As chamadas club drugs compreendem um vasto grupo de substâncias frequentemente utilizadas em bares, festas e raves, com a finalidade de intensificar o contato social e a estimulação sensorial. Englobam desde substâncias sintéticas comumente conhecidas, como a anfetamina, a metanfetamina, o MDMA, até moléculas de surgimento mais recente, denominadas novas substâncias psicoativas. Isoladas ou associadas a outras drogas, é possível que sejam causa de morte per se, ou que predisponham o usuário a envolver-se em situações potencialmente fatais, sendo necessário que os órgãos de Perícia Criminal (Institutos Médico Legais e Institutos de Criminalística) estejam aptos a detectar e quantificar essas substâncias em amostras biológicas. O presente trabalho teve como objetivo desenvolver um método analítico para identificação e quantificação de club drugs em sangue total, utilizando cromatografia líquida acoplada a espectrometria de massas com analisador híbrido quadrupolotempo de voo (LC-QTOF). Após o desenvolvimento do método, este foi validado utilizando as diretrizes do guia de validação do Scientific Working Group for Forensic Toxicology (SWGTOX), sendo analisados de linearidade, limite de detecção, limite de quantificação, efeito matriz, precisão intradia, precisão interdia, exatidão e integridade de diluição, além de recuperação e eficiência do processo. O método desenvolvido compreendeu a determinação de MDA, MDMA, 2C-B, DOB, cetamina, mCPP, cocaína e cocaetileno. Amostras provenientes de casos reais de morte não natural, oriundas do Instituto Médico Legal Aristoclides Teixeira de Goiânia - GO foram analisadas pelo método desenvolvido. 56 casos foram selecionados, em sua maioria com histórico de morte por projétil de arma de fogo e acidente de transito. Das 56 amostras analisadas, 28,5% (n=16) foram positivas para cocaína e/ou cocaetileno. As demais substâncias pesquisadas não foram encontradas nas amostras. / Club drugs are a large group of substances consumed in pubs, parties and raves, aiming to intensify social contact and sensorial stimulation. The term comprises largely known substances such as amphetamine, methamphetamine, 3,4-methylenodioxymethamphetamine (MDMA), as well as so-called new psychoactive substances, which are synthetic drugs recently developed or recently introduced in drug market. Club drugs can be taken alone, combined with each other or, most frequently, with alcohol or other commonly abused drugs such as cocaine. In any of these situations, club drugs can possibly be the cause of death or potentialize the involvement of the user with crime and potentially fatal behavior. Thus, official organisms in charge of criminal investigation must be capable of identifying and quantifying these substances in biological samples. The present work aimed the development of an analytical method to identify and quantify club drugs in whole blood, using liquid chromatography - mass spectrometry with hybrid analyzer quadrupole - time of flight (LC-QTOF). After analytical development, the method was validated according to do Scientific Working Group for Forensic Toxicology (SWGTOX) guidelines, evaluating linearity, limit of detection, limit of quantification, matrix effect, precision, intermediate precision, bias and dilution integrity, besides recovery and process efficiency. The developed method comprised MDA, MDMA, 2C-B, DOB, ketamine, mCPP, cocaine and cocaethylene determination. Real samples related to non-natural deaths were collected at Institute of the Legal Medicine Aristoclides Teixeira, Goiânia, Goiás, Brazil, and analyzed by the developed method. 56 cases were selected, most of them related to fire gun injury and traffic events, 28,5% (n=16) of them being positive for cocaine and/or cocaethylene. None of the other drugs comprised in the analysis were detected in these samples.

