Separation of polyunsaturated fatty acid radicals by high-performance liquid chromatography with electron spin resonance and ultraviolet detection.

In the reaction of soybean lipoxygenase (EC 1.13.11.12) with polyunsaturated fatty acids such as linoleic, linolenic and arachidonic acids, some radical species were detected using the electron spin resonance (ESR) spin-trapping technique. The radical species derived from the three polyunsaturated fatty acids were not distinguishable because the ESR spectra of the spin adducts of nitrosobenzene with their three radical species showed no difference in their hyperfine splittings. To overcome this defect of the spin-trapping technique, these spin-adducts were separated by employing high-performance liquid chromatography (HPLC) combined with ESR spectroscopy. The spin adducts were eluted from a C18 reversed-phase column in the order linolenic acid, linoleic acid and arachidonic acid. The half-lives of the spin adducts separated by HPLC-ESR were determined as linoleic acid 600 min, linolenic acid 360 min and arachidonic acid 160 min. The use of an ultraviolet detector together with the HPLC-ESR technique resulted in a 500-fold increase in sensitivity in the detection of the radical species.