Targeted Hydrolysis of -Lactam Antibiotics in Dry

water

Communication Targeted Hydrolysis of β-Lactam Antibiotics in Dry Suspension Residue: A Proposed Method to Reduce Ecological Toxicity and Bacterial Resistance

Arne Brahms and Christian Peifer *

Institute of Pharmacy, Christian-Albrechts-University of Kiel, Gutenbergstraße 76, D-24116 Kiel, Germany; [email protected] * Correspondence: [email protected]; Tel.: +49-431-880-1137

Abstract: Within our therapeutic drug arsenal, antibiotics are of significant importance and are widely used in huge amounts to medicate, e.g., bacterial infections in humans and animals. Regarding the more than 10 types of antimicrobial drugs, the highly important orally taken β-lactams typically include dry suspension formulations. In many cases for this formulation, even after usage according to specification, residues remain in the prepared dry suspension bottle, which is often cleaned at home and the contents are flushed down into domestic wastewater. This plausible practice adds to the fact that, e.g., amoxicillin can be found in river waters, and is to be monitored in the EU, as given by resolution 2008/105/EG article 8b. When imported into the environment, β-lactam antibiotics can cause severe ecological problems, and equally importantly, therapeutic applications of these antibiotics are endangered by the forced development of pathogenic resistance. To avoid these issues, we developed and validated a fast, simple, robust, and cost-effective method using a 1 M sodium hydroxide solution to effectively hydrolyze and inactivate β-lactam residues. In this Citation: Brahms, A.; Peifer, C. paper, we strongly propose a procedure involving pharmacists to take back residue of β-lactam dry Targeted Hydrolysis of β-Lactam β Antibiotics in Dry Suspension suspension formulations. Subsequently, qualified pharmaceutical staff could inactivate -lactam Residue: A Proposed Method to residue in the laboratory by the proposed method, and then dispose of the mixture into wastewater. Reduce Ecological Toxicity and Bacterial Resistance. Water 2021, 13, Keywords: β-lactam antibiotics; targeted hydrolysis; environmental benefit 2225. https://doi.org/10.3390/ w13162225

Academic Editor: Rama Pulicharla 1. Introduction Anthropogenic pollution of the environment has recently gained increased public Received: 9 June 2021 interest and is triggering more and more concern [1,2]. Examples of bio-accumulative Accepted: 9 August 2021 pollutants include microplastics [3,4], toxic/synthetic chemicals [5], and drugs (pharma- Published: 16 August 2021 cologically highly active pharmaceutical ingredients, APIs) that can be detected in the environment, even in remote areas [1,2]. APIs are actually used in therapeutic approaches Publisher’s Note: MDPI stays neutral to treat human and animal diseases but can also cause severe collateral side effects when with regard to jurisdictional claims in such biologically highly active compounds survive wastewater cleanup and reach aquatic- published maps and institutional afﬁl- or ecosystems beyond [6,7]. Human and veterinary drugs are used in massive quantities iations. worldwide [8], and the effect of APIs and/or their metabolites after excretion into the environment is, in most cases, not fully understood [9]. Suggested solutions to the complex problem of persistent synthetic compounds such as APIs in the environment have already been put forward, including approaches towards green chemistry [10], technological up- Copyright: © 2021 by the authors. grades of sewage water plants regarding photolytic and oxidative degradation [11], and Licensee MDPI, Basel, Switzerland. sociological approaches [12]. This article is an open access article In general, the excretion via urine or feces from patients using intended dosage distributed under the terms and schemes can be considered to be a major source of APIs and their metabolites in wastew- conditions of the Creative Commons ater [10]. Other pathways of pollution may include improper domestic disposal [13] Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ and from the industrial waste of API manufacturing plants [10]. However, most APIs 4.0/). are speciﬁcally designed for optimal bioavailability and strong metabolic degradation

