Philosophiae Doctor - PhD / South African Carpobrotus species have been found to contain hydrolysable tannins, various tlavonoids e.g. rutin and hyperoside, phytosterols and aromatic acids which have a diverse range of pharmacological properties including antimicrobial and, antioxidant activities. The main aim of the thesis was to determine the natural products in C. mellei and C. quadrifidus using chromatographic techniques and electrochemical analysis. The antioxidant activity of both Carpobrotus species was determined by using a superoxide dismutase (SOD) biosensor. ESI-LC-MS was used to separate and determine tlavonoids in C. mellei and C. quadrifidus. 8 tlavonoid compounds: catechin, epicatechin, epicatechin-epicatechin, coumarylquinic acid, isorhamnetin, quercetin-hexose (hyperoside), rutin and myricetin-deoxyhexose were identified. Cyclic and square wave voltammetry were used to detect tlavonoids from C. mellei and C. quadrifidus. Catechin was detected in the ethyl acetate extract of C. mellei and C. quadrifidus. The oxidation potential of the plant extracts were observed at +150.6 mV to +1072.6 mV. The oxidation mechanism proceeds in sequential steps, related to the catechol moiety, -OH groups in C ring and the resorcinol group. The oxidation process of the catechol moiety involves a two electron - two proton reversible reaction and forms o-quinone. This occurs first at low potential and is a reversible reaction. The hydroxyl group in the C ring and resorcinol group oxidise there after and undergo an irreversible reaction. UV-vis and FTIR spectroscopy were used to confirm the presence of catechin in the ethyl acetate extract of both plants. UV -visible spectroelectrochemistry confirmed the oxidation process of catechin at constant potential. Since C. mellei and C. quadrifidus were confirmed to contain flavonoids by ESI-LC-MS and electrochemical analysis, the antioxidant activity was further investigated using a SOD biosensor. The superoxide dismutase (SOD) enzyme was immobilised with 1% Nafion on a platinum electrode. Detection limit and sensitivity of the SOD biosensor were found to be 0.03918 umol L-' and 1.44 !lA (umol i.'):', respectively. The results showed that C. mellei and C. quadrifidus have antioxidant activity, with relative antioxidant capacity (RAC) of 24% and 42%, respectively.

As of today, synthetic cannabinoids are one of the biggest groups of new psychoactive substances.These substances can be used as substitutes for the psychoactive drug cannabis, avoiding the legalrestrictions on cannabis. Furthermore, a variety of synthetic cannabinoids are synthesized with eithersignificant or very minor structural differences, making the detection of said novel drugs hard to keepup with and is therefore of great importance to have standards which help in the identification of theintake of the parent synthetic cannabinoid. In this project, several metabolites ofsynthetic cannabinoids with indole/indazole cores with differentside chains was synthesized. The general strategy used in this project was to N-alkylate the desiredcore followed by amide coupling with L-tert-leucine methyl ester or L-Valine methyl esterhydrochloride which resulted in 8 potential synthetic cannabinoid metabolites.

This master thesis deals with a monitoring of a content of the trichothecene mycotoxins deoxynivalenol, nivalenol, T-2 toxin and HT-2 toxin in malting barley using the LC-MS/MS method. The theoretical part describes general characteristics of mycotoxins and their significant producer filamentous fungus of Fusarium species. Further, important trichothecene mycotoxins and mycotoxins generally which are commonly found in malting barley were also characterized. In the theoretical part of the thesis possibilities for a determination of the mycotoxins by the chromatographic methods were presented too; the immunochemical methods were also mentioned. In the experimental section an analysis of the B type trichothecenes was optimized by LC/APCI-MS/MS and of the A type trichothecenes by LC/ESI-MS/MS. When analyzing 57 samples of different barley varieties the deoxynivalenol reached the highest values (up to 945,2 µg.kg-1), namely in the case of the Sebastian variety with corn as the fore-crop. The highest values of nivalenol, T-2 toxin and HT-2 toxin (138,4 µg.kg-1; 21,8 µg.kg-1 and 68,7 µg.kg-1 respectively) were found in the Prestige variety of barley with winter wheat as the fore-crop. Subsequently a second set of four experimental samples of the Sebastian variety of barley and malt produced from the variety with corn as the fore-crop were analysed. In this group three samples were artificially infected with the filamentous fungi of Fusarium species; the fourth sample was not artificially infected and served as a control sample. Even in the case of the artificially infected samples the deoxynivalenol reached the highest values. The master thesis was implemented in the Research Institute of Brewing and Malting, Plc. in Brno.

