Characterization of the initial carbon-centered pentadienyl radical and subsequent radicals in lipid peroxidation: identification via on-line high performance liquid chromatography/electron spin resonance and mass spectrometry

The previously reported combination of an on-line high-performance liquid chromatography (LC)/electron spin resonance (ESR) system with mass spectrometric analysis (MS) created a unique technique to identify a variety of lipid-derived radicals (•Ld) formed from in vitro lipid peroxidation (Iwahashi et al. [20]). To improve the sensitivity, resolution, and reliability of this method for in vitro and in vivo studies, we have investigated the effects of mobile phase pH, modifiers, and columns on the chromatographic separation of linoleic acid-derived radical adducts. Using tetrahydrofuran (THF) and 0.1% glacial acetic acid (HOAc) in an H2O/acetonitrile (ACN) mobile phase greatly increased the resolution and retention reproducibility of lipid radical adducts in LC/ESR. In addition, these modifications allowed the elimination of an ESR tuning problem and the synchronization of UV and ESR detection of radical adducts in on-line LC/ESR, neither of which had been possible previously. Analyte purity was therefore increased, thus increasing the reliability of radical detection via on-line LC/ESR as well as radical identification via MS analysis. For the first time, POBN adducts of linoleic carbon-centered pentadienyl radicals (L•) were detected and identified. The optimization of chromatography in the LC/ESR and MS combination provided a reliable and sensitive way for the detection and identification of expected radical adducts in vitro and in vivo.