Untargeted Metabolomics Analysis using UPLC-QTOF

Chromatograms were acquired with a UPLC I-Class System coupled to a Xevo G2-XS QToF Mass Spectrometer (Waters®, Milford, MA, USA) using a modified protocol described previously [73 (link)]. Samples were injected at a concentration of 0.1 mg/mL with an injection volume of 1 µL. The separation was performed using a HSS T3 column, at 40 °C, and at a flow rate of 0.6 mL/min. A gradient of H₂O + 0.1% formic acid (FA) for the mobile phase A, and MeCN + 0.1% FA, for the mobile phase B. The solvent system was as follows: from 99:1 to 0:100 in 11.5 min followed by washing and reconditioning of the column over 3.5 min. The mass range was set from m/z 190 to 1200, the capillary voltage was 3.0 kV, the cone and the desolvation gas flow were at 50 and 1200 L/h, respectively. The source temperature was 150 °C and the desolvation temperature 550 °C, and the sampling cone and source offset were set at 40 V and 80 V, respectively. Data-dependent acquisition (DDA) mode was used to select the five most intense precursor ions of each MS spectrum (excluding the peaks corresponding to the isotopic contribution of the ¹³C). Those ions were then fragmented using a collision energy (CE) ramp: low CE from 15–25 eV to high CE of 40–60 eV. Solvent (MeOH) and non-inoculated media extracts were injected under the same conditions and used as controls.