Massiver wirtschaftlicher Schaden und der Verlust des Verbrauchervertrauens hervorgerufen durch die BSE - Krise führten zu einer radikalen Änderung in der Futtermittel- und Lebensmittelgesetzgebung. Neben diversen anderen Maßnahmen wurden u. a. das Gehirn und Rückenmark von über 12 Monate alten Wiederkäuern vom Gesetzgeber als so genanntes spezifiziertes Risikomaterialien (SRM) definiert. Die spezielle Entsorgung und das Verarbeitungsverbot dieser Materialien in Lebens- und Futtermitteln hatte zum Ziel die Infektkette zu unterbrechen und dadurch das Risiko einer Infektion mit dem BSE-Erreger für den Verbraucher zu senken. Um die Einhaltung des Verarbeitungsverbotes von SRM in Lebensmitteln zu überprüfen, wurden diverse ZNS-Nachweisverfahren entwickelt. Jedoch ist keines, der hierfür entwickelten molekularbiologischen und immunochemischen Verfahren in der Lage sowohl Spezies, als auch Tieralter des nachgewiesenen ZNS zu bestimmen und damit eine potentielle Einordnung zum SRM zu ermöglichen. Darüber hinaus hat sich für fast alle ZNS-Marker der Einfluss von hohen Prozesstemperaturen als nachteilig für die Nachweisbarkeit erwiesen. NIEDERER und BOLLHALDER (2001) entwickelten ein auf Fettsäureanalytik basierendes ZNS-Nachweisverfahren, welches am Institut für Lebensmittelhygiene der Universität Leipzig weiterentwickelt wurde. Vorteile dieses Verfahrens sind die hitzestabilen Marker und die Möglichkeit mit Hilfe von spezifischen Fettsäureverhältnissen Spezies und Tieralters des nachgewiesenen ZNS zu ermitteln. Somit ist es mit diesem Verfahren erstmals möglich das nachgewiesene ZNS der Gruppe der spezifizierten Risikomaterialien zuzuordnen. Ziel dieser Arbeit war es, das am Institut für Lebensmittelhygiene weiterentwickelte Verfahren auf Praxistauglichkeit anhand eines externen Blindversuchs zu testen. Hierbei sollten mögliche Schwachstellen identifiziert und im weiterführenden Verlauf dieser Arbeit Lösungen erarbeitet werden. Für den Blindversuch wurden vom Max-Rubner-Institut, Standort Kulmbach, insgesamt 72 Brühwurstproben zur Verfügung gestellt, welche mittels des nach LÜCKER et al. (2005) beschriebenen Verfahrens untersucht wurden. Die Ergebnisse des Blindversuchs deuten auf eine grundsätzliche Eignung des Verfahrens für den Nachweis von spezifiziertem Risikomaterial in Fleischerzeugnissen hin. Jedoch konnte bei der Tierartdifferenzierung und vor allem bei der Altersbestimmung ein Optimierungsbedarf ermittelt werden. Somit ergaben sich für diese Arbeit folgende weiterführende Aufgabenstellungen: 1. Die Optimierung der Tierartbestimmung auch im Hinblick auf Erweiterung des Speziesspektrums und 2. die Optimierung der Tieraltersbestimmung von Rinder- und Schaf-ZNS. Insgesamt 257 ZNS-Proben der Spezies Rind, Schaf, Schwein, Ziege und Geflügel wurden einer Fettsäureanalyse unterzogen. Aus allen analysierten Fettsäuren wurden 67 Fettsäureverhältnisse gebildet. Zur Identifikation für die Speziesdifferenzierung geeigneter Fettsäureverhältnisse wurde das statistische Verfahren der Partial Least Square – Discriminant Analysis (PLS-DA) eingesetzt. Hierbei ergab sich, dass von den 67 untersuchten Fettsäureverhältnissen insgesamt 14 für die Differenzierung zwischen den Tierarten Rind, Schwein, Schaf, Ziege und Geflügel geeignet sind. Für die Optimierung der Tieraltersbestimmung wurden 37 ZNS-Proben vom Rind und elf ZNS-Proben vom Schaf untersucht. Die analysierten Fettsäuren wurden auf ihre Korrelation mit dem Alter untersucht. Stark korrelierende Fettsäuren und deren Verhältnisse wurden mit Hilfe der Regressionsanalyse auf ihre Vorhersagefähigkeit geprüft. Hierbei gelang die Identifikation von vier neuen Fettsäureverhältnissen. Für die Altersschätzung bei der Tierart Rind scheinen sich die FS-Verhältnisse 2OH-C24:0/2OH-C25:0 und 2OH-C24:1(n-7)/2OH-C25:0 am besten zu eignen. Für die Tierart Schaf sollten die Verhältnisse 2OH-C25:0/2OH-C26:0 und 2OH-C25:0/2OH-C26:1(n-7) bevorzugt eingesetzt werden. Im Vergleich zur bisherigen Verfahrensweise ist mit Hilfe dieser Fettsäureverhältnisse und deren Regressionsformeln eine deutlich präzisere Altersschätzung für die Tierarten Rind und Schaf möglich. Der neue Ansatz bietet die Möglichkeit flexibel auf zukünftige Änderungen der Altersgrenze von SRM zu reagieren. / The immense economical damage and the loss of consumer trust caused by the bovine spongiform encephalopathy (BSE) - crisis resulted in a radical alteration of the feed- and foodlegislation. Besides several other measures, the legislator defined the brain and spinal cord of ruminants older than 12 month as specified risk material (SRM). The special disposal and the processing prohibition of these materials in food and feedstuffs aimed to interrupt the infection chain and to reduce the risk of an infection caused by the BSE pathogen for the consumer. In order to control the processing prohibition of SRM in food, several CNS detection methods were developed. But none of the designed molecular biological or immuno chemical methods has the ability to detect species and age of the CNS. Therefore, a classification of the detected CNS as SRM is not possible. Furthermore, high process temperatures influence nega-tively the detection of almost all CNS markers. NIEDERER and BOLLHALDER (2001) developed a CNS detection procedure based on the analysis of fatty acids, which was improved at the Institute of Food Hygiene, University Leipzig. Advantages of this procedure are the heat stability of the markers and the possibility to identify species and