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.