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

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Abstract

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 N2. 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.

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Acknowledgments

This work was supported by RM2012-00004-00-00. J.M. Landete has a postdoctoral contract with the research program “Ramón y Cajal” (MINECO, Spain). We are grateful to the ICTAN Analysis Services Unit for providing chromatography and mass spectrometry facilities.

Conflict of Interest

Pilar Gaya declares that she has no conflict of interest. Juan Luis Arqués declares that he has no conflict of interest. Margarita Medina declares that she has no conflict of interest. Inmaculada Álvarez declares that she has no conflict of interest. José Mª Landete declares that he has no conflict of interest.

Ethical Approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Informed Consent

Informed consent was obtained from all individual participants included in the study.

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Authors and Affiliations

  1. Departamento de Tecnología de Alimentos, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Carretera de La Coruña Km 7.5, 28040, Madrid, Spain

    Pilar Gaya, Juan Luis Arqués, Margarita Medina & José Maria Landete

  2. Unit Service of Analytical Techniques, Instrumentation and Microbiology (USTA), Institute of Food Science and Nutrition (ICTAN-CSIC), Jose Antonio Novais, 10, 28040, Madrid, Spain

    Inmaculada Álvarez

Authors
  1. Pilar Gaya
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  2. Juan Luis Arqués
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  4. Inmaculada Álvarez
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  5. José Maria Landete
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Correspondence to José Maria Landete.

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Gaya, P., Arqués, J.L., Medina, M. et al. A New HPLC-PAD/HPLC-ESI-MS Method for the Analysis of Phytoestrogens Produced by Bacterial Metabolism. Food Anal. Methods 9, 537–547 (2016). https://doi.org/10.1007/s12161-015-0226-3

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