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Os frutos de S. saponaria, espécie bastante abundante em São Carlos e outras regiões do Brasil são ricos em glicosídeos anfifílicos, ou seja, compostos por uma parte polar e outra apolar. Devido a esta peculiaridade tendem a formar espuma mostrando propriedades químicas semelhantes às dos tensoativos. Alguns destes glicosídeos pertencem à classe das saponinas e são constituídos por uma aglicona de estrutura carbônica a qual está ligada a uma ou duas cadeias de açúcar. O interesse pelas propriedades tensoativas deste glicosídeos motivou o estudo destas substâncias para uso em eletroforese capilar. Os frutos foram extraídos com metanol e fracionados em coluna cromatográfica preparativa utilizando sephadex LH-20 como fase estacionária. Após a eluição, as frações foram analisadas por espectrometria de massas e estudadas por eletroforese capilar. A eletroforese capilar de zona mostrou-se uma técnica viável para o estudo das frações obtidas. Embora um grau de pureza elevado não tenha sido alcançado, a fração B foi utilizada como aditivo para tampão em cromatografia eletrocinética micelar (MEKC) e uma interação diferenciada foi observada do nitrobenzeno com o tampão aditivado em relação ao tampão com SDS puro. / Sapindus saponaria is a very abundant species in São Carlos and others regions of Brazil. Fruits of S. saponaria have a high content of glycosides which possess well-defined regions of hydrophobic and hydrophilic feature denominated amphiphilic molecule. Thus, this can form a foam showing chemical proprieties equals to surfactants. Some glycosides belong to class of saponins and are composed of carbonic structure designates aglycone, which is linked in one or two sugar chains. The purpose of the present study was the use of glycosides due to their surfactants properties in further applications of capillary electrophoresis. The extraction of crude fruits was carried out with methanol and this extract was fractionated in Sephadex LH-20. The fractions were analyzed for mass spectrometry and studied in capillary electrophoresis (CE). This is a feasible technique for the study of fractions obtained. Although a high degree of purity was not reached, the fraction B were used as additive for the electrolyte background in MEKC. The nitrobenzene showed different interaction with micellar system in the electrolyte background with fraction B.

A ezetimiba e a sinvastatina são fármacos hipolipidêmicos. A ezetimiba pertence à nova classe das 2 - azetidinona, inibidores e bloqueadores do colesterol intestinal. A sinvastatina pertence à classe dos inibidores competitivos da hidroxi-3-metilglutarilcoenzima A redutase (HMG-CoA), que é a última etapa regulada na síntese do colesterol. O objetivo do trabalho foi desenvolver e validar métodos por cromatografia liquida de alta eficiência (HPLC) e eletroforese capilar (CE), rápidos, seletivos e confiáveis para determinação dos hipolipidêmicos em formulações farmacêuticas. A separação cromatográfica foi realizada usando coluna Nano separation technologies (NST) Cianopropril (CN) (150 mmx 4,6 mm, com partícula 3,5 µm), e eluição isocrático usando água purificada: acetonitrila (48:52, v/v); vazão 0,8 mL/min e volume de injeção de 20 µL. A temperatura da coluna foi de 35 ºC e a detecção foi realizada com detector na região do UV em 238 nm. O método por cromatografia eletrocinética micelar (MEKC) foi desenvolvido utilizando capilar de sílica fundida 30 cm (comprimento efetivo) x 50 µm d.i. e eletrólito constituído de tetraborato de sódio (TBS) 20 mmol L-1: dodecil sulfato de sódio (SDS) 30 mmol L-1, pH 9,0 ajustado com 10% ácido fosfórico: acetonitrila 12% v/v. O tempo de injeção foi de 3 segundos com pressão hidrodinâmica de 20 mbar, voltagem aplicada de 30 kV e detecção no UV em 238 nm. Os métodos analíticos foram validados de acordo com os requerimentos vigentes da ANVISA, ICH e Farmacopéia Americana. Portanto, os métodos propostos demonstraram ser lineares, precisos, exatos e adequados para quantificação simultânea da ezetimiba e sinvastatina em formas