Separation of molecular species of ceramides as benzoyl andp‐nitrobenzoyl derivatives by high performance liquid chromatography

Ceramides were subjected to direct separation and structural analysis by high performance liquid chromatography after derivatization with benzoyl andp‐nitrobenzoyl chloride. The structures of benzoyl andp‐nitrobenzoyl derivatives of a representative ceramide, N‐heptadecanoyl D‐sphingosine, were fully characterized by mass spectrometry. Excellent separation of molecular species of ceramides was achieved with a 10‐μ LiChrosorb RP18 column by isocratic elution. Both the acyl group and the long‐chain base moiety were separated on this reverse‐phase column. The method has been used to determine quantitatively molecular species of nonhydroxy ceramides derived from bovine brain and egg yolk. Comparison of gas liquid chromatographic and high performance liquid chromatographic assays of intact ceramides was made.