<p>Oxidative stress can be defined as a state of disturbance in the pro-oxidant/antioxidant balance in favour of the former, leading to potential damage. Processes associated with oxidative stress involve reactive oxygen species and radicals and can result in elevated levels of oxidatively modified or toxic molecules that can cause cellular malfunction, and even cell death. Destruction of membrane lipids, lipid peroxidation, caused by reactive oxygen species and radicals has been coupled to many diseases and also normal ageing. </p><p>The measurement of low molecular weight biomarkers of oxidative stress present in complex matrices such as brain tissue, plasma, urine or cerebrospinal fluid is a delicate and difficult task and there is a need for improved analytical tools in this field of research. </p><p>The major foci of this thesis and the work underlying it are the development of analytical techniques and methods for determining biomarkers for oxidative stress and lipid peroxidation. Aspects of particular concern include the effects of sample treatments prior to analysis, evaluation of the developed methods with respect to possible artefacts, and the scope for results to be misinterpreted. The specific research goals and issues addressed are detailed in five papers, which this thesis is based upon.</p><p><b>Paper I</b> focuses on malondialdehyde, describing and evaluating two new simplified sample pre-treatment regimes for the determination of malondialdehyde in rat brain tissue by capillary electrophoresis with UV detection. The effects of sample storing and handling are also considered.</p><p><b>Paper II</b> describes the synthesis, characterization and implementation of a new internal standard for the determination of malondialdehyde in biological samples using electrophoretic or chromatographic separation techniques. The usefulness of the internal standard is demonstrated in analyses of rat brain tissue samples.</p><p><b>Paper III</b> presents a method for the determination of 4-hydroxynon-2-enal in brain tissue from rats employing micellar electrokinetic chromatography separation and laser-induced fluorescence detection. </p><p><b>Paper IV</b> is focused on the development of a new methodology for determining the stereoisomeric F2-isoprostanes in human urine samples employing chromatographic separation on porous graphitic carbon and detection by electrospray ionization-tandem mass spectrometry. The results from this study conflict with the hypothesis that peripheral isoprostanes are elevated in patients with Alzheimer’s disease.</p><p><b>Paper V</b> describes porous graphitic carbon chromatography-tandem mass spectrometry for the determination of isoprostanes in human cerebrospinal fluid. A new simplified sample pre-treatment regime, involving a column switching technique, is presented that allows direct injection of a relatively large volume of CSF into the chromatographic system.</p>

<p>Infrared absorption spectra can provide analytically useful information on a large variety of compounds, ranging from small ions to large biological molecules. In fact, all analytes that possess a dipole moment that changes during vibration are infrared-active. The infrared (IR) spectrum can be subdivided into far-, mid- and near- regions. The focus of attention in this thesis is the mid-IR region, in which the fundamental vibrations of most organic compounds are located, thus providing scope for positive structural identification. However, while such near-ubiquitous signals can be very useful for monitoring simple molecules in simple systems, they can be increasingly disadvantageous as the number of analytes and/or the complexity of the sample matrix increases. Thus, hyphenation to a separation system prior to detection is desirable. Paper I appended to this thesis presents (for the first time) the on-line hyphenation between Fourier transform infrared spectroscopy, FTIR, and capillary zone electrophoresis, CZE. CZE is a highly efficient separation technique that separates ionic analytes with respect to their charge-to-size ratio. It is most commonly performed in aqueous buffers in fused silica capillaries. Since these capillaries absorb virtually all infrared light an IR-transparent flow cell had to be developed. In further studies (Paper II) the applicability of CZE is expanded to include neutral analytes by the addition of micelles to the buffer, and micellar electrokinetic chromatography, MEKC, was successfully hyphenated to FTIR for the first time. Paper III describes an application of the on-line CZE-FTIR technique in which non-UV-absorbing analytes in a complex matrix were separated, identified and quantified in one run.</p><p>Measuring aqueous solutions in the mid-IR region is not straightforward since water absorbs intensely in this region, sometimes completely, leaving no transmitted, detectable light. For this reason, quantum cascade lasers are interesting. These lasers represent a new type of mid-IR semiconducting lasers with high output power due to their ingenious design. The laser action lies within one conduction band (intersubband) and can be tailored to emit light in the entire mid-IR region using the same semiconducting material. To investigate their potential to increase the optical path length in aqueous solutions, these lasers were used with an aqueous flow system (Paper IV), and the experience gained in these experiments enabled hyphenation of such lasers to a CZE system (Paper V).</p>

