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Development of Techniques and Methods for the Quantitative Analysis of Endogenous Substances by Microcolumn Liquid Chromatography Coupled to Mass Spectrometry
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
2004 (English)Doctoral thesis, comprehensive summary (Other academic)Alternative title
Utveckling av tekniker och metoder för kvantitativ analys av endogena substanser med mikrokolonn vätskekromatografi sammankopplad med masspektrometri (Swedish)
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. , p. 48
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 936
Keyword [en]
Analytical chemistry
Keyword [sv]
Analytisk kemi
National Category
Analytical Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-3990ISBN: 91-554-5869-6 (print)OAI: oai:DiVA.org:uu-3990DiVA, id: diva2:164038
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
List of papers
1. Comparison between different sheathless electrospray emitter configurations regarding the performance of nanoscale liquid chromatography time-of-flight mass spectrometry analysis
Open this publication in new window or tab >>Comparison between different sheathless electrospray emitter configurations regarding the performance of nanoscale liquid chromatography time-of-flight mass spectrometry analysis
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2004 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1033, no 2, p. 257-266Article in journal (Refereed) Published
Abstract [en]

Four different sheathless electrospray ionization (ESI) configurations were investigated for a nano liquid chromatography (LC) system. The studied configurations were: a column with an integrated emitter, with the ESI potential applied before or after the column, and a column with separate emitter, with the ESI voltage applied at a union before the emitter or at the emitter tip. The results indicates that the efficiency of the LC system is rather independent of the configuration when using 95 μm i.d. columns, acetic mobile phase and standard peptides as a sample. Introduction of post column dead volume seems not to be a critical issue at least with flow rates down to 600 nl/min.

Keyword
Electrospray ionization, Band broadening, Principal component analysis, Instrumentation, Peptides
National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-91889 (URN)10.1016/j.chroma.2004.01.063 (DOI)
Available from: 2004-05-25 Created: 2004-05-25 Last updated: 2017-12-14Bibliographically approved
2. Gold-coated fused-silica sheathless electrospray emitters based on vapor-deposited titanium adhesion layers
Open this publication in new window or tab >>Gold-coated fused-silica sheathless electrospray emitters based on vapor-deposited titanium adhesion layers
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2003 In: Rapid Communication Mass Spectrometry, Vol. 17, no 14, p. 1535-1540Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-91337 (URN)
Available from: 2004-01-30 Created: 2004-01-30Bibliographically approved
3. Interference of the electrospray voltage on chromatographic separations using porous graphitic carbon columns
Open this publication in new window or tab >>Interference of the electrospray voltage on chromatographic separations using porous graphitic carbon columns
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2004 (English)In: Journal of Mass Spectrometry, ISSN 1076-5174, E-ISSN 1096-9888, Vol. 39, no 2, p. 216-222Article in journal (Refereed) Published
Abstract [en]

The electrospray ionization (ESI) voltage is shown to interfere with liquid chromatographic separations performed with packed porous graphitic carbon (PGC) capillary columns. This interference is ascribed to the presence of an electric field over the conductive column in the absence of an earth point between the column and the ESI emitter. The current evolved alters the chromatographic behavior of the catecholamine metabolite 3-O-methyl-DOPA significantly, as both peak splitting and a dramatic decrease in the retention time were observed. Furthermore, the response from the mass spectrometer was decreased by 33% at the same time. A related compound, tyrosine, exhibited decreased retention times but no peak splitting, whereas no shifts in the retention times (or peak splitting) were seen for the less retained dopamine and noradrenaline. When the current through the PGC column was eliminated by the use of an earth point between the column and the ESI emitter, the chromatographic behavior of the column was found to return slowly to normal after hours of equilibration with 60 : 40 (v/v) methanol-ammonium formate buffer of pH 2.9. The behavior of the PGC column with and without the earth point was found to be highly reproducible during a period of 1 month. We propose that the effect of the ESI voltage on the chromatographic behavior of the PGC column is due to associated redox reactions affecting both the PGC particles and the analytes. It is concluded that (for analytical reasons), care should be taken to ensure that no current is flowing through the chromatographic system when interfacing PGC columns, and conducting parts in general, to ESI mass spectrometry.

Keyword
catecholamine, dopamine, electrochemistry, porous graphitic carbon, electrospray, mass spectrometry, liquid chromatography
National Category
Inorganic Chemistry
Identifiers
urn:nbn:se:uu:diva-91888 (URN)10.1002/jms.589 (DOI)
Available from: 2004-05-25 Created: 2004-05-25 Last updated: 2017-12-14Bibliographically approved
4. A feasibility study of solid supported enhanced microdialysis
Open this publication in new window or tab >>A feasibility study of solid supported enhanced microdialysis
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2004 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 76, no 6, p. 1678-1682Article 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.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-93181 (URN)10.1021/ac035305l (DOI)15018567 (PubMedID)
Available from: 2005-05-10 Created: 2005-05-10 Last updated: 2017-12-14Bibliographically approved
5. Quantitation of tryptophan, kynurenine and kynurenic acid in human plasma by capillary liquid chromatography - electrospray
Open this publication in new window or tab >>Quantitation of tryptophan, kynurenine and kynurenic acid in human plasma by capillary liquid chromatography - electrospray
2002 In: Journal of Chromatography B, Vol. 780, no 2, p. 381-387Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-91340 (URN)
Available from: 2004-01-30 Created: 2004-01-30 Last updated: 2011-03-21
6. Beta-interferon effects the tryptophan metabolism in the plasma of multiple sclerosis patients
Open this publication in new window or tab >>Beta-interferon effects the tryptophan metabolism in the plasma of multiple sclerosis patients
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Manuscript (Other academic)
Identifiers
urn:nbn:se:uu:diva-91341 (URN)
Available from: 2004-01-30 Created: 2004-01-30 Last updated: 2011-03-21

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