Presented work is focused on application of combined instrumental method RP-HPLC/ESI-MS in analysis of several groups of natural compounds with positive physiological activities. Especially some antioxidants were studied in commonly and abundantly consumed food. Moreover some other substances than standard compounds were observed in complex dies. In conclusion the HPLC/ESI-MS method is comparatively advantageous in phenolic analysis, while another type of ionisation would be used in case of carotenoid compound.

The presented thesis deals with the issue of mycotoxins in brewing materials and beer. Attention was devoted mainly to the selected fusarium mycotoxins (deoxynivalenol, zearalenol, T-2 toxin, and HT-2 toxin) ochratoxin A and aflatoxins B1, B2, G1, and G2. The aim of the thesis was to optimize and validate analytical methods for the determination of the above mentioned mycotoxins in the brewing materials and beer. Analytes were separated using high-performance liquid chromatography with mass – spectrometric detection (HPLC-MS/MS) and ultra-performance liquid chromatography with fluorescence detection (UPLC/FLR). These analytical methods were then applied for mapping the occurrence of fusarium mycotoxins in malting barley crops in the Czech Republic and monitoring the level of contamination with mycotoxins in malting and brewing industries. In addition, experiments studying over-foaming of beer were conducted as primary gushing – over-foaming of beer – is connected, similarly as mycotoxins, with the presence of microscopic filamentous fungi in the raw materials for beer production. Studies describing in detail these methods are part of this thesis (Annex I – V). From all published results, it is evident that the occurrence of mycotoxins in cereals including barley is natural and cannot be completely prevented, not even if all conditions of correct agricultural practice are observed. It is known that some mycotoxins present in contaminated malting barley pass to the final product – beer due to their chemical and physical properties. However, the mycotoxin concentrations found do not mean any significant health risk for consumers.

Malaria is a disease caused by the protozoan parasite Plasmodium where the species that causes the most severe form of malaria in humans is known as Plasmodium falciparum. At least 40% of the global population is at risk of contracting malaria with 627 000 people dying as a result of this disease in 2012. Approximately 90% of all malaria deaths occur in sub-Saharan Africa, where approximately every 30 seconds a young child dies, making malaria the leading cause of death in children under the age of five years old. The malaria parasite has a complex life cycle utilising both invertebrate and vertebrate hosts across sexual and asexual stages. The erythrocyte invasion stage of the life cycle in the human whereby the invasive merozoite form of the parasite enters the erythrocyte is a central and essential step, and it is during this stage that the clinical symptoms of malaria manifest themselves. Merozoites invade erythrocytes utilising multiple, highly specific receptor-ligand interactions in a series of co-ordinated events. The aim of this study was to better understand the interactions occurring between the merozoite and erythrocyte during invasion by using modern, cutting-edge proteomic techniques. This was done in the hope of laying the foundation for the discovery of new key therapeutic targets for antimalarial drug and vaccine-based strategies, as there is currently no commercially available antimalarial vaccine and no drug to which the parasite has not at least started showing resistance. In this study healthy human erythrocytes were treated separately with different protein-altering enzymes and chemicals being trypsin, the potent oxidant sodium periodate (NaIO4), the amine cross-linker tris(2-chloroethyl)amine hydrochloride (TCEA) and the thiol cross-linker 1,11-bis(maleimido)triethylene glycol (BM(PEG)3). The resulting erythrocyte protein alterations were visualised as protein band differences on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), where treated and untreated control erythrocyte ghost protein fingerprints were visually compared to one another. The protein bands showing differences between treated and control samples were in-gel digested using trypsin