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Made available in DSpace on 2014-06-12T16:30:54Z (GMT). No. of bitstreams: 2 arquivo5971_1.pdf: 1816878 bytes, checksum: f1018babade0b0f2c775129b5ef2b317 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2003 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Capraria biflora L. é uma espécie pertencente à família Scrophulariaceae, originária das Antilhas e América do Sul, sendo amplamente distribuída no continente americano. Dentre os usos populares, C. biflora L. pode ser utilizada contra dores no estômago, febres, gonorréia, como diurética, calmante e para o tratamento de problemas urinários. Dentre os constituintes químicos, pode-se isolar a biflorina, iridóides glicosídeos e sesquiterpenos. Os objetivos deste trabalho foram: realizar a padronização farmacognóstica da espécie; realizar a caracterização térmica e estudo cinético de degradação térmica da biflorina; validar um método de doseamento da biflorina em HPLC; e verificar o efeito do extrato hidroalcoólico das folhas e da biflorina em processos de marcação com tecnécio-99m. A padronização botânica foi realizada segundo os procedimentos usuais, envolvendo o estudo histológico e histoquímico de raízes, caule e folhas. A partir do isolamento da biflorina do extrato bruto das raízes, procedeu-se a sua caracterização térmica, por análise das curvas TG e DSC, bem como foi realizado estudo cinético utilizando-se curvas TG, pelos métodos isotérmico e não-isotérmico. A validação do método de doseamento da biflorina foi realizado de acordo com as especificações da Internacional Conference of Harmonization, através da avaliação dos parâmetros linearidade, exatidão, precisão, e limites de detecção e quantificação. Para os ensaios de radiomarcação, avaliou-se o efeito da biflorina na marcação in vitro de elementos sanguíneos com Tc-99m, bem como o efeito do extrato hidroalcoólico das folhas de C. biflora L. na marcação in vitro e in vivo de elementos sanguíneos com Tc-99m, na biodistribuição do radiofármaco, e na fragilidade osmótica e morfologia de hemácias marcadas com o radiofármaco. A caracterização farmacobotânica evidenciou características que foram consideradas importantes para o diagnóstico da espécie, tais como a presença de conspícuas células pétreas no caule e cutícula estriada nas folhas. Na caracterização térmica da biflorina, a curva TG revelou a presença de três estágios de decomposição, e a curva DSC apresentou cinco picos referentes à fases de transição. No estudo de degradação, em ambos os métodos, foi observado uma cinética de ordem zero, bem como determinou-se para cada método os parâmetros ordem da reação, energia cinética envolvida e fator de freqüência. O método de doseamento proposto apresentou curto tempo de análise, uma linearidade satisfatória, exatidão próxima a 100% e coeficientes de variação menores que 6%. Os estudos in vitro comprovaram que a biflorina reduz a percentagem de radioatividade em células sanguíneas e proteínas plasmáticas, e a maior concentração utilizada não alterou a radioatividade em linfócitos. Foi observado que o extrato das folhas alterou in vitro a percentagem de radioatividade nas células sanguíneas e proteínas plasmáticas; in vivo alterou apenas as proteínas celulares; promoveu alterações morfológicas nas células sanguíneas incubadas in vitro e in vivo; e sua maior concentração utilizada não alterou a biodistribuição do Tc-99m e a radiomarcação em linfócitos. Os resultados obtidos neste trabalho com C. biflora L. serão de grande importância na padronização de futuras formas farmacêuticas contendo extratos ou princípios ativos isolados desta planta

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Chloroperoxidase (CPO), secreted by marine fungus Caldariomyces fumago, is the most versatile catalyst among known heme enzymes. Chloroperoxidase can catalyze epoxidation reactions with high enantioselectivity and high yield, which makes CPO an attractive candidate for both industrial and medicinal chiral synthesis. Toward this end, we have constructed two CPO mutants, F103A and N74V. Chiral HPLC was used to evaluate the enantioselectivity and yield of CPO and the mutants toward the epoxidation of styrene and its derivatives. Both of the mutants show dramatically changed epoxidation profiles compared to the parent protein. This information provided fresh insight into the mechanism through which CPO achieves its enantioselectivity. Furthermore, effort was made to understand the biological function of CPO through characterization of CPO catalyzed oxidation of dimethylsulfoniopropionate (DMSP), a secondary metabolite of many marine algal species that plays a pivotal role in marine ecology and global climate.

