Monitoring pharmaceuticals in the aquatic environment using enzyme-linked immunosorbent assay (ELISA)—a practical overview

Abstract The presence of pharmaceuticals, which are considered as contaminants of emerging concern, in natural waters is currently recognized as a widespread problem. Monitoring these contaminants in the environment has been an important field of research since their presence can affect the ecosystems even at very low levels. Several analytical techniques have been developed to detect and quantify trace concentrations of these contaminants in the aquatic environment, namely high-performance liquid chromatography, gas chromatography, and capillary electrophoresis, usually coupled to different types of detectors, which need to be complemented with time-consuming and costly sample cleaning and pre-concentration procedures. Generally, the enzymelinked immunosorbent assay (ELISA), as other immunoassay methodologies, is mostly used in biological samples (most frequently urine and blood). However, during the last years, the number of studies referring the use of ELISA for the analysis of pharmaceuticals in complex environmental samples has been growing. Therefore, this work aims to present an overview of the application of ELISA for screening and quantification of pharmaceuticals in the aquatic environment, namely in water samples and biological tissues. The experimental procedures together with the main advantages and limitations of the assay are addressed, as well as new incomes related with the application of molecular imprinted polymers to mimic antibodies in similar, but alternative, approaches.


Introduction
Environmental pollution is an issue with multidisciplinary impacts and implications and water pollution is one of the most relevant problems of the current century. All living organisms depend on water and its contamination worsen even more the scarcity of potable water resources. A new global water quality threat is related to the so-called emerging contaminants, which are not commonly monitored but have the potential to enter the environment and cause known or suspected adverse ecological and/or human health effects [1]. Pharmaceuticals are an important group amongst these emerging contaminants that have been under increasing scientific scrutiny [1][2][3]. In general, their occurrence in the environment ranges between the nanograms to micrograms per litre [1,4,5].
They have been detected in influents and effluents from Sewage Treatment Plants (STPs), groundwater, surface water, and even drinking water [10,[16][17][18][19][20][21][22]. The low concentrations of pharmaceuticals in the aquatic environment implies the need for techniques with adequate sensitivity for its monitoring. In this context, conventional chromatographic techniques are 9 show that this technique has potential for environmental applications and can present some advantages relatively to the traditional chromatographic techniques.
The main advantages shown by ELISA are the low detection limits without sample pre-treatment, the analysis of several samples simultaneously, and the low cost of the equipment. ELISA is also a good tool to assess the spatial and temporal distribution of a single analyte [41] or to be used in a first approach in large-scale environmental screenings.
The main limitations of ELISA include the use of antibodies, which sometimes could be expensive; the possible need of coupling sample pre-treatment procedures; crossreactivity (CR) effects due to the presence of compounds similar in structure to the target analyte; and, as for other analytical techniques, matrix effects (mainly, pH, natural organic matter, and salinity) [67]. Also, the use of organic solvents can be problematic due to enzyme denaturation. Also, ELISA is known to be mostly a single analyte technique, which is a disadvantage relatively to other chromatographic techniques that possess multiplex capacities. To overcome this, there have been an increase trend in the development of multiplex immunoassays using antibody microarrays chips and bead-based assays [68,69].
Studies on the application of multiplexed assays for the determination of pharmaceuticals in real aquatic samples are scarce. An interesting example is the work by Carl et al. [7[]0] who successfully applied a multiplexed ELISA for the determination of caffeine, carbamazepine, diclofenac and isolithocholic acid in real wastewater samples, with subsequent validation with LC/MS/MS. However, these techniques also present their own disadvantages mainly related with CR that tend to increase with increasing number of target analytes [68]. In fact, the referred limitations of ELISA, CR is one of the most relevant in what concerns environmental samples, being an indicator of the assay specificity. CR is related to the response of the antibody when in presence of other compounds usually similar to the analyte [67]. CR is defined as the mass or concentration of interferent required to displace 50% of the label. So, the percentage of CR is equal to 100 times the concentration of analyte (S) at 50% response divided by the concentration of interferent (I) at 50% response (equation 3).

% =
[ ] 50 [ ] 50 × 100 (equation 3) The presence of structurally similar compounds presenting significant CR can influence the specificity of the immunoassay and overestimate the concentrations of the target analyte or even generate false positives. For that reason, it is important to assess the specificity of the antibody and the evaluation of possible cross-reactants present in samples in order to adequately use ELISA, as a quantification/screening method, for environmental monitoring. Yet, the selection of possible cross-reactants might be very challenging. Crossreactants with unexpectedly different chemical structure can appear, especially when the matrices are as complex as environmental samples.

