Esta tese foi dividida em três capítulos. O capítulo I descreve o desenvolvimento do método Column switching UHPLC-MS/MS para a determinação simultaneamente de fármacos psicotrópicos em amostras de plasma de pacientes esquizofrênicos. A politerapia é uma prática comum no tratamento da esquizofrenia. Portanto, a monitorização terapêutica destes fármacos tem sido realizada para o ajuste das doses e individualização da terapia farmacológica. O método Column switching UHPLC-MS/MS apresentou linearidade na faixa de concentração de 0,025 a 1,25 ng mL-1 com R2 acima de 0,9950 e a falta de teste de ajuste (p > 0,05); precisão com coeficientes de variação inferiores a 12% e exatidão com erro padrão relativo inferior a 14%. Este método foi aplicado com sucesso para determinação de fármacos em amostras de plasma de pacientes esquizofrênicos para fins de monitorização terapêutica. No capítulo II, o desempenho cromatográfico de colunas C18 superficialmente e totalmente porosas com diferentes tamanhos de partícula foi avaliado para a análise de fármacos psicotrópicos por LC-MS/MS e LC-DAD. Com o sistema LC-MS/MS foram avaliados os seguintes parâmetros cromatográficos: altura do prato reduzido vs velocidade linear reduzida, impedância vs velocidade linear reduzida, tempo da corrida cromatográfica vs vazão, pressão vs vazão, resolução, capacidade de pico, assimetria e fator de retenção. Já com o sistema LC-DAD foram avaliados a hidrofobicidade, atividade silanol e impurezas metálicas também foram avaliadas. As colunas com superfície carregada apresentaram maior eficiência cromatográfica para os fármacos em sua forma ionizada. Já as colunas com partículas menores que 2 µm (Cortecs 1,6 µm, Acquity 1,7 µm, e Kinetex 1,7 µm) apresentaram maior eficiência cromatográfica para os fármacos na forma parcialmente ionizada. Os modelos matemáticos gerados foram capazes de prever a pressão e o tempo da corrida cromatográfica em diferentes vazões para todas as colunas. Considerando a eficiência, impedância, resolução, capacidade de pico, fator de retenção e hidrofobicidade, as colunas Cortecs 1,6 µm e Acquity 1,7 µm apresentaram melhor desempenho durante a análise dos fármacos em amostra de plasma. O capítulo III descreve o desenvolvimento e validação dos métodos SPME-UHPLC-MS/MS e Bio-SPME-Nano-ESI-MS/MS para a determinação dos endocanabinóides (AEA e 2-AG) em amostras biológicas. Para a otimização do processo SPME foram avaliadas as fases SPME (C18, C30 e HLB) e os solventes para dessorção (metanol, acetonitrila e isopropanol). Os aditivos modificadores de matriz, como cloridrato de guanidina, ácido trifluoroacético e acetonitrila foram avaliados por planejamento experimental. Os métodos SPME-UHPLC-MS/MS e Bio-SPME-Nano-ESI-MS/MS, com a fase HLB biocompatível, apresentaram para ambos endocanabinóides valores de LOQs de 1 ng mL-1 e 50 ng mL-1, respectivamente. O método Bio-SPME-Nano-ESI-MS/MS permitiu o direto acoplamento da fibra SPME ao espectrômetro de massas via dessorção/ionização nanoeletrospray que resultou em rápida determinação quantitativa dos endocanabinóides em amostras biológicas. / This thesis is divided into three chapters. Chapter I describes the development of a column switching UHPLCMS/MS method to determine psychotropic drugs in schizophrenic patients plasma samples simultaneously. Polytherapy is a common practice in schizophrenia treatment. Therefore, therapeutic drug monitoring has been applied to adjust doses and to customize pharmacological therapy. The column switching UHPLCMS/MS method developed here is linear at concentrations ranging from 0.025 to 1.25 ng mL-1 with R2 above 0.9950 and presents lack of fit test (p > 0.05), precision with coefficients of variation lower than 12%, and accuracy with relative standard error lower