Conselho Nacional de Desenvolvimento Científico e Tecnológico / This work describes the development and application of analytical methods to establish parameters to the quality control of fruit and trunk latex of H. speciosa using High Performance Liquid Chromatography (HPLC) with diode array detect and evaporative light scattering detector (ELSD). As a first step chromatographic profile analytical method was developed and validated for the analysis of authentic sample of trunk latex of H. speciosa, and then the optimized method was used for the analysis of commercial samples of trunk latex sold in open markets from Sergipe, Brazil. The visual comparison of the chromatographic profiles of different samples of trunk latex allowed checking the authenticity and dissimilarities of chemical profiles of these samples. Seven chemical markers were purified by semi-preparative HPLC-DAD from the trunk latex of H. speciosa, the characterization by 1H, 13C NMR allowed the identification of the following substances cyclohexylethanoid glucoside cornoside, cyclohexylethanoid glucoside dihydrocornoside and (7,8)-treo-4,7,9,9 -tetrahydroxy-3,3 -dimethoxy-8-O-4 -neolignan-7-O-β-D-glucopyranoside. As a second step of our work chromatographic method for qualitative analysis of chemical markers lupeol, α-amyrin, β-amyrin e 3- β-O-acyl lupeol esters in the fruit latex of H. speciosa and in the mangaba commercial pulp was developed, the optimized method showed itself appropriate to the identification of such substances with adequate separation. In the end one analytical method for the quantification of the lupeol ester content in fruit latex and commercial pulp was developed and validated using the HPLC-DAD-ELSD. In the validation study were evaluated the figures of merit selectivity, linearity, limit of quantification and detection, precision, accuracy, stability and robustness according to the standards described in RE nº 899/03 (ANVISA). The quantification of lupeol ester by both detectors was significantly similar (259.44 μg/mg DAD and 269.58 μg/mg ELSD) with one coefficient of variation of 2.7%. This paper presents a contribution to the quality control of H. speciosa samples. / O presente trabalho apresenta o desenvolvimento e aplicação de métodos analíticos para o controle de qualidade do látex do fruto e do caule de H. speciosa Gomes utilizando cromatografia líquida de alta eficiência com os detectores de arranjo de diodos e evaporativo por espalhamento de luz (HPLC-DAD-ELSD). No primeiro momento, um método analítico para a obtenção do perfil cromatográfico foi desenvolvido e validado para a análise de uma amostra autêntica do látex do caule de H. speciosa; em seguida foi utilizado o método otimizado para a análise de amostras do látex do caule comercializadas em feiras livres do Estado de Sergipe. A comparação visual dos perfis cromatográficos das diferentes amostras do látex do caule permitiu averiguar a autenticidade e as dissimilaridades dos perfis químicos dessas amostras. Utilizando uma coluna semi-preparativa, sete marcadores químicos foram purificados a partir do látex do caule de H. speciosa; a caracterização por RMN 1H, 13C possibilitou a identificação das seguintes substâncias: ciclohexiletanóide glicosilado cornosídeo, ciclohexiletanóide glicosilado dihidrocornosídeo e (7,8)-treo-4,7,9,9 -tetrahidroxi-3,3 -dimetoxi- 8-O-4 -neolignana-7-O-β-D-glicopiranosídeo. No segundo momento, um método cromatográfico para análise qualitativa dos marcadores químicos lupeol, α-amirina, β-amirina e ésteres 3- β-O-acil lupeol no látex dos frutos de H. speciosa e em polpa comercial de mangaba foi desenvolvido. Por fim, um método analítico para a quantificação do teor de ésteres de lupeol em látex dos frutos de H. speciosa e em polpa comercial foi desenvolvido e validado utilizando HPLC-DAD-ELSD. No estudo da validação foram avaliadas as figuras de mérito seletividade, linearidade, limite de quantificação e detecção, precisão, exatidão, estabilidade e robustez conforme as normas descritas na RE nº 899/03 (ANVISA). A quantificação do teor de ésteres de lupeol por ambos os detectores se mostraram significativamente similares (259,44 μg/mg para o DAD e 269,58 μg/mg para o ELSD) com coeficiente de variação de 2,7 %. Este trabalho apresenta uma contribuição ao controle de qualidade de amostras de H. speciosa.

