Mass spectrometry is being increasingly used in biomarker research mainly due to its ability to achieve high selectivity coupled with high sensitivity. This dissertation focuses on quantitative mass spectrometric investigations of two protein biomarkers i.e. serum thymidine kinase 1 (TK1) and human osteopontin (OPN). First part of this research was focused on developing a liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method for measuring the activity of TK1 in serum by monitoring the conversion of a TK1 specific exogenous substrate, 3’-deoxy-3’-fluorothymidine (FLT), to its mono-phosphorylated form 3’-deoxy-3’-fluorothymidine monophosphate (FLT-MP). A method to quantify FLT-MP on LC-MS/MS was developed and validated. The method was linear over the range of 2.5-2000 ng/mL with a mean correlation coefficient of 0.9935. Using the developed method, serum TK1 activity was measured in serum from hepatocellular carcinoma (HCC) patients and age-matched controls under standardized conditions. A sub-population of the HCC patient samples showed an almost 20-fold enhanced TK1 activity compared to the controls. A method was developed and validated for quantifying human osteopontin from plasma using immunoaffinity isolations coupled with microflow liquid chromatography and tandem mass spectrometry (MFLC-MS/MS). A biologically relevant tryptic peptide ‘GDSVVYGLR’ which is unique to hOPN was identified and used as a signature peptide for this method. The method was validated over a range of 25-600 ng/mL. The performance of the method was compliant with USFDA validation guidance. In addition, a stable isotope labeled (SIL) peptide GDSVVYGLR* and an extended SIL peptide TYDGRGDSVV*YGLRSKSKKF’ were evaluated as internal standards (IS) to account for signature peptide digestion instability and variability. In the digestion variability studies, the use of extended SIL peptide as internal standard limited the total variability within ±30%. Alternatively, when SIL peptide was used as internal standard the variability ranged from -67.4% to +50.6 %. The applicability of the validated method was demonstrated by analyzing plasma samples obtained from 10 healthy individuals and 10 breast cancer patients. More than 9-fold increase in the mean plasma hOPN concentration was seen in 30% of the breast cancer patient samples (n=10) in comparison to the healthy volunteer samples. In a proof of concept investigation, a stable isotope labeled signature peptide was evaluated as an internal standard to compensate for immunocapture variability during quantification of human osteopontin (hOPN) by immunoaffinity coupled LC-MS/MS. Immunocapture variability was induced by varying the antibody amount per well. The immunocapture variability ranged from -80.9 % to +77.0 % when the IS was added after immunocapture and from -37.5% to +20.3% when the IS was added before immunocapture. The lower variability demonstrates the ability of SIL-IS peptide to compensate for variation during immunocapture.

Numerous chemical species are important to the health of biological systems. Some species can be beneficial at low doses and harmful at high doses. Other species are highly reactive and trigger serious cell damage. Improved methods to detect the presence and activity of such species are needed. In this work, several biologically important species were studied using appropriate analytical techniques. Fluoride is an important species in human physiology. It strengthens teeth and gives protection against dental caries. However, elevated concentrations of fluoride in the body can lead to health problems such as dental and skeletal fluorosis. Reported fluoride sensors used fluorescence quenching methods in determining fluoride concentration. Our study explored synthesis and characterization of 1,8-bis(phenylthioureido) naphthalene (compound 1) as a fluoride sensing molecule. Compound 1 showed a remarkable 40 fold enhancement in fluorescence with 5 eq of fluoride addition. Compound 1 also showed possibility of visual colorimetric sensing with fluoride. Free radical mediated oxidations of biomolecules are responsible for different pathological conditions in the human body. Superoxide is generated in cells and tissues during oxidative burst. Moderately reactive superoxide is converted to peroxyl, alkoxyl and hydroxyl radicals by various enzymatic, chemical, and biochemical processes. Hydroxyl radical imparts rapid, non specific oxidative damage to biomolecules such as proteins and lipids. Superoxide also reacts with nitric oxide in cells to yield peroxynitrite, which is highly reactive and damages biomolecules. Both hydroxyl radical and peroxynitrite readily react with amino acids containing aromatic side chains. Low density lipoprotein (LDL) carries cholesterol in the human body. Elevated concentration of LDL is a potential risk factor for atherosclerosis. Previous research drew a strong correlation between oxidized low density lipoprotein (ox-LDL) and plaque formation in the arterial wall. More importantly, oxidative damage causes structural changes to the LDL protein (apo B-100) which might facilitate the uptake of LDL by macrophages. In this study LDL was exposed to various concentrations of hydroxyl radical peroxynitrite and hypochlorite. Thereafter oxidized amino acid residues in apo B-100 were mapped by LC-MS/MS methods. We found widely distributed oxidative modifications in the apo B-100 amino acid sequence.

