Liquid Chromatography – High Resolution Mass Spectrometry Method for Monitoring of 17 Mycotoxins in Human Plasma for Exposure Studies

Mycotoxins are secondary metabolites produced by filamentous fungi. Primary route of human exposure to mycotoxins is the intake of the contaminated food. Minimizing mycotoxin exposure is important for population health, as their chronic toxic effects have been associated with kidney and liver diseases, some types of cancer and immunosuppression. The objective of this work was to develop and validate a multi-class mycotoxin method suitable for exposure monitoring of mycotoxins in human plasma. A sensitive liquid chromatography – mass spectrometry method was developed for 17 mycotoxins: nivalenol (NIV), deoxynivalenol, fusarenon X, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, T-2 toxin, HT-2 toxin, aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, zearalenone, α-zearalenol (α-ZOL), β-zearalenol, zearalanone, α-zeranoland, and β-zeranol. The method relies on three-step liquid-liquid extraction with ethyl acetate to eliminate the need for immunoaffinity extraction and minimize ionization matrix effects. Chromatographic separation of mycotoxins, including all isomers, was achieved with pentafluorophenyl column and water/methanol mobile phase. Mycotoxin detection and quantitation were performed using high-resolution mass spectrometry on LTQ Velos Orbitrap, in both positive and negative electrospray ionization (ESI(+) and (ESI(−)). The use of 0.02% acetic acid as mobile phase additive for ESI(−) resulted in significant increase in ionization efficiency ranging from 1.7 to 26 times for mycotoxins that ionize better in ESI(−). The optimized method was validated according to FDA guidance procedures. LOQs of all mycotoxins ranged from 0.1 to 0.5 ng/ml, except NIV which resulted in LOQ of 3 ng/ml because of low extraction recovery of this highly polar mycotoxin. Mean intra-day accuracy ranged from 85.8% to 116.4%, and intra-day precision (n = 6) ranged from 1.6% to 12.5% RSD for all mycotoxins except α-ZOL where mean accuracy ranged from 72.9% to 97.2%. Inter-day accuracy and precision were 85.6% to 111.5% and 2.7 to 15.6% RSD respectively, showing good analytical performance of the method for biomonitoring

Multi-Class Liquid Chromatography-High Resolution Mass Spectrometry Methods for Monitoring of Mycotoxins and Metabolites in Human Plasma for Exposure Studies