then sequenced by liquid chromatography tandem mass spectrometry (LC-MS/MS) and identified using proteomics-based software. In this way, the erythrocyte proteins altered by each enzyme/chemical treatment were identified. Malaria invasion assays were performed where each treatment group of erythrocytes as well as the control erythrocytes were incubated separately with schizont stage malaria parasites for the duration of one complete life cycle. Using fluorescent staining and flow cytometry, the invasion inhibition efficiency for each treatment group was evaluated. By utilising these methods, the identification and the relative importance of the erythrocyte proteins involved in the invasion process were determined. Protein fingerprints of control and treated erythrocyte ghosts were visualised and optimised on SDS PAGE where induced protein band differences were successfully identified by LC-MS/MS. It was found that each treatment altered erythrocyte proteins with changes found in Band 3, actin, phosphoglycerate kinase 1, spectrin alpha, spectrin beta, ankyrin, haemoglobin, Bands 4.1 and 4.2, glycophorin A and stomatin. The invasion assays revealed that TCEA inhibited invasion to the greatest extent as compared to the other treatments, followed by BM(PEG)3 and trypsin. Sodium periodate-treated erythrocytes could not be assessed using the invasion assay due to auto-haemolysis. Band 3, glycophorin A, Band 4.1 and stomatin appear to be of higher relative importance in the invasion process as compared to the other altered erythrocyte proteins. These results confirmed the known roles of spectrin alpha, spectrin beta, glycophorin A, Band 3 and Band 4.1 in invasion, and suggested that ankyrin, Band 4.2 and stomatin may also be involved. This study highlighted the potential that modern, cutting-edge proteomic techniques provide when applied to previous comparative studies found in older literature, as previously unidentified proteins that can be involved in invasion were revealed. These results can be used as a foundation in future studies in order to identify new key targets for the development of new antimalarial drug- and vaccine-based strategies, with the hope of preventing the suffering of the millions of malaria-inflicted people worldwide, and ultimately eradicating this deadly disease. / Dissertation (MSc)--University of Pretoria, 2014. / tm2015 / Pharmacology / MSc / Unrestricted

Candidate: Nikola Kollárová Title of diploma thesis: Detection of Sap2 in the secretome of Candida albicans cell wall and secretory mutants Charles University, Faculty of Pharmacy in Hradec Králové, Department of Biological and Medicinal Sciences Complutense University of Madrid, Faculty of Pharmacy, Department of Microbiology II Study program: Pharmacy Backgound: The aim of this diploma thesis was to search for C. albicans proteins involved in the secretion of the secreted aspartyl proteinase 2 enzyme (Sap2) evaluating the ability to degrade BSA (bovine serum albumin) as a source of nitrogen in several cell wall and secretory mutants of C. albicans. The work was carried out at the Department of Microbiology II, Faculty of Pharmacy, Complutense University of Madrid. Methods: The supernatant samples of several Candida albicans mutants were tested by SDS-PAGE electrophoresis and stained. Bands corresponding to BSA were observed and compared to controls. The other method was counted with 96-well plate. Results: The correlation between optical density and degradation of BSA was observed. Some mutants with disability to degrade BSA were found in a pilot screening of the ability to degrade BSA using 96-well plate method. That fact was confirmed by SDS-PAGE electrophoresis. C. albicans mutants showing...

