FundaÃÃo de Amparo à Pesquisa do Estado do Cearà / Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / AtravÃs da cromatografia lÃquida de alta eficiÃncia com detecÃÃo por espectrometria de massa no modo MS/MS, desenvolveu-se um mÃtodo sensÃvel e altamente seletivo para determinar os nÃveis de acetato de megestrol no plasma humano, usando a betametasona como padrÃo interno. O analito e o padrÃo interno foram extraÃdos das amostras do plasma atravÃs da soluÃÃo de extraÃÃo hexano-acetato de etila (1:1 v/v), e cromatografados em uma coluna C18. A fase mÃvel consistiu de acetonitrila-Ãgua (80:20 v/v), contendo 0,1% de Ãcido fÃrmico. A detecÃÃo foi realizada em um triplo-quadrupolo, atravÃs de um espectrÃmetro de massa no modo de monitoramento de reaÃÃo mÃltipla via eletrospray. O mÃtodo tem um tempo de corrida de 3 minutos e limite de quantificaÃÃo de 2 ng/mL. As curvas de calibraÃÃo foram obtidas utilizando uma faixa de concentraÃÃo de 2 a 150 ng/mL. As precisÃes intralote foram 3,16%, 4,65% e 2,68%, e a acurÃcia intralote foi de 6,77%, 6,23% e 5,73% para 6, 60 e 120 ng/mL, respectivamente. As precisÃes interlote foram 7,76%, 6,23% e 6,37%, e a acurÃcia interlote foi de 0,08%, 1,55% e 2,11% para as mesmas concentraÃÃes. Este mÃtodo validado foi aplicado com sucesso para a determinaÃÃo dos parÃmetros farmacocinÃticos dos comprimidos de acetato de megestrol administrados a 30 voluntÃrios sadios. / A sensitive and highly selective liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed to determine megestrol acetate in human plasma using betamethazone as the internal standard. The analyte and internal standard were extracted from plasma samples by hexane/ethyl acetate (1:1 v/v), and chromatographed on a C18 column. The mobile phase consisted of acetonitrila-water (80:20 v/v) including formic acid 0.1%. Detection was performed on a triple quadropole tandem mass spectrometer by multiple reaction mode via electrospray source. The method has a chromatographic run time of 3 min and a limit of quantification of 2 ng/mL. The linear calibration curves were obtained in the concentration range 2 â 150 ng/mL. The intra-batch precisions were 3.16%, 4.65%, and 2.68%; and the intra-batch accuracy was 6.77%, 6.23%, and 5.73% for 6, 60 and 120 ng/mL, respectively. The inter-batch precision was 7.76%, 6.23%, and 6.37% and the inter-batch accuracy was 0.08%, 1.55%, and 2.11% for the same concentrations. This validated method was successfully applied for the determination of pharmacokinetic profiles of megestrol acetate tablets administered to 30 healthy volunteers.

