Selective and sensitive UHPLC-ESI-MS/MS method for simultaneous quantification of protein oxidation products in food systems

Protein oxidation affects structural, functional, and sensorial properties of foods. However, the mechanism and health consequences of dietary protein oxidation are not well understood, mainly due to lack of reliable analytical methods to quantify specific protein oxidation products in complex food matrices. The aim of this work was to develop a chromatographic method for simultaneous analysis of individual protein-bound oxidation products in foods upon total protein hydrolysis.1 Accordingly, a targeted parallel reaction monitoring-based quantitative method was developed using a high-resolution Orbitrap mass spectrometer coupled to a UHPLC system. Eighteen different protein oxidation markers, namely o-Tyr, m-Tyr, 3-nitrotyrosine, dityrosine, 3,4-dihydroxyphenylalanine, 3-chlorotyrosine, 5-hydroxytryptophan, kynurenine, kynurenic acid, 3-hydroxykunurenine, N’-formylkynurenine, dioxindolylalanine isomers, tryptamine, Met, Tyr, Trp, and Phe were chromatographically separated using a reversed phase column and subsequently quantified using stable isotopically labelled internal standards. The developed method was validated for linearity, selectivity, recovery, precision, matrix effects, carry over, and stability parameters by following US FDA guidelines. The limit of detection, accuracy and precision of the target analytes were in the range of 0.2–15 ng/mL, 1–20% of nominal concentration, and 1–17% coefficient of variation, respectively. Incorporation of isotopically labelled internal standards effectively compensated matrix effects and autosampler degradation of analytes. The method was applied to quantify target analytes in milk, infant formula, pork liver pâté, chicken and fish, with results showing kynurenine, o-Tyr and dioxindolylalanine isomers as the major oxidation products (1-30 mg/100 g protein). In oxidizing environment containing excess hydroxyl radical (•OH), 20–120 mg/100 g protein of specific oxidation products were formed. The results have also shown that Fenton chemistry favors the formation of o-Tyr ...