the continued use of ecstasy and the appearance of bad mood and lacks in memory. All three groups reported positive mood at the dance club (on-drug). Nevertheless two days after, the ecstasy users felt significantly more depressed, abnormal, unsociable, unpleasant, and less good tempered, than the controls. The cognitive performance such as verbal recall and visual scanning was significantly reduced on regular users. Memory recall show also worst results compared to regular users (Parrott and Lasky, 1998). This occurs because persistent drug use induced neuroadaptations in molecular, cellular and neural system levels that are critical in the transition to addition. These 17 neuroadaptations occur, in the brain, to support the constant presence of drugs and decrease the strong stimulant response caused by drug use. In consequence, psychological function is also changed. Initially, occurs the decreasing of the pleasure and the beginning of the unpleasant withdrawal symptoms (Koob and Moal, 2001; Robinson and Berridge, 2003). The withdrawals are the symptoms of reversing the development of neuroadaptation to a drug. The drug users feel physical discomfort, psychological pain and other symptoms that differ according to the drug, and discourage people from trying to escape their addiction (Koob and Moal, 2001; Robinson and Berridge, 2003). With the continued consumption of drugs, the drug users become drug abusers, as drug induce sensitization of brain systems and the users start to have a compulsive behaviour to take addictive drugs. Furthermore, drug abusers have dysfunction of frontal cortical systems. The frontal cortical system regulates decision making and has an inhibitory control over behavior. The presence of drugs leads to impaired judgment and promotes impulsivity in addicts (Robinson and Berridge, 2003). 6. Tolerance and sensitization in drug of abuse Tolerance is a decreased sensitivity to a drug that develops as a result of repeated exposure to it. In this case the drug has fewer effects following repeated exposures. Therefore a higher dose of drug is required to produce the same effect (Figure 9)(Brust, 2004). Tolerance is an expected accompaniment of regular drug use that reflects the adaptation of the receptor site to the presence of the drug. The function of tolerance is to maintain homeostatic balance functioning in spite of the stimulating effects of a foreign substance (drug abuse). Dopamine and other neurotransmitters are present in the brain at higher levels than usual, so the brain tries to adjust, by turning off some of the receptors (locks) into which that molecular key fits. The system may also change the level of neurotransmitters which it produces in order to compensate for the increased levels of whatever drug is being taken. Depending on the drug, these processes can happen in a matter of minutes or over several weeks (Miller and Gold, 1991). Sensitization or reverse tolerance is an increase in a drug effect that occurs after repeated exposures to the drug. In this case the drug has more effects than that before exposure it. Therefore a lower dose of drug is required to produce the same effect (Figure 9) (Swift and Lewis, 2009). This term is well explained with incentive sensitization theory. It refers to neurobiological changes that occur in brain mesolimbic dopamine systems and other structures that belong to the same brain circuit that mediate the psychological function of incentive salience (“wanting”) (Berridge and Robinson, 2011). 18 Figure 9. Shifts in a dose-response curve with tolerance and sensitization. Drug abuse causes tolerance in the body of the drug users by many mechanisms. One of them is natural or innate tolerance. Natural tolerance occurs when preexist interindividual variations in sensitivity to the drug, before the first administration. It can arise from genetic variation of receptors at which the drug acts or differences among individuals in drug absorption, metabolism, or excretion. Genetic variability is strongly influenced by the environment. Innate tolerance is observed with alcohol. People who have low innate sensitivity, in young adults are of higher risk for alcoholism later (Golan et al., 2012). Whereas innate tolerance has already preexisted in our body, acquired tolerance is determined by an individual's experiences that results from exposure to drug abuse. This kind of tolerance can develop during a single drug exposure (acute tolerance) or during from repeated exposure (chronic tolerance) (Goudie and Young, 1995). Pharmacologists divide tolerance into two broad categories: dispositional or pharmacokinetic tolerance, which reduce the concentration of a drug or its duration of action in a target system; and functional or pharmacodynamic tolerance, which reduce the sensitivity of drug-sensitive systems to a given drug concentration (Krasnegor, 1978; Goudie and Young, 1995). Dispositional tolerance occurs when the body speeds up the metabolism of the drug in order to eliminate it. There is a physiological change in absorption, distribution, metabolism, or excretion that diminish the concentration of a drug at effectors sites. For example, an increase in the production of enzymes in the liver that breaks down the drug (Krasnegor, 1978). Functional tolerance is described for changes in sensitivity that results from adaptive changes in drug-sensitive systems that diminish the initial effects of a drug. It occurs when the brain learns to compensate for the effects of the drug by using parts of the brain that are not affected. This happens in chronic alcohol and marijuana users. The brain manages to function quite well despite levels of intoxication that would incapacitate people who are less accustomed to the drug. Tolerance of psychoactive drugs is largely functional (Krasnegor, 1978). 19 6.1 Alcohol Both types of tolerance occur with the consumption of alcohol. Initially occurs the pharmacokinetic tolerance and then pharmacodynamic tolerance. The pharmacokinetic tolerance is recognizable through a slight increase in both ADH activity and in the liver microssomal ethanol-oxidizing system (MEOS). For this reason, the elimination of alcohol becomes more rapid in chronic users. Functional tolerance result of a direct adaptation of CNS tissues to alcohol, so it is necessary drink more quantity of alcohol for have the same effect (Sommer and Spanagel, 2012). 6.2 Opiates Tolerance develops rapidly to most opioids, particularly with the more potent analgesics. After repeated uses of opiates, it produces complete tolerance to their euphoric effects, as well as sedation and respiratory depression. In chronic users, it causes pupils constriction, producing the pinpoint pupils, as well as slow bowel function. Because of this differential tolerance to specific opiates effects, long term opiate users have little risk of overdose (Dupont, 1997). Cross-tolerance is also common among the opioids, with a predictable variability depending on the opioid receptor type most prominently affected (Marc and Schuckit, 2006). 6.3 Cannabis The users of cannabis can develop tolerance, if they consume high doses of cannabis for a sustained period of time. The phrase “Less is more” was the slogan used in 1976 to deter recreational consumption of cannabis: “The less frequently a person users cannabis, the less likely is that person to develop tolerance to the original dose, and the less likely is that person develop cannabis tolerance”. In fact, little tolerance is observed when the doses of cannabis are small, and infrequent for limited duration (Mathre, 1997). 6.4 Cocaine Cocaine has optimal conditions to develop tolerance, mainly acute tolerance. Due to the short action of cocaine, acute tolerance develops within 24h (Grabowski, 1994). Cross tolerance occurs when cocaine users consume the drug with other psychostimulants as amphetamines and methamphetamines. Repeated administration of cocaine induced tolerance to the effects of methamphetamines, and repeated exposure to 26 Cocaine withdrawal is also characterized by electroencephalogram (EEG) changes, usually involving an excess of alpha power along with a decrease in the faster delta and theta frequency bands, with at least one report of a continuation of these findings in some individuals for 6 months. This phenomenon is related to changes in dopamine (DA) transmission, in several brain areas, including the amygdala. Additional changes are also observed in cerebral glucose utilization and changes in the normal hormonal alterations, including prolactin (Alper, 1999). 