Formation of Free and Protein-Bound Heterocyclic Amines in Roast Beef Patties Assessed by UPLC-MS/MS

The effect of different roasting temperatures on the amounts of 17 heterocyclic amines (HAs) from seven categories of both free and protein-bound states in roast beef patties was assessed using an ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method. There were increased amounts and more types of HAs detected at higher roasting temperatures. Nine free HAs were detected at 250 °C, including PhIP (14.34 ± 0.36 ng/g), DMIP (1.02 ± 0.07 ng/g), 1,5,6-TMIP (1.70 ± 0.08 ng/g), MeIQ (0.36 ± 0.01 ng/g), IQx (0.37 ± 0.04 ng/g), MeIQx (9.94 ± 0.61 ng/g), 4,8-DiMeIQx (0.90 ± 0.05 ng/g), norharman (6.03 ± 0.30 ng/g), and harman (2.60 ± 0.09 ng/g). Also, 37.32 ng/g of total free HAs was generated. Twelve protein-bound HAs were detected in roast beef patties at 250 °C, including PhIP (1.70 ± 0.13 ng/g), DMIP (2.33 ± 0.25 ng/g), 1,5,6-TMIP (3.62 ± 0.49 ng/g), MeIQ (5.47 ± 0.18 ng/g), IQ[4,5-b] (0.70 ± 0.03 ng/g) MeIQx (4.03 ± 0.41 ng/g), 4,8-DiMeIQx (0.67 ± 0.09 ng/g), MeAαC (19.51 ± 1.12 ng/g), AαC (2.91 ± 0.45 ng/g), norharman (1304.96 ± 110.73 ng/g), harman (400.85 ± 25.29 ng/g), and Phe-P-1 (0.81 ± 0.06 ng/g). The highest amount of protein-bound HAs was 2913.31 ng/g at 175 °C. PhIP tended to exist in a free state, whereas MeIQ, harman, and norharman tended to exist in a protein-bound state. Furthermore, Phe-P-1, MeAαC, and AαC were detected only in a protein-bound state. These results could be useful for evaluating the exposure to HAs in a daily diet.