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 Δ9-tetrahidrocanabinol (THC), 11-hidroxi-Δ9-tetrahidrocannabinol (THC-OH) e 11-nor-Δ9-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 Δ9-tetrahydrocannabinol (THC), 11-hydroxy-Δ9-tetrahydrocannabinol (THC-OH), and 11-nor-Δ9-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.

Le bisphénol A (BPA), ubiquitaire dans l'environnement, est reconnu comme étant un perturbateur endocrinien (PE). La forte réactivité du BPA avec le chlore entraine la formation de dérivés chlorés du BPA (ClxBPA), possédant une activité perturbatrice endocrinienne supérieure au BPA. En santé environnementale, l'évaluation du risque chez l'homme implique notamment la mise en place d'études de biomonitoring et de toxicocinétique nécessitant l'analyse des micropolluants environnementaux dans les milieux biologiques.Afin d'estimer l'exposition des populations au BPA et ClxBPA, une méthode d'analyse par LC-MS/MS du BPA et des ClxBPA non conjugués a été validée dans l'urine. Cette méthode a ensuite été modifiée afin d'améliorer la rapidité et la sensibilité du dosage, et a été appliquée à la mesure de l'exposition aux PE dans une cohorte de femmes enceintes.L'étude de la toxicocinétique du BPA montre la formation de dérivés conjugués, éliminés par voie urinaire. Classiquement, pour la mesure des dérivés conjugués, des méthodes indirectes utilisant la déconjugaison enzymatique sont proposées. Dans ce cadre, nous avons développé une méthode originale permettant de valider l'efficacité de la déconjugaison. Si la mesure indirecte des métabolites après déconjugaison semble plus aisée, il reste néanmoins plus rationnel de doser directement l'analyte recherché. C'est pourquoi, après synthèse des substances étalons, nous avons pu développer, pour la 1ère fois, une méthode de dosage des dérivés glucuronides et sulfates du BPA et du Cl2BPA dans l'urine.Ce travail propose, à travers notamment le développement de méthodes d'analyse, des outils fiables pour la mesure de l'exposition aux PE. / Bisphenol A (BPA), a well-known endocrine disruptor (ED), is ubiquitously found in the environment. The high reactivity of BPA with chlorine results in the formation of chlorinated derivatives of BPA (ClxBPA), having a higher endocrine disrupting activity than BPA. In the field of environmental health sciences, human risk assessment implies, in particular, to perform biomonitoring and toxicokinetic studies requiring the analysis of environmental micropollutants in biological matrices. To estimate population exposure to BPA and ClxBPA, a LC-MS/MS method to assay BPA and ClxBPA was validated in urine. This method was then modified to improve the speed and sensitivity of the assay, and was applied to the measurement of ED exposure in a cohort of pregnant women.Toxicokinetic studies of BPA report the formation of conjugates, excreted in urine. Conventionally, for the measurement of conjugated derivatives, indirect methods using enzymatic deconjugation are proposed. In this context, we have developed an original method for validating the deconjugation efficiency.If the indirect measurement of metabolites after deconjugation appears easier, it is more rationale to assay directly the metabolite. Therefore, after synthesis of analytical standards, we were able to develop, for the first time, a method for assaying glucuronides and sulfates BPA and Cl2BPA derivatives in urine.This work offers, especially through the development of analytical methods, reliable tools for assessing human exposure to ED.

