LC-HRMS-based identification and quantification of novel 2,5-diketopiperazines in roasted coffee.

2,5-Diketopiperazines (DKPs), the simplest cyclic dipeptide derivatives, are formed via thermal intramolecular condensation of amino acids and characterized by the distinctive bitterness in foods such as roasted coffee. This study employed liquid chromatography-high resolution mass spectrometry (LC-HRMS)-based methods to identify and quantify novel DKPs in coffee samples. A total of 33 DKPs were identified using electron-spray ionization tandem mass spectrometry (ESI-MS/MS) in positive-ion mode based on their characteristic fragment ions, with 23 newly reported. DKPs with polar residues, such as glutamic acid and lysine, were identified in their bicyclic forms. A quantitative analytical method was developed using authentic standards to quantify the target compounds in powdered commercial coffee samples and in coffee infusions. Cyclo(Pro-Leu), cyclo(Pro-Val), and cyclo(Pro-Tyr) were found to be the most abundant DKPs across various roasting conditions, with concentrations ranging between 5 and 50 μg/mL and 10-250 μg/mL in the tested commercial samples. DKP concentrations determined in aqueous infusions of coffee were in the range of reported bitterness threshold concentrations. Roasting kinetics at two selected temperatures were determined.