L'ypérite est une arme chimique de guerre de la famille des vésicants. Sa facilité de synthèse et l'existence de stocks importants dans le monde en font une menace à la fois pour les populations civiles et les militaires. Cette menace est renforcée par le fait qu'à l'heure actuelle il n'existe pas d'antidote efficace contre ce toxique de guerre. L'alkylation de l'ADN par l'ypérite aboutit à la formation d'adduits. L'objectif de cette thèse a consisté à mettre au point une méthode de détection quantificative de ces adduits par chromatographie liquide haute performance couplée à la spectrométrie de masse en tandem (HPLC-MS/MS) et d'appliquer cette méthode à l'étude de leur formation et de leur persistance après une exposition cutanée chez la souris SKH-1. Les résultats ont montré dans la peau exposée que la fréquence des adduits était maximale dès 6h post-exposition. Toutefois, leur persistance était relativement longue puisqu'ils étaient toujours détectables 3 semaines après exposition. Une diffusion radiale de l'ypérite a été mise en évidence par la détection des adduits qu'elle forme dans des échantillons de peau non directement exposés. Les adduits ont été également détectés dans plusieurs organes internes. La fréquence maximale des adduits a été mesurée 6h ou J1 post-exposition. Ils ont été décelés jusque J21 post-exposition. Les résultats ont montré qu'il se formait plus d'adduits dans le cerveau et les poumons que dans les reins, la rate et le foie. La persistance des adduits dans le cerveau et les poumons était moindre après la détersion de la peau exposée, illustrant ainsi la constitution dans cette dernière d'un réservoir d'ypérite. La mesure des activités de réparation de l'ADN a montré que l'ypérite exerçait une double action génotoxique, à savoir formation de dommages à l'ADN et inhibition des activités de réparation. / Sulphur mustard is a chemical warfare which belongs to the vesicants family. Its easy synthesis and the existence of important stocks in the world make it a threat for both the general population and militaries. This threat is reinforced by the fact that currently there is not efficient antidote against this war toxic. DNA alkylation by sulphur mustard leads to adducts formation. The objective of this thesis consisted in developing a method of quantification of these adducts by high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) and to apply this method to the study of the formation and persistence of the adducts after cutaneous exposure in SKH-1 mouse. Results have shown in exposed skin that adducts frequency was maximal as soon as 6h post-exposure. A radial diffusion of sulphur mustard was highlighted by the detection of adducts it forms in skin samples non-directly exposed. Adducts were also detected in several internal organs. Maximal frequency was measured at 6h or d1 post-exposure. They were detected until d21 post-exposure. Results have shown that adducts were produced in larger amount in brain and lungs than in kidneys, spleen and liver. The persistence of adducts was lower in brain and lungs after the detersion of exposed skin, thus illustrating the constitution of a reservoir of sulphur mustard in this tissue. Measurement of DNA repair activities showed that suilphur mustard behave as a two-edge sword genotoxic, namely formation of DNA damages and inhibition of repair activities.

