Accurate mass 6220 tof

A Cogent Diamond Hydride Type C column (MicroSolv) was used for normal phase chromatography on 1200 LC system (Agilent Technologies) coupled to an Accurate Mass 6220 TOF (Agilent Technologies) mass spectrometer fitted with an MultiMode ion source. Metabolite extracts were mixed with solvent A 1:1 and separated using mobile phase of solvent gradient A and B: 0–2 min, 85% B; 3–5 min, 80% B; 6–7 min, 75%; 8–9 min, 70% B; 10–11.1 min, 50% B; 11.1–14:10 min 20% B; 14:10-18:10 5% B; 18:10–19 85% B. Solvent A was acetonitrile with 0.2% acetic acid and solvent B was ddH2O with 0.2% acetic acid. Reference mass solution (G1969-85001, Agilent Technologies) was used for continuous mass axis calibration. Analytical amino acids standards (Fluka A9906) was used for retention time match. Ions were identified based on their accurate mass, retention time and spectral information, yielding errors below five ppm. Spectra were analysed using MassHunter Qualitative Analysis B.07.00 and MassHunter Profinder B.08.00 software. Statistical validation of samples/runs were performed using principal component analysis, using Mass Profiler Professional (B.07.01).

For separation and detection of metabolites, LC-MS analysis was conducted using an Agilent 1200 LC system containing a Cogent Diamond Hydride Type C silica column (150 mm × 2.1 mm; Microsolv Technologies) coupled to an Agilent Accurate Mass 6220 TOF as described (Eoh and Rhee, 2013 (link)). The mobile phase consisted of solvent A (double-distilled H₂O with 0.2% formic acid) and solvent B (ACN with 0.2% formic acid) at a flow rate of 0.4 ml/min with the following gradient: 0–2 min, 85% B; 3–5 min, 80% B; 6–7 min, 75% B; 8–9 min, 70% B; 10–11.1 min, 50% B; 11.1–14 min, 20% B; and 14.1–24 min, 5% B; followed by a 10-min equilibration period at 85% solvent B before injection of the next sample. Dynamic mass axis calibration was accomplished by continuous infusion of a reference mass solution. Electrospray ionization capillary and fragmentor voltages were set at 3,500 V and 135 V, respectively. The nebulizer pressure was set to 40 psig and nitrogen drying gas was maintained at 250°C, set to a flow rate of 10 liter/min. The MS acquisition rate was 1.5 spectra/s and m/z data ranging from 50 to 1,700 was stored. Data were analyzed using Profinder B.08.00 software, and ions were assigned as specific metabolites based on mass accuracy within 5 ppm and retention times within 1 min of those determined for chemical standards.