I den här studien har metoder för att separera immunoglobulin (IgG) utvecklats med hjälp av kapillärelektrofores (CE) och Ultra högupplösande vätskekromatografi-Mass spektrometri (UHPLC-MS). Vid analysen av peptider från IgG spjälkat av trypsin (IgG TD) detekterades 26 glykopepeptider med UHPLC-MS genom att använda olika gradienter och mobilfaser. Dessutom analyserades fyra olika koncentrations av IgG TD för att testa detektionsgränsen. Resultatet visade att glykopeptider kunde detekteras i alla fyra koncentrationer men antalet detekterade glykopeptider minskade med minskande koncentration. Den sista delen av UHPLC-MS analysen var en selektivitetsanalys där en blandning av bovint serumalbumin spjälkat av trypsin (BSA TD) och IgG TD analyserades i olika koncentrationer. Selektivitetsanalysen visade att ungefär samma antal IgG glykopeptider detekterades som vid analys av IgG TD. Vid CE-analysen användes tre olika kapillärer, varav två av dem var polyvinylalkohol-coated och olika buffertlösningar (BGE) användes. De slutgiltiga resultaten visade 5 toppar som skulle kunna representera IgG gykopeptider men eftersom ingen MS utfördes kunde inga slutsatser dras. Slutligen, användes Matrix-Assisted Laser desorption/ionisation-time-of-flight (MALDI-TOF) för att analysera proverna innan analys och för att jämföra resultaten med UHPLC-MS. / In this study, methods to separate immunoglobulin G (IgG) glycopeptides were developed using capillary electrophoresis (CE) and ultra-high-performance liquid chromatography-Mass spectrometry (UHPLC-MS). In the UHPLC-MS- analysis, 26 glycopeptides were detected during the analysis of trypsin digested IgG (IgG TD) using different gradients and mobile phases. Moreover, a limit of detection study with four different concentrations (0.01, 0.1, 0.5 and 2mg/mL) was performed. Although the number of detected glycopeptides decreased with decreasing concentration, glycopeptides were detected in the 0.01 mg/mL IgG TD sample. Lastly, a selectivity study was performed, in which two trypsin digested Bovine serum albumin (BSA TD) and IgG TD mixtures with different concentrations were analysed. The results showed that approximately the same number glycopeptides were detected as in the IgG sample suggesting that the BSA sample did not interfere with the detection of glycopeptides. Moreover, the CE-analysis included three different capillaries and several background electrolytes (BGEs). Two of the capillaries were polyvinyl alcohol (PVA) coated. The final electropherogram showed five peaks possibly representing IgG glycopeptides but no MS were performed on the CE part leading to no conclusions for the CE-part. Matrix-Assisted Laser Desorption/Ionisation- time-of flight (MALDI-TOF) was performed throughout the project to control the samples and for comparison to the UHPLC-MS analysis.

O sequenciamento genético do DNA humano permitiu maior compreensão da funcionalidade dos seres vivos e principalmente a causa de muitas doenças. Entretanto, os estudos em genética têm se limitado a resolverem os problemas da ciência, e atualmente, a solução para o avanço nessa área tem se atribuído à proteômica. Dessa forma, a pesquisa em química analítica intensificou-se na busca de estratégias melhores para a caracterização de proteomas, em três aspectos principais: preparo de amostra, desenvolvimento da instrumentação analítica e bioinformática. Verifica-se a possibilidade da aplicação de muitas técnicas, atualmente, destaca-se a análise de peptídeos (proteômica shotgun) por cromatografia líquida multidimensional acoplada à espectrometria de massas sequencial (LC/LC-MS/MS), devido à possibilidade de automatização, minimização dos problemas e resultados satisfatórios na análise de amostras biológicas complexas. Portanto, neste trabalho desenvolveu-se um método LC/LC-MS/MS (modalidade on-line column switching) o qual se constitui de coluna trocadora catiônica (homemade), trap de aprisionamento e limpeza, coluna capilar hidrofóbica e separação e detecção por espectrometria de massas sequencial automatizada. Com a proposta de uma instrumentação analítica aperfeiçoada, realizamos a confecção de um trap com partículas de elevada retenção dos peptídeos, o que permite ótima recuperação de amostra. Por se tratar de uma técnica de elevada complexidade instrumental, devido a difícil compatibilidade entre as dimensões cromatográficas, possíveis perda de analito no processo e elevado tempo de análise, propomos uma nova abordagem de otimização, por meio de estudos quimiométricos. Assim, neste trabalho, foram avaliados doze parâmetros instrumentais e destes houve uma simplificação de apenas dois fatores, responsáveis por 95% da resposta ótima do método. Este fato permitiu valores da cobertura da proteína de BSA (82,54%), número de peptídeos (65) e score (2134,05) superiores aos reportados na literatura, os quais apresentam tempos de análise maiores. Este estudo fornece informações do comportamento químico dos peptídeos em relação ao método proposto, por meio de uma superfície de resposta e equação matemática que pode contribuir para a aplicação em diferentes proteomas. / The genetic sequence of human DNA has helped the comprehension of life and principally the cause of various diseases. However, genetic studies have limited to resolve science problems and currently solutions to advance in this field have been attributed to proteomics. Thus, the analytical chemistry has intensified on the search for a