Pharmaceuticals most frequently identified in the aquatic environment include beta-blocker drugs, non-steroidal anti-inflammatory drugs, antibiotics, and female sex hormones [
28]. Sex hormones include natural estrogens E1, E2 and E3 used in hormone replacement therapy and as a component of contraceptives. In the rivers of Europe, the estimated concentration of these hormones is 0.3–3.5 ng L
−1 [
29,
30] Due to their high biological activity, it is necessary to develop analytical methods that would allow determination of their concentration not only as a sum of estrogens, but also of particular species, E1 or E2. The solubility of estrogens in water ranges from 0.8 to 30 mg L
−1 (
Table S1).
3.2. Analysis
3.2.1. FT-IR Spectra
At each stage of the synthesis, structure of the products was examined with FT-IR method. The obtained magnetic and non-magnetic adsorbent materials differ in their internal structure. Mag-MIPs/mag-NIPs are core–shell materials with a Fe3O4@SiO2 core, while MIPs/NIPs are coreless polymers also used as the outer shell. In the FT-IR spectrum of pure Fe3O4, the Fe-O bond absorption band appeared at 580 cm−1. The characteristic absorption of bare Fe3O4 for Fe3O4@SiO2 was observed at 576 cm−1 (Fe-O vibrations). The spectrum of Fe3O4@SiO2 showed strong peaks at 1085 and 796 cm−1. These peaks could be assigned to the asymmetric and symmetric linear stretching vibrations of the Si-O-Si bonds. The bending vibration absorption peaks of Si-O-Si and Si-OH were observed at 462 and 962 cm−1, respectively. These are indicative of the silica layer on Fe3O4. However, these bands were not detected for the core–shell materials. This is understandable, as, in the FT-IR analysis, signals from the surface of a material are more prevalent. The MIPs/NIPs and the core–shell materials have the same polymeric surface; therefore, their FT-IR spectra are analogous.
FT-IR analyses of E1-MIP, E2-MIP, NIP (
Figure 3a), E1-mag-MIP, E2-mag-MIP, and mag-NIP were performed (
Figure 3b). The majority of the signals can be attributed to the polymeric surface of the materials. After polymerization, C=C double bond stretching (1635 cm
−1) and C=O double bond bending (555 cm
−1) signals were observed and ascribed to the double bond in the MAA monomer. Moreover, the O-H stretching at 2935 cm
−1 and the O-H bending vibration at 1383 cm
−1 confirmed the presence of carboxylic groups. The presence of peaks at 1717 (C=O stretching) and 1140 cm
−1 (C-O stretching) show the existence of EGDMA cross-linker. The C-O-C asymmetric and symmetric group signals at 1256 and 1041 cm
−1 can be derived from the monomer chain. The stretching vibrations at 1294–1293 cm
−1 for C-O bond and stretching vibrations at 1076 cm
−1 for C-H bonds are characteristic of these groups. Asymmetrical stretching absorption at 655–650 cm
−1 for C-O-C, bending vibration at 626–622 cm
−1 for C-O-H, and bending absorption at 566–564 cm
−1 for C-C=O are observed in the spectra.
The NIP/mag-NIP and MIPs/mag-MIPs spectra showed only slight differences in the intensity and positioning of the signals. The hydrogen bond and the van der Waals interactions between polymers and templates resulted in observable, small changes in spectra at 2700–3700 cm−1 range.
A strong and broad absorbance peak assigned to the stretching vibration of hydroxyl groups was found at 3300 cm
−1. Hydroxyl groups in the hormone and monomer molecule formed intra- and/or inter-molecular hydrogen bonds O-H. In general, steroidal estrogens containing only a phenolic hydroxyl, e.g., E1, produced an O-H band at a higher wavelength than those containing only an alcoholic hydroxyl, e.g., E2 [
31,
32,
33]. The least intense band from hydrogen bonds is observed in NIP and mag-NIP reference samples, stemming from the monomer interactions in the polymer structure. These results show that carbonyl and hydroxyl groups are the dominant functional groups in the imprinted polymer and play a crucial role in the interactions between templates and polymers.
3.2.2. Thermal Analysis
Thermogravimetric curves of the materials studied are shown in
Figure 4a,b.
