Abstract
Denatonium is widely recognized as an intensely bitter synthetic compound used to prevent accidental ingestion of toxic or undesirable products. Here, we report the natural occurrence of denatonium in seed-derived tissues of Camellia japonica . Direct chewing of the seeds produced exceptional bitterness, consistent with LC-MS/MS detection. To confirm reproducibility, samples from different sources or growth conditions, including germinated sprouts and oil-removed seed cake, were analyzed. Denatonium was detected in the radicle, plumule, cotyledons, and seed cake. Its identity was confirmed by comparison with an authentic denatonium benzoate standard, based on retention time at 5.63 min, precursor ion, and diagnostic MS/MS fragment ions. Quantitative analysis revealed high levels of denatonium, reaching approximately 3 g/kg dry weight in cotyledons and 2 g/kg dry weight in seed cake. These findings demonstrate that C. japonica seeds naturally accumulate denatonium, suggesting a role in seed-associated chemical defense.