A METHOD FOR THE SIMULTANEOUS DETERMINATION OF ALKYLACYLGLYCEROL, DIACYLGLYCEROL, MONOALKYLGLYCEROL, MONOACYLGLYCEROL, AND CHOLESTEROL BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

We describe a method for the quantitative analysis of the individual subclasses (1-O-alkyl and 1-acyl) of diradylglycerols and monoradylglycerols. These lipids, along with cholesterol, were separated from other neutral and polar lipids on silica columns and analyzed by normal-phase high-performance liquid chromatography (HPLC) as their benzoate derivatives. Cholesterylbenzoate, alkylacylglycerolbenzoate, diacylglycerolbenzoate, monoalkylglyceroldibenzoate, and monoacylglyceroldibenzoate eluted from HPLC in five distinct zones. The derivatives of diradylglycerols and monoradylglycerols were further separated within each discrete zone on the basis of the total number of aliphatic carbons at the sn-1 and sn-2 positions. Radiolabeled cholesterol and dihexadecanoylglycerol were used to monitor recovery. Amounts of synthetic alkylacylglycerol, diacylglycerol, monoalkylglycerol, and monoacylglycerol as low as 0.2 nmol per subclass could be accurately quantified. The technique was used to determine the content of diradylglycerol and monoradylglycerol subclasses in Madin-Darby canine kidney and CFTL-12 mast cells. This method should prove useful for the quantitation of lipid second messengers in cultured cells. © 1991.