Störungen des Lipidstoffwechsels sind entscheidend an der Entstehung atherosklerotisch bedingter kardiovaskulärer Erkrankungen (CVD) beteiligt. Insbesondere erhöhte Plasmakonzentrationen von Low-density-lipoprotein Cholesterin (LDL-C) tragen zur Entstehung von Atherosklerose bei. Apolipoproteine sind strukturelle und funktionelle Bestandteile von Lipoproteinen und damit als mögliche Biomarker und therapeutische Angriffspunkte der CVD Gegenstand aktueller Forschung. Zur experimentellen Untersuchung von kardiovaskulären Erkrankungen ist die Maus das bevorzugte Tiermodell. Im Mausmodell basiert quantitative Proteinanalytik bislang auf Antikörper-abhängigen Methoden (z.B. Western Blot), welche teils ungenau und materialaufwendig sind. Ein Verfahren zur effizienten und quantitativen Analyse von Apoliproteinen (Apos) in Mäusen, welches Flüssigchromatographie gekoppelt mit Massenspektrometrie (LC-MS/MS) nutzt, wurde bislang noch nicht beschrieben. In der vorliegenden Arbeit wurde eine LC-MS/MS Methode zur simultanen Quantifizierung von apoA-I, apoA-II, apoA-IV, apoB total, apoB-100, apoC-I, apoE und apoJ aus murinen Plasmaproben (3 µL) etabliert und validiert. Ausgehend von einer für humane Proben bestehenden Methode wurden die analytischen Bedingungen (z.B. Prüfung der Peptide, Dauer des tryptischen Verdaus, Linearität, Reproduzierbarkeit, Vergleich mit immunologischen Methoden) im Mausmodel ermittelt und optimiert. Anschließend wurde das Verfahren angewendet, um Apo-Plasmakonzentrationen in normocholesterinämischen C57BL/6, sowie in hypercholesterinämischen LDL-Rezeptor-defizienten (LDLR0) und apoE-defizienten Mäusen (ApoE0) zu charakterisieren. Dabei ermittelten wir moderate Unterschiede der Apo-Plasmakonzentrationen zwischen gefastetem und postprandialem Zustand, wobei apoA-IV und apoC-I postprandial erhöht waren und apoJ erniedrigt war. ApoE war in LDLR0 Mäusen 6-fach höher konzentriert als in C57BL/6 Mäusen und wurde erwartungsgemäß in ApoE0 Tieren nicht detektiert. Apo-B48 zeigte die höchste relative Konzentration in Apo-E0 Mäusen (93% des apoB-total), verglichen mit 61% in LDLR0-Mäusen. Zudem wurden Apo-Konzentrationen auf isolierten HDL-Partikeln bestimmt und zwischen den Mauslinien verglichen. Das entwickelte Verfahren kann zur weiteren Charakterisierung von Apos in verschiedenen Mausmodellen eingesetzt werden und damit als Grundlage für weitere Arbeiten zum Verständnis der Pathophysiologie und Funktionsweise von Apos dienen.

Vitamin K belongs to the family of fat-soluble vitamins, which is not determinated in clinical laboratories. It is a cofactor necessary for posttranslational γ-carboxylation of glutamyl residues in selected proteins such as the osteocalcin, and procoagulation factors II, VII, IX, X. Vitamin K deficient individuals appear to have more undercarboxylated proteins, which are functionally defective. Lack of this vitamin has been associated with risk of developing osteoporosis and cardiovascular diseases. The aim of this work was to develop and validate the HPLC method and the LC-MS/MS method for determination of three vitamin K's forms - vitamin K1, MK-4 and MK-7 in serum. After successful validation of both methods, patient samples and healthy population samples were measured. There were measured 350 patient samples by HPLC method. These samples were divided into two groups - patients with diagnostic of osteoporosis and patients without osteoporosis. We measured 946 samples by LC-MS/MS method. Samples were divided into groups: patients with osteoporosis, patients without osteoporosis, healthy population, patients with osteopenia and patients with cystic fibrosis. The reference range of vitamin K in healthy population was obtained by LC-MS/MS method. The next aim was to compare the effectiveness of...

La myéloperoxydase (MPO) est une enzyme pro-oxydante qui est capable de causer des dommages aux biomolécules, comme les acides nucléiques, en les chlorant spécifiquement par le biais de l’acide hypochloreux (HClO) qu’elle produit. La MPO étant présente au sein de tissus prostatiques et impliquée dans divers mécanismes physiopathologiques (incluant les cancers), il nous semblait important d’évaluer dans ce travail l’impact de sa présence dans le développement de lésions prostatiques ainsi que celui de la présence de nucléosides spécifiquement modifiés par la MPO sur la réplication, la transcription et la traduction.Dans un premier temps, des méthodes ont été développées afin de permettre l’analyse quantitative des nucléosides de l’ADN, de l’ARN, du pool cytoplasmique et du plasma. Des protocoles d’extraction et d’hydrolyse de l’ADN et de l’ARN ainsi que des protocoles de purification et de déphoshorylation des nucléosides libres (pool cytoplasmique et plasma) ont été développés ou adaptés afin de pouvoir ensuite séparer et quantifier les nucléosides par chromatographie liquide à ultra haute performance couplée à un spectromètre de masse Triple Quadupôle (LC/MSMS). La méthode analytique LC/MSMS a été développée et validée pour l’étude