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Design and application of molecularly imprinted polymers for adsorption and environmental assessment of anti-inflammatory drugs in wastewater samples

In this study, a series of molecularly imprinted polymers (MIPs) have been synthesized using separately diclofenac, naproxen, and ibuprofen as templates with three different polymerization approaches. Two functional monomers, methacrylic acid (MAA) and 2-vinylpyridine (2-VP), were tested and ethylene glycol dimethacrylate (EGDMA) was used as crosslinker; also, template-free polymers (NIPs) were synthesized. It was found that the MIP with the highest retention percentage for diclofenac was the one prepared by the emulsion approach and with MAA (98.3%); for naproxen, the one prepared by the bulk polymerization with MAA (99%); and for ibuprofen, the one synthesized by bulk with 2-VP (97.7%). These three MIPs were characterized by scanning electron microscopy, thermogravimetric test, Fourier transform infrared, specific area measurements, and surface charge. It was found that the emulsion method allowed particle size control, while the bulk method gave heterogeneous particles. The three evaluated MIPs exhibited thermal stability up to 300 °C, and it was observed that 2-VP confers greater stability to the material. From the BET analysis, it was demonstrated that the MIPs and NIPs evaluated are mesoporous materials with a pore size between 10 and 20 nm. In addition, the monomer influenced the surface charge of the material, since the MAA conferred an acidic point of zero charge (PZC), while the 2-VP conferred a PZC of basic character. Through adsorption isotherms, it was determined that there is a higher adsorption capacity of the MIPs at acidic pH following a pseudo-second-order kinetic model. Finally, the MIPs were used to determine the non-steroidal anti-inflammatory drugs (NSAIDs) understudy in San Luis Potosí, México, wastewater, finding concentrations of 0.642, 0.985, and 0.403 mg L-1 for DCF, NPX, and IBP, respectively.


Introduction
Pharmaceutical compounds represent an environmental concern because of their presence in aquatic ecosystems, leading to ecotoxicological effects (Parolini 2020). In addition, due to their extensive use for diseases treatments in humans and animals, wastewater discharges from municipal systems present high concentrations of these compounds and their metabolites, leading to harmful effects on the environment shortly (Eslami et al. 2015).
Among pharmaceuticals, non-steroidal anti-inflammatory drugs (NSAIDs) diclofenac (DCF), naproxen (NPX), and ibuprofen (IBP) are the most frequently detected and distributed drugs in the environment (Świacka et al. 2021;Wojcieszyńska and Guzik 2020). For example, for DCF, Bonnefille et al. reported a review of the presence of this compound in water and aquatic organisms, finding concentrations in water ranging from 50 ng L −1 to 3000 ng L −1 (Bonnefille et al. 2018). Several adverse effects have been reported from exposure to NSAIDs in aquatic species. For example, IBP was reported to cause an increase in the activity of some antioxidant and biotransformation enzymes in zebrafish (Bartoskova et al. 2013). In addition, an alteration in biochemical biomarkers and DNA damage in Daphnia magna species has also been reported by exposure to DCF, NPX, and IBP (Gómez-Oliván et al. 2014). It has been reported that the elimination of NSAIDs in wastewater treatment plants (WWTP) is inefficient; as a consequence, they are discharged directly into water resources, reaching levels that produce effects on aquatic organisms such as hormonal effects on their reproduction, oxidative stress, and genotoxicity (Gani et al. 2021;Islas-Flores et al. 2017;Madikizela and Ncube 2021;Parida et al. 2021).
There are significant challenges in removing and monitoring NSAIDs in complex environmental matrices such as wastewater. Regarding monitoring, the most commonly used analytical methodologies for identification and quantification are high-performance liquid chromatography (HPLC) and spectrophotometric methods (Kot-Wasik et al. 2007). In most cases, the determination of NSAIDs involves a pre-concentration step followed by separation and quantification usually performed by liquid chromatographic methods (Paíga et al. 2015).
Research in the environmental field increasingly relies on specific and sensitive analytical methods, which must detect and quantify trace and ultra-trace pollutants in very complex matrices; sample preparation is a key step for the assessment of pollutants; this step includes the pre-concentration of the analyte and the removal of the matrix effect which allows to obtain lower detection limits and increase sensibility and selectivity. Studies on the quantification of NSAIDs in different environmental matrices have focused on the development of methods such as solid phase extraction (Zhou et al. 2021), solid phase microextraction (Shamsayei et al. 2018), and ultrasoundassisted dispersive liquid-liquid microextraction (Qiao et al. 2016), among others.
Among these techniques, solid phase extraction is one of the most widely used sample pre-concentration techniques due to the diversity of adsorbents, high pre-concentration capacity and recovery. According to the literature, the use of molecularly imprinted polymers (MIPs) as a highly selective adsorbent material is a promising approach for the quantification of emerging contaminants in complex matrices (Madikizela et al. 2018).
These adsorbents are three-dimensional synthetic materials with pores or cavities to specifically retain a molecule of interest; MIPs are synthesized in the presence of this template (analyte of interest), which gives the polymer the specific shape, arrangement, orientation, and bonds to selectively retain it, which after being removed from the binding sites leaves the empty cavities to be re-occupied by the analyte or a highly related compound (chemical family of the analyte). Although MIPs have been used in areas that require high selectivity such as chromatographic methods, sensors, and contaminant removal, the use as highly selective extraction materials is the most widely used application (Azizi and Bottaro 2020), because it exhibits low cost, easy preparation, reversible adsorption and desorption, thermal, mechanical, and chemical stability (de Leon-Martinez et al. 2020).
Therefore, this work's main objective was to synthesize and characterize molecularly imprinted polymers to selectively remove IBP, NPX, and DCF in an aqueous solution. Furthermore, the adsorption process was studied at different solution pH to evaluate the adsorption mechanism, and an analysis and quantification method of the three compounds is also proposed. Finally, the materials developed were used to pre-concentrate and quantify the presence of NSAIDs in wastewater from the city of San Luis Potosí, México.

