The phosphorylation-dependent PPIase (peptidyl prolyl isomerase), Pin1 (Protein interacting with NIMA#1), has been found to regulate cell cycle through a simple conformational change, the cis-trans isomerization of phospho-Ser/Thr-Pro amide bonds. A variety of key cell cycle regulatory phosphoproteins, including Cdc25 phosphatase,Cdc27, p53 oncogene, c-Myc oncogene, Wee1 kinase, Myt1 kinase, and NIMA kinas, have been confirmed as substrates of Pin1. Pin1 was also observed to be overexpressed in a variety of cancer cell lines, and the inhibitors of Pin1 showed antiproliferative activities towards these cancer cells. These results implied that Pin1 might serve as a potential anti-cancer drug target. Besides, Pin1 has an important neuroprotective function and represents a potential new therapeutic agent for Alzheimer's disease. In order to understand the interaction between Pin1 and Cdc25c and the role of Pin1 in the mechanism for the regulation of mitosis, two amide isosteres, Ser-Ψ[(Z)CH=C]-Pro-OH and Ser-Ψ[(E)CH=C]-Pro-OH were incorporated into two peptidomimetics derived from human Cdc25c. Phosphorylation of these two peptidomimetics by the incubation with Cdc2 was studied using LC-MS/MS technique. It was found that Cdc2 kinase was conformationally specific to its Cdc25c substrate. Only the trans conformer of Cdc25c at its Ser168-Pro position can be recognized and phosphorylated by Cdc2 kinase, thereby creating the binding site for Pin1. In an effort to improve the cell permeability of the charged inhibitors of Pin1, bisPOM (pivaloyloxymethyl) prodrug moiety was introduced to mask the phosphate group of Fmoc-pSer-Ψ[(Z)CH=C]-Pro-(2)-N-(3)-ethylaminoindole, which is one inhibitor of Pin1. Fmoc-pSer-Ψ[(Z)CH=C]-Pro-(2)-N-(3)-ethylaminoindole and its bisPOM prodrug were synthesized efficiently starting with Boc-Ser-Ψ[(Z)CH=C]-Pro-OH in 24% and 12% yields respectively. The charged inhibitor showed a moderate inhibition towards Pin1 (IC50 = 28.3 μM). Its antiproliferative activity towards A2780 ovarian cancer cells (IC50 = 46.2 μM) was significantly improved by its bisPOM prodrug (IC50 = 26.9 μM), which is comparable to the IC50 of the charged inhibitor towards Pin1 enzymatic activity. These results not only established the bisPOM strategy as an efficient prodrug choice for Pin1 inhibitors, but also added additional evidence for Pin1 as a potential anticancer drug target. / Ph. D.

La consommation des composés pharmaceutiques est en constante croissance à travers le monde et leur utilisation peut entraîner une accumulation dans l’environnement d’où leur désignation à titre de contaminants émergents. De nombreux groupes de recherche se penchent donc sur les questions de leurs effets sur des organismes vivants, sur la santé de certains écosystèmes ou encore sur l’efficacité de différents types de traitement des eaux usées. Or, peu de données sont disponibles quant aux concentrations présentes dans différentes matrices telles que les eaux usées ou l’eau de surface dans lesquelles elles sont déversées ou encore l’impact que peuvent avoir les hôpitaux sur l’occurrence des composés pharmaceutiques. La recherche dans ce domaine peut s’avérer un outil clé dans l’établissement de normes et de limites pour différents contaminants puisque celles-ci sont encore marginales dans la plupart des pays du monde, surtout au niveau des médicaments. Dans le cadre de cette thèse, différentes méthodes analytiques ont été développées pour faire l’analyse de classes ciblées de médicaments par chromatographie liquide couplée à la spectrométrie de masse afin de quantifier ceux-ci dans diverses matrices environnementales. Un des objectifs du présent ouvrage est donc de détailler les travaux de développement et de validation de méthodes analytiques robustes. Une première méthode d’extraction en phase solide en ligne avec la chromatographie liquide ultra-haute performance couplée à la spectrométrie de masse en tandem (SPE en ligne UPLC-MS/MS) a été développée afin de faire l’analyse de huit différentes classes de composés pharmaceutiques susceptibles d’être retrouvés dans les eaux usées. Afin de déterminer la distribution et l’adsorption des composés ciblés sur la phase solide de différentes matrices, une méthode d’extraction et de purification a également été optimisée de telle sorte que les extraits de matière particulaire et de sédiments soient compatibles avec cette méthode analytique. Ces méthodes ont été appliquées sur de nombreuses eaux usées d’hôpitaux et d’usine de traitement des eaux au Québec (Canada). Les produits de chimiothérapies présentent un défi analytique supplémentaire puisqu’ils sont polaires comparativement à la plupart des autres classes de composés pharmaceutiques. Il y a donc peu d’études sur la présence et le comportement de ces contaminants bien que ceux-ci présentent un risque écotoxicologique potentiellement important. Un deuxième objectif de cette thèse a donc été de comparer différentes alternatives pour la préconcentration et la séparation chromatographique, notamment la chromatographie à interaction hydrophile (HILIC). Celle-ci a mené à la validation de deux méthodes analytiques dont la sensibilité est de l’ordre des ng/L pour ces composés dans des eaux usées brutes. Ces méthodes de SPE en ligne UPLC-MS/MS ont été appliquées à de nombreux échantillons d’effluents d’hôpitaux ainsi qu’à des usines de traitement des eaux usées au Québec (Canada). D’autre part, la méthode SPE en ligne UPLC-MS/MS pour l’analyse des multiples classes de composés pharmaceutiques a été validée pour l’eau de surface enfin d’atteindre un dernier objectif de cet ouvrage qui consiste à évaluer différentes sources de contamination de cette matrice à proximité de zones densément peuplées. Durant de larges campagnes d’échantillonnage sur le fleuve Saint-Laurent, échelonnées sur une période de cinq ans et ayant couvert une zone géographique de près de 700 km entre le Lac Ontario et l’estuaire, plus de 400 échantillons ont été prélevés et analysés. Parmi ceux-ci figurent des échantillons provenant de 56 rivières tributaires au fleuve prélevés afin de déterminer l’impact de celles-ci en termes de pollution en composés pharmaceutiques et de les comparer avec des points de rejet d’eau usée de villes telles que Montréal ou Québec. Enfin, ces mêmes méthodes ont été appliquées lors de projets en collaboration avec des chercheurs se penchant sur les effets écotoxicologiques des composés pharmaceutiques sur des organismes aquatiques dans des matrices d’eau de surface ainsi que pour de développement de technologies alternatives pour le traitement des eaux usées. / A constant increase in pharmaceutical compounds worldwide can accentuate problems related to these pollutants of emerging concern in environmental matrices. Therefore, many researchers are studying their effects on living organisms, the health of impacted ecosystems, and the efficiency of different types of wastewater treatment systems. However, there is a lack of available data on the occurrence and concentrations of pharmaceuticals in different matrices, such as wastewaters or receiving surface waters, and the relative importance of hospital effluents on the load of these contaminants. Research in this field can be a key tool on different drugs since only a few countries have implemented norms and guidance, especially for medications. As part of this thesis, different analytical methods have been developed for the analysis of selected drugs, and liquid chromatography coupled with mass spectrometry was used to quantify multi-class pharmaceuticals occurring in various environmental matrices. One objective of this manuscript is to develop and validate robust analytical methods. A first extraction method on-line with liquid chromatography coupled to tandem mass spectrometry (on-line SPE UPLC-MS/MS) was developed to analyze eight different pharmaceutical classes susceptible to be found in wastewaters. With the aim of evaluating the partitioning and adsorption of target compounds onto a solid phase, an extraction and purification method was optimized to ensure that particulate matter and sediment extracts were