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Apesar da rápida expansão das aplicações da cromatografia líquida acoplada à espectrometria de massas em química clínica, a análise de metabólitos de baixo peso molecular e alta polaridade em matrizes biológicas ainda permanece como um grande desafio analítico. Dentre os compostos de grande importância no diagnóstico de doenças metabólicas que ainda carecem de melhores alternativas bioanalíticas destacam-se os aminoácidos. O presente estudo descreve o desenvolvimento e a validação de um novo método para a quantificação de 24 aminoácidos em plasma explorando a cromatografia líquida acoplada a espectrômetros de massas em tandem. Foi construído um método de detecção baseado em SRM (múltiplas reações selecionadas) com duas transições de massas para cada um dos 24 aminoácidos e os 19 padrões internos marcados com isótopos estáveis. Foram avaliadas três estratégias de separação cromatográfica e o melhor desempenho foi obtido com fase reversa com octadecilsilano (C18) com pareamento iônico com o ácido perfluoropentanoico. O método cromatográfico final permitiu a separação dos 24 aminoácidos, com resolução completa dos isômeros: leucina, isoleucina e allo-isoleucina, em 11 minutos incluindo o tempo de re-estabilização da coluna cromatográfica. Os limites de quantificação variaram em 113 fmol a 6 pmol injetados na coluna cromatográfica. A imprecisão obtida nos níveis testados para todos os aminoácidos foi inferior a 14%. O método apresentou linearidade para os intervalos testados chegando a 1,5 mmol.L-1 para vários compostos. Os ensaios de arraste mostraram que os limites máximos obtidos na linearidade não geram nenhuma interferência subsequente. A exatidão do método foi avaliada com amostras provenientes do programa de referência interlaboratorial European Research Network for evaluation and improvement of screening, Diagnosis and treatment of Inherited disorders of Metabolism (ERNDIM) e com o material de referência certificado do National Institute of Standards and Technology (NIST). Todos os analitos mostraram equivalência estatística com o método desenvolvido. / Despite the widespread use of liquid chromatography coupled to mass spectrometry applications in clinical chemistry, the analysis of low molecular weight and high polar metabolites in biological matrices remains as a major analytical challenge. Notwithstanding the key role played by amino acids in the diagnosis of metabolic diseases, there is still need for improvements in bioanalytical process of these analytes. The present study describes the development and validation of a new method for quantification of 24 amino acids in plasma based on liquid chromatography coupled to tandem mass spectrometry. Detection and quantification were achieved building a selected reaction monitoring method two mass transitions for each 24 amino acids and 19 stable isotope internal standards. Three chromatographic strategies for separation were evaluated, and best performance was achieved using reversed-phase octadecylsilane with perfluropentanoic acid as ion pairing agent. The separation method allowed separation of 24 amino acids with full resolution for isomers leucine, isoleucine and alloisoleucine in 11 minutes, including column equilibration time. The limits of quantification ranged from 113 fmol to 6 pmol (on column injection). Imprecisions for all evaluated levels and amino acids were less than 14%. The method is linear in all clinical intervals and extending up to 1.5 mmol.L-1. Carryover evaluation demonstrated absence of interference in the following injection throughout the analytical interval. Method accuracy was evaluated analyzing reference samples from European Research Network for evaluation and improvement of screening, Diagnosis and treatment of Inherited disorders of Metabolism (ERNDIM) and National Institute of Standards and Technology (NIST). Statistical equivalence was demonstrated for all analytes using the present method.

