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

Solid-phase microextraction as sample preparation method for metabolomics

The main objective of the emerging field of metabolomics is the analysis of all small molecule metabolites present in a particular living system in order to provide better understanding of dynamic processes occurring in living systems. This type of studies is of interest in various fields including systems biology, medicine and drug discovery. The main requirements for sample preparation methods used in global metabolomic studies are lack of selectivity, incorporation of a metabolism quenching step and good reproducibility. The efficiency of metabolism quenching and stability of analytes in selected biofluid or tissue dictate how accurately the analytical results represent true metabolome composition at the time of sampling. However, complete quenching of metabolism is not easily accomplished, so sample preparation can significantly affect metabolome's composition and the quality of acquired metabolomics data. In this research, the feasibility of the use of solid-phase microextraction (SPME) in direct extraction mode for global metabolomic studies of biological fluids based on liquid chromatography-mass spectrometry (LC-MS) was investigated for the first time. Initial research presented in this thesis focused on resolving several outstanding issues regarding the use of SPME for the analysis of biological fluids. SPME was not simultaneously capable to provide high-sample throughput and high degree of automation when coupled to LC-MS. This was successfully addressed through the development and evaluation of a new robotic station based on a 96-well plate format and an array of 96 SPME fibres. The parallel format of extraction and desorption allowed increased sample throughput of >1000 samples/day which represents the highest throughput of any SPME technique to date. This exceeds sample throughput requirements for a typical metabolomics study whereby ~100 samples/day are processed. SPME can also be used for direct in vivo sampling of flowing blood of an animal without the need to isolate a defined sample volume. This format of SPME is particularly attractive for metabolomic studies as it decreases the overall number of steps and also eliminates the need for metabolism quenching step because only small molecular weight species are extracted by the device, whereas large biological macromolecules such as proteins are not extracted by the coating. In current work, in vivo SPME sampling was successfully applied for sampling of mice for the first time. The proposed sampling procedure was fully validated against traditional terminal and serial sampling approaches for a pharmacokinetic study of carbamazepine and its metabolite. Excellent agreement of pharmacokinetic parameters such as systemic clearance, steady-state volume of distribution and terminal half-life was found for all three methods, with no statistically significant differences (p>0.05). The performance of new prototype commercial SPME devices based on hypodermic needle was also evaluated within the context of the study. The availability of such single-use devices with excellent inter-fibre reproducibility (<10% RSD) presents an important step forward in order to gain wider acceptance of in vivo SPME sampling. Finally, existing SPME coatings were not suitable for the simultaneous direct extraction of both hydrophilic and hydrophobic species, which is one of the requirements for a successful global metabolomics study. To address this issue, a systematic study of 40 types of commercially available sorbents was carried out using a metabolite standard test mixture spanning a wide molecular weight (80-777 Da) and polarity range (log P range of -5 to 7.4). The best performance for balanced extraction of species of varying polarity was achieved by (i) mixed-mode coating containing octadecyl or octyl group and benzenesulfonic acid ion exchange group, (ii) polar-enhanced polystyrene-divinylbenzene polymeric coatings and (iii) phenylboronic acid coatings. The second aspect of the research focused on the evaluation of SPME for a global metabolomics study of human plasma using two complementary LC-MS methods developed on benchtop Orbitrap MS system: reverse-phase method using pentafluorophenyl LC stationary phase and HILIC method using underivatized silica stationary phase. The parameters influencing overall method sensitivity such as voltages, mass ranges and ion inject times into C-trap were optimized to ensure best instrument performance for global metabolomic studies. Orbitrap system provided a powerful platform for metabolomics because of its high resolution and mass accuracy, thus helping to distinguish between metabolites with same nominal mass. The acquisition speed of the instrument at the highest resolution setting was insufficient for use with ultrahigh performance liquid chromatography (UHPLC), so all methods were developed using conventional LC. However, overall metabolite coverage achieved in current study compared well or even exceeded metabolite coverage reported in literature on different LC-MS or UHPLC-MS platforms including time-of-flight, quadrupole time-of-flight and hybrid Orbitrap instruments. The performance of SPME was fully compared versus traditional methods for global metabolomics (plasma protein precipitation and ultrafiltration). The main findings of this systematic study show that SPME provides improved coverage of hydrophobic metabolites versus ultrafiltration and reduces ionization suppression effects observed with both plasma protein precipitation and ultrafiltration methods. Using SPME, <5% and <20% of peaks showed significant matrix effects in reverse phase and HILIC methods, respectively and the observed effects were mostly correlated to elution within retention time window of anticoagulant for the majority of metabolites showing this effect. This improves overall quality of collected metabolomics data and can also improve metabolite coverage. For example, the highest number of metabolite features (3320 features) was observed using SPME in combination with negative ESI reverse-phase LC method, while in positive ESI mode plasma protein precipitation with methanol/ethanol mixture provided the most comprehensive metabolite coverage (3245 features versus 1821 features observed for SPME). Method precision of SPME method was excellent as evaluated using median RSD (11-18% RSD) of all metabolites detected. A proof-of-concept in vivo SPME study was also performed on mice to study the effects of carbamazepine administration and shows that SPME can be used as successful sample preparation method for global metabolomic studies in combination with unsupervised statistical data analysis techniques. This study highlights important advantages of in vivo sampling approaches including the ability to capture short-lived and/or unstable metabolites, to achieve truer representation of the metabolome at the time of sampling than achievable by blood withdrawal methods and the ability to use smaller animal cohorts while obtaining highly-relevant data sets. The experimental results provide new and useful insight into the effects of different sample preparation methods on the collected metabolomics data, and establish both in vitro and in vivo SPME as a new tool for global LC-MS metabolomics analysis for the first time

