The purpose of this project was to investigate if retention of polar compounds that are given to treat tuberculosis, diabetes, inflammatory bowel disease and childhood leukemia could be obtained with HILIC separation. By varying different parameters for different types of columns the compounds were analyzed with the aim of finding guidelines for future method optimizations. To perform these analyzes three different columns were tested – ZIC-HILIC (silica-based with zwitterions), ZIC-pHILIC (polymer-based with zwitterions) and XBridge Amide (amide functions). The results were evaluated with selected quality measures. The parameters being varied were pH, temperature, flow rate, type of buffer and ionic strength. In addition, comparisons between isocratic and gradient separations were performed.   Over 1 000 analyzes were conducted in which retention of 16 of total 18 substances were successfully obtained with HILIC. The columns that generated the best results in terms of greatest number of identified compounds were ZIC-HILIC and XBridge Amide. ZIC-pHILIC offered a wide pH range but generally gave inferior chromatography. The influence of the different parameters on the results has not been investigated in sufficient scope. This means that no specific methods for the different drug groups can be reported.

Hydrophilic interaction liquid chromatography is a frequently used separation method for analysis of polar compounds. It is an alternative method to reversed-phase chromatography, where these compounds show insufficient or very weak retention. A high number of stationary phases are currently available for HILIC and new ones are still being developed. The aim of this diploma thesis was to characterize and compare three relatively new HILIC columns containing an unmodified silica gel (HILIC-A), aminopropyl modified silica (HILIC-B) and polyhydroxyl chain modified silica (HILIC-N) as stationary phase. Based on the study of the effect of acetonitrile content in mobile phase on the retention of a model set of peptides, a multimodal retention mechanism was demonstrated. Analysis of 18 model analytes with different pKa values showed, that the composition of aqueous part of mobile phase (buffer), more specifically it's concentration and pH value, has a significant impact on retention of ionized analytes and peptides on the studied stationary phases. A significant contribution of ionic interactions to retention was observed on HILIC-B and HILIC-A columns. The retention of basic compounds on the HILIC-B column increased with increasing ionic strength, while it decreased on the HILIC-A column. Increasing the...