better strategy to proteomic characterization in three principal aspects: sample preparation, development of analytical instrumentation, and bioinformatics. Proteomics involves the application of many techniques, currently; the peptide analyses (shotgun proteomics) by multidimensional liquid chromatography coupled to tandem mass spectrometry (LC/LC-MS/MS) is the state of the art. The main reasons are because it allows full system automation, less problems in repeatability, and adequate results in analysis of highly complex biological samples. Therefore, this dissertation developed the method LC/LC-MS/MS (modality on-line column switching), this has a cation exchange column (homemade), trap column to clean, hydrophobic capillary column and separation and detection by tandem mass spectrometry. We have proposed an improved analytical instrumentation, with a homemade trap that particles have high retention of peptide, which permit great recuperation of samples. Because it is an instrumental technique difficult, such as, obtain compatibility between the chromatography dimensions, can lose samples in the process and long time analysis, we have proposed a new optimization approach with chemometric analysis of data. On that, were evaluated twelve instrumental parameters and there was a simplification only two factors, these were responsible for 95% of greater response of method. As a result, the coverage of BSA protein was 82,54%, number of peptides 65 and score of 2134,05 values of high significance if compare from that there are in literature that presented greater time analysis. This work describes information about chemistry of peptides to method proposed through a surface of response and math equation that can contribute to different proteomes.

Les acides docosahexaénoïque (DHA) et arachidonique (ArA), qui appartiennent respectivement aux familles n-3 et n-6, sont présents en grande quantité dans les tissus cérébraux, en particulier dans les phospholipides membranaires. Ces deux acides gras polyinsaturés (AGPI) jouent des rôles essentiels dans le fonctionnement cérébral, notamment dans le neurodéveloppement et la neuroinflammation. De plus, ces AGPI sont des précurseurs de plusieurs médiateurs lipidiques oxygénés impliqués dans divers processus physiologiques et pathologiques.Dans ce contexte, l'objectif de cette thèse était de caractériser les métabolites oxygénés dérivés du DHA et de l'ArA dans le cerveau, étape cruciale pour une meilleure compréhension de leurs rôles biologiques dans cet organe. Ainsi, une technique d'analyse à haute performance a été utilisée, la chromatographie liquide couplée à de la spectrométrie de masse en tandem (UHPLC-MS/MS), pour identifier les différents métabolites oxygénés dérivés du DHA et de l'ArA présents dans les cerveaux de rats exsanguinés et non exsanguinés, que ce soit en conditions basales ou inflammatoires. Ainsi, plusieurs produits oxygénés dérivés du DHA et de l'ArA ont été identifiés et quantifiés dans les cerveaux exsanguinés et non-exsanguinés, à la fois à l'état libre dans le cerveau mais également estérifiés dans les phospholipides. Les métabolites mono-hydroxylés sont les principaux dérivés oxygénés du DHA et de l'ArA, cependant, des quantités mesurables de produits di-hydroxylés, tels que le 8,15-diHETE et la protectine DX, ont également été détectées. L'exsanguination permet de différencier les métabolites oxygénés du tissu cérébral de ceux présents dans le sang. De plus, le métabolisme oxygéné du DHA et de l'ArA est impacté lors d'une inflammation cérébrale. En effet, dans ces conditions, la synthèse de certains métabolites est augmentée, notamment celle de la protectine D1, molécule connue pour ces fortes propriétés anti-inflammatoires. Ces résultats méritent de plus amples recherches dans des conditions pathophysiologiques, notamment lors de maladies neurodégénératives, afin d'observer leur impact sur le métabolisme oxygéné de ces AGPI / Docosahexaenoic (DHA) and arachidonic (ArA) acids, two PUFA which belong to n-3 and n-6 families respectively, are both present at high amount in brain tissues, and especially in membrane phospholipids. These two polyunsaturated fatty acids (PUFA) play important roles in brain functioning, notably in neurodevelopment and neuroinflammation. Moreover, these PUFA are precursors of various oxygenated lipid mediators involved in diverse physiological and pathological processes. In this context, the aim of this work was to characterize oxygenated metabolites derived from DHA and ArA in brain, crucial step for better understanding their biological roles in this organ. For this purpose, a high performance analytical approach was usd, the liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS), in order to identify the different oxygenated metabolites derived from DHA and ArA in exsanguinated and non-exsanguinated brains, both in basal and inflammatory conditions. Several oxygenated products from DHA and ArA were identified and measured in exsanguinated and non-exsanguinated brain, both in the free form and esterified in phospholipids. Mono-hydroxylated metabolites were the main oxygenated derivatives from DHA and ArA, however, measurable amounts of di-hydroxylated products such as 8,15-diHETE and protectin DX, were also detected. Exsanguination allowed discriminating oxygenated metabolites from brain tissue against those in blood brain. Moreover, DHA and ArA oxygenated metabolism was impacted during brain inflammation. Indeed, in these conditions, the synthesis of some metabolites was increased, including that of protectin D1, molecule known for its strong anti-inflammatory properties. These results deserve further research in pathophysiological conditions, especially in neurodegenerative diseases, in order to observe their impact on the oxygenated metabolism of these PUFA

