O presente estudo objetivou avaliar a microbiota fúngica e a ocorrência de micotoxinas por Cromatografia Líquida de Alta Eficiência acoplada à Espectrometria de Massas em amostras de grãos de trigo recém-colhidas e armazenadas. Os resultados revelaram a predominância do gênero Alternaria nas amostras de todas as coletas, seguido de Epicoccum (12,6%), Fusarium (8,3%) e mais 16 outros gêneros de fungos filamentosos. As espécies de Fusarium e Alternaria foram identificadas utilizando métodos moleculares, sendo F. graminearum e A. alternata as espécies mais frequentes. De 70 amostras, 69 (98,6%) estavam contaminadas com a toxina DON (210-2910 ppb) e 3 (4,3%) amostras contaminadas com a toxina ZEA (20-30,1 ppb). O nível de contaminação por DON é inferior aos limites estabelecidos recentemente pela legislação brasileira (3000 ppb), porém superam os limites da Comunidade Européia (1750 ppb). É prudente que se realize um monitoramento contínuo de grãos de trigo a fim de avaliar a exposição dos consumidores às contaminações e estabelecer diretrizes de segurança alimentar. / The present study aimed to evaluated the mycoflora and occurrence of mycotoxins by high performance liquid chromatography coupled to tandem mass in freshly harvested and stored wheat samples. The results showed the predominance of genus Alternaria in all samples, followed by Epicoccum (12.6%), Fusarium (8.3%) and over 16 other genera of filamentous fungi. The species of Fusarium and Alternaria were identified using molecular methods, and F. graminearum and A. alternata were the most frequent species. Of 70 samples, 69 (98.6%) were contaminated with toxin DON (210-2910 ppb), and 3 (4.3%) samples contaminated with the toxin ZEA (20-30.1 ppb). The level of DON contamination is below of the limits recently established by Brazilian legislation (3000 ppb), but exceed the limits of the European Community (1750 ppb). It is prudent to perform a continuous monitoring of wheat to assess consumer exposure to contaminants and establish guidelines for food safety.

Orientador: Marcelo Alexandre Prado / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-21T09:52:53Z (GMT). No. of bitstreams: 1 MirandaeSilva_LigiaMaria_M.pdf: 148256 bytes, checksum: 789cac2002bb2e8dcb1bf70832d395b6 (MD5) Previous issue date: 2012 / Resumo: O crescente aumento populacional em escala mundial, tornou necessário um grande esforço por parte da agricultura para aumentar, a cada ano, a produção de alimentos para atender as necessidades do mercado externo e interno do Brasil. Recursos técnicos e científicos passaram então, a serem aplicados em busca da melhoria na produção dos cultivos,principalmente mediante o uso de fertilizantes e praguicidas. Com isso, a sociedade se deparou com problemas de ordem de equilíbrio ambiental e saúde pública, pois devido à contínua diversificação dos fitoparasitas, surgem, a todo momento, reduções do período de tempo entre aplicações consecutivas, e mais importante talvez, usos de doses mais altas e emprego simultâneo de diferentes pesticidas, por parte dos agricultores, objetivando complementar ações específicas ou alcançar efeitos sinérgicos para maiores rendimentos na produção. Tal situação traz como conseqüência óbvia e direta, o aumento, inaceitável, dos riscos de contaminação do meio ambiente com resíduos químicos de defensívos da área agropecuarista prejudiciais à saúde, o que leva a inúmeros problemas relativos à segurança alimentar dos produtos consumidos, e à uma preocupação de âmbito nacional evidenciada pela criação do Programa de Análise de Resíduos de Agrotóxicos em alimentos (PARA) da ANVISA. O aumento na necessidade de detecção e quantificação destes compostos, acarretou o desenvolvimento de pesquisas no setor, a fim de atingir uma melhoria na eficiência,qualidade e rapidez de resposta nas análises. A possibilidade do estudo de não apenas um de cada vez, mas de até 300 compostos sendo extraídos, detectados e quantificados simultâneamente se tornou a saída mais viável, tanto qualitativa quanto economicamente, facilitando o monitoramento contínuo do fornecimento de produtos do setor alimentício pelos chamados