1. Introduction
The majority of vital functions of the human body can be altered by endocrine disruptors (EDs), which interfere with the natural production, release, or elimination of hormones. Therefore, they disturb the performance of the endocrine system and can cause adverse health effects in humans and other mammals [
1,
2,
3,
4]. It has been observed that particularly during fetal development and early childhood, exposure to low doses of EDs (ppb) can have serious effects on human life and reproduction, causing decreased mental capacity, genital abnormalities, and cancers [
5,
6]. Many EDs such as phenolic endocrine disruptors can often be found in rivers and ground waters as they are widely used in the plastic industry and regularly released to the environment. Further, they can migrate closer, contaminating food and drinking water [
4,
7,
8]. Therefore, it is extremely important to develop a suitable technique that enables the fast and easy detection of low concentrations of EDs, particularly in aqueous media.
This article provides preliminary studies on the extraction and preconcentration of two important EDs, bisphenol A (BPA) and progesterone (PG). They have been identified as some of the most common hazardous substances in the human environment, often released from various plastics (e.g., baby or beverage bottles). Much evidence has shown that BPA has toxic properties, inducing estrogenic endocrine disruption and the acceleration of tumorigenic progression [
9]. In 2018, the European Commission issued a new regulation on the use of BPA in packaging. The Specific Migration Limit (SML), which is the maximum amount of the chemical allowed to migrate from packaging into food, was lowered from 0.6 mg to 0.05 mg of BPA per kg of food. The new Tolerable Daily Intake (t-TDI) was set at 4 µg/kg body weight per day [
10]. Additionally, since 2011, BPA has been forbidden in the manufacture of baby bottles and any food contact material intended for children up to 3 years of age. To protect aquatic life and mammalian consumers, the concentration of BPA in water has been set at 1.4 µg/L and in sediment at 9.9 µg/kg dry weight [
11]. This has raised a great concern about the utilisation of BPA and its potential harmful effects on human health. Therefore, it is very important to monitor even trace amounts of BPA [
12]. The major part of our work focused on the development of a new, sufficient method for the detection of BPA, which can further be applied in the determination of other highly dangerous EDs, e.g., progesterone (PG). PG is a hormone which stimulates and regulates the activity of cells and organs. Its secretion is controlled by the adrenal glands [
13]. In the last 20–30 years, the knowledge about progesterone has significantly increased, leading to great progress in progestational therapy [
14]. It has been reported that PG has revealed biological activity already at 123 ng/L, affecting human health, causing cancer, and disrupting natural hormonal activity. Currently, there are no laws that regulate the level of PG in the environment [
15].
Over the years, scientists have been trying to develop new methods to detect EDs in environmental samples. Generally, the process involves lengthy pretreatment steps of the samples to reduce matrix interferences and to enhance the detection of the target hazardous compounds, particularly their trace amounts. Only after those steps it is possible to start proper extraction and analysis. Recently, the popular leading technique for the extraction of pollutants from environmental liquid samples has been Solid Phase Extraction (SPE), which allows relatively fast and easy measurements [
16]. In the last decade, molecularly imprinted polymers (MIPs) combined with SPE have attracted much attention, especially in the extraction of trace amounts of analytes from large sample volumes and complex matrices. Moreover, MIPs are low-cost and very stable [
17,
18]. The principle of molecular imprinting is to create recognition sites for a target analyte in the polymeric matrix. During this process, functional and cross-linking monomers are polymerised or co-polymerised in the presence of an imprint molecule, called a molecular template [
19]. During the synthesis, functional monomers form a complex with template molecule while the highly cross-linked polymeric backbone helps to keep functional groups in specific positions. After polymerisation, imprinted molecules are subsequently removed from the matrix, leaving cavities with an affinity to a chosen template. In that way, the polymer matrix keeps the molecular memory of the analytes and it can be used in their molecular recognition [
20,
21,
22,
23,
24,
25,
26,
27,
28,
29]. As a reference, non-molecularly imprinted polymers (NIPs) without the template are used, following the same synthesis procedure as for MIPs, except in the absence of a template. Therefore, NIPs have the same composition as MIPs but without the presence of any specific cavity. Therefore, the nature of the interactions developed between MIPs and the template is the same as those developed between NIPs and the template, while the difference between these two types of polymers is the strength of these interactions. If the cavities created during the polymerisation are well defined, the strength of the interactions in MIPs should be significantly better than those in NIPs. The retention properties of the synthesised polymers can be established based on the adsorption capacity and the mechanism of the adsorption of target analytes onto the surface of the polymeric material. One of the methods widely used to define these binding properties is the determination of adsorption isotherms. The Langmuir-Freundlich isotherms (LF) can model the adsorption of an adsorbate in MIPs and NIPs along with homogeneous and heterogeneous distributions in high and low concentrations of analytes [
30,
31].