9. Toxic reactions and overdose in drug of abuse Toxic reactions caused by psychoactive substances consist in toxic physiological, psychological, behavioral manifestations and life-threatening reactions resulting from intentional and unintentional (accidental) overdose. A drug overdose is the use of drug in an amount that is higher than is normally used, or when the drug is taken in combination with other drugs inclusive alcohol (Khantzian and McKenna, 1979). All drugs have the potential to produce toxic effects and cause overdose by a variety of mechanisms such as: Toxic effects that are direct and predictable following a drug overdose occur due to the drug itself or it may reflect a change in the metabolism of the drug, resulting in a toxic metabolite; Toxic effects may be direct and predictable following repeated dosing of the drug, are mediated by metabolites, pharmacologic in nature or immunologic in mechanism; Toxic effects may be direct and unpredictable, either following one dose or just a few doses occur due to a idiosyncratic response to drugs and may be immunologic or pharmacologic in mechanism. This type of toxic effects appear in only a very few patients and have no good predictors for their occurrence; The last one are toxic effects that results of some drug interaction or may be caused by a change of tolerance due to temporary drug abstinence (Khantzian and McKenna, 1979; Kjelsberg et al., 1995). With the exception of using opioid antagonists, overdose conditions are usually treated by supporting the vital signs, so that the body can metabolize and excrete the drugs and return to normal. After that is necessary to control of symptoms regardless of the specific drug involved. If the drugs involved in overdose were depressants, the patient will require control of respirations and bolstering of blood pressure, because the vital signals were depressed. If the drugs involved in overdose were stimulants or opioids, the emergency treatment depends upon the vital sign changes. The mainly treatment in these cases includes a controlling high blood pressure and elevated body temperature. If the drug involved was opioid, the clinical should administer an opioid antagonist (e.g., naloxone). Another general rule is to consider gastric lavage, followed by the possible use 27 of activated charcoal if there is evidence of recent oral administration of the drugs (Marc and Schuckit, 2006). 9.1 Alcohol An overdose of alcohol occurs when a person has a blood alcohol concentration (BAC) sufficient to produce impairments that increase the risk of harm. The severity of toxic reaction could vary among individual characteristics such as: age, drinking experience, gender and the amount of drinker food eaten (Li et al., 2003). There are two forms of ethanol intoxication: pathological intoxication, also called idiosyncratic intoxication and acute alcoholic paranoid state. The acute alcoholic paranoid intoxication consists of sudden extreme excitement, sometimes with delusions, hallucinations, and violent behavior, even homicide. In some cases after minutes to hours, there is amnesia for the episode. The pathological intoxication is characterized by psychological dissociative reactions resulting of paradoxical excitation (Brust, 2004). Acute overdose with ethanol presents primarily as CNS depression. The most common symptoms of acute alcoholic overdose are: signs of vasodilatation (flushing, tachycardia, hypotension, and hypothermia), depressed consciousness, hyponatremia, hypovolemia, electrolyte imbalance, hypoglycemia and abnormal temperature. In chronic ethanol intoxication is common to appear hepatotoxicity and cardiotoxicity, causing potential cirrhosis and cardiomypathy progressing to failure, respectively (Malcolm and Alkana, 1983; Brust, 2004; Waring et al., 2008). The treatment of severe ethanol poisoning is similar to that of other depressant drugs. Death is from respiratory depression, and so patients require artificial ventilation in an intensive care unit. In chronic ethanol ingestion is necessary to make the detoxification with employ of chlordiazepoxide, to prevent delerium tremens and other signs of ethanol withdrawal (Brust, 2004). 9.2 Opiates The opioid overdose is usually an acute, life-threatening event that is most often accidental but that could represent a deliberate suicide. At least one overdose occurs during the course of 50% of heroin users. The user is likely to be found in a semicomatose condition with evidence of a recent intravenous injection. The risk of overdose depends on many factors such as: the high-quality heroin (increase the risk) or use of a more potent opioid (e.g. fentanyl), the price of the drug (less cost, high risk of overdose), the use of quinine for dilute the drug can decrease the activity of the cardiac pacemaker, 28 decrease cardiac electrical conductivity, and thereby induce a prolonged cardiac electrical refractory period that increases the risk for ventricular fibrillation (Marc and Schuckit, 2006). Acute overdose causes depression, especially of the respiratory centre. The symptoms associated with opioids overdose include: reduced levels of consciousness, lethargy, miosis (pinpoint pupils), flaccid muscle tone, cool skin, hypotension, bradycardia, hypothermia, cyanosis, hypoventilation, apnea and coma. Death is usually due to respiratory failure (Darke and Zador, 1996). Opioid overdose is treated by administration of opioid antagonist such as naloxone or naltrexone. Naloxone is mainly used in the treatment of opioid-overdose-induced respiratory depression, in ultra-rapid detoxification and in combination with buprenorphine for maintenance therapy to prevent intravenous abuse. However the administration of naloxone in opioid-dependent patient has some implications that include: the occurrence of vomiting and aspiration is potentially life threatening, and in patients treated for severe pain with high-dose naloxone may cause catecholamine release and consequently pulmonary edema and cardiac arrhythmias. So the administration of naloxone imply an adequate monitoring of the cardiorespiratory status of the patient (Dorp et al., 2007). 9.3 Cannabis Cannabis has acute and chronic effects on mental health. Acute effects vary among individuals and the degree and severity of these effects is related to the dosage, method of administration, environment and personality of the user. Chronic effects could cause serious psychiatric illness such as: depression, anxiety, low motivation, psychosis and schizophrenia (Cho et al., 2005). Although cannabis cause less clinically relevant effects, when used in higher quantities causes adverse toxic reactions. High doses of cannabis cause auditory and visual illusions or hallucinations that consist of flashes of light or color, geometric figures, human faces, or complex pictures. Also, it can cause bizarre illusions include loss of depth perception, the appearance of people talking with their mouths, voices unsynchronized, and “streaking” (moving light sources in a dark environment becoming long streaks as in a time-exposed photograph).The patients can describe fantastic complex hallucinations with extraordinary dilation of subjective time, and there are some reports that propose that marijuana improves night vision. If increasing more the higher doses of cannabis, we could observe confusion, disorientation, anxiety, psychotic depression or excitement, bradycardia and hypotension occur. Nowadays, fatal overdose has not been documented (Hollister, 1986; Brust, 2004; Cho et al., 2005; Calafat et al., 2012). 29 Cannabis severe symptoms are treated by administration of benzodiazepines, haloperidol or other anxiolytic. This medication is adequate for treatment of anxiety and feature of cannabis withdrawal (Piomelli, 2004). 9.4 Cocaine Overdose of cocaine occurs in users with the following characteristics: female gender, injectors of cocaine, to have a longer cocaine use careers, to have used more cocaine in the preceding month and preceding 6 months, to have higher levels of cocaine dependence and more extensive polydrug use (Kaye and Darke, 2004). Cocaine acts in CNS and in cardiovascular system cause biphasic response. In lower doses it tends to improve motor performance and to produce a decrease in heart rate via actions on the vagus nerve. In high doses it causes deterioration in CNS, with subsequent severe tremors and possible convulsions, and in cardiovascular system causes an increased heart rate and vasoconstriction, with a resulting elevation in blood pressure (Marc and Schuckit, 2006). The most common symptoms of overdose are: palpitations, nausea, vomiting, dilated pupils, an increased body temperature, intense sweating, seizures, muscle contractions, arrhythmias due to catecholamine release. Fatal cocaine overdose has also occurred due to brain hemorrhage, stroke and kidney failure, myocardial infarction, hyperthermia, or ventricular arrhythmias (Gerada and Ashworth, 1997; Kaye and Darke, 2004). The time course of effects differs with the route of administration. Intravenous use of cocaine has an onset of about 3–5 min, with peak effects at 10–20 min, and a fading high by 45 min or less. The risk of overdosing increases when it is administered in a way that causes a rapid increase in brain levels of the drug, as happens with injected cocaine (Kaye and Darke, 2004; Marc and Schuckit, 2006). Actually there is no specific antidote for cocaine overdose. The treatment options are supportive and symptomatic in nature, and will depend on which clinical features are present. If the patient is unconscious should be ensured the adequate ventilation and volume depletion, cardiac arrhythmias, seizures, hypertension, agitation, and hyperthermia should be managed symptomatically. Benzodiazepines are the medication of choice for the management of patients with agitation, seizures, tachycardia, and hypertension. If hypertension persists after the administration of benzodiazepines specific antihypertensive therapy (e.g. intravenous nitrates or calcium-channel blockers) can be given. Beta-blockers should be avoided due to the risk of coronary vasoconstriction and paradoxical hypertension. However, has been described recently, that the administration of intravenous lipid is an effective treatment for cardiovascular complications associated 30 with local anaesthetic-induced toxicity. This medication is also acts as an effective antidote to the overdose of lipid-soluble drugs including beta-receptor antagonists, calcium channel blockers and antidepressants (Carrera et al., 2005; Jakkala-Saibaba et al., 2011). 