Background: Measurement of plasma metanephrine, normetanephrine and 3-methoxytyramine is useful in the diagnosis of phaeochromocytomas, but many assays require a large volume of plasma due to poor assay sensitivity, and often require lengthy sample preparation. Our aim was to develop a method for measurement of plasma metanephrines using a small sample volume with minimal hands-on preparation. Methods: Samples were deproteinised using 10 K spin filters prior to online solid phase extraction using a Waters Acquity UPLC Online SPE Manager (Waters, Manchester, UK) coupled to a Waters Xevo TQ-S mass spectrometer (Waters, Manchester, UK). The assay was validated and results compared to a previously published method. Results: We achieved a limit of quantification of 37.5 pmol/L for metanephrine and 3-methoxytyramine and 75 pmol/L for normetanephrine using only 150 mL of sample. The assay was linear up to 30,000 pmol/L for all analytes and in a method comparison study results showed good agreement with a previously published LC-MS/MS assay. Conclusions: We have developed a simple method for measurement of plasma metanephrine, normetanephrine and 3-methoxytyramine using only 150 mL of sample. There is minimal hands-on sample preparation required and the assay is suitable for routine use in a clinical laboratory.

Das übergeordnete Ziel dieser Promotion war die Untersuchung und Kompensation des Matrixeffekts für die Analytik von Arzneimitteln in komplexen Probenmatrices mit LC-ESI-MS/MS-Technik. Zunächst konnte eine einfache analytische Methode für eine breite analytische Anwendbarkeit entwickelt werden. Es zeigte sich jedoch, dass die Matrixeffektkompensation zu einem Mehraufwand bei der Probenvorbereitung führt. Deshalb wurde anschließend der Mechanismus des Matrixeffektes auf die LC-ESI-MS/MS-Technik genauer untersucht. Die gewonnenen Erkenntnisse wurden anschließend eingesetzt, um eine einfache alternative Quantifizierungsmethode mittels der PCI eines internen Standards zu entwickeln. Im ersten Teilprojekt wurde eine LC-ESI-MS/MS-Methode für die Analytik von häufig verschriebenen Antibiotika in Abwasserproben der Stadt Dresden entwickelt. Da weder Vergleichsmatrix für Abwasser zur Verfügung stand, noch für alle Zielanalyte isotopenmarkierte Standards erhältlich sind, wurde der stark variierende Matrixeffekt der Abwasserproben mittels der Standardaddition kompensiert. Die Ergebnisse der Methodenentwicklung zeigen, dass eine genaue und flexible Methode entwickelt werden konnte, die Matrixkompensation jedoch zu einem erhöhten Zeit- und Materialaufwand führt. Es wurde deutlich, dass neben bisher genutzten Kompensationsmethoden für den Matrixeffekt, wie Standardaddition und interner isotopenmarkierter Standards, neue alternative Strategien getestet werden müssen. In dem zweiten Teilprojekt wurde daher der Matrixeffektmechanismus von Urin-, Plasma- und verschiedenen Abwasserproben bei der Messung von verschiedenen Arzneimitteln mittels LC-ESI-MS/MS analysiert. Die Ergebnisse der Untersuchungen mittels „post-column infusion“ konnten bisherige Erkenntnisse zu Matrixeffektmechanismen bestätigen und das Verständnis vertiefen. Matrixeffekte sind von der jeweiligen Zusammensetzung der Probenmatrix abhängig, aber auch substanzspezifisch. Dabei kommt es zwischen Analyt und Begleitsubstanzen zu einer Konkurrenz um freie Ladungsträger oder zu einer veränderten Anordnung/Verteilung innerhalb der ESI-Spray-Tröpfchen. Gleichzeitig zeigten die Ergebnisse, dass es auch andere Mechanismen, wie z. B. Ladungstransfers zwischen Analyt und Begleitsubstanzen, geben muss. Schließlich wurden die Ergebnisse des zweiten Teilprojekts in einer innovativen Methodenentwicklung zur Matrixkompensation und zur Quantifizierung von 16 Arzneimitteln in Urinproben verwendet. Der Matrixeffekt der Substanzen mit vergleichbarer Signalsuppression konnte über einen einzelnen nachsäuleninfundierten