age of the detected CNS. Therefore, this procedure is the first, which facilitates the potential of identifying SRM. The aim of this work was to test the practicability of the improved CNS detection procedure in an external blind trial. During this, possible weak points should be identified and solutions for their elimination presented. Furthermore, the sample preparation of the method should be optimized with regard to cost and time reduction. For the external blind trial the Max-Rubner-Institute, Kulmbach, produced 72 emulsion type sausages. These sausages were analysed according to the procedure described by LÜCKER et al. 2005. The results of the blind trial show the suitability of the procedure for the detection of SRM in meat products in principle. However, some results revealed that the species and age identification required further enhancement. Therefore, the following additional topics of this work were: 1. Optimization of species identification especially with regard to other species 2. Optimization of animal age prediction for cattle and sheep CNS. Selected fatty acids of total 257 CNS samples from cattle, pig, sheep, goat and several poultry species were analysed. These fatty acids were combined to 67 fatty acid ratios. Afterwards, the application of these ratios for the species detection was tested by using the statistical method of Partial Least Square – Discriminant Analysis (PLS-DA). In result, 14 out of 67 fatty acid ratios are suitable for differentiation between the species cattle, pig, sheep, goat and poultry. For the optimization of animal age prediction 37 CNS samples from cattle and eleven CNS samples from sheep were examined. All analyzed fatty acids were checked for their correlation with age. Afterwards strong correlating fatty acids and their fatty acid ratios were examined for their predictive ability by the application of regression analysis. In result, four new fatty acid ratios for age prediction could be identified. For the age prediction of cattle CNS the fatty acid ratios 2OH-C24:0/2OH-C25:0 and 2OH-C24:1(n-7)/2OH-C25:0 are the best choice. In sheep CNS the ratios 2OH-C25:0/2OH-C26:0 and 2OH-C25:0/2OH-C26:1(n-7) should be preferred for prediction of age. In comparison to the previous age prediction method, the application of these fatty acid ratios and their regression formula led to more accurate results. Furthermore, it offers the possibility to adopt to possible variations of the age limits within the SRM definition in future.

No description available.

The use of chemicals, which generates a lot off benefits, is responsible for the contamination of soil, water and foods. The concern about contamination of surface and ground water systems by pesticides has grown in scientific circles, especially by the presence of pesticides and other compounds in water sources. Thus, rapid analytical methods, sensitive and efficient must be developed in order to verify the concentration levels of pesticides and other organic compounds in water samples agree with those established by law to protect human health and the environment media. This study aimed to optimize, validate and implement an extraction method to determine residues of mononitro, ketones, trifluralin, oxirane, lindane, 2,4-D ester, chlorpyrifos, bromoketal, α-endosulfan, β-endosulfan and endosulfan sulfate in samples of tap water, industrial wastewater and groundwater by Dispersive Liquid-Liquid Microextraction (DLLME) modified and Gas Chromatography with Mass Spectrometry Tandem using Triple Quadrupole type (GC-(TQ)MS/MS). The method is based on the rapid injection, with a syringe, of a mixture of an extractor solvent (50 μL of carbon tetrachloride) and an dispersor solvent (2.0 mL acetone) previously cooled (ice batch, 0 - 4 °C) in an aqueous sample (5.0 mL), contained in a 10 mL graduated glass tube with conical bottom, with no pH correction and cooled (ice batch, 0 - 4 °C). It is not necessary to stir the sample in this step. Remove 25 μL of the sedimented phase separated by simple sedimentation for 10 min. from the bottom of conical tube with a microsyringe and transferred to a vial of 200 μL for GC- (TQ)MS/MS determination. The preconcentration factor obtained was 100 times. The method was validated by determining the limits of detection and quantification limits (LOD and LOQ), linearity, precision and accuracy. The calibration curves showed adequate linearity between 0.05 e 10 μg L-1 with coefficients of determination higher than 0.993. The method showed good recovery values between 70 and 115%, RSD(%) ranged from 10.9 to 17.2%, LOQs between 0.02 and 0.09 μg L-1, and was considered adequate for the analysis of pesticide residues and other organic compounds evaluated in water samples. After validation, the method was applied to samples of drinking water, industrial wastewater and groundwater. No pesticide residues and other organic compounds evaluated were found in the water samples tested. / A utilização de substâncias químicas, que por um lado gera benefícios, por outro é responsável pela contaminação do solo, água e alimentos.A preocupação com a contaminação de sistemas aquáticos superficiais e subterrâneos por pesticidas tem crescido no meio científico, em especial pela presença de pesticidas e outras susbtâncias químicas em mananciais de água. Dessa forma, métodos analíticos rápidos, sensíveis e eficientes têm de ser desenvolvidos para verificar se os níveis de concentração dos pesticidas e outras substâncias químicas em