31 PART II: General and specific objectives of the thesis 32 33 1. Objectives of the thesis The general objective of this work was to evaluate through surveys and collection/analysis of biological samples, the drug abuse patterns in Coimbra recreational nightlife. The specific objectives of this work were: - To investigate the presence of psychoactive substances in biological samples ceded by willing participants of this study; - To analyze qualitatively and quantitatively the components of these samples by GC-MS, to be implemented in the present studies; - To confront the answers in the surveys with the chemical results of analyzed samples; - The final objective is to contribute for a better risk management concerning drug abuse among young people, by comparing their perception of the risk abuse patterns. 34 35 PART III: Experimental Part 42 43 Chapter I I Materials and Methods Identification and quantification of psychoactive drugs in biological samples 44 45 1. Identification and quantification of psychoactive drugs in biological samples 1.1 Ethics statement This research was approved by the following institutions: National Commission of Data Protection, Ethics Commission of Faculty of Pharmacy University of Porto, City Hall of Coimbra and ARS-IP. A written informed consent was signed by all participants of this study. 1.2 Reagents and standards All the chemicals used for the ethanol quantification were of analytical grade: ethanol (> 99.9%, Panreac, Barcelona, Spain), 1-propanol (> 99%, Sigma-Aldrich Co., St. Louis,MO), Triton X-100 (Sigma-Aldrich Co.). The analytical standard 9-Tetrahydrocannabinol, molecular mass 314.45 g mol-1 and the internal standard benzophenone, molecular mass 182.22 g mol−1 were obtained from Sigma-Aldrich (St Louis, MO, USA). Methanol and N,Obis(trimethylsilyl)trifluoroacetamide/trimethylchlorosilane (BSTFA+1%TMCS) were purchased from Sigma-Aldrich (St Louis, MO, USA). Hexane, acetonitrile and ethyl acetate were purchased from Merck (Darmstadt, Germany). Potassium dihydrogen phosphate and di-potassium hydrogen phosphate were purchased from Merck (Darmstadt, Germany). Nitrogen (99.99% purity) and helium (99.99%) were obtained from Gasin (Portugal). The analytical standard cocaine hydrocloride, molecular mass 339.81 g mol-1 and the internal standard benzophenone, molecular mass 182.22 g mol−1 were obtained from Sigma-Aldrich (St Louis, MO, USA). The cocaine screening test was obtained from commercial brand First Check@ Test. - All the reagents used were of analytical grade or from the highest available grade. 1.3 Biological specimens Blood and oral fluid samples were collected from young adults attending recreational nightlife, in Coimbra, at least, once per week. Blood samples were collected from the median cubital vein. The collection method used in this study was through a commercial vacuum-sampling system, decreasing the risk of contamination. Initially, a tourniquet was used to distend the vein prior to sampling. After that, the skin was cleaned with disinfectant swabs containing alcohol. In view of the requirement to prevent stress on patients, only 5 to 10 mL of blood was collected. These samples were kept in heparin tubes at 7ºC. The oral fluid samples were collected through a spitting method (around 1-2 46 mL) for an appropriate collection container and were kept at 7ºC. After the collection, all samples were stored and transported, in refrigerator containers, to the Laboratory of Toxicology, Faculty of Pharmacy University of Porto. For validation of the analytical method for THC, drug-free blood was used, to which was added the appropriate volume of THC standards. All samples were stored at -20 ºC prior to analysis. 1.4 Analysis of ethanol in blood and oral fluid samples Sixty nine blood samples, 17 oral fluid samples, and 46 exhaled air samples were obtained from these participants. Blood and oral fluid analysis were performed according to a previously published method (Pontes et al., 2009). 1.4.1 Preparation of stock solution A sock solution containing ethanol was prepared in commercial deionized water from the purchased sample (> 99.9%, Panreac, Barcelona, Spain), at the concentration of 12g/L. 1.4.2 Preparation of calibration standard solution For blood sample analysis, calibration standard solutions of 0; 0.15; 0.30; 0.60; 1.20; 2.40 g/L were freshly prepared for each analysis from the stock solution of 12g/L ethanol, by adding the appropriate volume to drug-free blood. For oral fluid sample analysis, calibration standard solutions of 0; 0.0375; 0.075; 0.30; 0.60 g/L were freshly prepared for each analysis from the stock solution of ethanol 12g/L by adding the appropriate volume to drug-free oral fluid. 1.4.3 Preparation of internal standard (IS) A working solution of 1-propanol was prepared in deionized water from the purchased solution of 1-propanol (> 99%, Sigma-Aldrich Co., St. Louis,MO), at the concentration of 2,2 g/L. This solution was used as internal standard (IS). - All working and stock solutions were prepared fresh daily and stored at 7 ºC prior use. 1.4.4 Sample preparation for gas-chromatography flame ionization detector 1.4.4.1 Drug-free samples One drug free blood and oral fluid samples were tested with the same protocol of ethanol blood/oral fluid samples respectively, in order to verify the selectivity of the method. 1.4.4.2 Blood samples 47 Two hundred microliters of blood samples were mixed with 40 μL of IS. The samples were diluted to 780 μL with the Triton X-100 solution. All samples were vortex mixed and centrifuged at 13.000 rpm for 3 min. 0.5 μL of the supernatant were directly injected into the GC-FID system. 1.4.4.3 Oral fluid samples Two hundred microliters of oral fluid samples were mixed with 40 μL of IS. The samples were diluted to 780 μL with the Triton X-100 solution. All samples were vortex mixed and centrifuged at 13.000 rpm for 3 min. 0.5 μL of the supernatant were directly injected into the GC-FID system. 1.4.5 Gas-chromatography flame ionization detector conditions The GC used was a ThermoFinnigan Model Focus GC equipped with a FID. The injection port of the chromatograph was installed with a glass liner (5-mmi.d.) appropriated for split analysis, to prevent the contamination of the GC column with non-volatile material from the tested matrices. For blood and oral fluid samples, the liner was replaced after 50 injections. The analyses were performed under the following chromatographic conditions: Column, CPWax 57 CB (WCOT Fused Silica), 25 m × 0.25 mm i.d., DF = 0.2 μm, from Varian (Palo Alto, CA). The temperature of the FID was 220ºC, and the injector temperature was 220°C. The oven temperature was programmed to 40°C (for 5 min), followed by an increase of 10ºC/min until 150ºC. After that, the oven temperature increase of 2ºC/min until 210ºC. The carrier gas was helium with a flow of 1.5 mL/min. The injection of blood and oral fluid samples were performed by means of a 10 μL Hamilton syringe (Model 701 RN) with a removable needle (needle gauge 22S), cleaned under vacuum between each injection with the Triton X-100 solution. The volume of injection was 0.5 μL, with a ratio of 1:180 min corresponding to a split flow of 120 mL/min for blood and oral fluid samples. The representative chromatogram was reprocessed using the following retention times for each analyte, presented in Table 1. Table 1. Retention times of ethanol, and IS analyzed by GC-FID. Analytes Retention time (minutes) Ethanol 3.1 IS 5.2 48 1.5 Analysis of 9-THC in blood and oral fluid samples 1.5.1 Preparation of stock solution A 1mg/mL methanolic solution containing 9-THC was prepared from the purchased solution of 9-THC Sigma-Aldrich (St Louis, MO, USA). 1.5.2 Preparation of working solutions Four working solutions of 10; 100; 1000; 10000 ng/mL were prepared from the stock solution (1mg/mL) in methanol. 1.5.3 Preparation of calibration standard solution Calibration standard solutions of 0, 1, 5, 10, 25, 50, 100, 500, 1000 ng/mL were freshly prepared for each analysis from working solutions by adding the appropriate volume to drug-free blood. 1.5.4 Preparation of internal standard (IS) A working solution of benzophenone was prepared in acetonitrile from the purchased solution of benzophenone Sigma-Aldrich (St Louis, MO, USA), at the concentration of 40ug/mL. This solution was used as internal standard (IS). - All working and stock solutions were prepared fresh daily and stored at -20 ºC prior use. 1.5.5 Sample preparation for gas-chromatography mass spectrometry 1.5.5.1 THC extraction from biological samples Different methods of LLE and SPE were tested to determine the optimal conditions of extraction. The method described is the one that resulted in higher recoveries. Hence, liquid-liquid extraction (LLE) was performed in falcon tubes of 15 mL. To each falcon 1 mL of blood sample and 50μL of I.S were added. All tubes were vortex mixed and added 1 mL of phosphate buffer (pH 4.1 M). The tubes were vortex mixed again and 5 mL of hexane/ethyl acetate (5/1) were added subsequently. The tubes were shaken on a rotary mixer for 10 min and centrifuged at 2500 rpm for 20 min. The organic layer was transferred to GC vials. A second aliquot of 5 mL hexane/ethyl acetate (5/1) was added to the remaining aqueous layer and the process was repeated to maximize recovery. The solvent extracts were totally evaporated under N2 at 50 ºC. 49 1.5.5.2 Derivatization procedure In this study, THC was derivatized by silylation, reacting with BSTFA and 1% TMCS. BSTFA is the silylation reagent that reacts with 9-THC replacing active hydrogens by a –Si(CH3)3 (trimethylsilyl) group. TMCS increases the reactivity of BSTFA. 