O quadro evolutivo da agricultura brasileira resulta em benefícios à população exigindo crescentes avanços tecnológicos no setor. Constantemente, novos agrotóxicos são introduzidos estimulando estudos científicos com a finalidade de determinar e avaliar os impactos na população e no meio ambiente. No presente trabalho, a matriz avaliada foi o lodo gerado no processo de tratamento de água para consumo humano, coletado na região do Vale do Ribeira, SP. A técnica empregada foi a cromatografia líquida de fase reversa acoplada à espectrometria de massas triploquadrupolar em tandem com ionização por electrospray. Os compostos foram extraídos previamente da matriz. O desenvolvimento da metodologia exigiu tratamento dos dados para que esses pudessem ser utilizados e transformados em informações confiáveis. Os processos envolvidos foram avaliados usando o conceito da validação de ensaios químicos. Os indicadores avaliados foram seletividade, linearidade, intervalo de trabalho, sensibilidade, exatidão, precisão, limite de detecção, limite de quantificação e robustez. Esses indicadores produziram valores quantitativos e qualitativos que foram estatisticamente evidenciados de forma objetiva. A metodologia desenvolvida e validade é simples. Como resultado, mesmo explorando a sensibilidade da técnica, os compostos estudados não foram encontrados no lodo da ETA de Registro. Isso leva a crer que esses compostos podem estar presentes em concentrações muito baixas, podem sofrer degradação durante o tratamento da água ou não são retidos completamente pela ETA. 7 / The evolving scenario of Brazilian agriculture brings benefits to the population and demands technological advances to this field. Constantly, new pesticides are introduced encouraging scientific studies with the aim of determine and evaluate impacts on the population and on environment. In this work, the evaluated sample was the sludge resulted from water treatment plant located in the Vale do Ribeira, São Paulo, Brazil. The technique used was the reversed phase liquid chromatography coupled to electrospray ionization tandem mass spectrometry. Compounds were previously liquid extracted from the matrix. The development of the methodology demanded data processing in order to be transformed into reliable information. The processes involved concepts of validation of chemical analysis. The evaluated parameters were selectivity, linearity, range, sensitivity, accuracy, precision, limit of detection, limit of quantification and robustness. The obtained qualitative and quantitative results were statistically treated and presented. The developed and validated methodology is simple. As results, even exploring the sensitivity of the analytical technique, the work compounds were not detected in the sludge of the WTP. One can explain that these compounds can be present in a very low concentration, can be degraded under the conditions of the water treatment process or are not completely retained by the WTP.

A avicultura de corte brasileira tem apresentado elevados índices de crescimento. No intuito de garantir uma maior eficiência da produção de aves, medicamentos veterinários são administrados para tratamento e prevenção de doenças. A coccidiose é uma dessas doenças, a qual é causada por várias espécies do protozoário do gênero Eimeria. Os poliéteres ionóforos são antibióticos largamente administrados no controle desses microrganismos. No entanto, a administração contínua de doses subterapêuticas dessa classe de fármacos pode acarretar no acúmulo de resíduos nos tecidos dos frangos de corte. No intuito de garantir a segurança dos consumidores, são definidos Limites Máximos de Resíduos (LMRs) por órgãos internacionais como: Food and Drug Administration (FDA), Commission Regulation, Codex Alimentarius; e nacionais como Ministério da Agricultura Pecuária e Abastecimento (MAPA). Dessa forma, este estudo teve por objetivo o desenvolvimento, validação e aplicação de um método para determinação em peito de frango de resíduos de Lasalocida (LAS), Monensina (MON), Salinomicina (SAL) e Narasina (NAR) por cromatografia líquida acoplada a espectrometria de massas sequencial com ionização por eletrospray (LC-ESI-MS/MS). Nigericina (NIG) foi utilizada como padrão interno do método analítico. As amostras foram liofilizadas, trituradas e tamisadas, sendo utilizadas no método amostras com tamanho de partículas entre 125 ?m e 250 ?m. Os resíduos foram extraídos com metanol e uma alíquota do extrato foi evaporada e reconstituída para análise em LC-ESI-MS/MS no modo positivo de monitoramento