Improved methods for the extraction and analysis of isoflavones from soy-containing foods and nutritional supplements by reversed-phase high-performance liquid chromatography and liquid chromatography–mass spectrometry

An improved method for extraction and analysis of isoflavones from soy protein, soy foods and nutritional supplements is presented. The method uses acetonitrile extraction without acidification, with apigenin as internal standard. Samples extracted in acetonitrile–water are diluted to 50% acetonitrile and directly injected for gradient HPLC separation on a C18 reversed-phase column. This method saves significant time during sample preparation and improves accuracy and precision. Conventional and rapid HPLC analysis methods compatible with the extraction scheme were developed. During development of the methods, unexpected minor forms of malonyl and acetyl isoflavones were discovered in extracts of soy proteins and in pure isoflavone standard preparations. By LC–triple MS, these peaks have identical composition to the respective 6″-O-malonyl- and 6″-O-acetyl-isoflavones from which they form. These minor forms are believed to be malonyl and acetyl isoflavones where the site of attachment is a hydroxyl other than the 6′-OH of the glucose. These compounds can represent significant minor isoflavone components of foods, which contain high concentrations of malonyl or acetyl isoflavones.