The major non-volatile reaction products formed from free amino acids during the early stage of coffee roasting were investigated using biomimetic in-bean experiments with labeled and unlabeled free amino acids. Comprehensive untargeted screening by ultra-high performance liquid chromatography-electrospray-ionization-quadrupole time-of-flight-tandem mass spectrometry (UHPLC-ESI-QToF-MS) in data-independent acquisition (DIA) mode was carried out and modeling by orthogonal partial least-squares discriminant analysis (OPLS-DA) helped in revealing 11 pyrazine structures identified in coffee for the first time. 2-(2',3',4'-Trihydroxybutyl)-(5/6)-methyl-pyrazine (1) and 2,(5/6)-bis(2',3',4'-trihydroxybutyl)-pyrazine (2) were the most prominent compounds, while 2-(3',4'-dihydroxybutyl)-(5/6)-methyl-pyrazine (5) and 2-(2',3',4'-trihydroxybutyl)-(5/6)-(2'-hydroxyethyl)-pyrazine (10) were not even previously identified in other food matrices. The structures could be verified by means of additional biomimetic in-bean experiments with labeled sucrose leveraging the carbon module labeling (CAMOLA) approach. Based on these results, plausible formation pathways could be formulated fitting into the known Maillard reaction mechanisms. Sucrose was highlighted as the predominant precursor of the carbon backbone of all identified pyrazines butonly 33-55% of the nitrogen atoms originated from free amino acids.
Keywords: CAMOLA; Maillard reaction; coffee flavor formation; pyrazines; roasting.