Hydroxylation and dimerization of zearalenone: comparison of chemical, enzymatic and electrochemical oxidation methods

2017 ◽  
Vol 10 (4) ◽  
pp. 297-307 ◽  
Author(s):  
J. Keller ◽  
H. Haase ◽  
M. Koch
Keyword(s):  
Mass Spectrometry ◽  
High Resolution Mass Spectrometry ◽  
Liver Microsomes ◽  
Transformation Products ◽  
In Vitro Assays ◽  
Rat Liver Microsomes ◽  
Boron Doped Diamond ◽  
Natural Processes ◽  
Increasing Demand

Investigations of the metabolic pathway of mycotoxins by microsomal techniques are often laborious, causing an increasing demand for easy and rapid simulation methods. Thus, the non-microsomal oxidation technique of electrochemistry coupled online to mass spectrometry (EC/MS) was applied to simulate phase I biotransformation of the Fusarium mycotoxin zearalenone (ZEA). The obtained transformation products were identified by high resolution mass spectrometry (FT-ICR) and HPLC-MS/MS. Transformation products (TPs) from EC/MS were compared to those of other oxidative methods, such as Fenton-like and Ce(IV) reactions and metabolites derived from in vitro assays (human and rat liver microsomes). Electrochemical oxidisation of ZEA was achieved by applying a potential between 0 and 2,500 mV vs Pd/H2 using a flow-through cell with a boron-doped diamond working electrode. Several mono-hydroxylated TPs were generated by EC/MS and Fenton-like reaction, which could also be found in microsomal in vitro assays. EC and Ce(IV) led to the formation of structurally different ZEA dimers and dimeric quinones probably connected over covalent biaryl C-C and C-O-C bonds. Although the dimerization of phenolic compounds is often observed in natural processes, ZEA dimers have not yet been reported. This is the first report on the formation of stable ZEA dimers and their related quinones. The tested non-microsomal methods, in particular EC/MS, could be useful in order to predict the biotransformation products of mycotoxins, even in cases where one to one simulation is not always feasible.

scholarly journals Nontargeted Metabolomics by High-Resolution Mass Spectrometry to Study the In Vitro Metabolism of a Dual Inverse Agonist of Estrogen-Related Receptors β and γ, DN203368

Pharmaceutics ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 776
Author(s):  
Sin-Eun Kim ◽  
Seung-Bae Ji ◽  
Euihyeon Kim ◽  
Minseon Jeong ◽  
Jina Kim ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Resolution ◽  
Human Liver ◽  
High Resolution Mass Spectrometry ◽  
Liver Microsomes ◽  
Human Liver Microsomes ◽  
Rat Liver Microsomes ◽  
High Resolution Mass ◽  
Resolution Mass

DN203368 ((E)-3-[1-(4-[4-isopropylpiperazine-1-yl]phenyl) 3-methyl-2-phenylbut-1-en-1-yl] phenol) is a 4-hydroxy tamoxifen analog that is a dual inverse agonist of estrogen-related receptor β/γ (ERRβ/γ). ERRγ is an orphan nuclear receptor that plays an important role in development and homeostasis and holds potential as a novel therapeutic target in metabolic diseases such as diabetes mellitus, obesity, and cancer. ERRβ is also one of the orphan nuclear receptors critical for many biological processes, such as development. We investigated the in vitro metabolism of DN203368 by conventional and metabolomic approaches using high-resolution mass spectrometry. The compound (100 μM) was incubated with rat and human liver microsomes in the presence of NADPH. In the metabolomic approach, the m/z value and retention time information obtained from the sample and heat-inactivated control group were statistically evaluated using principal component analysis and orthogonal partial least-squares discriminant analysis. Significant features responsible for group separation were then identified using tandem mass spectra. Seven metabolites of DN203368 were identified in rat liver microsomes and the metabolic pathways include hydroxylation (M1-3), N-oxidation (M4), N-deisopropylation (M5), N,N-dealkylation (M6), and oxidation and dehydrogenation (M7). Only five metabolites (M2, M3, and M5-M7) were detected in human liver microsomes. In the conventional approach using extracted ion monitoring for values of mass increase or decrease by known metabolic reactions, only five metabolites (M1-M5) were found in rat liver microsomes, whereas three metabolites (M2, M3, and M5) were found in human liver microsomes. This study revealed that nontargeted metabolomics combined with high-resolution mass spectrometry and multivariate analysis could be a more efficient tool for drug metabolite identification than the conventional approach. These results might also be useful for understanding the pharmacokinetics and metabolism of DN203368 in animals and humans.

