Determination of major phenolic compounds using HPLC

Sample extracts were centrifuged for 10 min at 13,000 rpm and subjected to chromatographic analysis using an Agilent 1260 Infinity Series reverse phase HPLC, equipped with DAD 1100 diode array detector (Agilent Technologies, Palo Alto, CA). A gradient elution method, involving 10 mM phosphoric acid (pH 2.5; Solvent A) and 100% methanol (Solvent B), was used. Sample extracts were eluted on a C-18 analytical column (Agilent Supelco SB-C18 250 × 4.6 mm internal diameter) with a packing material particle size of 5 μm, at a flow rate of 0.7 mL/min at ambient temperature, with a total run time of 25 min. Pure standards of phenolic acids in 100% methanol were used to calibrate retention times on the standard curve. Chromatograms obtained were then analyzed using Agilent Chemstation integration software. Phenolic acid concentrations were expressed in micrograms per gram FW of barley sprouts.