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.

Made available in DSpace on 2014-07-29T15:25:22Z (GMT). No. of bitstreams: 1 Tese Carina-pos defesa.pdf: 485460 bytes, checksum: 2ca505db4ca73a05349dd92fbfc7df1d (MD5) Previous issue date: 2011-03-18 / Montelukast is a potent reversible selective inhibitor of cysteinilleukotrien- 1 receptor, avoiding that these mediators promote the asthmatic response. Its commercialization in Brazil, as a terminated product, is protected by patent up to 2010. Once the active ingredient Montelukast is recent in the pharmaceutical market and there is no methodology description in official compendiums capable to assure the quality of new formulations, the objective of this paper was the pharmaceutics of montelukast film coated tablets, the development and the validation of analytical and bioanalytical methodologies foreseeing the pharmaceutical equivalence and bioequivalence with the reference medication of the market. With this purpose, some physicalchemical parameters were characterized, assay and dissolution methodologies were developed and validated per high performance liquid chromatography with ultraviolet detection (HPLC-UV) for the quantification of montelukast present in 10.0mg film coated tablets. The quantification of montelukast sodium in human plasma was performed using Loratadine as internal standard and high performance liquid chromatography attached to mass detector (HPLC - MS / MS). The active ingredient was extracted from the human plasma using precipitation extraction. The results found for the parameters of specificity, linearity, accuracy, precision, quantification and detection limits and stability in the methodologies validation confirm they were adequate for the objective proposed. The analytical methodologies developed and validated were applied in the pharmaceutics of the tablets for the determination of the formulation similar to the market reference medication Singulair®. This formulation was submitted to stability assays to assure its quality and to allow the performance of pharmaceutical equivalence and bioequivalence with the purpose of registering as a generic medication. / O Montelucaste é um potente inibidor seletivo reversível do receptor cisteinil-leucotrieno-1, evitando que os mediadores provoquem a resposta asmática. Sua comercialização no Brasil, na forma de produto acabado, é protegida por patente até 2010. Uma vez que o fármaco Montelucaste é recente no mercado farmacêutico e não há descrição de metodologia em compêndios oficiais capaz de assegurar a qualidade das novas formulações, o objetivo deste trabalho foi o desenvolvimento farmacotécnico de comprimidos revestidos de montelucaste, desenvolvimento e validação de métodos analítico e bioanalítico visando obtenção de equivalência farmacêutica e bioequivalência com o medicamento referência de mercado. Com esta finalidade, foram caracterizados alguns parâmetros físico-químicos, desenvolvidos e validados métodos de doseamento e dissolução por cromatografia líquida de alta eficiência com detecção ultravioleta (HPLC-UV) para quantificação de montelucaste presente em comprimidos revestidos de 10,0 mg. A determinação de montelucaste sódico em plasma humano foi realizada utilizando-se loratadina como padrão interno e cromatografia líquida de alta eficiência acoplada a detector de massas (HPLC MS/MS). O fármaco foi extraído do plasma humano utilizando extração por precipitação. Os resultados encontrados dos parâmetros de especificidade, linearidade, exatidão, precisão, limites de quantificação e detecção e estabilidade nas validações dos métodos confirmam que os mesmos foram adequados para a finalidade proposta. Os métodos analíticos desenvolvidos e validados foram aplicados no desenvolvimento farmacotécnico dos comprimidos para determinação de uma formulação próxima ao medicamento referência de mercado Singulair®. Esta formulação foi submetida a ensaios de estabilidade para assegurar sua qualidade e permitir a realização de equivalência e bioequivalência farmacêutica com o intuito de registro como medicamento genérico.

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.

