Agilent 6520 quadrupole time of flight mass spectrometer

LC-MS analyses were performed on an 1200 HPLC system coupled to an Agilent 6520 Quadrupole-Time of Flight (Q-TOF) mass spectrometer (Agilent Technologies, USA). A Zorbax Eclipse Plus C18 column with dimensions of 150 mm × 2.1 mm and 5.0 μm particle size was used. The temperature of column was set at 30 °C. A gradient elution was applied using 0.1% formic acid in water as solvent (A) and 0.1% formic acid in acetonitrile as solvent (B). The following solvent gradient was applied: from 2% B to 12% B within 2 min, to 15% B within 6 min, to 60% B within 9 min, to 100% B within 9 min, to 2% B within 1 min, and hold for 5 min. The flow rate was set at 0.25 mL min⁻¹ and 1–5 μL of samples were injected. Mass resolution was set to 10 000. The electrospray and fragmentor voltages were set at 3500 and 120 V, respectively. The gas temperature was maintained at 350 °C. The drying gas (nitrogen) flow rate and nebulizer gas (nitrogen) pressure were 8 L min⁻¹ and 30 psi, respectively. MS/MS product ion scans were carried out at a collision energy of 5–45 V. Ultra-high-purity nitrogen was used as the collision gas. The scan was performed in positive mode in the m/z range 25–1000 with the scan time of 0.77 second.