than 14%. This method was successfully applied to determine drugs in schizophrenic patients plasma samples for therapeutic drug monitoring. In chapter II, the chromatographic performance of C18 superficially porous columns and of C18 fully porous columns with different particle sizes were evaluated for analysis of psychotropic drugs by LC-MS/MS and LC-DAD. Within the LC-MS/MS system, the following chromatographic parameters were assessed: reduced plate height vs reduced linear velocity, impedance vs reduced linear velocity, chromatographic run time vs flow rate, backpressure vs flow rate, resolution, peak capacity, asymmetry, and retention factor. Within the LC-DAD system, hydrophobicity, silanol activity, and metal impurities were also examined. Columns with charged surface displayed improved chromatographic efficiency for drugs in the ionized form. Columns with particles smaller than 2 µm (Cortecs 1.6 µm, Acquity 1.7 µm, and Kinetex 1.7 µm) presented higher chromatographic efficiency for the drugs, which were in their partially ionized form. The generated mathematical models were able to predict the backpressure and the chromatographic run time at different flow rates for all the columns. Considering efficiency, impedance, resolution, peak capacity, retention factor, and hydrophobicity, columns Cortecs 1.6 µm and Acquity 1.7 µm provided the best performance during analysis of drugs in plasma samples. Chapter III describes the development and validation of the SPME-UHPLC-MS/MS and the Bio-SPME-Nano-ESI-MS/MS methods for determination of endocannabinoids (AEA and 2-AG) in biological samples. To optimize the SPME process, SPME coatings (C18, C30, and HLB) and solvents for desorption (methanol, acetonitrile, and isopropanol) were evaluated. Matrix modifier additives, such as guanidine hydrochloride, trifluoroacetic acid, and acetonitrile, were assessed by experimental design. The SPME-UHPC-MS/MS and the Bio-SPME-Nano-ESI-MS/MS methods with HLB biocompatible coating provided LOQ values of 1 ng mL-1 and 50 ng mL-1, respectively, for both endocannabinoids. The Bio-SPME-Nano-ESI-MS/MS method allowed direct coupling of SPME fibers to the mass spectrometer by desorption/ionization nanoelectrospray, which resulted in rapid quantitative determinations of endocannabinoids in biological samples.

Per- and polyfluorinated substances (PFAS) are used in the production of ski wax to reduce the friction between the snow and the ski. In this occupational study of ski wax technicians’ exposure to PFAS and particulate aerosol we have collected whole blood (wb) (n =94), air (n =84) and aerosol (n =159) samples at World Cup events from 2007-2011. We have analysed the blood, air and aerosol with respect to 13 perfluorocarboxylic acids (PFCAs), 4 perfluorosulfonic acids (PFSAs), 3 fluorotelomer alcohols (FTOHs), 3 fluorotelomer acids (FTCAs) and 3 unsaturated fluorotelomer acids (FTUCAs). Further, we assessed the exposure to 3 particulate aerosol fractions (inhalable, respirable and total aerosol) in air. In comparison to a general population, several of the PFCA blood levels are elevated in the technicians’, primarily  erfluorooctanoate (PFOA) and perfluorononate (PFNA) with concentrations up to 628 and 163 ng/mL wb, respectively. Further,  we detected FTUCAs and FTCAs in the blood, suggesting biotransformation of FTOHs to PFCAs. The metabolites 5:3 and 7:3 FTCA were detected in all blood samples at levels up to 6.1 and 3.9 ng/mL wb. Levels of perfluorohexadecanoic acid PFHxDA) and perfluorooctadecanoic acid (PFOcDA) were detected in the technician’s blood at mean concentration up to 4.22 ng/mL wb and 4.25 ng/mL wb. The FTOH levels in air of the wax cabin during work ranged up to 997 000 ng/m3 (average=114 000 ng/m3 ) and PFOA up to 4 890 ng/m3 (average= 526 ng/m3 . FTOHs were not detected in aerosols but PFOA showed average levels of 12 000 ng/m3 (range=1 230- 46 900 ng/m3 ). The occupational exposure limit (OEL) of 2 mg/m3 was exceeded in 37% of the personal measurements with aerosol  concentrations up to 15 mg/m3 . Keywords : Perfluorinated, polyfluorinated, FIS, occupational exposure, ski wax,  iotransformation, metabolism, fluorotelomer alcohol, fluorotelomer acid, aerosol, dust, UPLC/MS-MS, GC/MS-MS