A microextração por sorbente empacotado (MEPS) é uma técnica de preparo de amostras ainda pouco utilizada no âmbito da toxicologia, em que os mesmos princípios da extração em fase sólida convencional são adaptados para uma escala miniaturizada. As principais vantagens da técnica estão associadas ao pequeno volume de amostra e de solventes utilizados, à possibilidade de realizar múltiplas extrações com um mesmo cartucho e à facilidade de automação. Os benzodiazepínicos possuem grande relevância na toxicologia dada sua ampla utilização e seus efeitos que podem, por exemplo, comprometer a capacidade de dirigir, além do uso abusivo, e como drogas facilitadoras de crimes. Neste trabalho, um método de MEPS foi desenvolvido e otimizado para a determinação de sete benzodiazepínicos e seus produtos de biotransformação (diazepam, clonazepam, flunitrazepam, alprazolam, bromazepam, 7-aminoflunitrazepam e nordiazepam) utilizando 100 µL de amostra de sangue total post mortem. Após a extração, os eluatos foram analisados por cromatografia líquida em fase reversa acoplada a espectrometria de massas. O método foi validado de acordo com as recomendações do Scientific Working Group for Forensic Toxicology, apresentando linearidade adequada de 5 a 500 ng.mL-1 . Os valores de exatidão (90,4 a 109,5%), precisão intra-dia (2,5 a 10,7 %CV) e inter-dia (1,1 a 8,0 %CV) também foram satisfatórios. MEPS foi realizada mais de 60 vezes com a mesma fase extratora sem evidências de contaminação cruzada. Dez amostras reais fornecidas pelo Instituto Médico Legal de São Paulo foram analisadas. Foram quantificados diazepam, nordiazepam, clonazepam e bromazepam. Os resultados encontrados em cada uma das amostras foram comparados com dados da literatura. / Microextraction by packed sorbent (MEPS) is a sample preparation technique still little used in toxicology, where the same principles of conventional solid phase extraction are adapted to a miniaturized scale. The main advantages of the technique are associated with the small volume of sample and solvents required, the possibility of performing multiple extractions with the same cartridge and ease process automation. Benzodiazepine drugs are relevant in toxicology because of their widespread use, and effects (which may, for example, compromise the ability to drive vehicles), abuse and records as crime-facilitating drugs. In this work, a MEPS method was developed and optimized for a determination of seven benzodiazepines and their metabolites (diazepam, nordiazepam, clonazepam, flunitrazepam, 7-aminoflunitrazepam, alprazolam, and bromazepam) using 100 µL of post mortem whole blood. After extraction, the eluates were analyzed by reversed-phase liquid chromatography coupled to mass spectrometry. The method was validated according to the recommendations of the Scientific Working Group for Forensic Toxicology, presenting adequate linearity from 5 to 500 ng.mL-1 . The values of accuracy (90.4 to 109.5%), intra-day precision (2.5 to 10.7 %CV) and inter-day (1.1 to 8.0 %CV) also presented satisfactory results. MEPS was performed more than 60 times with the same extractive phase without compromising the results with the evidence of carryover. Institute of Legal Medicine were submitted to analysis by MEPS-LC-MS/MS. In these samples, the following analytes were quantified: diazepam, nordiazepam, clonazepam and bromazepam. The results found in each of the samples were compared with data from the literature.

In recent years, the abuse of synthetic cathinones or ‘bath salts’ has become a major public health concern. Although these compounds were initially sold legally and labeled “not for human consumption”, the ‘bath salts’ are psychostimulants, with similar structures and pharmacologic mechanisms to cocaine, the amphetamines, and 3,4 methylendioxymethamphetamine (MDMA, Molly, or Ecstasy). The reported use of these substances by women of child-bearing age highlights the necessity of studies seeking to delineate risks of prenatal exposure. Three popular drugs of this type are methylone, mephedrone, and 3, 4-methylenedioxypyrovalerone (MDPV). Unfortunately, there is currently no information available on the teratogenicity of these compounds, or of the extent to which they cross the placenta. As such, the purpose of this study was to examine the pharmacokinetic profile of the ‘bath salts’ in a pregnancy model. Pregnant mice (E17.5 gestation) were injected intraperitoneally with a cocktail of 5mg/kg methylone, 10mg/kg mephedrone, and 3mg/kg (MDPV) dissolved in sterile saline. Maternal brain, maternal plasma, placenta, and fetal brain were collected at 30s, 1min, 5min, 10min, 15min, 30min, 1h, 2h, 4h, and 8h following injection. Methylone, mephedrone, and MDPV were extracted from tissue by solid phase extraction, and concentrations were determined using a previously validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Interestingly, all 3 cathinones reached measurable concentrations in the placenta, as well as the fetal brain; in fact, for MDPV, the maximal concentration (Cmax) was highest in fetal brain, while mephedrone's highest Cmax value was achieved in placenta. Additionally, the total drug exposure for all 3 compounds (as represented by area under the curve, AUC) was higher in fetal matrices (placenta and fetal brain) than in maternal matrices (maternal brain and plasma), and the half-lives for the drugs were longer. Given the extensive presence of methylone, mephedrone, and MDPV in the fetal brain following prenatal exposure, fetal risk is definitely a concern. As there are currently no prenatal studies available on the teratogenicity of these agents, pregnant patients should be informed about the potential risks that these substances may have.