Synthesis of molecularly imprinted polymers
The synthesis of the polymers was carried out with slight modifications of those reported in previous works of our research group (Diaz de León-Martínez et al. 2018), under three non-covalent approaches.
(i) Emulsion polymerization First, PVA (1.55 g) and SDS (0.1078 g) were dissolved in hot water (75 mL) in a flat-bottomed flask with stirring by a magnetic stir bar at 250 rpm under an inert nitrogen atmosphere (99.9% purity) and hermetically sealed. Then, in another flask, the pre-polymerization mixture was prepared, template molecule (DCF 60 mg, NPX 50 mg, or IBP 40 mg) and functional monomer (MAA 85 µL or 2-VP 110 µL) dissolved in methanol (2 mL) and porogen (toluene, 4 mL). Crosslinker (EDGMA, 2 mL) and initiator (AIBN, 150 µL) were added to this mixture and stirred with a magnetic stir bar at 250 rpm until complete dissolution. Subsequently, the pre-polymerization mixture was added droplet by droplet by syringe to the above mixture; the reaction was kept at a constant temperature of 70 °C in a glycerin bath with continuous stirring at 100 rpm for 24 h. Finally, the polymer was obtained and washed with hot water to remove PVA and SDS residues.
(iii) Co-precipitation polymerization This method consists of the same procedure and reaction conditions as the bulk polymerization, but with the addition of 5 times the porogen (toluene, 20 mL) and solvent (methanol 10 mL), with constant magnetic stirring at 100 rpm at 70 °C for 24 h.
The non-imprinted polymers (NIPs) were prepared in the same way as the corresponding MIPs but without the presence of the template molecule.
The obtained polymers were crushed in a mortar and sieved to a particle size of 250 μm. For cleaning of the template molecule in the MIPs, ten washing cycles were used with 10 mL of methanol in 0.1% 0.1 N HCl; each cycle was sonicated for 10 min and centrifuged at 2500 rpm for 5 min; the polymers were recovered and weighed to establish the reaction yield.

Determination of NSAIDs by HPLC-DAD
The determination of DCF, NPX, and IBP was performed by diode-array high-performance liquid chromatography (HPLC-DAD) (Agilent 1260 Infinity Series) at a wavelength of 220 nm. The separation was carried out through an Eclipse XDB C 18 column (4.6 × 250 mm, 5 µm). The mobile phase consisted of 40% ortho-phosphoric acid (pH 2.6) and 60% acetonitrile in an isocratic flow rate at 1 mL min −1 for 10 min with a sample injection volume of 20 μL.
The validation of the analytical method was carried out based on the guide for the validation of analytical methods for the determination of organic compounds at trace level (AOAC/FAO/IAEA/IUPAC 2000) by assessing the following parameters: limit of detection (LOD) and quantification (LOQ), linearity (R 2 ), sensibility, and precision. Linearity was expressed by the correlation coefficient (R 2 ) and sensitivity determined by the slope of the working range curve (LOQ-2 mg L −1 ) obtained from the average of seven curves; precision was obtained by repeatability (evaluating three curves in one day) and reproducibility (obtained from 7 calibration curves over three days). The limit of detection (LOD) was determined using the blank analysis method using the following formula: LOD = X + 3SD blank . The limit of quantification was calculated in concentration units as LOQ = LOD = X + 10SD blank .

Determination of the retention percentages
NPX, DCF, and IBP retention assays on the synthesized MIPs were performed by packing 20 mg of each MIP into 6 mL solid-phase extraction cartridges, and then, 1 mL of loading solution was added at a concentration of 2 mg L −1 of the corresponding NSAIDs used as the template for each imprinted polymer. The eluent of each loading was analyzed by HPLC-DAD, and the retention percentage (%R) was calculated with the following equation: where C o is the initial concentration of each NSAID in the load (mg L −1 ) and C f is the eluent concentration in each load (mg L −1 ). Subsequently, the MIP with the highest retention percentage for each NSAID was selected for further analysis.

Morphological characterization of MIPs/NIPs
Micrographs of the MIPs and NIPs were obtained using a field emission scanning electron microscope (FE-SEM) FEI model Inspect F50 to evaluate the surface and morphology of all the polymers obtained.

Thermogravimetric analysis
The thermogravimetric analysis was performed using a thermogravimetric analyzer (TGA), PerkinElmer, model Pyris Diamond TGA/DTA in the presence of nitrogen (60 mL min −1 ). The temperature profile programmed to the TGA consisted of two processes that occurred in series, first a drying process of the sample at 105 °C and then the pyrolysis process that took place by increasing the sample temperature up to 600 °C at 10 °C min −1 .

Infrared spectroscopy
The functional groups of the materials were evaluated by FT-IR Fourier transform infrared spectroscopy (Thermo Scientific, Nicolet iS10 model). Infrared spectra were collected in a spectral range from 500 to 4000 cm −1 .