This dissertation describes the elucidation and implementation of derivatisation in the quantification of biologically active vitamin D metabolites in limited volume serum and dry blood spot samples (DBS) using the liquid chromatography tandem-mass spectrometry (LC-MS/MS) analytical technique. This manuscript describes in detail the development and validation of an analytical methodology, highlighting the role derivatisation and mass spectrometry plays in the structural characterisation and quantification of vitamin D metabolites. The first chapter reviews comprehensively, the history of vitamin D biosynthesis discovery as an anti-rickets agent, the biochemistry of vitamin D, its metabolic pathway, functions in the different biological systems and the consequences of its deficiency in the body. The second chapter reviews the current methods and techniques utilised for the detection and characterization of vitamin D metabolites, with specific emphasis based on the contribution made by derivatisation and mass spectrometry. A brief introduction to derivatisation is provided, with specific focus on PTAD and Amplifex diene reagents (Cooksontype reagents) used in this study. The importance of sensitivity and selectivity of targeted analytes is described first in detail for underivatised analytes, followed by PTAD and Amplifex derivatised samples. Chapter 2 also describes the importance of vitamin D quantification using liquid chromatography, the strengths and limitations of LC-MS/MS when used in isolation and after derivatisation. Also discussed, is how combining these techniques can overcome inherent limitations in LCMS/MS and enhance analytical performance. In Chapter 3 the materials and methods used and the study design is laid out, describing a brief introduction of the routinely used clinical diagnostics assay enzyme-linked immunosorbent assay (ELISA) as a reference method and is compared to an LC-MS/MS assay, to ascertain discrepancies and agreement between both methodologies from the same volunteer samples. Chapters 3 and 4 describes the comprehensive development, optimisation and validation of the highly sensitive PTAD derivatives LC-MS/MS assay for the quantification of active vitamin D metabolites, as well as the development of method using Amplifex diene derivatisation. Also discussed, is sample preparation optimisation of DBS and Mitra micro-samples. A holistic approach was taken to the development of the methodologies to provide data from which the required analytical information can be obtained for method evaluation and statistical analysis. The validated PTAD derivatives method is applied to the quantification of vitamin D metabolites in limited volume (100 μL) clinical human serum samples from 30 volunteers compared to results obtained using the clinical diagnostics ELISA technique. In Chapter 4 data analysis is described and the results are further discussed and a conclusion made based on the findings from the study. This study envisaged that combination of limited sample volume and DBS, derivatisation and LCMS/ MS is a powerful tool in vitamin D metabolite analysis and provided evidence of a positive increase in sensitivity and selectivity between derivatised compared to underivatised samples. A 10-fold increase in signal-to-noise-ratio (S/N) was observed when comparing PTAD derivatised, and Amplifex diene derivatised versus underivatised samples. Chapter 5 presents suggested future directions and considerations in the areas of vitamin D metabolite derivatisation and DBS sampling technique analysis using LC-MS/MS research based on the results presented in this dissertation. / Dissertation (MSc)--University of Pretoria, 2017. / Pharmacology / MSc / Unrestricted

Ziel der vorliegenden Promotionsarbeit war die Untersuchung von Sepsis-assoziierten Veränderungen des Arachidonsäure (AA)-Metabolismus und die Identifikation differentiell regulierter AA-Metabolite mit Prüfung ihres diagnostischen Potentials bei Patienten mit Sepsis unter Anwendung eines in-vitro Lipopolysaccharid (LPS) Vollblutaktivierungs-Modells. In Zellüberständen von nicht-aktiviertem und LPS-aktiviertem Heparinblut (25 Sepsis- Patienten, 15 Gesunde) wurden AA-Metabolite mittels Flüssigkeitschromatographie-Tandem- Massenspektrometrie analysiert. In einer unabhängigen Kohorte (10 Sepsis-Patienten, 3 Gesunde) wurden nach RNA-Isolation aus Zellmaterial zusätzlich Target-Gene des AAMetabolismus (Cyclooxygenase (COX)-2 und mikrosomale Prostaglandin-E-Synthase (mPGES)-1 mittels quantitativer Reverse Transkriptase-Polymerase Kettenreaktion (RT-PCR) untersucht. Es konnte eine differentielle Freisetzung von AA, AA-Analoga und der COX-assoziierten Metabolite Prostaglandin (PG) E2, 11-Hydroxyeicosatetraensäure (HETE) und Thromboxan (TX) B2 zwischen Patienten und gesunden Kontrollpersonen gezeigt werden. Sepsis-Patienten wiesen dabei gegenüber Gesunden eine deutlich reduzierte Freisetzung von AA und den COXassoziierten Metaboliten 11-HETE und PGE2 auf. Das Ausmaß der reduzierten Mediatorenfreisetzung bei Sepsis-Patienten war mit der Schwere der Erkrankungssymptomatik und dem klinischen Outcome assoziiert. Auf Genexpressionsebene zeigte sich eine reduzierte Induzierbarkeit der COX-2 mRNA-Expression bei Sepsis-Patienten gegenüber Gesunden, jedoch eine erhaltene Induzierbarkeit auf der Ebene der mPGES-1.:I Bibliographische Beschreibung………………………………………………………2 II Abkürzungen……………………………..……………………………………………4 III Einleitung 1. Epidemiologie und Definition der Sepsis……………………………………………..…6 2. Pathophysiologie der Sepsis……………………………………………………………..7 3. Stellenwert und Limitationen labordiagnostischer Marker bei Sepsis…………………..8 4. Eicosanoide und Sepsis…………………………………………………………………10 5. In-vitro LPS Vollblutaktivierungs-Modell für die Prüfung der Eicosanoidantwort auf Genexpressions- und Mediatorenebene……………..................12 6. Bestimmung von Eicosanoiden mittels LC-MS/MS........................................................16 7. Zielstellung der Arbeit……………………………………………………………….....18 IV Publikation…………………………………………………………….......................19 V Zusammenfassung der Arbeit………………………………………………………29 VI Literatur……………………………………………………………………………...32 VII Erklärung über die eigenständige Abfassung der Arbeit.………..…………….....35 VIII Lebenslauf………………………………………………………………………........36 IX Spezifizierung des wissenschaftlichen Beitrages zur Publikation...........................38 X Danksagung………………………………………………………………………......39

