HPLC-PDA-MSn Analysis of Compounds

HPLC-PDA-MSⁿ mass spectra was performed through a ThermoFinnigan (Thermo Electron Corporation, Austin, TX, USA) LC system coupled with a mass spectrometer (LCQ-Duo ion trap) having an ESI source (ThermoQuest, Thermo Scientific, Waltham, MA, USA) [15 (link)]. The injection process, flow rate, elution solvents, resolution, and negative MS operating parameters were described previously [17 (link)]. In brief, a Zorbax Eclipse XDB-C18, rapid resolution, 150 × 4.6 mm, 3.5 μm column was used (Agilent, Santa Clara, CA, USA). A gradient consisting of water and acetonitrile (ACN), each having 0.1% formic acid, was applied, and ACN was increased from 5% to 30% within 60 min and then to 90% within the next 30 min at a flow rate of 1 mL/min and a 1:1 split before the ESI source [17 (link)].