Objective: The aim of this study was to investigate inherent in vitro permeability, metabolism, and cytotoxicity of idebenone - an active used to protect skin as an anti-aging agent -and compare it to idebenone linoleate. Methods: Idebenone and idebenone linoleate were investigated in pig ear skin and melanoma (B16: F10 mouse) cells. Diffusion experiments were conducted at 37 °C (bath temperature) using Franz diffusion cells. Authentic metabolite samples were synthetically prepared. Samples were analyzed using liquid chromatography-mass spectrometry/mass spectrometry. Cell viability was determined via the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. Results: Idebenone was shown to permeate across viable porcine ear tissue; there was no evidence that idebenone linoleate permeated across porcine ear tissue after 4 h. Idebenone was metabolized to idebenone acid in both pig ear and mouse melanocytes; only minor idebenone linoleate metabolism was observed. Idebenone displayed delayed in vitro toxicity (via MTT assay) in melanocytes, while idebenone linoleate displayed no such in vitro toxicity. Conclusions: The in vitro metabolism and cytotoxicity results suggest that metabolic activation of idebenone is the likely culprit that activates the skin irritation mechanism via idebenone in vivo usage. An idebenone ester (e.g. idebenone linoleate) appears to provide a superior in vitro safety profile over idebenone.

Raloxifene is a highly insoluble, highly metabolized serum estrogen receptor modulator approved for use in the treatment of osteoporosis. Hydroxybutenyl-beta-cyclodextrin (HBenBCD) is a novel solubility enhancer previously demonstrated to increase the oral bioavailability of tamoxifen, letrozole, and itraconazole. The current study evaluated the pharmacokinetics of raloxifene in oral and intravenous formulations with HBenBCD in male Wistar-Hannover rats. Analytical methodology to measure raloxifene and its metabolites was developed by measuring raloxifene metabolism in vitro. Formulation with HBenBCD significantly increased raloxifene oral bioavailability. Mean ± S.D. oral bioavailabilities were 2.6 ± 0.4% for raloxifene formulated with microcrystalline cellulose, 7.7 ± 2.1% for a solid capsule formulation of raloxifene:HBenBCD complex, and 5.7 ± 1.3% for a liquid-filled capsule formulation containing raloxifene:HBenBCD/PEG400/H2O. Relative to raloxifene/microcrystalline filled capsules, the presence of HBenBCD in the solid capsule formulation afforded: (i) a decrease in raloxifene Tmax (2.5 ± 0.5 h versus 4.0 ± 0.5 h); (ii) a two-fold increase in raloxifene Cmax and a three-fold increase in raloxifene AUC; and (iii) a 12-fold increase in raloxifene glucuronide Cmax and a 6.5-fold increase in raloxifene glucuronide AUC. Hence, these studies demonstrate that raloxifene formulations containing HBenBCD significantly increased the oral bioavailability in rats relative to formulations that did not contain HBenBCD.

