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A feasibility study of solid supported enhanced microdialysis
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
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2004 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 76, no 6, 1678-1682 p.Article in journal (Refereed) Published
Abstract [en]

For the first time, a solid supported enhanced microdialysis methodology for analysis of neuropeptides is described. The microdialysis samples were, in this study, subsequently collected in fractions, dissolved from the solid particles, dried, and resolved in a formic acid buffer in order to make them suitable for capillary liquid chromatography-mass spectrometry. Different microdialysis flow profiles were evaluated where air-gapped continuous flow was considered most suitable for the solid supported microdialysis mode. Six endogenous neuropeptides were initially used to investigate the feasibility of this enhanced microdialysis methodology. The improved relative recovery obtained from the solid supported enhanced microdialysis was varying from no effect to 10 times higher as compared to ordinary microdialysis. The most efficient enrichment was obtained for luteinizing hormone releasing hormone, which was the largest but also the most hydrophilic of the peptides. In contrast, no significant difference in recovery was observed for Leu-enkephalin being the smallest and the most hydrophobic peptide tested. These results indicate an increased flux and selective uptake of hydrophilic peptides across the membrane and enrichment on the particles in solid supported microdialysis.

Place, publisher, year, edition, pages
2004. Vol. 76, no 6, 1678-1682 p.
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:uu:diva-93181DOI: 10.1021/ac035305lPubMedID: 15018567OAI: oai:DiVA.org:uu-93181DiVA: diva2:166581
Available from: 2005-05-10 Created: 2005-05-10 Last updated: 2013-06-14Bibliographically approved
In thesis
1. Microscale Tools for Sample Preparation, Separation and Detection of Neuropeptides
Open this publication in new window or tab >>Microscale Tools for Sample Preparation, Separation and Detection of Neuropeptides
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Mikroskaliga verktyg för provpreparering, separation och detektion av neuropeptider
Abstract [en]

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.

Place, publisher, year, edition, pages
Uppsala: Kemiska institutionen, 2005. 62 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 64
Keyword
Analytical chemistry, Neuropeptides, Microchip, Enhanced microdialysis, Poly(dimethylsiloxane) (PDMS), Electrospray ionization (ESI), Multidimensional separation, Electrochemical manipulation, Mass spectrometry (MS), Capillary electrophoresis (CE), Microdevice, Microfabrication, Micro total analysis system (μ-TAS), Analytisk kemi
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-5838 (URN)91-554-6279-0 (ISBN)
Public defence
2005-06-03, Room B42, BMC, Uppsala, 10:15
Opponent
Supervisors
Available from: 2005-05-10 Created: 2005-05-10 Last updated: 2011-12-08Bibliographically approved
2. Development of Techniques and Methods for the Quantitative Analysis of Endogenous Substances by Microcolumn Liquid Chromatography Coupled to Mass Spectrometry
Open this publication in new window or tab >>Development of Techniques and Methods for the Quantitative Analysis of Endogenous Substances by Microcolumn Liquid Chromatography Coupled to Mass Spectrometry
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Utveckling av tekniker och metoder för kvantitativ analys av endogena substanser med mikrokolonn vätskekromatografi sammankopplad med masspektrometri
Abstract [en]

Liquid chromatography-mass spectrometry (LC-MS) is a powerful technique for the analysis of endogenous compounds. The introduction of electrospray ionization (ESI) as an interface between LC and MS has contributed strongly to a trend towards miniaturization of LC, due to the possibility to perform ESI at low flow rates. In this thesis, several aspects regarding the design of miniaturized LC systems and electrospray emitters were investigated. In addition miniaturized LC-ESI-MS have been used for the qualitative and quantitative analysis of endogenous polar compounds, peptides and protein digests.

The performance of miniaturized LC-MS was compared using different electrospray emitter configurations. The results indicated that the efficiency of the LC system is rather independent of the configuration of the emitter.

The lifetime of gold-coated fused silica electrospray emitters based on vapor deposited adhesion layers of titanium were investigated. The long lifetime of the emitter facilitates the use in LC-MS experiments, exemplified LC-MS by analysis of neuropeptides.

The ESI voltage is shown to interfere with liquid chromatographic separations performed in packed porous graphitic carbon capillary column. This interference is ascribed to the presence of an electric field over the conductive column in absence of a ground point between the column and the ESI emitter.

The solid supported enhanced microdialysis for analysis of neuropeptides were compared with conventional microdialysis. The difference between the two methodologies were evaluated by LC-MS analysis of the microdialysates. The solid supported method gave in general higher relative recoveries.

Finally, a method of standard addition was developed to determine total level of tryptophan and two of its metabolites in human plasma by capillary LC-ESI tandem mass spectrometry. The method was applied in a clinical study of multiple scleroses patients treated with cytokines (IFN Beta 1a, 1b). The results show that the intervention effects the tryptophan metabolism.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2004. 48 p.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 936
Keyword
Analytical chemistry, Analytisk kemi
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-3990 (URN)91-554-5869-6 (ISBN)
Public defence
2004-02-20, B 41, BMC, Husargatan 3 75 237, Uppsala, 10:15
Opponent
Supervisors
Available from: 2004-01-30 Created: 2004-01-30 Last updated: 2013-06-14Bibliographically approved
3. Quantitative Bioanalysis: Liquid separations coupled to targeted mass spectrometric measurements of bioactive compounds
Open this publication in new window or tab >>Quantitative Bioanalysis: Liquid separations coupled to targeted mass spectrometric measurements of bioactive compounds
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2008. 61 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 412
Keyword
Analytical chemistry, Method development, Enhanced microdialysis, Column switching, Liquid chromatography, Capillary electrophoresis, Electrospray ionization, Mass spectrometry, Triple quadrupole, Time-of-flight, Quantitation, Validation, Analytisk kemi
National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-8581 (URN)978-91-554-7135-4 (ISBN)
Public defence
2008-04-10, B22, BMC, Husargatan 3, Uppsala, 10:15 (English)
Opponent
Supervisors
Available from: 2008-03-17 Created: 2008-03-17 Last updated: 2013-06-20Bibliographically approved

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