<![CDATA[Rubber seeds (Hevea brasiliensis) are underutilized plant by-products with potential for food and feed applications, but cyanogenic glycosides remain an important safety concern. This study investigated processing-associated changes in the metabolite profile of rubber-seed flour using untargeted liquid chromatography–high-resolution mass spectrometry (LC–HRMS). We evaluated four treatments: unprocessed whole seeds (A), soaked and boiled seeds without the seed coat (B), soaked and boiled seeds followed by oil pressing (C), and soaked and boiled seeds with the seed coat retained (D), with three biological replicates per treatment. After spectral-quality assessment, contaminant removal, and plausibility review, 39 putative metabolite annotations were retained for multivariate analysis. Principal component analysis separated the unprocessed samples from the processed groups; PC1 and PC2 explained 35.0% and 19.6% of the variance, respectively. A two-component partial least-squares discriminant analysis model showed Q² = 0.630 and R² = 0.918, but leave-one-out cross-validation classified only 50% of samples correctly, indicating limited predictive performance in this small dataset. The processed groups showed substantial overlap, suggesting that their profiles were more similar to one another than to the unprocessed control. VIP-ranked features included glycerolipids, amino-acid-related compounds, and fatty acids; these were treated as putative annotations rather than definitive identifications. Linamarin was assigned in only one sample and lacked MS/MS confirmation, so its presence and any effect of processing on cyanogenic risk remain unresolved. Overall, soaking and boiling were associated with changes in the measured metabolite profile. Targeted quantification of linamarin and hydrogen cyanide, confirmation of key metabolite annotations with authentic standards and MS/MS, and validation in a larger factorial experiment are required before practical safety or nutritional claims can be made. Contribution to Sustainable Development Goals (SDGs):SDG 2- Zero Hunger SDG 12 - Responsible Consumption and Production SDG 13 - Climate Action]]>
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