Há fortes evidências dos efeitos negativos da exposição gestacional a poluentes atmosféricos. No entanto, mecanismos de atuação de poluentes não são bem compreendidos. Alterações fisiológicas anômalas na progenitora, durante o período de gravidez, podem causar mudanças permanentes na prole, que podem desencadear futuras doenças na vida adulta. Portanto, o estudo dessas alterações maternas é importante. A metabolômica é definida como a análise global do metaboloma de um organismo em experimentos comparativos, com o objetivo de observar mudanças relativas da abundância dos metabólitos, o aparecimento ou desaparecimento de metabólitos, e pode fornecer uma melhor compreensão dos mecanismos de funcionamento celular dos organismos a nível molecular. Nesse trabalho, um estudo experimental de exposição gestacional materna, ao material particulado fino (MP2,5), foi realizado, para avaliar os efeitos dessa exposição no metabolismo, por meio da análise metabolômica global da urina de camundongos fêmeas progenitoras expostas ao MP2,5 (grupo teste) ou a ar filtrado (grupo controle) durante a gestação. Um método cromatográfico e de preparo de amostra para metabolômica urinária por HILIC-MS foram otimizados. Para a otimização da condição cromatográfica, foram investigados a influência de aditivos, concentração de sal e pH da fase móvel, bem como, a rampa do gradiente. A melhor condição foi escolhida por meio da avaliação do formato de pico, da intensidade relativa e do CV do tempo de retenção para 15 m/z selecionados, assim como, pelo número total de molecular features e CV da intensidade desses molecular features. A melhor condição obtida apresenta 20 mmol/L de formiato de amônio em sua composição do solvente B da fase móvel e 95% acetonitrila e 5% solução aquosa 400 mmol/L de formiato de amônio na composição do solvente A. Para o preparo de amostra, foram examinados diferentes solventes orgânicos e suas misturas, para a precipitação de proteínas da urina. O isopropanol foi o solvente apresentou os melhores resultados para o preparo de amostra. Dessa forma, com o método analítico otimizado, as amostras de urina de camundongos fêmeas prenhas foram submetidas à analise metabolômica global por HILIC-MS. O metaboloma dos animais foi bastante alterado pela exposição gestacional ao material particulado. Observou-se alteração dos níveis de carnitinas, aminoácidos, peptídeos, entre outros. Há também indícios de que a poluição atmosférica alterou a microbiota intestinal dos animais, devido ao aumento de N-óxido de trimetilamina, um metabólito que também é relacionado ao processo de aterosclerose. Níveis de metabólitos relacionados ao metabolismo da histidina também foram alterados devido a exposição ao MP2,5. Níveis de carnitina e acilcarnitinas foram aumentados no teste, sugerindo alteração da produção de energia na mitocontria. / There are strong evidences regarding negative effects of gestational exposure to air pollution. However, the mechanisms of action of air pollutants are not well established. Maternal anomalous physiological changes during pregnancy may cause permanent changes in offsprings, that might initiate future diseases in adult life. Therefore, the study of those maternal changes during pregnancy is important. Metabolomics is defined as the global analysis of the metabolome of an organism in comparative studies, for the measurement of relative changes in the metabolite abundance, appearance or disappearance. Metabolomics might provide a better understanding of cellular functioning at the molecular level. In this work, an experimental study of maternal gestational exposure to fine particulate matter (PM2.5) was accomplished to evaluate the effects of this exposure to the metabolism, by an untargeted metabolomics analysis of urine from pregnant mice exposed to PM2.5 or to filtered air during pregnancy. A chromatographic and sample preparation methods for urinary untargeted metabolomics analysis by HILIC-MS were optimized. For the chromatography optimization, the influence of mobile phase additives, salt concentration and pH, as well as, the gradient ramp were investigated. The best condition was chosen by the evaluation of peak shape, relative intensity and retention time CV of 15 selected m/z, as well as, the total number of molecular features and the intensity CV of those molecular features. The best condition comprises of 20 mmol/L of ammonium formate as solvent B, and 95% acetonitrile and 5% 400 mmol/L of ammonium formate as solvent A, in the composition of the mobile phase. For the sample preparation, different solvents, along with, their mixtures were examined for the urine protein precipitation. Isopropanol was the solvent that presented the best results for sample preparation. Thus, after the analytical method optimized, urine samples from the progenitors were submitted to untargeted metabolomics analysis by HILIC-MS. The animals\' metabolome were significantly changed by the gestational exposure to particulate matter. It was observed changes in the levels of carnitines, amino acids, peptides, among others. There is some indication that the air pollution has altered the gut microbiota, due to the enhancement of trimethylamine N-oxide (TMAO), a metabolite that is also related to the atherosclerosis process. The level of metabolites related to histidine metabolism were also altered due to PM2.5 exposure. Carnitine and acylcarnitine levels were also increased in the test group, suggesting an altered energy production in the mitochondria.