métodos multiresíduos. O presentre trabalho teve como princípio a validação de um método multiresíduo para análise de 14 analitos usando uma técnica de alto poder de concentração e limpeza do extrato como o GPC (Gel Permeation Chromatography) e detecção e quantificação por GC-MS/MS e LC-MS/MS. Os pesticidas investigados englobam classes como: acaricidas, inseticidas, fungicidas, nematicidas e formicidas de aplicação foliar, em sementes ou em solo, sendo que o acefato, metamidofós, acetamiprido e o thiamethoxan foram extraídos de amostras de batata e feijão e analisados por LC-MS/MS e a azoxistrobina, bifentrina, carbofuran, chlorotalonil, clorpirifós, clorfenapir, etofenprox, famoxadone,metalaxil, procimidone e o tebuconazole em amostras de batata e tomate e analisados por GCMS/MS. Os limites de detecção (LD) encontrados variaram de 0,06 a 2,89µg/L, e os coeficientes de variação (CV), de 0,036 a 2,036%. As recuperações foram determinadas em cada tipo de amostras, e os valores encontrados estavam entre 93,34% e 109,67%. Nenhuma das matrizes utilizadas apresentaram resultados insatisfatórios e o método utilizado mostrouse robusto e de fácil aplicação para todos os analitos testados / Abstract: The growing population worldwide, has required a great effort on the part of agriculture to increase each year, the production of food to meet the needs of external and internal market of Brazil. Technical and scientific resources spent then, to be applied in pursuit of improved crop production, mainly through the use of fertilizers and pesticides.With this, the company encountered problems in the balance of environmental and public health, since due to the continuous diversification of plant parasites, arise at any moment,reductions in the time period between consecutive applications, and perhaps most important,uses more doses high and simultaneous use of different pesticides by farmers, aiming to complete specific actions or to achieve synergistic effects in producing higher yields. This situation brings obvious and direct consequence, the increase unacceptable risk of environmental contamination with chemical residues from pesticides in farms are harmful to health, which leads to numerous problems relating to food safety of the products consumed, and to a concern nationwide evidenced by the creation of the Program Analysis of Pesticide Residues in Food (TO) of ANVISA. The increase in the necessity for detection and quantification of these compounds, led the development of research in the sector in order to achieve an improvement in efficiency, quality and responsiveness in the analyzes. The possibility of studying not just one at a time, but up to 300 compounds being extracted,detected and quantified simultaneously output became more viable, both qualitatively and economically, facilitating continuous monitoring of the supply of products by the food industry called methods multiresidue. The principle presentre work was the validation of a multiresidue method for analysis of 14 analytes using a technique of high power concentration and cleanup of the extract as GPC (Gel Permeation Chromatography) and detection and quantification by GC-MS/MS and LC- MS / MS. The pesticides investigated include classes such as acaricides, insecticides, fungicides, insecticides and nematicides foliar, seed or soil,and acephate, methamidophos, and Acetamiprid thiamethoxan were extracted from samples of potatoes and beans and analyzed by LC-MS / MS and azoxystrobin, bifenthrin, carbofuran,chlorothalonil, chlorpyrifos, chlorfenapyr, etofenprox, famoxadone, metalaxyl, procymidone and tebuconazole in samples of potato and tomato and analyzed by GC-MS/MS. The limits of detection (LOD) ranged from 0.06 to 2.89 mg / L, and the coefficients of variation (CV), 0.036 to 2.036%. The recoveries were determined for each type of samples, and the values were between 93.34% and 109.67%. None of the arrays used had unsatisfactory results and method proved to be robust and easy to apply for all analytes tested / Mestrado / Ciência de Alimentos / Mestra em Ciência de Alimentos

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).

