Agilent 6545 q tof mass spectrometer

For the metabolomic study, cells under the same treatment conditions were harvested, and metabolites were extracted using a cold methanol-acetonitrile-water solution (2:2:1 v/v/v). The extracts were then analyzed using an Agilent 1290 Infinity II LC system coupled with an Agilent 6545 Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, United States). The chromatographic separation was carried out on a Waters ACQUITY UPLC BEH Amide column (2.1 mm × 100 mm, 1.7 μm). The raw data were processed and analyzed using Agilent MassHunter Qualitative Analysis software (B.06.00).

Qualitative analysis was performed using an Agilent 1290 LC system (Agilent Technologies, Santa Clara, CA, USA) connected to an Agilent 6545 Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). Agilent Mass Hunter Qualitative Analysis software (B.07.00) was used for MS control, data acquisition, and data analysis.
The separation column was ACQUITY UPLC HSS T3 (1.8 µm, 2.1 mm × 100 mm), with mobile phase was composed of water containing 0.1 % formic acid (solvent A) and acetonitrile containing 0.1 % formic acid (solvent B). The gradient elution program was set as follows: 0–5 min, 98 % A; 5–27 min, 94 % A; 27–30 min, 90 % A; 30–32 min, 85 % A; 32–38 min, 75 % A; 38–41 min, 48 % A; 41–51 min, 36 % A; 51–85 min, 2 % A. The flow rate was 0.2 ml/min, with column temperature was 30℃, and the injection volume was 2 μl.
MS/MS analyses were performed in both positive and negative ESI modes with the mass range set to m/z 50–1500 in full scan resolution mode at a scan rate of 30 scans per second. The ESI parameters were set as follows: sheath gas temperature 350℃, drying gas flow 12 L/min, capillary voltage 3.5 kV, fragmentor voltage 130 V, skimmer 65 V, and data were recorded in centroid mode. The ion scan was performed with a collision energy value of 40 eV under the same operating conditions as the primary MS scan.

Silica gel (75–120 mesh), RP-C₁₈ silica (38–63 μm), and organic solvents were purchased from Wako Pure Chemical Industries (Osaka, Japan). Sephadex LH-20 was purchased from GE Healthcare (Uppsala, Sweden). Analytical TLC was performed on precoated Silica gel 60 GF₂₅₄ (20 $\times$ 20 cm $\times$ 0.2 mm thick) or precoated RP-C₁₈ F₂₅₄ plates (5 $\times$ 7.5 cm $\times$ 0.2 mm thick) on aluminium sheets, from Merck Co., Darmstadt, Germany. Ultraviolet (UV) spectra were obtained using UV-visible spectrophotometer (Shimadzu 1601 PC, model TCC240, Kyoto, Japan). Optical rotations were measured with a Jasco DIP-370 polarimeter. Then, 1D and 2D spectra were obtained using a Bruker DRX 600 NMR spectrometer (Bruker Daltonics Inc., Billerica, MA, USA), using TMS as an internal standard. Mass analysis (HRMS) was performed with an Agilent 6545 Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA).