Trace analysis of estrogens in milk samples by molecularly imprinted solid phase extraction with genistein as a dummy template molecule and high-performance liquid chromatography-tandem mass spectrometry

Dummy molecularly imprinted polymer microspheres (DMIPMS) towards estrogens were synthesized by Pickering emulsion polymerization employing genistein (GEN) as a dummy template molecule. The FTIR analysis indicated the successful preparation of the imprinted polymers, and the characterization results of scanning electron microscopy and nitrogen adsorption desorption measurement indicated that the obtained DMIPMS are in possess of regular spherical shapes, porous structures and narrow diameter distribution, a BET surface area of 402.74 m² g^-1, a total pore volume of 0.568 cm³ g^-1 and a pore diameter of 3.62 nm. The binding capacity and selectivity of DMIPMS were investigated in equilibrium binding experiments and chromatographic evaluation experiments through scatchard analysis and molecular imprinting factor (IF) analysis, respectively. The MIPs showed high binding capacity and excellent selectivity towards seven selected natural and synthetic estrogens, which are Estrone (E1), 17β-estradiol (βE2), estriol (E3), ethinylestradiol (EE2), dienestrol(DS), diethylstilbestrol (DES), and hexestrol (HEX). A method for selective determination of seven estrogens in milk samples via dummy molecularly imprinted solid phase extraction coupled with HPLC-MS/MS was developed, which showed good linearity from 2 to 500 µg L^-1 with a correlation coefficient (R2) of more than 0.999. The detection limits were within the range of 0.10-0.35 µg L^-1 and the recoveries of the seven estrogens at three spiking levels (10,100,250 µg L^-1) ranged from 88.9% to 102.3% with relative standard deviation (RSD, n = 5) for intra-day and inter-day assays varied from 0.8% to 4.5%. The developed method is thus proven to be efficient and reliable for regular monitoring of trace estrogens in complex matrices such as milk samples.