Dissertations / Theses on the topic 'Lc-hrms'

Le micotossine sono contaminanti significativi negli alimenti e nei mangimi e sono causa di gravi effetti tossici sulla salute umana e animale. Diversi studi hanno dimostrato l’esistenza di una moltitudine di metaboliti fungini che contaminano alimenti e mangimi. Recentemente, le autorità sanitarie internazionali hanno espresso preoccupazione per la presenza di micotossine "emergenti" negli alimenti. Tra le specie fungine responsabili della produzione di tali metaboliti, la specie Alternaria ha la capacità di produrre più di 70 tossine, come Alternariol (AOH), Alternariol monomethyl ether (AME), Tenuazonic Acid (TeA) e Tentoxin (TEN), presenti nei cereali, in prodotti a base di pomodoro, nell’olio d'oliva, nella frutta e nella verdura fresca. Queste micotossine sono state recentemente oggetto di pareri scientifici dell'Autorità Europea per la Sicurezza Alimentare (EFSA), che ha espresso preoccupazione per la scarsa disponibilità di dati sull'incidenza negli alimenti destinati al consumo umano e animale. Esiste, infatti, un alto grado di incertezza legato alla rappresentatività degli alimenti attualmente testati. Inoltre, la mancanza di informazioni sul metodo analitico utilizzato contribuisce ulteriormente a creare incertezza sui livelli di tossine da Alternaria. Sono necessari ulteriori studi per aggiornare la valutazione del rischio da parte dell'EFSA e, pertanto, l'obiettivo specifico di questo progetto era incentrato sullo sviluppo di metodi LC-MS/MS e LC/HRMS accurati per l'analisi delle tossine da Alternaria. Il progetto è stato sviluppato in collaborazione con l'Istituto Zooprofilattico Sperimentale della Sicilia. Il metodo LC-MS/MS è stato utilizzato per l'analisi target delle micotossine emergenti nelle matrici alimentari. In particolare sono state svolte le seguenti attività: • ottimizzazione dei parametri del metodo per lo screening simultaneo di Alternariol (AOH), Alternariol monometiletere (AME), Acido tenuazonico (TeA), Ocratossine e Zearalenone (ZEA), Fumonisina B1, B2, B3, tossina T-2, HT-2 tossina e deossinivalenolo (DON) (vomitossina); • sviluppo di un protocollo di preparazione del campione, da applicare a cereali e frutta secca; il protocollo di preparazione del campione si è basato su una doppia estrazione (estrazione liquida) e purificazione mediante estrazione in fase solida (SPE). • validazione del metodo secondo le linee guida comunitarie (Decisione della Commissione 2002/657/CE); il metodo è stato ottimizzatoo valutando i seguenti parametri: specificità, precisione, linearità strumentale, recupero, limite di decisione (CCα), capacità di rilevamento (CCβ), effetto matrice e robustezza. La cromatografia accoppiata alla spettrometria di massa ad alta risoluzione (UHPLC-Q-HRMS) è stata utilizzata per la determinazione, sensibile e specifica, delle tossine da Alternaria. Le prestazioni ottenute sono state confrontate con quelle ottenute mediante cromatografia liquida ad alte prestazioni. Tutti e due i metodi hanno mostrato una buona linearità e ripetibilità. Si dice che lo spettrometro di massa Q-Exactive sia più adatto per il rilevamento in tracce rispetto ai metodi MS/MS basati sul triplo quadrupolo, perché, nonostante abbia prestazioni comparabili, ha una migliore selettività. Il progetto di ricerca avrebbe dovuto fornire un'ampia indagine sul contenuto emergente di micotossine in vari prodotti alimentari, contribuendo a una stima dell'esposizione al rischio di micotossine. Le difficoltà legate alla pandemia in corso hanno reso difficile la ricerca, inizialmente prevista in questo progetto. Nonostante tutto, lo scopo di questo lavoro è stato colmare il vuoto di dati e fornire informazioni rilevanti per migliorare la sicurezza dei prodotti locali.<br>Mycotoxins are significant contaminants in food and feeds. These molecules have demonstrated serious effects in both human and animal health. Different studies showed the presence of a multitude of fungal metabolites that contaminate food and feed. Recently, the international health authorities have expressed concerns about the presence of "emerging" mycotoxins in foodstuffs. The term “emerging mycotoxins” is commonly referred to those compounds that are currently under the spotlight of the scientific community and the policy-makers, due to their toxicological profile. Among the fungal species responsible for the metabolite production, Alternaria species have the ability to produce more than 70 toxins, such as Alternariol (AOH), Alternariol monomethyl ether (AME), Tenuazonic Acid (TeA) e Tentoxin (TEN), found in cereals, tomato products, olive oil, fresh fruits and vegetables. These mycotoxins have been recently covered by scientific opinions from the European Food Safety Authority (EFSA), who has expressed concern about the low availability of incidence data in food for human and animal consumption. There is, indeed, a high degree of uncertainty related to the representativeness of the food currently tested because it contains too many inaccuracies. Furthermore, the lack of information on the analytical method used further contributes to the uncertainty of the reported Alternaria toxin levels. More comprehensive studies are required to update the risk evaluation by the EFSA and, therefore, the specific aim of this project was focussed on the development of accurate LC-MS/MS and LC/HRMS methods for the analysis of emerging Alternaria toxins. The protocol was employed in collaboration with the Istituto Zooprofilattico Sperimentale della Sicilia. LC-MS/MS method was used for the target analysis of emerging mycotoxins in food matrices. In particular, the following activities were carried out: • optimisation of the method parameters for simultaneous screening of Alternariol (AOH), Alternariol monomethyl ether (AME), Tenuazonic Acid (TeA), Ochratoxins and Zearalenone (ZEA), Fumonisin B1, B2, B3, T-2 toxin, HT-2 toxin and deoxynivalenol (DON) (vomitoxin); • development of a sample preparation protocol, apply to cereals and dry fruits; the sample preparation protocol was based on a double extraction (Liquid extraction) and purification through solid-phase extraction (SPE). • validation of the method according to EU guidelines (Commission Decision 2002/657/EC); the method was performed by evaluating the following parameters: specificity, precision, instrumental linearity, recovery, decision limit (CCα), detection capability (CCβ), matrix effect and ruggedness. The ultrahigh-performance chromatography coupled to quadrupole-orbitrap high-resolution mass spectrometry (UHPLC-Q-HRMS) was applied to the sensitive and specific determination of the emerging Alternaria toxins. Performances were compared to those obtained by high-performance liquid chromatography detection. All two methods showed good linearity and repeatability. The Q-Exactive mass spectrometer is said to be better suitable for trace detection than state-of-the-art MS/MS methods based on the triple quadrupole, because, despite having performance comparable, it has better selectivity. The research project should have provided a broad investigation of the emerging mycotoxin content in various food products, contributing to an estimate of mycotoxin risk exposure. The difficulties linked to the pandemic in progress made difficult research, initially planned within this project. Despite everything, the purpose of this work was to fill the data gap and provide relevant information to improve the safety of local products.

Le taux de mortalité associé aux infections par le S. aureus résistant à la méticilline (SARM) est le plus élevé parmi les infections staphylococciques. Le SARM est aussi la plus fréquente des bactéries multirésistantes, plaçant souvent le médecin en situation d'impasse thérapeutique. Une meilleure compréhension des phénomènes de résistance aux antibiotiques permettrait de mieux détecter ces bactéries et concevoir de nouveaux antibiotiques. Dans ce contexte, ces travaux de thèse visaient à étudier le phénotype de résistance à la méticilline de S. aureus par une approche métabolomique.La première partie de ce travail a été dédiée au développement des outils nécessaires aux analyses métabolomiques globales de S. aureus. Ces développements ont impliqué la mise place d'un protocole de préparation d'échantillon spécifique et multi-étapes, de méthodes de chromatographie liquide couplée à la spectrométrie de masse à haute résolution (LC/HRMS) et l'implémentation d'une base de données spectrales dédiée.Dans ces conditions, nous avons pu extraire et identifier jusqu'à 210 métabolites intracellulaires, ce qui représente la plus large couverture métabolique de S. aureus obtenue à ce jour. Dans un second temps, nous avons appliqué l'approche développée à l'étude de souches SARM et de souches sensibles à la méticilline (SASM). Après optimisation rigoureuse des conditions de culture, l'étude approfondie de 24 souches cliniques nous a permis de dégager une signature métabolique du phénotype de la résistance à la méticilline, signant notamment l'implication des voies de biosynthèse de la paroi et de la capsule bactérienne.Le séquençage complet du génome de ces 24 souches combiné à l'étude complémentaire de 16 souches de SARM de fonds génétiques différents nous a ensuite permis de souligner la pertinence de cette signature métabolique vis-à-vis du phénotype étudié. Enfin, l'étude de souches isogéniques en présence d'antibiotique a également confirmé l'implication de la synthèse de la paroi dans la distinction SARM/SASM<br>The mortality rate associated with Methicillin resistant S. aureus (MRSA) is the highest among the staphylococcal infections. A better understanding of antibiotic resistance phenomena would lead to better detect these bacteria and design new antibiotics. In this context, this PhD work aimed to study the MRSA phenotype by using a metabolomics approach. The first part of this work has been dedicated to developing a global metabolomic analysis of S. aureus: i.e., the setting up of a reliable sample preparation protocol, the development of liquid chromatography-high resolution mass spectrometry methods, and the implementation of a dedicated spectral database. Under these conditions, 210 metabolites have been extracted and identified in bacterial cell extracts, which is the widest metabolic coverage of S. aureus obtained to date.Secondly, we have performed a metabolomic study of MRSA and methicillin susceptible S. Aureus (MSSA) strains. The analysis of 24 clinical strains has allowed us to highlight a metabolic signature of MRSA phenotype of which metabolites involved in bacterial wall and capsule biosynthesis pathways were part. The complete genome sequencing of these 24 strains combined with the complementary study of 16 MRSA strains of different genetic backgrounds have reinforced the relevance of this metabolic signature. Finally, the study of isogenic strains in the presence of an antibiotic has confirmed the involvement of metabolites of the wall and capsule biosynthesis pathways in the distinction of MRSA/MSSA phenotypes

Novel psychoactive substances (NPS) represent a great challenge to toxicologists due to the ability of illicit drug manufacturers to alter NPS chemical structures quickly and with ease to circumvent legislation regulating their use. Each time a new structure is introduced, there is a possibility that it has not been previously recorded in law enforcement or scientific databases. Many toxicology laboratories use targeted analytical methods that rely on libraries of known compounds to identify drugs in samples. However, these libraries do not include large numbers of NPS which could result in non-identification or detection. High-resolution mass spectrometry (HRMS) has been suggested as a method for screening a wide variety of analytes due to its higher sensitivity and mass accuracy as compared to some other forms of mass spectrometry. This technique can generate characteristic MS/MS spectral data for use in compound identification. The main goal of this research was to create a high-resolution mass spectrometry (HRMS) library of NPS and metabolites, as well as validate a method for screening and confirmation of these substances. The study consisted of three main tasks which included; the development of a large high-resolution MS/MS spectral library and database, validation of a method for screening and confirmation of over 800 NPS and metabolites, and screening of blind-spiked and authentic urine specimens to determine real-world applicability of the HRMS library and method. During validation, several isomeric and structurally related NPS were observed which could not be adequately separated using traditional LC methods. A fourth task was therefore added to investigate improved separation using two-dimensional liquid chromatography (2D-LC). Increased resolving power is achieved in 2D-LC through the coupling of multiple orthogonal separation systems. Ultimately, an on-line, comprehensive method was developed using orthogonal reversed-phase columns in each dimension (RP x RP) for improved separation of co-eluting and isomeric synthetic cannabinoids. This work can aid laboratories in the identification of NPS through the use of a validated LC-QTOF-MS method for screening and confirmation and HRMS spectral library. In instances where isomeric and structurally related NPS are not sufficiently separated, RP x RP methods can be explored.

