Development and validation of LC-MS/MS method for determination of very long acyl chain (C22:0 and C24:0) ceramides in human plasma

Ceramide is a key metabolite in both anabolic and catabolic pathways of sphingolipids. The very long fatty acyl chain ceramides N -(docosanoyl)-sphing-4-enine (Cer(22:0)) and N -(tetracosanoyl)-sphing-4-enine (Cer(24:0)) are associated with multiple biological functions. Elevated levels of these sphingolipids in tissues and in the circulation have been associated with insulin resistance and diabetes. To facilitate quantification of these very long chain ceramides in clinical samples from human subjects, we have developed a sensitive, accurate, and high-throughput assay for determination of Cer(22:0) and Cer(24:0) in human plasma. Cer(22:0) and Cer(24:0) and their deuterated internal standards were extracted by protein precipitation and chromatographically separated by HPLC. The analytes and their internal standards were ionized using positive-ion electrospray mass spectrometry, then detected by multiple-reaction monitoring with a tandem mass spectrometer. Total liquid chromatography–tandem mass spectrometry (LC-MS/MS) runtime was 5 min. The assay exhibited a linear dynamic range of 0.02–4 and 0.08–16 μg/ml for Cer(22:0) and Cer(24:0), respectively, in human plasma with corresponding absolute recoveries from plasma at 109 and 114 %, respectively. The lower limit of quantifications were 0.02 and 0.08 μg/ml for Cer(22:0) and Cer(24:0), respectively. Acceptable precision and accuracy were obtained for concentrations over the calibration curve ranges. With the semi-automated format and short LC runtime for the assay, a throughput of ∼200 samples/day can easily be achieved. Figure LC-MS/MS chromatograms for Cer(22:0) and Cer(24:0) in LLOQ, in which the analyte and internal standard are shown in blue and red, respectively