Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: See item for full text / AFRIKAANSE OPSOMMING: Sien item vir volteks

published_or_final_version / abstract / Chemistry / Doctoral / Doctor of Philosophy

The Supercritical Fluid Extraction (SFE) technique was used to fractionate Suwannee River reference fulvic acid (FA). The fractions were characterized by gas chromatography (GC) and reversed-phase high performance liquid chromatography (RP-HPLC). A SFE fractionation method was developed using stepwise gradient of supercritical CO₂ and methanol. Three FA fractions were separated. The average mass recovery was 102% with the coefficient of variation of 2.8%. The fractionation dynamics and the difference in the ratios of UV absorption to fluorescence emission indicate the real fractionation of the FA. The HPLC chromatographic peak patterns and the spectra of the corresponding peaks were almost indistinguishable. The overall results of this research support the argument that FA exhibits polymer-like molecular structure.

High performance liquid chromatography was used to measure ribonucleoside triphosphates in microbial samples. Anion exchange columns in a radial compression module were used to separate and quantify purine and pyrimidine ribonucleotides. Endogenous ribonucleoside triphosphates were extracted from Escherichia coli and pseudomonas aeruginosa using three different solvents, namely trifluorocetic acid (TFA; 0.5M), trichloroacetic acid (TCA; 6 per cent w/v) and formic acid (1.0M) Extracts were assayed for uridine 5'-triphosphate (ATP), and guanosine 5'-triphosphate (GTP) by using anion exchange radial compression high performance (pressure) liquid chromatography. The three extraction produres were compared for yield of triphosphates. E. coli, the TFA extraction procedure was more sensitive and reliable than TCA and formic acid extraction procedures, but , in P. aeruginosa, the best yields of ATP and GTP were obrained following extraction with TFA. Yields of UTP and CTP increased when extraction was performed in TCA. These data illustrate that different extraction produres produce different measures for different triphosphates, a point often overlooked.

High Performance Liquid Chromatography (HPLC) was used to investigate the utility of this technique for the analytical and preparative separation of components of aquatic fulvic acids (FA). Three modes of HPLC namely adsorption, anion exchange and reversed phase were evaluated. Aquatic fulvic acids were either extracted from surface water and sediment samples collected from the Southwest of the U.S., or were provided in a high purity form from the USGS. On the adsorption mode, a major fraction of aquatic fulvic acid was isolated on a semipreparative scale and subjected to Carbon-13 NMR and FAB Mass Spectroscopy. Results indicated that (1) The analyzed fraction of fulvic acid contains more aliphatic than aromatic moieties; (2) Methoxy, carboxylic acids, and esters are well-defined moieties of the macromolecule; (3) Phenolic components of the macromolecules were not detected in the Carbon-13 NMR spectrum possibly because of the presence of stable free radicals. Results of the anion exchange mode have shown that at least three types of acidic functionalities in aquatic fulvic acid can be separated. Results also indicated that aquatic fulvic acid can be progressively fractionated by using subsequent modes of HPLC. Results of reversed phase mode have shown that (1) The fractionation of aquatic fulvic acid by RP-HPLC is essentially controlled by the polarity and/or pH of the carrier solvent system; (2) Under different RP-HPLC conditions aquatic fulvic acid from several locations are fractionated into the same major components; (3) Fulvic acid extracted from water and sediment from the same site are more similar than those extracted from different sites; (4) Cationic and anionic ion pair reagents indicated the presence of amphoteric compounds within the polymeric structure of fulvic acid. Each mode of HPLC provided a characteristic profile of fulvic acid. The results of this research provided basic information on the behavior of aquatic fulvic acids under three modes of HPLC. Such informations are prerequisite for further investigation by spectroscopic methods.

