LC-MS Metabolite Separation Protocol

The samples were maintained at 10 °C in the auto sampler, and the column was thermostated to 25 °C. The reversed-phase separation was performed on a C18 column (3.5 µm; 2.1 × 150 mm, XBridge®, Waters, Milford, MA, USA) coupled to a C18 precolumn (4 × 3 mm, Phenomenex, Torrance, CA, USA). The injection volume was 2 µl. The eluent system consisted of (A) H₂O + 0.1 % FA and (B) MeOH + 0.1 % FA that was passed through the column at a flow rate of 0.25 ml/min with a chromatographic gradient according to [1 (link)]. Here, a linear gradient was utilized. The initial mobile phase composition was 10% of eluent B, which was held constant for 2 min. In the next 30 min, eluent B was increased linearly to 100% and maintained at 100% for another 4 min. Lastly, the column was recalibrated for 8 min with 10% eluent B.