Characterisation of Compounds by High Performance Liquid Chromatography-Mass Spectrometry

EA extract was subjected to LC-MS analysis using the Bruker Amazon ion trap mass spectrometer equipped with a Dionex ultimate 3000 RS Diode array detector (Billerica, MA, USA). The heated capillary and spray voltage were maintained at 250°C and 4.5 kV, respectively. Additionally, nitrogen was operated at 80 psi for sheath gas flow rate and at 20 psi for the auxiliary gas flow rate. The MS³ collision gas used was helium with a collision energy of 30% of the 5 V end-cap maximum tickling voltage. The mass spectra were scanned from m/z 100-2,000 in both the positive and negative ion modes with a scan speed at one scan per second. Each fraction was dissolved in methanol at 1 mg/ml and filtered through a 0.45 μl RC membrane. During the HPLC run, 1 μl of fraction was injected into the Develosil diol column (4.6 x 250 mm, Batch: 250612). The mobile phase consists of solvent A, 2% acetic acid in acetonitrile and solvent B, acidic aqueous methanol (CH₃OH: H₂O: HOAc, 95:3:2 v/v/v). Elution programme started with 7% B for 3 min, ramping up to 37.6% at 60 min, 100% at 63 min and holding for 7 min at a flow rate at 1.0 ml/min at 25°C.