<p>This is to my knowledge the first time a ten-year follow-up study of salivary cortisol concentrations measured by immunoassays in relation to posttraumatic symptoms according to the Impact of Event Scale (IES) is made. The study was performed on 78 Swedish UN soldiers after a 6-months mission in the former republic of Yugoslavia. Follow-up investigations were performed six months, twelve months and ten years after their return to Sweden. Morning and evening salivary cortisol concentrations were determined by radioimmunoassay (RIA) and enzyme-linked immunoassay (EIA) and subjective posttraumatic avoidance and intrusion symptoms were measured with the IES (see Appendix I).</p><p> </p><p>This study concerns the methodological description of the EIA for determination of salivary cortisol and the comparison of the results from all three follow-up investigations. Post-traumatic stress symptoms according to IES (intrusion subscale and total score) increased significantly over ten years of time. There was an significant interrelationship between the change in both morning and evening salivary cortisol concentrations, measured with immunoassays, and changes in self-rated posttraumatic intrusive symptoms, according to IES, during ten years follow-up, after a six months mission in Bosnia in the way that salivary cortisol concentrations showed a tendency to decrease over ten years of time in subjects with a higher IES score. The rise in morning salivary cortisol, from awakening until 30 minutes later, was significantly correlated with the ratings of posttraumatic stress symptoms according to the IES ten years after the mission.   </p>

<p>Today there are no fully general analytical techniques available for detection and confirmation of known and unknown substances in toxicological screening, further tools are therefore needed. The development of mass spectrometry with time-of-flight (TOF) detection is promising but there are still areas to be further developed and evaluated, both instrumentation and applications.</p><p>During 2009 The National Board of Forensic Medicine-Department of Forensic Genetics and Forensic Toxicology, (RMV) started cooperation with the instrumentation company Waters (Manchester, UK) and the Department of Clinical Pharmacology (KI, Solna) evaluating a new TOF-instrument for toxicological screening. My assignment as a part of this project has been to create a limited and relevant database of drugs and toxics in Excel, including monoisotopic mass, used when screening for pharmaceutical substances and their metabolites most probable to be found in Swedish autopsy material.</p><p>A limited database has been developed based on information from several sources, it ended up in 875 analytes and metabolites. A limited but complete database is more reliable in practise than a big database, by means of a lower frequency of isobars and more information included (e.g. retention time from liquid chromatography) making analysis faster. Commercial databases are generally theoretical, lacking information about for example retention time that often is an important criterion for identification.</p>

Analysis of biological samples can be a difficult task. This thesis covers a broad aspect of the analytical areas of capillary electrophoresis (CE) and capillary electrochromatography (CEC) in combination with mass spectrometry (MS) that are of great importance for achieving fast, accurate and sensitive bioanalyses. A significantly time reduced and automated system for sample cleanup was developed to greatly simplify the pretreatment process of biological samples with a complex matrix. Desalting and preconcentration of species in urine was conducted and the limit of detection for the antidepressant escitalopram was lowered 10 times. This extraction devise was also successfully incorporated in a chip based platform for the possibility to be a part of multidimensional separation systems. The reduced risk of sample loss leads to improved detection limits, which are usually one the most challenging parts when working with bioanalyses. In the area of separation, a monomer surface with tailored hydrophobicity was developed to achieve rapid, high efficient separations of complex mixtures. Within five minutes a tryptic digest of a protein could be separated and then identified by a Mascot search. The applications addressed have been focused on medical conditions which are of highest interest for both physicians and patients. A high throughput analysis of the kynurenine metabolites with CE-MS offers a new method to rapidly examine samples from patients with neurological disorders. A screening study of possible biomarkers for the two different types of appendicitis, gangraenous and phlegmonous was conducted. Indicative patterns were found for both pre and post surgery of the two types of inflammation as well as between them. The divergences were traced back to the MS peaks obtained in the CE- and CEC-MS setups as possible biomarkers for the two forms of appendicitis. A preliminary study of polycystic ovary syndrome also offered some valuable results for future biomarker identification.

Development and improvement of analytical techniques are vital in analytical chemistry research. This thesis describes the development and use of tailored surfaces for bioanalytical applications. In sample preparation, solid phase extraction is often used and the development of a protocol for extraction on a molecular imprinted polymer (MISPE) directly from plasma sample is presented. Molecular imprinted polymers (MIP) offer selective sorbents for the imprinted analyte. MISPE has mainly been used in organic phase but in this thesis the development of a protocol for direct extraction of the analyte form an aqueous phase is described. For analysis of complex samples a separation step is often needed. The growing interest in analysis of biological samples and analysis of the human proteome and potential biomarkers has increased the interest in developing new separation techniques. Capillary electrophoresis (CE) has evolved into an important technique for use in analysis of body fluids. In this thesis a novel polyamine coating named PolyE 323 tailored for minimizing the adsorption of basic proteins to the surface is introduced. A straightforward coating protocol, with four simple rinsing steps, was developed. The coating was highly reproducible and useable over a wide pH range. Successful protein separations on PolyE-323-coated capillaries coupled to electrospray ionization mass spectrometry (ESI-MS) were demonstrated. The coated capillaries were also used in studies of protein content of aqueous humor samples from cataract patients as a complement to capillary liquid chromatography. In the studies presented the protein content of aqueous humor samples from two clinical groups was compared. By using capillary liquid separation techniques coupled to matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and MS/MS in combination with isobaric tags for relative and absolute quantitation (iTRAQ) the identity and relative concentrations of proteins in the samples were evaluated. Earlier studies of the proteins in these kinds of samples have mainly been done with techniques using immunological detection where the proteins of interest were chosen in advance. In this thesis it was shown that liquid-based separation techniques are a good complement and by using the mass spectrometry approach presented the protein content of the samples could be evaluated without bias.