Les phytochélatines (PC) sont des polypeptides ayant la structure générale, (alpha-Glu-Cys)n-Gly, où n = 2 à 11. Leur synthèse est induite par un grand nombre de végétaux en réponse à une élévation de la concentration du milieu en métaux, en particulier le cadmium (ci-après, Cd). Le but de cette étude a été de développer un outil pour évaluer la biodisponibilité du Cd dans les eaux douces. Pour ce faire, une méthode analytique a été réalisée afin de déterminer les phytochélatines induites dans les algues C. reinhardtii. Celle-ci consiste à utiliser la chromatographie liquide couplée à la spectrométrie de masse en tandem (LCHP-SM/SM) "on-line". L’ionisation des molécules est celle faite par électronébulisation (IEN) (traduction de electrospray ionisation). L’objectif principal de ce mémoire est la validation de cette méthode : la détermination des courbes de calibration et des limites de détection et l'identification d'interférences potentielles. L’utilisation de dithiothreitol (DTT) à une concentration de 25 mM a été nécessaire à la conservation de la forme réduite des phytochélatines. En effet, suite à la validation de la méthode d’analyse des phytochélatines il a été démontré qu’elle représente un potentiel d’application. Ceci dans la mesure où l’induction des phytochélatines (PC2, PC3 et PC4) dans les algues C. reinhardtii a été possible à deux concentrations de Cd (1 x 10-7 M et 1 x 10 6 M) et ce, après plusieurs temps d'induction (1, 2, 4, et 6 h). Ainsi, l’étude de la stabilité des phytochélatines a été réalisée et toutes les températures examinées ont démontré une diminution des phytochélatines analysées par HPLC-ESI-MS/MS. Il se pourrait que la cause de la dégradation des phytochélatines soit physique ou chimique plutôt que bactérienne. Toutefois, un approfondissement au niveau de la purification de la solution d’extraction serait nécessaire à la mise au point de la dite méthode analytique afin de quantifier les phytochélatines dans l’algue C. reinhardtii. / Phytochelatins (PC) are polypeptides having the general structure, (alpha-Glu-Cys)n-Gly, where n = 2 to 11. Many plants respond to an elevated concentration of metals in environment, particularly Cd, by synthesizing PC. The purpose of this study was to develop a tool to assess the bioavailability of the Cd in fresh water by determining phytochelatins in algae, C. reinhardtii, by online HPLC-ESI-MS/MS. The gold of this work was the validation of the analytical method i.e. the determination of the calibration curves and the limits of detection. The addition of dithiothreitol (DTT), 25 mM, was found to be necessary to maintain the PC in their reduced form for analysis. It was shown that the liquid chromatography coupled to tandem mass spectrometry (HPLC-ESI-MS/MS) technique has excellent potential for PC analysis, however, it will still requires some more work with respect to sample purification. Furthermore, the stability of the PC was evaluated for different sample storage temperatures. At all temperatures studied, some degradation of PC was observed possibly due to physical rather than chemical or bacterial reasons. Finally, the induction of phytochelatins (PC2, PC3 and PC4) was observed in C. reinhardtii for two Cd concentrations (10-7 M and 10-6 M) and for several induction times (1, 2, 4, and 6 h).

