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Analytical surface sampling tools enable direct chemical studies of solid biological samples, including morphologically diverse tissue, and provide both fast chemical analysis with minimum sample preparation and information about molecular spatial distribution. Spatially resolved investigations of tissue sections could help to reveal pathological mechanisms at specific tissue locations. Mass spectrometry imaging (MSI) is a valuable method for direct tissue analysis that can give a full molecular profile of heterogeneous biological in a short period of time. However, MSI is not always capable of identifying isomeric and isobaric species, especially for low-weight metabolites. Moreover, the complex chemical matrix of biological samples significantly influences the performance of MSI, including introducing artefacts such as ion suppression. Therefore, separation by liquid chromatography or capillary electrophoresis prior mass spectrometric detection is beneficial to mitigate such artefacts and increase sensitivity. Recently, the new setup for surface sampling capillary electrophoresis mass spectrometry (SS-CE-MS) was presented that enables direct sampling from a tissue location followed by separation and mass spectrometric detection. This thesis is aimed to show the developments and improvements of SS-CE-MS along with applications of quantitative spatial metabolomics. Paper I demonstrates SS-CE-MS using a sheath liquid interface to the MS and its robustness and reproducibility both for the injection process and the connection to mass spectrometer. Analysis of small polar molecules, lipids, and proteins in both tissue and blood samples is described in addition to an increased throughput using multisegmented electrokinetic injection. In Paper II five quantitative approaches for SS-CE-MS were developed and evaluated. Off-line one-point calibration was found to be optimal for tissue analysis and further used for mapping metabolites, including isomeric species, in four regions of rat brain. Specifically, aromatic amino acids were found decreased in cortex and the isomers valine and leucine were more abundant than their isomers betaine and isoleucine, respectively. Overall, this thesis shows SS-CE-MS as promising new quantitative method for future applications to healthy and pathological tissue investigation.

A number of nitroxide adducts and N-acyloxytrialkylammonium salts were prepared, isolated, characterized and evaluated as initiators for the controlled and living free radical polymerization of methacrylate and dimethacrylate monomers under mildly thermal and photochemical conditions. The initiators and polymerization methods that were developed could potentially be used for improving resins employed in dental applications. Using very easy synthesis strategies, the following nitroxide initiators were prepared in high purity, isolated and characterized: 1-Benzoylperoxy-2,2,6,6-tetramethyl-piperidine (BPO/TEMPO), 1-(2'-Cyano-2'-propoxy)-2,2,6,6-tetramethylpiperidine (AIBN/TEMPO), 1,1-ditertbutyl-1-(1-methyl-1-cyanoethoxy)-amine (AIBN/DBN), 1,1-ditertbutyl-1-(benzoylperoxy)-amine (BPO/DBN) and 2,2,6,6,-tetramethyl-4-oxo-1-(1-methyl-1-cyanoethoxy)-piperidine (AIBN/4-OXO-TEMPO). Using H2SO4 additive and an improved unimolecular initiation in nitroxide mediated polymerization, living and controlled polymerization of methyl methacrylate (MMA), tri-ethylene glycol dimethacrylate (TEGDMA) and ethoxylated bisphenol A dimethacrylate (EBPADMA) were accomplished, for the first time ever for most of the initiators. Linear polymers (PMMA) were produced in high yield (93 %) under mildly thermal conditions (T = 70 oC) and with excellent attributes: (PDI = 1.04-1.26), Mn (87000), Tg (122-128 oC), Td (290-410 oC). Highly crosslinked polymers, poly(TEGDMA) and poly(EBPADMA), were produced in high yield (100 %) with Td (350-400 oC). The initiators were stable for a year and half at 0 oC. Two routes were investigated for the preparation of N-acyloxytrialkylammonium salts. The more efficient of these routes was used to make several novel analogs of the salts. The salts were evaluated for the free radical polymerization of MMA, TEGDMA and EBPADMA under mildly thermal (T = 60 oC) conditions with and without H2SO4 additive. Polymerization rate, yield and polymer attributes all improved upon application of H2SO4 additive. PMMA was produced with excellent attributes (PDI = 1.01-1.06), Mn (96,000-122000), Td (330-385) and Tg (127-134). Highly crosslinked poly(TEGDMA) and poly(EBPADMA) were produced with Td ranges of 300-374 oC and 375-411 oC respectively. / Chemistry

