Aqua c18 column

Flavonols were analyzed by HPLC using the same HPLC system described above according to the method of Cho et al. (2005) [32 (link)]. Separation was performed at room temperature on a 4.6 mm × 250 mm Aqua C₁₈ column (Phenomenex, Torrance, CA, USA) preceded by a 3.0 mm × 4.0 mm ODS C₁₈ guard column. The mobile phase was a linear gradient of 2% acetic acid (A) and 0.5% acetic acid in water and acetonitrile (50:50 v/v) (B) from 10% B to 55% B in 50 min and from 55% B to 100% B in 10 min at a flow rate of 1 mL/min. The system was equilibrated for 20 min at the initial gradient prior to each injection. A detection wavelength of 360 nm was used to monitor flavonol peaks. Flavonols were quantified as rutin equivalents using an external calibration curve (3.3, 6.6, 13.2, 26.4, 52.8, 105.6, 211.2 mg/L; R² = 0.9999) of an authentic standard (Sigma-Aldrich, St. Louis, MO, USA), with results expressed as mg of rutin equivalents per g of WBB powder.

Optical rotations were measured with a Jasco DIP 140 polarimeter. ECD spectra were taken on a Jasco J-810 CD spectropolarimeter. UV and IR spectra were obtained using Perkin-Elmer Lambda 40 and Perkin-Elmer Spectrum BX FTIR instruments, respectively. All NMR spectra were recorded in MeOH-d₄ using a Bruker Avance 300 DPX spectrometer. Spectra were referenced to residual solvent signals with resonances at δ_H/C 3.35/49.0. HRESIMS were recorded on a LTQ Orbitrap mass spectrometer.
HPLC was performed on a Waters HPLC system equipped with a 1525µ binary pump, a 2998 PDA detector, Breeze 2 software and a Rheodyne 7725i injection system. A Macherey-Nagel Nucleoshell C₁₈ column (250 mm × 4.6 mm; 5 µm), Nucleodur PolarTec column (250 mm × 4.6 mm; 5 µm), Pyramid C₁₈ column (250 mm × 4.6 mm; 5 µm) and Phenomenex Kinetex C₁₈ column (250 mm × 4.6 mm; 5 µm) as well as a Phenomenex Aqua C₁₈ column (250 mm × 10 mm; 5 µm) were used.

Protein gel identification was performed by liquid chromatography/mass spectrometry in MONITOR HELIX (shanghai, China). Briefly, the SDS-PAGE gel of the corresponding location was cut, reduced, alkylated, and digested into peptides with trypsin (10 ng/μl). 2 μl of the peptide solution was separated using a Phenomenex aqua C18 column with a 2 μm particle size in a 200-mm length 75 μm internal diameter, 100 Å pore size on UltiMate 3000 HPLC unit (UltiMate 3000, Dionex, USA) and identified using Q Exactive HF hybrid quadrupole-orbitrap mass spectrometer (Q Exactive HF, Thermo Fisher, USA). Raw data were analyzed with MaxQuant software (version 1.6.0.16) and screened according to FDR <0.01. High-confidence peptides were blasted with the UniProt human protein database (https://www.uniprot.org/) for protein identification.

A high-performance liquid chromatography system (HPLC, Agilent Technologies, CA) coupled in series to either a diode array detector (DAD; Agilent Technologies) or a time-of-flight mass spectrometer (TOF/MS; Agilent Technologies) was used for quantitative and qualitative analysis, respectively.
Compounds (20 µl injection per sample) were separated and eluted at 25°C on an Aqua C18 column (5 µm particle size, 250 × 4.60 mm; Phenomenex, CA) under various isocratic conditions of acetonitrile in water (containing formic acid at a final concentration of 0.1%) as a mobile phase at various flow rates (Supplementary Table S1). Dimethyl phthalate, propanil, monalide, and chlorpropham and their degradation products (monomethyl phthalate, 3,4-dichloroaniline, 4-chloroaniline, and 3-chloroaniline, respectively) were quantified using authentic standards with the DAD operating at appropriate wavelengths (Supplementary Table S1). The identities of all test chemicals and, where possible, their transformation products were confirmed with the TOF/MS operating as described previously (Pandey et al. 2010 (link)).

The chromatographic system consisted of a binary LC pump (L-6200A Intelligent Pump) and an Autosampler (CTC Combi Pal) set to +4 °C. The separation was achieved on a Phenomenex Aqua C18 column (5 µm, 50 × 4.6 mm) using isocratic elution at ambient temperature with 0.1% formic acid in water (90%), methanol (5%) and tetrahydrofuran (5%). Detection was performed using a SCIEX API 3000 triple quadrupole mass spectrometer equipped with a turbo ion spray interface (SCIEX, Concord, ON, Canada). High-purity nitrogen gas was used as a nebulizer, curtain, auxiliary, and collision gas. The spectrometer was operated in the positive ion mode for the detection of caffeine (m/z 195 → 138), paraxanthine (m/z 181 → 124), theophylline (m/z 181 → 124), theobromine (m/z 181 → 138), and the internal standard (m/z 198 → 140). Data acquisition was performed using RAD (version 2.6, PE Sciex, Thornhill, ON, Canada) and data processing using MacQuan (version 1.6, Perkin Elmer, Toronto, ON, Canada, 1991–1998). The precision of the spiked quality control samples for all compounds was below 10%, with an accuracy between 85% and 115%.

Separations used either a Synergi Fusion-RP C₁₈ column (ID: 2 mm, length: 150 mm, particle size: 4 µm, pore size: 80 Å, Phenomenex) or Aqua C₁₈ column (ID: 2 mm, length: 150 mm, particle size: 3 µm, pore size: 125 Å, Phenomenex) using a System Gold HPLC equipped with a 507e autosampler (100 µL injection volume), a 125NM binary gradient pump, and a 166 UV/VIS detector. Eluents were water (eluent B1) and aqueous acetonitrile (60% v/v, eluent B2 or 40% v/v, eluent B3) containing sodium or potassium phosphate buffer or ammonium acetate (10 mmol/L). Eluents were prepared from aqueous stock solutions (0.1 mol/L) adjusted to pH 7.2, in case of ammonium acetate with ammonia. Solvents were filtered (pore size, 0.2 µm; Pall Corp., Ann Arbor, MI) and sonicated for 15 min prior to use. Peptides were eluted using a linear acetonitrile gradient from either 5 to 95% eluent B2 in 30 min, 5 to 70% eluent B2 in 65 min, or 7.5 to 95% eluent B3 in 58 min. Unless otherwise indicated, separations were performed at 60 °C with a flow rate of 0.2 mL/min and the absorbance was recorded at 214 nm. Fractions were analyzed by matrix-assisted laser desorption/ionization time-of-flight MS (MALDI-TOF/TOF–MS) on a 5800 proteomic analyzer (ABSciex GmbH, Darmstadt, Germany) operating in reflector mode and using a solution of α-cyano-4-hydroxycinnamic acid (4 g/L) in eluent A2 as matrix.