Textural properties
The textural properties of the materials were obtained from nitrogen adsorption-desorption measurements at 77 K using an automatic adsorption instrument (Autosorb-IQ, Quantachrome Instrument Corp., Boynton Beach, FL, USA). The specific surface area (S BET ) was calculated using the BET method. Barrett, Joyner, and Halenda's (BJH) method was used to calculate the pore size distributions.

Surface charge distribution and point of zero charge
The surface charge of MIPs and NIPs was determined by the acid-base titration proposed by Kuzin and Loskutov (1996). For this purpose, 50 mL of neutralizing solutions with pH between 2 and 12 was prepared by adding various volumes between 0.2 and 10 mL of titrated solutions of HCl and NaOH, both at a concentration of 0.1 N. The solutions were then volumetrically titrated with a 0.1 N NaCl solution.
In a centrifuge tube, 0.1 g of MIP or NIP and 25 mL of the different neutralizing solutions were added; likewise, 25 mL of each solution was placed in centrifuge tubes that were left as blanks. Every 30 min, each tube was agitated until 2 h, and at the end, the pH was evaluated, and then, the potentiometric curves of the blank, MIP, and NIP were plotted.
The surface charge was calculated with the equation where C s is the surface charge of the adsorbent at a given pH value (C g −1 ), C N is the concentration of the neutralizing (2) solution (mol L −1 ), V A is the volume of 0.1 N NaOH or HCl solution used to reach a given pH value in the experiment using the adsorbent (L), V B is the volume of 0.1 N NaOH or HCl solution to reach a given pH value in the experiment using the adsorbent (L), V B is the volume of 0.1 N NaOH or HCl solution used to reach a given pH value in the experiment without adsorbent (L), m is the mass of MIP or NIP (g), and F is the Faraday constant (96,485 C mol −1 ).

Adsorption kinetics and isotherms
The Freundlich and Langmuir isotherm models have been widely used in fitting adsorption equilibrium data of organic compounds in the aqueous phase. Redlich-Peterson R is a three-parameter isotherm model that results from a combination of the Freundlich and Langmuir model (Soto et al. 2011). These models were used to fit the adsorption equilibrium data and their equations are shown below.
where C (mg L −1 ) is the adsorbate concentration at equilibrium, q m (mg L −1 ) is the maximum adsorption capacity, K (L mg −1 ) is the affinity constant related to the adsorption energy, k (mg 1−1/n L 1/n g −1 ) is a constant related to the adsorption capacity of the adsorbent, the constant n indicates the adsorption intensity, and a (L g −1 ), b (L mg −1 ) β , and b are constants of the Redlich-Peterson isotherm. Concentrations in the range of 1 to 500 mg L −1 were used for the isotherms, and all experiments were performed at 24 °C. The equilibrium adsorption capacity, q (mg g −1 ), was calculated according to the equation where C o (mg L −1 ) is the initial adsorbate concentration and V (L) and W (g) are the volume of the standard solutions and the weight of the polymer, respectively.
The kinetic study was performed following the retention assay methodology and determining the concentration of each free NSAID in the supernatant at time intervals with an initial concentration of 10 mg L −1 .
The kinetic test data were analyzed using the pseudofirst and pseudo-second-order equations. The pseudofirst-order (PFO) and pseudo-second-order (PSO) equations are shown below, respectively: where q e and q t (mg g −1 ) are the amounts of adsorbate bound to the adsorbent at equilibrium and at time t (min), respectively, and k 1 (min −1 ) and k 2 (g mg −1 min −1 ) are the PFO and PSO model constants, respectively. To fit the experimental data to the analyzed mathematical models, Statistica® version 7.0 software was used, and the best-fitting model was chosen because of the correlation coefficient (R 2 ) closest to 1.

Imprinting factor
To verify the selectivity of each of the MIPs against the template for which they were designed, a mixture containing DCF, NPX, and IBP, all at a concentration of 2 mg L −1 in Milli-Q water pH 6, was prepared. 1 mL of this mixture was then passed through each of the MIPs following the retention assay methodology. Similarly, the NIPs were analyzed to compare them to their corresponding MIP.
The specificity of MIPs and NIPs was estimated by the partition coefficient of the NSAIDs between polymer and solution. The partition coefficient was calculated by the following equation: where C p (mg L −1 ) is the amount of analyte bound to the MIP or NIP and C s (mg L −1 ) is the concentration of each analyte remaining in the loading eluent. Also, the imprinting factor (IF) was used to evaluate the selectivity of each MIP against the template for which it was synthesized in the presence of the other NIPs similar in structure. The IF was calculated with the following equation: where K MIP and K NIP represent the partition coefficients of the compounds understudy for the MIP and NIP, respectively. Additionally, the addition of sulfamethoxazole and metronidazole at 1 mg L −1 was performed to verify interferences with organic molecules not similar to NSAIDs.

MIPs for the determination of NSAIDs in wastewater
For the pilot study, five raw wastewater samples were obtained at the following locations: Camino a San Juanico el Chico, Periférico Norte, San Luis Potosí, coordinates 22°13′24.6″N 100°59′19.1″W. These water samples came from the locations' communities, and 1 L of raw water was collected at 1 m depth. Temperature (25 °C) and pH were recorded at the sampling site (7.1), and then, pH was adjusted to 2 with HCl to prevent the growth of microorganisms. Finally, the sample was frozen until analysis. Columns of 50 mL were packed with 50 mg of each MIP to pre-concentrate DCF, NPX, and IBP. 40 mL of wastewater was passed through each column, and then, 4 mL of MeOH was added as elution. Finally, this eluent was filtered with 0.45 µm PTFE filters, and each elution was analyzed by HPLC-DAD. In parallel, the same methodology was followed, but sampling 40 mL of synthetic water containing 1 mL of a mixture of 1 mg L −1 of each NSAID at pH 6. Each experiment was performed in triplicate at 24 °C.