A ondansetrona (1,2,3,9-tetrahidro-9-metil-3-[(2-metil-1H-imidazol-1-il)metil]-4H-carbazol-ona) é o primeiro fármaco da classe dos antagonistas seletivos dos receptores de serotonina 5-HT3. É utilizada na prevenção de náusea e vômito induzidos por agentes quimioterápicos. O objetivo deste estudo foi avaliar a equivalência terapêutica através da análise da bioequivalência de dois produtos contendo 8 mg de ondansetrona, sendo um sob a forma de comprimidos de liberação convencional e outro sob a forma de comprimidos de desintegração oral, produzidos por laboratórios distintos. O ensaio de bioequivalência entre o produto teste (Vonau® flash) e o produto referência (Zofran®) foi do tipo randomizado, cruzado e aberto. Os produtos foram administrados por via oral aos voluntários em dose única de 8 mg de ondansetrona. Amostras de sangue foram coletadas até 24 horas após a administração e analisadas através de método de cromatografia líquida de alta eficiência acoplada a um detector de massas. As curvas médias de decaimento plasmático dos produtos teste (Vonau® flash) e referência (Zofran®) e as médias dos parâmetros farmacocinéticos Cmax (referência: 31,88 ng/mL; teste: 30,42 ng/mL); tmax (referência: 1,99 h; teste: 2,15 h), ASC0-t (referência: 227,66 ng×h/mL; teste: 223,68 ng×h/mL) e ASC0- (referência: 252,76 ng×h/mL; teste: 248,22 ng×h/mL) apresentaram-se semelhantes. O intervalo de confiança 90 % para a razão de Cmax (87,5 % - 103,8 %), ASC0-t (89,3 % - 107,2 %) e ASC0- (89,7 106,0 %) encontram-se entre 80 125 %, dentro dos limites propostos pela ANVISA. Conclui-se que os produtos teste e referência são bioequivalentes, podendo ser administrados de forma intercambiável, sem prejuízo do efeito terapêutico. / Ondansetron (1,2,3,9-tetrahydro-9-methyl-3-[2-methyl-1H-imidazol-1yl)methyl]-4H-carbazol-one is the first drug of the class of antagonists selective receptor 5-HT3. It is used in the prevention of nausea and vomiting caused by chemotherapy agents. The purpose of this study was to evaluate the therapeutic equivalence examining the bioequivalence of two products containing 8 mg ondansetron, one in the conventional release tablet, and other in oral disintegration tablet produced by different laboratories. The bioequivalence assay between the test product (Vonau® flash) and reference product (Zofran®) was randomized, crossover and open study. The medication was administered in a single dose of 8 mg of ondansetron. Blood samples were collected until 24 hours after administration and analyzed using a validated high-performance liquid chromatographic method with mass spectrometer detection. The average plasmatic decay curves obtained for the test product (Vonau® flash) and reference product (Zofran®) and the averages of pharmacokinetics parameters Cmax (reference: 31.88 ng/mL; test: 30.42 ng/mL); tmax (reference: 1.99 h; test: 2.15 h), AUC0-t (reference: 227.66 ng×h/mL; test: 223.68 ng×h/mL) and AUC0- (reference: 252.76 ng×h/mL; test: 248.22 ng×h/mL) has been similar. The 90 % confidence intervals for the ratio of Cmax (87.5 % - 103.8 %), AUC0-t (89.3 % - 107.2%) and AUC0- (89.7 % - 106.0 %) values for the test and reference products are within the 80 125 % interval proposed by ANVISA. It was concluded that the test and reference products are bioequivalent and can be considered interchangeable in medical practice, without prejudice to the therapeutic effect.

