Original Contribution 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 STEVEN YUE QIAN,* KENNETH B. TOMER, † GUI-HUA YUE, † QIONG GUO,* MARIA B. KADIISKA,* and RONALD P. MASON* *Laboratory of Pharmacology and Chemistry; and † Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA (Received 15 May 2002; Revised 13 June 2002; Accepted 13 June 2002) Abstract—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 (  L d ) 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 H 2 O/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. Published by Elsevier Science Inc. Keywords—Lipid-derived radicals, POBN adducts of linoleic-derived radicals, On-line LC/ESR separation, ESI and tandem mass spectrometries, Soybean lipoxygenase-dependant lipid peroxidation, Free radicals INTRODUCTION Free radicals play important roles in living systems and have been implicated in aging and in the pathology of many human diseases, including heart disease and cancer [1– 6]. As the starting point in understanding the mech- anism and role of radicals in human disease, we seek to identify and quantify each radical produced. However, in living systems, most radicals such as superoxide, hy- droxyl radical, and lipid- and protein-derived radicals are highly reactive species with very short lifetimes. Even electron spin resonance (ESR), the only technique that can specifically detect free radicals, is not sensitive enough to detect those short-lived radical species. To overcome this difficulty, the ESR spin trapping technique was developed in the 1970s [7–9]; in this technique, a spin trap reacts with short-lived radicals to form rela- tively stable radical adducts. Since then, ESR spin trap- ping has been widely exploited in chemical, biochemical, and medical research to detect many kinds of free radi- cals. Unfortunately, the ESR study of spin trapped radicals has a major weakness: the identification of the radical structure is based on the hyperfine couplings of the ESR spectra alone, and these couplings provide information only about the radical center, not comprehensive struc- tural information such as the molecular mass of the radical adduct. Thus, alternative structures cannot be distinguished when the radical adducts have similar cou- pling constants. Address correspondence to: Steven Yue Qian, Laboratory of Phar- macology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, P.O. Box 12233, Research Triangle Park, NC 27709, USA; Tel: (919) 541-0495; Fax: (919) 541-1043; E-Mail: qian1@niehs.nih.gov. Free Radical Biology & Medicine, Vol. 33, No. 7, pp. 998 –1009, 2002 Published by Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/02/$–see front matter PII S0891-5849(02)00992-9 998