|Metabolite analysis by GC-MS
In all organisms the number of intra-cellular metabolites vastly outweighs the number of extra-cellular ones. A direct analysis and quantitation of intra-cellular metabolites by 1H-NMR spectroscopy is therefore usually not possible. On the one hand the complexity of the mixture means that the signals will overlap and thus prevent identification, while on the other, the concentration of many metabolites will be too low to be detected by this method. In these circumstances the method of choice is GC-MS both because of its high fractionation ability and because of the high information content of the spectra. Using our current instrument it is possible to separate and analyse around 200 different metabolites. A relative quantitation is possible using internal standards and gives information about the relative metabolite concentrations in different samples. Individual calibration curves must be constructed for the absolute quantitation of individual metabolites. This quantitation has been established for all amino acids which can be incorporated into proteins and for around 50 further metabolites including organic acids, non-protein amino acids, carbohydrates and fatty acids.
Since the majority of metabolites are too polar for a direct separation by gas chromatography they must be silylated in a preliminary step.
Metabolite concentration: in the nano-molar to pico-molar range