<p>Advances in life science require knowledge of active molecules in complex biological systems. These molecules are often only present for a certain time and at limited concentrations. Integrated micro-analytical tools for sampling, separation and mass spectrometric (MS) detection would meet these requests and are therefore continuously gaining interest. An on-line coupling of analytical functions provides shorter analysis time and less manual sample handling. In this thesis, improved compatibility of microdialysis sampling and multidimensional separations coupled to MS detection are developed and discussed.</p><p>Microdialysis was used <i>in vitro</i> for determination of the non-protein bound fraction of the drug ropivacaine. The sampling unit was coupled on-line to capillary column liquid chromatography (LC) followed by ultraviolet or MS detection. For MS detection, the system was extended with a desalting step and an addition of internal standard. A method for MS screening of microdialysates, collected <i>in vivo,</i> was also developed. The method involved sampling and measurements of the chemical pattern of molecules that generally are ignored in clinical investigations. Chemometric tools were used to extract the relevant information and to compare samples from stimulated and control tissues.</p><p>Complex samples often require separation in more than one dimension. On-line interfaces for sample transfer between LC and capillary electrophoresis (CE) were developed in soft poly(dimethylsiloxane) (PDMS). MS detection in the LC-CE system was optimised on frequent sampling of the CE peak or on high resolution in mass spectra using time-of-flight (TOF)MS or Fourier transform ion cyclotron resonance (FTICR)MS, respectively. Aspects on electrode positioning in the LC-CE interface led to development of an on-column CE electrode. A successful method for deactivation of the PDMS surface using a polyamine polymer was also developed. The systems were evaluated using peptides and proteins, molecules that are gaining increased attention in bioscience, and consequently also in chemical analysis. </p>

Capillary Electrophoresis (CE) was explored as a means for physicochemical and biopharmaceutical characterisation of small drug molecules. Special attention was paid to the characterisation of acid-base and lipophilic properties of drug compounds by analysing their migration behaviour in different CE systems. The thesis comprises an overview of the field together with separate studies on the different topics. The utility of CE for the determination of pKa of labile drug compounds was investigated. A general methodology was developed comprising key steps such as the use of a stabilising sample diluent, electromigration injection, and analyte characterisation by UV-Vis spectroscopy. The methodology was successfully applied for two sets of drug compounds, labile at low and high pH, respectively. CE was also evaluated for experimental modelling of passive intestinal membrane permeability by studying analyte migration in liposomal, microemulsion and micellar electrolytes. Good correlation is reported between CE migration and Caco-2 cell absorption estimates and for in vitro inhibition of thrombin. Interestingly, a slightly better correlation was obtained for liposomal electrolytes. The utility of liposomes in CE was further extended by developing a novel procedure for immobilising liposomes inside fused silica capillaries. This approach enabled direct on-line coupling of liposome CE to high sensitivity mass spectrometry. The utility of liposome-coated capillaries is demonstrated for estimating drug passive intestinal membrane permeability. Its use in biopharmaceutical drug profiling is discussed. Utilising advanced molecular descriptors, commonly applied to in silico prediction of passive intestinal membrane permeability, migration of analytes in micellar CE systems could be well predicted. The novel approach was based on hierarchical multivariate analytics and use of molecular descriptors for both analytes and micellar media surfactants. Demonstrated results propose that the CE format could be useful to validate how representative molecular descriptors are for describing molecular behaviour in complex liquid media, e.g. physiological systems.

