Determination of anti-SARS-CoV-2 virustatic pharmaceuticals in the aquatic environment using high-performance liquid chromatography high-resolution mass spectrometry.

The Covid-19 pandemic has affected the global population since 2019. The rapid development and approval of vaccines has brought relief. Yet, effective cures are still being researched. Even if the pandemic situation may end, SARS-CoV-2 will remain and, thus, continued application of the drugs will lead to emissions of the active ingredients into the aquatic environment, as with other anthropogenic micropollutants. However, a general method for trace analysis of antiviral drugs is still missing. To this purpose, favipiravir, remdesivir, its active metabolite GS-441524, molnupiravir and its active metabolite EIDD-1931 were selected as representative analytes. A method was developed based on solid phase extraction and high-performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight high-resolution mass spectrometry. Optimization comprised the choice of chromatographic columns, elution gradient, mass spectrometry and tandem mass spectrometry parameters. Solid phase extraction proved suitable for increase in limits of detection and quantitation. amelioration of the limits of detection and quantitation. Matrix effects were investigated applying the optimized method to a wastewater sample with added virustatics. All five compounds could be separated with reversed phase chromatography, whereas EIDD-1931 profited from hydrophilic interaction liquid chromatography. The optimized method yielded limits of detection and quantification of 2.1·10-1, 6.9·10-1 µg·L-1 for favipiravir, 1.8·10-3, 5.5·10-3 µg·L-1 for remdesivir, 1.9·10-3, 7.6·10-3 µg·L-1 for GS-441524, 2.9·10-3, 8.7·10-3 µg·L-1 for molnupiravir, and 1.3·10-1, 3.8·10-1 µg·L-1 for EIDD 1931. The method was first applied to compound stability testing at pH 2.8 and 9.7. At pH 2.8, remdesivir, GS-441524 and molnupiravir proved stable, whereas about 14% of EIDD-1931 and favipiravir were degraded. All five antiviral compounds were almost completely decomposed at pH 9.7. The application of the method was further demonstrated for potential transformation product detection on favipiravir ozonation monitoring.

Metoprolol and Its Degradation and Transformation Products Using AOPs-Assessment of Aquatic Ecotoxicity Using QSAR.

Pharmaceuticals are found in waterbodies worldwide. Conventional sewage treatment plants are often not able to eliminate these micropollutants. Hence, Advanced Oxidation Processes (AOPs) have been heavily investigated. Here, metoprolol is exposed to UV irradiation, hydrogen peroxide, and ozonation. Degradation was analyzed using chemical kinetics both for initial and secondary products. Photo-induced irradiation enhanced by hydrogen peroxide addition accelerated degradation more than ozonation, leading to complete elimination. Degradation and transformation products were identified by high-performance liquid-chromatography coupled to high-resolution higher-order mass spectrometry. The proposed structures allowed to apply Quantitative Structure-Activity Relationship (QSAR) analysis to predict ecotoxicity. Degradation products were generally associated with a lower ecotoxicological hazard to the aquatic environment according to OECD QSAR toolbox and VEGA. Comparison of potential structural isomers suggested forecasts may become more reliable with larger databases in the future.

Enzymatic degradability of diclofenac ozonation products: A mechanistic analysis.

The treatment of waterborne micropollutants, such as diclofenac, presents a significant challenge to wastewater treatment plants due to their incomplete removal by conventional methods. Ozonation is an effective technique for the degradation of micropollutants. However, incomplete oxidation can lead to the formation of ecotoxic by-products that require a subsequent post-treatment step. In this study, we analyze the susceptibility of micropollutant ozonation products to enzymatic digestion with laccase from Trametes versicolor to evaluate the potential of enzymatic treatment as a post-ozonation step. The omnipresent micropollutant diclofenac is used as an example, and the enzymatic degradation kinetics of all 14 detected ozonation products are analyzed by high-performance liquid chromatography coupled with high-resolution mass spectrometry (HPLC-HRMS) and tandem mass spectrometry (MS2). The analysis shows that most of the ozonation products are responsive to chemo-enzymatic treatment but show considerable variation in enzymatic degradation kinetics and efficiencies. Mechanistic investigation of representative transformation products reveals that the hydroxylated aromatic nature of the ozonation products matches the substrate spectrum, facilitating their rapid recognition as substrates by laccase. However, after initiation by laccase, the subsequent chemical pathway of the enzymatically formed radicals determines the global degradability observed in the enzymatic process. Substrates capable of forming stable molecular oxidation products inhibit complete detoxification by oligomerization. This emphasizes that it is not the enzymatic uptake of the substrates but the channelling of the reaction of the substrate radicals towards the oligomerization of the substrate radicals that is the key step in the further development of an enzymatic treatment step for wastewater applications.

Enzymatic post-treatment of ozonation: laccase-mediated removal of the by-products of acetaminophen ozonation.

Ozonation is a powerful technique to remove micropollutants from wastewater. As chemical oxidation of wastewater comes with the formation of varying, possibly persistent and toxic by-products, post-treatment of the ozonated effluent is routinely suggested. This study explored an enzymatic treatment of ozonation products using the laccase from Trametes versicolor. A high-performance liquid chromatography coupled with high-resolution mass spectrometry (HPLC-HRMS) analysis revealed that the major by-products were effectively degraded by the enzymatic post-treatment. The enzymatic removal of the by-products reduced the ecotoxicity of the ozonation effluent, as monitored by the inhibition of Aliivibrio fischeri. The ecotoxicity was more effectively reduced by enzymatic post-oxidation at pH 7 than at the activity maximum of the laccase at pH 5. A mechanistic HPLC-HRMS and UV/Vis spectroscopic analysis revealed that acidic conditions favored rapid conversion of the phenolic by-products to dead-end products in the absence of nucleophiles. In contrast, the polymerization to harmless insoluble polymers was favored at neutral conditions. Hence, coupling ozonation with laccase-catalyzed post-oxidation at neutral conditions, which are present in wastewater effluents, is suggested as a new resource-efficient method to remove persistent micropollutants while excluding the emission of potentially harmful by-products.