A aquicultura teve um aumento significativo em todo o mundo nos últimos anos. Muitas classes de antimicrobianos são usadas na aquicultura para o tratamento de infecções causadas por bactérias patogênicas. Entretanto, a contaminação do ambiente, do alimento e a ocorrência de resistência microbiana decorrentes da intensa utilização dos antimicrobianos são motivos de preocupação. Com o objetivo de se saber a ocorrência de antimicrobianos e possíveis formação de resistência microbiana em pisciculturas paulistas, um método rápido, sensível e simples de extração em fase sólida acoplada à cromatografia líquida e espectrometria de massas sequencial (SPE-LC-MS/MS), foi desenvolvido e validado para a determinação simultânea de 12 antimicrobianos (oxitetraciclina, tetraciclina, clortetraciclina, ciprofloxacina, enrofloxacina, sarafloxacina, norfloxacina, florfenicol, cloranfenicol, sulfatizol, sulfadimetoxina e sulfametazina) em água superficial e sedimento. Outro método, utilizando LC-MS/MS, foi elaborado para a determinação dos antimicrobianos em peixes. Paralelamente, também, foi avaliada a seleção de resistência microbiana dessas classes de antimicrobianos em peixe. Os antimicrobianos foram extraídos do sedimento com acetonitrila e tampão citrato, a fase orgânica foi eliminada e a purificação do extrato realizada com cartuchos SPE Strata SAX 500 mg Phenomenex. Os extratos de sedimento e as amostras de água (sem pré-tratamento) foram injetadas em um sistema analítico, pela primeira vez utilizado no Brasil, que consistia em uma pré-concentração com um amostrador automático equipado com um loop de 900 ?L, uma válvula usada para alternar entre os modos de carregamento ou eluição, duas bombas e um sistema de MS/MS. Os extratos de peixe foram purificados por filtração utilizando cartuchos Captiva ND. Sulfadimetoxina-d6 foi utilizado como padrão interno para aferir a precisão dos resultados. Os métodos desenvolvidos foram validados baseados na decisão da União Europeia 2002/657/CE. As amostras foram coletadas de 4 pisciculturas localizadas na represa da usina hidrelétrica de Ilha Solteira, Brasil. Foram feitas 4 amostragens no período de abril de 2013 a janeiro de 2014, totalizando 144 amostras de água, 144 de sedimento e 126 amostras de peixe. Resíduos de oxitetraciclina, tetraciclina e clortetraciclina foram encontrados em sedimentos e oxitetraciclina, tetraciclina e florfenicol foram identificados nas amostras de água e peixe; à medida que aumentava a distância dos tanques e o tamanho do peixe as quantidades encontradas nas amostras diminuíam. Isolou-se bactérias resistentes a quinolonas, tetraciclinas, sulfonamidas em 36 cepas e o índice de resistência múltipla a antibióticos (MAR), variou entre 0 e 0,86, ou seja, cepas sensíveis a 100% dos antimicrobianos testados e outras resistentes a 86%. De acordo com os resultados encontrados considera-se que ações mais restritivas são necessárias quanto ao uso intensivo de antibióticos na produção de peixes / The aquiculture has had a sharp increase worldwide in the last years. Many classes of antibiotics have been used in aquaculture to treat infections caused by a number of pathogenic bacteria. However, environmental and food contamination and bacterial resistance are the main concerns arisen by these intense uses. In order to know the occurrence of antibiotics and possible antimicrobial resistance in fish farms in São Paulo, a fast, sensitive, and simple on-line solid phase extraction to liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) was developed and validated for simultaneous assessment of 12 drugs (chloramphenicol, florfenicol, oxytetracycline, tetracycline, chlortetracycline, sulfadimethoxine, sulfathiazole, sulfamethazine, enrofloxacin, ciprofloxacin, norfloxacin, and sarafloxacin) in surface water and sediment. Another method using LC-MS/MS was elaborated to determine antibiotics in fish. In parallel, the selection of antimicrobial resistance of these classes of antibiotics in fish was evaluated. The antibiotics were extracted from sediment with acetonitrile and citric buffer, the organic phase was eliminated and the clean-up was made by Strata SAX 500 mg of Phenomenex. The water phase of sediment and water samples (without pre-treatment) was injected in the analytical system, which consisted of a pre-concentration with an automated liquid sampler fitted with a 900 ?L injection loop; a valve is used to switch between the load or elution modes, a pair of pumps and a MS/MS system, it is the first time that this system is used in Brazil. The fish extracts were cleaned by filtration by Captiva cartridges. Sulfadimethoxine-d6 was used as an internal standard to obtain more reliable results. The developed method was validated based in the European Union Decision 2002/657/EC. The samples were collected from 4 fish farms located in Ilha Solteira hydroelectric dam, Brazil. Four sampling were made in the period April/2013 until January/2014, totalizing 144 samples of water and sediment and 126 fish samples. Residues of oxytetracycline, tetracycline and chlortetracycline, were found in sediment and oxytetracycline, tetracycline, and florfenicol have been identified in water and fish samples, with increasing distance from the tanks and the size of the fishs the quantities of residue found in the samples decreased. Bacteria were resistant to quinolones, tetracyclines, sulfonamides in 36 strains and the multiple antibiotic resistance index (MAR) values ranged between 0 and 0.86, that is, strains with a sensitivity of 100% to the tested antimicrobials and others resistant of 86% to the tested antimicrobials. According to the results it is believed that more stringent measures are needed concerning the intensive use of antibiotics in fish production