amostras de água estão de acordo com os estabelecidos pela legislação, a fim de resguardar a saúde humana e o meio ambiente. Este estudo teve como finalidade otimizar, validar e aplicar um método de extração e determinação de resíduos de mononitro, ketona, trifluralina, oxirano, lindano, 2,4-D-éster, clorpirifós, bromoketal, α-endosulfan, β-endosulfan e endosulfan sulfato em amostras água de torneira, efluente industrial e água subterrânea, aplicando a técnica DLLME modificada e quantificação por Cromatografia Gasosa acoplada à Espectrometria de Massas Sequencial tipo Triplo Quadrupolo (GC-(TQ)MS/MS). A técnica de extração DLLME está baseada na injeção rápida, com auxílio de uma seringa, de uma mistura dos solventes extrator (50 μL de tetracloreto de carbono) e dispersor (2,0 mL de acetona) previamente resfriada em banho de gelo a uma temperatura entre 0 e 4 °C, em uma amostra aquosa (5,0 mL) contida em um tubo de vidro, com fundo cônico, graduados com volume de 10 mL, sem correção de pH e mantida resfriada em banho de gelo entre 0 e 4 °C. O procedimento não requer agitação da amostra. Retira-se a fase sedimentada separada por sedimentação simples após 10 min., do fundo do tubo cônico com auxílio de uma microseringa de 25 μL e transfere-se para um frasco de 200 μL, para determinação no sistema GC-(TQ)MS/MS. O fator de pré-concentração obtido é de 100 vezes. Na validação do método determinaram-se os limites de detecção e limites de quantificação (LOD e LOQ), linearidade, precisão e exatidão. As curvas analíticas apresentaram linearidade adequada entre 0,05 e 10 μg L-1 com valores de coeficiente de determinação superior a 0,993. O método apresentou bons valores de recuperação, entre 70 e 115% e RSD(%) entre 10,9 e 17,2%, com LOQs entre 0,02 e 0,09 μg L-1. O método foi considerado adequado para a análise de resíduos dos pesticidas em estudo em amostras de água. Após a validação, o método foi aplicado em amostras de água de potável, efluente industrial e água subterrânea. Não foram encontrados resíduos dos pesticidas e outras substâncias químicas estudados nas amostras analisadas.

Les résines végétales sont des sécrétions de végétaux qui ont été utilisées par l’homme de l’Antiquité à nos jours dans de nombreuses applications (pharmaceutique, cosmétique et artistique). Ces exsudats sont composés majoritairement de terpènes. L'identification et la quantification de l'ensemble de ces composés dans les extraits végétaux reste un défi du fait de leur très grande diversité structurale. L’objectif de ce travail a été de développer de nouvelles approches analytiques pour identifier et quantifier les composés terpéniques présents dans ce matériel végétal afin d’en assurer le contrôle qualité et la certification. Deux méthodes séparatives ont été sélectionnées: la TLC et la GC. Pour ces deux techniques on s’est intéressé à toutes les potentialités de leur couplage avec la spectrométrie de masse. Le développement en TLC-1D et TLC-2D a permis le « screening » rapide des résines végétales et la faisabilité du couplage avec le MALDI-TOF-MS a été mise en évidence pour l’identification des marqueurs majoritaires (acides triterpéniques). La GC a permis une caractérisation plus aboutie des résines en mettant en place une méthode d’analyse exhaustive des terpènes des plus volatils au non-volatils. L’optimisation des différentes étapes de la méthodologie GC-MS s’est effectuée en se basant sur la méthode des plans d’expérience ainsi que sur des analyses statistiques tels que l’ACP et la CAH. Dans un souci d’apporter des éléments plus précis pour distinguer les résines les plus proches, la quantification de leurs marqueurs majoritaires a été établie après une validation complète de la méthode GC. L’ensemble de ce travail a permis de développer des outils pour une caractérisation rapide des extraits de résines permettant de différencier les espèces même les plus proches. / Resins are hydrocarbon secretions of many plants and well known for their protective benefits. They have been used as raw materials for a wide range of applications (pharmaceutic, cosmetic and artistic). Plant resins are complex mixtures of organic substances mainly terpenoid compounds which constitute the most abundant and structurally diverse group of plant secondary metabolites. The chemical characterization of this material results in long and difficult separation due to the wide range of polarity and volatility of its constituents. The aim of this work was to develop new analytical approaches to improve the identification of resins certifying their origin and ensuring the quality control. For that purpose two analytical methods were selected: TLC and GC approaches hyphenated to mass spectrometry. TLC-1D and TLC-2D allow a rapid screening and first visual differences of resins. The innovating TLC coupling to MALDI-TOF-MS gives a clear identification of major markers (triterpenic acids). In order to have complementary information about the composition of resins, a gas chromatography-mass spectrometry (GC-MS) method was developed to analyze volatile to non-volatile compounds. The various stages of optimization were based on experimental design and statistical (PCA and HAC) approaches. For closely related resins, a quantitative approach was investigated based on a complete validation for major markers. This work allows the development of two complementary techniques that give a powerful approach for fast and reliable differentiation of various resins even the closest ones.

