Performing quantitative analysis of targeted bioactive compounds in biological samples, such as blood plasma, cerebrospinal fluid or extracts from pig liver, put high demands on the ruggedness of the method acquiring the results. In addition to the complexity of the sample matrix, the bioactive compounds targeted for analysis usually have low levels of natural abundance, further increasing the demand on the analytical method sensitivity. Furthermore, quantitation of analytes at trace levels in the presence of large amounts of interfering species in biofluids must aim for repeatable precision, high accuracy ensuring the closeness to the true values, a linear response spanning over several orders of magnitude and low limits of quantitation to be valid for monitoring disease states in clinical analysis. An analytical method most commonly follow a certain order of events, called the analytical chain, which includes; experimental planning, sampling, sample pre-treatment, separation of species, detection, evaluation, interpretation and validation, all equally important for the outcome of the results. In this thesis, the scope has been to create novel methods, or to refine already existing methods, in order to achieve even better performances of the different events in the analytical chain. One of the aspects has been to sample and enrich analytes in vivo by the use of solid supported microdialysis, giving the advantage of almost real-time monitoring of analyte levels within a living host with targeted selectivity due to the analyte affinity for solid particles. Another aspect to selectively clean and enrich analytes in a complex matrix has been developed and automated on-line by the use of a column-switching technique before the analytical separation. By using several steps of extraction and separation coupled on-line to selected detection by the use of a triple quadrupole mass spectrometer facilitates great selectivity of species. The mass spectrometer also gives the ability to distinguish between isotopically labelled analogues coeluting with the analytes yielding the necessary accuracy for quantitative evaluation. Both development of analytical methods and clinical applications has been performed, as well as improvements of existing techniques, all to improve the quantitation of trace levels of targeted analytes in biofluids.

<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>

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.