ELISA as a tool for monitoring pharmaceuticals in the aquatic environment
In the past 20 years, a substantial amount of work has been done to determine the occurrence, fate, effects, and risks of pharmaceuticals in the environment [71]. In that sense, since ELISA has been applied to monitor the presence of these organic contaminants in water systems and also in biological tissues, some of them from organisms used as indicators of environmental pollution [22,23,41,49,66,[72][73][74][75][76][77], the following two sections present the literature related to this subject. 11 ELISA has been used alone as an analytical tool to detect and/or quantify pharmaceuticals in the environment, however there are also some reports about the use of ELISA combined with extraction and preconcentration methods to enhance the detection signal or to clean-up the samples. Tables 2 and 3 Table 2) and endocrine disruptors and hormones (presented in Table 3), in environmental aqueous matrices. Those studies are discussed below in detail.

Direct determination of pharmaceuticals in untreated samples
One of the most commonly found pharmaceuticals in surface waters is carbamazepine (CBZ). CBZ has been proposed as a marker of anthropogenic pollution [66,77] and the screening of this pharmaceutical in environmental samples through ELISA has been studied by some authors [41, 66,79] ( Table 2). The sensitivities of the assays used for the detection of CBZ are in the order of the low micrograms per litre, the lowest being the quantification limit achieved by Bahlmann et al.
[41] (~0.025 µg L -1 ), all obtained without sample preconcentration. In these studies, CR effects, pH, ionic strength, and matrix effects were analysed in order to optimize the method and the validation was performed using LC-MS/MS as reference method. Indeed, the validation of ELISA by a reference technique has allowed for the detection of overestimation due to matrix or CR effects. In a work by Bahlmann et al. [79], it was considered that two CBZ metabolites -epoxycarbamazepine (EP-CBZ) and 2-hydroxycarbamazepine (2OH-CBZ), with molar CRs of 83% and 14%, respectively -were the main responsible for the overestimation of the ELISA results, since they were probable to be found in the environment (CBZ can be excreted as 3% EP-CBZ and 5-6% 2OH-CBZ). Interestingly, the metabolite 10,11-dihydro-10,11-dihydroxy-CBZ (DiOH-CBZ) is excreted by humans at higher rates than the parent compound itself and, thus commonly occurs in the environment often at higher concentrations than CBZ [41].
However, this metabolite is not a relevant cross-reactant of this ELISA and in this sense hardly influences the CBZ determination [79]. A few years later, the same authors carried out an in-deep and comprehensive analysis on the role of CBZ metabolites in the determination of this pharmaceutical by ELISA. For that purpose, Bahlmann et al. [96], recurring to LC-ELISA, concluded that a 30% overestimation of the ELISA results for CBZ was not only due to the CR of the previously referred metabolites, seeing that the concentration that they presented in the samples were too low for such an overestimation.
Surprisingly, the observed 30% overestimation in the concentration of CBZ in water samples, was mainly due to the cross-reaction of cetirizine (CET), an anti-histaminic drug, which does not have structural similarities with CBZ. The identification of this crossreactant was possible through LC fractionation of the sample before ELISA analysis followed by LC-MS/MS analysis. The authors performed an investigation of the CRs of CET and its derivatives/metabolites, namely norchlorcyclizine, that indicated that these two compounds showed high CR, especially at low pH values (4.5), probably due to the protonation of the amine group in a distance of four atoms from the azepine's nitrogen.
Also, the crystal structures of CBZ and norchlorcyclizine were analyzed (since, at that time, the CET crystal structure was not known) allowing to conclude that the antibody possessed a "blind spot" on the atomic junction between the CBZ phenyl rings, which was the main cause of the CR for other compounds such as CET and 10,11-dihydro-10,11epoxycarbamazepine [96]. In the same work, it was shown that pH also influenced the enantio-selectivity, where antibody's affinity showed to be higher for (S)-CET than for (R)- 13 CET increasing from 4 times to more than 30 times from pH 4.5 to 10.5. This study revealed that the high environmental concentrations of the anti-histaminic pharmaceutical CET clearly justified the impact of this pharmaceutical in the CBZ determination using this assay; the highest overestimations were found in the spring and summer due to the seasonality of CET prescription [96].
