Synthesis, tandem MS- and NMR-based characterization, and quantification of the carbon 13-labeled advanced glycation...

6-N-carboxymethyllysine (CML), generated by the glycation and/or oxidation of lysine residues, has been measured in biological materials and food products using techniques such as ELISA, HPLC with fluorescence detection and mass spectrometry methods. Only limited information has been reported regarding the preparation of standards labeled with either deuterium, (13)C or (15)N atoms to be used as internal standards. In the present paper, a synthesis of carbon-13 labeled CML is described using l,2-(13)C(2)-glyoxylic acid and 2-N-acetyllysine as starting materials. The resulting labeled 2-N-acetyl-CML was purified by HPLC-UV as a dibutyl ester. After a deprotection step, the yield was evaluated to be 53% when the reaction was conducted 17 h at 37 degrees C. CML was extensively studied by (1)H- and (13)C-NMR and the fragments observed in the collision induced dissociation (CID) spectrum were also assigned. Finally, the standards of CML and carbon-13 labeled CML were accurately quantified based on (1)H-NMR and tandem MS using lysine as an internal reference.