A hidroxicloroquina (HCQ) é um importante fármaco quiral usado, principalmente, no tratamento de artrite reumatóide, lupus eritematoso sistêmico e malária e cujas propriedades farmacocinéticas e farmacodinâmicas são estereosseletivas. Em relação às propriedades farmacocinéticas, alguns estudos prévios indicam que o metabolismo estereosseletivo parece ser função da espécie estudada, o que implica na necessidade de métodos seletivos para a determinação de seus enantiômeros em materiais biológicos. Assim, propôs-se o desenvolvimento e a validação de métodos para análise dos enantiômeros do fármaco inalterado e de seus principais metabólitos em frações microssomais de homogeneizados de fígado de ratos e camundongos. Para tanto foram empregadas as técnicas de eletroforese capilar (CE) e de cromatografia líquida de alta eficiência (HPLC). Inicialmente foi desenvolvido um método por HPLC, em uma etapa, para a quantificação dos enantiômeros de dois metabólitos da HCQ, desetilcloroquina (DCQ) e desetilhidroxicloroquina (DHCQ) em microssomas de fígado de ratos e camundongos. A separação foi efetuada utilizando-se a coluna Chiralpak AD-RH e hexano:isopropanol (92:8, v/v) acrescido de 0,1% de dietilamina como fase móvel. O procedimento de extração líquido-líquido foi utilizado para a preparação das amostras. A metodologia desenvolvida resultou na completa resolução dos enantiômeros da HCQ, DCQ e DHCQ e pode ser considerada adequada, visto que os parâmetros de validação mostraram valores dentro dos limites exigidos na literatura. O segundo método desenvolvido permitiu a quantificação dos enantiômeros dos três metabólitos da HCQ: DCQ, DHCQ e bisdesetilcloroquina (BDCQ) em microssomas de fígado de camundongos. A separação foi efetuada utilizando-se um tubo capilar de sílica fundida e solução do eletrólito tris(hidroximetil)aminometano 100 mmol/L, ajustada a pH 9,0 com ácido fosfórico e acrescida de 1% (m/v) de β –CD - sulfatada e 30 mmol/L de β –CD - hidroxipropilada. O procedimento de extração líquido-líquido foi eficiente para remover interferentes e os parâmetros de validação mostraram valores dentro dos limites exigidos na literatura. Os métodos desenvolvidos foram aplicados no estudo in vitro do metabolismo da HCQ em frações microssomais de fígado dos animais, verificando-se que o principal metabólito formado é o (-)-(R)-DHCQ, para ambas espécies estudadas. / Hydroxychloroquine (HCQ) is an important chiral drug used specially in the treatment of rheumatoid arthritis, systemic lupus erythematosus and malaria, with stereoselective pharmacokinetic and pharmacodinamic properties. Concerning these properties, some previous studies indicate that the stereoselective metabolism seems to be a function of the studied species, therefore selective methods are required for the determination of its enantiomers in biological matrix. Thus, the present work reports the development and validation of methods for the analysis of the enantiomers of HCQ and its main metabolites in microsomal fraction of rats and mice liver homogenates. Capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) were used for this purpose. Initially, a one-step HPLC method was developed for the quantification of the enantiomers of two HCQ metabolites, desethylchloroquine (DCQ) and desethylhydroxychloroquine (DHCQ) in microsomal fraction of rats and mice liver homogenates. The separation was performed on a Chiralpak AD-RH column using hexane:isopropanol (92:8, v/v) plus 0.1% diethylamine as the mobile phase. Liquid-liquid extraction procedure was used for sample preparation. The developed methodology resulted in the complete resolution of HCQ, DCQ and DHCQ enantiomers and can be considered suitable because the validation parameters are in accordance with the limits established in the literature. The second developed method allowed the quantification of the enantiomers of the three HCQ metabolites, DCQ, DHCQ and bisdesethylchloroquine (BDCQ) in microsomal fraction of mice liver homogenates. The separation was performed using a fused-silica capillary tube and tris (hydroxymethyl) aminometane 100 mmol/L electrolyte solution, adjusted at pH 9.0 with phosphoric acid, containing 1% (w/v) sulfated- β -CD and 30 mmol/L hydroxypropyl- β -CD. The liquid-liquid extraction procedure was efficient to remove interferents and the validation parameters showed values within accordance to the literature. The developed methods were applied in the in vitro metabolism study of HCQ in microsomal fractions of the liver of the animals and it was verified that the main metabolite formed is (-)-(R)-DHCQ for both animal species studied.

