Submitted by Ana Hilda Fonseca (anahilda@ufba.br) on 2014-09-19T18:04:41Z No. of bitstreams: 1 Tese_Sarah_Rocha_LSGT.pdf: 3229640 bytes, checksum: af1acce9c5d43271ea20530b0fd1f510 (MD5) / Approved for entry into archive by Fatima Cleômenis Botelho Maria (botelho@ufba.br) on 2014-09-22T12:43:20Z (GMT) No. of bitstreams: 1 Tese_Sarah_Rocha_LSGT.pdf: 3229640 bytes, checksum: af1acce9c5d43271ea20530b0fd1f510 (MD5) / Made available in DSpace on 2014-09-22T12:43:20Z (GMT). No. of bitstreams: 1 Tese_Sarah_Rocha_LSGT.pdf: 3229640 bytes, checksum: af1acce9c5d43271ea20530b0fd1f510 (MD5) / Um método de extração em ponto nuvem de hidrocarbonetos policíclicos aromáticos foi desenvolvido, utilizando tensoativos octilfenol polietoxilados com derivatização do coacervato, como etapa que antecede a detecção por cromatografia a gás acoplada à espectrometria de massa. Foi realizado um estudo da influência da presença de aditivos na redução da temperatura de ponto nuvem do OPEO30 e do OPEO35, surfactantes com temperaturas de ponto nuvem superiores a 100 oC, através adição de sais, álcoois, OPEO7.5 e misturas de sais e álcoois. Observou-se em todos os casos diminuição da temperatura, sendo o sulfato de sódio o aditivo que resultou maior redução da temperatura. Selecionou-se o OPEO30 para as demais etapas do trabalho. Fez-se a otimização das condições da reação de derivatização do coacervato por meio de planejamento fatorial 23, utilizando-se a resposta global para avaliar os resultados. Selecionou-se as condições do ponto central do planejamento fatorial, ou seja, 125 L de BSTFA, 100 L de piridina e banho de água à 70 oC por 45 min, para a derivatização de 30 L de fase rica em surfactante. O procedimento de ponto nuvem foi otimizado, sendo avaliadas as variáveis concentração de surfactante, temperatura e tempo de banho de água, sendo proposta a seguinte metodologia: 1,0 mL de OPEO30 10% m v-1, 10,0 mL de amostra, 1,25 g de sulfato de sódio anidro, banho de água à 70 oC por 10 min, centrifugação à 3000 rpm por cinco min. O método mostrou-se seletivo, sendo observado efeito de matriz intenso, o qual foi corrigido. Os limites de quantificação obtidos estão entre 39 e 176 ng L-1. O método proposto também foi comparado com o método USEPA 3510C, estando as concentrações obtidas, quando analisada uma amostra real de água subterrânea, bem próximas, com recuperações variando entre 90,0 e 97,5 %. O método proposto foi aplicado a amostras de água subterrânea coletadas em poços localizados em postos de revenda de combustíveis. Em todas as amostras foram detectados HPA. / A method for cloud point extraction of polycyclic aromatic hydrocarbons was developed using surfactants octylphenol polyethoxylated with the coacervate derivatization as step prior to gas chromatography-mass spectrometry detection. An influence of additives study on the reduction of cloud point temperature OPEO30 and OPEO35, surfactants with cloud point temperatures above 100 ° C, was conducted, by addition of salts, alcohols, and mixtures OPEO7.5 salts and alcohols. It was observed in all cases cloud point reduction and sodium sulfate was the additive that result in a most effective. The OPEO30 was selected for others work steps. The factorial design 23 was used to optimize the reaction of derivatization, using the global response to evaluate the results. Were selected the central point experimental design condition, 125 L of BSTFA, 100 L of pyridine and water bath at 70 oC for 45 min for 30 L of surfactant-rich phase derivatization. The cloud point extraction procedure was optimized and surfactant concentration, heating bath temperature and time variables, were evaluated. Was proposed the following method: 1,0 mL of OPEO30 10% v m-1 solution, 10,0 mL of sample, 1,25 g of anhydrous sodium sulfate, water bath at 70 °C for 10 min. The centrifugation was performed at 3000 rpm for five min, removing the aqueous layer after each centrifugation step. A 30L coacervate aliquot was withdrawn, and performed derivatization and subsequent GC-MS analysis. The method was validated. The method was selective, an intense matrix effect was observed and was corrected. The quantification limits obtained were between 39 and 176 ng L-1.

