2.5. Quantification of phenolic acids by HPLC
Phenolic acids were extracted using the method reported by Xu et al. (2009) and Burgos et al. (2013) with slight modifications. Briefly, 0.100 g of sample was extracted with 3.5 mL of a methanol: water: acetic acid solution (80:19.5:0.5) using sonication for 20 min. After centrifugation of the sample at 8000 rpm for 10 min, the residue was extracted again under the same conditions, and the sample was heated to 80 °C for 5 min in the third extraction. The supernatants were collected, evaporated, adjusted to 5 mL with water and cleaned by passed through a 0.45 μm filter.
The analysis was performed using a Waters model 2995 separation system (Waters, Corp., Milford, USA) using a C18 reversed-phase column (symmetry Waters; 5 mm, 4.6 mm, 250 mm) and a gradient elution of 1% acetic acid in water (eluent A) and acetonitrile containing 0.1% acetic acid (eluent B). The gradient started with 3% B for 7 min, was increased to 5–40% B between 7 and 45 min, reached 100% B at 46 min, was maintained at this level to 51 min and was then decreased to 3% B within 6 min. The flow rate was set to 0.7 mL/min with a re-equilibration time of 13 min. Phenolics were detected with a UV–visible photodiode array detector (Waters model 2696) at the maximum absorption wavelength. Standard solutions were prepared by dissolving chlorogenic acid, caffeic acid, p-coumaric acid, and ferulic acid at concentrations ranging from 10 μg/mL to 50 μg/mL.