Rp 18 gp250 column

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.

One gram of the sample was extracted for 24 hours with 20 mL of ethanol in a 35°C water bath. The solution was filtered and prepared for further HPLC analysis. A Hitachi HPLC system with L-2130 pump, L-2200 autosampler, and UV-detector (214 nm and 350 nm, L-2400 UV detector) was used. A RP-18 GP250 column (250 mm × 4.6mm inner diameter, 5 μm; Mightysil, Kanto Chemical Co.) was used, with a mobile phase of phosphate buffer (H₂O: 85% phosphoric acid 99.7:0.3, v/v)/acetonitrile/methanol at a flow rate of 1.0 mL/min. The injection volume of the sample was 20 μL. The analytic condition was performed using Fuleki’s method [9 ]. First, the standard products of quercetin and rutin were dissolved in methanol for establishment of a calibration curve, then calibration curves were constructed using the peak area (Y axis) and the concentration (mg/mL; X axis) of quercetin or rutin standards. The application of HPLC analysis of A. chinense extracts was performed, and the content of quercetin and rutin was determined under different processes associated with A. chinense.