Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / This work presents multi-method for pesticide residues determination in fruit jams. Slurries of the fruit jams and ultrapure (u.p.) water were prepared to yield homogeneous samples and to facilitate the sample handling. The modified QuEChERS method was used for the sample preparation. Due the high detectability of the micro flow liquid chromatography triple quadrupole-linear ion trap mass spectrometry equipment (μLC-QTRAP-MS/MS) and to minimize the matrix effect (%) the acetonitrile extracts were diluted 30-fold before analysis. The method validation was performed analyzing spiked samples at the concentrations of 9 and 45 μg kg-1 (n=5). The method met validation criteria of 70 120% recovery and relative standard deviation (RSD) ≤ 20% for 93% (99) of the 107 pesticides evaluated. The reporting limit (RL) was 9 and 45 μg kg-1 for, respectively, 66% and 26% of the analytes, 5% of the compounds did not fulfill the requirements for validation and 3% were not detected at the studied concentrations. The validated method was applied to the analysis of 51 different fruit jam samples from Brazil and from Spain. Pesticide residues were detected in 80% of the samples, 51% of which contained at least one pesticide at concentration higher than 10 μg kg-1. Moreover, the stability of five pesticides was evaluated in the preparation of home-made fruit jams from spiked fruits. Thereunto, five types of fruits (orange, apple, strawberry, pear and peach, with n=2 for each type of fruit) were grinded and spiked at 500 μg kg-1 with a mixture of carbendazim, chlorpyrifos, imidacloprid, iprodione and propargite. Home-made jams were prepared with these spiked samples by cooking, over medium heat, the grinded spiked fruit with sugar and u.p. water (ratio, 5:5:2, w/w/w), in an open pan for 30 min. The modified QuEChERS extraction method was applied either to blank fruits, to the fruits spiked at 500 μg kg-1 and to the home-made jams (prepared from spiked fruits at 500 μg kg-1). The extracts of home-made jams and blank fruits were analyzed by liquid chromatography quadrupole-time of flight mass spectrometry (LC-QTOF-MS) to ascertain the occurrence of the known metabolites of the spiked pesticides and to determine the number of co-extracted matrix components from the fruits and from the jams. All samples were also analyzed by μLC-QTRAP-MS/MS for estimation of the pesticides processing factors. So the pesticide concentrations found in the spiked fruits were compared to the concentrations found in the home-made jams (pesticide concentration measured in the jams/pesticide concentration measured in the raw fruits). / Este trabalho apresenta um método multirresidual para determinação de resíduos de agrotóxicos em doces de fruta em pasta. Para obter-se amostras homogêneas e de fácil manipulação preparou-se uma mistura (slurry) dos doces de frutas e água ultrapura (u.p.). O preparo das amostras foi feito utilizando o método QuEChERS modificado. Com o objetivo de diminuir o efeito matriz (%) das amostras e pela alta detectabilidade do equipamento de cromatografia de micro vazão a líquido acoplada à espectrometria de massas híbrida triplo quadrupolo-armadilha de íons linear (μLC-QTRAP-MS/MS) os extratos de acetonitrila foram diluídos na razão de 1:30 (v/v) antes da análise. A validação do método analítico foi feita pela análise de amostras de doces de uva fortificadas nas concentrações de 9 e 45 μg kg-1 (n=5). Dos 107 agrotóxicos avaliados 93% (99) obtiveram recuperações de 70 a 120% e desvio padrão relativo (RSD) ≤ 20%. Os limites de notificação (RL) foram de 9 e 45 μg kg-1 para, respectivamente, 66% e 26% dos agrotóxicos avaliados, 5% dos compostos não alcançaram os pré-requisitos necessários para a validação e 3% não foram detectados nas concentrações estudadas. Após a validação do método, foram analisadas 51 amostras de doces de frutas provenientes do Brasil e da Espanha. Em 80% amostras foram detectados resíduos de agrotóxicos, 51% destas continham no mínimo um agrotóxico em concentração maior que 10 μg kg-1. Além disso, avaliou-se a estabilidade de cinco agrotóxicos no preparo de doces de frutas caseiros. Para isso, cinco tipos de frutas (laranja, maçã, morango, pera e pêssego, com n=2 para cada tipo de fruta) foram trituradas e fortificadas com uma mistura de carbendazim, clorpirifós, imidacloprido, iprodiona e propargito a 500 μg kg-1. Essas amostras foram utilizadas no preparo de doces de frutas caseiros. Para isso, as frutas trituradas e fortificadas foram cozidas com açúcar e água u.p. (proporção de 5:5:2, m/m/m) em panela aberta sob fogo médio durante 30 min. O método QuEChERS modificado foi aplicado às frutas não fortificadas, às frutas fortificadas (500 μg kg-1) e aos doces de frutas caseiros (preparados com frutas fortificadas a 500 μg kg-1). Os extratos dessas amostras foram analisados por cromatografia a líquido acoplada à espectrometria de massas híbrida quadrupolo-de tempo de voo (LC-QTOF-MS), com o objetivo de verificar a presença dos metabólitos conhecidos dos agrotóxicos e determinar o número de componentes coextraídos das matrizes. Todas as amostras foram analisadas também por μLC-QTRAPMS/ MS com o objetivo de comparar a concentração dos agrotóxicos nas frutas fortificadas com as concentrações nos doces de frutas correspondentes. A partir dessa relação foi possível estimar um fator de processamento (FP) para os agrotóxicos no preparo dos doces de frutas caseiros (concentração do agrotóxico no doce de fruta/concentração do agrotóxico na fruta fortificada).