compatible with the analytical method. These optimized methods were then applied to several hospital effluents and wastewater treatment plants samples in the province of Quebec (Canada). Chemotherapy agents present an additional analytical challenge since these drugs are relatively polar compared with most of other pharmaceutical classes. This explains the low number of publications relating to their occurrence and behaviour even though they present a high ecotoxicological hazard. A second objective of this thesis was to compare different potential alternatives for enrichment and chromatographic separation of these pollutants, notably the use of hydrophilic interaction chromatography (HILIC). This work led to the validation of two analytical methods for which the sensitivity reached ng/L levels for these compounds in raw sewage. These methods were successfully applied to samples from numerous hospital effluents and wastewater treatment plants from the province of Quebec (Canada). Also, a multi-class pharmaceutical method was validated for surface water in order to achieve the last objective of this thesis which consisted in the determination of the different contamination sources of this matrix near densely populated areas. During major sampling campaigns on the Saint-Lawrence River, spanning over five years and covering a geographical area of approx. 700 km between Lake Ontario and the estuary, a total of more than 400 samples were collected and analyzed. Among those, 56 different rivers tributary to the Saint-Lawrence were sampled to establish the impact they may have on pharmaceutical pollution. Data were compared with wastewater rejection points from cities, such as Montreal and Quebec. Finally, these methods were applied in the context of collaboration projects with researchers focusing on measuring ecotoxicological effects of pharmaceutical compounds on living organisms in surface water matrices and on the development of alternative wastewater treatment technologies.

Venezuelan equine encephalitis virus (VEEV) is an alphavirus that causes disease in humans and equines eliciting both an agricultural and public health threat. In humans, the disease typically presents as a febrile illness with common signs of fever and malaise. Four to fourteen percent of Venezuelan equine encephalitis (VEE) cases are associated with severe neurological complications due to encephalitis caused by VEEV's propensity to infect the brain. Public health concerns are exacerbated by VEEV's aerosolization capabilities, low infectious dose and affordability to mass produce. These qualities drove interest in the pathogen as a bioweapon by the US and the former Soviet Union during the cold war. As a precautionary response to VEEV's notoriety as a biothreat, the National Institute of Allergies and Infectious Diseases has classified VEEV as a category B priority pathogen, and the Human Health Services and United States Department of Agriculture list live virulent strains of VEEV as a select agent and require the pathogen to be manipulated in highly regulated biosafety level 3 (BSL3) facilities. There are currently no FDA approved vaccines or antivirals to target VEEV or other closely related alphaviruses associated with clinical disease in humans. The research performed in this dissertation aimed to elucidate new antiviral targets and treatments to help bridge gaps in current understanding of alphaviruses. The current market lacks available antibodies for E1 specific isolation. In response, a recombinant VEEV TC-83 was produced with a V5 tag at the C-terminal of the E1 sequence to enable VEEV E1 detection. Sequencing was used to verify V5 insertion in the plasmid and immunoprecipitation was used to verify V5 insertion within the E1 glycoprotein. Replication kinetics experiments verified the virus replicated similarly to the parental VEEV TC-83 strain, while passaging experiments verified the tag was highly stable for up to 10 passages. This research produced a cost-effective and highly efficient means to probe and isolate the E1 glycoprotein without modifying the viability of the virus. Knowledge of host protein interactions with VEEV E1 glycoprotein has been limited, with most E1 research focusing on its fusion capabilities. Utilizing 293-T cells infected with E1-V5 TC-83, co-immunoprecipitation was performed to isolate E1 and associated interactors. A total of 486 host and 5 viral protein interactors of E1 were discovered after normalization to the negative control. The top peptide spectrum matches (PSMs) revealed a number of chaperone proteins and ubiquitin proteins as top interactors of VEEV E1. These results effectively revealed a number of previously unknown alphavirus interactions that can be targeted by antivirals and explored further for implications in viral replication. LC-MS/MS results showed that protein disulfide isomerase family A member 6 (PDIA6) interacted with E1. High PSMs, presence in all 3 replicates, similar cellular localization to E1 and known associations between other viruses and protein disulfide isomerase (PDI) family members made this protein an optimum target for further analysis. Co-immunoprecipitation and co-localization experiments were used to validate the LC-MS/MS results. Involvement of PDIs in VEEV replication were explored utilizing two known PDI inhibitors, LOC14 and Nitazoxanide. LOC14, a non-FDA approved broad-spectrum PDI inhibitor, showed broad-spectrum alphavirus antiviral potential, decreasing titers of VEEV TC-83, VEEV Trinidad Donkey strain, eastern equine encephalitis virus (EEEV), chikungunya virus (CHIKV) and Sindbis (SINV) virus in a dose dependent manner. Nitazoxanide, an FDA approved drug known to inhibit PDIA3, was shown to have minimal toxicity and effectively reduced VEEV TC-83 and EEEV titers at concentrations with 100% cell viability. Time of addition assays, E1 expression time course studies, and early event assays showed PDI inhibition with these drugs effects early viral production events. RNA quantification, confocal microscopy and biotin switch assay experiments show that the drugs also prevented proper folding of the E1 glycoprotein and decreased expression of E1 on the peripheral membrane. With no current treatments for alphaviruses, these data provide an effective broad-spectrum target that affects viral replication at multiple stages in-vitro. Nitazoxanide also presents as a promising, non-toxic drug that could be repurposed to combat a number of clinically relevant alphaviruses. Valosin containing protein (VCP) was also shown to interact with the E1 glycoprotein. Exploration of VCP's interaction with alphavirus E1 has never been explored, yet it was previously shown to be involved in alphavirus replication. Co-localization and co-immunoprecipitation experiments were performed validating the interaction between VCP and E1. siRNA knockdown of VCP in 293-T cells and U87-MG cells showed a significant reduction in VEEV TC-83 titers. The allosteric VCP inhibitor, NMS-873, also reduced VEEV TC-83 titers, but was shown to be less effective against CHIKV, SINV and EEEV, suggesting the NMS-873 mechanism is more selective for VEEV. Mechanism experiments showed that reduction of VCP with NMS-873 inhibits early events of VEEV replication. These results elucidate VCP's association with E1 and show that VCP can be targeted to decrease VEEV viral replication. / Doctor of Philosophy / Venezuelan equine encephalitis virus (VEEV) causes disease in humans, as well as horses, donkeys and other closely related animals. In humans, the virus causes a flu-like disease and sometimes swelling of the brain. This can be associated with symptoms such as light sensitivity, confusion and sometimes coma. Prior to the Cold War, VEEV was researched by the US and previous Soviet Union's militaries in hopes to deploy the virus as a bioweapon. Current treaties prevent active production of such weapons, yet allows for defensive research to continue in preparation for a worst-case scenario. Currently no FDA approved medications or vaccines exist to combat the virus further exacerbating concerns. In order to protect laboratorians