OBJECTIVETo establish a high-performance liquid chromatography-tandem mass spectrometry (HPLC-ESI-MS) method for simultaneous quantitative analysis of different ceramide species in cells.

METHODSThe analysis was performed on an Agilent 1290 HPLC system with a ZORBAX Eclipse XDB-C8 (150.0 mm × 2.1 mm, 3.5 μL) column and a temperature of 35 ℃. Methanol with 1 mmol/L ammonium formate and 0.2% formic acid was used as mobile phase A and 100% methanol was used as mobile phase B. And the ceramides were separated by gradient elution at a flow rate of 0.3 mL/min. Electrospray ionization (ESI) with multiple reaction monitoring (MRM) was used in the analysis.

RESULTSFour ceramide species all had a good linear response in the determination ranges (R² ≥ 0.9987). The average recoveries (n = 9) were 99.1%,99.9%,100.5% and 98.2% with RSDs of 5.6%, 5.1%, 4.7% and 5.5%, respectively. In addition, the levels of ceramides in FL cells were relatively stable, while the C24-ceramide had the highest level.

CONCLUSIONThe HPLC-ESI-MS method for simultaneous analysis of ceramides has high accuracy, reproducibility and linearity, which may be used for quantification of ceramide species in cells.