UPLC Identification of Polyphenols

An ultra performance liquid chromatography (UPLC) system (Waters, Milford, MA, USA) equipped with a binary solvent delivery pump, auto-sampler, and a photodiode array (PDA) detector was used to identify individual polyphenols, as described previously [22 (link)]. The UPLC settings used in this study are summarized in Table 1. The mobile phase consisted of a binary solvent system comprising water (Solvent A) and acetonitrile (Solvent B) supplemented with 0.1% formic acid. A total of 17 phenolic compounds such as, gallic acid, 3,4-dihydroxybenzoic acid, 4-hydroxybenzoic acid, 2,4-dihydroxybenzoic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, sinapic acid, rutin, quercetin 3-β-D-glucoside, benzoic acid, kaempferol 3-O-β-rutinoside, quercetin, cinnamic acid, and kaempferol were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used as standards. For each standard compound, a methanol solution was prepared, resulting in a final concentration of 1000 ng/mL. A mixture of all standard compounds was then utilized to establish the calibration curve. The polyphenol content of each sample was calculated using the method described previously [22 (link)] with minor modification.