Spherisorb 5 μm ods2 4.6 150 mm analytical column

The total phenolic content (TPC) was determined using the Folin–Ciocalteu reagent according to a previous literature [16] . HPLC was used for the chromatographic analysis of individual polyphenols as described below. Nine hundred microliters of 0.5% acetic acid in methanol were added to each sample (100 μL) of polyphenolic extract and filtered using 0.45 μm polypropylene membrane into HPLC vials. The system was Breeze™ 2 HPLC and included 2707 autosampler, 1525 Binary HPLC pump, 2998 photodiode detector, and Empower 3 chromatography data analysis software from Waters Canada (Montreal, QC). The separation (20 μL) was done using Waters Spherisorb^® 5 μm ODS2 4.6 × 150 mm analytical column at a flow rate of 0.80 mL/min. A binary mobile phase of 0.5% acetic acid in water (A) and 0.5% acetic acid in methanol (B) was used to elute the phenolic compounds from the column. The linear gradient conditions were for 0–5 min A:B (100:0), 5–30 min A:B (80:20), 30–35 min A:B (50:50), 35–55 min A:B (20:80), and 55–60 min A:B (0:100). The column was equilibrated for 10 min between injections. Peaks were detected at 280 and 315 nm and were identified by matching retention times of peaks in samples to those of standards and by comparison with literature data [16] .