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 alpha-(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 x 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