Analysis of positional isomers of monounsaturated fatty acids by high performance liquid chromatography of 2,4-dinitrophenylhydrazones of reduced ozonides

A method is described for quantifying the positional isomers in monounsaturated fatty acid methyl ester (FAME) fractions. The procedure involves the preparation of 2,4-dinitrophenylhydrazones (DNPH) of the fragments generated during reductive ozonolysis of FAME, class isolation of the aldehyde and aldehyde ester DNPH, and separation of the aldehyde ester derivatives by high performance liquid chromatography (HPLC). The high extinction coefficient of the DNPH provides for a sensitive assay which is linear for a large range of components over a concentration range of 0.075-5 nmol/component, and the stability of the DNPH permits the independent analysis of the aldehyde and aldehyde ester fragments generated during reductive ozonolysis. The reductive ozonolysis-DNPH-HPLC method developed is as sensitive, reproducible, and accurate as reductive ozonolysis-gas-liquid chromatography and does not suffer from some of the drawbacks of the classical procedure.