Simplified absolute metabolite quantification by gas chromatography–isotope dilution mass spectrometry on the basis of commercially available source material

In the field of metabolomics, GC–MS has rather established itself as a tool for semi-quantitative strategies like metabolic fingerprinting or metabolic profiling. Absolute quantification of intra- or extracellular metabolites is nowadays mostly accomplished by application of diverse LC–MS techniques. Only few groups have so far adopted GC–MS technology for this exceptionally challenging task. Besides numerous and deeply investigated problems related to sample generation, the pronounced matrix effects in biological samples have led to the almost mandatory application of isotope dilution mass spectrometry (IDMS) for the accurate determination of absolute metabolite concentrations. Nevertheless, access to stable isotope labeled internal standards (ILIS), which are in many cases commercially unavailable, is quite laborious and very expensive. Here we present an improved and simplified gas chromatography–isotope dilution mass spectrometry (GC–IDMS) protocol for the absolute determination of intra- and extracellular metabolite levels. Commercially available 13C-labeled algal cells were used as a convenient source for the preparation of internal standards. Advantages as well as limitations of the described method are discussed.

► It was demonstrated that GC–IDMS is suitable for the accurate absolute quantification of metabolites in microbial cultures.
► The described method exhibits certain attractive advantages over established LC–IDMS protocols.
► Certain metabolites were subjected to absolute quantification by GC–IDMS for the first time.
► Examination of derivatization conditions and sample matrix effects resulted in distinct improvements as well as practical recommendations.
► First-time use of commercially available U–13C-labeled algal cells as a convenient source for the generation of isotope labeled internal standards.