Simplified fluorometric assay for sphingosine bases.

Abstract

A simplified procedure for fluorometric determination of sphingosine bases is presented. Nitrogenous bases are reacted with fluorescamine and then extracted into a lower phase solvent, obviating the need to transfer the reaction products prior to quantitation. Consequently, the entire procedure may be carried out in a single set of screw-capped reaction tubes. Chloroform was found to be the best solvent of those tested for the maximal extraction of derivatized sphingosine bases with negligible extraction of nitrogenous contaminants derived from phospholipids and amino sugars. The simplified method retains excellent sensitivity and selectivity and allows for simultaneous processing of large numbers of samples. Furthermore, quantitation of glycolipid-derived hexosamine can be obtained by measuring the fluorescence of the aqueous phase after partitioning.