L'objectiu principal d'aquestaTesi Doctoral és el desenvolupament de diferents estratègies per disminuir els límits de detecció de l’electroforesicapil•lar per a la determinació de compostos farmacèutics i els productes de cura personal. Aquestes estratègies es basen en les tècniques de preconcentració electroforètiques i cromatogràfiques, i l'ús de l’espectrometria de masses com a sistema de detecció. Com a tècniques de preconcentració electroforètiques s'han estudiat les tècniques de samplestacking i sweeping, i com a tècnica de preconcentració cromatogràficas’ha avaluat l'acoblament en línia entre l'extracció en fase sòlida i l'electroforesicapil•lar (In-line SPE-CE). Entre elsPPCPs, aquesta tesi doctoral es centra específicament en els antiinflamatoris no esteroïdals(AINE), els parabens i els filtres ultraviolats. Un altre dels objectius d'aquestaTesi Doctoral és estudiarl’aplicabilitat de les metodologies desenvolupades per a l'anàlisi de mostres ambientals per determinar PPCP. / The main objective of this Doctoral Thesis is the development of different strategies to decrease the detection limits of capillary electrophoresis for the determination of pharmaceutical and personal care products. These strategies are based on electrophoretic and chromatographic preconcentration techniques, and the use of mass spectrometry as a detection system. The electrophoretic preconcentration techniques studied included sample stacking techniques and sweeping while the chromatographic preconcentration technique evaluated was in-line coupling between solid phase extraction and capillary electrophoresis. With respect to PPCPs, this Doctoral Thesis focuses specifically on non-steroidal anti-inflammatory drugs (NSAIDs), parabens and UV-filters. Another objective of this Doctoral Thesis is to study the suitability of the developed methodologies for the determination of PPCPs in environmental samples.

The analysis of low abundant biological molecules is often challenging due to their chemical properties, low concentration and limited sample volumes. Neuropeptides are one group of molecules that fits these criteria. Neuropeptides also play an important role in biological functions, which makes them extra interesting to analyze. A classic chemical analysis involves sampling, sample preparation, separation and detection. In this thesis, an enhanced solid supported microdialysis method was developed and used as a combined sampling- and preparation technique. In general, significantly increased extraction efficiency was obtained for all studied peptides. To be able to control the small sample volumes and to minimize the loss of neuropeptides because of unwanted adsorption onto surfaces, the subsequent analysis steps were miniaturized to a micro total analysis system (µ-TAS), which allowed sample pre-treatment, injection, separation, manipulation and detection. In order to incorporate these analysis functions to a microchip, a novel microfabrication protocol was developed. This method facilitated three-dimensional structures to be fabricated without the need of clean room facilities. The sample pre-treatment step was carried out by solid phase extraction from beads packed in the microchip. Femtomole levels of neuropeptides were detected from samples possessing the same properties as microdialysates. The developed injection system made it possible to conduct injections from a liquid chromatographic separation into a capillary electrophoresis channel, which facilitated for advanced multidimensional separations. An electrochemical sample manipulation system was also developed. In the last part, different electrospray emitter tip designs made directly from the edge of the microchip substrate were developed and evaluated. The emitters were proven to be comparable with conventional, capillary based emitters in stability, durability and dynamic flow range. Although additional developments remain, the analysis steps described in this thesis open a door to an integrated, on-line µ-TAS for neuropeptides analysis in complex biological samples.

Advances in life science require knowledge of active molecules in complex biological systems. These molecules are often only present for a certain time and at limited concentrations. Integrated micro-analytical tools for sampling, separation and mass spectrometric (MS) detection would meet these requests and are therefore continuously gaining interest. An on-line coupling of analytical functions provides shorter analysis time and less manual sample handling. In this thesis, improved compatibility of microdialysis sampling and multidimensional separations coupled to MS detection are developed and discussed. Microdialysis was used in vitro for determination of the non-protein bound fraction of the drug ropivacaine. The sampling unit was coupled on-line to capillary column liquid chromatography (LC) followed by ultraviolet or MS detection. For MS detection, the system was extended with a desalting step and an addition of internal standard. A method for MS screening of microdialysates, collected in vivo, was also developed. The method involved sampling and measurements of the chemical pattern of molecules that generally are ignored in clinical investigations. Chemometric tools were used to extract the relevant information and to compare samples from stimulated and control tissues. Complex samples often require separation in more than one dimension. On-line interfaces for sample transfer between LC and capillary electrophoresis (CE) were developed in soft poly(dimethylsiloxane) (PDMS). MS detection in the LC-CE system was optimised on frequent sampling of the CE peak or on high resolution in mass spectra using time-of-flight (TOF)MS or Fourier transform ion cyclotron resonance (FTICR)MS, respectively. Aspects on electrode positioning in the LC-CE interface led to development of an on-column CE electrode. A successful method for deactivation of the PDMS surface using a polyamine polymer was also developed. The systems were evaluated using peptides and proteins, molecules that are gaining increased attention in bioscience, and consequently also in chemical analysis.

