Introduction: Post-mortem whole blood samples differ greatly in quality, lipemia is one cause of concern in toxicological analyses. Around 4 % of all samples sent to RMV are given a notation of lipemic content. The aim of the thesis was to study the effects of lipemia on the quantification of 14 benzodiazepines and 5 similar sedative and antianxiety drugs as well as evaluate the pre-analytical process aiming to reduce the effects of lipemia.  Methods: Blood samples were simulated with bovine blood, analyte spiking, and lipid spiking with either the nutrition emulsion Intralipid or with a mixture of post-mortem lipids from authentic samples. The outset was the by RMV currently used LLE method followed by UPLC- MS/MS and the extraction method was altered and evaluated. Matrix effects were also studied.  Results: Lipemia were found to be a great interference when quantifying benzodiazepines. For most analytes, internal standard could compensate for the loss of analyte but there was a problem with analytes not having their own IS. The 7-amino-compounds were greatly affected by lipemia and propiomazine and dihydropropiomazine showed extreme losses. Equilibration of IS did not result in similar loss as analyte. Dilution of sample reduced losses caused by lipemic content. SPE resulted in extracts free from lipids and high yields but there were analyte losses similar to LLE. No matrix effects from the lipids were found. Samples spiked with Intralipid gave poorer analyte yields than those spiked with post-mortem lipids.  Conclusion: Dilution is the most successful method to reduce pre-analytical matrix effects as long as the concentration is not so low that it risks getting lower than the analytical limits when doing so. Not homogenising samples before sampling is giving incorrect results. SPE could, if optimised for the analyte retention and elution, remove lipids from samples and obtain accurate analyte concentrations. Pooling lipids from post-mortem samples is a possible method for simulating lipemic whole blood. Intralipid and the PM-mix gave the same indications, but to different extents. Further studies where the ability to mimic authentic lipids are needed for both Intralipid and PM-mix.

Peptidoglycan (PGN) är en bakteriecellväggskomponent och känd för att känna igen olika receptorer eller enzymer för att leda aktiveringsimmunsystemet. Den allmänna strukturen för PGN består av sockerkedjor inklusive N-acetylglutamin (GlcNAc), N-acetylmuraminsyra (MurNAc) och tvärbundna peptidkedjor. PGN-fragment med D-Lac-ändpeptider har hittats från vankomycinresistenta enterokocker men ett kemiskt syntetiserat PGN-fragment med en D-Lac-ändpeptid har inte undersökts i detalj. Således fokuserade vi på syntesen av PGN-fragmentstrukturer som inkluderar en D-Ala-D-Lac-rest vid den terminala delen av peptidkedjan. För att syntetisera dessa fragmentstrukturer planerade vi att kombinera fastfassyntes (för Lac-peptiddelen) och lösningsfassyntes (för glykanberedning och kondensation). Detta tillvägagångssätt är fördelaktigt för framställning av peptidoglykanfragment med en komplex grenad peptiddel. Först beredde vi sockerdelen MurNAc-derivatet i lösningsfassyntes från ett glukosderivat. Medan den Lac-innehållande peptiden framställdes med fastfas-peptidsyntes med användning av 2-klortritylkloridharts. Med denna förening gav kondensationen av dessa två föreningar det önskade D-Lac-avslutade peptidoglykanfragmentet. / Peptidoglycan (PGN) is a bacterial cell wall component and known to be recognized by various receptors or enzymes to lead the activation immune system. The general structure of PGN consists of sugar chains including N-acetylglutamine (GlcNAc), N-acetylmuramic acid (MurNAc) and cross-linked peptide chains. PGN fragments having D-Lac terminus peptides have been found from vancomycin-resistant enterococcus, but a chemically synthesized PGN fragment having a D-Lac terminus peptide has not been examined in detail. Thus, we focused on the synthesis of PGN fragment structures that include a D-Ala-D-Lac residue at the terminal part of the peptide chain. In order to synthesize these fragment structures, we planned to combine solid-phase synthesis (for the peptide- Lac part) and solution-phase synthesis (for glycan preparation and the condensation). This approach is advantageous for the preparation of peptidoglycan fragments having complex branched peptide moiety. First, we prepared the sugar moiety MurNAc derivative in solution-phase synthesis from a glucose derivative. While, the Lac-containing peptide was prepared with solid-phase peptide synthesis using 2-chlorotrityl chloride resin. Having this compound, the condensation of these two compounds gave the desired D-Lac-terminated peptidoglycan fragment.

Peptide drugs are used to treat a broad spectrum of diseases such as cancer and HIV and have many more promising applications, such as new vaccines against SARS-CoV-2. The most popular manufacturing method for peptides is solid-phase peptide synthesis (SPPS). The main drawback of SPPS is that it is a costly and wasteful process.  SpinChem is a company that provides technology solutions for chemical processes. Recently, SpinChem has started investigating if their Rotating Bed Reactor (RBR) is suitable for peptide synthesis. The goal of this project is to investigate how the RBR can make processes like SPPS more resource-efficient. The idea is that the RBR-system can maximize the solid-phase to liquid ratio (STL). The STL is the ratio of the volume of solid-phase material and the volume of liquid. By maximizing the STL, it is possible to manufacture peptides using less solvents and chemicals. The main quest of the project is formulated into a single question:  How does a high STL affect the efficiency of the RBR-system?  To answer the question, Minitab's statistical software and design of experiments (DOE) will be used to plan and perform experiments in both lab- and industrial scales. DOE factorial experiments are used to gain as much information as possible about the new RBR-system. The results are analyzed and summarized to make a solid foundation for the continued work on the new RBR application.  Peptide synthesis efficiency in the RBR-system is measured using ionic adsorption. The ionic adsorption rate is measured in both lab-scale and industrial-scale experiments. In the lab-scale experiments, the decrease of ions was on average 86,5% after just 15 s with an average STL of 0,936. The industrial-scale experiments showed a similar result where the average decrease in ions was 92,9% after 20 s with an average STL of 0,947. It was concluded that the RBR-system can reduce the consumption of washing-solvent in SPPS by up to 82%.

