Extraction of liver lipids was performed according to the method described by Folch et al.30 (link). Dihydroceramides and ceramides and were isolated by solid phase extraction chromatography using C12:0 dihydroceramide and C17:0 ceramide (Avanti Polar Lipids, Alabaster, Al, USA) as internal standards. Samples were analysed by liquid chromatography-mass spectrometry (LC–MS) using a Thermo Exactive Orbitrap mass spectrometer (Thermo Scientific, Hemel Hempsted, UK) equipped with a heated electrospray ionization (HESI) probe and coupled to a Thermo Accela 1250 ultra-high-pressure liquid chromatography (UHPLC) system. Samples were injected onto a Thermo Hypersil Gold C18 column (2.1 mm by 100 mm; 1.9 μm) maintained at 50 °C. Mobile phase A consisted of water containing 10 mM ammonium formate and 0.1% (vol/vol) formic acid. Mobile phase B consisted of a 90:10 mixture of isopropanol-acetonitrile containing 10 mM ammonium formate and 0.1% (vol/vol) formic acid. The initial conditions for analysis were 65% mobile phase A, 35% mobile phase B and the percentage of mobile phase B was increased from 35 to 65% over 4 min, followed by 65% to 100% over 15 min, with a hold for 2 min before reequilibration to the starting conditions over 6 min. The flow rate was 400 μl/min and samples were analyzed in positive ion mode. The LC–MS data were processed with Thermo Xcalibur v2.1 (Thermo Scientific) with signals corresponding to the accurate mass-to-charge ratio (m/z) values for dihydroceramide and ceramide molecular species extracted from raw data sets with the mass error set to 5 ppm. Quantification was achieved by relating the peak area of the dihydroceramide and ceramide lipid species to the peak area of their respective internal standard. All values were normalised to the wet weight of liver.
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