Este trabalho descreve o desenvolvimento de novos métodos cromatográficos em conjunto com técnicas de microextração para determinação de parabenos em produtos cosméticos e leite humano e rifampicina em amostras de plasma. Destaca-se também o desenvolvimento da fase extratora molecularmente impressa para extração em fase sólida (SPE) de parabenos em amostras de leite humano. O capítulo I descreve a avaliação da extração sortiva em barra de agitação (SBSE) em conjunto com a cromatografia líquida (LC) com detecção espectrofotométrica (LC-UV) para análises de parabenos em produtos cosméticos para fins de controle de qualidade. As variáveis do processo de extração SBSE, tais como tempo e temperatura de extração, pH e força iônica da amostra e condições de dessorção foram otimizadas para aumentar a sensibilidade analítica do método e diminuir o tempo de análise. O método SBSE/LC-UV desenvolvido apresentou faixa linear correspondente ao limite de quantificação (LQ) a 2,50 µg. mg-1 e coeficientes de determinação maiores que 0,993. Os valores de coeficiente de variação resultantes das análises de precisão interensaios foram 5,0% para todos os parabenos analisados e a exatidão variou de 90 a 100%. O método desenvolvido foi aplicado para análises de amostras de produtos cosméticos comerciais (cremes hidratantes, antitranspirantes em creme e filtros solares). O método SBSE/LC-UV padronizado e validado resultou nas seguintes vantagens em relação aos métodos convencionais: simplicidade no preparo de amostra, redução do volume de solvente orgânico e da quantidade de amostra utilizada. O capítulo II descreve o desenvolvimento da fase extratora molecularmente impressa para SPE de parabenos em amostras de leite humano e análises por cromatografia em fase líquida com detecção espectrofotométrica (SPE/LC-UV). Os polímeros de impressão molecular, materiais sintéticos baseados em sistemas biomiméticos, foram sintetizados através do processo sol-gel tendo como molde, o benzilparabeno. Após a remoção do molde, cavidades seletivas (sítios de reconhecimento molecular) ao benzilparabeno e análogos estruturais foram reveladas. O método SPE/LC desenvolvido apresentou faixa linear correspondente ao limite de quantificação a 150 ng. mL-1 e coeficientes de determinação 0,992. Os valores de coeficiente de variação resultantes das análises de precisão interensaios foram 13% e a exatidão variou de 86 a 117%. A aplicabilidade do método SPE/LC foi demonstrada através das análises de amostras de leite humano de lactantes. Segundo os parâmetros de validação analítica avaliados, o método padronizado SPE/LC-UV é adequado para análises de parabenos em amostras de leite humano. O capítulo III descreve a avaliação da microextraçao em fase sólida no capilar (in-tube SPME) acoplada à cromatografia líquida (LC) com detecção espectrofotométrica (LC-UV) para análises de rifampicina em amostras de plasma para fins de monitorização terapêutica. As variáveis in-tube SPME, tais como fase estacionária da coluna capilar, volume de amostra, ciclos aspirar/dispensar e vazão foram otimizadas visando obter maior eficiência do processo. O método in-tube SPME/LC desenvolvido apresentou faixa linear correspondente ao limite de quantificação (0,1 µg. mL-1) a 100 µg. mL-1 e coeficientes de determinação igual a 0,998. Os valores de coeficiente de variação resultantes das análises de precisão interensaios foram 2% e a exatidão variou de 83 a 92%. A aplicabilidade do método in-tube SPME/LC, padronizado e validado foi comprovada através das análises de amostras de plasma de pacientes em tratamento com rifampicina. O método in-tube SPME/LC resultou na automação do processo de análise, maior precisão analítica, menor tempo de análise, menor volume de solvente orgânico e de fluido biológico. / This work describes the development of chromatographic methods in combination with microextraction techniques for the determination of parabens in cosmetic and human milk samples and of rifampicin in plasma samples. The design of a molecularly imprinted extraction phase for the solid-phase extraction (SPE) analysis of parabens in human milk samples is also reported. Chapter I describes the evaluation of the stir bar sorptive extraction (SBSE) in combination with liquid chromatography (LC) with UV detection (LC-UV) for the analysis of parabens in cosmetics aiming at quality control. The SBSE variables such as extraction time, temperature, pH of the matrix, ionic strength, and desorption conditions have been optimized, in order to establish the parabens partition equilibrium in a short analysis time. The