Analysis of sphingoid bases by reversed-phase high performance liquid chromatography

A reversed-phase high performance liquid chromatography (HPLC) technique was developed for the analysis of sphingoid bases as their biphenylcarbonyl derivatives. The sphingoid bases (0.3-500 nmol) obtained from sphingolipids after hydrolysis were quantitatively derivatized with biphenylcarbonyl chloride at room temperature for 90 min. After removal of excess reagent by partitioning, the derivatized products were quantitatively analyzed on a reversed-phase HPLC column, with detection at 280 nm. A mixture of biphenylcarbonyl C18-5-hydroxysphinganine, C18-erythro and threo sphingenine, and sphinganine, C18-5-O-methyl and 3-O-methyl sphingenine, C20-sphingenine, sphingosyl phosphorylcholine, and psychosine and were well resolved from one another. The method was employed for the analysis of less than nanomole quantities of sphingoid bases from sphingomyelin and cerebrosides.