Tinidazol, 1-[2-(ethylsulphonyl)ethyl]-2-methyl-5-nitroimidazole, é um membro da classe dos nitroimidazóis que apresenta atividade amebicida, giardicida, tricomonicida e anaeróbica. O objetivo deste estudo foi avaliar a bioequivalência de duas marcas comerciais de comprimidos contendo 500 mg de tinidazol em voluntários sadios. O ensaio de bioequivalência entre o produto teste (Amplium® - FARMASA) e o produto referência (Pletil® - Pharmacia do Brasil Ltda) foi do tipo randomizado, cruzado e aberto. O medicamento foi administrado em dose única de 500 mg de tinidazol a 24 voluntários sadios. Amostras de sangue foram coletadas até 72 horas após a administração e analisadas através de método de cromatografia líquida de alta eficiência validado com detecção UV. As curvas médias de decaimento plasmatico obtidas para o produto teste (Amplium® - FARMASA) e para o produto referência (Pletil® - Pharmacia do Brasil Ltda) foram semelhantes, da mesma forma que os parâmetros farmacocinéticos Cmax (referência: 11,34 µg/mL; teste: 11,11 µg/mL), t<SUB.max (referência: 1,67 h; teste: 1,71 h), AUC0-t (referência: 201,92 µgxh/mL; teste: 198,15 µgxh/mL), AUC0-&#8734; (referência: 208,25 µg/mL; teste: 203,80 µgxh/mL) e t(½)el (referência = 14,05 h; teste = 13,91 h). A análise multivariada realizada através da análise de variância (ANOVA), para avaliação dos efeitos produto, grupo e período, revelou a ausência destes efeitos no estudo, indicando que o delineamento do estudo foi apropriado. Os valores do intervalo de confiança 90% para a razão de Cmax (93.9 % - 102.6 %), AUC0-t (94.9 % - 101.1 %) e AUC0-&#8734; (94.6 % -100.8 %) encontram-se entre 80 - 125 %, intervalo proposto pelo FDA e ANVISA. A comparação estatística dos parâmetros AUCo-t , AUC0-&#8734; e Cmax indicam claramente não haver diferença significativa entre os dois produtos contendo 500 mg de tinidazol. Baseado nos resultados farmacocinéticos e estatísticos deste, pode-se concluir que os dois produtos são bioequivalentes e podem ser considerados intercambiáveis na terapêutica. / Tinidazole, 1-[2-(ethylsulphonyl)ethyl]-2-methyl-5-nitroimidazole, is a member of the 5-nitroimidazole class of antimicrobial agents with amoebicidal, giardicidal, trichomonicidal and anaerobic activity. The purpose of this study was to evaluate the bioequivalence of two brands of tinidazole 500mg tablets in healthy human volunteers. The procedure of bioequivalence between the test product (Amplium® - FARMASA) and reference product (Pletil® - Pharmacia do Brasil Ltda) was a randomized, crossover and open study. The medication was administered in a single dose of 500 mg of tinidazole to 24 healthy volunteers. Blood samples were collected until 72 hours after administration and analised using a validated high-performance liquid chromatographic method with UV detection. The average plasmatic decay curves obtained for the test product (Amplium® - FARMASA) and reference product (Pletil® - Pharmacia do Brasil Ltda) were similar, in the same way as were the pharmacokinetic parameters Cmax (reference: 11.34 µg/mL; test: 11.11 µg/mL), tmax (reference: 1.67 h; test: 1.71 h), AUC0-t (reference: 201.92 µgxh/mL; test: 198.15 µgxh/mL), AUC0-&#8734; (reference: 208.25 µg/mL; test: 203.80 µgxh/mL) and t(½)el (reference = 14.05 hours, test = 13.91 hours). The multivariate analysis accomplished through analysis of variance (ANOVA), for assessment of product, group and period effects, revealed the absence of any of these effects in the present study, indicating that the crossover design was properly performed. j The 90% confidence intervals for the ratio of Cmax (93.9 % - 102.6 %), AUC0-t (94.9 % - 101.1 %) and AUC0-&#8734; (94.6 % - 100.8 %) values for the test and reference products are within the 80 - 125 % interval proposed by FDA and ANVISA. Statistical comparison of AUC0-t , AUC0-&#8734; and Cmax clearly indicated no significant difference between the two brands of tinidazole 500 mg tablets. Based on the pharmacokinetic and statistical results of this study, we can conclude that the two products are bioequivalent, and can be considered interchangeable in medical practice.