linear range of the SBSE/LC method lay from LOQ to 2.5 µg. mg-1, with a coefficient of determination higher than 0.993. The interday precision of the SBSE/LC method presented a coefficient of variation lower than 5%, and the accuracy ranged from 90 to 99%. The effectiveness of the proposed method for the analysis of commercial cosmetic products such as body creams, antiperspirant creams, and sunscreens has been proven. Therefore, the combination of SBSE with liquid desorption, followed by LC analysis, provides a simple, and selective tool for the analysis of parabens in cosmetic products using minimized volume of organic solvent as well as very small amount of the target sample. Chapter II presents the development of a molecularly imprinted extraction phase for the SPE analysis of parabens in human milk samples. The molecularly imprinted polymers, which are synthetic materials based on biomimetic systems, were synthesized by the sol-gel technology in the presence of the template (benzilparaben). After template removal, selective cavities (benzilparaben) were revealed for parabens selective extraction. The linear range of the SPE/LC method lay from LOQ to 150 ng. mL-1, with a coefficient of determination higher than 0.992. The interday precision of the SPE/LC method presented a coefficient of variation lower than 13%, and the accuracy ranged from 86 to 117%. The effectiveness of the proposed method for the analysis of human milk samples has been demonstrated. According to the evaluated analytical validation parameters, the SPE/LC-UV method is suitable for the analysis of parabens in human milk samples. Chapter III depicts the evaluation of solid-phase microextraction in the capillary (in-tube SPME) in combination with liquid chromatography (LC) with UV detection (LC-UV) for the analysis of rifampicin in plasma samples for therapeutic drug monitoring. The in-tube SPME/LC method was linear over the LOQ (0.1) to 100 g. mL-1 range, with a linear coefficient value (r2) of 0.998. The inter-assay precision presented coefficient of variation 2%, and the accuracy ranged from 83 to 92%. The effectiveness and practicability of the proposed method have been by analysis of plasma samples from ageing patients undergoing therapy with rifampicin. The in-tube SPME/LC method allowed for automated continuous sample preparation (extraction, concentration, desorption, and injection of analytes) and minimized the analysis time as well as the volumes of organic solvent and biological fluid.

Orientador: Susanne Rath / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-10T22:20:57Z (GMT). No. of bitstreams: 1 Pereira_LeandroAlves_M.pdf: 1182437 bytes, checksum: 9e5bb63e48650f93fbbd8632bfc95f93 (MD5) Previous issue date: 2008 / Resumo: Resíduos de agrotóxicos presentes em alimentos podem exceder os limites máximos de resíduos, quando as boas práticas agrícolas não são respeitadas. Conseqüentemente, a determinação dos resíduos de agrotóxicos é extremamente importante para avaliar o uso apropriado dessas substâncias e os riscos à saúde humana decorrentes da sua presença nos alimentos. A determinação de resíduos de agrotóxicos em alimentos requer etapas de preparo de amostras que consistem, geralmente de etapas de extração, remoção de interferentes e concentração do analito anterior análise cromatográfica. Para tanto, tem sido largamente empregada a extração em fase sólida. O objetivo do presente trabalho foi a síntese e caracterização de polímeros de impressão molecular (MIP) para uso em cartuchos de extração em fase sólida, visando a determinação de fenitrotiona (FNT) em tomates. O polímero foi sintetizado a partir do ácido metacrílico (monômero funcional), etileno glicoldimetilacrilato (reagente de ligação cruzada), 2'2azo-bis-iso-butironitrila (iniciador radicalar) em diclorometano (solvente porogênico), usando a FNT como molécula molde. Também foi sintetizado um polímero de controle (NIP). O MIP foi caracterizado por técnicas de infravermelho de transformada de Fourier, ressonância magnética nuclear do estado sólido, microscopia de varredura eletrônica e porosimetria de sorção de nitrogênio. O modelo de Langmuir-Freundlich foi o que apresentou o melhor ajuste para descrever a adsorção da FNT no polímero. O MIP foi empregado em cartuchos de extração em fase sólida e as etapas de condicionamento, carregamento, lavagem e eluição otimizadas. A extração em fase sólida molecularmente impressa foi aplicada na determinação de FNT em amostras de tomates, usando a cromatografia líquida de alta eficiência associada a um detetor de arranjo de diodos. A eficiência