and prevent unintentional or intentional introduction of the virus into the community, the virus is only manipulated in highly secure facilities with barriers that separate the virus from personnel and the outside environment. A component of the virus called E1, allows for the virus to be released from a structure, called an endosome, that transports the virus into the cell. Currently, E1 is mostly known for this function, yet our research found that E1 interacts with 486 protein components of the host cell, suggesting a more elaborate role of E1 than previously understood. This list of interactors provides numerous new targets for potential medications to combat VEEV and other closely related viruses. Discovered E1 interactors, protein disulfide isomerase family A member 6 (PDIA6) and valosin containing protein (VCP), were validated through extensive experimentation and their function in viral replication was further explored. Protein disulfide isomerases (PDI), such as PDIA6, play an important role in folding proteins, which are cellular components made of organic building blocks called amino acids. PDIs do so by creating organic pillars, called disulfide bonds, between two cysteine amino acid residues. These disulfide bonds contribute to the 3D shape of the proteins they fold which are essential for the protein's function. E1 of VEEV has a total of eight disulfide bonds within its structure, highlighting that disulfide bonds are likely essential for the protein's structure, and therefore, function. We verified that E1 could not properly fold without PDI function by using two compounds that prevented PDI from forming or breaking disulfide bonds, specifically LOC14 and FDA approved drug nitazoxanide. Cells treated with one of either compound before and after infection with VEEV, were found to produce E1 protein with significantly less disulfide bonds therefore producing less viable virus. Further experiments also showed that the compounds also affected early stages in the virus production cycle. These two mechanisms explain the significant reduction in production of VEEV and related viruses when PDI is inhibited. These results provide a new VEEV drug target, PDIs, as well as two compounds that can potentially be used to combat VEEV and other related viruses that have no current treatment options. Another host interactor, VCP, functions throughout the cell and is known for unfolding of numerous substrates, including proteins. It is involved in numerous cellular functions thus making this interactor a promising target for drug treatment. Cells with reduced VCP function were shown to produce less progeny VEEV. Cells treated with NMS-873, a compound that reduces VCP function was also shown to reduce VEEV production. NMS-863 inhibition of VCP was shown to effect early events in VEEV replication. These results further emphasize the E1 interactors discovered are invaluable novel targets for VEEV drug treatment.

No / Nanoscale reversed-phase liquid chromatography (LC) combined with electrospray ionization-tandem mass spectrometry (ESI-MS/MS) has been used as a method for the direct identification of multiple cytochrome P450 (P450) isoforms found in male and female rat liver. In this targeted proteomic approach, rat liver microsomes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by in-gel tryptic digestion of the proteins present in the 48- to 62-kDa bands. The resultant peptides were extracted and analyzed by LC-ESI-MS/MS. P450 identifications were made by searching the MS/MS data against a rat protein database containing 21,576 entries including 47 P450s using Sequest software (Thermo Electron, Hemel Hempstead, UK). Twenty-four P450 isoforms from the subfamilies 1A, 2A, 2B, 2C, 2D, 2E, 3A, 4A, 4F, CYP17, and CYP19 were positively identified in rat liver.

No / Surfactants are commonly used as cleansing agents and yet there are concerns they may also have a role in skin irritation. Presently, the lack of suitable methods for quantitative and qualitative analysis of surfactant deposition on skin has hindered the in-depth investigation of such effects. Here, we report the application of reverse phase liquid chromatography electrospray ionisation mass spectrometry (LC/ESI-MS/MS) assays for two surfactants commonly used in consumer products, namely sodium lauryl ether sulphate (SLES) and laurylamidopropyl betaine (LAPB), to a baseline study aiming to assess deposition levels on human skin. The linearity of the assays was established at 3-20 ng, with coefficient of variation below 5%. Detection limits were 100 pg for LAPB and 1 ng for SLES; quantitation limits were 500 pg for LAPB and 2.5 ng for SLES. The baseline study was conducted using a panel of 40 healthy volunteers. Skin extract samples were taken in triplicate from forearms, using ethanol. SLES was detected on most volunteers, with 75% of them having SLES deposits in the range of 100-600 ng/cm2. LAPB was detected on the skin of all volunteers with 85% of them having deposit levels within the concentration range of 1-100 ng/cm2. These results demonstrate the extent to which commonly used surfactants remain on the skin during the day. The analytical methods reported here can be applied to the investigation of surfactants in relation to general skin condition and the development and optimisation of new consumer wash products. / EPSRC

No / Surfactants are commonly used as cleansing agents and yet there are concerns that they may also have a role in skin irritation. The lack of suitable methods for the quantitative and qualitative analysis of surfactant deposition on skin has hindered the in-depth investigation of such effects. Here, we report the application of reversed-phase liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI-MS/MS) assays for two surfactants commonly used in consumer products, namely sodium lauryl ether sulfate (SLES) and laurylamidopropyl betaine (LAPB), to a baseline study aiming to assess deposition levels on human skin. The linearity of the assays was established at 3-20 ng, with coefficient of variation below 5%. The detection limits were 100 pg for LAPB and 1 ng for SLES; quantitation limits were 500 pg for LAPB and 2.5 ng for SLES. The baseline study was conducted using a panel of 40 healthy volunteers. Skin extract samples were taken in triplicate from forearms, using ethanol. SLES was detected on most volunteers, with 75% of them having SLES deposits in the range of 100-600 ng/cm(2). LAPB was detected on the skin of all volunteers with 85% of them having deposit levels within the concentration range of 1-100 ng/cm(2). These results demonstrate the extent to which commonly used surfactants remain on the skin during the day. The analytical methods reported here can be applied to the investigation of surfactants in relation to general skin condition and to the development and optimisation of new consumer wash products. / EPSRC-DTA award / School Life Sciences

No / Resin found within Canaanite amphorae from the Late Bronze Age shipwreck discovered off the coast of southwest Turkey at Uluburun has previously been identified as Pistacia sp. Although evidence from Egypt suggests that this resin was in high demand and typically transported in such amphorae, it has also been proposed that the amphorae contained wine, with the resin used to seal the interior surfaces and to flavour and/or preserve the wine. To attempt to resolve this question, we have analysed five samples of pistacia resin found in amphorae from the shipwreck using a range of analytical techniques which have used in the past for the analysis of wine residues: spot tests, FT-IR, and HPLC-MS-MS. As well as the archaeological samples, we have analysed modern samples of pistacia resin, leaves and fruit to determine the effectiveness of each technique and to exclude the possibility of false positive results. In addition to the analyses for wine we also detail analysis (GC-MS) of the terpenoids for the purpose of further molecular characterisation of the resin. Bulk stable isotope analysis was used in comparison with similar resins to attempt to identify the geographical origin of the resin.