A ciclobenzaprina é um relaxante muscular de ação central estruturalmente similar aos antidepressivos tricíclicos. O objetivo deste trabalho foi avaliar a bioequivalência de comprimidos contendo 10 mg de cloridrato de ciclobenzaprina em voluntários sadios. O estudo de bioequivalência entre o produto teste (Miosan®) e referência (Flexeril®) foi do tipo randomizado, aberto e cruzado. Os produtos foram administrados por via oral aos voluntários em dose única de 10 mg de cloridrato de ciclobenzaprina. Amostras de sangue foram coletadas até 240 horas após a administração do fármaco e quantificadas por método previamente validado através de cromatografia líquida de alta eficiência acoplada a um detector de massas. As curvas médias de decaimento plasmático dos produtos teste (Miosan®) e referência (Flexeril®) foram semelhantes ASC0-t (teste: 193,00 ngxh/mL; referência: 191,66 ngxh/mL) e ASC0∞ (teste: 211,34 ngxh/mL; referência: 209,35 ngxh/mL). Assim como os parâmetros farmacocinéticos relativos à absorção de ciclobenzaprina, Cmax (teste: 7,16 ng/mL; referência: 6,95 ng/mL), tmax (teste: 4,61 h; referência: 4,48 h), Ka (referência: 0,79; teste: 0,67) e t(1/2)a (referência: 1,79 h; teste: 2,02 h). Os parâmetros farmacocinéticos relativos à eliminação plasmática de ciclobenzaprina Cl (teste: 31,15 L/h; referência: 31,73 L/h), Vd (teste: 1378,54 L e referência (1357,87 L), kß (referência: 0,08; teste: 0,08), t(1/2)ß (referência: 9,43 h; teste: 9,20 h), k&#947 (referência: 0,02; teste: 0,02) e t(1/2)&#947 (referência: 32,92 h; teste: 31,67 h) também apresentaram-se semelhantes entre os dois produtos. A análise multivariada realizada por meio da análise de variância (ANOVA), para a avaliação dos efeitos produto, grupo e período, revelou a ausência destes efeitos, indicando que o delineamento do estudo foi adequado. Os resultados do intervalo de confiança (I.C. 90 %) para a razão de Cmax (93,0 % - 112,0 %), ASC0-t(92,6 % - 111,1 %) e ASC0&#8734 (93,1 % - 110,4 %), encontram-se dentro dos limites estabelecidos pela ANVISA e FDA (80 - 125 %). A análise estatística dos parâmetros Cmax, ASC0-t e ASC0-&#8734 indicam que não há diferenças entre os dois produtos contendo 10 mg de cloridrato de ciclobenzaprina. Com base nos resultados deste estudo, conclui-se que os produtos avaliados são bioequivalentes e podem ser considerados intercambiáveis na terapêutica. / Cyclobenzaprine is a centrally acting muscle relaxant that has similarity with a tricyclic antidepressant. The purpose of this study was to evaluate the bioequivalence of two brands of cyclobenzaprine 10 mg tablets in healthy volunteers. The procedure of bioequivalence between test product (Miosan®) and reference product (Flexeril®) was a randomized, open and crossover study. The products were administered in a single oral dose of 10 mg of cyclobenzaprine hydrochloride to healthy volunteers. Blood samples were collected until 240 hours after administration and quantified by validated method using high-pressure liquid chromatography with mass spectrometric detection. The average plasmatic decay curves of test (Miosan&#174) and reference (Flexeril&#174) products were similar ASC0-t (test: 193,00 ngxh/mL; reference: 191,66 ngxh/mL), in the same way that absorption parameters Cmax (test: 7,16 ng/mL; reference: 6,95 ng/mL), tmax (test: 4,61 h; reference: 4,48 h), Ka (reference: 0,79; test: 0,67) e t(1/2)a (reference: 1,79 h; test: 2,02 h). The elimination parameters Cl (test: 31,15 L/h; reference: 31,73 L/h), Vd (test: 1378,54 L e reference (1357,87 L), k&#914 (reference: 0,08; test: 0,08), t(1/2)&#946 (reference: 9,43 h; test: 9,20 h), k&#947 (reference: 0,02; test: 0,02) e t(1/2)&#947 (reference: 32,92 h; test: 31,67 h) were similar between products too. The multivariate analysis accomplished trough analysis of variance (ANOVA), for assessment of product, group and period effects, revealed the absence of any of these effects in the present study, indicating that the crossover design was properly performed. The 90 % confidence intervals for the ratio of Cmax(93,0 % - 112,0 %), AUC0-t(92,6 % - 111,1 %) and AUC0&#8734 (93,1 % - 110,4 %) values for the test and reference products are within the 80 - 125 % interval proposed by ANVISA e FDA. Statistical analysis of Cmax, AUC0-t e AUC0-&#8734 parameters indicated no significant difference between two brands of 10 mg cyclobenzaprine hydrochloride products. Based in the results of this study, we can conclude that the two products are bioequivalent and can be considered interchangeable in the medical practice.

