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Mass Spectrometry of Biologically Active Small Molecules: Focusing on polyphenols, alkaloids and amino acids
Stockholm University, Faculty of Science, Department of Analytical Chemistry. (Department of Analytical Chemistry, Leopold L. Ilag)
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The foci of this dissertation are on advanced liquid chromatography (LC) separation and mass spectrometry (MS) techniques for the analysis of small bioactive molecules. In addition to discussing general aspects of such techniques the results from analyses of polyphenols (PPs), alkaloids and amino acids published in five appended studies are presented and discussed. High efficiency and well understood principles make LC the method of choice for separating analytes in many kinds of scientific investigations. Moreover, when LC is coupled to an MS instrument, analytes are separated in two stages: firstly they are separated and pre-concentrated in narrow bands using LC and then separated according to their mass-to-charge (m/z) ratios in the MS instrument. Some MS instruments can provide highly accurate molecular weight measurements and mass resolution allowing identification of unknown compounds based purely on MS data, thus making prior separation unnecessary. However, prior separation is essential for analyzing substances in most complex matrices – especially useful is the ultra-high performance LC (UHPLC). The advantages of using UHPLC rather than HPLC for the analysis of PPs in tea and wine were evaluated in one of the studies this thesis is based upon. The phenolic composition of red wine was also examined, using a novel LDI technique, following solid phase extraction (SPE). A class of small aromatic molecules (medicinally important alkaloids) also proved to be amenable to straightforward analysis, by thin layer chromatography (TLC) work-up followed by LDI-MS. Finally, a LC-MS method for monitoring neurotoxins (β-N-methyl-amino-L-alanine and 2,3-diaminobutyric acid) in complex biological matrices was developed and applied. Overall, the studies show that careful attention to the physicochemical properties of analytes can provide insights that can greatly facilitate the development of alternative methods to analyze them, e.g. by LDI.

Place, publisher, year, edition, pages
Stockholm: Department of Analytic Chemistry, Stockholm University , 2010. , 61 p.
Keyword [en]
LC-MS, LDI, Mass spectrometry, HPLC, UHPLC, polyphenols, phenolic acids, BMAA
National Category
Analytical Chemistry Analytical Chemistry
Research subject
Analytical Chemistry
Identifiers
URN: urn:nbn:se:su:diva-33233ISBN: 978-91-7155-982-1 (print)OAI: oai:DiVA.org:su-33233DiVA: diva2:282767
Public defence
2010-01-29, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 13:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: In press. Paper 5: Manuscript.

Available from: 2010-01-07 Created: 2009-12-21 Last updated: 2015-06-08Bibliographically approved
List of papers
1. Analysis of phenolic compounds by high performance liquidchromatography and ultra performance liquid chromatography
Open this publication in new window or tab >>Analysis of phenolic compounds by high performance liquidchromatography and ultra performance liquid chromatography
2008 (English)In: Talanta: The International Journal of Pure and Applied Analytical Chemistry, ISSN 0039-9140, Vol. 76, no 1, 189-199 p.Article in journal (Refereed) Published
Abstract [en]

Two novel chromatographic methods both based on utilization of sub-2-micron particle columns were developed for the analysis of phenolic compounds in this work. An HPLC system was equipped with C18 silica-based analytical column (50 mm × 4.6 mm, 1.8 μm) and a UPLC system with ethylene-bridged hybrid C18 analytical column (100 mm × 2.1 mm, 1.7 μm).

In total 34 phenolic substances were divided into groups of phenolic acids, flavonoids, catechins and coumarins and were analysed in sequence using different gradient methods. System suitability test data, including repeatability of retention time and peak area, mean values of asymmetry factor, resolution, peak capacity and the height equivalent of a theoretical plate were determined for each gradient method by 10 replicate injections. The developed methods were applied in the analysis of real samples (grape wines, teas).

Keyword
UPLC, HPLC, Phenolic compounds, Gradient analysis
Identifiers
urn:nbn:se:su:diva-33253 (URN)10.1016/j.talanta.2008.02.021 (DOI)
Available from: 2009-12-21 Created: 2009-12-21 Last updated: 2009-12-22Bibliographically approved
2. Matrix-less laser desorption/ionisation mass spectrometry of polyphenols in red wine
Open this publication in new window or tab >>Matrix-less laser desorption/ionisation mass spectrometry of polyphenols in red wine
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2009 (English)In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 23, no 12, 1834-1840 p.Article in journal (Refereed) Published
Abstract [en]

Matrix-assisted laser desorption/ionisation (MALDI) of small molecules is challenging and in most cases impossible due to interferences from matrix ions precluding analysis of molecules <300-500 Da. A common matrix such as ferulic acid belongs to an important class of compounds associated with antioxidant activity. If the shared phenolic structure is related to the propensity as an active MALDI matrix then it follows that direct laser desorption/ionisation should be possible for polyphenols. Indeed matrix-less laser desorption/ionisation mass spectrometry is achieved whereby the analyte functions as a matrix and was used to monitor low molecular weight compounds in wine samples. Sensitivity ranging from 0.12-87 pmol/spot was achieved for eight phenolic acids (4-coumaric, 4-hydroxybenzoic, caffeic, ferulic, gallic, protocatechuic, syringic, vanillic) and 0.02 pmol/spot for trans-resveratrol. Additionally, 4-coumaric, 4-hydroxybenzoic, caffeic, ferulic, gallic, syringic, vanillic acids and trans-resveratrol were identified in wine samples using accurate mass measurements consistent with reported profiles based on liquid chromatography (LC)/MS. Minimal sample pre-treatment make the technique potentially appropriate for fingerprinting, screening and quality control of wine samples. Copyright © 2009 John Wiley & Sons, Ltd.