internen Standard kompensiert werden. Die Ergebnisse zeigen einen deutlichen Vorteil der entwickelten Methode gegenüber Matrix-Kalibrierung in Präzision und Richtigkeit oder dem Einsatz von isotopenmarkierten internen Standards in Aufwand der Methodenentwicklung und Verbrauch von Standardsubstanzen. Die Ergebnisse der vorliegenden Arbeit zeigen die Bedeutung, Komplexität und den Einfluss der Matrixeffekte in der Anwendung der LC-ESI-MS/MS-Technik. Einerseits sind geeignete Methoden für die Minimierung von Matrixeffekten wie Probenvorbereitung und Chromatographie nötig, andererseits müssen Ionisierungsmechanismen, insbesondere die Wechselwirkungen von Zielanalyten und Begleitsubstanzen, zukünftig Gegenstand weiterer Untersuchungen sein. Die Ergebnisse dieser Arbeit liefern wichtige Beiträge zur Verbesserung der Analytik von komplexen Proben mittels der LC-ESI-MS/MS-Technik. / The overall goal of this Ph.D. thesis was to investigate and compensate the matrix effect of the analysis of drugs in complex sample matrices with LC-ESI-MS/MS technique. First, a simple analytical method for a broad analytical applicability was developed for wastewater analysis. However, the matrix effect compensation embraced the main part effort in sample preparation. Therefore, the mechanism of the matrix effect on the LC-ESI-MS/MS technique was examined in more detail. The findings were used to develop an alternative quantification method using post-column infusion of an internal standard substance. In the first project, a LC-ESI-MS/MS method was developed to analyze commonly prescribed antibiotics in wastewater samples of Dresden. Since neither comparison matrix for wastewater nor all isotopically-labeled analogs for the target analytes were available, the strongly varying matrix effect of the wastewater samples was compensated by standard addition. The results show that the developed method is precise and flexible, but the matrix effect compensation leads to an increased expenditure of time and materials. Besides previously used matrix effect compensation methods, such as standard addition and internal isotopically-labeled standard, new alternative strategies need to be tested. Therefore, the matrix effect mechanism of various drugs and sample matrix combinations was examined in the second project using post-column infusion. The results confirmed previous findings on matrix effect mechanisms and deepened our understanding that matrix effects not only depend on the composition of the sample matrix but are also substance-specific. This results to a competition of free charge carriers between analyte and accompanying substances or to an alternated distribution within the ESI spray droplets. Furthermore, the results indicate that there are other mechanisms, such as charge transfer between analyte and concomitant substances. The results of the second project were used to invent a method for matrix effect compensation and quantification of 16 drugs in urine samples. The matrix effects of the substances with comparable signal suppression were compensated by a single post-column infused internal standard. The developed method has a significant advantage over the matrix calibration regarding precision and accuracy as well as the use of isotopically-labeled internal standards in effort of method development and consumption of standard substances. Finally, the results of this work show the importance, complexity and influence of the matrix effects in the application of the LC-ESI-MS/MS-technique. Suitable methods for minimizing matrix effects such as sample preparation and chromatography are needed and ionization mechanisms, in particular the interactions of target analytes with accompanying substances, should be investigated in future studies. The work of this Ph.D. project contributes to the improvement of the analysis of complex samples using the LC-ESI-MS/MS-technique.

