Ods am 301 column

For the preparation of polyphenol extracts, 1 g of ground materials was mixed with 20 mL of 80% methanol. The mixture was shaken for 24 hours at room temperature, filtered through a Whatman number 42 filter paper, freeze dried at −40°C until use. The polyphenol profile of tested extract was determined according to the method of Kurata et al [18 ]. The polyphenol extract was filtered through a cellulose acetate membrane (0.20 μm; Advantec, Tokyo, Japan), and 10 μL of the filtrate was injected into a high-performance liquid chromatography (HPLC) system (Waters 2996; Newark, Delaware, USA) using an ODS column (ODS-AM 301 column, 4.6 mm × 150 mm, 5 μm particles; YMC, Kyoto, Japan). The temperature was set at 40°C. The mobile phase consisted of water containing 0.2% (v/v) formic acid (A) and methanol (B). Elution was performed with the linear gradient as follows: 2% B from 0 minutes to 15 minutes, 2%–45% B from 15 minutes to 50 minutes, and 45% B from 50 minutes to 65 minutes. The flow rate was 1 mL/minute. Polyphenolic compounds were then compared with the obtained standards.

For high-performance liquid chromatography (HPLC) analysis, 1 g of freeze-dried materials was mixed with 20 mL of 800 g kg⁻¹ methanol. The mixture was shaken for 24 hours at room temperature, filtered through a Whatman No. 42 filter paper, and was freeze-dried at −40°C until use. Polyphenols profile of SPLE and GCBE samples was determined according to the method of Jeng et al [15 ]. The polyphenols sample was filtered through a cellulose acetate membrane (0.20 μm, Advantec, Tokyo, Japan), and 10 μL of the filtrate was injected into HPLC (Waters 2996, Waters, Milford, MA, USA) using a ODS (Output Delivery System) column (ODS-AM 301 column, 4.6 × 150 mm, 5-μm particles; YMC, Kyoto, Japan). The temperature was set at 40°C. The mobile phase consisted of water containing 0.2% (v/v) formic acid (A) and methanol (B). Elution was performed with the linear gradient as follows: 2% B from 0 minutes to 15 minutes, 2% to 45% from 15 minutes to 50 minutes, and 45% B from 50 minutes to 65 minutes. The flow rate was 1 mL min⁻¹. Polyphenolic compounds were then compared with the obtained standards.