High-performance liquid chromatographic determination of linoleic acid peroxide-derived radicals using electrochemical detection

High-performance liquid chromatography–electrochemical detection (HPLC–ED) was applied to detect 13-hydroperoxide octadecadienoic acid (13-HPODE)-derived radicals such as the pentyl radical and octanoic acid radical. The 13-HPODE-derived radicals were successfully detected using HPLC–ED by the combined use of the spin-trapping technique with α-(4-pyridyl-1-oxide)-N-tert.-butylnitrone (4-POBN). The 4-POBN-pentyl radical adduct was detected at the retention time of 18.2±0.3 min on the elution profile of HPLC–ED with an ODS column (15 cm×4.6 mm I.D.) using a flow-rate of 1.0 ml/min with 50 mM ammonium acetate in 29% (v/v) aqueous acetonitrile. The 4-POBN–octanoic acid radical adduct was also detected at the retention time of 13.7±0.7 min using a flow-rate of 1.0 ml/min with 50 mM ammonium acetate in 14% (v/v) aqueous acetonitrile. The concentrations of the 4-POBN radical adducts were determined using HPLC–ED without an internal standard. HPLC–ED is 100 times as sensitive as HPLC–electron spin resonance (ESR) under the ESR and ED conditions employed here. Even 1.8 pmol of the 4-POBN–pentyl (or octanoic acid) radical adduct was detectable using HPLC–ED.