scholarly journals Liquid Chromatography-High-Resolution Mass Spectrometry-Based In Vitro Toxicometabolomics of the Synthetic Cathinones 4-MPD and 4-MEAP in Pooled Human Liver Microsomes

Metabolites ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 3
Author(s):  
Sascha K. Manier ◽  
Florian Schwermer ◽  
Lea Wagmann ◽  
Niels Eckstein ◽  
Markus R. Meyer
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
High Resolution ◽  
Hierarchical Clustering ◽  
Human Liver ◽  
High Resolution Mass Spectrometry ◽  
Liver Microsomes ◽  
Synthetic Cathinones ◽  
Resolution Mass

Synthetic cathinones belong to the most often seized new psychoactive substances on an international level. This study investigated the toxicometabolomics, particularly the in vitro metabolism of 2-(methylamino)-1-(4-methylphenyl)-1-pentanone (4-MPD) and 2-(ethylamino)-1-(4-methylphenyl)-1-pentanone (4-MEAP) in pooled human liver microsomes (pHLM) using untargeted metabolomics techniques. Incubations were performed with the substrates in concentrations ranging from 0, 12.5, and 25 µM. Analysis was done by means of high-performance liquid chromatography coupled to high-resolution mass spectrometry (HPLC-HRMS/MS) in full scan only and the obtained data was evaluated using XCMS Online and MetaboAnalyst. Significant features were putatively identified using a separate parallel reaction monitoring method. Statistical analysis was performed using Kruskal-Wallis test for prefiltering significant features and subsequent hierarchical clustering, as well as principal component analysis (PCA). Hierarchical clustering or PCA showed a distinct clustering of all concentrations with most of the features z-scores rising with the concentration of the investigated substances. Identification of significant features left many of them unidentified but revealed metabolites of both 4-MPD and 4-MEAP. Both substances formed carboxylic acids, were hydroxylated at the alkyl chain, and formed metabolites after combined hydroxylation and reduction of the cathinone oxo group. 4-MPD additionally formed a dihydroxy metabolite and a hydroxylamine. 4-MEAP was additionally found reduced at the cathinone oxo group, N-dealkylated, and formed an oxo metabolite. These findings are the first to describe the metabolic pathways of 4-MPD and to extend our knowledge about the metabolism of 4-MEAP. Findings, particularly the MS data of the metabolites, are essential for setting up metabolite-based toxicological (urine) screening procedures.

scholarly journals Identification of in vitro and in vivo potential metabolites of novel cardiovascular and adrenolytic drugs by liquid chromatography-mass spectrometry with the aid of experimental design

2019 ◽  
Vol 18 (2) ◽  
pp. 179-194
Author(s):  
Malgorzata Szultka-Mlynska ◽  
Katarzyna Pauter ◽  
Boguslaw Buszewski
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Phase Ii ◽  
Liver Microsomes ◽  
Biologically Active ◽  
Rat Liver Microsomes ◽  
Gas Temperature ◽  
Liquid Chromatography Tandem Mass

Abstract Drug metabolism in liver microsomes was studied in vitro using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Relevant drug was incubated with dog, human and rat liver microsomes (DLMs, HLMs, RLMs) along with NADPH, and the reaction mixture was analyzed by LC-MS/MS to obtain specific metabolic profile. GRACE analytical C18 column, Vision HT (50 × 2 mm, 1.5 μm) was implemented with acetonitrile and water (+ 5 mM ammonium acetate) in a gradient mode as the mobile phase at a flow 0.4 mL.min−1. Different phase I and phase II metabolites were detected and structurally described. The metabolism of the studied drugs occurred via oxidation, hydroxylation and oxidative deamination processes. Conjugates with the glucuronic acid and sulfate were also observed as phase II biotransformation. The central composite design (CCD) showed that factors, such as time incubation, liver microsomal enzymes concentration and NADPH concentration, along with drying gas temperature, nebulizer gas pressure and capillary voltage significantly affected the final response of the method. This study describes the novel information about the chemical structure of the potential metabolites of selected biologically active compounds, which provide vital data for further pharmacokinetic and in vivo metabolism studies.

