Développement d’outils analytiques et méthodologiques pour l’analyse et le suivi de composés vétérinaires et stéroïdes hormonaux à l’état de traces dans l’eau et le sol

Several chemical products are dispersed in the environment and the consequences can be sometimes harmful for humans and the ecosystems. Among these substances appear the antibiotics and the hormonal steroids. Nowadays, only few data are available on the presence and the fate of these substances in the environment in particular for solid matrices, mainly due to a lack of methodologies. Consequently, methods to analyze traces of « emergent » contaminants in water and soil were carried out. Therefore, multi-residues and inter-families procedures based on LC-MS/MS analysis were established. Concerning the aqueous samples, 23 analytes are extracted with the SPE technique (OASIS HLB). MLQs are between 0.09 and 34 ng/L. For the solid matrix, the extraction procedure of 31 compounds is inspired from the method called QuEChERS and followed by a purification step. This methodology was validated and MLQs between 0.013 and 3 ng/g were obtained. Two antibiotics families, tetracyclines and fluoroquinolones, were studied separately as they have specific physical/chemical properties and are therefore difficult to extract from soil with an inter-families method. Then, the method developed for the soil matrix allowed a statistic study which showed the impact of the soil parameters on the recoveries and matrix effects. Finally, the methodologies were applied to a soil column study which allowed obtaining data on the transfer, accumulation and degradation of the substances in soilDe nombreux produits chimiques se retrouvent dispersés dans l'environnement avec des conséquences parfois néfastes pour les hommes et les écosystèmes. Parmi ces substances figurent les antibiotiques et les stéroïdes hormonaux. Peu de données sont disponibles quant à la présence et le devenir de ces substances dans l'environnement notamment pour le sol, par manque de méthodologies. Nous avons donc mis au point des procédures d'analyse de traces de ces contaminants émergents, dans l'eau et le sol. Nous avons développé des méthodes multi-résidus et inter-familles basées sur des analyses LCMS/ MS. Pour les échantillons aqueux, l'extraction est menée sur phase solide (SPE, OASIS HLB). Les MLQs sont comprises entre 0.09 et 34 ng/L. Pour la matrice solide, la procédure d'extraction est inspirée de la méthode appelée QuEChERS suivie d'une purification SPE. Elle a été validée et des MLQs entre 0.013 et 3 ng/g ont été atteintes. Les tétracyclines et les fluoroquinolones, ont été étudiées séparément car elles ont des propriétés physico-chimiques bien spécifiques les rendant difficiles à extraire correctement du sol avec une méthode inter-familles. La méthode développée sur la matrice sol a permis une étude statistique mettant en exergue l'impact de certains paramètres du sol sur les rendements d'extraction et les effets matrice. Les méthodes ont été appliquées à une étude en colonnes de sol pour obtenir des données sur le transfert, l'accumulation et la dégradation des composés dans le so

NMR analysis of Nile Blue (C. I. Basic Blue 12) and Thionine (C. I. 52000) in solution

The dyes Nile Blue (C. I. Basic Blue 12, NB) and Thionine (C. I. 52000, TH) were examined in both ionic and neutral forms in different solvents using NMR and UV-visible Spectroscopy to firmly establish the structures of the molecules and to assess the nature and extent of their aggregating characteristics. 1H and 13C NMR assignments and chemical shift data were used along with (for NB) nuclear Overhauser effect information enabling a structure for self-assembly to be proposed. In both cases these data were supplemented with variable temperature, dilution and diffusion-based experimental results using 1H NMR spectroscopy thereby enabling the extended aggregate structures to be assessed in terms of the relative strength of self-association and the extent to which extended aggregates could form. Full and detailed solution phase NMR analysis of such dyes, especially the two studied in this context, have either not been widely reported or have not been studied to the depth presented here. The data and their interpretation form an important addition to the analysis of this class of dye compounds and provide additional insight into the effects of self-assembly on the behaviour of such molecules in various solution-phase environments

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

Development of methodological and analytical tools for the analysis and the monitoring of veterinary antibiotics and hormonal steroids at trace levels in water and soil

De nombreux produits chimiques se retrouvent dispersés dans l'environnement avec des conséquences parfois néfastes pour les hommes et les écosystèmes. Parmi ces substances figurent les antibiotiques et les stéroïdes hormonaux. Peu de données sont disponibles quant à la présence et le devenir de ces substances dans l'environnement notamment pour le sol, par manque de méthodologies. Nous avons donc mis au point des procédures d'analyse de traces de ces contaminants émergents, dans l'eau et le sol. Nous avons développé des méthodes multi-résidus et inter-familles basées sur des analyses LCMS/ MS. Pour les échantillons aqueux, l'extraction est menée sur phase solide (SPE, OASIS HLB). Les MLQs sont comprises entre 0.09 et 34 ng/L. Pour la matrice solide, la procédure d'extraction est inspirée de la méthode appelée QuEChERS suivie d'une purification SPE. Elle a été validée et des MLQs entre 0.013 et 3 ng/g ont été atteintes. Les tétracyclines et les fluoroquinolones, ont été étudiées séparément car elles ont des propriétés physico-chimiques bien spécifiques les rendant difficiles à extraire correctement du sol avec une méthode inter-familles. La méthode développée sur la matrice sol a permis une étude statistique mettant en exergue l'impact de certains paramètres du sol sur les rendements d'extraction et les effets matrice. Les méthodes ont été appliquées à une étude en colonnes de sol pour obtenir des données sur le transfert, l'accumulation et la dégradation des composés dans le solSeveral chemical products are dispersed in the environment and the consequences can be sometimes harmful for humans and the ecosystems. Among these substances appear the antibiotics and the hormonal steroids. Nowadays, only few data are available on the presence and the fate of these substances in the environment in particular for solid matrices, mainly due to a lack of methodologies. Consequently, methods to analyze traces of « emergent » contaminants in water and soil were carried out. Therefore, multi-residues and inter-families procedures based on LC-MS/MS analysis were established. Concerning the aqueous samples, 23 analytes are extracted with the SPE technique (OASIS HLB). MLQs are between 0.09 and 34 ng/L. For the solid matrix, the extraction procedure of 31 compounds is inspired from the method called QuEChERS and followed by a purification step. This methodology was validated and MLQs between 0.013 and 3 ng/g were obtained. Two antibiotics families, tetracyclines and fluoroquinolones, were studied separately as they have specific physical/chemical properties and are therefore difficult to extract from soil with an inter-families method. Then, the method developed for the soil matrix allowed a statistic study which showed the impact of the soil parameters on the recoveries and matrix effects. Finally, the methodologies were applied to a soil column study which allowed obtaining data on the transfer, accumulation and degradation of the substances in soi