El uso de sustancias medicamentosas como promotores de crecimiento en la alimentación de animales de granja y en acuicultura es una práctica ilegal en la Unión Europea. Dada la incidencia que puedan tener las sustancias administradas a los animales para la salud de los consumidores, la Unión Europea estableció un reglamento para todos los países miembros que corresponde a la voluntad de proteger la salud pública. La prohibición de dichas sustancias promotoras del crecimiento ha motivado un mercado negro a nivel europeo que precisa de estrictos controles sistemáticos para su detección. Los medicamentos veterinarios más utilizados en estos casos fraudulentos son aquellos que tienen una acción anabolizante, hormonal o tireostática. El objetivo principal de este trabajo consiste en el desarrollo de métodos analíticos rápidos y la evaluación de su aplicación como técnicas de "screening" o criba en el control sanitario de residuos medicamentosos y de sustancias promotoras del crecimiento en animales de granja. Concretamente, se desarrollan protocolos de análisis rápidos y específicos para detectar residuos de ?-agonistas (clembuterol y mabuterol), lactonas del ácido resorcílico (zeranol), antimicrobianos (carbadox), agentes antitiroideos (metil-tiouracilo) y corticoides (dexametasona) en muestras de agua de beber, orina y pienso de distintas especies animales. Se han utilizado los métodos inmunológicos tipo ELISA debido a su especificidad, rapidez y gran número de muestras que se pueden analizar de forma simultánea. La cromatografía líquida de alta resolución ha sido también una herramienta muy eficaz para la detección de varias sustancias. Todos los métodos puestos a punto y optimizados para los analitos y respectivas matrices estudiados demostraron buena sensibilidad y robustez y han podido ser validados conforme a la normativa vigente que afecta a todos los países miembros de la Unión Europea (Decisión 2002/657/EC). / Reig Riera, MM. (2010). Desarrollo de métodos rápidos de detección de residuos medicamentosos en animales de granja [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/8644 / Palancia

High Performance Liquid Chromatography is currently used for the purpose of analytical evaluation of drugs. This is mainly because it allows the separation method both, a qualitative and a quantitative, analysis of high selectivity mixture evaluation and sensitivity. The diploma thesis deals with the issue of purity evaluation of pharmaceutical substance Nalbufin hydrochloride. The aim of the experimental part of the diploma thesis deals with the development and validation of the analytical method for assessing the purity of Nalbufin hydrochloride.An HPLC method was developed on a Nova-Pak C18 column. The mobile phase consisted of two components A and B. MF A composition was as follows: 0.97 g of sodium octane sulfonate was dissolved in 900 mL of water to which were added 100 mL of acetonitrile and 2 mL of triethylamine. Created solution was treated with phosphoric acid to pH 2.5. MF B had the following composition: 0.86g of sodium octanesulfonate was dissolved in 800 mL of water to which 200 mL was added acetonitrile of 2 mL of TEA. The resulting pH was adjusted to pH 2.5 with phosphoric acid. gradient MF had the following composition: From zero minutes from 100% A to 30 min. to 0% A. The 30-60min. 0% A, 60-61 min. with a linear change to 100% A, 61-70 min. equilibrium into the original conditions to 100% A at a flow rate of 1 mL/min. In the following section we evaluated the basic validation parameters: linear dynamic range 0.3 - 4.5 g/mL, we calculated the linear regression equation for Nalbuphine in R2 (0.9999), Oxycodone R2 (0.9999) and Noroxycodone R2 (0.9998). The method gave us detection limits for Nalbuphine 0.069 g/mL. Oxycodone had a detection limit of 0.053 g/mL and Noroxycodone 0.048mg / mL. The limist of quantification in these cases were 0.209 g / mL for Nalbuphine, 0.161 g/mL for Oxycodone and 0.147 g/mL for Noroxycodone. Repeatability for the limit of quantification was also set expressed by the relative standard deviation. For Nalbuphine - RSD = 0.40%, Oxycodone - RSD = 2.39% and Noroxycodone - RSD = 1.25% (RSD 7.0%). The following validation parameter was accuracy. The resulting RSD was 0.44% (RSD 5.0%). The last evaluated parameter was robustness. For pH 2.4, the change value was resolution of 1.5% and repeatability of RSD = 0.85%. The change of resolution value for pH 2.6 was 2.8% and repeatability RSD = 1.29% (max. 5% limit). The second factor observed for the robustness was the temperature change of the column. The arithmetic average was calculated from the individual peak areas and relative standard deviation RSD = 3.45% was evaluated with the change of resolution, which had a value of 3.34%. Thus, we can state that the developed chromatographic method has been verified in the givenvalidation parameters and is suitable for determining the purity of Nalbuphine hydrochloride.