Immunoassays basieren auf der Anwendung von Antikörpern, welche selektiv den zu messenden Analyten binden. Die Richtigkeit der erhaltenen Ergebnisse hängt maßgeblich von der Selektivität der Antikörper ab und kann durch Interferenzen gestört werden. In dieser Arbeit wurde eine Methode entwickelt, bei der die Probe mittels Hochleistungsflüssigkeitschromatographie (LC) in Fraktionen aufgetrennt wird und diese Fraktionen anschließend mittels Enzyme-linked Immunosorbent Assay (ELISA) vermessen werden. Dieses Verfahren wurde als LC-ELISA bezeichnet. Das erhaltene Profil aus im ELISA gemessener Analytkonzentration in Abhängigkeit von der Elutionszeit wurde als LC-ELISAgramm bezeichnet und bietet die Möglichkeit, Interferenzen zu erkennen, welche beim ELISA unentdeckt bleiben. Als Modellanalyten für die zu untersuchenden ELISAs dienten Sulfamethoxazol (SMX), Carbamazepin (CBZ) und Estron (E1). Dabei wurden verschiedene Umweltmatrices wie Oberflächenwasser und Abwässer mit dem jeweiligen ELISA vermessen. Es wurde ein Ansatz zur Unterscheidung von spezifischen und unspezifischen Interferenzen in Umweltproben aufgezeigt. Durch diesen Ansatz und Anwendung der sauren Hydrolyse der Probe war es möglich, einen bisher unbekannten SMX-Metaboliten zu detektieren und dessen wahrscheinliche Kreuzreaktivität mit 460 ± 150 % abzuschätzen. Es wurde zudem ein neuer Tracer in einer linearen 13-Stufen-Synthese entwickelt, wobei neuartig die Konjugation der Peroxidase an der N1-Position des SMX erfolgte. / Immunoassays are based on the use of antibodies that selectively bind the analyte. The trueness of the results obtained depends to a great extent on the selectivity of the antibodies and can be affected by interferences. In this study, a method was developed in which the sample is separated into fractions by using high-performance liquid chromatography (LC) and these fractions are measured using an enzyme-linked immunosorbent assay (ELISA). This method was referred to as LC-ELISA. The profile obtained from the measured analyte concentration by ELISA as a function of the elution time was referred to as LC-ELISAgram and offers the possibility to detect interferences which otherwise remain undetected during the ELISA. Sulfamethoxazole (SMX), carbamazepine (CBZ) and estrone (E1) were used as model analytes for the ELISA and LC-ELISA measurements. Various environmental matrices such as surface water and wastewater were examined for their interference in the respective ELISA. The good quantification properties of the validated LC-ELISA have been used to demonstrate an approach to distinguish between specific and non-specific interferences from environmental samples. By this approach and application of acidic hydrolysis of the sample, it was possible to detect a previously unknown metabolite of SMX and estimate its cross-reactivity to probably 460 ± 150%. Furthermore, a new tracer was developed in a linear 13-step synthesis, which resulted in the novel conjugation of the peroxidase at the N1-position of SMX. The new hapten was also used for the synthesis of a novel immunogen.

