LC-MS/MS analysis of small molecules

LC-MS/MS analysis was operated in both positive and negative ion modes with the parameters optimized according to previously published procedures with some modifications (17 (link)). LC-MS/MS analyses were performed using a Vanquish UHPLC system (Thermo Fisher) coupled with an Orbitrap Q Exactive series mass spectrometer (Thermo Fisher). Samples were injected into the Hyperil Gold column (100 × 2.1 mm, 1.9 μm) using a 16-min linear gradient at a flow rate of 0.2 mL/min. The eluents for the positive polarity mode were eluent A (0.1% FA in Water) and eluent B (Methanol). The eluents for the negative polarity mode were eluent A (5 mM ammonium acetate, pH 9.0) and eluent B (Methanol). The solvent gradient was set as follows: 1.5 min, 2% B; 12.0 min, 100% B; 14.0 min, 100% B; 14.1 min, 2% B; 16 min, 2% B. The flow rate was 0.2 mL/min. Q Exactive mass series spectrometer was operated in positive/negative polarity mode with a spray voltage of 3.2 kV, a capillary temperature of 320°C, a sheath gas flow rate of 35 arb, and an aux gas flow rate of 10 arb.