2.4. Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC)

Samples were analyzed with reversed-phase chromatography using a Poroshell 120 EC C-18 column (4.6 × 100 mm, 2.7 μm), a Poroshell 120 EC-C18 4.6 mm guard-column, and an instrument 1260 Infinity II, with a quaternary pump, a PDA detector, and a vialsampler (Agilent technology, Santa Clara, CA, USA). The separation was achieved by using 0.1% H₃PO₄ (mobile phase A), and 100% methanol (mobile phase B) with a gradient 0 min 5% B, 5 min 25% B, 14 min 34% B, 25 min 37% B, 30 min 40% B, 34 min 49% B, 35 min 50% B, 58 min 51% B, 60 min 55% B, 62 min 80% B, 65 min 80% B, 67 min 5% B, and 72 min 5% B [20]. The flow was 0.5 mL/min, and each sample was injected in 10 μL. To identify phenolic compounds, samples were spiked with authentic standards. In addition, phenolic compounds were identified by comparing UV/Vis spectra and retention time with those of authentic standards. The quantification was conducted using calibration curves. Quercetin-3-xyloside, quercetin derivative, and p-coumaroylquinic acid were identified with the help of the literature and quantified using quercetin and p-coumaric acid calibration curves.