9-THC measurement was performed with the addition of 60 μL of BSTFA +1%TMC. The samples were vortex mixed and heated for 60 min at 70 ºC. After cooling to room temperature, the samples were injected into the GC-MS system. 1.5.6 Gas-chromatography mass spectrometry conditions Quantitative GC-MS analysis was performed on a Varian CP-3800 gas chromatograph (USA) equipped with an ion-trap Varian GC-MS Saturn 4000 mass detector. Chromatographic separation was achieved using a capillary column VF-5ms (30 m × 0.25 mm i.d. × 0.25 μm) and a high-purity helium C-60 carrier gas. An initial temperature of 100 ºC was maintained for 1 min, increased to 300 at 15 ºC/min, and held for 1 min, giving a total run time of 24.33 min approximately. The flow of the carrier gas was maintained at 1.0 mL/min. The injector port was set at 250 ºC. Analyses were performed in full scan in splitless injection mode. The obtained full scan chromatogram was reprocessed using the following selected qualifier ions and retention times for each analyte, presented in Table 2. The underlined ions were used for quantification. Table 2. Retention times and m/z ions of 9-THC and IS by GC-MS. Analytes Retention time (minutes) Fragments (m/z) 9-THC 12.32 315; 371;386 IS 7.76 105; 182 The integration of the chromatographic peaks for quantitative analysis was performed by monitoring the fullscan chromatogram with specific selected m/z ions allowing more precise peak integration. 50 1.6 Method validation of 9-THC Selective and sensitive analytical methods for the quantitative evaluation of psychoactive substances and their metabolites are critical for a successful conduction of analytical studies. Bioanalytical method validation includes all of the procedures that demonstrate that a particular method used for quantitative measurement of analytes in a given biological matrix such as blood, plasma and oral fluid is reliable and reproducible for the intended use. The Food and Drug Administration guidance, that is actually accepted by the biopharmaceutical industries as the gold standard method validation approach, established the fundamental parameters for method validation that include: accuracy, precision, selectivity, sensitivity, reproducibility, and stability (Department of Health and Human Services, 2001). In this work, the validation methods of 9-THC for blood samples were based on the determination of selectivity, linearity, LOD, LLOQ, precision, accuracy, recovery and reproducibility. In order to obtain these validation data, calibration curves were prepared by spiking blank whole blood with appropriate concentrations of 9-THC standard. 1.6.1 Selectivity Six blank samples with no analytes or IS added were extracted by LLE as described previously and analyzed by GC-MS to detect possible chromatographic interferences with THC. Chromatographic selectivity was evaluated by the presence or absence of co-eluting peaks at the retention times of the analytes. Three independent experiments were performed. 1.6.2 Linearity The method linearity was determined by evaluation of the regression curve (ratio of analyte peak area and IS peak area versus analyte concentration) and expressed by the determination coefficient (r2) using spiked samples. The calibration curve (y = mx + b) was obtained using nine different concentrations (0, 1, 5, 10, 25, 50, 100, 500, 1000 ng/mL). The mean slopes were obtained for calculating the concentration of real samples (unknown concentrations). These concentrations were prepared daily as mentioned before. 1.6.3 Limit of detection and lower limit of quantification In this work, LOD and LLOQ were obtained based on the standard deviation of the response and the slope of the calibration curve. The LOD and LLOQ are expressed accordingly to the following equations respectively. The σ is the standard deviation of the response and S is the slope of the calibration curve. 51 1.6.4 Precision The precision of an analytical procedure is defined as the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions (Department of Health and Human Services, 2001). It is expressed as the coefficient of variation (%CV). Intraday precision data was quantified by analyzing the areas of three replicates of three concentrations (low, 10; medium, 100; and high, 1000 ng/mL) and calculating the %CV. The areas of the same three concentrations, injected on three consecutive days, were used to calculate the interday repeatability (%CV). A %CV value of ≤15% for interday and intraday analysis was considered satisfactory. 1.6.5 Accuracy The accuracy of an analytical method as the closeness of agreement between the conventional true value and the value found. It is expressed as a percentage. In this work, the accuracy of the THC method was determined by spiking blank matrix with three different THC concentrations (low, 10; medium, 100; and high, 1000 ng/mL) and through the calculation of the percentage deviation between the calculated value and the nominal value [accuracy (%) = (experimental concentration/theoretical concentration) × 100]. A deviation percentage of ≤15% was considered satisfactory. 1.6.6 Recovery The recovery was determined by analyzing two sample groups of the same concentrations (10, 100 and 1000 ng/mL) in triplicate. In the first group, THC and the internal standard were added before the liquid-liquid extraction as following mentioned above. In the second group, THC and internal standard were added after the liquid-liquid extraction, before drying. The recovery was evaluated by the comparison of the mean response of the two groups. The response of the unextracted group represents 100% recovery. A deviation percentage of ≤20% was considered satisfactory. 1.6.7 Reproducibility The reproducibility of an analytical method is determined by analyzing the same concentration of the sample five times. The objective of reproducibility is to verify that the same method will provide the same result. In this work, the reproducibility was determined by analyzing the same concentration (100ng/mL) five times. 58 Figure 15. Residence of participants Figure 16. Personal habitation Figure 17. Professional status 59 1.2 Recreational nightlife habits Data about recreational nightlife habits revealed an average of 2,09 nights out per week and 8,01 nights out per month. The inquiries visit in average 3,08 recreational spaces in each night out (Table 5). In relation to the money that the participants spent per night, it varies among less than 5€ and more than 50€. However, the majority of participants spend 10€ per night (Figure 18). Table 5. Recreational nightlife habits Figure 18. Money spent per night Recreational nightlife habits N Min Max Mean SD Mode Median Nights out per week 78 0 7 2,09 1,531 1 2,00 Nights out per month 78 0 30 8,01 5,669 4 8,00 Recreational spaces 78 0 10 3,08 1,618 3 3,00 N: number of answers; Min: minimum; Max: maximum; SD: standard deviation 60 1.3 Health information The participants of this study weigh, in average, 69,71 kilograms and height, in average, 1,7372 meters (Table 6). The majority of participants (78,2%) do not have health problems. Among the participants that have health problems (21,8%), about half of them were not taking medication. All participants who were taking medication said that it was prescribed by his doctor (Table 7). The last food intake by participants was, in average, 4,85 per hours ago (Table 6). Table 6. Health information Table 7. Health problems and use of medication Use of medication Health Problems Use Prescription Medication No Yes Total No Yes Total No Count 61 8 69 0 0 0 % of Total 78,2% 10,3% 88,5% 0% 0% 0% Yes Count 0 9 9 0 9 9 % of Total 0% 11,5% 11,5% 0% 100% 100% Total Count 61 17 78 0 9 9 % of Total 78,2% 21,8% 100,0% 0% 100% 100% 1.4 Problems due to use of psychoactive substances Regarding to health problems related to the use of drugs, 19 (6,8%) of the individuals affirmed that already had problems with the use of psychoactive substances. The majority of the participants, 8 (42,11%), were injured and 4 (21,05%) got sick. Only 2 people (10,53%) had a sexual regret, 1 (5,26%) had family or friends problems, 1 (5,26%) had a crisis anxiety or a mental disturbance, 1 (5,26%) had a road accident and 2 (10,53%) have already experienced all of these problems. Health information N Min Max Mean SD Mode Median Weight 78 45 115 69,71 13,234 70 70,00 Height 78 1,52 1,93 1,7372 0,08842 1,8 1,7400 Last feed administrated (hours ago) 78 1 9 4,85 2,330 8 5,00 N: number of answers; Min: minimum; Max: maximum; SD: standard deviation 61 In respect to the influence of psychoactive substances in social behavior of the 78 participants, 12,82% answered that the use of drugs interfered to their friendship; 25,6% had already driven under the effect of psychoactive substances and 14,1% had being involved in conflicts with authority due to drug use (Table 8). In relation to the major risk associated with going out at night, only 7,69% of the inquiries answered “no risk”, 17,95% mentioned the alcohol effects, 16,67% traffic accidents, 14,10% drug effects, 11, 54% alcohol and drug effects and physical violence, 7,69% sexual violence, 5,13% to get unconsciousness, 3,85% stealing or robbery, 2,56% the drug users and 1,28% the police (Figure 19). Table 8. Problems due to use of psychoactives substances Frequency Percent Valid Percent Cumulative Percent Influence of psychoactive substances in friends relations No 68 87,2 87,2 87,2 Yes 10 12,8 12,8 12,8 Total 78 100,0 100,0 Drive under psychoactives