de six résidus modifiés :les 5‐chloro-(2’-désoxy)cytidines, les 8-chloro-(2’-désoxy)guanosines et les 8-oxo-(2’-désoxy)guanosines. Les quatre premiers étant des marqueurs spécifiques de la MPO et la formation des oxoguanosines pouvant être catalysée par l’enzyme (marqueur du stress oxydatif). Cette méthode a notamment l’avantage de présenter des limites de quantification plus faibles que dans la littérature, abaissant de 4 à 25 fois la sensibilité de la méthode selon le nucléoside.Deuxièmement, nous nous sommes attardés sur l’implication de la MPO dans le développement de lésions prostatiques, en recherchant la présence de ces nucléosides modifiés au sein de biopsies prostatiques de patients et de cultures cellulaires prostatiques traitées in vitro par la MPO, à l’aide des méthodes développées précédemment. Néanmoins, il s’est avéré qu’aucun chloronucléoside n’a été détecté dans ces cas de figure. Une méthode d’immunomarquage par fluorescence a également été développée afin d’essayer de colocaliser la présence de la MPO et son activité au sein de tissus prostatiques de patients souffrant de lésions prostatiques. Ce marquage avait pour but d’estimer l’activité réelle de la présence de MPO, et donc son rôle au sein de ces lésions prostatiques mais un phénomène d’autofluorescence n’a pas permis d’aboutir à des résultats.Finalement, l’étude du plasma de patients hémodialysés et de la pénétration intracellulaire de nucléosides modifiés depuis les liquides extracellulaires, tel que le plasma, vers le compartiment cellulaire ont été abordées. Ces expériences ont également été rendues possibles grâce aux méthodes développées et validés précédemment. Puisqu’il s’avère que les échantillons de plasma de donneurs sains et de patients contiennent de la 5-chlorocytidine et que cette dernière est capable de pénétrer in vitro dans les cellules prostatiques, l’aspect de l’incorporation de ces nucléosides au sein de l’ADN et l’ARN a également été approfondi. Il en résulte que seule la 5-chlorocytidine s’incorpore dans l’ARN lors de la transcription en présence de nucléosides modifiés dans le milieu alors qu’aucun nucléoside modifié ne s’incorpore dans l’ADN lors de sa réplication. Ce dernier point est probablement dû à une perte partielle ou complète de l’activité de certaines enzymes impliquées dans la réplication face aux chloronucléosides. De plus, l’étude du rendement de la traduction a montré que cette incorporation spécifique était accompagnée d’une importante réduction de la traduction de l’ARN en protéines.En conclusion, bien que ce travail ait permis d’établir une méthode LC/MSMS sensible, précise et exacte pour l’analyse de nucléosides modifiés (chlorés et oxydés) dans différentes matrices, il n’a pas permis de mettre en évidence des chloronucléosides dans les tissus prostatiques de patients et d'impliquer la MPO dans le développement de lésions prostatiques. Le rôle de la MPO dans les tissus prostatiques reste donc encore à élucider. De plus, ce travail a mis pour la première fois en évidence la présence de 5-chlorocytidine dans le plasma humain ;ce même nucléoside est capable de s’incorporer spécifiquement dans l’ARN, influençant fortement le rendement de la production de protéines, ce qui soulève encore des questions quant à son impact sur les fonctions et la viabilité des cellules en présence de 5-chlorocytidine dans le milieu extracellulaire. / Doctorat en Sciences biomédicales et pharmaceutiques (Pharmacie) / info:eu-repo/semantics/nonPublished

The purpose of this report is to compare seven different methods for biomarker detection and quantification based on previously published papers. The methods investigated are ELISA, LC-MS/MS, UPLC-MS/MS, LC-IM/MS, IA-LC-MS/MS, MSIA-HR/AM, HTRF and AlphaLISA ® . The focus lies on biomarkers relevant for diabetes, obesity and cardiovascular diseases.Namely insulin, proinsulin, glucagon and C-peptide. Particular significance is assigned to the comparison of the currently widest used method, ELISA, with various types of LC-MS/MS. The report concludes ELISA being superior to LC-MS/MS methods in terms of recovery and precision, while LC-MS/MS is superior in accuracy, multiplexing, specificity, throughput and sample cost. This suggests that different types of LC-MS/MS has the potential to gain momentum in the field of biomarker quantification if they become more available.

