Sample preparation as the first step in an analytical procedure has an important role, particularly in bioanalysis, because of the complexity of biological samples (blood plasma and urine). Biological matrix such as plasma and blood contains proteins, organic and inorganic salts, acids, bases and various organic compounds with similar chemistry to the analytes of interest. Thus the basic concept of a sample preparation method is to convert a real matrix into a format that is suitable for analysis by an analytical technique. Therefore the choice of an appropriate sample preparation method greatly influences the reliability and accuracy of the analysis results. The aim of this thesis was to develop and validate of microextraction by packed syringe (MEPS) as a fast, selective, accurate and fully automated sample preparation technique for determination of BAM peptides in human plasma and local anaesthtics in human plasma and urine samples using silica and polymer sorbents. First work presents use of MEPS technique online with LC-MS/MS as a tool for the quantification of BAM peptide in plasma samples. MEPS technique provides significant advantages such as the speed and the simplicity of the sample-preparation process. Compared with other extraction techniques, such as protein precipitation and ultrafiltration, MEPS gave cleaner samples and higher recovery. In the second work, MEPS technique was developed by using synthesized molecularly imprinted polymer (MIP) as a sorbent for selective quantification of a homologous series of local anaesthetics, containing lidocaine, ropivacaine, mepivacaine, and bupivacaine in human plasma and urine samples. Compared with other conventional sorbent, the use of MIP provides high selectivity of the extraction and decrease the matrix effect.

Beer is a fermented alcoholic beverage that spans back to over 5000 years years ago and is consumed all over the world. Beer is usually made from malted barley but it can also be made from different cereals and starches. Beer contains both saturated and unsaturated fatty acids (FA) which are derived from the malt that is used. Unsaturated fatty acids (FA) such as linoleic and linolenic acid are sensitive to oxidation which can affect foam, smell and taste of a beer. The purpose of this study was to extract and analyse FA profile of three different types of beer, Bryggmästaren Bästa Ekologiska, 5,2:an and Premium Gold to assess the durability of these types of beer.  Beer, fermented solution, malt and wort was provided by Åbro Bryggeri. Lipid extraction was performed on the samples, lipids were extracted with soxtec for malted product and liquid-liquid extraction for beer, fermented solution and wort. The lipids were analysed with Gas-Chromatography Flame Ion Detector.  The malted products contained unsaturated FA’s where linoleic acid was the dominating FA while the beers, fermented solutions and worts contained both unsaturated FAs and saturated FAs. The unsaturated FA were erucic acid while the saturated FAs were lauric acid, myristic acid and palmitic acid. The amount of FA’s detected in the beer was extremely low which is due to the method used as some fat might have been lost out during the liquid-liquid extraction or during an evaporation step due to a leakage in the selected rotary evaporation system. The conclusion of this study was that although fatty acids were present in malt and liquids from the brewing process, the amount was too low to cause risk of either an altered taste of the beer or increased risk of oxidation of unsaturated FAs. However, due to a limitation of data from extracted- and analysed samples, this reduces the outcome of this work and a quantitative study is needed in order to determine more exact FA profiles from the analysed samples.

The nitrogenous metabolite urea is an important biomarker that indicates cows’ nutritional intake. Today, determining the urea concentration from milk samples requires analysis techniques in a lab which is time-consuming and expensive. It would be favourable to have an on-site measurement method to achieve a fast detection allowing farmers to quickly adjust the feed of individual cows, which might make it possible to reduce costs, increase milk production, and/or reduce the amount of nitrogen emission to the environment. This thesis is a part of a collaboration project between Linköping University and the Swedish University of Agricultural Sciences named “On-farm measurement of milk urea - development of a sensor”. The thesis investigated the activity and stability of the urease in an electrochemical biosensor, in which the urease is immobilised via encapsulation in a gel of poly(carbamoyl sulfonate) on a screenprinted electrode coated with a metal catalyst (copper), a cation exchanger (Nafion) and a conductive polymer (polyaniline). The linear range of the biosensor was successfully extended up to 2500 μM urea with a diffusion barrier composed of chitosan and polyvinyl butyral, enabling higher urea concentrations measurement than without the barrier (680 μM). Reproducibility, reusability, and storage stability measurements of the urease immobilised electrodes were performed and evaluated. A comparison between free enzyme, immobilised enzyme on the electrode surface and immobilised enzyme on an electrode surface that had been stored for a few days, was conducted to determine urease activity and stability. In addition, five components of milk (casein, lactose, ammonium, iron, and ascorbic acid) were measured separately to evaluate their interferences during milk urea detection. Interference was observed in several cases. A final evaluation of the present electrochemical biosensor was done by analysis of real milk samples giving promising results for future development. / On-farm measurement of milk urea - development of a sensor