High-throughput solid-phase microextraction in multi-well-plate format

This review summarizes different formats of high-throughput, multi-well, solid-phase microextraction (SPME), including fiber, thin-film and in-tip configurations, with the particular focus on its fit within regulated analysis. New developments of the devices, such as monolithic and biocompatible extraction phases, are covered. Finally, selected applications of the technique, including the analysis of whole-blood samples and automated binding studies, are presented

Systematic Assessment of Seven Solvent and Solid-Phase Extraction Methods for Metabolomics Analysis of Human Plasma by LC-MS

The comparison of extraction methods for global metabolomics is usually executed in biofluids only and focuses on metabolite coverage and method repeatability. This limits our detailed understanding of extraction parameters such as recovery and matrix effects and prevents side-by-side comparison of different sample preparation strategies. To address this gap in knowledge, seven solvent-based and solid-phase extraction methods were systematically evaluated using standard analytes spiked into both buffer and human plasma. We compared recovery, coverage, repeatability, matrix effects, selectivity and orthogonality of all methods tested for non-lipid metabolome in combination with reversed-phased and mixed-mode liquid chromatography mass spectrometry analysis (LC-MS). Our results confirmed wide selectivity and excellent precision of solvent precipitations, but revealed their high susceptibility to matrix effects. The use of all seven methods showed high overlap and redundancy which resulted in metabolite coverage increases of 34–80% depending on LC-MS method employed as compared to the best single extraction protocol (methanol/ethanol precipitation) despite 7x increase in MS analysis time and sample consumption. The most orthogonal methods to methanol-based precipitation were ion-exchange solid-phase extraction and liquid-liquid extraction using methyl-tertbutyl ether. Our results help facilitate rational design and selection of sample preparation methods and internal standards for global metabolomics

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

Amino Acid Starvation Induced by Invasive Bacterial Pathogens Triggers an Innate Host Defense Program

SummaryAutophagy, which targets cellular constituents for degradation, is normally inhibited in metabolically replete cells by the metabolic checkpoint kinase mTOR. Although autophagic degradation of invasive bacteria has emerged as a critical host defense mechanism, the signals that induce autophagy upon bacterial infection remain unclear. We find that infection of epithelial cells with Shigella and Salmonella triggers acute intracellular amino acid (AA) starvation due to host membrane damage. Pathogen-induced AA starvation caused downregulation of mTOR activity, resulting in the induction of autophagy. In Salmonella-infected cells, membrane integrity and cytosolic AA levels rapidly normalized, favoring mTOR reactivation at the surface of the Salmonella-containing vacuole and bacterial escape from autophagy. In addition, bacteria-induced AA starvation activated the GCN2 kinase, eukaryotic initiation factor 2α, and the transcription factor ATF3-dependent integrated stress response and transcriptional reprogramming. Thus, AA starvation induced by bacterial pathogens is sensed by the host to trigger protective innate immune and stress responses

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

Metabolomics 2022 workshop report: state of QA/QC best practices in LC-MS-based untargeted metabolomics, informed through mQACC community engagement initiatives.

IntroductionThe Metabolomics Quality Assurance and Quality Control Consortium (mQACC) organized a workshop during the Metabolomics 2022 conference.ObjectivesThe goal of the workshop was to disseminate recent findings from mQACC community-engagement efforts and to solicit feedback about a living guidance document of QA/QC best practices for untargeted LC-MS metabolomics.MethodsFour QC-related topics were presented.ResultsDuring the discussion, participants expressed the need for detailed guidance on a broad range of QA/QC-related topics accompanied by use-cases.ConclusionsOngoing efforts will continue to identify, catalog, harmonize, and disseminate QA/QC best practices, including outreach activities, to establish and continually update QA/QC guidelines

Sensitivity Analysis of the Transit Boardings Estimation and Simulation Tool (TBEST) Model

Public transportation, although modest in the United States carrying about 2 percent trips, still serves millions of people as the main and only means of transportation. Recently released data set by Census, the 2006 American Community Survey (ACS) shows the main mode of travel for work commute is not surprisingly the automobile with over 86 percent and public transportation with nearly 5 percent users. Transit agencies strive to provide effective, convenient, and desirable transport. Because of the constant changes in our environment, being able to predict the response of riders to different network or system changes is extremely useful. Ridership can be described as a function of the amount of service supplied such as frequency, span of service, and travel time. One of the methods for estimating ridership forecasts and evaluating ridership response is to use the new state-of-art software TBEST. TBEST stands for Transit Boardings Estimation and Simulation Tool and is the third generation of such transit models sponsored by the Florida Department of Transportation (FDOT). Designed for comprehensive transit network and short term transit planning, it offers great benefits to its users. TBEST is a user friendly, yet very advanced transit ridership forecasting graphical software which is interfaced with ArcGIS. This paper evaluates different sensitivity tests and compares the results to known industry used elasticities. Because the current TBEST experience is modest, the results will provide users with a general idea of the model\u27s sensitivity and help in the process of model refinements. Sensitivity tests such as service frequency, span of service, service allocation, and travel time will be carried out in a systematic order for all six time periods as defined by TBEST. Results showed that TBEST Model is overestimating and is highly sensitive to headway changes, specifically headway decrease. The opposite effect of almost no sensitivity is shown for the in-vehicle travel times

High-throughput solid-phase microextraction in multi-well-plate format

This review summarizes different formats of high-throughput, multi-well, solid-phase microextraction (SPME), including fiber, thin-film and in-tip configurations, with the particular focus on its fit within regulated analysis. New developments of the devices, such as monolithic and biocompatible extraction phases, are covered. Finally, selected applications of the technique, including the analysis of whole-blood samples and automated binding studies, are presented