MIPs have already proved their efficiency in selective preconcentration and measurements of endocrine disruptors in complex samples, such as ground water, milk, and serum [
32,
33,
34,
35]. In the detection of BPA, MIPs were used in several different formats, e.g., as a stationary phase in the capillary electrophoresis [
31], superparamagnetic surface modified nanoparticles [
36], or bulk materials combined with liquid chromatography-mass detection [
37]. Despite many attempts of the synthesis and application of MIPs, they always suffered from low performance, complicated data analysis, and high batch variability. Similar problems were related to the separation and quantification of PG, e.g., using MIPs combined with SPE and HPLC [
33], as hydrogels [
38], or in the application of MIPs in electrochemical sensors, hydrogels [
39,
40], and optical biosensors [
41]. Therefore, in our work we tried to enhance the synthesis and the performance of MIPs by the optimisation of different parameters during polymerisation, such as the concentration of the cross-linker, solvent, and template ratio. The preliminary work was performed in order to detect and quantify BPA and PG in water samples. Additionally, our objective was to contribute to the growing area of research involving imprinting polymers. Therefore, the purpose of the work was to use MIPs as a new highly efficient sorbent which could be applied in solid phase extraction. First part of the work involved testing a variety of functional monomers to select that most suitable for the polymerisation of the MIPs for BPA and PG (see
Supplementary Materials). After that, it was necessary to perform a brief experimental design in order to optimise all variables that affect the process of the retention of target molecules, based on the maximum adsorption capacity using adsorption isotherms and kinetic studies.
The optimal designs were generated in MODDE software, which enabled us to screen and optimise experimental variables due to the integrated cross-validated model fitting, model fit visualisations, and predictive capabilities [
42,
43,
44].
4. Conclusions
Using radical polymerisation, it was possible to obtain high yields of different polymers (up to 96.6%). The optimal conditions for the synthesis of MIPs for BPA and PG were defined following experimental design, which helped to eliminate all insignificant factors for the polymerisation and therefore reduced the large number of syntheses.
The limits of detection and quantification of the analytes in water were obtained using HPLC with a fluorescence detector for BPA and a UV-visible detector for PG. For BPA, the LOD was 0.015 mg/mL and the LOQ was 0.045 mg/mL. For PG, the LOD was 11.3 mg/mL and the LOQ was 34.0 mg/mL.
The kinetic studies showed rapid BPA and PG retention, in less than 15 min of contact. The adsorption isotherms were analysed using Langmuir and Freundlich models. Results indicated that each cavity allowed binding only a single target molecule, which created a monolayer and later a multilayer on the surface of the polymer.
Overall, significant differences between retention capacities for MIPs and NIPs were observed. The Qmax of the retention of BPA for MIP 2 was 9.88 times higher than that for the control (NIP 2), and for MIP 3 was it 7.53 times higher than that for the control (NIP 3). The Qmax of the retention for PG for MIP 2 was 8.28 times higher than that for the control (NIP 2), and for MIP 3 it was 9.03 times higher than that for the control (NIP 3). Additionally, there was no significant cross-reactivity reported between MIPs and other related molecules. When the MIPs were applied in real water samples the results were practically identical to those of ultrapure water. This proved the great efficiency of the proposed materials and their potential application in environmental samples. Therefore, further work should include the testing of different mobile phases for the batch experiments, e.g., by decreasing the polarity of a media we could try to decrease the non-specific interactions between analytes and the active sites of imprinted polymers.