La présence d’hydrocarbures dans l’environnement que ce soit suite à un déversement accidentel de pétrole en mer ou suite à des apports chroniques, est une préoccupation majeure en raison de leur écotoxicité et de leur potentiel à être bioaccumulés, et ainsi, pénétrer la chaîne alimentaire. Par conséquent, ces molécules sont sous haute surveillance et il est nécessaire de disposer de méthodes analytiques permettant de les identifier et de les quantifier, et ceci, pour des concentrations allant du mg/L au ng/L. Cependant et dans la plupart des cas, les analyses en laboratoire des hydrocarbures, notamment aromatiques, se limitent à la quantification des 16 hydrocarbures aromatiques polycycliques (HAP) identifiés par l’Agence américaine de Protection Environnementale (US-EPA) comme étant dangereux pour l’environnement du fait de leur caractère cancérigène. Or, les hydrocarbures aromatiques polycycliques soufrés (HAPS) ainsi que leurs homologues substitués sont, sur le plan chimique, structurellement proches des HAP, et peuvent donc présenter des risques environnementaux similaires, à savoir être cancérigènes ou mutagènes. Le travail de recherche réalisé s’inscrit dans ce contexte avec pour objectif des développements méthodologiques permettant l’extraction et l’analyse d’une gamme plus large de HAP, des HAPS et leurs dérivés alkylés directement dans les produits pétroliers ou dissous en phase aqueuse. Des protocoles d’extraction par "stir bar sorptive extraction" (SBSE) et par microextraction sur phase solide (SPME), et des méthodes d’analyse par chromatographie en phase gazeuse couplée à la spectrométrie de masse simple (GC-MS) et en tandem (GC-MS-MS) ont été développés. Les résultats obtenus plaident en faveur de ces méthodologies aussi bien en termes de linéarité de la réponse qu’en termes de sensibilité, méthodologies qui ont été appliquées avec succès pour la détermination des analytes d’intérêt dans des fractions solubles préparées au laboratoire (WAF et WSF). Pour la CG-MS-MS, si elle se révèle particulièrement adaptée car elle apporte un degré de certitude élevé, elle n’en reste pas moins une technique délicate à mettre en oeuvre, notamment dans le cas des composés alkylés pour lesquels des solutions étalons ne sont pas disponibles. Pour pallier cette difficulté, un produit de référence contenant l’ensemble des analytes d’intérêt a été caractérisé. Pour les composés alkylés non disponibles commercialement, des appro-ximations ont été effectuées par MS simple en se basant sur une analyse comparée des coefficients de réponse en mode MRM et SIM. La méthodologie ainsi mise au point a permis de caractériser le fioul de l’Erika avec une faible variabilité des résultats. Ce produit peut servir de référence pour l’analyse quantitative de l’ensemble des familles de composés identifiés dans cette étude. / The presence of hydrocarbons in the environment either as a result of oil spills at sea or due to chronic discharge is a major concern because of their ecotoxicity and their potential to bioaccumulate and thus enter the food chain. Therefore, these molecules are closely monitored and reliable analytical methods are required to identify and quantify them, for concentrations ranging from mg/L to ng/L. However, in most cases, laboratory analyses of hydrocarbons, especially aromatic hydrocarbons, are limited to the quantification of 16 polycyclic aromatic hydrocarbons (PAHs) identified by the U.S. Environmental Protection Agency (U.S. EPA) as hazardous to the environment due to their carcinogenic nature. However, polycyclic aromatic sulphur heterocycles (PASHs) and their substituted homologs are, in chemical terms, structurally similar to PAHs, and therefore can pose similar environmental risks, i.e. they can be carcinogenic or mutagenic. In this context, the research work carried out aims to develop methodologies for the extraction and analysis of a wider range of PAHs, PASHs and their alkyl derivatives directly in oil or dissolved in the aqueous phase. Extraction protocols by stir bar sorptive extraction (SBSE) and solid phase microextraction (SPME), and methods of analysis by gas chromatography coupled with mass spectrometry (GC-MS) and with tandem mass spectrometry (GC-MS-MS) have been developed. The results argue in favor of these methodologies both in terms of linearity of the response and in terms of sensitivity. These methodologies that have been successfully applied for the determination of analytes of interest in the water accommodated fraction and water soluble fraction prepared in the laboratory (WAF and WSF). For GC-MS-MS, while it is particularly suitable because it provides an additional level of selectivity, it is a difficult technique to implement, in particular in the case of molecules for which no calibration solutions are available. To overcome this difficulty, a reference oil containing all the target molecules was characterized. For alkylated compounds that are not commercially available, approximations were made by simple MS, based on comparative analysis of response coefficients in MRM (Multiple Reaction Monitoring) and SIM (Single Ion Monitoring) modes. The finalized method was used to characterize the Erika fuel oil, with low variability in the results. This product can be used as a reference for the quantitative analysis of all the families of molecules identified in this study.

