Open metabolic profiling by UPLC/QTof-MS

Mice were treated with cefoperazone 0.5 mg/mL for one, three, or five days before the contents from the small intestines were collected, immediately snap frozen, and stored at -80°C for future analysis.

To prepare samples for LC/MS analysis, intestinal contents (~250 mg) were mixed with extraction solvent (1:1 MeOH: water) at a ratio of 1:5 (w/v). The mixture was vortexed for 2 min, followed by 10 min sonication. After centrifugation at 13,000 rpm for 15 min at 4 °C, the supernatant was then transferred to autosampler vials for analysis.

A 5 μL aliquot of the extracted supernatant was introduced into a Waters Acquity Ultra Performance Liquid Chromatography (UPLC) system (Waters, Milford, MA) equipped with a Waters bridged ethyl hybrid (BEH) C8 column with a dimension of 2.1 mm × 10 cm and 1.7 μm particle size. The column was held at 40°C. The UPLC mobile phase consisted of 0.1% formic acid in water (solution A) and 0.1% formic acid in acetonitrile (solution B). While maintaining a constant flow rate of 0.4 mL/min, the plasma metabolites were eluted using linear gradients of 2–80% solution B from 0 to 15 min, and 80–98% solution B from 15 to 17 min. The final gradient composition was held constant for 2 min, followed by a return to 2% solution B at 19.1 min. Mass spectrometric data were collected with a Waters QTof Premier mass spectrometer (Waters, Milford, MA) operated in positive and negative ionization electrospray modes, as reported previously. Briefly, MS^E analysis was performed on a QTof mass spectrometer set up with 5 eV for low collision energy and a ramp collision energy ranging from 20 to 30 eV. Full scan mode from m/z 80 to 1000, and from 0 to 22 min was used for data collection. The cefoperazone intensity was obtained using Micromass MarkerLynx XS Application Version 4.1 (Waters, Milford, MA) with extended statistical tools and normalized to the total intensity of each run.