Giokas Dimosthenis L, Vlessidis Athanasios G, Evmiridis Nicholaos P
Laboratory of Analytical Chemistry, Department of Chemistry, University of Ioannina, 451 10 Ioannina, Greece.
Anal Chim Acta. 2007 Apr 18;589(1):59-65. doi: 10.1016/j.aca.2007.02.041. Epub 2007 Feb 22.
This study establishes a new method to analyze the radical scavenging activity of antioxidants based on the luminol-H(2)O(2)-Co(II)/EDTA chemiluminescence and flow injection analysis. The method is based on the catalytic oxidation of hydrogen peroxide by Co(II)/EDTA complex, forming a free radical flux that can produce a stable chemiluminescence signal which is attenuated in the presence of antioxidants. A properly designed FIA manifold and the appropriate regulation of the chemiluminescence-reagent mixture enabled the establishment of a reaction-sensitive analytical procedure that minimizes oxidant-antioxidant interactions while favors the inhibition effect of antioxidants on the free radicals flux. In that manner, the uncontrolled experimental variability induced by side-reactions occurring antagonistically is reduced. The method was examined in-vitro for the continuous monitoring of the generation of oxygen-derived free radicals and antioxidants, which is closer to in-vivo conditions, with three common antioxidants (ascorbic acid, glutathione and uric acid). All three antioxidants were found to inhibit the luminescent signal with strict logarithmic linear mode, yielding calibration curves rectilinear in the range of 5 x 10(-8) - 5 x 10(-5) molL(-1) and detection limits at the 10(-8) molL(-1) levels. The F-statistic was employed to assess the ability of the method to detect differences in the activity of the examined antioxidants. The results suggest that the proposed method can be used efficiently for the detection of free radical activity in real samples.
本研究基于鲁米诺-H₂O₂-Co(II)/EDTA化学发光和流动注射分析建立了一种分析抗氧化剂自由基清除活性的新方法。该方法基于Co(II)/EDTA络合物催化氧化过氧化氢，形成自由基通量，该通量可产生稳定的化学发光信号，在抗氧化剂存在下该信号会减弱。精心设计的流动注射分析流路和对化学发光试剂混合物的适当调节，使得能够建立一种反应灵敏的分析程序，该程序可将氧化剂-抗氧化剂相互作用降至最低，同时有利于抗氧化剂对自由基通量的抑制作用。通过这种方式，减少了由拮抗发生的副反应引起的不受控制的实验变异性。该方法在体外用于连续监测氧衍生自由基和抗氧化剂的生成，这更接近体内条件，使用了三种常见的抗氧化剂（抗坏血酸、谷胱甘肽和尿酸）。发现所有三种抗氧化剂均以严格的对数线性模式抑制发光信号 在5×10⁻⁸ - 5×10⁻⁵ molL⁻¹范围内产生线性校准曲线，检测限为10⁻⁸ molL⁻¹水平。使用F统计量来评估该方法检测所检查抗氧化剂活性差异的能力。结果表明，所提出的方法可有效地用于检测实际样品中的自由基活性。
