Electrospray Ionization and samples complexity in Meta-metabolomics: a biomarker or a suppressed ion?

International audienceIntroductionElectrospray Ionization is one of the most utilized ionization techniques for LC-MS-based metabolomics [1]. However, it presents several drawbacks, e.g. the ion suppression phenomenon, causing ion intensity decrease [2]. The occurrence of the phenomenon is higher if the sample is more complex. Thus, studying samples with different complexities may lead to consider some non-significant molecular features as markers of discrimination. This is due to ion suppression in samples with higher complexity.Material and MethodsThe issue is reported in an environmental context [3,4]. The study is performed on control non-spiked sediment samples and sediments spiked with a complex biopesticide; Bacillus thuringiensis israelensis. Meta-metabolome (endometabolome + xenometabolome) is extracted with QuEChERS method, then analyzed by LC-QToF in order to perform untargeted metabolic profiling, to discover the biomarkers of exposure. This to understand the pesticide impact on spiked sediments compared to control sediments.Results and DiscussionResults revealed several markers with lower intensity in the spiked group. They were co-eluting with multi-charged xenometabolites. Hence, these markers are either less concentrated due to a biological impact, or suppressed by the co-eluted molecules. Thus, to discriminate between biomarkers and suppressed ions, samples are diluted and analyzed. In fact, as dilution decreases the ion suppression, suppressed features are no more significantly discriminant between the two groups of samples.References[1] Bedair et al. 2008. Trends Anal. Chem. 27(3):238–250[2] Antignac et al. 2005. Anal. Chim. Acta. 529(1–2):129–136[3] Patil et al. 2016. Sci. Total Environ. 566–567:552–558[4] Salvia et al. 2018. Environ. Sci. Pollut. Res. 25(30):29841–2984

Développement d’un nouveau proxy universel pour évaluer le devenir et l’impact environnemental de (bio)pesticides complexes par métabolomique basée sur la spectrométrie de masse