de reação selecionada (SRM). A etapa de clean up foi realizada por meio de filtração do extrato em membrana de PTFE 0,22 ?m. O método apresentou-se linear no intervalo de concentração de 0,0 a 40,0 ?g kg-1 para LAS, de 0,0 a 20,0 ?g kg-1 para MON, de 0,0 a 200,0 ?g kg-1 para SAL e de 0,0 a 30,0 ?g kg-1 para NAR. Para todos os antibióticos o coeficiente de correlação linear (r) esteve de acordo com as diretrizes para métodos bioanalíticos (r >= 0,98). A repetitividade foi avaliada em três concentrações de fortificação para cada antibiótico ionóforo, sendo realizada em três dias consecutivos. Foi obtida boa reprodutibilidade intralaboratorial, com coeficiente de variação (CV) de 4,5% para NAR (22,5 ?g kg-1) e 15,2% para LAS (10,0 ?g kg-1). A recuperação ficou acima de 90,0% para todos os antibióticos, sendo de 91,5% para LAS (10,0 ?g kg-1) e 99,1% para MON (15 ?g kg-1). Valores satisfatórios para o limite de decisão (CC?) e capacidade de detecção (CC?) foram obtidos, sendo CC? de 11,8 ?g kg-1 e 115,2 ?g kg-1 para MON e SAL, respectivamente, e CC? de 13,6 ?g kg-1 e 130,3 ?g kg-1 para os mesmos antibióticos. Os limites de detecção (LD) e os limites de quantificação (LQ) foram determinados a partir de dados da curva de calibração. Todos os antibióticos apresentaram LQ inferior ao seu respectivo LMR. O método foi aplicado em 18 amostras disponíveis comercialmente em Ribeirão Preto, SP. De todas as amostras analisadas apenas uma apresentou resíduo de NAR com concentração acima do seu respectivo LD. / The Brazilian poultry industry has shown high rates of growth. In order to ensure greater efficiency of poultry production, veterinary drugs are administered for the treatment and prevention of diseases. Coccidiosis is one of those diseases, which is caused by various species of protozoa of the genus Eimeria. Polyether ionophore antibiotics are widely administered to control these microorganisms. Continuous administration of subtherapeutic doses of this class of drugs may result in the accumulation of residues at low concentration levels in the tissues of broiler. In order to ensure consumer safety, are set Maximum Residue Limits (MRLs) by international organizations such as Food and Drug Administration (FDA), Commission Regulation (EU), Codex Alimentarius; and national such as Ministério da Agricultura e Pecuária (MAPA). Thus, this study aimed to develop, validate and apply a simple method for determination of residues of Lasalocid (LAS), Monensin (MON), Salinomycin (SAL) and Narasin (NAR) in chicken meat by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Nigericin (NIG) was used as qualitative internal standard. Samples were lyophilized, powdered and sieved. Particles size between 125 ?m and 250 ?m were used. Residues were extracted with methanol and aliquots of the extracts were evaporated and reconstituted for injection in the LC-ESI-MS/MS operated in positive selected reaction monitoring (SRM). From the molecular ion were obtained two fragment ions and that of higher relative abundance was used in quantification process. Clean-up step was performed by filtration on 0.22 ?m PTFE membrane. In the validation process, this method showed linearity over the concentration range from 0.0 to 40.0 ?g kg-1 for LAS; 0.0 to 20.0 ?g kg-1 for MON; 0.0 to 200.0 ?g kg-1 for SAL and 0.0 to 30.0 ?g kg-1 for NAR. For all antibiotics the coefficient of linear correlation (r) was in accordance with the guidelines for bioanalytical methods (r >= 0.99). The intra-day precision was evaluated at three spiked concentrations for each ionophore antibiotic and was determined in three consecutive days. Good inter-day precision was obtained, with relative standard deviations from 4.5% for NAR (22.5 ?g kg-1) and 15.2% for LAS (10.0 ?g kg-1). For all antibiotics recovery above 90.0% was obtained, which were 91.5% for LAS (10.0 ?g kg-1) and 99.1% for MON (15.0 ?g kg-1). Satisfactory results of decision limit (CC?) and detection capability (CC?) were determined, which were CC? 11.8 ?g kg-1 and 115.2 ?g kg-1 for MON and SAL, respectively; CC? 13.6 ?g kg-1 and 130.3 ?g kg-1 for the same drugs. The limit of detection (LD) and limit of quantification (LQ) were determined from data of the calibration curve. All the LQs were remarkably below the MLRs for each antibiotic. The method was applied to 18 samples acquired in Ribeirão Preto, SP. Of all analyzed samples only one exhibited traces of NAR with concentration above its respective LD. Thus, the proposed method can be applied in routine analysis on samples of commercial chicken meat.

