Cnwbond carbon gcb spe cartridge

The freeze-dried sample was pulverized using a mixer mill (MM 400, Retsch) and zirconia beads at 30 Hz for 1.5 min. Weigh 100 mg powder and extract it with 0.6 ml 70 % methanol solution at 4 °C overnight. After centrifugation of 10,000g for 10 min, the extract solution was passed through a CNWBOND Carbon-GCB SPE Cartridge (250 mg, 3 ml; ANPEL, Shanghai, China) was absorbed and used with 0.22 μm filter membrane (SCAA-104; ANPEL, Shanghai, China) was filtered and then analyzed by LC-MS.
The liquid chromatography was performed on Waters ACQUITY UPLC HSS T3 C18 column (1.8 μm diameter, 2.1 mm inner diameter × 100 mm length) with ultra-pure water (A phase) containing 0.04 % acetic acid and acetonitrile (B phase) containing 0.04 % acetic acid as mobile phases. The elution gradient starts from 5 % B phase at 0.00 min, increases linearly to 95 % B phase within 10.00 min and maintains for 1 min, then decreases to 5 % B phase within 0.00 min and completes equilibrium at 14.00 min. The experimental Settings included a flow rate of 0.35 mL/min, a column temperature of 40 °C, and an injection volume of 4 μL. The mass spectrum conditions mainly include:ESI temperature 550 °C, mass spectrum voltage 5500 V, CUR 30 psi. The quantitative monitoring mode for the metabolites was set to multiple reaction monitoring. DAMs between two cultivars were determined on the basis of the VIP ≥1 and fold change ≥2 or fold change ≤0.5.

C1, C2, D1, and D2 samples were analyzed using a broadly targeted metabolomics approach. A total of 840 metabolites were divided into 11 categories, including amino acids and their derivatives, saccharides and organic acids (Supplementary Table 3). Referring to the method of Chen et al. [87 (link)], the specific experimental methods were as follows: (1) The freeze-dried seeds were crushed using a mixer mill (MM 400, Retsch) with a zirconia bead for 1.5 min at 30 Hz. 100 mg powder was weighted and extracted overnight at 4℃ with 1.0 ml 70% aqueous methanol. Following centrifugation at 10, 000 g for 10 min, the extracts were absorbed (CNWBONDCarbon-GCB SPE Cartridge, 250 mg, 3 ml; ANPEL, Shanghai, China, www.anpel.com.cn/cnw) and filtrated (SCAA-104, 0.22 μm pore size; ANPEL, Shanghai, China, http://www.anpel.com.cn/) before LC-MS analysis. (2) The sample extracts were analyzed using an LC-ESI-MS/MS system (HPLC, Shim-pack UFLC SHIMADZU CBM30A system, www.shimadzu.com.cn/; MS, Applied Biosystems 6500 Q TRAP, www.appliedbiosystems.com.cn/). The analytical conditions were as follows: HPLC: column, Waters ACQUITY UPLC HSS T3 C18 (1.8 μm, 2.1 mm*100 mm); solvent system: water (0.04% acetic acid): acetonitrile (0.04% acetic acid); gradient program: 95:5 V/V at 0 min, 5:95 V/V at 11.0 min, 5:95 V/V at 12.0 min, 95:5 V/V at 12.1 min, 95:5 V/V at 15.0 min; flow rate, 0.40 ml/min; temperature: 40 °C; injection volume: 2 µl. (3) The effluent was alternatively connected to an ESI-triple quadrupole-linear ion trap (Q TRAP)-MS. LIT and triple quadrupole (QQQ) scans were acquired on a triple quadrupole-linear ion trap mass spectrometer (Q TRAP), API 6500 Q TRAP LC/MS/MS System, equipped with an ESI Turbo Ion-Spray interface, operating in a positive ion mode and controlled by Analyst 1.6.3 software (AB Sciex). (4) The ESI source operation parameters were as follows: ion source, turbo spray; source temperature 500 °C; ion spray voltage (IS) 5500 V; ion source gas I (GSI), gas II(GSII), curtain gas (CUR) were set at 55, 60, and 25.0 psi, respectively; instrument tuning and mass calibration were performed with 10 and 100 µmol/L polypropylene glycol solutions in QQQ and LIT modes, respectively. QQQ scans were acquired as MRM experiments with collision gas (nitrogen) set to 5 psi. DP and CE for individual MRM transitions were adopted with further DP and CE optimization. A specific set of MRM transitions were monitored for each period according to the metabolites eluted within this period.