Vitamin-D-Mangel und -Unterversorgung haben sich zu einem großen Problem für die öffentliche Gesundheit entwickelt, das vor allem auf eine unzureichende Sonnenlichtexposition und eine begrenzte Zufuhr dieses essenziellen Nährstoffs über die Nahrung zurückzuführen ist. Diese "Pandemie" hat zu einem bemerkenswerten Anstieg der Nachfrage nach der Bestimmung des zirkulierenden 25-Hydroxyvitamin-D-Spiegels (25(OH)D) geführt. Die meisten Studien fokussieren sich in erster Linie auf 25(OH)D und betrachten es als Biomarker für den Vitamin-D-Status. Es besteht jedoch ein wachsendes Interesse an der gleichzeitigen Messung mehrerer klinisch bedeutsamer Vitamin-D-Metaboliten wie dem nativen Vitamin D, 25(OH)D-Epimeren, 1,25-Dihydroxyvitamin D und 24,25-Dihydroxyvitamin D. Die Flüssigchromatographie/Tandem-Massenspektrometrie (LC-MS/MS) gilt als "Goldstandard" für die Bestimmung des Vitamin-D-Spiegels und ermöglicht die gleichzeitige Analyse mehrerer Analyten, die für das Verständnis der physiologischen Rolle von Vitamin D und seiner klinischen Auswirkungen unerlässlich sind. In der vorliegenden Doktorarbeit soll verschiedene Aspekte der Vitamin-D-Landschaft erforschen, umfassende Einblicke geben und Herausforderungen innerhalb des Forschungsthemas besprechen. Zunächst lag der Schwerpunkt auf dem Vergleich mehrerer Derivatisierungsreagenzien für die Vitamin-D-Analyse und der Frage, wie sie sich auf die Nachweisempfindlichkeit der Methode und die chromatographische Trennung der getesteten Vitamin-D-Metaboliten auswirken. Ein zweiter Schwerpunkt lag auf der Untersuchung der Stabilität der Derivatisierungsprodukte in Serumextrakten. Der letzte Versuch war die Entwicklung einer LC-MS/MS-Methode, die die Ergebnisse der vorangegangenen Untersuchungen nutzen sollte. Als Ergebnis wurde eine neue Methode vorgestellt, die meines Wissens zum ersten Mal FMP-TS für die chemische Derivatisierung von Vitamin-D-Metaboliten verwendet. / Vitamin D deficiency has emerged as a significant public health concern, attributed largely to insufficient exposure to sunlight and limited dietary sources rich in this essential nutrient. This “pandemic” has led to a notable increase in the demand for assessing circulating levels of 25-hydroxyvitamin D (25(OH)D). Most studies have primarily focused on 25(OH)D, considered it as the vitamin D status biomarker. However, there is a growing interest in simultaneously measuring multiple clinically significant vitamin D metabolites such as the native vitamin D, 25(OH)D epimer, 1,25-dihydroxyvitamin D, 24,25-dihydroxyvitamin D and others. Thus, there is an urgent need to develop analytical methods which will be able to separate and quantify the metabolites of interest accurately and precisely. Liquid chromatography/tandem mass spectrometry (LC-MS/MS) stands out as the “gold standard” technique for vitamin D analysis. Its versatility enables simultaneous analysis of multiple analytes, thereby facilitating the generation of comprehensive vitamin D profiles essential for understanding its physiological role and clinical implications. The present doctoral thesis is designed to explore diverse aspects of vitamin D landscape, providing comprehensive insights and addressing challenges within the research topic. Initially there was a focus on comparing multiple derivatization reagents for vitamin D analysis and discovering how they impact the method’s detection sensitivity and the chromatographic separation of the tested vitamin D metabolites. Moreover, there was a second focal point on examining the stability of the derivatization products in serum extracts. The final attempt was the development of an LC-MS/MS method that would utilize the outcomes of the investigations conducted before. As a result, a new method was introduced utilizing FMP-TS for the chemical derivatization of vitamin D metabolites, a reagent which has not been reported before for vitamin D analysis.

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.

Ce travail de thèse rentre dans le cadre d’un projet de recherche appliquée mené à l’Institut National de Criminalistique et de Criminologie s’intitulant « développement d’un système intégral de détection et d’identification de résidus de tir » visant à doter le laboratoire de balistique chimique d’un outil performant pour la recherche et la caractérisation des résidus de tir aussi bien inorganiques qu’organiques. Comme nous l’expliquons dans le chapitre 1, l’analyse des résidus de tir est aujourd’hui essentiellement basée sur la mise en évidence par microscopie électronique à balayage des particules métalliques provenant de l’amorce. Cette pratique a des limites. Introductif, ce chapitre met en lumière la relation entre criminologie et criminalistique, donne un aperçu de la classification des armes et décrit les relations entre les armes de poing, les munitions, les résidus de tir et la criminalistique. La motivation de l’analyse de résidus organiques et plus particulièrement des stabilisants pour poudres propulsives y est aussi expliquée. Le but principal de ce travail étant le développement d’une méthode d’analyse des stabilisants par spectrométrie de masse en tandem couplée à la chromatographie liquide à haute performance (LC-MS/MS), ce premier chapitre aborde de façon détaillée le principe de fonctionnement d’un spectromètre de masse quadripolaire, qu’il soit avec simple quadripôle ou en tandem, et traite avec une attention particulière les aspects relatifs à la production d’ions, l’acquisition des données et la détection des ions. Le chapitre 2 est consacré aux résultats et discussions. Comme point de départ de tous les travaux ultérieurs, l’optimisation du fonctionnement du spectromètre et plus particulièrement la détermination des « ions précurseurs » et des « ions produits » y est traitée en détails. De plus, l’étude de la fragmentation des ions précurseurs y occupe une place prépondérante et conditionne l’établissement d’une méthode de détection et d’acquisition des données. Dans ce même chapitre sont successivement traités la mise au point de la méthode chromatographique pour l’analyse des stabilisants par LC-MS/MS, le couplage LC-MS/MS, la collecte et préparation des échantillons, dont l’extraction sur phase solide (SPE). Disposant des procédures de traitement des échantillons et d’une méthode LC-MS/MS, il nous a été possible de mener l’étude de faisabilité de l’analyse des stabilisants dans les échantillons prélevés sur les mains d’un tireur. Cette étude fait l’objet d’une proposition de publication jointe en annexe. Pour être exploitable en criminalistique, ce travail est complété par une étude sur les fréquences d’observation des stabilisants pour poudres propulsives sur les mains des non-tireurs. L’ensemble des résultats nous permet de dresser un bilan et proposer des perspectives décrites dans le chapitre 3. A cet égard, nous proposons de poursuivre les essais relatifs aux tests de persistance des résidus de stabilisant sur les mains des tireurs et d’entamer une étude sur la caractérisation des résidus de tir de la nitrocellulose. Du point de vue de l’exploitation des acquis de ce travail, nous suggérons que les méthodes et procédures développées soient mises à l’épreuve dans les affaires nécessitant l’analyse des prélèvements effectués sur les vêtements d’un suspect ou d’une victime. Cette mise à l’épreuve permettra de comparer l’efficacité de la méthode LC-MS/MS à celle de la technique SEM/EDX habituellement utilisée. Le chapitre 4 est consacré aux matériels et méthodes détaillant notamment les procédures de collectes et de traitement des échantillons, les paramètres de fonctionnement du spectromètre de masse et les méthodes analytiques. <br> <br> This thesis deals with a research project entitled "Development of an integral system of detection and identification of gunshot residues”, carried out at the Belgian National Institute of Criminalistics and Criminology (NICC). This project aims at providing the Laboratory of Chemical Ballistics, a powerful tool to detect and identify organic gunshot residues. For instance, the analyses of gunshot residues is presently performed mainly for characterizing metallic particles originating from the primer, using scanning electron microscopy coupled to energy dispersive X-ray microanalysis (SEM-EDX). However this technique has its limits and the goal of the thesis was the development of a method for the analysis of the stabilizers (and their derivatives) present in propellant powders with the use of tandem mass spectrometry coupled to high performance liquid chromatography (LC-MS/MS). The introduction highlights the relations between Criminology and Criminalistics; it gives an outline of the classification of weapons, and describes how handguns, ammunitions, gunshot residues and Criminalistics can be linked when investigating criminal cases. The motivation of the analysis of organic gunshot residues and in particular the propellant powder stabilizers is also explained. Chapter 1 deals in detail with the working principle