Synthesis of imprinted and non-imprinted polymers
A total of 18 MIPs and 6 NIPs were obtained (6 MIPs per template molecule) (Table 1). Between 1.5 and 2 g of polymer were obtained for the co-precipitation and bulk methods, and between 0.8 and 1 g were obtained for the emulsion method. The cleanup of the MIPs was analyzed by HPLC to verify the absence of each one of the NSAIDs. Figure 1 presents a chromatogram of the fifth wash of the MIP for DCF synthesized by emulsion using MAA as monomer and the chromatogram in the tenth wash, where it is observed the absence of the DCF peak that corroborates that the MIP was free of the template; this was proven for each NSAID, found that at wash 7 and 8 it was free of NXP and IPB, respectively. This result is interesting as it indicates the appropriate conditions for recovery for MIP as a function of the concentration of the NSAIDs; thus, lower concentrations require less solvent volume for MIP regeneration. This has been reported in MIPs of MAA with a β-cyclodextrin template where they observed that in 3 washes of 30 mL of MeOH and water, regeneration of the polymer is achieved (Wang et al. 2019).
The validation of the method was obtained from the calibration curves for each NSAID obtained from 0.05 to 2 mg L −1 . In addition, linearity for all NSAIDs was verified with the R 2 value being greater than 0.999; a limit of detection of 0.029 mg L −1 , 0.018 mg L −1 , and 0.057 mg L −1 for DCF, NPX, and IBP, respectively; and a limit of quantification of 0.18, 0.11, and 0.33 mg L −1 for DCF, NPX, and IBP, respectively. Figure 2 shows an example chromatogram showing the retention times for NPX at 4.8 min, DCF at 7.9 min, and IBP at 9.1 min. Figure 3a shows the retention percentages obtained for the MIPs of DCF, NPX, and IBP. The MIP synthesized by the emulsion method for DCF (ME-DCF-01) and bulk for NPX (MB-NPX-03), both prepared with methacrylic acid (MAA) as a monomer, obtained the highest retention percentages of 98.3% and 99.0%, respectively. Concerning their NIPs (NIP-E1, NIP-B3), they reached between 50 and 58%.

Retention percentages
The MIP with the IBP template with the highest percentage retention was synthesized in bulk using 2-VP as the monomer (MB-IBP-04), with an IBP retention of   Figure 3b shows the comparison of the retention percentages of the MIPs with their corresponding NIPs (NIP-E1, NIP-B3, and NIP-B4); it can be observed a difference in the results obtaining retention values of the NIPs, due to the imprinting of the template molecules. In other studies, it has already been observed that NIPs have adsorption capacity; however, the recovery of the analyte is generally lower than that of the imprinted polymer, which proves the effect of molecular imprinting (Liu et al. 2020;Zheng et al. 2018). Thus, high retention percentages were considered as selection factors of the MIPs for the following tests.

Morphological characterization by scanning electron microscopy
Micrographs obtained for DCF MIP and NIP, both synthesized by emulsion, are shown in Fig. 4a, b. The images show spherical particles for both MIP and NIP, with a size up to 1 μm. This particle shape is commonly obtained in emulsion-synthesized polymers; this technique allows to control how the polymer is created within the micelles in solution (Chen et al. 2015).
In Fig. 4c, d, micrographs are presented for the polymers MB-NPX-03 and MB-IBP-04, both mass-synthesized, for which agglomerated particles (cauliflower-shaped) of irregular and heterogeneous size were observed. These characteristics were also present in their corresponding NIPs.
Since both MIPs and NIPs have similar morphology when synthesized by the same method, this may indicate that the presence of DCF, NPX, or IBP in the polymer matrix does not interfere with the obtained morphology of the polymers. The morphologies are due to monomer-crosslinker-porogen interaction; Renkecz et al. (2014) showed that in copolymerization using MAA-EGDMA and toluene as solvent, the growth of cauliflower-shaped microparticles probably proceeds with the enthalpic precipitation mechanism that is usually observed in low solvency media (Renkecz et al. 2014). Thermogravimetric analysis Figure 5 shows the mass loss (ML) curves for each evaluation. All the polymers lose water in the initial step, up to approximately 100 °C. The MB-NPX-04 polymer was the least stable, presenting the highest weight loss up to 75% at 400 °C. It was observed that MB-IBP-04 was the most stable MIP with a weight loss of 60% at 400 °C; this result can be explained by the 2-vinylpyridine monomer that provides greater stability to the material compared to the polymers synthesized with methacrylic acid. For all three MIPs, thermal stability up to around 300 °C was observed, thus confirming that these materials withstand critical working conditions. The derivative of mass loss (dML) curves are presented in Fig. 6. It is observed that the thermal degradations take place in one or two steps for the polymeric materials. For ME-DCF-01, the main thermal event starts around 200 °C, has the maximum degradation rate at 363 °C with a weight loss of 35%, and stops around 460 °C. MB-NPX-03 polymer has a maximum degradation rate at 345 °C with a weight loss of 42%; this single thermal degradation step starts around 250 °C and ends around 440 °C. Finally, for MB-IBP-04, thermal degradation occurs in two steps. The main step starts around 250 °C, having a maximum degradation rate at 319 °C with a weight loss of 32%, and ends around 440 °C. The second step has a maximum degradation rate of around 400 °C.
The MIP of DCF and NPX (ME-DCF-01 and MB-NPX-03, respectively) exhibit very close maximum degradation rates because both polymers were synthesized with MAA as the monomer. This compound in a temperature range of 270 °C to 450 °C decomposes by dehydration of the acid groups forming anhydrides, which then decompose by decarboxylation (Cervantes-Uc et al. 2006;Dima et al. 2012). For MB-IBP-04, the weight loss in the 300 °C to 400 °C regions is mainly attributed to cleavages in the crosslinker-monomer bonds and thermooxidative degradation of the vinylpyridine chain, respectively (Jaymand 2011; Wu et al. 2003).

