An LC/MS/MS method for the simultaneous determination of individual sphingolipid species in B cells

• An LC-ESI–MS/MS method was developed to investigate the profile of sphingolipids.
• The extraction method was modified to ensure the high recovery of all sphingolipids.
• 33 molecular species were simultaneously analyzed in a single chromatographic run.
• The distribution of sphingolipids were different in healthy and cancerous cells.
• The method is easily adaptable to other biological sample types.

Comprehensive profiling of sphingolipids is of great importance for clinical and pharmaceutical studies. An LC/MS/MS method was established for the simultaneous separation and quantification of individual sphingolipid species including ceramides, dihydroceramides, glucosylceramides, sphingosine, sphingosine-1-phosphate, sphinganine and sphinganine-1-phosphate. All target individual sphingolipid species were separated and quantified in a single chromatographic run of <20 min. Method validation results indicated that calibration curves were linear in the range of 2.5–10,000 nM for ceramides and glucosylceramides, 10–10,000 nM for dihydroceramides, 5–10,000 nM for sphingosine, sphingosine-1-phosphate, sphinganine and sphinganine-1-phosphate, respectively. The limits of detection ranged from 0.5 nM to 5 nM. Accuracies of 92.5–113% with precisions of 0.3–8.0% RSD were obtained for all of the standards over a wide range of concentrations. The application of this method was demonstrated using B cells collected from Chronic Lymphocytic Leukemia patients (n = 5) and healthy donors (n = 4). 17 sphingolipid species were successfully characterized and quantified in the lipid extract. This is a rapid method that could be readily adapted to lipidomic investigations of sphingolipids in other bio-fluids and tissues.