Os pesticidas têm sido largamente empregados na agricultura para controle de pragas, doenças e ervas daninhas. O uso intensivo de pesticidas nas culturas de tomates, desrespeitando as boas práticas agrícolas, tem causado preocupações quanto à provável contaminação do produto final e muitos métodos multirresíduos têm sido empregados para avaliar e determinar os níveis de resíduos em amostras de alimentos. Para tanto, o objetivo deste estudo foi a validação do método usando QuEChERS no preparo de amostra e LC-MS/MS para a quantificação de resíduos de 61 pesticidas de diferentes classes químicas em tomate. A detecção foi realizada utilizando espectrômetro de massas no modo MRM dinâmico, o tempo de análise foi de 13 min com coluna analítica recheada com partículas de 1,8 \'mü\'m de partícula. Dos 61 pesticidas estudados, 46 estão de acordo com os parâmetros de validação da Comissão Européia e Anvisa, 15% dos pesticidas validados apresentaram efeito matriz médio e as recuperações ficaram entre 87 e 116% e coeficiente de variação de 5 a 17%. Mais de 85% dos compostos investigados apresentaram limites de detecção igual ou menor que 5 \'mü\'g kg-1 e de quantificação igual ou menor que 10 \'mü\'g kg-1. Foram analisadas 58 amostras de tomate coletadas em supermercados da cidade de Piracicaba, SP, Brasil. Doze compostos foram detectados em trinta e cinco amostras (60% do total analisado), todos abaixo do limite máximo de resíduos permitido no Brasil para acefato, acetamiprido, azoxistrobina, benalaxil, bromuconazol, diflubenzurom, imidacloprido, iprodiona, procloraz e tiametoxam, e 15 amostras positivas para metamidofós e 1 para oxamil, que não possuem uso autorizado para a cultura de tomate. Foi realizado um estudo de lavagem de tomates contaminados com produtos formulados (8 pesticidas) com água, solução com 10% de vinagre e com 10% de bicarbonato de sódio e analisada a casca e polpa, para avaliar a capacidade de remoção de cada procedimento. Todos os tratamentos de lavagens (n=3) diferiram estatisticamente para todos os pesticidas avaliados (n=8), com exceção do fipronil, para o qual as lavagens com solução de 10% de bicarbonato de sódio e água não apresentaram diferença no nível de significância de 5%. A lavagem com água ou outras soluções antes do consumo é indicada para a redução de resíduos de pesticidas em tomate e a retirada da casca também contribui para essa redução / Pesticides have been widely used in agriculture to control pests, diseases and weeds. The intensive use of pesticides in tomato crops disrespecting good agricultural practices have been causing concerns about the possible contamination of the final product and many multiresidue methods have been used in order to evaluate and determine the levels of residues in food samples. Therefore, the aim of this study was validate the method using QuEChERS sample preparation and LC-MS/MS for quantification of 61 pesticides residues from different chemical classes in tomato. Detection was performed using mass spectrometry in dynamic MRM mode, run time had 13 min and analytical column packed with 1,8 \'mü\'m particles. Of the 61 pesticides studied, 46 are in accordance with the validation parameters of the European Commission and ANVISA, 15% of the validated pesticides presented matrix effect, recoveries were between 87 and 116% and coefficient of variation 5 to 17%. More than 85% of the compounds investigated showed limits of detection less or equal than 5 \'mü\'g kg-1 and the limits of quantification less or equal than 10 \'mü\'g kg-1. 58 real samples of tomato were analyzed and collected in supermarkets in Piracicaba, SP, Brazil. Twelve compounds were detected in thirty-five samples (60% of the total analyzed), all below the maximum residue limit allowed in Brazil for acephate, acetamiprid, azoxystrobin, benalaxyl, bromuconazole, diflubenzuron, imidacloprid, iprodione, prochloraz and thiamethoxam, and 15 positive samples of methamidophos and 1 for oxamyl, which are not authorized to use at the culture of tomatoes. A wash study was conducted with spiked tomatoes using formulated products (8 pesticides) with water, 10% acetic acid, 10% sodium bicarbonate solution and analyzed concentration in the peel and pulp, in order to evaluate the capacity to remove pesticides in each procedure. All wash treatments (n= 3) differed significantly for all pesticides evaluated (n= 8), with the exception of fipronil, which the washing with 10% of sodium bicarbonate solution and water no showed difference in the level of significance 5%. The washing with water or other solutions before consumption is indicated for the reduction of pesticide residues in tomatoes and the peeling also contributes to this reduction