La métabolomique est une approche émergente des technologies «omiques». Initialement, les études de métabolomique ont été menées dans les domaines de la médecine, des biotechnologies ou de la biologie des systèmes, mais récemment, son application s’est étendue à de nouveaux ressorts tels que le contrôle du dopage. L'objectif de notre étude est d'évaluer la pertinence d'une telle approche pour le dépistage des stéroïdes anabolisants chez le cheval, à travers le cas du stanozolol. Ce travail de thèse est orienté autour de 3 axes principaux : i) l’évaluation de la méthodologie en mettant l'accent sur le traitement de l'urine de cheval, un échantillon biologique complexe. Ii) l'identification des facteurs confondants à travers l’étude d’une cohorte de 16 chevaux évoluant dans leur environnement habituel pendant un an. Iii) le développement d’une méthode capable de discriminer 2 groupes de sujets : traités et non traités au stanozolol. Les échantillons d'urine ont été analysés par chromatographie en phase liquide couplée à la spectrométrie de masse à haute résolution (HPLC/HRMS). Le traitement des données a été réalisé avec le logiciel XCMS sous R et les analyses statistiques avec le logiciel SIMCA P12. Le profilage métabolique urinaire non ciblé, qui prend en compte l’ensemble des perturbations détectées suite à l’administration de stanozolol, a été développé pour mettre en évidence son utilisation chez le cheval. Une fois la détermination des biomarqueurs et leur identification terminée, il peut être étendu à d’autres anabolisants stéroïdiens et appliqué dans leur dépistage. La métabolomique apparaît comme un outil de dépistage encourageant et prometteur pour la lutte anti-dopage<br>Metabolomic is an emerging field of “omics” research. Initially, metabolomic studies were conducted in the fields of medicine, biotechnologies or system biology, but nowadays its application is spread out on new areas such as doping control. The objective of this study is to evaluate the relevance of such approach for the global screening of anabolic steroids in the horse, through the case of stanozolol, an exogenous anabolic steroid. The present PhD thesis work has three principle axes of study: i) methodology evaluation with emphasis on the sample treatment of biological matrix such as horse urine; ii) identification of confounding factors through one year cohort study of sixteen horses in their accustomed environment; iii) development of method able to discriminate two groups of subjects: stanozolol-treated and untreated population through eight months case-control study of sixteen horses by multivariate data analysis. Urine samples were analyzed by liquid chromatography coupled to high resolution mass spectrometry (HPLC/HRMS). Data treatment was carried out with XCMS R package and multivariate statistical analysis with SIMCA P12 software. A metabolic signature that is characteristic of stanozolol exposure obtained by developed method could be used to single out putative drug metabolites. After hypothesis-free determination of biomarkers and their identification, they can be applied for the screening of drug abuse. The potential of non-targeted metabolomics, as powerful screening tool, is encouraging and gives large-scale opportunities in discovering novel and/or emerging drugs

Les chloroparaffines (CP) sont des chaînes de n-alcanes polychlorées, utilisées en tant que plastifiants, retardateurs de flamme ou lubrifiants. Alors que leur ubiquité dans l’environnement a été démontrée par diverses études, l’évaluation du risque associé à l’exposition de l’homme à ces contaminants en Europe demeure très limitée. Le manque de données s’expliquant notamment par de nombreux défis analytiques, la présente thèse a été initiée pour développer une stratégie analytique innovante puis pour fournir des données nouvelles sur le comportement des CP le long de la chaîne alimentaire et sur leur prévalence dans les aliments. Une stratégie analytique basée sur un couplage LC-HRMS a d’abord été mise en place, avec une attention particulière portée à leur séparation chromatographique et à leur ionisation. La stratégie analytique finalisée, incluant une préparation d’échantillon optimisée et un traitement de données automatisé, permet d’analyser les CP de C10 à C36 à l’état de traces (ppb) dans des matrices biologiques complexes. Cette méthode a été appliquée à l’étude du comportement des CP chez la poule pondeuse en fonction de la longueur de chaîne et du degré de chloration, après exposition alimentaire. Il a pu ainsi être démontré que tous les CP sont biodisponibles. De plus, les CP ont été retrouvés dans les organes internes de la poule démontrant leur circulation dans l’organisme, à l’exception des chaînes les plus longues à taux de chlore élevé. Enfin, l’analyse d’un jeu d’aliments représentant le panier de la ménagère a permis de présenter une première évaluation de l’exposition alimentaire en France<br>Chlorinated paraffins (CPs) are polychlorinated n-alkane chains, used as plasticizers, flame retardants or lubricants. While their ubiquity in the environment has been evidenced in various studies, the risk assessment related to the human exposure to those contaminants in Europe remains incomplete. As the lack of data arises from many analytical challenges, this thesis was initiated to develop an innovative analytical strategy and to provide new insight on the behaviour of CPs along the food chain and their occurrence in food. An analytical strategy based on LC-HRMS coupling was first implemented, with particular attention paid to their chromatographic separation and ionization efficiency. The finalised analytical strategy, including an optimized sample preparation and automated data processing, enables trace analysis (ppb) of CPs from C10 to C36 in complex biological matrices. This method was applied to study the behaviour of CPs in the laying hen according to chain length and degree of chlorination after dietary exposure. It was thus demonstrated that all CPs are bioavailable. Moreover, the CPs were found in the internal organs of the hen demonstrating their circulation in the organism, with the exception of the longest chains with a high chlorine content. Last, the analysis of various foodstuffs representing the household basket has allowed to present a first assessment of dietary exposure in France

Le profilage métabolomique global de matrices biologiques dans de larges séries d'échantillon est un enjeu majeur. Dans ce contexte, notre travail vise à développer des approches LC-HRMS et outils bioinformatiques pour les analyses métabolomique et lipidomique de larges cohortes. Dans un premier temps, nous avons développé puis évalué la pertinence de 4 méthodes LC-HRMS dans l'annotation du métabolome/lipidome sérique humain. Ainsi, une base de données spectrales a été implémentée à l'aide de spectres MS, MS/MS et les temps de rétention de composés de référence afin d'assurer l'annotation de jeux de données. La combinaison de méthodes RP, HILIC et PFPP-HRMS a permis l'identification de 266 métabolites et 706 espèces lipidiques sériques répartis sur 20 et 24 classes chimiques respectivement dont 27% d'espèces isomères. Ces outils ont été appliqués, dans un second temps, à la stratification de 78 patients diabétiques. Outre le syndrome métabolique marqué (perturbation du métabolisme énergétique), nos analyses ont montré l'impact délétère de facteurs physiologiques confondants -âge et IMC-. Nous en avons évalué l'influence sur une cohorte de 227 salariés du CEA. Les empreintes lipidomiques sont robustes, néanmoins l'impact de l'IMC est marqué pour les lipides neutres. L'effet du genre démontre un catabolisme masculin important. L'effet de l'âge se manifeste par des activités enzymatiques altérées. Ces études combinent une analyse globale métabolomique et lipidomique des mêmes échantillons humains. Elles visent à construire une base de données relationnelle incluant données spectrales et biologiques servant à la caractérisation de biomarqueurs dans le cas d'études cliniques<br>Global metabolomic profiling of biological media in large sample sets is a major challenge. In this context, our work aims to develop LC-HRMS approaches and data mining tools for metabolomics and lipidomics analysis of large cohorts. We have first developed and evaluated the reliability of four LC-HRMS methods in the annotation of human serum metabolome and lipidome. Thus, spectral database was implemented using MS spectra, MS/MS and retention times of reference compounds to further ensure datasets annotation. The combination of RP, PFPP and HILIC-HRMS methods allowed identification of 266 metabolites and 706 lipid species in human serum over 20 to 24 chemical classes respectively including 27% of isomeric species. These analytical tools were then applied for the stratification of 78 diabetic patients. Unsurprisingly, we highlighted a metabolic syndrome (energy metabolism disruption), moreover our analyses have shown the deleterious impact of confounding physiological factors on diabetes biomarker discovery –age and BMI-. We finally evaluated their influence on a cohort of 227 CEA employees. Lipidomic fingerprints are robust, however BMI impact is marked for neutral lipids. Gender effect shows significant male catabolism and age altered enzyme activities. These studies combine an overall metabolomics and lipidomics analyses of the same human samples. They aim to build up a relational database including spectral and biological data for biomarker characterization in clinical studies

Un des axes de recherche porté par le laboratoire Mer-Molécules-Santé de l’Université de Nantes consiste en l’étude de la chimiodiversité des métabolites produits par des souches fongiques marines pour la mise en évidence de composés potentiellement valorisables en thérapeutique. Les travaux réalisés lors de cette thèse ont porté sur deux souches fongiques marines. L’étude du métabolome d’une souche de l’espèce Penicillium ubiquetum en couplant approche OSMAC et étude métabolomique par analyses LC-HRMS/MS a mis en évidence le milieu CYA-EDM comme induisant particulièrement la production de métabolites d’intérêt. Une purification guidée par spectrométrie de masse à partir de cultures à grande échelle a conduit à l’isolement de neuf mérosesquiterpènes dont deux originaux, de deux nouveaux analogues dans la série rare des C25 stéroïdes et de l’acide dimorphécolique. En parallèle, une souche marine de Penicillium expansum a été étudiée pour sa production de communésines, série d’alcaloïdes indoliques complexes. La mise au point d’un protocole de purification a permis d’isoler de façon sélective les communésines A et B majoritaires, matériaux de départ pour l’hémisynthèse de neuf analogues dont sept nouveaux. Ceci a permis de confirmer la structure de certaines communésines naturelles minoritaires détectées par LC-HRMS/MS mais non isolables. L’évaluation biologique de ces composés a été réalisée (cytotoxicité sur lignées cancéreuses humaines KB et MCF-7 et effet sur duodénum isolé de rat) et a permis d’établir les premières relations structures-activité de la série communésines<br>One of the research areas carried out by the Mer-Molecules-Santé laboratory of the University of Nantes concerns the study of the chemodiversity of metabolites produced by marine fungal strains for the detection of potentially valuable compounds in therapeutics. The work carried out during this thesis focused on two marine fungal strains. The study of the metabolome of a Penicillium ubiquetum strain by coupling the OSMAC approach and a metabolomics study by LC-HRMS/MS revealed the CYA-EDM medium as particularly inducing the production of metabolites of interest. Mass spectrometry-guided purification from large scale cultures led to the isolation of nine merosesquiterpenes including two new compounds, two new analogs of the rare C25 steroids and dimorphecolic acid. In parallel, a marine strain of Penicillium expansum has been studied for its production of communesins, a series of complex indole alkaloids. The development of a purification protocol made possible to selectively isolate the major communesin A and B, starting materials for the semisynthesis of nine analogs, seven of which were new. This allowed to confirm the structure of some natural minor communesins detected by LC-HRMS/MS but not isolable. The biological evaluation of these compounds was carried out (cytotoxicity on human cancer lines KB and MCF-7 and effect on rat isolated duodenum) and allowed to establish the first structure-activity relationships in the communesin series

