Uplc beh amide

Liquid chromatography‒mass spectrometry (LC‒MS) (Novogene, China) was utilized for mass spectrometry analysis. Cell samples were ground and mixed with an extraction solution, then centrifuged to collect the supernatant, which was dried using a nitrogen blower. The dried sample was reconstituted in a mixed solvent of acetonitrile and water, followed by sonication and centrifugation in an ice-water bath. Mass spectrometric analysis utilized a Waters UPLC BEH Amide chromatographic column (55°C) with a 5 μL injection volume. Electrospray ionization (ESI) source conditions included an ion source temperature of 600°C and a voltage set at -4500V or 5500V. Multiple reaction monitoring (MRM) was employed for scanning. Data processing included peak alignment, retention time correction, and peak area extraction using SCIEX OS software. Metabolite structures were matched utilizing primary and secondary mass spectra and database retrieval, followed by standardization and statistical analysis of the acquired data.

Samples were extracted from the cell pellets and separated using two types of columns. A UPLC BEH Amide 2.1 × 100 mm, 1.7 µm analytic column (Waters, Eschborn Germany) with a 400 µL min⁻¹ flow rate for hydrophilic interaction liquid chromatography (HILIC) and a Kinetex XB18 2.1 ×100 mm, 1.7 µm (Phenomenex, Aschaffenburg Germany) for reverse phase chromatography (RP) with a 300 µL min⁻¹ flow rate. A volume of 5 µL per sample was injected. The autosampler was cooled to 10 °C, and the column oven heated to 40 °C. MS settings in the positive mode were as follows: Gas 1 55 psi, Gas 2 65 psi, Curtain gas 35 psi, temperature 500 °C, Ion Spray Voltage 5500 V, declustering potential 80 V. The mass range of the TOF MS and MS/MS scans were 50–2000 m/z and the collision energy was ramped from 15–55 V. MS settings in the negative mode were as follows: Gas 1 55 psi, Gas 2 65 psi, Cur 35 psi, temperature 500 °C, Ion Spray Voltage –4500 V, declustering potential –80 V. The mass range of the TOF MS and MS/MS scans were 50–2000 m/z and the collision energy was ramped from –15–55 V. The data was collected in the data-dependent-acquisition mode. A more detailed description of the procedure and data analysis can be found in Supplementary Note S16.

UHPLC-QTOF-MS analyses were conducted on the UHPLC system (Agilent 1290, Agilent Technologies, Santa Clara, CA, USA) with UPLC BEH Amide column (1.7 μm, 2.1 mm × 100 mm, Waters, Milford, CT, USA) coupled to the AB Sciex Triple TOF 6600 system (Framingham, MA, USA). The mobile phase was prepared using two solvents. Solvent A consisted of 25 mM of NH₄Ac and 25 mM of NH₄OH in water (pH = 9.75), and solvent B was acetonitrile. Gradient elution was carried out as follows: 0 min, 95% B; 0.5 min, 95% B; 7 min, 65% B; 8 min, 40% B; 9 min, 40% B; 9.1 min, 95% B; and 12 min, 95% B. The injection volume for all samples was 1 μL, and the flow rate of the mobile phase was 0.5 mL min⁻¹. The Triple TOF mass spectrometer assisted by information-dependent acquisition method was used to collect MS/MS spectra. The MS data were acquired via Analyst TF 1.7 software (AB Sciex, Framingham, MA, USA). During data collection, 12 precursor ions with an intensity of more than 100 were selected for fragmentation at a collision energy of 30 V (15 MS/MS events at an ion accumulation time of 50 ms each). The instrumental parameters of the electrospray ionization source were set as follows: ion source gas 1 = 60 Psi; ion source gas 2 = 60 psi; curtain gas = 35 psi; source temperature = 600 °C; and ion spray voltage floating = 5000 V and −4000 V for positive and negative modes, respectively.