UPLC-ESI-TOF-MS Analysis of Metabolites

Ultra performance liquid chromatography (UPLC) with time-of-flight mass spectrometry (ESI-TOF-MS) was performed at our outsourcing facility by a well-established method [58 (link),59 (link)]. In brief, the sample separation was performed using UltiMate 3000LC combined with Q Exactive mass spectrometry (Thermo) followed by screening with ESI-MS. The LC system is a combination of a two-system unit with Thermo hyper gold C18 (100×2.1mm 1.9 μm) combined with the UltiMate 3000LC system. The mobile phase comprises two solvents—solvent A and solvent B. Solvent A comprises 0.1% formic acid, 5% acetonitrile, and water, and solvent B is a mixture of 0.1% formic acid and acetonitrile with a gradient elution of 0–1.5 min, 0–20% B; 1.5–9.5 min, 20–100% B; 9.5–14.5 min, 100% B; 14.5–14.6 min, 100–0% B; 14.6–18.0 min, 0% B. The flow rate for the mobile phase was fixed at 0.3 mL/min. The column temperature was maintained at 40°C, and the sample manager temperature was set at 4 °C. Mass spectrometry parameters in ESI+ and ESI- modes were set. For positive ion mode (ESI+), the experimental parameters were the following: heater temperature set at 300 °C, flow rate of the sheath gas set at 45 arb, auxiliary gas flow rate set at 15 arb, sweep gas flow rate set at 1 arb with a spray voltage of 3.0 kV, capillary temperature set at 350 °C, and the S-Lens RF level adjusted to 30%.