O sequenciamento genético do DNA humano permitiu maior compreensão da funcionalidade dos seres vivos e principalmente a causa de muitas doenças. Entretanto, os estudos em genética têm se limitado a resolverem os problemas da ciência, e atualmente, a solução para o avanço nessa área tem se atribuído à proteômica. Dessa forma, a pesquisa em química analítica intensificou-se na busca de estratégias melhores para a caracterização de proteomas, em três aspectos principais: preparo de amostra, desenvolvimento da instrumentação analítica e bioinformática. Verifica-se a possibilidade da aplicação de muitas técnicas, atualmente, destaca-se a análise de peptídeos (proteômica shotgun) por cromatografia líquida multidimensional acoplada à espectrometria de massas sequencial (LC/LC-MS/MS), devido à possibilidade de automatização, minimização dos problemas e resultados satisfatórios na análise de amostras biológicas complexas. Portanto, neste trabalho desenvolveu-se um método LC/LC-MS/MS (modalidade on-line column switching) o qual se constitui de coluna trocadora catiônica (homemade), trap de aprisionamento e limpeza, coluna capilar hidrofóbica e separação e detecção por espectrometria de massas sequencial automatizada. Com a proposta de uma instrumentação analítica aperfeiçoada, realizamos a confecção de um trap com partículas de elevada retenção dos peptídeos, o que permite ótima recuperação de amostra. Por se tratar de uma técnica de elevada complexidade instrumental, devido a difícil compatibilidade entre as dimensões cromatográficas, possíveis perda de analito no processo e elevado tempo de análise, propomos uma nova abordagem de otimização, por meio de estudos quimiométricos. Assim, neste trabalho, foram avaliados doze parâmetros instrumentais e destes houve uma simplificação de apenas dois fatores, responsáveis por 95% da resposta ótima do método. Este fato permitiu valores da cobertura da proteína de BSA (82,54%), número de peptídeos (65) e score (2134,05) superiores aos reportados na literatura, os quais apresentam tempos de análise maiores. Este estudo fornece informações do comportamento químico dos peptídeos em relação ao método proposto, por meio de uma superfície de resposta e equação matemática que pode contribuir para a aplicação em diferentes proteomas. / The genetic sequence of human DNA has helped the comprehension of life and principally the cause of various diseases. However, genetic studies have limited to resolve science problems and currently solutions to advance in this field have been attributed to proteomics. Thus, the analytical chemistry has intensified on the search for a better strategy to proteomic characterization in three principal aspects: sample preparation, development of analytical instrumentation, and bioinformatics. Proteomics involves the application of many techniques, currently; the peptide analyses (shotgun proteomics) by multidimensional liquid chromatography coupled to tandem mass spectrometry (LC/LC-MS/MS) is the state of the art. The main reasons are because it allows full system automation, less problems in repeatability, and adequate results in analysis of highly complex biological samples. Therefore, this dissertation developed the method LC/LC-MS/MS (modality on-line column switching), this has a cation exchange column (homemade), trap column to clean, hydrophobic capillary column and separation and detection by tandem mass spectrometry. We have proposed an improved analytical instrumentation, with a homemade trap that particles have high retention of peptide, which permit great recuperation of samples. Because it is an instrumental technique difficult, such as, obtain compatibility between the chromatography dimensions, can lose samples in the process and long time analysis, we have proposed a new optimization approach with chemometric analysis of data. On that, were evaluated twelve instrumental parameters and there was a simplification only two factors, these were responsible for 95% of greater response of method. As a result, the coverage of BSA protein was 82,54%, number of peptides 65 and score of 2134,05 values of high significance if compare from that there are in literature that presented greater time analysis. This work describes information about chemistry of peptides to method proposed through a surface of response and math equation that can contribute to different proteomes.