Em uma sociedade que lida com pressão diariamente para completar suas tarefas, a privação de sono é uma consequência comum. Como artifício para manter-se apto a trabalhar ou se divertir à noite, algumas pessoas utilizam a cocaína, cujo consumo é estudado há décadas, mas cuja associação com a privação de sono ainda não foi avaliada pela toxicologia. Este estudo utiliza a metabolômica para gerar um mapa de alterações metabólicas associadas a estas condições e sua iteração. Utilizando análise cromatográfica líquida no modo HILIC e espectrometria de massas com um analisador do tipo \"tempo de voo\" (TOF), os cromatogramas e espectros urinários de 60 ratos Wistar machos foram analisados utilizando o pacote XCMS (Bioconductor) na plataforma R. Os tratamentos estatísticos de dados (PCA, OPLS-DA, MANOVA) foram realizados através dos programas SIMCA 11 P+ e IBM SPSS, culminando em atribuições putativas dos metabólitos discriminantes nas condições estudadas (efeito da cocaína, efeito da privação total de sono e seu efeito combinado). Foram então evidenciados cinco marcadores biológicos de dano associados à cocaína, três associados à privação de sono e dois à sua iteração. Estes metabólitos foram identificados putativamente através de busca em banco de dados (Metlin, MassTrix, HMDB, Lipidmaps) e tiveram suas rotas metabólicas associadas através do banco de rotas metabólicas KEGG. Há diferenças metabólicas estatisticamente evidenciáveis e inclusive duradouras nas vias do ciclo da tirosina e do sistema dopaminérgico, além do ciclo do citrato. / In a society that deals daily with pressure to complete its tasks, sleep deprivation is a common consequence. As an excuse to keep oneself fit to work but to have fun at night some people use cocaine, whose consumption is studied for decades, but the association with sleep deprivation had not yet been evaluated by toxicology. This study uses metabolomics to generate a map of metabolic abnormalities associated with these conditions and its iteration. Using liquid chromatographic in HILIC mode and \"time of flight\" mass spectrometry (TOF), mass chromatograms of urine from 60 male Wistar rats were analysed using XCMS package (Bioconductor) running on R platform. Statistical data treatment (PCA, OPLS-DA, MANOVA) were performed using the programs SIMCA P + 11 and IBM SPSS, culminating in putative metabolites assignments that discriminate the conditions in the study (cocaine effect, total sleep deprivation effect and their combined effect). We highlighted five biological markers of damage associated with cocaine, three associated with sleep deprivation and their iteration. These metabolites were putatively identified by public databases (Metlin, MassTrix, HMDB, Lipidmaps) and their associated metabolic pathways were assessed through KEGG database. There are significant differences on metabolic pathways of the tyrosine cycle, the dopaminergic system and the citrate cycle.

Em uma sociedade que lida com pressão diariamente para completar suas tarefas, a privação de sono é uma consequência comum. Como artifício para manter-se apto a trabalhar ou se divertir à noite, algumas pessoas utilizam a cocaína, cujo consumo é estudado há décadas, mas cuja associação com a privação de sono ainda não foi avaliada pela toxicologia. Este estudo utiliza a metabolômica para gerar um mapa de alterações metabólicas associadas a estas condições e sua iteração. Utilizando análise cromatográfica líquida no modo HILIC e espectrometria de massas com um analisador do tipo \"tempo de voo\" (TOF), os cromatogramas e espectros urinários de 60 ratos Wistar machos foram analisados utilizando o pacote XCMS (Bioconductor) na plataforma R. Os tratamentos estatísticos de dados (PCA, OPLS-DA, MANOVA) foram realizados através dos programas SIMCA 11 P+ e IBM SPSS, culminando em atribuições putativas dos metabólitos discriminantes nas condições estudadas (efeito da cocaína, efeito da privação total de sono e seu efeito combinado). Foram então evidenciados cinco marcadores biológicos de dano associados à cocaína, três associados à privação de sono e dois à sua iteração. Estes metabólitos foram identificados putativamente através de busca em banco de dados (Metlin, MassTrix, HMDB, Lipidmaps) e tiveram suas rotas metabólicas associadas através do banco de rotas metabólicas KEGG. Há diferenças metabólicas estatisticamente evidenciáveis e inclusive duradouras nas vias do ciclo da tirosina e do sistema dopaminérgico, além do ciclo do citrato. / In a society that deals daily with pressure to complete its tasks, sleep deprivation is a common consequence. As an excuse to keep oneself fit to work but to have fun at night some people use cocaine, whose consumption is studied for decades, but the association with sleep deprivation had not yet been evaluated by toxicology. This study uses metabolomics to generate a map of metabolic abnormalities associated with