HPLC Analysis of Phenolic Compounds

Extracted phenolic compounds were analyzed by the method of Ayala-Soto et al. (24 (link)) and adjusted by Zavala-López and García-Lara (23 (link)) using a HPLC system (Agilent 1100 Santa Clara, CA) coupled with a photodiode array (PDA) detector (Agilent G1315D, Santa Clara, CA). Linear gradient elution was performed using HPLC-grade water (CAS: 7732-18-5, BDH, West Chester, PA) acidified (pH = 2) with trifluoroacetic acid (CAS: 76-05-1, Sigma-Aldrich, St. Louis, MO) and acetonitrile (CAS: 75-05-8, BDH, West Chester, PA), at a flow rate of 0.6 mL/min at 25°C. Phenolic compounds were separated with a Zorbax SB-Aq, 4.6 mm ID × 150 mm (3.5 um) reverse phase column. The Chemstation software (for LC Copyright© Agilent Technologies, 1990–2003) was used to process the data and command the equipment. Peak identification of trans-ferulic acid (CAS: 537-98-4, Sigma-Aldrich, St. Louis, MO) and p-coumaric acid (CAS: 501-98-4, Sigma-Aldrich, St. Louis, MO) was based on retention time and absorption spectra of these standards. Identification of diferulic acids was performed according to retention times and absorption spectra reported by Ayala-Soto et al. (24 (link)) and expressed as equivalents of ferulic acid.