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Due to the complexity of its matrix, more efficient methods have been studied for the determination of pesticide residues in maize. The use of QuEChERS method coupled to low temperature clean up technique has showed advantages to reduce lipids during sample preparation in matrix with high fat content. It was optimized and validated in this study a multiresidue method for the determination of pesticides in maize employing QuEChERS and GC-MS/MS. The extraction procedure was optimized though the factorial design, and consisted in weighing 2.5 g of sample and adding 10 mL of ultrapurified water followed by vortex shaking for 1 min. After this, It was added 10 mL of acetonitrile and the tube was stirred for 1 h in a platform shaker at 200 rpm. Partition step was performed using 1 g of NaCl and 6 g of MgSO4, followed by manual shaking for 1 min and centrifugation at 3400 rpm for 8 min, at 10 ºC. An aliquot of 4 mL of the extract was transferred to a 15 mL tube for clean up step at low temperature for 12 h around -20 ºC. Finally, another clean up stage was realized applying dispersive SPE, 1 mL of the extract was transferred to a tube containing 150 mg of MgSO4, 50 mg of C18 and 50 mg of PSA followed by shaking and centrifugation. The final extract was filtered and analyzed by GC-MS/MS. Analytical parameters evaluated in this work were linearity of the analytical curve, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy and matrix effect for the 12 compounds studied. The method showed efficient, with recovery results in a range of 55.3 to 110.1% in the spiked levels of concentration of 40, 100 and 200 μg kg-1 and RSD ≤ 20%. Values of instrument LOD and LOQ were 1.5 and 5.0 μg L-1, respectively. Method LOQ was 100 μg kg-1 for most compounds. Thereby, It was concluded that the proposed method demonstrated to be adequate for the analysis of most pesticide residues studied in maize, since the parameters evaluated were in according to the limits required for chromatographic methods validation applied in residues analysis. / Devido à complexidade da sua matriz, métodos mais eficientes para a determinação de resíduos de agrotóxicos em milho têm sido estudados. O uso do método QuEChERS, juntamente com a técnica de precipitação a baixa temperatura tem apresentado vantagens para a redução de lipídios durante o preparo de amostra de matrizes com alto teor de gordura. Neste estudo, otimizou-se e validou-se um método multirresíduo para determinação de agrotóxicos em milho empregando QuEChERS e GC-MS/MS. O procedimento de extração, otimizado através de planejamento fatorial, constituiu na pesagem de 2,5 g da amostra, adição de 10 mL de água purificada, seguida de agitação em vórtex por 1 min e posterior adição de 10 mL de acetonitrila seguindo de agitação em agitador pendular por 1 h a 200 rpm. A partição utilizando sais foi realizada com 1 g de NaCl e 6 g de MgSO4 e agitação manual por 1 min, seguida de centrifugação a 3400 rpm por 8 min a 10 ⁰C. Uma alíquota de 4 mL do extrato foi retirada e transferida para um tubo de 15 mL para a etapa de limpeza do extrato a baixa temperatura por 12 h a aproximadamente -20 ⁰C. Após, efetuou-se a etapa final de limpeza do extrato com SPE dispersiva onde retirou-se 1 mL deste extrato e transferiu-se para um tubo contendo 150 mg de MgSO4, 50 mg de C18 e 50 mg de PSA, repetindo a agitação por 1 min e centrifugação a 3400 rpm por 8 min. O extrato final foi filtrado e analisado por GC-MS/MS. Os parâmetros analíticos avaliados neste trabalho foram a linearidade da curva analítica, limite de detecção (LOD), limite de quantificação (LOQ), precisão, exatidão e efeito matriz para os 12 compostos estudados. O método demonstrou-se eficiente, com resultados de recuperação na faixa de 55,3 a 110,1% nos níveis de fortificação 40, 100 e 200 μg kg-1 e com RSD ≤ 20%. Os valores de LOD e LOQ do instrumento foram de 1,5 e 5,0 μg L-1, respectivamente, e o valor de LOQ do método foi de 100 μg kg-1para a maioria dos compostos. Desta forma, conclui-se que o método otimizado mostrou-se adequado para análise da maioria dos resíduos de agrotóxicos estudados em milho, pois os parâmetros avaliados encontram-se dentro dos limites exigidos para validação de métodos cromatográficos aplicados a análise de resíduos.

