antibiotics Article Pitfalls in the Immunochemical Determination of β-Lactam Antibiotics in Water Alexander Ecke 1,2 and Rudolf J. Schneider 1,3,* 1 BAM Federal Institute for Materials Research and Testing, 12205 Berlin, Germany; [email protected] 2 Department of Chemistry, Humboldt-Universität zu Berlin, 12489 Berlin, Germany 3 Faculty III Process Sciences, Technische Universität Berlin, 10623 Berlin, Germany * Correspondence: [email protected]; Tel.: +49-30-8104-1151 Abstract: Contamination of waters with pharmaceuticals is an alarming problem as it may support the evolution of antimicrobial resistance. Therefore, fast and cost-effective analytical methods for potential on-site analysis are desired in order to control the water quality and assure the safety of its use as a source of drinking water. Antibody-based methods, such as the enzyme-linked immunosorbent assay (ELISA), can be helpful in this regard but can also have certain pitfalls in store, depending on the analyte. As shown here for the class of β-lactam antibiotics, hydrolysis of the β-lactam ring is a key factor in the immunochemical analysis as it influences antibody recognition. With the antibody used in this study, the limit of detection (LOD) in the immunoassay could be significantly reduced by hydrolysis for the five tested penicillins, with the lowest LOD for carbenicillin (0.2 nmol/L) and the greatest impact on penicillins G and V (reduction by 85%). In addition to enhanced quantification, our strategy also provides access to information about the degree of hydrolysis in water samples as shown for the most abundant penicillin amoxicillin. Keywords: β-lactams; penicillins; hydrolysis; ELISA; immunoassay Citation: Ecke, A.; Schneider, R.J. Pitfalls in the Immunochemical Determination of β-Lactam Antibiotics in Water. Antibiotics 2021, 1. Introduction 10, 298. https://doi.org/10.3390/ The overuse of antibiotics in human and veterinary medicine contributes to an increas- antibiotics10030298 ing discharge of pharmaceuticals and their metabolites into the environment via excretion and disposal [1–4]. The major concern about antibiotics in the environment and especially Academic Editor: Nicholas Dixon in (drinking) water is the potential evolution of antibiotic resistance, which poses a severe health risk to humans and animals [5–9]. From all classes of antibiotics, β-lactam antibiotics Received: 20 January 2021 are prescribed most frequently, and particularly the aminopenicillin amoxicillin (AMX, Accepted: 9 March 2021 Figure1) is found among the top 25 of all prescribed drugs per year in each of the past Published: 12 March 2021 20 years [10–13]. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Figure 1. Chemical structure of amoxicillin (AMX). Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. As a consequence, AMX is traceable in many countries in hospital effluents, wastewa- This article is an open access article ter treatment plant influents as well as effluents, and eventually in surface waters which of- distributed under the terms and ten serve as a source of drinking water [14–21]. In addition to the parent drug, further risk conditions of the Creative Commons arises from the hydrolysis products of β-lactams, which can be formed after prolonged Attribution (CC BY) license (https:// residence time in water. Even though antibiotic activity is lost upon hydrolysis, the formed creativecommons.org/licenses/by/ hydrolysis products may still cause biological effects, e.g., allergenicity or ecotoxicity, 4.0/). that have not yet been investigated in detail [22,23]. Nevertheless, studies to identify Antibiotics 2021, 10, 298. https://doi.org/10.3390/antibiotics10030298 https://www.mdpi.com/journal/antibiotics Antibiotics 2021, 10, 298 2 of 12 those compounds and elucidate the mechanisms and kinetics of their formation have been carried out for the example of AMX [23–28]. In view of the numerous potential harmful metabolites that can be formed from β-lactams in the aquatic environment, effective screening for these compounds together with the unaltered parent antibiotic is required, especially since the Commission of the European Union has added AMX to a watch list of substances for Union-wide monitoring in the field of water policy [29]. Generally, mass spectrometry (MS)-based methods can be useful for this purpose as they enable highly accurate and sensitive determination of pharmaceutical compounds in water [30–34]. However, facing a large number of analytes, i.e., hydrolysis products of β-lactams, MS-based methods would be highly time-consuming and costly. Moreover, these instrumental methods require specially trained personnel as well as they are immobile and therefore not suitable for on-site testing. In contrast to this, immunochemical methods using specific antibodies allow for cost-effective on-site analysis with high throughput of samples and ease of experimentation. The standard technique in immunoassays can be considered the enzyme-linked immunosorbent assay (ELISA) which often serves