Metabolite Structural Identification by HPLC, MS, and NMR

The analyses and structural identification of metabolites were performed by the combinational use of HPLC, high resolution electrospray ionization mass spectroscopy (HR-ESI-MS), and nuclear magnetic resonance (NMR), as described previously [3 (link),11 (link),23 (link),25 (link),28 (link)]. Briefly, analytical HPLC was performed in an Agilent HPLC 1200 system (Agilent, Waldbronn, Germany) equipped with a SilGreen C18 column (250 × 4.6 mm id, 5 μm particle size). The mobile phase was composed of 0.1% trifluoroacetic acid in H₂O (solvent A) and acetonitrile (solvent B). The gradient elution was as follows: 0–5 min, 15% B; 5–20 min, 50% B; 20–28 min, 100% B. The flow rate was 1.0 mL/min. The injection volume was 50 μL and the effluents were monitored at 25 °C by a DAD detector at 243 nm. Glucosylated products were collected on an SEP LC-52 system (SEP. Co. Ltd., Beijing, China) with a YMC C18 preparative column (250 × 10.0 μm ID, 5 μm; YMC Co. Ltd., Kyoto, Japan). The data collection for HR-ESI-MS were carried out on a Thermo Scientific Exactive Orbitrap LC-Mass spectrometer (Thermo Scientific, Waltham, MA, USA). ¹H-NMR (600 MHz), ¹³C-NMR (151 MHz), and 2D-NMR spectrometric data were recorded with AVANCE III HD 600 NMR spectrometer (Bruker, Rheinstetten, Germany). Chemical shifts are given in δ (ppm) with the solvent (CD₃OD-d4) peaks as references.