A New HPLC-PAD/HPLC-ESI-MS Method for the Analysis of Phytoestrogens Produced by Bacterial Metabolism

Phytoestrogens (isoflavones, ellagitannins, and lignans) are polyphenols with an estrogenic/antiestrogenic function present in fruits, vegetables, and foods derived from them. Some microorganisms are capable of transforming isoflavones, ellagitannins, and lignans into equol, urolithins, and enterolignans, respectively. These compounds are more bioavailable, they have more estrogenic/antiestrogenic and antioxidant activity than their precursors, and they are associated with beneficial effects for human health. Extracts of soybean and lignans and ellagic acid were incubated with feces, or bacteria isolated from feces, and later the phytoestrogens were extracted twice with diethyl ether and twice with ethyl acetate, and the solvents were evaporated at room temperature under a N-2. Subsequently, phytoestrogens were detected by HPLC-PAD and chromatographic peaks were identified by HPLC-ESI/M, and they were confirmed by comparison of retention times and characteristics of UV spectra with those of standards. Analysis of phytoestrogens by means of HPLC-PAD allowed the simultaneous detection of isoflavones, ellagitannins, and lignans metabolized, or not, by intestinal microbiota with high sensitivity (LOD: equol 0.11 mg/L; urolithin A 0.17 mg/L, enterodiol 0.15 mg/L). Assays were linear up to at least 20 mg/L in all standards with a high correlation (equol > 0.999; urolithin A > 0.998, enterodiol > 0.999). Therefore, the described method constitutes an affordable and easy alternative method for the detection of phytoestrogen metabolism. Moreover, this method could be applied to the detection of isoflavones, lignans, and ellagitannins in food.