HPLC analysis and quantification of phenolic compounds

Phenolic compounds were identified using high performance liquid chromatography (HPLC) Technique at Food Safety and Quality Control Laboratory (FSQC 0911-0915/2019), Faculty of Agriculture, Cairo University, Egypt. HPLC analysis was carried out according to Lu, Yuan⁶³ using an Agilent 1260 infinity HPLC Series (Agilent, USA), equipped with a quaternary pump. Phenolic substances were separated on a Kinetex 5 µm EVO C18 100 mm × 4.6 mm, (Phenomenex, USA) equipped with a variable-wavelength detector (VWD detector) set at 284 nm. Injection volume of 20 μl was carried out with auto-sampling injector. The separation was achieved using a ternary linear elution gradient with (A) HPLC grade water 0.2% H₃PO₄ (v/v), (B) methanol and (C) acetonitrile. Retention time and peak spectra of standard phenolic compounds (pyrogallol, quinol, gallic acid, 3-hydroxytyrosol, catechol, p- hydroxy benzoic acid, catechin, chlorogenic, vanillic acid, caffeic acid, syringic acid, p- coumaric acid, benzoic acid, ferulic acid, rutin, ellagic acid, o- coumaric acid, resveratrol, cinnamic acid, quercetin, rosmarinic acid, naringenin, myricetin and kaempferol) were used for identification. All phenolic compounds were quantified and expressed as µg/ g extract.