National Category
Natural Sciences
Identifiers
urn:nbn:se:su:diva-33255 (URN)10.1002/rcm.4062 (DOI)
Available from: 2009-12-21 Created: 2009-12-21 Last updated: 2011-08-31Bibliographically approved
3. Matrix-free thin-layer chromatography/laser desorption ionization mass spectrometry for facile separation andidentification of medicinal alkaloids
Open this publication in new window or tab >>Matrix-free thin-layer chromatography/laser desorption ionization mass spectrometry for facile separation andidentification of medicinal alkaloids
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2009 (English)In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 23, no 23, 3655-3660 p.Article in journal (Refereed) Published
Abstract [en]

Quaternary protoberberine alkaloids belong to a pharmaceutically important class of isoquinoline alkaloids associated with bactericidal, fungicidal, insecticidal and antiviral activities. As traditional medicine gains wider acceptance, quick and robust analytical methods for the screening and analysis of plants containing these compounds attract considerable interest. Thin-layer chromatography (TLC) combined with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a powerful technique but suffers from dilution of the TLC bands resulting in decreased sensitivity and masking of signals in the low-mass region both due to addition of matrix. This study integrates for the first time conventional silica gel TLC and laser desorption ionization mass spectrometry (LDI-MS) thus eliminating the need for any external matrix. Successful separation of berberine (Rf = 0.56) and palmatine (Rf = 0.46) from Berberis barandana including their identification by MS are demonstrated. Furthermore, a robust electrospray ionization (ESI)-MS method utilizing residual sample from TLC for quantification of berberine applying selected reaction monitoring and standard addition method is presented. The amount of berberine in the plant root prepared for the study was determined to be 0.70% (w/w). Copyright © 2009 John Wiley & Sons, Ltd.

Identifiers
urn:nbn:se:su:diva-33258 (URN)10.1002/rcm.4297 (DOI)
Available from: 2009-12-21 Created: 2009-12-21 Last updated: 2011-08-31Bibliographically approved
4. Analytical protocol for identification of BMAA and DAB in biologicalsamples
Open this publication in new window or tab >>Analytical protocol for identification of BMAA and DAB in biologicalsamples
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2010 (English)In: The Analyst, ISSN 0003-2654, E-ISSN 1364-5528, Vol. 135, 127-132 p.Article in journal (Refereed) Published
Abstract [en]

 

b

-N-methylamino-L-alanine (BMAA) is a non-protein amino acid, thought to be inflicting neurodegenerative diseases related to ALS/PDC in human beings. Due to conflicting data concerning the presence of BMAA in various biological matrixes, we present a robust and sensitive method for high confidence identification of BMAA after derivatization by 6-aminoquinolyl-N

-hydroxysuccinimidyl carbamate (AQC). The efficient sample pretreatment in combination with LC-MS/MS SRM enables chromatographic separation of BMAA from the isomer 2,3-diaminobutyric acid (DAB). The method is applicable for selective BMAA/DAB detection in various biological samples ranging from a prokaryotic cyanobacterium to eukaryotic fish.

Place, publisher, year, edition, pages
The Royal Society of Chemistry, 2010
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-33260 (URN)10.1039/b921048b (DOI)
Available from: 2009-12-21 Created: 2009-12-21 Last updated: 2011-01-10Bibliographically approved
5. Transfer of a cyanobacterial neurotoxin within a temperate aquatic ecosystem suggests pathways for human exposure
Open this publication in new window or tab >>Transfer of a cyanobacterial neurotoxin within a temperate aquatic ecosystem suggests pathways for human exposure
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2010 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 107, no 20, 9252-9257 p.Article in journal (Refereed) Published
Abstract [en]

beta-methylamino-L-alanine (BMAA), a neurotoxic nonprotein amino acid produced by most cyanobacteria, has been proposed to be the causative agent of devastating neurodegenerative diseases on the island of Guam in the Pacific Ocean. Because cyanobacteria are widespread globally, we hypothesized that BMAA might occur and bioaccumulate in other ecosystems. Here we demonstrate, based on a recently developed extraction and HPLC-MS/MS method and long-term monitoring of BMAA in cyanobacterial populations of a temperate aquatic ecosystem (Baltic Sea, 2007-2008), that BMAA is biosynthesized by cyanobacterial genera dominating the massive surface blooms of this water body. BMAA also was found at higher concentrations in organisms of higher trophic levels that directly or indirectly feed on cyanobacteria, such as zooplankton and various vertebrates (fish) and invertebrates (mussels, oysters). Pelagic and benthic fish species used for human consumption were included. The highest BMAA levels were detected in the muscle and brain of bottom-dwelling fishes. The discovery of regular biosynthesis of the neurotoxin BMAA in a large temperate aquatic ecosystem combined with its possible transfer and bioaccumulation within major food webs, some ending in human consumption, is alarming and requires attention.

Keyword
beta-methylamino-L-alanine, Baltic Sea, cyanobacteria, liquid chromatography/mass spectrometry, bioaccumulation
National Category
Biological Sciences
Research subject
Plant Physiology
Identifiers
urn:nbn:se:su:diva-50529 (URN)10.1073/pnas.0914417107 (DOI)000277822600043 ()
Note

authorCount :8

Available from: 2010-12-28 Created: 2010-12-28 Last updated: 2017-12-11Bibliographically approved

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