Separation of Representative Lipid Compounds of Biological Membranes and Lipid Derivatives from Peroxidized Polyunsaturated Fatty Acids by Reversed Phase High-Performance Liquid Chromatography

Abstract

A complex mixture of different lipid compounds, including phosphatidylcholine, phosphatidylserine, all-trans-retinol, 15(S)-hydroperoxyeicosatetraenoic acid, D-α-tocopherol, saturated and unsaturated fatty acids can be separated by reversed phase HPLC by using a C-18,120 mm × 4 mm, 3 pm particle size column and a step gradient from acetonitrile/water (1:1; v:v) to 100%, ace-tonitrile at a flow rate of 0.8 ml/min. By applying this elution condition, separation of various groups of lipid hydroperoxides and lipid derivatives, each one originating from a different in vitro peroxidized polyun-saturated fatty acid, can be obtained. Simultaneous detection is carried out by a diode array detector at a wavelength accumulation range set up between 195 and 400 nm. The possibility of simultaneously having such a large number of measurements renders this chromato-graphic method particularly suitable in studies concerning lipid peroxidation where, in addition to the detection of free radical-induced lipid hydroperoxides, data on some key antioxidant molecules, i.e. vitamin A and E, as well as that of structural compounds of biological membranes, i.e. phosphatidylcholine and phosphatidylserine, can be achieved.