substances No 58 74,4 74,4 74,4 Yes 20 25,6 25,6 25,6 Total 78 100,0 100,0 Problems with authority due to use of psychoactive substances No 67 85,9 85,9 85,9 Yes 11 14,1 14,1 14,1 Total 78 100,0 100,0 Figure 19. Perception of risk 62 1.5 Drug abuse patterns The psychoactive substance more used by young people in recreational settings was alcohol (87,2%), followed by tobacco (64,1%). Regarding to illegal psychoactive substances, cannabis was the most used drug (19,2%), followed by cocaine and amphetamines (2,6%), LSD and smartshop substances (1,3%). In this study, it was not detected the use of other illicit drugs such as: ecstasy, psilocybin, mescaline, heroin, GHB, or flunitrazepam. On the other hand, cathinones or synthetic cannabinoids may fall in the 1,3% use of smartshop substances (Table 9). The 87,2% of participants who used alcohol, in average, consumed 5,40 fermented drinks and 3,16 distilled drinks (Table 9). All of the 64,1% of participants that used tobacco, consumed smoked tobacco. These young people smoke in average 14,84 cigars when go out at night. The brand of cigarettes more common was Marlboro with 20,51%, following Camel with 12,8%, and 10, 28% used other brands of cigars (Figure 20). Among the 19,2% individuals who used cannabis, 6,41% do not smoke one complete cannabis cigarette, but only smoke, in average, 4,2 raisins. The rest of them, smoked in average 2,73 cigarettes (Figure 21). The 2,6% of people who used cocaine, in average sniffed 2 lines, and 1,3% shared material. All users of cocaine sniffed this psychoactive substance. About the 2,6% users of amphetamines, 1,3% ingested in average 1 pill, and 1,3% smoked in average 2 raisins. The only one user of LSD (1,3%) used one stamp in that week. The only one user of smartshop substances (1,3%) ingested one pill named creatine (Table 9). 85,9% of the participants do not use substances for relaxing in the end of the night, although 6,41% use cannabis, 5,13% use medicines (2,56% valium, 1,28% guronsan 1,28% alprazolam),1,28% use cocaine and 1,28% use tobacco for sleeping well (Figure 22). 63 Table 9. Psychoactive substances used by participants during that week Figure 20. Brands of cigarettes used by participants Use of Psychoactive Substances Frequency (Total) Percent (Total) Route Administ. Min Max Mean SD Tobacco Cigarettes 50 64,1% Smoked 2 70 14,84 15,403 Alcohol Fermented Distilled 68 87,2% Ingested Ingested 0 0 20 13 5,40 3,16 5,303 3,732 Cocaine Lines 2 2,6% Sniffed 1 3 2,00 1,414 Amphetamines Pills Raisins 2 2,6% Ingested Smoked 1 2 1 2 1,00 2,00 0 0 Ecstasy 0 0% 0 0 0 0 Cannabis Cigarettes Raisins 15 19,2% Smoked Smoked 0 2 10 8 2,73 4,20 3,348 2,280 LSD Stamps 1 1,3% Ingested 1 1 __ __ Ketamine 0 0% 0 0 0 0 Psilocybin 0 0% 0 0 0 0 Mescaline 0 0% 0 0 0 0 Heroin 0 0% 0 0 0 0 GHB 0 0% 0 0 0 0 Flunitrazepam 0 0% 0 0 0 0 Cathinones 0 0% 0 0 0 0 Synthetic Cannabinoids 0 0% 0 0 0 0 Smartshops Pills Substances 1 1,3% Ingested 1 1 __ __ Administ: Administration Min: minimum; Max: maximum; SD: standard deviation 64 Figure 21. Number of cannabis cigarettes Figure 22. Use of psychoactive substances for sleeping well 1.6 Drug abuse patterns and socio-demographic characteristics With the objective of analyzing the influence of gender on drug abuse patterns, it was used the non parametric test of Pearson Chi-Square. For the psychoactive substance tobacco, 28(35,9%) are no smokers, being 8 (38,1%) females and 20(35,1%) males. On other hand, 50(64,1%) of the total participants are smokers, being 13(61,9%) females and 37 (64,9%) males. Using the non parametric test of Pearson Chi-Square p=0,806, there are no statistically significant differences between gender and tobacco smoking (Table 10). For alcohol, 10 participants (10,8%) do not consume alcohol, being 3 (14,3%) females and 7(12,3%) males. On the other hand, 68(87,2%) of the total participants 65 consume alcohol, being 18(87,5%) females and 50 (87,7%) males. Using the non parametric test of Pearson Chi-Square p=0,814 there are no statistically significant differences between gender and consumption of alcohol (Table 11). Concerning cannabis, 15(19,2%) of the total participants used cannabis, being 1(4,8%) females and 14 (24,6%) males. Although it is not possible to determine statistically the influence of gender in cannabis use, it is possible to observe that there is an association between male gender and cannabis use (Table 12). When analyzed the number of tobacco cigarettes, we can observe that males (average 17,22) consume more cigarettes than females (average 8,08). The same occurs with alcohol, the mean of fermented and distilled drinks being higher in males (average 6,43 and 3,76 respectively) than in females (average 2,61 and 1,56 respectively) (Table 13). Table 10. Gender vs Tobacco Non parametric test of Pearson Chi-Square p=0,806 Gender Total Female Male Tobacco No Count 8 20 28 % within Gender 38,1% 35,1% 35,9% Yes Count 13 37 50 % within Gender 61,9% 64,9% 64,1% Total Count 21 57 78 % within Gender 100,0% 100,0% 100,0% Table 11. Gender vs Alcohol Non parametric test of Pearson Chi-Square p=0,814 Gender Total Female Male Alcohol No Count 3 7 10 % within Gender 14,3% 12,3% 12,8% Yes Count 18 50 68 % within Gender 85,7% 87,7% 87,2% Total Count 21 57 78 % within Gender 100,0% 100,0% 100,0% 66 Table 12. Gender vs Cannabis Table 13. Gender vs number of cigarettes or drinks Comparing the drug abuse patterns with the professional situation of the participants, we can conclude that, for tobacco and alcohol, the results obtained using the test of Pearson Chi-Square did not provide statistically significant differences between the drug and the professional situation (p=0,478). However, when consumption of cannabis and professional situation were compared, we observed that the majority, 9 (60%), were students and the others are musician 1(10%), DJ 1(10%) and other professions. Applying the same probabilistic test, the results are statistically significant with p=0,029. For cocaine due to the small size of the samples (2 individuals) is not possible to determine the statistic probability (Table 14). Gender Total Female Male Cannabis No Count 20 43 63 % within Gender 95,2% 75,4% 80,8% Yes Count 1 14 15 % within Gender 4,8% 24,6% 19,2% Total Count 21 57 78 % within Gender 100,0% 100,0% 100,0% Gender N Mean Std. Deviation Std. Error Mean NºCigarettes Female 13 8,08 4,536 1,258 Male 37 17,22 17,139 2,818 NºFermented Female 18 2,61 2,933 ,691 Male 49 6,43 5,624 ,803 NºDestilled Female 18 1,56 2,148 ,506 Male 49 3,76 4,024 ,575 67 Table 14. Professional Situation vs use of psychoactive substances Tobacco Alcohol Cocaine Cannabis Total No Yes No Yes No Yes no yes Professional Situation Bartender Count 0 2 0 2 2 0 2 0 2 % within Tobacco ,0 ,0 ,0 ,0 ,0 ,0 ,0 ,0 ,0 DJ Count 0 3 1 2 3 0 2 1 3 % within Tobacco ,0 ,1 ,1 ,0 ,0 ,0 ,0 ,1 ,0 Factory of electric motors Count 1 0 0 1 1 0 1 0 1 % within Tobacco ,0 ,0 ,0 ,0 ,0 ,0 ,0 ,0 ,0 Housekeeping Count 2 0 0 2 2 0 2 0 2 % within Tobacco ,1 ,0 ,0 ,0 ,0 ,0 ,0 ,0 ,0 Locksmith Count 0 1 0 1 1 0 0 1 1 % within Tobacco ,0 ,0 ,0 ,0 ,0 ,0 ,0 ,1 ,0 Musician Count 1 0 0 1 0 1 0 1 1 % within Tobacco ,0 ,0 ,0 ,0 ,0 ,5 ,0 ,1 ,0 Painter Count 0 1 0 1 1 0 1 0 1 % within Tobacco ,0 ,0 ,0 ,0 ,0 ,0 ,0 ,0 ,0 Public works Count 0 1 0 1 1 0 1 0 1 % within Tobacco ,0 ,0 ,0 ,0 ,0 ,0 ,0 ,0 ,0 Real Estate Agent Count 0 1 0 1 1 0 0 1 1 % within Tobacco ,0 ,0 ,0 ,0 ,0 ,0 ,0 ,1 ,0 Security Count 1 0 0 1 1 0 1 0 1 % within Tobacco ,0 ,0 ,0 ,0 ,0 ,0 ,0 ,0 ,0 Soldier Count 0 1 0 1 1 0 1 0 1 % within Tobacco ,0 ,0 ,0 ,0 ,0 ,0 ,0 ,0 ,0 Student Count 21 32 5 48 52 1 44 9 53 % within Tobacco ,8 ,6 ,5 ,7 ,7 ,5 ,7 ,6 ,7 Student/Worker Count 2 4 3 3 6 0 6 0 6 % within Tobacco ,1 ,1 ,3 ,0 ,1 ,0 ,1 ,0 ,1 Unemployed Count 0 2 0 2 2 0 0 2 2 % within Tobacco ,0 ,0 ,0 ,0 ,0 ,0 ,0 ,1 ,0 Worker Count 0 2 1 1 2 0 2 0 2 % within Tobacco ,0 ,0 ,1 ,0 ,0 ,0 ,0 ,0 ,0 Analyzing the household where the participants live with the drug abuse patterns, in relation to tobacco there are no statistically significant differences (p=0,358). However, when we analyse alcohol consumption, 48 (70%) who drink alcohol live with family and 11(20%) live with friends, being the P value 0,73. Therefore, there is not a positive 74 75 1. Implementation of methodologies for the identification and quantification of psychoactive substances in biological samples 1.1 Analysis of ethanol 1.1.1 Pre-treatment of samples Ethanol is a volatile compound whose detection and quantification in biological matrices can be made with a direct injection, using a capillary column, in the gas chromatography with flame ionization detection (GC–FID). In this work, the analysis of ethanol in blood samples, and in oral fluid samples, were realized according to method previously published method (Pontes et al., 2009). This validated method was chosen because is a rapid and efficient method that requires only the addiction of triton-x100 solution (containing acetonitrile in its composition) for the pre-treatment of the samples. This solution was used to dilute the blood and oral fluid matrices. 1.1.2 Detection by gas-chromatography flame ionization detector Gas-chromatography flame ionization detector has a high sensitivity, uniform response to hydrocarbons, and a broad linear range that have made the flame ionization detector (FID) one of the most widely used detector in gas chromatography. The FID response of hydrocarbons is proportional to the mass of carbon present in the sample (Jorgensen et al., 1990). In this work, interferences were ruled out by verifying the absence of peaks in the retention time of ethanol. The retention time for ethanol and IS (1propanol) were 3.06 min and 5.08 respectively. The acetonitrile, integrant part of triton-x 100 solution has as retention time of 3.71 min (Figure 24). Figure 24. Representative chromatograms obtained from blood sample. The retention time for ethanol, IS (1propanol) and acetonotrile were 3.06, 5.08 and 3.71 min respectively. 