HPLC Quantification of Flavonoid Phytochemicals

In this study, the HPLC system consisting of an autosampler, Varian (Les Ulis, France) Prostar 230 pump, as well as a Varian Prostar 335 photodiode array detector was used, and controlled by Galaxie software (Varian v1.9.3.2). The separation was performed at 35 °C using C18 core-shell column with iso-butyl side chains with TMS endcapping (Kinetex 5 µm XB-C18, 100 Å, LC Column 150 × 4.6 mm, core-shell silica, Phenomenex Le Pecq France). The mobile phase was composed of a methanol (solvent A) and HPLC grade water (solvent B) mixture, both being acidified with 0.05% formic acid. The linear gradient was applied for the mobile phase variation from a 5:95 (v/v) to a 100:0 (v/v) mixture of solvents A and B, respectively. Flow rate was 1.30 mL/min, and injection volume was 3 µL. Maximum back pressure was 110 bar. The detection was conducted using a PDA (photodiode array detector), and the quantification was performed at 320 nm (the maximum absorbance wavelength of the studied flavonoids). Flavonoid phytochemicals were then identified by means of comparison with the authentic standards (Extrasynthese, Genay, France), as described previously [36 (link)].