these conditions and its iteration. Using liquid chromatographic in HILIC mode and \"time of flight\" mass spectrometry (TOF), mass chromatograms of urine from 60 male Wistar rats were analysed using XCMS package (Bioconductor) running on R platform. Statistical data treatment (PCA, OPLS-DA, MANOVA) were performed using the programs SIMCA P + 11 and IBM SPSS, culminating in putative metabolites assignments that discriminate the conditions in the study (cocaine effect, total sleep deprivation effect and their combined effect). We highlighted five biological markers of damage associated with cocaine, three associated with sleep deprivation and their iteration. These metabolites were putatively identified by public databases (Metlin, MassTrix, HMDB, Lipidmaps) and their associated metabolic pathways were assessed through KEGG database. There are significant differences on metabolic pathways of the tyrosine cycle, the dopaminergic system and the citrate cycle.

Apesar da rápida expansão das aplicações da cromatografia líquida acoplada à espectrometria de massas em química clínica, a análise de metabólitos de baixo peso molecular e alta polaridade em matrizes biológicas ainda permanece como um grande desafio analítico. Dentre os compostos de grande importância no diagnóstico de doenças metabólicas que ainda carecem de melhores alternativas bioanalíticas destacam-se os aminoácidos. O presente estudo descreve o desenvolvimento e a validação de um novo método para a quantificação de 24 aminoácidos em plasma explorando a cromatografia líquida acoplada a espectrômetros de massas em tandem. Foi construído um método de detecção baseado em SRM (múltiplas reações selecionadas) com duas transições de massas para cada um dos 24 aminoácidos e os 19 padrões internos marcados com isótopos estáveis. Foram avaliadas três estratégias de separação cromatográfica e o melhor desempenho foi obtido com fase reversa com octadecilsilano (C18) com pareamento iônico com o ácido perfluoropentanoico. O método cromatográfico final permitiu a separação dos 24 aminoácidos, com resolução completa dos isômeros: leucina, isoleucina e allo-isoleucina, em 11 minutos incluindo o tempo de re-estabilização da coluna cromatográfica. Os limites de quantificação variaram em 113 fmol a 6 pmol injetados na coluna cromatográfica. A imprecisão obtida nos níveis testados para todos os aminoácidos foi inferior a 14%. O método apresentou linearidade para os intervalos testados chegando a 1,5 mmol.L-1 para vários compostos. Os ensaios de arraste mostraram que os limites máximos obtidos na linearidade não geram nenhuma interferência subsequente. A exatidão do método foi avaliada com amostras provenientes do programa de referência interlaboratorial European Research Network for evaluation and improvement of screening, Diagnosis and treatment of Inherited disorders of Metabolism (ERNDIM) e com o material de referência certificado do National Institute of Standards and Technology (NIST). Todos os analitos mostraram equivalência estatística com o método desenvolvido. / Despite the widespread use of liquid chromatography coupled to mass spectrometry applications in clinical chemistry, the analysis of low molecular weight and high polar metabolites in biological matrices remains as a major analytical challenge. Notwithstanding the key role played by amino acids in the diagnosis of metabolic diseases, there is still need for improvements in bioanalytical process of these analytes. The present study describes the development and validation of a new method for quantification of 24 amino acids in plasma based on liquid chromatography coupled to tandem mass spectrometry. Detection and quantification were achieved building a selected reaction monitoring method two mass transitions for each 24 amino acids and 19 stable isotope internal standards. Three chromatographic strategies for separation were evaluated, and best performance was achieved using reversed-phase octadecylsilane with perfluropentanoic acid as ion pairing agent. The separation method allowed separation of 24 amino acids with full resolution for isomers leucine, isoleucine and alloisoleucine in 11 minutes, including column equilibration time. The limits of quantification ranged from 113 fmol to 6 pmol (on column injection). Imprecisions for all evaluated levels and amino acids were less than 14%. The method is linear in all clinical intervals and extending up to 1.5 mmol.L-1. Carryover evaluation demonstrated absence of interference in the following injection throughout the analytical interval. Method accuracy was evaluated analyzing reference samples from European Research Network for evaluation and improvement of screening, Diagnosis and treatment of Inherited disorders of Metabolism (ERNDIM) and National Institute of Standards and Technology (NIST). Statistical equivalence was demonstrated for all analytes using the present method.