Introdução: A administração de morfina através de bomba de infusão controlada pelo paciente (ACP) no tratamento da dor pós-cirurgica e traumática tem-se mostrado promissora e faz parte da rotina terapêutica de muitos hospitais. No entanto, doses altas ou repetidas deste opióide estão associadas a efeitos adversos dose dependentes, dentre eles, a depressão respiratória. No caso de pacientes submetidos a cirurgias de tórax, além da analgesia pós-operatória, devem também ser considerados como parâmetros relevantes a anestesia regional (intratecal) no intra-operatório, a qual pode contribuir para melhora da função pulmonar pós-operatória e extubação precoce no pós-operatório e a circulação extracorpórea (CEC), potencial fator de alteração na cinética de fármacos. Objetivos: Investigar a influência da morfina intratecal e da circulação extracorpórea (CEC) sobre o consumo de morfina ACP, área sob a curva e escores de dor no período pós-operatório, bem como propor modelo farmacocinético-farmacodinâmico (PK-PD) para correlação dessas variáveis. Adicionalmente, foi desenvolvido método analítico para quantificação da morfina plasmática. Métodos: 59 pacientes submetidos à cirurgia eletiva de revascularização com CEC e sem CEC, na presença ou não de morfina intratecal intra-operatória foram distribuídos em grupos com base na combinação das intervenções acima mencionadas. No período pós-operatório, todos os pacientes receberam bolus IV de 1mg de morfina, e então o dispositivo ACP foi instalado na unidade de terapia intensiva, através de cateter venoso após a extubação orotraqueal. A morfina ACP foi liberada através de livre demanda solicitada pelo paciente (bolus de 1 mg), lock-out de 5 min até 36 horas do pós-operatório. Coletaram-se amostras seriadas de sangue de cateter venoso no período (3,6,12,18,24,36 horas) e a morfina plasmática foi determinada através da cromatografia líquida - espectrometria de massas (LC-MS/MS ESI+) após a purificação das amostras de plasma. A intensidade da dor foi monitorada no mesmo período pela escala análoga visual (EAV). A modelagem PK-PD foi investigada pelo GraphPad Prism 5.0. Resultados: O consumo de morfina e a intensidade da dor diferiram entre os grupos. O modelo do EMAX e a curva de histerese foram propostos pela modelagem PK-PD. Conclusões: O método analítico mostrou-se adequado na determinação da morfina plasmática. O consumo de morfina os escores de dor EAV no pós-operatório diferiram pela comparação dos grupos de pacientes investigados. Menores doses de morfina ACP foram requeridas pelos pacientes que receberam morfina intratecal intra-operatória. Demonstrou-se através do modelo do EMAX correlação não linear entre os parâmetros consumo de morfina e AUC0-36, e curva de histerese foi obtida quando se plotou consumo de morfina versus escore de dor. / Introduction: Morphine administration using patient controlled analgesia (PCA) for treatment of post surgical and traumatic pain has been a current practice in many hospitals. However, large or repeated doses of this opioid are associated to dose dependent adverse events, including, respiratory depression. Considering patients submitted to thoracic surgery, in addition to the postoperatory analgesia, two other relevant parameters must be considered: regional anesthesia (intrathecal) in the intra-operatory period, which should contribute to the respiratory function improvement and decrease in the extubation time; and the cardiopulmonary bypass (OPCAB), that potentially alters the drugs\' kinetics. Objectives: To investigate the influence of intrathecal morphine administration and cardiopulmonary bypass (OPCAB) in the morphine PCA drug requirements, area under the curve of morphine plasma concentration versus time and pain scores in the postoperative period, and to choose a pharmacokinetic-pharmacodynamic model to correlate these variables. In addition, an analytical method was developed to quantify morphine in plasma. Methods: 59 patients submitted to elective coronary artery bypass grafting (CABG) with (CPB) and without cardiopulmonary bypass (OPCAB), with and without intrathecal morphine in the intra-operative period were distributed by the combination of the above mentioned interventions. In the postoperative period, all the patients were given an IV bolus of 1mg of morphine, and then PCA device was installed in the intensive care unit by a venous catheter after the orotracheal extubation. Morphine PCA was delivered on demand (boluses of 1 mg), lock-out of 5 min until 36 hours of the postoperative period. A serial of blood samples were collected from venous catheter of patients at the postoperative period (3,6,12,18,24,36 hrs) and morphine plasma concentrations were determined by Liquid Chromatography-Mass Spectrometry ((LC-MS/MS ESI+)) after the purification of plasma samples. Pain scores were monitored during the same period by a visual analogue scale, VAS or 1-2-3 pain scale. PK-PD modeling was investigated by applying the GraphPad Prism 5.0. Results: Drug dose requirements and analgesia were significant different in patients of groups investigated. EMAX model and the hysteresis curve were proposed by PK-PD modeling to correlate drug requirements and AUC 0-36 or VAS. Conclusions: LC-MS/MS (ESI+) method was adequate for drug measurements in plasma. Morphine dose requirements and analgesia were different by comparison of groups. Lower doses of morphine by PCA were required for the groups that have received intrathecal morphine intraoperatively. It was demonstrated a non linear correlation between parameters by EMAX model when drug requirements and AUC0-36 were plotted, and the hysteresis curve was obtained when analgesia dose requirements was plotted against pain score.

