Investigation of Maillard reaction products using 15N isotope studies and analysis by electrospray ionization-mass spectrometry

Structural characterization of reaction products of Maillard Chemistry has proved to be rather challenging. This can be directly attributed to the extreme complexity of product reaction mixtures that are known to range from small organic molecules to extremely large polymers. In addition, contemporary analytical techniques have been significantly challenged by the hydrophilic nature of advanced glycation endproducts (AGEs). Recently, methods such as on-line capillary electrophoresis-electrospray ionization-mass spectrometry (CE-MS) have emerged to provide a means of structurally characterizing molecules of extreme hydrophilicity. However, while this technique provides a means to identify such molecules, it is often difficult to differentiate products of ion/molecule reactions that can occur in the interface from authentic Maillard reaction products (MRPs). These artifacts are misleading, and lead to erroneous conclusions regarding solution composition. In the present study, we have prepared MRPs by reaction of 5-hydroxymethylfurfural (5-HMF) separately with 14N-glycine and 15N-glycine. CE-MS analysis of a 50:50 (vv) mixture of these solutions readily identifies product of ion/molecule reactions by a multiplicity of isotopes that fit a simple binomial expansion statistical output. By contrast, the mass spectrum of a MRP present in a preformed mixture of isotopically pure reaction products is much simpler. Such spectra display two responses (one for each isotope) that are separated by a mass number that directly corresponds to the number of nitrogen atoms present in the MRP. In addition, tandem mass spectrometric analysis of both of the isotopically pure MRPs aids the structural characterization of molecules of interest. Each fragment ion increases in mass by the number of nitrogen atoms it contains in the spectrum of the 15N labeled MRP. This strategy is demonstrated by the detection and structural characterization of 2-formyl-5-(hydroxy-methyl)pyrrole-1-acetic acid.