Characterization and direct quantitation of ceramide molecular species from lipid extracts of biological samples by electrospray ionization tandem mass spectrometry.
A rapid, simple, and reliable method has been developed for the characterization and quantitation of ceramide molecular species directly from chloroform extracts of biological samples by electrospray ionization tandem mass spectrometry (ESI/MS/MS). By exploiting the differential fragmentation patterns of deprotonated ceramide ions, individual 2-hydroxy and nonhydroxy ceramide molecular species were readily identified by ESI/MS/MS with the neutral loss of fragments of mass 256.2 and 327.3 which correspond to sphingosine derivatives. The ions generated from the neutral loss of 256.2 (i.e., [M - H - 256.2](-)) are unique for ceramides with N-acyl sphingosine with the 18-carbon homolog. However, the sensitivity for nonhydroxy ceramides in ESI/MS/MS with the neutral loss of 256.2 is approximately threefold higher than that for 2-hydroxy ceramides. The ions resulting from the neutral loss of 327.3 (i.e., [M - H - 327.3](-)) are specific for 2-hydroxy ceramides. Additionally, all ceramides including both 2-hydroxy and nonhydroxy forms can be confirmed and accurately quantitated by ESI/MS/MS with the neutral loss of 240.2 after correction for (13)C isotope factors. This methodology demonstrated a 1000-fold linear dynamic range and a detection limit at the subfemtomole range and was applied to directly quantitate ceramide molecular species in chloroform extracts of biological samples including brain tissues and cell cultures.
Han
Xianlin
X
Division of Bioorganic Chemistry and Molecular Pharmacology, Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
D000818:Animals; D001923:Brain Chemistry; D002518:Ceramides / Q000032:analysis*; D008055:Lipids / Q000737:chemistry* / Q000302:isolation & purification; D051381:Rats; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D002518:Ceramides; D008055:Lipids