[pt] Os glicocorticóides sintéticos tais como a triancinolona acetonido e a prednisolona não possuem fluorescência natural e nem são induzidos a fluorescer por meio do procedimento padrão para glicocorticóides naturais, que consiste no tratamento com ácido sulfúrico concentrado. No presente trabalho, um procedimento de derivação fotoquímica para triancinolona acetonido e prednisolona foi desenvolvido e cuidadosamente otimizado com o intuito de se obter derivados fluorescentes estáveis que permitiram elaborar um método espectrofluorimétrico para determinação dos mesmos em formulações farmacêuticas. Este procedimento consistiu na exposição à radiação ultravioleta (300 nm) de soluções ácidas dos glicocorticóides sintéticos em reator fotoquímico. Parâmetros experimentais, tais como tipo e concentração do ácido, temperatura e tempo de aquecimento, sistema de solventes e tempo de exposição ao UV se mostraram críticos e por isso foram cuidadosamente estudados. As características do reator também se mostraram importantes para o sucesso do procedimento. A intensidade da fluorescência (excitação em 240 nm e emissão em 350 nm) e a estabilidade dos fotoprodutos se mostraram apropriados para permitir o desenvolvimento de um método espectrofluorimétrico. Parâmetros instrumentais importantes, tais como banda espectral de passagem e a velocidade de varredura foram otimizados, visando à obtenção da melhor razão sinal do analito/sinal do branco e da melhor resolução espectral. Parâmetros analíticos de mérito, obtidos a partir das curvas analíticas, revelaram limites de detecção de 5 e 6 ng mL-1 para triancinolona acetonido e prednisolona, respectivamente; e limites de quantificação de 17 ng mL-1 para a triancinolona acetonido e 19 ng mL-1 para a prednisolona. As faixas lineares dinâmicas, nos dois casos, se estenderam por três ordens de grandeza com coeficientes de linearidade (r2) de 0,99. Este método espectrofluorimétrico foi testado em formulações farmacêuticas, obtendo-se os melhores resultados para os comprimidos e soluções injetáveis. Estudos para avaliar o efeito de dois potenciais interferentes (polimixina B e benzocaína) também foram realizados. Resultados de recuperação foram avaliados tomando como referência os valores indicados na bula do medicamento e também valores obtidos com o procedimento de referência (HPLC com detecção fotométrica UVvis) utilizado no Jockey Club Brasileiro, obtendo resultados entre 97 ± 14 e 104 ± 6 %. / [en] The synthetic glucocorticoid such as triamcinolone acetonide and prednisolone neither present natural fluorescence nor fluorescence can be induced by means of the standard procedure used for natural glucocorticoids which consists on a treatment with concentrated sulfuric acid. In the present work, a photochemical derivatization procedure for these two synthetic glucocorticoids was developed and carefully optimized aiming the obtantion of fluorescent derivatives stable enough to allow the development of a spectrofluorimetric method for determination of these analytes in pharmaceutical formulations. The photochemical derivatization procedure consisted on the exposure of acidic solutions of these synthetic glucocorticoids to the ultraviolet radiation (300 nm) in a photochemical reactor. Experimental parameters such as type and concentration of the acid, temperature and heating time, solvent system and UV exposition time have shown to be critical and therefore they were carefully studied. The design of the photochemical reactor) has also shown to be relevant for the success of the procedure. The intensity of the fluorescence (with maximum excitation and emission wavelenghts at 240 nm and 350 nm respectively) and the stability of these photoproducts, have shown to be appropriated for the development of a spectrofluorimetric method. Important instrumental parameters such as spectral bandpass and scan velocity have been optimized aiming the achievement of best signal-to-blank ratio and spectral resolution. Analytical parameters of merit, obtained from the analytical curve, have indicated limits of detention of 5 and 6 ng mL-1 for triamcinolone acetonide and prednisolone, respectively, and limits of quantification of 17 ng mL-1 for triamcinolone acetonide and 18 ng mL-1 for prednisolone. The linear dynamic range for both analytes extended over three orders of magnitude with linear coefficients (r2) of 0,99. The spectrofluorimetric method was tested by the analysis of pharmaceutical formulations, with best performance for tablets and injectable solutions. A study to evaluate the effect of two potential interferents (polimixine B and benzocaine) has also been made. Recovery results were evaluated using both, the reference values indicated in the medicine instructions and concentration values obtained using the reference procedure (HPLC with UV-visible photometric detection) used in the Brazilian Jockey Club. Satisfactory recovery results were achieved (between 97 ± 14 and 104 ± 6 %).

