<p>The objective of this master thesis was to develop an analytical method for the quantification of the cancer drug Imatinib and its main metabolite CGP74588 in plasma. Imatinib is used in the treatment of chronic myeloid leukemia and gastrointestinal stroma tumors. A quantitative analytical method was developed where reversed-phase columns with different stationary phases were studied and the sensitivity was tested with both UV detectors and a mass spectrometric detection. Since the substances were measured in plasma a solid-phase extraction was developed to purify the samples before analysis. The column chosen for the separation was the Max-RP C12 column (100 x 3 mm, 4 μm particle size) manufactured by Phenomenex with a gradient mobile phase with 1% formic acid in methanol and water. The gradient was as follows; 0 min 15:85, 7 min 60:40, 9 min 60:40 with a total runtime of 13.5 min. The internal standard chosen was Opipramol. Mass spectrometric detection using a sonic spray ionization interface in positive mode proved to be about as sensitive as UV detection at 261 nm. The generated (M+H+)+ ions were isolated and fragmented with the use of three mass spectrometric methods; one for Imatinib (transition 494 —› 394), one for CGP74588 (transition 480 —› 394) and one for Opipramol (transition 364 —› 171). For the purification of the plasma samples an Oasis HLB solid-phase extraction cartridge was selected and the recoveries were close to 100%.</p><p>The developed method was partially validated and showed coefficients of variation (CV) for intra-and inter-day precision between 0.4 and 5.4% with UV detection. The validation results for the mass spectrometer were inconclusive.</p>

<p>Anabolic androgenic steroids are testosterone and its derivates. Testosterone is the most important naturally existing sex hormone for men and is used for its anabolic effects providing increased muscle mass. Testosterone is taken orally or by intramuscular injection in its ester form and are available illegally in different forms of esters. Anabolic androgenic steroids are today analyzed only in urine. To differentiate between the human natural testosterone and exogenous supply the quote natural testosterone and epitestosterone is used. Detection of testosterone esters in serum is an unmistakable proof of exogenous supply of testosterone. The aim of this thesis was to find a method for determining testosterone esters in serum and to study an extraction method possible for quantification of testosterone esters in serum.</p><p>The technique used to separate and identify the Testosterone esters was Liquid Chromatography Tandem Mass Spectrometry Electro Spray Ionisation. Parameters for chromatography and mass detection were optimized for nine testosterone esters and evaluated according to selectivity, resolution and intensity. A method that could be used for determination of testosterone esters in serum was found. The MS-method was set and at least three possible transitions for each testosterone ester were found. The best choice of column proved to be the C18 column where all the esters were separated and seven of them were base-line separated. The C18 column along with methanol and ammonium acetate buffer, 5 mM, pH 5 showed the highest sensitivity for Multiple Reaction Monitoring-detection. A gradient profile for a total runtime of 5.6 minutes was established. Two alternative extraction procedures were tested, with <em>tert</em>-butylmethylether or diethyl ether/ethyl acetate and both seemed to work satisfactory. Analysis of serum proved to work well and no severe interference occurred. Results from the linearity tests indicate that future quantification method in serum will be possible.</p>

This thesis describes the development of liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for the trace analysis of active pharmaceutical ingredients (APIs) and their metabolites in aqueous environmental matrices. The research was focused on the development of chiral LC-MS/MS methods for the analysis of fluoxetine and metoprolol, as well as their chiral metabolites in environmental water samples. A method was also developed for the achiral compounds, diazepam and nordiazepam. The LC-MS/MS methods were validated by the use of the isotope-labeled compounds. As these isotope-labeled compounds were not found in the wastewater samples, the validation could be assessed at trace level concentrations in the actual matrices in which the analytes were detected. The analytes were extracted from the water samples using solid phase extraction methods. Different types of solid phase extraction sorbents were evaluated. Fluoxetine and norfluoxetine were extracted through the use of a mixed mode polymeric based extraction sorbent. A hydrophilic and lipophilic balanced sorbent was employed for the simultaneous extraction of metoprolol and its metabolites, the base α-hydroxymetoprolol and the acidic metabolite deaminated metoprolol. Moreover, silica based C18 extraction discs were applied for the sample preparation of diazepam and nordiazepam. The chromatographic separations were conducted in reversed phase LC with MS compatible mobile phases. The enantiomers of fluoxetine and norfluoxetine were simultaneously separated using the chiral stationary phase (CSP), α1-acid glycoprotein (AGP). The Chiral AGP column was also applied for the separation of the enantiomers of deaminated metoprolol. For the simultaneous separation of the metoprolol enantiomers and the four stereoisomers of α-hydroxymetoprolol, the cellobiohydrolase (CBH) protein based CSP was used. An octadecyl silica based LC column was applied for the separation of diazepam and nordiazepam. The analytes were detected by the use of tandem quadrupole mass spectrometry operating in selective reactive monitoring mode. High resolution MS, employing a quadrupole time-of-flight (QqTOF) mass analyzer, was utilized for the identification of an unknown compound in wastewater samples. The APIs and their metabolites, as well as their respective enantiomers, were quantified in raw and treated wastewater from Uppsala, Sweden along with surface water from the River Fyris in Uppsala.

