Abstract
N-Acetylglucosamine is a key component of bacterial and fungal cell walls and of the extracellular matrix of animal cells. It plays a variety of roles at the cell surface structure and is under discussion to be involved in signaling pathways. The presence of a number of N-acetylhexosamine stereoisomers in samples of biological or biotechnological origin demands for dedicated high efficiency separation methods, due to identical exact mass and similar fragmentation patterns of the stereoisomers. Gas chromatography offers high sample capacity, separation efficiency, and precision under repeatability conditions of measurement, which is a necessity for the analysis of low abundant stereoisomers in biological samples. Automated online derivatization facilitates to overcome the main obstacle for the use of gas chromatography in metabolomics, namely, the derivatization of polar metabolites prior to analysis. Using alkoximation and subsequent trimethylsilylation, carbohydrates and their derivatives are known to show several derivatives, since derivatization is incomplete as well as highly matrix dependent inherent to the high number of functional groups present in carbohydrates. A method based on efficient separation of ethoximated and trimethylsilylated N-acetylglucosamines was developed. Accurate absolute quantification is enabled using biologically derived 13C labeled internal standards eliminating systematic errors related to sample pretreatment and analysis. Due to the lack of certified reference materials, a methodological comparison between tandem and time-of-flight mass spectrometric instrumentation was performed for mass spectrometric assessment of trueness. Both methods showed limits of detection in the lower femtomol range. The methods were applied to biological samples of Penicillium chrysogenum cultivations with different matrices revealing excellent agreement of both mass spectrometric techniques.
Highlights
N-Acetylglucosamine is a key component of bacterial and fungal cell walls and of the extracellular matrix of animal cells
As a matter of fact, derivatization efficiency was roughly estimated to be in the range 25%−110% for amino acids, 60%−90% for organic acids, and 80%−115% for monosaccharides and sugar alcohols, which are less complex molecules exhibiting a lower number of functional groups than N-acetylhexosamines.[13]
Since both the 4TMS and the 5TMS derivatives can be detected for N-acetylhexoasmines,[19] the use of isotope labeled internal standards or mathematical correction strategies for quantitation[26] are required
Summary
N-Acetylglucosamine is a key component of bacterial and fungal cell walls and of the extracellular matrix of animal cells. For TOFMS analysis, the following exact masses were chosen as quantifier ions: m/z 537.2662 (low concentrations in samples), 538.2676 (high concentrations in samples), and the fully 13C labeled isotopologue m/z 548.3007 as the internal standard ion.
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