HPLC-DPPH• Radical Scavenging Analysis

HPLC-DPPH^•-scavenging online analysis was carried out on a Waters HPLC system (Waters Corporation, Milford, MA, USA) as previously described [21 (link)] with minor changes. The Hypersil C18 analytical column (250 × 0.46 cm, 5 µm; Supelco Analytical, Bellefonte, PA, USA) was used for compound separation. The mobile phase consisted of 0.4% formic acid in water (A) and acetonitrile (B). The elution profile was at the start point 90% A, then changing to 60% in 45 min, after that, in 50 min A was decreased to 5%, then in 53 min increased to 90% A, then it was held at 90% for 3 min, and in 2 min it was returned to initial conditions and the column was equilibrated for 5 min. The decrease of absorbance after the reaction of radical scavengers with DPPH^• was measured at 515 nm with the variable-wavelength Shimadzu SPD–20A UV detector (Shimadzu Corporation, Kyoto, Japan), while the identification of compounds was performed by using the Waters Acquity UPLC system (Milford, MA, USA).