Academic literature on the topic 'LC-MS/MS and LC/HRMS'

Palytoxin (PlTX) and analogues are produced by certain dinoflagellates, sea anemones, corals and cyanobacteria. PlTX can accumulate in the food chain and when consumed it may cause intoxication with symptoms like myalgia, weakness, fever, nausea, and vomiting. The analysis of PlTXs is challenging, and because of the large molecular structure, it is difficult to develop a sensitive and selective liquid chromatography-mass spectrometry (LC-MS/MS) method. In this work, an LC-MS/MS method was developed to analyse PlTXs with use of lithium iodine and formic acid as additives in the mobile phase. For method development, initially, LC-hrMS was used to accurately determine the elemental composition of the precursor and product ions. The main adduct formed was [M + H + 2Li]3+. Fragments were identified with LC-hrMS and these were incorporated in the LC-MS/MS method. A method of 10 min was developed and a solid phase extraction clean-up procedure was optimised for shellfish matrix. The determined limits of detection were respectively 8 and 22 µg PlTX kg−1 for mussel and oyster matrix. Oysters gave a low recovery of approximately 50% for PlTX during extraction. The method was successfully in-house validated, repeatability had a relative standard deviation less than 20% (n = 5) at 30 µg PlTX kg−1 in mussel, cockle, and ensis, and at 60 µg PlTX kg−1 in oyster.

Rituximab is a chimeric immunoglobulin G1-kappa (IgG1κ) antibody targeting the CD20 antigen on B-lymphocytes. Its applications are various, such as for the treatment of chronic lymphoid leukemia or non-Hodgkin’s lymphoma in oncology, and it can also be used in the treatment of certain autoimmune diseases. Several studies support the interest in therapeutic drug monitoring to optimize dosing regimens of rituximab. Thus, two different laboratories have developed accurate and reproductive methods to quantify rituximab in human plasma: one using liquid chromatography quadripolar tandem mass spectrometer (LC-MS/MS) and the other, liquid chromatography orbitrap tandem mass spectrometer (LC-MS/HRMS). For both assays, quantification was based on albumin depletion or IgG-immunocapture, surrogate peptide analysis, and full-length stable isotope-labeled rituximab. With LC-MS/MS, the concentration range was from 5 to 500 µg/mL, the within- and between-run precisions were <8.5%, and the limit of quantitation was 5 µg/mL. With LC-MS/HRMS, the concentration range was from 10 to 200 µg/mL, the within- and between-run accuracy were <11.5%, and the limit of quantitation was 2 µg/mL. Rituximab plasma concentrations from 63 patients treated for vasculitis were compared. Bland–Altman analysis and Passing–Bablok regression showed the interchangeability between these two methods. Overall, these methods were robust and reliable and could be applied to routine clinical samples.

Ligand-binding assay (LBA) and LC–MS have been the preferred bioanalytical techniques for the quantitation and biotransformation assessment of various therapeutic modalities. This review provides an overview of the applications of LBA, LC–MS/MS and LC–HRMS for the bioanalysis of complex protein therapeutics including antibody–drug conjugates, fusion proteins and PEGylated proteins as well as oligonucleotide therapeutics. The strengths and limitations of LBA and LC–MS, along with some guidelines on the choice of appropriate bioanalytical technique(s) for the bioanalysis of these therapeutic modalities are presented. With the discovery of novel and more complex therapeutic modalities, there is an increased need for the biopharmaceutical industry to develop a comprehensive bioanalytical strategy integrating both LBA and LC–MS.

Ciguatera poisoning is a global health concern caused by the consumption of seafood containing ciguatoxins (CTXs). Detection of CTXs poses significant analytical challenges due to their low abundance even in highly toxic fish, the diverse and in-part unclarified structures of many CTX congeners, and the lack of reference standards. Selective detection of CTXs requires methods such as liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) or high-resolution MS (LC–HRMS). While HRMS data can provide greatly improved resolution, it is typically less sensitive than targeted LC–MS/MS and does not reliably comply with the FDA guidance level of 0.1 µg/kg CTXs in fish tissue that was established for Caribbean CTX-1 (C-CTX-1). In this study, we provide a new chemical derivatization approach employing a fast and simple one-pot derivatization with Girard’s reagent T (GRT) that tags the C-56-ketone intermediate of the two equilibrating C-56 epimers of C-CTX-1 with a quaternary ammonium moiety. This derivatization improved the LC–MS/MS and LC–HRMS responses to C-CTX-1 by approximately 40- and 17-fold on average, respectively. These improvements in sensitivity to the GRT-derivative of C-CTX-1 are attributable to: the improved ionization efficiency caused by insertion of a quaternary ammonium ion; the absence of adduct-ions and water-loss peaks for the GRT derivative in the mass spectrometer, and; the prevention of on-column epimerization (at C-56 of C-CTX-1) by GRT derivatization, leading to much better chromatographic peak shapes. This C-CTX-1–GRT derivatization strategy mitigates many of the shortcomings of current LC–MS analyses for C-CTX-1 by improving instrument sensitivity, while at the same time adding selectivity due to the reactivity of GRT with ketones and aldehydes.

