HPLC analysis of isoflavonoids and other phenolic agents from foods and from human fluids

Abstract

A fast, precise and selective diode array HPLC method is presented for the extraction and analysis of soy isoflavonoids from foods and from human urine, plasma, end breast milk in support of mechanistic and epidemiologic studies assessing the potential cancer protective role of soya or isoflavones. Solid phase or solvent erection was chosen for isolation, and enzymatic or acid hydrolysis procedures were used for aglycone production depending on the matrix to be analyzed. C-18 reversed-phase HPLC was applied to selectively separate and quantitate daidzein (1),2 glycitein (3),2 and genistein (4),2 including their malonyl (a)2 and acetyl (b)2 esters, and their mammalian metabolites equol (6)2 and O-desmethylangolensin (7),2 as well as formononetin (2),2 biochanin-A (5),2 and coumestrol (8)2 using a gradient elution system. UV absorbance scans and authentic standards were applied for identification purposes, additional to fluorometric monitoring, electrochemical detection, and GC/MS analysis after trimethyl silylation. Detection limits of 20-μl injections were found to be 1.09, 0.53, 3.28, and 1.00 pmoles for daidzein, genistein, equol, and O-desmethyl-angoiensin (DMA), respectively, by monitoring at the individual compound's absorption maximum. The proposed method was applied to monitor isoflavone levels in soy foods and in human plasma, urine and breast milk after challenge with roasted soybeans. Implications of the presented results on the potential activity of isoflavones to prevent cancer by exposing newborn infants to these agents are discussed.