LC-MS-Based Metabolomic Characterization of an Unsaponifiable Bixa orellana Fraction Before and After Tocotrienol Enrichment

The industrial processing of Bixa orellana L. seeds for bixin production generates large amounts of lipid-rich residues with potential as sources of bioactive compounds. These by-products contain tocotrienols and other lipophilic metabolites with antioxidant, anti-inflammatory, and antiproliferative properties. Untargeted metabolomics is a valuable strategy for characterizing these complex matrices and supporting their valorization within circular economy approaches. This study compared the metabolomic profiles of two fractions obtained from industrial processing: TT, the unsaponifiable residual fraction from bixin production, and T3, a tocotrienol-enriched fraction produced from TT by molecular distillation. Samples (10 mg/mL) were dissolved in hexane, diluted in methanol, and analyzed in triplicate by UPLC-QTOF-MS using MSE acquisition in positive and negative electrospray ionization modes (m/z 100–1000). Data were processed in Progenesis™ QI v2.4 and analyzed in MetaboAnalyst 5.0 after sum normalization, log transformation, and Pareto scaling. Differential features were selected using fold change ≥2.0 and FDR-adjusted p < 0.05. Initially, 2444 positive-mode and 1578 negative-mode features were detected. After quality filtering (RSD <25%), 1677 and 864 features remained, respectively. Comparative analysis identified 453 differential features in positive mode and 163 in negative mode. Principal Component Analysis showed clear metabolic discrimination between TT and T3, demonstrating that molecular distillation substantially altered the metabolomic composition of the original residue. Putative annotation indicated enrichment of fatty acids and derivatives, sesquiterpenes, diterpenes, carotenoids/apocarotenoids, sphingolipids, and complex lipids in T3, whereas TT showed higher abundance of terpenoids, oxylipins, phospholipids, steroids/sterols, lipophilic phenolics, and nitrogen-containing lipids. Overall, 79 differential features were plausibly annotated. These findings demonstrate that molecular distillation selectively enriches tocotrienol-associated metabolites and highlight the biotechnological and pharmaceutical potential of B. orellana industrial by-products, supporting untargeted LC-MS metabolomics as a powerful tool for their characterization and valorization.