De nos jours, environ 3000 huiles essentielles sont produites et utilisées dans le monde avec des champs d'application aussi variés que la cosmétique, la parfumerie l'agro-alimentaire, la pharmacie et l'aromathérapie. Ces huiles sont extraites des hespéridés ou des plantes aromatiques et médicinales. Étant donné que la culture de ces matières premières implique généralement l'application de pesticides, la présence de tels résidus dans les huiles essentielles ne peut pas être écartée. Compte tenu du nombre important de pesticides employés et des nombreux champs d'application des huiles essentielles, il apparaît nécessaire, afin d'assurer la santé du consommateur, de disposer d'une méthode d'analyse multi-résidus capable de doser les pesticides dans les huiles essentielles. L'état de l'art des méthodes d'analyse lié à cette problématique a révélé un manque évident de performance des méthodes actuelles aussi bien en termes de limite de détection que du nombre de pesticides analysés. Ce mémoire propose donc la mise au point d'une méthode d'analyse multi-résidus de pesticides dans les huiles essentielles par couplage de la chromatographie liquide et de la spectrométrie de masse, technologie la plus à même de répondre à la problématique au vue de la bibliographie. Après avoir mis en évidence les performances du nouveau mode d'acquisition Scheduled SRM, disponible sur le spectromètre de masse 4000 QTrap, pour la détection et la quantification de 250 pesticides dans un solvant déterminé, l'importance de considérer la nature de la matrice, aussi bien dans les méthodes de préparation que lors de l'analyse de l'échantillon, a ensuite été démontrée en étudiant deux huiles essentielles représentatives (lavandin et citron). Enfin, la méthode d'analyse multi-résidus LC-MS/MS a été appliquée à la recherche de pesticides dans des échantillons réels d'huiles essentielles. Elle a démontré sa capacité à détecter, quantifier et identifier les pesticides dans ces matrices au travers de l'utilisation d'un mode d'acquisition couplé SRM-EPI faisant appel aux spécificités du spectromètre de masse hybride et notamment de sa trappe d'ion linéaire. Ce travail a de plus révélé l'importance de disposer d'une telle méthode au regard du nombre de pesticides détectés dans les échantillons et de leurs concentrations relativement élevées. Celles-ci peuvent en effet atteindre des teneurs supérieures au milligramme par litre dans les huiles essentielles analysées.

Le foisonnement filamenteux est un problème récurant dans de nombreuses stations d'épuration à boues activées. L'objectif de ces travaux est d'améliorer la compréhension des mécanismes physiologiques et moléculaires impliqués dans la filamentation des microorganismes, afin de pouvoir orienter de futures stratégies de lutte contre le phénomène de bulking. Sphaerotilus natans, qui peut croître réversiblement sous forme monocellulaire ou filamenteuse, a été utilisée comme bactérie modèle pour cette étude. Différents types de cultures, ainsi que des suivis par cytométrie en flux et marquage au cFDA/SE, ont montré que les diverses souches de S. natans adoptent des morphologies différentes et que les filaments croissent par divisions cellulaires successives et non par un chaînage des bactéries. Une analyse par RT-QPCR a mis en évidence que l'expression du gène sthA augmente fortement après induction de la filamentation et reste ensuite à un niveau élevé. Une comparaison de l'expression protéique des formes monocellulaire et filamenteuse, par LC-MS-MS, a permis d'identifier des protéines impliquées dans la filamentation, et notamment dans la synthèse de la gaine. La concentration intracellulaire en ARNr, mesurée par RT-QPCR, varie durant la croissance de S. natans et d'autres microorganismes, entraînant une diminution importante de l'intensité du marquage FISH, mesurée par cytométrie en flux. L'utilisation de la technique FISH pour quantifier des microorganismes est donc remise en question, d'autant plus dans des matrices aussi complexes que les boues activées. Ces observations mettent également en doute l'hypothèse, émise en utilisant ce mode de quantification, d'une déstructuration des filaments consécutive à un retour à des conditions de culture plus favorables.

