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).