Anabolic androgenic steroids are testosterone and its derivates. Testosterone is the most important naturally existing sex hormone for men and is used for its anabolic effects providing increased muscle mass. Testosterone is taken orally or by intramuscular injection in its ester form and are available illegally in different forms of esters. Anabolic androgenic steroids are today analyzed only in urine. To differentiate between the human natural testosterone and exogenous supply the quote natural testosterone and epitestosterone is used. Detection of testosterone esters in serum is an unmistakable proof of exogenous supply of testosterone. The aim of this thesis was to find a method for determining testosterone esters in serum and to study an extraction method possible for quantification of testosterone esters in serum. The technique used to separate and identify the Testosterone esters was Liquid Chromatography Tandem Mass Spectrometry Electro Spray Ionisation. Parameters for chromatography and mass detection were optimized for nine testosterone esters and evaluated according to selectivity, resolution and intensity. A method that could be used for determination of testosterone esters in serum was found. The MS-method was set and at least three possible transitions for each testosterone ester were found. The best choice of column proved to be the C18 column where all the esters were separated and seven of them were base-line separated. The C18 column along with methanol and ammonium acetate buffer, 5 mM, pH 5 showed the highest sensitivity for Multiple Reaction Monitoring-detection. A gradient profile for a total runtime of 5.6 minutes was established. Two alternative extraction procedures were tested, with tert-butylmethylether or diethyl ether/ethyl acetate and both seemed to work satisfactory. Analysis of serum proved to work well and no severe interference occurred. Results from the linearity tests indicate that future quantification method in serum will be possible.

The objective of this master thesis was to develop an analytical method for the quantification of the cancer drug Imatinib and its main metabolite CGP74588 in plasma. Imatinib is used in the treatment of chronic myeloid leukemia and gastrointestinal stroma tumors. A quantitative analytical method was developed where reversed-phase columns with different stationary phases were studied and the sensitivity was tested with both UV detectors and a mass spectrometric detection. Since the substances were measured in plasma a solid-phase extraction was developed to purify the samples before analysis. The column chosen for the separation was the Max-RP C12 column (100 x 3 mm, 4 μm particle size) manufactured by Phenomenex with a gradient mobile phase with 1% formic acid in methanol and water. The gradient was as follows; 0 min 15:85, 7 min 60:40, 9 min 60:40 with a total runtime of 13.5 min. The internal standard chosen was Opipramol. Mass spectrometric detection using a sonic spray ionization interface in positive mode proved to be about as sensitive as UV detection at 261 nm. The generated (M+H+)+ ions were isolated and fragmented with the use of three mass spectrometric methods; one for Imatinib (transition 494 —› 394), one for CGP74588 (transition 480 —› 394) and one for Opipramol (transition 364 —› 171). For the purification of the plasma samples an Oasis HLB solid-phase extraction cartridge was selected and the recoveries were close to 100%. The developed method was partially validated and showed coefficients of variation (CV) for intra-and inter-day precision between 0.4 and 5.4% with UV detection. The validation results for the mass spectrometer were inconclusive.

