Separation of representative lipid compounds of biological membranes and lipid derivatives from peroxidized polyunsaturated fatty acids by reversed phase high-performance liquid chromatography

A complex mixture of different lipid compounds, including phosphatidylcholine, phosphatidylserine, all-trans-retinol, 15(S)-hydroperoxyeicosatetraenoic acid, D-alpha-tocopherol, saturated and unsaturated fatty acids can be separated by reversed phase HPLC by using a C-18, 120 mm x 4 mm, 3 mu m particle size column and a step gradient from acetonitrile/water (1:1; v:v) to 100% acetonitrile 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 polyunsaturated 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 chromatographic 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.