Despite the ecological and sanitary awareness, worldwide consumption of pesticides is increasing. As these chemical products represent several adverse effects on human health and environment, measures should be taken in order to limit their impacts. Biocontrol products are proposed as an alternative solution of the synthetic products. In fact, these “biopesticides” are presumed to be less harmful and relatively less persistent. However, this a priori must be examined and strict risk assessment of those new substances should be considered.The development of biocontrol solutions proceeds first of all through the proposed protocols to study their activity and their environmental fate and impact. Currently, half-life (DT50) is used in order to evaluate the environmental fate of synthetic pesticides. However, DT50 approach gives only information about pesticides' persistence in the environment, but no indications concerning the formation of degradation products or its impact on biodiversity are provided. Furthermore, biocontrol products are complex (bio)chemical mixes. The DT50 is not applicable for such complex products. Therefore, novel analytical approaches should be considered in order to overcome these difficulties.A novel approach based on meta-metabolomics and Mass Spectrometry; the “Environmental Metabolic Footprinting” (EMF), was recently introduced. It affords a novel universal and integrative proxy; the “resilience time”, dedicated to assess the environmental fate and impact of complex (bio)pesticides in environmental matrices (e.g. soil, sediment). Nonetheless, the development of such Mass Spectrometry-based untargeted meta-metabolomics approach needs to be in-depth studied. Several tasks should be addressed: 1) performant extraction protocols and GC/LC-(HR)MS-based analytical methods should be set up, 2) suitable data processing and chemometric tools should be developed to deal with the complexity of the generated datasets, 3) the impact of xenometabolome complexity on MS-based analyses should be assessed, and 4) the study of the volatile residues should be considered and thus needs new analytical methodologies to be developed.The work was carried out following 3 main axes. The first axis addressed 1) the development of extraction protocols and LC-HRMS methods to analyze both pesticides xenometabolites and soil endometabolites, and 2) the development of a novel chemometric approach to assess the extraction performance. Novel extraction protocols have been proven optimal for the EMF, and the chemometric approach was thus validated. The second axis assessed the impact of xenometabolome complexity on the determination of environmental biomarkers. Ion suppression was revealed and thus a pragmatic strategy has been developed to overcome its influence. The third axis aimed to set-up a novel methodology in order to analyze the volatile residues of complex pesticides. HS-SPME-GC-MS analyses were coupled to chemometrics in order to perform kinetics studies and to follow the transformation of the volatile residues. The chemometric workflow proved its reliability to explain pesticide’s transformation and novel xenometabolites and by-products were identified.In conclusion, significant advances were carried to the EMF. It has been consolidated for laboratory and field applications that must be investigated in order to improve the proxy and to validate it as a reliable approach for pesticides risk evaluation.Malgré la prise de conscience écologique et sanitaire, la consommation mondiale de pesticides est en augmentation. Étant donné que ces produits chimiques présentent de nombreux effets néfastes sur la santé humaine et l'environnement, des mesures doivent être prises afin de limiter leurs effets. Les produits de biocontrôle sont proposés comme une solution alternative aux produits synthétiques. En effet, ces « biopesticides » sont présumés être moins nocifs et relativement moins persistants. Toutefois, cet a priori doit être examiné et une évaluation stricte des risques de ces nouvelles substances doit être envisagée.Le développement de solutions de biocontrôle passe d'abord par les protocoles proposés pour étudier leur activité, leur devenir et leur impact environnemental. Actuellement, le temps de demi-vie (t½) est utilisé pour évaluer le devenir environnemental des pesticides synthétiques. Cependant, l'approche t½ ne donne que des informations sur la persistance des pesticides dans l'environnement, mais aucune indication concernant la formation de produits de dégradation ou son impact sur la biodiversité n'est apportée. De plus, les produits de biocontrôle sont des mélanges (bio)chimiques complexes. La t½ n'est pas applicable pour ces produits complexes. Par conséquent, de nouvelles approches analytiques doivent être envisagées afin de surmonter ces difficultés.Une nouvelle approche basée sur la méta-métabolomique et la Spectrométrie de Masse; « Empreinte Métabolique Environnementale » (EMF), a été récemment introduite. Elle offre un nouveau proxy universel et intégratif; le « temps de résilience », dédié à l'évaluation du devenir environnemental et de l'impact des (bio)pesticides complexes dans des matrices environnementales (ex. sol, sédiments). Néanmoins, le développement d'une telle approche de méta-métabolomique non ciblée basée sur la Spectrométrie de Masse doit être étudié en profondeur. Plusieurs tâches doivent alors être abordées: 1) les protocoles d'extraction performants et les méthodes analytiques basées sur la GC/LC-(HR)MS doivent être mis en place, 2) le traitement de données et les outils chimiométriques appropriés doivent être développés pour maitriser la complexité des ensembles des données générées, 3) l'impact de la complexité du xénométabolome sur les analyses basées sur la MS doit être évalué, et 4) l'étude des résidus volatiles doit être envisagée et nécessite donc le développement de nouvelles méthodologies analytiques.Le travail a été mené sur 3 axes principaux. Le premier axe portait sur 1) le développement de protocoles d'extraction et des méthodes LC-HRMS pour analyser à la fois les xénométabolites des pesticides et les endométabolites du sol, et 2) le développement d'une nouvelle approche chimiométrique pour évaluer les performances d'extraction. De nouveaux protocoles d'extraction se sont avérés optimaux pour l'EMF, et l'approche chimiométrique a donc été validée. Le deuxième axe a évalué l'impact de la complexité du xénométabolome sur la détermination des biomarqueurs environnementaux. La suppression d'ion a été révélée et une stratégie pragmatique a donc été élaborée pour surmonter son influence. Le troisième axe visait à mettre en place une nouvelle méthodologie pour analyser les résidus volatils de pesticides complexes. Des analyses HS-SPME-GC-MS ont été couplées à la chimiométrie afin de réaliser des études cinétiques et de suivre la transformation des résidus volatils. Le workflow chimiométrique a prouvé sa fiabilité pour expliquer la transformation du pesticide et de nouveaux xénométabolites et sous-produits ont été identifiés.En conclusion, une avancée significative a été apportée à l’EMF. Elle a été consolidée pour les applications en laboratoire et sur le terrain qui doivent être étudiées afin d'améliorer le proxy et de le valider comme une approche fiable pour l'évaluation des risques des pesticides

Headspace-Solid Phase Micro Extraction-Gas Chromatography-Quadrupole Mass Spectrometry-based metabolomics for kinetics tracking of natural herbicides’ volatile residues: a simple non-destructive method