as reference method for other assay formats. ELISAs for the determination of β-lactam antibiotics and particularly AMX have been developed previously but these studies focused on the determination of the unaltered drug and did not comprise studies on degradation products [35–37], even though the influence of β-lactam hydrolysis on antibody recognition has been reported before [38,39]. In this work, we present the first example of utilizing hydrolysis of β-lactams for the improved immunochemical determination of these compounds and their hydrolysis products, providing a method for the quick evaluation of drinking water safety and quality in terms of contamination with pharmaceuticals and therefore the risk of potential microbiological resistance development. 2. Results and Discussion 2.1. Hydrolysis of AMX While developing and optimizing an indirect competitive ELISA for the determination of AMX, we observed a significant shift in the calibration curves obtained for differently aged standard solutions (calibrators) of AMX in water. Interestingly, the curves for aged standard solutions were shifted to lower IC50 values, tantamount to a lower limit of detection. This shift reached its end point after two months of storage at 4 ◦C and could be ascribed to hydrolysis of the parent drug by hydrolyzing standard solutions of AMX in 0.1 M NaOH, which yielded a similar calibration curve in the ELISA (see Figure2). Under these conditions (pH 12.5), it was found that hydrolysis had proceeded to a certain level because no further shift in the calibration curve was observable after reaction times of 18 h at 4 ◦C or 3 h at RT, respectively. In order to discover the species that is recognized better by the antibody and therefore leads to the decreased IC50, several known hydrolysis products of AMX were synthesized or purchased as pure compounds and tested in the ELISA to compare their calibration curves. It was found that only the primary hydrolysis product amoxicilloic acid (HP1), which is formed by hydrolytic cleavage of the β-lactam ring, yields a complete inhibition curve in the examined concentration range (Figure3). It is noteworthy that the IC 50 of HP1 (7.48 ± 0.58 nM) is lower compared to hydrolyzed AMX, indicating that at least one additional species is formed in the hydrolyzed standards which shifts the calibration curve to an increased IC50 value again. Under the applied basic conditions, the formation of amoxicillin piperazine-2,5-dione (HP2, IC50 = 28,700 ± 1400 nM) by intramolecular ring closure of HP1 is feasible as well. The generation of the decarboxylation product amoxilloic acid (HP3) and 3-(4-hydroxyphenyl)-pyrazine-2-ol (HP4), which is considered a stable end product of AMX hydrolysis, are rather unlikely as their formation is favored under acidic conditions and elevated temperatures [23]. Antibiotics 2021, 10, 298 3 of 12 Figure 2. ELISA calibration curves obtained for standard solutions series of AMX after different treatments, (a) freshly prepared (reference), (b) aged for 2 months at 4 ◦C in a refrigerator, and (c) ◦ hydrolyzed during 24 h at 4 C, also in a fridge. IC50 values: (a) 1470 ± 120 nM, (b) 52.2 ± 4.4 nM, and (c) 38.1 ± 3.6 nM. Figure 3. ELISA calibration curves of selected AMX hydrolysis products and their structures. Given that the employed anti-AMX antibody shows an approximately 200-fold higher affinity towards HP1 than to the parent drug AMX, it appears probable that this antibody had been raised against HP1 rather than AMX. Depending on the immunogen synthesis strategy, i.e., the coupling of AMX to a carrier protein, the β-lactam ring could have been hydrolyzed prior to the immunization procedure. The producer of the antibody was contacted for details on the immunogen synthesis but classified this information as proprietary. However, as known from other immunogen syntheses, the coupling is often performed at alkaline pH allowing hydrolysis of the β-lactam ring to occur [36,38,40,41]. 2.2. Cross-Reactivity of Related Penicillins A similar trend was observed for related penicillins that were tested for cross-reactivity. Regarding their structure, the following β-lactams were chosen: (1) ampicillin (AMP) which is lacking the aromatic hydroxyl group of AMX; (2) carbenicillin (CRB) with the α-amino group of AMP exchanged for a carboxyl group; (3) penicillin G (PenG) lacking any functional group in α-position; and (4) penicillin V (PenV) with an additional oxygen atom linking the phenyl ring to the α-carbon. ELISA calibration curves were recorded Antibiotics 2021, 10, 298 4 of 12 for each compound before and after alkaline hydrolysis of the respective standards and compared to fresh and hydrolyzed AMX standards (see Figure4). Figure 4. (a) ELISA calibration curves of structurally similar β-lactams before, and (b) after hy- drolysis, and their structures (right). IC50 values (before; after hydrolysis): AMP (1350 ± 130 nM; 36.2 ± 3.7 nM), CRB (11.0 ± 0.4 nM; 3.8 ± 0.5 nM), PenG (700 ± 70 nM; 11.1 ± 1.7 nM), and PenV (702 ± 66 nM; 11.6 ± 2.5 nM).