The Butia odorata has received special attention from the scientific community because of their nutritional and functional properties that have been revealed, in addition to its rightdown sensory characteristics. The aim of this study was to characterize different genotypes from Butia odorata fruit collected in Santa Maria - RS (SM) and Santa Rosa - RS (SR) regions during three consecutive harvests in relation to various physical and chemical properties. Many analyses were performed like: biometric, color and quality parameters, fatty acid composition in the Butia pulp. An HS-SPME method was developed for the isolation and subsequent quantification (GC-FID) and identification (GC/MS) of volatile compounds (VCs). The biometric parameters results showed pulp yields between 40.38 and 73.91%. The color of the pulp ranged between yellow and orange shades. TSS ranged between 12.39 and 15.50 °Brix, acidity between 0.69 and 2.26 g% citric acid and pH between 3.17 and 3.95. The fruit had humidity between 78.04 and 85.85 g%; ashes between 0.47 and 0.77 g%; protein between 0.57 and 0.93 g%, crude fiber between 0.84 and 4.02 g%; lipid between 0.12 and 2.27 g%. The fatty acids such as palmitic acid, linoleic and linolenic acids were found in abundance. It was possible to observe the partial discrimination between the two regions in the PCA using the physico-chemical variables. The samples from SR characterized by high scores for lipids and palmitic acid, while SM samples showed a higher humidity and TSS. The method employed use DVB/CAR/PDMS fiber for the VCs extraction of the centrifuged Butia pulp , added with 30% NaCl, and maintaining the system at a temperature of 35 °C for 45 minutes, under stirring. The volatile fraction was analyzed in GC and were identified 77 compounds among acids (5), alcohols (14), aldehydes (9), ketones (12), lactones (3), terpenes (2) and esters (34). The ethyl hexanoate compound was the major compound in almost samples. The PCA from VCs enabled to realize grouping of samples into two groups according to region of origin. The fruits from Santa Maria showed a high content of esters and the fruits from Santa Rosa had higher concentrations of alcohols and aldehydes. An exploratory analysis of the variables of physico-chemical composition and volatile of the samples was observed using PCA. In the graphs analysis also realized a partial separation of samples according to their region: the samples SM2-10 and RS1-11 distanced themselves from the other and it indicates greater particularity in their chemical compositions. Therefore the fruits (Butia odorata) showed great variability in biometric characteristics, physico-chemical and volatile profiles according to region of origin. It indicates a strong influence of soil and climatic factors. In this study, the nutritional characteristics and high yield pulp of some Butia fruits demonstrated the high potential for fresh consumption or for the manufacture of processed products / O butiá (Butia odorata) tem recebido especial atenção da comunidade científica, em virtude de suas propriedades nutritivas e funcionais que estão sendo reveladas, além das suas características sensoriais peculiares, ainda pouco exploradas. Desta forma, o objetivo do presente trabalho foi caracterizar diferentes genótipos de frutos de butiazeiros coletados nas regiões de Santa Maria RS (SM) e Santa Rosa RS (SR) em três safras consecutivas, quanto as diversas propriedades físicoquímicas. Foram realizadas análises biométricas, de cor, dos parâmetros de qualidade, composição centesimal e ácidos graxos da polpa do butiá, além do desenvolvimento de método de isolamento por SPME com posterior quantificação (GC-FID) e identificação (GC/MS) dos compostos voláteis (CVs). Os resultados dos parâmetros biométricos revelaram rendimentos de polpa, entre 40,38 e 73,91%. A cor da polpa oscilou em tons de amarelo e laranja. Os SST variaram entre 12,39 e 15,50 °Brix, a acidez entre 0,69 e 2,26 g% em ácido cítrico e o pH entre 3,17 e 3,95. As frutas apresentaram umidade entre 78,04 e 85,85 g%; cinzas entre 0,47 e 0,77 g%, proteína entre 0,57 e 0,93 g%, fibra bruta entre 0,84 e 4,02 g% e lipídios de 0,12 a 2,27 g%. Os ácidos graxos encontrados em maior abundância foram o ácido palmítico, linoléico e linolênico. Na Análise dos Componentes Principais (PCA) empregando as variáveis físico-químicas observou-se a discriminação parcial entre as duas regiões, sendo as amostras da região de SR caracterizadas por elevados escores para lipídios e ácido palmítico, enquanto que as da região de SM apresentaram, em geral, maior umidade e teor de SST. O método desenvolvido emprega o uso da fibra DVB/CAR/PDMS para a extração dos CVs da polpa de butiá centrifugada, adicionada de 30% de NaCl, e mantendo o sistema a uma temperatura de 35 ºC por 45 minutos, sob agitação. A fração volátil foi analisada em GC e nela identificados 77 compostos entre ácidos (5), alcoóis (14), aldeídos (9), cetonas (12), lactonas (3), terpenos (2) e, principalmente, ésteres (34). Entre estes se destacou o hexanoato de etila como composto majoritário. A PCA dos CVs possibilitou ver o agrupamento das amostras em dois grupos conforme a região de origem, onde, em geral, os butiás de Santa Maria apresentaram elevado teor de ésteres, enquanto os de Santa Rosa obtiveram maiores concentrações de alcoóis e aldeídos. Uma análise exploratória entre as variáveis de composição físico-química e volátil das amostras estudadas foram observadas através de uma PCA. Na visualização dos gráficos notou-se também uma parcial separação das amostras quanto à região de origem, sendo que as amostras SM2-10 e SR1-11 distanciaram-se das demais, indicando maior particularidade em suas composições químicas. Sendo assim, os butiás apresentaram grande variabilidade em suas características biométricas, físicoquímicas e nos perfis voláteis em função da região de origem e safra e, portanto, apresentou forte influência de fatores edafoclimáticos. Sobretudo, as características nutricionais e o alto rendimento de polpa de alguns frutos de butiás evidenciaram, neste estudo, o alto potencial para consumo in natura ou mesmo para a fabricação de produtos processados.