A aquicultura teve um aumento significativo em todo o mundo nos últimos anos. Muitas classes de antimicrobianos são usadas na aquicultura para o tratamento de infecções causadas por bactérias patogênicas. Entretanto, a contaminação do ambiente, do alimento e a ocorrência de resistência microbiana decorrentes da intensa utilização dos antimicrobianos são motivos de preocupação. Com o objetivo de se saber a ocorrência de antimicrobianos e possíveis formação de resistência microbiana em pisciculturas paulistas, um método rápido, sensível e simples de extração em fase sólida acoplada à cromatografia líquida e espectrometria de massas sequencial (SPE-LC-MS/MS), foi desenvolvido e validado para a determinação simultânea de 12 antimicrobianos (oxitetraciclina, tetraciclina, clortetraciclina, ciprofloxacina, enrofloxacina, sarafloxacina, norfloxacina, florfenicol, cloranfenicol, sulfatizol, sulfadimetoxina e sulfametazina) em água superficial e sedimento. Outro método, utilizando LC-MS/MS, foi elaborado para a determinação dos antimicrobianos em peixes. Paralelamente, também, foi avaliada a seleção de resistência microbiana dessas classes de antimicrobianos em peixe. Os antimicrobianos foram extraídos do sedimento com acetonitrila e tampão citrato, a fase orgânica foi eliminada e a purificação do extrato realizada com cartuchos SPE Strata SAX 500 mg Phenomenex. Os extratos de sedimento e as amostras de água (sem pré-tratamento) foram injetadas em um sistema analítico, pela primeira vez utilizado no Brasil, que consistia em uma pré-concentração com um amostrador automático equipado com um loop de 900 ?L, uma válvula usada para alternar entre os modos de carregamento ou eluição, duas bombas e um sistema de MS/MS. Os extratos de peixe foram purificados por filtração utilizando cartuchos Captiva ND. Sulfadimetoxina-d6 foi utilizado como padrão interno para aferir a precisão dos resultados. Os métodos desenvolvidos foram validados baseados na decisão da União Europeia 2002/657/CE. As amostras foram coletadas de 4 pisciculturas localizadas na represa da usina hidrelétrica de Ilha Solteira, Brasil. Foram feitas 4 amostragens no período de abril de 2013 a janeiro de 2014, totalizando 144 amostras de água, 144 de sedimento e 126 amostras de peixe. Resíduos de oxitetraciclina, tetraciclina e clortetraciclina foram encontrados em sedimentos e oxitetraciclina, tetraciclina e florfenicol foram identificados nas amostras de água e peixe; à medida que aumentava a distância dos tanques e o tamanho do peixe as quantidades encontradas nas amostras diminuíam. Isolou-se bactérias resistentes a quinolonas, tetraciclinas, sulfonamidas em 36 cepas e o índice de resistência múltipla a antibióticos (MAR), variou entre 0 e 0,86, ou seja, cepas sensíveis a 100% dos antimicrobianos testados e outras resistentes a 86%. De acordo com os resultados encontrados considera-se que ações mais restritivas são necessárias quanto ao uso intensivo de antibióticos na produção de peixes / The aquiculture has had a sharp increase worldwide in the last years. Many classes of antibiotics have been used in aquaculture to treat infections caused by a number of pathogenic bacteria. However, environmental and food contamination and bacterial resistance are the main concerns arisen by these intense uses. In order to know the occurrence of antibiotics and possible antimicrobial resistance in fish farms in São Paulo, a fast, sensitive, and simple on-line solid phase extraction to liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) was developed and validated for simultaneous assessment of 12 drugs (chloramphenicol, florfenicol, oxytetracycline, tetracycline, chlortetracycline, sulfadimethoxine, sulfathiazole, sulfamethazine, enrofloxacin, ciprofloxacin, norfloxacin, and sarafloxacin) in surface water and sediment. Another method using LC-MS/MS was elaborated to determine antibiotics in fish. In parallel, the selection of antimicrobial resistance of these classes of antibiotics in fish was evaluated. The antibiotics were extracted from sediment with acetonitrile and citric buffer, the organic phase was eliminated and the clean-up was made by Strata SAX 500 mg of Phenomenex. The water phase of sediment and water samples (without pre-treatment) was injected in the analytical system, which consisted of a pre-concentration with an automated liquid sampler fitted with a 900 ?L injection loop; a valve is used to switch between the load or elution modes, a pair of pumps and a MS/MS system, it is the first time that this system is used in Brazil. The fish extracts were cleaned by filtration by Captiva cartridges. Sulfadimethoxine-d6 was used as an internal standard to obtain more reliable results. The developed method was validated based in the European Union Decision 2002/657/EC. The samples were collected from 4 fish farms located in Ilha Solteira hydroelectric dam, Brazil. Four sampling were made in the period April/2013 until January/2014, totalizing 144 samples of water and sediment and 126 fish samples. Residues of oxytetracycline, tetracycline and chlortetracycline, were found in sediment and oxytetracycline, tetracycline, and florfenicol have been identified in water and fish samples, with increasing distance from the tanks and the size of the fishs the quantities of residue found in the samples decreased. Bacteria were resistant to quinolones, tetracyclines, sulfonamides in 36 strains and the multiple antibiotic resistance index (MAR) values ranged between 0 and 0.86, that is, strains with a sensitivity of 100% to the tested antimicrobials and others resistant of 86% to the tested antimicrobials. According to the results it is believed that more stringent measures are needed concerning the intensive use of antibiotics in fish production

