Sphingolipid Profiling by HPLC-MS/MS

Sphingolipid mass spectrometry analysis was carried out as previously reported (90 (link)). Briefly, a Thermo Accela high-performance liquid chromatography (HPLC) system (San Jose, CA) was used to separate the dried extracts dissolved in 150 μL of ammonium formate (1 mM) with 0.2% of formic acid in methanol. A Peeke Scientific Spectra C8 (Redwood City, CA) HPLC column (150 × 3 mm) was used, into which 10 μL of samples were injected. The HPLC was coupled to the HESI source of a Thermo TSQ Quantum Ultra triple quadrupole mass spectrometer (San Jose, CA). The sphingolipid profile was performed using positive ion mode, with the high voltage set to 3.5 kV, vaporizer temperature at 400°C, sheath gas pressure at 60, auxiliary gas pressure at 15, and a capillary temperature of 300°C. The collision cell was operated at 1.5 mTorr of argon. For the duration of the run, transitions for each lipid species were monitored at 100- or 50-ms dwell time. A total of 20 lipid standards for our profile from Avanti (Alabaster, AL) were used to develop calibration curves, and these curves were then used for lipid species to be monitored. Processing of the samples was done using Thermo Xcalibur 2.2 Quan browser software and exported to Excel for reporting results. Sphingolipid concentration determined by mass spectrometry was further normalized by the P_i abundance (91 (link)).