Organic aerosol is a major constituent of atmospheric fine particulates (PM2.5), which adversely affect human health and change the Earth’s radiative energy balance. Primary organic aerosol is directly emitted from sources and secondary organic aerosol (SOA) is formed in the atmosphere following oxidation of volatile organic compounds (VOC) from anthropogenic and biogenic sources. Biogenic SOA is enhanced by anthropogenic pollutants such as sulfate and NOx that mainly come from fossil fuel combustion. However, the extent to which the anthropogenic pollutants enhance biogenic SOA in different environments is unknown. The central hypothesis of this thesis is that organosulfates, organic compounds containing a sulfate ester group, are useful as tracers for anthropogenically-influenced biogenic SOA. This research aims to provide a better understanding of the sources of PM2.5 organic carbon (OC), particularly secondary organic carbon (SOC), through the inclusion of organosulfates in an organic tracer-based source apportionment model. The specific objectives of this research include 1) development of a highly sensitive and accurate method to quantify highly polar organosulfates in atmospheric aerosols, 2) identification and quantification of major organosulfate species in the ambient air, and 3) determination of anthropogenic and biogenic sources and their contributions to PM2.5 OC using an organic tracer-based positive matrix factorization (PMF) model. A highly sensitive and accurate method was developed and validated for the quantification of highly polar organosulfates using hydrophilic interaction liquid chromatography (HILIC) and tandem mass spectrometry (MS/MS). The developed method shows excellent retention of carboxylic acid and hydroxyl containing organosulfates. The HILIC-MS/MS method was applied to PM2.5 samples collected in summer 2013 at a rural site in Centreville, AL. Quantified organosulfates accounted for approximately 0.3% of PM2.5 OC. Other major organosulfates, for which standards are not available, were monitored by their fragmentation to the bisulfate anion and/ or sulfate ion radical. The major organosulfates were determined to be 2-methyltetrol sulfate and other isoprene-derived organosulfates. Eight sources of the PM2.5 OC in Centreville, AL were identified using PMF model through the application of organosulfates and commonly used organic tracers measured in samples collected during the daytime and nighttime: vehicle emissions (8%), prescribed burning (11%), isoprene SOC formed under low-NOx (13%) and high-NOx conditions (11%), SOC formed by photochemical reactions (9%), oxidatively aged biogenic SOC (6%), sulfuric acid-influenced SOC (21%), and monoterpene SOC formed under high-NOx conditions (21%). The organosulfates enabled organic tracer-based PMF to resolve sulfuric acid-influenced SOC, while the daytime and nighttime measurements enabled organic tracer-based PMF to resolve SOC formation pathways with diurnal variations (e.g. SOC formed by photochemical reactions). The PM2.5 OC in Centreville was mainly secondary in origin (81%) and was influenced by NOx, ozone (a product of photochemical reactions of NOx and VOC), and sulfuric acid. Together, primary and secondary OC influenced by the fossil fuel use was 76%. Thus, the majority of the PM2.5 OC in Centreville during summer can be controlled by the reduction of fossil fuel use. The HILIC-MS/MS method was also applied to daily PM2.5 samples collected from an urban site in Atlanta, GA during August 2015. The major organosulfate species identified in Atlanta were dominated by 2-methyltetrol sulfate and other isoprene-derived organosulfates, similar to Centreville. They contributed 16% of PM2.5 OC and accounted for the majority of the isoprene-derived SOA that had not previously been identified at the molecular level. The concentrations of the major isoprene-derived organosulfates in Atlanta were two to six times higher than in Centreville. The greatest enhancement was obtained for 2-methylglyceric acid sulfate, a known isoprene SOA tracer formed under high-NOx conditions, reflecting the 15 times higher average NOx concentration in Atlanta during August 2015 compared to Centreville in summer 2013. These results indicate that NOx had a stronger influence on isoprene-derived organosulfate formation in urban Atlanta compared to rural Centreville. Overall, these results indicate that organosulfates are useful tracers for anthropogenically-influenced biogenic SOA. Thus, it is important to quantify them for use in organic tracer-based PMF modeling to determine the anthropogenically-influenced biogenic SOC in PM2.5 OC.