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are specifically designed for optimal bioavailability and strong metabolic degradation re‐ sistance,resistance, and and these these can canshow show significant signiﬁcant persistence persistence in the in aquatic the aquatic environment, environment, thus thusac‐ cumulatingaccumulating and and yielding yielding trace trace quantities quantities in inrivers, rivers, lakes, lakes, and and even even drinking drinking water water [14]. [14 ]. ProminentProminent examples examples of of environmentally environmentally critical critical APIs APIs include include endocrine endocrine disruptors disruptors such such asas ethinylestradiol ethinylestradiol [15], [15 the], the non non-steroidal‐steroidal pain pain killer killer and and antiphlogistic antiphlogistic diclofenac diclofenac [16], the [16], antiepilepticthe antiepileptic carbamazepine carbamazepine [17], and [17], contrast and contrast agents agents such as such iomeprol as iomeprol [18] used [18 ]in used diag in‐ nosticsdiagnostics (Scheme (Scheme 1). 1).

ethinylestradiol carbamazepine

diclofenac iomeprol

SchemeScheme 1. 1. ChemicalChemical structures structures of of prominent prominent examples examples of of environmentally environmentally critical critical APIs. APIs.

Furthermore,Furthermore, antibiotics antibiotics such such as as the the bulk bulk drugs drugs ciprofloxacin ciprofloxacin and and amoxicillin amoxicillin are are amongamong the the most most confirmed confirmed substances substances in in environmental environmental waters waters often often exceeding exceeding “safe” “safe” levelslevels [19]. [19]. These These data data correlate correlate to to the the fact fact that, e.g., inin 20192019 inin Germany,Germany, 666 666 metric metric tons tons of ofantibiotics antibiotics were were used used for for human human therapy, therapy, and and 742 metric742 metric tons tons for veterinary for veterinary use, of use, which, of which,in both in cases, both βcases,-lactams β‐lactams accounted accounted for the for largest the largest part (e.g., part veterinary (e.g., veterinary penicillins penicillins used to usedtreat to livestock treat livestock totaled totaled 279 metric 279 metric tons) [ 20tons)]. [20]. TheThe ecological ecological relevance relevance of of critical critical APIs APIs is emphasized by resolutionresolution 2008/105/EG2008/105/EG articlearticle 8b, 8b, a a guideline guideline for for agencies agencies to monitor ethinylestradiol, erythromycin, erythromycin, azithromycin, azithromy‐ cin,ciprofloxacin, ciprofloxacin, and and amoxicillin amoxicillin (among (among other other critical critical APIs) APIs) inin water systems systems of of the the Eu Eu-‐ ropeanropean Union. Union. However, However, while while one one might might expect expect that that the the highly highly metabolic metabolic resistant resistant ciprofloxacinciprofloxacin [21] [21 ]can can be be detected detected in in water water samples samples [22], [22], to to our our surprise, surprise, the the relatively relatively metabolicallymetabolically and and chemically chemically reactive reactive β‐βlactam-lactam antibiotic antibiotic amoxicillin amoxicillin [23] [23 can] can also also be be de de-‐ tectedtected at at high high concentrations concentrations in in river river waters waters [24]. [24]. Obviously, Obviously, amoxicillin amoxicillin is is widely widely used used againstagainst many many bacterial bacterial infections infections and, and, additionally, additionally, it it is is administered administered in in high high dosages dosages (e.g.,(e.g., more more than than 3000 3000 mg/adult mg/adult patient patient daily). daily). Consequently, Consequently, it itis is troubling troubling to to find find such such highhigh amounts amounts of of amoxicillin amoxicillin in in river river waters, waters, suggesting suggesting significant significant deposition deposition pathways pathways intointo the the aquatic aquatic environment environment [25,26]. [25,26]. When When considering considering plausible plausible sources, sources, a afraction fraction of of therapeutically employed amoxicillin could be excreted non-metabolized from patients, as therapeutically employed amoxicillin could be excreted non‐metabolized from patients, outlined above [27]. This entry path requires the amoxicillin/β-lactam antibiotic to appar- as outlined above [27]. This entry path requires the amoxicillin/β‐lactam antibiotic to ap‐ ently survive, at least partially [28], the harsh biodegradation processes in sewage water parently survive, at least partially [28], the harsh biodegradation processes in sewage wa‐ treatment plants [19]. Secondly, raw wastewater may not have been treated sufficiently [29]. ter treatment plants [19]. Secondly, raw wastewater may not have been treated sufficiently In line with this notion, amoxicillin originating from veterinary approaches [30] is more [29]. In line with this notion, amoxicillin originating from veterinary approaches [30] is likely to make its way directly into water systems without passing through any sewage more likely to make its way directly into water systems without passing through any sew‐ water treatment [31]. Furthermore, we hypothesize an alternative amoxicillin/β-lactam age water treatment [31]. Furthermore, we hypothesize an alternative amoxicillin/β‐lac‐ source for pollution since, in the vast majority of European countries (as well as in other tam source for pollution since, in the vast majority of European countries (as well as in developed countries), pharmaceutical waste must be returned to community pharmacies.