Obesity is a serious problem in society today [1,2]. It might seems to have been caused simply by excess consumption of food compared to energy expenditure but obesity is actually a complex metabolic disorder centred on adipose lipid metabolism and cellular signalling systems linked to it [3]. Understanding the biology of adipose tissue (AT) is very important for the identification of novel and potential therapeutic targets in order to prevent and treat obesity-related disorders [4]. We utilized analytical approach liquid chromatography coupled to mass spectrometry (LC-MS) to study adipose tissue metabolism. Also, we were especially interested in the effect of omega-3 polyunsaturated fatty acids (PUFA) on that metabolism. Rodent and cell line experiments were performed and analyses were done of white adipose tissue (WAT), serum/plasma samples or cells as well as milk samples from mothers. At first, we established several ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) methods for analysis of acylcarnitines (AC), amino acids (AmA) and other metabolites. Importantly, these methods were able to distinguish isobaric species of AC which is not usually possible. Using these approaches we uncovered several acylcarnitines, i.e. long chain AC, carnitine, acylcarnitine C4 and...

In forensic toxicology, opioids are frequently associated with drug abuse or drug-related death cases. An optimal method for use in the identification and quantification of opioids in a complex blood matrix is of paramount importance. Along with the ability to identify and quantitate opioids, this method should be accurate, sensitive, and selective. The application of sample pre-treatment and solid-phase extraction are common to purify and concentrate the target analytes before analyzing with liquid chromatography-tandem mass spectrometry. The purpose of this study was to compare the performance of two standard operating procedures, adopted by the Massachusetts State Police Crime Laboratory Toxicology and the Biomedical Forensic Sciences– Toxicology Laboratory at Boston University School of Medicine, for detecting opioids in blood. A total of eight drugs were analyzed: 6-monoacetylmorphine, codeine, fentanyl, hydrocodone, morphine, norhydrocodone, oxycodone, and oxymorphone. Comparison was performed using the parameters studied as part of method validation, including calibration model, bias, precision, carryover, interferences, ionization suppression/enhancement, and recovery. The results indicated that the method from Massachusetts State Police provided a better performance with between-run precision, interferences from matrix and other commonly encountered drugs, matrix effect at high concentration (250 ng/mL) and matrix recovery. Meanwhile, the method from Biomedical Forensic Sciences showed less bias, within-run precision, and matrix effect at low concentrations. Carryover and internal standard interference were comparable in both standard operating procedures. The calibration models were adjusted by altering the selection of regression model for improved quantification method performance. The volume of solvents, sample matrix, as well as time, were taken into consideration in accessing the overall performance of identification and quantitation. Both procedures were comparable yet the one from Massachusetts State Police was more beneficial in identifying the target analytes with greater sensitivity and selectivity and the one from Biomedical Forensic Sciences was more economical and efficient.

