The initial goal of the project was to develop a more effective method for the purifications of oligonucleotides. This was achieved by developing a method of ion-exchange based on-column deprotection. The resulting method was compared to preestablished methods using LC-MS fraction analysis evaluated according to a purityscore with the acceptance criteria of ≥ 0.45. The on-column deprotection was shownto achieve a purity score > 0.6 for both of the fully thiolated and the partially thiolatedoligonucleotides while the other methods of regular ion-exchange chromatography,ion-pair chromatography and reversed phase chromatography only achieved a purityscore of ≥ 0.6 for only the fully thiolated oligonucleotides. The ion-exchange methodsbecame dependent on more chaotropic agents than expected, but the results gainedduring the resin screening portion of the project may provide a remedy. During theresin screening, the effects of two factors, pore size and ligand density, were investigated. Showing that the pore size did not affect the oligonucleotides to any extent,resulting in the ligand density being the major affecting factor. When studying thedifferent resins, a trend of lower retention with lower ligand density was observed.These results provide a foundation to use lower ligand density resins compatible withless harsh conditions when applying the on-column deprotection in future optimization for the purification of pharmaceutical oligonucleotides. / Syftet med projektet var att utveckla en mer effektiv metod för upprening av oligonukleotider. Detta uppnåddes genom att utveckla en metod för direkt avlägsning av denskyddande gruppen direkt på kolonnen baserat på jonbytes kromatografi. Den resulterande metoden jämfördes med tidigare etablerade metoder genom LC-MS fraktionsanalys och utvärderades enligt ett renhetsbetyg med acceptanskriterier på ≥ 0,45.Metoden visade sig uppnå ett renhetsbetyg > 0,6 för både de fullständigt tiolieradeoch delvis tiolierade oligonukleotider, medan andra metoder som vanlig jonutbyteskromatografi, jonpars-kromatografi och reversed phase-kromatografi endast uppnådde ett renhetsbetyg ≤ 0,6 för fullständigt tiolierade oligonukleotider. Jonutbytesmetoderna blev beroende av fler kaotropiska kemikalier än förväntat, men resultatenfrån resin undersökningen under projektet kan ge en lösning på detta. Under resinundersökningen utreddes effekterna av två faktorer, porstorlek och liganddensitet. Detvisades att porstorleken inte påverkade oligonukleotiderna i någon större utsträckningoch att liganddensiteten var den avgörande faktorn. Vid studier av olika resiner observerades en trend mot lägre retention vid lägre liganddensitet. Dessa resultat ger engrund för att använda resiner med lägre liganddensitet i den utevecklade metoden.Vilket bör tillåta mindre hårda förhållanden vid fortsatt optimering av uppreningenför farmaceutiska oligonucleotider.

<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.</p><p>The utility of CE for the determination of pK_a 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.</p><p>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.</p><p>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.</p><p>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.</p>

<p>Studies of protein and peptide expression are vital in order to understand complex biological systems. As demonstrated in this thesis, on-line packed capillary liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR MS) is a useful analytical tool for such studies.</p><p>A proteomics method, based on global tryptic digestion and subsequent separation and detection of the peptides by LC-FTICR MS, was developed for qualitative analysis of body fluids. Initial experiments on cerebrospinal fluid (CSF) provided results that were comparable or superior to those achieved by more time- and sample-consuming techniques. The method was also successfully applied on plasma and amniotic fluid. One of the major challenges in proteomics is the broad dynamic range of proteins in biological matrices. The advantages of removing high-abundant components from CSF and plasma prior to MS were demonstrated.</p><p>In order to search for potential biomarkers, mass chromatograms of CSF from patients suffering from amyotrophic lateral sclerosis (ALS) and controls were compared using an in-house constructed pattern recognition program. ALS-specific patterns were observed, and four out of five unknown samples were correctly assigned. Alternative strategies to quantitatively compare two pools of samples rely on differential chemical labeling. The performance of one such method, quantification-using-enhanced-signal-tags, was investigated in complex sample analysis. The experimental intensity ratios were proven to be consistent with the prepared concentration ratios of abundant proteins in CSF.</p><p>Finally, the thesis reports on the first experiments where electron capture dissociation (ECD) was successfully incorporated in on-line LC-MS experiments. ECD and nozzle-skimmer fragmentation were applied to a sample of endocrine peptides extracted from mouse pancreatic islets. The two fragmentation methods provided complementary information. However, the method needs further optimization before it can be applied in the analysis of more complex samples, such as body fluids.</p>

<p>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. </p><p>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. </p><p>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.</p>

<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.

This thesis focuses on the implementation of species-specific isotope dilution (SSID) methodology and stable isotope tracers to determine mercury species occurrence and transformation processes in-situ and during sample treatment. Isotope enriched tracers of methyl-, ethyl- and inorganic mercury were synthesised and applied in different combinations to marine and biological samples. Experimental results were obtained using gas chromatography-inductively coupled plasma-mass spectrometry (GC-ICP-MS). Mercury methylation and methylmercury demethylation processes in surface sediments were studied in the brackish Öre River estuary, Bothnian Bay. Uni- and multivariate data evaluation identified the organic material content and mercury methylation potential in the sediments as important factors controlling incipient methylmercury levels. Mercury species distribution in mice treated with the pharmaceutical preservative Thimerosal (ethylmercurithiosalicylate) was studied. The ethylmercury moiety of Thimerosal was observed to rapidly convert to inorganic mercury in the mice during the treatment period as well as during sample treatment, hence necessitating SSID methodology for accurate ethylmercury determinations in biological samples. To facilitate the introduction of SSID as a routine quantitative method in mercury speciation, a methylmercury isotopic certified reference material (ICRM) was produced. Prior to certification, the stability of the material was examined in conventional and isochronous stability studies spanning 12 months, which permitted uncertainty estimation of the methylmercury amount content for two years of shelf-life. Finally, a field-adapted SSID method for methylmercury determinations in natural water samples was developed. The proposed analytical protocol significantly simplified sample storage- and treatment procedures without sacrifices in analytical accuracy.

Studies of protein and peptide expression are vital in order to understand complex biological systems. As demonstrated in this thesis, on-line packed capillary liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR MS) is a useful analytical tool for such studies. A proteomics method, based on global tryptic digestion and subsequent separation and detection of the peptides by LC-FTICR MS, was developed for qualitative analysis of body fluids. Initial experiments on cerebrospinal fluid (CSF) provided results that were comparable or superior to those achieved by more time- and sample-consuming techniques. The method was also successfully applied on plasma and amniotic fluid. One of the major challenges in proteomics is the broad dynamic range of proteins in biological matrices. The advantages of removing high-abundant components from CSF and plasma prior to MS were demonstrated. In order to search for potential biomarkers, mass chromatograms of CSF from patients suffering from amyotrophic lateral sclerosis (ALS) and controls were compared using an in-house constructed pattern recognition program. ALS-specific patterns were observed, and four out of five unknown samples were correctly assigned. Alternative strategies to quantitatively compare two pools of samples rely on differential chemical labeling. The performance of one such method, quantification-using-enhanced-signal-tags, was investigated in complex sample analysis. The experimental intensity ratios were proven to be consistent with the prepared concentration ratios of abundant proteins in CSF. Finally, the thesis reports on the first experiments where electron capture dissociation (ECD) was successfully incorporated in on-line LC-MS experiments. ECD and nozzle-skimmer fragmentation were applied to a sample of endocrine peptides extracted from mouse pancreatic islets. The two fragmentation methods provided complementary information. However, the method needs further optimization before it can be applied in the analysis of more complex samples, such as body fluids.

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.