Made available in DSpace on 2016-08-19T12:56:31Z (GMT). No. of bitstreams: 1 Raphael Teixeira Verbinnen.pdf: 4011751 bytes, checksum: 6a77c544380be3240e2586a2ad753bff (MD5) Previous issue date: 2009-04-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Given the humanity perception of necessity to preserve the environment and the incessant search for sustainable development, today the preservation of natural water sources is by far one of the most worrying factors for the preservation of life. Besides the most common aquatics pollutants, also there is one called emerging pollutants, which in recent decades have been detected due the technological advances. These involve mainly the organic micropollutants, which present the endocrine disrupters (ED's) main group. Among the ED's, the natural and synthetic sex steroids hormones have significant importance because their daily use in medicine, in the replacement therapy and contraceptive methods, which has contributed to the continuous release in domestic sewage. There are several consequences of this effect, including fertility declining, occurrence of various cancers and disorders in the development and homeostasis of organisms. The domestic and industrial sewage and leaching from agricultural fields fertilized with sludge from sewage treatment plants (STP's) are the main sources of contamination. This study allowed the chromatographic separation of the hormones estriol (E3), 17 β-estradiol (E2), estrone (E1) and 17 α-ethynylestradiol (EE2) in relatively short duration (16,5 minutes), using the acetonitrile:water (ACN: H2O) mixture as mobile phase in gradient mode, constant flow of 1 mL min-1, injection volume of 20 μL, λ = 280 nm and temperature of 27 ºC. The method was developed in two laboratories, involving different instruments. Four procedures and two adsorbents (C18 e OPT) of SPE were tested. The sample preparation method, including dechlorination and the SPE procedure with C18 cartridge, resulted in the recovery values within the acceptable limit. The developed method was validated according to guidelines of the ANVISA`s Guide for Validation, and bioanalytical methods was adapted for this study. The method was then applied in the analysis of the natural (17β-estradiol, estrone and estriol) and the synthetic (17α-ethinylestradiol) hormone in drinking water from the city of São Luís Peaks were not identified for the substances studied, probably substances concentrations below the limits of detection and quantification of the method were found. / Diante da percepção do homem sobre a necessidade de se preservar o ambiente e da busca incessante pelo desenvolvimento sustentável, hoje a proteção das fontes de água natural é de longe um dos fatores mais preocupantes para a conservação da vida. Além dos poluentes aquáticos mais comuns, existem também os chamados poluentes emergentes, que nas últimas décadas vêm sendo detectados em função do aprimoramento de métodos e técnicas de detecção. Estes envolvem principalmente os micropoluentes orgânicos, que por sua vez apresentam os desreguladores endócrinos (DEs) como principal grupo. Dentre os DEs, os hormônios sexuais esteróides, naturais e sintéticos, têm significativa importância, devido ao seu uso diário na medicina, em terapias de reposição e métodos contraceptivos, o que tem provocado o contínuo lançamento em esgotos sanitários. Várias são as conseqüências do efeito destas substâncias, dentre elas: diminuição da fertilidade, ocorrência de cânceres diversos e perturbações no desenvolvimento e na homeostase de organismos. Os esgotos sanitários e industriais, além da lixiviação de campos de agricultura adubados com lodo proveniente de estações de tratamento de esgotos (ETEs) são as principais fontes de contaminação. Este trabalho possibilitou a separação cromatográfica dos hormônios estriol (E3), 17 β-estradiol (E2), estrona (E1) e 17 α-etinilestradiol (EE2) em tempo relativamente curto (16,5 min), utilizando como fase móvel a mistura acetonitrila:água (ACN:H2O), em modo gradiente, fluxo de 1 mL min-1, λ = 280 nm e temperatura de 27 ºC. O método foi desenvolvido em dois laboratórios, envolvendo dois instrumentos diferentes. Foram testados 4 procedimentos e 2 adsorventes de extração em fase sólida (C18 e OPT). O método de preparo da amostra, incluindo a descloração e o procedimento de EFS, com cartucho C18 resultou em valores de recuperação dentro dos limites considerados adequados. O método desenvolvido foi validado segundo as orientações do Guia para Validação de Métodos Analíticos e Bioanalíticos publicado pela ANVISA, tomando-se por base os procedimentos para os métodos bioanalíticos, os quais foram adaptados para este estudo. O método foi, então, aplicado na análise dos hormônios naturais (17β-estradiol, estrona e estriol) e o sintético (17α- etinilestradiol) em água potável distribuída na cidade de São Luís. Para as amostras analisadas, não foram identificados picos referentes às substâncias de interesse, portanto pode-se afirmar que estas foram encontradas em concentrações inferiores aos limites de detecção e de quantificação do método.