<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 long bio-polymère d'ADN est condensé à l’intérieur du noyau des cellules eukaryotes à l'aide de petites protéines appelées histones. En plus de leurs fonctions condensatrices,ces histones sont également la cible de nombreuses modifications post-traductionnelles(MPT), particulièrement au niveau de leur section N-terminale. Ces modifications réversibles font partie d’un code d’histones épi-génétique transmissible qui orchestre et module dynamiquement certains événements impliquant la chromatine, tels l’activation et la désactivation de gènes ainsi que la duplication et la réparation d’ADN. Ces modifications sont impliquées subséquemment dans la signalisation et la progression de cancers, tels que la leucémie. En conséquence, l'élucidation des modifications d’histones est importante pour comprendre leurs fonctions biologiques. Une méthodologie analytique a été mise au point en laboratoire pour isoler, détecter, et quantifier les MPT d’histones en utilisant une approche rapide à deux volets à l’aide d’outils bioinformatiques spécialisés. La méthodologie développée en laboratoire a été validée en utilisant des histones de souche sauvage ainsi que deux types d’histones mutants déficients en enzymes acétyltransferase. Des trois sources d’histones utilisées, la seule MPT qui a démontré un changement significatif est l’acétylation de l’histone H3 à lysine 56 (H3K56ac). L’expression et la stoechiométrie de cette MPT, issue de cellules de souche sauvage et de cellules mutantes, ont été déterminées avec précision et comparées. Les fonctions de balayage polyvalentes d'un instrument à trappe ionique quadrupôle linéaire hybride ont été utilisées pour améliorer la détection de protéines intactes. Le mode de balayage « enhanced multiply charged » (EMC) a été modifié pour contenir et détecter les ions de protéines intactes situées dans la trappe ionique linéaire. Ce mode de balayage nommé « targeted EMC » (tEMC) a permis de quadrupler le niveau de sensibilité (signal/interférence), et quintupler la résolution du mode de balayage conventionnel. De plus, la capacité de séparation des charges du tEMC a réduit de façon significative les effets de « space charge » dans la trappe ionique linéaire. La résolution supérieure du mode tEMC a permis de différencier plusieurs isoformes modifiées, particulièrement pour l’histone H3. L’analyse des peptides d’histones trypsiques à l’aide du mode de balayage « MRM » a permis le séquençage et la quantification de MPT avec un haut degré de précision. La seule MPT qui était sous-exprimée entre l’histone de souche sauvage et le mutant DOT1L fut la méthylation de l’histone H3 lysine 79(H3K79me1). Les effets de deux inhibiteurs d’enzymes HDAC (HDACi) sur l’expression de MPT d’histone ont été évalués en utilisant la méthodologie analytique mentionnée. Les histones extraites de cellules normales et cancéreuses ont été exposées à du Vorinostat(SAHA) ou du Entinostat (MS-275) pour une période de 24 à 72 heures. Deux histones furent principalement affectées, soit H3 et H4. Étonnamment, les mêmes effets n'ont pas été détectés lorsque les cellules normales ont été traitées avec le HDACi pour une période de 48 à 72 heures. Une méthode absolue de quantification avec une courbe d’étalonnage a été développée pour le peptide H3K56ac. Contrairement à certaines publications, nos résultats démontrent que cette MPT est présente dans les cellules mammifères avec une stoechiométrie très basse (< 0,1%) et n'est pas surexprimée de façon significative après le traitement au HDACi. / In eukaryotic cells, the lengthy DNA biopolymer is condensed into the cell nucleus with the aid of small packaging proteins called histones. In addition to their packing functions,histones are also targets of numerous post translational modifications (PTMs), especially on their N-terminus. These reversible modifications are believed to be constituents of a heritable epigenetic “histone code” that dynamically orchestrate and modulate chromatin based events such as gene activation and silencing, DNA replication and repair, and are also involved in the downstream signaling and progression of cancers, such as leukemia. Thus, the elucidation of histone PTMs is important in understanding their biological function. An analytical workflow was designed and set-up in the laboratory to isolate, detect, and quantitate histone PTM, using a two-pronged, unbiased, and rapid approach with specialized bioinformatic tools. The workflow was validated using histones from wildtype, and 2 mutants deficient in acetyltransferase activity. Between the three histone sources, the only PTM that demonstrated any change was acetylation at histone H3 lysine 56 (H3K56ac). The down-regulation and stoichiometry of this PTM was accurately assessed between wild-type and mutant cells. The versatile scan functions of a hybrid quadrupole-linear ion trap instrument were exploited to enhance the detection of intact histone proteins. The enhanced multiply charged (EMC) scan was modified in order to contain and detect intact protein ions within the linear ion trap. This targeted EMC (or tEMC) resulted in not only a 4-fold increase in signal-to-noise, but also a 5-fold increase in resolution. Furthermore, the charge separation capability of the tEMC dramatically reduced space charge effects within the linear ion trap. The superior resolution of the tEMC mode allowed for the discimination of many modified histone isoforms, especially for histone H3. Using the bottom-up strategy with multiple reaction monitoring (MRM), histone peptides were quantified and sequenced with a high degree of precision. The only PTM that was down-regulated between wild-type and DOT1L mutant histones was methylation at histone H3 lysine 79 (H3K79me1). The effects of two clinically relevant small molecule HDAC inhibitors (HDACi) on histone PTMs patterns were assessed using the analytical workflow developed. Histones derived from both normal and cancer cells were exposed to either Vorinostat (SAHA) or Entinostat (MS-275) over a 24- to 72 hour period. The two core histones primarily affected were H3 and H4. Surprisingly, the same effects were not observed when normal cells were treated with three doses of SAHA at 24-hour intervals over a 72-hour period. An absolute quantitation method using a calibration curve was developed for H3K56ac. In opposition to other published literature, our findings demonstrate that this PTM is present in very low stoichiometry (< 0.1%) in mammalian cells, and exhibits no significant up-regulation in different cell lines treated with several types of HDACi.

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.