LC-MS/MS analysis
Samples were prepared by adding 180 µl acetonitrile (containing 1 nM loperamide as internal standard) to 20 µl mouse plasma or fecal extract. Samples were vortexed for 5 minutes at room temperature and centrifuged at 16,000 x g for 10 minutes at 4°C. After transfer of the supernatant to a glass vial, 8µl was injected into a Thermo Hypersil C18 column (100 x 2.1mm, 1.9um) on a Shimadzu Nexera UHPLC system and the separation of compounds was performed using a gradient elution from 25% to 95% acetonitrile (with 0.1% formic acid) in 5 minutes at a flow rate of 0.2 ml/min and oven temperature of 50°C. The effluent was ionized using positive ion electrospray on a Thermo TSQ Quantiva triple-quadrupole mass spectrometer. The integrated peak areas for these transitions were calculated for each sample using Skyline software (version 21.0, University of Washington).