Photo degradation of contaminants of emerging concern (CECs) under simulated solar radiation: Implications for their environmental fate

Contaminants of emerging concern (CECs) are continuously being released into the environment mainly because of their incomplete removal in the sewage treatment plants (STPs). The CECs selected for the study include antibiotics (macrolides, sulfonamides and ciprofloxacin), sucralose (an artificial sweetener) and dioctyl sulfosuccinate (DOSS, chemical dispersant used in the Deepwater Horizon oil spill). After being discharged into waterways from STPs, photo degradation is a key factor in dictating the environmental fate of antibiotics and sucralose. Photodegradation efficiency depends on many factors such as pH of the matrix, matrix composition, light source and structure of the molecule. These factors exert either synergistic or antagonistic effects in the environment and thus experiments with isolated factors may not yield the same results as the natural environmental processes. Hence in the current study photodegradation of 13 CECs (antibiotics, sucralose and dicotyl sulfosuccinate) were evaluated using natural water matrices with varying composition (deionized water, fresh water and salt water) as well as radiation of different wavelengths (254 nm, 350 nm and simulated solar radiation) in order to mimic natural processes. As expected the contribution of each factor on the overall rate of photodegradation is contaminant specific, for example under similar conditions, the rate in natural waters compared to pure water was enhanced for antibiotics (2-11 fold), significantly reduced for sucralose (no degradation seen in natural waters) and similar in both media for DOSS. In general, it was observed that the studied compounds degraded faster at 254 nm, while when using a simulated sunlight radiation the rate of photolysis of DOSS increased and the rates for antibiotics decreased in comparison to the 350 nm radiation. The photo stability of the studied CECs followed the order sucralose > DOSS > macrolides > sulfonamides > ciprofloxacin and a positive relationship was observed between photo stability and their ubiquitous presence in natural aquatic matrices. An online LC-MS/MS method was developed and validated for sucralose and further applied to reclaimed waters (n =56) and drinking waters (n = 43) from South Florida. Sucralose was detected in reclaimed waters with concentrations reaching up to 18 μg/L. High frequency of detection (> 80%) in drinking waters indicate contamination of ground waters in South Florida by anthropogenic activity.^

The effect of iron oxide nanoparticles on the fate and transformation of arsenic in aquatic environments

Iron oxides and arsenic are prevalent in the environment. With the increase interest in the use of iron oxide nanoparticles (IONPs) for contaminant remediation and the high toxicity of arsenic, it is crucial that we evaluate the interactions between IONPs and arsenic. The goal was to understand the environmental behavior of IONPs in regards to their particle size, aggregation and stability, and to determine how this behavior influences IONPs-arsenic interactions. ^ A variety of dispersion techniques were investigated to disperse bare commercial IONPs. Vortex was able to disperse commercial hematite nanoparticles into unstable dispersions with particles in the micrometer size range while probe ultrasonication dispersed the particles into stable dispersions of nanometer size ranges for a prolonged period of time. Using probe ultrasonication and vortex to prepare IONPs suspensions of different particle sizes, the adsorption of arsenite and arsenate to bare hematite nanoparticles and hematite aggregates were investigated. To understand the difference in the adsorptive behavior, adsorption kinetics and isotherm parameters were determined. Both arsenite and arsenate were capable of adsorbing to hematite nanoparticles and hematite aggregates but the rate and capacity of adsorption is dependent upon the hematite particle size, the stability of the dispersion and the type of sorbed arsenic species. Once arsenic was adsorbed onto the hematite surface, both iron and arsenic can undergo redox transformation both microbially and photochemically and these processes can be intertwined. Arsenic speciation studies in the presence of hematite particles were performed and the effect of light on the redox process was preliminary quantified. The redox behavior of arsenite and arsenate were different depending on the hematite particle size, the stability of the suspension and the presence of environmental factors such as microbes and light. The results from this study are important and have significant environmental implications as arsenic mobility and bioavailability can be affected by its adsorption to hematite particles and by its surface mediated redox transformation. Moreover, this study furthers our understanding on how the particle size influences the interactions between IONPs and arsenic thereby clarifying the role of IONPs in the biogeochemical cycling of arsenic.^