Grâce aux récents progrès en terme d'instrumentation analytique, la protéomique, en tant que science qui étudie le protéome d'un organisme ou d'un milieu, a connu un véritable tournant et a permis d'étendre le champ des connaissances sur le fonctionnement du vivant dans son ensemble (structure, fonction, métabolisme, dynamisme). Néanmoins, l'étude des protéomes représente un challenge pour de nombreux biologistes, chimistes et biochimistes, en raison notamment de la complexité des échantillons étudiés. De nombreux protocoles analytiques ont d'ores et déjà été développés. Cependant, dans leur ensemble, ces stratégies sont relativement longues et multi-étapes et le plus souvent ciblées sur une protéine donnée.Dans ce contexte, ce travail de thèse a pour objectif de simplifier les protocoles expérimentaux existants afin de limiter les pertes en protéines et ainsi amplifier leurs signaux au cours d'une analyse « Bottom-up » par LC-HRMS (analyse « hors gel »). Chaque étape du processus a été décortiquée et optimisée. Dans un premier temps, les paramètres UPLC-HRMS/MS ont été optimisés afin d'améliorer la détection et la quantification des protéines présentes à des concentrations très variables dans les milieux étudiés. Ensuite, une approche de purification simplifiée qui repose sur une seule et unique étape de lavage et solubilisation des protéines a été mise au point. La démonstration de son efficacité « chimique » et « biologique » a ensuite été réalisée via une étude mécanistique au cours de laquelle les changements de conformation des protéines ont été étudiés à chacune des étapes de purification proposées. Enfin, certains paramètres influençant l'extraction des protéines à partir de ces mêmes matrices ont été étudiés afin de proposer à terme un protocole d'extraction à la carte compatible avec une analyse directe par LC-HRMS/MS<br>Recent advances in proteomics have been spurred by the rapid development of hybrid and/or high-resolution mass analysers (HRMS/MS). These powerful instrumentations have led to significant improvements in « Bottom-up » approach and have enabled to deepen our knowledge on the functionality of biological systems (structure, function, metabolism, dynamic, etc).Despite their high sensibilities, the potential of such instruments could be significantly lessened by an imperfect sample pre-treatment. In this context, current sample pretreatments follow multi-steps experimental workflows, which alternatively lead to low recoveries of proteins. In this line, this study aims at developing a simple and versatile strategy in order to reduce protein losses and enhance their detection in gel-free LC-MS analysis. First, an analytical method based on liquid chromatography and tandem mass spectrometry was developed to detect and quantify complex peptides mixture. Secondly, a universal, simple and fast purification approach was designed with the aim to purify protein extracts in only one-step. For this purpose, the molecular reactivity, dynamics and conformational changes of proteins at each development step were comprehensively investigated with a set of spectroscopic techniques, in order to select the best strategy. Finally, different factors influencing extraction of proteins were investigated with the goal in the long term to propose an on-demand extraction protocol for direct analysis of proteins by LC-HRMS/MS

Le tossine PSP (paralytic shellfish poison) sono composti altamente tossici prodotti principalmente da dinoflagellati marini appartenenti ai generi Alexandrium, Gymnodinium e Pyrodinium. Le tossine fitoplanctoniche possono essere accumulate nei molluschi filtratori e in altri frutti di mare. Le tossine PSP si legano ai canali del sodio e impediscono il flusso di ioni attraverso la membrana. Di conseguenza, le tossine causano sintomi di avvelenamento (ad esempio di natura neurologica) nell'uomo. In passato il test biologico sui topi (MBA) era un metodo utilizzato per rilevare le tossine PSP nei molluschi bivalvi. Secondo il regolamento (UE) 2021/1709, il contenuto di tossine PSP dei molluschi bivalvi deve essere determinato utilizzando la norma EN 14526:2017, metodo di rilevamento in fluorescenza mediante derivatizzazione pre-colonna, vietando qualsiasi metodo che implichi l'uso di un animale vivo. Sono stati studiati metodi alternativi per l'analisi delle tossine PSP per ottenere un metodo più rapido e semplice rispetto al metodo HPLC/FLD ufficiale. Abbiamo testato campioni fortificati e campioni naturalmente contaminate per l'ottimizzazione sia del test competitivo in vitro su colture cellulari che del metodo LC-HRMS come metodo di screening per la valutazione della presenza di tossine algali. I risultati mostrano la possibilità di utilizzare LC-HRMS e i saggi basati sull'utilizzo di colture cellulari (Cell Based Assays) come metodi alternativi alla sperimentazione animale per evidenziare la presenza di tossine PSP.<br>Paralytic shellfish poisoning (PSP) toxins are highly toxic compounds mainly produced by marine dinoflagellates belonging to Alexandrium, Gymnodinium and Pyrodinium genera. The phytoplanktonic toxins can be accumulated in filter feeding shellfish and other seafood. PSP toxins bind to voltage-gated sodium channels and prevent the flux of ions across the membrane. As a result, the toxins cause paralytic shellfish poisoning (e.g. neurological distress) in human. In the past the mouse bioassay (MBA) was method used for detecting PSP toxins in bivalve mollusks. According to Regulation (EU) 2021/1709, PSP toxins content of bivalve mollusks shall be determined using EN 14526:2017, Fluorescence Detection method using pre-column derivatization, by prohibiting any method entailing the use of a live animal. Alternative methods for analysis of PSP toxins were studied to obtain faster, quicker and easier method than official HPLC/FLD method. We tested spiked and naturally incurred samples for the optimization of both competitive in vitro assay on cell cultures and HPLC-HRMS method as screening method for the assessment of the presence of algal toxins. The results show the possibility of using LC-HRMS or assays based on the use of cell cultures (Cell Based Assays) as alternative methods to animal experiments to highlight the presence of PSP toxins.

The increasing number of foodomics studies based on non-targeted methods shows that this approach is considered by scientists to be an efficient way of evaluating food safety and quality. In the last few years, high resolution mass spectrometry (HRMS) has indeed gained wider acceptance thanks to the high selectivity and sensitivity achievable during analysis. In contrast to the classic unit-mass-resolution MS/MS approach, HRMS provides more information on sample composition through collection of full-scan spectra and thanks to the possibility of performing retrospective data analysis. Consequently, even without defining compound-specific tuning, HRMS data can be used for identification of suspect compounds or for structural elucidation of unknowns. HRMS can only compete with classic MS/MS methods using the targeted approach, even if it allows the simultaneous detection of a higher number of compounds. In contrast, HRMS is a more promising approach when suspect and non-targeted screening analysis is performed, not only because full-scan and retrospective analysis is feasible, but also because the accurate mass of both precursor and product ions and their isotope patterns are provided. Furthermore, a non-targeted approach leads to specific profiling of biological systems through a wide selection of chemical descriptors, and provides the fingerprint of the system under investigation, useful for more easily identifying potential adulteration. The aim of this work was to extend comprehension of the three different HRMS approaches (nontargeted, suspect and targeted screening), examining both their potential and limitations in relation to the analysis of the compounds of interest in different matrices. Initially, the objectives concerned the possibility of developing new methods – one for each HRMS screening approach – for the analysis of glycosides and phenolic compounds, in order to furnish innovative and well-performing analytical tools for food safety and quality control at all stages of food production, processing and distribution. Furthermore, they regarded the possibility of investigating the nature and occurrence of glycosides and phenolic compounds in widely consumed beverage and food commodities, such as grape, wine, spirits, cocoa and honeys. The thesis, which includes both published and in litteris papers, describes newly developed analytical methods and their technological applications in the study of different matrices, focusing on: - Investigation of Neutral Loss experiments as an instrument for non-targeted screening analysis of glycosides, and performance evaluation of this analytical approach in relation to the glycosidic profiling of international monovarietal wines; - Investigation of the distribution of free and glycosylated low-molecular-weight phenolic compounds in skin and seeds of color-rich Vitis vinifera grapes cultivated in southern Uruguay, combining Neutral loss experiment and suspect screening analysis; - Investigation of the selectivity and sensitivity of the HRMS approach for targeted analysis of free and glycosylated low-molecular-weight phenolic compounds and suspect screening analysis of the latter, together with evaluation of the best sample clean-up procedure for reducing matrix interference; - Investigation of the distribution of free and glycosylated low-molecular-weight phenolic compounds in skin, pulp and seeds, focusing on both Vitis vinifera and hybrid grapes; - Investigation of the impact of alcoholic fermentation on the free and glycosylated phenolic profile of wines produced from grapes of hybrid varieties; - Study of the occurrence of glycosylated low-molecular-weight phenolic compounds in tannins of different botanical origin, in order to evaluate the alteration of the phenolic profile of wines after tannin addition; - Study of the free phenolic composition of wood barrels, in order to evaluate phenolic enrichment during ageing, and investigation of the possible impact of different barrel sanitation treatments on the phenolic transfer from wood to wine; - Study of the free and glycosylated low-molecular-weight phenolic profile in Primitivo di Manduria and Negroamaro wines of different vintages and evaluation of the effect of wine ageing; - Investigation of the possibility of considering free or glycosylated low-molecular-weight phenolic compounds as new markers for beverages and food characterization and their geographical traceability, focusing on wine, spirits, vinegar, food tannins, cocoa beans and honeys; - Implementation of investigative methods of suspect screening analysis using naturally rich matrices as available sources of compounds of interest. This approach was applied on plant products for alkaloid identification; - Investigation of the selectivity and sensitivity of the HRMS approach for suspect screening analysis of flavonoids in flowering plant (C. pareira) extracts.

