2.7. Extraction and determination of phenolic acid composition
The phenolic compounds present in parboiled rice were determined by the method of Butsat and Siriamornpun (2010) with some modifications. Briefly, samples were extracted with 80% aqueous methanol (1:10, w/v) at 25 °C using a shaking incubator at 150 rpm for 16 h. The mixtures were centrifuged at 2500 rpm for 20 min and the supernatants were collected. The pellet was re-extracted under identical conditions, and supernatants from both extractions were combined. The solvent was removed under vacuum at 40 °C and the resulting concentrated slurries were lyophilized. For HPLC analysis, residue was dissolved in 5 ml methanol and then was passed through a 0.45 μm filter. HPLC analysis was performed using Shimadzu LC-20AC pumps, SPD-M20A Diode-array detection; chromatographic separations were performed on a LUNA C-18 column (4.6 × 250 mm i.d., 5 μm). The elution system was performed using 1% acetic acid in water (A) and acetonitrile (B) as mobile phase. Gradient elution was performed as follows: 0–40 min, from 0–70% B with a flow rate 1 ml/min; 40–45 min, from 70–80% B with a flow rate 1 ml/min; 45–55 min, from 80– 85% B with a flow rate 1.2 ml/min; 55–57 min, from 85–90% B with a flow rate 1.2 ml/min; 57–75 min 90% B with a flow rate 1.2 ml/min. Operating conditions were as follows: column temperature, 40 °C, injection volume, 20 μL, UV–Diode Array detection at 280 nm (hydrobenzoic acids) and 320 nm (hydroxycinnamic acids). Phenolic compounds in the samples were identified by comparing their relative retention times and UV spectra with authentic compounds and were detected using an external standard method.