Quantitative analysis of brain galactosylceramides by high performance liquid chromatography of their perbenzoyl...

A high performance liquid chromatographic (HPLC) method for analysis of galactosylceramides as their benzyol derivates has been devised. Samples containing 10-150 nmoles of monohexoslceramides are benzoylated by heating for 60 min at 60 degree C in 0.5 ml of 10% (v/v) Benzoyl chloride in pyridine. The products are purified by solvent distribution and analyzed by SPLC. The benzoylated cerebrosides with nonhydroxy fatty acids are separated from those with hydroxy fatty acids on a Zipax column with 7% ethyl acetate in hexane as a solvent and UV absorption at 280 nm is recorded. This isocratic procedure can be applied directly to chloroform-methanol procedure can be applied directly to chloroform-methanol extracts of adult brain with a relative standard deviation of 3.0% for cerebrosides with nonhydroxy fatty acids and 4.0% for cerebrosides with hydroxy fatty acids. Sulfatides do not interfere in the assay and can be converted to cerebrosides after desulfation by mild acid methanolysis. Benzoylated glucosyl- and galactosylceramides can be separated on a MicroPak NH2 column with 1.5% 2-propanol in cyclopentane as the chromatographic solvent.