A eletroforese capilar (CE) é uma técnica poderosa de separação que explora as diferenças na mobilidade de espécies iônicas sob efeito do campo elétrico. Não permite, contudo, separação de misturas de moléculas neutras, possível mediante formação de complexos com carga, derivatização química ou cromatografia eletrocinética micelar (MEKC). A derivatização eletroquímica tem sido usada em combinação com HPLC, entre outras técnicas, mas não ainda com CE e para fins quantitativos, como proposto e demonstrado nesta dissertação, em que se enfoca, como sistemas modelo, álcoois primários de cadeia curta e se recorre a sistema em fluxo designado de EC-CE-C4D, que consiste de uma célula eletroquímica acoplada com equipamento de eletroforese capilar provido de detector de condutividade sem contato direto com os eletrodos. O sistema EC-CE-C4D, inicialmente concebido para efetuar pré-concentração e redissolução eletroquímica de metais seguida de separação eletroforética, possibilitou também o monitoramento de produtos com carga, formados em processos eletrocatalíticos, fato que inspirou a investigação da aplicabilidade, também do sistema à derivatização eletroquímica de analitos neutros em iônicos ou ionizáveis. Inicialmente, a oxidação de etanol foi avaliada por voltametria cíclica em eletrodos de ouro, paládio e platina policristalinos, nos meios ácido, neutro e alcalino. Maior formação de ácido acético se deu sobre platina em meio ácido (pH = 2,3). Entre os tampões avaliados por simulação (PeakMaster 5.1) para a separação dos carboxilatos, o TRIS/HCl (pH = 8,6) foi favorável em termos de resolução, rapidez e sensibilidade. Após a eletrooxidação da amostra sobre Pt a 1,6 V vs. Ag/AgCl por 60 s seguida de transferência de nanolitros dos produtos formados para o capilar por injeção hidrodinâmica, o meio ácido é trocado pelo tampão de corrida com auxílio de microbombas controladas por computador. A eficiência da conversão eletroquímica dos álcoois em carboxilatos, de 16% para etanol e propanol nas condições adotadas, apesar de não muito eficiente, proporcionou boa repetibilidade e limites de quantificação ao redor de 5 10-5 mol L-1 para etanol, 1-propanol, 1-butanol e 1-pentanol, ou seja, cerca de 20 vezes melhores que os reportados para MEKC. As curvas analíticas para os 4 álcoois apresentaram boa linearidade (R > 0,996) e a boa separação entre os picos permitindo determinações quantitativas simultâneas. Como exemplo de aplicação a amostras reais, demonstrou-se a determinação quantitativa de etanol em cerveja Pilsen convencional (teor alcoólico 4,5% vol.) e sem álcool (teor alcoólico <0,5% vol.), diluídas de 1000 vezes e 100 vezes, respectivamente. A repetibilidade das análises foi muito boa (~6% DPR, n = 40), indicando a estabilidade do sistema automático e ausência de envenenamento da superfície do eletrodo. Resultados ainda melhores foram obtidos utilizando 1-propanol como padrão interno para a determinação de etanol. Comparação dos resultados da dosagem alcoólica obtida com o sistema EC-CE-C4D e por CG-FID não apresentou diferença significativa utilizando um intervalo de confiança de 95 %, demonstrando a efetividade e concordância da nova combinação de técnicas. Este trabalho pioneiro, aliando derivatização eletroquímica com a eletroforese para a quantificação de espécies neutras, abre perspectivas de pesquisa não só para aprofundamento e otimização da análise de álcoois, como para a derivatização de outras espécies neutras na faixa de pH comum em CE, como aldeídos e açúcares. / Capillary