Ces travaux de thèse s’insèrent dans le contexte de la contamination du milieu marin par les contaminants émergents, tels que les produits pharmaceutiques (PP), et de leurs effets sur les organismes marins. L’étude des liens de cause à effet entre l’exposition à un ou plusieurs contaminants et la réponse de la moule méditerranéenne, Mytilus galloprovincialis, a été l’axe central des recherches présentées dans cette thèse. Afin de renseigner les mécanismes d’action et les potentiels effets toxiques des PP, données manquantes dans la littérature, les approches –omiques telles que la métabolomique et la protéomique ont été favorisées. Les effets de l’antiépileptique carbamazépine (CBZ), un PP fréquemment détecté dans le milieu marin, ont dans un premier temps été investigués sur M. galloprovincialis à travers une approche intégrée de la métabolomique et de la protéogénomique. La stratégie de fusion des données appliquée a mis en évidence des signatures protéiques et métaboliques corrélées entre elles en réponse à l’exposition. L’utilisation d’outils bioinformatiques remettant les protéines et métabolites dans leur contexte biologique a ainsi révélé des changements concernant la synthèse des protéines, la dégradation des acides gras, la métabolisation des acides aminés et des glucides, et la programmation de la mort cellulaire. Bien que l’étude des effets d’un seul contaminant soit essentielle pour obtenir des informations d’ordre mécanistique, elle s’éloigne en revanche de la pertinence d’une exposition environnementale, les organismes étant exposés simultanément à une multitude de contaminants. Dans le but d’intégrer cette complexité, les moules M. galloprovincialis ont été exposées à un rejet de station d’épuration (STEP), voie d’entrée principale des PP dans le milieu marin. L’analyse des empreintes métaboliques générées a d’abord été effectuée sur les moules mâles afin d’écarter la variabilité biologique liée au sexe (susceptible de masquer la réponse à l’exposition). Plusieurs voies métaboliques ont ainsi été révélées comme impactées (ex. acides aminés, bases puriques et pyrimidiques, cycle de Krebs, neurohormones, etc.) pouvant perturber plusieurs fonctions et processus biologiques (ex. métabolisme énergétique, système immunitaire, osmorégulation, reproduction, formation du byssus, etc.) et avoir des conséquences néfastes sur l’organisme. En se basant sur la littérature, des hypothèses de relation de cause à effet ont pu être établies entre certains contaminants détectés dans le rejet de STEP (38 PP et 4 pesticides) et les effets observés. A partir de cette même expérimentation, le facteur sexe a ensuite été intégré dans le traitement des données issues des individus mâles/femelles et exposés/non-exposés afin de comprendre le rôle du sexe dans la réponse à l’exposition. Pour cela, l’approche statistique Analyse of variance Multiblock Orthogonal Partial Least Square s’est révélée pertinente pour ce genre de design expérimental multifactoriel. Cette approche a ainsi pu associer la variabilité des données métabolomiques à leur(s) facteur(s) d’origine(s). Une réponse commune entre les deux sexes, reliée au facteur exposition, a été mise en évidence à travers la modulation de plusieurs lysophospholipides induite par un stress oxydant. Tandis qu’une réponse sexe-dépendante, reliée à l’interaction entre les facteurs sexe et exposition, a été décrite suite à une modulation de certains lipides polaires selon le sexe et une perturbation de la voie de la kynurénine uniquement chez les mâles. Ces travaux de thèse ont pu renforcer les connaissances sur les effets d’un PP préoccupant pour l’environnement, la CBZ, exclue de tout cadre réglementaire, ainsi que sur les effets d’une exposition proche des conditions environnementales reconstituées à travers un rejet de STEP. Des approches originales d’investigation des effets et d’analyses des données ont été pertinemment appliquées<br>This PhD thesis takes place in a context of the contamination of marine environment by emerging contaminants, such as pharmaceutical active compounds (PhAC), and their effects on marine organisms. The study of causal relationships between exposure to one or more contaminants and the response of the Mediterranean mussel, Mytilus galloprovincialis, was the central focus of the research presented in this thesis. In order to provide information on the mechanisms of action and potential toxic effects of PhAC, data lacking in the literature, -omic approaches such as metabolomics and proteomics were applied. The effects of the antiepileptic carbamazepine (CBZ), a PhAC frequently detected in the marine environment, were first investigated on M. galloprovincialis through an integrated approach of metabolomics and proteogenomics. The data fusion strategy applied revealed correlated protein and metabolic signatures in response to exposure. The use of bioinformatics tools that put proteins and metabolites into their biological context thus highlighted changes in protein synthesis, fatty acid degradation, amino acid and carbohydrate metabolism, and cell death programming. Although the study of the effects of a single contaminant is essential to obtain mechanistic information, it is far removed from the relevance of environmental exposure, since organisms are exposed simultaneously to a multitude of contaminants. In order to integrate this complexity, mussels M. galloprovincialis were exposed to a wastewater treatment plant (WWTP) effluent, the main pathway of PhAC into the marine environment. Analysis of the metabolic fingerprints generated was first performed on male mussels to rule out gender-related biological variability (which could hide the response to exposure). Several metabolic pathways were thus revealed to be impacted (e.g. amino acids, purines and pyrimidines, Krebs cycle, neurohormones, etc.) which can disrupt several biological functions and processes (e.g. energy metabolism, immune system, osmorregulation, reproduction, byssal formation, etc.) and have adverse consequences on the organism. Based on the literature, hypotheses of causal relationships have been established between certain contaminants detected in the WWTP effluent (38 PPs and 4 pesticides) and the effects observed. Based on the same experiment, the gender factor was then integrated into the processing of data acquired from male/female and exposed/unexposed individuals in order to understand the role of gender in the response to exposure. To this end, the statistical approach of Analysis of Variance Multiblock Orthogonal Partial Least Square proved to be relevant for this kind of multifactorial experimental design. This approach was thus able to characterize and relate the variability of metabolomics data to its different factors of origin. A common response between the two genders, related to the exposure factor, was demonstrated through the modulation of several lysophospholipids induced by oxidative stress. While a gender-specific response, related to the interaction between gender and exposure factors, has been described following a modulation of certain polar lipids according to gender and a disruption of the kynurenin pathway only in males. This thesis work was able to strengthen knowledge on the effects of a PhAC of concern for the environment, CBZ, excluded from any regulatory framework, as well as on the effects of an exposure close to the environmental conditions reconstituted through a WWTP effluent. Original approaches to effects investigation and data analysis have been pertinently applied

Les anti-inflammatoires non stéroidiens (AINS) sont l’un des médicaments les plus utilisés dans le monde, accessibles sur ordonnances ou en vente libre. Plusieurs articles ont fait état d’une forte consommation d’AINS par des coureurs pendant les ultratrails. Plusieurs études montrent qu’environ 50% des coureurs utillisent des AINS sans ordonnance médicale, sans forcément connaître leurs effets indésirables et leur toxicité. A ce jour, seulement des données basées sur des questionnaires ont été rapportées sur la prévalence des AINS chez les ultratrailers. Or, en raison de la méconnaissance des coureurs concernant les AINS, l’utilisation du questionnaire peut entraîner des résultats faux positifs ou faux négatifs. Dans cette étude, nous avons étudié la prévalence des AINS chez les utratrailers via des prélèvements de salive et sang capillaire. Des protocoles de préparation d’échantillon et une méthode LC-MS/HRMS ont été développés puis validés pour 19 AINS dans la salive et pour 18 AINS dans le sang capillaire. La méthode a été appliquée avec succès sur des échantillons collectés auprès de participants de l’Ultratrail du Mont Blanc® (UTMB®) 2021. Les résultats ont montré une prévalence des AINS supérieure dans les prélèvements de sang capillaire (46,6%) que dans les prélèvements salivaires (18,5%) ainsi qu’à partir des données du questionnaire (13,8%). En conclusion, ce travail est le premier rapportant l’identification et la quantification d’autant d’AINS dans la salive et le sang capillaire. De plus, l’étude menée lors de l’UTMB® 2021 a montré que la recherche d’AINS dans le sang capillaire est le moyen le plus pertinent pour étudier la prévalence dans l’ultratrail<br>Non-steroidal anti-inflammatory drugs (NSAIDs) are one of the most used drugs worldwide accessible through medical prescription or over-the-counter (OTC). Several published articles have reported a high consumption of NSAIDs by runners during ultratrail. Pain relief and prevention are the main reasons for these drugs consumption by runners. Several studies reported that around 50% runners used NSAIDs without medical prescription, these data show that half of runner may have low information or even lack of information about NSAIDs adverse effects. Up to date, only questionnaire-based data reported about NSAIDs prevalence by the runners. Therefore, using questionnaire may not provide the right information and leads to false positive or false negative results. In this study, we used oral fluid (OF) and dried blood spots (DBS), as biological samples for the study of NSAIDs consumption prevalence by the runners. Sample preparation workflows and a LC-MS/HRMS analytical method were developed and validated according to European Medicines Agency guideline for 19 and 18 different NSAIDs in OF and DBS, respectively. The method was successfully applied on OF and DBS samples collected from Ultramarathon du Mont Blanc® 2021. Results showed a higher prevalence of NSAIDs using DBS (46.6%) than OF (18.5%) and questionnaire (13.8%). To conclude, this work is the first one that reported a LC-MS/HRMS method for identification and quantification of as many NSAIDs in oral fluid and dried blood spots. Moreover, the prospective study conducted on UTMB® 2021 showed that the analysis of drugs directly in DBS is the most relevant tool to determine the prevalence in ultratrail events

Face à l’expansion géographique des biotoxines marines, à l’émergence de nouvelles toxines et compte tenu du risque avéré pour la santé humaine, il est essentiel de disposer d’outils suffisamment versatiles et performants pour détecter une gamme, la plus large possible, de toxines connues ou émergentes de manière à garantir la sécurité des consommateurs. Cette thèse s’inscrit dans la démarche de surveillance de la qualité sanitaire des produits de la pêche. Elle a pour finalité de contribuer à l’évolution du dispositif de veille d’émergence par le développement d’une approche non ciblée reposant sur l’utilisation de la spectrométrie de masse haute résolution comme alternative à la pratique controversée du bio-essai sur souris.Les travaux entrepris ont permis dans un premier temps de développer et caractériser une méthode par chromatographie liquide couplée à la spectrométrie de masse haute résolution pour l'analyse ciblée de 32 toxines marines avec une gamme étendue de polarités, utilisant un spectromètre de masse haute résolution. Deux types de séparations chromatographiques, en phase inverse et à interactions hydrophiles, ont été mises en place pour la séparation des toxines lipophiles et hydrophiles. Ensuite une stratégie décrivant les différentes étapes d’une approche non ciblée allant de l’acquisition au traitement des données par des outils chimiométriques a été développée. Le traitement des données acquises en mode non ciblé a été réalisé au moyen de deux types de logiciels différents : une suite logicielle commerciale (Sciex) et un logiciel open source (XCMS). Cette stratégie a été testée avec succès dans le cadre d’une preuve de concept sur des échantillons d’huîtres et de moules supplémentés avec certaines toxines et analysés en aveugle. Elle a ensuite été appliquée sur des échantillons impliqués dans des cas de toxi-infections alimentaires collectives liés à la consommation de violets du genre Microcosmus, selon les deux approches différentes, le suspect screening et l’analyse sans a priori<br>Considering the globalisation of marine biotoxin distribution, the emergence of new toxins and the proven risk to human health, it is essential to have tools that are sufficiently versatile and effective to detect the widest possible range of known or emerging toxins in order to guarantee consumers’ safety. This PhD falls within the scope of the monitoring of marine toxins. It aims to contribute to the evolution of the vigilance system by implementing a non-targeted approach based on the use of high-resolution mass spectrometry as an alternative to the use of the controversial mouse bioassay.First, a liquid chromatography high-resolution mass spectrometry method was developed and characterized for the analysis of 32 marine toxins with a wide range of polarities, using a hybrid quadrupole time-of-flight (QTOF) mass spectrometer. Reversed-phase and hydrophilic interactions columns have been used for the separation of targeted lipophilic and hydrophilic toxins. A strategy describing the different steps of a non-targeted approach, from acquisition to data processing, by chemometric tools was then developed using two different softwares (the SCIEX software suite, commercially available, and XCMS, an open source software). The developed workflow was succefully tested as part of a proof, on blindly analysed spiked samples (mussels and oysters) with different toxins. The strategy was then applied to naturally contaminated samples involved in human intoxications and related to the consumption of sea squirts of the Microcosmus genus using two approaches to process the data generated by the non-targeted analysis; the first one consists in screening a list of suspects (suspect screening) and the second one is an analysis without a priori

