Towell J, Kalyanaraman B
Department of Chemistry, Carroll College, Waukesha, Wisconsin.
Anal Biochem. 1991 Jul;196(1):111-9. doi: 10.1016/0003-2697(91)90126-e.
Although high-performance liquid chromatography with electrochemical detection (HPLC-EC) has been previously used to analyze radicals trapped by 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), these techniques omitted the critical use of an internal standard. To improve reliability we have incorporated 3-carbamoyl-2,2,5,5-tetramethylpyrrolidin-1-yloxy free radical (CTPO) as a stable, oxidizable, and paramagnetic internal standard. Radicals generated during the uv photolysis of hydrogen peroxide were trapped by DMPO, analyzed by HPLC-EC, and quantified by relative comparison of peak areas to those of CTPO. DMPO adducts of hydroxyethyl, hydroxymethyl, and carbon dioxide anion radicals have been further characterized by ESR analysis of their 13C isotopes sampled from the HPLC eluant. Studies of the voltage dependence of the electrochemical signal demonstrate how this can be used to further confirm the identity of artifactual HPLC-EC peaks with similar retention times.
尽管高效液相色谱-电化学检测法（HPLC-EC）此前已用于分析由5,5-二甲基-1-吡咯啉-N-氧化物（DMPO）捕获的自由基，但这些技术忽略了内标的关键用途。为提高可靠性，我们引入了3-氨基甲酰基-2,2,5,5-四甲基吡咯烷-1-氧基自由基（CTPO）作为一种稳定、可氧化且顺磁性的内标。过氧化氢紫外光解过程中产生的自由基被DMPO捕获，通过HPLC-EC进行分析，并通过与CTPO的峰面积进行相对比较来定量。羟乙基、羟甲基和二氧化碳阴离子自由基的DMPO加合物已通过对从HPLC洗脱液中采样的13C同位素进行ESR分析得到进一步表征。对电化学信号的电压依赖性研究表明，这可用于进一步确认保留时间相似的人为HPLC-EC峰的身份。