Elemental analysis of biological matrices by laser ablation high resolution inductively coupled plasma mass spectrometry (LA-HR-ICP-MS) and high resolution inductively coupled plasma mass spectrometry (HR -ICP -MS)

The need for elemental analysis of biological matrices such as bone, teeth, and plant matter for sourcing purposes has emerged within the forensic and geochemical laboratories. Trace elemental analyses for the comparison of materials such as glass by inductively coupled plasma mass spectrometry (ICP-MS) and laser ablation ICP-MS has been shown to offer a high degree of discrimination between different manufacturing sources. Unit resolution ICP-MS instruments may suffer from some polyatomic interferences including 40Ar16O+, 40Ar 16O1H+, and 40Ca 16O+ that affect iron measurement at trace levels. Iron is an important element in the analysis of glass and also of interest for the analysis of several biological matrices. A comparison of the analytical performance of two different ICP-MS systems for iron analysis in glass for determining the method detection limits (MDLs), accuracy, and precision of the measurement is presented. Acid digestion and laser ablation methods are also compared. Iron polyatomic interferences were reduced or resolved by using dynamic reaction cell and high resolution ICP-MS. MDLs as low as 0.03 μg g-1 and 0.14 μg g-1 for laser ablation and solution based analyses respectively were achieved. The use of helium as a carrier gas demonstrated improvement in the detection limits of both iron isotopes (56Fe and 57Fe) in medium resolution for the HR-ICP-MS and with a dynamic reaction cell (DRC) coupled to a quadrupole ICP-MS system. ^ The development and application of robust analytical methods for the quantification of trace elements in biological matrices has lead to a better understanding of the potential utility of these measurements in forensic chemical analyses. Standard reference materials (SRMs) were used in the development of an analytical method using HR-ICP-MS and LA-HR-ICP-MS that was subsequently applied on the analysis of real samples. Bone, teeth and ashed marijuana samples were analyzed with the developed method. ^ Elemental analysis of bone samples from 12 different individuals provided discrimination between individuals, when femur and humerus bones were considered separately. Discrimination of 14 teeth samples based on elemental composition was achieved with the exception of one case where samples from the same individual were not associated with each other. The discrimination of 49 different ashed plant (cannabis) samples was achieved using the developed method. ^

Assessment of the occurrence and potential risks of antibiotics and their metabolites in South Florida waters using liquid chromatography tandem mass spectrometry

An automated on-line SPE-LC-MS/MS method was developed for the quantitation of multiple classes of antibiotics in environmental waters. High sensitivity in the low ng/L range was accomplished by using large volume injections with 10-mL of sample. Positive confirmation of analytes was achieved using two selected reaction monitoring (SRM) transitions per antibiotic and quantitation was performed using an internal standard approach. Samples were extracted using online solid phase extraction, then using column switching technique; extracted samples were immediately passed through liquid chromatography and analyzed by tandem mass spectrometry. The total run time per each sample was 20 min. The statistically calculated method detection limits for various environmental samples were between 1.2 and 63 ng/L. Furthermore, the method was validated in terms of precision, accuracy and linearity. ^ The developed analytical methodology was used to measure the occurrence of antibiotics in reclaimed waters (n=56), surface waters (n=53), ground waters (n=8) and drinking waters (n=54) collected from different parts of South Florida. In reclaimed waters, the most frequently detected antibiotics were nalidixic acid, erythromycin, clarithromycin, azithromycin trimethoprim, sulfamethoxazole and ofloxacin (19.3-604.9 ng/L). Detection of antibiotics in reclaimed waters indicates that they can't be completely removed by conventional wastewater treatment process. Furthermore, the average mass loads of antibiotics released into the local environment through reclaimed water were estimated as 0.248 Kg/day. Among the surface waters samples, Miami River (reaching up to 580 ng/L) and Black Creek canal (up to 124 ng/L) showed highest concentrations of antibiotics. No traces of antibiotics were found in ground waters. On the other hand, erythromycin (monitored as anhydro erythromycin) was detected in 82% of the drinking water samples (n.d-66 ng/L). The developed approach is suitable for both research and monitoring applications.^ Major metabolites of antibiotics in reclaimed wates were identified and quantified using high resolution benchtop Q-Exactive orbitrap mass spectrometer. A phase I metabolite of erythromycin was tentatively identified in full scan based on accurate mass measurement. Using extracted ion chromatogram (XIC), high resolution data-dependent MS/MS spectra and metabolic profiling software the metabolite was identified as desmethyl anhydro erythromycin with molecular formula C36H63NO12 and m/z 702.4423. The molar concentration of the metabolite to erythromycin was in the order of 13 %. To my knowledge, this is the first known report on this metabolite in reclaimed water. Another compound acetyl-sulfamethoxazole, a phase II metabolite of sulfamethoxazole was also identified in reclaimed water and mole fraction of the metabolite represent 36 %, of the cumulative sulfamethoxazole concentration. The results were illustrating the importance to include metabolites also in the routine analysis to obtain a mass balance for better understanding of the occurrence, fate and distribution of antibiotics in the environment. ^ Finally, all the antibiotics detected in reclaimed and surface waters were investigated to assess the potential risk to the aquatic organisms. The surface water antibiotic concentrations that represented the real time exposure conditions revealed that the macrolide antibiotics, erythromycin, clarithromycin and tylosin along with quinolone antibiotic, ciprofloxacin were suspected to induce high toxicity to aquatic biota. Preliminary results showing that, among the antibiotic groups tested, macrolides posed the highest ecological threat, and therefore, they may need to be further evaluated with, long-term exposure studies considering bioaccumulation factors and more number of species selected. Overall, the occurrence of antibiotics in aquatic environment is posing an ecological health concern.^