76 1.1.3 Selectivity of the method The GC-FID chromatograms of spiked samples were compared with the chromatograms obtained with a blank blood/oral fluid sample. No interference peaks were detected in the retention times of ethanol neither in blood sample (Figure 25) nor in oral fluid sample (Figure 26). Figure 25. Representative chromatograms obtained from blood sample. The figure A represents a blank blood sample without ethanol. The figure B represents a blood sample containing ethanol. Figure 26. Representative chromatograms obtained from oral fluid sample. The figure A represents a blank oral fluid sample without ethanol. The figure B represents an oral fluid sample containing ethanol. 77 1.1.4 Calibration curve In this work, the calibration curve of ethanol for blood samples was evaluated in the range of 0 g/L to 2.4 g/L. This calibration curve was obtained with six concentrations (0; 0.15; 0.30; 0.60; 1.20; 2.40 g/L). For oral fluid samples, the calibration curve was evaluated in the range of 0 mg/L to 0.60 g/L, and the calibration curve was obtained with five concentrations (0; 0.0375; 0.075; 0.30; 0.60 g/L). All blood/oral fluid samples were analyzed according to the procedure previously described (Chapter I) respectively. The weighted least squares regression equations and coefficients of correlation were calculated from these curves. The GC-FID chromatogram peak area ratios of ethanol/IS were determined to establish calibration equation (Figure 27). Figure 27. Calibration curves for blood and oral fluid samples. Plotted peak areas of the ethanol/IS peak areas versus concentrations (0; 0.15; 0.30; 0.60; 1.20; 2.40 g/L) for blood samples and (0; 0.0375; 0.075; 0.30; 0.60 g/L) for oral fluid samples The method was linear at the concentration range established, with determination coefficients (r2) greater than 0.99 for the calibration curves of THC for blood and oral fluid samples (Table 19). Table 19. Blood and oral fluid linear regressions analysis of ethanol standard solutions. Type of samples Y=mx+b R2 Concentration range (g/L) Blood Y=8.5735x +0.1575 0.9989 0-2.4 Oral Fluid Y=8.127x +0.0896 0.9987 0-0.6 78 2.1 Analysis of 9-THC 2.1.1 Sample preparation for gas-chromatography mass spectrometry 2.1.1.1 Extraction of samples In toxicological analysis, plasma or blood are the ideal samples for the detection and quantification xenobiotics. The concentrations of many xenobiotics, and their metabolites, tend to be higher in blood, thereby facilitating detection (Dinis-Oliveira et al., 2010). However, blood is a complex biological matrix containing many interferents, such as proteins, hormones and blood cells. The separation and elimination of this interferents is an important step for obtain good results. Actually the analyses of cannabinoids in biological samples are continually being developed in forensic toxicology. The mainly methods that are described for the extraction of cannabinoids from blood include solid phase extraction (SPE) (REF) and liquid–liquid extraction (LLE) (Andrews and Paterson, 2012). In this case the liquid-liquid extraction was quick, efficient and more favorable in 9-THC analysis over SPE due to the nature of the sample matrix. Using SPE method to extract 9-THC in blood samples, it was obtained a bad peak resolution and the compounds were not well separated (Figure 28(1)). However, in LLE the best results were obtained in THC analysis. (Figure 28(2)). The chosen LLE method for this study proved to be simple and rapid in the preparation of samples prior to analysis by GC-MS. Figure 28. Different methods of 9-THC extraction in blood samples. 1. Chromatogram of SPE method showing a bad peak resolution. 2. Chromatogram of LLE method showing a good peak resolution and good separation of the compounds. 79 2.1.1.2 Derivatization 9-THC has a tri-cyclic 21carbon structure without nitrogen and with two chiral centers in transconfiguration. It is a volatile viscous oil with high lipid solubility and low aqueous solubility and a pKa of 10.6 (Sharma et al., 2012). Due to the presence of only one hydroxyl group and its long chemical structure, it was necessary to test different compounds, times and temperatures of derivatization for obtaining the best peak resolution. In this study, 9-THC was derivatized by silylation, reacting with BSTFA and TMCS. BSTFA is the silylation reagent that reacts with 9-THC and replaces active hydrogens with a –Si(CH3)3 (trimethylsilyl) group (Figure 29). TMCS increases the reactivity of BSTFA. The derivatization process using BSTFA +1%TMC for 1h at 70 ºC, allowed an increase in 9-THC volatility, improving the thermal stability and consequently the detectability of the derivative (Figure 30) Figure 30. 9-THC with and without the derivatization step. 1. Chromatogram of 9-THC without derivatization step showing a bad peak resolution. 2. Chromatogram of 9-THC with derivatization step showing a good peak resolution. Figure 29. Derivatization reaction of THC 80 2.2 Method Validation of 9-THC 2.2.1 Detection by gas-chromatography mass spectrometry Preliminary tests were performed to determine the best conditions of chromatographic separation and detection in order to obtain the best peak resolution and separation of 9-THC. In the chromatogram, it is possible to identify the peak of 9-THC. Figure 31. Mass spectrum of 9-THC without derivatization. Figure 32. Mass spectrum of 9-THC derivatized. 81 Based on mass spectrum of each peak, for 9-THC the first peak represents 9THC without derivatization (Figure 31) the second peak shows 9-THC derivatized (Figure 32). For 9-THC, three ions were used. The most abundant ion m/z 386 was used for quantification and the other ions m/z 371 and 315 were used for the proper identification. 2.2.2 Selectivity The GC-MS chromatograms of spiked samples were compared with the chromatograms obtained with a blank blood sample. No interference peaks were detected in the retention times of 9-THC (Figure 33) or in the IS and selected ions (Figure 34). Figure 33. Reconstructed GC-MS (SIM mode) chromatogram of a blank blood sample (1000 ng/mL) and THC (m/z 386+371+315). Figure 34. Reconstructed GC-MS (SIM mode) chromatogram of a blank blood sample (1000 ng/mL) and internal standard (m/z 105+182). 82 2.2.3 Linearity In this work, the 9-THC linearity studies were evaluated in the range of 0 ng/mL to 1000 ng/mL. This calibration curve was obtained with nine concentrations (0, 1, 5, 10, 25, 50, 100, 500, 1000 ng/mL). The blood samples were analyzed according to a previously described procedure (Chapter I). The weighted least square regression equation and coefficient of correlation were calculated from this curve. The GC-MS chromatogram peak area ratios of 9-THC/IS were determined to establish calibration equation (Figure 35). Figure 35. 9-THC calibration curve. Plotted peak area of the 9-THC/IS peak area versus concentrations (0, 1, 5, 10, 25, 50,100, 500, 1000 ng/mL). The method was linear at the concentration range established, with determination coefficients (r2) greater than 0.99 for the calibration curve of 9-THC (Table 20). Table 20. Blood linear regression analysis of 9-THC standard solutions (0-1000 ng/mL). Psychoactive substance Y=mx+b R2 Concentration range (ng/mL) LOD (ng/mL) LLOQ (ng/mL) THC Y=0.0006x -0.0001 0.9996 0-1000 0.23 0.69 LOD, limit of detection; LLOQ, lower limit of quantification y = 0,0006x - 0,0001 R² = 0,9996 -0,1 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0 200 400 600 800 1000 1200 THC calibration curve ng/mL area THC/IS 83 2.2.4 Limit of detection and lower limit of quantification The LOD is 0.23 ng/mL and LLOQ is 0. 69 ng/mL for 9-THC (Table 20). These results show a good capacity of this method for the quantification of 9-THC analytes, although these analytes were in low concentrations. 2.2.5 Precision The %CV values calculated for 9-THC intra and inter-day precision studies did not exceed 15%, so that the developed method was considered precise (Table 21). 2.2.6 Accuracy The accuracies of 9-THC were determined in the range of 98,36-106,59% (Table 21), which are within the proposed acceptance limits for this parameter (100 ± 15%). 2.2.7 Recovery At three different concentrations of 9-THC (10, 100, 1000 ng/mL), the results obtained indicated an efficient clean-up procedure, with extraction recoveries in the range of 102.95 to 108.68%. The recoveries are within the proposed acceptance limits for this parameter (100 ± 20%) (Table 21). 