Il est reconnu que le benzène, le toluène, l’éthylbenzène et les isomères du xylène, composés organiques volatils (COVs) communément désignés BTEX, produisent des effets nocifs sur la santé humaine et sur les végétaux dépendamment de la durée et des niveaux d’exposition. Le benzène en particulier est classé cancérogène et une exposition à des concentrations supérieures à 64 g/m3 de benzène peut être fatale en 5–10 minutes. Par conséquent, la mesure en temps réel des BTEX dans l’air ambiant est essentielle pour détecter rapidement un danger associé à leur émission dans l’air et pour estimer les risques potentiels pour les êtres vivants et pour l’environnement. Dans cette thèse, une méthode d’analyse en temps réel des BTEX dans l’air ambiant a été développée et validée. La méthode est basée sur la technique d’échantillonnage direct de l’air couplée avec la spectrométrie de masse en tandem utilisant une source d’ionisation chimique à pression atmosphérique (APCI-MS/MS directe). La validation analytique a démontré la sensibilité (limite de détection LDM 1–2 μg/m3), la précision (coefficient de variation CV < 10%), l’exactitude (exactitude > 95%) et la sélectivité de la méthode. Des échantillons d’air ambiant provenant d’un site d’enfouissement de déchets industriels et de divers garages d’entretien automobile ont été analysés par la méthode développée. La comparaison des résultats avec ceux obtenus par la technique de chromatographie gazeuse on-line couplée avec un détecteur à ionisation de flamme (GC-FID) a donné des résultats similaires. La capacité de la méthode pour l’évaluation rapide des risques potentiels associés à une exposition aux BTEX a été prouvée à travers une étude de terrain avec analyse de risque pour la santé des travailleurs dans trois garages d’entretien automobile et par des expériences sous atmosphères simulées. Les concentrations mesurées dans l’air ambiant des garages étaient de 8,9–25 µg/m3 pour le benzène, 119–1156 µg/m3 pour le toluène, 9–70 µg/m3 pour l’éthylbenzène et 45–347 µg/m3 pour les xylènes. Une dose quotidienne environnementale totale entre 1,46 10-3 et 2,52 10-3 mg/kg/jour a été déterminée pour le benzène. Le risque de cancer lié à l’exposition environnementale totale au benzène estimé pour les travailleurs étudiés se situait entre 1,1 10-5 et 1,8 10-5. Une nouvelle méthode APCI-MS/MS a été également développée et validée pour l’analyse directe de l’octaméthylcyclotétrasiloxane (D4) et le décaméthylcyclopentasiloxane (D5) dans l’air et les biogaz. Le D4 et le D5 sont des siloxanes cycliques volatils largement utilisés comme solvants dans les processus industriels et les produits de consommation à la place des COVs précurseurs d’ozone troposphérique tels que les BTEX. Leur présence ubiquitaire dans les échantillons d’air ambiant, due à l’utilisation massive, suscite un besoin d’études de toxicité. De telles études requièrent des analyses qualitatives et quantitatives de traces de ces composés. Par ailleurs, la présence de traces de ces substances dans un biogaz entrave son utilisation comme source d’énergie renouvelable en causant des dommages coûteux à l’équipement. L’analyse des siloxanes dans un biogaz s’avère donc essentielle pour déterminer si le biogaz nécessite une purification avant son utilisation pour la production d’énergie. La méthode développée dans cette étude possède une bonne sensibilité (LDM 4–6 μg/m3), une bonne précision (CV < 10%), une bonne exactitude (> 93%) et une grande sélectivité. Il a été également démontré qu’en utilisant cette méthode avec l’hexaméthyl-d18-disiloxane comme étalon interne, la détection et la quantification du D4 et du D5 dans des échantillons réels de biogaz peuvent être accomplies avec une meilleure sensibilité (LDM ~ 2 μg/m3), une grande précision (CV < 5%) et une grande exactitude (> 97%). Une variété d’échantillons de biogaz prélevés au site d’enfouissement sanitaire du Complexe Environnemental de Saint-Michel à Montréal a été analysée avec succès par cette nouvelle méthode. Les concentrations mesurées étaient de 131–1275 µg/m3 pour le D4 et 250–6226 µg/m3 pour le D5. Ces résultats représentent les premières données rapportées dans la littérature sur la concentration des siloxanes D4 et D5 dans les biogaz d’enfouissement en fonction de l’âge des déchets. / It is known that benzene, toluene, ethylbenzene and xylene isomers, volatile organic compounds (VOCs) commonly called BTEX, have toxic health effects on humans and plants depending on duration and levels of exposure. Benzene in particular is classified carcinogenic, and exposure to benzene at concentrations above 64 g/m3 can be fatal within 5–10 minutes. Therefore, real-time monitoring of BTEX in ambient air is essential for the early warning detection associated with their release and in estimating the potential exposure risks to living beings and the environment. In this thesis, a real-time analysis method for BTEX in ambient air was developed and validated. The method is based on the direct-air sampling technique coupled with tandem mass spectrometry using atmospheric pressure chemical ionization (direct APCI-MS/MS). Validation of the method has shown that it is sensitive (limit of detection LOD 1–2 μg/m3), precise (relative standard deviation RSD < 10%), accurate (accuracy > 95%) and selective. Ambient air samples from an industrial waste landfill site and various automobile repair shops were analyzed by the developed method. Comparison of results with those obtained by online gas chromatography coupled with a flame ionization detector (GC-FID) technique exhibited similar results. The capacity of the method for the fast evaluation of potential risks associated with an exposure to BTEX has been