Rice is socially and economically important because it is the main component of the diet of more than half of the world s population. Brazil is one of the main rice producers in the world and Rio Grande do Sul state yields about 60% of the national production. This grain has been recently recognized by its functional characteristics; therefore, the demand for special cultivars, such as cateto rice, black rice and red rice, has increased lately, due to their complex nutritional composition and higher value, even though the number of consumers is still low. The quality of these grains must be monitored to guarantee food safety. A fundamental parameter of the quality is the investigation of pesticide residue. This study has developed and validated a method for pesticide multiresidue determination in different types of rice by Liquid Chromatography coupled with tandem Mass Spectrometry (LC-MS/MS) employing the acetate QuEChERS method in sample preparation. In the cleanup step, the type and the quantity of sorbent were optimized. Besides the sorbents which are frequently used, like PSA and C18, others that had never been tested so far, such as chitosan and diatomaceous earth, were also evaluated. After the optimization, 150 mg of C18 as sorbent was chosen. The efficiency and the reliability of the method were evaluated according to some parameters, such as linearity, limits of detection and quantification, accuracy and precision (repeatability and intermediate precision), matrix effect and robustness. Analytical curves showed adequate linearity (r2 ≥ 0.99) for all analytes. Recovery ranges were 54-112; 50-112; 48-116 and 51-122% for red, whole, cateto and white rice, respectively, with RSD lower than 20%. The limits of quantification ranged from 5 to 25 μg kg-1. The study of the matrix effect has shown low values (< 20%) for most of analytes. Supression was the predominant effect for 80% of the analytes. This effect was compensated by matrix-matched calibration. The method analytes has shown the robustness of the different types of rice with accuracy and precision. Therefore, the proposed method can be efficiently applied to determine pesticides in different types of rice because all validation parameters comply with the limits recommended for the validation of chromatographic methods. / O arroz tem grande importância social e econômica, pois é um dos componentes principais da alimentação de mais da metade da população mundial. O Brasil está entre os principais produtores mundiais de arroz, sendo que o estado do Rio Grande do Sul contribui com cerca de 60% da produção nacional. Atualmente, este cereal vem sendo reconhecido pelas suas características funcionais, neste sentindo, apesar de um grupo ainda restrito de consumidores, cresce a demanda por cultivares especiais, como arroz cateto, arroz negro e arroz vermelho, pois este tem uma composição nutricional bastante complexa e maior valor agregado. É de extrema importância para a segurança alimentar que seja monitorado a qualidade desses grãos. Um parâmetro determinante nessa qualidade é a investigação de resíduos de agrotóxicos. Nesse trabalho foi desenvolvido e validado um método multirresíduos para determinação de agrotóxicos em diferentes tipos de arroz por Cromatografia Líquida acoplada à Espectrometria de Massas em série (LC-MS/MS) empregando o método QuEChERS acetato no preparo de amostra. Na etapa de limpeza do extrato, foram otimizados o tipo e a quantidade de sorvente, sendo avaliados além dos sorventes geralmente empregados, como PSA e C18, outros materiais, até então nunca investigados para este fim, como a quitosana e terra diatomácea. Para esse estudo após otimização foi escolhido 150 mg de C18 como sorvente. A eficiência e confiabilidade do método foram avaliadas através de alguns parâmetros de validação como linearidade, limites de detecção e quantificação, exatidão e precisão (repetibilidade e precisão intermediária), efeito matriz e robustez. As curvas analíticas apresentaram linearidade (r2 ≥ 0,99) para todos analitos no solvente e para 80% dos analitos considerando a curva trabalho. As recuperações foram entre 54-112; 50-112; 48-116 e 51-122% para o arroz vermelho, integral, cateto e branco, respectivamente, com RSD menor que 20%. Os limites de quantificação do método foram na faixa de 5 a 25 μg kg-1. Em relação ao efeito matriz, o efeito foi baixo (< 20%) para maioria dos analitos, ocorrendo supressão para 80% dos analitos. Esse efeito foi compensado com calibração por superposição na matriz. O método apresentou robustez para a variação de matriz, uma vez que pode ser aplicado em diferentes tipos de arroz mantendo a exatidão e precisão. Portanto, concluiu-se que o método proposto pode ser aplicado de forma eficiente para a determinação de resíduos de agrotóxicos em diferentes tipos de arroz, uma vez que todos os parâmetros de validação encontram-se de acordo com os limites sugeridos para validação de métodos cromatográficos.