This pH feature of the above-mentioned assay was used by Calisto et al. [66] to simultaneously determine the concentration of CBZ and CET (the main cross-reactant). As previously referred, the selectivity of the monoclonal antibody towards CET and CBZ was proven to be highly dependent on pH. Hence, two pH values, 4.5 (maximum CET selectivity) and 10.5 (maximum CBZ selectivity), were selected to study the CR and the concentrations of both pharmaceuticals using a system of equations considering the CR and the assay signal at both pH [66]. An overestimation of the concentration of CBZ (about 2-29%) was still observed when comparing with the LC-MS/MS results, possibly due to the cumulative contributions of matrix effects with the presence of the identified CBZ metabolites, EP-CBZ or 2OH-CBZ, also recognized by the used antibody. These two examples [66,96] show how CR effects, usually considered a disadvantage, might also open the possibility of using ELISA for the determination of more than one pharmaceutical by just changing the pH of the assay. However, the application of multi-analyte ELISA for the specific and individual quantification of different pharmaceuticals in the aquatic environment is scarce. In a different way, multi-analyte ELISA has been used for the recognition and quantification of pharmaceutical families. This is the case of Adrian et al. [97] who performed a multi-analyte detection for three families of antibiotics (sulfonamides, fluoroquinolones and ß-lactams) in milk samples. On the other hand, multianalyte ELISA has been also applied for the quantification of some organic contaminants in 14 the aquatic environment, such as pesticides [98][99][100][101], which are out of the scope of this review.
Caffeine (CAF) is another compound that, along with CBZ, has been proposed as anthropogenic marker for wastewater contamination of surface waters. In Table 2 [83] which developed an ELISA method with a pre-step of LC fractionation (LC-ELISA) to quantify SMX in water samples ( Table 2). The application of a LC fractionation prior to the ELISA measurements had also been applied by Bahlmann et al. [96] in order to identify cross-reactants of CBZ, as mentioned earlier in this section.
However, in the case of Hoffmann et al. [83], the application of the LC fractionation was intended to overcome general matrix effects. With this LC-ELISA, which allowed for a 1000-fold enrichment of the samples, a quantification limit of 1 ng L -1 was theoretically possible, considering no matrix interference. The effects of CR of 24 similar compounds was investigated, as well as other matrix effects. It was disclosed that succinimidyl-sulfamethoxazole (SMX-Succ), N-acetyl-SMX and sulfamethizole were the most problematic compounds with high CR, the last one presenting a higher antibody affinity than SMX itself. The high affinity of the antibody for these three compounds is related with the similarity of their spatial structure and the high electron density in the aromatic ring of the molecule. Another interesting aspect has to do with the fact that cross-reactant compounds with relevant structural similarities can present low CRs as a consequence of having their structure sterically hindered, as it was the case of SMX-β-D-glucuronide. The authors concluded that the fractionation of the environmental samples before the ELISA allowed to eliminate most of the interferences. This LC-ELISA was validated by LC-MS/MS and despite the good agreement between the methods, LC-ELISA presented some disadvantages such as the need of pre-concentration and LC fractionation steps, which eliminate the operational simplicity typical of ELISA, and high CR for similar compounds that occur in environmental water samples [83].
Diclofenac is a therapeutic agent that has been proposed as a priority hazardous substance [2,102]. Among the most sensitive methods used for the quantification of diclofenac is a GC-MS method with a LOQ of 6 pg L -1 , yet involving a relatively laborious sample pre-treatment and a derivatization step [103]. Other relevant methods were reported, namely, a SPE-UPLC-QqQMS/MS method with a LOD of 0.1 pg L -1 and LOQ of 0.2 ng L -pure water, a LOD of 6 ng L -1 , achieved with any sample preparation which, in this context, is a very interesting result when compared with the LOD and LOQ values achieved by conventional methods (Table 2). In this work, the metabolite 5-hydroxydiclofenac presented 100% of CR for the assay, due to its dichlorophenyl ring, while the other tested metabolites showed CRs below 2% [49]. After verifying an overestimation of the results in wastewater samples, and in order to disclose the influence of diclofenac glucuronide (a product of the metabolization of diclofenac in the human body), the authors applied enzymatic or acidic treatments to transform the conjugate into the original unconjugated form. With this experiment, and the simultaneous analysis of both treated and untreated samples by ELISA and GC-MS, it was found that the probable cause of the overestimation could, in fact, be attributed to the presence of diclofenac glucuronide, not due to the high concentration of this metabolite in the environment but to the higher affinity of the antibody for the conjugate when compared with the parent drug [49]. While some authors refer that glucuronide conjugates are not prone to be probable CRs (as the structure of the parent compound is sterically hindered [83]), the opposite trend was suggested in this study [49].