The utilization of chiral ion mobility spectrometry for the detection of enantiomeric mixtures and thermally labile compounds

This dissertation utilized electrospray ion mobility mass spectrometry (ESI-IMS-MS) to develop methods necessary for the separation of chiral compounds of forensic interest. The compounds separated included ephedrines and pseudoephedrines, that occur as impurities in confiscated amphetamine type substances (ATS) in an effort to determine the origin of these substances. The ESI-IMS-MS technique proved to be faster and more cost effective than traditional chromatographic methods currently used to conduct chiral separations such as gas and liquid chromatography. Both mass spectrometric and computational analysis revealed the separation mechanism of these chiral interactions allowing for further development to separate other chiral compounds by IMS. Successful separation of chiral compounds was achieved utilizing a variety of modifiers injected into the IMS drift tube. It was found that the modifiers themselves did not need to be chiral in nature and that achiral modifiers were sufficient in performing the required separations. The ESI-IMS-MS technique was also used to detect thermally labile compounds which are commonly found in explosive substances. The methods developed provided mass spectrometric identification of the type of ionic species being detected from explosive analytes as well as the appropriate solvent that enhances detection of these analytes in either the negative or positive ion mode. An application of the developed technique was applied to the analysis of a variety of low explosive smokeless powder samples. It was found that the developed ESI-IMS-MS technique not only detected the components of the smokeless powders, but also provided data that allowed the classification of the analyzed smokeless powders by manufacturer or make. ^

Comprehensive forensic toxicological analysis of designer drugs

New designer drugs are constantly emerging onto the illicit drug market and it is often difficult to validate and maintaincomprehensive analytical methods for accurate detection of these compounds. Generally, toxicology laboratories utilize a screening method, such as immunoassay, for the presumptive identification of drugs of abuse. When a positive result occurs, confirmatory methods, such as gas chromatography (GC) or liquid chromatography (LC) coupled with mass spectrometry (MS), are required for more sensitive and specific analyses. In recent years, the need to study the activities of these compounds in screening assays as well as to develop confirmatory techniques to detect them in biological specimens has been recognized. Severe intoxications and fatalities have been encountered with emerging designer drugs, presenting analytical challenges for detection and identification of such novel compounds. The first major task of this research was to evaluate the performance of commercially available immunoassays to determine if designer drugs were cross-reactive. The second major task was to develop and validate a confirmatory method, using LC-MS, to identify and quantify these designer drugs in biological specimens.^ Cross-reactivity towards the cathinone derivatives was found to be minimal. Several other phenethylamines demonstrated cross-reactivity at low concentrations, but results were consistent with those published by the assay manufacturer or as reported in the literature. Current immunoassay-based screening methods may not be ideal for presumptively identifying most designer drugs, including the "bath salts." For this reason, an LC-MS based confirmatory method was developed for 32 compounds, including eight cathinone derivatives, with limits of quantification in the range of 1-10 ng/mL. The method was fully validated for selectivity, matrix effects, stability, recovery, precision, and accuracy. In order to compare the screening and confirmatory techniques, several human specimens were analyzed to demonstrate the importance of using a specific analytical method, such as LC-MS, to detect designer drugs in serum as immunoassays lack cross-reactivity with the novel compounds. Overall, minimal cross-reactivity was observed, highlighting the conclusion that these presumptive screens cannot detect many of the designer drugs and that a confirmatory technique, such as the LC-MS, is required for the comprehensive forensic toxicological analysis of designer drugs.^