2.2.8 Reproducibility At five equal concentrations of 9-THC (100 ng/mL), the results obtained did not exceed 15% of coefficient of variation. The reproducibility of 9-THC developed method was considered reproducible (Table 21). Table 21. Precision, accuracy, recovery and reproducibility (%) for THC. Analytes Concentration (ng/mL) Intra-day precision (%, n=3) Inter-day precision (%,n=3) Accuracy (%, n=3) Recovery (%) Reproducibility (%) THC 10 100 1000 4.25 0.34 0.64 7.03 1.61 3.29 98.35 101.17 106.59 108.68 102.95 105.09 6.98 90 difficulty to handle the sample in the laboratory; it may also be contaminated with food and other debris from the mouth. For these reasons, oral fluid cannot be seen as a substitute for blood or urine drug testing (Drummer, 2006). According to Jones (1979) (Jones, 1979) there is a relationship between ethanol concentration determined in oral fluid and in blood samples. This relationship is translated by the regression equation y=0.109+1.071x, where y is saliva ethanol concentration (mmol/l) and x is blood ethanol concentration (mmol/l). However, the results obtained in the present work, shows that oral fluid samples have lower concentration of alcohol when compared to blood samples. These results could be explained due to intraindividual reasons. Oral fluid can vary in flow rate, depending on several factors as emotional or hunger. The dry mouth syndrome could be caused by anxiety, lack of proper hydration of the individual and tobacco or THC smoke after drink, can also infuence. Hence, if the volume of oral fluid is less than 1 mL it is required the use of sensitive detection techniques. The stimulated production of oral fluid by citric acid candy, chewing gum or other agents will change the pH and concentration of drug in the oral fluid. This is scientifically proven for lower concentrations (Drummer, 2006). Other important factor that should be taken into account is the local where the blood was collected. While in study realized by Jones, 1979, he collected capillary blood samples, in the present study venous blood samples were collected. Works from other authors showed that the ethanol present in venous blood is less concentrated than that in capillary blood, until onset of the post-absorptive phase of ethanol metabolism (Jones, 1979). This could explain, in part, the results obtained in the present work. Finally, the evaporation of ethanol during the sampling procedure, as happens with blood samples could also contribute for the final result. In conclusion, there is a lot of intra and inter-subject variations due to the technique used, the individual physiology, the characteristic of the compounds and the local of the sample collection, that influence the concentration of alcohol in oral fluid samples. 1.2 Levels of ethanol in biological samples and its effects Although the concentration of ethanol in biological samples and its respective effects depends on many intra and inter individual factors previously described, the levels greater than 3-4 g/L can be fatal due to respiratory depression. The effects of alcohol ranging from minor impairment of motor coordination and sensation, to amnesia, loss of consciousness and coma when blood levels exceeding 300 mg alcohol/100mL blood (30 mg%) (Marc and Schuckit, 2006)(Table 23). 91 Table 23. Concentrations of ethanol in blood samples and its effects (adapted from emedicine.medscape.com). BAC (g/L) Effects of ethanol Less than 0.5 No loss coordination and slight euphoria. 0.6 – 0.8 Relaxation, lower inhibition, slight impairment of balance, speech and vision. 0.9 – 1.2 Significant impairment of motor coordination, loss of good judgment and euphoria. 1.3 – 3 Dysphoria, anxiety, nausea, needs of assistance and total mental confusion. 3 – 4 Loss of consciousness. More than 4 Onset of coma, possible death due to respiratory depression. Analyzing the ethanol results obtained in biological samples, it is possible to observe that among the 78 individuals, only 6 did not drink alcohol. Among 72 individuals, 17 participants have less than 0.5 g/L in biological samples, 10 individuals have values of ethanol between 0.6 g/L and 0.8 g/L, 9 individuals have values between 0.9 g/L and 1.2 g/L, 27 participants have values between 1.3 g/L and 3 g/L, and 9 individuals have values higher than 3g/L. With this investigation it was possible to find high levels of alcohol consumption in recreational spaces, by young people. The majority of them had high blood alcohol concentrations that could put them at severe risk. In spite of knowing the bad consequences of alcohol, some of them expected to consume high quantities of alcohol across the course of their night out. This situation is very worrying, mainly when there is an increased risk of social harm associated to alcohol comsumption , particularly violence, intoxication, DST´s, and other risk factors. Nowadays, some studies evidence the increased effectiveness of strategies to reduce alcohol-related harm in drinking environments, mainly in countries outside Europe. The measures for reducing the availability of alcohol, should focus on the restrictions of alcohol outlet density and increase alcohol price. These measures have significant effect, but are rarely used in practice (Hughes et al., 2011). 2. Application of GC-MS for detecting 9-THC in biological samples The method validated for 9-THC was applied to analyze THC in blood and oral fluid samples collected from young adults who were in nightclubs and affirmed having used cannabis on that night or on that week. Thirteen blood samples and two oral fluid samples were obtained from these participants. All blood samples were positive and the only two oral fluid samples were negative for 9-THC. 92 Analysis of blood samples revealed concentrations ranging from 4,69 to 62,40 ng/mL for THC. Reconstructed full scan chromatogram of real blood sample (subject number 14) is shown in Figure 38. Figure 38. Reconstructed GC-MS Full Scan chromatograms obtained from blood sample (1) positive for THC (subject number 14) and the respective THC standard (2). THC m/z = 386+371+315. As it happens with alcohol, for the correct interpretation of the THC results is important to take into account some aspects: i) the intra and inter individual physiology of each individual (age, gender, weight, height), ii) their behavioral (mixture of THC with other drugs, route of administration of THC), and iii) genetic features (that caused alterations in toxicokinetic and toxicodinamic of THC). Beyond these interindividual factors, there are also other variables such as: the origin of the THC and the precise quantity that the individuals put in their cigarettes. These factors were not possible to control, but could affect the concentration of Δ9-THC in biological samples. Behavioral features as mixture of THC with other psychoactive substances (alcohol or tobacco) could be dangerous, because this mixture becomes stronger and produces more unpredictable effects than if they were used separately. Some of the individuals that participated in this study, had smoked cannabis and ingested alcohol. Cannabinoids and alcohol activate the same reward pathways (dopaminergic system), and CB1 endocannabinoid receptor plays an important role in regulating the positive reinforcing properties of alcohol. When both are used together, its effects become stronger and could cause inhibit vomiting, dizziness and increased paranoia (Mechoulam and Parker, 2003). The route of administration used by THC consumers may influence the concentration of 9-THC in blood samples. When the drug is administrated orally, over 90 93 per cent of the dose was absorbed, and the plasma levels of metabolites of Δ9-THC peaked at three hours. When the drug is inhaled the metabolites peack from 10 to 140 minutes, and the physiologic effects are felt in this moment (Lemberger et al., 1972). 2.1 THC levels in blood samples and its effects 9-THC has higher lipophilicity, and therefore it is rapidly absorbed and distributed to tissues, passing through the mucosal epithelium into the bloodstream. When the ratio between blood/tissue is in equilibrium, it is possible to determinate a direct correlation of THC blood concentration and effect (Cone and Huestis, 1993; Huestis and Cone, 2004). The study realized by Schwope et al., 2012 (Schwope et al., 2012) demonstrated the concentrations of THC in whole blood, after some volunteers smoked one single cigarette, that contained 54mg of 9-THC. After 0,25h, they had, in mean, 150μg/L of THC in whole blood (ranging 93 to 250 μg/L), after 4h about 13μg/L (ranging 0-32 μg/L) and after 6h about 8ug/L (ranging 0-27μg/L). The effects that they felt over the time were: “high” (6685 μg/L of THC in blood), good drug effects, stoned, stimulated, sedated and anxious (Schwope et al., 2012). The author Mattes et.al, 1994 (Huestis and Cone, 2004) reported that 10-15mg of THC resulted in the production of 2,5ng/mL of THC in plasma after 2h following administration. Also, other authors reported that THC plasma levels were about 14 ng/mL, in six volunteers who consumed 20mg of THC (Huestis and Cone, 2004). Other studies, show that an estimated concentration of THC in blood samples, in a range of 7-29 ng/ml, causes 50% of the high effects expected for THC (Cone and Huestis, 1993). Comparing these studies, for the range of THC from 4,69 to 62,40 ng/mL in whole blood, and knowing that the ratio between blood to plasma of THC concentration is 0,5, it is possible to conclude that the concentration of THC found in the present study, is sufficient for causing 50% of maximal subjective high effects. In fact, among all THC users that participated in this study, only 2 individuals have THC blood concentrations lower than 7 ng/mL. Cannabis is currently one of the most widely used illegal drugs in Coimbra’ recreational spaces, an important information for those involved in risk management in Coimbra night life. 94 95 Chapter VI Comparison of participants answers with the identification of psychoactive substances in collected biological samples 96 97 1. Alcohol analyzed in biological samples vs answers about use of alcohol For the question about use of alcohol during that week, 87,18% of the participants refered to have used alcohol while 12,82% indicated absence of alcohol consumption during that week (Figure 39). Following analyses of biological samples, we observed that 92,31% of the participants gave positive results for ethanol and only 7,69% gave negative results (Figure 40). Consulting the table 24, we observe that subject #64 and #39, reported to have used alcohol, but the results where negative for ethanol. This could happen due to individual factors (shown in chapter V) or possible evaporation of ethanol during the sampling procedure. It is important to note that all of these dubious samples were analyzed two times separately in different days. For these reasons is very probable that some individuals have hidden the consumption of alcohol. Although the questionnaire is confidential, several of the participants showed reluctance and were afraid that the study becomes public, or to be shown by media. Figure 39. Answers given by participants about alcohol consumption. Figure 40. Biological samples analyzed by GC-FID method. 