The aim of this study was to investigate the utility of direct analysis in real time quadrupole time-of-flight mass spectrometry and gas chromatography quadrupole time-of-flight mass spectrometry in the analysis of 162 different synthetic cannabinoids. Direct analysis in real time quadrupole time-of-flight mass spectrometry is shown to be a rapid and accurate analytical method for synthetic cannabinoids. Spectra can be generated with less than 1.5 ng of the drug in under a minute and be successfully searched against previously generated ESI-QTOF libraries in most cases (118/130 drugs tested) as well as can also be applied to the identification of synthetic cannabinoids in a mixture. Gas chromatography quadrupole time-of-flight mass spectrometry, while requiring a much longer analysis time, is shown to accurately distinguish all but 19 compounds (140/159). These two instruments have proven to be viable alternatives in synthetic cannabinoid analysis and will greatly benefit forensic laboratories.

Background: Background of this work was the investigation of the endocannabinoid system (ECS) in the Fmr1 knock- out (KO) mouse. The Fmr1- KO mouse is a mouse model for fragile X syndrome (FXS). FXS is the leading monogenic cause for autism spectrum disorders (ASD) in humans. The Fmr1- KO mouse displays autistic behavior such as an impaired social interaction, repetitive behavior, cognitive deficits, increased anxiety and aggressiveness. Alterations of the ECS have been suggested to play a key role in the etiopathology of a variety of neuropsychiatric disorders. Until today, little has been described about the involvement of the ECS in ASD. Interrogation: 1. Evaluating the manifestation of typical cannabinoid- induced effects in the Fmr1- KO mouse 2. Investigating the influenceability of autistic symptoms with THC treatment in the Fmr1- KO mouse 3. Analyzing the signaling cascade of the stimulated and unstimulated ECS in different brain regions of the Fmr1- KO mouse Material and Methods: Experiments were carried out on adult (12±1 weeks old) male Fmr1- KO and Fmr1- wild- type (WT) mice from the C57BL/6J- (B6)- background. N= 15 mice received THC (10mg/kg bodyweight) and N= 16 received WIN55,212 (3mg/kg bodyweight). 30min after injection, the body temperature was measured and the distance animals moved in an open field during 15min was recorded (locomotion). Then, animals were placed with their forepaws onto a horizontally fixed bar and the time remaining in this position (catalepsy) was measured. Finally animals were placed on a preheated plate and the temperature at which a pain stimulus occurred was determined (testing analgesia). All 4 experiments are called tetrad experiment. Afterwards changes in body temperature, locomotion, catalepsy and analgesia of the animals was evaluated. To explore long-term effects of THC after the tetrad, N= 15 animals were tested in a social interaction test with a female contact mouse, 10 and 20 days after THC treatment. Therefore, the tested mouse and the contact mouse were placed together into a cage and the time mice spent in social interaction (nose, body and anogential sniffing, allogrooming and body contact) was manually quantified during 6min of recorded testing time. Another group of N= 19 received a premedication of rimonabant (Cannabinoid- receptor 1 (CB1) antagonist, 3mg/kg bodyweight) 30min prior to THC treatment. Rimonabant prevents THC from binding to CB1 and therefore allows the assessment of the involvement of CB1 in mediating social behavior. Furthermore the suggestibility of context-dependent fear conditioning with THC treatment has been tested on N= 13 mice. Animals were placed into a conditioning chamber that delivered 6 short electric shocks with a 30sec pause to their paws (conditioning phase). Immediately afterwards mice received THC or placebo. 