L’une des principales contraintes dans l’analyse de produits naturels est l’accès au standard analytique de molécules considérées comme marqueurs d’une plante ou faisant l’objet de restrictions car potentiellement dangereuses. Les problématiques qui en découlent ont tissé le fil conducteur de l’ensemble des travaux de cette thèse. Le premier objectif a été d’étudier les performances qu’offre le couplage entre l’HPTLC et la spectrométrie de masse via l’Interface MS 2 dans le domaine de l’identification de marqueurs de plantes employées dans les compléments alimentaires. Il apparaît que dans le cadre de contrôle de routine, ce couplage a toute sa place car les informations obtenues pourraient permettre l’identification de plantes selon des monographies issues de la pharmacopée européenne. Cependant, dans le cadre de la recherche de nouveaux composés, les résultats obtenus restent malgré tout aléatoires. Le deuxième objectif a été d’étudier la CPC dans le cadre de l’isolement de marqueurs à l’échelle préparative. Pour cela, les valépotriates, molécules soumises à diverses réglementations à travers le monde et dont l’accès au standard analytique est difficile ont été choisie comme analytes modèles. Après un développement de méthode à l’échelle analytique, un transfert d’échelle non linéaire se basant sur le concept de l’« espace libre entre les pics » a été évalué. Pour la première fois ce modèle a été utilisé pour isoler non pas une, mais deux molécules qui en plus, coéluent. La CPC a permis d’isoler en une étape, deux molécules structurellement proches à un niveau de pureté supérieur à 95% et avec un taux de recouvrement de 90%. De plus, cette étude a permis de confirmer la structure du 7-homovaltrate qui faisait, jusqu’alors, l’objet de discussion. Le troisième et dernier objectif de cette thèse a été d’évaluer l’apport de l’HRMS pour la caractérisation et le dosage de familles de composés. Pour illustrer cette étude, les alcaloïdes pyrrolizidiniques (AP), qui sont des molécules responsables de dizaines de milliers d’intoxication à travers le monde, ont été sélectionnés. L’emploi d’une HRMS a permis de mettre en perspective la pertinence des résultats issus d’un dosage des seuls AP dont le standard analytique est disponible car elle a permis d’identifier d’autres AP dans une mauvaise herbe fréquemment incriminée dans la contamination de denrées alimentaires<br>Principal limitation to natural product analysis is the access to the analytical standards of molecules considered as markers or potentially dangerous. Their emerging issues are the common thread of this doctoral work. The first objective was to study the performances of HPTLC-MS coupling via Interface MS 2 in the field of plants markers identifications that are commonly used in dietary supplements elaboration. It seems that this coupling is really usefull for routine control, because resulting mass spectrometric informations may permit plants identifications in accordance with pharmacopoeias monographs. But, in case of structural elucidation of new active compounds, the obtained results are extremely uncertains. The second objective was to evaluate CPC performances in the field of markers isolation at an industrial scale. To illustrate this study, valepotriates have been chosen. These secondary metabolites are regulated all over the world but the access to their analytical standards remains problematic. After a chromatographic method development at an analytical scale, a non lineary scale up based on the « free space between peak » concept has been performed. For the first time, this concept has been applied to two molecules which, in addition, are co-eluting. CPC allowed a one step isolation of two structurally close molecules at over 95% purity and with 90% recovery. More over, this study permitted to confirm the structure of 7-homovaltrate which was ambiguous. The third and last objectif was to evaluate HRMS for the characterization and the quantification of family of compounds. To illustrate this study, pyrrolizidine alkaloids family (PA) has been selected. These molecules are responsible of tens of thousands deaths around the world. HRMS afforded identification of AP which analytical standard is not available in weed commonly incriminated in food contamination. This put into perspective the relevance of the results obtained through a method that only quantifies available analytical standards of AP. Furthermore, HRMS informations allowed to discriminate a molecule wich had the same fragment ions as the AP but which wasn’t an AP

L’exposition chronique à des mélanges complexes de contaminants chimiques (xénobiotiques) est suspectée de contribuer à la survenue de certaines maladies chroniques. Encouragées par le développement de la spectrométrie de masse à haute résolution (SMHR) et l’émergence du concept d’exposome, des méthodes analytiques non-ciblées commencent à voir le jour pour caractériser l’exposition humaine aux xénobiotiques sans a priori. Ces méthodes innovantes pourraient ainsi permettre un changement d’échelle pour identifier de nouveaux facteurs de risque chimiques dans des études épidémiologiques. Ces approches présentent néanmoins plusieurs verrous, en lien, entre autres, avec la présence des contaminants à l’état de trace dans des matrices biologiques. Une optimisation de chaque étape analytique (préparation d’échantillon) et bio-informatique (prétraitement des données, annotation) est donc indispensable pour surmonter ces limites. L’objectif principal de ce travail est d’implémenter un workflow non-ciblé applicable aux études épidémiologiques pour apporter une solution opérationnelle à la caractérisation de l’exposome chimique interne à large échelle. Les développements effectués ont permis de proposer un workflow de préparation d’échantillon simple à mettre en œuvre et s’appuyant sur deux méthodes complémentaires pour élargir significativement l’espace chimique visible (jusqu’à 80% de marqueurs spécifiques à une méthode). L’optimisation de logiciels de prétraitement des données, réalisée pour la première fois dans un contexte exposomique, a permis de démontrer la nécessité d’ajuster certains paramètres pour assurer la détection des xénobiotiques à l’état de trace. Le développement d’un logiciel pour automatiser les approches de profilage de suspects avec des prédicteurs MS1, ainsi que le développement d’indices de confiance a permis de prioriser les marqueurs pertinents pour la curation manuelle. Une application à large échelle sur 125 échantillons de sérum de la cohorte Pélagie a permis de démontrer la robustesse et la sensibilité de ce nouveau workflow, ainsi que d’enrichir l’exposome chimique documenté avec la mise en évidence de nouveaux biomarqueurs d’exposition<br>Chronic exposure to complex mixtures of chemical contaminants (xenobiotics) is suspected to contribute to the onset of chronic diseases. The technological advances high-resolution mass spectrometry (HRMS), as well as the concept of exposome, have set the stage for the development of new non-targeted methods to characterize human exposure to xenobiotics without a priori. These innovative approaches may therefore allow changing scale to identify chemical risk factors in epidemiological studies. However, non-targeted approaches are still subject to a number of barriers, partly linked to the presence of these xenobiotics at trace levels in biological matrices. An optimization of every analytical (i.e. sample preparation) and bioinformatical (i.e. data processing, annotation) step of the workflow is thus required. The main objective of this work is to implement an HRMS-based non-targeted workflow applicable to epidemiological studies, to provide an operational solution to characterize the internal chemical exposome at a large scale. The undertaken developments allowed proposing a simple sample preparation workflow based on two complementary methods to expand the visible chemical space (up to 80% of features specific to one method). The optimization of various data processing tools, performed for the first time in an exposomics context, allowed demonstrating the necessity to adjust key parameters to accurately detect xenobiotics. Moreover, the development of a software to automatize suspect screening approaches using MS1 predictors, and of algorithms to compute confidence indices, allowed efficiently prioritizing features for manual curation. A large-scale application of this optimized workflow on 125 serum samples from the Pélagie cohort allowed demonstrating the robustness and sensitivity of this new workflow, and enriching the documented chemical exposome with the uncovering of new biomarkers of exposure