Fourier transform infrared spectroscopy (FT-IR)
The FT-IR spectra for DCF, NIP-E1, and ME-DCF-01 are observed in Fig. 7a. Two characteristic bands are seen at 1573 and 1397 cm −1 , related to C = OCOO-stretching vibrations and O-CO-CH3 bending vibrations; respectively, these are attributed to the functional groups' diclofenac. The broad bands at 3401 and 3235 cm −1 are attributed to the N-H bond-related bending and stretching vibrations, which may be due to template and polymer matrix effects. The sawtooth peaks in 700-800 cm −1 can be attributed to aromatic substitutions at positions 1, 2, and 3 of the DCF. Therefore, all these bands are associated with the DCF molecule, confirming that the template molecule was imprinted on the MIP. Figure 7b shows the FT-IR spectra of the NPX, NIP-B3, and MB-NPX-03 samples. The peaks confirm the presence of the template molecule (NPX) in the MIP at 1586, 1370, and 800-900 cm −1 , corresponding to the vibrations of the aromatic C═C, aromatic ─CH 3 , and adjacent aromatic 1,2-hydrogen bonds, respectively. The similarity in peaks between MIP and NIP spectra of NPX at 2983 and 2998 cm −1 (CH 3 bond vibration), 1723 cm −1 (corresponding to C═O bond), 1631 and 1625 cm −1 (C═C bond stretching vibration), and 1450 and 1454 cm −1 (C-O bond vibration) is attributed to the functional groups of the polymeric matrix, which is the same for MIP and NIP.
The spectra of IBP, NIP-B4, and MB-IBP-04 are shown in Fig. 7c; the characteristic bands of IBP corresponding

Specific area and pore diameter
The specific area and average pore diameter of MIPs and NIPs were calculated using the BET and BJH methods. Table 2 shows the obtained values, where specific area values in the range of 111 to 410 m 2 g −1 are observed, indicating that both MIPs and NIPs are materials with high porosity. In addition, the obtained pore diameters evidenced that all the materials are mesoporous. The specific area of MIPs is larger than that of NIPs; this result is explained by the effectiveness of stencil printing (Tabaraki and Sadeghinejad 2020). In addition, it was found that the mass polymerization method improves pore formation and specific area since values of the specific area and pore volume are higher than the values obtained from the polymers obtained by emulsion.
It has been reported that in polymerizations, toluene is a good pore former in the presence of MAA and EDGMA (Sellergren and Shea 1993). These results were confirmed by Renkecz et al. (2014), where they observed a difference in pore size of up to fourfold of DCF MIPs synthesized with toluene with respect to chloroform (Renkecz et al. 2014). On the other hand, when comparing the results of surface area for NPX (S BET : 17 m 2 g −1 ) and DFC (S BET : 24 m 2 g −1 ) MIPs of this author, we observed that our MIPs present from 4.6 to 17 times greater surface area with respect to their MIPs; it should be noted that they used 4-vinylpyridine as monomer.

Point of zero charges (PZC) of the adsorbent
The surface charge of material in an aqueous solution is generated from interactions between ions in solution (H + and HO −) and the surface functional groups of the adsorbent.  Surface charge and point of zero charges (PZC) are essential features in adsorbent surface science and play a key role in the adsorption process. The surface charge of the adsorbent with acidic and basic groups is highly dependent on the pH of the surrounding solution, and the interaction between the adsorbent surface sites and the electrolyte species determines the PZC (Madrakian et al. 2013).
Depending on the pH of the solution, the surface-active sites of an adsorbent may undergo protonation or deprotonation, and thus, a charged surface will be formed. Figure 8a shows the potentiometric curves for the blank and the solution with the ME-DCF-01 polymer. It is observed that the curves intersect at a pH value of 3.35, indicating that the PZC for MIP is acidic, similarly observed for NIP-E1, where the curves intersect at 3.15 (Fig. 8b). Concerning the polymers MB-NPX-03 and NIP-B3, acidic PZC of 3.18 and 3.12 were obtained, respectively. These results can be attributed to the monomer used in the synthesis of the polymers since they were all synthesized with methacrylic acid (weak acid pKa 4.7).
On the contrary, for the polymers MB-IBP-04 and NIP-B4, the PZC was 8.89 and 7.98, respectively, so the PZC is basic. The above is attributed to 2-vinylpyridine, which is a weak base of pKa 4.9.