<p>Electrospray ionization (ESI) mass spectrometry (MS) in combination with liquid chromatography (LC) is an excellent tool for the identification of drug metabolites. Utilizing this hyphenated technique in combination with proper sample pretreatment, the metabolic pathways of the analgesic drug ketobemidone were investigated in human urine and rat microdialysate from blood and brain. Two novel phase I metabolites (ketobemidone N-oxide and meta-hydroxymethoxyketobemidone) and three novel phase II metabolites (glucuronic acid conjugates of ketobemidone, norketobemidone and hydroxymethoxyketobemidone) were identified in human urine. Further, norketobemidone and ketobemidone N-oxide were identified in rat microdialysate from brain after regional distribution of ketobemidone in striatum. This indicates that the brain itself has the possibility to metabolize ketobemidone. </p><p>Synthetic ketobemidone metabolites were used for comparison of retention times and tandem MS spectra with the possible metabolites recovered from the biological samples. The conjugated metabolites were identified by accurate mass measurements and tandem MS spectra of the aglycones. The accuracy of the estimated masses was better than 2.1 ppm for two out of three conjugates in presence of internal standard.</p><p>On-line micro-SPE was successfully used for trapping and desalting of the microdialysates. The small SPE pre-column made it possible to inject approximately 100 times more sample on the analytical column compared to injection without pre-column. Selective trapping was demonstrated for the polar catechol amine metabolite, dihydroxyketobemidone, which forms covalent complexes with phenylboronic acid (PBA). A fluorinated silica type stationary phase was the only column out of several tested that was able to separate ketobemidone and all relevant phase I metabolites. </p><p>Liquid chromatography and mass spectrometry are independently valuable tools in the field of analytical pharmaceutical chemistry. The present study showed that the combination of LC-MS, with its excellent selectivity and sensitivity, offers an outstanding tool in the qualitative analysis of drugs and metabolites in biological fluids. </p>

Electrospray ionization (ESI) mass spectrometry (MS) in combination with liquid chromatography (LC) is an excellent tool for the identification of drug metabolites. Utilizing this hyphenated technique in combination with proper sample pretreatment, the metabolic pathways of the analgesic drug ketobemidone were investigated in human urine and rat microdialysate from blood and brain. Two novel phase I metabolites (ketobemidone N-oxide and meta-hydroxymethoxyketobemidone) and three novel phase II metabolites (glucuronic acid conjugates of ketobemidone, norketobemidone and hydroxymethoxyketobemidone) were identified in human urine. Further, norketobemidone and ketobemidone N-oxide were identified in rat microdialysate from brain after regional distribution of ketobemidone in striatum. This indicates that the brain itself has the possibility to metabolize ketobemidone. Synthetic ketobemidone metabolites were used for comparison of retention times and tandem MS spectra with the possible metabolites recovered from the biological samples. The conjugated metabolites were identified by accurate mass measurements and tandem MS spectra of the aglycones. The accuracy of the estimated masses was better than 2.1 ppm for two out of three conjugates in presence of internal standard. On-line micro-SPE was successfully used for trapping and desalting of the microdialysates. The small SPE pre-column made it possible to inject approximately 100 times more sample on the analytical column compared to injection without pre-column. Selective trapping was demonstrated for the polar catechol amine metabolite, dihydroxyketobemidone, which forms covalent complexes with phenylboronic acid (PBA). A fluorinated silica type stationary phase was the only column out of several tested that was able to separate ketobemidone and all relevant phase I metabolites. Liquid chromatography and mass spectrometry are independently valuable tools in the field of analytical pharmaceutical chemistry. The present study showed that the combination of LC-MS, with its excellent selectivity and sensitivity, offers an outstanding tool in the qualitative analysis of drugs and metabolites in biological fluids.

