Combination of mass spectrometry and site-specific NMR isotope analyses in the characterisation of amino acids

Experimental strategies have been developed which make amino acid probes accessible to the NMR study of site‐specific natural isotope fractionation. A multi‐isotope approach, based on combined mass spectrometry determination of hydrogen‐, carbon‐ and nitrogen‐overall isotope contents and on 2H‐NMR determination of site‐specific hydrogen isotope ratios, shows that relatively wide ranges of variation are exhibited by the isotopic parameters of glutamic acid, aspartic acid, alanine, proline and lysine samples from different origins. Moreover, relatively large deviations with respect to a random distribution of deuterium among the molecular sites are quantified and compared. A noticeable enantiomeric imbalance at the methylenic site adjacent to the chiral centre is also detected in natural samples of L ‐glutamic and L ‐aspartic acids in particular. The results are analysed in terms of filiation criteria and technological effects. It is shown in particular that the C3 or C4 metabolic origin of the raw materials utilised in fermentation processes can be recognised. Principal component analyses involving the two mean isotopic variables δ13C and δ15N and the three site‐specific variables (D/H)x (D/H)β and (D/H)γ associated with positions α, β and γ of glutamic acids, for example, are helpful for interpreting the main discriminating factors. This approach provides the basis for new applications to origin recognition of fundamental constituents and metabolites of plant or animal species.