de extração, para amostras branco de tomates fortificadas com 5mg g de FNT, foi de 65 %. Nenhum interferente foi observado nos cromatogramas no tempo de retenção da FNT, indicando a seletividade do polímero / Abstract: Pesticide residues present in food can exceed maximum residue level when good agricultural practices are not followed. Consequently, the determination of pesticide residues is extremely important to evaluate the appropriate use of theses substances and risks to human health due to their presence in food. The determination of pesticide residues in food requires sample preparation steps that consist mainly of extraction, clean-up, and concentration of the analyte prior to chromatographic quantitation.. For this purpose, solid-phase extraction has been widely employed. The aims of this work were the synthesis and characterization of a molecularly imprinted polymer (MIP) to be employed as a sorbent in solid phase extraction cartridges for the determination of fenitrothiom (FNT) in tomatoes. The polymer was synthesized using FNT as the template molecule, methacrylic acid as the functional monomer, ethylene glycol dimethacrylate as the cross-linking monomer, and 2,2-azobis-isobutyronitrile as the initiator. Dichloromethane was used as the porogenic solvent. Also, a non-imprinted polymer was synthesized. The MIP was characterized using the following techniques: FT-IR, NMR, electronic microscopy and BET. The adsorption of FNT on the polymer was fitted to the Langmuir-Freundlich model. The polymer was used in the solid-phase extraction cartridges and the conditioning, sample loading, clean-up and elution steps were optimized. The molecularly imprinted solid phase extraction was used for the determination of FNT in tomatoes, using high performance liquid chromatography with a diode array detector. For extraction efficiency, for a fortified blank tomato sample (5 mg g FNT), was 65 %. No interferences were observed in the chromatograms at the retention time of FNT, indicating the selectivity of the polymer / Mestrado / Quimica Analitica / Mestre em Química

Este trabalho descreve o desenvolvimento de novos métodos cromatográficos em conjunto com técnicas de microextração para determinação de parabenos em produtos cosméticos e leite humano e rifampicina em amostras de plasma. Destaca-se também o desenvolvimento da fase extratora molecularmente impressa para extração em fase sólida (SPE) de parabenos em amostras de leite humano. O capítulo I descreve a avaliação da extração sortiva em barra de agitação (SBSE) em conjunto com a cromatografia líquida (LC) com detecção espectrofotométrica (LC-UV) para análises de parabenos em produtos cosméticos para fins de controle de qualidade. As variáveis do processo de extração SBSE, tais como tempo e temperatura de extração, pH e força iônica da amostra e condições de dessorção foram otimizadas para aumentar a sensibilidade analítica do método e diminuir o tempo de análise. O método SBSE/LC-UV desenvolvido apresentou faixa linear correspondente ao limite de quantificação (LQ) a 2,50 µg. mg-1 e coeficientes de determinação maiores que 0,993. Os valores de coeficiente de variação resultantes das análises de precisão interensaios foram 5,0% para todos os parabenos analisados e a exatidão variou de 90 a 100%. O método desenvolvido foi aplicado para análises de amostras de produtos cosméticos comerciais (cremes hidratantes, antitranspirantes em creme e filtros solares). O método SBSE/LC-UV padronizado e validado resultou nas seguintes vantagens em relação aos métodos convencionais: simplicidade no preparo de amostra, redução do volume de solvente orgânico e da quantidade de amostra utilizada. O capítulo II descreve o desenvolvimento da fase extratora molecularmente impressa para SPE de parabenos em amostras de leite humano e análises por cromatografia em fase líquida com detecção espectrofotométrica (SPE/LC-UV). Os polímeros de impressão molecular, materiais sintéticos baseados em sistemas biomiméticos, foram sintetizados através do processo sol-gel tendo como molde, o benzilparabeno. Após a remoção do molde, cavidades seletivas (sítios de reconhecimento molecular) ao benzilparabeno e análogos estruturais foram reveladas. O método SPE/LC desenvolvido apresentou faixa linear correspondente ao limite de quantificação a 150 ng. mL-1 e coeficientes de determinação 0,992. Os valores de coeficiente de variação resultantes das análises de precisão interensaios foram 13% e a exatidão variou de 86 a 117%. A aplicabilidade do método SPE/LC foi demonstrada através das análises de amostras de leite