HILIC-MS Metabolome Profiling Protocol

The LC–MS method involved hydrophilic interaction chromatography (HILIC) coupled with negative mode electrospray ionization to the Q Exactive PLUS hybrid quadrupole-orbitrap mass spectrometer (Thermo Scientific). The LC separation was performed on a XBridge BEH Amide column (150 mm × 2.1 mm, 2.5 μm particle size, Waters, Milford, MA) using a gradient of solvent A (95%:5% H₂O:Acetonitrile with 20 mM Ammonium Bicarbonate), and solvent B (100% Acetonitrile). The gradient was 0 min, 85% B; 2 min, 85% B; 3 min, 80% B; 5 min, 80% B; 6 min, 75% B; 7 min, 75% B; 8 min, 70% B; 9 min, 70% B; 10 min, 50% B; 12 min, 50% B; 13 min, 25% B; 16 min, 25% B; 18 min, 0% B; 23 min, 0% B; 24 min, 85% B; 30 min, 85% B. The flow rate was 150 μl min⁻¹. Injection volume was 5 μL and column temperature 25 °C. The MS scans were in negative ion mode with a resolution of 140,000 at m/z 200 unless specified otherwise. The automatic gain control (AGC) target was 5e5 unless specified otherwise. The maximum injection time was 30 ms. Scan range was 75–1000 unless specified otherwise.