An HPLC Method for the Determination of Isoflavones and the Evaluation of Their Antioxidant Capacity in Both Homogeneous and Microheterogeneous Systems

Abstract

Abstract In this work, we developed an HPLC method to simultaneously quantify and hence evaluate the stability, distribution, and antioxidant capacity of six isoflavones: genistein, genistin, daidzein, daidzin, glycitin, and biochanin A. Isoflavones have been described as having an important estrogenic activity to treat menopausal symptoms and can reduce postmenopausal bone loss and also participate in the prevention of cardiovascular diseases. These beneficial properties are believed derived from their capacity to act as free-radical scavengers. Isoflavones are formulated in capsules and creams and also can be used as antioxidants in liposomes. HPLC separation was achieved on an Agilent Hypersil ODS C18 column. The mobile phase consisted of 0.02–0.2% orthophosphoric acid in water–acetonitrile with gradient elution. The diode array detector was operated at 260 nm. The hydrophobicity of isoflavones was determined through their distribution in octanol–buffer. These results allowed us to establish a relation between chemical structure, pKa, lipophilicity, and the characteristics of the dispersion medium. Photolysis of hydrogen peroxide was used to measure the HO• scavenging capability of isoflavones. In liposomes, the order of reactivity of the studied compounds was genistein > biochanin A > genistin > daidzein > daidzin > glycitin.