We report the use of ultra pressure liquid chromatography (UPLC), coupled to a tandem mass spectrometer operated in multiple reaction monitoring mode to determine the advanced glycation endproduct, Nepsilon-(carboxymethyl)lysine (CML). The procedure was applied to acid hydrolyzates of protein isolated from a range of foods (milks processed at different temperatures, butter, cheese, infant formulae, bread, raw and cooked minced beef and olive oil). Highest levels of CML were determined in white bread crust (15.2 +/- 0.63 mmol/mol Lys), wholemeal bread crust (13.1 +/- 0.61 mmol/mol Lys) and evaporated full-fat milk (4.86 +/- 0.77 mmol/mol Lys). Lowest levels of CML were measured in raw minced beef beef (0.03 +/- 002 mmol/mol Lys), raw full-fat cow's milk (0.08 +/- 0.03 mmol/mol Lys) and pasteurized skimmed cow's milk (0.09 +/- 0.002 mmol/mol Lys). CML could not be detected in olive oil.

Modification of protein by carbonyl compounds under in vitro physiological conditions is site-directed. There are few reports of the site specificity of glycation of proteins using heating conditions of relevance to food processing. The aim of this study was to determine the site specificity of modification of beta-casein (betaCN) by glucose and methylglyoxal (MGO). betaCN (1.33 M, 3.2%) was heated with either glucose (1.345 M, 4.6%) or MGO (1 mM) at 95 degrees C for up to 4 h. Tryptic digests were prepared and analysed by ultra performance liquid chromatography electrospray ionisation mass spectrometry (UPLC-ES/MS). The sites of formation of the Amadori product, N(epsilon)-(fructosyl)lysine (FL), and the advanced glycation end-products, N(epsilon)-(carboxymethyl)lysine (CML), MGO-derived dihydroxyimidazolidine (MG-DH) and MGO-derived hydroimidazolone (MG-HI), were located. FL and CML were detected at K107 and K176 residues in betaCN/glucose incubations. Indigenous N ( epsilon )-(lactulosyl)lysine was detected at K107 only. MG-DH and MG-HI were detected at R202 and possibly R183 residues in both betaCN/glucose and betaCN/MGO incubations. Glycation of betaCN by glucose and MGO resulted in similar site specificity for MG-DH and MG-HI formation.