This dissertation describes the advantages and feasibility of newly synthesized near-infrared (NIR) bis-heptamethine cyanine (BHmC) dyes for non-covalent labeling schemes. The NIR BHmCs were synthesized for biomolecule assay. The advantages of NIR BHmCs for biomolecule labeling and the instrumental advantages of the near-infrared region are also demonstrated. Chapter 1 introduces the theory and applications of dye chemistry. For bioanalysis, this chapter presents covalent and non-covalent labeling. The covalent labeling depends on the functionality of amino acids and the non-covalent labeling relies on the binding site of a protein. Due to the complicated binding process in non-covalent labeling, this chapter also discusses the binding equilibria in spectroscopic and chromatographic analyses. Chapter 2 and 3 evaluate the novel BHmCs for non-covalent labeling with human serum albumin (HSA) and report the influence of micro-environment on BHmCs. The interesting character of BHmCs in aqueous solutions is that the dyes exhibit non- or low-fluorescence compared to their monomer counterpart, RK780. It is due to their H-type closed clam-shell form in the solutions. The addition of HSA or organic solvents opens up the clam-shell form and enhances fluorescence. The binding equilibria are also examed. Chapter 4 provides a brief introduction that summaries the use of capillary electrophoresis (CE), and offers a detailed instrumentation that discusses the importance and advantage of a detector in NIR region for CE separation. Chapter 5 focuses on the use of NIR cyanine dyes with capillary electrcophoresis with near-infrared laser induce fluorescence (CE-NIR-LIF) detection. The NIR dyes with different functional groups show that RK780 is a suitable NIR dye for HSA labeling. The use of BHmCs with CE-NIR-LIF reduces signal noises that are commonly caused by the interaction between NIR cyanine dyes and negatively charged capillary wall. In addition, bovine carbonic anhydrase II (BCA II) is applied to study the influence of hydrophobicity on non-covalent labeling. Finally, chapter 6 presents the conformational dependency of BHmCs on the mobility in capillary and evaluates the further possibility of BHmCs for small molecule detection. Acridine orange (AO) is used as a sample and it breaks up the aggregate and enhances fluorescence. The inserted AO into BHmC changes the mobility in capillary, owing to the conformational changes by AO.

Doenças infecciosas parasitárias consideradas negligenciadas representam um grande problema de saúde pública em muitos países e regiões. Os fármacos disponíveis na terapêutica são, em geral, tóxicos e de eficácia discutível. Portanto, a descoberta e o planejamento de novos quimioterápicos são extremamente necessários. Neste contexto, os pró-fármacos dendriméricos podem ser úteis. Porém, é necessário esforço adicional para viabilizar os custos, simplificar as estratégias de síntese e investigar os comportamentos de liberação. Ademais, é importante a melhoria dos métodos analíticos, dos métodos de purificação e identificação dos produtos de síntese, para a determinação das propriedades físico-químicas e atividade biológica, visando à efetiva aplicação desta tecnologia. Face ao exposto, o objetivo deste trabalho foi estudar a identidade, pureza e liberação de dois potenciais pró-fármacos dendriméricos, baseados em 3- hidroxiflavona, planejados para serem ativos em doença de Chagas e leishmaniose. O primeiro, estruturalmente, contendo inositol como núcleo e ramos constituídos por éster da 3- hidroxiflavona com ácido málico e o segundo, estruturalmente contendo o dendrímero PAMAM-G0 (poliamidoamina