electrophoresis (CE) is a powerful separation technique which exploits the differences in the mobility of ionic species under the effect of electric field. However, it does not allow the separation of mixtures of neutral molecules, and alternatives like formation of charged complexes, chemical derivatization and micellar electrokinetic chromatography (MEKC) have been proposed. Electrochemical derivatization has been combined before with HPLC, among other techniques, but not yet with CE and for quantitative purposes, as proposed and demonstrated in this Masters Thesis, focusing as model systems, short chain primary alcohols, using a flow system, designated EC-EC-C4D, composed of an electrochemical cell coupled with a capillary electrophoresis equipment provided with conductivity detector without direct contact with the electrodes. The EC-CE-C4D system, originally proposed to perform electrochemical pre-concentration and stripping of metals followed by electrophoretic separation, also enabled the monitoring of charged products formed in electrocatalytic processes, inspiring the application of the system to electrochemical derivatization of uncharged analytes into ionic or ionizable ones. Initially, the ethanol oxidation was evaluated by cyclic voltammetry on gold, palladium and platinum polycrystalline electrodes, in acid, neutral and alkaline medium. Larger formation of acetic acid was observed on platinum in acid medium (pH = 2.3). Among the buffers evaluated by simulation (PeakMaster 5.1) for separation of carboxylates, aiming to compromise between resolution, speed and sensitivity, TRIS / HCl (pH 8.6) was favorable. After the sample electrooxidation on Pt at 1.6 V vs. Ag/AgCl for 60 s is replaced automatically (by computer controlled micropumps) by running buffer, followed by the hydrodynamic injection into the capillary of products formed, the acid medium is replaced by running buffer with help of computer controlled micropumps. The efficiency of the electrochemical conversion of alcohols into carboxylates, 16% for ethanol and propanol at the given conditions, although not very efficient, but showed good repeatability and rendered quantification limits around 5 10-5 mol L-1 for ethanol, 1-propanol, 1-butanol and 1-pentanol the 4 alcohols, about 20 times better for MEKC. The analytical curves for the four alcohols, showed good linearity (r > 0.996) and the separation between peaks allowed simultaneous quantitative determinations. As an example of application to real samples, the quantitative determination of ethanol in Pilsen conventional beer (alcohol content 4.5% vol.) and non-alcoholic beer (alcohol content <0.5% vol.) was carried out after 1000-fold and 100 fold dilution, respectively. The repeatability of the analysis was very good (~6% RSD, n = 40), indicating the stability of the system and no poisoning of the electrode. Even better results were obtained using 1-propanol as an internal standard for the determination of ethanol. Comparison of results obtained with the CE-CE-C4D system and by GC-FID showed no significant difference for a confidence interval of 95%, demonstrating the effectiveness and consistency of the new combination of techniques. This proof of concept work of the combination of electrochemical derivatization with electrophoresis for quantification of neutral species opens perspectives not only for the optimization of the analysis of alcohols, but for research on the derivatization of other neutral species in the pH range typical for CE, like aldehydes and sugars.

