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Quantitative Bioanalysis: Liquid separations coupled to targeted mass spectrometric measurements of bioactive compounds
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
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. , p. 61
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 412
Keywords [en]
Analytical chemistry, Method development, Enhanced microdialysis, Column switching, Liquid chromatography, Capillary electrophoresis, Electrospray ionization, Mass spectrometry, Triple quadrupole, Time-of-flight, Quantitation, Validation
Keywords [sv]
Analytisk kemi
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-8581ISBN: 978-91-554-7135-4 (print)OAI: oai:DiVA.org:uu-8581DiVA, id: diva2:171664
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
List of papers
1. Online capillary solid phase extraction and liquid chromatographic separation with quantitative tandem mass spectrometric detection (SPE-LC-MS/MS) of ximelagatran and its metabolites in a complex matrix.
Open this publication in new window or tab >>Online capillary solid phase extraction and liquid chromatographic separation with quantitative tandem mass spectrometric detection (SPE-LC-MS/MS) of ximelagatran and its metabolites in a complex matrix.
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2009 (English)In: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376X, Vol. 877, no 3, p. 291-297Article in journal (Refereed) Published
Abstract [en]

This work presents the development and validation of a fully automated quantitative analysis method of melagatran, its prodrug ximelagatran, and its major metabolites for the study of drug behavior in biofluids. The method involves online sample clean-up and enrichment on a C4 capillary column followed by separation on a capillary C18 column. Electrospray ionization tandem mass spectrometric detection in positive ion mode was performed with multiple reactions monitoring of eight different transients, divided into two time segments with four transients each. The structural similarity, the complexity of the matrix (pig liver extract) and the formation of isobaric fragment ions, made efficient chromatographic separation necessary. The analysis method provides valid accuracy (<9%; RSD%), precision (<8%; RSD%), linearity (<1.2 nM–1 μM; R2 > 0.999), limit of quantitation (<3.6 nM), retention repeatability (<1.2%; RSD%), selectivity, as well as analyte and column stabilities over a wide concentration range.

Keywords
Online, solid phase extraction, liquid chromatography, mass spectrometry, ximelagatran, metabolites, pig liver
National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-88170 (URN)10.1016/j.jchromb.2008.12.017 (DOI)000262877500026 ()19117807 (PubMedID)
Available from: 2009-01-22 Created: 2009-01-22 Last updated: 2017-12-14Bibliographically approved
2. Interferon-beta affects the tryptophan metabolism in multiple sclerosis patients
Open this publication in new window or tab >>Interferon-beta affects the tryptophan metabolism in multiple sclerosis patients
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2005 (English)In: European Journal of Neurology, ISSN 1351-5101, E-ISSN 1468-1331, Vol. 12, no 8, p. 625-631Article in journal (Refereed) Published
Abstract [en]

Tryptophan and its metabolites are of great interest in understanding the pathogenesis of multiple sclerosis (MS). The total levels of tryptophan and its metabolites, kynurenine and kynurenic acid were determined in plasma by capillary liquid chromatography electrospray ionisation tandem mass spectrometry. This is the first report of the plasma levels of these analytes in healthy controls and relapsing-remitting MS patients receiving long-term and acute interferon-beta (IFN-beta) treatment. Twenty-four hours post-administration increased kynurenine levels (first IFN MS versus healthy, P = 0.042) and kynurenine/tryptophan ratio (K/T; first IFN MS versus healthy, P =0.027; first IFN MS versus long-term IFN MS, P = 0.036) were found. The long-term IFN MS group had higher K/T ratios at 4 and 12 h post-administration (P = 0.015 and 0.009, respectively). The increase of K/T ratio in the first IFN MS group indicate an induction of the enzyme indolamine-2,3-dioxygenase (IDO), as reported earlier in experimental allergic encephalomyelitis. As IDO is participating in both inflammatory and neurodegenerative processes, further knowledge of its involvement in the pathogenesis of MS is of great importance.