Damp building materials are subjected to degradation processes due to moisture and also microbial growth, with both of these giving rise to emissions of volatile organic compounds (VOCs) that may contribute to indoor air health problems. The overall aim of this thesis was to investigate emissions of reactive and non-reactive VOCs from damp building materials and from the microorganisms growing on them, and also to investigate the possible health impact of these compounds. Three studies were carried out in order to study emissions of VOCs. The first investigated emissions from a mixture of five fungi (Aspergillus versicolor, Fusarium culmorum, Penicillium chrysogenum, Ulocladium botrytis and Wallemia sebi) and the second emissions from the bacterium Streptomyces albidoflavus. In both studies the microorganisms were cultivated on three different building materials (pine wood, particle board and gypsum board) and one synthetic media, MEA and TGEA respectively. The bacterium was also cultivated on sand. Air samples from the cultures were collected on six different adsorbents and chemosorbents to sample a wide range of compounds such as VOCs, aldehydes, amines and light-weight organic acids. The samples were analyzed with gas chromatography, high-pressure liquid chromatography and ion chromatography. Mass spectrometry was used for identification of the compounds. Alcohols and ketones were the predominant compound groups identified. The bacterial culture growing on TGEA emitted ammonia, methylamine, diethylamine and ethylamine. The third study dealt with secondary emissions collected from buildings with moisture and mould problems. Samples were taken when the materials were dry and also after they had been wet for a week. Most alcohols and ketones could be identified from the wet materials. Trimethylamine and triethylamine, were identified from sand contaminated by Bacillus. One study looked at the development of a method for analysis of primary and secondary amines with LC-MS/MS. A three-step process was developed, with the first step screening the samples for NIT derivatives with selected reaction monitoring, SRM. In the second step a precursor ion scan gave the [M+H]+ ion, and the last step involved fragmentation with a product ion scan. It was possible to separate and identify all the investigated amines, which showed that the method was both specific and selective and therefore well suited for the analysis of amines in complex environments. The last study comprised two exposure studies. In study 1 each participant took part in two exposure conditions, one with air from mouldy building materials and one with blank air for a 60 minute period. In study 2 each participant was exposed four times (for a period of 10 min) at random to air from mouldy building materials and blank air, with and without nose-clip. The participants rated air quality and symptoms before, during and after each exposure. Exposure to moderate VOC levels resulted in reports of perceived poor air quality, but no such results were received when exposing the participants to low VOC levels.

L'évaluation des risques environnementaux et sanitaires liés à la présence de résidus de médicaments dans l'environnement, représente un enjeu majeur en particulier au regard de la gestion du cycle des usages de l'eau. Les effluents des établissements de soins représentent une source non négligeable de pollution et justifient le développement de techniques spécifiques de mesures des émissions de résidus de médicaments dans leurs eaux usées. Dans le cadre de ces développements de méthode, l'échantillonnage représente une des difficultés majeures car la matrice brute des eaux en sortie des hôpitaux est très chargée en matières organiques, les débits sont extrêmement variables, les sites de prélèvement sont difficiles d'accès et il existe une forte variabilité des dispensations des traitements jour/nuit et semaine/week-ends. Les échantillonneurs intégratifs apparus récemment, offrent une alternative intéressante aux stratégies d'échantillonnage existantes, permettant d'effectuer un suivi moyenné sur de longues périodes d'observation associé à une simplicité d'usage et une réduction des coûts. Ce travail porte sur l'étude de l'applicabilité des échantillonneurs intégratifs POCIS (Polar Organic Chemical Integrative Sampler) au dosage des résidus de médicaments dans les effluents hospitaliers. Ce dispositif a principalement été utilisé dans les eaux de surface et les effluents de STEP et son application dans les eaux usées reste rare. 6 molécules déjà identifiées comme représentatives des grandes familles de médicaments utilisés à l'hôpital (Aténolol, Prednisolone, Méthylprednisolone, Sulfaméthoxazole, Ofloxacine, Kétoprofène) ont été retenues.Les cinétiques d'adsorption des molécules sur les POCIS ont été suivies en laboratoire en condition de maitrise des paramètres les plus influents : température, vitesse de l'eau, charge en matière organique, colmatage...Ce calibrage a pour but de déterminer le coefficient d'échantillonnage Rs (L/j) spécifique à chaque molécule et nécessaire au calcul de la concentration dans le milieu d'exposition du POCIS. Nous avons observé une augmentation des Rs quand la vitesse de l'eau augmente ou quand la température augmente. Dans les eaux usées, la valeur de Rs est plus faible et la durée de la phase linéaire est réduite comparée à l'eau du robinet. Pour ce type d'application, la période d'exposition ne devra pas dépasser 4 à 5 jours en raison du colmatage des membranes et d'une teneur élevée en particules organiques dissoutes. Après calibrage dans l'eau du robinet et dans l'eau usée, les POCIS ont été exposés in situ dans un effluent hospitalier pour mesurer les concentrations en 6 molécules ciblées. Cinq ont pu être quantifiées et les concentrations calculées à partir des extraits des POCIS sont concordantes avec celles obtenues par prélèvement direct d'un échantillon moyenné d'eaux usées. Le facteur limitant réside dans les difficultés d'accès au site et la présence de solides dans le collecteur d'eaux usées. Ce travail ouvre des perspectives quant à l'application des POCIS pour les effluents hospitaliers et pourra à terme contribuer à l'acquisition de données pour une meilleure surveillance des rejets.