A apramicina é um antibiótico aminoglicosídeo produzido por uma cepa de Streptomyces Tenebrarius, utilizada na medicina veterinária na forma de sulfato para tratar e prevenir doenças infecciosas produzidas por bactérias gram-negativas em porcos, bezerros e aves domésticas. As preparações comercialmente disponíveis são injetável, premix para adição em rações e pó oral solúvel, sendo esta última objeto de estudo deste trabalho. A Farmacopéia Britânica é o único compêndio oficial que descreve a apramicina (tanto a matéria-prima quanto as preparações) e recomenda o método microbiológico turbidimétrico para o seu doseamento. Embora os métodos microbiológicos ainda sejam os métodos de escolha na determinação da potência de antibióticos, há uma tendência crescente em substituí-los pelos métodos físico-químicos, sobretudo a cromatografia líquida de alta eficiência (CLAE), a qual proporciona sensibilidade, especificidade, exatidão e precisão; ela já vem sendo usada com sucesso na determinação do teor de antibióticos, embora a sua potência possa estar sujeita a confirmação por um método microbiológico. Considerando-se as vantagens da CLAE, bem como a inexistência de métodos físico-químicos oficiais para a análise de apramicina, o objetivo do presente trabalho foi desenvolver e validar um método empregando a cromatografia líquida de alta eficiência para a quantificação de apramicina em pó oral solúvel, bem como correlacioná-lo com o método turbidimétrico. Foi desenvolvido um método por CLAE empregando fase reversa com o uso de uma coluna C18 de 150 mm x 4,6 mm x 4 &#181;m, derivatização pré-coluna com o-ftalaldeído (OPA) e detecção por UV em 332 nm. A metodologia proposta foi validada de acordo com normas dos compêndios oficiais e apresentou: boa robustez; linearidade no intervalo de 0,02 a 0,05 mg/mL com obtenção de uma curva de calibração de equação igual a y = 132887304,980x + 127223,837 e coeficiente de correlação linear de 0,9999; boa precisão, sendo o DPR<1,0% para a precisão intra-dia e DPR = 1,1% para a precisão inter-dia; 99,33% de recuperação média, demonstrando exatidão; sensibilidade, com limites de detecção e de quantificação de 0,08 e 0,25 &#181;g/mL, respectivamente. Três lotes de apramicina pó oral solúvel foram analisados pelo método por CLAE e pelo método microbiológico oficial e a comparação estatística entre os resultados obtidos através do Teste T demonstrou que não houve diferença significativa entre ambos no nível de &#945; = 0,05. / Apramycin is an aminoglycoside antibiotic produced by Streptomyces tenebrarius strain, widely used in veterinary medicine under sulfate form to treat and prevent infectious diseases induced by gram-negative bacteria in swines, calves and poultry. The commercially available preparations consist of injections, additive feed (premix) and soluble oral powder, being the latter the aim of this work. British Pharmacopoeia is the only official compendium that describes apramycin (bulk pharmaceutical and its formulations) recommending the microbiological turbidimetric method for its dosage. Although microbiological methods are still of chosen for the determination of antibiotics potency, there is an increasing tendency of replacing them by physico-chemical methods, mainly high performance liquid chromatography (HPLC), which offers sensitivity, specificity, accuracy, and precision; it has been successfully used in the determination of antibiotic assay, although its potency may be subjected to confirmation by a microbiological method. Taking into account the advantages of HPLC as well as the absence of official physicochemical methods to analyze apramycin, the aim of the present work was to develop and validate a method employing high performance liquid chromatography to the quantification of soluble oral powder, as well as to compare it with the turbidimetric method. A HPLC method was developed employing reverse phase with a C18 column of 150 mm x 4,6 mm x 4 &#181;m, pre-column derivatization with OPA (o-phthalaldehyde) and UV detection at 332 nm. The proposed method was validated according to official compendia guidelines, having demonstrated: good robustness; linearity in the concentration range of 0,02 to 0,05 mg/mL with linear equation of y = 132887304,980x + 127223,837 and correlation coeficient of 0,999; good precision, being RSD<1,0% for intra-day precision and = 1,1% for inter-day precision; average recuperation of 99,33%, what demonstrates accuracy; sensitivity, with quantification and detection limits of 0,08 and 0,25 &#181;L/mL, respectively. Three batches of apramycin soluble oral powder were analyzed by HPLC method and by the official microbiological method having the statistical comparison between the obtained results by T test demonstrated that there are no significant differences between both of them at &#945; = 0,05 level.