Keywords
electrospray ionisation, interferon-beta, kynurenic acid, kynurenine, mass spectrometry, multiple sclerosis, plasma, tryptophan
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-96923 (URN)10.1111/j.1468-1331.2005.01041.x (DOI)000230841800009 ()
Available from: 2008-03-17 Created: 2008-03-17 Last updated: 2017-12-14Bibliographically approved
3. High throughput analysis of tryptophan metabolites in a complex matrix using capillary electrophoresis coupled to time-of-flight mass spectrometry
Open this publication in new window or tab >>High throughput analysis of tryptophan metabolites in a complex matrix using capillary electrophoresis coupled to time-of-flight mass spectrometry
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2007 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1159, no 1-2, p. 154-158Article in journal (Refereed) Published
Abstract [en]

A capillary electrophoresis method for separation and detection with time-of-flight mass spectrometry is described for tryptophan metabolites in the kynurenic pathway. Tryptophan metabolites are usually difficult to detect with electrospray mass spectrometry since they have low surface activity and occur in low nanomolar to micromolar range in body fluids. Modification of the silica-wall with 1-(4-iodobutyl)4-aza-1-azoniabicyclo[2,2,2]octane iodide, also named M7C4I, has successfully been used to deactivate the fused silica wall and generate a stable reversed electroosmotic flow. Utilizing this advantage together with electrospray ionization time-of-flight mass spectrometry, which generates high resolution and fast acquisition monitoring of species, proved to be successful even for such a complex matrix like human cerebrospinal fluid.

Keywords
Tryptophan, Kynurenine, Kynurenic acid, Quinolinic acid, Picolinic acid, Capillary electrophoresis, Mass spectrometry, Time-of-flight, Complex matrix, Cerebrospinal fluid
National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-96907 (URN)10.1016/j.chroma.2007.04.044 (DOI)000248466800018 ()17477928 (PubMedID)
Available from: 2008-05-13 Created: 2008-05-13 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. Rapid capillary electrophoresis time-of-flight mass spectrometry separations of peptides and proteins using a monoquaternarized piperazine compound (M7C4l) for capillary coatings
Open this publication in new window or tab >>Rapid capillary electrophoresis time-of-flight mass spectrometry separations of peptides and proteins using a monoquaternarized piperazine compound (M7C4l) for capillary coatings
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2008 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 29, no 8, p. 1619-1625Article in journal (Refereed) Published
Abstract [en]

A monoquaternarized piperazine, 1-(4-iodobutyl) 4-aza-1-azoniabicyclo[2,2,2] octane iodide (M7C4I), has been evaluated as a surface derivatization reagent for CE in combination with TOF MS for the analysis of proteins, peptides, and protein digests. The M7C4I piperazine, at alkaline pH, forms a covalent bond via alkylation of the ionized silanols producing a cationic surface with a highly stable and reversed EOF. The obtained surface yields rapid separations (less than 5 min) of peptides and proteins at acidic pH with high separation efficiencies (up to 1.1 X 10(6) plates/m for peptides and up to 1.8 x 10(6) plates/m for proteins) and no observed bleeding of the coating reagent into the mass spectrometer. The simplicity of the coating procedure also enables fast (2 min) regeneration of the surface, if necessary. This is useful in the analysis of complex samples in order to prevent possible memory effects. The potential of using M7C4I-coated capillaries for MS analysis of complex samples is demonstrated by the separation of peptides, proteins, and protein digests. Even more, the spectacular thing in which large intact proteins with molecular masses over 0.5 MDa could be separated. The coating showed good ability to handle these large proteins with high efficiency and retained peak shape as demonstrated by separation of IgG(1) (150 kDa) and thyroglobulin (669 kDa).

Keywords
Capillary electrophoresis, M7C4l, Peptides, Proteins, Protein digests, Time-of-flight
National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-16217 (URN)10.1002/elps.200700737 (DOI)000255703100005 ()
Available from: 2008-05-13 Created: 2008-05-13 Last updated: 2017-12-08Bibliographically approved

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