farmacêuticas sólidas. / Ezetimibe and simvastatin are hipolipidemic drugs. Ezetimibe belongs to a new class of 2 - azetidione, inhibitors and blockers of intestinal chrolesterol. Simvastatin belongs a class of competitive inhibitors of 3-hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase, which is the last regulated step in the cholesterol synthesis. The aim of this project was to develop and validate fast, selective and reliable chromatographic and electrophoretic methods, to determine hypolipidemic drugs in pharmaceutical formulations. The chromatographic separation was carried out on a Nano separation technologies (NST) Cyanpropyl (CN) (150 mm x 4,6 mm, 3,5 µm), isocratic elution using purified water: acetonitrila (48:52 v/v), the flow rate was 0,8 mL/min and the injection volume was 20 µL. The column temperature was kept at 35 ºC and detection wavelength was set at 238 mn. The micellar electrokinetic chromatographic method was developed using a fused silica capillary column 30 cm (effective length) x 50 µm i.d, the electrolyte was constituted of 20 mmol L-1 tetraborate buffer solution: 30 mmol L-1 sodium dodecyl sulphate (SDS), pH 9.0 adjusted with 10% phosphoric acid: 12 % v/v acetonitrile. The injection time was 3 s at 20 mbar, the applied voltage was 30 kV and detection was set at 238 nm. The both methods were developed and validated according to ANVISA, ICH and US Pharmacopeia guidelines. Therefore, the proposed methods proved to be linear, precise, accurate and suitable for simultaneous quantitation of ezetimibe and simvastatin in solid pharmaceutical formulations.

The research presented in this thesis involves the application of chiral cationic and anionic surfactants for simultaneous enantioseparation of structurally similar compounds in capillary electrophoresis (CE) and CE coupled to mass spectrometry (CE-MS). The first chapter briefly introduces the fundamentals of CE and CE-MS, emphasizing the micellar electrokinetic chromatography (MEKC) and MEKC-MS techniques, as well as ionic liquids (ILs) and affinity CE (ACE). In chapter 2, a mixture of five racemic profen (PROF) drugs are simultaneously separated with the combined use of 2,3,6-tri-O-methyl-β-cyclodextrin (TM-β-CD) and IL-type surfactant, N-undecenoxycarbonyl-L-leucinol bromide (L-UCLB). Enantioseparations of these PROFs are optimized using a standard recipe containing 35.00 mM TM-β-CD, 5.00 mM sodium acetate at pH 5.0, and varying the concentration as well as chain length of the IL surfactants. The batch-to-batch reproducibility of L-UCLB is found to be acceptable in terms of enantiomeric resolution, and migration time. A competitive inhibition mechanism is proposed to investigate the ternary interactions among TM-β-CD, ILs, and PROFs. The apparent binding constant of TM-β-CD to L-UCLB is estimated by nonlinear and linear plotting methods. The binding constants of one representative PROF (e.g., fenoprofen) to TM-β-CD and to L-UCLB are estimated by a secondary plotting approach. The R- and S-fenoprofen having different binding constant values, resulting in the enantioseparation due to the synergistic effect of TM-β-CD and L-UCLB. The R- and S-configurations of barbiturates display differences in potency and biological activity. In Chapter 3, a multivariate MEKC-ESI-MS approach for the simultaneous analysis of the racemic mixture of three barbiturates is presented. The chiral selector employed is the polymeric surfactant polysodium N-undecenoxycarbonyl-L-isoleucinate. The central composite design is used to optimize the chiral resolution, decrease the total analysis time, and improve the ESI-MS signal-to-noise ratio for these barbiturates. In preliminary experiments, the ranges of the factors investigated in the multivariate approaches are determined. Then the multivariate optimizations are conducted to determine the best overall chiral resolution with shortest possible run times for barbiturates. The limit of detection of ESI-MS is several folds higher compared to the UV detection. The predicted optimum results are in good agreement with the experimental data.