The polymers are stable to about 100 °C. For E2-MIP, the loss of mass was observed at lower temperature, which is related to its melting point, 178 °C for E2. The melting point of E1 is slightly higher and amounts to 260 °C. Therefore, around this temperature, a loss of mass for E1-MIP was observed. It confirms that during the process of polymer synthesis, the templates were stable. We successfully obtained polymers with various cavities binding E1 or E2 molecules. The greatest mass loss was observed in the range 180–300 °C, and it was more pronounced for E1-/E2-MIP and E1-/E2-mag-MIP than for NIP and mag-NIP. It is interpreted as the release of hormones from the materials’ structures. In the range of 178–300 °C the most intensive desorption of E2 was observed. For E1, the range was 260–300 °C. These ranges, 178–300 °C for E2 and 260–300 °C for E1, were applied for thermal desorption of the analyte from E1-/E2-MIP and E1-/E2-mag-MIP in FAPA-MS analysis. Twenty percent of residual material in the case of magnetic polymers may result from the presence of a magnetic core that has not decomposed within the temperature range.
3.2.3. SEM Images
All of the obtained materials were characterized with SEM. The SEM images (low (500×) and high (30,000×) magnification) are shown in
Figure S2. It is visible that the surface of E1-MIP/E2-MIP after the template removal is more homogenous in comparison the polymer before the template removal. Images reveal that the obtained magnetic particles have a spherical morphology and a diameter less than 100 nm. Due to their small size, these nanoparticles agglomerate easily. Moreover, the presence of the core ensures specific particle size and shape. The polymer builds up around the spherical core to form spherical particles whose area is greater than that of MIPs. Moreover, the recognition sites are located on the surface of the material, facilitating the analyte binding. Due to their magnetic and surface properties, mag-MIPs are more efficient than MIPs obtained by traditional synthesis methods, as indicated in
Section 3.2.5. These advantages of mag-MIPs render them highly attractive for a wide variety of applications in separation techniques.
3.2.4. Analysis of Estrogens in Aqueous Solution Using the ESI-MS Technique
The studies were carried out for aqueous solutions of E1 and E2 in the range from 0.27 μg L
−1 to 2.7 mg L
−1. In the ESI-MS analysis (positive ions mode) of the freshly prepared E1 solution, a signal was observed at
m/z 271 [M+H]
+ and a low intensity fragmentation signal
m/z 253 [M-18]
+ (
Figure S3). This mode of fragmentation of the E1 molecule was confirmed by the ESI-MS
2 spectra (
Figure S4). In the case of E2, we observed a signal at
m/z 273 [M+H]
+ and a fragmentation signal at
m/z 255 [M-18]
+ (
Figure S5). This fragmentation pathway of the E2 molecule was confirmed by the ESI-MS
2 spectra (
Figure S6). The relationship between the signal intensity and the concentration of analyte was plotted (
Figure S7). It was found that, for the samples prepared immediately prior to the measurement, the range of linearity in the E1 and E2 determination ranged from 0.135 to 2.7 mg L
−1, while the LOD of E1 and E2 in the ESI-MS method was 27 and 13.6 μg L
−1, respectively.
Under the influence of environmental factors (pH, temperature, and oxygen), estrogens undergo chemical transformation. The rate of this process in water is strongly dependent on the concentration of hormones. In laboratory conditions, the stability of E1 and E2 in water at pH = 7 was tested (
Figure 5b) as a function of time: measurements were performed after 5, 10, 20, 30, 60, 120 min, 24 h, and 7 days for two concentrations of each of the hormones: 5.4 and 1.35 mg L
−1.
E1 signal gradually disappeared in water samples, which was related to its inter-conversion into E2. The concentration drop was the most pronounced up to 30 min after the dissolution of E1 in water at pH = 7. After 120 min, its concentration remained constant. The concentration of E1 in water after this time was similar, 24 μg L−1 for both initial concentrations of E1 studied. This value decreased slightly after 24 h. It can be concluded that, in solutions with high dilutions of estrogens, compounds degrade more slowly. However, after a week, no signals from E1 were detected. The concentration of E2 was most intensively reduced in up to 10 min after its dissolution in water. Then, the concentration of E2 decreased slowly in direct proportion to the initial concentration. The E2 concentration after 120 min was 1.64 mg L−1 for the higher initial concentration and 0.41 mg L−1 for the lower one. Within 24 h, the concentrations slightly decreased, but, after a week, the signals from E2 were also not detected. The rate of decomposition of estrogens in water is an additional factor hindering the determination of this group of compounds. The development of selective MIPs/mag-MIPs, and their use for pre-concentration in chemical analysis using various mass spectrometry techniques, allows the development of a new analytical procedure for the qualitative and quantitative determination of E1 and E2 in aqueous solutions. Furthermore, the use of MIPs/mag-MIPs is a simple and effective method for stabilizing and storing analytes. Binding of E1 and E2 in polymer structures prevents their conversion and degradation.