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Typically, soil is the final destination of pesticides applied in agriculture and of veterinary drugs employed in animal production. The global concern about the possible consequences of the increased use of these compounds is reflected in increasingly stringent regulations on the levels of residues in agricultural products, animal products and in the environment. This results in intensifying the demand for environmental certification, reduction of Maximum Residue Levels in food and the introduction of procedures for traceability in production processes. In this study, we compared different extraction methods: mechanical agitation, ultrasound and QuEChERS for the determination of 76 pesticides and 9 veterinary drugs in soil using Ultra High Performance Liquid Chromatography coupled to tandem Mass Spectrometry (UHPLC-MS/MS). The extraction method that showed the best results consisted of weighing 10 g of soil followed by the addition of 10 mL of water and kept at rest for 10 min. Then added 10 mL of acetonitrile containing 1% (v/v) acetic acid and proceeded with mechanical shaking for 15 min. Then, it was added 4 g of anhydrous magnesium sulfate and 1.7 g of anhydrous sodium acetate and shaked manually and vigorously for 1 min. Subsequently, the tube was centrifuged for 8 min (3400 rpm) and the extract was filtered and diluted (1:4, v/v) in water before analysis by UHPLC-MS/MS. The method was validated by evaluating several parameters, such as linearity of analytical curves, limits of detection and quantification (LOD and LOQ), matrix effect, as well as accuracy an precision in terms of percentage of recovery and RSD. The analytical curves prepared in solvent and in the blank matrix extract showed linearity for most compounds, with coefficients of determination greater than 0.99, and the standard curves prepared in "blank" matrix extract were used to evaluate the matrix effect. To verify the accuracy and precision of the method, fortification in four concentration levels (10, 25, 50 and 100 μg kg-1) we performed, and extracted in replicate (n = 6), resulting in recovery values between 70 to 120%, with RSD lower than 20% for most of the compounds. Values of method LOQ and LOD were from 3.0 to 7.5 μg kg-1 and from 10 to 25 μg kg-1, respectively. After validation, the method was applied to the determination of pesticides and veterinary drugs in real samples, it showed to be very efficient to be applied in routine analysis. / Normalmente, o solo é o destino final dos agrotóxicos utilizados na agricultura e dos medicamentos veterinários utilizados na criação de animais. A preocupação mundial com as possíveis consequências do aumento do uso destes compostos se reflete em regulamentações cada vez mais exigentes sobre os níveis de resíduos nos produtos agrícolas, produtos de origem animal e no ambiente. Isso resulta na intensificação da demanda por certificação ambiental, redução dos limites máximos de resíduos em alimentos e a introdução de procedimentos que permitam a rastreabilidade nos processos produtivos. Neste estudo, comparou-se diferentes métodos de extração, sendo eles, agitação mecânica, ultrassom e QuEChERS para a determinação de 76 agrotóxicos e 9 medicamentos veterinários em solo utilizando Cromatografia Líquida de Ultra Eficiência acoplada à Espectrometria de Massas em Série (UHPLC-MS/MS). O método de extração que apresentou os melhores resultados consistiu na pesagem de 10 g de solo, seguido da adição de 10 mL de água e mantido em repouso por 10 min. Em seguida adicionou-se 10 mL de acetonitrila contendo 1% (v/v) de ácido acético e procedeu-se a agitação mecânica por 15 min. Logo após, acrescentou-se 4 g de sulfato de magnésio anidro e 1,7 g de acetato de sódio anidro e agitou-se manual e vigorosamente por 1 min. Posteriormente, o tubo foi centrifugado por 8 min (3400 rpm) e o extrato foi filtrado e diluído (1:4, v/v) em água antes da análise por UHPLC-MS/MS. O método foi validado avaliando-se vários parâmetros, como linearidade das curvas analíticas, limites de detecção e quantificação (LOD e LOQ), efeito matriz, assim como exatidão e precisão, em termos de percentual de recuperação e RSD. As curvas analíticas preparadas no solvente e no extrato branco da matriz apresentaram linearidade adequada para a maioria dos compostos, com valores de coeficiente de determinação maiores que 0,99, sendo que as curvas analíticas preparadas no extrato branco da matriz foram utilizadas para compensar o efeito matriz. Para verificar a exatidão e precisão do método, efetuou-se a fortificação em quatro níveis de concentração (10, 25, 50 e 100 μg kg-1), e extração em replicata (n = 6), obtendo-se valores de recuperação entre 70 e 120%, com RSD menores que 20% para a grande maioria dos compostos. Os valores de LOD e LOQ do método variaram entre 3,0 e 7,5 μg kg-1 e 10 e 25 μg kg-1, respectivamente. Após a validação, o método foi aplicado para a determinação de agrotóxicos e medicamentos veterinários em amostras reais, mostrando-se bastante eficiente para ser aplicado em análises de rotina.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Soy beverages are aqueous extracts of soybeans which are a source of high quality protein and no contains cholesterol or lactose, can be considered functional foods. These beverages have a large acceptance among consumers and due to they are extracted from the grain, the beneficial components are preserved, but may also contain residues of pesticides used to control pest infestation during the growing of the grains. This study aimed to develop and validate a multiresidue method based on QuEChERS extraction for the simultaneous determination of 41 pesticides in soy-based beverages using ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). In the sample preparation step, it was used 10 mL of sample and 10 mL of acetonitrile and addition of salts. The clean-up procedure was optimized by experimental design, evaluating different quantities of the sorbents primary secondary amine (PSA) and octadecylsilane (C18).The clean-up step was performed with magnesium sulfate anhydrous and C18. To