Micellar electrokinetic chromatography (MEKC) and capillary electrochromatography (CEC) are two of the major capillary electrophoresis (CE) modes that have been interfaced to mass spectrometry (MS) for sensitive and selective analysis of chiral compounds. This research combines these two modes and expands their applications in chiral CE analysis. Chapter 1 is a review of amino acid based molecular micelles used in MEKC-MS for enantioselective analysis over the past five years. In this chapter, a typical MEKC-MS experiment setup as well as detailed standard operating procedure in synthesis of molecular micelles and running a typical MEKC-MS experiment using the molecular micelles is discussed. Chapter 2 described a multivariate MEKC-MS optimization for the simultaneous analysis of two negatively charged model chiral compounds in negative ion mode with molecular micelles. In this chapter, a central composite design (CCD) is used to first construct a series of experiments to optimize all the important MEKC-MS parameters. Next, response surface methodology (RSM) was used to analyze the interactions between the factors, picking up the best separation and detection conditions, predicting the result of the chiral separation/MS detection, and finally running the actual experiment and comparing the chromatographic results with the predicted parameters. Chapter 3 demonstrates a similar multivariate MEKC-MS optimization for analysis of a positively charged model chiral compound in a positive ion mode. The same CCD and RSM methods were used to optimize the separations and MS sensitivity. Chapter 4 describes a chiral analysis of four neutral benzoin derivatives (hydrobenzoin, benzoin, benzoin methyl ether, and benzoin ethyl ether) using MEKC coupled to atmospheric pressure photo-ionization mass spectrometry (APPI-MS). The same multivariate experimental design strategy was used to optimize the MEKC as well as APPI-MS parameters. Simultaneous chiral separation of all four benzoin derivatives was achieved with high detection sensitivity compared to UV-detection. Chapter 5 introduces a novel one-pot synthesis scheme for an acryloyl-terminated, carbamate-linked surfactant-bound monolith with leucine head group and different chain lengths. The method promises to open up the discovery of new amino acid based polymeric monoliths for chiral separations and enhanced chemoselectivity for simultaneous chiral separations and enhanced detection in CEC and CEC-MS. In Chapter 6, five amide-linked surfactant-bound monoliths with different chain lengths and head groups (leucine, valine, and phenylalanine) were synthesized and characterized. Enantioseparation of several test compounds was achieved by CEC using the monolithic columns. One of the chiral surfactant, sodium 11-acrylamidoundecanoyl-L-leucinate (SAAUL), was polymerized in aqueous solution under 60Co radiation to form molecular micelle poly-SAAUL. MEKC experiments were carried out with the poly-SAAUL molecular micelle to separate ten cationic chiral compounds. The result was compared with the CEC separation using the AAUL monolithic column. This study is the first comparison of chiral CEC and MEKC with the same surfactant monomer, which has the capability of forming both chiral stationary phase for CEC and chiral pseudophase for MEKC.

Os frutos de S. saponaria, espécie bastante abundante em São Carlos e outras regiões do Brasil são ricos em glicosídeos anfifílicos, ou seja, compostos por uma parte polar e outra apolar. Devido a esta peculiaridade tendem a formar espuma mostrando propriedades químicas semelhantes às dos tensoativos. Alguns destes glicosídeos pertencem à classe das saponinas e são constituídos por uma aglicona de estrutura carbônica a qual está ligada a uma ou duas cadeias de açúcar. O interesse pelas propriedades tensoativas deste glicosídeos motivou o estudo destas substâncias para uso em eletroforese capilar. Os frutos foram extraídos com metanol e fracionados em coluna cromatográfica preparativa utilizando sephadex LH-20 como fase estacionária. Após a eluição, as frações foram analisadas por espectrometria de massas e estudadas por eletroforese capilar. A eletroforese capilar de zona mostrou-se uma técnica viável para o estudo das frações obtidas. Embora um grau de pureza elevado não tenha sido alcançado, a fração B foi utilizada como aditivo para tampão em cromatografia eletrocinética micelar (MEKC) e uma interação diferenciada foi observada do nitrobenzeno com o tampão aditivado em relação ao tampão com SDS puro. / Sapindus saponaria is a very abundant species in São Carlos and others regions of Brazil. Fruits of S. saponaria have a high content of glycosides which possess well-defined regions of hydrophobic and hydrophilic feature denominated amphiphilic molecule. Thus, this can form a foam showing chemical proprieties equals to surfactants. Some glycosides belong to class of saponins and are composed of carbonic structure designates aglycone, which is linked in one or two sugar chains. The purpose of the present study was the use of glycosides due to their surfactants properties in further applications of capillary electrophoresis. The extraction of crude fruits was carried out with methanol and this extract was fractionated in Sephadex LH-20. The fractions were analyzed for mass spectrometry and studied in capillary electrophoresis (CE). This is a feasible technique for the study of fractions obtained. Although a high degree of purity was not reached, the fraction B were used as additive for the electrolyte background in MEKC. The nitrobenzene showed different interaction with micellar system in the electrolyte background with fraction B.