3.2.5. E1-MIP, E2-MIP, E1-Mag-MIP and E2-Mag-MIP Selectivity for E1 or E2 in Hormone Aqueous Solutions
The synthesis of two types of magnetic and non-magnetic MIP allowed the use of two different isolation procedures of the analyte: centrifugation and magnetic separation. However, the main goal was to compare the performance of the two materials. For this purpose, 10 mg of empty E1-MIP and 10 mg of empty E1-mag-MIP were added to two separate, 10 mL aqueous solutions of 2.7 mg L
−1 E1. The concentration of E1 in water after the addition of the empty E1-MIP and E1-mag-MIP was examined after 2 h (
Figure 6a).
Based on the amount of E1 in the solution after 2 h, in the samples to which E1-MIP and E1-mag-MIP were added, the sorption capacity of the materials was determined. E1-MIP bound 0.02 g of E1 per 1 g of the polymer. In comparison, while 1 g of a E1-mag-MIP bound 0.03 g of E1.
During the measurement at time t = 0 h for E1 solution, the low intensity signal assigned to E2 was observed. It is a result of the rapid conversion of E1 into the more stable E2 in the aqueous environment. Two hours after the addition of E1-mag-MIP, no signals from E1 in the solutions were detected, indicating the complete binding of the analyte to the selective cavities in the polymer structure. In the sample with E1-MIP, a low intensity signal, confirming the presence of small amounts of E1, was recorded. This confirms that the mag-MIPs adsorb analyte more efficiently. After 2 h, in both cases, the presence of E2 was found, indicating rapid conversion of E1 after the release from the polymer structure (as a result of the equilibrium state), occurring before the non-converted molecules were bound again. In the E1-mag-MIP solution, a signal of lower intensity was recorded. The detected signals from E2 indicate that it was not adsorbed within the E1-MIP and E1-mag-MIP structure, which confirms the selectivity of the obtained material.
To determine the selectivity of the polymers in relation to E1 and E2, the change in the concentration of E1 in water after addition of empty E2-MIP and E2-mag-MIP was examined (
Figure 6b).
After 2 h of conducting the experiment, the E1 concentration decreased as a result of its inter-conversion into E2, which was successively bound by E2-MIP or E2-mag-MIP. E2-mag-MIP adsorbs newly formed E2 more quickly than E2-MIP. The state of chemical equilibrium shifts towards E2, and, as a result, E1 inter-converts faster in the sample with the addition of E2-mag-MIP. These studies confirmed that the polymers obtained have selective cavities for particular types of estrogens. In the first stage, E1 is transformed into E2, and, in the second stage, E2 is selectively adsorbed by E2-MIP and E2-mag-MIP.
The change in the concentration of E2 aqueous solution after addition of the empty E2-MIP and E2-mag-MIP after 2 h was also studied (
Figure 7a).
Immediately after sample preparation, the signal for E2 was recorded. In this case, E1 was not found in the solution. After 2 h, the concentration of E2 significantly decreased in both samples. In the solution, E2-mag-MIP was observed to bind the analyte more efficiently than E2-MIP. Comparing the decrease in the concentration of E2 in the solution after the addition of E2-MIP and E2-mag-MIP, it was found that 1 g of E2-MIP bound 0.02 g of E2, while 1 g of E2-mag-MIP bound 0.03 g.
To determine the selectivity of the polymers towards E1 and E2, the change in the concentration of E2 in water after the addition of empty E1-MIP and E1-mag-MIP was examined (
Figure 7b).
As E2 does not inter-convert into E1, only the signals from E2 were recorded. The decrease in the signal intensity after 2 h is associated with the rapid degradation in aqueous solutions, containing high concentration of the hormone. The loss of E2 after 2 h corresponds to the previously demonstrated hormone instability over time. E1-MIP and E1-mag-MIP do not absorb E2 in their E1 dedicated cavities.
The empty E1-MIP/E1-mag-MIP (10 mg) added to the aqueous solution of E1 or E2-MIP/E2-mag-MIP added to the aqueous solution containing E2 in the concentration range of 0.027–27 μg L−1 resulted in binding of about 95% of the analytes within the first 5 min. The difference in the concentrations of the analytes is a measure of selectivity of the polymers. The selectivity of mag-MIPs, as measured by the amount of adsorbed estrogens, was E1/E2 of 100/10 for E1-mag-MIP and 100/5 for E2-mag-MIP.
The addition of the polymers with no selective cavities, NIP and mag-NIP, to the E1 and E2 water solutions caused no changes in the hormones’ concentrations, due to the lack of binding properties. The decrease in the hormones’ concentrations was associated with the process of their degradation in aqueous solutions.
3.2.6. The Effect of pH on the Estrogens Release from E1-Mag-MIP and E2-Mag-MIP
To determine the stability of estrogens in mag-MIPs structures and the release kinetics of mag-MIPs analytes, their release was tested at various environmental pHs (
Figure 8a,b).