assess the accuracy of the method, blank samples were spiked at 10, 25 and 50 μg L-1 with n = 6. To evaluate the matrix effect, inclinations of curves in acetonitrile and blank extract of the matrix were compared. The results of the validation are satisfactory, since the method presented optimized recoveries between 70 and 112% with RSD less than 19%. Curves were prepared in the matrix extract, due to some compounds presented pronounced matrix effect. The curves showed r² ≥ 0,99 for all compounds in the range between the respective limit of quantification and 50 μg L-1. The method proved to be suitable for the determination of the compounds and may be used for routine analysis. / Bebidas à base de soja são extratos aquosos do grão de soja, que representam uma fonte de proteínas de alta qualidade e não contém colesterol ou lactose, podendo ser consideradas alimentos funcionais. Essas bebidas têm grande aceitabilidade entre consumidores e por serem extraídas do grão, os componentes benéficos são preservados, mas também podem conter resíduos de agrotóxicos empregados para controlar a infestação de pragas durante o cultivo dos grãos. O presente trabalho teve por objetivo desenvolver e validar um método multirresíduo baseado na extração por QuEChERS para a determinação simultânea de 41 agrotóxicos em bebidas à base de soja por meio de cromatografia líquida de ultra alta eficiência acoplada à espectrometria de massas em série (UHPLC-MS/MS). Na etapa de preparo de amostra, utilizou-se 10 mL de amostra e 10 mL de acetonitrila e adição de sais. O procedimento de limpeza dos extratos foi otimizado a partir de planejamento experimental, avaliando diferentes quantidades dos sorventes amina primária secundária (PSA) e octadecilsilano (C18). A etapa de limpeza dos extratos foi realizada com sulfato de magnésio anidro e C18. Para avaliar a exatidão do método, amostras branco foram fortificadas nos níveis de 10, 25 e 50 μg L-1, com n = 6. Para avaliação do efeito matriz foram comparadas as inclinações de curvas em acetonitrila e no extrato branco da matriz. Os resultados da validação foram satisfatórios, já que o método otimizado apresentou recuperações entre 70 e 112%, com RSD menor que 19%. As curvas foram preparadas no extrato da matriz, devido a alguns compostos apresentarem efeito matriz acentuado. Foi obtido r² ≥ 0,99 para todos os compostos, na faixa entre os respectivos limites de quantificação e 50 μg L-1. O método provou ser adequado para a determinação dos compostos e pode ser utilizado para análises de rotina.

Submitted by Cristhiane Guerra (cristhiane.guerra@gmail.com) on 2017-02-03T13:55:25Z No. of bitstreams: 1 arquivototal.pdf: 3872528 bytes, checksum: 5e60729b8ed99a17f54ca9ea2044f022 (MD5) / Made available in DSpace on 2017-02-03T13:55:25Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 3872528 bytes, checksum: 5e60729b8ed99a17f54ca9ea2044f022 (MD5) Previous issue date: 2016-10-14 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Apocynaceae is a botanical family that includes between 3700 and 5100 species are distributed almost worldwide, but mainly in tropical and subtropical regions. In Brazil there are about 90 genera and 850 species, inhabiting different vegetation types. Within this large botanical family is gender Hancornia, which is considered monotypic and therefore its unique species is Hancornia speciosa Gomes, and is abundant in the northeastern cerrado. In view of this, this paper aims to study fitoquimicamente Hancornia speciosa thus contributing to the expansion of the chemical knowledge of the species. In addition to evaluating the antifungal activity of the extract and isolated compounds. For the study, the plant material after drying and pulverizing, was subjected to extraction and chromatography processes for isolation of chemical constituents. Techniques such as Cromatrografia Liquid High efficiency and Ultra-high Performance Liquid Chromatography were employed at work. The chemical structure of the substances was determined by 1H Nuclear Magnetic Resonance, 13C and dimensional comparisons with literature data. Chromatographic fractionation of the chloroform phase resulted in the isolation of caffeate acetate, the ethyl acetate phase gave the isoquecetrina beyond the narigenina, and a mix of acid 4,5-O-E- caffeoyl-quinic acid with 3,5-di –O-E- caffeoyl-quinic the butanol phase. All these compounds were first reported in Hancornia speciosa. Characterization of the extract of Hancornia speciosa leaves by Ultra-high Performance Liquid Chromatography coupled to Mass Spectrometry also provided the identification of twenty-four compounds. Since the study of the antifungal activity of the extracts provide a positive result, but compounds isolated did not provide a positive result, not being able to inhibit the growth of the strains in any concentration tested. This work contributed indicating that this species is bioprodutora phenolic compounds, as demonstrated in this phytochemical study. / Apocynaceae é uma família botânica que incluem entre 3700 e 5100 espécies, estando distribuídas praticamente no mundo todo, mas principalmente nas regiões tropicais e subtropicais. No Brasil ocorrem aproximadamente 90 gêneros e 850 espécies, habitando diversas formações vegetais. Dentro dessa grande família botânica está o gênero Hancornia, que é considerado monotípico e, por isso, sua única espécie é Hancornia speciosa Gomes, abundante no cerrado nordestino. Diante