Fast derivatization procedure for the analysis of phytoestrogens in soy milk by gas chromatography tandem mass spectrometry

In this work an optimized GC–MS/MS method, based on a simple and fast derivatization procedure, is proposed for the determination of five phytoestrogens (formononetin, daidzein, coumestrol, genistein and biochanin A) in soy milk. A systematic study of the instrumental analysis conditions was carried out, optimizing different MS acquisition parameters. At the same time, the derivatization procedure was optimized testing three silylating reagents. Among them, N,O-bis-(trimethylsilyl) trifluoroacetamide (BSTFA) was chosen as the most suitable derivatization reagent and an experimental design was performed to find the best conditions for the silylation reaction. The results showed the independence of the derivatization efficiency from the heating treatment applied before analysis, enabling the derivatization directly in the injection port; moreover, the use of a temperature programmed injection enhanced sensitivity and precision of the analysis. The described optimization steps allowed a significant gain in the sensitivity of the overall method, leading to detection limits of 0.1–17.7 μg L− 1, which are lower or comparable to values reported in the literature for GC–MS analysis of these analytes. High specificity was reached as well, thanks to the careful study of the conditions for tandem MS detection. Precision was good for formononetin, daidzein and coumestrol, with coefficients of variance of 4.7–6.1%; on the contrary, the analysis of genistein and biochanin A was characterized by low precision, probably due to poor stability of the corresponding derivatives. The method was tested on some soy milk samples from the Italian market, proving its suitability for the analysis of phytoestrogens in this complex matrix.