Mycotoxins are secondary metabolites produced by fungi that can pose a serious threat to human and animal health due to their toxicity. The assessment of human chronic exposure to mycotoxins requires reliable and highly sensitive multi-analyte assay(s) enabling simultaneous measurements of common toxicologically important mycotoxins and their metabolites in human plasma. The first goal of the thesis was to develop sensitive liquid chromatography – high-resolution mass spectrometry (LC-HRMS) multi-mycotoxin method(s) for the detection and quantification of common toxicologically important mycotoxins frequently occurring in Canada and emerging mycotoxins of interest. Based on the results of extraction recoveries and chromatographic separation, two LC-HRMS methods were required to cover the full mycotoxin panel of interest. The first method combined liquid-liquid extraction with pentafluorophenyl reversed-phase LC-HRMS for the quantification of 17 mycotoxins, aflatoxins B1, B2, G1 and G2, zearalenone, 7-α-hydroxy-zearalenol (α-ZOL), 7-β-hydroxy-zearalenol, zearalanone, 7-α-hydroxy-zearalanol, 7-β-hydroxy-zearalanol, T-2 toxin, HT-2 toxin, deoxynivalenol, nivalenol, 15-acetyldeoxynivalenol, 3-acetyldeoxynivalenol and fusarenon X. The method was validated using procedures described in the Food and Drug Administration (FDA) guidance for Industry Bioanalytical Method Validation. Lower limits of quantification (LLOQs) ranged from 0.1 to 0.5 ng/ml, except for nivalenol (3 ng/ml). The method (intra-day and inter-day) accuracy and precision ranged from 85.6% to 116.4% and from 1.6% to 15.6% RSD, respectively, excluding α-ZOL for which an accuracy of 72.9 % to 97.2% was observed. The second method covered ten mycotoxins, fumonisin B1, fumonisin B2, ochratoxin α (OTα), citrinin, ochratoxin A, beauvericin, enniatin A, enniatin A1 (ENNA1), enniatin B (ENNB) and enniatin B1, and combined methanol protein precipitation with C18 reversed-phase chromatography and polarity-switching LC-HRMS. LLOQs ranged from 1.25 to 4 ng/ml. Absolute recovery ranged from 86.6% to 127.7% in individual plasma samples. Significant matrix effects were observed for OTα (77.5%) in one out of ten individual plasma samples and fumonisins (134.8% to 167.8%), ENNB (69.7% to 79.4%) and ENNA (69.3% to 79.2%) in all individual plasma samples. The rest of the mycotoxins showed negligible matrix effects ranging from 87.2% to 112.2% in all lots of plasma tested. Excellent LLOQs, negligible matrix effects and accurate quantitation capability of the first method coupled with the lower cost of analysis per sample make the method suitable for large-scale analysis of human plasma samples. The second method is also simple and low cost but requires additional modification to further improve LLOQs and reduce the matrix effect before full validation and implementation. Both methods are versatile and can be applied for retrospective analysis and other applications such as metabolism studies due to the use of HRMS and superior chromatographic separation. To show this capability, the first method was successfully applied for the in-depth metabolism studies of 17 mycotoxins. The method showed excellent suitability and advantages for the detection of various mycotoxin metabolites from Phase I metabolism and glucuronidation obtained from human microsomal incubations. Two ppm mass accuracy with internal mass calibration reduced the number of possible elemental formulas for a measured m/z value. Data-dependent acquisition in combination with collision-induced dissociation or higher energy collisional dissociation was used to ensure adequate fragmentation and to study the structure of the mycotoxin metabolites. The Compound Discoverer 2.1 software, which contains extensive libraries of common metabolic pathways and mass spectral libraries, was used to streamline the identification and the characterization of the metabolites. In total, 188 mycotoxin metabolites were generated, characterized and used to build an extensive in-house library of human mycotoxin metabolites. One hundred metabolites were reported for the first time, showing the power and sensitivity of the approach. For these 17 mycotoxins, 92 metabolites were previously described in literature, and among these known metabolites only four could not be generated using our approach. Currently, this is the most comprehensive LC-MS library of human mycotoxin metabolites. In conclusion, both LC-MS methods and the in-house mycotoxin metabolite library will allow the monitoring of 27 mycotoxins and their 188 metabolites in large-scale biomonitoring studies. In the long-term, this will help to prioritize metabolites that should be routinely included during exposure monitoring studies and will provide important new data on mycotoxin exposure of the Canadian population

Characterization of Phase I and Glucuronide Phase II Metabolites of 17 Mycotoxins Using Liquid Chromatography—High-Resolution Mass Spectrometry

Routine mycotoxin biomonitoring methods do not include many mycotoxin phase I and phase II metabolites, which may significantly underestimate mycotoxin exposure especially for heavily metabolized mycotoxins. Additional research efforts are also needed to measure metabolites in vivo after exposure and to establish which mycotoxin metabolites should be prioritized for the inclusion during large-scale biomonitoring efforts. The objective of this study was to perform human in vitro microsomal incubations of 17 mycotoxins and systematically characterize all resulting metabolites using liquid chromatography–high-resolution mass spectrometry (LC-HRMS). The results obtained were then used to build a comprehensive LC-MS library and expand a validated 17-mycotoxin method for exposure monitoring to screening of additional 188 metabolites, including 100 metabolites reported for the first time. The final method represents one of the most comprehensive LC-HRMS methods for mycotoxin biomonitoring or metabolism/fate studies

Metabolism and pharmacokinetics of a potent N -acylindole antagonist of the OXE receptor for the eosinophil chemoattractant 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) in rats and monkeys

We previously identified the indole 264 as a potent in vitro antagonist of the human OXE receptor that mediates the actions of the powerful eosinophil chemoattractant 5-oxo-ETE. No antagonists of this receptor are currently commercially available or are being tested in clinical studies. The lack of a rodent ortholog of the OXE receptor has hampered progress in this area because of the unavailability of commonly used mouse or rat animal models. In the present study, we examined the feasibility of using the cynomolgus monkey as an animal model to investigate the efficacy of orally administered 264 in future in vivo studies. We first confirmed that 264 is active in monkeys by showing that it is a potent inhibitor of 5-oxo-ETE-induced actin polymerization and chemotaxis in granulocytes. The major microsomal metabolites of 264 were identified by cochromatography with authentic chemically synthesized standards and LC-MS/MS as its ω2-hydroxy and ω2-oxo derivatives, formed by ω2-oxidation of its hexyl side chain. Small amounts of ω1-oxidation products were also identified. None of these metabolites have substantial antagonist potency. High levels of 264 appeared rapidly in the blood following oral administration to both rats and monkeys, and declined to low levels by 24 h. As with microsomes, its major plasma metabolites in monkeys were ω2-oxidation products. We conclude that the monkey is a suitable animal model to investigate potential therapeutic effects of 264. This, or a related compound with diminished susceptibility to ω2-oxidation, could be a useful therapeutic agent in eosinophilic disorders such as asthma