disto, este trabalho tem como objetivo estudar fitoquimicamente Hancornia speciosa contribuindo com o conhecimento químico da espécie, além de avaliar a atividade antifúngica do extrato e compostos isolados. Para realização do estudo, o material vegetal, após secagem e pulverização, foi submetido a processos de extração e cromatografia para isolamentos dos constituintes químicos. Técnicas como Cromatrografia Líquida de Alta eficiência e Cromatrografia Líquida de Eficiência Ultra Elevada foram empregadas no trabalho. A estrutura química das substâncias foi determinada por Ressonância Magnética Nuclear de 1H, 13C e bidimensionais em comparações com dados da literatura. O fracionamento cromatográfico da fase clorofórmica resultou no isolamento do cafeato de etila, da fase acetato de etila obteve-se a isoquecetrina, além da narigenina, e uma mistura do ácido 4,5-di-O-E- cafeoil-quínico com o ácido 3,5-di-O-E- cafeoil-quínico da fase butanólica. Todos esses compostos foram relatados pela primeira vez em Hancornia speciosa. A caracterização do extrato das folhas desta espécie por Cromatografia Líquida de Eficiência Ultra Elevada acoplada à Espectrometria de Massas forneceu ainda a identificação de vinte e quatro compostos. Já o estudo da atividade antifúngica do extrato forneceu um resultado positivo, porém o estudo dos compostos isolados não forneceu, não sendo capaz de inibir o crescimento das cepas em estudo em nenhuma concentração testada. Este trabalho contribuiu evidenciando que esta espécie é bioprodutora de compostos fenólicos, como demonstrado neste estudo fitoquímico.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The emerging pollutants are substances that enter continuously in the environment and are being more recognize due to the advancement of instrumental and sample preparation techniques. These contaminants include several chemical classes, such as pharmaceuticals, hormones, personal care products, flame retardants and others. Water is one of the most susceptible matrix since the sewage discharge is one of the main routes of environmental pollution. Bar adsorptive microextration (BAμE) has shown great analytical capacity for the analysis of organic compounds in residual level and has become a well-established analytical tool in sample preparation. In this work, a simple and low cost method was validated for the analysis of 13 emerging contaminants in water using BAμE with polymeric sorbent followed by ultra high performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS). The preparation, stability tests and development of BAμE devices are also discussed. Eleven different coating phases were evaluated. In order to select the best combination of experimental conditions for extraction and back extraction, central composite design (CCD) with four variables was applied. Validation results were satisfactory, since the method presented recoveries between 74% and 118% with relative standard deviations (RSD) < 19%. The analytical performance presented detection and quantification limits of 0.012 to 0.6 and 0.04 to 2 μg L-1, respectively. The proposed method combines a simple and effective sample preparation for the determination of emerging contaminants in water using a microextraction technique (BAμE) followed by UHPLC-MS/MS analysis. The method applicability was evaluated using real samples of surface, drinking and tap water and 6 positive samples were found indicating the presence of bisphenol A (0.08-0.665 μg L-1) and paracetamol (0.104-4.2 μg L-1). / Os contaminantes emergentes são substâncias que entram continuamente no meio ambiente e que estão sendo reconhecidas com maior profundidade devido ao avanço de técnicas instrumentais e de preparo de amostra. Estes compostos englobam diversas classes químicas, como por exemplo, fármacos, hormônios, produtos de higiene e cuidado pessoal, retardantes de chama, dentre outros. Uma das matrizes mais suscetíveis à contaminação é a água tendo em vista que a descarga de esgoto é uma das principais vias de poluição ambiental. A microextração adsortiva em barra (BAμE) tem demonstrado grande capacidade analítica para a análise de compostos orgânicos em nível residual e vem se tornando uma ferramenta analítica bem estabelecida no âmbito de preparo de amostras. Neste estudo foi desenvolvido e validado um método simples e de baixo custo para a determinação de 13 contaminantes emergentes em água utilizando BAμE e empregando sorvente polimérico com posterior análise por cromatografia liquida de ultra eficiência acoplada à espectrometria de massas em série (UHPLC-MS/MS). O preparo das barras bem como os testes de estabilidade foram avaliados. Onze sorventes foram testados para revestir os dispositivos. A fim de avaliar a melhor combinação de parâmetros para a extração e dessorção dos analitos, utilizou-se um planejamento do composto central (CCD) com 4 variáveis. Os resultados da validação foram satisfatórios uma vez que as recuperações obtidas ficaram entre 74% e 118% com desvio padrão relativo RSD < 19%. Os limites de detecção e quantificação foram de 0,012 a 0,6 e 0,04 a 2,0 μg L-1, respectivamente. O método proposto combina uma etapa de preparo de amostra simples e eficaz para a determinação de contaminantes emergentes em água. A aplicabilidade do método foi avaliada utilizando amostras reais de água de superfície, de torneira e mineral e destas, 6 amostras apresentaram bisfenol A (0,08-0,665 μg L-1) e paracetamol (0,104-4,2 μg L-1).