International audienceDespite their known risks, herbicides are still essential for agriculture. Thus, natural herbicides are increasingly recommended to replace synthetic ones. They are supposed less harmful on human health and ecosystem. However, there are still limitations in studying the environmental fate of several of them. The main reason is the lack of methods dedicated for this type of herbicides. In fact, they usually consist of complex mixtures of substances, of which several are unknown.Hence, in the framework of the Environmental Metabolic Footprinting (EMF) approach, the current work presents a method dedicated to analyze the volatile residues of herbicides applied on soil. The aim is to track the evolution of the volatile compounds issued from the herbicide by an untargeted metabolomics-based kinetics, in order to determine the “resilience time” of the gaseous phase above the soil. The approach aims to explore the environmental fate of these herbicides by combining it to other methods of the EMF. Moreover, it can estimate the exposure of farmers, insects and plants to potential toxic volatile substances.The method is based on Headspace-Solid Phase Micro Extraction-Gas Chromatography-Quadrupole Mass Spectrometry (HS-SPME-GC-Q MS). The HS-SPME provides a non-destructive extraction. This allows reducing the number of samples. The GC separation technique provides high reproducible analysis for volatile compounds. In addition, it allows the calculation of the Retention Index (RI) as a tool for the molecular characterization. On the other hand, despite the low resolution of the Quadrupole mass analyzer, the Electronic Impact provides reproducible MS fragmentation patterns used for spectral library search and putative identification of unknown compounds.The method was experimented for a pilot study. It was applied on a natural herbicide: the extract of Myrica gale, containing the Myrigalone A active substance. The setup of the analytical tool was performed by optimizing the system parameters. Then, a 38 days kinetics study was applied on control and spiked soil samples. Results proved the reliability of the method and demonstrated the robustness of the system and low matrix effect. Thus, new high scale experiments are planned. The integration of other natural and synthetic herbicides will be also considered

Simultaneous untargeted and targeted profiling of underivatized primary metabolites on sulfur-deficient barley

International audienceMetabolomics based-mass spectrometry are increasingly applied in diverse scientific domains, notably agronomy and plant biology, to understand plants’ behaviors under biotic/abiotic stress conditions. In fact, these stress conditions are able to disrupt many biosynthetic pathways that include mainly primary metabolites. Profiling and quantifying primary metabolites remain a challenging task because they are poorly retained in reverse phase columns due to their high polarity. The aim of our method which is to simultaneously perform an untargeted/targeted metabolite profiling in order to understand the nutrient deficiency effect on plants. Two fast and accurate methods were developed to detect and quantify amino acids, organic acids, sulfur metabolites, and secondary metabolites using an UPLC coupled to a QTOF mass spectrometer. An HSS T3 column was used to analyze amino acids and sulfur containing metabolites in positive ionization mode, and a Luna R Omega PS C18 column was used for organic acids profiling in negative mode. Ionization was achieved using an electrospray ion source (ESI). Methods were successfully applied allowing to detect, quantify and discriminate primary metabolites in short-runs without any additional sampling step such as derivatization or ion pairing. On the other hand, untargeted analysis was conducted using Progenesis QI performing alignment, peak picking, normalization, metabolite identifications and multivariate analysis. The simultaneous analysis provided cumulative information allowing to discriminate between two plant batches. Thus, discriminant biomarkers were identified and validated. A fast and innovated simultaneous untargeted/targeted method has successfully been developed and applied to sulfur deficiency on Barley

Simultaneous untargeted and targeted metabolomics profiling of underivatized primary metabolites in sulfur-deficient barley by ultra-high performance liquid chromatography-quadrupole/time-of-flight mass spectrometry

