Esi q tof ms ms 6520 instrument

For the metabolomic analysis, an Agilent 1290 LC system coupled to an ESI-Q-TOF MS/MS 6520 instrument (Agilent Technologies, Barcelona, Spain) was used. In all cases, 2 μL of extracted sample were applied onto a reversed-phase column (Zorbax SB-Aq 1.8 μm 2.1 × 50 mm; Agilent Technologies, Barcelona, Spain) equipped with a precolumn (Zorba-SB-C8 Rapid Resolution Cartridge 2.1 × 30 mm 3.5 μm; Agilent Technologies, Barcelona, Spain) with a column temperature of 60 °C. The flow rate was 0.6 mL/min. Solvent A was composed of water containing 0.2% acetic acid, and solvent B was composed of methanol 0.2% acetic acid. The gradient started at 2% B and increased to 98% B in 13 min and held at 98% B for 6 min. Pot-time was set at 5 min. Data were collected in positive electrospray mode, with the TOF operated in full-scan mode at 50–1600 m/z in an extended dynamic range (2 GHz) using N2 as the nebuliser gas (10 L/min, 350 °C). The capillary voltage was 4000 V, with a scan rate of 1.5 scan/s. The ESI source used a separate nebuliser for the continuous, low-level (10 L/min) introduction of reference mass compounds: 121.050873 and 922.009798 (positive ion mode) and 119.036320 and 966.000725 (negative ion mode), which were used for continuous, online mass calibration.