UHPLC-HRMSn Characterization of Compounds

The UHPLC-HRMSn system consists of a LTQ Orbitrap XL MS with an Accela 1250 binary Pump, a PAL HTC Accela TMO autosampler, an Accela PDA detector (Thermo Fisher Scientific, San Jose, CA), and an Agilent G1316A column compartment (Agilent, Santa Clara, CA). The operating conditions of the electrospray (ESI) source were similar to our previous work [25 (link)] with the differeces were as follows: sheath gas at 70 (arbitrary units), spray voltage at 4.5 kV; capillary temperature, 250 °C; capillary voltage, 50 V; tube lens offset, 120 V. MSⁿ activation parameters used an isolation width of 2.0 amu, max ion injection time of 200 ms, and normalized collision energy at 35%. The UHPLC separation was carried out on a Hypersil Gold C₁₈ column (200 mm × 2.1 mm, 1.9 μm) (Thermo Fisher Scientific, San Jose, CA) with a flow rate of 0.3 mL/min. The mobile phase consisted of a combination of A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile). A gradient elution program was employed as follows: from 5% to 25% B over 0–30.0 min, to 30% B at 40.0min, to 85% B at 60.0 min, to 95 % B at 65.0 min, and then a return to the initial conditions. The column temperature was set at 50 °C.