A ezetimiba e a sinvastatina são fármacos hipolipidêmicos. A ezetimiba pertence à nova classe das 2 - azetidinona, inibidores e bloqueadores do colesterol intestinal. A sinvastatina pertence à classe dos inibidores competitivos da hidroxi-3-metilglutarilcoenzima A redutase (HMG-CoA), que é a última etapa regulada na síntese do colesterol. O objetivo do trabalho foi desenvolver e validar métodos por cromatografia liquida de alta eficiência (HPLC) e eletroforese capilar (CE), rápidos, seletivos e confiáveis para determinação dos hipolipidêmicos em formulações farmacêuticas. A separação cromatográfica foi realizada usando coluna Nano separation technologies (NST) Cianopropril (CN) (150 mmx 4,6 mm, com partícula 3,5 µm), e eluição isocrático usando água purificada: acetonitrila (48:52, v/v); vazão 0,8 mL/min e volume de injeção de 20 µL. A temperatura da coluna foi de 35 ºC e a detecção foi realizada com detector na região do UV em 238 nm. O método por cromatografia eletrocinética micelar (MEKC) foi desenvolvido utilizando capilar de sílica fundida 30 cm (comprimento efetivo) x 50 µm d.i. e eletrólito constituído de tetraborato de sódio (TBS) 20 mmol L-1: dodecil sulfato de sódio (SDS) 30 mmol L-1, pH 9,0 ajustado com 10% ácido fosfórico: acetonitrila 12% v/v. O tempo de injeção foi de 3 segundos com pressão hidrodinâmica de 20 mbar, voltagem aplicada de 30 kV e detecção no UV em 238 nm. Os métodos analíticos foram validados de acordo com os requerimentos vigentes da ANVISA, ICH e Farmacopéia Americana. Portanto, os métodos propostos demonstraram ser lineares, precisos, exatos e adequados para quantificação simultânea da ezetimiba e sinvastatina em formas farmacêuticas sólidas. / Ezetimibe and simvastatin are hipolipidemic drugs. Ezetimibe belongs to a new class of 2 - azetidione, inhibitors and blockers of intestinal chrolesterol. Simvastatin belongs a class of competitive inhibitors of 3-hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase, which is the last regulated step in the cholesterol synthesis. The aim of this project was to develop and validate fast, selective and reliable chromatographic and electrophoretic methods, to determine hypolipidemic drugs in pharmaceutical formulations. The chromatographic separation was carried out on a Nano separation technologies (NST) Cyanpropyl (CN) (150 mm x 4,6 mm, 3,5 µm), isocratic elution using purified water: acetonitrila (48:52 v/v), the flow rate was 0,8 mL/min and the injection volume was 20 µL. The column temperature was kept at 35 ºC and detection wavelength was set at 238 mn. The micellar electrokinetic chromatographic method was developed using a fused silica capillary column 30 cm (effective length) x 50 µm i.d, the electrolyte was constituted of 20 mmol L-1 tetraborate buffer solution: 30 mmol L-1 sodium dodecyl sulphate (SDS), pH 9.0 adjusted with 10% phosphoric acid: 12 % v/v acetonitrile. The injection time was 3 s at 20 mbar, the applied voltage was 30 kV and detection was set at 238 nm. The both methods were developed and validated according to ANVISA, ICH and US Pharmacopeia guidelines. Therefore, the proposed methods proved to be linear, precise, accurate and suitable for simultaneous quantitation of ezetimibe and simvastatin in solid pharmaceutical formulations.