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / In this study, was developed and validated a multi-residue method for the determination of 40 pesticides in green coffee beans. To obtain a very homogeneous sample, the coffee beans were first milled, followed by a slurry preparation of coffee and water (ratio 1:4, w/w). For the extraction procedure modifications of the QuEChERS method were developed. Acetonitrile containing 1% acetic acid and the procedure internal standard (P.I.S.) was used as extraction solvent. After the acetonitrile addition, 3 g of anhydrous magnesium sulfate were added, to promote phase separation between the organic phase (acetonitrile) and the aqueous phase. For the dispersive solid-phase extraction (dSPE) clean-up step, two sorbents were tested in separate and in different amounts and were also tested mixtures of them. Ergo the best results were obtained using 500 mg C18-bonded silica together with 600 mg of magnesium sulfate, for drying. The pesticides were determined by gas chromatography tandem mass spectrometry (GC-MS). Considering the characteristics of the pesticides and the complexity of the coffee matrix the GC-MS had to be operated in negative chemical ionization mode (NCI) and single ion monitoring mode (SIM) which provided high sensitivity and selectivity to pesticide detection. The validation was performed by analyzing spiked samples at three different concentrations (10, 20 and 50 μg kg-1), with 6 replicates (n=6) at each spike concentration. For each one of the 51 pesticides determined by GC-MS (NCI-SIM), the linearity (r) of calibration curves, accuracy (as recovery percent), instrument and method limits of detection and quantification (LOD and LOQ), precision (as RSD percent) and matrix effects (percent) were determined. From the 51 pesticides studied, approximately 76% showed average recoveries between 70-120% and 75% RSD ≤ 20% for the spike concentration of 10 μg kg-1. For the spike concentrations of 20 and 50 μg kg-1, the recoveries (%) and RSD (%) values were even better. The method LOQ was 10, 20 and 50 μg kg-1, for respectively 33, 3 and 6 pesticides, 4 pesticides could only be detected via their degradation products and 5 compounds did not presented recoveries between 70-120% and RSD ≤ 20% thus the method LOQ could not be determined for those pesticides. The matrix effect was present for all the pesticides even with the use of a clean-up step, requiring therefore matrixmatched calibration standards for application in routine analysis. / Neste estudo, foi desenvolvido e validado um método multirresidual para determinação de 40 pesticidas em grãos de café verde. Para se obter uma amostra bem homogênea, os grãos de café verde foram previamente moídos e em seguida preparou-se um slurry de café e água (razão 1:4, m/m). Para o procedimento de extração utilizou-se o método QuEChERS, no qual foram introduzidas algumas modificações. Como solvente extrator, utilizou-se acetonitrila contendo 1% de ácido acético e padrão interno do procedimento (P.I.P.). Após a adição da acetonitrila, para promover o particionamento entre as fases orgânica e aquosa, adicionou-se 3 g de sulfato de magnésio anidro. Para a purificação do extrato de café, foram testados dois adsorventes, separadamente e em diferentes quantidades e também misturas entre os dois adsorventes, em uma etapa de extração em fase sólida dispersiva (dSPE), sendo que os melhores resultados foram obtidos com a utilização de 500 mg de C18 juntamente com 600 mg de sulfato de magnésio, para remoção de água. A determinação dos pesticidas foi feita em um sistema de cromatografia gasosa acoplado a um espectrômetro de massas (GC-MS). Em função das características dos pesticidas e da complexidade da matriz, o GC-MS foi operado no modo de ionização química negativa (NCI) e monitoramento seletivo de íons (SIM), que forneceu grande sensibilidade e seletividade na determinação dos pesticidas. A validação foi realizada pela análise de amostras de café fortificado em três diferentes concentrações (10, 20 e 50 μg kg-1), com 6 replicatas (n=6) para cada concentração. Para cada um dos pesticidas, avaliou-se a linearidade da curva analítica (r), exatidão (recuperação%), limite de detecção e quantificação (LOD e LOQ) do instrumento e do método, precisão (RSD%) e efeito matriz (%). Dos 51 pesticidas estudados, aproximadamente 76% obtiveram recuperações médias entre 70-120% e 75% apresentaram RSD ≤ 20% para amostras fortificadas na concentração de 10 μg kg-1. Para as fortificações de 20 e 50 μg kg-1 os resultados foram ainda melhores. O LOQ do método foi estabelecido em 10, 20 e 50 μg kg-1, respectivamente para 33, 3 e 6 pesticidas, 4 pesticidas somente puderam ser detectados via seus produtos de degradação e 5 compostos não apresentaram recuperações entre 70-120% e RSD≤ 20%, assim o LOQ do método não pode ser estabelecido para esses pesticidas. Mesmo com a utilização de uma etapa de purificação do extrato de café o efeito matriz manteve-se presente para todos os pesticidas, dessa forma, para que o método possa ser implantado em análises de rotina, a confecção das curvas analíticas deve ser feita em matriz (matrix-matched calibration).