24h later contextdependent fear was evaluated by quantification of the time mice spent freezing in the conditioning-chamber (fear) without receiving foot shocks. Intraneuronal signaling of the ECS was analyzed with N= 29 animals using western blots. Quantities of phosphorylated (“activated”) protein kinases (ERK, AKT and S6) from different brain homogenates (hippocampus, striatum, cortex and cerebellum) were therefore measured after THC or placebo injection (30 minutes prior to sacrificing). Results: Cannabinoids induced hypothermia, hypolocomotion, analgesia and catalepsy in WTmice. These effects were significantly less detectable in Fmr1- KO mice. Effects of both cannabinoids, THC and WIN55,212, were comparable with a slightly greater but not significant efficiency of THC. THC treated WT- mice exhibited further reduced social interaction 10 days after treatment, an effect that was partially prevented by premedication with rimonabant. THC increased social interaction in Fmr1- KO mice comparable to the level of untreated WT- mice. THC had no effect on behavior of WT- mice in context-dependent fear conditioning. Fmr1- KO mice showed significant less contextdependent fear conditioning compared to WT- mice. THC facilitated the recognition of an anxiety-correlated context in Fmr1- KO mice comparable to untreated WT- mice. In western blots significant changes in the THC- induced signaling cascade were detectable and depending on genotype, brain-region and analyzed protein-kinase. In the hippocampus there were no changes in untreated Fmr1- KO mice compared to WT- mice. THC had no effect on activation of protein-kinases in WT- and Fmr1- KO mice. In the striatum there were no changes in untreated Fmr1- KO mice compared to WTmice. THC significantly increased activity of ERK, AKT and S6 in WT-mice and not in Fmr1- KO mice. In the cortex of untreated Fmr1- KO mice AKT showed a significantly increased activity compared to WT- mice. THC significantly increased AKT activity in WT- mice without having an effect on KO- mice. In the cerebellum there were no changes in untreated Fmr1- KO mice compared to WT- mice. THC significantly increased ERK- activity in Fmr1- KO mice but had no effect on protein kinase activity in WT- mice. Conclusion: We observed physiological cannabinoid effects in WT- mice after treatment with THC and WIN55,212. These effects are significantly attenuated in Fmr1- KO mice. This may be interpreted as a desensitization of the ECS in the Fmr1- KO mouse. At the same time it was demonstrated that THC has the potential to improve context dependent memory consolidation and to increase social interaction in the Fmr1- KO mouse. In particular the influence of THC on impaired social interaction should be a target of further investigations to find possible therapeutic options for this typical symptom of Autism. Underlying molecular mechanisms remain unclear and the analysis of THC stimulated intraneuronal signaling gave no clear indication of possible molecular alterations in the Fmr1- KO mouse.

Anhand einer prospektiven epidemiologischen Verlaufsstudie an einer repräsentativen Bevölkerungsstichprobe von ursprünglich 14- bis 17jährigen Jugendlichen (N=1395) werden die Häufigkeit und die Verlaufsmuster des Konsums, des Mißbrauchs und der Abhängigkeit von Cannabis untersucht. Die Jugendlichen wurden persönlich mit Hilfe eines Interviews befragt, wobei als diagnostisches Instrument das M-CIDI mit seinen DSM-IV Algorithmen verwendet wurde. Die Ergebnisse aus der Basiserhebung ergaben, daß 1995 etwa 20% aller 14- bis 17jährigen - in der Mehrzahl wiederholt - Cannabis probiert hatten und 6% der Population einen regelmäßigen Gebrauch aufweisen. Die Einjahres-Inzidenz (= Auftreten neuer Fälle) für den Cannabisgebrauch der nun 15- bis 19jährigen betrug zum Zeitpunkt der Nachuntersuchung 20,1% für die Männer und 16,3% für die Frauen. Für den regelmäßigen Gebrauch wurden Inzidenzraen von 12% (Männer) und 8,4% (Frauen) ermittelt. Die Stabilität des Konsumverhaltens zwischen der Basis- und der Nachuntersuchung war relativ hoch un stieg mit höherem Initialkonsum an. Angesichts der hohen Gebrauchsrate von 32,4% bei den 15- bis 19jährigen bei der Nachuntersuchung ergab sich im Vergleich zu anderen psychotropen Substanzen eine relativ niedrige Prävalenz für Mißbrauch und Abhängigkeit (4%). Das Vorliegen einer Diagnose bei der Erstuntersuchung weist allerdings eine relativ hohe Stabilität über die Nachuntersuchungskette auf. / The paper examines the prevalence, incidence