Rapid separation and quantitation of major phospholipids in biological samples by combined high-performance liquid chromatography and automated phosphorus analyzer.

Phospholipids extracted from tissue samples were separated by an isocratic high-performance-liquid-chromatographic (HPLC) method and simultaneously quantitated by an automated phosphorus analyzer. Results from various tissues were compared with previously published data obtained by thin-layer chromatography (TLC). Liver, heart, skeletal muscle, kidney and brain cortex synaptosomes from rats were examined. Optimal separation of major phospholipids of these tissues was achieved in a single HPLC run using a mobile phase of acetonitrile, methanol and sulfuric acid 100:2.1:0.05 (v:v:v). Recoveries of pure phospholipids injected onto the column averaged 75-80%. Similar recoveries were obtained with heart and skeletal muscle phospholipids, whereas liver, kidney and synaptosomes yielded lower recoveries (50-66%), suggesting the presence of other phospholipids in these tissues which did not elute from the column. The composition of total and individual phospholipids varied among the tissues and was generally similar to previously reported findings with TLC. The intraassay coefficients of variation ranged from 5 to 11%. We conclude that this technique is a reliable, rapid, and reproducible method for separation and quantitation of the major phospholipid species of tissues and subcellular fractions.