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Amino acids are organic compounds abundant in animal tissues, especially in meat and meat products, in free form or polymers, participating in biochemical reactions and may be precursors of compounds of interest in the quality control of these foods. The aim of this study was to optimize and validate a method for determination of free amino acids by gas chromatography in Italian dry fermented sausage subjected to ultrasonic bath for 3, 6 and 9 min. Free amino acids were evaluated in salami pieces fermented/seasoned (initial time), at finished product (28 days) and 120 days after manufacture. The extraction was performed using 0.1 N HCl. An aliquot of samples were subjected to vacuum drying, followed by derivatization with MTBSTFA, which has been optimized with respect to time, temperature and reaction solvents (pyridine and acetonitrile). The instrumental and chromatographic conditions of injector temperature, constant pressure and column temperature program was optimized to ensure the method selectivity and efficiency. The use of a mixture of acetonitrile/MTBSTFA in the ratio 1:1 (v/v) using a temperature of 100 °C for 2 h promoted derivatizing all L-amino acids contained in the analytical standards.The injector temperature of 320 °C and 15 psi constant pressure gave the best efficiency in mass transfer of the analytes into the column, while also providing higher resolution and accuracy. The determination coefficients obtained for amino acids were higher than 0.99, except for glycine (0.98). Fourteen amino acids showed a linear range of from 1.0 to 160.0 mg L-1 with LOD and LOQ ranged between 0.3 and 10 g ml-1 and 1 and 20 mg L-1 and inter-day coefficients of variation accurately less than 15% for most analytes.The increase in the concentrations of most amino acids was observed over time processing/storage, indicating that there was an intense proteolytic activity with the release of free amino acids. In the finished product (28 days), treatment with ultrasound affected significantly (p <0.05) concentration of some amino acids. At this point, the amino acids alanine, glycine, valine, leucine, proline and methionine (non-polar) and tyrosine (polar uncharged) showed lower concentrations to control, on the other hand there was an increase of glutamic acid and arginine. The free amino acids found in salami are important compounds because through chemical reactions of decarboxylation or deamination can be precursors of volatile compounds responsible for characteristic processed meat product flavor/aroma. Thus, the employment of no conventional analytical techniques such as gas chromatography for determination of free amino acids allows obtaining information of great importance for the quality control and product development, correlating the technological and nutritional properties with the properties sensory involving amino acids as precursors. / Os aminoácidos são compostos orgânicos abundantes em tecidos animais, sobretudo em carne e produtos cárneos, na forma livre ou de polímeros, participando de reações bioquímicas, podendo ser precursores de compostos de interesse no controle de qualidade destes alimentos. O objetivo do presente trabalho foi otimizar e validar um método para determinação de aminoácidos livres por cromatografia em fase gasosa em salame tipo Italiano submetido a banho ultrassônico por 3, 6 e 9 min. Os aminoácidos livres foram avaliados na massa de salame não fermentada/maturada (tempo inicial), no produto acabado (28 dias) e após 120 dias da fabricação. A extração dos analitos foi realizada com a utilização de HCl 0,1 N. Uma alíquota das amostras foram submetidas à secagem a vácuo seguida de derivatização com MTBSTFA, a qual foi otimizada em relação ao tempo, temperatura e solventes de reação (piridina e acetonitrila). As condições instrumentais e cromatográficas de temperatura do injetor, pressão constante e programação de temperatura da coluna foram otimizadas para garantir a seletividade e eficiência do método. O emprego da mistura de acetonitrila/MTBSTFA na proporção de 1:1 (v/v), utilizando a temperatura de 100 °C por 2 h promoveu a derivatização de todos os L- aminoácidos contidos nos padrões analíticos. A temperatura do injetor a 320 °C e pressão constante de 15 psi proporcionou a melhor na eficiência na transferência de massa dos analitos para coluna, proporcionando também maior resolução e precisão. Os coeficientes de determinação obtidos para os aminoácidos foram maiores que 0,99, com exceção da glicina (0,98). Quatorze aminoácidos apresentaram faixa linear entre 1,0 160,0 μg mL-1, com LOD e LOQ variando entre 0,3 e 10 μg mL-1 e 1 e 20 μg mL-1 e precisão intra-dia com coeficientes de variação menores que 15% para a maioria dos analitos. O aumento nas concentrações da maioria dos aminoácidos foi observado com decorrer do tempo do processamento/armazenamento, indicando que houve uma intensa atividade proteolítica com liberação de aminoácidos livres. No produto pronto (28 dias), os tratamentos com ultrassom afetaram significativa (p<0,05) a concentração de alguns aminoácidos. Nesse ponto, os aminoácidos alanina, glicina, valina, leucina, prolina e metionina (apolares) e tirosina (polar sem carga) apresentaram concentrações inferiores ao controle, por outro lado foi observado um aumento dos de ácido glutâmico e arginina. Os aminoácidos livres encontrados nas amostras de salame são compostos importantes, pois através de reações químicas de descarboxilação ou desaminação podem ser precursores de compostos voláteis responsáveis pelo sabor/aroma característicos do produto cárneo processado. Dessa forma, o emprego de técnicas analíticas não convencionais, como a cromatografia em fase gasosa, para a determinação de aminoácidos livres, permite a obtenção de informações de grande importância no controle de qualidade e desenvolvimento de produtos, correlacionando as propriedades tecnológicas e nutricionais com as propriedades sensoriais que envolvem os aminoácidos como precursores.