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Specialized companies manage the collected pharmaceutical waste. In spite of this, it is apparent that antibiotics are found in water efﬂuents.

Residue of β-Lactam Dry Suspension Formulation as a Potential Source of Pollution For amoxicillin and further β-lactam antibiotics, a unique dosage situation applies, especially for children. From a pharmaceutical and technological point of view, in addition to classic, rather large β-lactam API-containing tablets, amoxicillin is often marketed in special taste-masked, dry suspension formulations suitable mainly for the treatment of infections in pediatrics [32]. Commercial dry suspension formulations come as dry mixtures that, prior to usage, require the addition of water, yielding a suspension, which is ready for about 2 weeks of usage when stored in the fridge [33]. Regarding this particularly convenient dosage form, the total amount of amoxicillin (and other β-lactam antibiotics respectively) in the prepared bottle covers a wide range of dosage correlating with the body weight of small children up to that of teens or adults. Typically, even after application according to specification, e.g., for smaller children, a significant residue of the prepared suspension remains in the bottle. Finally, after a relatively short therapy, this residue may then be cleaned at home and the content flushed down into domestic wastewater [34], whereas the empty bottle is disposed of as glass waste. A similar scenario could be true for procedures in hospitals [35]. In some cases, the package instructions of dry suspension pharmaceuticals advise asking a pharmacist how to proceed regarding the disposal of the remaining formulation, but without giving any details about professional waste management. Accordingly, to the best of our knowledge, there is no general guideline for pharmacists regarding how to handle such dry suspension remains. The rinsing of prepared dry suspension bottles after therapeutic usage, may quite plausibly contribute to the fact that amoxicillin, as well as other β-lactams (and potentially additional APIs from liquid formulations), are present in high concentrations in river waters. In addition to antibiotics potentially causing severe ecological issues in general [36], a forced development of striking bacterial resistance [37] may occur, thus diminishing their highly valuable antibiotic efficacy for future applications [38].

2. Materials and Methods 2.1. Reactivity of β-Lactams towards Hydrolysis The molecular mechanism of action of β-lactam antibiotics, including penicillins, cephalosporins, and β-lactamase inhibitors, typically involves a nucleophilic attack from a catalytic serine residue within the active site of penicillin-binding proteins (PBPs such as transpeptidases) [39]. The reaction leads to ring opening of the β-lactam and thus pseudo- irreversible covalent acylation of the target protein (Figure1A). Likewise, in addition to the required electrophilic properties of the β-lactam carbonyl atom, this pharmacophore “warhead” is highly susceptible also to, e.g., hydroxylamine or hydroxide ions [27]. The alkaline hydrolytic reaction leads to immediate β-lactam ring opening and degradation towards penicillic acid derivatives, and subsequently towards a variety of further products, all inactive against bacteria due to the missing key β-lactam moiety (Figure1B) [ 39]. The unwanted reactivity of β-lactam antibiotics in an alkaline environment is the subject of studies [40] typically performed by the pharmaceutical industry in the context of quality control activities to assess API stability [41].

2.2. Targeted Hydrolysis of the β-Lactam Ring In contrast to these stability applications, we aimed to exploit alkaline conditions for the targeted quantitative hydrolysis of β-lactam dry suspension residue. Given a potentially relatively quick hydrolysis and thus pharmacological inactivation of the β- lactam ring [23], we set out to develop a fast, robust, cost-effective, and suitable method for the alkaline degradation of amoxicillin and other β-lactam antibiotic dry suspension residues. In order to demonstrate the efﬁcacy of the method, we performed analytical studies on the sodium hydroxide mediated hydrolysis of four different β-lactam antibiotic Water 2021, 13, x FOR PEER REVIEW 4 of 10

A B

Figure 1. (A) Molecular mechanism of action of β‐lactams towards acylation of a key serine residue within the active site of PBPs. (B) Hydrolytic reaction of the β‐lactam ring induced by a hydroxide ion to yield antibiotically inactive penicillic acid, and downstream further polar degradation products.