Orientadores: Susanne Rath, Sonia Claudia do Nascimento de Queiroz / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-26T03:44:24Z (GMT). No. of bitstreams: 1 Oliveira_RenataCabrerade_D.pdf: 3973991 bytes, checksum: 53109a8e29cba1ca926627e521d33fd2 (MD5) Previous issue date: 2014 / Resumo: O uso frequente e indiscriminado de agrotóxicos na agricultura tem acarretado a presença de resíduos nos alimentos e contaminação ambiental, principalmente em países com grande potencial agrícola, como o Brasil. Para avaliar a presença de resíduos de agrotóxicos no ambiente, as abelhas e os produtos apícolas têm sido apontados como potenciais bioindicadores, podendo ser utilizados para monitorar grandes áreas devido às longas distâncias percorridas. Assim sendo, o potencial do uso do pólen apícola como bioindicador da contaminação ambiental por agrotóxicos foi avaliado neste trabalho. Para isso, foi necessário desenvolver e validar um método analítico para determinação de multirresíduos em pólen apícola, utilizando cromatografia a gás acoplada à espectrometria de massas sequencial (GC-MS/MS). Para definição das condições ótimas de extração, dois processos foram avaliados: o QuEChERS, e o de partição com acetonitrila. O QuEChERS mostrou ser mais eficiente (melhor seletividade e eficiência de extração) e foi validado para a determinação de 27 agrotóxicos. Estudos de sorção mostraram que os agrotóxicos são fortemente sorvidos no pólen. Nas amostras provenientes do apiário experimental na Embrapa em Jaguariúna/SP não foram encontrados níveis quantificáveis dos agrotóxicos pesquisados, enquanto a presença de resíduos de agrotóxicos nas amostras fornecidas por apicultores de Ribeirão Preto/SP foi confirmada e quantificada. Os métodos desenvolvidos e validados mostraram ser eficientes e podem ser utilizados no monitoramento ambiental quanto à presença de resíduos de agrotóxicos. Os resultados confirmam o potencial do pólen apícola como bioindicador de contaminação ambiental por agrotóxicos / Abstract: The extensive use of pesticides in agriculture crop has led to the presence of residues in food and environmental contamination, especially in countries with great agricultural potential, such as Brazil. To assess the presence of pesticide residues in the environment, honeybees and bee products have been mentioned as potential bioindicators, which can be used to monitor large areas due to long distances travelled. Therefore, the potential use of bee pollen as a bioindicator of environmental pesticides contamination has been reported in this work. For this it was necessary to develop and validate an analytical method for the determination of multiresidues in pollen, using gas chromatography coupled to tandem mass spectrometry (GC-MS/MS). For the definition of optimum extraction conditions, two procedures were evaluated: QuEChERS, and partition with acetonitrile. The QuEChERS proved to be more efficient (improved selectivity and extraction efficiency), and was validated for the determination of 27 pesticides. Sorption studies showed that pesticides are strongly sorbed in pollen. Unquantifiable levels of pesticides surveyed were found in the samples from experimental apiary at Embrapa in Jaguariúna/SP, while the presence of pesticide residues in samples provided by apiarists from Ribeirão Preto/SP was confirmed and quantified. The validated analytical methods proved to be efficient and can be used in environmental monitoring for the presence of pesticide residues. The results confirm the potential of bee pollen as a bioindicator of environmental pesticides contamination / Doutorado / Quimica Analitica / Doutora em Ciências

