BA metabolomics analysis by LC-MS

Human stool samples (~400 mg) were weighed into 7 mL bead tubes containing 1.4 mm ceramic beads (Omni International) and extracted using 80% methanol containing deuterated labeled internal standards [ISTDS: chenodeoxycholic acid–d4 (CDCA-d4), taurochenodeoxycholic acid-d4 (T-CDCA-d4), glycochenodeoxycholic acid-d4 (G-CDCA-d4), cholic acid-d4 (CA-d4), taurocholic acid-d4 (T-CA-d4), glycocholic acid-d4 (G-CA-d4), deoxycholic acid-d4 (DCA-d4), taurodeoxycholic acid-d4 (T-DCA-d4), glycodeoxycholic acid-d4 (G-DCA-d4), lithocholic acid-d4 (LCA-d4), tauro-lithocholic acid (T-LCA-d4), glycolithocholic acid-d4 (G-LCA-d4), ursodeoxycholic acid-d4 (UDCA-d4), T-UDCA-d4, and β-muricholic acid-d5 (β-MCA-d5)] for a final concentration of 100 mg/m. The ISTDs were purchased from Cambridge Isotope Laboratories (Tewksbury, MA); except β-MCA-d5 that was purchased from Isosciences (Ambler, PA). Samples were homogenized using a Bead Ruptor (Omni International, Kennesaw, GA) at 6 m/s for 6 cycles of 30 s at 4°C, then stored at −80°C overnight. Stool extracts were then vortexed, transferred to 5 mL Eppendorf tubes, centrifuged at 20,000 × g for 20 min at 4°C and diluted with Milli-Q water to 50% methanol content. Samples were then filtered using 96-well Acroprep Advance plates (Pall Corporation, Port Washington, NY) and analyzed by LC-MS analysis BA were separated using two chromatographic methods: 1.) Agilent 1290 Infinity LC system coupled to an Agilent 6550 iFunnel Q-TOF with a 1.6 μm, 2.1 × 50 mm CORTECS T3 column (Waters) and 2.) Agilent 1290 Infinity II LC system coupled to an Agilent 6546 Q-TOF with a 1.7 μm, 2.1 × 150 mm ACQUITY UPLC BEH Shield RP18 column (Waters). Active reference mass correction was used according to the manufacturer’s instructions. Mobile phase A was 0.1% formic acid in water, mobile phase B was 0.1% formic acid in acetone. LC gradient for Method 1: 30% B to 65% B over 14 min (flow rate: 0.4 mL/min) and for Method 2: 30% B to 80% B over 30 min (flow rate: 0.3 mL/min). Both methods ended with 100% B for 2 min and re-equilibration for 6 min. Acquisition was from m/z 50–1,700 at 2 Hz. Mass spectrometer source conditions for both methods: gas temperature: 240°C; drying gas flow: 10 L/min; nebulizer: 60 psi; sheath gas temperature and flow: 400°C and 10 L/min, respectively. The voltages used were: fragmentor: 175 V; skimmer: 45 V; capillary: 4000 V; and nozzle: 500 V. Sample injection volume was 5 μL, and data was collected in negative ionization mode. A pooled sample was injected regularly throughout the batch to monitor instrument performance. Data files were converted to Agilent SureMass format and analyzed in Agilent MassHunter Quantitative Analysis software (version 10.1, Agilent Technologies, Santa Clara, CA). Concentrations were estimated by referencing peak areas to specified ISTDs of known concentration, except for sulfated-BAs and 3-oxo-LCA, which used an external calibration curve.