Muchas de las actividades humanas contribuyen al deterioro del medio ambiente debido a la gran variedad de residuos químicos vertidos. Algunas de estas sustancias químicas son bastante persistentes y causan serios efectos en los animales y en la salud humana en un largo período de tiempo, incluso cuando están presentes en concentraciones muy bajas (Kuramitz et al, 2002), como ocurre con los disruptores endocrinos (DEs). Un disruptor endocrino (DE), conforme a la definición de la Comisión de las Comunidades Europeas, es una sustancia exógena o una mezcla que altera la(s) función(es) del sistema endocrino y consecuentemente causa efectos adversos a la salud en un individuo o en su descendencia o en parte de la población (CEC, 1999). Como efectos adversos a la salud, los estudios médicos han demostrado en los últimos años un deterioro en la capacidad reproductiva humana en los países más industrializados; un aumento en la incidencia de alteraciones en el desarrollo de los órganos genitales; la aparición de la menstruación en edades cada vez más precoces (menarquia precoz); mayor frecuencia de la endometriosis; y el cáncer en los órganos que dependen de las hormonas, como es el caso de la mama, de la próstata, del testículo y del ovario. A propósito, el cáncer de ovario es una de las principales causas de mortandad en el mundo occidental (Aranda, 2004). Debido a la actividad estrogénica, tanto los detergentes como los esteroides han sido incluidos en las diversas listas preliminares de los compuestos DEs. El nonilfenol (NP) (compuesto sintético que posee actividad estrogénica, actúa como DE y es ampliamente usado como materia bruta para la fabricación de detergentes, anti-oxidantes de plástico, pesticidas y como un agente suplementario y estabilizador del cloruro de polivinilo (PVC) (Kuramitz et al, 2002)) ha sido clasificado como sustancia peligrosa prioritaria en el campo de la política del agua por la Estructura Directiva del Agua de la Comunidad Europea 2000/60/EC y por la decisión final de la Unión Europea nº 2455/2001/EC. La Directiva 2008/105/CE establece que el NP deberá presentar, como valor medio anual máximo, la concentración de 0,3 μg/L y como concentración máxima admisible, el valor de 2,0 μg/L, tanto para las aguas superficiales continentales como para otras aguas superficiales. La Agencia de Protección Ambiental (EPA, 2006) establece, en el criterio de calidad del agua, los valores límites de concentración de NP en agua dulce: 28 μg/L (a corto plazo) y de 6,6 μg/L (media de 4 días), para proteger la vida acuática de los efectos adversos. La estrona (E1) (estrógeno natural, excretado por el hombre y por la mujer a través de la orina y de los excrementos, está presente en muchas aguas y efluentes domésticos, presenta alta persistencia en el ambiente y alto potencial como DE (Schäfer and Waite, 2002)) está en la lista nº 3 de contaminantes químicos (CCL 3 List) de la EPA (2001), pero no existen reglamentaciones que limiten la concentración de este contaminante en el agua. Lo que existe es una indicación de que concentraciones de E1, en el rango entre 0,01 y 0,10 μg/L, ejercen efectos estrogénicos en peces (Routledge et al., 1998; Petrovic et al., 2004). Como la cuestión de la contaminación del agua por los DEs es un problema emergente y muy grave, por la perturbación que provoca en el sistema hormonal humano, el objetivo de este trabajo es comparar la eficiencia de la eliminación del NP y de la E1 de las aguas superficiales de un río brasileño, mediante tratamientos, a escala de laboratorio, como: las técnicas de membrana de nanofiltración, de ultrafiltración, el tratamiento convencional (coagulación, floculación, sedimentación y filtración en arena) y el carbón activo en polvo y en grano, como adsorbentes, buscando contribuir con conocimientos para la mejoría de la calidad del agua potable y consecuentemente a la mejoría de la calidad de vida / Many human activities contribute to environmental degradation due to the wide variety of chemical waste dumped. Some of the chemicals are quite persistent and cause serious effects on animal and human health over a long period of time, even when present in very low concentrations (Kuramitz et al, 2002), like endocrine disruptors (EDs). An endocrine disruptor (ED) as defined by the Commission of the European Communities, is an exogenous substance