High-performance liquid chromatography analysis of molecular species of sphingolipid-related long chain bases and long chain base phosphates in Saccharomyces cerevisiae after derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate

The molecular species of dihydrosphingosines and phytosphingosines and their 1-phosphates with carbon chain lengths from 16 to 20 have been tagged with the fluorescent amino group reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. All these derivatives could be resolved by reversed phase HPLC on a CIS column. A convenient one-pot method is described whereby lipid extracts from strains of Saccharomyces cerevisiae containing carbon chain length homologs of sphingolipid long chain bases and their phosphorylated derivatives were directly reacted with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, ester lipids were deacylated, and the reaction mixtures were subjected to liquid chromatography. Five molecular species of both sphingolipid long chain bases and their phosphorylated derivatives are for the first time separated and analyzed. The procedure is quite sensitive, requiring only similar to 10(8) wild-type cells. (C) 2001 Academic Press.