Effect of protein on the detection of stilbene estrogens in milk

Abstract

Fluorescence spectrometry was used to investigate the binding interactions of bovine serum albumin (BSA) with three stilbene estrogens (hexestrol, diethylstilbestrol and dienoestrol). Further research into the binding ratios between stilbene estrogens and actual milk samples was carried out by equilibrium dialysis method. Meanwhile, the effect of protein on the extraction efficiency of stilbene estrogens in milk samples was investigated in detail. The results show that stilbene estrogens strongly bound with the milk samples. In 70% (v/v) ethanol–water extracting solution, a slow but full denaturation of the protein matrix of the sample takes place, which causes the drugs bonded with protein to be released. Then an appropriate amount of K₂HPO₄ was added to the above extraction solution to form a stable aqueous two phase system. Following on, the fat-soluble stilbene estrogen residues were extracted into the upper phase with high extraction efficiency. Purification steps were omitted in this work because the fat-soluble impurities were extracted less in 70% (v/v) ethanol–water solution than in hydrophobic solvents, such as liquid–liquid extraction procedure. The proposed approach was satisfactorily applied to the quick determination of stilbene estrogen residues in milk by high performance liquid chromatography (HPLC). Overall recoveries were 83.2–93.8% with RSD values less than 4.52%, and the detection limits were in the range of 11.7 ng g⁻¹ to 20.7 ng g⁻¹. The sample preparation method was straightforward, efficient, economically advantageous and environmentally-friendly.