The authors also referred an important obstacle in what concerns the evaluation of metabolites as cross-reactants: in most cases, the metabolites are not commercially available [49]. This also has direct implications on the environmental quantification of metabolites, which would allow to shed some light on the different forms in which the original drug can occur and the fate of those derivatives.
Another study by Huebner et al. [80] developed an ELISA based on a monoclonal antibody for diclofenac. The method proved to be reliable, without the need of a preconcentration step, and stable to potential matrix interferences, namely, pH, calcium chloride concentration and humic acids (HA). It presented CR up to 10% for diclofenac metabolites, but under 1% for other NSAIDs. The results compared very favourably with the reference technique SPE-LC-MS, presenting only differences of about 12% and 3% for wastewater and surface water, respectively, with the results of ELISA being slightly higher ( Table 2).
The presence of indomethacin, a NSAID, in water samples was studied by Huo et al. [81]. The method developed by Huo et al. [81] was an indirect competitive ELISA presenting a LOD 500 times lower than the validation technique SPE-HPLC-UVIS, which needed a pre-concentration procedure by 100-fold. The correlation coefficient between ELISA and HPLC was 0.988, however, the ELISA presented an CR of 92.3% for acemetacin. The results obtained by ELISA (Table 2) were similar to the ones obtained with SPE-HPLC, however, due to the highest LOD, this last method was not capable of detecting indomethacin in one river sample. For other samples, the authors obtained an overestimation of the concentrations determined by ELISA (around 30%, in average).
Steroid hormones such as 17β-estradiol (E2) and 17α-ethinylestradiol (EE2) are amongst the most commonly estrogens found in wastewater [55] which belongs to a class of compounds that may interfere with the normal function of the endocrine system of humans and wildlife. As for the other contaminants addressed in this review, chromatographic methods are generally used to the detection of these compounds in environmental samples [107]. However, as compared with other pharmaceuticals, it is interesting to note the relatively large number of publications referring the use of ELISA as a screening method for estrogens (Table 3). Between the publications where ELISA is used to detect/quantify hormones, at least seven describe the use of commercial kits [84][85][86][88][89][90][91] (Table 3). These kits present LODs in the range of ng L -1 to µg L -1 , however, most of them present high CR for similar compounds and are affected by the matrix, being, therefore, more useful as screening tool than as quantitative methods. Indeed, ELISA kits, generally and when not coupled to sample treatment procedures, present higher LODs or LOQs than specifically developed ELISA methods.
In what concerns the development of ELISA and respective application in environmental samples for the detection of E2 and EE2, some interesting works have been published with particular focus on the enhancement of selectivity and sensitivity of the assay. For instance, Schneider et al. [92] used a chemiluminescence ELISA (CLEIA) to analyse EE2 in surface water and wastewater from STP effluents at sub-ppt levels ( Table   3). The validation of the method was performed by LC-MS/MS, which involved a preconcentration step by SPE to achieve a LOD as low as the one of ELISA. The results between CLEIA and LC-MS/MS were consistent showing the applicability of the method.
Silva et al.
[55] also studied the presence of estrogens in environmental samples (complex aqueous matrices) quantifying E2 and EE2 in surface and wastewaters using an ELISA without any sample clean-up procedures (Table 3). In this study, organic matter, represented by HA, was revealed to interfere more in the quantification of E2 than of EE2.
The authors considered that this interference was probably related to the denaturation of proteins and enzymes in the presence of HA, which can bind to the Ab and/or to the tracer (unspecific binding). These matrix effects were overcome using a BSA sample buffer that was added to the wells prior to the addition of the analyte. The method allowed to quantify E2 in two wastewater samples, after primary treatment and after biological treatment, respectively, and in a surface water sample ( Table 3).
Some of these studies addressed the importance of metabolites as CRs of these steroid hormones. In general, the main metabolites of E2 and EE2 mentioned as presenting CR for the developed ELISA methods are sulphates and glucuronides derivates (conjugated 20 at ring position 3) [87,92,93], similarly to other examples previously mentioned in this review.

