Quantification of p-Cresol in fecal samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS)

Mice were individually placed in a clean empty plastic cage and feces were collected over 10 min and immediately snapped-frozen in liquid nitrogen and stored at − 80 °C until analysis. Thawed fecal pellets were extracted as previously described in [63]. Briefly fecal pellets were homogenized in ice-cold water (1:1 w:v ratio), vortexed 30 s, ultrasonicated for 2 min, then subjected to ultracentrifugation (175,000×g, 30 min, 4 °C). For the determination of unconjugated p-Cresol by LC-MS/MS, fecal supernatant preparation included addition of an internal standard (1 ng/mL of p-Cresol-d8, Eurisotop, St-Aubin, France) and derivatization with dansyl chloride according to [64]. Dansylation was shown to improve signal intensity in LC-MS/MS analysis of low-abundance phenolic compounds [65]. Briefly, 100 μL of fecal supernatant were mixed with 300 μL of an acetonitrile solution vortexed, incubated at − 20 °C for 20 min and centrifuged (12,000×g, 7 min, 4 °C). Two hundred microliters of the resulting supernatant were mixed with 50 μL of 0.1 M carbonate-bicarbonate solution (pH = 10), 125 μL of water, and 125 μL of dansyl chloride at a final concentration of 0.5 mg/mL. After vortexing, the mixture was incubated for 10 min at 60 °C and extracted with 2.5 mL of hexane. Hexane residues were air-dried and reconstituted with 1 mL of acetonitrile-water (1:1, v/v). Fifteen microliters of sample were injected and analyzed using a Waters ACQUITY ultraperformance liquid chromatography (UPLC) system equipped with a binary solvent delivery manager and sample manager (Waters Corporation, Milford, MA, USA) and coupled to a tandem quadrupole-time-of-flight (Q-TOF) mass spectrometer equipped with an electrospray interface (Waters Corporation). Quantifications were performed by referencing calibration curves obtained with internal standards. Unconjugated p-Cresol was identified by comparing with the accurate mass and the retention time of the reference standard (p-Cresol, Sigma-Aldrich) in our in-house library.