Made available in DSpace on 2016-08-19T12:56:32Z (GMT). No. of bitstreams: 1 VERONICA ALVES GONCALVES DA SILVA.pdf: 6289705 bytes, checksum: 1c73081d0d3faa6d1cdc03eef3a8bc0c (MD5) Previous issue date: 2008-06-16 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Honey is a natural substance produced by bees and by your nature it is considered a healthy product. Presence of additives and/or preserve agents is not permitted. On apiculture, sulfonamydes are largely used on prevention and control of beehive disease, such as American foulbrood caused by Paenibacillus larvae. A consequence of this practice is the presence of these residues in honey, that should cause allergy reactions on consumers. In the last years, some publicated works is discussing the presence of antibiotic and sulfonamides residues in honey. In Brazil, the presence of antibiotics residues in food is controled by the Agência Nacional de Vigilância Sanitária (ANVISA) an by the Ministério da Agricultura, Pecuária e do Abastecimento (MAPA). The realization of monitoring contaminants residues in food programs, as well as the Plano Nacional de Controle de Resíduos (PNCR) from MAPA are essentials to the sanitary vigilance actions development, preventing and controlling possible hazard to the consumer health. This work has the objective the development and validation of multiresidue analytical method to the sulfadiazine, sulfathiazole, sulfadimedine and sulfadimethoxine determination in bee honey using high performance liquid chromatography (HPLC) with diode array detector (DAD). The established methodology consisted on such conditions: solid phase extraction with Nexus sorbent, elution with acetonitrile 5 mL, and reconstitution with sodium acetate buffer (0,05 mol L-1) pH 4,5, with 1% methanol addition, separation on analytical column C18, acetonitrile and water mobile phase, on gradient elution mode and detection on 278 nm. The sample preparation consisted on the acid hydrolysis with hydrochloric acid 2 mol L-1 of a honey rate and adjust to pH 4,5 with sodium acetate buffer solution 0,05 mol L-1 pH 4,5. The method was validated and the results demonstrated accuracy and precision between the limits established by the current legislation. Good linearity range was encountered, obtaining recovery values varying from 41 to 89% and relative standard deviation estimative varying between 2,36 and 14% to the four studied compounds. The four sulfonamides detection limits in honey sample varying from 20 to 30 ^g kg-1 and the quantification limits varied from 60 to 90 ^g kg-1. Considering 100 ^g kg-1 the residue maximum limit established by the Brazilian legislation to the SDZ, STZ, SMZ and SDM total concentration, the described method permit suitable determination of the four sulfonamides in bee honey. / O mel é uma substância natural, produzida pelas abelhas e por sua natureza é considerado um produto saudável, não sendo permitido a presença de aditivos e/ou agentes conservadores. Na apicultura as sulfonamidas são muito utilizadas na prevenção e controle de doenças em colméias, como a cria pútrida americana causada pelo Paenibacillus larvae. Uma conseqüência desta prática é a presença destes resíduos no mel, o que pode causar reações alérgicas nos consumidores. Nos últimos anos, alguns trabalhos na literatura vêm discutindo a presença de resíduos de antibióticos e sulfonamidas no mel. No Brasil, a presença de resíduos de antibióticos nos alimentos é controlada pela Agência Nacional de Vigilância Sanitária (ANVISA) e pelo Ministério da Agricultura, Pecuária e do Abastecimento (MAPA). A realização de programas de monitoramentos de resíduos de contaminantes em alimentos, assim como o Plano Nacional