2.5. Anthocyanin HPLC Analysis

Anthocyanin HPLC analysis was performed according to the procedure described in our previous study, which reported the identification of cyanidin 3-diglucoside (C3DG), cyanidin 3-sambubioside (C3S), and cyanindin-3-glucoside (C3G) with their mass spectra m/z 318 [M+H]+, 287 MSⁿ for C3DG, m/z 581 [M+H]+, 287 MSⁿ for C3S, and m/z 449 [M+H]+, 287 MSⁿ for C3G in L. radiata flowers [19]. Powders (0.1 g) of L. radiata flowers at different developmental stages were extracted using 1.5 mL water/methanoic acid (95:5, v/v) and then mildly sonicated for 20 min at 25 °C. After centrifugation at 8000 rpm for 30 min, supernatants were filtered through a Polytetrafluorethylene (PTFE) hydrophilic syringe. The HPLC system (Agilent Technologies, Palo Alto, CA, USA) including a Synergy 4-μm Polar-RP 80A column (250 × 4.6 mm id; Phenomenex, Torrance, CA, USA) with a Security Guard Cartridge Kit (AQ C18, 4 × 3 mm id; Phenomenex, Torrance, CA, USA) was for the isolation of anthocyanins. The analytical conditions and identification and quantification of the anthocyanin content was carried out based on our previous study [19]. The linear equations were y = 6.042x − 4.204 for C3G and the concentrations of C3DG and C3S were calculated as equivalents of C3G.