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The optimization and validation of an analytical method for 51 pesticides determination in tomato, using UPLC-MS/MS, was developed in order to fulfill the requirement 5.4.5 (Method Validation) of the NBR ISO/IEC 17025:2005 and following the orientation document DOQ-CGCRE-008 (Orientation of analytical methods validation). This item is very important to a laboratory to be accredited by INMETRO (Brazilian accreditation body). The method validation was carried out following all the validation parameters, such as linearity of the analytical curves, limits of detection and quantification, matrix effects, accuracy and precision of the method. The studied compounds in this work were determined by UPLC-MS/MS in the positive ESI ionization mode. The stock solutions of the pesticides were prepared in organic solvent compatible with the compound solubility. These solutions were prepared by two different analysts in order to validate the preparation by comparing the areas of the injections (n=7) of each individual solution. After evaluation, 43 from 51 studied solutions showed results between the acceptable range of ± 10% demonstrating the satisfactory quality of solutions preparation. The evaluated pesticides were extracted from tomatoes applying the mini Luke method with a mixture 1:1:1 (v/v/v) of each acetone/petroleum ether/dichloromethane solvent as extraction solvent. After extraction, the organic solvent extract was evaporate and reconstituted in appropriate solvent (acidified methanol with 0.1% acetic acid) for UPLC-MS/MS analysis. The recovery experiments were done at 10, 20 and 50 μg kg-1 spike levels, by two analysts in different days, and 7 replicates (n=7) for each spike level plus the blank matrix (without pesticides). The limits of detection and quantification for the instrument and method were estimated, as well as the linearity of the analytical curves evaluated based on determination coefficient (r²), dynamic linear range, accuracy by recovery experiments (%), precision (RSD%) and matrix effect. From the 51 studied compounds, 46 showed recoveries between the range of 81 115% for all the evaluated spike levels. About precision, 92% of the compounds showed RSD% below 18.7% in the lowest spike level. The quantification limit of the method, for 82% from all evaluated compounds, was the lowest spike level studied (10 μg kg-1). The matrix effect observed was between ± 20% for all evaluated compounds, showing that there is not considerable suppression or enhancement in the analytes signal. For all evaluated pesticides the intermediate precision was below 20% of RSD showing the good repeatability of the method. / O desenvolvimento e validação do método analítico para determinação de 51 agrotóxicos em tomate por UPLC-MS/MS foi realizado a fim de atender ao requisito 5.4.5 (Validação de Métodos) da norma NBR ISO/IEC 17025:2005 e seguindo o documento orientativo DOQ-CGCRE-008 (Orientação sobre validação de métodos analíticos). Esse item descrito na norma é de fundamental importância para que o laboratório possa ser acreditado pelo INMETRO. A validação do método seguiu todos os parâmetros de validação, tal como a linearidade das curvas analíticas, limites de detecção e quantificação, efeito matriz, exatidão e precisão do método. Os agrotóxicos estudados neste trabalho foram determinados por UPLC- MS/MS no modo de ionização ESI positivo. As soluções estoque dos agrotóxicos foram preparadas em solvente orgânico compatível com a solubilidade de cada substância. Essas soluções foram preparadas por dois analistas distintos a fim de validar o preparo dessas a partir da comparação das áreas das injeções realizadas (n=7) de cada solução individual. Após avaliação, 43 das 51 soluções estudadas mostraram resultados dentro da faixa de ± 10% demonstrando a excelente qualidade no preparo dessas soluções. Os agrotóxicos estudados foram extraídos das amostras de tomate utilizando o método mini Luke o qual faz o uso de uma mistura 1:1: 1 (v/v/v) de acetona/éter de petróleo/diclorometano como solvente extrator. Após extração, o extrato em solvente orgânico foi evaporado e ressuspendido em solvente apropriado (metanol acidificado com 0,1% ácido acético) para análise por UPLC-MS/MS. O estudo de fortificação e recuperação dos analitos foi realizado nas concentrações de 10, 20 e 50 μg kg-1, por dois analistas, e 7 replicatas (n=7) para cada concentração, além das amostras branco (sem conter os agrotóxicos). Os limites de detecção e quantificação do instrumento e do método foram determinados, bem como a linearidade das curvas analíticas avaliadas através do coeficiente de determinação (r²), faixa linear dinâmica de trabalho, exatidão através do estudo de fortificação e recuperação (%) dos analitos, precisão (RSD%) e efeito matriz. Dos 51 agrotóxicos estudados, 46 apresentaram recuperação média na faixa de 81 a 115 % para todas as concentrações avaliadas. Com relação à precisão, 92% dos agrotóxicos demonstraram RSD% abaixo de 18,7% na menor concentração de fortificação. O limite de quantificação do método, para 82% dos agrotóxicos, foi a concentração de fortificação mais baixa estudada (10 μg kg-1). O efeito matriz observado foi inferior a ± 20% para todos os agrotóxicos avaliados, demonstrando assim que não há supressão ou aumento considerável no sinal do analito. Para todos os agrotóxicos estudados os resultados de precisão intermediária (onde foram avaliados diferentes analistas em diferentes dias) ficaram abaixo de 20% demonstrando a repetitividade satisfatória do método proposto.