of a quadrupole mass spectrometer, whether it is with a single quadrupole or composed of a tandem mass spectrometer. Important aspects connected with the ion production, the data acquisition and the ion detection are also considered. Chapter 2 presents the results and discussions. It begins with the optimization of the operating conditions of the spectrometer, and focuses more specifically on the determination of “precursor ions" and "daughter ions”. The study of the precursor ion is more important because it is required for the choice of parameters involved in the data acquisition mode. The development of a liquid chromatography (LC) method and the procedures for collecting and preparing the samples are explained. Moreover, Chapter 2 summarizes the results of a feasibility study of the analyses of stabilizers in samples collected from the hands of a shooter. This study was published in a peer reviewed journal [1]. A complementary study on the frequencies of observation of stabilizers on the hands of non-shooter persons is reported at the end the Chapter. The results allow also proposing perspectives which are described in Chapter 3. In this respect, the characterization of nitrocellulose residues and the study of the persistence of stabilizer residues on the hands of shooters should be undertaken. From the practical point of view, the developed methods and procedures should be tested in real cases which involve the analyses of the samples taken from the clothing of suspects and/or victims. These tests should allow an assessment of the LC-MS/MS method compared to the efficiency of the SEM/EDX technique currently used. Chapter 4 is dedicated to the materials and methods; the sample collection and treatment procedures, as well as the working parameters of the mass spectrometer and the analytical methods are explained in detail. [1]. Désiré Laza, Ph.D.; Bart Nys, Ph.D.; Jan De Kinder, Ph.D.; Andrée Kirsch - De Mesmaeker, Ph.D.; and Cécile Moucheron, Ph.D. Development of a Quantitative LC-MS/MS Method for the Analysis of Common Propellant Powder Stabilizers in Gunshot Residue. J. Forensic Sci, July 2007, Vol. 52, N° 4, 842-850.

An acetonitrile based protein extraction method was developed that demonstrated high efficient and effective removal of high abundant proteins from both human and murine serum. The protein content of the extract was characterised using gel electrophoresis, the Bradford assay and liquid chromatography tandem mass spectrometry (LC-MS/MS) with database searching. Selected reaction monitoring (SRM) analysis was used to quantify the levels of high abundant serum proteins to further validate the extraction methodology. The ACN depletion method, in combination with artificial neural networks (ANNs) data mining software, was applied to a murine growth hormone (GH) gene doping study with the aim of identifying biomarker ions capable of detecting gene doping. The LC-MS and ANNs analysis approach failed to conclusively identify a biomarker to gene doping in the mouse model. However, the application of the same technique to serum from a rhGH administration study in humans, returned models capable of discriminating between rhGH treated placebo states. The ion identified as being the most discriminatory was characterised using mass spectrometry, and was derived from the protein leucine-rich a-2-glycoprotein (LRG). Multiple LRG related tryptic peptides were identified as being up-regulated upon dosing with recombinant human GH (rhGH). A high throughput LC-MS/MS and SRM approach was developed to quantify proteins in human serum. The approach was validated by comparison of LC-MS/MS derived APO A1 concentrations with those obtained using established clinical analyser technologies. The LC-MS/MS methodology was applied to a large cohort of 257 serum samples from two rhGH administration studies performed at Royal Free Hospital . The two administrations included serum samples from 15 individuals who had been dosed daily with rhGH. Serum concentrations of the established rhGH biomarker insulin-like growth factor-I (IGF-I) were quantified by LC-MS/MS and compared well with those determined using two different immunoassay-based methodologies. Serum concentrations of the LRG protein were measured simultaneously with IGF-I and appeared to increase in 14 of the 15 rhGH dosed individuals. Combining the LRG and IGF-I data further increased the separation of rhGH treated and placebo states within each individual, and the application of ANNs analysis showed that the combination of the two proteins increased the discrimination characteristics over using IGF-I alone. The murine equivalent of the LRG protein was identified and SRM transitions for a tryptically derived peptide were developed, along with transitions for monitoring a peptide from the murine IGF-I protein. These transitions were used to quantify the two proteins in the remaining aliquots from a murine GH gene doping experiment, however neither protein appeared to increase in the GH +ve plasmid samples that were analysed.