Личностные детерминанты повышения эффективности занятий спортом представителей молодежи Московской области

The article focuses on the issue of improving athletes’ performance. The subject of the research is personal determinants of improving the effectiveness of sports activities among young people. The purpose is to identify personal determinants that predefine the effectiveness of sports among young people. In the process of studying personal determinants that predefine the effectiveness of sports among young people we applied the methods of logical, theoretical and comparative analysis, content analysis, interpretation and scientific data interpretation, generalisation and modelling. On the basis of theoretical analysis the study identified the most significant personal determinants for the increase in the effectiveness of sports among youth, developed a theoretical structural and functional psychological “Personal determinants of improving the effectiveness of sports among young people” model and determined the interrelation of intrapersonal structures mediating the process of the influence of biological, social and individual personal determinants to increase the effectiveness of sports among young peopleВ статье уделено внимание проблеме повышения эффективности спортсменов. Предметом исследования служат личностные детерминанты повышения эффективности занятий спортом представителей молодежи. Цель работы – выявление личностных детерминант, предопределяющих повышение эффективности занятий спортом представителей молодежи. В процессе исследования личностных детерминант, предопределяющих повышение эффективности занятий спортом представителей молодежи, были использованы логический, теоретический и сравнительный виды анализа, контент-анализ, интерпретация научных данных, обобщение, моделирование. В результате проведенного исследования на основе теоретического анализа выделены наиболее значимые личностные детерминанты повышения эффективности занятий спортом представителей молодежи, разработана теоретическая структурно-функциональная психологическая модель «Личностные детерминанты повышения эффективности занятий спортом представителей молодежи», выявлена взаимосвязь внутриличностных структур, опосредующих процесс влияния биологических, социальных, индивидуально-личностных детерминант личности на повышение эффективности занятий спортом представителей молодеж

Novel Highly Potent and Metabolically Resistant Oxoeicosanoid (OXE) Receptor Antagonists That Block the Actions of the Granulocyte Chemoattractant 5‑Oxo-6,8,11,14-Eicosatetraenoic Acid (5-oxo-ETE)

5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a potent lipid mediator that induces tissue eosinophilia via the selective OXE receptor (OXE-R), which is an attractive therapeutic target in eosinophilic diseases. We previously identified indole OXE-R antagonists that block 5-oxo-ETE-induced primate eosinophil activation. Although these compounds possess good oral absorption, their plasma levels decline rapidly due to extensive oxidation of their hexyl side chain. We have now succeeded in dramatically increasing antagonist potency and resistance to metabolism by replacing the hexyl group with phenylpentyl or phenylhexyl side chains. Compared with our previous lead compound <b><i>S</i>-230</b>, our most potent antagonist, <b><i>S</i>-C025</b>, has an IC₅₀ (120 pM) over 80 times lower and a substantially longer plasma half-life. A single major metabolite, which retains antagonist activity (IC₅₀, 690 pM) and has a prolonged lifetime in plasma was observed. These new highly potent OXE-R antagonists may provide a novel strategy for the treatment of eosinophilic disorders like asthma

Pharmacokinetics and Metabolism of Selective Oxoeicosanoid (OXE) Receptor Antagonists and Their Effects on 5‑Oxo-6,8,11,14-eicosatetraenoic Acid (5-Oxo-ETE)-Induced Granulocyte Activation in Monkeys

The potent eosinophil chemoattractant 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a 5-lipoxygenase product that acts via the selective OXE receptor, which is present in many species, but not rodents. We previously reported that the indole <b>230</b> is a potent human OXE receptor antagonist. The objective of the present study was to determine whether the monkey would be a suitable animal model to investigate its pharmaceutical potential. We found that monkey leukocytes synthesize and respond to 5-oxo-ETE and that <b>230</b> is a potent antagonist of the OXE receptor in monkey eosinophils. Pharmacokinetic studies revealed that <b>230</b> appears rapidly in the blood following oral administration. Using chemically synthesized standards, we identified the major microsomal and plasma metabolites of <b>230</b> as products of ω2-hydroxylation of the alkyl side chain. These studies demonstrate that the monkey is a promising animal model to investigate the drug potential of OXE receptor antagonists