and stability patterns of cannabis use and dependence in a prospective epidemiological study of a random community sample of adolescents, aged 14 to 17 years (N=1395) at time 1. Assesments are based on personal interviews by trained clinical psychologists using the computerized DSM-IV lifetime and 12-month change version of the M-CIDI. Results indicate that in 1995 every fifth person aged 14 to 17 years had used cannabis at least once and 6% reported regular use. The 12-month incidence rates (i.e. proportion of the sample reporting first cannabis use in the last year) among subjects, 15 to 19 years of age at time 2, was high, with 20,1% of males and 16,3% of females reporting first use. The incidence of regular use was 12,0% (males) and 8,4% (females). The stability of consumption patterns from time 1 to time 2 was high. Increasing with higher initial consumption. Given the reexamined samples overall high cannabis consumption rates of 32,4%, the prevalence of clinically manifest DSM-IV abuse and dependence was low in comparison to other psychologic drugs (4%). However there was remarkable diagnostic stability over time in this age group of those with an initial diagnosis at time 1 receiving a diagnosis again at time 2.

The thesis was divided into two parts. The first part deals with selected long-term impacts of cannabis use and basic concepts such as history, methods of application, use or legislation. In order to understand the whole issue, the basic components of the cannabis plant and their effects. The main part of the theory was then devoted to the possibilities of social work in the field of addiction, but also health complications and therapeutic effects of cannabis. The first part was built on a professional basis literature that addresses this topic. As the work dealt with a very current topic, the latest HARP treatment program was also mentioned. In the second part of the work semi-structured interviews were performed in order to confirm the theoretical part of the work and bring a certain authenticity to the work. The goal was to offer to the reader comprehensive information about cannabis in one place, clearly and transparently. The negatives and the positives that this plant can cause as a result of use were analyzed.

Factors that confer predisposition and vulnerability for alcoholism and other substance abuse disorders may be described usefully within the gene-environment interplay framework. Thus, it is postulated that heritability provides a major contribution not only to alcohol but also to other substances of abuse. Studies of evoked potential amplitude reduction have provided a highly suitable and testable method for the assessment of both environmentally-determined and heritable characteristics pertaining to substance use and dependence. The different personal attributes that may co-exist with parental influence or exist in a shared, monozygotic relationship contribute to the final expression of addiction. In this connection, it appears that personality disorders are highly prevalent co-morbid conditions among addicted individuals, and, this co-morbidity is likely to be accounted for by multiple complex eti-ological relationships, not least in adolescent individuals. Co-morbidity associated with deficient executive functioning may be observed too in alcohol-related aggressiveness and crimes of violence. The successful intervention into alcohol dependence and craving brought about by baclofen in both human and animal studies elucidates glutamatergic mechanisms in alcoholism whereas the role of the dopamine transporter, in conjunction with both the noradrenergic and serotonergic transporters, are implicated in cocaine dependence and craving. The role of the cannabinoids in ontogeny through an influence upon the expression of key genes for the development of neurotransmitter systems must be considered. Finally, the particular form of behaviour/ characteristic outcome due to childhood circumstance may lie with biological, gene-based determinants, for example individual characteristics of monoamine oxidase (MAO) activity levels, thereby rendering simple predictive measures both redundant and misguiding.