La presenza di residui chimici negli alimenti, costituiti sia da composti farmaceutici che da contaminati ambientali, è un argomento di crescente interesse e preoccupazione per la sanità pubblica. L’Unione Europea periodicamente sancisce leggi e rapporti aggiornati, con lo scopo di condurre piani di monitoraggio e linee guida sull’ impiego di tali composti a livello industriale, agricolo e terapeutico e livelli massimi residuali (LMR e ML) negli alimenti, al fine di prevenire il rischio per il consumatore. Sulla base di queste considerazioni, il presente elaborato ha lo scopo di studiare la presenza di residui chimici in diversi alimenti di origine animale, al fine di caratterizzare il rischio per il consumatore. In primo luogo, ci siamo focalizzati sullo studio di alimenti provenienti dal settore ittico, che è un'ottima fonte di nutrienti, con importanti benefici per la salute umana. Ci siamo concentrati su cozze e vongole, animali filtratori e bioindicatori adatti per le loro caratteristiche a bioaccumulare un'ampia gamma di inquinanti ambientali. Lo scopo del primo elaborato è stato quello di valutare il rischio per il consumatore dei principali metalli (Hg, Cd, Pb, Ni, Cre As), attraverso il consumo di molluschi sulla base dei limiti massimi dichiarati dall’Unione Europea o, ove disponibili, sulla base dei valori soglia dichiarati dall’EFSA. Dal primo lavoro è emerso che vi è un basso rischio per il consumatore medio; tuttavia, i consumatori ai percentili superiori, possono essere soggetti a lesioni cutane e/o neoplasie polmonari, cutanee e vescicali per l’elevata assunzione di As. Soggetti Ni sensibili, possono invece essere soggetti a dermatiti allergiche. Il secondo lavoro di ricerca si è invece concentrato sulla ricerca dei medesimi metalli nel tonno, il cui consumo è in aumento secondo i dati della commissione europea, per le sue capacità di bioaccumulo. Sono stati, così, analizzati 131 campioni provenienti da diverse zone FAO. Dai risultati, è emerso che solo un tonno rosso, proveniente dal mare Adriatico e 11 tonni gialli hanno superato i livelli massimi residuali di Pb; tre tonni rossi provenienti da diverse sottozone del 7 Mediterraneo hanno superato i livelli massimi consentiti per il mercurio. La valutazione degli effetti tossicologici cumulativi ha indicato un rischio trascurabile sia per i medi che alti consumatori. L’obiettivo del terzo studio è stato quello di studiare la presenza di inquinanti organici persistenti e di antimicrobici nei salmoni selvatici e di allevamento di diverse aree geografiche. I salmoni d'allevamento hanno mostrato una presenza di contaminanti ambientali superiore a quelli selvatici, probabilmente a causa di un maggiore impatto demografico. Il rischio legato ai composti organofosforati, agli idrocarburi policiclici aromatici, ai policlorobifenili e ai pesticidi organoclorurati derivanti dall'assunzione di salmone si è rilevato molto basso, mentre la presenza di polobromodifenilietere congenere 99 (PBDE99) e acido perfluoroottanoico (PFOA) suscita maggiore preoccupazione. Gli antibiotici sono stati riscontrati con bassa frequenza solo nel salmone allevato. Da questi lavori, sulla base dei dati ottenuti, possiamo confermare che vi è basso rischio per il consumatore medio Il consumo regolare di carne e prodotti a base di carne fornisce un significativo apporto di proteine e micronutrienti essenziali. La carne suina, ad esempio, è impiegata in molti paesi per produrre prodotti derivati (prosciutti e salumi) ad alto valore qualitativo. Anche il consumo di carne di selvaggina, pur essendo un prodotto di nicchia, è in costante aumento e i cacciatori, le loro famiglie e le persone a loro strettamente legate possono essere considerati una sottopopolazione ad alto consumo. Nel quarto lavoro abbiamo cosi studiato la presenza di polibromodifenilietere e sostanze perfluoroalchiliche provenienti da otto Stati membri dell'UE (Austria, Danimarca, Francia, Germania, Olanda, Italia, Polonia e Spagna). La commissione Europea non ha definito limiti massimi per tali composti e dai nostri risultati non sono state rilevate sostanze perfluoroalchiliche ad eccezione dell’acido perfluoroottanoico in un solo campione austriaco. I polibromodifenilietere sono stati rilevati solo in 3 dei 77 campioni investigati. I risultati mostrano che i campioni analizzati non rappresentano un rischio per il consumatore. Recentemente una successiva relazione EFSA ha richiesto di porre maggiore attenzione sulla presenza dei perfluoroalchilici, i cui valori soglia sono stati ridotti drasticamente per il loro rischio tossicologico. Nel quinto lavoro ci siamo focalizzati su quattro diverse specie selvatiche (camoscio, capriolo, capriolo, cervo e cinghiale) con abitudini alimentari differenti. Gli animali selvatici sono considerati 8 specie sentinelle e quindi ottimi indicatori ambientali. Campioni muscolari di settantanove animali sono stati raccolti durante la stagione venatoria in una zona montana dell'Italia settentrionale. Nei campioni non sono stati trovati polibromodifenilieteri. Al contrario i pesticidi organoclorurati e organofosforati e i policlorobifenili sono stati rilevati in quasi tutti i campioni a diversi intervalli di concentrazione, mostrando una frequenza maggiore nelle specie di ungulati rispetto al cinghiale. I PFA ,invece, sono stati riscontrati solo nei cinghiali. Tra gli idrocarburi, antracene e benzopirene, sono stati trovati solo nel camoscio a basse concentrazioni. Possiamo nuovamente concludere che per il frequente ritrovamento a basse concentrazioni dei contaminati, ad eccezione di singoli composti riscontrati ad alte concentrazioni, e del basso consumo di carne di selvaggina rapportata ad altre tipologie di carne, vi è un basso rischio per il consumatore italiano. Un ulteriore tema di crescente interesse per la sanità pubblica è stato lo studio dei contaminati ambientali persistenti nel miele, in particolare il miele biologico. Infatti, nonostante l'apicoltura biologica escluda (o consenta in modo restrittivo) l'impiego di farmaci o pesticidi, molti inquinanti possono contaminare api, miele e polline. Pertanto, l'attenzione si è concentrata sullo studio di un ampio spettro di analiti quali, pesticidi, inquinanti organici persistenti e antibiotici in mieli organici raccolti in diverse aree produttive con diverso impatto agricolo, zootecnico o antropico per verificare il potenziale trasferimento di xenobiotici nella catena di approvvigionamento da fonti diverse rispetto alle pratiche apistiche. È stata confermata la presenza di diversi composti, come policlorobifenili, i polibromodifenilietere e gli idrocarburi policiclici aromatici non solo nelle arnie in prossimità di centri altamente urbanizzati, dove le concentrazioni erano più elevate, ma in tutti i contesti ambientali, confermando la possibilità di trasferimento da fonti ambientali e l’ubiquità di tali composti. Il mancato ritrovamento di antibiotici nei campioni analizzati esclude la possibilità di trasferimento accidentale delle molecole dall’ambiente in cui sono posizionate le arnie. Per ottenere una così ampia e diversificata ricerca, ogni lavoro è stato approciato in modo differente per il pretrattamento dei campioni, l’ottimizzazione del metodo analitico, l’estrazione degli analiti e il loro successivo clean up prima dell’analisi con cromatografia liquida in spettrometria di massa tandem (LC-MS/MS) o gas spettrometria (GC-MS/MS). L'approccio di natura analitico-strumentale ha richiesto per ogni ricerca un’accurata e ampia ricerca per ottenere l'ottimizzazione delle prestazioni strumentali e delle fasi di pretrattamento dei campioni, al fine di raggiungere buoni livelli di sensibilità, specificità e robustezza dei metodi analitici impiegati per poi fare 9 considerazioni di natura qualitativa, quantitativa e statistica. La pianificazione delle prove, l'ottimizzazione e la convalida dei metodi sono state eseguite secondo la Commissione SANTE/10553/2018 (SANTE 2018). I risultati di questo lavoro suggeriscono che il rischio per la salute media dei consumatori è basso. Le concentrazioni ambientali dei composti organoclorurati persistenti sembrano diminuite negli ultimi due decenni, probabilmente grazie ai progressi nella rilevazione analitica e al miglioramento dei controlli europei. I PCB sono ancora presenti nell'ambiente a causa del loro ampio impiego a livello industriale nel secolo scorso e delle loro peculiarità chimico fisiche, anche se il loro uso, oggi, è stato vietato in molte applicazioni. Per quanto riguarda i composti emergenti, i PFAs destano preoccupazione a causa del loro ampio uso e del loro possibile ruolo tossicologico. Recentemente la Commissione Europea ha infatti drasticamente diminuito i livelli soglia per queste classi per salvaguardare la salute umana. Gli antibiotici sono ancora motivo di preoccupazione e necessitano di uno stretto controllo per garantire la sicurezza umana e ridurre le resistenze, tema tuttora più che attuale.<br>The presence of xenobiotic residues, both drugs and environmental contaminants, in food is a cause for concern and therefore the European Authorities issue reports or laws in order to propose monitoring plans, Health-based Guidance Values (HBGV) and maximum residue levels or maximum levels (MRLs and MLs). Based on these considerations, this doctoral thesis studies the presence of residues in different foods of animal origin, aimed at a characterization of the risk for the consumer. Firstly, we studied seafood, which is an excellent source of nutrients, with important human health benefits. We focused on mussels and clams, filter feeders animals, suitable bio indicator organisms due to their bioaccumulation ability of a wide range of environmental pollutants. In the first research study, we evaluated the Italian consumer risk related to metal exposition through molluscs, on the basis on the MLs stated by the European Union, where available, or, otherwise, based on the HBGV stated by EFSA. About our results, regarding the human metal exposure, we conclude that there is a low risk for the average consumer; however, high percentile consumers, may be subjected to skin lesions, and lung, skin and bladder cancer due to high intake of As, while Ni sensitive individuals can undergo allergic dermatitis due to constant Ni presence in the studied molluscs. Subsequently, we focused on most consumed fish like salmon, tuna which consumption has consistently risen. In the second study about salmon, the aim was to investigate the presence of persistent organic pollutants (POPs) and antimicrobials in wild and farmed salmons from different geographic areas. Farmed salmons showed slightly higher presence of environmental contaminants than wild ones, likely due to the decreased possibility of a constant exposition. Antibiotics were seldom found only in farmed salmon. Risk related to organophosphate compounds (Ops), polycyclic aromatic hydrocarbons (PAHs), polychlorobiphenyls (PCBs) and organochlorine pesticides (OCPs) deriving from salmon intake were of is very low concern, while the presence of polybromodiphenyl ether (PBDE99) and perfluorooctanoic acid (PFOA), is a cause for a bit higher concern. The substantial lack of data about the detected antibiotics in salmon did not allow an extrapolation from MRLs of terrestrial animals and a risk characterization In the third work on tuna, a long-living fish with high biomagnification ability, we studied the presence of metals with high toxicological importance for public health (Hg, Pb, Cd, As, Cr, Ni). One hundred thirty-one samples were analysed. One red tuna from the Adriatic Sea and 11 yellow tunas exceeded Pb maximum levels (MLs); three red tunas from different Mediterranean sub-areas exceeded Hg MLs. The evaluation of cumulative effects indicated that only a negligible health hazard could derive from the ingestion of tuna, for both average and high consumers. The risk of carcinogenicity from Cr is still under debate at the concentrations detectable in food. In these two works, we confirm a low risk, related to the studied compounds, for average consumer health due to fish consumption. The regular consumption of meat and meat products provides a significant intake of proteins and essential micronutrients. Pork meat, for example, is used in many countries to produce derivative products (hams and cured meats) with high qualitative value. Also, game animal meat consumption, though being a niche product, is constantly increasing and hunters, their families and persons closely associated with them can be regarded as a high consumption subpopulation. Furthermore, game animals are a suitable indicator about environmental pollutant such as PCBs, PBDEs, PAHs and brominated flame retardants (BFRs). In the fourth work we studied the occurrence of PBDEs and perfluoroalkyl substances (PFASs) from eight EU Member States (Austria, Denmark, French, Germany, Holland, Italy, Poland and Spain). The European Commission has not stated maximum limits (MLs) for some environmental pollutants such as polybrominated diphenyl ether PBDEs and PFASs; no perfluoroalkyl substances were detected, except PFOA, in only one Austrian sample. PBDEs were detected in three out of 77 samples: the one coming from Germany showed the presence of all congeners analyzed the ones from Netherland and Italy, respectively PBDE 153 and PBDE 100. The results show that the analyzed samples do not pose a risk for human beings about PFASs and PBDEs. A following report from EFSA, requires a new attention on PFAS, with HBGV being drastically reduced. In the fifth work we studied four different animal species (chamois, roe deer, red deer and wild boar) that have different nutrition habits. Game animals are a suitable sentinel species to have a picture of the environment. Muscle samples from seventy-nine animals were collected during the hunting season in a Northern Italy mountain area. No PBDEs were found in the samples. OCPs, OPs and PCBs were detected in almost all samples at different concentration ranges, showing higher frequency in ungulate species than in wild boar. PFAs were found only in wild boar. Anthracene and benzopyrene, among PAHs, were found only in chamois at low concentrations. A low risk for consumers can be indicated due to the frequent detection of contaminants at trace levels, to the scarce prevalence of high concentrations of some contaminants and to the low consumption of game animal meat. An important topic in the researches carried out in my doctorate was the investigation of POPs in organic honey. However, even if organic beekeeping excludes (or restrictively allows) the use drugs or pesticides many pollutants may contaminate bee matrices, comprising bee, honey and pollen. Therefore, the focus was the investigation of a broad spectrum of analytes, pesticides, persistent organic pollutants and antibiotics in organic honeys collected in different productive areas with different agricultural, zootechnical or anthropic impact to verify the potential transfer of xenobiotics into supply chain from different sources than beekeeping practices. The presence of several compounds, such as PCBs, PBDE and PAHs was confirmed, not only in proximity to highly urbanised centres, where the concentrations were higher, but in all environment contexts, confirming the theory that these are ubiquitous contaminants. No antibiotics were found in samples analysed suggesting that presence of antibiotics is from beekeeping practices. The analytes in the different matrices required different approaches for sample pretreatment, extraction, clean up and fractionation before the analysis with liquid chromatography–tandem mass spectrometry (LC-MS/MS) or – gas mass spectrometry (GC-MS/MS). The approach of analytical-instrumental nature has provided for the optimisation of instrumental performances as well as of the steps of sample pretreatment, in order to achieve good levels of sensitivity, specificity and robustness of the method to then make considerations of qualitative, quantitative and statistical nature. The trials planning, optimisation and validation of the methods were performed according to Commission SANTE/10553/2018 (SANTE 2018). The results of this manuscript suggest that there is a low risk for the average consumer health. Environmental concentrations of persistent organochlorine compounds have been decreasing over the past two decades, and this correlates with remarkable advances in the detection of exceedingly low levels of these compounds in human populations and the improvement of European control. PCBs still are present in environment due to their industrial source even if their use was banned in many industries application. Regarding emerging compounds, PFAs still need to be concern due to their wide use and their possible toxicological role. Recently European commission decreased the HBGVs for these classes to safeguard human health. Antibiotics still are a matter of concern and need a close control to ensure human safety and decrease antimicrobial resistance.

<p>The two additive brominated flame retardants, tetrabromoethylcyclohexane (TBECH) and hexabromocyclododecane (HBCD) are used to prevent fire to start and spread. They are simply mixed with material and are most likely to leach out in the environment, because of non-covalently binding to the material. TBECH can exist as four pairs of enantiomers, α-, β-, γ- and δ-TBECH. The technical HBCD can exist as three pairs of enantiomers, α-, β- and γ-HBCD and two meso forms δ- and ε-HBCD. None of these compounds are produced in Sweden, but they are imported to industries. TBECH has been found in Beluga blubber and can accumulate in zebrafish. HBCD has been found in water environments and can be toxic to and bioaccumulate in water-living animals.</p><p>In this study, a method was developed for separation and detection of α-, β-, γ- and δ-TBECH on HRGC/HRMS. All TBECH-isomers could be separated with the developed method. How much of the TBECH isomers that were recovered after applying existing extraction and clean-up procedures, normally applied for clean-up and extraction of PCBs and PCDD/Fs, was evaluated. Low recovered amounts (6.8-35.5 %) of TBECH-isomers added in known amounts to three different whale samples indicate severe evaporation losses and possibly photolytic degradation. None of the four enantiomers were detected in the three whale samples. For HBCD analysis, both the chromatography and MS/MS parameters were optimised for δ- and ε- HBCD yielding good chromatography and sensitivity. However, due to technical difficulties during the time-period of this project, no whale samples could be analysed for HBCD on UPLC/MS/MS. </p>