Introdução: A administração de morfina através de bomba de infusão controlada pelo paciente (ACP) no tratamento da dor pós-cirurgica e traumática tem-se mostrado promissora e faz parte da rotina terapêutica de muitos hospitais. No entanto, doses altas ou repetidas deste opióide estão associadas a efeitos adversos dose dependentes, dentre eles, a depressão respiratória. No caso de pacientes submetidos a cirurgias de tórax, além da analgesia pós-operatória, devem também ser considerados como parâmetros relevantes a anestesia regional (intratecal) no intra-operatório, a qual pode contribuir para melhora da função pulmonar pós-operatória e extubação precoce no pós-operatório e a circulação extracorpórea (CEC), potencial fator de alteração na cinética de fármacos. Objetivos: Investigar a influência da morfina intratecal e da circulação extracorpórea (CEC) sobre o consumo de morfina ACP, área sob a curva e escores de dor no período pós-operatório, bem como propor modelo farmacocinético-farmacodinâmico (PK-PD) para correlação dessas variáveis. Adicionalmente, foi desenvolvido método analítico para quantificação da morfina plasmática. Métodos: 59 pacientes submetidos à cirurgia eletiva de revascularização com CEC e sem CEC, na presença ou não de morfina intratecal intra-operatória foram distribuídos em grupos com base na combinação das intervenções acima mencionadas. No período pós-operatório, todos os pacientes receberam bolus IV de 1mg de morfina, e então o dispositivo ACP foi instalado na unidade de terapia intensiva, através de cateter venoso após a extubação orotraqueal. A morfina ACP foi liberada através de livre demanda solicitada pelo paciente (bolus de 1 mg), lock-out de 5 min até 36 horas do pós-operatório. Coletaram-se amostras seriadas de sangue de cateter venoso no período (3,6,12,18,24,36 horas) e a morfina plasmática foi determinada através da cromatografia líquida - espectrometria de massas (LC-MS/MS ESI+) após a purificação das amostras de plasma. A intensidade da dor foi monitorada no mesmo período pela escala análoga visual (EAV). A modelagem PK-PD foi investigada pelo GraphPad Prism 5.0. Resultados: O consumo de morfina e a intensidade da dor diferiram entre os grupos. O modelo do EMAX e a curva de histerese foram propostos pela modelagem PK-PD. Conclusões: O método analítico mostrou-se adequado na determinação da morfina plasmática. O consumo de morfina os escores de dor EAV no pós-operatório diferiram pela comparação dos grupos de pacientes investigados. Menores doses de morfina ACP foram requeridas pelos pacientes que receberam morfina intratecal intra-operatória. Demonstrou-se através do modelo do EMAX correlação não linear entre os parâmetros consumo de morfina e AUC0-36, e curva de histerese foi obtida quando se plotou consumo de morfina versus escore de dor. / Introduction: Morphine administration using patient controlled analgesia (PCA) for treatment of post surgical and traumatic pain has been a current practice in many hospitals. However, large or repeated doses of this opioid are associated to dose dependent adverse events, including, respiratory depression. Considering patients submitted to thoracic surgery, in addition to the postoperatory analgesia, two other relevant parameters must be considered: regional anesthesia (intrathecal) in the intra-operatory period, which should contribute to the respiratory function improvement and decrease in the extubation time; and the cardiopulmonary bypass (OPCAB), that potentially alters the drugs\' kinetics. Objectives: To investigate the influence of intrathecal morphine administration and cardiopulmonary bypass (OPCAB) in the morphine PCA drug requirements, area under the curve of morphine plasma concentration versus time and pain scores in the postoperative period, and to choose a pharmacokinetic-pharmacodynamic model to correlate these variables. In addition, an analytical method was developed to quantify morphine in plasma. Methods: 59 patients submitted to elective coronary artery bypass grafting (CABG) with (CPB) and without cardiopulmonary bypass (OPCAB), with and without intrathecal morphine in the intra-operative period were distributed by the combination of the above mentioned interventions. In the postoperative period, all the patients were given an IV bolus of 1mg of morphine, and then PCA device was installed in the intensive care unit by a venous catheter after the orotracheal extubation. Morphine PCA was delivered on demand (boluses of 1 mg), lock-out of 5 min until 36 hours of the postoperative period. A serial of blood samples were collected from venous catheter of patients at the postoperative period (3,6,12,18,24,36 hrs) and morphine plasma concentrations were determined by Liquid Chromatography-Mass Spectrometry ((LC-MS/MS ESI+)) after the purification of plasma samples. Pain scores were monitored during the same period by a visual analogue scale, VAS or 1-2-3 pain scale. PK-PD modeling was investigated by applying the GraphPad Prism 5.0. Results: Drug dose requirements and analgesia were significant different in patients of groups investigated. EMAX model and the hysteresis curve were proposed by PK-PD modeling to correlate drug requirements and AUC 0-36 or VAS. Conclusions: LC-MS/MS (ESI+) method was adequate for drug measurements in plasma. Morphine dose requirements and analgesia were different by comparison of groups. Lower doses of morphine by PCA were required for the groups that have received intrathecal morphine intraoperatively. It was demonstrated a non linear correlation between parameters by EMAX model when drug requirements and AUC0-36 were plotted, and the hysteresis curve was obtained when analgesia dose requirements was plotted against pain score.

