Identification of phytoestrogens in bovine milk using liquid chromatography/electrospray tandem mass spectrometry

In an international context of promoting scientific research on food safety, the interest in molecules having potential hormonal disrupting effects is growing. While industrial endocrine disruptors (phthalates, alkylphenols, PCBs, etc.) have been studied for several years, natural compounds like phytoestrogens remain less investigated. Accordingly, a research project was initiated with its main objectives to develop efficient analytical methods for a wide range of phytoestrogens in various food matrices, and to evaluate their occurrence in food products. Electrospray ionization with tandem mass spectrometric (MS/MS) analysis of isoflavones (genistein, daidzein, equol, formononetin, biochanin A), lignans (enterolactone, enterodiol), and coumestans (coumestrol) was investigated. This study revealed the formation of a large number of fragment ions in both positive and negative modes, corresponding to specific cleavages of the hydroxyl, carbonyl, and/or methoxy groups, and to Retro-Diels-Alder reactions. An LC/ESI-MS/MS method was developed consistent with the 2002/657/EC European decision criteria. An extraction and clean-up method was developed for milk samples. The identification limit for the proposed method appears to be under 1 ng/mL. The developed methodology was applied to various milk samples, and the occurrence of isoflavones (particularly equol) was demonstrated in the concentration range 1-30 ng/mL. The efficiency of the proposed analytical method permitted evaluation of a new and promising approach to a global risk assessment of natural estrogenic active substances including phytoestrogens and their metabolites.