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
Iwahashi
H
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Department of Chemistry, Wakayama Medical College, Japan. chem1@wakayama-med.ac.jp
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004563:Electrochemistry; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals; D008041:Linoleic Acids / Q000737:chemistry*; D008054:Lipid Peroxides / Q000737:chemistry*
D005609:Free Radicals; D008041:Linoleic Acids; D008054:Lipid Peroxides; C038649:13-hydroperoxy-9,11-octadecadienoic acid