Adsorption isotherms and kinetics
The obtained kinetic data were analyzed by the pseudofirst-order (PFO) and pseudo-second-order (PSO) models. Table 3 shows the kinetic adsorption parameters of DCF, NPX, and IBP on the corresponding MIP and NIP with an initial concentration of 10 mg L −1 . It is observed that all the tested polymers were best fit to the PSO model due to the R 2 value close to 1. These results could indicate that the target molecules are adsorbed on the binding sites via chemisorption bonds, which involve a chemical reaction or chemicallike force bonding between the functional groups of the adsorbent and the template. The best fit to the PSO model indicated that the adsorption mechanism could depend on both the adsorbate and the adsorbent, and the rate-limiting step is chemisorption involving valence forces through the exchange of electrons (Surikumaran et al. 2014).
For all MIPs and NIPs, adsorption increased rapidly in the first 20 min and reached equilibrium at 30 min. This type of polymeric adsorbent generally shows very fast equilibrium times, representing an excellent methodological advantage. With other adsorbents, the equilibrium time can take several days (Guo et al. 2018). On the other hand, the MIPs presented higher adsorption capacities compared to  Table 3 Comparison of pseudo-first-order and pseudo-second-order kinetic parameters for MIPs and NIPs 20 mg of MIP or NIP, initial concentration of 10 mg L −1 , pH 6, 24 °C q e,exp and q e,cal are the amount of adsorbate (mg g −1 ) on adsorbent at experimental and calculated equilibrium, respectively k 1 and k 2 constants of the pseudo-first-order and pseudo-second-order model, respectively Pseudo-second-order MIP/NIP q e,exp (mg g −1 ) q e,cal (mg g −1 ) k 1 (min −1 ) R 2 q e,cal (mg g −1 ) k 2 (g mg −1 min −1 ) the non-imprinted polymers. As shown in Fig. 9, for MB-IBP-04, q e was 0.514 mg g −1 , exhibiting a higher adsorption capacity than NIP-B4 (0.264 mg g −1 ) and obtaining faster adsorption due to the higher value of the k 2 constant. This phenomenon was observed for all 3 MIPs evaluated and their corresponding NIPs; thus, this confirms the positive effect of molecular imprinting on the adsorption capacity.
The MIPs show similar adsorption mechanisms, due to similarities in adsorbent synthesis, both in terms of polymerization reagents and methodology, and to the physicochemical properties of the analytes, mainly the very close pKa of the 3 NSAIDs.
Equilibrium data were fitted to the Freundlich, Langmuir, and Redlich-Peterson models. The best fit of the isotherm models was chosen by the value of the correlation coefficient (R 2 ) closest to 1. The constants of the adsorption isotherms at different pHs for the MIPs/NIPs are shown in Table 4. The polymers ME-DCF-01 and MB-NPX-03 fitted the Langmuir model best at pH 6 and 24 °C (R 2 = 09,846 and 0.9896, respectively), which indicates that adsorption can occur in a monolayer and is considered a homogeneous surface with a specific number of binding sites where a molecule can be adsorbed. It should be noted that in ME-DCF-01 an R 2 value closer to 1 for the Redlich-Peterson model was obtained; however, because β equals 1, the isotherm is transformed to the Langmuir isotherm; therefore, the model is adequately described by the two isotherms mentioned. In comparison, the corresponding NIPs, NIP-E1 and NIP-B3, were better fitted to the Freundlich and Langmuir model, respectively.
The imprinted polymer MB-IBP-04 best fits the Freundlich model at pH 6 and 24 °C (R 2 = 0.9896), indicating that adsorption occurs in multilayer and the value of n > 1 Fig. 9 Pseudo-second-order kinetic models for MB-IBP-04 and NIP-B4 (20 mg MIP or NIP, initial concentration of 10 mg L −1 , pH 6, 24 °C) Table 4 Adsorption isotherm constants for the MIPs/NIPs evaluated at different pH (20 mg MIP/NIP, 30 min contact, 1 mL solution, medium temperature 24 °C) 20 mg of MIP or NIP, 24 °C. q m is the maximum amount of adsorbate (mg g −1 ) on adsorbent at equilibrium. K is the Langmuir constant, k is the Freundlich constant, and n is the exponent of the Freundlich isotherm. a, b, and β are the constants of the R-P isotherm and R is the correlation coefficient MIP/NIP pH Langmuir Freundlich Redlich-Peterson q m (mg g −1 ) K (L mg −1 ) R 2 k (mg 1−1/n L 1/n /g) n R 2 a (L g −1 ) b (L mg −1 ) β β R 2 ME-DCF-01 3 14. indicates a favorable adsorption condition in IBP MIP. This model describes that most of the surfaces are heterogeneous, and multiple sites are available for adsorption. The obtained values of k indicate that the molecules are adsorbed with an intensity characteristic for a chemisorption process. As for NIP-B4, it fitted best to the Langmuir isotherm, following the characteristics mentioned above for this model. When analyzing the influence of pH in the medium, it was found that the adsorption capacity of the MIPs against each NSAID is higher in the acidic region than in the alkaline region. As can be observed in Fig. 10a, b with the imprinted polymers, adsorption at pH 3 and 6 has a higher removal of DCF and NPX compared to adsorption at pH 9. This is because both MIPs at pH 3 have a surface charge close to zero since the PZC is 3.35 for the DCF MIP and 3.18 for the NPX MIP. Furthermore, at pH 6, good removal of each template molecule is obtained because the surface charge, although negative, is very low, so there is no noticeable repulsion between the adsorbent and the adsorbate.
In addition, because the pKa of DCF is 4.2 and that of NPX is 4.3, these pollutants exist mainly as their conjugate base forms at basic pH (Fig. 10). Consequently, the electrostatic repulsion is lower at acidic pH in solution so that, at pH 9, the adsorption of both analytes decays. These results suggest that attractive hydrogen bonds and other interactions promote adsorption.
For MB-IBP-04, higher IBP removal efficiency was obtained in a pH range of 3 to 6 (Fig. 10c). This is because the surface charge of the MIP is almost neutral in that pH range; in addition, the pKa for 2-VP is 4.9, which promotes Van der Waals force interactions between the IBP molecules and the adsorbent since at pH < 4.9 IBP is in its protonated form. The removal efficiency decreased at pH 9 due to repulsive interactions between negative IBP molecules and the MIP surface. The results obtained in the isotherms by the influence of the pH of the medium suggest that the main interactions between DFC/NPX/IBP and each MIP or NIP were mainly related by electrostatic or Van der Waals forces. A good imprinting effect was also demonstrated for MIP having a higher adsorption efficiency than NIP over the whole pH range evaluated.
A comparison of the values obtained with other MIPs synthesized with different methods was performed; it is observed that the retention percentages are similar in all the studies (Table 5). In the study carried out by Madikizela and Chimuka (2016a), a MIP was designed by the bulk method with NPX, IBP, and DCF as templates, 2-vinyliridine as functional monomer, and EDGMA as crosslinker. Fig. 10 Effect of pH on the best-fit adsorption isotherms for each NSAID on the corresponding MIP at 24 °C. a Redlich-Peterson isotherms for ME-DCF-01. b Langmuir isotherms for MB-NPX-03. c Redlich-Peterson isotherms for MB-IBP-04. 20 mg of MIP or NIP, 30 min contact, 1 mL solution, medium temperature 24 °C In the study, the MIP fits the Langmuir model better, and equivalent maximum adsorption capacities were obtained compared to our material.

