Hss t3 column

Samples preparation and metabolites extraction were proceed as our previous described [30 (link)]. In brief, fresh biomass (50 mg) was crushed in liquid nitrogen, and then metabolites were extracted with MeOH:water (1:1) in Eppendorf tubes. Extracts were dried in a vacuum centrifuge, and then polar metabolites were analyzed using GC–MS as described, and UPLC-QE orbitrap/MS as follows. Analyses in HILIC and the RPLC mode proceeded using Waters Acquity UPLC BEH-Amide and HSS T3 columns (Waters Corp, Milford, MA, USA), respectively. Data were acquired by MS/MS via data-dependent acquisition (DDA) in the range of 70–1050 for both the positive and negative ion modes. The RAW files from GC–MS and LC-QE orbitrap/MS were generated using MSDIAL 3.7 and Compound discovery software [73 (link), 74 (link)], respectively. The lipidomic analysis, including sample preparation, lipid extraction, and identification, proceeded as described [75 ]. Additional details of the metabolomic and lipidomic analyses are available in Additional file 6.

ELG powder (250 mg) was dissolved in 25 mL of 75% ethanol. To facilitate dissolution, this solution underwent sonication twice at 300 W and 40 kHz, followed by centrifugation at a speed of 12, 000 rpm for 5 min. The UPLC was performed using UPLC/Q-TOF–MS (Sciex Triple TOF 4600 high resolution mass spectrum coupled with an Agilent 1290 UPLC system; AB Sciex, Darmstadt, Germany; Agilent, Los Angeles, CA, USA). For separation, the UPLC system was utilized with HSS T3 columns from Acquity (2.1 × 100 mm, 1.8 μm; Waters, Milford, CT, USA), and 2 µL of the supernatant was injected. A solvent system comprising 0.1% formic acid-acetonitrile (phase A) and 0.1% formic acid–water (phase B) was used for gradient elution with a flow rate of 0.3 mL/min. The elution program was as follows: 10% A-20% A 0–5 min, 20% A 5–9 min, 20% A-30% A 9–16 min, 30% A-45% A 16–26 min, 45% A-95% A 26–32 min, 95% A 32–36 min, 95% A-10% A 36–37 min, and 10% A 37–40 min. The specific gradient elution program of the mobile phase was illustrated in Additional file 1: Table S1.

LC-MS/MS analysis was performed using a Waters ACQUITY I-Class ultra-performance liquid chromatograph coupled to a SCIEX QTRAP 6500 mass spectrometer with standard electrospray ionization source (ESI). Analytes were separated using a Waters HSS T3 column (2.1 mm × 100 mm × 1.8 µm) with column temperature at 40 °C. Mobile phase A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in acetonitrile. Samples (5 or 10 µl) were injected using an autosampler at 8 °C. Analytes were eluted at a flow rate of 0.3 ml/min with a gradient of increasing mobile phase B concentration, as follows: 0.2% for 1 min, 0.2% to 10% for 1 min, 10% to 25% for 4 min, and 25% to 90% for 2 min. The column was cleaned with 90% mobile phase B for 1.5 min and then equilibrated with 0.2% mobile phase B for 5 min. Analytes were detected in the mass spectrometer using MRM in positive mode under the following conditions: ion spray voltage: 5500 V, temperature: 550 °C, curtain gas: 40 psi, collision gas: high, ion source gas 1: 45 psi, ion source gas 2: 50 psi, entrance potential: 10 V, collision cell exit potential: 11 V. The detailed conditions for MRM transitions, optimized declustering potential (DP), and collision energy (CE) are listed in Table 1. All LC-MS/MS systems were controlled by Analyst® software version 1.6.2 (SCIEX) and MRM data were acquired using the same software.