Progesterone in the mare declines to almost undetectable concentrations in late gestation. It’s metabolized into several pregnanes, some circulating at very high concentrations. Although the function of many pregnanes remains unclear, 5α-dihydroprogesterone and allopregnanolone are bioactive. Measurements of pregnanes in late gestation are typically by immunoassay, although results are confounded by cross-reactivity with related pregnanes. Conversely, liquid chromatography tandem mass spectrometry (LC-MS/MS) allows differentiation of individual pregnanes. The purposes of these studies were: 1) to evaluate the ability of a 5α-reductase inhibitor, dutasteride, to alter pregnane metabolism and pregnancy outcome, 2) to evaluate changes in target pregnanes in late gestation by LC-MS/MS in mares with ascending placentitis, and 3) compare immunoassay and LC-MS/MS detection of pregnanes in late gestation. Our findings suggest that dutasteride significantly altered pregnane metabolism without effects on pregnancy outcome. Pregnane measurement by LC-MS/MS resulted in a significant (p<0.05) differences in assay results, while correlation was observed between immunoassay measurements and actual progesterone concentrations by LC-MS/MS. These studies demonstrate the complexity of pregnane metabolism in late gestation in the mare and the necessity of LC-MS/MS to detect specific changes that immunoassays cannot differentiate.

Introduction: Cytochromes P450 (CYPs) are the major family of enzymes responsible for detoxification and metabolism of a wide range of both endogenous and xenobiotics chemicals in living organisms. The use of CYPs to activate prodrugs to cytotoxins selectively in tumours has been explored including AQ4N, Phortress and Aminoflavone. CYP1A1, CYP1B1, CYP2W1, and CYP4F11 have been identified as expressed in tumour tissue and surrounding stroma at high frequency compared to most normal tissues. Aim is to investigate the differential metabolism of novel chloromethylindoline by high frequency expressed CYPs in tumours. This differential may be exploited to elicit a selective chemotherapeutic effect by metabolising inert small molecules to potent cytotoxins within the tumour environment. Materials and Methods: Sensitive and specific LC/MS/MS techniques have been developed to investigate the metabolism of chloromethylindolines. Recombinant enzymes and transfected cell lines were used to investigate the metabolic profiles with a focus on production of the cytotoxic derivatives of chloromethylindolines. Results: Detailed metabolic studies show that (1-(Chloromethyl)-1,2-dihydropyrrolo [3,2-e]indol-3(6H)-yl)(5-methoxy-1H-indol-2-yl) methanone (ICT2700) and other chloromethylindolines are converted by CYP1A1 mediated hydroxylation at the C-5 position leading to highly potent metabolites. In vitro cytotoxicity studies showed differentials of up to 1000-fold was achieved between CYP1A1 activated compared to the non-metabolised parent molecules. The reactivity of metabolites of ICT2700 was also explored using glutathione as a nucleophile. The metabolites were identified by a combination of LC/MS and LC MS/MS techniques. Investigations using mouse and human liver microsomes show that a large number of metabolites are created though none were shown to be associated with a potential anticancer effect. Studies focused on CYP2W1 show that this isoform metabolised ICT2706 to a cytotoxic species and a pharmacokinetic study showed a good distribution of ICT2706 into mouse tissues including tumour. However metabolism of ICT2726 by CYP2W1 resulted only in a non-toxic metabolite profile and may have potential as a biomarker for functional CYP2W1 in tissues. Preliminary studies show that palmitic acid hydroxylation is a useful marker of functional CYP4F11. Summary and conclusion: The in vitro results show that the chloromethylindolines are a novel class of agent with potential as prodrugs that following specific hydroxylation by CYP1A1 and CYP2W1 are converted to ultra-potent cytotoxins. Other metabolites are also evident which are not cytotoxic. Studies in vivo show that selected chloromethylindolines possess a good pharmacokinetic profile and show potential as prodrug anticancer agents that require activation by CYP1A1 or CYP2W1. The methods, results, progress and suggestions for future work are presented in this thesis.