Combining ELISA with extraction and pre-concentration procedures
Few papers refer the use of extraction and pre-concentration methods applied to ELISA. References to extraction methods are most commonly found due to the complexity of the sample matrix, which can also imply a concentration factor of the analyte. Preconcentration procedures are not extensively applied because ELISA is already a sensitive method and does not rely on these procedures to achieve very low detection limits.
However, when needed, the use of organic solvents in such procedures interferes with ELISA, since they can affect the conformation of the antibody [108], implying an extra step associated to the evaluation of the effects of organic solvents on the assay sensitivity. An example of the use of pre-concentration and purification methods applied to ELISA is the one described in Huang and Sedlak [85] which investigated the use of commercially available ELISA for the quantification of estrogenic hormones, namely, E2 and EE2, in  (Table 3).
Lima et al. [75] also analysed E2 and EE2 in potable, surface and wastewater samples by direct competitive ELISA after applying a dispersive liquid-liquid microextraction (DLLME). The DLLME procedure lead to overestimation of the results, however it proved to be reliable if both standard and samples were subjected to the same extraction procedure. Working ranges of 1.2-8000 ng L -1 for E2 and 0.22-1500 ng L -1 for EE2 were obtained, which implied the decrease of the lower LOQ for both E2 and EE2 for about 30 and 100 times, respectively, comparing with ELISA without DLLME pretreatment. Despite the good results, no reference analytical technique was applied to confirm the quantification of the hormones in water samples. Also, and as referred above, for the particular case of EE2, the pre-treatment step did not improve the LOD of the method when comparing with the one obtained by Schneider et al. [92] without preconcentration step but with a chemiluminescent detection (Table 3).
Hintemann et al. [87] also applied an extraction/purification procedure to surface and waste waters samples to quantify the compounds E2 and EE2 through ELISA ( Table   3). The procedure involved an adjustment of pH and the use of several organic solvents (methanol, hexane, and acetone); however, the optimization of the assay concerning the It is noteworthy that, in a large amount of studies, ELISA is applied without being preceded by complex extraction and/or pre-concentration procedures, as it is perceptible, in particular, in Table 2 (note that for the quantification of hormones (Table 3), the use of extraction and pre-concentration is much more often applied). Most of the times, and even for wastewater and surface waters, the QR range of the assay is adequate without recurring to pre-concentration factors. In these complex matrices, the main concern usually lies in matrix effects that influence the recognition of the pharmaceutical by the antibody, namely dissolved organic matter and salinity. Apart from an initial filtration of the samples, which is thoroughly applied in literature studies (e.g. [41,49,66,79,92]), diluting the samples and/or applying a sample buffer are commonly used strategies to overcome significant interferences. Dilution is more frequently applied in wastewaters, particularly influent wastewaters. Huebner et al. [80], in the quantification of diclofenac in wastewater, diluted the samples by 20-fold due to the high concentration found for this pharmaceutical. Also in the quantification of diclofenac, Deng et al. [49], tested several dilution factors (from 10-to 200-fold) and concluded that a 10-fold dilution with ultrapure water was enough to eliminate matrix effects. Again, dilution is only a possibility as a way of solving matrix interferences, due to the low quantification limits typical of these assays. In what concerns the use of sample buffer, it is introduced in assays to level out complex matrix characteristics that can affect the binding between the antibody and the antigen, and it is added to all samples and calibrators [79]. Its composition may vary depending on specific applications. Yet, most commonly, it includes a buffering agent (such as citrate, glycin or tris(hydroxymethyl) aminomethane (TRIS), depending on the optimal pH for the assay); high concentrations of salt (NaCl) and EDTA (applied to saline samples or to counter the ionic composition of the samples) and bovine serum albumin (BSA) (which is thought to inactivate contaminants that might cause the denaturation of the antibodies, such as dissolved organic matter) [79].

Pharmaceuticals analysis in biological tissues of aquatic organisms
The presence of contaminants in the aquatic environment can directly affect some aquatic species (metabolism, oxidative stress, etc.), possibly resulting in bioaccumulation in the organisms' tissues. In this context, the determination of trace levels of organic contaminants in biological tissues is extremely relevant to better understand the impact of such contamination. Some studies have been made on the utilization of ELISA for the analysis of pharmaceuticals in biological samples (Table 4). These studies are important since some aquatic species can be used as indicators of environmental contamination [109].