Conselho Nacional de Desenvolvimento Científico e Tecnológico / The use of pesticides always has been associated with the effective control of pests or invasive weeds to ensure an increase in the food production. However, the indiscriminate use of these substances has caused the degradation of water resources. In Brazil, the Ministry of Health through Ordinance 2914 defines several parameters of potability, among them, the maximum limits allowed for same pesticides. In this work it was developed and validated a method for the determination of residues of 70 pesticides in drinking water using (SPE) for sample preparation and determination by Gas and Liquid Chromatography coupled to tandem mass spectrometry, triple quadrupole analyzer (GC-(TQ)MS/MS and LC- (TQ)MS/MS). It was evaluated different sample volume, sorbents and solvent of elution. The best results were obtained using 100 mL sample acidified at pH 2.5, Oasis® SPE cartridge HLB 60 mg/3 mL and dichloromethane/methanol as eluent. Analytical curves were linear between 10 and 250 Sg L-1, with r2 values greater than 0.99 for all compounds. The values of method LOQ were 0.02 Sg L-1 for aldrin, dieldrin and chlordane and 0.5 Sg L-1 for the other compounds. To evaluate accuracy the blank samples ware fortified at 0.5, 1.5 and 4.0 Sg L- 1 and an extra level at 0.02 Sg L-1 for aldrin, dieldrin and chlordane. The method showed good precision, with RSD values below to 20% and good accuracy, with recoveries between 70 and 120%. Only the compounds methamidophos, aldicarb, benfuracarb, terbufos, benomyl and thiophanate methyl were not recovered adequately. The matrix effect was evaluated, showing upper 10% for the most compounds. In order to compensate this effect, analytical curves were obtained with standarts prepared in blank extracts of the matrix. The validated method was applied to 12 samples of drinking water of different characteristics (river, shed, well and treated water), and just one of the river samples presented residues of lambda-cyhalothrin. The results indicate that the proposed method is suitable for analysis of pesticides residues in drinking water, since all the validation parameters met the suggested limits and parameters for validation of chromatographic methods. / O uso de agrotóxicos sempre esteve associado à efetividade no controle de pragas ou plantas invasoras para aumentar a produção de alimentos. No entanto, o uso indiscriminado dessas substâncias vem causando a degradação dos recursos hídricos. No Brasil, o Ministério da Saúde através da Portaria 2914 define diversos parâmetros de potabilidade, entre eles, os limites máximos permitidos para alguns agrotóxicos. No presente trabalho foi desenvolvido e validado um método para a determinação de resíduos de 70 agrotóxicos em água potável, utilizando Extração em Fase Sólida (SPE) para o preparo de amostra e determinação por Cromatografia Gasosa e Líquida, acopladas à Espectrometria de Massas sequencial, com analisador triplo quadrupolo (GC-(TQ)MS/MS e LC- (TQ)MS/MS). Foram avaliados diferentes volumes de amostra, sorventes e solventes de eluição. Os melhores resultados foram obtidos com 100 mL de amostra acidificada em pH 2,5, cartucho SPE Oasis® HLB 60 mg/3 mL e diclorometano/metanol como eluente. As curvas analíticas apresentaram linearidade entre 10 e 250 Sg L-1, com valores de r2 maiores que 0,99 para todos os compostos. Os valores de LOQ do método foram 0,02 Sg L-1 para aldrin, dieldrin e clordano e de 0,5 Sg L-1 para os demais compostos. Para avaliação da exatidão as amostras branco foram fortificadas em 0,5, 1,5 e 4,0 Sg L-1 e um nível extra em 0,02 Sg L-1 para os compostos aldrin, dieldrin e clordano. O método apresentou boa precisão, com valores de RSD inferiores a 20% e boa exatidão, com recuperações entre 70 e 120%. Apenas os compostos metamidofós, aldicarbe, benfuracarbe, terbufós, benomil e tiofanato metílico não foram recuperados de forma adequada. O efeito matriz foi avaliado, mostrando-se superior a 10% para a maioria dos compostos. A fim de compensar este efeito, utilizou-se curvas analíticas preparadas no extrato branco da matriz. O método validado foi aplicado em 12 amostras de água potável de diferentes características (rio, vertente, poço e água tratada), sendo que apenas uma das amostras de água de rio apresentou resíduos de lambda-cialotrina. Os resultados indicam que o método proposto é adequado à análise de resíduos de agrotóxicos em água potável, visto que todos os parâmetros de validação atenderam os limites e parâmetros sugeridos para validação de métodos cromatográficos.