2.2. Targeted Hydrolysis of the β‐Lactam Ring In contrast to these stability applications, we aimed to exploit alkaline conditions for the targeted quantitative hydrolysis of β‐lactam dry suspension residue. Given a poten‐ tially relatively quick hydrolysis and thus pharmacological inactivation of the β‐lactam ring [23], we set out to develop a fast, robust, cost‐effective, and suitable method for the Water 2021, 13, 2225 4 of 10 alkaline degradation of amoxicillin and other β‐lactam antibiotic dry suspension residues. In order to demonstrate the efficacy of the method, we performed analytical studies on the sodium hydroxide mediated hydrolysis of four different β‐lactam antibiotic APIs com‐ monlyAPIs marketed commonly as marketed dry suspensions, as dry suspensions, namely amoxicillin, namely amoxicillin, cefaclor, cefadroxil, cefaclor, and cefadroxil, cefurox and‐ imecefuroxime (Scheme 2). (Scheme It is significant2). It is signiﬁcant that, for analytical that, for purposes, analytical both purposes, the pure both APIs the and pure their APIs preparedand their dry prepared suspension dry suspension formulations, formulations, respectively, respectively, were stirred were with stirred 1 M with NaOH 1 M solu NaOH‐ tionsolution at room at temperature room temperature and the and progress the progress of the ofhydrolytic the hydrolytic reaction reaction was monitored was monitored by HPLCby HPLC and LC/MS and LC/MS analysis analysis (n = 6 (nfor = validation; 6 for validation; n = 3 for n = hydrolysis 3 for hydrolysis experiments; experiments; for de‐ for tailsdetails see Supplementary see Supplementary Materials). Materials). As a suitable As a suitable endpoint endpoint for the for reaction, the reaction, we defined we deﬁned the timethe point time at point which at the which quantitative the quantitative hydrolyzation, hydrolyzation, and thus and 100% thus degradation 100% degradation of the β‐ of Water 2021, 13, x FOR PEER REVIEW 4 of 10 lactam,the β occurred.-lactam, occurred. A more detailed A more kinetics detailed of kinetics the alkaline of the hydrolyzation alkaline hydrolyzation has been reported has been elsewherereported [42]. elsewhere [42]. The dilution solution of amoxicillin was prepared from 20.0 mg paracetamol and 2.5 A mL acetic acid in 197.5 mL filtered andB distilled water. For diluting the cefaclor probes, a solution of 20.0 mg paracetamol and 308 mg ammonium acetate was diluted in 4 mL ace‐ tonitrile and 169 mL bidistilled water. By adding acetic acid dropwise to the solution, a pH value of 4 was obtained. For cefadroxil and cefuroxime, the same dilution solution was used, and consisted of, 20.0 mg paracetamol and 308 mg ammonium acetate in 10 mL acetonitrile and 190 mL filtered and bidistilled water. The pH was adjusted to pH 5 by adding acetic acid dropwise.

2.3. Preparation of HPLC Samples The amoxicillin and cefaclor probes were prepared by adding 1.0 mL of the alkaline dry suspension reaction mixture to 9.0 mL of the dilution solution. After mixing, the so‐ Figure 1.1. ((A)) MolecularMolecular mechanismmechanismlution was of of filtered action action of ofthroughβ β‐-lactamslactams a towardsLLG towards filter. acylation acylation The cefadroxil of of a keya key serine serine and residue cefuroxime residue within within probes the the active active were site site ofpre ‐ PBPs.of PBPs. (B )( HydrolyticB) Hydrolytic reaction reactionpared of by the of addingtheβ-lactam β‐lactam 0.2 ring mL ring induced of induced the by alkaline a by hydroxide a hydroxide dry suspension ion toion yield to yield antibiotically reaction antibiotically mixture inactive inactive to penicillic 10.0 penicillic mL acid, of the andacid, downstream and downstream further furtherdilution polar polar degradation solution. degradation products. The products.probe was then mixed and filtered through an LLG filter.

