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Abstract BACKGROUND There is no clear conclusion for the quantitative detection of melanoidins. The present study aimed to characterize the melanoidins formed from d ‐fructose and l ‐alanine at different treatments (hydrothermal and dry‐heating treatment) using the three‐dimensional excitation‐emission matrix (3‐DEEM) and high‐performance liquid chromatography (HPLC). RESULTS The Maillard reaction products gradually increased and the content of melanoidins ( A 420 nm) gradually increased with the prolongation of reaction time. The 3‐DEEM spectrum and fluorescence area integration revealed the change of fluorescence products, with the melanoidins mainly concentrating in the region V (humic acid‐like organics). In addition, the HPLC system equipped with a fluorescence detector also effectively separated melanoidins from Maillard reaction products. Fourier transform infrared spectroscopy results showed that the polymerization of melanoidins is related to aldol condensation accompanied by a decrease of CO and OH, as well as an increase of CO. The aromatic ring and conjugated double bond in the melanoidins structure enhanced the fluorescence intensity. The linearity of melanoidins was confirmed over the range 0.01–10 mg mL −1 , and fluorescence quenching occured when the concentration was greater than 10 mg mL −1 . CONCLUSION The 3‐DEEM combined with HPLC effectively characterized melanoidins, and the melanoidin concentrations showed a linear relationship in the range 0.01–10 mg mL −1 . This study provides a new idea for the determination of melanoidins content in a complex food matrix. © 2025 Society of Chemical Industry.
Wu et al. (Sun,) studied this question.