Electrochemistry-mass spectrometry bridging the gap between suspect and target screening of valsartan transformation products in wastewater treatment plant effluent.

Degradation of xenobiotics in wastewater treatment plants may lead to the formation of transformation products with higher persistence or increased (eco-)toxic potential compared to the parent compounds. Accordingly, the identification of transformation products from wastewater treatment plant effluents has gained increasing attention. Here, we show the potential of electrochemistry hyphenated to liquid chromatography and mass spectrometry for the prediction of oxidative degradation in wastewater treatment plants using the antihypertensive drug valsartan as a model compound. This approach identifies seven electrochemical transformation products of valsartan, which are used to conduct a suspect screening in effluent of the main wastewater treatment plant in the city of Münster in Germany. Apart from the parent compound valsartan, an electrochemically predicted transformation product, the N-dealkylated ETP336, is detected in wastewater treatment plant effluent. Subsequently, a targeted liquid chromatographytandem mass spectrometry method for the detection of valsartan and its electrochemical transformation products is set up. Here, electrochemical oxidation is used to generate reference materials of the transformation products in situ by hyphenating electrochemistry online to a triple quadrupole mass spectrometer. Using this setup, multiple reaction monitoring transitions are set up without the need for laborious and costly synthesis and isolation of reference materials for the transformation products. The targeted method is then applied to extracts from wastewater treatment plant effluent and the presence of ETP336 and valsartan in the samples is verified. The presented workflow can be used to set up targeted analysis methods for previously unknown transformation products even without the need for expensive high-resolution mass spectrometers.

Laser desorption/ionization-mass spectrometry for the analysis of interphases in lithium ion batteries.

Laser desorption/ionization-mass spectrometry (LDI-MS) is introduced as a complementary technique for the analysis of interphases formed at electrode|electrolyte interfaces in lithium ion batteries (LIBs). An understanding of these interphases is crucial for designing interphase-forming electrolyte formulations and increasing battery lifetime. Especially organic species are analyzed more effectively using LDI-MS than with established methodologies. The combination with trapped ion mobility spectrometry and tandem mass spectrometry yields additional structural information of interphase components. Furthermore, LDI-MS imaging reveals the lateral distribution of compounds on the electrode surface. Using the introduced methods, a deeper understanding of the mechanism of action of the established solid electrolyte interphase-forming electrolyte additive 3,4-dimethyloxazolidine-2,5-dione (Ala-N-CA) for silicon/graphite anodes is obtained, and active electrochemical transformation products are unambiguously identified. In the future, LDI-MS will help to provide a deeper understanding of interfacial processes in LIBs by using it in a multimodal approach with other surface analysis methods to obtain complementary information.

Benfluorex metabolism complemented by electrochemistry-mass spectrometry.

The hypolipidemic and hypoglycemic drug benfluorex was widely applied to treat type 2 diabetes mellitus and metabolic syndrome in overweight patients since 1976. However, benfluorex was connected to multiple cases of valvular heart disease and pulmonary arterial hypertension later on. Similar adverse drug reactions were previously found to be associated to the structurally related drug fenfluramine, which was attributed to the formation of its N-deethylated metabolite norfenfluramine. Even though norfenfluramine was known to be a common metabolite of fenfluramine and benfluorex, only fenfluramine was withdrawn from European and United States markets in 1997 while benfluorex remained available until 2009. In this work, the metabolism of benfluorex is simulated by an online hyphenation of electrochemistry and mass spectrometry and the observed transformation products are further characterized using liquid chromatography and high-resolution tandem mass spectrometry. Using this approach, norfenfluramine is found to be the main electrochemical transformation product of benfluorex. Considering the knowledge about norfenfluramine toxicity, rapid metabolite screening using electrochemistry hyphenated to mass spectrometry could have been used to predict the potential of benfluorex for adverse drug reactions early on, showcasing the value of electrochemical metabolism mimicry for rapid drug safety evaluation.

Comparison of in vitro approaches for predicting the metabolism of the selective androgen receptor modulator RAD140.

The identification of metabolites allows for the expansion of possible targets for anti-doping analysis. Especially for novel substances such as selective androgen receptor modulators (SARMs), information on metabolic fate is scarce. Novel approaches such as the organ on a chip technology may provide a metabolic profile that resembles human in vivo samples more closely than approaches that rely on human liver fractions only. In this study, the SARM RAD140 was metabolized by means of subcellular human liver fractions, human liver spheroids in an organ on a chip platform, and electrochemical (EC) conversion. The resulting metabolites were analyzed with LC-HRMS/MS and compared to a human doping control urine sample that yielded an adverse analytical finding for RAD140. A total of 16 metabolites were detected in urine, while 14, 13, and 7 metabolites were detected in samples obtained from the organ on a chip experiment, the subcellular liver fraction, and EC experiments, respectively. All tested techniques resulted in the detection of RAD140 metabolites. In the organ on a chip samples, the highest number of metabolites were detected. The subcellular liver fractions and organ on a chip techniques are deemed complementary to predict metabolites of RAD140, as both techniques produce distinct metabolites that are also found in an anonymized human in vivo urine sample.

Electrochemistry/mass spectrometry (EC/MS) for fast generation and identification of novel reactive metabolites of two unsymmetrical bisacridines with anticancer activity.