Oxidative stress can be defined as a state of disturbance in the pro-oxidant/antioxidant balance in favour of the former, leading to potential damage. Processes associated with oxidative stress involve reactive oxygen species and radicals and can result in elevated levels of oxidatively modified or toxic molecules that can cause cellular malfunction, and even cell death. Destruction of membrane lipids, lipid peroxidation, caused by reactive oxygen species and radicals has been coupled to many diseases and also normal ageing. The measurement of low molecular weight biomarkers of oxidative stress present in complex matrices such as brain tissue, plasma, urine or cerebrospinal fluid is a delicate and difficult task and there is a need for improved analytical tools in this field of research. The major foci of this thesis and the work underlying it are the development of analytical techniques and methods for determining biomarkers for oxidative stress and lipid peroxidation. Aspects of particular concern include the effects of sample treatments prior to analysis, evaluation of the developed methods with respect to possible artefacts, and the scope for results to be misinterpreted. The specific research goals and issues addressed are detailed in five papers, which this thesis is based upon. <b>Paper I</b> focuses on malondialdehyde, describing and evaluating two new simplified sample pre-treatment regimes for the determination of malondialdehyde in rat brain tissue by capillary electrophoresis with UV detection. The effects of sample storing and handling are also considered. <b>Paper II</b> describes the synthesis, characterization and implementation of a new internal standard for the determination of malondialdehyde in biological samples using electrophoretic or chromatographic separation techniques. The usefulness of the internal standard is demonstrated in analyses of rat brain tissue samples. <b>Paper III</b> presents a method for the determination of 4-hydroxynon-2-enal in brain tissue from rats employing micellar electrokinetic chromatography separation and laser-induced fluorescence detection. <b>Paper IV</b> is focused on the development of a new methodology for determining the stereoisomeric F2-isoprostanes in human urine samples employing chromatographic separation on porous graphitic carbon and detection by electrospray ionization-tandem mass spectrometry. The results from this study conflict with the hypothesis that peripheral isoprostanes are elevated in patients with Alzheimer’s disease. <b>Paper V</b> describes porous graphitic carbon chromatography-tandem mass spectrometry for the determination of isoprostanes in human cerebrospinal fluid. A new simplified sample pre-treatment regime, involving a column switching technique, is presented that allows direct injection of a relatively large volume of CSF into the chromatographic system.

Infrared absorption spectra can provide analytically useful information on a large variety of compounds, ranging from small ions to large biological molecules. In fact, all analytes that possess a dipole moment that changes during vibration are infrared-active. The infrared (IR) spectrum can be subdivided into far-, mid- and near- regions. The focus of attention in this thesis is the mid-IR region, in which the fundamental vibrations of most organic compounds are located, thus providing scope for positive structural identification. However, while such near-ubiquitous signals can be very useful for monitoring simple molecules in simple systems, they can be increasingly disadvantageous as the number of analytes and/or the complexity of the sample matrix increases. Thus, hyphenation to a separation system prior to detection is desirable. Paper I appended to this thesis presents (for the first time) the on-line hyphenation between Fourier transform infrared spectroscopy, FTIR, and capillary zone electrophoresis, CZE. CZE is a highly efficient separation technique that separates ionic analytes with respect to their charge-to-size ratio. It is most commonly performed in aqueous buffers in fused silica capillaries. Since these capillaries absorb virtually all infrared light an IR-transparent flow cell had to be developed. In further studies (Paper II) the applicability of CZE is expanded to include neutral analytes by the addition of micelles to the buffer, and micellar electrokinetic chromatography, MEKC, was successfully hyphenated to FTIR for the first time. Paper III describes an application of the on-line CZE-FTIR technique in which non-UV-absorbing analytes in a complex matrix were separated, identified and quantified in one run. Measuring aqueous solutions in the mid-IR region is not straightforward since water absorbs intensely in this region, sometimes completely, leaving no transmitted, detectable light. For this reason, quantum cascade lasers are interesting. These lasers represent a new type of mid-IR semiconducting lasers with high output power due to their ingenious design. The laser action lies within one conduction band (intersubband) and can be tailored to emit light in the entire mid-IR region using the same semiconducting material. To investigate their potential to increase the optical path length in aqueous solutions, these lasers were used with an aqueous flow system (Paper IV), and the experience gained in these experiments enabled hyphenation of such lasers to a CZE system (Paper V).