La protéine kinase TOR (Target Of Rapamycin) a été identifiée chez la levure et les mammifères comme participant à deux complexes protéiques qui servent de carrefour entre la perception des facteurs endogènes et exogènes et la stimulation de la croissance cellulaire. Depuis la découverte de la kinase AtTOR chez Arabidopsis thaliana, des études ont été menées afin de mieux caractériser son rôle chez les plantes et l’influence de son niveau d’expression sur la régulation du métabolisme et du développement.Au cours de ce travail, j’ai contribué à l’étude de cette kinase en étudiant l’influence de l’inactivation de TOR sur la composition du ribosome au niveau protéique et sur le niveau de phosphorylation de ces protéines, ainsi que sur l’organisation du méristème au niveau moléculaire et cytologique Au cours de cette étude, j’ai montré que certaines protéines constitutives du ribosome pourraient être des cibles de l’activité TOR au niveau de leur abondance et/ou de leur état de phosphorylation. Ainsi, l’inactivation de TOR entraine une diminution du niveau de phosphorylation des protéines RPS6 et pourrait influencer l’abondance des protéines acides constitutives du stalk ribosomal, une structure importante dans la régulation de la traduction. Les résultats obtenus suggèrent également que l’activité TOR est nécessaire au maintien du méristème à l’état fonctionnel en régulant les voies importantes contrôlant la division et la différentiation au sein de cette structure. / The TOR (Target Of Rapamycin) kinase has first been identified in yeast and mammals as being part of two different protein complexes that are implicated in the stimulation of cell growth in response to endogenous and exogenous stimuli. Since the discovery of this kinase in Arabidopsis, some studies have been led to characterize its role in plants and the influence of its expression level on the metabolism and development regulation.In this study, I worked on the influence of the TOR inactivation on the composition of the ribosome on its protein composition and on the phosphorylation status of these proteins and also on the organisation of the meristem at a molecular and cellular level.Regarding to the results I have obtained, I showed that TOR may regulate the abundance and/or the phosphorylation status of some proteins involved in the ribosome composition. Hence, TOR inactivation leads to a decrease of the phosphorylation level of RPS6 proteins and could regulate the abundance of acid proteins constitutive of the ribosomal stalk, a structure important for the translation regulation. The results obtained also suggest that TOR activity may be necessary to keep the meristem functional by the regulation of the main important pathways controlling division and differentiation in that structure.

Les antibiotiques sont des polluants présents dans les écosystèmes aquatiques, réceptacles ultimes des substances anthropiques. L’étude de ces composés porte sur leur rémanence dans le milieu ou leurs effets sur des organismes naturels. De nombreux efforts ont été faits à l’échelle mondiale pour l’évaluation de la qualité environnementale des différentes ressources en eau pour la survie des espèces aquatiques mais aussi pour la consommation humaine et le risque sanitaire lié. Dans ce but, l’optimisation des techniques analytiques pour ces composés dans les systèmes aquatiques demeure une nécessité. Notre objectif est de développer des méthodes d’extraction et de détection pour 12 molécules appartenant à la famille des aminoglycosides et de la colistine dans les eaux des stations d’épuration et les eaux hospitalières. L’absence des méthodes d’analyse pour ces composés ainsi que le manque des études permettant leur détection dans l’eau sont les raisons de leur étude. L’Extraction sur Phase Solide (SPE) en mode classique (hors ligne) ou en ligne, suivie d’une analyse par la Chromatographie Liquide couplée à la Spectrométrie de Masse (LC/MS/MS) est la méthode la plus couramment employée pour ce type d’analyse. Les paramètres sont optimisés et validés afin d’assurer les meilleures conditions utilisées dans les analyses environnementales. Cette technique a été appliquée sur des échantillons réels des eaux des stations d’épuration à Bordeaux et au Liban. / Antibiotics are pollutants present in aquatic ecosystems ultimate receptacles of anthropogenic substances. These compounds are studied as their persistence in the environment or their effects on natural organisms. Numerous efforts have been made worldwide to assess the environmental quality of different water resources for the survival of aquatic species, but also for human consumption and health risk related. Towards goal, the optimization of analytical techniques for these compounds in aquatic systems remains a necessity. Our objective is to develop extraction and detection methods for 12 molecules of aminoglycosides and colistin in sewage treatment plants and hospitals waters. The lack of analytical methods for analysis of these compounds and the deficiency of studies for their detection in water is the reason for their study. Solid Phase Extraction (SPE) in classic mode (offline) or online followed by Liquid Chromatography analysis coupled with Mass Spectrometry (LC/MS/ MS) is the most method commonly used for this type of analysis. The parameters are optimized and validated to ensure the best conditions for the environmental analysis. This technique was applied to real samples of wastewater treatment plants in Bordeaux and Lebanon.

