Sensitive method for the analysis of phospholipid subclasses and molecular species as 1-anthroyl derivatives of their diglycerides

Abstract

A sensitive high-performance liquid chromatographic (HPLC) method for the separation and quantitation of phospholipid subclasses and molecular species has been developed. Phospholipids for analysis are hydrolyzed to the diradyl glycerols (DGs) with phospholipase C and the resulting DGs reacted with a molar excess of 1-anthroyl nitrile in the presence of quinuclidine or 4-dimethylaminopyridine to form a stable adduct. The anthroyl-DGs were separated into alkenylacyl, alkylacyl, and diacyl subclasses either by using normal-phase HPLC or by thin-layer chromatography on silica gel G plates. Molecular species within alkenylacyl, alkylacyl, and diacyl subclasses were separated using reversed-phase HPLC. Separation of the individual subclasses was achieved for ethanolamine phosphoglycerides from bovine brain, as well as choline and ethanolamine phosphoglycerides from human neutrophils. Separation and quantitation of individual molecular species were carried out for alkenylacyl, alkylacyl, and diacyl subclasses of bovine brain ethanolamine phosphoglycerides by their absorbance at 254 nm with correction for recoveries as normalized to the internal standard 1,2-dipentadecanoyl-3-phosphatidylcholine added before the hydrolysis of phospholipids with phospholipase C or 1,2-dipentadecanoyl-3-anthroyl glycerol added after complete derivatization. The extinction coefficient of the 1-anthroyl derivatives were greater than 68,000 permitting the generation of concentration-dependent determinations which were linear to less than 1 pmol when monitored at 254 nm. Thus, this procedure provides a new and very sensitive method for the quantitation of picomole quantities of phospholipids or DGs by HPLC techniques.