or mixture that alters the function(s) of the endocrine system and consequently causes adverse health effects in a person or their progeny or part of the population (CEC, 1999). As adverse health effects, medical studies have shown in recent years a decline in human reproductive capacity in most industrialized countries; an increase in the incidence of abnormalities in the development of the genitals; the appearance of menstruation increasingly earlier (early menarche); increased frequency of endometriosis; and cancers in organs that depend on hormones, such as breast, prostate, testicle and ovary. By the way, ovarian cancer is a major cause of death in the Western world (Aranda, 2004). Due to estrogenic activity both detergents and steroids have been included in the preliminary lists of various ED compounds. Nonylphenol (NP)(synthetic compound that has estrogenic activity, acts as a ED and is widely used as raw material for manufacturing detergents, plastic anti-oxidants, pesticides and as an additional agent and stabilizer of polyvinyl chloride (PVC) (Kuramitz et al, 2002)) has been classified as priority hazardous substance in the field of water policy by the European Community Water Framework Directive 2000/60/EC and by the final decision of the European Union N. 2455/2001/EC. Directive 2008/105/EC provides that NP shall be limited, as an annual average, to the maximum concentration of 0.3 μg/L and to a maximum allowable concentration of 2.0 μg/L, both for inland surface and other surface waters. The Environmental Protection Agency (EPA, 2006) provides, regarding water quality criterion, the NP concentration limits in fresh water: 28 μg/L (short term) and 6.6 μg/L (average of four days) to protect aquatic life from adverse effects. Estrone (E1) (natural estrogen, excreted by men and women in urine and feces, is present in many waters and domestic wastewater, has high environmental persistence and high potential as ED (Schäfer and Waite, 2002)) is include in EPA Contaminant Candidate List 3 (CCL 3) (2001), but there are no regulations that limit concentration of this pollutant in the water. What exists is an indication that E1 concentrations in the range between 0.01 and 0.10 μg/L exert estrogenic effects in fish (Routledge et al., 1998, Petrovic et al., 2004). Since the issue of water pollution by EDs is a very serious emerging problem, for the disturbance it causes in the human hormonal system, the objective of this study is to compare the efficiency of the elimination of NP and E1 from surface waters of a river in Brazil by treatments in laboratory scale, such as the techniques of nanofiltration and ultrafiltration membrane, conventional treatment (coagulation, flocculation, sedimentation and sand filtration) and activated carbon powder and grain as adsorbents, seeking to contribute with knowledge to improve drinking water quality and consequently quality of life.

Die Verwendung anthropogener organischer Spurenstoffe wie beispielsweise Pharmazeutika, Lifestyle-Produkte, Biozide und Pestizide als Indikatoren für die Bewertung der Wasserqualität hat großes Interesse in der Wissenschaftsgemeinde geweckt, und die Verwendung dieser Substanzen als Indikatoren für die Prozessoptimierung, Quellzuordnung und zur Abschätzung des Ausmaßes einer möglichen Kontamination (z. B. den Abwasseranteil von Oberflächen- und Grundwasser) besitzt ein sehr großes Anwendungspotential. Die hier präsentierte Arbeit ist die erfolgreiche und konsequente Weiterführung bestehender Forschungsaktivitäten zur Eignung ausgewählter Spurenstoffe als Indikatoren für die Bewertung der Wasserqualität, ihrem Vorkommen und Verhalten in der Umwelt sowie ihrer Redox-spezifischen Transformation. Um eine Substanz als Indikator verwenden zu können, müssen sensitive und selektive Analysenmethoden verfügbar sein. In der vorliegenden Arbeit wird die Entwicklung einer Multimethode für den Nachweis von 46 basischen, neutralen und sauren Analyten mittels der Hochleistungs-Flüssigchromatographie und Elektronenspray-Ionisation (ESI) mit anschließender Tandem-Massenspektrometrie (HPLC-ESI-MS/MS) beschrieben. Das ausgewählte Analytenspektrum deckt einen weiten Bereich hinsichtlich der Polarität der Stoffe (log Kow <0–5,9) sowie ihrer repräsentierten Kontaminationsquellen ab. Die Besonderheit der entwickelten Methode stellt die simultane