DETERMINATION OF PHOSPHATIDYLCHOLINE, PHOSPHATIDYLGLYCEROL AND THEIR LYSO FORMS FROM LIPOSOME DISPERSIONS BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY USING HIGH-SENSITIVITY REFRACTIVE-INDEX DETECTION

An assay for quantitative analysis of phosphatidylcholine and phosphatidylglycerol, and their corresponding hydrolysis products lysophosphatidylcholine and lysophosphatidylglycerol using high-performance liquid chromatography with high-sensitivity refractive index detection was developed. The separation of the phospholipids of interest was achieved on a Zorbax NH2 column (25 cm x 4.6 mm I.D.)with a mobile phase consisting of acetonitrile-methanol 10 mM ammonium dihydrogen phosphate solution pH 4.8 (64:28:8, v/v/v) at a flow-rate of 1.5 ml/min. The response of the refractive index detector to different types of phosphatidylcholine with varying degrees of unsaturation was constant, while the ultraviolet detector response was strongly dependent on the degree of unsaturation. This makes refractive index detection suitable for the determination of natural phospholipids which show a wide variety of fatty acid composition. The method was validated for the determination of phosphatidylcholine, phosphatidylglycerol, lysophosphatidylcholine and lysophosphatidylglycerol in a model liposome dispersion. Synthetic phospholipids of high purity served as external standards and quantitation was based on peak areas. Calibration curves were linear over two orders of magnitude, and detection limits of phosphatidylcholine, phosphatidylglycerol, lysophosphatidylcholine and lysophosphatidylglycerol were 22, 29, 30 and 50-mu-g/ml, respectively. The method precision for a standard phospholipid mixture and for a phosphatidylcholine phosphatidylglycerol containing liposome dispersion was in the range of 0.6-4.5% relative standard deviation.