Electrospray ionization source

Two different ultra-performance liquid chromatography-mass spectrometers were used: Agilent 1290 Infinity UHPLC system coupled to an Agilent 6530 Accurate-Mass Quadrupole Time-of-Flight Mass Spectrometer with Dual Electrospray Ionization Source (Agilent, CA, United States); and Waters ACQUITY UPLC I-Class PLUS System coupled to a Waters SQ Detector 2 with Electrospray Ionization Source (Waters, MA, United States). The Nuclear Magnetic Resonance (NMR) spectra were performed using a Bruker AVANCE-500 (Bruker Corporation, Germany), and the chemical shifts were referenced to tetramethylsilane (TMS). Column chromatography (CC) was run on silica gel 60 (230–400 mesh, Merck, Germany), and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Sweden). Thin layer chromatography (TLC) analysis was carried out using silica gel 60 F₂₅₄ (TLC) plates (Merck, Germany). MS-grade methanol (MeOH) and acetonitrile (ACN) were obtained from RCI Labscan, Ltd. (Bangkok, Thailand). Formic acid was purchased from Thermo scientific, Inc. (Rockford, IL, United States). Ultrapure water was prepared with a Milli-Q system (Millipore, France).

Collected soil samples (about 10 g) were mixed individually with 10 mL of acetonitrile and were extracted using the QuEChERS method [87 (link)]. The content of an ECQUEU750CT-MP Mylar pouch (United Chemical Technologies, Levittown, PA, USA) was added to each sample, shaken immediately for more than 2 min, and centrifuged for 5 min at ≥3000 rcf. The supernatant was filtered directly into a sample vial through a 0.2 μm PVDF syringe filter. Ten microliters of each sample were injected into the Liquid Chromatography Mass-Spectrometry (LCMS) instrument [88 (link)], consisting of a 1260 Infinity II pump (model G7111B) coupled to a triple quadrupole (model G6465B) mass spectrometer with an Electrospray Ionization source (Agilent Technologies Inc., Santa Clara, CA, USA). The column was Kinetex XB-C18, 3.0 mm × 100 mm, 2.6 μm (Phenomenex, Torrance, CA, USA). The flow rate was 0.2 mL/min, and the gradient was as follows: 1 min at 50% MeOH; 5 min increase to 95% MeOH; 3 min at 95% MeOH; 1 min decrease to 50% MeOH; 1 min 50% MeOH.

Structural characterization of flavonols in bayberry leaves was conducted by HPLC, LC-MS, and Nuclear Magnetic Resonance (NMR) Spectroscopy. The operated HPLC system was equipped with a e2695 pump, a 2998 diode array detector, and a SunFire C18 analytical column (5 μm, 4.5 × 250 mm) (Waters, USA). The mobile phase solution was the same as that of pre-HPLC. Gradient program was as follows: 0-40 min, 10%-38% of B; 40-60 min, 38%-48% of B; 60-70 min, 48%-100% of B; 70-75 min, 100%-10% of B; and 75-80 min, 10% of B. Flow rate was set at 1 mL/min, and injection value was 10 μL.
LC-MS identification was carried out as our previous report [11 (link)]. Chromatographic separations were performed under the same gradient procedure as HPLC using an Agilent 1290 Infinity system (Agilent Technologies, USA) equipped with an X-Bridge C18 analytical column (4.6 × 250 mm). MS analysis was conducted by an Agilent 6460 triple quadrupole mass spectrometer coupled to an electrospray ionization source (Agilent Technologies, Santa Clara, CA, USA) and operated in the negative ionization mode.
The ¹³C-NMR spectra were measured in DMSO-d6 at 25°C on an Agilent 600 MHz instrument. Solvent residual peak δ 39.52 was used to calibrate all ¹³C chemical shifts in the present study. The chemical shifts of NMR were calculated and extracted by MestReNova (version 6.1.0).