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

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 <20min. Method validation results indicated that calibration curves were linear in the range of 2.5-10,000nM for ceramides and glucosylceramides, 10-10,000nM for dihydroceramides, 5-10,000nM for sphingosine, sphingosine-1-phosphate, sphinganine and sphinganine-1-phosphate, respectively. The limits of detection ranged from 0.5nM to 5nM. 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.