Chez l’homme, les erreurs innées du métabolisme des lipides sont dues à des déficits enzymatiques, entraînant une accumulation intracellulaire de substrats lipidiques. Il en résulte un large éventail de symptômes tels que des atteintes viscérales, osseuses et dans certains cas neurologiques. En outre, de nombreux patients atteints de ce type de maladie présentent des anomalies hématologiques et vasculaires attribuées à des anomalies rhéologiques du globule rouge (GR). Ces observations ont conduit à l’hypothèse de l’existence d’un lien entre les propriétés anormales du GR et sa composition lipidique. Or actuellement, le profil lipidique du GR normal reste méconnu. Cependant, le diagnostic précoce de ces troubles est d’une importance capitale pour la prise en charge des patients, notamment dans les cas où un traitement correctif est disponible. La maladie de Gaucher (MG) de type 1, qui est une maladie lysosomale caractérisée par un déficit en β-glucocérébrosidase et pour laquelle un traitement enzymatique substitutif (ERT) est proposé, en est le meilleur exemple. D’où l’intérêt de disposer d’un outil simple et rapide de diagnostic de ce type de maladie.Dans le cas de la MG, le diagnostic repose encore sur la mise en évidence, laborieuse, du déficit enzymatique. Néanmoins, des travaux récents suggèrent que les anomalies rhéologiques du GR pourraient être dues à l’accumulation de quatre sphingolipides, le glucosylcéramide, la glucosylsphingosine, la sphingosine et la sphingosine-1-phosphate, qui seraient de bons candidats biomarqueurs. Or, les méthodes actuelles de dosage de ces sphingolipides nécessitent au moins deux étapes chromatographiques, avec pour chacune une étape longue et fastidieuse de préparation de l’échantillon, ce qui ne facilite guère une approche lipidomique de ce sujet. En outre, seul le glucosylcéramide a été dosé dans le GR tandis que les trois autres sphingolipides n’ont été dosés que dans le plasma. Ces candidats biomarqueurs restent donc à valider.Dans cette thèse, nous avons développé et validé une méthode simple et rapide, par UHPLC-MS/MS, de dosage simultané des 4 sphingolipides impliqués dans la MG. L’application de cette méthode à des GR provenant de patients atteints de la MG, en collaboration avec l’Institut National de Transfusion Sanguine et la société Shire, nous a permis de : 1- valider un biomarqueur parmi les quatre proposés et de montrer que les trois autres n’étaient pas suffisamment spécifiques ; 2- vérifier l’efficacité du traitement ERT actuellement proposé et 3- confirmer l’hypothèse de départ reliant les anomalies rhéologiques du GR à sa composition lipidique.De même, une étude systématique des conditions opératoires nous a permis de généraliser la méthode proposée à l’identification et au dosage de l’ensemble des sphingolipides présents dans un GR ainsi que des phospholipides, constituants majoritaires de sa membrane. Appliquée à la quantification simultanée d’une trentaine de sphingolipides et de phospholipides dans le GR normal et celui de la MG, cette méthode nous a permis de mettre en évidence l’implication d’autres lipides polaires dans la maladie de Gaucher, outre les 4 sphingolipides jusqu’alors proposés. De même, il est prévu de l’adapter à moyen terme pour le profilage total, par classe, de tous les lipides présents dans le GR.Enfin, nous avons évalué d’autres techniques de SM telles que la haute résolution et la mobilité ionique (TWIMS et DIMS) dans le but d’affiner la recherche de nouveaux biomarqueurs, notamment par l’identification des lipides isomères non discriminables par les techniques de MS conventionnelles. Grâce à une collaboration avec le Laboratoire de Chimie Physique (LCP, CNRS UMR 8000) nous avons montré la faisabilité de cette approche en séparant en DIMS deux isomères : la galactosylsphingosine 18:1 et la glucosylsphingosine 18:1 et nous poursuivons actuellement cette étude pour séparer d’autres couples d’isomères. / In humans, hereditary disorders of lipid metabolism are due to enzyme deficiencies, resulting in intracellular accumulation of lipid substrates. This results in a wide range of symptoms such as visceral, bone and in some cases neurological disorders. Furthermore, many patients suffering such diseases have hematologic and vascular symptoms attributed to red blood cell (RBC) rheological abnormalities. These observations led to a hypothesis linking RBC abnormal properties to its lipid composition. However, the lipid profile of normal RBC remains unknown to date. Early diagnosis of these conditions is of importance notably when a therapy is available. This is the case for Gaucher disease (GD) type 1, a lysosomal disorder characterized by β-glucocerebrosidase deficiency, where an enzyme