de Controle de Resíduos (PNCR) do MAPA são essenciais para que ações de vigilância sanitária possam ser desenvolvidas, prevenindo e controlando possíveis danos à saúde do consumidor. Este trabalho tem por objetivo desenvolver e validar um método analítico multiresidual para a determinação de sulfadiazina, sulfatiazol, sulfadimidina e sulfadimetoxina em mel de abelhas usando cromatografia líquida de alta eficiência (CLAE) com detector de arranjo de fotodiodos (DAD). A metodologia estabelecida consistiu nas seguintes condições: extração em fase sólida com sorvente Nexus, eluição com 5 mL de acetonitrila e reconstituição com solução-tampão acetato de sódio (0,05 mol L-1) pH 4,5, com adição de 1% de metanol, separação em coluna analítica C18, fase móvel acetonitrila e água, no modo de eluição em gradiente e detecção a 278 nm. O preparo de amostra consistiu na hidrólise ácida com ácido clorídrico 2 mol L-1 de uma alíquota de mel e ajuste para pH 4,5 com solução tampão acetato de sódio 0,05 mol L-1 pH 4,5. O método foi validado e os resultados demonstraram exatidão e precisão dentro dos limites estabelecidos pela legislação vigente. Foi encontrada boa faixa de linearidade, obtendo-se valores de recuperação que variaram de 41 a 89% e estimativa do desvio padrão relativo variando entre 2,36 e 14% para os quatro compostos estudados. Os limites de detecção das quatro sulfas em amostra de mel variaram de 20 a 30 ^g kg-1 e os limites de quantificação variaram de 60 a 90 ^g kg-1. Considerando o limite máximo de resíduo estabelecido pela legislação brasileira de 100 ^g kg-1 para as concentrações totais de SDZ, STZ, SMZ e SDM, o método descrito permite adequada determinação das quatro sulfas em amostras de mel de abelhas.

Neste trabalho utilizou-se a cromatografia líquida de alta eficiência (CLAE) e a eletroforese capilar (EC) como ferramentas analíticas para avaliação de flavonoides em extratos hidrolisados de folhas de Rubus e de Morus. Os extratos foram preparados por hidrólise ácida em ultrassom e analisados por CLAE-DAD e EC-DAD. Os métodos elaborados foram validados e aplicados. Quercetina e canferol foram identificados nestes extratos por CLAE-DAD, EC-DAD e CLUE-DAD/EM. Em Rubus erythrocladus quantificou-se 848,43 ± 66,68 μg.g-1 e 304,35 ± 17,29 μg.g-1 de quercetina e canferol, respectivamente, por CLAE-DAD e 836,37 ± 149,43 μg.g-1 de quercetina por EC-DAD. Em Rubus idaeus quantificou-se 698,32 ± 1,29 μg.g-1 e 184,20 ± 4,34 μg.g-1 de quercetina e canferol, respectivamente, por CLAE-DAD. Em Morus nigra quantificou-se 2323,90 ± 145,35 μg.g-1 e 1446,36 ± 59,00 μg.g-1, de quercetina e canferol, respectivamente, por CLAE-DAD e 2552,82 ± 275,30 μg.g-1 e 1188,67 ± 99,21 μg.g-1 de quercetina e canferol, respectivamente, por EC-DAD. Não foi observada diferença significativa entre a quantificação por CLAE-DAD e por EC-DAD, porém o método por CLAE-DAD se mostrou muito mais sensível do que o método por EC-DAD. Os mesmos compostos também foram identificados nestes extratos por CLUE-DAD/EM. Por esta ferramenta analítica se pode observar uma diferença entre o perfil químico das amostras de Rubus e de Morus. Quatro amostras comerciais de folhas de amora foram analisadas, observando grandes variações nas concentrações de quercetina e de canferol presentes nestes extratos. Além disso, através de um screening de atividade antifúngico verificou-se que os extratos não hidrolisados de Rubus erythrocladus e Rubus idaeus apresentaram ação contra três espécies de fungos filamentosos patógenos humanos: Thichophyton rubrum 51, Microsporum gypseum 01 e Microsporum canis 40. / In this work we used the high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) as analytical tools