A Sensitive, Quantitative Analysis of Molecular Species of Phospholipid by High-performance Liquid Chromatography Using Dinitrobenzoyl Derivatives

A high-performance liquid chromatographic (HPLC) method for quantitative microanalysis of phospholipid molecular species was studied. Diacylglycerols were prepared from phospholipids by phospholipase C treatment and converted to the corresponding dinitrobenzoyl derivatives (DNB-DG), which could be sensitively detected at 254 nm. The DNB-DGs were resolved by HPLC using a reverse column with an isocratic solvent. Peak areas of DNB-DGs prepared from dipalmitoyl and dioleoyl phosphatidylcholines were proportional to the amounts of the derivatives. The quantification could be done at pmole level. The molecular species profiles of phosphatidylcholine of rat lung and liver obtained by the DNB-DG method were almost identical with those determined by DG-acetate RI method reported by Itoh et al. These results indicate that the peak areas of DNB-DG obtained at 254 nm are not affected by differences in the structure of molecular species, such as the numbers of the isolated double bond. This sensitive DNB-DG HPLC method was applied for the microdetermination of molecular species of acidic phospholipids, i. e., phosphatidylglycerol and phosphatidylinositol, in human bronchoalveolar lavage fluids. Their contens in the fluids seemed to change according to the state of alveolar proteinosis. It was found that the molecular species of phosphatidylglycerol and phosphatidylinositol were distinctly different from each other in human bronchoalveolar lavage fluids