replacement therapy (ERT) is proposed. Hence, the availability of a simple and rapid tool of diagnosis of such a disorder is of great importance, notably for a better patient care and monitoring.To the best of our knowledge, standard diagnosis procedures and monitoring of GD patients are still based on the tedious evaluation of enzyme deficiency. Nevertheless, recent works suggest that these rheological disorders may be due to the accumulation of four sphingolipids, glucosylceramide, glucosylsphingosine, sphingosine and sphingosine-1-phosphate, which could be considered as relevant biomarkers. However, most of current determination methods of these sphingolipids require at least two liquid chromatographic runs, each with a time-consuming sample preparation step that does not facilitate a lipidomic approach. In addition, only glucosylceramide was quantified in RBC while the other three sphingolipids were quantified only in plasma. Thus, these biomarker candidates remain to be validated.In this PhD, we describe a simple and rapid UHPLC-MS/MS method of simultaneous determination of the 4 sphingolipids involved in GD in both plasma and RBC. The application of this method to RBC from GD patients, in collaboration with the Institut National de Transfusion Sanguine and Shire (USA), allowed us: 1- to validate one biomarker among the four proposed candidates and to show that the other three candidates are not specific; 2- to check the efficiency of the proposed ERT and 3- to confirm the initial hypothesis linking the RBC rheological abnormalities to its lipid composition.Also, a systematic study of the operating conditions allowed us to generalize the proposed method to the determination of not only all the sphingolipids present in RBC but also all phospholipids, which are the major constituents of its membrane. The application of the later method to the simultaneous quantification of thirty sphingolipids and phospholipids in normal and GD RBCs, allowed us to validate it and to unravel the involvement of other candidate biomarkers of GD, different from the 4 previous sphingolipids. Providing appropriate modifications, this method is intended to be used for the profiling of all lipid classes in plasma and RBC. This is our main objective in the medium-term.Finally, we evaluated other modern MS techniques such as high resolution (HRMS) and ion mobility (TWIMS and DIMS) in order to refine the investigation of new biomarker candidates, including the separation of lipid isomers that cannot be discriminated by conventional MS techniques. Indeed, in collaboration with the Laboratoire de Chimie Physique (LCP, CNRS UMR 8000), we here show the feasibility of this approach by achieving the separation of two isomers, by the DIMS technique: galactosylsphingosine 18:1 and glucosylsphingosine 18:1, which cannot be separated by conventional methods. We are currently pursuing these investigations in order to separate other isomers.

Perfluoroalkyl substances (PFASs) are anthropogenic compounds that have been classed as persistent organic pollutants (POPs) and are found in both commercial and industrial products. PFASs have been detected in different environmental matrices and have been found to bioaccumulate in all trophic levels. The adverse effects that are associated with PFAS exposure include reduced body weight, increased liver weight, hepatocellular hypertrophy, a decrease in serum cholesterol and triglycerides. This project aims to characterize the metabolic changes in lipid metabolism in the liver after exposure to one of the well-studied PFASs, the perfluorooctane sulfonate (PFOS), during the embryo development using the domestic chicken as a model organism. The characterization of the metabolic changes was done by conducting both quantitative lipidomic analysis and semi-quantitative global profiling on extracted lipids from liver homogenates from a former related project looking at fatty acid profiles. The extracted lipids were analyzed using UHPLC/Q-TOF-MS. In the quantitative analysis, the PFOS-treated groups (0.1 ug/g and 1.0 ug/g)exhibited higher lipid concentrations when compared with the solvent control group (5% DMSO) and the untreated group leading to the conclusion that PFOS exposure disrupts the lipid metabolism. When comparing the lipid concentrations between the two PFOS-treated groups (0.1 ug/g and 1.0 ug/g), the majority of the lipids exhibited higher lipid concentrations in the 1.0 ug/g PFOS-treated groups leading to the conclusion that the effect PFOS has on the lipid metabolism is dose dependent. In the global profiling analysis, 63 lipids showed significant differences when comparing the solvent control group with samples either treated with 0.1 ug/g PFOS or 1.0 ug/g PFOS.