Anatoxin-a (ANTX) is a neurotoxin produced by several freshwater cyanobacteria and has been implicated in the death of livestock and domestic animals from consumption of tainted surface waters. ANTX is unstable under normal conditions and is somewhat problematic to extract and study. Accelerated solvent extraction (ASE) combined with liquid chromatography-mass spectrometry (LC/MS) was used to develop an efficient extraction and analytical method for both ANTX and the more commonly encountered hepatotoxic microcystins produced by cyanobacteria. The effects of nitrogen supply on the cellular production and release of ANTX was investigated in Aphanizomenon issatschenkoi (Ussaczew) Proschkina-Lavrenko (Nostocales). In contrast to the predictions of the carbonnutrient balance hypothesis, the maximum production was observed under moderate N stress. In addition, steady state fugacity-based models were employed to investigate ANTX’s distribution and fate in freshwater ecosytems. ANTX was not found to be very persistent in aquatic ecosystems and did not appear to bioaccumulate in fish, at least not from the dissolved phase.

Idiosyncratic drug reactions (IDRs) represent a significant medical problem and pose a great challenge to drug development. Circumstantial evidence suggests that, in most cases, reactive metabolites of the drug are responsible. The major focus of this thesis is the identification of reactive metabolites and the synthesis of analogs required to test several hypotheses related to involvement of metabolism and covalent binding in the mechanisms of IDRs. Minocycline is unique among tetracyclines in causing a significant incidence of a lupus-like syndrome and autoimmune hepatitis. In this study, we demonstrated that minocycline is oxidized to reactive intermediates by myeloperoxidase/H2O2/Cl-, HOCl, horseradish peroxidase/H2O2, or hepatic microsomes. When trapped with N-acetylcysteine (NAC), two adducts with protonated molecular ions at m/z 619 were isolated and analyzed by NMR. One represents attack of the aromatic D ring by NAC meta to the N, N-dimethylamino group, implying that the reactive intermediate was a quinone iminium ion. The other adduct, which was not observed when minocycline was oxidized by hepatic microsomes, indicates that the NAC is attached at the junction of the B and C rings, suggesting that the HOCl added across the double bond of the B ring leading to a reactive molecule, and then NAC displaced the chloride ion. Nevirapine, an anti-HIV drug, is associated with idiosyncratic skin rashes in humans. The goal of this project was to investigate whether the 12-hydroxylation pathway is responsible for the skin rash. To test a part of this hypothesis, 12-trideuteronevirapine, 12-OH-NVP sulfate, and several other analogs of nevirapine were synthesized. D-penicillamine is known to cause idiosyncratic autoimmune reactions in humans. The goal of this project was to test whether D-penicillamine covalently binds to macrophages and triggers downstream events leading to autoimmunity. To test a part of this hypothesis, D-penicillamine conjugated to biotin was synthesized. In summary, reactive metabolites of minocycline were found that likely explain why minocycline has an IDR profile unique among the tetracyclines. In addition, analogs of nevirapine and D-penicillamine required for mechanistic studies of nevirapine and D-penicillamine-induced IDRs were synthesized. These studies provide additional support for the involvement of reactive metabolites in the mechanisms of IDRs.

GnRH peptides have been discovered in a wide variety of vertebrate and invertebrate organisms, and work is ongoing to characterize additional unique isoforms. This dissertation describes the investigation of reversed-phase chromatographic and mass spectrometric behavior of GnRH peptides, the development and application of an LC-MS/MS method for qualitative identification of GnRH peptides, and the comprehensive validation of an LC-MS/MS method for simultaneous, quantitative measurement of hydroxyproline9GnRH (Hyp9GnRH) and mammalian GnRH (mGnRH) in rat brain tissues. Chromatographic and mass spectrometric behavior of GnRH isoforms was characterized for six GnRH model peptides. Using reversed-phase high performance liquid chromatography (HPLC), nearly complete separation of the model GnRH peptides was achieved. Evaluation of electrospray source conditions indicated that certain parameters can be adjusted to affect the abundance of selected charge states and improve response. Using the conditions found to be optimal for GnRH peptides in general, a method was developed to facilitate characterization of novel GnRH isoforms or confirm the identity of known isoforms. Fragmentation patterns for six model GnRH isoforms were examined to determine what portion of the primary sequence could be elucidated by de novo sequencing, and a simple solid phase extraction protocol was developed to isolate the model GnRH compounds from tissue samples. Application of the method to rat brain samples resulted in successful isolation and structural confirmation of hydroxyproline9GnRH and mammalian GnRH. A quantitative method for the determination of concentrations of hydroxyproline9GnRH and mammalian GnRH in rat brain tissue was developed and rigorously validated. Guinea pig brains were found to be a suitable substitute matrix for rat brains, and accuracy and precision were determined after four validation runs. Stability of both peptides in samples over long-term storage and under experimental conditions were evaluated, and the LC-MS/MS method was compared to an enzyme-linked immunoassay. Thirty-one brains from Sprague-Dawley rats were analyzed using the LC-MS/MS procedure and compared to published results for Hyp9GnRH and mGnRH.