Ractopamine hydrochloride (RAC) is a β-agonist additive that has been used in many countries as a repartitioning agent, redirecting nutrients in order to increase leanness and decrease lipid deposition in pigs. Countries from the European Union and Asia question their safety, while American countries and Australia allow their controlled use as an additive added to the feed of pigs in the finishing phase. In Brazil, the Ministry of Agriculture, Livestock and Food Supply together with the national production sector, developed a Program called \"SplitSystem\" to ensure a safe product without RAC in order to meet international sanitary requirements. However, co-products used in animal feed may contain RAC, such as meat and bone meal (MBM), one of the main feed ingredients used in many countries which can partially replace soybean meal to lower costs. As the level of RAC in this protein source has not been established an experiment was under taken to examine the impact on pig tissues of increasing amounts of meat and bone meal (MBM) in four dietary groups: 0, 7, 14 and 21% w/w of MBM-containing RAC (53.5 µg kg-1) in the diet. The purpose was to verify if ractopamine residues remain in pig tissues (muscle, liver, kidneys, and lungs) and how much is eliminated through urine. To address these concerns, gilts were fed RAC via MBM daily, from weaning until slaughter. RAC was determined in muscle, liver, kidneys, and lungs with a limit of detection (LOD) = 0.15, 0.5, 0.5 and 1.0 µg kg-1, respectively), and no RAC residues were quantified above the limit of quantification (LOQ) = 0.5, 2.5, 2.5 and 2.5 µg kg-1, respectively). In urine, RAC concentration remained below 1.35 µg L-1. These values are below the maximum residue limits (MRLs) established by legislation. Therefore, MBM (53.5 µg kg-1 of RAC) can be used up to 21% in pig diets, however when considering restrictive markets, it is recommended not to use MBM. / O cloridrato de ractopamina (RAC) é um aditivo β-agonista que tem sido usado em muitos países para redirecionar nutrientes a fim de aumentar a deposição de tecido muscular e diminuição de lipídios em suínos. Países da União Européia e Ásia questionam sua segurança, enquanto os países da América e a Austrália permitem seu uso controlado como um aditivo adicionado à ração na fase de terminação em suínos. No Brasil, o Ministério da Agricultura, em conjunto com o setor produtivo nacional, desenvolveu um programa chamado \"SplitSystem\" para garantir um produto seguro sem RAC, a fim de atender aos requisitos sanitários internacionais. No entanto, os co-produtos utilizados na ração animal podem conter RAC, como farinha de carne e ossos (FCO), um dos principais ingredientes utilizados em muitos países para substituir parcialmente o farelo de soja, a fim de reduzir os custos de produção. Como o nível de RAC nessa fonte de proteína não foi estabelecido, um experimento foi conduzido para examinar o impacto sobre os tecidos suínos que receberam níveis crescentes de FCO, divididos em quatro grupos: 0, 7, 14 e 21% de FCO contendo 53,5 µg kg-1 de RAC, na dieta dos animais. O objetivo foi verificar se resíduos de RAC permanecem nos tecidos suínos (lombo, fígado, rim e pulmão) e o quanto é eliminado através da urina. Para atender a essas preocupações, as leitoas foram alimentadas com RAC via FCO diariamente, desde o desmame até o abate. A RAC foi determinada em lombos, rins, fígados e pulmões com um limite de detecção (LOD) = 0,15; 0,5; 0,5 e 1,0 µg kg-1, respectivamente, e nenhum resíduo de RAC foi quantificado acima do limite de quantificação (LOQ) = 0,5; 2,5; 2,5 e 2,5 µg kg-1, respectivamente. Na urina, a concentração de RAC permaneceu abaixo de 1,35 µg L-1. Estes valores são inferiores aos limites máximos residuais (LMRs) estabelecidos pela legislação. Concluindo que a FCO (53.5 µg kg-1 de RAC) pode ser utilizada com até 21% em rações para suínos, entretanto, ao considerar mercados restritivos, recomenda-se não usar a FCO.

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.