Apesar da rápida expansão das aplicações da cromatografia líquida acoplada à espectrometria de massas em química clínica, a análise de metabólitos de baixo peso molecular e alta polaridade em matrizes biológicas ainda permanece como um grande desafio analítico. Dentre os compostos de grande importância no diagnóstico de doenças metabólicas que ainda carecem de melhores alternativas bioanalíticas destacam-se os aminoácidos. O presente estudo descreve o desenvolvimento e a validação de um novo método para a quantificação de 24 aminoácidos em plasma explorando a cromatografia líquida acoplada a espectrômetros de massas em tandem. Foi construído um método de detecção baseado em SRM (múltiplas reações selecionadas) com duas transições de massas para cada um dos 24 aminoácidos e os 19 padrões internos marcados com isótopos estáveis. Foram avaliadas três estratégias de separação cromatográfica e o melhor desempenho foi obtido com fase reversa com octadecilsilano (C18) com pareamento iônico com o ácido perfluoropentanoico. O método cromatográfico final permitiu a separação dos 24 aminoácidos, com resolução completa dos isômeros: leucina, isoleucina e allo-isoleucina, em 11 minutos incluindo o tempo de re-estabilização da coluna cromatográfica. Os limites de quantificação variaram em 113 fmol a 6 pmol injetados na coluna cromatográfica. A imprecisão obtida nos níveis testados para todos os aminoácidos foi inferior a 14%. O método apresentou linearidade para os intervalos testados chegando a 1,5 mmol.L-1 para vários compostos. Os ensaios de arraste mostraram que os limites máximos obtidos na linearidade não geram nenhuma interferência subsequente. A exatidão do método foi avaliada com amostras provenientes do programa de referência interlaboratorial European Research Network for evaluation and improvement of screening, Diagnosis and treatment of Inherited disorders of Metabolism (ERNDIM) e com o material de referência certificado do National Institute of Standards and Technology (NIST). Todos os analitos mostraram equivalência estatística com o método desenvolvido. / Despite the widespread use of liquid chromatography coupled to mass spectrometry applications in clinical chemistry, the analysis of low molecular weight and high polar metabolites in biological matrices remains as a major analytical challenge. Notwithstanding the key role played by amino acids in the diagnosis of metabolic diseases, there is still need for improvements in bioanalytical process of these analytes. The present study describes the development and validation of a new method for quantification of 24 amino acids in plasma based on liquid chromatography coupled to tandem mass spectrometry. Detection and quantification were achieved building a selected reaction monitoring method two mass transitions for each 24 amino acids and 19 stable isotope internal standards. Three chromatographic strategies for separation were evaluated, and best performance was achieved using reversed-phase octadecylsilane with perfluropentanoic acid as ion pairing agent. The separation method allowed separation of 24 amino acids with full resolution for isomers leucine, isoleucine and alloisoleucine in 11 minutes, including column equilibration time. The limits of quantification ranged from 113 fmol to 6 pmol (on column injection). Imprecisions for all evaluated levels and amino acids were less than 14%. The method is linear in all clinical intervals and extending up to 1.5 mmol.L-1. Carryover evaluation demonstrated absence of interference in the following injection throughout the analytical interval. Method accuracy was evaluated analyzing reference samples from European Research Network for evaluation and improvement of screening, Diagnosis and treatment of Inherited disorders of Metabolism (ERNDIM) and National Institute of Standards and Technology (NIST). Statistical equivalence was demonstrated for all analytes using the present method.