Phospholipids are an important group of polar lipids constituting the main component of cell membranes. Their proportion may vary depending on many factors of the surrounding environment in which the cell is located. Determination of membrane phospholipids is essential in many scientific, industrial and economic sectors. The aim of this work has been to develop a separation method for determination of membrane phospholipids by which it was possible to analyze phosphorylated parts of phospholipids and fatty acids from one sample. Comprehensive gas chromatography with mass detection (GC×GC-MS) was chosen for the assay. This method allows the separation of the entire sample on two serially connected different columns, among which is the interface called modulator. The preparation of the sample includes a cleavage of the phospholipid molecule by the enzyme phospholipase C, which released the phosphorylated polar headgroups. These polar parts had to be derivatized before analysis. The principle of the chosen derivatization consisted in the use of two different silylation agents (hexamethyldisilazane and N,O-Bis(trimethylsilyl) trifluoracetamide) in two steps. Conditions were selected for efficient separation of the silylderivatives of phosphorylated headgroups using GC×GC-MS using a cryogenic...

Estrogens, alkylphenols and bisphenol-A, enter the environment through waste water systems and waste disposal of manufactured products e.g. detergents, paints, polycarbonates and flameretardants. These analytes disrupt the endocrine function of living organisms affecting their reproductive health and those of future generations. Gas phase low molecular- mass aldehydes and amines are typically eye, nose, and throat irritants. Formaldehyde is classified as a probable human carcinogen. Given their negative impact on human health it is urgent to monitor pollutants at extremely low levels in both air and water. The aqueous pollutants are often concentrated using solid phase extraction cartridges or liquid-liquid extraction followed by derivatization. Methods that can most effectively and selectively pre-concentrate aldehydes and amines involve in situ derivatization. Unfortunately, the derivatizing reagents as well as their associated solvents or adsorbents, are responsible for problems encountered with these methods. Polydimethylsiloxane (PDMS) has emerged as the ideal concentration and reaction medium for trace analysis. However the expensive commercial devices such as SPME and SBSE both require the samples to be returned to the laboratory for concentration. Due to the open tubular nature of the PDMS multichannel trap (MCT), developed in our laboratory, it is ideally suited for on-site and online sampling. The MCTs have a high analyte capacity owing to the large volume of PDMS available for concentration. The derivatization reaction can be performed in situ providing a “onepot concentration and reaction device”. This allows for reduced risk of contamination of / or losses of the sample and a sampling method that can cater for both air and water samples. To demonstrate the versatility of the PDMS MCT, two approaches for concentration in PDMS were investigated in this study, namely, 1) the on-line concentration and in situ derivatization of volatile polar analytes from air followed by REMPI-TOFMS detection, and 2) the concentration of phenolic lipophilic analytes from water requiring derivatization prior to analysis by GC/MS. 1) Analyte and derivatizing reagent were simultaneously introduced into the PDMS trap using a ypress- fit connector. The reaction occurs in situ followed by thermal desorption using a thermal modulator array alone or in conjunction with a thermal desorption unit. The aldehydes and amine derivatives were successfully detected by the REMPI-TOFMS. Reaction efficiencies were determined at room temperature without catalysts. Formaldehyde yielded a low reaction/concentration efficiency of 41 % with phenylhydrazine in PDMS, while acetaldehyde, acrolein and crotonal displayed much improved values of 92, 61 and 74 % respectively. Both propylamine and butylamine yielded 28 % reaction/concentration efficiency with benzaldehyde in the PDMS matrix. Detection limits obtained with this technique were significantly lower than the permissible exposure limits set by the Occupational Safety and Health Administration. It should be noted that the detection limits were not determined by actual measurement but by extrapolation from a larger signal. 