HPLC Analysis of Phenolic Compounds

Ethanolic extracts (20 μL), previously filtered using a 0.45 μm nylon membrane filter, were injected in the HPLC system (Hitachi Chromaster, Tokyo, Japan). The chromatographic separation was carried out with a NUCLEODUR^® C₁₈ HTec column (250 × 4.6 mm, the particle size of 5 μm, Macherey-Nagel, Düren, Germany). The mobile phase consisted of deionized water containing 0.1% trifluoroacetic acid (A) and methanol (B). The gradient was programmed in the following order: 90% A at 0–3 min, 70% A at 20 min, 60% A at 30 min, 40% A at 50 min, and 80% A at 60 min. The flow rate was 1.0 mL/min, and the column temperature was maintained at 25 °C. Phenolic compounds were detected by comparing their chromatographic behavior and monitoring UV absorption at 320 nm with authentic standards and reported data [5 (link),10 (link),19 (link)].