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 Δ9-tetrahidrocanabinol (THC), 11-hidroxi-Δ9-tetrahidrocannabinol (THC-OH) e 11-nor-Δ9-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 Δ9-tetrahydrocannabinol (THC), 11-hydroxy-Δ9-tetrahydrocannabinol (THC-OH), and 11-nor-Δ9-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.

Malgré une sensible baisse des ventes depuis quelques années, la France reste le premier utilisateur européen de phytosanitaires. Ceci n’est pas sans conséquences sur l’environnement et les organismes non visés par ces produits. De nombreuses études ont permis d’une part de mieux comprendre les phénomènes de transfert des pesticides dans les compartiments eau-air sols et d’autre part de mettre en évidence leur contamination. Les données concernant l’eau sont plus nombreuses du fait de son statut de réceptacle ultime des pollutions.et des normes établies par les textes réglementaires. La Directive Cadre européenne sur l’Eau (DCE) adoptée en 2000 vise à atteindre le bon état chimique et écologique des eaux d’ici 2015 avec une obligation de résultats. Le bon état chimique dépend du respect des normes de qualité environnementales (NQE) établies notamment pour 24 pesticides. En 2012, 5% des points de mesure en cours d’eau et 19% des points de mesure en eaux souterraines ne respectent pas ces NQE.Le Languedoc Roussillon est depuis de nombreuses années la première région viticole de France et souffre d’une pression phytosanitaire importante. En 2014, un rapport de l’Agence Régionale de Santé (ARS) fait même état de l’utilisation encore récente de pesticides interdits (simazine, terbuthylazine…) depuis le début des années 2000. L’ARS a de plus affirmé que la plupart des cas de non-conformité avérés pouvaient être rattachés à la présence d’activité agricole dans le bassin versant du captage.Remédier à ces contaminations passe par le suivi de la qualité des eaux et la caractérisation de la pollution. Les outils actuels s’avèrent ne pas être toujours représentatifs de cette pollution ;de nouveaux outils sont donc mis au point. L’étude présentée ici porte sur l’utilisation de l’échantillonnage passif de type POCIS (Polar Organic Chemical Integrative Sampler) pour le suivi de la contamination en pesticides de trois bassins versants languedociens : Paulhan,Puissalicon, Roujan.Dans un premier temps, l’étude des limites possibles du dispositif POCIS a été faite pour évaluer son applicabilité dans les conditions d’exposition imposées par le terrain. Le comportement des molécules au sein de l’outil a été étudié pour une meilleure compréhension des phénomènes de diffusion qui s’y produisent. Ces études ont mené à la mise en place de calibrations en laboratoire en conditions contrôlées. [...] La mise en place des dispositifs POCIS sur les trois sites d’études a mis en évidence la prédominance de pesticides utilisés en viticulture et a montré tout l’intérêt de l’outil d’un point de vue qualitatif. La comparaison de l’outil avec l’échantillonnage automatisé montre des disparités entre concentration réelle et concentration estimée via le POCIS. Ce dernier reste à améliorer d’un point de vue quantitatif pour obtenir un échantillonneur fiable et robuste pour ce type de milieu d’exposition. / Despite a noticeable sales decrease since few years, France remains the first european user of phytosanitary products. This is not without consequences on the environment and the untargeted organisms. Several studies allowed on one hand to better understand phenomena of pesticides transfer in water-air and soils compartments and on the other hand to highlight their contamination. Data concerning the water are more numerous cause of its status of ultimate receptacle of pollution and standards established by regulatory texts. The european Water Framework Directive (WFD) adopted in 2000 aims to achieve good chemical and ecological status of water bodies by 2015 with an obligation of results. The good chemical status depends on respect for the Environmental Quality Standards (EQS) established, for instance, for 24 pesticides. In 2012, 5% of river measurement points and 19%of groundwater measurement points do not respect these EQS. For many years, Languedoc Roussillon is the first wine-producing region of France and suffers from a significant pesticide pressure. In 2014, a report from the Regional Health Agency (RHA) has mentioned a relatively recent use of banned pesticides since the early 2000’s like simazin and terbutylazin. Moreover, RHA stated that most of recognized cases of non-compliance could be linked to the presence of agricultural activity in the catchment basin.[...] The establishment of POCIS devices on the three study sites showed the predominance ofpesticides used in viticulture and showed all the interest of the tool on a qualitative point ofview. The comparison of the tool with the automated sampling shows disparities between real concentration and concentration estimated with the POCIS. However, it remains to improvethe tool on a quantitative way to obtain a reliable and robust sampler for this type of exposure medium.