L’étude du lien entre l’exposition de l’Homme aux substances chimiques, notamment via son alimentation,et sa santé est un sujet de préoccupation majeure dans notre société qui présente des challenges multiples.Nous nous sommes attelés à l’un d’entre eux par la production de données analytiques fiables relatives à la contamination des denrées alimentaires et des fluides biologiques humains. Le développement de méthodes analytiques reposant sur des technologies en rupture, à même de s’adresser aux paris de la mesure de composés chimiques émergents (e.g. les retardateurs de flamme bromés) ou historiques mais sous le prisme de la sensibilité et du haut débit (e.g. les stéroïdes, les pesticides organochlorés, les dioxines et les polychlorobiphényles) a été au cœur de ce travail. Le couplage GC/APCI/MS sur système triple quadripolaire s’est imposé au cours de nos évaluations. L’association de la chromatographie en phase gazeuse et de l’ionisation chimique à pression atmosphérique a apporté un gain en sélectivité remarquable comparée aux approches traditionnellement retenues dans le domaine et a autorisé l’exploration des ultra-traces dans des matrices biologiques complexes. Cette géométrie nous a permis d’innover en pratiquant des séparations rapides sur colonne capillaire ultra-courte de 2,5 m. La vitesse de balayage et l’excellente sensibilité de l’analyseur de masse nous ont en outre donné accès à une analyse quantitative fiable et multi-paramètres. Ce travail ouvre d’excellentes perspectives vis-à-vis de la production de données d’exposition externe et interne élargies en réponse aux défis à venir entourant la caractérisation de l’exposome Humain. / The study of the link between Human exposure tochemical substances (notably via his food the intake) and Health, is a major concern in our society and poses many challenges. We endeavoured to address one of them by producing reliable analytical data on foodstuffs and human biological fluids contamination. The development of analytical methods based on breakthrough technologies, capable of challenging theemerging (e.g. brominated flame retardants) or historical compounds measurement but through the prism of sensitivity and high throughput (e.g. steroids,organochlorine pesticides, dioxins andpolychlorobiphenyls), was at the heart of our work.Using a GC/APCI/MS with a triple quadripole systememerged as a favorable choice as our evaluations progressed. The association of gas chromatography and atmospheric pressure chemical ionisation brought in a remarkable gain in terms of selectivity, compared to the approaches traditionally selected in this field, andauthorized ultra-trace exploration in complex biologicalmatrices. This geometry allowed us to innovate by performing fast separations on an ultra-short 2.5 mcolumn. Besides, the mass analyser scan speed and high sensitivity gave us access to a reliable and multiparameters quantitative analysis. This work opens up excellent perspectives for the production of expanded external and internal exposure data to meet the future challenges surrounding Human exposome characterisation.

Pesticides are the substances designed for destroying, preventing and controlling of pests and protection of plants. Their wide usage in agriculture causes contamination of ground, surface and drinking water, because wastewater treatment plants are not able to remove them. Mostly used pesticides are triazine based pesticides. However their usage is worldwide reduced because of their negative influences on human health and contamination of environment especially with their metabolites, about their toxicity are no information. A method for determination of triazine pesticides atrazine, cyanazine, propazine, simazine, terbuthylazine and their metabolites hydroxytrazine, desethylatrazine, desisopropylatrazine, hydroxyterbuthylazine a desethylterbuthylazine was developed and validated in this diploma work. The determination was realized by LC-MS/MS with electrospray ionization. LOD ranged between 0.5-3.2 pg/ml and LOQ between 1.5-9.6 pg/ml. A test of stability of the maternal substances of these pesticides was performed, in which temperature was found to have the biggest influence on their degradation. A monitoring of the presence of triazine pesticides and their metabolites in water was carried out in Czech Republic. Low concentrations of the maternal substances in surface water and ground water were...