La carne e i prodotti a base di carne sono le principali fonti di diete umane. La salute degli animali da produzione alimentare dovrebbe essere adeguatamente monitorata e controllata al fine di ridurre i rischi per la catena alimentare. Gli studi presentati in questa tesi includevano le strategie di (i) valutare l'esito della spedizione del rapporto patologico agli allevamenti di suini di provenienza e (ii) sviluppare il metodo di rilevazione e studiare la prevalenza di contaminanti ambientali e farmaci veterinari nel maiale, vitello, uova di gallina e alimenti per bambini. Nel capitolo 3, abbiamo raccolto i record delle ispezioni delle carni a livello nazionale. Il numero di grandi allevamenti rappresenta il 9% del totale ma hanno prodotto il 48,5% di suini da macello. Per la percentuale di lesioni patologiche nelle carcasse, i suoi coefficienti di variazione (CV) sono del 42% nella classe dei grandi allevamenti. Ciò suggerisce che il livello di salute nei grandi allevamenti era più omogeneo rispetto agli quelli piccoli e medi. Al termine dello studio, abbiamo analizzato le influenze delle lesioni patologiche dopo aver inviato il risultato post mortem agli allevamenti di provenienza. I risultati evidenziano che le percentuali di fegato e polmone sono gradualmente ridotte dello 0,02% al mese. Il feedback sui risultati post mortem migliora la trasparenza delle informazioni governative, la collaborazione tra produttori e veterinario ufficiale e la salute della mandria per alimenti più sicuri di origine animale. Nei capitoli 4, 5 e 6, abbiamo sviluppato I metodi di rilevazione altamente sensibili su sostanze perfluoroalchiliche (PFAS), bifenili policlorurati (PCB), idrocarburi policiclici aromatici (IPA), eteri difenilici polibromurati (PBDE), pesticidi e antibiotici. Il limite di quantificazione (LOQ) è 0,015-0,15 ng g-1 in PFAS, 0,5 ng g-1 in PBDE, il che è conforme alla decisione 2002/657/CE della Commissione. Abbiamo applicato i nostri metodi per lo studio di diverse matrici animali: maiale, vitello e alimenti per bambini. I risultati suggeriscono che la prevalenza di contaminanti ambientali in carne di maiale, vitello e alimenti per bambini sono bassi e non comportano rischi per la salute umana. Nel capitolo 7, abbiamo sviluppato un metodo rapido e facile da applicare per rilevare il fipronil e il suo metabolita e l'amitraz dalle uova di gallina. Il LOQ è 0,89 ng g-1 in fipronil e 2,4 ng g-1 in amitraz. I risultati descritti in questa tesi consistono nella chiara comprensione dei metodi di rilevazione di contaminanti ambientali e farmaci veterinari nei suini, vitelli e pollami. Inoltre, tramite la mail di feedback, i produttori di suini hanno ricevuto le osservazioni continue dal macello, quindi hanno intrapreso azioni per ridurre le lesioni patologiche. I risultati introdotti in questa tesi possono essere il modo futuro di mantenere la sicurezza lungo tutta la catena alimentare.<br>Meat and meat products are the principal sources of human diets. The health of food-producing animals should under proper monitoring and control in order to reduce risks to the food supply chain. The studies presented in this thesis included the strategies of (i) Evaluate the outcome of feeding back the pathological report to the origin pig farms, and (ii) Build up the detection method and investigate the prevalence for environmental contaminants and veterinary drugs in pork, veal, chicken eggs and baby foods. In Chapter 3, we collected meat inspection records at a national level. The number of large farms account for 9% of the total but produced 48.5% slaughtering pigs. About the percentage of pathological lesions in the carcass, its coefficients of variation (CVs) is of 42% in the class of large farms. It suggests that the health level in large farms were more homogenous than in small and medium ones. At the final of the study, we analysed the influences of pathological lesions after having sent the post-mortem result to pig producers. The results highlight that the percentages of liver and lung had gradually reduced by 0.02% per month. The feedback of post-mortem result improves the transparency of government information, the close collaboration between producers and official veterinarian, and the herd health, for safer food of animal origin. In Chapter 4, 5 and 6, we developed highly sensitive detection methods on perfluoroalkyl substances (PFASs), polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), polybrominated diphenyl ethers (PBDEs), pesticides, and antibiotics. The limit of quantification (LOQ) is 0.015-0.15 ng g-1 in PFASs, 0.5 ng g-1 in PBDE, which complied with Commission Decision 2002/657/EC. We applied our methods for the investigation of several animal matrices: pork, veal, and baby food. The results suggest that the prevalence of environmental contaminants in pork, veal, and baby food are low and do not post risks to human health. In Chapter 7, we developed a quick and easy-to-apply method to detect Fipronil and its metabolite and Amitraz from chicken eggs. The LOQ is 0.89 ng g-1 in Fipronil, and 2.4 ng g-1 in Amitraz. The outputs described in this thesis consists of clear understanding of detection methods for environmental contaminants and veterinary drugs in swine, calves and poultry. Besides, via the feedback mail, the pig producers received continuous observations from the slaughterhouse. Thus they took actions to reduce pathological lesions. The results introduced in this thesis can be the future way to keep food safety throughout the food chain.

Chemometrics is a chemical discipline that uses mathematical and statistical methods in order to extract useful information from multivariate chemical data. Moreover, chemometrics is applied to correlate quality parameters or physical properties to analytical instrument data such as calculating pH from a measurement of hydrogen ion activity or a Fourier transform interpolation of a spectrum. Aim of this thesis project is to develop chemometrical strategies for the elaboration and the interpretation of non-selective complex data in order to solve real problems in food, industry and environmental fields.

Biopharmaceuticals are becoming one of the most promising drugs on the market mainly due to their successful treatment of a vast array of serious diseases, such as cancers, immune disorders, and infections. Structurally, biopharmaceuticals are proteins and it is important to mention that more than 60 % of biopharmaceuticals are glycosylated. Glycosylation is one of the most common posttranslational modifications. It is also the most demanding and the most complex posttranslational modification. The research showed that glycosylation can significantly impact on the safety, efficiency, and quality of the therapeutic glycoproteins. In the first part of the introduction of the present thesis, the development of the therapeutic glycoproteins and their classification were reviewed. Glycosylation process and nomenclature were also discussed. The second part of the introduction revealed current issues in the field of the production and the characterization of the therapeutic glycoproteins. In the context of the doctoral thesis, we introduced new approach, namely hydrophilic interaction liquid chromatography coupled to a high-resolution mass spectrometer (HILIC-HR-MS) combined with Principal Component Analysis (PCA) and classification through Soft Independent Modelling by Class Analogy (SIMCA) data treatment. Accordingly, N-glycans were first enzymatically released using peptide-N-glycosidase F (PNGase F) and reduced using sodium borohydride. Then those N-glycans were separated by HILIC and detected by HR-MS. PCA and SIMCA simplified interpretation of the MS data collected in the huge tables. PCA was applied to test whether it is possible to visualize N-glycosylation differences between samples and to help identifying within which N-glycans changes occurred. SIMCA, which is a more complex data analysis technique, was applied to build and validate a classification models. SIMCA was also applied to verify whether it is possible to use built models to classify real samples. Described approach enabled us to detect small changes in N-glycosylation of the therapeutic glycoproteins (a change of only 1% in relative glycan abundance). It was applied to assess changes in N-glycosylation of therapeutic glycoproteins. Accordingly, we tested N-glycosylation consistency between batches of infliximab, trastuzumab, and bevacizumab and monitored the N-glycosylation of bevacizumab over storage time in plastic syringes.Furthermore, we worked on the faster sample preparation technique, where online-solid-phase extraction (SPE)-LC was combined to the previously mentioned HILIC-MS-PCA/SIMCA method. Online-SPE-LC allowed us to faster the sample preparation in terms of avoiding time-consuming cleaning steps.<br>Doctorat en Sciences<br>info:eu-repo/semantics/nonPublished

Les plantes médicinales constituent une source inexhaustible de composés (des produits naturels - PN) utilisé en médecine pour la prévention et le traitement de diverses maladies. L'introduction de nouvelles technologies et méthodes dans le domaine de la chimie des produits naturels a permis le développement de méthodes ‘high throughput’ pour la détermination de la composition chimique des extraits de plantes, l'évaluation de leurs propriétés et l'exploration de leur potentiel en tant que candidats médicaments. Dernièrement, la métabolomique, une approche intégrée incorporant les avantages des technologies d'analyse moderne et la puissance de la bioinformatique s’est révélé un outil efficace dans la biologie des systèmes. En particulier, l'application de la métabolomique pour la découverte de nouveaux composés bioactifs constitue un domaine émergent dans la chimie des produits naturels. Dans ce contexte, le genre Acronychia de la famille des Rutaceae a été choisi sur la base de son usage en médecine traditionnelle pour ses propriétés antimicrobienne, antipyrétique, antispasmodique et anti-inflammatoire. Nombre de méthodes chromatographiques modernes, spectrométriques et spectroscopiques sont utilisées pour l'exploration de leur contenu en métabolites suivant trois axes principaux constituant les trois chapitres de cette thèse. En bref, le premier chapitre décrit l’étude phytochimique d’Acronychia pedunculata, l’identification des métabolites secondaires contenus dans cette espèce et l'évaluation de leurs propriétés biologiques. Le deuxième chapitre vise au développement de méthodes analytiques pour l'identification des dimères d’acétophénones (marqueurs chimiotaxonomiques du genre) et aux stratégies utilisées pour la déréplication de ces différents extraits et la caractérisation chimique des composés par UHPLC-HRMSn. Le troisième chapitre se concentre sur l'application de méthodologies métabolomique (RMN et LC-MS) pour l'analyse comparative (entre les différentes espèces, origines, organes), pour des études chimiotaxonomiques (entre les espèces) et pour la corrélation des composés contenus avec une activité pharmacologique<br>Medicinal plants constitute an unfailing source of compounds (natural products – NPs) utilised in medicine for the prevention and treatment of various deceases. The introduction of new technologies and methods in the field of natural products chemistry enabled the development of high throughput methodologies for the chemical composition determination of plant extracts, evaluation of their properties and the exploration of their potentials as drug candidates. Lately, metabolomics, an integrated approach incorporating the advantages of modern analytical technologies and the power of bioinformatics has been proven an efficient tool in systems biology. In particular, the application of metabolomics for the discovery of new bioactive compounds constitutes an emerging field in natural products chemistry. In this context, Acronychia genus of Rutaceae family was selected based on its well-known traditional use as antimicrobial, antipyretic, antispasmodic and anti-inflammatory therapeutic agent. Modern chromatographic, spectrometric and spectroscopic methods were utilised for the exploration of their metabolite content following three basic axes constituting the three chapters of this thesis. Briefly, the first chapter describes the phytochemical investigation of Acronychia pedunculata, the identification of secondary metabolites contained in this species and evaluation of their biological properties. The second chapter refers to the development of analytical methods for the identification of acetophenones (chemotaxonomic markers of the genus) and to the dereplication strategies for the chemical characterisation of extracts by UHPLC-HRMSn. The third chapter focuses on the application of metabolomic methodologies (LC-MS &amp; NMR) for comparative analysis (between different species, origins, organs), chemotaxonomic studies (between species) and compound-activity correlations

Charles Univesity, Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Candidate: Jana Tomanová Supervisor: doc. PharmDr. Radim Kučera, Ph.D. Consultant: PharmDr. Vít Šesták, Ph.D. Title of the Diploma Thesis: LC-HRMS analysis of selected drugs in biological material I. High performance liquid chromatography is one of the most widespread separation analytical techniques. It is used in various medical, pharmaceutical and industrial laboratories. It uses different affinities of the substances in the analysed mixture to the mobile and stationary phases. Mass spectrometry is one of the instrumental techniques by which charged particles are separated in the gas phase based on the mass- to-charge ratio. Nowadays, great emphasis is placed on the fact that the individual analytical methods which are being developed are subsequently validated according to a guideline issued by national or international authorities. Validation is a process that verifies that a method is suitable for its intended use. In this thesis, the conditions for the determination of warfarin in patient's serum by means of mass spectrometer with LTQ XL linear ion trap were optimized and validated. The optimization of the method was based on the already developed and validated...