Abstract Background Metabolomics based on mass spectrometry analysis are increasingly applied in diverse scientific domains, notably agronomy and plant biology, in order to understand plants’ behaviors under different stress conditions. In fact, these stress conditions are able to disrupt many biosynthetic pathways that include mainly primary metabolites. Profiling and quantifying primary metabolites remain a challenging task because they are poorly retained in reverse phase columns, due to their high polarity and acid–base properties. The aim of this work is to develop a simultaneous untargeted/targeted profiling of amino acids, organic acids, sulfur metabolites, and other several metabolites. This method will be applied on sulfur depleted barley, in order to study this type of stress, which is difficult to detect at early stage. Also, this method aims to explore the impact of this stress on barley’s metabolome. Results Ultra-high performance liquid chromatography–high resolution mass spectrometry-based method was successfully applied to real samples allowing to discriminate, detect, and quantify primary metabolites in short-runs without any additional sampling step such as derivatization or ion pairing. The retention of polar metabolites was successfully achieved using modified C18 columns with high reproducibility (relative standard deviation below 10%). The quantification method showed a high sensitivity and robustness. Furthermore, high resolution mass spectrometry detection provided reliable quantification based on exact mass, eliminating potential interferences, and allowing the simultaneous untargeted metabolomics analysis. The untargeted data analysis was conducted using Progenesis QI software, performing alignment, peak picking, normalization and multivariate analysis. The simultaneous analysis provided cumulative information allowing to discriminate between two plant batches. Thus, discriminant biomarkers were identified and validated. Simultaneously, quantification confirmed coherently the relative abundance of these biomarkers. Conclusions A fast and innovated simultaneous untargeted/targeted method has successfully been developed and applied to sulfur deficiency on barley. This work opens interesting perspectives in both fundamental and applied research. Biomarker discovery give precious indication to understand plant behavior during a nutritional deficiency. Thus, direct or indirect measurement of these compounds allows a real time fertilization management and encounter the challenges of sustainable agriculture

Simultaneous untargeted and targeted profiling of underivatized primary metabolites on sulfur-deficient barley

International audienceMetabolomics based-mass spectrometry are increasingly applied in diverse scientific domains, notably agronomy and plant biology, to understand plants’ behaviors under biotic/abiotic stress conditions. In fact, these stress conditions are able to disrupt many biosynthetic pathways that include mainly primary metabolites. Profiling and quantifying primary metabolites remain a challenging task because they are poorly retained in reverse phase columns due to their high polarity. The aim of our method which is to simultaneously perform an untargeted/targeted metabolite profiling in order to understand the nutrient deficiency effect on plants. Two fast and accurate methods were developed to detect and quantify amino acids, organic acids, sulfur metabolites, and secondary metabolites using an UPLC coupled to a QTOF mass spectrometer. An HSS T3 column was used to analyze amino acids and sulfur containing metabolites in positive ionization mode, and a Luna R Omega PS C18 column was used for organic acids profiling in negative mode. Ionization was achieved using an electrospray ion source (ESI). Methods were successfully applied allowing to detect, quantify and discriminate primary metabolites in short-runs without any additional sampling step such as derivatization or ion pairing. On the other hand, untargeted analysis was conducted using Progenesis QI performing alignment, peak picking, normalization, metabolite identifications and multivariate analysis. The simultaneous analysis provided cumulative information allowing to discriminate between two plant batches. Thus, discriminant biomarkers were identified and validated. A fast and innovated simultaneous untargeted/targeted method has successfully been developed and applied to sulfur deficiency on Barley

Online HS-SPME-GC-MS-based Untargeted Volatile Metabolomics for Studying Emerging Complex Biopesticides: a Proof of Concept: W4M Workflow and Parameters