Orientador: Mary Rosa Rodrigues de Marchi / Coorientador: Marisa Veiga Capela / Banca: Eduardo Bessa Azevedo / Banca: Sandro José de Andrade / Resumo: A análise da literatura aponta para um crescente número de trabalhos em matrizes ambientais que buscam identificar e quantificar diversas substâncias poluentes. O estudo destas substâncias resulta em grandes conjuntos de dados, que necessitam ser devidamente analisados para a interpretação mais confiável possível. Por outro lado, os métodos analiticos utilizados nestas determinações envolvem diversas etapas e parâmetros que podem influenciar na confiabilidade analítica. Resultados analíticos sem confiabilidade identificada podem levar a tomada de decisões totalmente equivocadas na área ambiental. Neste contexto, é fundamental que os laboratórios disponham de meios e critérios objetivos para demonstrar que os métodos de ensaio que executam conduzem a resultados confiáveis e adequados à qualidade pretendida. Este trabalho teve como objetivo o estudo de procedimentos estatisticos e quimiométricos na determinação dos hidrocarbonetos policíclicos aromáticos (HPAs) empregando a cromatografia líquida de alta performance com detecção por fluorescência (HPLC-FLU), enfocando a otimização estatística de procedimentos para validação do método para obtenção e tratamento de sinais analíticos dos HPAs, bem como o estudo do comportamento dos analitos em diferentes matrizes ambientais. Neste sentido foram utilizadas ferramentas quimiométricas, como análise de componentes principais, analise hierárquica de agrupamentos e técnicas de planejamento experimental para analise exploratória dos dados e indicação das similaridades cromatográficas entre os HPAs e do perfil destes em matrizes ambientais. Também foram realizados testes estatísticos inferenciais e descritivos cujas ferramentas foram implementadas em planilhas de cálculo para utilização em procedimentos necessários a parâmetros de validação, como estabilidade de soluções, linearidade, limites de detecção e quantificação, recuperação e efeito matriz / Abstract: The literature review points to a growing number of studies in environmental matrices which identify and quantify various pollutants. The study of these substances results in large data sets that need to be properly considered for the interpretation to be as more reliable as possible. On the other hand, the analytical methods used in these determinations involve several steps and parameters that can influence the analytical reliability. Analytical results that do not identify reliability can lead to completely flawed decision-making in the environmental area. In this context, it is essential laboratories to have adequate and objective criteria to demonstrate that the test methods performed lead to reliable results and appropriate to the desired quality. This work aimed to study the statistical and chemometric procedures for the determination of polycyclic aromatic hydrocarbons (PAHs) by liquid chromatography high performance with fluorescence detection (HPLC-FLU). It focuses on the optimization of statistical procedures for validating the method for obtaining and processing signals from analytical PAH as well as the study of the behavior of different analytes in environmental matrices. In this sense were used chemometric tools, such as principal component analysis, hierarchical cluster analysis and experimental design techniques for exploratory data analysis, indicating the similarities between PAH and chromatographic profile in environmental matrices. Tests were made descriptively and use of inferential statistical tools which have been implemented in spreadsheets for use in procedures required for validation parameters, such as stability of solutions, linearity, limits of detection and quantification, recovery and matrix effect. For studies on PAHs was determined as the optimal use of 5 replicates, the use of labor standards for a maximum period of 30 days and the development of analytical curves in the matrix / Mestre