Festphasenanreicherung (SPE), Trennung und Detektion aller Analyten dar. Um dieses realisieren zu können, wird das ESI-Interface in beiden möglichen Operationsmodi (+/−) verwendet, so dass pro Probe nur eine Injektion notwendig ist. Die Bestimmungsgrenzen der Methode in Fluss- und Meerwasser liegen im Bereich weniger ng/L. Im weiteren Verlauf der Arbeit wird die hohe Flexibilität der Methode (Integration zusätzlicher Analyten und Anpassung an andere Wassertypen) demonstriert. Im darauf folgenden Abschnitt werden die Ergebnisse eines intensiven Fluss-Monitorings vorgestellt. Der Fokus liegt dabei auf der Korrelation von 41 Spurenstoffen mit Kalium (K+) und deren räumlichen und zeitlichen Varianz. Da Urin je nach K+-Hintergrundkonzentration des Gewässers eine signifikante K+-Quelle darstellen kann, ist in Gewässern mit hohem Abwasseranteil eine positive Korrelation von abwasserbürtigen Stoffen und K+ zu erwarten. Diese Korrelation ist für Stoffe mit folgenden Charakteristika bestätigt worden: 1) Kläranlagenabläufe sind die Hauptquelle der Substanz; 2) Die Fracht der Substanz in der Kläranlage ist nur geringen zeitlichen Schwankungen unterworfen; und 3) Hohe Persistenz der Verbindung bei der Abwasserbehandlung und in der Umwelt. Neben anderen Spurenstoffen zeigen Carbamazepin, Sulfamethoxazol und Tolyltriazol die beste Korrelation. Darüber hinaus sind die K+-Äquivalente der einzelnen Stoffe offensichtlich abhängig von Landnutzung und Bevölkerungsstruktur im Einzugsgebiet des untersuchten Flussabschnitts. Eine Korrelation mit K+ zeigt, dass die Konzentration des korrelierenden Spurenstoffs nur vom Abfluss des Fließgewässers abhängig ist. Nach diesem Konzept könnte die Vorhersage der Konzentration entsprechender Spurenstoffe an bestimmten Flussabschnitten erheblich vereinfacht werden. Analog zu den genannten Charakteristika 1–3 kann der Ansatz zur Quellidentifizierung neu auftretender/identifizierter Substanzen genutzt werden. Darüber hinaus könnten Eintragsfunktionen für die korrelierenden Spurenstoffe hinsichtlich Oberflächenwasser/Grundwasser-Interaktion hergeleitet werden. Dies würde eine realistischere Bewertung der Reinigungsleistung von Anlagen zur (künstlichen) Grundwasseranreicherung ermöglichen. Anschließend wird eine Methode präsentiert, mit Hilfe derer sich das Volumen von schnell transportiertem, unbehandeltem Abwasser in einem Karstaquifer abschätzen lässt. Eine Kontamination mit unbehandeltem Abwasser und die damit verbundene bakterielle Belastung stellen eine ernsthafte Bedrohung für die Trinkwasserqualität und die öffentliche Gesundheit dar. Das Ausmaß einer Kontamination quantifizieren zu können ist allerdings meist problematisch. Daher wurde ein bereits bekannter Massenbilanzansatz der aktuellen Fragestellung angepasst. In die Berechnung der Abwassermenge fließen ein: Die Coffein-Fracht an der Quelle, die übliche Coffein-Belastung in unbehandeltem Abwasser und der tägliche durchschnittliche Trinkwasserverbrauch pro Person im beobachteten Quelleinzugsgebiet. Der entwickelte Ansatz wurde zur Berechnung der täglich zuströmenden Abwassermenge an einem bereits gut charakterisierten Karstaquifer (Gallusquelle, Deutschland) angewendet. Weiterhin werden die Ergebnisse einer Mikrokosmos-Studie zur Transformation des Antibiotikums Sulfamethoxazol (SMX) unter denitrifizierenden Bedingungen vorgestellt. Ein selektiver Reaktionsmechanismus mit den unter denitrifizierenden Bedingungen gebildeten N-Spezies Stickstoffmonoxid (NO) und Nitrit (NO2−) ist die zugrunde liegende Arbeitshypothese und die Bildung der daraus abgeleiteten Transformationsprodukte (TP) 4-Nitro-N-(5-methylisoxazol-3-yl)-benzenesulfonamid (4-Nitro-SMX) und N-(5-methylisoxazol-3-yl)-benzenesulfonamid (Desamino-SMX) während des zeitlichen Verlaufs eines Wasser/Sediment-Batchversuchs wird dargestellt. Beide TPs können auch in Umweltproben nachgewiesen werden. Unter geeigneten Reaktionsbedingungen kann das TP 4-Nitro-SMX zudem zu SMX retransformiert werden. Dies zeigt die hohe Relevanz der vorliegenden Arbeit hinsichtlich des Vorkommens und Verhaltens dieses Antibiotikums in der Umwelt und für das Monitoring der Wasserqualität. Darüber hinaus können Redox-spezifische TPs als Indikatoren für den reaktiven Stofftransport verwendet werden.

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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.

