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Microbial metabolomics with gas chromatography/mass spectrometry

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Author: Koek, M.M. · Muilwijk, B. · Werf, M.J. van der · Hankemeier, T.
Institution: TNO Kwaliteit van Leven
Source:Analytical Chemistry, 4, 78, 1272-1281
Identifier: 239131
doi: doi:10.1021/ac051683+
Keywords: Biology · Biotechnology · Amines · Amino acids · Bacteria · Escherichia coli · Fatty acids · Gas chromatography · Mass spectrometry · Substrates · Bacillus subtilis · Metabolite classes · Product concentrations · Propionibacterium freudenreichii · Metabolism · alcohol derivative · aldehyde · amine · amino acid · aromatic compound · carboxylic acid · purine · pyrimidine · sugar acid · sugar amine · sugar phosphate · unclassified drug · analytic method · article · Bacillus subtilis · bacterial cell · bacterial growth · chemical analysis · chemical modification · controlled study · dry weight · Escherichia coli · gas chromatography · mass spectrometry · metabolomics · nonhuman · oximation · Propionibacterium freudenreichii · quantitative analysis · silylation · validation process · Bacillus subtilis · Escherichia coli · Gas Chromatography-Mass Spectrometry · Propionibacterium · Reference Standards · Reproducibility of Results · Sensitivity and Specificity


An analytical method was set up suitable for the analysis of microbial metabolomes, consisting of an oximation and silylation derivatization reaction and subsequent analysis by gas chromatography coupled to mass spectrometry. Microbial matrixes contain many compounds that potentially interfere with either the derivatization procedure or analysis, such as high concentrations of salts, complex media or buffer components, or extremely high substrate and product concentrations. The developed method was extensively validated using different microorganisms, i.e., Bacillus subtilis, Propionibacterium freudenreichii, and Escherichia coli. Many metabolite classes could be analyzed with the method: alcohols, aldehydes, amino acids, amines, fatty acids, (phospho-) organic acids, sugars, sugar acids, (acyl-) sugar amines, sugar phosphate, purines, pyrimidines, and aromatic compounds. The derivatization reaction proved to be efficient (>50% transferred to derivatized form) and repeatable (relative standard deviations <10%). Linearity for most metabolites was satisfactory with regression coefficients better than 0.996. Quantification limits were 40-500 pg on-column or 0.1-0.7 mmol/g of microbial cells (dry weight). Generally, intrabatch precision (repeatability) and interbatch precision (reproducibility) for the analysis of metabolites in cell extracts was better than 10 and 15%, respectively. Notwithstanding the nontargeted character of the method and complex microbial matrix, analytical performance for most metabolites fit the requirements for target analysis in bioanalysis. The suitability of the method was demonstrated by analysis of E. coli samples harvested at different growth phases. © 2006 American Chemical Society.