Imprinting factor and selectivity of MIPs and NIPs
To evaluate the selectivity of the imprinted polymers against the molecule for which it was designed, the imprinting factor (IF) was calculated for each MIP; these values are presented in Table 6. It was observed that the polymer ME-DCF-01 and MB-NPX-03 with methacrylic acid monomer showed a high adsorption capacity (q e ) to adsorb DCF and NPX and low for IBP; similarly, the K MIP partition coefficient values are higher for DCF and NPX than for IBP; these results were similar in the study of Dai et al. (2013), where they indicate the similarity of extraction between carbamazepine and DCF; this study explains the imprinting between the template molecule with the monomer. Carbamazepine and DCF have a benzene ring and carboxylic group that could have printed similarly with the 2-vinylpyridine monomer used in this study, which affected the recognition and resulted in high extraction capacities but low selectivity (Dai et al. 2013). Although IBP has a similar structure to DCF and NPX, the extraction capacity is lower than DCF and NPX, indicating that the memory of the specific functional group plays an important role in conformation memory (An et al. 2008). Thus, MIP recognition was dominated not only by the size of the molecule but also by the distribution of the functional groups themselves.
The imprinted polymer MB-NPX-03 obtained a higher IF towards NPX; however, the retention rate towards DCF and IBP is high, 97.6% and 89.8%, respectively. This shows that the polymers ME-DCF-01 and MB-NPX-03 can adsorb in high percentages the 3 studied NSAIDs, since the molecules are homologous and have very similar physicochemical properties, such as pKa (4-5), so the mole fraction of protonated species and conjugated bases will be practically the same at any pH in the medium. In contrast, MB-IBP-04 obtained a higher IF towards IBP compared to those obtained towards DCF and NPX, so this MIP is selective towards its template molecule; possibly, the success in molecular imprinting in MIP is due to the simpler and less sterically hindered structure of IBP. This result was similar to that obtained by Madikizela and Chimuka, obtaining a percentage of recovery between 85 and 103% for the same NSAIDs evaluated in this study, synthesizing a polymer by bulking using 2-VP as  (Farrington and Regan 2007) monomer and the 3 NSAIDs in combination as a template (Madikizela and Chimuka 2016b). On the other hand, an interference assay was performed with sulfamethoxazole and ciprofloxacin, and retention of less than 30% of these compounds was observed, indicating that the MIPs of NSAIDs are specific because they are similar molecules. Additionally, the values of partition coefficients determined for MIPs (K MIP ) and NIPs (K NIP ) and shown in Table 6 ranged from 430.77 to 2980.3 and from 97.93 to 197.52 mL g −1 , respectively. According to Parra-Marfíl et al. (2020), partition coefficient values higher than mL g −1 are considered adequate, indicating that MIPs are highly affine to DCF, NPX, and IBP. The %R or q e values give a measure of the adsorbate removed, while the partition coefficient is a direct measurement of the affinity of the adsorbent for adsorbate under the conditions it is tested (Parra-Marfíl et al. 2020).

