Ceramide quantification by LC-MS/MS

Sample solutions (0.8 mL) were transferred into a screw-capped glass test tube and mixed with chloroform (2 mL), methanol (1 mL), and 0.25 nmol of internal standards (C17 ceramide, C12 GlcCer, C17 SM), and test tubes were incubated at 48°C for 2h in shakers. After incubation, 10 M KOH (0.15 mL) was added and mixed by vortexing, and test tubes were incubated at 37°C for 2 h in shakers. Chloroform (1 mL) and water (1 mL) were added, test tubes were centrifuged at 1000 × g for 5 min, and the lower phases were collected and placed in fresh test tubes. Chloroform (2 mL) was added to the upper phases, mixed by vortexing, test tubes were centrifuged at 1000 × g for 5 min, and the lower phases were re-collected, placed in fresh test tubes, dried under a nitrogen stream, and dissolved in 100 μL of acetonitrile: methanol (19:1, v:v) for analysis by LC-MS/MS.

LC-MS/MS analysis was carried out using a Triple TOF 5600 system (AB SCIEX, Foster City, CA, USA) in electrospray ionization (ESI)-positive mode. A 10 μL volume of sample solution was injected onto an InertSustain NH2 hydrophilic interaction chromatography column (particle size 5 μm; diameter 2.1 mm; length 100 mm; GL Science, Tokyo, Japan). Solvent A was acetonitrile: methanol: formic acid (95:5:0.2, v:v:v) containing 5 mM ammonium formate, while solvent B was methanol: formic acid (100:0.2, v:v) containing 5 mM ammonium formate. Samples were eluted at 0.2 mL/ min over a 45 min gradient (0−5 min 0% B, 5−10 min from 0% to 20% B, 10−12 min 20% B, 12–15 min from 20% to 50% B, 15−22 min 50% B, 22−27 min from 50% to 80% B, 27−30 min, 30–45 min from 80% to 0% B).