Thermo orbitrap qexactive

The analysis was performed on a Dionex UltiMate 3000 RSLC system (Thermo Fisher Scientific, Germering, Germany) coupled to a Thermo Orbitrap Q Exactive (Thermo Fisher Scientific, Bremen, Germany). The metabolites were separated using hydrophilic interaction liquid chromatography (HILIC) with a ZIC-pHILIC column (150 mm × 4.6 mm, 5 μm column, Merck Sequant, Darmstadt, Germany). The column was maintained at 25°C, and samples were eluted with a linear gradient (A: 20 mM ammonium carbonate in water; B: 100% acetonitrile) from 80% A and 20% B to 95% A and 5% B. The flow rate was 0.3 ml/min. Orbitrap Q Exactive instrument was operated in positive and negative mode at mass resolution of 70,000 and the full scan of m/z range 70–1,050. The source voltage was +3.8 kV for the positive mode and −3.8 kV for the negative mode, sheath gas 40 (arbitrary units), auxiliary gas 5 (arbitrary units), and capillary temperature of 320°C. A standard mix (kindly provided by Glasgow Polyomics, United Kingdom) consisted of about 150 compounds (reference compounds for metabolite identification) and was run together with the samples. The quality control samples were extracts obtained from beer and human urine (kindly provided by Glasgow Polyomics, United Kingdom) that were used to check the signal reproducibility and the quality of the chromatography.

The experiments were run at different time points (Table 1) using the same LC-MS platform: Thermo Orbitrap QExactive (Thermo Fisher Scientific) mass spectrometer coupled with a Dionex UltiMate 3000 RSLC system (Thermo Fisher Scientific, Hemel Hempstead, UK) using a ZIC-pHILIC column (150 mm × 4.6 mm, 5 μm column, Merck SeQuant). While the same flow rate was used for all three datasets, the length of the run differed for D_VL, which lasted longer than the other two datasets. Data from both positive (+ve) and negative (−ve) electrospray ionisation (ESI) mode was obtained.