This diploma thesis deals with optimization of separation conditions for the four groups of analytes related to 7-deazaadenosine (each group was composed of four derivatized nucleosides) using hydrophilic interaction liquid chromatography. All the sixteen analytes were synthesized as potentially cytostatically active compounds. The effect of the type of stationary phase in the chromatographic column, the ratio of organic and aqueous parts of the mobile phase, pH of the buffer used as the mobile phase component and the concentration of ammonium acetate in the buffer in the range of 5-50 mM were tested during the optimization process. Three stationary phases were tested - bare silica (Spherisorb® Silica column), silica- bonded amide (TSKgel Amide-80 column) and silica-bonded native cyclofructan 6 (Frulic-N column). The dimensions of all columns were 2504.6 mm i.d.; particle size 5 µm. During the optimization of separation the mechanism of HILIC was studied. It was found that the distribution of analytes between the aqueous layer partially coated on the surface of the stationary phase and the mobile phase and also the adsorption of analytes on the stationary phase participated in the retention and separation mechanism in all tested chromatographic systems. The three groups of analytes (SK1, SK3, SK4)...

(EN) This thesis deals with the determination of nucleotides in bacterial cells. Nucleotides play crucial role in most of the metabolic pathway. Determining their concentrations could give us important clues about the influence of internal and external conditions on the bacterial metabolism. Previously published papers dealing with the analysis of nucleotides and other intracellular metabolites can be divided into two groups according to the analytical approach: a) metabolomic approach and b) targeted approach dealing with narrow group of target analytes. In the case a) most authors use the state-of-the-art LC-MS/MS technique, whereas in the case b) robust UV detection coupled mainly to IP-LC is widely used. The aim of this study was to combine both approaches to obtain a method for routine analysis that would take advantages of mass detection, such as sensitivity and selectivity, while avoiding the need of demanding optimization of MS/MS transitions and expert service. The main purpose of the newly developed HILIC-MS method is its universal applicability in most biological and biochemical laboratories.

A nanoHILIC-MS platform was developed to provide a means for top-down and middle-up analysis of glycoproteins. A mechanistic approach was taken in evaluating characteristics of the stationary phase of the chromatography column. Results from this evaluation informed the selection of a polyacrylamide brush layer-based bonded phase, which was applied to nonporous silica nanoparticles. Fluorescence microscopy was used for direct analysis of labeled proteins during nanoHILIC separations, with the platform subsequently adapted for mass spectrometry-based detection of intact and semi-intact glycoproteins. It was found that the standard technique of linking a hollow needle emitter to the capillary chromatography column for mass spectrometry-based detection yielded an unacceptable level of band broadening, necessitating the development of a column with an integrated needle emitter. The resulting nanoHILIC-MS method yielded peak widths as sharp as 3.5 seconds, enabling ultra-sensitive detection and identification of 28 glycoforms of an IgG1κ monoclonal antibody standard.

No description available.