The analytical determination of pharmaceuticals in biological tissues of relatively small organisms is quite challenging, mainly due the size of the sample, which makes difficult the application of the most common pre-concentration techniques. Most of the studies in literature focus on the effect of the drugs CBZ, CET, and CAF in several aquatic species, namely, in mussels [110], clams [54, [111][112][113][114][115][116], and polychaetes [116,117]. In these studies, the application of ELISA allowed the quantification of the pharmaceuticals in biological tissues from organisms subjected to environmentally relevant concentrations of pollutants. For instance, several authors [73,118,119] [122] referred that antibodies might be the Achilles' heel of ELISA. Concerning the production of specific antibodies, this is particularly challenging for low molecular weight compounds, as it is the case of pharmaceuticals, which are not able to directly induce an immune response in animals and, thus, antibody production. The production of antibodies for this type of compounds is achieved by its conjugation (and sometimes only after chemical modification) with carrier proteins that, as a whole, act as the antigen. Thus, this extra step for low molecular weight compounds is critical in antibody production, with possible influence in the affinity and specificity of the resulting antibody [44]. Also, as seen in section 3.1, and still concerning where it is highlighted that sensitive and specific detection of low molecular weight molecules by these binders is still a challenge in real-world samples.
In the particular case of MIPs, these have been considered synthetic analogues of biological antibody-antigen systems, operating as a lock and key mechanism, allowing a specific selectivity to the molecules that were templated during their production [126].
Antibody mimic by molecular imprinting and its use in binding assays was proposed twenty years ago [127] and, since then, some studies have pointed to the advantages of using MIPs as substitutes of antibodies in immunoassays [127,128]. First, and perhaps the most important advantage, is the fact that they can be synthetized, which allows to have a broader range of specific molecules to bind to specific target compounds. Also, MIPs can be produced for target small molecules, unlike antibodies, which are only easily produced for macromolecules. Secondly, MIPs have higher durability than antibodies regarding storage conditions, such as temperature. Finally, production of MIPs is a low cost approach when compared with the production of antibodies [129]. These advantages give MIPs a great potential as substitutes for antibodies in ELISA, although limitations, such as the low sensitivity and specificity when compared with the immunoassay-grade antisera, cannot be disregarded [128].
The synthesis of MIPs and their analytic application, including their utilization in the so designated biomimetic ELISA-like assays (BELISAs) or pseudo-ELISA, has been addressed by some authors, as referred in the reviews by Bedwell and Whitcombe [126] and Chen et al. [130]. Regarding the specific application of MIP-based ELISA, some studies may be found in the literature. For example, a MIP-based ELISA was used for the quantification of vancomycin in buffer and blood samples, which presented a linear range of quantification between 0.001 and 70 nM [126,131]. Smolinska-Kempisty et al. [122] successfully compared the performance of nanoMIPs with antibodies for four small molecule targets, including L-thyroxine, a medication used to treat thyroid hormone deficiency. In this study, the nanoMIPs-based assay showed comparable sensitivities to ELISA using mono-or polyclonal antibodies [122].
The application of MIPs-based ELISA for the detection of pharmaceuticals in the food [132][133][134] and clinical areas [135-137] has been widely developed in the last decade.
Although such application in environmental samples is still very incipient, this field is certainly worth exploring and will probably undergo great progress in the near future.
Indeed, promissory results were already obtained by Wang et al. [138], who conceived a fast and direct competitive BELISA for the determination of estrone in environmental 28 water. These authors synthetized an imprinted film of controlled thickness to be used as artificial antibody and their method exhibited excellent performance, with recoveries ranging from 80 to 95%, in the quantitative determination of estrone in river and lake water samples. Despite the low LOD achieved by the BELISA (8.0 ± 0.2 µg L -1 ), the method also showed high CRs for other five estrogenic compounds, namely, 17β-estradiol, estriol, diethylstilbestriol, and progesterone, showing that it was not very selective towards estrone.
Nevertheless, no significant differences were present when comparing the analysis of spiked water samples by HPLC and BELISA. Altogether, it may be said that MIPs-based ELISA can be an interesting variation to be implemented for the monitoring of pharmaceuticals in the environment, presenting several advantages related with the stability of the assays and the possibility of having larger spectrum of small molecules that can be detected.