to evaluate the hydrolyzed flavonoids in extracts of Rubus and Morus leaves. The extracts were prepared by acid hydrolysis in ultrasound and analyzed by HPLC- DAD and CE -DAD. The developed methods were validated and applied. Quercetin and kaempferol were identified in these extracts by HPLC-DAD, EC-DAD and UPLC-DAD/MS. In Rubus erythrocladus were quantified 848.43 ± 66.68 μg.g-1 and 304.35 ± 17.29 μg.g-1 of quercetin and kaempferol, respectively, by HPLC-DAD and 836.37 ± 149.43 μg.g-1 of quercetina by CE –DAD. In Rubus idaeus were quantified 698.32 ± 1.29 μg.g-1 and 184.20 ± 4.34 μg.g-1 of quercetin and kaempferol, respectively, by HPLC-DAD. In Morus nigra were quantified 2323.90 ± 145.35 μg.g-1 and 1446.36 ± 59.00 μg.g-1 of quercetin and kaempferol, respectively, by HPLC-DAD and 2552.82 ± 275.30 μg.g-1 and 1188.67 ± 99.21 μg.g-1 of quercetin and kaempferol, respectively, by CE -DAD. No significant difference in quantification by HPLC-DAD and CE-DAD was observed, but the HPLC-DAD method proved to be more sensitive than EC-DAD method. The same compounds were also identified in these extracts by UPLC-DAD/EM. Using this analytical tool, it was possible to observe a difference in the chemical profile of Rubus and Morus extracts. Commercial samples of blackberry leaves tea were analyzed by HPLC-DAD and it was observed big variation in the concentrations of quercetin and kaempferol in these extracts. Moreover, through a screening antifungal activity, it was found that no hydrolyzed extracts of Rubus idaeus and Rubus erythrocladus showed action against three species of filamentous fungal human pathogens: Thichophyton rubrum 51, Microsporum gypseum 01 and Microsporum canis 40.

Masters of Science / The aim of this study was therefore to evaluate the antioxidant and anti-diabesity potential of a hot water extract of C. maculata in non-cell based assays and correlate the activities with phenolic composition. Total antioxidant capacity (TAC) was assessed in terms of free radical scavenging and iron reducing ability. The DPPH, ABTS, ORAC and FRAP assays were employed. Anti-diabesity potential was assessed in terms of the inhibition of the digestive enzymes, α-glucosidase and pancreatic lipase

This diploma thesis deals with the natural antioxidants present in cereals, especially in barley (Hordeum vulgare). A close attention is paid to the study of carotenoids determination was conducted. In the experimental part, the method of beta-carotene determination was optimized using high-performance liquid chromatography, diode array detector and mass detector (HPLC/DAD/APCI-MS). The method was used for the beta-carotene and lutein determination in the samples of barleycorn, malt and green barley. This method involves the sample saponification, extraction by diethylether, followed by separation on ODS Hypersil 250x4,6 mm, 5m column, using MTBE/MeOH (20:80) as mobile phase and spectrophotometric detection (450 nm). Quantitative analysis was implemented in the HPLC/DAD system. The MS detector was used for identification of analytes. A number of still unpublished data about the content of beta-carotene and lutein in several varieties of malting barley, malt and green barley are stated in this thesis. The highest content of beta-carotene was found in the green barley sample of variety Malz, harvested in first grow phase (8,49 mg/kg of the dry matter). The content of beta-carotene in barleycorn is relatively low (0,07-0,14 mg/kg of the dry matter). The content of beta-carotene is several times higher in the malt produced from barleycorn (0,24-0,56 mg/kg of the dry matter). The diploma thesis was implemented in the Research Institute of Brewing and Malting, Plc. in Brno.