scholarly journals In Vitro Toxicokinetics and Phase I Biotransformation of the Mycotoxin Penitrem A in Dogs

Toxins ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 293 ◽  
Author(s):  
Silvio Uhlig ◽  
Lada Ivanova ◽  
Pauline Voorspoels ◽  
Christiane Kruse Fæste
Keyword(s):  
Phase I ◽  
High Resolution Mass Spectrometry ◽  
Liver Microsomes ◽  
Absorbed Dose ◽  
Metabolite Profile ◽  
Oxidation Products ◽  
In Vitro Assays ◽  
Penicillium Species

The tremorgenic mycotoxin penitrem A is produced by Penicillium species as a secondary metabolite on moldy food and feed. Dogs are sometimes exposed to penitrem A by consumption of spoiled food waste or fallen fruit. The lipophilic toxin crosses the blood-brain barrier and targets neuroreceptors and neurotransmitter release mechanisms in the central and peripheral nervous systems. Typical symptoms of penitrem A intoxication are periodical or continuous tremors, which can be passing, persistent or lethal, depending on the absorbed dose. There is presently no information on the biotransformation and toxicokinetics of penitrem A in dogs. The aim of the present study was therefore to identify potential metabolites of the toxin by performing in vitro biotransformation assays in dog liver microsomes. Analyses by liquid chromatography coupled to high-resolution mass spectrometry led to the provisional identification of eleven penitrem A phase I metabolites, which were tentatively characterized as various oxidation products. Furthermore, elimination parameters determined in in vitro assays run under linear kinetics were used for in vitro-to-in vivo extrapolation of the toxicokinetic data, predicting a maximal bioavailability of more than 50%. The metabolite profile detected in the in vitro assays was similar to that observed in the plasma of an intoxicated dog, confirming the predictive capability of the in vitro approach.

scholarly journals In vitro biotransformation of pyrrolizidine alkaloids in different species: part II—identification and quantitative assessment of the metabolite profile of six structurally different pyrrolizidine alkaloids

2020 ◽  
Vol 94 (11) ◽  
pp. 3759-3774
Author(s):  
Ina Geburek ◽  
Dieter Schrenk ◽  
Anja These
Keyword(s):  
Human Liver ◽  
Pyrrolizidine Alkaloids ◽  
High Resolution Mass Spectrometry ◽  
Liver Microsomes ◽  
Metabolite Profile ◽  
Human Liver Microsomes ◽  
Rat Liver Microsomes ◽  
Metabolite Profiles ◽  
Fragmentation Patterns

Abstract Pyrrolizidine alkaloids (PA) exert their toxic effects only after bioactivation. Although their toxicity has already been studied and metabolic pathways including important metabolites were described, the quantification of the latter revealed a large unknown portion of the metabolized PA. In this study, the qualitative and quantitative metabolite profiles of structurally different PAs in rat and human liver microsomes were investigated. Between five metabolites for europine and up to 48 metabolites for lasiocarpine were detected. Proposals for the chemical structure of each metabolite were derived based on fragmentation patterns using high-resolution mass spectrometry. The metabolite profiles of the diester PAs showed a relatively good agreement between both species. The metabolic reactions were summarized into three groups: dehydrogenation, oxygenation, and shortening of necic acid(s). While dehydrogenation of the necine base is considered as bioactivation, both other routes are considered as detoxification steps. The most abundant changes found for open chained diesters were dealkylations, while the major metabolic pathway for cyclic diesters was oxygenation especially at the nitrogen atom. In addition, all diester PAs formed several dehydrogenation products, via the insertion of a second double bond in the necine base, including the formation of glutathione conjugates. In rat liver microsomes, all investigated PAs formed dehydropyrrolizidine metabolites with the highest amount formed by lasiocarpine, whereas in human liver microsomes, these metabolites could only be detected for diesters. Our findings demonstrate that an extensive analysis of PA metabolism can provide the basis for a better understanding of PA toxicity and support future risk assessment.

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