The major objectives of this study were to investigate the antioxidant properties of flaxseed lignans secoisolariciresinol (SECO 2) and secoisolariciresinol diglycoside (SDG 1) and their major oxidative compounds using 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH 47) in an in vitro model of lipid peroxidation. This investigation was facilitated by the structural elucidation of the major oxidative compounds and the ability of flaxseed lignans to delay the onset of oxidation in two model systems. <p>This study showed that SECO 2 oxidation occurs at the aromatic (4-OH) and aliphatic (9-OH) hydroxyl groups. Conversely for SDG 1, only compounds derived from the oxidation of aromatic hydroxyl groups were obtained because the 9-OH position is glucosylated. <p>SECO 2 oxidation with AAPH 47 showed that the intermediate 2a is most likely involved in the generation of early-forming (48 and 52) and 2c for the formation of late-forming (49, 50 and 51) oxidation compounds. Compound 48 is formed from dimerization of 2a that is converted to 52 and then to 51. Compound 50 was formed by the addition of a carbon-centre free radical of AAPH (AP radical) to 2c. Compounds 50 and 51 trap carbon-centered AP radicals supporting SECO 2 as a chain-breaking antioxidant and AAPH 47 as a proper model for study of SECO 2 oxidation in vitro. <p>SDG 1 oxidation with AAPH 47 indicated that intermediates 1b and 1c are most likely involved for the formation of early forming compounds (55 and 58) and 1a leads to the late forming compounds (56 and 57). Compound 55 is a result of dimerization. Compound 56 may be directly formed via intermediate radical 1a by adding AP free radicals. Compound 56 was a stable non-radical compound that could trap AP free radicals, thereby supporting SDG 1 as a chain-breaking antioxidant. Hydrogen abstraction from 4-hydroxyl yielded the radical 1a and hydroxyl radical addition to 1a yielded 57. Compound 58 formed from the addition of OH or H2O to 1c. <p>This study demonstrated that AAPH 47 produces carbon-centred AP radicals upon thermal decomposition and mimics the formation of lipid peroxyl radicals. Interaction of carbon-centred AP radicals with SECO 2 and SDG 1 provides a good model to study the antioxidant reactions of SECO 2 in vitro. p*The relative antioxidant capacity of the flaxseed lignans versus BHT 17, in two model systems, was determined. The stoichiometric ratio for SECO 2 and SDG 1 were 1.5 and 1.1-1.2, respectively, compared to BHT 17 (2.0). The induction time by Rancimat analyzer measured inhibition of autoxidation mediated by flaxseed lignans SECO, SDG and SDG polymer in comparison with BHT 17. The induction time data demonstrated that SECO 2 protected canola oil better than either SDG 1 or SDG polymer 3. <p>These results are important for better understanding about the chemistry behind flaxseed lignan antioxidant activities. This study provided useful evidence that flaxseed lignans can be used as natural antioxidants.

Fragment-based drug discovery is an emerging process that has gained popularity in recent years. The process starts from small molecules called fragments. One major step in fragment-based drug discovery is fragment screening, which is a strategy to screen libraries of small molecules to find hits. The strategy in theory is more efficient than traditional high-throughput screening that works with larger molecules. As fragments intrinsically possess weak affinity to a target, detection techniques of high sensitivity to affinity are required for fragment screening. Furthermore, the use of different screening methods is necessary to improve the likelihood of success in finding suitable fragments. Since no single method can work for all types of screening, there is a demand for new techniques. The aim of this thesis is to introduce weak affinity chromatography (WAC) as a novel technique for fragment screening. WAC is, as the name suggests, an affinity-based liquid chromatographic technique that separates compounds based on their different weak affinities to an immobilized target. The higher affinity a compound has towards the target, the longer it remains in the separation unit, and this will be expressed as a longer retention time. The affinity measure and ranking of affinity can be achieved by processing the obtained retention times of analyzed compounds. In this thesis, WAC is studied for fragment screening on two platforms. The first system comprised a 24-channel affinity cartridge that works in cooperation with an eight-needle autosampler and 24 parallel UV detector units. The second system was a standard analytical LC-MS platform that is connected to an affinity column, generally called WAC-MS or affinity LC-MS. The evaluation criteria