5c18 ms 2 cosmosil packed column

The high-performance liquid chromatography (HPLC) system (Waters, Milford, MA) consisted of a series 600 controller, a series 717 plus autosampler, and a series 996 photodiode-array detector, connected to a cartridge column (GL Sciences, Tokyo, Japan; average particle size of 5 μm) and a Cosmosil packed 5C₁₈-MS-II column (Nacalai, San Diego, CA; internal diameter of 4.6×250 mm; average particle size of 5 μm). The mobile phase consisted of buffer A (2.5% methanol in 0.01 M ammonium dihydrogen phosphate, pH 5.3) and buffer B (20% methanol in 0.01 M ammonium dihydrogen phosphate, pH 5.1). Elution started with 100% buffer A and consisted of the following linear gradient steps: 0–10 min, 0–25% buffer B; 10–20 min, 25–40% buffer B; 20–60 min, 40–100% buffer B. A flow rate of 0.9 ml/min and an injection volume of 20 μl was used. Temperature of the column was maintained at 25°C. Detection was done at a wavelength of 260 nm. Deionised water used for preparation of the HPLC mobile phase and sample dilution was prepared with the Milli-Q purification system (Millipore, Bedford, MA). Nitrogenous bases, nucleosides, HPLC-grade methanol, and ammonium dihydrogen phosphate were obtained from Sigma-Aldrich (St. Louis, MO).

Shimadzu LC‐40 HPLC system was used for chromatographic analysis of EPI and icariin. Reversed phase C18 ODS column (10ID × 250 mm, Cosmosil 5C18‐MS‐II Packed column, Nacalai Tesque) was used for the column. Column temperature 30°C, The flow rate was set to 2.0 mL/min and the detection wavelength was 210 nm. The mobile phase was 0.1% phosphoric acid solution (A)‐acetonitrile (B), the linear elution procedure was 0–50 min, 10%–100% B.

Chromatographic analysis of phenolic compounds in the BD and AD samples was carried out according to Sęczyk et al. [18 (link)]. Samples were analyzed with a Varian ProStar HPLC system (Varian, Palo Alto, CA, USA) equipped with a ProStar diode array detector (DAD). The analytical column was a 250 mm × 4.6 mm COSMOSIL 5C18-MS-II Packed Column (Nacalai Tesque, Inc., Kyoto, Japan). The mobile phase consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). Elution mode was programmed as follows: 0–5 min, 5% B; 5–20 min, 5–100% B; 20–25 min, 100% B; 25–30 min 100–5% B. The flow rate was 1 mL/min and the column thermostat was set at 30 °C. The spectrophotometric detection was performed at wavelengths: 270 nm for GA, 280 nm for CAT, 325 nm for FA and CGA, and 350 nm for Q and A. Quantification of phenolic compounds was carried out using external standard calibration curves.