Evaluation of intermediate compounds in β-carboline alkaloids formation using model system including isotope labeled glycerol

β-carbolines (βCs) such as norharman and harman are thermally formed in foods and have been associated with neurotoxic and mutagenic effects. This study explores glycerol-derived intermediates in βC formation, identifying 3-vinylindole as a key intermediate via isotope labeling. Model systems with labeled glycerol were used for carbon tracing. Quantitation and structural analysis were performed by HPLC, LC-MS/MS, and ESI-qTOF-MS/MS. In applied model system, formaldehyde levels increased from 33.77 to 77.12 mg/kg. Correspondingly, norharman concentrations rose from 98.59 to 680.19 μg/kg, demonstrating a dose-dependent relationship between glycerol content and βC formation. Time-resolved profiling showed 3-vinylindole peaked at 10 min and declined, while norharman concentration concurrently increased, supporting its role as a transient intermediate. Isotope tracing further confirmed a + 2 Da mass shift in 3-vinylindole, indicating the incorporation of glycerol-derived carbons. These findings elucidate a novel pathway for βC formation under thermal conditions and highlight glycerol's contribution to this process during food processing.