This diploma thesis deals with design and experimental study of transdermal transport of pharmaceutically active agents from gel matrices, which contain humic substance in its structure. A model absorption membrane was represented by the skin of pig´s earlobes. The study of the release of active substances and Lignohumte was performed due to the vertical diffusion cells. The amount of released humic substance was characterized by UV-VIS method and the amount of released active agent from gel matrix was determined by HPLC-DAD. The most interesting finding of this diploma thesis was that Lignohumate enhances transdermal transport of active agents and supports their release from gel samples to the particular environment.

No description available.

This diploma thesis deals with macerates of comfrey (Symphytum officinale) in selected fats (food lard, cosmetic lard and almond oil). Selected fats were first characterized by dry matter content, saponification, acid, iodine number, peroxide value. At the same time, total and free fatty acids were determined using GC-FID. Macerates were prepared by extraction of comfrey roots with selected fats. In prepared macerates the change in acid and peroxide value was monitored, as well as the content of selected bioactive compounds. The content of total phytosterol and total carotenoid content was determined by UV-VIS spectrometry, phytosterols and carotenoids were also analysed using HPLC-DAD. Total phenolic content was measured using Folin-Ciocalteu reagent and antioxidant activity by ABTS assay. The measured properties were then compared with industrially produced comfrey ointment. In macerated fats the increase in acid and peroxide value was determined. Furthermore, an increase in the content of total phytosterols and total carotenoids was observed. Using HPLC-DAD the content of -sitosterol and stigmasterol was determined, but carotenoids were not detected. Of the tocopherols, only DL--tocopherol acetate was detected. During maceration, the content of total phenolic compound in fat increased, which caused a change in antioxidant activity.

<p>In this thesis some multivariate spectroscopic methods for the analysis of solutions are proposed. Spectroscopy and multivariate data analysis form a powerful combination for obtaining both quantitative and qualitative information and it is shown how spectroscopic techniques in combination with chemometric data evaluation can be used to obtain rapid, simple and efficient analytical methods. These spectroscopic methods consisting of spectroscopic analysis, a high level of automation and chemometric data evaluation can lead to analytical methods with a high analytical capacity, and for these methods, the term high-capacity analysis (HCA) is suggested. It is further shown how chemometric evaluation of the multivariate data in chromatographic analyses decreases the need for baseline separation. </p><p>The thesis is based on six papers and the chemometric tools used are experimental design, principal component analysis (PCA), soft independent modelling of class analogy (SIMCA), partial least squares regression (PLS) and parallel factor analysis (PARAFAC). The analytical techniques utilised are scanning ultraviolet-visible (UV-Vis) spectroscopy, diode array detection (DAD) used in non-column chromatographic diode array UV spectroscopy, high-performance liquid chromatography with diode array detection (HPLC-DAD) and fluorescence spectroscopy. The methods proposed are exemplified in the analysis of pharmaceutical solutions and serum proteins.</p><p>In Paper I a method is proposed for the determination of the content and identity of the active compound in pharmaceutical solutions by means of UV-Vis spectroscopy, orthogonal signal correction and multivariate calibration with PLS and SIMCA classification. Paper II proposes a new method for the rapid determination of pharmaceutical solutions by the use of non-column chromatographic diode array UV spectroscopy, i.e. a conventional HPLC-DAD system without any chromatographic column connected. In Paper III an investigation is made of the ability of a control sample, of known content and identity to diagnose and correct errors in multivariate predictions something that together with use of multivariate residuals can make it possible to use the same calibration model over time. In Paper IV a method is proposed for simultaneous determination of serum proteins with fluorescence spectroscopy and multivariate calibration. Paper V proposes a method for the determination of chromatographic peak purity by means of PCA of HPLC-DAD data. In Paper VI PARAFAC is applied for the decomposition of DAD data of some partially separated peaks into the pure chromatographic, spectral and concentration profiles. </p>