<p>U okviru ovog rada izvr&scaron;eno je ispitivanje različitih procesa ekstrakcije herbe industrijske konoplje. Za ekstrakciju određenih bioaktivnih jedinjenja primenjene su klasične tehnike ekstrakcije, maceracija i Soxhlet ekstrakcija, dok su od savremenih metoda primenjene ultrazvučna ekstrakcija, mikrotalasna ekstrakcija, ekstrakcija vodom u subkritičnom stanju i ekstrakcija ugljendioksidom u superkritičnom stanju. Praćenjem uticaja ulaznih promenjivih (procesnih parametara) na odabrane izlazne promenjive (sadržaj bioaktivnih jedinjenja ili antioksidativnu aktivnost) za svaku tehniku ekstrakcije pojedinačno utvrđeni su optimalni uslovi ekstrakcije. Nakon izvr&scaron;enog poređenja odabrana je najadekvatnija tehnika ekstrakcije za pripremu tečnog ekstrakta koji će biti transformisan u suvi ekstrakt primenom metode su&scaron;enja raspr&scaron;ivanjem. Prilikom dobijanja suvog ekstrakta ispitan je uticaj različite koncentracije nosača na kvalitet ekstrakata i efikasnost su&scaron;enja. Dobijeni ekstrakti su okarakterisani u pogledu fizičkih i hemijskih osobina. Sadržaj kanabinoida u tečnim ekstraktima suve herbe industrijske konoplje ispitanih u ovoj doktorskoj disertaciji je bio u opsegu od 0,4314 mg/mL CBD-a i 0,0364 mg/mL THC-a (u optimalnom ekstraktu dobijenom maceracijom) do 1,0420 mg/mL CBD-a i 0,0430 mg/mL THC-a (u optimalnom ekstraktu dobijenom mikrotalasnom ekstrakcijom). U ekstraktima koji su nepolarnog karaktera i koji su dobijeni primenom Soxhlet ekstrakcije sadržaj CBD-a i THC-a je bio 64,40 i 2,90 mg/g, dok je primenom superkritične ekstrakcije dobijen sadržaj CBD-a i THC-a u opsegu od 76,1193 do 163,1111 mg/g i od 4,1746 do 6,5803 mg/g. Tečni ekstrakti dobijeni primenom vode i etanola kao ekstragensa bili su bogati flavonoidima i fenolima, a utvrđeno je i da imaju značajnu antioksidativnu aktivnost, dok su ekstrakti dobijeni Soxhlet ekstrakcijom i ekstrakcijom ugljendioksidom u superkritičnom stanju bili bogati lipofilnim jedinjenjima i terpenima. Ovako dobijeni ekstrakti predstavljaju visokovredne proizvode koji imaju veliki potencijal za upotrebu u farmaceutskoj industriji.</p> / <p>Various extraction processes of the aerial parts of industrial hemp were examined. From classic extractions, maceration and Soxhlet extraction were applied, while from novel methods, ultrasound-assisted extraction, microwave-assisted extraction, subcritical water extraction and supercritical carbon dioxide extraction were applied. By monitoring the influence of the input variables on the selected output variables for each extraction, the optimal extraction conditions were determined separately. After a comparison of all the extraction processes tested, microwave-assisted extraction was selected to prepare the liquid extract for spray drying. The effect of different carrier concentrations was examined during the preparation of the dry extract. The extracts obtained were characterized in terms of physical and chemical properties. The content of cannabinoids in liquid extracts of dry Cannabis sativa herba examined ranged from 0.4314 mg/mL for CBD and 0.0364 mg/mL for THC in the optimum extract obtained by maceration to 1.0420 mg/mL for CBD and 0.0430 mg/mL for THC in the optimum extract obtained by microwave-assisted extraction. The CBD and THC content in extract obtained by Soxhlet extraction was 64.4000 mg/mL and 2.9000 mg/mL, respectively, while the highest content of CBD and THC in the supercritical carbon dioxide extract was 163.11 and 6.5803 mg/g. In addition to cannabinoids, the liquid extracts obtained were rich in phenols and flavonoids, and have considerable antioxidant activity, while the extracts obtained by Soxhlet extraction and supercritical carbon dioxide were rich in lipophilic compounds and terpenes. The extracts mentioned above are high value products which have great potential for implementation in the pharmaceutical industry.</p>

No description available.

