LC-MS metabolomics analysis and metabolites identification

100 µl of fecal water or urine samples were thawed and filtered using 0.22 µm pore size membrane (Jiménez-Girón et al. 2015). A blank with methanol is prepared. Five microliters of 4 mg/mL ribitol dissolved in MilliQ water was added to every sample as an internal standard to correct for any loss of metabolite during the extraction process. Metabolomic analysis of filtered solution was performed using Agilent 6550 iFunnel Q-TOF LC/MS system (Agilent Technologies, Santa Clara, CA, USA), operated in both positive and negative ion mode. Six times urine and six times fecal sample were run before the actual samples for conditioning of column.2 µl of samples were injected into an Agilent ZORBAX Rapid Resolution HD SB C18 (2.1 × 100 mm, 1.8 μm) maintained at 45 °C. The flow rate was set at a constant 0.4 ml/min and the pressure was 600 bar. The gradient mobile phase was composed of phase A (water containing 0.1% formic acid) and phase B (acetonitrile containing 0.1% formic acid). The gradient started with 95% A from 0 to 1 min and decreased to 5% from 1 min to 13 min, holding at 5% A till 16 min then turned to 95% in next 10 minutes and holding at 95% A for 4 minutes.

The parameters were the following: capillary voltage 3500 V, nozzle voltage 1000 V, skimmer voltage 65 V, drying gas temperature 200, sheath gas temperature 350, fragmentor voltage 175 V, drying gas flow rate 14 l/min, sheath Gas flow rate 11 l/min, nebulizer pressure 35 psi. MS data were recorded across the range of 50− 1700 m/z at 1.5 spectra/s. Each sample was injected and analysed two times.

All raw data extracted and processed using Agilent MassHunter Qualitative Analysis B.07.00 software. A list of peak areas, retention time and mass to charge (m/z) were obtained and metabolites were identified by comparing the data to selected databases, namely, KEGG, HMDB, and METLIN.