Identification of Compounds in Subfraction

The identification of the compounds in IV and VI sub-fractions was obtained by liquid chromatography mass spectrometry (LC-MS/MS) on a Q-Tof Premier mass spectrometer (Waters Corporation, Milford, MA, USA) coupled to an Alliance 2695 HPLC system (Waters Corporation, Milford, MA, USA) using the same parameters of a validated method previously described and employed for A. glabra samples [10 (link)]. In particular, a negative ionization mode was used to acquire the electrospray mass spectra data for a mass range m/z 100 to m/z 1000 in according to the present literature [67 (link),68 (link)]. An Atlantis T3 C18 column was used (Waters Corporation, Milford, USA, 100.00 × 2.10 mm; 3.00 µm particle size) at 40 °C. The mobile phases were 0.10% aqueous formic acid (solvent A) and 0.10% formic acid in acetonitrile (solvent B). The stepwise gradient from 10% to 90% solvent B was applied at flow rate of 300 µL/min for 25 min. Cone voltage and capillary voltage were set at 30 V and 3 kV, respectively. Argon was used as collision gas and the collision induced fragmentation (CID) of the analytes was achieved using 12 to 30 eV energy. The injection volume for all the samples was 3.00 µL.