Xevo g2 tof ms

Each sample (2 μl) was injected onto a Waters Acquity Ultra-performance liquid chromatography (UPLC) system in a randomized order with a pooled quality control (QC) injection after every six sample injections and separated using a Waters Acquity UPLC ethylene bridged hybrid Amide column (1.7 μM, 2.1 by 30 mm) at a constant flow rate of 0.4 μl min -1 . The elution solvents were A, acetonitrile with 0.1% (v/v) formic acid; B, water with 10 mM ammonium formate and 0.1% formic acid. The solvent gradient started with 0.1% B at 0 min, increased to 80% B at 1.5 min, held at 80% B until 2.0 min, reequilibrated to 0.1% B at 2.25 min, held at 0.1% B for 2.5 min. The total run time was 4.75 min. The column and samples were held at 45 and 4 ˚C, respectively. The column eluent was infused into a Waters Xevo G2 TOF-MS with an electrospray source in positive mass spectrometry (MS) fullscan mode with target enhancement at 133 m/z. The dwell time was 0.2 s. The collision energy was set at 0. Calibration was performed using sodium iodide with 0.001 mg ml -1 mass accuracy. The capillary voltage was held at 2,200 V, source temperature at 150 ˚C, and N desolvation temperature at 350 ˚C with a flow rate of 800 L h -1 . The ions used for quantification are listed in Table 1.

Extracts (0.5 µL, comprising 175 µg equivalent of faeces) were injected onto a Waters nanoAcquity UHPLC and separated using a Waters nanoAcquity HSS-T3 (100mm x 100 µm x 1.8 µm, 100 Å) column. Chromatographic separation was carried out at 700 nL/min using UHPLC grade (>99.99% purity) water and acetonitrile as mobile phases A and B respectively, both modified with 0.01% formic acid. A gradient elution was used: 0 mins 10% B, 4 mins 30% B, 18 mins 50% B, 30 mins 100% B, 100% B maintained for 10 minutes then equilibrated in initial conditions for a further 15 minutes. Metabolites were detected in positive and negative nESI modes using a Waters Xevo G2
TOFMS tuned to a mass resolution of 20,000 and equipped with a nano ESI source using homemade pulled fused silica emitters. [26] A sample from each group and a QC sample was also analysed using MS E for collisional-induced dissociation with an energy ramp of 10-40 eV to obtain fragment information for identification of metabolite structures.