98 Table 24. Concentration of ethanol in blood, oral fluid and exhaled air samples, compared to responses given by the participants for the question about alcohol. Nº of samples Concentration blood samples (g/L) Concentration oral fluid samples (g/L) Concentration exhaled air (g/L) Answer about use of alcohol (Yes/No) Results obtained by GCFID 1 1,56 Nc 1.74 Yes Yes 2 1,19 Nc 1.27 Yes Yes 3 0,139 Nc Nc No Yes 4 Nd Nc Nc No No 5 0,604 Nc Nc Yes Yes 6 1,84 Nc Nc Yes Yes 7 1,34 Nc Nc Yes Yes 8 1,75 Nc Nc Yes Yes 9 2,15 Nc Nc Yes Yes 10 0,956 Nc 1.14 Yes Yes 11 0,770 Nc 0.93 Yes Yes 12 1,55 Nc 1.64 Yes Yes 13 1,19 Nc 1.63 Yes Yes 14 0,843 Nc Nc Yes Yes 15 Nc 0,016 Nc Yes Yes 16 0,053 Nc Nc Yes Yes 17* 0,369 0,049 1.14 Yes Yes 18 Nc 0,116 Nc Yes Yes 19* 1,01 0,013 Nc No Yes 20* 0,141 0,011 Nc No Yes 21 Nc Nd Nc No No 22 0,136 Nc Nc Yes Yes 23 Nc 0,022 Nc No Yes 24 Nc 0,019 Nc Yes Yes 25* 1,02 0,020 1.14 Yes Yes 26* 0,145 0,012 Nc Yes Yes 27* 1,48 0,014 Nc Yes Yes 28* 0,165 0,012 Nc Yes Yes 29* 0,049 0,013 Nc Yes Yes 30* 0,154 0,011 Nc Yes Yes 31 0,903 Nc Nc Yes Yes 32 0,572 Nc 1.72 Yes Yes 33 1,83 Nc Nc Yes Yes 34 0,105 Nc 0.70 No Yes 35 2,07 Nc Nc Yes Yes 99 36 0,696 Nc 2.33 Yes Yes 37 1,59 Nc Nc Yes Yes 38 1,11 Nc Nc Yes Yes 39 Nd Nc Nc Yes No 40 0,782 Nc Nc Yes Yes 41 0,890 Nc 1.52 Yes Yes 42 1,70 Nc 2.17 Yes Yes 43 0,181 Nc 0,69 Yes Yes 44 0,284 Nc 1.52 Yes Yes 45 0,136 Nc 0.63 Yes Yes 46 2,05 Nc 3.27 Yes Yes 47 0,041 Nc 0.66 Yes Yes 48 1,70 Nc 3.48 Yes Yes 49 0,059 Nc 0.39 Yes Yes 50 0,750 Nc 3.12 Yes Yes 51 1,46 Nc 3.88 Yes Yes 52 0,808 Nc 3.57 Yes Yes 53 0,389 Nc 1.12 Yes Yes 54 0,639 Nc 1.66 Yes Yes 55 0,611 Nc 1.59 Yes Yes 56 0,686 Nc 1.88 Yes Yes 57 2,07 Nc 3.12 Yes Yes 58 0,678 Nc 1.49 Yes Yes 59 1,33 Nc 3.27 Yes Yes 60 1,87 Nc Nc Yes Yes 61 0,122 Nc 1.20 Yes Yes 62 0,512 Nc 0.9 Yes Yes 63 0,856 Nc 1.71 Yes Yes 64 Nd Nc Nc Yes No 65 Nd Nc Nd No No 66 Nd Nc Nd No No 67 0,043 Nc 0,17 Yes Yes 68 0,620 Nc 2,05 Yes Yes 69 Nc 1,18 3.30 Yes Yes 70 1,27 Nc 2,07 Yes Yes 71 1,16 Nc Nc Yes Yes 72 1,19 Nc 2,72 Yes Yes 73 Nc 0,026 0,18 Yes Yes 74 Nc 0,055 Nc No Yes 75 Nc Nc 0,19 Yes Yes 106 frequency of drunkenness increases (Calafat et al., 2008). Also in our study, was possible to observe a discrepancy among genders in relation to recreational habits. Males have more tendencies for going out more times, using more than one type of psychoactive substance in the same night, and use more illegal drugs than females. Studies realized by EMCDDA, 2005 corroborate that the number of females in relation to males is generally lower for illegal drugs and have lower incidence of recent or frequent patterns of drug use. These discrepancies between genders tend to be lower over the years (EMCDDA, 2005). Another study realized in two different Spanish islands (Ibiza and Mallorca), reported that cannabis and cocaine were more commonly used by males in both locations (Hughes et al., 2009). Relatively to the frequency of nights out per week, Lomba, 2008, affirmed that inquired youngsters go out about seven days per month, mainly on weekends. This is concordant with our study. However, we observed a light increased in nights out per month, with an average of 8,01 (Lomba et al., 2008). In respect of problems due to use of psychoactive substances, this study clearly showed that the participants who used more harmful substances, had more problems in all sectors. Relatively to health problems due to use of psychoactive substances, our study revealed higher incidence in cocaine users (odds ratio of 3,22), followed by cannabis (2,56). The use of alcohol was also a positive relation. In fact, a study realized by Hughes, 2009 in two different locations (Malorca and Ibiza) reported that illness, discussions, physical fighting, unintentional injuring and needing to go to hospital, were more commonly reported by users of illicit drugs, especially in Ibiza. This fact could occur due to drug patterns in Ibiza have revealed to be relatively high, when compared with studies in other places, as in Portugal or whole Spain. In contrast, the drug patterns in Malorca had more concordance to our study. The number of cocaine users was 7,5% and ecstasy was 4% (Hughes et al., 2009). Another subject that is associated with the consumption of psychoactive substances is the risky sexual behavior. Dowing, 2010 reported a comparative crosssectional between Germany, British and Spanish users. She concluded that the main factor for 34,1% of the participants contributing for sex on holiday, was high levels of drunkenness (Dowing et al., 2010). However, in your study this data does not have statistic relevance. Only one user reported to have had a sexual regret due to use of psychoactive substances. This could happen due Coimbra is not a characteristic touristic city. In fact, the number of touristic people in our study was much reduced. Driving under influence of psychoactive substances and related driving-related problems, are also relevant subjects in our study. It is known that alcohol and cannabis users have an increased risk of 1,5x and 1,6x of having driving-related problems, 107 respectively, while cocaine users have a 3x increased risk. In our study, we verified that 25,6% of the participants have already driven under influence of psychoactive substances.. These results are in partial agreement with the study published by Calafat, 2008, in which he observed a higher number of participants that had already driven drunk, or drugged (65,2%) (Calafat et al., 2008). Nowadays, several authors have addressed some interventions that could be implemented in recreational spaces, with objective to reduce harm associated with recreational nightlife venues. Calafat, 2012, is one of these authors that highlight the importance of regulating standards that should be implemented in European recreational settings. For this author, the use of preventive interventions including venue management, underage checkouts, staff training and collaboration with the police, are some of prevention standards that could exist in nightlife industry (Calafat et al., 2012). In fact, all of these measures could implemented; however for many countries, including Portugal, it means more costs. Nevertheless, to ensure promotion of health and safety in nightlife spaces, risk assessment and risk control are lifesaving issues that should become an incentive to each and every one of us. 108 109 Part V Conclusion and future perspectives 110 111 1. Conclusion I. Drug abuse patterns in Coimbra recreational nightlife was monitored in this work through the use of a survey and collection/analysis of biological samples. II. Although this is a pilot study with only 78 samples, it was possible to obtain significantly different data. III. In general males go out with more frequency, using more psychoactive substances, both in quantity as in diversity, than females. IV. Alcohol was the most used psychoactive substance (87,2%), followed by tobacco (64,1%), cannabis (19,2%), cocaine and amphetamines (2,6%) and finally LSD and smartshop substances (1,3%). V. Males have more tendencies to be polyusers. VI. Cocaine users have the highest risk for health problems perceptive to the users, followed by cannabis, alcohol and finally tobacco. VII. Relatively to biological samples, ethanol was the substance that has more discordance, comparing to the surveys. 92,31% of the participants gave positive results for ethanol while only 87,18% of them answered positively for the consumption of alcohol. VIII. The THC results show more concordance, when compared to the answers of the surveys. All samples were positive for THC, except two oral fluid samples. IX. A sensitive, reproducible, precise, accurate and inexpensive GC-MS method was developed and validated to identify and quantify THC in whole blood samples. X. The proposed GC-MS method was successfully applied in the quantification of THC in real blood samples and shown to be appropriate for routine analysis. Blood samples represent the most used matrix with relevant importance in toxicological analysis. XI. Liquid-liquid extraction revealed to be the best extraction, allowing to obtain the best peak resolution and separation of the compounds. 112 XII. The use of exhaled air to analyze the concentration of ethanol revealed to be the best method. However, the use of more than one matrix to quantify and qualify ethanol is essential for having a precise and better result. 113 2. Future Perspectives I. Using screening methods, it was not possible to detect amphetamines and cocaine. The next step will be analyzing these samples with high sensitive methods. II. 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