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Comprehensive vitamer profiling of folate mono- and polyglutamates in baker's yeast (Saccharomyces cerevisiae) as a function of different sample preparation procedures.
Metabolites 10:301 (2020)
Folates are a group of B(9)vitamins playing an important role in many metabolic processes such as methylation reactions, nucleotide synthesis or oxidation and reduction processes. However, humans are not able to synthesize folates de novo and thus rely on external sources thereof. Baker's yeast (Saccharomyces cerevisiae) has been shown to produce high amounts of this vitamin but extensive identification of its folate metabolism is still lacking. Therefore, we optimized and compared different sample preparation and purification procedures applying solid phase extraction (SPE). Strong anion exchange (SAX), C18 and hydrophilic-lipophilic-balanced (HLB) materials were tested for their applicability in future metabolomics studies. SAX turned out to be the preferred material for the quantitative purification of folates. Qualification of several folate vitamers was achieved by ultra-high pressure liquid chromatography quadrupole time of flight mass spectrometry (UHPLC-Q-ToF-MS) measurements and quantification was performed by liquid chromatography tandem mass spectrometry (LC-MS/MS) applying stable isotope dilution assays (SIDAs). The oxidation products-pyrazino-triazine (MeFox) was included into the SIDA method for total folate determination and validation. Applying the best protocol (SAX) in regard to folate recovery, we analyzed 32 different vitamers in different polyglutamate states up to nonaglutamates, of which we could further identify 26 vitamers based on tandem-MS (MS2) spectra. Total folate quantification revealed differences in formyl folate contents depending on the cartridge chemistry used for purification. These are supposedly a result of interconversion reactions occurring during sample preparation due to variation in pH adjustments for the different purification protocols. The occurrence of interconversion and oxidation reactions should be taken into consideration in sample preparation procedures for metabolomics analyses with a focus on folates.
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Publication type
Article: Journal article
Document type
Scientific Article
Keywords
Folate Metabolism ; Folate Polyglutamates ; Baker's Yeast ; Solid Phase Extraction ; S-pyrazino-triazine (mefox); Performance Liquid-chromatography; Ionization Mass-spectrometry; Solid-phase Extraction; One-carbon Metabolism; Folic-acid; 5-methyltetrahydrofolic Acid; Hplc Analysis; Human Serum; Lc-ms/ms; Metabolomics
ISSN (print) / ISBN
2218-1989
e-ISSN
2218-1989
Journal
Metabolites
Quellenangaben
Volume: 10,
Issue: 8,
Article Number: 301
Publisher
MDPI
Publishing Place
St Alban-anlage 66, Ch-4052 Basel, Switzerland
Non-patent literature
Publications
Reviewing status
Peer reviewed