Method for evaluating the bioconversion of radioactive polyunsaturated fatty acids by use of reversed-phase liquid chromatography

Reversed-phase high-performance liquid chromatography on a thermostatted octadecylsilyl column was used to separate mixtures of labelled polyunsaturated fatty acids (as their methyl esters) formed by successive desaturations and elongations of labelled linoleic (18:2 n - 6) or linolenic (18:3 n - 3) acid by rat liver microsomes. Acetonitrile-water mixtures were used for elution of the esters. Unsaturated and saturated esters were detected by their refractive indices. The order of elution of fatty acid methyl esters in complex mixtures varies as a function of the chain length and unsaturation, analysis temperature, water concentration and solvent flow-rate. The peak areas vary as a function of the unsaturation. Specific radioactivities of 14C-labelled fatty acids and the percentage distribution of radioactivity among fatty acids from complex mixtures can be efficiently determined by collection and direct measurement of the radioactivity in the solvent by liquid scintillation counting. The method can be applied to complete compositional analysis, but is especially useful for determination of specific radioactivities during studies on the metabolic conversion of labelled polyunsaturated fatty acids.