de geração inicial) como transportador e ácido succínico como espaçante. Desenvolveram-se métodos adequados à determinação da 3-hidroxiflavona por HPLC-UV (Cromatografia líquida de alto desempenho, com detecção no ultravioleta) e MEKC (Cromatografia eletrocinética micelar). Comparando-se esses métodos, o método por HPLC foi mais sensível, preciso e exato na quantificação da 3-hidroflavona, enquanto o método por eletroforese capilar foi mais rápido e de menor custo. O éster da 3-hidroxiflavona com o ácido málico mostrou-se instável em soluções orgânicas, aquosas em diferentes pH e nas condições reacionais de diversas estratégias de síntese avaliadas, o que impediu a obtenção do dendrímero baseado em inositol como núcleo conforme proposto. Já o dendrímero PAMAM-G0 funcionalizado com 3-hidroxiflavona foi sintetizado, purificado e caracterizado com sucesso. Não se observou liberação da 3-hidroxiflavona a partir desse dendrímero em solução gástrica simulada (pH 1,2) e a mesma foi lenta em soluções tampão com pH entre 5,0 e 8,5, a 37,0 ºC. Ensaios de atividade biológica do PAMAM-G0-SUC-3-OH-FLAV em amastigotas de Trypanosoma cruzi, cepas Y(Curitiba) e Y(SS), comparativamente ao benznidazol e ao nifurtimox, mostraram atividade moderada e baixa seletividade. / Infectious parasitoses considered neglected diseases represent a great health problem for many countries and areas. Drugs available in the therapeutics are, generally, toxics and do not have good efficacy. So, the discovery and design of new chemotherapeutic agents are extremely needed. In this context, dendrimeric prodrugs may be useful. However, additional effort is required to make the costs accessible, to simplify the synthetic strategies and to investigate the behavior of cleavage. The improvement of analytical methods, purification methods and identification of synthetic products, in order to determine the physicochemical properties and bioactivity aiming to effectively implement this technology, is also required. Based on foregoing considerations, the objective of this work was to study the identity, purity and drug release of two potential dendrimeric prodrugs, based on 3-hydroxyflavone, designed to be active in leishmaniasis and Chagas disease. The first structurally contains myo-inositol as the core and branches consisting of esters from 3-hydroxyflavone with malic acid. The second structurally contains PAMAM-G0 dendrimer (initial generation polyamidoamine) as carrier and succinic acid as spacer. Suitable analytical methods for determining 3-hydroxyflavone by HPLC-UV (High Performance Liquid Chromatography) and MEKC (Micellar Electrokinetic Chromatography) have been developed. Comparing these methods, HPLC method showed more sensitivity, precision and accuracy in the quantification of 3-hydroxyflavone, while the capillary electrophoresis method was faster and less expensive. The ester of 3-hydroxyflavone with malic acid showed to be unstable in organic and aqueous solutions, at different pH and at reaction conditions of synthetic strategies evaluated, which prevented the obtaining of dendrimer based on mio-isositol as core. Notwithstanding, PAMAM-G0 dendrimer funcionalized with 3- hydroxyflavone was synthesized, purified and characterized successfully. There were no 3- hydroxyflavone releases from this dendrimer in simulated gastric fluid (pH 1.2) and a slow release was observed in buffer solutions with pH between 5.0 and 8.5, at 37.0 ºC. Submitted to biological assays in amastigotes of two strains of T. cruzi, Y(Curitiba) and Y(SS), compared to benznidazole e nifurtimox, PAMAM-G0-SUCC-3-OH-FLAV showed moderated activity and low selectivity index.