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Candidate: Kristýna Švarcová Consultant: RNDr. Martin Mžik, Ph.D. Supervisor: doc. PharmDr. Radim Kučera, Ph.D. Title: LC-HRMS analysis of selected drugs in biological material II. Anxiety disorders affect up to 25 % of the population in their life, the lifetime prevalence is 13,6 %. Insomnia affect at least one third of the population in their life, the prevalence is 15-40 %. Drugs used to treat anxiety and insomnia easily cross the blood-brain barrier and affect the CNS. It is therefore necessary to determine the correct dosing schedule and monitor treatment. The result is optimization of treatment, reduction of side effects and increased patient adherence to treatment. The rapid qualitative and quantitative analysis for urgent cases of intoxication is also needed. This diploma thesis deals with development and optimization of an extraction method for selected benzodiazepines (alprazolam, bromazepam, diazepam, chlordiazepoxide, clonazepam, midazolam, oxazepam) and zolpidem in human serum. Subsequently, the whole analytical method was validated for use in clinical practice using the UHPLC-HRMS. Protein precipitation with acetonitrile was the most suitable sample preparation...

5 ABSTRACT Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Candidate: Anna Tláskalová Supervisor - specialist: Mgr. Martin Mžik Supervisor: doc. PharmDr. Radim Kučera, Ph.D. Title: LC-HRMS analysis of selected antihypertensive drugs in biological material for compliance assessment About 40 % of Czech population between the ages of 25 and 64 suffer from arterial hypertension. Although an array of effective antihypertensives is available nowadays, optimal blood pressure during treatment is reached by mere 30 % of the patients. This is mainly due to the patients' poor adherence to the treatment, who use their medicaments incorrectly or not at all. The adherence of a patient to the treatment, as well as pharmacokinetics, represent the most significant source of variability of the answer to the treatment and notably influences its outcome. The monitoring of plasmatic levels of antihypertensives is one of the methods of modern medicine which enables both an effective supervision of the incorrectly compensated patients, and the adjustment of dosage scheme. This diploma thesis focuses on development and optimisation of extraction procedures for selected antihypertensives (amiloride, amlodipine, betaxolol, bisoprolol, carvedilol, celiprolol,...

L’abuso di sostanze stupefacenti è ancora oggi un problema sempre crescente che affligge tutte le fasce di età. Molti dei consumatori non sono avvisati dei danni e dei problemi correlati ai rischi nell’assumerle. Nei recenti le Nuove Sostanze Psicoattive (o NPS dall’acronimo inglese) sono spesso vendute online e conosciute come “Legal Highs”: queste sostanze sono nuove molecole di origine naturale o sintetica, le quali vengono vendute negli smart shops come incensi, sali da bagno o standard ad uso non umano, spesso appartenenti a definite classi chimiche. L’introduzione di così tanti analoghi strutturali pensati per aggirare le legislazioni esistenti è costante: ogni volta che una sostanza viene schedata ed inserita nelle tabelle della normativa nazionale, un nuovo analogo con modifiche alla struttura viene immesso sul mercato. Per questi motivi le NPS hanno una priorità elevata per l’Unione Europea e i suoi stati membri, inclusa l’Italia. È anche riportato come l’uso di queste sostanze sia associato a disturbi mentali e stati alterati della psiche. A causa della mancanza di controllo legislativo, queste sostanze sono facilmente reperibili negli Smart Shops o nel mercato online. Gli effetti farmacologici e la difficoltà di trovare in matrice biologica la molecola non metabolizzata pone l’accento sull’importanza dell’identificazione dei metaboliti per lo sviluppo di metodi analitici per investigazioni cliniche e forensi. Queste sostanze sono spesso largamente metabolizzate ed escrete con le urine, ma per molte di loro i dati riguardanti il metabolismo umano sono limitati. Il maggior traguardo che il nostro progetto si prefissa, è sviluppare e testare metodi analitici efficaci per la rivelazione di legal highs basati sulla cromatografia liquida accoppiata alla spettrometria di massa tandem (LC-MS/MS) o spettrometria di massa ad alta risoluzione (LCHRMS) utilizzando differenti approcci con idonei strumenti analitici e bioanalitici. In questo lavoro sono state sviluppate diverse tecniche estrattive su campioni di interesse per la tossicologia forense come sangue, urina e capelli, risultando essere strumenti con un’alta sensibilità e specificità. Questi metodi permettono di ottenere informazioni qualitative e quantitative sulle NPS analizzate. In particolare la spettrometria di massa ad alta risoluzione ha permesso la determinazione di sostanze psicoattive e l’identificazione dei suoi metaboliti nei casi in cui è stata applicata adeguatamente. Nella parte finale del progetto ci si è concentrati sull’identificazione nuovi metaboliti di NPS sconosciuti in letteratura avvalendosi non solo delle tecniche LC-MS/MS e LC-HRMS sviluppate in precedenza ma anche di tecniche di chimica farmaceutica computazionale e bioinformatica, affiancate a metodologie di incubazione in vitro delle sostanze. 7 Questo ha permesso di identificare pattern metabolici fino adesso sconosciuti che sono stati caratterizzati tramite una metodologia LC-HRMS. Confrontando i dati delle analisi in vitro con i dati informatici ottenuti dagli esperimenti in silico si è riusciti a sviluppare un protocollo applicabile ad ogni nuova sostanza psicoattiva che compare sul mercato in modo da identificare velocemente il pattern metabolico. L’efficacia del metodo è stata confermata confrontando i risultati su campioni di urina provenienti da studi farmacologici condotti sulla sostanza in esame. Tutti i metodi sviluppati sono stati validati, in accordo con le linee guida internazionali.

Tese de mestrado, Química, Universidade de Lisboa, Faculdade de Ciências, 2019<br>Este trabalho foi realizado no âmbito de um protocolo entre a Faculdade de Ciências da Universidade de Lisboa (FCUL), o Laboratório de Polícia Científica da Polícia Judiciária (LPC/PJ) e o Instituto Superior Técnico (IST), que tem como objetivo a análise, determinação de padrões e investigação científica de Novas Substâncias Psicoativas (NSP) em Portugal. Nos últimos anos, o ‘’cenário’’ do mundo das drogas mudou devido ao crescente desenvolvimento e consumo de NSP. NSP designa-se por ‘’uma substância na forma pura ou numa preparação que não está abrangida pela Convenção Única das Nações Unidas sobre os Estupefacientes, de 1961, alterada pelo Protocolo de 1972, nem pela Convenção das Nações Unidas sobre as Substâncias Psicotrópicas, de 1971, mas que pode colocar riscos sociais ou para a saúde semelhantes aos colocados pelas substâncias abrangidas pelas referidas convenções’’. Devido ao rápido crescimento das NSP no mercado, a resposta das autoridades é dificultada em termos de estudo toxicológicos e de desenvolvimento de metodologias analíticas adequadas para a identificação e quantificação de NSP e dos seus metabolitos em amostras biológicas. Este conhecimento é essencial para a atuação das autoridades forenses e antidoping. Assim, é fundamental a aproximação entre as autoridades judiciais e os grupos de investigação, para a identificação de metabolitos de NSP que entram no mercado, assim como de outros que se antecipam que poderão brevemente surgir no mercado. As catinonas sintéticas constituem um dos maiores grupos de NSP, são análogos estruturais da catinona, um dos principais compostos psicoativos da planta Catha edulis. Este trabalho teve como objetivo a identificação metabolitos de Fase I e de Fase II das catinonas 4'-metil-N,N-dietilcatinona (4-MDEC, também conhecida por 4Me-anfepramona), 4’-metil-N,N-dimetilcatinona (4-MDMC), 4’-cloropirrolidinovalerofenona (4Cl-PVP) e 4'-cloroetilcatinona (4-CEC), que apesar de já ter sido reportado o aparecimento no mercado de substâncias recreativas são ainda desconhecidos os seus metabolitos. Para tal, estas catinonas foram incubadas in vitro, em microssomas de rato e humano (RLM e HLM, respetivamente) e fração S9 de fígado de rato, na presença de cofatores adequados e os metabolitos gerados foram posteriormente identificados por Cromatografia Líquida acoplada à espetrometria de massa de alta resolução de tandem com ionização por Electrospray (LC-ESI-HRMS/MS). A utilização desta técnica de alta resolução foi essencial para elucidação estrutural dos metabolitos identificados. Este procedimento possibilitou a identificação, pela primeira vez, de dois metabolitos de Fase I e um de Fase II da 4Me-anfepramona, 1 metabolito de Fase II da 4-MDMC, 4 metabolitos de Fase I e três de Fase II da 4Cl-PVP e dois de Fase I e um de Fase II da 4-CEC. Para além destes, foi também identificado um novo metabolito de Fase II da alfa-pirrolidinovalerofenona (α-PVP). Esta cationona serviu de controlo positivo nas incubações, por já serem conhecidos alguns dos seus metabolitos. Os resultados obtidos serão de grande utilidade para as entidades judiciais, tornando agora possível atestar o consumo destes NSP por identificação em amostras biológicas dos metabolitos identificados no âmbito desta tese. Este estudo constitui ainda o primeiro passo para o conhecimento mais profundo da atividade/toxicidade dos vários metabolitos identificados.<br>This work was carried out under the scope of a protocol established between Faculdade de Ciências da Universidade de Lisboa (FCUL), Laboratório de Polícia Científica da Polícia Judiciária (LPC/PJ) and Instituto Superior Técnico (IST), which is aimed at the analysis, standard preparation and scientific investigation of New Psychoactive Substances (NPS) in Portugal. In recent years, the drug scene has changed with the increasing development and consumption of New Psychoactive Substances (NPS). NPS means '' a substance in pure form or in a misture that is not covered by the 1961 United Nations Single Convention on Narcotic Drugs, as amended by the 1972 Protocol, or by the United Nations Convention on Psychotropic Substances, 1971, but which may pose social or health risks similar to those posed by substances covered by those Conventions ''. Due to the rapid growth of NPSs on the market, the authorities' response is hampered in terms of toxicological study and of the development of suitable analytical methodologies for the identification and quantification of NPSs and their metabolites in biological samples. This knowledge is pivotal for the work of forensic and anti-doping authorities. Therefore, the collaborative work between judicial authorities and research groups is crucial for identifying the metabolites of NPS new in the market as well as of others which are anticipated to appear in the market in a near future. Synthetic cathinones, which constitute one of the largest groups of NPS, are structural analogues of cathinone, one of the main psychoactive compounds of the Catha edulis plant. The aim of this work was to identify Phase I and Phase II metabolites of the cathinones 4Me-anfepramone, 4-MDMC, 4Cl-PVP and 4-CEC, although their appearance on the recreational substances market has already been reported, their metabolites are still unknown.. To this end, these cathinones were incubated in vitro in rat and human microsomes (RLM and HLM, respectively) and rat liver S9 fraction, in the presence of appropriate cofactors, and the generated metabolites were further identified by Liquid Chromatography coupled to Electrospray ionization tandem high resolution mass spectrometry (LC-ESI-HRMS/MS). The use of this high-resolution technique was essential for structural elucidation of the identified metabolites. This strategy allowed the first-hand identification of two Phase I and one Phase II metabolites of 4Me-anphepramone, one Phase II metabolite of 4-MDMC, four Phase I metabolites and three Phase II metabolites of 4Cl-PVP, two Phase I and one Phase II of 4-CEC. In addition to these, a new α-PVP Phase II metabolite was also identified. This cathionone was used as a positive control in incubations, taking into consideration that some of its metabolites are already reported in literature. The results obtained will be very useful for the judicial entities, making now possible to attest the consume of these NPS upon detection of the metabolites identified in the scope of this thesis, in biological samples. This study constitutes also the first step towards a deeper understanding of the activity/toxicity of the multiple metabolites identified.