A method of high-performance liquid chromatography with evaporative light-scattering detector (HPLC-ELSD) has been optimalized for the determination of neutral and polar lipids. Column filled by silica with chemically bonded diol has been used as stationary phase. As mobile phase, a ternary gradient composed from A: hexan-tetrahydrofuran 99:1 (v/v), B: isopropanol-chloroform-acetic acid 82:20:0,01 (v/v/v), C: isopropanol-water-triethylamine 47:47:6 (v/v/v) was used. Calibration curves have been measured in the range 2-200 μg of the injected amount; for individual lipid classes, optimal interlay of experimental data corresponded to the following functions: triacylglycerols - third order polynom (R=0,998), cholesterol esters - exponential dependence (R=0,998), free cholesterol - third order polynom (R=0.9998), ceramid - exponential dependence (R=0,992), cardiolipin - square dependence (R=0,998), phosphatidylethanolamine - exponential dependence (R=0,999), phosphatidylcholine - square dependence (R=0,997), phosphatidylserine - third order polynom (R=0,9985), sphingomyelin - third order polynom (R=0,9997), lysophosphatidylcholine - exponential dependence (R=0,9986). Analysis of the synthetic control sample showed recovery in the range of 82-95%. On the basis od these measurements, concentration of...

Silica-based monolithic HPLC columns contain a novel chromatographic support in which the traditional particulate packing has been replaced with a single, continuous network (monolith) of porous silica. The main advantage of such a network is decreased backpressure due to macropores (2 μM) throughout the network. This allows high flow rates, and hence fast analyses that are unattainable with traditional particulate columns. The Chromolith SpeedROD™ (EM Science, Gibbstown NJ) is a commercially available silica-based monolithic column. This work investigated the chromatographic properties of the 50x4.60 mm (ODS) SpeedROD™. Data fit to the van Deemter equation (mean square error=0.834) indicated that the van Deemter model was valid for monolithic columns. An effective particle size of 4 μM for the SpeedROD™ column was assigned by comparing the minimum of van Deemter curves with a series of particulate columns having various particle diameters. Separation Impedance (E), an empirically derived measure of column performance, was calculated as an alternate method of evaluating column efficiency. Data collected using this model confirmed monolithic columns behaves as a (more efficient) 3 μM column. A series of experiments were designed to compare the effects of mobile phase strength and mobile phase viscosity between the SpeedROD™ column and a particulate column. The results indicated both solvent strength and viscosity have effects on the monolithic column at the optimum linear velocity. A fast (90 s) HPLC method was developed using the SpeedROD™ column and a seven-component test mixture with a large range of hydrophobicities. The precision for both retention time and peak area was measured at high linear velocities (8 mL/min) and the percent relative standard deviation (RSD) calculated. Column to column reproducibility (n=6) was measured. The overall percent RSD ranged from 0.25% to 4.56% for retention time and from 1.08% to 6.77% for peak area. Run to run reproducibility (n=15) was measured for all six columns. Averages ranged for retention time from 0.89% to 5.09% RSD and for peak area from 4.65% to 6.18% RSD. Applications for the SpeedROD™ column with various sample types were developed and discussed. These methods demonstrated the effectiveness of the SpeedROD™ at fast flow rates. / Ph. D.