Pilot study: concentrations of NSAIDs in wastewater from the city of San Luis Potosí, Mexico
A pilot study was conducted using the characterized MIPs in wastewater samples from San Luis Potosí city to evaluate the feasibility of applying our technology. Table 7 shows the average concentrations of each NSAID found in raw wastewater and fortified water samples at 1 mg L −1 . With the use of the imprinted polymers, the following amounts of each template molecule were concentrated: with ME-DCF-01 0.642 mg L −1 ± 0.068 mg L −1 , with MB-NPX-03 0.987 mg L −1 ± 0.184 mg L −1 , and with MB-IBP-04 0.403 mg L −1 ± 0.071 mg L −1 . Figure 11 shows examples of the chromatograms in fortified water and problem sample (wastewater); it is observed that the 3 MIPs present excellent retention of NSAIDs, but we consider that the most important result in this section was the evaluation of the fortified water, demonstrating the high pre-concentration of NSAIDs, but also the decrease of the matrix effect in a complex sample.
Higher concentrations were obtained for the template molecules using their corresponding MIP, while extraction of molecules different from the MIP template obtained lower concentrations; this indicates that the evaluation of NSAIDs should focus on the corresponding MIP for each target molecule.
In our country, there are no regulations that determine the concentrations and discharges of pharmaceuticals in water; in addition, these types of compounds are not eliminated in their entirety in the conventional wastewater treatment process. The NSAIDs evaluated in the present study are the most consumed and therefore discarded worldwide; now, in the context of COVID-19, the use of antipyretics and anti-inflammatory drugs increased up to 27% (O'Flynn et al. 2021), as well as their presence in wastewater treatment plants (Yan et al. 2021). From the pharmacological point of view, NSAIDs are primarily designed to carry out physiological processes and resist inactivation before exerting the therapeutic effect for which they were developed. These properties are also responsible for their low biodegradability and potential toxic effects in aquatic and terrestrial ecosystems, which is increased by the continuous introduction into these environments (Joachim et al. 2021;Wang et al. 2021).
To compare NSAID concentrations in the study, research conducted in Mexico, where the concentrations of these compounds have been determined in wastewater treatment plants, were evaluated. Rivera et al. evaluated the concentration of different drugs in the influent and effluent of a wastewater treatment plant in Cuernavaca, Morelos, finding in the influent a concentration of DCF of 0.0023 mg L −1 , 0.0026 mg L −1 of NPX, and 0.0019 mg L −1 of IBP, while in the effluent they reported a concentration of DCF of 0.002 mg L −1 , 0.00026 mg L −1 of NPX, and no IBP was detected (Rivera-Jaimes et al. 2018). In another study, 12  . 11 Chromatograms of samples of fortified water (1 mg L −1 ) and wastewater. a MB-IBP-04/IBP-fortified water. b MB-IBP-04/IBP-wastewater. c ME-DCF-01/DCF-fortified water. d ME-DCF-01/DCF-wastewater. e MB-NPX-03/NPX-fortified water. f MB-NPX-03/NPX-wastewater effluents from three wastewater treatment plants in the Metropolitan Zone of the Valley of Mexico were analyzed: University City (CU), Coyoacán, and Cerro de la Estrella. For IBP, the concentration in the effluents ranged from 0.0002 to 0.0028 mg L −1 . NPX was the analyte quantified in the highest concentration in the three treatment plants; in the effluents, its concentration ranged from 0.0028 to 0.054 mg L −1 (Peña-Álvarez and Castillo-Alanís 2015). In developing countries, monitoring of the pollutants in this study is not routinely performed; therefore, there are no regulations that establish permissible limits in environmental matrices that protect human and ecosystem health . ). This may be due to the lack of availability of highly sensitive instrumentation such as liquid chromatography coupled to time-of-flight and quadrupole mass spectrometry and lack of access to purchase or/and maintain the operating costs associated with these technologies. To address this challenge, research must focus on the development of preparative techniques that can improve the sensitivity of laboratory instruments. We demonstrated that the synthesized materials show similar retention and reproducibility values to various studies; however, no significant applications have been demonstrated in wastewater in developing countries. Thus, research in the environmental area is of great relevance in our country, with the purpose of providing information to the authorities for the establishment of pertinent regulations for the protection of the environment and human health.

Conclusion
A highly selective and efficient material was developed for the quantification of DCF, NPX, and IBP in wastewater samples. 10 min of contact with the solution and 20 mg of MIP were sufficient to obtain high percentages of DCF, NPX, and IBP recovery of 98.3, 99, and 97.7% respectively. The three evaluated MIPs exhibited thermal stability up to 300 °C, and it was observed that 2-VP confers greater stability to the material; in addition, we obtained mesoporous materials with a pore size between 10 and 20 nm. Additionally, it was observed that the material charge is influenced by the functional monomer; in the case of the DCF and NPX MIPs, the PZC value was 3.35 and 3.15 using the MAA monomer, and for the IBP MIP, it was 9.89 using 2-VP; this is related to the release of the compounds from the material observing that at pH 3 a higher release occurs regarding pH 9 for DFC and NPX; in the case of IBP, higher IBP removal efficiency was obtained in a pH range of 3 to 6; this is because the surface charge of the MIP is almost neutral in that pH range; in addition, the pKa for 2-VP is 4.9, which promotes Van der Waals force interactions between the IBP molecules and the adsorbent since at pH < 4.9 ibuprofen is in its protonated form.
Higher concentrations were obtained for the template molecules using their corresponding MIP, while extraction of molecules different from the MIP template obtained lower concentrations; this indicates that the evaluation of NSAIDs should focus on the corresponding MIP for each target molecule. Finally, the MIPs were used to determine the NSAIDs understudy in San Luis Potosí, México, wastewater, finding concentrations of 0.642, 0.985, and 0.403 mg L -1 for DCF, NPX, and IBP, respectively. In our country, there are no regulations that determine the concentrations and discharges of pharmaceuticals in water; in addition, these types of compounds are not eliminated in their entirety in the conventional wastewater treatment process. The NSAIDs evaluated in the present study are the most consumed and therefore discarded worldwide; now, in the context of COVID-19, the use of antipyretics and anti-inflammatory drugs increased up to 27% (O'Flynn et al. 2021), as well as their presence in wastewater treatment plants (Yan et al. 2021). Drug extraction using MIPs can effectively replace traditional extraction methodologies due to the multiple advantages that they present, such as reusability, low cost, and stability. Consequently, molecularly imprinted polymers could be an alternative for the determination of this type of contaminants in complex environmental matrices.