Synthetic cannabinoids (SCs), present a multitude of problems in terms of maintaining up-to-date, reliable, specific, and sensitive methods of detection. Synthetic cannabinoids are novel psychoactive substances originally synthesized for medical use and research purposes. Abuse of these compounds, however, has demonstrated a variety of effects ranging from euphoria to aggressive behavior and loss of consciousness. The most dangerous reported result of synthetic cannabinoids use has been death. The number of synthetic cannabinoid compounds detected drastically increased from two to over 80 compounds within six years. The marketing of these compounds, similar naming, and described pharmacological interactions, create the dangerous and very false perception that SCs are similar to, or the same as, tetrahydrocannabinol in cannabis products. This research focused on the development of a method to detect and quantify seven synthetic cannabinoids in urine, plasma, and gummy bears. The seven synthetic cannabinoids studied include XLR-11, AB-PINACA 5-pentanoic acid metabolite, UR-144 5-pentanoic acid metabolite, 5F-PB-22, AM-2201 4-hydroxypentyl metabolite, JWH-018, and JWH-018 5-hydroxypentyl metabolite. Sample preparation methods and a two dimensional liquid chromatography tandem mass spectrometry method were optimized and developed for analysis of the seven SCs in each matrix. The method was successfully applied to 17 authentic urine case samples previously screened positive for synthetic cannabinoids and a calibration curve for each matrix was generated from spiked samples at varying concentrations. Utilizing two-dimensional (2D) chromatography for the analysis of synthetic cannabinoids allowed for a novel approach to be employed. With this method, 100% organic samples were analyzed with improved resolution and increased sensitivity. The sample preparation method for the urine and plasma samples included a protein precipitation technique with acid followed by solid phase extraction (SPE) on a mixed-mode reversed phase strong anion exchange sorbent. The spiked gummy bear samples were prepared in 50% methanol in water, dissolved by heating, and extracted with SPE on the same sorbent used for the urine and plasma samples. A 200µL injection of the 100% MeOH extracts was injected into 2D-LC-MS/MS for analysis with a loading and diluting solvent consisting of water and 2% ammonium hydroxide and elution solvents containing water or methanol with 0.5% formic acid. These conditions were optimized with an automated method development protocol assessing various conditions such as mobile phase solvents, pH additives, and trap column chemistries. The final chromatography method utilized an ACQUITY ultra performance liquid chromatography (UPLC) ethylene bridged hybrid (BEH) C8 2.1 x 30mm, 10µm trap column and an ACQUITY UPLC high strength silica with tri-functional C18 bonding (HSS T3) analytical column 2.1 x 150mm, 1.7µm. The urine calibration curve produced had a linear dynamic range (LDR) of 0.05-2.5ng/mL for UR-144 5-COOH and AB-PINACA 5-COOH and 0.05-5ng/mL for the other five synthetic cannabinoids. R2 values included 0.992 and 0.993 for UR-144 5-COOH and AB-PINACA 5-COOH, respectively and 0.995 or above for the other five compounds. Synthetic cannabinoids were detected at varying concentrations in all 17 urine case samples. Analysis of plasma and gummy bear samples was also successfully carried out. Plasma calibration curves had a LDR 0.05-10ng/mL with all R2 values above 0.995. Gummy bear calibration curves produced a LDR of 0.05-10ng/mL or 0.05-2.5ng/mL with R2 values over 0.995. All extraction recovery values were greater than 80% with the exception of 63% recovery for AB-PINACA 5-COOH in the gummy bear matrix. Suppression effects of 8%, 19%, and 6.6% were observed for urine, plasma, and gummy bears, respectively. Relatively low recovery values, reduced linear dynamic ranges, and suppression matrix effects for the carboxylic acid analytes assessed in this research suggested an alternative approach may be more successful for the analysis of these particular compound types in all three matrices. Overall, a sensitive, specific, and reliable method was developed with low limits of detection and quantification for efficient and rapid analysis of compounds at trace levels utilizing 2D-LC-MS/MS.

No description available.

Eastern Cistern B on the Ad-Deir Plateau is unusual in its breadth and variety of ceramic vessels represented by thousands of sherds. These sherds raised significant questions about the purpose and use of the vessels that they originally came from. This thesis argues that the ceramic vessels that were deposited in Eastern Cistern B represent the deposited remains of an ancient Nabataean ritual feast. Furthermore, this thesis also helps to answer the questions surrounding what the ceramic containers once held and their relationship to that ritual meal. This thesis identified residues absorbed within the ceramic fragments through the use of a gas chromatographer-mass spectrometer analysis. The data demonstrated that the vessels once contained remains of medicinal plants, such as crocus, oleander, and others as well as post deposition contaminants throughout the majority of the samples. Those contaminations and other limitations mentioned in this thesis may provide a basis for future investigations for absorbed residue analysis in Petra, Jordan. In turn, the relationship between the medicinal plants and the feast at Eastern Cistern B suggests that a healing ritual possibly accompanied a meal suggesting archaeologists begin examining this and other associated events that accompanied ritual feasts in and among the Nabataeans and in the ancient Near East more broadly.