After 10 min of running the experiment, E1 release from the polymer structure was low. The maximum concentration of E1 in all pH tested occurred after 30 min. The process of analyte recovery was most efficient at pH = 5. A characteristic feature of the release curves is that it reaches a plateau, as the maximum value is reached. However, not in this case. At pH = 5, E1 degradation was the fastest, which is unfavorable from an analytical point of view. At pH = 9.5, less E1 was released; however, degradation was much slower. At pH = 7, the smallest amount of analyte was released, which persisted over time, but did not give reliable quantitative results. For E1 analysis, pH plays a significant role. E1 is the most stable at pH = 7, but it is difficult to recover it from the polymer structure at this pH.
E2 release from E2-mag-MIP was much faster than E1 from E1-mag-MIP. Due to the minimal sample preparation time of 10 min after introducing E2-mag-MIP into buffer solutions, in two cases, maximum values of E2 concentration were recorded at the beginning of the measurement. At pH = 5, E2 was released at the highest concentration. At pH = 7, the amount of E2 released was lower. Regarding degradation rates, in an acidic environment, the concentration dropped rapidly, signifying the fastest degradation. At the neutral pH, E2 degradation occurred more slowly. At pH = 9.5, the maximum release of E2 from E2-mag-MIP occurred after 30 min, followed by degradation. The amount of E2 released was comparable to the amount of E2 released at pH = 7. In all samples, E2 degraded very quickly, which practically prevented its quantitative determination after release from the polymer matrix.
The estrogens bound in the structure of mag-MIPs are stable over time, and the release can be controlled by the means of pH or by temperature change after separation from the solution. In the aqueous solution, when E1 and E2 are released from MIP or mag-MIP, their chemical transformation and degradation takes place. The solution to this problem is direct analysis of estrogens from polymeric structures by the FAPA-MS method.
3.2.7. Analysis of Estrogens in an Aqueous Solution Using E1-/E2-Mag-MIP and FAPA-MS Technique
The analysis of estrogens was carried out using a plasma stream as an ionizing agent for the analytes. The scheme of the FAPA-MS measuring system is shown in
Figure 9a,b.
The FAPA-MS spectra of estrogens using plasma ionization are shown in
Figure 10a,b and
Figure 11a–c.
The FAPA-MS spectrum of E1 shows a signal at
m/z 271 [M+H]
+, whereas the FAPA-MS
2 spectrum reveals the signals at
m/z 133,
m/z 157,
m/z 197, and
m/z 253 characteristic for E1 as a result of ion fragmentation
m/z 271 [M+H]
+. The FAPA-MS spectra of E1 using plasma ionization depend on the temperature of the heating table used for thermal desorption of the compound (260-300 °C). A signal with intensity changing from
m/z 269 [M-2H]
+ to 273 [M+H]
+ is observed in the E2 spectrum, while the FAPA-MS
2 spectrum reveals the signals at
m/z 135,
m/z 159,
m/z 173, and
m/z 255 characteristic of E2 as a result of ion fragmentation
m/z 273 [M+H]
+ and the signal at
m/z 251 (
Figure 11b) as a result of ion fragmentation
m/z 269 [M+H]
+ (
Figure 11c).
FAPA-MS analysis successfully detected the presence of E1 and E2 in samples. In addition, the source of FAPA ions utilizes mild ionization and provides less intense signals from fragmentation ions in comparison to ESI-MS. In the FAPA-MS
2 spectra, the most intense fragmentation signal comes from the water molecule cleavage. In accordance with the procedure described in the experimental part
Section 2.4.5, the obtained mag-MIPs were used to bind and concentrate estrogens occurring at low concentrations in water solutions and then to determine the estrogens content in polymers by means of thermal desorption and plasma ionization. The results are presented in
Figure 12a,b and
Figure 13a,b.
The results of the conducted research show that the technique applied allows determination of estrogens in water at a concentration of 0.271 μg L−1 and that estrogens bound in MIPs/mag-MIPs do not undergo transformation in up to 72 h. This was confirmed by testing the material after 24, 48, and 72 h. The spectra were identical to the one obtained at T = 0. It was demonstrated that bound hormones are stable in polymeric structures, which enables their quantitative analysis and gives the possibility of stable storage of the analyte for a short time (three days). The method involving the use of mag-MIPs to concentrate, transport, and then determine estrogens by the FAPA-MS technique makes it possible to reduce the limit of detection of these compounds to 0.136 μg L−1 and allows measurements to be made during the time when the conversion of E1/E2 bound in mag-MIPs structures is inhibited. In addition, in the FAPA-MS technique, the analyte is directly and completely recovered from the polymer structure. This solves the problem related to the pH of the water sample, estrogen inter-conversion, and degradation.