Rapid and comprehensive metabolites identification of 5-demethylnobiletin in rats using UPLC/Triple-TOF-MS/MS based on multiple mass defect filter and their neuroprotection against ferroptosis

5-Demethylnobiletin (5-deNOB) is a hydroxylated polymethoxyflavone (PMF) from Citrus plants known for its neurotrophic, anti-tumor, and antioxidant bioactivities. An ultra-high performance liquid chromatography coupled with triple-time of flight tandem mass spectrometry (UPLC/Triple-TOF-MS/MS) analysis combining with multiple mass defect filter (MMDF) and MetabolitePilotTM was employed to detect and characterize the metabolites of 5-deNOB in rats. A total of 130 metabolites were identified in rats, with 100, 25, 34, and 52 metabolites found in urine, plasma, bile, and feces, respectively. The major metabolic pathways involved demethylation, hydroxylation, dehydroxylation, glucuronidation, and methylation. In a bioassay of evaluating neuroprotection against ferroptosis in PC12 cells, most of the metabolites exhibited superior activity compared to 5-deNOB. These results provide valuable insights into the in vivo pharmacodynamic properties of 5-deNOB and offer potential active small molecules for neuroprotective therapy. Furthermore, the findings demonstrate the effectiveness of UPLC/Triple-TOF-MS/MS combined with MMDF and MetabolitePilotTM for rapid discovery and identification of the in vivo metabolites of natural products.