demonstrated through a field study with health risk assessment for workers at three automobile repair shops and through experiments under simulated atmospheres. Concentrations measured in the ambient air of the garages were in the ranges of 8.9–25 µg/m3 for benzene, 119–1156 µg/m3 for toluene, 9–70 µg/m3 for ethylbenzene, and 45–347 µg/m3 for xylenes. A total environmental daily dose of 1.46 10-3–2.52 10-3 mg/kg/day was determined for benzene. The estimated cancer risk due to the total environmental exposure to benzene was between 1.1 10-5 and 1.8 10-5 for the workers studied. A novel APCI-MS/MS method was also developed and validated for the direct analysis of octamethylcyclotetrasiloxane (D4) and decamethylcyclopentasiloxane (D5) in air and biogases. D4 and D5 are cyclic volatile siloxanes widely used in industrial processes and consumer products as replacement solvents for the tropospheric ozone forming VOCs, such as BTEX. Their ubiquitous presence in ambient air samples, due to the growing consumption, raises the need for toxicity studies which require qualitative and quantitative trace analysis of these compounds. Furthermore, the presence of trace amounts of these substances in a biogas hampers its use as a source of renewable energy by causing expensive damages to the equipment. Thus, siloxane analysis of the biogas is essential in determining if purification is needed before the use for energy production. The method developed in this study for these aims has good sensitivity (LOD 4–6 μg/m3), good precision (RSD < 10%), good accuracy (> 93%) and high selectivity. It was also shown that by using this method with hexamethyl-d18-disiloxane as an internal standard, detection and quantification of D4 and D5 in real biogas samples can be done with a better sensitivity (LOD ~ 2 μg/m3), high precision (RSD < 5%), and high accuracy (> 97%). Various biogas samples collected from the landfill site of the Complexe Environnemental de Saint-Michel in Montreal have been successfully analyzed by this new method. Concentrations measured were in the ranges of 131–1275 µg/m3 for D4 and 250–6226 µg/m3 for D5. These results represent the first primary-literature-reported data on siloxanes D4 and D5 contents of landfill-derived biogases as a function of the refuse age.

<p>Insekticidi novije generacije, neonikotinoidi, odlikuju se specifičnim načinom &nbsp;delovanja na&nbsp;nervni sistem insekata. Radi dobijanja &scaron;to brže i kvalitetnije informacije o izloženosti životne sredine&nbsp;ovim insekticidima i količinama njihovih ostataka u hrani potrebno je raspolagati odgovarajućim&nbsp;instrumentalnim metodama za njihovo određivanje. Razvijene su i optimizovane analitičke metode&nbsp;zasnovane na tečnoj hromatografiji za određivanje sedam odabranih neonikotinoida (dinotefurana,&nbsp;nitenpirama, tiametoksama, klotianidina, imidakloprida, acetamiprida &nbsp;i tiakloprida) u medu i likeru od&nbsp;meda. Ispitivana je mogućnost određivanja klotianidina pomoću tečne hromatografije visoke efikasnosti&nbsp;sa detektrorom od niza dioda (HPLC-DAD) primenom kombinacije tečno-tečne i ekstrakcije na čvrstoj&nbsp;fazi iz uzoraka meda. Na osnovu preliminarnih rezultata može se zaključiti da kori&scaron;ćenje &nbsp;faznih-čvrsto&nbsp;kolona u kombinaciji sa tečno-tečnom ekstrakcijom dihlormetanom rezultira prihvatljivim prinosom&nbsp;klotianidina u uzorcima meda pri koncentraciji od oko 0,5 &micro;g g^-1&nbsp;klotianidina. Radi dobijanja većih&nbsp;prinosa odabrana je disperzna tečno-tečna mikroekstrakcija (DLLME) kao tehnika pripreme uzoraka&nbsp;meda. Testirana je upotreba acetonitrila kao disperznog sredstva. Pored hloroforma, kori&scaron;ćen je i&nbsp;dihlormetan kao drugo ekstrakciono sredstvo, kako bi se &nbsp;uporedila efikasnost ekstrakcije. Zabeleženi su&nbsp;prinosi klotianidina od 69,7 i 68,3% &nbsp;u zavisnosti da li je kori&scaron;ćen hloroform, odnosno DHM kao rastvor&nbsp;za ekstrakciju. Može se zaključiti da je prinos ekstrakcije bio povoljniji pri odnosu 0,5 mL ACN i 2,0&nbsp;mL DHM. Prinosi su se kretali od 68,4% do 92,1%, &scaron;to je ukazalo da su parametri DLLME ekstrakcije optimalni. Kako bi se detaljnije ispitali ključni parametri DLLME tehnike, kori&scaron;ćena je metodologija povr&scaron;ine odziva (RSM), kao i detekcija na osetljivijem kuplovanom masenom detektoru (MS/MS). Optimizovani su HPLC-MS/MS &nbsp;parametri kako bi se obezbedilo zadovoljavajuće hromatografsko &nbsp;razdvajanje i niske granice detekcije (GD, 0,5&ndash;1,0 &mu;g kg^-1) i određivanja (GO, 1,0&ndash;2,5 &mu;g kg^-1) ispitivanih neonikotinoida u medu. Upotrebom centralno kompozitnog dizajna konstruisani su kvadratni modeli ispitivanih faktora: zapremine ekstrakcionog (DHM, 1,0&ndash;3,0 mL) i disperznog (ACN, 0,0&ndash;1,0 mL) sredstva, izračunati statistički parametri i optimizovan proces DLLME upotrebom <em>Derringer</em>-ove funkcije poželjnih odgovora. Upotrebom MMC i SC krivih u opsegu GO&ndash;100,0 &mu;g kg^-1&nbsp;ispitan je uticaj matriksa pri čemu zaključeno je da je najveći uticaj matriksa bio na odziv analitičkog signala nitenpirama, dinotefurana i klotianidina. Ispitani su prinosi odabranih neonikotinoida (R, 74,3&ndash;113,9%), kao i preciznost metode u uslovima ponovljivosti (RSD, 2,74&ndash; 11,8%) i intermedijerne reproduktivnosti (RSD, 6,64&ndash;16,2%). Brza (retenciona vremena 1,5&ndash;9,9 min) i osetljiva metoda, koja tro&scaron;i malu količinu rastvarača, primenjena je za ispitivanje 15 realnih uzoraka meda različitog cvetnog porekla. Rezultati su pokazali da ispitivani med nije sadržao ostatke ispitivanih neonikotinoida u koncentracijama iznad GD. Dalje istraživanje je bilo usmereno ka&nbsp; razvijanju i optimizaciji HPLC-DAD analitičke