In this final thesis, an existing method for analysis of chlorophenols (CP) in bottom sediments has been updated and adjusted for analysis of chlorophenols in soil. The covalent bonds between the chlorophenols and the soil matrix were broken through basic hydrolysis and the chlorophenols were then separated from the water phase through addition of sulphuric acid followed by ether extraction. The chromatography was improved through extractive acetylation of the chlorophenols. The updated method was then applied on soil samples from a contaminated area (a former sawmill in Hyttsjö, Östergötland, Sweden). The analyse was preformed by GC/MS with respect to 2-MonoCP, 4-MonoCP, 2,4-DiCP, 2,6-DiCP, 2,4,6-TriCP, 2,3,4,6-TetraCP and pentachlorophenol (PCP). Contamination of chlorophenols in nature can be explained by the former use of wood preservative chemicals based on chlorophenols. In the 1960s and the 1970s these chemicals were used in Sweden, but due to their toxicity they were banned by the Swedish government in 1978. In Hyttsjö a pentachlorophenol-based product named Santobrite was used for several years. The concentration of PCP in the soil samples from Hyttsjö varied from 0.2-&gt;1.8 ng/mg dry substance. 2,3,4,6- Tetrachlorophenol was also detected in some of the soil samples.

Liquid chromatography is an analytical technique that is constantly facing new challenges in the separation of small molecules and large biomacromolecules. Recently the development of ultra high pressure liquid chromatography has increased the demand on sturdy particles as stationary phase. At the same time the particle size has decreased to sub-2 µm and packed into shorter analytical columns. This thesis deals with the development of new ways of preparing particulate polymer materials using divinylbenzene (DVB) as crosslinker. It includes a novel procedure for synthesizing monodisperse polymer particles by photoinitiated precipitation polymerization. A 150 W short arc xenon lamp was used to initiate the polymerizations. The synthesized particles are monodisperse and have an average particle size ranging from 1.5 to 4 μm depending on reaction conditions and have subsequently been used as grafting templates. The surface of DVB particles contains residual vinyl groups that serve as anchoring points for further functionalization via a variety of grafting schemes. Copolymerization with incorporation of 2,3-epoxypropyl methacrylate yielded pendant oxirane groups on the particle surface. Atom transfer radical polymerization (ATRP) was used to graft methacrylates from the surface resulting in a core-shell type material. A “grafting to” scheme was used to attach pre-made sulfopropyl methacrylate telomers onto particles containing oxirane rings. / Populärvetenskaplig sammanfattning på svenska: Vätskekromatografi är en analytisk kemisk teknik som ständigt står inför nya utmaningar när det gäller att separera allt från små organiska föreningar till stora makro¬molekyler. Denna avhandling beskriver tillverkning av polymera partiklar med exceptionellt jämn storleksfördelning och ytmodifiering av dessa, för användning som stationärfas i kromatografi¬kolonner. Polymeriserings¬tekniken som används är utfällnings¬polymerisering där lösningen UV-bestrålas av en 150 W xenonlampa. Monomeren (byggstenen) löses tillsammans med en intiator i ett lösningsmedel och efterhand som polymeriseringen fortskrider faller polymerpartiklarna ut. Polymerpartiklarna är gjorda av monomeren divinylbensen som fungerar som en tvärbindare, dvs att den länkar ihop flera kedjor till ett hårt litet nystan. Partiklarna växte till en storlek på 1,5 till 4 µm under två till fyra dygn. Efter tillverkningen är partiklarnas yta täckta av vinylgrupper som kan användas för att fästa funktionella polymerkedjor. Genom att tillföra monomeren 2,3-epoxipropyl¬metakrylat i polymeriseringen kunde man desutom få en partikelyta som innehöll epoxigrupper. Epoxigrupperna användes för att fästa positivt laddade polymerkedjor av bestämd längd. Materialet packades i en kromatografikolonn och användes för att separera en testlösning bestående av fyra proteiner. Partiklarna användes även som bas för ymppolymerisering där den vinyltäckta ytan fått reagera med vätebromid. Detta gör att partiklarna blir stora makroinitiatorer som kan användas för att på ett kontrollerat sätt låta polymerkedjor växa från ytan. I en undersökning ympades 2,3-epoxypropylmetakrylat från ytan på partiklarna och resultatet blev ett tjockt ytskikt. Epoxigrupperna kunde sedan hydrolyseras till dioler vilket gjorde partiklarna mer hydrofila.