Determination of less than a nanomol of cerebrosides by high performance liquid chromatography with gradient elution analysis

A gradient elution high performance liquid chromatography method with detection at 230 nm for the analysis of perbenzoylated cerebrosides containing hydroxy and nonhydroxy fatty acids is described. The quantitative range of the method is 0.5-10 nmol of cerebrosides. The limit of detection for injected sample is about 10 picomol. The analysis time by liquid chromatography is less than 5 minutes and prior purification of the cerebrosides from other lipids in brain lipid extracts is not necessary. The cerebrosides are first perbenzoylated with 50 micronl of 10% benzoyl chloride in pyridine and then separated on a chromatographic column of Zipax. The gradient elution is with 2.8-5.5% of dioxane in hexane. This gradient elution micromethod is at least 10 times more sensitive than isocratic elution methods with detection at 280 nm. The method is applicable to other biological materials containing minute amounts of cerebrosides if the glycolipid fraction is first isolated from the lipid extracts. A further fourfold increase in the sensitivity is achieved by replacing air in the reference cell of the detector by gradient elution solvent.