Paralytic shellfish toxins (PSTs) are a complex class of analogs of the potent neurotoxin saxitoxin (STX). Since calibration standards are not available for many PSTs, including C-11 hydroxyl analogs called M-toxins, accurate quantitation by liquid chromatography–mass spectrometry (LC-MS) can be challenging. In the absence of standards, PSTs are often semiquantitated using standards of a different analog (e.g., STX), an approach with a high degree of uncertainty due to the highly variable sensitivity between analytes in electrospray ionization. Here, relative molar response factors (RMRs) were investigated for a broad range of PSTs using common LC-MS approaches in order to improve the quantitation of PSTs for which standards are unavailable. First, several M-toxins (M1-M6, M9 and dcM6) were semipurified from shellfish using preparative gel filtration chromatography and quantitated using LC-charged aerosol detection (LC-CAD). The RMRs of PST certified reference materials (CRMs) and M-toxins were then determined using selective reaction monitoring LC-MS/MS and full scan LC-high-resolution MS (LC-HRMS) methods in positive and negative electrospray ionization. In general, RMRs for PSTs with similar chemical structures were comparable, but varied significantly between subclasses, with M-toxins showing the lowest sensitivity. For example, STX showed a greater than 50-fold higher RMR than M4 and M6 by LC-HRMS. The MS instrument, scan mode and polarity also had significant impacts on RMRs and should be carefully considered when semiquantitating PSTs by LC-MS. As a demonstration of their utility, the RMRs determined were applied to the semiquantitation of PSTs in contaminated mussels, showing good agreement with results from calibration with CRMs.

[D-Leu1]MC-LY (1) ([M + H]+ m/z 1044.5673, Δ 2.0 ppm), a new microcystin, was isolated from Microcystis aeruginosa strain CPCC-464. The compound was characterized by 1H and 13C NMR spectroscopy, liquid chromatography–high resolution tandem mass spectrometry (LC–HRMS/MS) and UV spectroscopy. A calibration reference material was produced after quantitation by 1H NMR spectroscopy and LC with chemiluminescence nitrogen detection. The potency of 1 in a protein phosphatase 2A inhibition assay was essentially the same as for MC-LR (2). Related microcystins, [D-Leu1]MC-LR (3) ([M + H]+ m/z 1037.6041, Δ 1.0 ppm), [D-Leu1]MC-M(O)R (6) ([M + H]+ m/z 1071.5565, Δ 2.0 ppm) and [D-Leu1]MC-MR (7) ([M + H]+ m/z 1055.5617, Δ 2.2 ppm), were also identified in culture extracts, along with traces of [D-Leu1]MC-M(O2)R (8) ([M + H]+ m/z 1087.5510, Δ 1.6 ppm), by a combination of chemical derivatization and LC–HRMS/MS experiments. The relative abundances of 1, 3, 6, 7 and 8 in a freshly extracted culture in the positive ionization mode LC–HRMS were ca. 84, 100, 3.0, 11 and 0.05, respectively. These and other results indicate that [D-Leu1]-containing MCs may be more common in cyanobacterial blooms than is generally appreciated but are easily overlooked with standard targeted LC–MS/MS screening methods.

Staphylococcal food poisoning outbreaks are caused by the ingestion of food contaminated with staphylococcal enterotoxins (SEs). Among the 27 SEs described in the literature to date, only a few can be detected using immuno-enzymatic-based methods that are strongly dependent on the availability of antibodies. Liquid chromatography, coupled to high-resolution mass spectrometry (LC-HRMS), has, therefore, been put forward as a relevant complementary method, but only for the detection of a limited number of enterotoxins. In this work, LC-HRMS was developed for the detection and quantification of 24 SEs. A database of 93 specific signature peptides and LC-HRMS parameters was optimized using sequences from 24 SEs, including their 162 variants. A label-free quantification protocol was established to overcome the absence of calibration standards. The LC-HRMS method showed high performance in terms of specificity, sensitivity, and accuracy when applied to 49 enterotoxin-producing strains. SE concentrations measured depended on both SE type and the coagulase-positive staphylococci (CPS) strain. This study indicates that LC-MS is a relevant alternative and complementary tool to ELISA methods. The advantages of LC-MS clearly lie in both the multiplex analysis of a large number of SEs, and the automated analysis of a high number of samples.