Exposomics applies metabolomics methods and technologies to the comprehensive analysis of all low molecular weight molecules (< 1.5 kDa) in complex biological samples to characterize the interaction between cellular metabolism and exogenous lifestyle exposures that determine health and quality of life. To fully access the diverse classes of biological molecules related to an individual’s metabolic profile, metabolomics frequently requires the use of complementary analytical platforms, and employs targeted and untargeted molecular profiling strategies to identify biomarkers that are clinically relevant to an individual’s health status. Chapter 2 describes a quinoline-based boronic acid biosensor for N-acetylneuraminic acid that undergoes a striking binding enhancement under strongly acidic conditions. For the first time, this work allows for direct analysis of acidic sugars with high selectivity when using UV absorbance or fluorescence detection based on formation of a highly stable boronate ester complex with metabolites containing an α-hydroxycarboxylate moiety. Chapter 3 describes a targeted analysis of 24 different organic contaminants using GC-MS that can serve as biomarkers of recent smoke exposure following search-and-rescue training exercises by firefighters located at three different sites across the province of Ontario. Importantly, skin and possible respiratory uptake of various polycyclic aromatic hydrocarbons, methoxyphenols, and resin acids was confirmed by peak excretion of several wood smoke biomarkers in urine within 6 h following acute exposure. Chapter 4 applied a cross-platform metabolomics strategy based on CE-MS and GC-MS in order to identify and validate dietary biomarkers in matching plasma and urine samples collected from healthy participants in the pilot Diet and Gene Interaction Study (DIGEST). For the first time, we demonstrate that a panel of metabolites can serve as reliable biomarkers following contrasting Prudent and Western diets over 2 weeks of food provisions, which correlated well with self-reported diet records. This work paves the way for the development of objective biomarkers for accurate assessment of wood smoke exposures, as well as complex dietary patterns as required for new advances in occupational health and nutritional epidemiology. / Dissertation / Doctor of Philosophy (PhD) / Exposomics is an emerging multidisciplinary science aimed at deciphering the complex interactions that impact human health and gene expression, such as lifestyle choices (i.e., habitual diet) and lifelong environmental exposures. There is growing interest in identifying biomarkers that can be readily measured for chronic disease prevention given an alarming global prevalence of obesity and cardiometabolic disorders, including heart disease, type 2 diabetes and cancer. The research in this thesis focuses on developing new analytical methods for identifying and quantifying metabolites that may allow for better assessments of human health, and has contributed to the development of novel biosensors for the targeted analysis of N-acetylneuraminic (sialic) acid and related acidic sugars, as well as high resolution methods for broad spectrum analysis of biotransformed organic contaminants from smoke exposure by GC-MS, and plasma and urinary metabolites that differentiate contrasting Prudent and Western diets and correlate well with self-reported diet records.

La phosphorylation des protéines constitue l’une des plus importantes modifications post-traductionnelles (PTMs) et intervient dans de multiples processus physiologiques tels, la croissance, la différenciation cellulaire, l’apoptose, etc. En dépit de son importance, l’analyse des phosphoprotéines demeure une tâche difficile en raison de leur nature dynamique (car la phosphorylation des protéines est un processus réversible) et de leur faible abondance relative. En effet, la détermination des sites de phosphorylation est souvent difficile car les phosphopeptides sont souvent difficiles à détecter par des méthodes d’analyse chromatographique classique et par spectrométrie de masse (MS). De récentes études ont démontré que les nombreuses méthodes d’enrichissement de phosphopeptides existantes ne sont pas complètes, et que le nombre total de phosphopeptides détectés ne chevauchent pas complètement ces méthodes. C’est pour cela qu’il existe une nécessité de combler les lacunes des méthodes d’enrichissement existantes afin d’avoir des analyses phosphoprotéomiques plus complètes. Dans cette étude, nous avons utilisé les liquides