A microextração por sorbente empacotado (MEPS) é uma técnica de preparo de amostras ainda pouco utilizada no âmbito da toxicologia, em que os mesmos princípios da extração em fase sólida convencional são adaptados para uma escala miniaturizada. As principais vantagens da técnica estão associadas ao pequeno volume de amostra e de solventes utilizados, à possibilidade de realizar múltiplas extrações com um mesmo cartucho e à facilidade de automação. Os benzodiazepínicos possuem grande relevância na toxicologia dada sua ampla utilização e seus efeitos que podem, por exemplo, comprometer a capacidade de dirigir, além do uso abusivo, e como drogas facilitadoras de crimes. Neste trabalho, um método de MEPS foi desenvolvido e otimizado para a determinação de sete benzodiazepínicos e seus produtos de biotransformação (diazepam, clonazepam, flunitrazepam, alprazolam, bromazepam, 7-aminoflunitrazepam e nordiazepam) utilizando 100 µL de amostra de sangue total post mortem. Após a extração, os eluatos foram analisados por cromatografia líquida em fase reversa acoplada a espectrometria de massas. O método foi validado de acordo com as recomendações do Scientific Working Group for Forensic Toxicology, apresentando linearidade adequada de 5 a 500 ng.mL-1 . Os valores de exatidão (90,4 a 109,5%), precisão intra-dia (2,5 a 10,7 %CV) e inter-dia (1,1 a 8,0 %CV) também foram satisfatórios. MEPS foi realizada mais de 60 vezes com a mesma fase extratora sem evidências de contaminação cruzada. Dez amostras reais fornecidas pelo Instituto Médico Legal de São Paulo foram analisadas. Foram quantificados diazepam, nordiazepam, clonazepam e bromazepam. Os resultados encontrados em cada uma das amostras foram comparados com dados da literatura. / Microextraction by packed sorbent (MEPS) is a sample preparation technique still little used in toxicology, where the same principles of conventional solid phase extraction are adapted to a miniaturized scale. The main advantages of the technique are associated with the small volume of sample and solvents required, the possibility of performing multiple extractions with the same cartridge and ease process automation. Benzodiazepine drugs are relevant in toxicology because of their widespread use, and effects (which may, for example, compromise the ability to drive vehicles), abuse and records as crime-facilitating drugs. In this work, a MEPS method was developed and optimized for a determination of seven benzodiazepines and their metabolites (diazepam, nordiazepam, clonazepam, flunitrazepam, 7-aminoflunitrazepam, alprazolam, and bromazepam) using 100 µL of post mortem whole blood. After extraction, the eluates were analyzed by reversed-phase liquid chromatography coupled to mass spectrometry. The method was validated according to the recommendations of the Scientific Working Group for Forensic Toxicology, presenting adequate linearity from 5 to 500 ng.mL-1 . The values of accuracy (90.4 to 109.5%), intra-day precision (2.5 to 10.7 %CV) and inter-day (1.1 to 8.0 %CV) also presented satisfactory results. MEPS was performed more than 60 times with the same extractive phase without compromising the results with the evidence of carryover. Institute of Legal Medicine were submitted to analysis by MEPS-LC-MS/MS. In these samples, the following analytes were quantified: diazepam, nordiazepam, clonazepam and bromazepam. The results found in each of the samples were compared with data from the literature.