Polycyclic aromatic hydrocarbons (PAHs) are compounds that can be found in environmental compartments such as atmosphere, soil, sediment and water. Throughout bioaccumulation, they can also be incorporated into vegetables and animals tissues. They are mainly originated from anthropogenic sources such as fossil fuels and biomass burnings. Due they potential toxicity, carcinogenicity and mutagenicity, they must be carefully monitored. The main goal of this study is to compare two extraction methods (ultrasound and Soxhlet) and the optimization of a cleanup column and chromatographic conditions (GC-MS) for separation and determination of 16 polycyclic aromatic hydrocarbons (PAHs) established as priorities by the Environmental Protection Agency of the United States. Once optimized, the method was applied to evaluate HPAs in sediment samples collected in the Sergipe river estuarine system. The extracts were analyzed by GCMS, using a DB-5 capillary column, splitless injection and operating in SIM mode, with three ions being monitored for each PAH, one ion principal and two others as confirmation ions. We conducted a validation study of the methods. The analytical curves showed coefficients of determination (R2) above 0.99 for all 16 PAHs. For the ultrasound method, the average recovery obtained in two fortification levels (12.5 ng g-1 and 62.5 ng g-1) showed no significant differences, with a recovering average ranging from 74 to 125.4% and RSD <19.2% for all compounds analyzed, except for pyrene. The limits of detection and quantification by ultrasound ranged from 0.04 to 0.23 ng g-1 and 0.12 to 0.78 ng g-1, respectively, and the limits of detection and quantification by Soxhlet ranged from 0.03 to 0.3 ng g-1 and 0.11 to 1.0 ng g-1, respectively. On the other hand, the Soxhlet extraction did not show good recoveries for low molecular weight PAHs, especially naphthalene, acenaphthylene and acenaphthene, with values ranging from 9.5 to 98.4%. Therefore, the extraction method chosen for the analysis of PAHs in sediment was the ultrasound. It showed good recovery, speed, solvent and adsorbent economy and low extraction time. It also allows simultaneous extraction of several samples. The total concentrations of PAHs in the sediments studied ranged from 6.7 ng g-1 to 377.7 ng g-1 dry weight. The species found in higher concentrations were Benzo[ghi]Perylene (61.9 ng g-1), chrysene (52.7 ng g- 1), fluoranthene (47.9 ng g-1) and pyrene (34.1 ng g-1). The investigation of the PAHs sources suggests pyrogenic origin for most of the compounds. / Os hidrocarbonetos policíclicos aromáticos (HPAs) são compostos que podem ser encontrados em diversos compartimentos ambientais tais como atmosfera, solo, sedimento e água. Por bioacumulação podem também estar incorporados aos organismos vegetais e animais. São originados, principalmente, de fontes e/ou atividades antrópicas, como a queima de combustíveis fósseis e da biomassa. Devido ao seu potencial tóxico, carcinogênico e mutagênico, eles devem ser cuidadosamente monitorados. O presente trabalho tem como objetivo a comparação de dois métodos de extração (ultra-som e Soxhlet), otimização de uma coluna (clean up) e das condições cromatográficas (GC-MS) para a separação e determinação dos 16 hidrocarbonetos policíclicos aromáticos (HPAs), estabelecidos como prioritários pela Agência de Proteção Ambiental dos Estados Unidos, em amostras de sedimentos coletadas no estuário do rio Sergipe. Os extratos foram analisados por GC-MS utilizando coluna capilar DB-5, injeção no modo splitless e operando no modo SIM, selecionando um íon principal e dois de confirmação de cada HPA. Foi realizado um estudo de validação dos métodos. As curvas analíticas apresentaram valores de coeficiente de determinação (R2) acima de 0,99 para os 16 HPAs. Pelo método de ultra-som, os valores médios de recuperação obtidos em dois níveis de fortificação (12,5 ng g-1 e 62,5 ng g-1) não apresentaram diferenças significativas, obtendo recuperação média na faixa de 74 a 125,4% e RSD< 19,2% para os compostos analisados, com exceção do pireno. Os limites de detecção e quantificação por ultra-som variaram de 0,04 a 0,23 ng g-1 e de 0,12 a 0,78 ng g-1, respectivamente, enquanto que por Soxhlet variaram de 0,03 a 0,3 ng g-1 e de 0,11 a 1,0 ng g-1, respectivamente. Por outro lado, a extração por Soxhlet não apresentou boa recuperação para os HPAs de baixa massa molar, principalmente, o naftaleno, acenaftileno e o acenafteno, que variou de 9,5 a 98,4%. Portanto, o procedimento escolhido para análise de HPAs em sedimento foi o ultra-som, pois mostrou boa recuperação, rapidez, economia de solvente e adsorvente, baixo tempo de extração, além de permitir a extração simultânea de todas as amostras. As concentrações de HPAs totais nos sedimentos estudados variaram de 6,7 ng g-1 a 377,7 ng g-1 em peso seco. As espécies encontradas em maior concentração foram Benzo[ghi]Perileno (61,9 ng g-1), Criseno (52,7 ng g-1), Fluoranteno (47,9 ng g-1) e Pireno (34,1 ng g-1). A investigação da fonte dos HPAs sugere origem pirogênica para a maioria dos compostos.