<p>Ispitani su hemijski sastav i biolo&scaron;ka aktivnost &scaron;est samoniklih vrsta iz tribusa Scandiceae<br />(familija Apiaceae): Anthriscus sylvestris, Anthriscus cerefolium, Chaerophyllum bulbosum,<br />Chaerophyllum hirsutum, Chaerophyllum temulentum i Scandix pecten-veneris. LC-MS-MS<br />analizom ekstrakata identifikovano je vi&scaron;e desetina sekundarnih biomolekula iz klasa flavonoida,<br />fenilpropenskih kiselina, lignana i kumarina. GC-MS analiza pružila je uvid u sastav volatilnih<br />komponenti i njihov hemosistematski značaj. Potvrđeno je da sve ispitivane vrste imaju umereno<br />antioksidantno, antiinflamatorno i antiproliferativno dejstvo.</p> / <p> Chemical composition and biological activity of six wild-growing species from<br /> Scandiceae tribe (Apiaceae family) &ndash; Anthriscus sylvestris, Anthriscus cerefolium, Chaerophyllum<br /> bulbosum, Chaerophyllum hirsutum, Chaerophyllum temulentum and Scandix pecten-veneris &ndash; was<br /> examined. By LC-MS-MS analysis, a large number of secondary biomolecules was identified in<br /> extracts, including flavonoids, phenylpropenic acids, lignans and coumarins. GC-MS analysis<br /> provided insight into volatile components composition and chemosystematic significance. All<br /> investigated species exhibited moderate antioxidant, anti-inflammatory and antiproliferative<br /> activity.</p>

Le présent projet vise à documenter la nécessité d’augmenter notre connaissance de la présence des contaminants organiques tels que les médicaments dans l’environnement et d’évaluer leur devenir environnemental. On a étudié la présence de composés pharmaceutiques dans différents échantillons d'eau. On a focalisé nos efforts spécialement sur les échantillons d'eau de l'usine d'épuration de la Ville de Montréal et ses effluents, les eaux de surface avoisinantes et l’eau du robinet dans la région de Montréal. Pour ce faire, on a tout d’abord développé deux méthodes analytiques automatisées basées sur la chromatographie liquide avec extraction en phase solide (SPE) couplée à la chromatographie liquide couplée à la spectrométrie de masse en tandem (LC-MS/MS). On a également étudié les performances des trois techniques d'ionisation à pression atmosphérique (API), pour ensuite les utiliser dans la méthode analytique développée. On a démontré que l'ionisation par électronébulisation (ESI) est une méthode d'ionisation plus efficace pour l'analyse des contaminants pharmaceutiques dans des échantillons de matrices très complexes comme les eaux usées. Une première méthode analytique SPE couplée à la LC-MS/MS a été développée et validée pour l'étude des échantillons complexes provenant de l'usine d'épuration de la Ville de Montréal et des eaux de surface près de l'usine. Cinq médicaments de prescription ont été étudiés: le bézafibrate (un régulateur de lipides), le cyclophosphamide et le méthotrexate (deux agents anticancéreux), l'orlistat (un agent anti-obésité) et l’énalapril (utilisé dans le traitement de l'hypertension). La plupart de ces drogues sont excrétées par le corps humain et rejetées dans les eaux usées domestiques en faisant par la suite leur chemin vers les usines municipales de traitement des eaux usées. On a pu démontrer qu'il y a un faible taux d’élimination à l'usine d'épuration pour le bézafibrate et l'énalapril. Ces deux composés ont aussi été détectés dans les eaux de surface sur un site à proximité immédiate de la décharge de l’effluent de la station d'épuration. i En observant la nécessité de l'amélioration des limites de détection de la première méthode analytique, une deuxième méthode a été développée. Pour la deuxième méthode, un total de 14 contaminants organiques, incluant trois agents anti-infectieux (clarithromycin, sulfaméthoxazole et triméthoprime), un anticonvulsant (carbamazépine) et son produit de dégradation (10,11-dihydrocarbamazépine), l'agent antihypertensif (enalapril), deux antinéoplastiques utilisés en chimiothérapie (cyclophosphamide et méthotrexate), des herbicides (atrazine, cyanazine, et simazine) et deux produits de transformation de l’atrazine (deséthylatrazine et déisopropylatrazine) ainsi qu’un agent antiseptique (triclocarban). Ces produits ont été quantifiés dans les eaux de surface ainsi que dans l’eau du robinet. L'amélioration des limites de détection pour cette méthode a été possible grâce à la charge d'un volume d'échantillon supérieur à celui utilisé dans la première méthode (10 mL vs 1 mL). D'autres techniques de confirmation, telles que les spectres des ions produits utilisant une