A microextração por sorbente empacotado (MEPS) é uma técnica de preparo de amostras ainda pouco utilizada no âmbito da toxicologia, em que os mesmos princípios da extração em fase sólida convencional são adaptados para uma escala miniaturizada. As principais vantagens da técnica estão associadas ao pequeno volume de amostra e de solventes utilizados, à possibilidade de realizar múltiplas extrações com um mesmo cartucho e à facilidade de automação. Os benzodiazepínicos possuem grande relevância na toxicologia dada sua ampla utilização e seus efeitos que podem, por exemplo, comprometer a capacidade de dirigir, além do uso abusivo, e como drogas facilitadoras de crimes. Neste trabalho, um método de MEPS foi desenvolvido e otimizado para a determinação de sete benzodiazepínicos e seus produtos de biotransformação (diazepam, clonazepam, flunitrazepam, alprazolam, bromazepam, 7-aminoflunitrazepam e nordiazepam) utilizando 100 µL de amostra de sangue total post mortem. Após a extração, os eluatos foram analisados por cromatografia líquida em fase reversa acoplada a espectrometria de massas. O método foi validado de acordo com as recomendações do Scientific Working Group for Forensic Toxicology, apresentando linearidade adequada de 5 a 500 ng.mL-1 . Os valores de exatidão (90,4 a 109,5%), precisão intra-dia (2,5 a 10,7 %CV) e inter-dia (1,1 a 8,0 %CV) também foram satisfatórios. MEPS foi realizada mais de 60 vezes com a mesma fase extratora sem evidências de contaminação cruzada. Dez amostras reais fornecidas pelo Instituto Médico Legal de São Paulo foram analisadas. Foram quantificados diazepam, nordiazepam, clonazepam e bromazepam. Os resultados encontrados em cada uma das amostras foram comparados com dados da literatura. / Microextraction by packed sorbent (MEPS) is a sample preparation technique still little used in toxicology, where the same principles of conventional solid phase extraction are adapted to a miniaturized scale. The main advantages of the technique are associated with the small volume of sample and solvents required, the possibility of performing multiple extractions with the same cartridge and ease process automation. Benzodiazepine drugs are relevant in toxicology because of their widespread use, and effects (which may, for example, compromise the ability to drive vehicles), abuse and records as crime-facilitating drugs. In this work, a MEPS method was developed and optimized for a determination of seven benzodiazepines and their metabolites (diazepam, nordiazepam, clonazepam, flunitrazepam, 7-aminoflunitrazepam, alprazolam, and bromazepam) using 100 µL of post mortem whole blood. After extraction, the eluates were analyzed by reversed-phase liquid chromatography coupled to mass spectrometry. The method was validated according to the recommendations of the Scientific Working Group for Forensic Toxicology, presenting adequate linearity from 5 to 500 ng.mL-1 . The values of accuracy (90.4 to 109.5%), intra-day precision (2.5 to 10.7 %CV) and inter-day (1.1 to 8.0 %CV) also presented satisfactory results. MEPS was performed more than 60 times with the same extractive phase without compromising the results with the evidence of carryover. Institute of Legal Medicine were submitted to analysis by MEPS-LC-MS/MS. In these samples, the following analytes were quantified: diazepam, nordiazepam, clonazepam and bromazepam. The results found in each of the samples were compared with data from the literature.

In recent years, the abuse of synthetic cathinones or ‘bath salts’ has become a major public health concern. Although these compounds were initially sold legally and labeled “not for human consumption”, the ‘bath salts’ are psychostimulants, with similar structures and pharmacologic mechanisms to cocaine, the amphetamines, and 3,4 methylendioxymethamphetamine (MDMA, Molly, or Ecstasy). The reported use of these substances by women of child-bearing age highlights the necessity of studies seeking to delineate risks of prenatal exposure. Three popular drugs of this type are methylone, mephedrone, and 3, 4-methylenedioxypyrovalerone (MDPV). Unfortunately, there is currently no information available on the teratogenicity of these compounds, or of the extent to which they cross the placenta. As such, the purpose of this study was to examine the pharmacokinetic profile of the ‘bath salts’ in a pregnancy model. Pregnant mice (E17.5 gestation) were injected intraperitoneally with a cocktail of 5mg/kg methylone, 10mg/kg mephedrone, and 3mg/kg (MDPV) dissolved in sterile saline. Maternal brain, maternal plasma, placenta, and fetal brain were collected at 30s, 1min, 5min, 10min, 15min, 30min, 1h, 2h, 4h, and 8h following injection. Methylone, mephedrone, and MDPV were extracted from tissue by solid phase extraction, and concentrations were determined using a previously validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Interestingly, all 3 cathinones reached measurable concentrations in the placenta, as well as the fetal brain; in fact, for MDPV, the maximal concentration (Cmax) was highest in fetal brain, while mephedrone's highest Cmax value was achieved in placenta. Additionally, the total drug exposure for all 3 compounds (as represented by area under the curve, AUC) was higher in fetal matrices (placenta and fetal brain) than in maternal matrices (maternal brain and plasma), and the half-lives for the drugs were longer. Given the extensive presence of methylone, mephedrone, and MDPV in the fetal brain following prenatal exposure, fetal risk is definitely a concern. As there are currently no prenatal studies available on the teratogenicity of these agents, pregnant patients should be informed about the potential risks that these substances may have.