Method development for the analysis of smokeless powders and organic gunshot residue by ultra performance liquid chromatography with tandem mass spectrometry

The goal of this project was to develop a rapid separation and detection method for analyzing organic compounds in smokeless powders and then test its applicability on gunshot residue (GSR) samples. In this project, a total of 20 common smokeless powder additives and their decomposition products were separated by ultra performance liquid chromatography (UPLC) and confirmed by tandem mass spectrometry (MS/MS) using multiple reaction monitoring mode (MRM). Some of the targeted compounds included diphenylamines, centralites, nitrotoluenes, nitroglycerin, and various phthalates. The compounds were ionized in the MS source using simultaneous positive and negative electrospray ionization (ESI) with negative atmospheric pressure chemical ionization (APCI) in order to detect all compounds in a single analysis. The developed UPLC/MS/MS method was applied to commercially available smokeless powders and gunshot residue samples recovered from the hands of shooters, spent cartridges, and smokeless powder retrieved from unfired cartridges. Distinct compositions were identified for smokeless powders from different manufacturers and from separate manufacturing lots. The procedure also produced specific chemical profiles when tested on gunshot residues from different manufacturers. Overall, this thesis represents the development of a rapid and reproducible procedure capable of simultaneously detecting the widest possible range of components present in organic gunshot residue.^

Speciation, metabolism, toxicity, and protein-binding of different arsenic species in human cells

Despite of its known toxicity and potential to cause cancer, arsenic has been proven to be a very important tool for the treatment of various refractory neoplasms. One of the promising arsenic-containing chemotherapeutic agents in clinical trials is Darinaparsin (dimethylarsinous glutathione, DMA III(GS)). In order to understand its toxicity and therapeutic efficacy, the metabolism of Darinaparsin in human cancer cells was evaluated. With the aim of detecting all potential intermediates and final products of the biotransformation of Darinaparsin and other arsenicals, an analytical method employing high performance liquid chromatography inductively coupled mass spectrometry (HPLC-ICP-MS) was developed. This method was shown to be capable of separating and detecting fourteen human arsenic metabolites in one chromatographic run. The developed analytical technique was used to evaluate the metabolism of Darinaparsin in human cancer cells. The major metabolites of Darinaparsin were identified as dimethylarsinic acid (DMAV), DMA III(GS), and dimethylarsinothioyl glutathione (DMMTAV(GS)). Moreover, the method was employed to study the conditions and mechanisms of formation of thiol-containing arsenic metabolites from DMAIII(GS) and DMAV as the mechanisms of formation of these important As species were unknown. The arsenic sulfur compounds studied included but were not limited to the newly discovered human arsenic metabolite DMMTA V(GS) and the unusually highly toxic dimethylmonothioarsinic acid (DMMTAV). It was found that these species may form from hydrogen sulfide produced in enzymatic reactions or by utilizing the sulfur present in protein persulfides. Possible pathways of thiolated arsenical formation were proposed and supporting data for their existence provided. In addition to known mechanism of arsenic toxicity such as protein-binding and reactive oxygen formation, it was proposed that the utilization of thiols from protein persulfides during the formation of thiolated arsenicals may be an additional mechanism of toxicity. The toxicities of DMAV(GS), DMMTA V, and DMMTAV(GS) were evaluated in cancer cells, and the ability of these cells to take the compounds up were compared. When assessing the toxicity by exposing multiple myeloma cells to arsenicals externally, DMMTAV(GS) was much less toxic than DMAIII(GS) and DMMTAV, probably as a result of its very limited uptake (less than 10% and 16% of DMAIII(GS) and DMMTAV respectively).^

