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 (t_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 (r²) 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 Cajanus scarabaeoides and Rhynchosia minima. 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.