in studying WAC for fragment screening using these platforms were throughput, affinity determination and ranking, specificity, operational platform characteristics and consumption of target protein and sample. The model target proteins were bovine serum albumin for the first platform, thrombin and trypsin for the latter. Screened fragments were either small molecule drugs, a thrombin-directed collection of compounds, or a general-purpose fragment library. To evaluate WAC for early stages of fragment elaboration, diastereomeric mixtures from a thrombin-directed synthesis project were screened. Although both analytical platforms can be used for fragment screening, WAC-MS shows more useful features due to easy access to the screening platform, higher throughput and ability to analyze mixtures. Affinity data from WAC are in good correlation with IC50 values from enzyme assay experiments. The possibility to distinguish specific from non- specific interactions plays an important role in the interpretation of WAC results. In this thesis, this was achieved by inhibiting the active site of the target protein to measure off-site interactions. WAC proves to be a sensitive, robust, moderate in cost and easy to access technique for fragment screening, and can also be useful in the early stages of fragment evolution. In conclusion, this thesis has demonstrated the proof of principle of using WAC as a new tool to monitor affinity and to select hits in fragment-based drug discovery. This thesis has indicated the primary possibilities, advantages as well as the limitations of WAC in fragment screening procedures.  In the future, WAC should be evaluated on other targets and fragment libraries in order to realize more fully the potential of the technology.

The major objectives of this study were to investigate the antioxidant properties of flaxseed lignans secoisolariciresinol (SECO 2) and secoisolariciresinol diglycoside (SDG 1) and their major oxidative compounds using 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH 47) in an in vitro model of lipid peroxidation. This investigation was facilitated by the structural elucidation of the major oxidative compounds and the ability of flaxseed lignans to delay the onset of oxidation in two model systems. <p>This study showed that SECO 2 oxidation occurs at the aromatic (4-OH) and aliphatic (9-OH) hydroxyl groups. Conversely for SDG 1, only compounds derived from the oxidation of aromatic hydroxyl groups were obtained because the 9-OH position is glucosylated. <p>SECO 2 oxidation with AAPH 47 showed that the intermediate 2a is most likely involved in the generation of early-forming (48 and 52) and 2c for the formation of late-forming (49, 50 and 51) oxidation compounds. Compound 48 is formed from dimerization of 2a that is converted to 52 and then to 51. Compound 50 was formed by the addition of a carbon-centre free radical of AAPH (AP radical) to 2c. Compounds 50 and 51 trap carbon-centered AP radicals supporting SECO 2 as a chain-breaking antioxidant and AAPH 47 as a proper model for study of SECO 2 oxidation in vitro. <p>SDG 1 oxidation with AAPH 47 indicated that intermediates 1b and 1c are most likely involved for the formation of early forming compounds (55 and 58) and 1a leads to the late forming compounds (56 and 57). Compound 55 is a result of dimerization. Compound 56 may be directly formed via intermediate radical 1a by adding AP free radicals. Compound 56 was a stable non-radical compound that could trap AP free radicals, thereby supporting SDG 1 as a chain-breaking antioxidant. Hydrogen abstraction from 4-hydroxyl yielded the radical 1a and hydroxyl radical addition to 1a yielded 57. Compound 58 formed from the addition of OH or H2O to 1c. <p>This study demonstrated that AAPH 47 produces carbon-centred AP radicals upon thermal decomposition and mimics the formation of lipid peroxyl radicals. Interaction of carbon-centred AP radicals with SECO 2 and SDG 1 provides a good model to study the antioxidant reactions of SECO 2 in vitro. p*The relative antioxidant capacity of the flaxseed lignans versus BHT 17, in two model systems, was determined. The stoichiometric ratio for SECO 2 and SDG 1 were 1.5 and 1.1-1.2, respectively, compared to BHT 17 (2.0). The induction time by Rancimat analyzer measured inhibition of autoxidation mediated by flaxseed lignans SECO, SDG and SDG polymer in comparison with BHT 17. The induction time data demonstrated that SECO 2 protected canola oil better than either SDG 1 or SDG polymer 3. <p>These results are important for better understanding about the chemistry behind flaxseed lignan antioxidant activities. This study provided useful evidence that flaxseed lignans can be used as natural antioxidants.