Pinnacle c18 column

The biomarkers, luteolin 7-O-glucoside, chlorogenic acid, kaempferol and isorhamnetin were purchased from Phytolab (Vestenbergsgreuth, Germany). The analysis was performed by HPLC with UV detection (Alliance 2695 Separations Module, Waters Instruments, Inc., Milford, MA, USA) using a reverse-phase C18 column (Pinnacle C18 column, 250 × 4.6 mm, 5 µm, Shimadzu, Kyoto, Japan). The mobile phase was composed of different proportions of (A) 0.5% acetic acid in ultra-pure water (acidified water), and (B) acetonitrile and methanol (60:40, v/v) using a flow rate of 1 mL/min. The optimized gradient program was as follows: 0-5 min (0-55% B), 5-10 min (55-65% B), 10-20 min (65-90% B), 20-30 min (90-100% B), and 30-35 min (0% B). Samples were injected into the system at 20 µL. The detection was performed at a single wavelength (280 nm) and processed using EMPOWER software, version 2.

The phenolic acids were quantified by HPLC with UV detection (Alliance 2695 Separations Module, Waters Instruments, Inc., Milford, MA, USA). The analyses were carried out on a reverse-phase C18 column (Pinnacle C18 column, 250 × 4.6 mm, 5 µm, Shimadzu, Kyoto, Japan). The mobile phase was composed of (A) 2% acetic acid in ultra-pure water (acidified water), and (B) acetonitrile and methanol (65:35, v/v) using a flow rate of 1 mL/min. The optimized gradient program was as follows: 0-10 min (10-45% B), 10-20 min (45-90% B), 20-23 min (90-10% B), and 23-25 min (10% B). Samples were injected into the system as 10 µL, and the analysis was performed at a single wavelength of 280 nm.