humano de lactantes. Segundo os parâmetros de validação analítica avaliados, o método padronizado SPE/LC-UV é adequado para análises de parabenos em amostras de leite humano. O capítulo III descreve a avaliação da microextraçao em fase sólida no capilar (in-tube SPME) acoplada à cromatografia líquida (LC) com detecção espectrofotométrica (LC-UV) para análises de rifampicina em amostras de plasma para fins de monitorização terapêutica. As variáveis in-tube SPME, tais como fase estacionária da coluna capilar, volume de amostra, ciclos aspirar/dispensar e vazão foram otimizadas visando obter maior eficiência do processo. O método in-tube SPME/LC desenvolvido apresentou faixa linear correspondente ao limite de quantificação (0,1 µg. mL-1) a 100 µg. mL-1 e coeficientes de determinação igual a 0,998. Os valores de coeficiente de variação resultantes das análises de precisão interensaios foram 2% e a exatidão variou de 83 a 92%. A aplicabilidade do método in-tube SPME/LC, padronizado e validado foi comprovada através das análises de amostras de plasma de pacientes em tratamento com rifampicina. O método in-tube SPME/LC resultou na automação do processo de análise, maior precisão analítica, menor tempo de análise, menor volume de solvente orgânico e de fluido biológico. / This work describes the development of chromatographic methods in combination with microextraction techniques for the determination of parabens in cosmetic and human milk samples and of rifampicin in plasma samples. The design of a molecularly imprinted extraction phase for the solid-phase extraction (SPE) analysis of parabens in human milk samples is also reported. Chapter I describes the evaluation of the stir bar sorptive extraction (SBSE) in combination with liquid chromatography (LC) with UV detection (LC-UV) for the analysis of parabens in cosmetics aiming at quality control. The SBSE variables such as extraction time, temperature, pH of the matrix, ionic strength, and desorption conditions have been optimized, in order to establish the parabens partition equilibrium in a short analysis time. The linear range of the SBSE/LC method lay from LOQ to 2.5 µg. mg-1, with a coefficient of determination higher than 0.993. The interday precision of the SBSE/LC method presented a coefficient of variation lower than 5%, and the accuracy ranged from 90 to 99%. The effectiveness of the proposed method for the analysis of commercial cosmetic products such as body creams, antiperspirant creams, and sunscreens has been proven. Therefore, the combination of SBSE with liquid desorption, followed by LC analysis, provides a simple, and selective tool for the analysis of parabens in cosmetic products using minimized volume of organic solvent as well as very small amount of the target sample. Chapter II presents the development of a molecularly imprinted extraction phase for the SPE analysis of parabens in human milk samples. The molecularly imprinted polymers, which are synthetic materials based on biomimetic systems, were synthesized by the sol-gel technology in the presence of the template (benzilparaben). After template removal, selective cavities (benzilparaben) were revealed for parabens selective extraction. The linear range of the SPE/LC method lay from LOQ to 150 ng. mL-1, with a coefficient of determination higher than 0.992. The interday precision of the SPE/LC method presented a coefficient of variation lower than 13%, and the accuracy ranged from 86 to 117%. The effectiveness of the proposed method for the analysis of human milk samples has been demonstrated. According to the evaluated analytical validation parameters, the SPE/LC-UV method is suitable for the analysis of parabens in human milk samples. Chapter III depicts the evaluation of solid-phase microextraction in the capillary (in-tube SPME) in combination with liquid chromatography (LC) with UV detection (LC-UV) for the analysis of rifampicin in plasma samples for therapeutic drug monitoring. The in-tube SPME/LC method was linear over the LOQ (0.1) to 100 g. mL-1 range, with a linear coefficient value (r2) of 0.998. The inter-assay precision presented coefficient of variation 2%, and the accuracy ranged from 83 to 92%. The effectiveness and practicability of the proposed method have been by analysis of plasma samples from ageing patients undergoing therapy with rifampicin. The in-tube SPME/LC method allowed for automated continuous sample preparation (extraction, concentration, desorption, and injection of analytes) and minimized the analysis time as well as the volumes of organic solvent and biological fluid.