Há fortes evidências dos efeitos negativos da exposição gestacional a poluentes atmosféricos. No entanto, mecanismos de atuação de poluentes não são bem compreendidos. Alterações fisiológicas anômalas na progenitora, durante o período de gravidez, podem causar mudanças permanentes na prole, que podem desencadear futuras doenças na vida adulta. Portanto, o estudo dessas alterações maternas é importante. A metabolômica é definida como a análise global do metaboloma de um organismo em experimentos comparativos, com o objetivo de observar mudanças relativas da abundância dos metabólitos, o aparecimento ou desaparecimento de metabólitos, e pode fornecer uma melhor compreensão dos mecanismos de funcionamento celular dos organismos a nível molecular. Nesse trabalho, um estudo experimental de exposição gestacional materna, ao material particulado fino (MP2,5), foi realizado, para avaliar os efeitos dessa exposição no metabolismo, por meio da análise metabolômica global da urina de camundongos fêmeas progenitoras expostas ao MP2,5 (grupo teste) ou a ar filtrado (grupo controle) durante a gestação. Um método cromatográfico e de preparo de amostra para metabolômica urinária por HILIC-MS foram otimizados. Para a otimização da condição cromatográfica, foram investigados a influência de aditivos, concentração de sal e pH da fase móvel, bem como, a rampa do gradiente. A melhor condição foi escolhida por meio da avaliação do formato de pico, da intensidade relativa e do CV do tempo de retenção para 15 m/z selecionados, assim como, pelo número total de molecular features e CV da intensidade desses molecular features. A melhor condição obtida apresenta 20 mmol/L de formiato de amônio em sua composição do solvente B da fase móvel e 95% acetonitrila e 5% solução aquosa 400 mmol/L de formiato de amônio na composição do solvente A. Para o preparo de amostra, foram examinados diferentes solventes orgânicos e suas misturas, para a precipitação de proteínas da urina. O isopropanol foi o solvente apresentou os melhores resultados para o preparo de amostra. Dessa forma, com o método analítico otimizado, as amostras de urina de camundongos fêmeas prenhas foram submetidas à analise metabolômica global por HILIC-MS. O metaboloma dos animais foi bastante alterado pela exposição gestacional ao material particulado. Observou-se alteração dos níveis de carnitinas, aminoácidos, peptídeos, entre outros. Há também indícios de que a poluição atmosférica alterou a microbiota intestinal dos animais, devido ao aumento de N-óxido de trimetilamina, um metabólito que também é relacionado ao processo de aterosclerose. Níveis de metabólitos relacionados ao metabolismo da histidina também foram alterados devido a exposição ao MP2,5. Níveis de carnitina e acilcarnitinas foram aumentados no teste, sugerindo alteração da produção de energia na mitocontria. / There are strong evidences regarding negative effects of gestational exposure to air pollution. However, the mechanisms of action of air pollutants are not well established. Maternal anomalous physiological changes during pregnancy may cause permanent changes in offsprings, that might initiate future diseases in adult life. Therefore, the study of those maternal changes during pregnancy is important. Metabolomics is defined as the global analysis of the metabolome of an organism in comparative studies, for the measurement of relative changes in the metabolite abundance, appearance or disappearance. Metabolomics might provide a better understanding of cellular functioning at the molecular level. In this work, an experimental study of maternal gestational exposure to fine particulate matter (PM2.5) was accomplished to evaluate the effects of this exposure to the metabolism, by an untargeted metabolomics analysis of urine from pregnant mice exposed to PM2.5 or to filtered air during pregnancy. A chromatographic and sample preparation methods for urinary untargeted metabolomics analysis by HILIC-MS were optimized. For the chromatography optimization, the influence of mobile phase additives, salt concentration and pH, as well as, the gradient ramp were investigated. The best condition was chosen by the evaluation of peak shape, relative intensity and retention time CV of 15 selected m/z, as well as, the total number of molecular features and the intensity CV of those molecular features. The best condition comprises of 20 mmol/L of ammonium formate as solvent B, and 95% acetonitrile and 5% 400 mmol/L of ammonium formate as solvent A, in the composition of the mobile phase. For the sample preparation, different solvents, along with, their mixtures were examined for the urine protein precipitation. Isopropanol was the solvent that presented the best results for sample preparation. Thus, after the analytical method optimized, urine samples from the progenitors were submitted to untargeted metabolomics analysis by HILIC-MS. The animals\' metabolome were significantly changed by the gestational exposure to particulate matter. It was observed changes in the levels of carnitines, amino acids, peptides, among others. There is some indication that the air pollution has altered the gut microbiota, due to the enhancement of trimethylamine N-oxide (TMAO), a metabolite that is also related to the atherosclerosis process. The level of metabolites related to histidine metabolism were also altered due to PM2.5 exposure. Carnitine and acylcarnitine levels were also increased in the test group, suggesting an altered energy production in the mitochondria.

This work focuses on the determination of inositol in drugs and food supplements using high-performance liquid chromatography in HILIC mode. The work is divided into several sections, it discusses the characteristics of analyzed myo-inositol including methods for saccharides determination. A chapter is detached for high-performance liquid chromatography. One of the work goals was also chromatography columns comparison. The experimental part includes measurement conditions, methods of sample preparation evaluated data and results including the discussion. Two real samples were analyzed, in both of them the content of myo-inositol was declared by the producer.