ioniques (LI), plus particulièrement les sels d’imidazolium, comme une technique d’enrichissement alternative, dans le but de favoriser une extraction sélective de phosphopeptides présents en solution. Les sels d’imidazolium ont donc été utilisés en raison de leurs propriétés physico-chimiques "facilement" ajustables selon la nature des substituants sur le noyau imidazolium et la nature de l’anion. Les sels de monoimidazolium et de bis-imidazolium possédant respectivement des chaînes linéaires à 4, 12 et 16 atomes de carbone et ayant différents anions ont été synthétisés et utilisés pour effectuer des extractions liquide-liquide et solide-liquide des phosphopeptides en solution. Dans un premier temps, des extractions liquide-liquide ont été réalisées en utilisant un liquide ionique (LI) ayant une chaine linéaire de 4 atomes de carbone. Ces extractions réalisées avec le bis(trifluoromethanesulfonyl) amide de 3-butyl-1-methylimidazolium (BMIM-NTf2) et l’hexafluorophosphate de 3-butyl-1-methylimidazolium (BMIM-PF6) n’ont pas montré une extraction notable du PPS comparativement au PN. Dans un deuxième temps, des extractions solide-liquide ont été réalisées en fonctionnalisant des particules solides avec des sels d’imidazolium possédant des chaines linéaires de 12 ou 16 atomes de carbone. Ces extractions ont été faites en utilisant un phosphopentapeptide Ac-Ile-pTyr-Gly-Glu-Phe-NH2 (PPS) en présence de 2 analogues acides non-phosphorylés. Il a été démontré que les sels d’imidazolium à chaine C12 étaient meilleurs pour extraire le PPS que les deux autres peptides PN (Ac-Ile-Tyr-Gly-Glu-Phe-NH2) et PE (Ac-Glu-Tyr-Gly-Glu-Phe-NH2) L’électrophorèse capillaire (CE) et la chromatographie liquide à haute performance couplée à la spectrométrie de masse (LC-MS) ont été utilisées pour quantifier le mélange des trois peptides avant et après extraction ; dans le but de mesurer la sélectivité et l’efficacité d’extraction de ces peptides par rapport à la composition chimique du liquide ionique utilisé. / Protein phosphorylation is one of the most important post-translational modifications because it is involved in multiple physiological processes such as growth, differentiation, apoptosis, etc. Despite its importance, the analysis of phosphoproteins remains a difficult task due to their dynamic nature (phosphorylation of proteins is a reversible process) and their low abundance. Indeed, the determination of phosphorylation sites is difficult because phosphopeptides are often difficult to detect by conventional chromatographic analysis and by mass spectrometric (MS) methods. Recent studies have shown that the existing methods of enrichment of phosphopeptides are not complete, and the total number of phosphopeptides detected does not overlap completely with those detected by these methods. The gaps in existing enrichment methods need to be filled in order to have more complete phosphoproteomic analyses. In the current study, ionic liquids (IL), specifically imidazolium salts, have been used in an alternative enrichment technique with potential for selective extraction of phosphopeptides from solution. Imidazolium salts were chosen because their physicochemical properties are readily adjustable depending on the nature of the substituent attached to the imidazolium core and the counter-anion. Monoimidazolium and bis-imidazolium salts with linear chains having respectively 4, 12, and 16 carbon atoms and with different anions were synthesized and used to carry out liquid-liquid and solid-liquid extractions of a phosphorylated peptide from a solution. At first, liquid-liquid extractions were carried out using an ionic liquid (IL) with a linear chain of 4 carbon atoms. These extractions performed with bis (trifluoromethanesulfonyl) amide 3-butyl-1-methylimidazolium (BMIM-NTf2) and hexafluorophosphate 3-butyl-1-methylimidazolium (BMIM-PF6) did not show a considerable extraction of PPS comparatively to the PN. Secondly, solid-liquid extractions were done by first functionalizing solid-phase particles with the imidazolium salts. The extractions were carried out using the phosphopentapeptide Ac-pTyr-Ile-Gly-Glu-Phe-NH2 (PPS) and its acidic non-phosphorylated analogues. It has been shown that the C12 chain imidazolium salts were better to extract PPS than the other two peptides PN (Ac-Ile-Tyr-Gly-Glu-Phe-NH2) and PE (Ac-Glu-Tyr-Gly-Glu-Phe-NH2). The extraction efficiency of these peptides was estimated by capillary electrophoresis (CE) and high performance liquid chromatography coupled with mass spectrometry (LC-MS).