Multi-functional sample preparation procedure for measuring phytoestrogens in milk, cereals, and baby-food by liquid-chromatography tandem mass spectrometry with subsequent determination of their estrogenic activity using transcriptomic assay

Abstract

A method dedicated to the determination of a multiple range of phytoestrogens as endocrine disruptor compounds in infant food products was developed, with as double objective the specific measurement of 13 parameters and the evaluation of the estrogenic potency associated to this quantitative profile. A combined enzymatic and acidic chemical hydrolysis followed by a double purification on two successive C 18 and SiOH Solid Phase Extraction cartridges permitted to efficiently purify milk, cereals and baby-food samples while eliminating naturally occurring estrogen hormones. A specific liquid chromatography-tandem mass spectrometric measurement authorised unambiguous identification and quantification of the target compounds. The proposed methodology was fully validated and applied to a set of around 30 real samples, demonstrating the presence of phytoestrogens at levels globally ranging from several μg kg −1 (ppb) to several tens mg kg −1 (ppm). The prepared sample extracts were proven to be suitable and compatible with the evaluation of their induced biological transcriptional activity on MCF-7 cell lines. Because permitting to cope with difficult issues such as low-dose and mixture effects, this proposed methodology may appear of particular interest for further exposure assessment studies and hazard characterisation investigations related to this class of endocrine disruptor compounds.