The development of a new drug requires knowledge about its metabolic fate in a living organism, regarding the comprehensive assessment of both drug therapeutic activity and toxicity profiles. Electrochemistry (EC) coupled with mass spectrometry (MS) is an efficient tool for predicting the phase I metabolism of redox-sensitive drugs. In particular, EC/MS represents a clear advantage for the generation of reactive drug transformation products and their direct identification compared to biological matrices. In this work, we focused on the characterization of novel electrochemical products of two representative unsymmetrical bisacridines (C-2028 and C-2045) with demonstrated high anticancer activity. The electrochemical thin-layer flow-through cell µ-PrepCell 2.0 (Antec Scientific) was used here for the effective metabolite electrosynthesis. The electrochemical simulation of C-2028 reductive and C-2045 oxidative metabolism resulted in the generation of new products that were not observed before. The formation of nitroso [M-O+H]+ and azoxy [2M-3O+H]+ species from C-2028, as well as a series of hydroxylated and/or dehydrogenated products, including possible quinones [M-2H+H]+ and [M+O-2H+H]+ from C-2045, was demonstrated. For the latter, a glutathione S-conjugate (m/z 935.3130) was also obtained in measurements supplemented with the excess of reduced glutathione. For the identification of the products of interest, structural confirmation based on MS/MS fragmentation experiments was performed. Novel products of electrochemical conversions of unsymmetrical bisacridines were discussed in the context of their possible biological effect on the human organism.

Elucidation of the environmental reductive metabolism of the herbicide tritosulfuron assisted by electrochemistry and mass spectrometry.

The environmental impact of pesticides and other pollutants is, to a great extent, determined by degradation and accumulation processes. Consequently, degradation pathways of pesticides have to be elucidated before approval by the authorities. In this study, the environmental metabolism of the sulfonylurea-herbicide tritosulfuron was investigated using aerobic soil degradation studies, during which a previously unidentified metabolite was observed using high performance liquid chromatography and mass spectrometry. The new metabolite was formed by reductive hydrogenation of tritosulfuron but the isolated amount and purity of the substance were insufficient to fully elucidate its structure. Therefore, electrochemistry coupled to mass spectrometry was successfully applied to mimic the reductive hydrogenation of tritosulfuron. After demonstrating the general feasibility of electrochemical reduction, the electrochemical conversion was scaled up to the semi-preparative scale and 1.0 mg of the hydrogenated product was synthesized. Similar retention times and mass spectrometric fragmentation patterns proved that the same hydrogenated product was formed electrochemically and in soil studies. Using the electrochemically generated standard, the structure of the metabolite was elucidated by means of NMR spectroscopy, which shows the potential of electrochemistry and mass spectrometry in environmental fate studies.

Development of an electrochemical flow-through cell for the fast and easy generation of isotopically labeled metabolite standards.

In drug development, metabolite standards of new chemical entities are required for a comprehensive safety evaluation. Stable isotope-labeled internal metabolite standards at the milligram scale, which are difficult and expensive to synthesize in common bottom-up approaches, are necessary for metabolite quantification using liquid chromatography/mass spectrometry. A preparative electrochemical flow-through cell is presented here as a powerful tool for the cheap and straightforward synthesis of milligram amounts of isotopically labeled metabolite standards. The developed cell scales up established, so-called "coulometric" electrochemical cells. Problems like electrode fouling and cross contamination between syntheses are addressed by the use of exchangeable working electrodes. The applicability of the developed cell for the synthesis of metabolite standards is demonstrated using isotopically labeled acetaminophen as a model system for the generation of a biologically relevant phase II metabolite.

Microfluidic Electrochemistry Meets Trapped Ion Mobility Spectrometry and High-Resolution Mass Spectrometry-In Situ Generation, Separation, and Detection of Isomeric Conjugates of Paracetamol and Ethoxyquin.

Over the last 3 decades, electrochemistry (EC) has been successfully applied in phase I and phase II metabolism simulation studies. The electrochemically generated phase I metabolite-like oxidation products can react with selected reagents to form phase II conjugates. During conjugate formation, the generation of isomeric compounds is possible. Such isomeric conjugates are often separated by high-performance liquid chromatography (HPLC). Here, we demonstrate a powerful approach that combines EC with ion mobility spectrometry to separate possible isomeric conjugates. In detail, we present the hyphenation of a microfluidic electrochemical chip with an integrated mixer coupled online to trapped ion mobility spectrometry (TIMS) and time-of-flight high-resolution mass spectrometry (ToF-HRMS), briefly chipEC-TIMS-ToF-HRMS. This novel method achieves results in several minutes, which is much faster than traditional separation approaches like HPLC, and was applied to the drug paracetamol and the controversial feed preservative ethoxyquin. The analytes were oxidized in situ in the electrochemical microfluidic chip under formation of reactive intermediates and mixed with different thiol-containing reagents to form conjugates. These were analyzed by TIMS-ToF-HRMS to identify possible isomers. It was observed that the oxidation products of both paracetamol and ethoxyquin form two isomeric conjugates, which are characterized by different ion mobilities, with each reagent. Therefore, using this hyphenated technique, it is possible to not only form reactive oxidation products and their conjugates in situ but also separate and detect these isomeric conjugates within only a few minutes.