metode upotrebom DLLME i QuEChERS tehnika za &nbsp;pripremu uzoraka za određivanje 7 neonikotinoida u uzorcima meda. U ovom delu istraživanja optimizovani su i hromatografski parametri, upotrebom RSM sa Box-Behnken-ovim dizajnom i Derringer-ovom funkcijom poželjnih odgovora. Od &nbsp;ispitivanih neonikotinoida dinotefuran i imidakloprid su bili u najvećoj meri izloženi uticaju matriksa, bez obzira na proceduru pripreme uzoraka. Može se istaći da je uticaj matriksa na analitički signal dinotefurana bio izraženiji u slučaju MS/MS, apostrofirajući manju robusnost ove metode određivanja. Prinosi neonikotinoida su &nbsp;bili (R, 73,1&ndash;118,3%), preciznost u uslovima ponovljivosti (RSD, 3,28&ndash;10,40%) i intermedijerne reproduktivnosti (RSD, 6,45&ndash;17,70%), a granice detekcije (GD, 1,5&ndash;2,5 &micro;g kg^-1) i određivanja (GO, 5,0&ndash;10,0 &micro;g kg^-1). Metoda je primenjena za ispitivanje 7 neonikotinoida u 104 uzorkameda različitog cvetnog porekla sa &nbsp;teritorije Autonomne Pokrajine Vojvodine. Detektovano je prisustvo tiakloprida, imidakloprida i tiametoksama u količinama koje su bile ispod MDK RS i EU. Analizirani su uzorci likera od meda - medice. Upoređivane su dve tehnike pripreme uzoraka, DLLME i QuEChERS i primenjeni optimizovani hromatografski &nbsp;uslovi i MS/MS parametri. U slučaju nitenpirama, dinotefurana i tiametoksama uticaj matriksa bio je najizraženiji. Metoda je validovana određivanjem prinosa neonikotinoida (R, 69,2&ndash;113,4%), preciznosti u uslovima ponovljivosti (RSD, 3,21&ndash;12,81%) i intermedijerne reproduktivnosti (RSD, 9,11&ndash;16,63%), kao i granice detekcije (GD, 0,5&ndash;2,5 &micro;g kg^-1) i određivanja (GO, 1,0&ndash;10,0 &micro;g kg^-1). Analizom 10 komercijalno dostupnih likera od meda otkriveno je&nbsp;prisustvo klotianidina i tiakloprida, evčzokinot&scaron; z&nbsp; na neophodnost daljeg kontrolisanja ovog proizvoda na&nbsp;prisustvo neonikotinoida. Ispitana je mogućnost uklanjanja odabranih neonikotinoida (dinotefurana,&nbsp;klotianidina i tiakloprida) iz vodene sredine (reke Dunav). Ispitivanje efikasnosti 6 različitih vrsta&nbsp;uklanjanja odabranih neonikotinoida (u prisustvu prirodne insolacije u laboratorijskim uslovima, sa&nbsp;dodatkom H2O2, sa dodatkom MWCNT, sa dodatkom MWCN+H&nbsp;₂O₂, sa dodatkom Fe-MWCNT, sa dodatkom Fe-MWCNT+H₂O₂) vr&scaron;eno je upotrebom prethodno razvijene HPLC-MS/MS metode. Krive uklanjanja odabranih neonikotinoida, pokazale su da tokom 60 minuta pri prirodnoj insolaciji&nbsp; u laboratorijskim uslovima koncentracija smanjenje oko 25%. Analitički signal dinotefurana dobijen u prisustvu H₂O_2&nbsp;pod istim uslovima ukazuje na uklanjanje ciljnog analita od oko 40%, tiakloprida od oko 70%, a klotianidina u potpunosti. Testirana je adsorpcija ciljnog analita na vi&scaron;ezidnim ugljeničnim nanocevima (MWCNT). Ovim postupkom može da se ukloni oko 30% dinotefurana, oko 50% klotianidina i 60% tiakloprida. U kombinaciji sa H₂O_2&nbsp;, MWCNT pokazuju bolju sposobnost uklanjanja za 15&ndash;50% u zavisnosti od ispitivanog neonikotinoida. Upotreba Fe-MWCNT i njihova kombinacija sa H₂O₂ otvorila je mogućnost za dalja &nbsp;ispitivanja mehanizma uklanjanja. Ustanovljeno je nastajanje intermedijera kojima odgovaraju m/z od 117,5 i 140,6 u slučaju razgradnje dinotefurana u sistemima sa H₂O₂, MWCNT+H₂O₂, Fe-MWCNT+H₂O_2&nbsp;i klotianidina u sistemu Fe-MWCNT+H₂O₂.</p> / <p>Neonicotinoid insecticides, as one of the fastest growing new generation of insecticides, have&nbsp;contributed to a significant reduction of toxicity for the environment; therefore, monitoring and&nbsp;determination of trace levels of the neonicotinoids in honey are necessary and demands highly efficient,&nbsp;selective and sensitive analytical techniques. The objective of the present work was to develop a rapid,&nbsp;sensitive, optimized and accurate analytical method based on liquid chromatography for determining&nbsp;seven neonicotinoid insecticides, dinotefuran, nitenpyram, thiamethoxam, clothianidin, imidacloprid,&nbsp;acetamiprid and thiacloprid in honey and honey liqueur samples. The possibility for determination of&nbsp;clothianidin in honey samples was investigated by HPLC with a diode array detector (HPLC-DAD).&nbsp;Based on preliminary results, it can be concluded that the use of a solid-phase column in combination&nbsp;with a liquid-liquid extraction with dichloromethane results in an acceptable recovery of clothianidin in&nbsp;the samples with a clothianidin concentration of about 0.5 &micro;g g^-1. After obtaining low recovery of&nbsp;clothianidin, dispersed liquid-liquid microextraction (DLLME) was selected as a technique for the&nbsp;preparation of honey samples.. The adequacy of acetonitrile as a dispersing agent was investigated.&nbsp;Besides the chloroform, a dichloromethane was used as a second extracting agent , in order to compare&nbsp;the relative efficiency of the extraction solvents. It can be concluded that the extraction recovery (68.4&ndash;92.1%) was more favorable with the use of 0.5 mL ACN and 2.0 mL DHM. Furthermore, LC-MS/MS&nbsp;parameters were optimized to unequivocally provide good chromatographic separation, low detection&nbsp;(LOD, 0.5&ndash;1.0 &mu;g L^&minus;1) and quantification (LOQ, 1.0&ndash;2.5 &mu;g L^&minus;1) limits for acetamiprid, clothianidin,&nbsp;thiamethoxam, imidacloprid, dinotefuran, thiacloprid and nitenpyram in honey samples. Using different&nbsp;<br />types (chloroform, dichloromethane) and volumes of extraction (1.0&ndash;3.0 mL) and dispersive&nbsp;(acetonitrile; 0.0&ndash;1.0 mL) solvent and by mathematical modeling it was possible to establish the optimal&nbsp;sample preparation procedure. Matrix-matched calibration