Development of advanced capillary electrophoresis techniques with UV and mass spectrometry detection for forensic, pharmaceutical and environmental applications

Capillary electrophoresis (CE) is a modern analytical technique, which is electrokinetic separation generated by high voltage and taken place inside the small capillaries. In this dissertation, several advanced capillary electrophoresis methods are presented using different approaches of CE and UV and mass spectrometry are utilized as the detection methods. ^ Capillary electrochromatography (CEC), as one of the CE modes, is a recent developed technique which is a hybrid of capillary electrophoresis and high performance liquid chromatography (HPLC). Capillary electrochromatography exhibits advantages of both techniques. In Chapter 2, monolithic capillary column are fabricated using in situ photoinitiation polymerization method. The column was then applied for the separation of six antidepressant compounds. ^ Meanwhile, a simple chiral separation method is developed and presented in Chapter 3. Beta cycodextrin was utilized to achieve the goal of chiral separation. Not only twelve cathinone analytes were separated, but also isomers of several analytes were enantiomerically separated. To better understand the molecular information on the analytes, the TOF-MS system was coupled with the CE. A sheath liquid and a partial filling technique (PFT) were employed to reduce the contamination of MS ionization source. Accurate molecular information was obtained. ^ It is necessary to propose, develop, and optimize new techniques that are suitable for trace-level analysis of samples in forensic, pharmaceutical, and environmental applications. Capillary electrophoresis (CE) was selected for this task, as it requires lower amounts of samples, it simplifies sample preparation, and it has the flexibility to perform separations of neutral and charged molecules as well as enantiomers. ^ Overall, the study demonstrates the versatility of capillary electrophoresis methods in forensic, pharmaceutical, and environmental applications.^

Application of biomarkers and compound specific stable isotopes for the assessment of hydrology as a driver of organic matter dynamics in the Everglades ecosystem

The Everglades is a sub-tropical coastal wetland characterized among others by its hydrological features and deposits of peat. Formation and preservation of organic matter in soils and sediments in this wetland ecosystem is critical for its sustainability and hydrological processes are important divers in the origin, transport and fate of organic matter. With this in mind, organic matter dynamics in the greater Florida Everglades was studied though various organic geochemistry techniques, especially biomarkers, bulk and compound specific δ13C and δD isotope analysis. The main objectives were focused on how different hydrological regimes in this ecosystem control organic matter dynamics, such as the mobilization of particulate organic matter (POM) in freshwater marshes and estuaries, and how organic geochemistry techniques can be applied to reconstruct Everglades paleo-hydrology. For this purpose organic matter in typical vegetation, floc, surface soils, soil cores, and estuarine suspended particulates were characterized in samples selected along hydrological gradients in the Water Conservation Area 3, Shark River Slough and Taylor Slough. ^ This research focused on three general themes: (1) Assessment of the environmental dynamics and source-specific particulate organic carbon export in a mangrove-dominated estuary. (2) Assessment of the origin, transport and fate of organic matter in freshwater marsh. (3) Assessment of historical changes in hydrological conditions in the Everglades (paleo-hydrology) though biomarkes and compound specific isotope analyses. This study reports the first estimate of particulate organic carbon loss from mangrove ecosystems in the Everglades, provides evidence for particulate organic matter transport with regards to the formation of ridge and slough landscapes in the Everglades, and demonstrates the applicability of the combined biomarker and compound-specific stable isotope approach as a means to generate paleohydrological data in wetlands. The data suggests that: (1) Carbon loss from mangrove estuaries is roughly split 50/50 between dissolved and particulate carbon; (2) hydrological remobilization of particulate organic matter from slough to ridge environments may play an important role in the maintenance of the Everglades freshwater landscape; and (3) Historical changes in hydrology have resulted in significant vegetation shifts from historical slough type vegetation to present ridge type vegetation. ^

The Creation and Evaluation of Novel Canine Training Aids for Cocaine Using Molecularly Encapsulated Sol-Gel Polymers and an Investigation of Canine Field Accuracy