The glycosylation patterns of monoclonal antibodies (Mabs) have become very important in determining therapeutic abilities of many drugs. The thesis studied 3 cell lines producing humanized Mabs in the presence of variable concentrations of the reducing agent dithiothreitol (DTT) to artificially lower the CRP and affect glycan patterns. A new high-throughput hydrophilic interaction chromatography (HILIC) method was developed and used to show a decrease in the Galactosylation Index (GI) of NS0 IgG1 by as much as 50% in cultures with CRP values lower than -100 mV. The shift in GI was unique to NS0 cultures; CHO DP-12 indicated no significant change in GI but did have a 7% increase in fucosylated species in cultures with higher [DTT]. Furthermore no DTT related shifts were observed in any of the CHO EG2-hFc glycans. EG2-hFc did however have an exceptionally high GI of 0.625 compared to GIs of 0.245 in DP-12 and 0.314 in NSO. Another component of the trials determined, using S35 radiolabeling, that the assembly pathway of IgG1 progressed via HC→HC2→HC2LC→HC2LC2 and that the ratio of heavy chain dimer to heavy chain monomer increased greatly over time for cultures with higher DTT concentrations. The increase in heavy chain dimers and lower GI appear to be correlated, possibly due to disruption of the disulfide bonds between LC and HC within the Golgi. This disruption in disulfide bonds affecting galactosyltransferase (GalT) activity is supported by the findings that the partially reduced fragments of IgG1; HC and HC2, are less galactosylated than the HC2LC and whole IgG1 when treated with GalT. When native and agalactosylated EG2-hFc and IgG1 were treated with GalT in vitro, EG2-hFc exhibited an almost 10 fold higher activity level. The cause for the higher activity may be due to overall size difference or point mutations in the Fc region of EG2-hFc. Through the manipulation of CRP, glycan patterns can be influenced however the effect is not universal and must be determined on a per cell line basis. Furthermore, EG2-hFc’s higher GI value may translate into better in vivo activity as a therapeutic and determination of reasons for the high GI may lead to better means for future glycoengineering. / February 2015

Hydrophilic interaction liquid chromatography is in the present time very often used for separation of polar compounds which are complicated separated in reverse phase HPLC or they are not sufficiently retained. Various stationary phases suitable for HILIC are commercially available and new stationary phases are still developing. In this work bare silica and isopropyl cyclofructan modified silica stationary phases were compared. Chromatographic behavior of sets of four pentapeptides and five nonapeptides was tested in detail under hydrophilic interaction liquid chromatography. Additionally, mobile phase composition changes the types of interaction and their strengths were studied using linear free energy relationship method. Obtained results denoted that the main role in the retention mechanism on the isopropyl cyclofructan stationary phases play hydrogen bond acidity and dipolarity/polarizibility, while dispersion interactions are preferred in the mobile phase. On the other hand, bare silica stationary phase provides only one interaction type - hydrogen bond acidity. Key words: peptides, cyclofructan, LFER, HILIC

The main focus of this thesis is on hydrophilic interaction chromatography (HILIC) and the preparation of stationary phases for HILIC. The mechanism of HILIC is also discussed; a large part of the discussion has been adapted from a review written by me and professor Irgum for the Journal of Separation Science (ref 34). By reevaluating the literature we have revealed that the notion of HILIC as simply partitioning chromatography needed modification. However, our interest in the HILIC mechanism was mainly inspired by the need to understand how to construct the optimal HILIC stationary phase. The ultimate stationary phase for HILIC is still not found. My theory is that a non-charged stationary phase capable of retaining a full hydration layer even at extreme acetonitrile (> 85%) concentrations should give a HILIC stationary phase with a more pure partitioning retention behavior similar to that of a swollen C18 reversed phase. The preparation of a sorbitol methacrylate grafted silica stationary phase is one of our attempts at producing such a stationary phase. The preparation of such a grafted silica has been performed, but with huge difficulty and this work is still far from producing a column of commercial quality and reprodicibility. This thesis also discusses a new method for the initiation of atom transfer radical polymerization from chlorinated silica. This new grafting scheme theoretically results in a silica particle grafted with equally long polymer chains, anchored to the silica carrier by a hydrolytically stable silicon-carbon bond. The hydrolytic stability is especially important for HILIC stationary phases due to the high water concentration at the surface.

The aim of this diploma thesis is detailed characterization of new diol-based column, i.e. Torus DIOL designed for supercritical fluid chromatography, in hydrophilic interaction liquid chromatography. This stationary phase contains diol ligands bonded on BEH ("bridged ethylene hybrid") particles. The BEH sorbent is more stable at high pH, pressure and temperature than conventional silica sorbents. Five fluorinated pesticides were successfully separated on Torus DIOL column. Retention mechanism and effect of different parameters were tested by analysis of small polar compounds (nucleobases, uridine and its derivatives, acidic, neutral and basic compounds). Both partitioning of analytes between an aqueous layer deposited on the surface of the stationary phase and the mobile phase and adsorption of analytes on the stationary phase participate in the retention mechanism. Effect of the mobile phase aqueous part pH was investigated with the following buffering solutions: formic acid (pH = 2.1), 10mM ammonium acetate (pH = 4.7 and pH = 9.5). Obtained results showed that aqueous part pH significantly affects retention of acidic compounds according to their pKA values. Retention of all tested analytes on Torus DIOL column slightly increased with increasing ionic strength of mobile phase. The retention...

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