ID Unknowns

Identification of unknown metabolites from GC-MS experiments

GC-MS analysis using MCF derivatization typically results in a data set that will consist of 50-150 compounds. These compounds can be classified into four groups:

1)Identified metabolites. Identification are based on retention time and mass spectra obtained from authentic chemical standards.

2)Putatively annotated metabolites. The annotations are primarily based on library matching of the acquired MS spectra with the NIST library. The annotation might be incorrect; however, the compound will most likely be of similar structure.

3)Putatively characterized compound classes

4)Unknown compounds.


Compounds in groups 2 (and 3) can be confirmed by using authentic standards to confirm the retention time and the mass spectrum.


Group no. 4 with the unknown compounds often constitutes 20-50% of the compounds in the data set. The number of amino and carboxylic groups in unknown compounds can be identified by derivatization with stable isotope-coded agent (d3-MCF and d4-methanol) as described by Kvitvang et al. “Quantitative analysis of amino and organic acids by methyl chloroformate derivatization and GC-MS/MS analysis”, DOI 10.1007/978-1-4939-1258-2_10.

For identification of the unknown compounds high-end instruments like the GCxGC-Accurate mass qTOF (Agilent 7200) and the Waters GC-APCI-accurate mass qTOF (Synapt G2-Si) can be applied. The GCxGC with two dimensional separation of compounds will enable a better separation of co-eluting compounds as well as increase the sensitivity of the analysis. By coupling the GCxGC with an accutrate mass qTOF very detailed information about the composition of the unknown compound will be obtained. The mass spectra of compounds in GC analysis most often consists of fragments of the parent ion. By applying a very delicate ionization (atmospheric pressure chemical ionization or APCI) the parent ion will often remain intact and APCI in combination with an accurate mass qTOF will enable the precise measurement of the mass of the parent ion, from which the molecular mass of the compound in question can be determined. An accurate determination of the molecular mass will enable the determination of the elemental composition of the compound.


The molecular mass and the elemental composition of the compounds in combination with other experimental information like retention time, mass spectra etc. can be subjected to a variety of algorithms in order to determine the identity of the compound. The principle and a workflow is described by Kind and Fiehn: “Seven golden rules for heuristic filtering of molecular formulas obtained by accurate mass spectrometry” BMC Bioinformatics 2007, 8:105. An example of the combination of the standard GC-MS with electron ionization (EI) and GC-MS (APCI) is described by Strehmel et al. “Annotating unknown components from GC/EI-MS-based metabolite profiling experiments using GC/APCI(+)-QTOFMS” Metabolomics 2014, 10:324.