and blank honey sample spiked in the&nbsp;<span style="font-size: 12px;">concentration range of LOQ&ndash;100.0 &mu;g kg</span>^{<span style="font-size: 12px;">&minus;1&nbsp;</span>}<span style="font-size: 12px;">were used to compensate the matrix effect and to fulfill the&nbsp;</span><span style="font-size: 12px;">requirements of SANCO/12495/2011 for the accuracy (R 74.3&ndash;113.9%) and precision (expressed in&nbsp;</span><span style="font-size: 12px;">terms of repeatability (RSD 2.74&ndash;11.8%) and within-laboratory reproducibility (RSDs &nbsp;6.64&ndash;16.2%)) of&nbsp;</span><span style="font-size: 12px;">the proposed method. The rapid (retention times 1.5&ndash;9.9 min), sensitive and low solvent consumption&nbsp;</span><span style="font-size: 12px;">procedure described in this work provides reliable, simultaneous, and quantitative method applicable for&nbsp;</span><span style="font-size: 12px;">the routine laboratory analysis of seven neonicotinoid residues in 15 real honey samples. Neonicotinoid &nbsp;</span><span style="font-size: 12px;">residues were not detected in any of the investigated samples. The objective of next study was to&nbsp;</span><span style="font-size: 12px;">develop and optimize HPLC-DAD analytical method with dispersive liquid-liquid microextraction&nbsp;</span><span style="font-size: 12px;">(DLLME) and QuEChERS sample preparation procedures for the simultaneously analysis of seven&nbsp;</span><span style="font-size: 12px;">neonicotinoids in honey samples. The liquid chromatographic conditions were optimized by response&nbsp;</span><span style="font-size: 12px;">surface methodology with <em>Box-Behnken</em> design and the global <em>Derringer</em>&acute;s desirability. The optimized&nbsp;</span><span style="font-size: 12px;">method was &nbsp;validated to fulfill the requirements of SANCO/12495/2011 standard for both sample&nbsp;</span><span style="font-size: 12px;">pretreatment procedures providing results for accuracy (R, 73.1&ndash;118.3%), repeatability (RSD, 3.28&ndash;</span><span style="font-size: 12px;">10.40%) and within-laboratory reproducibility (RSD, 6.45&ndash;17.70%), limits of detection (LOD, 1.5&ndash;2.5&nbsp;</span><span style="font-size: 12px;">g&micro; kg</span>^{<span style="font-size: 12px;">-1</span>}<span style="font-size: 12px;">) and quantification (LOQ, 5.0&ndash;10.0 &micro;g kg</span>^{<span style="font-size: 12px;">-1</span>}<span style="font-size: 12px;">). For the first time, more than 100 honey samples&nbsp;</span><span style="font-size: 12px;">collected from all 7 counties of Autonomous Province of Vojvodina were analyzed. The presence of&nbsp;</span><span style="font-size: 12px;">thiacloprid, imidacloprid and thiametoxam was discovered in a small number of samples. The objective&nbsp;</span><span style="font-size: 12px;">of next study was to develop an optimized LC-MS/MS analytical method with DLLME and QuEChERS&nbsp;</span><span style="font-size: 12px;">procedures for analysis of 7 neonicotinoids in honey liqueur. The method was validated to fulfill the&nbsp;</span><span style="font-size: 12px;">requirements of SANCO/12495/2011 for both sample pretreatment procedures providing results for&nbsp;</span><span style="font-size: 12px;">accuracy (R, 69.2&ndash;113.4% for DLLME; 71.8&ndash;94.9% for QuEChERS), precision (RSD expressed in&nbsp;</span><span style="font-size: 12px;">terms of repeatability (3.21&ndash;10.20% for DLLME; 4.19&ndash;12.81% for QuEChERS) and within-laboratory&nbsp;</span><span style="font-size: 12px;">reproducibility (9.11&ndash;16.63% for DLLME; 11.32&ndash;16.40% for QuEChERS)), limits of detection (LOD,&nbsp;</span><span style="font-size: 12px;">0.5&ndash;1.5 g&micro; L</span>^{<span style="font-size: 12px;">-1&nbsp;</span>}<span style="font-size: 12px;">for DLLME; 1.0&ndash;2.5 g&micro; L</span>^{<span style="font-size: 12px;">-1&nbsp;</span>}<span style="font-size: 12px;">for QuEChERS) and quantification (LOQ, 1.0&ndash;5.0 g&micro; L</span>^{<span style="font-size: 12px;">-1&nbsp;</span>}<span style="font-size: 12px;">for&nbsp;</span><span style="font-size: 12px;">DLLME; 2.5&ndash;10.0 &micro;g L</span>^{<span style="font-size: 12px;">-1&nbsp;</span>}<span style="font-size: 12px;">for QuEChERS). Analysis of real honey liqueur samples obtained from local&nbsp;</span><span style="font-size: 12px;">markets showed the presence of clothianidin or thiacloprid in four of the analyzed samples, therefore&nbsp;</span><span style="font-size: 12px;">implicating the necessity of ongoing control of this type of traditional product. &nbsp;Removal of selected&nbsp;</span><span style="font-size: 12px;">neonicitinoid insecticides - dinotefuran, clothianidin and thiacloprid using MWCNT and H</span><span style="font-size: 12px;">₂O_2&nbsp;</span><span style="font-size: 12px;">from&nbsp;</span><span style="font-size: 12px;">Danube water matrix was investigated. &nbsp;Efficiency of different systems for neonicotinoids removal&nbsp;</span><span style="font-size: 12px;">(under natural insolation in laboratory, with H</span><span style="font-size: 12px;">₂O₂, with MWCNT, with MWCNT+ H</span><span style="font-size: 12px;">₂O₂, with Fe-MWCNT, with Fe-MWCNT+H₂O₂) was evaluated with developed LC-MS/MS method. Analysis of&nbsp;</span><span style="font-size: 12px;">degradation rates revealed loss of 25% of the initial neonicotinoid concentration under natural insolation in&nbsp;</span><span style="font-size: 12px;">the laboratory conditions during 60 min. Addition of chemical agent H₂O_2&nbsp;promoted loss of 40% of the&nbsp;</span><span style="font-size: 12px;">initial dinotefuran, 70% of thiacloprid concentration and total removal of clothianidin under same&nbsp;</span><span style="font-size: 12px;">conditions. With the addition &nbsp;of MWCNT concentration of dinotefuran, clothianidin and thiacloprid&nbsp;</span><span style="font-size: 12px;">decayed for 30, 50 and 60%, respectively. Iron modification of MWCNT in combination with H</span><span style="font-size: 12px;">₂O_2&nbsp;</span><span style="font-size: 12px;">increased the removal rate of selected neonicotinoid for 15&ndash;50%. Presence of intermediates was&nbsp;</span><span style="font-size: 12px;">discovered in systems of dinotefuran with H₂O₂, MWCNT+H</span><span style="font-size: 12px;">₂O₂, e-MWCNT+H₂O_2&nbsp;</span><span style="font-size: 12px;">and of&nbsp;</span><span style="font-size: 12px;">clothianidin in systems with Fe-MWCNT+H₂O_2&nbsp;</span><span style="font-size: 12px;">with m/z of 117,5 and 140,6.&nbsp;</span></p>