O presente trabalho, teve como objetivo, dar continuidade ao estudo apresentado na forma de dissertação de mestrado, utilizando a complexona EDTA, totalmente \"neutralizada\". na forma de Y4-. Na titulação de íons metálicos, a hidrólise acentuada deste ânion, Y4-, causa um aumento marcante de pH após o ponto estequiométrico. Esta variação de pH pode ser acompanhada potenciometricamente, utilizando um eletrodo de vidro, ou visualmente com um indicador ácido/base como a fenolftaleina, em substituição a indicadores metalocrômicos. Uma série de parâmetros foram considerados na preparação do reagente, sua armazenagem e padronização bem como os problemas ligados a percentagem de \"neutralização\". Vários equilíbrios foram considerados no desenvolvimento de um programa em linguagem BASIC, para interpretar a curva teórica da titulação de Mg2+ com EDTA \"tetraneutralizado\", na qual participam também espécies como MgHY-, antes do ponto estequiométrico. Um método complexométrico rápido e preciso foi desenvolvido com este titulante para determinar Mg2+ e SO42- simultaneamente (este por método indireto para o excesso de íons Ba2+) com viabilidade para ser aplicado em água do mar e em concentrados salinos. O método mostrou-se adequado, também, para a determinação da mistura Ca2+ e Mg2+, em substituição ao método clássico com tampão e indicador metalocrômico, eriocromo T. / This thesis has the purpose to give continuity to a former study from the master dissertation, about the use of the complexon EDTA, totally \"neutralized\", as the species Y4-. During the titration of metallic cations the marked hydrolysis of the y4- anion causes a marked pH increase after the stoichiometric point. This change of pH can be followed potentiometrically with the glass electrode or by visual end point indication by on acid/base indicator as phenolphtalein instead of metalochromic indicators. A serie of parameters were considered in the preparation of the titrant, its storage and standardization as well as problems related with the percent neutraIization\". Equilibria were considered and used in a computer program in BASIC language in order to interpret the theoretical titration curve. The species MgHY- was found in significant contribution before the end point. A fast and precise complexometric method was developed with this titrant for Mg2+ and SO42- simultaneously (this last by titration of excess of standard Ba2+ solution). The method has been found adequate to determine mixture of Ca2+ and Mg2+ instead of the classic method which uses a buffer and eriochrome T as indicator.

<p>The actual signal recorded with an analytical instrument is not always a true reflection of the analysed sample. In this thesis a further insight of the atmospheric pressure ionisation processes electrospray (ESI) and atmospheric pressure chemical ionisation (APCI) has been endeavoured, to provide a deeper understanding of and ways to minimize this bias.</p><p>A response model for ESI has been modified and used to study the influence of solvent composition on the observed mass spectrometric signal. The response model divides the response into an analyte partitioning coefficient and an instrumental response factor. A number of experimental parameters influencing the response were investigated including spray position relative to the orifice, spray potential, nebulizer and curtain gas flow rates, ionic strength and organic content of the sprayed solution. The history of the generated droplets turned out to be of significant importance to both the partitioning coefficients and the instrumental response factor. Furthermore, it was found that the total ionic strength and not only the electrolyte concentration will influence the instrumental response factor.</p><p>In addition, based on the importance of hydrophobicity and electrophoretic mobility, a model was proposed for the ion distribution within the electrosprayed droplets.</p><p>The coupling of an electrochemical (EC) cell to a mass spectrometric (MS) system has been evaluated. The coupling of the EC cell to the MS was made to decouple the cell from the high voltage circuit of the ESI. The feasibility for analyte ionisation, sample pre-concentration and solvent exchange as well as studying redox reaction products was shown.</p>

<p>Electrospray ionisation (ESI) coupled to mass spectrometry (MS) is one of the most important detection techniques for chemical analysis of small drugs as well as large biomolecules in life science today. In this thesis, aspects on improved compatibility between liquid based separation systems and mass spectrometric detection were investigated regarding buffers, sample preparation and analysis of polar compounds as well as peptides and protein digests for enhanced ESI-MS performance. </p><p>Capillary electrophoresis (CE) coupled to ESI-MS detection, was evaluated using both a sheath flow interface and a sheathless design. The separation of peptides and small, polar compounds was optimised for both CE-ESI interfaces. The effect of sheath liquid composition was also studied with the aim to improve sensitivity in the ESI-MS detection. </p><p>Polar compounds were retained and separated by capillary ion-pair chromatography coupled to ESI-MS detection. Since commonly used ion-pairing reagents are detrimental to the ESI process they were effectively removed before the ionisation by the use of a trapping column after the separation. Alternatively, the ion-pairing reagents were exchanged to volatile constituents. </p><p>A method for peptide mapping by liquid chromatography (LC)-ESI-MS was developed for lactate dehydrogenase. The method was further enhanced to involve the proteolysis on-line to the LC-ESI-MS. No manual sample handling was then needed and the total analysis time decreased from 7 to 1.5 hours. The amount of information was also shown to increase in the on-line system.</p><p>Finally, the on-line concept was extended to an innovative interface for direct coupling of a pumped liquid flow to an electroosmotically driven flow. This provided a valve-free sample transfer between capillary LC and CE, aiming towards increased peak capacity per unit time for the analysis of complex peptide samples. </p>