Biological detectors, such as canines, are valuable tools used for the rapid identification of illicit materials. However, recent increased scrutiny over the reliability, field accuracy, and the capabilities of each detection canine is currently being evaluated in the legal system. For example, the Supreme Court case, State of Florida v. Harris, discussed the need for continuous monitoring of canine abilities, thresholds, and search capabilities. As a result, the fallibility of canines for detection was brought to light, as well as a need for further research and understanding of canine detection. This study is two-fold, as it looks to not only create new training aids for canines that can be manipulated for dissipation control, but also investigates canine field accuracy to objects with similar odors to illicit materials. It was the goal of this research to improve upon current canine training aid mimics. Sol-gel polymer training aids, imprinted with the active odor of cocaine, were developed. This novel training aid improved upon the longevity of currently existing training aids, while also provided a way to manipulate the polymer network to alter the dissipation rate of the imprinted active odors. The manipulation of the polymer network could allow handlers to control the abundance of odors presented to their canines, familiarizing themselves to their canine’s capabilities and thresholds, thereby increasing the canines’ strength in court. The field accuracy of detection canines was recently called into question during the Supreme Court case, State of Florida v. Jardines, where it was argued that if cocaine’s active odor, methyl benzoate, was found to be produced by the popular landscaping flower, snapdragons, canines will false alert to said flowers. Therefore, snapdragon flowers were grown and tested both in the laboratory and in the field to determine the odors produced by snapdragon flowers; the persistence of these odors once flowers have been cut; and whether detection canines will alert to both growing and cut flowers during a blind search scenario. Results revealed that although methyl benzoate is produced by snapdragon flowers, certified narcotics detection canines can distinguish cocaine’s odor profile from that of snapdragon flowers and will not alert.

Development of a surface-enhanced raman spectroscopy method for the detection of benzodiazepines in urine

Benzodiazepines are among the most prescribed compounds for anti-anxiety and are present in many toxicological screens. These drugs are also prominent in the commission of drug facilitated sexual assaults due their effects on the central nervous system. Due to their potency, a low dose of these compounds is often administered to victims; therefore, the target detection limit for these compounds in biological samples is 10 ng/mL. Currently these compounds are predominantly analyzed using immunoassay techniques; however more specific screening methods are needed. ^ The goal of this dissertation was to develop a rapid, specific screening technique for benzodiazepines in urine samples utilizing surface-enhanced Raman spectroscopy (SERS), which has previously been shown be capable of to detect trace quantities of pharmaceutical compounds in aqueous solutions. Surface enhanced Raman spectroscopy has the advantage of overcoming the low sensitivity and fluorescence effects seen with conventional Raman spectroscopy. The spectra are obtained by applying an analyte onto a SERS-active metal substrate such as colloidal metal particles. SERS signals can be further increased with the addition of aggregate solutions. These agents cause the nanoparticles to amass and form hot-spots which increase the signal intensity. ^ In this work, the colloidal particles are spherical gold nanoparticles in aqueous solution with an average size of approximately 30 nm. The optimum aggregating agent for the detection of benzodiazepines was determined to be 16.7 mM MgCl2, providing the highest signal intensities at the lowest drug concentrations with limits of detection between 0.5 and 127 ng/mL. A supported liquid extraction technique was utilized as a rapid clean extraction for benzodiazepines from urine at a pH of 5.0, allowing for clean extraction with limits of detection between 6 and 640 ng/mL. It was shown that at this pH other drugs that are prevalent in urine samples can be removed providing the selective detection of the benzodiazepine of interest. ^ This technique has been shown to provide rapid (less than twenty minutes), sensitive, and specific detection of benzodiazepines at low concentrations in urine. It provides the forensic community with a sensitive and specific screening technique for the detection of benzodiazepines in drug facilitated assault cases.^

Analyse simultanée des hormones stéroïdiennes et leurs formes chimiques dans les matrices d'eau et d'urine par SPE-LC-MS/MS en ligne.

