10-hydroxy-11-12-epoxyeicosa-5-8-14-trienoic-acid and 12-hydroxy-5-8-10-heptadecatrienoic-acid

A method for profiling arachidonic acid metabolites by radio gas chromatography (GC) is described. The incubation mixture of rabbit platelets with [14C]arachidonic acid was purified on a Sep-Pak C18 cartridge and derivatized with diazomethane, O-methylhydroxylamine and dimethyl-isopropylsilylimidazole. The recovery of total 14C-radioactivity was 93.1 +/- 7.2%. Loss of radioactivity during derivatization was negligible. Baseline separations for [14C]arachidonic acid and its metabolites were obtained in a single run within 45 min by GC using a synchronized accumulating radioisotope detector (GC/SARD). The recovery of radioactivity from the GC column was virtually 100%. The chemical structures of the metabolites were confirmed by GC/mass spectrometry; peaks of arachidonic acid metabolites were assigned by comparison of the methylene unit values with those of radioactive peaks in GC/SARD analyses. The intra-assay coefficients of variation in GC/SARD analyses were less than 10%. The method was used to map the profile of arachidonic acid metabolites formed by rabbit platelets in the presence of indomethacin, baicalein or glutathione.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Blood Platelets; Chromatography, Gas; Fatty Acids, Unsaturated; Hydroxyeicosatetraenoic Acids; Leukotrienes; Rabbits