Quantification of Maillard Reaction Products

Acetonitrile (LC-MS grade) and methanol (HPLC grade) were obtained from VWR Prolabo (Darmstadt, Germany). Freshly double distilled water (Bi 18E double distillation system, QCS, Maintal, Germany) was used for solvents production for LC-MS analysis. Nonafluoropentanoic acid (NFPA) was from Sigma-Aldrich (Steinheim, Germany). Reference material for calibration of MRPs was synthesized in our laboratory as described before: N-ε-fructosyllysine [30 (link)], pyrraline [31 (link),32 (link)], CML [17 (link)], CEL [17 (link)], and MG-H1 [17 (link)]. Stable isotope labelled internal standards for HPLC-MS/MS analysis were synthesized similarly but using [¹³C₆,¹⁵N₂]lysine ([¹³C₆,¹⁵N₂]Pyr) and [¹³C₆]arginine ([¹³C₆]MG-H1) instead of the unlabeled compounds. The synthesis of [¹³C₃]CEL was described recently [29 (link)]. [²H₂]CML was purchased from PolyPeptide (Strasbourg, France), [¹³C₆,¹⁵N₂]lysine from Campro (Berlin, Germany), and [¹³C₆]arginine from Eurisotop (Saarbrücken, Germany). Enzymatic hydrolysis was performed by using pronase E (Merck, Darmstadt, Germany) and pepsin, aminopeptidase and prolidase (all from Sigma-Aldrich, Steinheim, Germany).