Department of Chemistry, Faculty of Engineering, Istanbul University, Avcilar 34320, Istanbul, Turkey.
Anal Chim Acta. 2010 Jul 26;674(1):79-88. doi: 10.1016/j.aca.2010.06.013. Epub 2010 Jun 16.
A novel on-line HPLC-cupric reducing antioxidant capacity (CUPRAC) method was developed for the selective determination of polyphenols (flavonoids, simple phenolic and hydroxycinnamic acids) in complex plant matrices. The method combines chromatographic separation, constituent analysis, and post-column identification of antioxidants in plant extracts. The separation of polyphenols was performed on a C18 column using gradient elution with two different mobile phase solutions, i.e., MeOH and 0.2% o-phosphoric acid. The HPLC-separated antioxidant polyphenols in the extracts react with copper(II)-neocuproine (Cu(II)-Nc) reagent in a post-column reaction coil to form a derivative. The reagent is reduced by antioxidants to the copper(I)-neocuproine (Cu(I)-Nc) chelate having maximum absorption at 450 nm. The negative peaks of antioxidant constituents were monitored by measuring the increase in absorbance due to Cu(I)-Nc. The detection limits of polyphenols at 450 nm (in the range of 0.17-3.46 microM) after post-column derivatization were comparable to those at 280 nm UV detection without derivatization. The developed method was successfully applied to the identification of antioxidant compounds in crude extracts of Camellia sinensis, Origanum marjorana and Mentha. The method is rapid, inexpensive, versatile, non-laborious, uses stable reagents, and enables the on-line qualitative and quantitative estimation of antioxidant constituents of complex plant samples.
一种新型的在线高效液相色谱-铜还原抗氧化能力（CUPRAC）方法被开发用于选择性测定复杂植物基质中的多酚（类黄酮、简单酚和羟基肉桂酸）。该方法结合了色谱分离、成分分析和植物提取物中抗氧化剂的柱后鉴定。多酚的分离在 C18 柱上进行，使用两种不同的流动相溶液（甲醇和 0.2%邻磷酸）进行梯度洗脱。在柱后反应盘管中，从提取物中分离出的抗氧化多酚与铜（II）-邻菲咯啉（Cu（II）-Nc）试剂反应，形成一种衍生物。抗氧化剂将该试剂还原为具有最大吸收峰在 450nm 的铜（I）-邻菲咯啉（Cu（I）-Nc）螯合物。通过测量由于 Cu（I）-Nc 而导致的吸光度增加，监测抗氧化成分的负峰。在柱后衍生化后，多酚在 450nm 处的检测限（在 0.17-3.46μM 范围内）与未经衍生化的 280nm UV 检测相当。该方法成功应用于茶叶、马郁兰和薄荷粗提物中抗氧化化合物的鉴定。该方法快速、廉价、多功能、不费力、使用稳定的试剂，并能够在线定性和定量估计复杂植物样品中的抗氧化成分。