Este estudo apresenta o desenvolvimento da metodologia de extração em fase sólida e separação em cromatográfica líquida acoplada a espectrometria de massas em sequencia, SPE-LC-MS/MS, para a determinação de 21 (vinte e um) fármacos pertencentes a diferentes classes terapeuticas, 1 (uma) droga de abuso e seu principal metabólito, em amostras de água superficial. A separação cromatográfica foi otimizada estudando o desempenho de fases estacionárias e fases móvies. A quantificação dos compostos selecionados foi realizada com a ionização por eletronebulização (electrospray ionization- ESI) e o espectrômetro de massas operando no modo de Monitoramento de Múltiplas Reações (Multiplas Reaction Monitoring- MRM). A validação da metodologia proposta foi realizada utilizando os parâmetros de seletividade, efeito de matriz, faixa de trabalho, linearidade, limites de detecção (LD) e quantificação (LQ), precisão, exatidão, recuperação e robustez. A validação da metodologia permitiu a sua aplicação na avaliação da distribuição dos 23 compostos selecionados, nas águas da represa Guarapiranga, um dos principais sistemas produtor de água potável da Região Metropolitana de São Paulo (RMSP). A presença desses poluentes nos ambientes aquáticos é proveniente da liberação direta do esgoto urbano das habitações do seu entorno, como consequência do precário sistema de saneamento básico. As águas da represa Guarapiranga foram avaliadas em 14 (quatorze) locais estrategicamente escolhidos e amostradas durante 3 (três) campanhas de coleta de amostra (agosto de 2011, setembro de 2012 e abril de 2013). Nessas amostras foram quantificados acetaminofeno (9,6 - 254 ng L-1), atenolol (8,5 177 ng L-1), benzoilegonina (7,9 139 ng L-1), cafeína (27 27386 ng L-1), carbamazepina (12 358 ng L-1), clortalidona (9,4 35 ng L-1), cocaína (12,8 2650 ng L-1), diclofenaco (8 35 ng L-1), enalapril (20 ng L-1), losartana (6,7 114 ng L-1) e valsartana (9,7 - 47 ng L-1). O ponto de coleta denominado de GU103-12 (23°4188.5S 46°4467.3W) foi a região que apresentou os valores mais elevados quanto ao nível de concentração dos compostos avaliados e ao índice de risco integrado de poluição química aquática (Integrated Risk Index of Chemical Aquatic Pollution IRICAP). O estudo também foi realizado em amostras de água de reservatórios das Unidades de Gerenciamento de Recursos Hídricos (UGRHI) 5 e 6 do Estado de São Paulo. Os resultados demonstraram que o uso e a ocupação do solo influenciam diretamente na qualidade da água dos reservatórios, evidenciando a necessidade de implementar melhorias no sistema de coleta de esgoto e de ocupação irregular para evitar a contaminação e o descarte inadequado em ambientes aquáticos. / This study presents the development of the methodology of solid phase extraction and liquid chromatography - tandem mass spectrometry, SPE-LC-MS/MS, for the determination of 21 (twenty one) pharmaceuticals belonging to different therapeutic groups, 1 (one) illicit drug and its major metabolite, in surface water samples. The chromatographic separation was optimized by studying the performance of different stationary and mobile phases. Quantitation of selected compounds was performed by electrospray ionization (ESI) and the mass spectrometer operating in a multiple reaction monitoring (MRM) mode. The validation of the proposed methodology was performed using the parameters of selectivity, matrix effect, dynamic range, linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, recovery and robustness. The validation of methodology allowed to apply the methodology in the evaluation of the distribution of the 23 (twenty one) selected compounds, in Guarapiranga Dam waters, an of the major producer system of drinking water of the Metropolitan Region of São Paulo (MRSP). The presence of these pollutants in aquatic environments is from the direct release of urban sewage from the homes of your surroundings, as a result of poor sanitation system. The waters of Guarapiranga dam were evaluated in 14 (fourteen) locations strategically chosen and sampled in 3 (three) campaigns of sample collection (August 2011, September 2012 and April 2013). In these samples were quantified acetaminophen (9.6 - 254 ng L-1), atenolol (8.5 - 177 ng L-1), benzoylegonine (7.9 - 139 ng L-1), caffeine (27 - 27386 ng L-1) carbamazepine (12 - 358 ng L-1), chlorthalidone (9.4 - 35 ng L-1), cocaine (12.8 - 2560 ng L-1), diclofenac (8 - 36 ng L-1), enalapril (20 ng L-1), losartan (6.7 - 114 ng L-1) and valsartan (9.7 - 47 ng L-1). The sample siting GU103-12 (23°4188.5S 46°4467.3W) was the region with the highest values in the level of concentration of the target compounds and the integrated risk index of chemical aquatic pollution (IRICAP). The study was also conducted on water samples from reservoirs of the UGRHI (Unit of Water Resources Management) 5 and 6, State o São Paulo. The results showed that the use and occupation of land directly influence the reservoir water quality highlighting the need to implement improvements in sewage collection system and illegal occupation to prevent contamination and the improper disposal in aquatic environments.