Coffee is one of the most consumed beverages in the world, with about 10 million tons of coffee consumed in 2021. There are a lot of steps between the coffee tree and served beverage, as there are several processing methods each contributing to differences in fragrance, aroma, and acidity. Coffee intake has shown to have health promoting effects and has been linked to lower mortality rate. The most well-known component of coffee is caffeine, a stimulant which has an awakening effect, but there are a lot more compounds which are less well known, like the chlorogenic acids. This is a family of related compounds which have antioxidative capacity and demonstrated anti-carcinogen, anti-obesity, and anti-diabetic effects in some studies. Specialty coffee is a category of coffee which has gone through a rigorous process where flavour, aroma, acidity, and a few other factors are taken into account before being graded. This poses as an extra income for some farmers, as it is more expensive than regular coffee. If climate keeps changing, this might lead to lesser opportunities for coffee farmers to grow and sell all kinds of coffee and working with higher quality, rather than higher quantity, might be a better option for the individual, as well as for the climate. RP-HPLC is a commonly used analysis method and will be used to conduct the analyses of this study. The aim of the study was to quantitatively determine the caffeine and 3-caffeoylquinic acid (3-CQA) content in artisanal processed specialty coffee in varieties with different origins and roasting grades. The coffee varieties were ground, and brewed using three methods, filter-funnel, French press and cold brew and thereafter analysed using RP-HPLC, developed from another study and configured to acquire quicker analyses. The development of the method yielded a standard curve which was used to calculate the caffeine and 3-CQA concentration in coffee samples. The results indicated that cold brews yielded higher concentrations of caffeine and 3-CQA in most cases. The least differences when comparing brewing methods within coffee varieties were seen between filter-funnel and French press coffee. The most notable difference was seen with the light roasted Ayla variety, which yielded much higher 3-CQA concentrations compared the other two varieties (Velo collective and Ureña). It can be concluded through other studies that the higher 3-CQA content could be explained by roasting grade, and that further analyses on raw coffee samples would be needed to test differences when accounting for origin, as different roasts affect the caffeine and 3-CQA content. / Kaffe är en av de mest konsumerade dryckerna i världen, med cirka 10 miljoner ton konsumerat år 2021. Det finns många steg från kaffeträd till kopp, då det finns olika sorters processning som alla bidrar till doft, smak och syrlighet. Intag av kaffe har associerats positiva hälsoeffekter och en lägre dödlighet. Den mest välkända komponenten i kaffe är koffein, en stimulant som har en uppiggande effekt, men det finns en del mindre kända komponenter, så som klorogensyrorna, som är en grupp av besläktade molekyler som har antioxidativ förmåga och uppvisat anti-carcinogena, anti-fetma och anti-diabetiska effekter i vissa studier. Specialitet-kaffe är en typ av kaffe som har gått igenom en gedigen process där smak, doft och syrlighet, samt ett flertal andra faktorer innan det betygsätts. Detta ger en möjlighet för kaffeodlare att tjäna mer pengar, då kaffet är dyrare. Om klimatförändringarna fortsätter i samma takt, så kan det leda till mindre möjligheter för kaffeodlare att försörja sig. Ett alternativ kan vara att satsa på att producera kaffe av hög kvalitet, snarare än hög kvantitet, vilket kan vara fördelaktigt även för miljön. RP-HPLC är en mycket vanlig analysmetod som användes för att utföra anlyser. Målet med denna studie var att kvantitativt bestämma koffein och 3-kaffeylkinasyra (3-CQA) koncentrationer i olika sorters hantverksmässigt processat kaffe med olika ursprung och rostgrad. De olika bryggmetoderna som utvärderades var filterkaffe, franskpress och kallbrygd (Cold Brew) och analyserades därefter genom en utvecklad RP-HPLC-metod som baserats från en tidigare studie och konfigurerats för att få snabbare analystider. En kalibreringskurva användes för att beräkna koffein- och 3-CQA-halt i de olika kaffeproverna. Resultaten indikerade att kallbryggt kaffe generellt gav högst koffein och 3-CQA koncentration i de flesta fallen. Minst skillnader mellan metoder fanns mellan filterbryggt och franskpresskaffe. Den största skillnaden sågs när man jämförde bryggmetoder på 3-CQA-koncentration, där Ayla-kaffet gav mycket högre halt gentemot de andra sorterna (Velo Collective och Ureña). Detta visade att ljusrostat kaffe ger en högre 3-CQA-halt vid bryggning, och detta kan bekräftas i andra studier som erhållit liknande resultat. Fler analyser skulle behöva göras på råa kaffebönor för att undersöka ifall ursprung påverkar halter utav koffein och 3-CQA, då rostgraden av bönan påverkar innehåll av båda.

