Isotope dilution ESI-LC-MS/MS for quantification of free and total Nε-(1-Carboxymethyl)-L-Lysine and free Nε-(1-Carboxyethyl)-L-Lysine: comparison of total Nε-(1-Carboxymethyl)-L-Lysine levels measured with new method to ELISA assay in gruel samples.

ESI-LC-MS/MS method with isotope dilution and SPE based on cation-exchange was developed for determination of free and total Nε-(1-Carboxymethyl)-L-Lysine (CML) and free Nε-(1-Carboxyethyl)-L-Lysine (CEL). The use of nonafluoropentanoic acid in mobile phase was omitted, SPE recoveries of 82±3% and 91±10% (n=6) for CML and CEL respectively and, calibration curves (R(2)>0.9985) were attained. The method was applied to gruel samples and LoQ for the method was 5 ng/ml, RSD <10% and accuracy was 115%. Total CML levels in the gruel samples varied from 103-408 mg/kg protein. Free CML levels which were 1000 times lower than total CML were three times higher than free CEL levels. CML in a gruel sample was 127±7, 84±9 and 253±28 mg/kg using the current ESI-LC-MS/MS, ELISA and GC-MS respectively. The described method has advantages over ELISA with respect to reproducibility and specificity and over GC-MS with respect to reproducibility.