Die Systematische Toxikologische Analyse (STA) stellt auf Grund der großen Vielfalt und der ständigen Zunahme an toxikologisch relevanten Substanzen eine der größten Herausforderungen in der chemischen Analyse dar. In der vorliegenden Arbeit wurde daher die Eignung der Flüssigchromatographie in Kombination mit der Hybrid-Quadrupol-Flugzeitmassenspektrometrie (LC-QTOF-MS) für diesen Zweck untersucht. Dazu wurden eine Datenbank mit über 7360 und eine CID-Spektrenbibliothek mit mehr als 2720 toxikologisch relevanten Substanzen erstellt und geeignete Probenvorbereitungsmethoden entwickelt. Die Erprobung der Methoden erfolgte an dotierten Blut- und Haarproben. Hierbei zeigte sich, dass die Analyse im Auto-MS/MS-Modus (Messzyklen von MS- und MS/MS-Spektren) eine Identifizierung basischer Substanzen mittels CID-Spektren zwischen 0,5 und 2 ng/ml im Blut ermöglichte. Die Nachweisgrenzen der für 24 Wirkstoffe validierten Methode in Haaren lagen bei 3 bis 15 pg/mg. Die Eignung der LC-QTOF-MS zur STA von Haarproben wurde an 30 Drogentodesfällen und 60 Todesfällen mit bekannter chronischer Medikamenteneinnahme zu Lebzeiten sowie an 77 Blutproben nachgewiesen. Für die Suche nach Metaboliten wurde ein Metaboliten-Tool entwickelt. In der praktischen Anwendung auf Datenfiles von Blut- und Haarproben erwies sich das Tool als wertvolles Hilfsmittel zur Identifizierung unbekannter Peaks und zur Bestätigung von Suchergebnissen in der Datenbank. Zur automatischen Konzentrationsabschätzung identifizierter Substanzen wurde ein Tool „Estimate Concentration“ geschaffen. Die Überprüfung des Verfahrens an realen Blut- und Haarproben durch Vergleich mit HPLC-DAD- und GC-MS-Ergebnissen wies eine gute Übereinstimmung der Konzentrationen auf. Insgesamt zeigten die Untersuchungen, dass die LC-QTOF-MS zurzeit die am besten geeignete Methode für die STA darstellt. Auch bei einem erst später aufkommenden Verdacht kann eine gezielte Suche in dem bereits gemessenen Datenfile durchgeführt werden. / Due to the large variety and the steady increase of toxicologically relevant substances, systematic toxicological analysis (STA) is one of the most difficult tasks in analytical chemistry and, therefore, a steady topic of research and methodical improvement. For this reason, the suitability of liquid chromatography in combination with hybrid quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) for STA was investigated. For this purpose, a database of more than 7360 and a CID spectra library of more than 2720 toxicologically relevant substances and suitable methods for sample preparation were developed. The application was evaluated at spiked blood and hair samples. It was found that the analysis in Auto-MS/MS mode (alternating measurement cycles of MS and MS/MS spectra) allowed substance identification in blood using CID spectra between 0.5 and 2 ng/ml for basic substances. The detection limits of the validated method in hair ranged from 3 to 15 pg/mg for 24 drugs. The suitability of LC-QTOF-MS for STA was tested for hair samples from 30 drug-related death cases and from 60 death cases with known chronic medication as well as for 77 blood samples. For the search of metabolites, a metabolite tool was developed. In the practical application to data files from blood and hair samples, the tool proved to be very helpful for identification of unknown peaks and for confirmation of results obtained only from the database without CID spectra. A tool "Estimate Concentration" was created for automatic estimation of concentrations of identified substances. The application to real blood and hair samples and the comparison of the concentrations with results from HPLC-DAD and GC-MS showed good agreement. Overall, these investigations showed that LC-QTOF-MS is currently the most favorable method for STA. Because of the comprehensive registration of all substances in a sample, the data files can be checked for the presence of certain poisons even later without new measurements.