Liver (25 mg) was extracted twice with 0.5 mL of pre-cooled isopropanol/water/ethyl acetate (30:10:60, v:v:v) containing 1:1000 Equisplash (Avanti) and 0.1 μM d7-C17-sphingosine. After homogenization (Precellys, Bertin Technologies, Rockville, MD) and centrifugation (Eppendorf, Hamburg, Germany), the supernatant was collected, evaporated to dryness (Thermo Scientific, Waltham, MA) and dissolved in 100 μL of isopropanol/acetonitrile/H2O (45:35:20, v:v:v). After centrifugation, supernatants were transferred to autosampler vials for LC-MS analysis.
Total lipids were analyzed by Vanquish UHPLC system coupled to an Orbitrap Fusion Lumos Tribrid™ mass spectrometer using a H-ESI™ ion source (all Thermo Fisher Scientific) with a Waters (Milford, MA) CSH C18 column (1.0 × 150 mm × 1.7 um particle size) as previously described(Tian et al. 2020 ). LC-MS data were analyzed by the open-source software MS-DIAL(Tsugawa et al. 2015 (link)), identifications were performed using the in-silico lipid library and the spectra was manually checked to confirm assignments. The experiment was processed in technical duplicate and the averages were used for statistical purposes.
Total lipids were analyzed by Vanquish UHPLC system coupled to an Orbitrap Fusion Lumos Tribrid™ mass spectrometer using a H-ESI™ ion source (all Thermo Fisher Scientific) with a Waters (Milford, MA) CSH C18 column (1.0 × 150 mm × 1.7 um particle size) as previously described(Tian et al. 2020 ). LC-MS data were analyzed by the open-source software MS-DIAL(Tsugawa et al. 2015 (link)), identifications were performed using the in-silico lipid library and the spectra was manually checked to confirm assignments. The experiment was processed in technical duplicate and the averages were used for statistical purposes.