L’utilisation croissante de pesticides dans l'agriculture peut entraîner de graves risques pour la santé humaine. En effet, des résidus de pesticides peuvent être retrouvés à l’état de trace dans de nombreux produits de grande consommation. Leur identification et leur analyse quantitative à l’état de trace dans des échantillons complexes, tel que les huiles végétales, constituent un défi analytique majeur. Malgré le potentiel élevé de méthodes analytiques comme la chromatographie en phase liquide couplée à la spectrométrie de masse (LC-MS/MS), l’introduction d’une étape d’extraction et de purification des extraits avant l’analyse chromatographique s’avère nécessaire. Afin d'augmenter la sélectivité de cette étape de traitement de l'échantillon, la synthèse des supports à empreintes moléculaires générant un mécanisme de reconnaissance moléculaire a été envisagé. Cette étude se concentre sur une famille de pesticides, les organophosphorés (OP), qui présentent des disparités structurales importantes et une gamme de polarité assez large (log P compris entre 0,7 et 4,7). Deux approches ont été envisagées pour la synthèse de ces supports imprimés. La première approche consiste à réaliser une polymérisation par voie radicalaire en utilisant des monomères organiques dans des solvants organiques peu polaires pour obtenir des polymères à empreintes moléculaires (MIP). La seconde approche consiste à produire les supports par voie sol-gel via l’hydrolyse puis la condensation d'organosilanes dans un milieu polaire pour produire des silices imprimées (MIS). Pour les deux approches, différentes conditions de synthèse ont été criblées en utilisant différentes molécules empreintes, monomères et solvants. La sélectivité des polymères imprimés résultants a d'abord été évaluée en étudiant les profils de rétention des OP en milieu pur. Les interactions non-spécifiques ont été évaluées en étudiant parallèlement la rétention des OP sur des supports non-imprimés (NIP/NIS) qui ont été synthétisés dans les mêmes conditions que les MIP/MIS mais sans introduire la molécule empreinte. Il est apparu que les supports MIP/MIS présentaient une complémentarité en termes d'extraction sélective des OP visés : les OP les plus polaires ont été extraits sélectivement par le MIS alors que les OP modérément polaires ont été extraits sélectivement par le MIP. La capacité de ces supports a été évaluée et se révèle adaptée à l'analyse des OP à l’état de traces dans des huiles végétales. Après avoir vérifié la répétabilité de la procédure d'extraction et des synthèses, les performances de ces supports ont été étudiées dans des milieux réels. / The increasing use of pesticides in agriculture causes serious health risks to humans. These pesticides may possibly be found in vegetable oils used as cosmetic ingredients. Their identification and reliable quantitative analysis at trace levels constitute a challenge for the safe use of such oils despite the high potential of analytical methods such as liquid chromatography coupled to mass spectrometry (LC-MS/MS). Their determination at low concentration levels in complex oil samples requires an extraction and a purification step. In order to increase the selectivity of the sample treatment step, the synthesis of imprinted sorbents can be considered. This study focusses on a group of pesticides, the organophosphorus (OPs) that present some structural disparity and belong to a wide range of polarity (log P values between 0.7 and 4.7). To produce imprinted sorbents, a first approach of synthesis consists in the radical polymerization of organic monomers in moderately polar organic solvents to obtain molecularly imprinted polymers (MIPs). The second one, the Sol-Gel approach, consists in the hydrolysis and then condensation of organosilanes in a polar medium to produce molecularly imprinted silicas (MIS). For both approaches, different conditions of synthesis were screened using different template molecules, monomers and solvents. The selectivity of the resulting imprinting polymers was first evaluated by studying the extraction profiles of OPs in pure media on MIP and MIS. The non-specific interactions were estimated by studying in parallel the retention of OPs on non-imprinted polymers synthesized in the same conditions as imprinted sorbents but in the absence of the template molecule. Both sorbents MIP/MIS present a complementarity in terms of selective extraction of the target OPs: polar OPs were extracted selectively using the MIS while moderately polar OPs were selectively extracted by the MIP. The capacity of these supports was evaluated and was consistent with the analysis of OPs at trace levels in real oil samples. After studying the repeatability of the extraction procedure and of the reliability of the syntheses, the performances of these supports were studied in real media. For this, MIP/MIS were applied to the selective extraction of OPs from different vegetable oils (almond, olive and sunflower oil) and similar results were obtained for the three different oils. Their potential in terms of ability to remove matrix interfering compounds were higher than those of the conventional method based on the use of C18 silica. The estimated limits of quantifications were lower than the Maximum Residue Levels (MRLs) established by EU Regulation 396/2005 for these compounds in oils.