The neural circuitry responsible for memory consists of complex components with dynamic interactions. In hippocampal area CA1, interactions between excitatory pyramidal cells and inhibitory interneurons shape ensemble activity which encodes sequential experience. An extremely diverse set of inhibitory interneurons, with variation in gene expression, synaptic targeting, state-dependent activity, and connectivity, contribute substantially to circuit activity, such as theta and sharp wave-ripple oscillations. The precise roles of each interneuron group is not well understood, though characterization of their activity reveals mechanisms underlying hippocampal circuit computation. In this dissertation, I aim to identify and disrupt interactions between pyramidal cells and local interneurons to clarify their role in shaping cell assembly activity. We characterized axo-axonic cell activity in sharp wave-ripples, and compared their control of pyramidal cell activity and ripple events to parvalbumin expressing neurons. We identified pyramidal cell-interneuron interactions during ripples, suggesting they serve as lateral inhibitors between cell assemblies. We additionally developed and implemented a novel neural device to explore the role of cannabinoid disruption of hippocampal oscillations and organization of assemblies in vivo in awake animals. We demonstrate that cannabinoid receptor type 1 within CA1 is responsible for suppression of theta and SPW-Rs. We also found that cannabinoid activation within CA1 circuitry, regardless of muted input from CA3, was sufficient to disrupt sharp wave-ripples, likely through interference of pyramidal cell-interneuron interactions. The work in this dissertation provides insight suggesting that interneuron activity must be studied at the spiking timescale to characterize their control over cell assembly activity. / Doctor of Philosophy / Understanding how the brain creates memory remains one of the greatest questions in the field of neuroscience. Coordinated brain activity serves to build communication on large and small scales, across brain regions and within circuits consisting of small groups of neurons. Precise coordination of activity and communication across neurons and regions is thought to build salient experience, which is achieved through the timing of neuron action potentials, or spikes. Neurons receive thousands of inputs that control their spiking activity. "Go and stop" signals from excitatory and inhibitory interneurons act to conduct synchronized activity, which is required for proper circuit function. Importantly, coordinated spiking across large groups of neurons is responsible for observed "brain waves", or oscillations, which reflect organized activity. In CA1 of the hippocampus, there are >20 subtypes of interneurons that all make distinct contributions to memory function, and the roles of these interneurons have not been fully studied within behaving animals. As engineers develop new tools, new methods become available to study and classify how unique groups of interneurons play a part in circuit activity. Thus, we sought to characterize the role of axo-axonic cells, a specialized interneuron with strong control over spiking activity, in hippocampal oscillations that are responsible for memory encoding and consolidation. We identified a new role for axo-axonic cells in the regulation of pyramidal cell spiking in sharp wave-ripple oscillations. Additionally, we developed a novel neural device that allowed us to investigate the mechanisms that underlie cannabinoids, molecules found in Cannabis sativa, and memory dysfunction. We leveraged the multifunctionality of our T-DOpE probe to focally deliver synthetic cannabinoid into the hippocampus in combination with optical control of circuits, with simultaneous recording of activity. We found that cannabinoids acting within CA1 sufficiently disrupt hippocampal oscillations, likely through hindering pyramidal cell-interneuron interactions. Together, these findings suggest that the spatial and temporal resolution required to study diverse roles of interneurons is high, and experiments designed to explore interneuron activity should especially emphasize fine time-scales.

