Sensitive analysis of phospholipid molecular species by high-performance liquid chromatography using fluorescent naproxen derivatives of diacylglycerols

Abstract

A sensitive high-performance liquid chromatographic (HPLC) method for the separation and determination of diacylglycerophospholipid and diacylglycerol (DAG) molecular species has been developed. Phospholipids are hydrolysed with phospholipase C and the resulting DAGs are reacted with naproxen chloride in the presence of 4-dimethylaminopyridine. The naproxen-DAGs were purified by thin-layer chromaography on silica gel G plates. Molecular species were separated using reversed-phase HPLC with isocratic elution and determined by measuring the absorbance at 230 nm or fluorescence at 352 nm (excitation at 332 nm). The method was applied to the determination of diacylglycerophosphoethanolamine in rat cerebrum and cerebellum. The molar absorption coefficient of the naproxen derivatives was 53 000 lmol −1 cm −1 at 230 nm, permitting the generation of linear concentration-dependent determinations down to less than 10 pmol. A ten-fold increase in sensitivity was obtained with a fluorescence detection system owing to the fluorescent properties of the proposed adduct.