A subtribo Lychnophorinae ocorre na região do Cerrado do Brasil e contém cerca de 120 espécies. Recentemente, a filogenia da subtribo Lychnophorinae, baseada em sequências de DNA e dados morfológicos foi capaz de fornecer novas informações sobre a subtribo e seus gêneros. Além disso, o Cerrado brasileiro possivelmente abriga uma parcela considerável da entomofauna neotropical. Os objetivos gerais deste projeto de pesquisa são obter perfis metabólicos de plantas da subtribo Lychnophorinae e utilizá-los como ferramenta quimiotaxonômica para auxiliar na resolução da classificação taxonômica dessa subtribo e ainda, obter perfis metabólicos de insetos que se alimentem dessas plantas, visando a identificação de possíveis interações inseto-planta. Foram analisadas 78 espécies de plantas por GC-MS e UHPLC-UV(DAD)-MS(ESI-Orbitrap) nos modos positivo e negativo de ionização. As coletas de insetos foram feitas em intervalos trimestrais e, em seguida, esses insetos foram analisados utilizando a mesma metodologia analítica das plantas. As \"impressões digitais\" metabólicas das plantas e dos insetos foram precessadas no MetAlignTM e no MSClust, e as matrizes geradas foram submetidas a análises multivariadas no SIMCA. As plantas foram submetidas a análise de componentes principais (PCA), análise de cluster hierárquico (HCA) e análise discriminante ortogonal por mínimos quadrados parciais (OPLS-DA), entretanto os insetos, juntamente com suas plantas hospedeiras, foram analisados por PCA com o intuito de determinar a correlação entre seus metabólitos secundários. Os resultados das análises metabolômicas apresentaram proximidade com a filogenia principalmente para os dois maiors gêneros, Eremanthus e Lychnophora, analisados separadamente. Portanto, os resultados sugerem que os dados gerados a partir das análises metabolômicas podem ser utilizados em estudos quimiotaxonômicos da subtribo Lychnophorinae, sobretudo como dados primários para a reconstrução filogenética de gêneros. No que diz respeito às análises de possíveis interações inseto-planta, foi possível observar que alguns espécimens apresentaram correlação significativa com as plantas hospedeiras, evidenciando que a abordagem metabolômica pode ser utilizada como ferramenta na investigação de interações inseto-planta. Nestas amostras, pôde-se observar a presença de triterpenos, flavonoides e lactonas sesquiterpênicas adquiridas nas plantas por meio da herbivoria. / The subtribe Lychnophorinae occurs in the Cerrado domain of the Brazilian Central Plateau. The relationships among its recognized genera, as well as the relationships between Lychnophorinae and other subtribes belonging in tribe Vernonieae have been recently investigated upon a phylogeny based on molecular and morphological data. In addition, a preliminar overview of insect diversity in Brazilian Cerrado suggests that it may harbor a considerable fraction of the neotropical. We here report the use of a comprehensive untargeted metabolomics approach, combining LC-MS and GC-MS data together, followed by multivariate analyses aiming to assess the congruence between metabolomics data and the phylogenetic hypothesis, as well as its potential as a chemotaxonomic tool. Also we report the use of untargeted metabolomics approach aiming to assess insect-plant interactions. We analyzed 78 species by GC-MS and LC-MS in both positive and negative ionization modes. The metabolic profiles obtained for these species were treated in MetAlign and in MSClust and the matrices generated were combined and used in SIMCA for hierarchical cluster analyses (HCA), principal component analyses (PCA) and orthogonal partial least square discriminant analysis (OPLS-DA). The insects were collected quarterly and analyzed by the same analytical methods as plants. Results show that metabolomics analyses are mostly congruent with the phylogenetic hypothesis especially at lower taxonomic levels. Therefore, our results suggest that data generated by metabolomics approaches provide valuable evidence for chemotaxonomical studies of Lychnophorinae subtribe, in particular as primary data for phylogenetic reconstruction of lineages as genera. Regarding to insects, it was possible to observe significative correlations between some insects and their host plants. In these samples, were able to identify triterpenes, flavonoids and sesquiterpene lactones.

Il est maintenant avéré que les contaminants présents dans les milieux aquatiques peuvent être toxiques à l’état de traces voire d’ultra traces. Il est donc important de développer des méthodes d’analyse performantes et sensibles pouvant atteindre ces niveaux de concentration. Dans cette optique, les techniques chromatographiques couplées à la spectrométrie de masse (GC-MS et LC-MS/MS) sont généralement utilisées pour l’analyse des polluants organiques. Les composés présents dans les milieux aquatiques possèdent des propriétés physico-chimiques très variées, d’apolaires à très polaires. Ainsi, développer une analyse simultanée pour toutes ces molécules représente un challenge analytique. Dans ce travail de thèse, plusieurs étapes du processus analytique ont été évaluées : la préparation de l’échantillon par extraction sur phase solide (SPE), la séparation par chromatographie et la détection par spectrométrie de masse. Une liste de composés modèles couramment retrouvés dans les milieux aquatiques a été établie pour conduire ces essais. Les méthodes d’extraction en phase solide hors ligne et en ligne ont été développées dans une optique d’analyse multirésidus à un niveau de traces. Les méthodes chromatographiques, gazeuse et liquide, couplées à la spectrométrie de masse ont été étudiées pour favoriser une analyse exhaustive et sensible. La dernière partie a consisté à appliquer les méthodes développées pour une approche d’analyse non ciblée. / It is now widely recognized that contaminants present in aquatic environments can be toxic at traces or even ultra-traces level. Therefore, it is important to develop efficient and sensitive analytical methods to reach these levels of concentration. In that respect, chromatographic techniques coupled to mass spectrometry (GC-MS and LC-MS / MS) are commonly used for the analysis of organic pollutants. The substances encountered in aquatic environments display a large range of physico-chemical properties, from apolar to very polar. Thus, developing a simultaneous analysis for all these molecules represents an analytical challenge. In this pHD work, several steps of the analytical process have been investigated: sample preparation by solid phase extraction (SPE), chromatographic separation and the detection by mass spectrometry. A list of model compounds commonly determined in aquatic environments was established to conduct the tests. Solid phase extraction methods, offline and online, were developed in a multiresidue analysis aim at traces level. Chromatographic methods, gaseous and liquid, coupled to mass spectrometry were studied to obtain an exhaustive and sensitive analysis. The last part consisted to apply the developed methods for a non-targeted analysis approach.