Thesis (MTech (Chemistry))--Cape Peninsula University of Technology, 2016. / Wines contain a number of phenolic compounds, belonging to non-flavonoid and flavonoid complexes. Phenolic compounds in wine are responsible for wine colour, astringency, and bitterness. Saccharomyces cerevisiae yeast is normally used in winemaking but it has been proved to decrease the phenolic content in wines. Current research on the use of non-Saccharomyces yeast in winemaking has produced better quality wines than S. cerevisiae yeast therefore improving the sensory profile of wine. This study evaluated effect of Torulaspora delbrueckii yeast on the phenolic content of experimental wines derived from Chenin blanc grapes. A reversed phase high-performance liquid chromatographic (RP-HPLC) method was used for the identification and quantitation of the phenolic compounds. The difference test method was used to determine the sensory attributes of wines. The data was subjected to analysis of variance to compare treatment differences between the wines and principal component analysis to establish possible correlation between the data sets. Furthermore, a gas chromatographic-flame ionization detection method (GC-FID) was used for the quantification of volatile compounds in the wines. In this work, wines made with T. delbrueckii strain M2/1 had high concentration of (+)-catechin, caffeic acid, ferulic acid and p-coumaric acid in all studied vintages. Wines made with VIN13 had higher concentrations of flavan-3-ols, compared to wines made with M2/1 and 654. In sensory evaluation, M2/1 wines were prominent in astringency and complexity. Yeast strain M2/1, also attributed to body and complexity of the wine. However, in this study no correlations were observed between the phenolic content and sensory attributes and vice versa. The quality of wine cannot be concluded by chemical or sensory analysis alone, but the data sets are complementary. Although the phenolic concentration of wines made with S. cerevisiae strain (VIN 13) and T. delbrueckii (M2/1) were similar in measured phenolic concentrations, they had different sensory attributes. Wines made during the 2013 vintage indicated the importance of the use of a strain with higher enzyme activity and high fermentation rate. There is minimal to no skin contact in white winemaking. Therefore, the use of a yeast strain with an increased enzyme activity can facilitate the extraction of phenolics from grape, resulting in wine with improved quality.

A diferença na atividade terapêutica, farmacocinética e / ou farmacodinâmica entre os enantiômeros de fármacos quirais impulsionou a necessidade de estudar e desenvolver métodos para determinação exata e precisa da pureza enantiomérica dos produtos farmacêuticos. Inicialmente, os enantiômeros foram separados em escala analítica. As condições analíticas foram adaptadas para a escala semi-preparativa para a obtenção enantiômeros puros. Os enantiômeros do bisoprolol foram separados através de Cromatografia Líquida de Alta Eficiência. Foi adotado o sistema direto de separação, fase normal, utilizando coluna Chiralcel OD (250 x 4,6 mm id). A fase móvel foi composta por hexano: etanol: dietilamina (80:20:0.2, v / v / v), vazão de 1mL/min e detecção em UV a 273 nm. A separação dos enantiômeros (R)-bisoprolol e (S)-bisoprolol foi obtida com sucesso em escala analítica e semi-preparativa. Considerando as características de uma separação quiral, podemos concluir que os resultados são eficazes, por ser uma separação rápida e seletiva. / The difference in therapeutic activity, pharmacokinetics, and / or pharmacodynamics between enantiomers of chiral drugs has raised the need to study and develop methods for accurate and precise determination of enantiomeric purity of pharmaceutical products. Initially, the enantiomers were separation in analytical scale. The analytical conditions were scaled up to semi-preparative level to obtain pure enantiomers. The enantiomers of bisoprolol were separated with high-performance liquid chromatography. Direct separation system was adopted in normal phase mode using Chiralcel OD column (250 x 4.6 mm id). The mobile phase was composed of hexane:ethanol:diethylamine (80:20:0.2, v/v/v), flow rate of 1mL/min and UV detection was made at 273 nm. The separation of enantiomers, (R)-bisoprolol and (S)-bisoprolol was successfully obtained in analytical and semi-preparative scale. Considering the characteristics of a chiral separation, we can conclude that the results are effective, because it is a fast and selective separation.