Indiana University-Purdue University Indianapolis (IUPUI) / Solid phase micro-extraction is a powerful and versatile technique, well-suited to the analysis of numerous samples of forensic interest. The exceptional sensitivity of the SPME platform, combined with its adaptability to traditional GC-MS systems and its ability to extract samples with minimal work-up, make it appropriate to applications in forensic laboratories. In a series of research projects, solid phase micro-extraction was employed for the analysis of explosives, commercial tire treatments, and entomological specimens. In the first project, the volatile organic compounds emanating from two brands of pseudo-explosive training aids for use in detector dog imprinting were determined by SPME-GC-MS, and the efficacy of these training materials was tested in live canine trials. In the second project, the headspace above various plasticizers was analyzed comparative to that of Composition C-4 in order to draw conclusions about the odor compound, 2- ethyl-1-hexnaol, with an eye toward the design of future training aids. In the third, automobile tires which had participated in professional race events were analyzed for the presence of illicit tire treatments, and in the fourth, a novel SPME-GC-MS method was developed for the analysis of blowfly (Diptera) liquid extracts. In the fifth and final project, the new method was put to the task of performing a chemotaxonomic analysis on pupa specimens, seeking to chemically characterize them according to their age, generation, and species.

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Improved analytical methods using novel cleanup techniques and inexpensive instrumentation for the determination of residue estrogens in municipal wastewater and swine lagoon wastewater have been developed. Presented approaches are less expensive, less time consuming, yet produce comparable detection limits and extraction efficiencies to existing methods. Chapter I provides an overview on environmental estrogens. Chapter II describes a novel method to quantify five estrogens including estriol, estrone, 17alpha-estradiol, 17beta-estradiol, and 17alpha-ethynylestradiol in influent and effluent municipal wastewater. The method includes sample preparation using solid-phase extraction followed by a “QuEChERS” cleanup, dansylation and LC/MS/MS detection. Hydrophilic-lipophilic balance solid phase extraction (SPE) was used for sample preconcentration and the extract was cleaned up using a dispersive SPE method using MgSO4, PSA and C-18. The resulting extract was then derivatized with dansyl chloride. Separation was achieved on a C-18 column and quantification was accomplished in the positive ion mode using multiple reaction monitoring. The method is capable of detecting below 1 ng/L. Chapter III describes improved approaches to quantify five estrogens and two conjugates, Estrone 3-glucuronide and beta-Estradiol 3-sulfate, in swine lagoon wastewater and storm water runoff. A considerable residue was collected when lagoon wastewater samples were centrifuged therefore both resulting residues and aqueous portions were analyzed separately. Analysis of the aqueous portions was carried out using a similar approach to the method described in Chapter II. However, a simple test-tube liquid-liquid extraction was used as an additional sample clean-up step. A modified QuEChERS method was utilized to efficiently extract the target analytes in the residue. Methods have 0.9 – 2 ng/L detection limits. Chapter IV describes an approach to quantify residue estrogens in municipal wastewater using a comprehensive two-dimensional gas chromatograph (GCxGC). This method requires no further cleanups after SPE and has detection limits ranging from 1.4 – 22.2 ng/L. All presented methods use relatively small initial sample volumes and produce negligible matrix interferences. The developed methods were validated by performing mini surveys on the estrogen levels in environmental aqueous matrices in north Mississippi.

No description available.

No description available.

Patman is a fluorescent membrane probe related to Laurdan. The structural distinctions between the two probes are the lengths of the aliphatic tails (eleven carbons in Laurdan and fifteen in Patman) and the presence of a trimethylammonium group on Patman that produces a positively-charged head. Preliminary studies exploring Patman as a probe to detect membrane properties during apoptosis revealed that the fluorescence intensity of two edges of the emission spectrum (435 and 500 nm) stabilizes at different rates as the probe binds to the cell membrane. To test whether these differences represent dissimilarities in probe binding to ordered and disordered domains, experiments were conducted to monitor Patman equilibration with bilayers composed of various mixtures of saturated and unsaturated phosphatidylcholines at temperatures above, at, and below the main thermotropic phase transition. In general, Patman equilibrated more rapidly with bilayers in the liquid-disordered phase than in the solid-ordered phase. With solid phase membranes, the fluorescence stabilized faster at 500 nm than at 435 nm. Similar, yet more subtle, results occurred in the lipid disordered phase. In contrast, the situation was reversed at the phase transition temperature; equilibration was faster at 435 nm than at 500 nm. To determine whether these results reflected specific properties of Patman, the experiments were repeated with Laurdan, and several distinctions were observed. First, equilibration with solid phase lipids was faster than for Patman and not different from equilibration with the fluid phase. Second, differences in rates between the two wavelengths were less than with Patman for solid phase membranes but greater than with Patman for melted bilayers. Third, at the phase transition temperature, the difference in equilibration rates was the opposite of the result obtained with Patman. Computer simulations were used to assist with interpretation of these results. The data suggest that both probes bind superficially to the membrane before incorporating among the lipid molecules. Once within the membrane, Patman localizes to at least two distinct depths within the bilayer. Probe molecules in the shallow, more hydrated position favor 500 nm emission and those occupying a deeper, dehydrated position emit primarily at 435 nm. Laurdan's equilibration additionally represents movement of the probe between leaflets and multiple bilayers.