Les nucléotides, terme regroupant les nucléosides monophosphate, diphosphate et triphosphate, sont impliqués dans de nombreux processus cellulaires. Ils représentent les éléments constitutifs des acides nucléiques, fournissent de l'énergie à des réactions métaboliques, jouent le rôle de transporteurs et seconds messagers. L'exploration des pools nucléotidiques apparaît indispensable afin de connaître leur rôle précis dans des situations physiologiques ou pathologiques. Nous avons développé une méthode de dosage des nucléotides endogènes (formes mono-, di- et triphosphate) par extraction en ligne sur colonne WAX couplée à la LC-MS/MS. La séparation analytique est réalisée sur colonne Hypercarb, sans agent de paire d'ions dans la phase mobile. Grâce à l'utilisation d'un triple quadripôle et une ionisation en mode positif, les nucléotides endogènes sont identifiés sans équivoque, y compris ceux possédant la même masse molaire. L'extraction et la séparation des nucléotides sont réalisées en 20 min. L'ensemble de la méthode, en comptant la ré- équilibration des colonnes, dure 37 min. La méthode de dosage a été validée pour les formes mono- et triphosphate et est applicable à des séries d'une vingtaine d'échantillons biologiques. D'autre part, dans une étude pré-analytique basée sur les plans d'expériences, nous avons comparé les conditions de préparation d'échantillons en vue du dosage de nucléotides intracellulaires dans 4 lignées cellulaires : 2 adhérentes (Messa et NCI-H292) et 2 en suspension (RL et L1210). Nous avons montré que les conditions pré-analytiques optimales dépendent de la lignée cellulaire soulignant ainsi l'importance de cette phase dans l'analyse des nucléotides. Enfin, l'expérience et les connaissances acquises lors du développement de la méthode de dosage des nucléotides ainsi que la large palette de molécules analysables avec cette méthode (nucléosides, nucléotides sucrés, autres métabolites), nous ont permis de développer des collaborations dans le domaine de la cancérologie avec différentes équipes de recherche. Par exemple, nous avons étudié l'implication des pools nucléotidiques dans le stress réplicatif induit par le stress oxydant et dans la reprogrammation cellulaire observée dans les cellules cancéreuses. Ainsi, les informations apportées par notre approche analytique, complémentaires des autres approches utilisées, ont montré l'implication des pools nucléotidiques dans la tumorigénèse. En conclusion, ce travail a permis de développer une technique analytique et de mettre en place une méthode de travail pour le dosage des nucléotides endogènes dans différents milieux biologiques / Nucleotides, term including nucleoside mono-, di- and triphosphates, are endogenous compounds playing various roles in biology. They are components of nucleic acids, provide energy to metabolic reactions and act as carriers or second messenger. The study of endogenous nucleotides has become of great interest in physiological and pathological conditions. We developed a method for the quantification of endogenous nucleotides, using an on-line extraction on a WAX column coupled with LC-MS/MS. Analytical separation is performed on a Hypercarb column, without ion pairing agent in the mobile phase. The use of a triple quadrupole mass spectrometer following positive mode ionization allows the unambiguous identification of nucleotides presenting the same mass. Extraction and separation of nucleotides are achieved within 20 min and the method including re-equilibration of the two columns within 37 min. The method was validated for the quantification of nucleoside mono- and triphosphates, and could be applied to series of more than twenty biological samples. Secondly, in a study based on design of experiments, pre-analytical parameters influencing results of intracellular nucleotides were compared in four cell lines. We demonstrated that optimal pre-analytical parameters depend on cell lines. This clearly highlights the importance of pre- analytical conditions for the quantification of intracellular nucleotides to be as representative as possible of the real levels in cells. Then, thanks to experience acquired during the development and the validation of the analytical method, scientific collaborations have been established with several cancer research teams. For example, implication of nucleotide metabolism in replicative stress induced by oxidative stress or in the metabolic reprogramming in cancer cells was studied. Results obtained by our analytical approach were complementary to those obtained by other techniques. To conclude, our work consisted on the study of the entire workflow for the analysis of endogenous nucleotides in various biological samples

