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

Highlights

• Molecularly imprinted polymers (MIPs) were prepared with genistein as dummy template.

• Regular spherical shape and narrow diameter distribution.

• The estrogens in milk were extracted by solid-phase extraction with MIPs as sorbents.

• High binding capacity and excellent selectivity for estrogens in aqueous environment.

• Trace analysis of estrogens in milk presented good recoveries and sensitivity.

Abstract

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.

Introduction

Generally, estrogens can be divided into endogenous and exogenous estrogens based on their sources [1]. Endogenous estrogens such as Estrone (E1), 17β-estradiol (βE2), 17α-estradiol (αE2) and estriol (E3) and their methylated and hydroxylated metabolites are formed naturally by humans and wildlife, while exogenous estrogens are foreign compounds, either naturally or synthetically produced, including ethinylestradiol (EE2), diethylstilbestrol (DES), hexestrol (HEX), and dienestrol (DS), etc, which have been widely used as a growth promoter for livestocks or as a treatment for estrogen-deficiency disorders in veterinary medicine [2]. These hormones can be secreted by the mammary gland, which synthesizes milk proteins and lactose during late pregnancy and lactation, and pass from the bloodstream into the milk [3], [4]. The effects of these estrogenic compounds have recently received considerable attention. They are considered as significant parts of the endocrine-disruptor compounds (EDCs) groups that interfere with the normal endocrine system and disrupt the physiologic compounds in the environment and food which are at low concentration may cause reproductive system disorders, decreases in sperm counts, disturbances in the balance of the ecosystem and even tumors such as breast cancer and ovarian cancer [5], [6], [7].

Nowadays estrogens have attracted increasing attentions in certain food matrices, especially in milk and its derivatives, which represents an important group of food commodities highly consumed by the world population. It is thus necessary to develop a sensitive and reliable analytical method for monitoring trace residues of estrogen in light of the complexity of milk matrixes.

In recent years, several analytical approaches have been developed for the determination of estrogens, including high-performance liquid chromatography (HPLC) [3], [8], [9], gas chromatography-mass spectrometry (GC–MS) [7], [10] and enzyme-linked immunosorbent assay [11]. Liquid chromatography coupled with tandem mass spectrometry is mostly employed for the analysis of estrogens [12], [13], [14], [15]. However, due to the complexity of the milk matrixes and the low concentrations, it is imperative to develop sample pre-treatment techniques with excellent clean-up and preconcentration efficiency for the selective extraction of estrogens before instrumental analysis. The technique of molecular imprinting polymers (MIP) coupled with solid-phase extraction (SPE), known as MIP-SPE, can achieve this purpose. SPE is a well-established technique most commonly employed for purification and preconcentration in sample pretreatment. The main drawback associated with conventional solid-phase extraction sorbents is their low selectivity resulting in the co-extraction of matrix interference [16]. Higher selectivity can be obtained using sorbents based on molecularly imprinted polymers (MIPs), which are highly cross-linked synthetic polymers that exhibit high affinity and selectivity for templates and its closely related analogues [17]. MIPs also have many other advantages such as physical robustness, chemical inertness towards organic solvents, acids or bases, resistance to elevated temperatures and pressures, low costs, and relative ease of preparation [18], [19]. MIPs have been proposed in recent years as sorbents for extracting estrogens in various complexity of matrixes [8], [17], [20], [21]. However, a significant drawback associated with reported MIPs lies in template bleeding which is caused by incomplete removal of the template from the polymer network. Thus, the possible bleeding of template molecules even after exhaustive washing steps will interfere analysis of the target objects to a certain extent, especially in the case of trace analysis [22]. This problem can be solved by a dummy template (a molecule of similar structure to analyte), which will not interfere with the analysis as any leakage of template may be separated in the subsequent chromatographic analysis [18]. As regard to the preparation of molecular-imprinted polymers (MIPs), bulk polymerization that inhabit rapidity and simplicity of its preparation was a approach commonly used. Nevertheless, the obtained monolithic polymers require time-consuming post-treatments such as crushing, grinding and sieving to produce desired fine particles, which are usually irregular with wide particle size distribution and low yields of polymers [23]. To overcome these shortcomings, various other techniques such as multi-step swelling polymerization, suspension polymerization and precipitation polymerization have been developed to prepare MIP microspheres. The method of multi-step swelling polymerization involves aqueous emulsions and the use of seed particles that will complicate the procedure and this method is not generally compatible to non-covalent imprinting systems [24]. As to suspension polymerization, the procedures usually involve stabilizers and/or surfactants that can interfere with the template monomer interactions. As water also involved, the aqueous media was not conducive to the synthesis of non-covalent MIPs that rely on hydrogen bonds [25]. Precipitation polymerization is an attractive stabilizer or surfactant-free method [24]. However, its needs for large volume of organic solvents as porogens may lead to the problems of environmental pollution and increased costs. In addition, MIP microspheres prepared by precipitation polymerization do not perform well under aqueous conditions. To improve its specificity in aqueous solution, more efforts need to be done, for example, by coating a hydrophilic layer on the MIP surfaces [26].

Consequently, a new promising alternative molecular imprinting method to synthesize water compatible MIPs with a high selectivity in aqueous solution using Pickering emulsion polymerization has been developed. Compared to the conventional polymerization approaches, Pickering emulsion polymerization that employ the solid nanoparticles as an emulsion stabilizer instead of organic surfactants is advantageous in terms of simplicity, high yields of polymers, good control of final particle sizes and obtaining water-compatible particles [27], [28]. In Pickering emulsion polymerization, firstly described by Pickering in 1907 [29], dispersed liquid droplets are stabilized by small solid particles attached to the droplet surfaces, preventing the coalescence of the droplets [23].

In this work, the structural analogs of estrogens, named Genistein (GEN) which is a group of phytoestrogens, was selected as the dummy template molecules to prepare MIP microspheres using the Pickering emulsion polymerization approach. The polymers with higher selectivity in aqueous solution were used as the selective extraction sorbents for estrogens from milk samples. A methodology for the simultaneous determination of E1, βE2, E3, EE2, DES, DS, and HEX by combining MIPSPE with HPLC-MS/MS detection was developed. The selectivity, accuracy and precision of the developed method were also evaluated. The present work describes for the first time the application of MIP microspheres imprinted with dummy template genistein to the extraction of estrogens from milk samples.