Candidate: Nikola Kollárová Title of diploma thesis: Detection of Sap2 in the secretome of Candida albicans cell wall and secretory mutants Charles University, Faculty of Pharmacy in Hradec Králové, Department of Biological and Medicinal Sciences Complutense University of Madrid, Faculty of Pharmacy, Department of Microbiology II Study program: Pharmacy Backgound: The aim of this diploma thesis was to search for C. albicans proteins involved in the secretion of the secreted aspartyl proteinase 2 enzyme (Sap2) evaluating the ability to degrade BSA (bovine serum albumin) as a source of nitrogen in several cell wall and secretory mutants of C. albicans. The work was carried out at the Department of Microbiology II, Faculty of Pharmacy, Complutense University of Madrid. Methods: The supernatant samples of several Candida albicans mutants were tested by SDS-PAGE electrophoresis and stained. Bands corresponding to BSA were observed and compared to controls. The other method was counted with 96-well plate. Results: The correlation between optical density and degradation of BSA was observed. Some mutants with disability to degrade BSA were found in a pilot screening of the ability to degrade BSA using 96-well plate method. That fact was confirmed by SDS-PAGE electrophoresis. C. albicans mutants showing...

In this research, electrochemical methods are used to synthesize the inorganic fraction of bone, hydroxyapatite, for application in biological implants and as a calibration material for elemental analysis in human bone. Optimal conditions of electrochemically deposited uniform apatite coatings on stainless steel were investigated. Apatite is a ceramic with many different phases and compositions that have beneficial characteristics for biomedical applications. Of those phases hydroxyapatite (HA) is the most biocompatible and is the primary constituent of the inorganic material in bones. HA coatings on metals and metal alloys have the ability to bridge the growth between human tissues and implant interface, where the metal provides the strength and HA provides the needed bioactivity. The calcium apatites were electrochemically deposited using a modified simulated body fluid adjusted to pH 4-10, for 1-3 hours at varying temperature of 25-65°C while maintaining cathodic potentials of -1.0 to -1.5V. It was observed that the composition and morphology of HA coatings change during deposition by the concentration of counter ions in solution, pH, temperature, applied potential, and post-sintering. The coatings were characterized by powder x-ray diffraction, Fourier transform infrared spectroscopy, and scanning electron microscopy. The precipitated powders from the experiment were also characterized, with results showing similarities to biological apatite. There is a need for quantitative elemental analysis of calcified biological matrices such as bone and teeth; however there are no suitable calibration materials commercially available for quantitative analysis. Matrix-matched standards are electrochemically synthesized for LA-ICP-MS analysis of human bone. The synthetic bioapatite is produced via a hydrothermal electrochemical process using a simulated body fluid solution to form hydroxyapatite. Additional bioapatite standards are synthesized containing trace amounts of metals. The x-ray diffraction of the synthesized standards shows an increase in cell volume for the crystal structure from 0.534 to 0.542 nm3 with the substitution of metals into the crystal structure. The analyte concentration and recoveries for the synthesized standards and reference materials were determined by ICP-MS with % RSD below 6.3% and limits of detection below 1.2 ng/mL for trace metals. The electrochemically synthesized bioapatite was also compared to standard reference materials with X-ray diffraction, FTIR, and Raman spectroscopy. Optimum laser ablation parameters were determined for the standards and human bone. The synthesized standards were homogeneous and the reproducibility for the isotope concentrations determined by LA-ICP-MS was between 3-10 % compared to 10-35% for SRM 1486 Bone Meal and SRM 1486 Bone Ash. A quantitative method has been developed for 2D mapping using LA-ICP-MS and the matrix-matched standards of metal-doped biopaptite to characterize metal concentrations in human bone. Laser ablation parameters for the method are refined resulting in concentration (ug/g) contour map measurements for each isotope measured in the human bone. Essential and non-essential metals, Al, Ca, Cu, Fe, Pb, and Zn are quantitatively mapped using these parameters. Limit of detection for the metals in the bone range from 0.001 to 0.08 ug/g. The LA-ICP-MS analysis method developed proves to be a straightforward and simple method for quantitative analysis of human bone.