0.45 mm polyvinylidene fluoride membrane filter

Sample powder (500 mg) was extracted with 50 mL 80% ethanol. This suspension was shaken for 45 minutes at room temperature and filtered through Whatman No. 4 filter paper. The residue was re-extracted five times with an additional 25 mL 80% ethanol. The combined filtrate was then rotary evaporated at 40°C and redissolved in deionized water to a final volume of 10 mL. The aqueous extract was filtered using a 0.45-mm polyvinylidene fluoride membrane filter (Millipore, Billerica, MA, USA) and analyzed using high-performance liquid chromatography (HPLC). The HPLC system consisted of a Hitachi L-2130 pump (Tokyo, Japan), a Rheodyne 7725i injector (Rohnert Park, CA, USA), a 20-mL sample loop, a Hitachi L-2400 UV detector, and an RP-18 GP250 column (4.6 mm × 250 mm; Mightysil, Kanto Chemical Co., Tokyo, Japan). The mobile phase was acetonitrile/deionizer water [75:25 (v/v)] at a flow rate of 0.8 mL/min, and UV detection was at 300 nm. Each organic acid was identified using the authentic organic acid (all from Sigma-Aldrich) and quantified by its respective calibration curve.