HPLC and LC/MScefadroxil analysis (n = 6 for validation; n =cefuroxime 3 for hydrolysis experiments; for de‐ tails see Supplementary Materials). As a suitable endpoint for the reaction, we defined the SchemeScheme 2. 2. ChemicalChemical structures structures of of the the four four β‐βlactam-lactam antibiotic antibiotic APIs APIs investigated investigated in in this this study. study. time point at which the quantitative hydrolyzation, and thus 100% degradation of the β‐ lactam, occurred. A more detailed kinetics of the alkaline hydrolyzation has been reported 3. ResultsThe dilution solution of amoxicillin was prepared from 20.0 mg paracetamol and 2.5elsewhere mL acetic [42]. acid in 197.5 mL filtered and distilled water. For diluting the cefaclor probes, In preliminary tests, we identified 1 M NaOH solution to be effective for the hydrol‐ a solutionThe dilution of 20.0 solution mg paracetamol of amoxicillin and 308 was mg prepared ammonium from 20.0 acetate mg was paracetamol diluted in and 4 mL 2.5 ysis of all four investigated β‐lactam APIs. Adding 1 M NaOH to the same volume of the acetonitrilemL acetic acid and in 169 197.5 mL mL bidistilled filtered water.and distilled By adding water. acetic For aciddiluting dropwise the cefaclor to the probes, solution, a respective prepared dry suspension formulation and stirring the mixture overnight at am‐ asolution pH value of 20.0 of 4 mg was paracetamol obtained. For and cefadroxil 308 mg ammonium and cefuroxime, acetate the was same diluted dilution in 4 solution mL ace‐ bient temperature accounted for a sufficient method. Consequently, all β‐lactams were wastonitrile used, and and 169 consisted mL bidistilled of, 20.0 mgwater. paracetamol By adding and acetic 308 acid mg ammonium dropwise to acetate the solution, in 10 mL a quantitatively destroyed, yielding a mixture of polar degradation products. A more de‐ acetonitrilepH value of and 4 was 190 obtained. mL filtered For and cefadroxil bidistilled and water. cefuroxime, The pH the was same adjusted dilution to pHsolution 5 by tailed analysis of the reaction kinetics actually showed quite a fast hydrolysis of the com‐ addingwas used, acetic and acid consisted dropwise. of, 20.0 mg paracetamol and 308 mg ammonium acetate in 10 mL monlyacetonitrile used amoxicillinand 190 mL (Figure filtered 2), and cefaclor bidistilled (Figure water. 3), and The cefadroxil pH was (seeadjusted Supplementary to pH 5 by Materials).2.3.adding Preparation acetic In contrast,acid of HPLC dropwise. cefuroxime Samples (see Supplementary Materials) turned out to be the most stable compound in these conditions. However, in order to also reach a robust and The amoxicillin and cefaclor probes were prepared by adding 1.0 mL of the alkaline quantitative hydrolysis for cefuroxime, stirring a mixture of 2 parts of 1 M NaOH and 1 dry2.3. Preparation suspension of reaction HPLC Samples mixture to 9.0 mL of the dilution solution. After mixing, the part of cefuroxime suspension formulation was effective in fully degrading cefuroxime solutionThe wasamoxicillin filtered and through cefaclor a LLG probes filter. were The prepared cefadroxil by andadding cefuroxime 1.0 mL of probes the alkaline were within 240 min (Table 1). dry suspension reaction mixture to 9.0 mL of the dilution solution. After mixing, the so‐ Key parameters for quantitative alkaline hydrolyzation reactions of the four β‐lactam lution was filtered through a LLG filter. The cefadroxil and cefuroxime probes were pre‐ APIs are summarized in Table 2. An additional experiment employing the conditions used pared by adding 0.2 mL of the alkaline dry suspension reaction mixture to 10.0 mL of the for the more robust cefuroxime, but subsequently applied to the mixture of all four com‐ dilution solution. The probe was then mixed and filtered through an LLG filter. bined dry suspension formulations, proved to quantitatively hydrolyze all β‐lactam APIs. Thus, as a robust and effective method to destroy the β‐lactam APIs, we propose to stir the collected suspension residue with twice the volume of 1 M NaOH at room temperature for at least 240 min.

