Liquid chromatography-tandem mass spectrometric determination of ceramides and related lipid species in cellular extracts.
A liquid chromatography-electrospray ionization tandem mass spectrometric (LC-MS/MS) method was developed for the simultaneous qualitative and quantitative determination of sphingolipid metabolites such as ceramides, sphingisine, sphinganine, sphingomyelins, and ceramide 1-phosphates in the extracts of human promyelocytic leukemia cells (HL-60). The assay uses C(4) ceramide as an internal standard; simple liquid extraction; a short XTerra MS C(18) (3 microm, 50 mm x2.0 mm) column; a gradient mobile phase of 5mM ammonium formate (pH 4.0)/methanol/tetrahydrofuran (5/2/3-->1/2/7); mass spectrometric detection using electrospray ionization. This LC-MS/MS method allowed the identification of 22 sphingolipid derivatives at pmol levels. In addition, this technique was successfully applied to analyze the changes of the sphingolipids profiles in cancer cells treated with apoptosis inducing agents, C(2) ceramide and H(2)O(2).
Yoo
Hye Hyun
HH
Bioanalysis and Biotransformation Research Center, Korea Institute of Science and Technology, P.O. Box 131, Chungryang, Seoul, South Korea.
Son
Junghyun
J
Kim
Dong-Hyun
DH
D017209:Apoptosis / Q000187:drug effects; D002518:Ceramides / Q000032:analysis*; D002853:Chromatography, Liquid / Q000379:methods*; D018922:HL-60 Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D002518:Ceramides; D006861:Hydrogen Peroxide