De nos jours, environ 3000 huiles essentielles sont produites et utilisées dans le monde avec des champs d'application aussi variés que la cosmétique, la parfumerie l'agro-alimentaire, la pharmacie et l'aromathérapie. Ces huiles sont extraites des hespéridés ou des plantes aromatiques et médicinales. Étant donné que la culture de ces matières premières implique généralement l'application de pesticides, la présence de tels résidus dans les huiles essentielles ne peut pas être écartée. Compte tenu du nombre important de pesticides employés et des nombreux champs d'application des huiles essentielles, il apparaît nécessaire, afin d'assurer la santé du consommateur, de disposer d'une méthode d'analyse multi-résidus capable de doser les pesticides dans les huiles essentielles. L'état de l'art des méthodes d'analyse lié à cette problématique a révélé un manque évident de performance des méthodes actuelles aussi bien en termes de limite de détection que du nombre de pesticides analysés. Ce mémoire propose donc la mise au point d'une méthode d'analyse multi-résidus de pesticides dans les huiles essentielles par couplage de la chromatographie liquide et de la spectrométrie de masse, technologie la plus à même de répondre à la problématique au vue de la bibliographie. Après avoir mis en évidence les performances du nouveau mode d'acquisition Scheduled SRM, disponible sur le spectromètre de masse 4000 QTrap, pour la détection et la quantification de 250 pesticides dans un solvant déterminé, l'importance de considérer la nature de la matrice, aussi bien dans les méthodes de préparation que lors de l'analyse de l'échantillon, a ensuite été démontrée en étudiant deux huiles essentielles représentatives (lavandin et citron). Enfin, la méthode d'analyse multi-résidus LC-MS/MS a été appliquée à la recherche de pesticides dans des échantillons réels d'huiles essentielles. Elle a démontré sa capacité à détecter, quantifier et identifier les pesticides dans ces matrices au travers de l'utilisation d'un mode d'acquisition couplé SRM-EPI faisant appel aux spécificités du spectromètre de masse hybride et notamment de sa trappe d'ion linéaire. Ce travail a de plus révélé l'importance de disposer d'une telle méthode au regard du nombre de pesticides détectés dans les échantillons et de leurs concentrations relativement élevées. Celles-ci peuvent en effet atteindre des teneurs supérieures au milligramme par litre dans les huiles essentielles analysées.

Le foisonnement filamenteux est un problème récurant dans de nombreuses stations d'épuration à boues activées. L'objectif de ces travaux est d'améliorer la compréhension des mécanismes physiologiques et moléculaires impliqués dans la filamentation des microorganismes, afin de pouvoir orienter de futures stratégies de lutte contre le phénomène de bulking. Sphaerotilus natans, qui peut croître réversiblement sous forme monocellulaire ou filamenteuse, a été utilisée comme bactérie modèle pour cette étude. Différents types de cultures, ainsi que des suivis par cytométrie en flux et marquage au cFDA/SE, ont montré que les diverses souches de S. natans adoptent des morphologies différentes et que les filaments croissent par divisions cellulaires successives et non par un chaînage des bactéries. Une analyse par RT-QPCR a mis en évidence que l'expression du gène sthA augmente fortement après induction de la filamentation et reste ensuite à un niveau élevé. Une comparaison de l'expression protéique des formes monocellulaire et filamenteuse, par LC-MS-MS, a permis d'identifier des protéines impliquées dans la filamentation, et notamment dans la synthèse de la gaine. La concentration intracellulaire en ARNr, mesurée par RT-QPCR, varie durant la croissance de S. natans et d'autres microorganismes, entraînant une diminution importante de l'intensité du marquage FISH, mesurée par cytométrie en flux. L'utilisation de la technique FISH pour quantifier des microorganismes est donc remise en question, d'autant plus dans des matrices aussi complexes que les boues activées. Ces observations mettent également en doute l'hypothèse, émise en utilisant ce mode de quantification, d'une déstructuration des filaments consécutive à un retour à des conditions de culture plus favorables.