Indiana University-Purdue University Indianapolis (IUPUI) / A version of this thesis was published as: Masters AR, McCoy M, Jones DR, and Desta Z. Stereoselective method to quantify bupropion and its three major metabolites, hydroxybupropion, erythro-dihydrobupropion, and threo-dihydrobupropion using HPLC-MS/MS. J Chromatography B Analyt Technol Biomed Life Sci 1015-1016:201-208, 2016. / Bupropion is a dual dopamine-norepinephrine uptake inhibitor and a nicotine receptor antagonist. Clinically, bupropion is given as a racemate for the management of depression, smoking cessation aid, and for the management of weight. Bupropion has also been targeted as a phenotypic probe of CYP2B6 activity. Bupropion metabolites are formed via oxidation (4-hydroxybupropion) through CYP2B6, and reduction (erythro- and threo-dihydrobupropion) through carbonyl reductases. These metabolites exhibit pharmacological activity, but little is known regarding their stereoselective disposition due to the lack of a chiral assay. A novel reversed phase chiral-HPLC-MS/MS method involving a simple liquid-liquid extraction procedure and a small plasma sample volume (50µL) was developed that allowed simultaneous separation and quantification of enantiomers of bupropion, 4-hydroxybupropion, and those of threo- and erythro-dihydrobupropion in human plasma. This method was successfully implemented to determine the unique stereoselective disposition of bupropion and its metabolites in 15 human volunteers administered a single 100 mg oral dose of racemic bupropion. Significant differences (p<0.05) in the stereoselective metabolism were observed for all of the enantiomers. The highest plasma exposure (AUC0-∞) was (2R, 3R)-4-hydoxybupropion, almost 65 fold higher, than (2S, 3S)-4-hydoxybupropion, and over 32 fold greater than the parent R-bupropion. The second highest plasma exposure was threo-dihydrobupropion A, which was almost 5 fold higher than threo-dihydrobupropion B. (Nomenclature of the enantiomers for erythro- and threo-dihydrobupropion was based on the chromatography of the first eluting peak as “A” and the second eluting peak as “B”.) Threo-dihydrobupropion A and B showed the most significant difference between the racemic and enantiomer profiles. Although the AUC was greater for threo-dihydrobupropion B, threo-dihydrobupropion A had a significantly (p<0.05) higher Cmax. The half-life for threo-dihydrobupropion A and erythro-dihydrobupropion A were the longest for all analytes, which could indicate accumulation in multiple dosing. The importance of this study was, for the first time, to be able to characterize the stereoselective metabolism of bupropion and its three major metabolites. This new method and subsequent pharmacokinetic data should enhance further research into bupropion stereoselective metabolism, drug interactions, and effect. / A version of this thesis was published as: Masters AR, McCoy M, Jones DR, and Desta Z. Stereoselective method to quantify bupropion and its three major metabolites, hydroxybupropion, erythro-dihydrobupropion, and threo-dihydrobupropion using HPLC-MS/MS. J Chromatography B Analyt Technol Biomed Life Sci 1015-1016:201-208, 2016.

Voxelotor (GBT440) is a haemoglobin S polymerization inhibitor used to treat anaemia in sickle cell disease. Due to an increase of arterial oxygen saturation as well as serum erythropoietin and haemoglobin, the World Anti-Doping Agency included voxelotor in the list of prohibited substances and methods in 2023. The objective of the present study was to identify and characterize metabolites of voxelotor to detect a potential misuse by athletes. The biotransformation was studied in vitro using the human hepatocellular cell line HepG2 and pooled human liver microsomes. The metabolites were analysed using high-performance liquid chromatography (high-resolution) mass spectrometry. In total, three phase I metabolites and six phase II metabolites (resulting from glucuro-conjugation and O-methylation) were formed by the HepG2 cells in a time-dependent manner, and two phase I metabolites were generated by the liver microsomes, among them one also found in the HepG2 incubations. A reduced metabolite and the glucuro-conjugate of a reduced metabolite were the most abundant formed by HepG2 cells. In addition, metabolites resulting from mono-hydroxylation, reduction and O-methylation in different combinations were identified. Voxelotor was also found as glucuro-conjugate with a low abundance. With the spectrometric behaviour of voxelotor and its in vitro metabolites described herein, an implementation in doping control screening and, consequently, a detection of an abuse in an athlete urine sample might be possible.

