Structural characterization and quantification of isomeric bioactive flavonoid C-glycosides utilizing high-performance liquid chromatography coupled with diode array detection and mass spectrometry

Flavonoid C-glycosides are secondary metabolites that occur selectively in plants. They are recognized for their antioxidant and broad range of biological properties. A recent study has reported that isoorientin regulates the GSK3β, NF-κB and Nrf2/HO-1 signalling pathways to exert neuroprotection. In this study, isomeric bioactive flavonoid C-glycosides were structurally characterized as isoorientin, orientin, vitexin, and isovitexin using tandem mass spectrometry. Furthermore, a simple isocratic, and robust RP-HPLC analytical method was developed and validated for the simultaneous quantification of these isomeric flavonoid C-glycosides in multiple plant matrices. The chromatographic separation was achieved on a C18 (150 × 4.6 mm, 3.5 μm) column at a flow rate of 1.2 mL min^-1. Acetonitrile and buffer (0.1% formic acid in a 95% water-methanol mixture) with 12 : 88 v/v were used as the mobile phase. The retention times (<i>t</i>_R) of isoorientin (9.23 ± 0.07 min), orientin (10.46 ± 0.09 min), vitexin (16.00 ± 0.13 min) and isovitexin (17.35 ± 0.10 min) were observed and quantified successfully. The limit of detection (LOD) and limit of quantification (LOQ) for the analytes were 2.53-4.31 and 7.66-13.08 μg mL^-1, respectively, while regression coefficients (<i>r</i>²) were found to be between 0.99989 and 0.99996. The abundance of isoorientin, orientin, vitexin and isovitexin was determined to be 13.25-94.85, 4.00-106.25, 2.16-25.03 and 7.56-14.14 mg g^-1 (w/w), respectively, in multiple matrices of <i>Cajanus scarabaeoides</i> and <i>Rhynchosia minima</i>. The developed isocratic HPLC method is the first to be reported and validated for its precision, accuracy, repeatability, and stability for the simultaneous quantification of flavonoid C-glycosides in six plant matrices.