The current workflow was applied to pre-process GC-MS data (Ghosson, H. et al. Anal. Chim. Acta (2020), 1134:58-74) using Galaxy Workflow4Metabolomics platform. The automated processing workflow used the metaMS package (Galaxy Version 2.1.1) dedicated for GC-MS data. In brief, a “matchedFilter” algorithm was used for peak piking, with a Full Width at Half Maximum (FWHM) of 5 (Gaussian model peak). In addition, GC-MS peaks were considered for peak piking only if: i) their pseudo-spectra contained a minimum of 5 m/z features, ii) if these peaks were present in at least 70 % of samples belonging to a defined condition. Between the different injections/runs, the similarity threshold between peaks pseudo-spectra was set to 0.7, and maximum peak Retention Time (RT) variation was set to 15 sec in order to prevent any potential splitting of a metabolite feature into two different features.This work in­tro­duces a novel on­line Head­space-Solid Phase Mi­croex­trac­tion-Gas Chro­matog­ra­phy-Mass Spec­trom­e­try-based un­tar­geted metabolomics ap­proach, sug­gested as an al­ter­na­tive tool to study the en­vi­ron­men­tal fate of volatile xenometabo­lites in emerg­ing com­plex biopes­ti­cides, e.g. the Myrica gale methano­lic ex­tract her­bi­cide con­tain­ing sev­eral un­known metabo­lites. A “liv­ing” mi­cro­cosm sam­ple was de­signed for non-de­struc­tive analy­sis by a 35-min HS-SPME au­to­mated ex­trac­tion and a 36-min GC-MS run. A 38-day ki­net­ics study was then ap­plied on two groups of soil sam­ples: con­trol and spiked. Sta­tis­ti­cal tools were used for the com­par­a­tive ki­net­ics study. The Prin­ci­pal Com­po­nent Analy­sis re­vealed and ex­plained the evo­lu­tion and the dis­si­pa­tion of the her­bi­cide volatile xenometabolome over time. The time-se­ries Heatmap and Mul­ti­vari­ate Em­pir­i­cal Bayes Analy­sis of Vari­ance al­lowed the pri­or­i­ti­za­tion of 101 rel­e­vant com­pounds in­clud­ing 22 degra­da­tion by-prod­ucts. Out of them, 96 xenometabo­lites were pu­ta­tively iden­ti­fied. They in­cluded 63 com­pounds that are iden­ti­fied as her­bi­cide com­po­nents for the first time. The Or­thog­o­nal Pro­jec­tions to La­tent Struc­tures Dis­crim­i­nant Analy­sis and its Cross-Val­i­da­tion test were used to as­sess the to­tal dis­si­pa­tion of the her­bi­cide volatile residues and method de­tec­tion limit. The re­pro­ducibil­ity of the method was also as­sessed. The high­est in­ter-sam­ples (n = 3) Peak Area RSD was 7.75 %. The high­est in­ter-sam­ples (n = 3) and in­ter-days (n = 8) Re­ten­tion Time SD were 0.43 sec and 3.44 sec, re­spec­tively. The work pre­sents a green, non-la­bo­ri­ous and high-through­put ap­proach. It re­quired a small num­ber of en­vi­ron­men­tal sam­ples (6 mi­cro­cosms) that were an­a­lyzed 8 times and were not de­stroyed dur­ing the study.Please refer to Ghosson, H. et al. Anal. Chim. Acta (2020), 1134:58-74. doi:10.1016/j.aca.2020.08.016

HS-SPME-GC-MS-based untargeted metabolomics for kinetics tracking of natural herbicides’ volatile residues: can we “footprint” the “Volatilome”??

International audienceDespite their known risks, herbicides are still essential for agriculture. Thus, natural herbicides are increasingly recommended to replace synthetic ones. However, there are still limitations in studying the environmental fate of several of them. The main reason is the lack of methods dedicated for this type of complex bio-herbicides. Hence, in the framework of the Environmental Metabolic Footprinting (EMF) approach, the current work presents a method dedicated to analyze the volatile residues of herbicides applied on soil. The aim is to track the evolution of the volatile compounds issued from the herbicide by an untargeted metabolomics-based kinetics, in order to determine the “resilience time” of the gaseous phase above the soil. The approach aims to explore the environmental fate of these herbicides. In fact, it will supplement other methods based on the EMF approach recently developed. Moreover, it can estimate the exposure of farmers, insects and plants to potential toxic volatile substances.The method is based on Headspace-Solid Phase Micro Extraction-Gas Chromatography-Electronic Impact Ionization-Quadrupole Mass Spectrometry (HS-SPME-GC-EI-Q MS). The HS-SPME provides a non-destructive extraction. This allows reducing the number of samples. The GC separation technique provides high reproducible analysis for volatile compounds. In addition, it allows the calculation of the Retention Index (RI) as a tool for the molecular characterization. On the other hand, despite the low resolution of the Quadrupole mass analyzer, the Electronic Impact Ionization provides reproducible MS fragmentation patterns used for spectral library search and fast putative identification of unknown compounds

LC-HRMS-based untargeted metabolomics as a tool for analytical development: In-depth assessment of exhaustive extraction protocols for pesticides-polluted soils

