Structure determination of marker metabolites by UHPLC ESI-QTOF-MS

The identity of marker metabolites described by metabolite fingerprinting was confirmed by MS/MS analysis. For that samples were analyzed by LC 1290 Infinity (Agilent Technologies, USA) coupled with an 6540 UHD Accurate-Mass Q-TOF LC MS instrument with Agilent Dual Jet Stream Technology as ESI source (Agilent Technologies, USA). For LC an ACQUITY UPLC HSS T3 column (2.1 x 100 mm, 1.8 μm particle size, Waters Corporation, USA) was used at a temperature of 40°C and a flow rate of 0.5 ml min^-1. The solvent system consists of solvent A (water/formic acid (100/0.1, v/v) and solvent B (acetonitrile/formic acid (100/0.1, v/v). The gradient was comparable as applied for UPLC TOF-MS analysis. The Q-TOF MS instrument was operated with a detection frequency of 2 GHz in the Extended Dynamic Range and the targeted MS/MS mode. The source conditions were: gas temperature: 250°C; drying gas flow: 8 l min^-1; nebulizer pressure: 35 psi; sheath gas temperature: 300°C; sheath gas flow: 8 l min^-1; VCap voltage: 3 kV; nozzle voltage: 200 V; fragmentor voltage: 100 V. Samples were ionized in negative and/or positive ESI mode with collision energy 10–30 eV. Isolation of precursor ions occurred within the narrow isolation width of 1.3 m/z. Data were acquired by Mass Hunter Workstation Acquisition software B.05.01 (Agilent Technologies, USA). Mass Hunter Qualitative Analysis B.06.01 (Agilent Technologies, USA) was used for data analysis.