Figure 2. (A). Reference HPLC chromatogram of prepared dry suspension of amoxicillin (AMX) with paracetamol as internal standard (int.std.). Retention times rt AMX = 7.4 min, rt int. std. = 11.0

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amoxicillin cefaclor

cefadroxil cefuroxime Scheme 2. Chemical structures of the four β‐lactam antibiotic APIs investigated in this study.

3. Results Water 2021, 13, 2225 5 of 10 In preliminary tests, we identified 1 M NaOH solution to be effective for the hydrol‐ ysis of all four investigated β‐lactam APIs. Adding 1 M NaOH to the same volume of the respective prepared dry suspension formulation and stirring the mixture overnight at am‐ preparedbient temperature by adding accounted 0.2 mL of thefor alkalinea sufficient dry method. suspension Consequently, reaction mixture all β‐ tolactams 10.0 mL were of thequantitatively dilution solution. destroyed, The probeyielding was a thenmixture mixed of andpolar filtered degradation through products. an LLG filter.A more de‐ tailed analysis of the reaction kinetics actually showed quite a fast hydrolysis of the com‐ 3.monly Results used amoxicillin (Figure 2), cefaclor (Figure 3), and cefadroxil (see Supplementary Materials).In preliminary In contrast, tests, cefuroxime we identified (see 1 MSupplementary NaOH solution Materials) to be effective turned for out the to hydrol- be the ysismost of stable all four compound investigated in theseβ-lactam conditions. APIs. However, Adding 1 in M order NaOH to toalso the reach same a volumerobust and of thequantitative respective hydrolysis prepared for dry cefuroxime, suspension stirring formulation a mixture and stirring of 2 parts the of mixture 1 M NaOH overnight and 1 atpart ambient of cefuroxime temperature suspension accounted formulation for a sufficient was effective method. in Consequently, fully degrading all cefuroximeβ-lactams werewithin quantitatively 240 min (Table destroyed, 1). yielding a mixture of polar degradation products. A more detailedKey analysis parameters of the for reactionquantitative kinetics alkaline actually hydrolyzation showed quite reactions a fast of hydrolysis the four β‐lactam of the commonlyAPIs are summarized used amoxicillin in Table (Figure 2. An2 additional), cefaclor (Figure experiment3), and employing cefadroxil the (see conditions Supplemen- used taryfor the Materials). more robust In contrast, cefuroxime, cefuroxime but subsequently (see Supplementary applied to Materials)the mixture turned of all four out to com be‐ thebined most dry stable suspension compound formulations, in these conditions. proved to quantitatively However, in orderhydrolyze to also all β‐ reachlactam a robust APIs. andThus, quantitative as a robust hydrolysis and effective for cefuroxime, method to stirringdestroy athe mixture β‐lactam of 2 APIs, parts ofwe 1 propose M NaOH to and stir 1the part collected of cefuroxime suspension suspension residue formulationwith twice the was volume effective of 1 in M fully NaOH degrading at room temperature cefuroxime withinfor at least 240 min 240 (Tablemin. 1).

FigureFigure 2. 2.( A(A).). Reference Reference HPLC HPLC chromatogram chromatogram of preparedof prepared dry dry suspension suspension of amoxicillin of amoxicillin (AMX) (AMX) with with paracetamol as internal standard (int.std.). Retention times rt AMX = 7.4 min, rt int. std. = 11.0 paracetamol as internal standard (int.std.). Retention times rt AMX = 7.4 min, rt int. std. = 11.0 min. (B). Sample mixture of AMX and int.std. was stirred for 20 min with the same volume of 0.5 M NaOH, which quantitatively hydrolyzed AMX, yielding penicillo acid derivate of amoxicillin as the

main degradation product (deg. prod., rt = 6.3 min; rt int. std. = 11.0 min). LC/MS analysis of the degradation products can be found in the Supplementary Materials.

