Abstract
Gas chromatography–mass spectrometry (GC-MS) is a widely used analytical technique in metabolomics. GC provides the highest resolution of any standard chromatographic separation method, and with modern instrumentation, retention times are very consistent between analyses. Electron impact ionization and fragmentation is generally reproducible between instruments and extensive libraries of spectra are available that enhance the identification of analytes. The major limitation is the restriction to volatile analytes, and hence the requirement to convert many metabolites to volatile derivatives through chemical derivatization. Here we compared the analytical performance of two derivatization techniques, silylation (TMS) and alkylation (MCF), used for the analysis of amino and non-amino organic acids as well as nucleotides in microbial-derived samples. The widely used TMS derivatization method showed poorer reproducibility and instability during chromatographic runs while the MCF derivatives presented better analytical performance. Therefore, alkylation (MCF) derivatization seems to be preferable for the analysis of polyfunctional amines, nucleotides and organic acids in microbial metabolomics studies.
Highlights
For analysis of volatile compounds, gas-chromatography (GC) coupled to mass spectrometry (MS)allows high analysis throughput at relatively low cost
We observed very similar quality of metabolite identification based on the overall score of spectra using either derivatization technique, the analytical performance of TMS derivatization presented here is alarming, but consistent with other recent reports of unsatisfactory analysis of TMS
Contrary to the results reported by Koek et al [7], we observed a very unsatisfactory analytical performance of several metabolites derivatized by silylation
Summary
For analysis of volatile compounds, gas-chromatography (GC) coupled to mass spectrometry (MS). Silylation of organic compounds is the classical and most widely used derivatization procedure for metabolome analysis by GC-MS (Figure 1) [1,2,3,4,5,6] Sugars and their derivatives (sugar alcohols, amino sugars, and others) are the class of metabolites most efficiently derivatized by silylation [1,2,6]. Silylation reactions require anhydrous reaction conditions, and the samples have to be completely free of water, and the reagent mixture in silylation reactions is not separated from the derivatives, the sample injected into the chromatographic column contains derivatives and residual reagents as well as non-derivatized involatile compounds This is especially problematic for complex biological samples such as extracellular culture media and body fluids, which contain large amounts of non-derivatizable compounds that may damage a capillary GC column. MCF derivatization is the best candidate to be used in parallel with silylation in order to achieve the goal of metabolomics that is the detection and analysis of as many metabolites as possible in biological samples
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