<p>Electrospray ionization (ESI) mass spectrometry (MS) in combination with liquid chromatography (LC) is an excellent tool for the identification of drug metabolites. Utilizing this hyphenated technique in combination with proper sample pretreatment, the metabolic pathways of the analgesic drug ketobemidone were investigated in human urine and rat microdialysate from blood and brain. Two novel phase I metabolites (ketobemidone N-oxide and meta-hydroxymethoxyketobemidone) and three novel phase II metabolites (glucuronic acid conjugates of ketobemidone, norketobemidone and hydroxymethoxyketobemidone) were identified in human urine. Further, norketobemidone and ketobemidone N-oxide were identified in rat microdialysate from brain after regional distribution of ketobemidone in striatum. This indicates that the brain itself has the possibility to metabolize ketobemidone. </p><p>Synthetic ketobemidone metabolites were used for comparison of retention times and tandem MS spectra with the possible metabolites recovered from the biological samples. The conjugated metabolites were identified by accurate mass measurements and tandem MS spectra of the aglycones. The accuracy of the estimated masses was better than 2.1 ppm for two out of three conjugates in presence of internal standard.</p><p>On-line micro-SPE was successfully used for trapping and desalting of the microdialysates. The small SPE pre-column made it possible to inject approximately 100 times more sample on the analytical column compared to injection without pre-column. Selective trapping was demonstrated for the polar catechol amine metabolite, dihydroxyketobemidone, which forms covalent complexes with phenylboronic acid (PBA). A fluorinated silica type stationary phase was the only column out of several tested that was able to separate ketobemidone and all relevant phase I metabolites. </p><p>Liquid chromatography and mass spectrometry are independently valuable tools in the field of analytical pharmaceutical chemistry. The present study showed that the combination of LC-MS, with its excellent selectivity and sensitivity, offers an outstanding tool in the qualitative analysis of drugs and metabolites in biological fluids. </p>

Electrospray ionization (ESI) mass spectrometry (MS) in combination with liquid chromatography (LC) is an excellent tool for the identification of drug metabolites. Utilizing this hyphenated technique in combination with proper sample pretreatment, the metabolic pathways of the analgesic drug ketobemidone were investigated in human urine and rat microdialysate from blood and brain. Two novel phase I metabolites (ketobemidone N-oxide and meta-hydroxymethoxyketobemidone) and three novel phase II metabolites (glucuronic acid conjugates of ketobemidone, norketobemidone and hydroxymethoxyketobemidone) were identified in human urine. Further, norketobemidone and ketobemidone N-oxide were identified in rat microdialysate from brain after regional distribution of ketobemidone in striatum. This indicates that the brain itself has the possibility to metabolize ketobemidone. Synthetic ketobemidone metabolites were used for comparison of retention times and tandem MS spectra with the possible metabolites recovered from the biological samples. The conjugated metabolites were identified by accurate mass measurements and tandem MS spectra of the aglycones. The accuracy of the estimated masses was better than 2.1 ppm for two out of three conjugates in presence of internal standard. On-line micro-SPE was successfully used for trapping and desalting of the microdialysates. The small SPE pre-column made it possible to inject approximately 100 times more sample on the analytical column compared to injection without pre-column. Selective trapping was demonstrated for the polar catechol amine metabolite, dihydroxyketobemidone, which forms covalent complexes with phenylboronic acid (PBA). A fluorinated silica type stationary phase was the only column out of several tested that was able to separate ketobemidone and all relevant phase I metabolites. Liquid chromatography and mass spectrometry are independently valuable tools in the field of analytical pharmaceutical chemistry. The present study showed that the combination of LC-MS, with its excellent selectivity and sensitivity, offers an outstanding tool in the qualitative analysis of drugs and metabolites in biological fluids.

Rhodobacter capsulatus is a versatile organism capable of growing on different growth conditions including photofermentation in the presence of carbon source, aerobic respiration, anaerobic respiration in the presence of an external electron acceptor such as DMSO. The photofermentative growth of R.capsulatus results in hydrogen production which stands out as an environmentally harmless method to produce hydrogen and accepted as one of the most promising process. Due to the serious problems such as as global climate change and environmental pollution caused by the fossil fuels, there is an increasing requirement for a clean and sustainable energy source. Furtherrmore, the ability of R.capsulatus to fix nitrogen, to use solar energy makes it a model to study various aspects of its metabolism. Thus the goal of this study is to increase the potential in biohydrogen production with the photofermentative bacteria and to investigate the proteins playing roles in different growth modes of the bacteria. In the present study, protein profiles of Rhodobacter capsulatus grown on respiratory, anaerobic respiratory and photofermentative growth modes were obtained. LC-MS/MS system is used to analyze the proteome as a high throughput technique. Physiological analysis such as HPLC for the analysis of the carbon source consumption, GC and analysis of pigments were carried out to state the environmental conditions. As a result, total of 460 proteins were identified with 17 proteins being unique to particular growth condition. Ratios of the proteins in different growth conditions were compared and important proteins were highlighted.