Determination of Tetracycline and Fluoroquinolone Antibiotics at Trace Levels in Sludge and Soil

This work describes the development of a sensitive analytical method to determine simultaneously traces of tetracycline and fluoroquinolone antibiotics in sludge and soil, based on PLE extraction, followed by SPE purification and finally an analysis by LC-MS/MS. Recoveries were greater than 87% in the case of fluoroquinolones and between 25.4 and 41.7% for tetracyclines. Low relative standard deviations (<15%) were obtained in both matrices. The limits of quantification were comprised between 1.1 and 4.6 ng/g and between 5 and 20 ng/g in soil and sludge, respectively. The method was then successfully applied to the analysis of the target antibiotics in sludge as well as soil that received spreading. The substances most frequently found and with the highest levels were fluoroquinolones with concentrations exceeding 1,000 ng/g in several samples of sludge and up to 16 ng/g in soil

Determination of tetracycline and fluoroquinolone antibiotics at trace levels in sludge and soil

International audienceThis work describes the development of a sensitive analytical method to determine simultaneously traces of tetracycline and fluoroquinolone antibiotics in sludge and soil, based on PLE extraction, followed by SPE purification and finally an analysis by LC-MS/MS. Recoveries were greater than 87% in the case of fluoroquinolones and between 25.4 and 41.7% for tetracyclines. Low relative standard deviations (<15%) were obtained in both matrices. The limits of quantification were comprised between 1.1 and 4.6 ng/g and between 5 and 20 ng/g in soil and sludge, respectively. The method was then successfully applied to the analysis of the target antibiotics in sludge as well as soil that received spreading. The substances most frequently found and with the highest levels were fluoroquinolones with concentrations exceeding 1,000 ng/g in several samples of sludge and up to 16 ng/g in soil

Statistical evaluation of the influence of soil properties on recoveries and matrix effects during the analysis of pharmaceutical compounds and steroids by quick, easy, cheap, effective, rugged and safe extraction followed by liquid chromatography-tandem mass spectrometry

International audienceNumerous chemical products are dispersed in our environment. Many of them are recognized as harmful to humans and the ecosystem. Among these harmful substances are antibiotics and steroid hormones. Currently, very few data are available on the presence and fate of these substances in the environment, in particular for solid matrices, mainly due to a lack of analytical methodologies. Indeed, soil is a very complex matrix, and the nature and composition of the soil has a significant impact on the extraction efficiency and the sensitivity of the method. For this reason a statistical approach was performed to study the influence of soil parameters (clay, silt, sand and organic carbon percentages and cation exchange capacity (CEC)) on recoveries and matrix effects of various pharmaceuticals and steroids. Thus, an analysis of covariance (ANCOVA) was performed when several substances were analyzed simultaneously, whereas a Pearson correlation was used to study the compounds individually. To the best of our knowledge, this study is the first time such an experiment was performed. The results showed that clay and organic carbon percentages as well as the CEC have an impact on the recoveries of most of the target substances, the variables being anti-correlated. This result suggests that the compounds are trapped in soils with high levels of clay and organic carbon and a high CEC. For the matrix effects, it was shown that the organic carbon content has a significant effect on steroid hormones and penicillin G matrix effects (positive correlation). Finally, interaction effects (first order) were evaluated. This latter point corresponds to the crossed effects that occur between explanatory variables (soil parameters). Indeed, the value taken by an explanatory variable can have an influence on the effect that another explanatory variable has on a dependent variable. For instance, it was shown that some parameters (silt, sand) have an impact on the effect that clay content has on recoveries. Besides, CEC and silt affect the influence that organic carbon percentage has on matrix effect. This original approach provides a better understanding of the complex interactions that occur in soil and could be useful to understand and predict the performance of an analytical method

Determination of Tetracycline and Fluoroquinolone Antibiotics at Trace Levels in Sludge and Soil

This work describes the development of a sensitive analytical method to determine simultaneously traces of tetracycline and fluoroquinolone antibiotics in sludge and soil, based on PLE extraction, followed by SPE purification and finally an analysis by LC-MS/MS. Recoveries were greater than 87% in the case of fluoroquinolones and between 25.4 and 41.7% for tetracyclines. Low relative standard deviations (<15%) were obtained in both matrices. The limits of quantification were comprised between 1.1 and 4.6 ng/g and between 5 and 20 ng/g in soil and sludge, respectively. The method was then successfully applied to the analysis of the target antibiotics in sludge as well as soil that received spreading. The substances most frequently found and with the highest levels were fluoroquinolones with concentrations exceeding 1,000 ng/g in several samples of sludge and up to 16 ng/g in soil

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

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