Les biocapteurs sont des moyens d'analyse en plein essor à la fois rapides, sélectifs et peu coûteux, applicables à des domaines très variés (environnement, santé, agroalimentaire…). La capacité de reconnaissance moléculaire extraordinaire de biomolécules telles que les enzymes ou les anticorps a été exploitée avec succès pour la réalisation de nombreux biocapteurs. Cependant, l'inconvénient majeur de ces récepteurs biologiques est qu'ils sont difficiles à produire et fragiles. Une manière de surmonter ces inconvénients consiste à les remplacer par des récepteurs artificiels présentant des propriétés de reconnaissance similaires. Parmi les matériaux biomimétiques prometteurs figurent les polymères à empreintes moléculaires (MIPs). Dans ce travail, nous nous sommes intéressés au développement de deux capteurs biomimétiques impédimétriques, le premier basé sur l'utilisation de poly(éthylène co-alcool vinylique) imprimé pour la détection de la créatinine et le deuxième sur des MIPs de polyméthacrylate pour la détection de la testostérone. Dans le premier cas, le polymère imprimé a été produit et déposé à la surface d'électrodes en or, soit par drop-coating, soit sous forme de nanofibres par la technique d'électrofilage. Dans le deuxième, le MIP a été synthétisé par polymérisation radicalaire de l'acide méthacrylique en présence d'éthylèneglycol diméthacrylate (réticulant), d'initiateur et de testostérone en utilisant la méthode du « grafting from » qui consiste à greffer d'abord l'initiateur sur la surface du transducteur mais pour la polymérisation on a utilisé deux approches (spin-coating d'une solution de prépolymérisation sur la surface du transducteur ou l'immersion de ce dernier dans la solution de monomère plus testostérone) suivie de l'exposition à une source d'energie pour effectuer la polymérisation. Les performances des capteurs (limite de détection, sélectivité, reproductibilité) ont ensuite été évaluées / Biosensors are rapid, selective and low-cost analytical devices of growing interest for a wide range of application fields (e.g. environment, food, health). The extraordinary molecular recognition capabilities of sensing biomolecules such as enzymes and antibodies have been successfully exploited in the elaboration of a number of biosensors. However, these biorecognition elements are often produced via complex and costful protocols and require specific handling conditions because of their poor stability. To circumvent these limitations, artificial receptors of similar recognition properties are now proposed as alternatives to natural receptors in sensor technology. Molecular imprinted polymers are among the most promising biomimetic materials reported. In this work, we developed two impedimetric biomimetic sensors. The first one is based on imprinted poly(ethylene co-vinyl alcohol) for creatinine detection and the second on polymethacrylate MIPs for testosterone analysis. In the first case, MIP was produced and deposited onto gold microelectrodes, either by spin-coating of a pre-polymerization solution, or by electrospinning. In the second case, MIPs were synthetized by photopolymerization of methacrylic acid in presence of ethyleneglycoldimethacrylate (cross-linker), an azo-initiator and testosterone as template using the “grafting from” method in which the initiator is first attached to the transducer surface but to effect polymerization we used two different approaches (dip-coating of a prepolymerization solution on the transducer surface functionalized with the initiator or immersing it in the solution of monomers and testosterone) followed by exposure to an energy source to effect polymerization. Then, analytical performances (linear range, detection limit, selectivity and reproducibility) of both creatinine and testosterone sensors were determined and compared