Dans les dernières années, les perturbateurs endocriniens ont été observés dans les rivières qui reçoivent des entrées importantes d’eaux usées. Parmi les perturbateurs endocriniens, les hormones stéroïdiennes naturelles et synthétiques sont des composés dont le potentiel d'imiter ou d'interférer avec les fonctions hormonales normales (développement, croissance et reproduction), est reconnu même au niveau ultra-traces (ng L-1). Bien que les hormones conjuguées soient moins actives que les hormones libres, elles peuvent être clivées et redevenir libres une fois exposées aux processus microbiens avant ou pendant le traitement des eaux usées. En raison de la nécessité d'identifier et de quantifier ces composés dans l'eau, une nouvelle méthode, entièrement automatisée, a été développée pour la détermination simultanée des deux formes de plusieurs hormones stéroïdiennes (conjuguées et libres) dans les matrices d'eau et dans l’urine des femmes. La méthode est basée sur l'extraction en phase solide couplée en ligne à la chromatographie liquide et la spectrométrie de masse en tandem (SPE-LC-MS/MS). Plusieurs paramètres ont été évalués dans le but d'optimiser l'efficacité de la méthode, tels que le type et le débit de la phase mobile, des différentes colonnes de SPE et de chromatographie, ainsi que différentes sources et modes d'ionisation des échantillons pour la MS. La méthode démontre une bonne linéarité (R2 > 0.993), ainsi qu'une précision avec un coefficient de variance inférieure à 10%. Les limites de quantification varient d’un minimum de 3 à 15 ng L-1 pour un volume d'injection entre 1 mL et 5 mL et le recouvrement des composés varie de 72 % à 117 %.

Development of Imaging Mass Spectrometry Analysis of Lipids in Biological and Clinically Relevant Applications

La spectrométrie de masse mesure la masse des ions selon leur rapport masse sur charge. Cette technique est employée dans plusieurs domaines et peut analyser des mélanges complexes. L’imagerie par spectrométrie de masse (Imaging Mass Spectrometry en anglais, IMS), une branche de la spectrométrie de masse, permet l’analyse des ions sur une surface, tout en conservant l’organisation spatiale des ions détectés. Jusqu’à présent, les échantillons les plus étudiés en IMS sont des sections tissulaires végétales ou animales. Parmi les molécules couramment analysées par l’IMS, les lipides ont suscité beaucoup d'intérêt. Les lipides sont impliqués dans les maladies et le fonctionnement normal des cellules; ils forment la membrane cellulaire et ont plusieurs rôles, comme celui de réguler des événements cellulaires. Considérant l’implication des lipides dans la biologie et la capacité du MALDI IMS à les analyser, nous avons développé des stratégies analytiques pour la manipulation des échantillons et l’analyse de larges ensembles de données lipidiques. La dégradation des lipides est très importante dans l’industrie alimentaire. De la même façon, les lipides des sections tissulaires risquent de se dégrader. Leurs produits de dégradation peuvent donc introduire des artefacts dans l’analyse IMS ainsi que la perte d’espèces lipidiques pouvant nuire à la précision des mesures d’abondance. Puisque les lipides oxydés sont aussi des médiateurs importants dans le développement de plusieurs maladies, leur réelle préservation devient donc critique. Dans les études multi-institutionnelles où les échantillons sont souvent transportés d’un emplacement à l’autre, des protocoles adaptés et validés, et des mesures de dégradation sont nécessaires. Nos principaux résultats sont les suivants : un accroissement en fonction du temps des phospholipides oxydés et des lysophospholipides dans des conditions ambiantes, une diminution de la présence des lipides ayant des acides gras insaturés et un effet inhibitoire sur ses phénomènes de la conservation des sections au froid sous N2. A température et atmosphère ambiantes, les phospholipides sont oxydés sur une échelle de temps typique d’une préparation IMS normale (~30 minutes). Les phospholipides sont aussi décomposés en lysophospholipides sur une échelle de temps de plusieurs jours. La validation d’une méthode de manipulation d’échantillon est d’autant plus importante lorsqu’il s’agit d’analyser un plus grand nombre d’échantillons. L’athérosclérose est une maladie cardiovasculaire induite par l’accumulation de matériel cellulaire sur la paroi artérielle. Puisque l’athérosclérose est un phénomène en trois dimension (3D), l'IMS 3D en série devient donc utile, d'une part, car elle a la capacité à localiser les molécules sur la longueur totale d’une plaque athéromateuse et, d'autre part, car elle peut identifier des mécanismes moléculaires du développement ou de la rupture des plaques. l'IMS 3D en série fait face à certains défis spécifiques, dont beaucoup se rapportent simplement à la reconstruction en 3D et à l’interprétation de la reconstruction moléculaire en temps réel. En tenant compte de ces objectifs et en utilisant l’IMS des lipides pour l’étude des plaques d’athérosclérose d’une carotide humaine et d’un modèle murin d’athérosclérose, nous avons élaboré des méthodes «open-source» pour la reconstruction des données de l’IMS en 3D. Notre méthodologie fournit un moyen d’obtenir des visualisations de haute qualité et démontre une stratégie pour l’interprétation rapide des données de l’IMS 3D par la segmentation multivariée. L’analyse d’aortes d’un modèle murin a été le point de départ pour le développement des méthodes car ce sont des échantillons mieux contrôlés. En corrélant les données acquises en mode d’ionisation positive et négative, l’IMS en 3D a permis de démontrer une accumulation des phospholipides dans les sinus aortiques. De plus, l’IMS par AgLDI a mis en évidence une localisation différentielle des acides gras libres, du cholestérol, des esters du cholestérol et des triglycérides. La segmentation multivariée des signaux lipidiques suite à l’analyse par IMS d’une carotide humaine démontre une histologie moléculaire corrélée avec le degré de sténose de l’artère. Ces recherches aident à mieux comprendre la complexité biologique de l’athérosclérose et peuvent possiblement prédire le développement de certains cas cliniques. La métastase au foie du cancer colorectal (Colorectal cancer liver metastasis en anglais, CRCLM) est la maladie métastatique du cancer colorectal primaire, un des cancers le plus fréquent au monde. L’évaluation et le pronostic des tumeurs CRCLM sont effectués avec l’histopathologie avec une marge d’erreur. Nous avons utilisé l’IMS des lipides pour identifier les compartiments histologiques du CRCLM et extraire leurs signatures lipidiques. En exploitant ces signatures moléculaires, nous avons pu déterminer un score histopathologique quantitatif et objectif et qui corrèle avec le pronostic. De plus, par la dissection des signatures lipidiques, nous avons identifié des espèces lipidiques individuelles qui sont discriminants des différentes histologies du CRCLM et qui peuvent potentiellement être utilisées comme des biomarqueurs pour la détermination de la réponse à la thérapie. Plus spécifiquement, nous avons trouvé une série de plasmalogènes et sphingolipides qui permettent de distinguer deux différents types de nécrose (infarct-like necrosis et usual necrosis en anglais, ILN et UN, respectivement). L’ILN est associé avec la réponse aux traitements chimiothérapiques, alors que l’UN est associé au fonctionnement normal de la tumeur.