Abstract: It is well established that ionizing radiation induces a variety of damage in DNA by direct effects that are mediated by one-electron oxidation and indirect effects that are mediated by the reaction of water radiolysis products, e.g., hydroxyl radicals (•OH). In cellular DNA, direct and indirect effects appear to have about an equal effect toward DNA damage. We have shown that ϒ-(gamma) ray irradiation of aqueous solutions of DNA, during which •OH is the major damaging ROS can lead to the formation several lesions. On the other hand, the methylation and oxidative demethylation of cytosine in CpG dinucleotides plays a critical role in the gene regulation. The C5 position of cytosine in CG dinucleotides is frequently methylated by DNA methyl transferees (DNMTs) and constitutes 4-5% of the total cytosine. Here, my PhD research work focuses on the analysis of oxidative base modifications of model compounds of methylated and non methylated oligonucleotides, isolated DNA (calf-thymus DNA) and F98 cultured cell by gamma radiation. In addition, we identified a series of modifications of the 2-deoxyribose moiety of DNA arising from the exposure of isolated and cellular DNA to ionizing radiation. We also studied one electron oxidation of cellular DNA in cultured human HeLa cells initiated by intense nanosecond 266 nm laser pulse irradiation, which produces cross-links between guanine and thymine bases (G*-T*). To achieve these goals, we developed several methods based on mass spectrometry to analyze base modifications in isolated DNA and cellular DNA. / Résumé : Les radiations ionisantes induisent une variété de dommages à l'ADN selon des effets directs, correspondant à une oxydation suite à l’éjection d’un électron, et indirecte, médiés par une réaction avec les produits issus de la radiolyse de l’eau environnante, tels que les radicaux hydroxyles (•OH). Au sein d’une cellule, l’importance relative des effets directs et indirects semble être quantitativement similaire en ce qui concerne les dommages induits à l'ADN cellulaire. Nous avons démontré que l'irradiation par rayons Υ-(gamma) de solutions aqueuses d'ADN, dont l’action délétère est principalement véhiculé e par l’intermédiaire des radicaux hydroxyles, peut induire sur l’ADN la formation de toute une palette de modifications. D'autre part, la méthylation et la déméthylation oxydative de la cytosine au sein de couples de dinucléotides CpG jouent un rôle essentiel dans la régulation des gènes. La position C5 de cette cytosine se retrouve fréquemment méthylée par les méthyltransférases (DNMTs) et constitue alors 4-5% de l’ensemble de la cytosine présente au sein de l’ADN. Mon projet de recherche est centralisé autour de l'analyse de la modification des bases de l’ADN suite à leur oxydation dans des composés modèles constitués d'oligonucléotides méthylés et non-méthylés, puis dans l'ADN isolé (extrait de cellules de thymus de veau) et enfin au sein de cultures cellulaires F98 ayant subies une irradiation par rayons Υ-(gamma). De plus, nous avons identifié une série de modifications spécifiques au groupement fonctionnel 2-désoxyribose de l'ADN résultant de l'exposition de l'ADN isolé et cellulaire aux rayonnements ionisants. Nous avons également étudié les conséquences de l’irradiation par des impulsions lasers nanoseconde à 266 nm de cultures cellulaires de lignée humaine (HeLa). Responsable d’une réaction d’oxydation suite à l’éjection d’un électron, l’identification des modifications induites à l’ADN cellulaire suite à l’irradiation laser a permis de mettre en évidence des pontages ADN-ADN caractéristiques entre les bases guanine et thymine (G*-T*). Pour atteindre ces objectifs, nous avons développé plusieurs méthodes d’analyse des modifications de bases au sein de l’ADN isolé et de l'ADN cellulaire basées sur la spectroscopie de masse.