<p>The thesis deals partly with methodology including construction</p><p>of hardware and new pulse sequences in the field of electrophoretic</p><p>NMR, and partly with practical use of ENMR and</p><p>diffusion NMR in the investigation of charged colloidal systems.</p><p>Several sources of artefacts are investigated, including gas production</p><p>at the electrodes, electroosmosis and Joule heating</p><p>effects that can cause convection. The electrophoretic double</p><p>stimulated-echo pulse sequence is introduced to suppress these</p><p>artefacts and to increase the feasible measuring range to higher</p><p>electric fields and conductivities.</p><p>The interaction between the non-ionic polymer poly(ethylene</p><p>oxide) PEO and differently charged surfactants is investigated</p><p>using the above mentioned methods. The investigated surfactants</p><p>are the anionic sodium dodecyl sulphate (SDS) and</p><p>potassium laurate (KC12), the cationic dodecyltrimethylammonium</p><p>bromide (CTAB) and the non-ionic octyl β-D-glucoside.</p><p>ENMR is also used to investigate two different mixed micelle</p><p>systems, with SDS as the charged surfactant component and</p><p>dodecyl malono-bis-N-methylglucamide (C12BNMG) respectively</p><p>tetra(ethylene oxide) dodecyl ether (C12EO4) as the nonionic</p><p>surfactant component. A method to calculate the degree</p><p>of counter-ion dissociation, αdissociation, as a function of composition</p><p>is demonstrated.</p><p>Finally diffusion NMR is used to compare transport dynamics</p><p>in gel electrolyte systems based on two differently grafted polymers;</p><p>one amphiphilic system containing polymethacrylate</p><p>grafted partly with polyethylene oxide and partly with fluorocarbons</p><p>and the corresponding nonamphiphilic system grafted</p><p>with only polyethylene oxide. Both systems contain the electrolyte</p><p>lithium bis(trifluoromethylsulfonyl) imide salt dissolved in</p><p>γ-butyrolactone. The results show that the system based on the</p><p>amphiphilic polymer has better transport dynamics and therefore</p><p>is more suited as material for battery</p>