Analyse simultanée des hormones stéroïdiennes et leurs formes chimiques dans les matrices d'eau et d'urine par SPE-LC-MS/MS en ligne

Dans les dernières années, les perturbateurs endocriniens ont été observés dans les rivières qui reçoivent des entrées importantes d’eaux usées. Parmi les perturbateurs endocriniens, les hormones stéroïdiennes naturelles et synthétiques sont des composés dont le potentiel d'imiter ou d'interférer avec les fonctions hormonales normales (développement, croissance et reproduction), est reconnu même au niveau ultra-traces (ng L-1). Bien que les hormones conjuguées soient moins actives que les hormones libres, elles peuvent être clivées et redevenir libres une fois exposées aux processus microbiens avant ou pendant le traitement des eaux usées. En raison de la nécessité d'identifier et de quantifier ces composés dans l'eau, une nouvelle méthode, entièrement automatisée, a été développée pour la détermination simultanée des deux formes de plusieurs hormones stéroïdiennes (conjuguées et libres) dans les matrices d'eau et dans l’urine des femmes. La méthode est basée sur l'extraction en phase solide couplée en ligne à la chromatographie liquide et la spectrométrie de masse en tandem (SPE-LC-MS/MS). Plusieurs paramètres ont été évalués dans le but d'optimiser l'efficacité de la méthode, tels que le type et le débit de la phase mobile, des différentes colonnes de SPE et de chromatographie, ainsi que différentes sources et modes d'ionisation des échantillons pour la MS. La méthode démontre une bonne linéarité (R2 > 0.993), ainsi qu'une précision avec un coefficient de variance inférieure à 10%. Les limites de quantification varient d’un minimum de 3 à 15 ng L-1 pour un volume d'injection entre 1 mL et 5 mL et le recouvrement des composés varie de 72 % à 117 %.