<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>Three chiral selectors (diketogulonic acid, benzoxycarbonylglycylproline and ketopinic acid) have been introduced for enantioseparation of pharmacologically active amines in non-aqueous capillary electrophoresis. The use of organic solvents, instead of aqueous buffers in the background electrolyte facilitated ion-pair formation between the analytes and the chiral selectors. The enantioresolution was strongly affected by the choice of selector and organic solvent but also depended on the other electrolytes. The most important parameter for the enantioresolution, apart from the choice of chiral selector, was the direction and magnitude of the electro-osmosis. Thus, covalently coated capillaries were used to suppress and to reverse this flow. Furthermore, the alkali metal hydroxide added to the background electrolyte had a great influence on the electro-osmosis. Exchanging LiOH for NaOH, was found to decrease the electro-osmotic flow. Interestingly, the flow was altered from cathodic to anodic, with KOH, RbOH or CsOH added to the ethanolic BGE. The occurrence of a reversed electro-osmosis had a great positive effect on the enantioresolution. An appropriate choice of solvent and electrolytes promoted also fast chiral separations, e.g., the enantiomers of isoprenaline were resolved within one minute. </p><p>The capillary electrophoresis systems developed within this work were applied for enantiomeric purity determinations of different pharmaceutical forms of drug products. A detection limit of 0.033 % was achieved for <i>1S,2R</i>-ephedrine, the enantiomeric impurity in Efedrin®, when diketogulonic acid was used as the selector. </p><p>By using the pre-concentration technique, transient isotachophoresis, the peak efficiency was enhanced for the enantiomers of timolol. This facilitated the introduction of a higher concentration of the sample into the capillary electrophoretic system containing ketopinic acid as the selector, and lowered the detection limit from 2.5 % to 0.2 % for the enantiomeric impurity <i>R</i>-timolol compared with injection without transient isotachophoresis.</p><p>The volatility of the non-aqueous media in capillary electrophoresis facilitated the hyphenation to mass spectrometry. The partial filling technique ensured that the selector did not contaminate the mass spectrometer, and the separated enantiomers of e.g., pronethalol were detected in the selector-free zone. </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.

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.

Three chiral selectors (diketogulonic acid, benzoxycarbonylglycylproline and ketopinic acid) have been introduced for enantioseparation of pharmacologically active amines in non-aqueous capillary electrophoresis. The use of organic solvents, instead of aqueous buffers in the background electrolyte facilitated ion-pair formation between the analytes and the chiral selectors. The enantioresolution was strongly affected by the choice of selector and organic solvent but also depended on the other electrolytes. The most important parameter for the enantioresolution, apart from the choice of chiral selector, was the direction and magnitude of the electro-osmosis. Thus, covalently coated capillaries were used to suppress and to reverse this flow. Furthermore, the alkali metal hydroxide added to the background electrolyte had a great influence on the electro-osmosis. Exchanging LiOH for NaOH, was found to decrease the electro-osmotic flow. Interestingly, the flow was altered from cathodic to anodic, with KOH, RbOH or CsOH added to the ethanolic BGE. The occurrence of a reversed electro-osmosis had a great positive effect on the enantioresolution. An appropriate choice of solvent and electrolytes promoted also fast chiral separations, e.g., the enantiomers of isoprenaline were resolved within one minute. The capillary electrophoresis systems developed within this work were applied for enantiomeric purity determinations of different pharmaceutical forms of drug products. A detection limit of 0.033 % was achieved for 1S,2R-ephedrine, the enantiomeric impurity in Efedrin®, when diketogulonic acid was used as the selector. By using the pre-concentration technique, transient isotachophoresis, the peak efficiency was enhanced for the enantiomers of timolol. This facilitated the introduction of a higher concentration of the sample into the capillary electrophoretic system containing ketopinic acid as the selector, and lowered the detection limit from 2.5 % to 0.2 % for the enantiomeric impurity R-timolol compared with injection without transient isotachophoresis. The volatility of the non-aqueous media in capillary electrophoresis facilitated the hyphenation to mass spectrometry. The partial filling technique ensured that the selector did not contaminate the mass spectrometer, and the separated enantiomers of e.g., pronethalol were detected in the selector-free zone.

The thesis focuses on the development of new homogeneous gels for the separation of drug enantiomers, peptides, DNA and virus by electrophoresis and electrochromatography in capillaries and microchips. This type of separation media offers high resolution and small zone broadening. Compared to particulate beds the resolution in this type of separation media is high because the eddy diffusion is zero and the resistance to mass transfer is small, since the diffusional distance between two polymer chains in the gel is considerably shorter than that between two beads in a packed bed. The gels have been characterized in terms of plate heights, plate numbers, resolution, etc. Gels of agarose, polyvinyl alcohol, albumin and polyacrylamide have been employed for electrochromatography or electrophoresis. N,N’-methylene-bisacrylamide, the most widely used crosslinker in polyacrylamide gels, was exchanged for allyl-β-cyclodextrin to get a multi-purpose gel, i.e., a separation medium the separation properties of which is determined not only by the polyacrylamide chains, but also by β-cyclodextrin with its complexation power. A cost-effective, hybrid microdevice has been designed for fast electrophoretic and electrochromatographic analyses as well as for microchromatography. It consists of a fused silica capillary mounted on a supporting plate which integrates most of the compartments necessary for automation and sensitive detection at short UV wavelengths.

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. 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. 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. 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.

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).