Ultimate 3000 dad

UV spectra were measured with a Thermo Ultimate 3000 DAD. IR spectra were measured with a PerkinElmer Spectrum One FTIR spectrometer. All NMR experiments were run on a Varian Unity spectrometer (500 MHz for ¹H), a Varian UNITY INOVA spectrometer (600 MHz for ¹H) equipped with a 5 mm triple resonance (HCN) cold probe, or a Bruker spectrometer (800 MHz for ¹H) outfitted with a 5 mm triple resonance (HCN) inverse cold probe. Residual solvent shifts for DMSO-d₆ or CD₃OD were defined as δ_H 2.50 or 3.31, respectively, for proton spectra and δ_C 39.52 or 49.00 for carbon spectra in accordance with reference spectra. Accurate mass measurements for molecular formula determinations were obtained on a Thermo Velos Pro Orbitrap ESI-FTMS. Prefractionation HPLC was performed using a Phenomenex Gemini-NX 5 µm C₁₈ (50 × 21.1 mm) column. Semipreparative HPLC fractions were generated using a Phenomenex Luna 5 µm C₁₈ (250 × 10 mm) column. Analytical LC-MS analysis was performed on samples at a concentration of approximately 0.5–10 mg/mL, using a reversed-phase 150 × 4.60 mm 5 µm C₁₈ Phenomenex Luna column in conjunction with a 4.0 × 3.0 mm C₁₈ (octadecyl) guard column and cartridge (holder part number: KJ0-4282; cartridge part number: AJ0-4287, Phenomenex, Inc.).

HPLC-DAD was performed with a Thermo Scientific HPLC system consisting of an Ultimate 3000 RS pump, an Ultimate 3000 RS autosampler, and an Ultimate 3000 DAD. The column used was a 4.6 mm i.d.×150 mm, 5 μm, Gemini C18 column (Phenomenex, Torrance, CA) to analyze the OPD/DNPH derivatives with MGO/MDA and the concentrations of EC, EGC, ECG, and EGCG in green tea with a flow rate of 1.0 mL/min. For the OPD/DNPH derivatives with MGO/MDA, column elution started with 20% solvent B (A: 100% water with 0.1% formic acid; B: 100% acetonitrile with 0.1% formic acid) for 5 mins, followed by a linear increase to 100% solvent B from 5 to 20 min, and then equilibrated to 20% B from 21 to 25 min for the next run. For green tea catechins, column elution started with 5% solvent B for 5 mins, followed by a linear increase to 35% solvent B from 5 to 25 min, and then a linear increase to 95% solvent B from 25 to 30 min, keep 95% solvent B for 3 min, then equilibrated to 5% B for the next run. The detection wavelength was 315 nm for MGO-OPD derivatives and 305 nm for MDA-DNPH derivatives, and 280 nm for green tea catechins. The injection volume of each sample was 80 μL. The temperature of autosampler was set to 8 °C. For contents of catechins in green tea, four catechins, EC, EGC, ECG, and EGCG were used as standards.