4.4. UPLC-QToF-MS/MS Analysis

An ACQUITY UPLC I-Class System coupled to the Xevo G2-XS QToF Mass Spectrometer (Waters^®, Milford, Massachusetts, USA) was used to analyze the crude MeOH extract, CHCl₃ subextract and C18 subfractions, which was equipped with an electrospray ionization (ESI) source operating with a positive polarity at a mass range of m/z 50–1600 Da. The samples were dissolved in methanol at a concentration of 0.1 mg/mL and filtered through a 0.2 μm PTFE syringe filter (Carl Roth, Karlsruhe, Germany) and then 2 µL of each sample was injected into the system. The separation of compounds was achieved using a binary LC solvent system controlled by MassLynx^® (version 4.1) to analyze MS and MS² data. An Acquity UPLC HSS T3 column (high-strength silica C18, 1.8 µm, 100 × 2.1 mm I.D., Waters^®) was operated at 40 °C and eluted with H₂O + 0.1% FA (A) and MeCN + 0.1% FA (B) ULC/MS grade at a flow rate of 0.6 mL/min with the following gradient: initial, 1%–100% B, 0–12 min; 100% B, 12–13 min and a column reconditioning phase until 15 min. The conditions of ESI were introduced as follows: capillary voltage: 0.8 kV, sample cone voltage: 40.0 V, source temperature: 150 °C, desolvation temperature: 550 °C, cone gas flow: 50 L/h, and desolvation gas flow: 1200 L/h. MS² setting was kept at a collision energy (CE) of 30 eV.