The drug salinomycin (SAL) is a polyether antibiotic and used in veterinary medicine as coccidiostat and growth promoter. Recently, SAL was suggested as a potential anticancer drug. However, transformation products (TPs) resulting from metabolic and environmental degradation of SAL are incompletely known and structural information is missing. In this study, we therefore systematically investigated the formation and identification of SAL derived TPs using electrochemistry (EC) in an electrochemical reactor and rat and human liver microsome incubation (RLM and HLM) as TP generating methods. Liquid chromatography (LC) coupled to high-resolution mass spectrometry (HRMS) was applied to determine accurate masses in a suspected target analysis to identify TPs and to deduce occurring modification reactions of derived TPs. A total of 14 new, structurally different TPs were found (two EC-TPs, five RLM-TPs, and 11 HLM-TPs). The main modification reactions are decarbonylation for EC-TPs and oxidation (hydroxylation) for RLM/HLM-TPs. Of particular interest are potassium-based TPs identified after liver microsome incubation because these might have been overlooked or declared as oxidated sodium adducts in previous, non-HRMS-based studies due to the small mass difference between K and O + Na of 21 mDa. The MS fragmentation pattern of TPs was used to predict the position of identified modifications in the SAL molecule. The obtained knowledge regarding transformation reactions and novel TPs of SAL will contribute to elucidate SAL-metabolites with regards to structural prediction.
Rationale: Lasalocid (LAS), an ionophore, is used in cattle and poultry farming as feed additive for its antibiotic and growth-promoting properties. Literature on transformation products (TP) resulting from LAS degradation is limited. So far, only hydroxylation is found to occur as the metabolic reaction during the LAS degradation. To investigate potential TPs of LAS, we used electrochemistry (EC) and liver microsome (LM) assays to synthesize TPs, which were identified using liquid chromatography high-resolution mass spectrometry (LC/HRMS).Methods: Electrochemically produced TPs were analyzed online by direct coupling of the electrochemical cell to the electrospray ionization (ESI) source of a Sciex Triple-TOF high resolution mass spectrometer. Then, EC-treated LAS solution was collected and analyzed offline using LC/HRMS to confirm stable TPs and improve their annotation with a chemical structure due to informative MS/MS spectra. In a complementary approach, TPs formed by rat and human microsomal incubation were investigated using LC/HRMS. The resulting data were used to investigate LAS modification reactions and elucidate the chemical structure of obtained TPs. Results:The online measurements identified a broad variety of TPs, resulting from modification reactions like (de-)hydrogenation, hydration, methylation, oxidation as well as adduct formation with methanol. We consistently observed different ion complexations of LAS and LAS-TPs (Na + ; 2Na + K + ; NaNH 4 + ; KNH 4 + ). Two stable methylated EC-TPs were found, structurally annotated, and assigned to a likely modification reaction. Using LM incubation, seven TPs were formed, mostly by oxidation/hydroxylation. After the identification of LM-TPs as Na + -complexes, we identified LM-TPs as K + -complexes. Conclusion:We identified and characterized TPs of LAS using EC-and LM-based methods. Moreover, we found different ion complexes of LAS-based TPs. This knowledge, especially the different ion complexes, may help elucidate the metabolic and environmental degradation pathways of LAS.
The knowledge of transformation pathways and transformation products of veterinary drugs is important for health, food and environmental matters. Residues (original veterinary drug and transformation products) are found in food products of animal origin and also in the environment (e.g., soil or surface water). Several transformation processes can alter the original veterinary drug, ranging from biotransformation in living organism to environmental degradation processes like photolysis, hydrolysis, or microbial processes. In this thesis, four veterinary drugs were investigated, three ionophore antibiotics Monensin, Salinomycin and Lasalocid and the macrocyclic lactone Moxidectin. Ionophore antibiotics are mainly used to cure and prevent coccidiosis in poultry especially prophylactic in broiler farming. Moxidectin is an antiparasitic drug and is used for the treatment of internal and external parasites in food‐ producing and companion animals. The main objective of this work was the usage of different laboratory approaches to generate and identify transformation products. The identification was conducted using high‐resolution mass spectrometry (HR‐MS). A major focus was put on the application of electrochemistry for simulation of transformation processes. The electrochemical reactor, equipped with a three‐electrode flow‐through cell, enabled the oxidation or reduction by applying a potential. Derived transformation products were analyzed by online coupling of the electrochemical reactor and a HR‐MS and offline by liquid chromatography (LC) combined with HR‐MS. The main modification reaction of the identified transformation products was different for each investigated veterinary drug. Monensin was showing decarboxylation and demethylation as main modification reactions, for Salinomycin mostly decarbonylation was occurring and for Lasalocid methylation was prevalent. For Moxidectin I observed oxidation (hydroxylation) reaction and adduct formation with solvent. In general, for salinomycin and Lasalocid more transient transformation products (online measurement) than stable transformation products (offline measurements) were detected. In contrast, the number of transformation products using online and offline measurements were identical for monensin and moxidectin. As a complementary approach, metabolism tests with rat or human liver microsomes were made for the ionophore antibiotics. Monensin was investigated by using rat liver microsomes and identified transformation products were based on decarboxylation and demethylation. Salinomycin and Lasalocid were converted by human and rat liver microsomes. For both substances were more transformation products found by using human liver microsomes. The transformation products of the rat liver microsome conversion were redundant, the transformation products were also found at the human liver microsome assay. Oxidation (hydroxylation) was found to be the main modification reaction for both. In addition, a frequent ion‐exchange between sodium and potassium was identified. The last two experiments were performed for one substance each, the hydrolysis of monensin and the photolysis of moxidectin was investigated. The transformation products of the pH‐ dependent hydrolysis were based on ring‐opening and dehydration. Moxidectin formed several transformation products by irradiation with UV‐C light and main modification reactions were isomeric changes, (de‐)hydration and changes of the methoxime moiety. In summary, transformation products of the four investigated veterinary drugs were generated by the different laboratory approaches. Most of the identified transformation products were identified for the first time. The resulting findings provide an understanding for clarifying the transformation behavior.
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