Uma das características de qualidade dos vinhos espumantes, e que também impõe a sua identidade, é a aparência das borbulhas. Tradicionalmente, acredita-se que a capacidade de formação e estabilização dessas borbulhas depende de macromoléculas do vinho, em especial das proteínas, devido a sua ação tensoativa. Este trabalho de doutorado visou o estudo proteômico do vinho espumante brasileiro a fim de identificar quais proteínas estão presentes nesses vinhos para entender melhor a influência dessas na estabilização da espuma (perlage e colarinho), e com o intuito de potencializar essa característica em nossos produtos. Foram utilizados os métodos de extração de proteínas clássico, ácido tricloroacético/acetona e de última geração, biblioteca combinatória de ligantes peptídicos, sendo estas separadas por SDS-PAGE, 2DE e OFFGEL. As proteínas extraídas foram digeridas com tripsina e a mistura de peptídeos analisada por nLC-MS/MS com metodologia shotgun. Os resultados iniciais obtidos por eletroforese 2DE e OFFGEL, mostraram a presença de três grupos de proteínas de massa molecular distintas, sendo duas próximas a 25 kDa e uma próxima a 70 kDa. Estas proteínas parecem estar presentes nos vinhos em mais de uma isoforma evidenciado pelo espalhamento de todas as bandas de mesma massa molecular em diferentes pH. Foram identificadas 40 proteínas, sendo 17 proteínas de organismos do sub-reino Viridiplantae e 23 proteínas pertencentes ao gênero Saccharomyces, onde 10 e 6 proteínas, respectivamente, estão presentes em pelo menos duas amostras de espumantes nacionais. Dessas, seis proteínas foram identificadas pela primeira vez em vinhos. Três proteínas originárias da levedura Saccharomyces cerevisiae estão presentes em todos os produtos analisados, podendo essas proteínas serem as responsáveis pela melhor formação de espuma observada em nossos produtos em relação ao Champagne (vinho espumante tradicional da França). / The type of fizzy bubbles is one of the aspects that characterizes the quality of sparkling wines and also helps defining their identity. Traditionally, it is believed that the ability of these bubbles to form and stabilize depends on the macromolecules found in the wine, particularly proteins, due to their surfactant action. The aim of this work is the proteomic study of the brazilian sparkling wines in order to identify which proteins are present to better understand the influence of these molecules in the foam formation (perlage and collar), in order to improve our products. The protein extraction methods used were the classical TCA/acetone precipitation and the modern combinatory peptide ligand library. Then, proteins were separated by SDS-PAGE, 2DE and OFFGEL. The protein extracted were digested with trypsin and the peptide mixture were analyzed with nLC-MS/MS using the shotgun method. The first results obtained by electrophoresis 2DE and OFFGEL showed the presence of three groups of proteins with different molecular mass, two of them close to 25 kDa and the other one close to 70 kDa. These proteins appear to be present in wine in more than one isoform evidenced by spreading in all bands of similar molecular weight at different pH. In total, 40 proteins were identified, 17 protein from Viridiplantae sub-kingdom organisms and 23 proteins belonging to Saccharomyces genus, where 10 and 6 proteins, respectively, are present in at least two samples of domestic sparkling wines. Six of those proteins were identified in wine for the first time. Three proteins originating from Saccharomyces cerevisiae are present in all analyzed products, and those may be responsible for a better foam formation observed in our products in comparison to Champagne (traditional French sparkling wine).