<p>The analysis of pesticide residues in fruit, vegetables, rape seed and water has been improved using developments in sample handling and analytical techniques. The method development is associated with analytical difficulties, since pesticides currently used in agriculture represent a variety of chemical classes having very different physico chemical properties. The method development also encounters difficulties when many various commodity classes with different characteristics are studied. The main task in pesticide residue analysis has been to provide multi residue methods, and traditionally GC has been the main analytical technique.</p><p>In order to regulate the use of hazardous pesticides, the EU commission introduces strict maximum residue levels (MRL). The need for improved sample handling and detection techniques are, however, high due to handling of lower detection limits, complex matrices and the need of more efficient sample throughput. Of the new techniques introduced as alternative techniques to the traditional extraction techniques, pressurised fluid extraction (PFE) has shown to be a promising technique in analysis of pesticide residues in fatty foodstuffs.</p><p>In water analysis, large sample volumes are needed due to low MRLs. The solid phase extraction (SPE) technique allows a concentration of large sample volumes and simplifies the tedious laboratory work with traditional separation funnels. A new approach was to use non-polar solvents for the sample extraction from the earlier used polymeric column. Both these techniques provide low solvent consumption, short extraction times and ability to automate the manual steps. </p><p>An LC-MS/MS multi residue method was finally developed for pesticide residues in fruit and vegetables. The technique is robust and sensitive and allows a simultaneous determination of 57 pesticides and metabolites in one single analysis and without any clean-up steps. The sensitivity was improved to achieve the maximum residue limits needed by EU. Several multi step methods, which involve more costly analysis, has been replaced by this technique.</p>

<p>This thesis exemplifies the strength in the combination of inductively coupled plasma (ICP) spectrometry and electrospray ionization tandem mass spectrometry ESI-MS/MS as detection techniques for liquid chromatography (LC) in the search for and investigation of compounds that bind or can bind hetero-atoms. Furthermore, some aspects involved in the coupling of LC and ICP spectrometry and quantification without identical standards have been studied.</p><p>The importance of using a separation step in combination with ICP spectrometry was shown for urine and blood plasma samples from patients treated with boron neutron capture therapy. In addition to the carrier molecule used in the therapy, one major and a few possible minor metabolites were found in the urine samples. One fragment mass of the major metabolite was obtained with LC-ESI-MS/MS. Liquid chromatography coupled to ICP-MS was also shown to be a valuable tool for fingerprinting metal-binding compounds in complex matrices, such as siderophores (iron-complexing compounds) in soil. The presence of at least two siderophores in a field soil solution sample could be revealed by LC-ICP-MS. Their identities could thereafter be determined by LC-ESI-MS/MS. </p><p>The non-UV-absorbing <i>o</i>-carboranylalanine could be quantified in relation to its degradation products by LC-ICP-AES, which provided information about the mechanism behind the degradation. Moreover, LC-ICP spectrometry was shown to provide an accurate quantification of biomolecules (bias < 10 %) when evaluated from external calibration graphs based on inorganic elemental standards. </p><p>Finally, the causes of the large decrease in boron signal seen when adding acetonitrile to the LC mobile phase in LC-ICP-MS was investigated in some detail. Space charge effects might explain a large part of the depression from carbon species on the boron signal.</p>

<p>Liquid chromatography-mass spectrometry (LC-MS) is a powerful technique for the analysis of endogenous compounds. The introduction of electrospray ionization (ESI) as an interface between LC and MS has contributed strongly to a trend towards miniaturization of LC, due to the possibility to perform ESI at low flow rates. In this thesis, several aspects regarding the design of miniaturized LC systems and electrospray emitters were investigated. In addition miniaturized LC-ESI-MS have been used for the qualitative and quantitative analysis of endogenous polar compounds, peptides and protein digests. </p><p>The performance of miniaturized LC-MS was compared using different electrospray emitter configurations. The results indicated that the efficiency of the LC system is rather independent of the configuration of the emitter. </p><p>The lifetime of gold-coated fused silica electrospray emitters based on vapor deposited adhesion layers of titanium were investigated. The long lifetime of the emitter facilitates the use in LC-MS experiments, exemplified LC-MS by analysis of neuropeptides.</p><p>The ESI voltage is shown to interfere with liquid chromatographic separations performed in packed porous graphitic carbon capillary column. This interference is ascribed to the presence of an electric field over the conductive column in absence of a ground point between the column and the ESI emitter.</p><p>The solid supported enhanced microdialysis for analysis of neuropeptides were compared with conventional microdialysis. The difference between the two methodologies were evaluated by LC-MS analysis of the microdialysates. The solid supported method gave in general higher relative recoveries.</p><p>Finally, a method of standard addition was developed to determine total level of tryptophan and two of its metabolites in human plasma by capillary LC-ESI tandem mass spectrometry. The method was applied in a clinical study of multiple scleroses patients treated with cytokines (IFN Beta 1a, 1b). The results show that the intervention effects the tryptophan metabolism.</p>