Drogpartiklar i utandningsluft är ett nytt forskningsområde med stora möjligheter att utvecklas för sjukdomsdiagnostik. En stor fördel är att provtagning kan utföras med större enkelhet och att proceduren kan upplevas som mindre integritetskränkande än till exempel blod- och urinprov. Det övergripande syftet med det här examensarbetet var att undersöka egenskaperna hos en apparat för provtagning vid namn SensAbues, som är framtagen för att samla in partiklar i utandningsluft för drogtestning. En del av syftet var att undersöka den variation som existerar i resultaten från tidigare forskning kring droganalys via utandningsprov. Examensarbetet utfördes utifrån litteraturstudier samt sex experiment. Utifrån teori och resultat ges rekommendationer på förändringar och förbättringar på provtagaren. Experiment I-III var relaterade till SensAbues egenskaper. De egenskaper som undersöktes var provtagarens flödesmotstånd (experiment I), hur partiklarna fördelar sig över provtagaren (experiment II) samt partikelstatistik gällande utandade partiklar samt provtagarens insamlingseffektivt (experiment III). I experiment IV-VI undersöktes anledningen till tidigare resultats variation, där det framförallt var tre hypoteser som låg som grund för experimentuppställningarna: 1) Att ökat flödesmotstånd skulle stimulera partikelbilandet och därmed öka koncentrationen av utandade drogpartiklar (experiment IV). 2) Att en speciell andningsmanöver skulle stimulera partikelbilandet och därmed öka koncentrationen av utandade drogpartiklar (experiment V). 3) Att partiklar från munhålan, som till exempel saliv, kontaminerar filtret vilket är anledningen till variationen i tidigare resultat (experiment VI). Resultaten från experiment I visar att det mesta flödesmotståndet sitter i provtagarens munstycke. I experiment II undersöktes spridningen av metadonpartiklar över provtagarens olika delar. Resultatet visar att det förekommer stor spridning och det verkar som att endast en liten del, cirka 3 % av metadonpartiklarna som andas ut fastnar i filtret, det vill säga den del som analyseras i laboratoriet. I experiment II går det inte att dra några slutsatser om provtagarens effektivitet, det vill säga hur stor andel av utandade metadonpartiklar som fångas upp av provtagaren, detta undersöks i experiment III. I experiment III undersöks provtagarens effektivitet och resultaten visar att provtagaren fångar upp cirka 99 % av utandade partiklar (alla utandade partiklar, ej drogpartiklar) Resultaten i experiment IV visade att ökat flödesmotstånd inte verkar ha någon betydelse för partikelkoncentrationen (generellt utandade partiklar, ej drogpartiklar). Resultaten i experiment V visade inte på att den speciella andningsmanövern bidrar till ökad koncentration av utandade drogpartiklar jämfört med vanlig provtagning. Det mest troliga skälet till tidigare forsknings varierande resultat är att partiklar från munhålan kontaminerar filtret, vilket visas i resultatet från experiment VI. Utifrån resultaten i experiment I-VI ges rekommendationer för utveckling av provtagaren. / Analysis of drug particles in exhaled air is a new research area with great potential for medical diagnoses. A major advantage is that sampling can be performed much easier and that the procedure may be perceived as less intrusive than, for example, blood or urine samples. The overall aim of this thesis work was to investigate some of the properties of a sampler named SensAbues, which is designed to collect particles in exhaled air. Another part of the purpose was to examine the variation in the results of previous research related to drugs through expiration. The work was based on literature studies and six separate experiments. Experiments I-III was related to SensAbues properties. The properties examined were the sampler's flow resistance (experiment I), how the particles are distributed over the sampler (experiment II) and particle statistics regarding exhaled particles and the sampler's collection effectiveness (experiment III). In experiments IV-VI previous research results variety was examined based on primarily three hypotheses: 1) Increased resistance would stimulate the amount of exhaled particles and thereby increase the concentration (experiment IV). 2) That a special breathing maneuver would stimulate the amount of exhaled particles and thereby increase the concentration (experiment V). 3) That particles from the oral cavity, such as saliva, would contaminate the filter and explain the variation in previous research results (experiment VI). The results from experiment I show that most of the flow resistance is located in the sampler's nozzle. In experiment II it shows that there is a great distribution of methadone particles over the sampler's different parts. Only a small part, about 3% of methadone particles exhaled trapped in the filter, which is the part that is analyzed in the laboratory. In experiment II, it is not possible to draw any conclusions about the sampler's efficiency, this examined in experiment III. In experiment III the sampler's effectiveness is examined and the results show that the sampler captures approximately 99% of exhaled particles (all exhaled particles, not drug particles). Results in experiment IV showed that the increased flow resistance does not seem to have any bearing on the particle concentration (generally exhaled particles, not drug particles). The results of experiment V did not show that the particular breathing maneuver contributes to increased concentration of exhaled drug particles compared with normal breathing. The most likely reasons for the variation in previous research are that particles from the mouth contaminate the filter, as shown in the results of experiment VI. Experiments I-VI leads to recommendations for further development of the sampler.

京都大学 / 0048 / 新制・課程博士 / 博士(薬学) / 甲第18209号 / 薬博第799号 / 新制||薬||237(附属図書館) / 31067 / 京都大学大学院薬学研究科創薬科学専攻 / (主査)教授 石濱 泰, 教授 松﨑 勝巳, 教授 加藤 博章 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

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