International audienceUntargeted metabolomics is an analytical chemistry approach dedicated for the analysis of small biomolecules called “metabolites”. One of its strengths is the ability of covering wide ranges of metabolic information in biological systems. However, exhaustive coverage of information is challenging from a chemical-analytical point of view. In fact, the widely different physical-chemical properties of molecules (e.g. polarity, acidity/basicity) make the study of various types of metabolites complicated. It demands critical methodological optimizations. Nevertheless, for untargeted approaches, “optimal conditions” are also hard to be defined and judged.The current work is focusing on the crucial step of meta-metabolome extraction. It seeks to develop an “exhaustive” extraction protocol able to extract different types of metabolites by covering a wide range of polarity. This is in the framework of developing the “Environmental Metabolic Footprinting” untargeted metabolomics-based approach [1-3], aiming to assess the environmental fate and impact of complex (bio)pesticides application on soil. Indeed, optimizing a method able to analyze diverse kind of metabolites can assure a wide range of information needed to assess the fate of the pesticide (xenometabolites) and its impact on soil’s biodiversity (endometabolites).Toward this objective, 5 extraction protocols based on solvents and mixtures with different polarities were developed and applied on 2 different types of soils, with 3 different environmental conditions (control, spiked with a synthetic pesticide, spiked with a natural pesticide). So far, 150 samples were extracted and analyzed with a broadband LC-HRMS method that was set-up for the purpose. The collected data were then handled and analyzed with various types of multivariate statistical analyses that were optimized in order to assess the “optimal” protocol, based on 4 main criteria, respectively: 1) the ability to widen the band of the extracted metabolites (qualitative), 2) the higher extraction yield (quantitative), 3) the repeatability of the extraction, and 4) the reliability in discriminating between spiked and control groups (the main aim of the EMF).The study showed that widening the polarity range for the extraction protocol, using a mix of solvents with different properties was applicable and demonstrated a significant ability in extracting a wide range of metabolites originating from both pesticide residues and soil endometabolome. It also showed comparable quantitative results and better repeatability comparing to other classical protocols. In addition, the study suggests multivariate analyses as a suitable tool not only for data processing for biological studies, but also for developing analytical methods and protocols, as it can give holistic explanations describing the large acquired datasets. Hence, in other words, untargeted metabolomics were used in order to improve the analytical method dedicated for an untargeted meta-metabolomics approach.[1]: Patil et al. 2016. Sci. Total Environ. 566–567:552–558[2]: Salvia et al. 2018. Environ. Sci. Pollut. Res. 25(30):29841–29847[3]: Ghosson 2020. Thesis. Université de Perpigna

Untargeted metabolomics as a tool to monitor biocontrol product residues' fate on field-treated Prunus persica

International audienceEvidence of chemical plant protection products' (PPPs) long-term impact has been found in all environmental compartments. Therefore, other types of PPPs are developed to complement chemical PPPs like PPPs from natural sources, namely biocontrol products (BPs). Little is known about those new BPs, and it is important to assess their potential long-term environmental impact. Recently, the Environmental Metabolic Footprinting (EMF) approach was developed. It permits studying sample's entire meta-metabolome (endometabolome and xenometabolome) through a kinetics tracking of metabolomes of treated and untreated samples. Those metabolomes are compared time-by-time to estimate the “resilience time” of the samples after treatment. The current study aims to investigate BP residues' dissipation on peach fruits (Prunus persica). For that, an untargeted Liquid Chromatography-Mass Spectrometry metabolomics approach based on the EMF was optimised to separate the xenometabolome of the PPP from the endometabolome of the fruits. This “new version” of the EMF approach is able to target the BP treatment residues' (xenometabolome) dissipation exclusively. Thus, it is able to determine the time needed to have no more residues in the studied matrix: the “dissipation interval”. Field experiment was conducted on peach tree orchard against brown rot treated with (i) a plant extract BP (Akivi); (ii) a reference mineral extract BP (Armicarb®); and (iii) a Chemical reference treatment campaign. Formulated Akivi and its by-products' dissipation was monitored, a degradation kinetics appeared but the sampling did not last long enough to allow the determination of the “dissipation interval”. Armicarb® and the Chemical reference's residues and by-products showed a persistence pattern along the sampling kinetics. These results indicate that the EMF approach, formerly developed on soil and sediment, is applicable for fruit matrices and can be used to investigate the fate of complex BP treatment on the matrix through the xenometabolome tracking on treated fruits