2) Aqueous analytes were concentrated in the PDMS MCT using a gravity flow rate of ~50 ìl/min. The trap was dried and 5 ìl derivatizing reagent added. At room temperature and without the presence of a catalyst, the reaction of alkylphenols with trifluoroacetic acid anhydride in the PDMS matrix was 100% complete after 5 minutes. Bisphenol-A reacted less than 50 % to completion during this period, but the amount of derivative formed remained constant. This study revealed that extraction efficiencies of the alkylphenols and bisphenol-A off the PDMS trap have poor batch-tobatch repeatability indicating that the PDMS matrix was not homogenous. For two different PDMS batches: tert-octylphenol displayed an extraction efficiency of 70 and 79%, nonylphenol displayed 84 and 43% while Bisphenol-A displayed 10 and 26% respectively. The thermally desorbed derivatives were analysed by GC/MS. Despite background contamination in the desorption unit, detection limits were at the ppt level. Detection limits were not determined by actual measurement but by extrapolation from a larger signal. / Thesis (PhD)--University of Pretoria, 2009. / Chemistry / unrestricted

Diploma thesis is focused on the choose of methods and optimalisation analysis procedure of selected pharmaceuticals (salicylic acid, ibuprofene, naproxene, ketoprofene and diclofenac) in surface and waste water by using gas chromatography with mass spectrometer (GC-MS). Solid-phase extraction (SPE) with Oasis HLB cartridges was used as an extraction method in this analysis. Extraction is followed by derivatization and their optimalization of selected pharmaceuticals. Derivatization and its optimalization were performed by two silylation reagens N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA). Different volumes of derivatization reagents, different temperatures and different times were used during the procedure. The best combination is then used for analysis of real samples. Real samples of waste water were collected in sewage treatment plants in Brno – Modřice and real samples of surface water were collected from several rivers and one pond in region Moravia. This thesis also presents and tests SPE methods for extraction and concentration selected sulfonamide residues (sulfamethazine, sulfamethoxazole, sulfapyridine and sulfathiazole) from the aquatic environment.

This master thesis deals with the potential influence of the waste dump Hrádek u Pacova and the wastewater treatment plant Brno – Modřice on surroundings using the optimized non-target screening method of organic pollutants in water and sediments. The experimental part is focused on the identification of organic compounds in samples of ground water from monitoring wells, the surface water from stream and pond and in sediment samples collected from streams in the vicinity of municipal waste dump. Organic compounds were also identified in water and sediment samples from the river Svratka near the municipal waste water treatment plant Modřice. The sequential liquid – liquid extraction was used for the isolation of organic compounds in water samples. Organic compounds in sediment samples were isolated by pressurized solvent extraction (PSE) with subsequent fractionation of the extract using column chromatography. Final analysis determination was performed using comprehensive orthogonal two-dimensional gas chromatography with mass spectrometric detection (GCxGC-TOF MS).

Mass spectrometry with electrospray ionization is an excellent method for structural analysis of coordination compounds with outstanding sensitivity and selectivity. However, it fails to detect some low-polar rhenium complexes. This master thesis describes derivatization method of non-ionizable rhenium complexes with 1,2-dihydroxybenzene and 2,3- dihydroxytoluenene. Fragmentation mechanisms and structure of prepared complexes was studied using high resolution mass spectrometry and collision-induced dissociation (CID). Furthermore, density functional theory (DFT) computational method was used for prediction of bond cleavage based on bond lengthening.