Os eicosanoides, produtos do metabolismo do ácido araquidônico, apresentam papel importante na homeostasia e na patogênese de diversas doenças humanas. A biossíntese desses compostos pode ser estimulada por agentes farmacológicos como ionóforos e inibidores da Ca2+-ATPase, e também por agonistas naturais como o formil-metionil-leucil-fenialanina (fMLP). Considerando os interesses em avaliar e comparar o perfil de mediadores lipídicos, como os leucotrienos (LTs), as prostaglandinas (PGs), os ácidos epoxieicosatrienoicos (EETs), os ácidos dihidroxitetraenoicos (DiHETEs) e os ácidos hidroxieicosatetraenoicos (HETEs), na saúde e na doença, o objetivo deste trabalho foi padronizar um método analítico para determinar do perfil de eicosanoides em plasma humano após estimulação do sangue total, e assim observar diferenças entre indivíduos saudáveis e doentes. Dessa forma, um método por cromatografia líquida de alta eficiência acoplada à espectrometria de massas sequencial (HPLC-MS/MS) foi validado para quantificação de 22 eicosanoides em plasma de indivíduos saudáveis. A análise por HPLCMS/ MS foi realizada em modo negativo pelo modo de varredura por monitoramento de reações múltiplas (MRM). A linearidade do método apresentou coeficiente de correlação (r) maior que 0,98 para todos os eicosanoides analisados. A precisão e exatidão intra e inter-ensaios tiveram desvio padrão e erro relativo menores que 15%, exceto para o limite inferior de quantificação cujos valores foram menores que 20%. Para estimulação das células do sangue total, quatro estímulos (fMLP, ionomicina, A23187 e tapsigargina) foram utilizados. A análise estatística mostrou que o A23187 e a tapsigargina foram os estímulos mais potentes na indução da produção de eicosanoides. Em seguida, comparamos o perfil de eicosanoides em amostras de plasma de indivíduos saudáveis com pacientes com anemia falciforme (AF), em tratamento com hidroxiureia (HU) ou transfusão sanguínea crônica. Os resultados demonstraram que o método é preciso para determinação de diferenças entre os pacientes e indivíduos saudáveis quanto à produção dos mediadores lipídicos 5-HETE, 12-HETE, LTB4, LTE4, TXB2 e PGE2. Portanto, nosso método analítico é sensível, específico e reprodutível para identificar e quantificar diferenças no perfil de eicosanoides em amostras de sangue estimuladas in vitro, e poderá contribuir para o estabelecimento do perfil de mediadores lipídicos em diferentes doenças inflamatórias e infecciosas. / Eicosanoids, products from arachidonic acid metabolism, play an important role in the homeostasis and in the pathogenesis of various human diseases. Pharmacological agents such as Ca2+ ionophores and Ca2+-ATPase inhibitors, as well as natural agonists such as fMet-leu-Phe (fMLP) can stimulate eicosanoid biosynthesis. Considering the interests in evaluate and compare the profile of lipid mediators, as leukotriens (LTs), prostaglandins (PGs), epoxyeicosatrienoic acids (EETs), dihydroxytetraenoic acids (DiHETEs) and hydroxyeicosatetraenoic acids (HETEs), in healthy and disease, the aim of this work was to standardize a method to determine the eicosanoid profile of human plasma samples after whole blood stimulation, and to assess differences between healthy and sick individuals. For this purpose, a liquid chromatographytandem mass spectrometry (LC-MS/MS) method was validated for the quantification of 22 eicosanoids using human plasma from healthy volunteers. In addition, we optimized a method for the stimulation of eicosanoids in human whole blood. LC-MS/MS analyses were performed by negative electrospray ionization and multiple reaction monitoring. An assumption of linearity resulted in a regression coefficient > 0.98 for all eicosanoids tested. The mean intra-assay and inter-assay accuracy and precision values had relative standard deviations and relative errors of < 15%, except for the lower limit of quantification, where these values were < 20%. For whole blood stimulation, four stimuli (fMLP, ionomycin, A23187, and thapsigargin) were used. Results of the statistical analysis showed that A23187 and thapsigargin were potent stimuli to induce the production of eicosanoids. We next compared the eicosanoid profiles of healthy volunteers to those of patients with sickle cell anemia (SCA) under treatment with hydroxyurea (HU) or after chronic red blood cell (RBC) transfusion. The results indicate that the method was sufficient to find a difference between lipid mediators released in whole blood of SCA patients compared to healthy subjects for 5-HETE, 12-HETE, LTB4, LTE4, TXB2, and PGE2. In conclusion, our analytical method is sensitive, specific and reproducible for indentify and quantify changes in eicosanoid profiles in whole blood stimulated in vitro, which can contribute to establishing the eicosanoid profiles associated with different inflammatory and infectious diseases.