This diploma thesis deals with the determination of glucosinolates in plant material by capillary electrophoresis and high performance liquid chromatography. The chemical structure, biosynthesis, degradation, and also biological effects of glucosinolates are described. One part of this work also deals with the methods, which glucosinolates in plant materials were determined by. The experimental part describes the separation of intact glucosinolates by capillary electrophoresis and high performance liquid chromatography. Two plant materials were available for the determination of glucosinolates, namely lyophilized rapeseed leaves and broccoli juice. Micellar electrokinetic chromatography using a cationic surfactant was used to determine intact glucosinolates by capillary electrophoresis. After finding the optimal conditions for the separation of intact glucosinolates, it was found that it is impossible to determine these substances in plant samples. The reason was interference from the matrix, which interfered with this determination. While using high performance liquid chromatography under optimal conditions, some of the intact glucosinolates were identified in a rapeseed plant sample. Furthermore, the calibration dependencies of individual glucosinolates were obtained and the recovery and...

Capillary electrophoresis (CE) is an analytical chemistry approach that allows for the efficient separation by charge of diverse classes of compounds for analysis, including secondary metabolites. The goal of this work was to optimize a buffer system for plant tissue analysis using micellar electrokinetic chromatography (MEKC), and by doing so to understand the role of buffer components in the performance of this form of capillary electrophoresis. In this experiment we implemented a factorial design to optimize buffer composition for separating plant tissue and secondary metabolites. The results of this experiment will be used to optimize a universal buffer for MEKC analysis that can be used on any variety of plant tissues. To determine the feasibility of this, a diverse set of plant secondary metabolite chemical standards in solution were tested as well as Helianthus annuus tissue to confirm the separation in a real biological sample. The results of this optimization yield insights into the utility of buffer components like electrolyte and pH for MEKC separation.

Micellar electrokinetic chromatography (MEKC), one of the major capillary electrophoresis (CE) modes, has been interfaced to mass spectrometry (MS) to provide high sensitivity and selectivity for analysis of chiral compounds. The research in this dissertation presents the development of novel polymeric glucopyranoside based molecular micelles (MoMs) (aka. polymeric surfactants) and their application in chiral MEKC-MS. Chapter 1 is a review of chiral CE-MS - in the period 2010-2015. In this chapter, the fundamental of chiral CE and CE-MS is illustrated and the recent developments of chiral selectors and their applications in chiral EKC-MS, CEC-MS and MEKC-MS are discussed in details. Chapter 2 introduces the development of a novel polymeric α-D-glucopyranoside based surfactants, n-alkyl-α-D-glucopyranoside 4,6-hydrogen phosphate, sodium salt. In this chapter, polymeric α-D-glucopyranoside-based surfactants with different chain length and head groups have been successfully synthesized, characterized and applied as compatible chiral selector in MEKC-ESI-MS/MS. or the enantioseparation of ephedrines and β-blockers. Chapter 3 continues to describe the employment of polymeric glucopyranoside based surfactants as chiral selector in MEKC-MS/MS. The polymeric β-D-glucopyranoside based surfactants, containing charged head groups such as n-alkyl β-D-glucopyranoside 4,6-hydrogen phosphate, sodium salt and n-alkyl β-D-glucopyranoside 6-hydrogen sulfate, monosodium salt were able to enantioseparate 21 cationic drugs and 8 binaphthyl atropisomers (BAIs) in MEKC-MS/MS, which promises to open up the possibility of turning an analytical technique into high throughput screening of chiral compounds. Physicochemical properties and enantioseparation capability of polymeric β-D-glucopyranoside based surfactants with different head groups and chain lengths were compared. Moreover, the comparison of polymeric α- and β-D-glucopyranoside 4,6-hydrogen phosphate, sodium salt were further explored with regard to enantioseparations of ephedrine alkaloids and b-blockers. The concept of multiplex chiral MEKC-MS for high throughput quantitation is demonstrated for the first time in scientific literature.