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.
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.

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.

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.

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. 

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.
Doctorat en Sciences
info:eu-repo/semantics/nonPublished

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
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

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
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 & NMR) for comparative analysis (between different species, origins, organs), chemotaxonomic studies (between species) and compound-activity correlations

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
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

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.
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.

The analysis of photographic developing solutions presents an interesting and challenging problem for the analytical chemist. The developing agents themselves contain a wide range of components of various different chemistries, both inorganic and organic components are present at a wide range of concentrations. The developing agents as well has having a complex chemistry of their own, on use, are further complicated by the formation of products which can further affect the developing solutions performance. This work uses a variety of chromatographic and mass spectrometric techniques to examine a range of compounds present in the black and white Kodak developer RA2000. Emphasis is given to the application of these techniques to solving the analytical problem. The first chapter gives a theoretical introduction to mass spectrometry and liquid chromatography, with a discussion on the general principles in interfacing the two techniques. Chapter two gives a theoretical introduction to photography as well as an in depth discussion of developing solutions, specifically the RA2000 black and white developing solution. Analysis of RA2000 by liquid chromatography and capillary electrophoresis is also described. Chapter three provides an in depth discussion of solid phase extraction techniques as an alternative sample preparation method to liquid / liquid solvent extraction. Analysis of RA2000 by solid phase extraction and liquid chromatography was carried out in an attempt to concentrate the components formed after use, as well as to try and separate out the heavily concentrated compounds. Chapter four describes the analysis of RA2000 by liquid chromatography-thermospray-mass spectrometry and liquid chromatography-particle beam-mass spectrometry. An in depth discussion of both techniques is given. A column switching arrangement was used to allow analysis of the lower concentration components.

Chlorinated paraffins (CPs) are high production volume chemicals composed of complex mixtures of thousands of compounds that have been applied widely as flame retardants and plasticizers. CPs have demonstrated toxic and bioaccumulative properties, while evidence suggests dietary intake to constitute a major pathway for human exposure. This study reports on the optimization and validation of an analytical method for the quantification of short- and medium-chained CPs (SCCPs and MCCPs, respectively) using gas chromatography-mass spectrometry (GC-MS) in fats and oils, and the development of liquid chromatography-high resolution mass spectrometry (LC-HRMS) methods for investigation of long chain CP (LCCP) occurrence. Extraction was performed by ultrasonication in n-hexane and dichloromethane followed by sulphuric acid and acidified silica cleanup and fractionation on neutral silica to remove potentially interfering organohalogen contaminants. Quantification of GC-MS results using a chlorine-content calibration procedure was assessed via repeated analysis (n=3) of olive oil fortified with SCCP and MCCP technical mixtures at two concentration levels and spiked lard samples from a recent European Union Reference Laboratory (EURL) interlaboratory study. The average accuracy ranged from 76 to 126% in the olive oil samples and from 57 to 150% in fortified lard, meeting the EURLs acceptability criteria for all tests, while the precision was < 15%. The applicability of the method was demonstrated by analysis of 26 fats and oil samples purchased in Belgium. SCCPs were detected in 31% of samples, ranging < LOQ to 19 ng/g, and MCCPs were present in 85%, ranging < LOQ to 190 ng/g. Each of four samples selected for homologue profiling by LC-HRMS were also found to contain LCCPs. This research demonstrates reliable methods for CP analysis in fats and oils and highlights the potential for contamination of these products by CPs. Fats and oils appear to be substantial contributors to overall human exposure to CPs.

This talk will discuss synergistic opportunities between the effects of recently explored MCPD mitigation concepts in palm oil and sunflower oil. The discussed laboratory concepts will include dry versus wet removal of residual sediments, various degumming conditions and the role the bleaching clay in mitigating or catalyzing the formation of MCPD. The effects of the discussed mitigation concepts were studied by integrating them into the physical refining of crude oil, including water-based degumming and sealed ampoule tests mimicking the thermal conditions of edible oil deodorization. Key experiments were also reproduced in laboratory benchtop deodorizer. The MCPD levels were monitored by both direct LC-HRMS and indirect GC-MS based approaches. Beyond reviewing the MCPD mitigation effects and pros/cons of the investigated concepts, particular emphasis will be put on assessing whether the observed effects are due to correlation or causality between the refining parameters and the MCPD levels.