<p>The combination of capillary electrophoresis (CE) and mass spectrometry (MS) constitutes a powerful microanalytical system in the fields of biology, medicine and chemistry. This thesis describes the development of three novel capillary coatings and demonstrates how these extend the utility of CE as a high-efficiency separation technique in protein analysis and biopharmaceutical drug screening.</p><p>Due to the rapidly growing interest in characterizing the human proteome, there is an increased need for rapid protein separations. The use of CE in protein analysis is, however, nontrivial due to problems with protein adsorption to the fused-silica capillary walls. In this thesis, this problem was addressed by developing two novel, physically adsorbed, cationic polymer surface coatings, denoted PolyE-323 and Q-agarose. By using simple rinsing protocols, highly reproducible coatings, stable over a wide range of pH 2-11 were generated. Successful protein separations using cationic-coated capillaries in CE-MS, equipped with either electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI), has been demonstrated.</p><p>In the pharmaceutical industry, favorable pharmacokinetic properties of a candidate drug, such as high bioavailability after oral administration, are crucial for a high success rate in clinical development. Tools for prediction of biopharmaceutically relevant drug properties are important in order to identify and discard poor candidate drugs as soon as possible. In this thesis, a membrane mimetic coating was developed by electrostatically immobilizing liposomes to the capillary wall, via an anchoring sublayer of Q-agarose. The liposome-coated capillaries were demonstrated in on-line CE-MS for prediction of drug membrane permeability.</p>

<p>In microscale separations, such as capillary electrophoresis and -liquid chromatography, the liquid flow rates are in the order of nanoliters per second. If such flow rates are to be interfaced with a mass spectrometer (MS) using electrospray (ES) ionization, without loss of separation efficiency, each fraction of the analyte zone must remain undisturbed by the high voltage contact necessary for ES. One design that accomplishes this is the pure sheathless approach, where a thin, vapor deposited metal film covers the outside of the electrospray emitter tip. </p><p>This thesis describes the development of such sheathless emitters. The lifetimes of polymer embedded gold (“fairy dust”) or graphite (“black dust”) emitters were shown to by far exceed those of previously used conductive films. In addition, the production of emitters with these coatings was substantially simplified. The increase in durability was found to be due to enhanced resistance towards the electrochemical processes associated with ES. In analogy, the reasons for the limited durability of previously used methods were correlated with their tendency to oxidize, or be mechanically removed, during electrochemical reactions. </p><p>Electrochemical processes associated with the electrospray potential were also found to seriously disturb analyses in which porous graphitic carbon was used as the separation medium. A proper choice of grounding point locations eliminated these disturbances.</p><p>At last, the differences regarding analytical performance of several sheathless interface configurations, used in capillary liquid chromatography, were examined. The best performance was obtained when a pure sheathless emitter with a conductive layer of polyimide and graphite was coupled to the LC column through a Teflon sleeve.</p>

<p>This thesis describes the utilization of the matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) and electrospray ionization (ESI) MS techniques for analysis of complex brain tissue samples. </p><p>Direct molecular profiling of biological samples using MALDI MS is a powerful tool for identifying phenotypic markers. MALDI MS-profiling of proteins and peptides directly on brain tissue sections was used for the first time to study experimental models of Parkinson’s disease (PD). The mass spectrometer was used to map the peptide and protein expression directly on 12 µm tissue sections in mass-to-charge (m/z) values, providing the capability of mapping specific molecules of the original sample, that is, localization, intensity and m/z ratio. Several protein and peptide expression profile differences were found in the dopamine denervated brains when compared to the corresponding controls, for example, calmodulin, cytochrome c, cytochrome c oxidase, and the neuroimmunophilin protein FKBP-12. The increased expression of FKBP-12 from the profiling experiments was supported by mRNA expression analysis and two-dimensional gel electrophoresis separation analysis. Multiple genetic deficits have linked impaired ubiquitin-conjugation pathways to various forms of familiar PD. This study showed for the first time an increased level of unconjugated ubiquitin specifically in the dorsal striatum of the dopamine depleted PD brain. The strength of the MALDI MS-profiling technique is that a minimum of sample handling and manipulation is necessary pre-analysis. This ensures preservation of the spatial localization of the biomolecules in the tissue section.</p><p>Biological liquid samples often contain high amounts of salt that is non-compatible with the ESI MS technique. A nano-flow capillary liquid chromatography (nanoLC) system coupled on-line with ESI-MS was used to study the metabolism of the peptide LVV-hemorphin-7 in the brain and blood using in vivo microdialysis. The microdialysis technique provides capabilities for very precise sampling in specific brain regions. The combination of on-line desalting and pre-concentration by nanoLC with ESI MS is a powerful tool to detect minute concentration of metabolic fragments and endogenous biomolecules.</p><p>The utilization of mass spectrometry in neuroscience applications provides a uniquely advantageous tool for the analysis of complex biochemical events that underlie the pathological symptoms expressed in different disease states. Furthermore, the MALDI-MS profiling technique shows great potential for the future with regards to proteome analysis and drug discovery.</p>