O preparo da amostra é uma das etapas mais importantes em toda a análise química. O isolamento e a concentração dos componentes da amostra são cruciais e busca-se sempre que essas etapas sejam as mais simples e consumam o mínimo possível de tempo e reagentes. Nos últimos anos, um tipo de material tem se mostrado bastante útil para análises químicas a partir de fluidos biológicos, os polímeros molecularmente impressos (MIPs). Os MIPs são sintetizados por reações de polimerização, na presença de uma molécula molde (template). A molécula molde se liga aos monômeros funcionais do polímero durante a reação de polimerização e permanece ligada à superfície das cadeias poliméricas quando a reação se completa. Terminada a polimerização, realiza-se a completa lavagem das moléculas molde, assim, restam na superfície polimérica cavidades tridimensionais complementares à molécula empregada como molde. Essas cavidades permitem a ligação reversível e preferencial da molécula molde ou outras com estrutura química semelhante. A Cannabis sativa é a droga ilícita mais consumida em todo o mundo e nos últimos anos muita atenção tem se voltado a seus efeitos toxicológicos no corpo humano e a aplicações medicinais. Nesta dissertação, foi sintetizado um MIP com a molécula molde catequina para a extração e posterior análise por LC-MS/MS dos canabinóides &Delta;9-tetrahidrocanabinol (THC), 11-hidroxi-&Delta;9-tetrahidrocannabinol (THC-OH) e 11-nor-&Delta;9-tetrahidrocannabinol-9-ácido carboxílico (THC-COOH) em amostras de urina. O MIP produzido foi empacotado em microdispositivo e empregado no preparo das amostras de urina por microextração por sorvente empacotado (MEPS). O método desenvolvido apresentou boa linearidade (valores de r de 0,977 para o THC e 0,994 para THC-OH e THC-COOH). Os limites de detecção e de quantificação foram respectivamente de 5 ng mL-1 e 20 ng mL-1, para os compostos THC e THC-OH, na faixa linear de 25 a 250 ng mL-1. Para o composto THC-COOH os limites de detecção e quantificação alcançados foram de 1 ng mL-1 e 5 ng mL-1, respectivamente, na faixa linear de 5 a 170 ng mL-1. O método apresentou valores razoáveis de precisão entre 3,2% (THC-COOH) e 25,1% (THC) e de exatidão, que variou entre -18,4 e 17,4 (ambos para o THC). O MIP empregado no preparo da amostra mostrou-se mais seletivo e específico do que materiais normalmente empregados para a extração dos canabinoides das amostras de urina, além de a técnica de extração por MEPS apresentar baixo consumo de solventes e amostra para a extração dos analitos e posterior análise por LC-MS/MS. / The sample preparation is one of the most important steps in every chemical analysis. The isolation and concentration of the sample components are crucial and it is always sought that these steps are simple and consume the lowest amount of time and reagents. In the recent years, a type of material has proved to be very useful for chemical analyzes of biological fluids, the molecularly imprinted polymers (MIPs). MIPs are synthesized by polymerization reactions in the presence of a template molecule. The template molecule binds to the functional monomers of the polymer during the polymerization reaction and remains bonded to the surface of the polymeric chains after the reaction is complete. After the polymerization is finished, the complete washing of the template molecules is carried out, thus, three-dimensional cavities, complementary to the molecule used as a template, remain on the polymer surface. These cavities allow the reversible and preferential bonding of the template molecule or others with similar chemical structure. Cannabis sativa is the most commonly consumed illicit drug in the world and in recent years much attention has focused on its toxicological effects on human body and for medical applications. In this master dissertation, a MIP was synthesized with the catechin molecule as template, for extraction and subsequent analysis by LC-MS/MS of the cannabinoids &Delta;9-tetrahydrocannabinol (THC), 11-hydroxy-&Delta;9-tetrahydrocannabinol (THC-OH), and 11-nor-&Delta;9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in urine samples. The MIP produced was packed in a microdevice and used in the preparation of the urine samples by microextraction by packed sorbent (MEPS). The developed method showed good linearity (r values of 0.977 for THC and 0.994 for THC-OH and THC-COOH). The detection and quantification limits were respectively 5 ng mL-1 and 20 ng mL-1 for THC and THC-OH in the linear range from 25 to 250 ng mL-1. For the compound THC-COOH the limits of detection and quantification achieved were 1 ng mL-1 and 5 ng mL-1, respectively, in the linear range from 5 to 170 ng mL-1. The method presented reasonable values of precision, between 3.2% (for THC-COOH) and 25.1% (for THC) and displayed accuracy ranging from -18.4 to 17.4 (both for THC). The MIP used in the sample preparation was more selective and specific than other materials usually employed for the extraction of the cannabinoids from the urine samples. The MEPS technique also showed low consumption of solvents and sample for sample preparation, extraction of analytes and subsequent analysis by LC-MS/MS.

