High-Performance Liquid Chromatography-Tandem Mass Spectrometry (HPLC-MS/MS) Analysis of Ceramides

Quantitation of ceramides was measured using HPLC-MS/MS (Hong et al., 2019). Cells were treated using same condition with Westen blot and PCR experiment. 10 ng of each ceramide: C14, C16, C18, C20, C22, C24, was added to each sample and calibrator as the internal standard solution. Bligh-Dyer method was used to extracted ceramides under acidic conditions. Briefly, 1 N HCl and 2:1 of methanol: chloroform (v/v) was mixed with calibrator and each sample. After being sonicated for 30 s, the samples were mixed with calibrators. Then the samples were stood on the rack for 5 min at room temperature and centrifuged for 5 min at 13,200 rpm. The organic phases were transferred to the new tubes and extracted again with 100% chloroform. After being dried using nitrogen gas, the samples were reconstituted in 100 μl of 100% ethanol. The samples were detected and identified using by HPLC-MS/MS analysis.