This research study successfully completed three objectives: 1) validate liquid-liquid, supported-liquid, and solid-phase extractions for the quantitation of three barbiturates (amobarbital, butalbital, and phenobarbital) in blood and urine using liquid chromatography-tandem mass spectrometry; 2) to compare the efficiency and effectiveness among methods in accomplishing extraction of barbiturates under the laboratory setting at Boston University School of Medicine; and 3) to report all the analytical data to RTI International for interlaboratory comparison. For the validation study, a six-point linear calibration model (20-2000 ng/mL) with inversely weighted concentration (1/x) was reproducible in all three sample preparation methods for both blood and urine with r2 greater than or equal to 0.994. Bias and precision evaluated from three controls throughout the range of the curve were within ±20% and ±20%CV, respectively. Neither carryover nor interference was observed. Detection limits were evaluated down to 5 ng/mL depending on the extraction procedure. Samples were able to be diluted up to 50 times prior to instrumental analysis. Samples were stable on autosampler at room temperature up to 72 hours after their initial analysis. Recovery of barbiturates from blood and urine all ranged from 45% to 86%. The effect of ionization suppression or enhancement was found to have minimal impact on the validation. For choosing the most suitable method quantifying barbiturates, efficiency and effectiveness were studied. Efficiency evaluates the time and ease of sample preparation required to prepare a sample for analysis. Supported-liquid extraction was found to be the most efficient method for extracting barbiturates as it required the least amount of time to perform and could be easily automated with minimal training. Effectiveness is an assessment of one’s ability to selectively recover target analyte at a reasonably low concentration. By considering a method’s recovery, extract cleanliness, detection limits, and reproducibility, liquid-liquid extraction was the best at quantifying barbiturates in blood and supported-liquid extraction was the most suitable method for extracting barbiturates from urine. For interlaboratory comparison, all the data collected has been reported to RTI International. These findings can be used for examining the overall reliability and reproducibility of the validated methods. Results obtained can also be used to explore the possibility for streamlining sample preparation in the forensic laboratory, and hence reducing the case backlog.

Despite efforts to control synthetic cannabinoids, clandestine manufacturers continue to modify their structures to avoid legal consequences, creating an ever-changing analytical target for forensic laboratories (1). Forensic toxicology laboratories often lack the needed resources or do not have the capabilities to test for these compounds and metabolites, requiring specimens to be submitted to reference laboratories (2). Drug stability can be affected by long storage times, temperature and preservatives (3). Although these factors can be controlled, systematic research is necessary to identify their impacts on the stability of these new synthetic cannabinoids that are continually emerging. The purpose of this research is to assess the stability of 17 synthetic cannabinoids in human whole blood and 10 synthetic cannabinoid metabolites in human urine using liquid chromatography-tandem mass spectrometry (LC-MS/MS) over thirty-five weeks. The analysis methods were validated in accordance to the Academy Standards Board (ASB) method validation guidelines for quantitative analysis and stability evaluation of the following analytes in blood: 4-cyano CUMYL-BUTINACA, ADB-PINACA, EMB-FUBINACA, JWH-250, MO-CHMINACA, 5-fluoro-3,5-ABPFUPPYCA, 5-fluoro ADB-PINACA, APP-PICA, CUMYL-THPINACA, PB-22, XLR11, 5-fluoro PY-PINACA, MDMB-FUBICA, MEP-CHMICA, NM2201, RCS-8, and UR144. The stability analysis in urine includes the following metabolites: 5-fluoro MDMB-PICA metabolite 7, 5-fluoro PB-22 3-carboxyindole, AB-FUBINACA metabolite 3, ADB-PINACA N-(4-hydroxypentyl), ADB-PINACA pentanoic acid, UR-144 Degradant N-pentanoic acid, PB-22 N-(5-hydroxypentyl), MDMB-FUBICA metabolite 3, UR-144 N-(5-hydroxypentyl), and JWH-250 N-pentanoic acid. Research samples were prepared by spiking with certified reference standards (Cayman Chemical, Ann Arbor, MI, USA) of each select synthetic cannabinoid in certified drug-free human whole blood (Boston Medical Center, Boston, MA, USA; Biological Specialty Corporation, Colmar, PA) and drug-free urine that was received as donations following the