Analysis and quantitation of free ceramide containing nonhydroxy and 2-hydroxy fatty acids, and phytosphingosine by high-performance liquid chromatography.

Reaction of ceramides containing nonhydroxy fatty acids with benzoyl chloride in pyridine at 70 degrees C for 1 hr resulted in N-benzoylation to form N,N-acyl,benzoyl derivatives; O-benzoylation also occurred. However with ceramides containing 2-hydroxy fatty acids and phytosphingosine only O-benzoylation occurred even on prolonged treatment. Only O-benzoylation occurred on reaction with benzoic an hydride. However, the benzoylation of ceramides with phytosphingosine could not be achieved with benzoic anhydride and this benzoylation was performed by reaction with benzoyl chloride at 70 degrees C for 4 hr. Because N,N-acyl,benzoyl derivatives of ceramides containing nonhydroxy fatty acids produced by treatment with benzoyl chloride overlap methyl benzoate on high-performance liquid chromatography, benzoic anhydride was preferable for benzoylation of ceramides with nonhydroxy and 2-hydroxy fatty acids. On the other hand, the reaction with benzoyl chloride at 70 degrees C for 4 hr was used for quantitation of benzoylated ceramides containing 2-hydroxy fatty acids and phytosphingosine. 3-(p-Phenylbenzoyl)estrone was used as an internal standard for both reactions and values for ceramides containing 2-hydroxy fatty acids obtained by the two reactions were in good agreement. This procedure was applied to measurement of the ceramide levels in the brain, liver, and kidney of rats during development. The levels of ceramides containing nonhydroxy and 2-hydroxy fatty acids in the brain, liver, and kidney increased to the adult levels and then remained unchanged. Ceramide with phytosphingosine was detected in the liver and kidney, where its concentration gradually increased with age, but it was not found in the brain. The composition of nonhydroxy fatty acids were also analyzed.