Pu 4180 rhplc pump

One-dimensional and two-dimensional NMR spectra were acquired on an AVANCE III (400 MHz) (Bruker BioSpin, Rheinstetten, Germany), with chemical shifts expressed in ppm. The NMR spectra were referenced to residual solvent peaks (CD₃OD: ¹H NMR 3.30 ppm, ¹³C NMR 49.0 ppm). HR-ESI-MS spectra were acquired on a Thermo Fisher Scientific Q-Exactive HR-ESI-Orbitrap-MS (Waltham, MA, USA). Medium pressure liquid chromatography (MPLC) was conducted using an AI-580 system equipped with an ULTRA PAK ODS-SM-50D (50 µm, 50 × 300 mm, Yamazen Corporation, Osaka, Japan). Reversed-phase (RP)-HPLC separations were performed with a recycling system comprising a PU-2086 Plus Intelligent prep pump (Jasco, Tokyo, Japan), UV-2075 detector (Jasco, Tokyo, Japan), Capcell Pak UG120 C18 column (5 μm, 20 × 250 mm, Osaka Soda, Osaka, Japan), Capcell Pak UG120 C18 column (5 μm, 10 × 250 mm, Osaka Soda, Osaka, Japan), and HPLC-grade solvents. For analytical HPLC, a PU-4180 RHPLC pump (Jasco, Tokyo, Japan), MD-4017 photodiode array detector (Jasco, Tokyo, Japan), and AS-4050 HPLC autosampler (Jasco, Tokyo, Japan) were used. Data were analysed using ChromNAV software v.2 (Jasco, Tokyo, Japan).

Eight phenolic acids, gallic acid, protocatechuic acid, vanillin, cinnamic acid, caffeic acid, ferulic acid, ρ-hydroxybenzoic acid, and salicylic acid were used as standards. The phenolic profile of the rice samples was identified and quantified using high-performance liquid chromatography (HPLC). The system included a PU-4180 RHPLC pump, LC-Net II/ADC controller, and a UV-4075 UV/Vis (Jasco, Tokyo, Japan) detector. The stationary phase was XBridge BEH Shield RP18 (Waters Corporation, Milford, MA, USA) while the mobile phase included solution A (0.5% aqueous acetic acid) and solution B (100% acetonitrile). The program was operated as 5% B from 0–2 min, 5 to 70% B from 2–12 min, 100% B from 12–16 min and maintained for 6 min, 100% B to 5% from 22–24 min; equilibration was for 10 min. A 5 µL aliquot of the sample was injected into the HPLC system, which was then operated with a flow rate of 400 µL/min for 35 min at room temperature. Phenolic compounds were identified and quantified based on the corresponding peaks and their areas on the HPLC chromatogram compared to those of standard chemicals.

A RP-HPLC system JASCO (Tokyo, Japan), equipped with AS-4150 RHPLC Autosampler, PU-4180 RHPLC Pump, LC-NetII/ADC Interface Box, CO-4060 Column Oven, MD-4010 Photo Diode Array Detector, and ChromNAV software, version 2.0 (JASCO, Tokyo, Japan) was used. For the quantification of the VES-GEM conjugate, a Zorbax Eclipse XDB-C18 column (5 μm, 4.6 mm × 250 mm, Agilent Technologies, Santa Clara, CA, USA) at 30 °C and methanol as the mobile phase, with a flow rate 1 mL/min, was used. The detection wavelength was set at 248 nm [39 (link)]. All samples were filtered before injection through a PTFE hydrophilic Scharlau syringe filter (13 mm, 0.22 µm). The injection volume was 20 µL. For the VES-GEM calibration curve, a stock solution of VES-GEM in ethanol (50 ppm, 10 mL) was progressively diluted to standard solution concentrations of 40 ppm, 30 ppm, 20 ppm, 10 ppm, 5 ppm, and 1 ppm. The calibration curve of the quantified VES-GEM concentration, as a function of peak area (absorbance detected) (Figure S1), was drawn and showed good linearity within a concentration range of 1–50 ppm: linear regression equation y = 20779x + 2003.5, R = 0.9995, LOD = 0.15 ppm, LOQ = 0.236 ppm. The retention time was t = 8.0 min (Figures S2 and S3).