Biogas, a gaseous mixture produced during decomposition of organic matter, is a renewable, easily generated and common byproduct of anaerobic digestion at wastewater treatment plants (WWTP), landfills and agricultural operations. There is growing interest in researching and utilizing the energy potential associated with its combustion. Siloxanes, a family of volatile organic silicon compounds, pose large impediments to biogas usage due to the formation and precipitation of silicon dioxide within combustion devices. Removal of siloxanes prior to combustion is therefore a growing endeavor. Research was performed to synthesize a representative gas stream produced from anaerobic digesters within WWTP. Methane, carbon dioxide and hydrogen sulfide were combined with humidity and gaseous siloxane in levels characteristically seen exiting anaerobic digesters. A methanol impinger train was utilized to sample the biogas composite. Gas chromatography-mass spectrometry (GC-MS) was used to determine gas-phase siloxane concentrations in the gas stream effluent for the purposes of confirming the generation of a consistent and reproducible biogas stream. / Master of Science

<p> This thesis focused on the development of comprehensive, rapid and simple methodologies for the analysis of fatty acids by gas chromatography mass spectrometry (GC/MS) and intact lipids by electrospray ionization tandem mass spectrometry (ESIIMS/MS). The methodologies were applied as a tool for functional genomics in the soil bacterium Sinorhizobium meliloti. The effects of inorganic phosphate (Pi)-starvation and acidity on lipid composition were studied. </p> <p> A micro-scale, one-vial method for the analysis of fatty acids as their fatty acid methyl esters by GC/MS was developed. The method required small sample sizes, involved minimum handling and avoided tedious extraction steps, which increased sample throughput. A series of quality controls were included to measure losses due to handling, derivatization efficiencies and the extent of side reactions. The method was suitable for the analysis of sensitive bacterial fatty acids such as cyclopropane fatty acids. </p> <p> A shotgun lipidomics approach was developed for the analysis of intact lipids by ESIIMS/MS. Fatty acid distributions were obtained for eight lipid classes and up to 58 individual lipids were identified in crude lipid extracts without sample cleanup or chromatography. For the first time, fatty acid distributions were provided for non-phosphorus containing lipids using shotgun lipidomics. Fatty acid distributions within lipid classes suggested that phospholipids and 1,2-diacylglyceryl-3-O-4'-(N,N,N-trimethyl)-homoserine lipids (TMHSs) were both synthesized from phosphatidic acid while sulfoquinovosyldiacylglycerol (SLs) had a different biosynthetic origin. </p> <p> The methodologies were applied to study knockout mutants of five genes thought to participate in lipid metabolism in S. meliloti. It was demonstrated that: (1) cfa2 gene coded for the main cyclopropane fatty acyl synthase; (2) the plsC gene coded for a fatty acy 1 transferase specific for C 16 fatty acids in the sn-2 position of phospholipids; (3) a metabolic phenotype was revealed for knockout mutants of dme and tme genes (DME and TME, malic enzymes) when succinate was the carbon source. </p> / Thesis / Doctor of Philosophy (PhD)

Apolipoprotein E (apoE) occurs on the majority of plasma lipoproteins and plays a major role in the lipid metabolism in the periphery and in the central nervous system. ApoE is a polymorphic protein with three common isoforms, apoE2, apoE3 and apoE4, derived from respective alleles '2, '3 and '4. The aim of this study was to develop a sample pretreatment protocol combined with rapid mass spectrometry (MS)-based assay for simultaneous apolipoprotein profiling and apoE phenotype identification. This assay was validated in 481 samples from patients with stable atherosclerotic cardiovascular disease (ASCVD) and applied to study association with mild cognitive impairment (MCI) in the LIFE Adult study, including overall 690 study subjects. Simultaneous quantification of 8–12 major apolipoproteins including apoA-I, apoB-100 and apoE could be performed within 6.5 min. Phenotyping determined with the developed MS assay had good agreement with the genotyping by real-time fluorescence PCR (97.5%). ApoE2 isoform was associated with the highest total apoE concentration compared to apoE3 and apoE4 (p < 0.001). In the subgroup of diabetic atherosclerotic cardiovascular disease (ASCVD) patients, apoE2 isoform was related to higher apoC-I levels (apoE2 vs. apoE3, p < 0.05), while in the subgroup of ASCVD patients under statin therapy apoE2 was related to lower apoB-100 levels (apoE2 vs. apoE3/apoE4, p < 0.05). A significant difference in apoE concentration observed between mild cognitive impairment (MCI) subjects and controls was confirmed for each apoE phenotype. In conclusion, this study provides evidence for the successful implementation of an MS-based apoE phenotyping assay, which can be used to assess phenotype effects on plasma lipid and apolipoprotein levels.

No description available.

No description available.