pente d’énergie de collision inverse dans un spectromètre de masse à triple quadripôle et la mesure des masses exactes par spectrométrie de masse à temps d’envol, ont été explorées. L'utilisation d'un analyseur de masse à temps d’envol a permis la confirmation de 6 des 14 analytes. Finalement, étant donné leur haute toxicité et pour évaluer leur persistance et leur transformation au niveau du traitement des eaux potables, la cinétique d'oxydation du cyclophosphamide et de méthotrexate avec l'ozone moléculaire et des radicaux OH a été étudiée. Les constantes de dégradation avec l'ozone moléculaire ont été calculées et la qualité de l'eau après traitement a pu être évaluée. Le rendement du processus d'ozonation a été amélioré pour la cyclophosphamide dans les eaux naturelles, en raison de la combinaison de réactions directes et indirectes. Cette étude a montré que l'ozone est très efficace pour oxyder le méthotrexate mais que le cyclophosphamide serait trop lent à s’oxyder pour un traitement efficace aux conditions usuelles de traitement de l’eau potable. / This project aims to document the need to increase our knowledge of organic contaminants such as pharmaceuticals in the environment and to assess their environmental fate. We studied the presence of pharmaceutical compounds in different water samples. We studied their presence in wastewater samples from the treatment plant of the City of Montreal, effluents, surface and tap water. To do this we developed two analytical methods based on solid phase extraction (SPE) coupled to liquid chromatography -tandem mass spectrometry (LC-MS/MS). . The performance of three atmospheric pressure ionization (API) techniques was also studied for their subsequent use in the developed method. It was demonstrated that electrospray ionization (ESI) is a more effective ionization method for the analysis of pharmaceutical contaminants in samples as complex as wastewaters. A first analytical LC-MS/MS method, was developed and validated for the investigation of samples from the wastewater treatment plant and surface waters near the plant of Montreal. Five prescription drugs were studied: bezafibrate (lipid regulator), cyclophosphamide, and methotrexate (two anticancer agents), orlistat (an anti-obesity agent) and enalapril used in the treatment of hypertension. Most of these drugs are excreted by the human body in high percentages and released into domestic wastewaters, making their way to the municipal wastewater treatment plants. It was demonstrated that there is a low rate of elimination at the wastewater treatment plant for bezafibrate and enalapril. These two compounds were also detected in surface waters on a site close to the discharge of the treatment plant effluents. For this first analytical method we observed the necessity of improvement of the detection limits of the method. A second method was then developed to improve the detection limits and to study a total of 14 organic contaminants, including three antiinfective agents (clarithromycin, sulfamethoxazole and trimethoprim), an anticonvulsant (carbamazepine) and its degradation product 10,11-dihydrocarbamazepine, the iii antihypertensive agent (enalapril), two antineoplastic (methotrexate and cyclophosphamide), herbicides (atrazine, cyanazine, and simazine) and two transformation products of atrazine (desethylatrazine and déisopropylatrazine) and an antiseptic agent (triclocarban). These products were quantified in surface water and tap water. The improvement of the detection limits of this method was possible due to the loading of a greater sample volume than that used in the first method (10 mL vs 1 mL). Other confirmation techniques, such as the data-dependent reverse energy ramp scan on a triple quadrupole and accurate mass measurements on a time-of-flight mass spectrometer were explored. Using time-of-flight mass spectrometer determinations allowed the confirmation of six of the 14 analytes. Finally, due to their toxic properties, the oxidation kinetics of cyclophosphamide and methotrexate with molecular ozone and OH radicals was studied in bench scale. The degradation constants with molecular ozone were calculated and the water quality after treatment was assessed. The overall process performance was improved for cyclophosphamide in natural waters, due to the combination of direct and indirect reactions. The study showed that ozone is very effective for the oxidation of methotrexate, but cyclophosphamide is too slowly oxidized for an effective drinking water treatment under usual conditions.