Key parameters for quantitative alkaline hydrolyzation reactions of the four β-lactam APIs are summarized in Table2. An additional experiment employing the conditions used for the more robust cefuroxime, but subsequently applied to the mixture of all four combined dry suspension formulations, proved to quantitatively hydrolyze all β-lactam APIs. Thus, as a robust and effective method to destroy the β-lactam APIs, we propose to stir the collected suspension residue with twice the volume of 1 M NaOH at room temperature for at least 240 min. Water 2021, 13, x FOR PEER REVIEW 6 of 10

min. (B). Sample mixture of AMX and int.std. was stirred for 20 min with the same volume of 0.5 M Water 2021, 13, 2225 NaOH, which quantitatively hydrolyzed AMX, yielding penicillo acid derivate of amoxicillin 6as of the 10 main degradation product (deg. prod., rt = 6.3 min; rt int. std. = 11.0 min). LC/MS analysis of the degradation products can be found in the Supplementary Materials.

Figure 3. (A). Reference HPLC chromatogram of prepared dry suspension of cefaclor (CfCl) with paracetamol as internal standard (int.std.). Further method details can be found in the SI. Retention timestimes rtrt CfClCfCl == 26.1 min, rt int. std. = 17.5 17.5 min. min. (B). Sample mixture of CfCl and int.std. was stirred for 20 min with the same volume of 0.4 M NaOH, which quantitatively (B). Sample mixture of CfCl and int.std. was stirred for 20 min with the same volume of 0.4 M NaOH, which quantitatively hydrolyzed cefaclor. LC/MS analysis of the degradation products can be found in the Supplementary Materials. hydrolyzed cefaclor. LC/MS analysis of the degradation products can be found in the Supplementary Materials. Table 1. Parameters for the alkaline hydrolyzation of the prepared cefuroxime β‐lactam dry suspen‐ sionTable formulation. 1. Parameters For each for thetime alkaline point, three hydrolyzation independent of experiments the prepared were cefuroxime performed.β-lactam * cv = coef dry‐ ficientsuspension of variation. formulation. For each time point, three independent experiments were performed. * cv = coefﬁcient of variation. Time for Quantitative c NaOH Ratio NaOH/ Degradation [%] cv * Time for Quantitative c NaOH Ratio NaOH/ Degradation Hydrolysis [min] [mol/L] Dry Suspension cv * Hydrolysis60 [min] [mol/L]0.5 Dry Suspension1:1 [%]93.6 0.21 6060 1.0 0.5 1:1 1:1 93.694.9 0.210.44 6060 0.5 1.0 2:1 1:1 94.995.6 0.440.35 60 0.5 2:1 95.6 0.35 6060 1.0 1.0 2:1 2:1 95.595.5 0.920.92 9090 1.0 1.0 2:1 2:1 99.899.8 0.150.15 120120 1.0 1.0 2:1 2:1 99.999.9 0.560.56 150150 1.0 1.0 2:1 2:1 99.999.9 0.700.70 180 1.0 2:1 99.9 0.70 180 1.0 2:1 99.9 0.70 210 1.0 2:1 100.0 0.22 210240 1.0 1.0 2:1 2:1 100.0100.0 0.600.22 240 1.0 2:1 100.0 0.60

Table 2. Parameters for quantitative alkaline hydrolyzation of prepared β‐β-lactamlactam dry suspension formulations.formulations. For For each each API, API, three three independent experiments were performed, yielding comparable 100% hydrolysis rates respectively.

c NaOH Ratio NaOH/PreparedRatio NaOH/Prepared Dry 100% β‐100%Lactamβ-Lactam Hydrolysis APIAPI c NaOH [mol/L] [mol/L] SuspensionDry Suspension HydrolysisReached Reached at at amoxicillinamoxicillin 0.5 0.51:1 1:120 20 min min cefaclor 0.4 1:1 20 min cefadroxil 1 2:1 60 min cefuroxime 1 2:1 240 min