De nombreux produits chimiques se retrouvent dispersés dans l'environnement avec des conséquences parfois néfastes pour les hommes et les écosystèmes. Parmi ces substances figurent les antibiotiques et les stéroïdes hormonaux. Peu de données sont disponibles quant à la présence et le devenir de ces substances dans l'environnement notamment pour le sol, par manque de méthodologies. Nous avons donc mis au point des procédures d'analyse de traces de ces contaminants émergents, dans l'eau et le sol. Nous avons développé des méthodes multi-résidus et inter-familles basées sur des analyses LCMS/ MS. Pour les échantillons aqueux, l'extraction est menée sur phase solide (SPE, OASIS HLB). Les MLQs sont comprises entre 0.09 et 34 ng/L. Pour la matrice solide, la procédure d'extraction est inspirée de la méthode appelée QuEChERS suivie d'une purification SPE. Elle a été validée et des MLQs entre 0.013 et 3 ng/g ont été atteintes. Les tétracyclines et les fluoroquinolones, ont été étudiées séparément car elles ont des propriétés physico-chimiques bien spécifiques les rendant difficiles à extraire correctement du sol avec une méthode inter-familles. La méthode développée sur la matrice sol a permis une étude statistique mettant en exergue l'impact de certains paramètres du sol sur les rendements d'extraction et les effets matrice. Les méthodes ont été appliquées à une étude en colonnes de sol pour obtenir des données sur le transfert, l'accumulation et la dégradation des composés dans le sol / Several chemical products are dispersed in the environment and the consequences can be sometimes harmful for humans and the ecosystems. Among these substances appear the antibiotics and the hormonal steroids. Nowadays, only few data are available on the presence and the fate of these substances in the environment in particular for solid matrices, mainly due to a lack of methodologies. Consequently, methods to analyze traces of « emergent » contaminants in water and soil were carried out. Therefore, multi-residues and inter-families procedures based on LC-MS/MS analysis were established. Concerning the aqueous samples, 23 analytes are extracted with the SPE technique (OASIS HLB). MLQs are between 0.09 and 34 ng/L. For the solid matrix, the extraction procedure of 31 compounds is inspired from the method called QuEChERS and followed by a purification step. This methodology was validated and MLQs between 0.013 and 3 ng/g were obtained. Two antibiotics families, tetracyclines and fluoroquinolones, were studied separately as they have specific physical/chemical properties and are therefore difficult to extract from soil with an inter-families method. Then, the method developed for the soil matrix allowed a statistic study which showed the impact of the soil parameters on the recoveries and matrix effects. Finally, the methodologies were applied to a soil column study which allowed obtaining data on the transfer, accumulation and degradation of the substances in soil

Tout au long du procédé d’élaboration d’un vin, des réactions d’oxydation peuvent se produire y compris au cours du vieillissement en bouteilles. Depuis quelques années, la profession viti-vinicole est confrontée au problème de l’oxydation prématurée des vins blancs.. A ce jour, peu d’études ont pu apporter des explications d’ordre chimique à ce phénomène, et les mécanismes réactionnels intervenant restent peu connus.L’objectif de ce travail de thèse est d’apporter, au travers d’une analyse moléculaire non ciblée en spectrométrie de masse à transformée de Fourier et à résonance cyclotronique des ions (FT-ICR-MS) couplée à une étude chimiométrique, des réponses originales aux questions posées par la problématique actuelle d’oxydation prématurée des vins blancs. Nous avons montré suite à l’étude d’une série de vins oxydés prématurément non seulement la grande diversité chimique des vins, mais aussi la présence d’un ensemble de masses typiques associées à ce phénomène. Pour une meilleure compréhension de l’origine de cette problématique, nous avons considéré deux autres types d’oxydation : une oxydation relative exclusivement à un apport contrôlé en oxygène et une autre relative à l'évolution naturelle de vins en bouteilles. Cette dernière consiste en le suivi de l'évolution des espaces chimiques de séries verticales de vins blancs et rouges en fonction du temps. Sur la série verticale des vins blancs allant de 1979 à 2006, une charnière à l’année 1990 a été observée avec des groupes de masses typiques de chacun des vins jeunes (1979-1990) et des vieux vins (1991-2006).La comparaison entre les espaces chimiques discriminants chacun de ces trois types d’oxydation ne révèle la présence que de trois masses en commun, ce qui appuie l'hypothèse de causes multiparamétriques à l’oxydation prématurée des vins blancs, qui ne serait donc pas un phénomène du exclusivement à une exposition non contrôlée à l'oxygène.Des essais d’élucidation structurale en FT-ICR-MS/MS des masses discriminantes des vins oxydés et de la série verticale ont été effectués et des schémas de fragmentation pour certaines masses sont proposés / During winemaking processes, many oxidation reactions may occur especially during the aging period. Recently, white wines are characterized by a problem of premature oxidation for which few studies have provided chemical