The combination of capillary electrophoresis (CE) and mass spectrometry (MS) constitutes a powerful microanalytical system in the fields of biology, medicine and chemistry. This thesis describes the development of three novel capillary coatings and demonstrates how these extend the utility of CE as a high-efficiency separation technique in protein analysis and biopharmaceutical drug screening. Due to the rapidly growing interest in characterizing the human proteome, there is an increased need for rapid protein separations. The use of CE in protein analysis is, however, nontrivial due to problems with protein adsorption to the fused-silica capillary walls. In this thesis, this problem was addressed by developing two novel, physically adsorbed, cationic polymer surface coatings, denoted PolyE-323 and Q-agarose. By using simple rinsing protocols, highly reproducible coatings, stable over a wide range of pH 2-11 were generated. Successful protein separations using cationic-coated capillaries in CE-MS, equipped with either electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI), has been demonstrated. In the pharmaceutical industry, favorable pharmacokinetic properties of a candidate drug, such as high bioavailability after oral administration, are crucial for a high success rate in clinical development. Tools for prediction of biopharmaceutically relevant drug properties are important in order to identify and discard poor candidate drugs as soon as possible. In this thesis, a membrane mimetic coating was developed by electrostatically immobilizing liposomes to the capillary wall, via an anchoring sublayer of Q-agarose. The liposome-coated capillaries were demonstrated in on-line CE-MS for prediction of drug membrane permeability.

The thesis deals partly with methodology including construction of hardware and new pulse sequences in the field of electrophoretic NMR, and partly with practical use of ENMR and diffusion NMR in the investigation of charged colloidal systems. Several sources of artefacts are investigated, including gas production at the electrodes, electroosmosis and Joule heating effects that can cause convection. The electrophoretic double stimulated-echo pulse sequence is introduced to suppress these artefacts and to increase the feasible measuring range to higher electric fields and conductivities. The interaction between the non-ionic polymer poly(ethylene oxide) PEO and differently charged surfactants is investigated using the above mentioned methods. The investigated surfactants are the anionic sodium dodecyl sulphate (SDS) and potassium laurate (KC12), the cationic dodecyltrimethylammonium bromide (CTAB) and the non-ionic octyl β-D-glucoside. ENMR is also used to investigate two different mixed micelle systems, with SDS as the charged surfactant component and dodecyl malono-bis-N-methylglucamide (C12BNMG) respectively tetra(ethylene oxide) dodecyl ether (C12EO4) as the nonionic surfactant component. A method to calculate the degree of counter-ion dissociation, αdissociation, as a function of composition is demonstrated. Finally diffusion NMR is used to compare transport dynamics in gel electrolyte systems based on two differently grafted polymers; one amphiphilic system containing polymethacrylate grafted partly with polyethylene oxide and partly with fluorocarbons and the corresponding nonamphiphilic system grafted with only polyethylene oxide. Both systems contain the electrolyte lithium bis(trifluoromethylsulfonyl) imide salt dissolved in γ-butyrolactone. The results show that the system based on the amphiphilic polymer has better transport dynamics and therefore is more suited as material for battery