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4. Discussion and Conclusions Therapeutically highly relevant β-lactams, such as amoxicillin and several cephalosporins, account for the most antibiotic usage in humans and, in particular, for the treatment of infections in children [43]. Thus, for future applications, it is necessary to keep the therapeutic efficacy of this long-used class of antibiotic. In line with this notion, it has been reported that only a few new antibiotic compounds or classes are currently being developed by the pharmaceutical industry [44]. Therefore, to help maintain the therapeutic effect of current β-lactam antibiotics, in this study, we provide evidence for a fast, simple, robust, and economic method for inactivating β-lactam residue from dry suspension formulations. The procedure could be used to effectively avoid environmental contamination by β-lactam antibiotics, adding further value by diminishing the potential for the development of bacterial resistance. Among the general methods used in modern wastewater plants to reduce the API load in the outlet [45], more specific methods were reported to tackle antibiotic residues. These include sophisticated electrochemical [46,47], photocatalytic [48–50], absorption [51], and oxidative [52] techniques, and these could be useful for addressing non-metabolized antibiotics subsequent to oral administration that are excreted by humans and animals. Obviously, these remedies cannot be easily addressed by the alkaline hydrolysis reported herein. However, our cost-effective method could help to avoid the introduction of β-lactams coming from the part of antibiotics contained in dry suspension formulation residue. Thus, the method could be helpful in reducing their total entry load into wastewaters. To sum up, we researched β-lactams typically used for dry suspension formulations and thus investigated four common β-lactam APIs and their respective dosage forms regarding their susceptibility to alkaline hydrolysis. When adding twice the volume of a 1 M NaOH solution to the remaining suspension formulation at ambient temperature, our analysis showed a fast hydrolysis of the four relevant β-lactam antibiotics within a 240 min reaction time, resulting in a mixture of polar degradation products. A valid alkaline hydrolysis and degradation of the combined dry suspension formulation residue containing the most used β-lactam APIs amoxicillin, cefaclor, cefadroxil, and cefuroxime can be achieved. Thus, this method is considered to be highly suitable for practical use regarding laboratories in pharmacies and in clinics. Furthermore, the proposed hydrolysis conditions should be investigated further to test the degradation of other approved β-lactam (and macrolide) suspension formulations in order to fully validate the described method. In general, it is not acceptable to have a poor or even non-existent waste management for APIs. In many developed countries, community pharmacies take back pharmaceutical waste, and the disposal is then managed by specialized companies. However, a particular situation applies to liquid formulations, which may often be disposed of into domestic wastewater. An effective and comprehensive disposal system should be established for the therapeutically and ecologically highly critical class of (β-lactam-) antibiotics present in dry suspension residue; it is in our own human interest to achieve this. Conceptually, it would be most effective to interfere early in the process of APIs, such as β-lactam antibiotics, draining unintentionally into the environment [13]. Thus, the data of this study could serve in the development of a future practice guideline, where pharmacists are involved to take back un-used residue of β-lactam dry suspension formulations from patients (clinic and public). Qualified pharmaceutical personnel could subsequently deactivate β-lactams in the laboratory by the described method using 1 M sodium hydroxide solution prior to rinsing the inactive mixture down the sink. A comparable process should be discussed for the use of β-lactam APIs in veterinary settings, in which antibiotics are often employed as pure substances; that is to say, without any formulation at all. The proposed method for the degradation of antibiotic residue prior to disposal could contribute to creating multiple benefits in order to significantly reduce the unwanted β-lactam load in the environment. Likewise, the procedure could help to reduce the emergence of collateral bacterial resistances against this highly needed class of effective antibiotics. Finally, given the strong growth of pathogenic/bacterial resistance and—as a direct consequence thereof— Water 2021, 13, 2225 8 of 10

the emergence of ineffective antibiotics, a responsible and suitable antibiotic API waste management program should be the focus of any serious public health program.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/w13162225/s1, Further details for Materials & Methods, HPLC analysis of amoxicillin, cefaclor, cefadroxil, cefuroxime, and validation parameters. Author Contributions: Conceptualization, C.P.; methodology, A.B.; validation, A.B.; formal analysis, A.B.; resources, C.P.; writing, C.P. Both authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Acknowledgments: The authors gratefully acknowledge the help of Martin Schütt, Sven Wichmann, and Ulrich Girreser for their excellent technical support. We acknowledge financial support by DFG within the funding programme “Open Access Publizieren”. Conflicts of Interest: The authors declare no conflict of interest.

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