UHPLC-QToF-MS metabolite profiling

Metabolites present in the extracts were analyzed using an Agilent 1290 HPLC system (Agilent, US) coupled to a Bruker Impact II HD Q-ToF-LC/MS (Bruker Daltonics GmbH, Germany). Metabolites were separated using a reversed-phase (RP) separation method. In RP mode, medium-polarity and non-polar metabolites were separated using an Eclipse Plus C18 column (50 mm × 2.1 mm ID) (Agilent, US). Chromatographic mobile phases consisted of MilliQ-H₂O + 0.2% formic acid (buffer A), Acetonitrile + 0.2% formic acid (Buffer C). The gradient started with 95% A and 5% C, with an initial gradient of 18 min to 100% C and a holding time of 2 min. Every run was followed by a 5 min washing step cycling from buffer C to buffer A to Isopropanol (buffer D) and back to the starting condition, where the column was equilibrated for another 2 min. Detection was carried out in positive and negative ionization modes with the following parameters: ESI settings: dry gas temperature = 220 °C, dry gas flow = 8.0 l/min, Nebulizer pressure = 2.2 bar, Capillary = 4500 V, end plate Offset = (-)500 V; MS-ToF setting: Funnel1 RF = 150 Vpp, Funnel 2 RF = 200 Vpp, hexapole RF = 50, Quadrupole = 1 eV, (Collision Energy for full scan MS = 7 eV, untargeted MSMS = stepping 30–50 eV); Acquisition Setting: mass range = 50–1300 m/z, Spectra rate = 6.0 Hz spectra/s, 1000 ms/spectrum.