Das Ziel dieser Arbeit war die Untersuchung des enantiomerenspezifischen Umweltverhaltens des Flammschutzmittels Hexabromcyclododecan (HBCD). Zu Beginn erfolgte daher die Optimierung und Validierung eines enantiomerenspezifischen Analysenverfahrens für die Bestimmung von HBCD in Biota. Die errechneten mittleren Wiederfindungen lagen im Bereich von 100-102 % und die Nachweisgrenzen zwischen 0,131 und 0,255 pg g-1. Untersuchungen zur ubiquitären Verteilung von HBCD erfolgten an Eiern der Silbermöwe deutscher Nord- und Ostseeinseln (Probenahme 1988-2008). In allen Fällen dominierte alpha-HBCD das Diastereomerenmuster, wobei eine bevorzugte Anreicherung von (-)-alpha-HBCD sowie ein zeitlicher Trend aller Enantiomeren-Gehalte festgestellt wurde. Zur Klärung der Frage einer Bioakkumulation sowie -isomerisierung der HBCD-Stereoisomere erfolgten Langzeit-Fütterungsversuche an Spiegelkarpfen. Die Untersuchungen ergaben eine signifikante Akkumulation des jeweils gefütterten HBCD-Enantiomers, jedoch konnte die Hypothese der Bioisomerisierung nicht bestätigt werden. Ein weiterer Schwerpunkt lag in Untersuchungen zur cytochromabhängigen enantiomerenspezifischen Biotransformation von HBCD im Rahmen des Metabolismus an Lebermikrosomen diverser Spezies. Hier konnte gezeigt werden, dass HBCD dem Phase I-Metabolismus unterliegt und hydroxyliert wird. Dabei weist jedes HBCD-Enantiomer ein spezifisches Metabolitenmuster auf, was eine Zuordnung der hydroxylierten Verbindungen zum entsprechenden HBCD-Enantiomer erlaubt. Anhand von Zeitreihen sowie der Berechnung von Halbwertszeiten konnte der Verdacht eines enantiomerenspezifischen Metabolismus in Richtung einer Anreicherung von (-)-alpha- und (+)-gamma-HBCD bestätigt werden. Inkubationsansätze mit reinen Cytochrom (CYP)-Isoformen sowie molekülmechanische Berechnungen legen die Vermutung nahe, dass dem CYP3A4 eine Schlüsselrolle bei der Metabolisierung von HBCD zukommt. / The main emphasis of this thesis was on the enantio-specific environmental behaviour of the polybrominated flame retardant hexabromocyclododecane (HBCD). Initially, an enantio-specific analytical method for the determination of HBCD in biota was optimised and validated. The calculated mean recoveries ranged from 100 to 102 % and the limits of detection are in the range of 0.131 to 0.255 pg g-1. First investigations of the ubiquitous environmental distribution of HBCD were performed using herring gull eggs from different islands in the North and Baltic Sea (sampling 1988 to 2008). In all cases alpha-HBCD was the predominant diastereomer. Significant deviations from the racemic mixture revealed a preferred enrichment of the first eluting (-)-alpha-HBCD. In addition, a temporal trend of HBCD levels was observed. To clarify the issue of accumulation as well as bioisomerisation of HBCD stereoisomers, a long-term feeding study with mirror carps was performed. The results showed an accumulation of each initially fed HBCD enantiomer, but hypothesis of a bioisomerisation could not be confirmed. Another important focus of this work was to study the cytochrome-dependent enantio-specific biotransformation of HBCD enantiomers in various species of liver microsomes. It was shown that HBCD is subject to phase I metabolism. In the course of this process, HBCD is metabolised to hydroxylated products, whereas each HBCD enantiomer results in a specific metabolite pattern allowing the allocation of the corresponding hydroxylated compounds. Investigation of time series as well as the calculation of half-lives, the hypothesis of an enantio-specific metabolism towards an enrichment of (-)-alpha- and (+)-gamma-HBCD could be confirmed. Incubation mixtures with pure cytochrome (CYP) isoforms, as well as molecular mechanic calculations suggest that CYP3A4 plays a key role in the biotransformation processes of HBCD.