O aleitamento materno está associado a benefícios de ordem nutricional, imunológica, afetiva, econômica e social. Grande parte das drogas lícitas e ilícitas consumidas pela lactante pode ser transferida para o leite, o que pode representar potenciais efeitos nocivos para a criança em curto e longo prazos. Embora o conhecimento a respeito de substâncias psicoativas durante a lactação venha sendo ampliado, ainda há poucas informações sobre as concentrações de substâncias excretadas por essa via e muitas dúvidas sobre os efeitos delas em crianças durante o período de amamentação. O leite materno é uma matriz não convencional que pode ser usada para avaliar a exposição de crianças a substâncias durante o aleitamento e sua principal vantagem é a coleta simples e não-invasiva. Contudo, a extração destas a partir do leite materno é um desafio analítico devido ao seu alto conteúdo lipídico e proteico, bem como a alteração na sua composição durante o período pós-parto. No presente trabalho, técnicas analíticas miniaturizadas foram desenvolvidas visando a detecção das substâncias psicoativas cocaína, cocaetileno, norcocaína, tetraidrocanabinol, canabinol, canabidiol em amostras de leite materno, utilizando cromatografia gasosa acoplada à espectrometria de massas (GC-MS). A microextração em fase líquida (LPME) foi aplicada para analitos de cocaína e a microextração em fase sólida (SPME) para canabinoides. Após o desenvolvimento e validação dos métodos, as análises foram aplicadas em amostras de leite coletadas no hospital municipal Dr. Arthur Ribeiro de Saboya (n=109). Além das técnicas terem se mostrado adequadas à extração e detecção dos analitos, confirmou-se a excreção de cocaína no leite materno em quantidades moderadas com resultado positivo para uma amostra (138 ng/mL). Bem como foi detectado tetraidrocanabinol nas concentrações de 20 ng/mL e 31 ng/mL em amostras de leite humano. / Maternal breastfeeding is related with nutritional, immunological, affective, economic, and social benefits. Most licit and illicit substances consumed by the nursing mother might be excreted in breast milk, which may cause potential harmful effects to the breastfed infant at short and long terms. Although the knowledge about the psychoactive substances during lactation is increasing, there is still few information on the levels in which these substances are excreted in breast milk and many questions about the possible effects of these substances in the infant during the nursing period still remain unanswered. Breast milk is an unconventional matrix that can be used to assess infant exposure to drugs, and its main advantage is its easy and non-invasive collection. However, the extraction of substances from breast milk is an analytical challenge because of its high protein and fat content and changing composition during the postpartum period. In this study, miniaturized analytical techniques will be carried out to determine psychoactive substances such as cocaine, cocaethylene, norcocaine, tetrahydrocannabinol, cannabinol, and cannabidiol in milk samples by using gas chromatography-mass spectrometry (GC-MS). Liquid-phase microextraction (LPME) was applied to cocaine analytes and solid-phase microextraction (SPME) was applied to cannabinoids. After the development and validation, the methods were applied in milk samples collected from municipal hospital Dr. Arthur Ribeiro de Saboya (n=109). These techniques have proved to be suitable for analyte extraction and detection. Cocaine excretion in breast milk has been confirmed given that one sample was positive to cocaine in moderate amounts (138 ng/mL), as well as, two samples could be confirmed positive to tetrahydrocannabinol at 20 ng/mL and 31 ng/mL.