explanation. To date, the involved mechanisms in this phenomenon remain poorly understood.The aim of this thesis project is to provide, through an untargeted molecular analysis using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) coupled to chemometric analysis, original clue to understand the premature oxidation of white wines. Based on the study of a series of premature oxidized white wines, we were able to elucidate the high complexity and the chemical diversity of wine, and got out typical masses characterizing the oxidation state. In order to better understand the origin of this phenomenon, we considered two alternative possibilities of oxidation: the first one induced by oxygen, and the second through a natural evolution of wines in bottles. The latter included the monitoring of the chemical evolution of white and red wines as a function of time. A very interesting result was obtained on the vertical series of white wines from 1979 to 2006, where two groups were separated at the 1990 vintage to provide a group of old wines (1979-1990) and a group of new wines (1991-2006). Typical discriminant masses were found for each group.A comparison between the chemical spaces discriminating each of the three types of oxidation (premature oxidation, oxidation with oxygen and natural evolution of wine in bottle) revealed very few common masses that may indicate that the phenomenon of premature oxidation is indeed influenced by multiple factors.Finally, a structural elucidation of the typical masses of the groups of oxidized and aged wines were established using FT-ICR-MS/MS. Possible fragmentations schemes of some of these masses were proposed

Les extraits naturels tels que les huiles essentielles, les absolues ou les résinoïdes utilisés en parfumerie, arômes et cosmétique sont produits à partir de matières premières naturelles. La culture de ces matières premières est souvent basée sur la monoculture et implique l'utilisation de pesticides. La réglementation pour les matières premières destinées à ces industries devient de plus en plus stricte en termes de niveaux résiduels autorisés de pesticides, en raison de leur impact sur la santé publique et sur l'environnement. Dans ce manuscrit, un état de l’art montrant la problématique pour l’analyse des pesticides dans les extraits naturels et les travaux dédiés à cette étude, est présenté. Une méthode d’analyse multi-résidus de pesticides a ensuite été mise au point dans les roses et les extraits (concrètes et absolues) issus de la rose. La préparation d’échantillon s’appuie sur l’approche QuEChERS utilisant l’extraction et la purification par SPE-dispersive. Les échantillons ont ensuite été analysés par GC-MS/MS. Les méthodes ainsi développées ont permis d’étudier les devenir des pesticides au cours du procédé d’extraction des roses. Le travail a été fait dans un premier temps sur des roses issues d’un champ bio puis dopées avec 57 pesticides. Chaque intermédiaire de transformation, excepté les cires, a été analysé mettant en évidence la répartition des pesticides. Il y est montré que les pesticides présents dans les roses sont co-extraits durant le procédé d’extraction et sont concentrés entre 100 et 300 fois dans l’absolue. Cette étude a ensuite été réalisée sur des roses et leurs extraits issus de champs avec des traitements phytosanitaires « classiques ». / Natural extracts in form of essential oils, concretes, resinoids, as well as absolutes used in perfume, aroma, and cosmetic industries are produced from natural raw materials. The cultivation of natural raw materials is often based on monoculture and involves the use of pesticides. Legal regulations for raw materials intended for these industries become increasingly stricter in terms of the allowed residual levels of chemicals used for treatment because of their impact on public health and on the environment. In this manuscript, a review showing pesticide analysis problems in natural extracts and works dedicated to this study, is presented. Next, a method for multi-residue pesticides analysis was developed in roses and extracts (concrete and absolute) from roses. The sample preparation is based on the QuEChERS approach using extraction and purification by SPE-dispersive. The samples were then analyzed by GC-MS / MS. Thus the developed methods were used to study pesticides behavior during the extraction process of the roses. In a first time, the work was done on roses from organic field and spiked with 57 pesticides. Each transformation intermediate, except waxes, was analyzed showing the distribution of pesticides. It showed that pesticides which were present in rose flowers were coextracted during extraction process and concentrated by factor between 100 and 300 in absolute. This study was then performed on roses and extracts from fields with pesticide "classic" phytosanitary treatments. The trend observed for these samples was the same as for the model: pesticides were concentrated in the absolute reaching concentrations of several milligrams per kilogram.