approved Institutional Review Board guidelines (Boston University School of Medicine, Boston, MA, USA). Blood samples were aliquoted into 6 mL BD Vacutainer Plastic Collection Tubes (Fisher Scientific, Waltham, MA, USA) and urine samples were stored in 15 mL Falcon Conical Centrifuge Tubes (Fisher Scientific, Waltham, MA, USA). Stability under room temperature (20ºC), refrigerator (4ºC), and freezer (-20ºC) at low and high concentrations were evaluated at select time points. A 5% solution of potassium oxalate and sodium fluoride or ethylenediaminetetraacetic acid (EDTA) was added to the preserved blood samples by the manufacturer prior to storage. The anticoagulant, potassium oxalate, was only added in solution to the preserved samples whereas none was added to the nonpreserved samples. Short-term urine samples were preserved with 1% of sodium fluoride prior to storage. Extraction of analytes was conducted using supported-liquid extraction (SLE) ISOLUTE 1 mL cartridges (Biotage, Charlotte, NC, USA) and reconstituted in 100 μL of 50:50 mixture of 0.1% formic acid in millipore deionized water and 0.1% formic acid in acetonitrile (Fisher Scientific, Waltham, MA, USA). Analysis was performed in triplicate using a reverse-phase C18 column (Waters XBridge C18 3.5 μM, 2.1 x 50 mm, Milford, MA, USA) on the Shimadzu Prominence Ultra-Fast Liquid Chromatography (UFLC, Kyoto, Japan) with SCIEX 4000 Q-Trap Electrospray Ionization Tandem Mass Spectrometry (ESI/MS/MS, Waltham, MA, USA) in positive ionization mode. The total run time was 8 minutes with a flow rate of 0.6 mL/min and injection volume of 10 μL. Linear calibration curves for each analyte with the exception of a quadratic regression for PB-22, all had acceptable R2 values > 0.99 using a weighting factor of 1/x. A linear dynamic range of 0.5 – 25 ng/mL was used for all analytes in blood except for NM2201 and APP-PICA with a limit of quantitation (LOQ) of 0.1 ng/mL and MO-CHMINACA with a working range of 0.5 – 15 ng/mL. A linear working range of 5 – 40 ng/mL was utilized for all metabolites in urine. No signs of carryover were observed. In general, analytes were considered stable if the average area ratio between the analyte and internal standard at the time point was within ± 20% of the average area ratio response at time point zero. In some cases, it was necessary to evaluate the complete picture of the stability data by reviewing analyte area, concentration, and overall stability data trend between timepoints at the low and high concentrations. In certain situations, an analyte was considered stable even if specific timepoints for a concentration were outside the ±20% range. For example, in cases where one concentration at a timepoint was within the ±20% range and the other concentration fell within ±30% range the analyte was considered stable overall. Long-term stability results revealed that all synthetic cannabinoids were stable at 21 to 35 weeks in frozen blood preserved with sodium fluoride except for APP-PICA. The preservatives are recommended to be added to blood to reduce the possibility of matrix inferences and minimize detrimental impacts on the stability of synthetic cannabinoids. Analytes experienced lower degradation in the order of samples that were kept frozen, refrigerated, and then at room temperature. Blood analytes that were stable up to 35 weeks in freezer generally had a core structure of a carbonyl substituent on a pyrazole or pyrrole with surrounding nonpolar groups; whereas compounds with two polar carbonyl functional groups present were found to experience degradation much earlier at 1 week or less in room temperature and refrigerator storage conditions. 5-fluoropentyl analogs, like XLR11 and 5-fluoro ADB-PINACA, in comparison to their counterpart analyte, UR144 and ADB-PINACA, were unstable at earlier time points under all storage conditions. Instability in the majority of the urine metabolites was not observed until after 9 weeks and was generally consistent across all storage conditions. The validated methods demonstrate a sensitive and reliable way to positively identify 17 different synthetic cannabinoids in human whole blood and 10 synthetic cannabinoid metabolites in urine for rapid time stability analysis at various storage conditions. The use of SLE improved sample preparation efficiency by decreasing the extraction time from 1 hour to 30 minutes compared to traditional extraction methods, such as solid-phase extraction (SPE) and liquid-liquid extraction (LLE). Further studies into additional matrices, such as oral fluid, longer storage times, and other emerging synthetic cannabinoid analytes would expand the scope of this research.