Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 145-155).

Many mixtures important to research consist of hundreds or even thousands of individual components of interest. These types of mixtures are far too complex to separate by a single chromatographic dimension in any reasonable amount of time. However, if a multidimensional approach is used, where a complex mixture is separated by an initial dimension, simpler fractions of that separation are collected and each of those fractions are analyzed individually, highly complex mixtures can be resolved in relatively short amounts of time. This dissertation serves as a guide to multidimensional chromatography, in particular, two-dimension liquid chromatography. There are many aspects of multidimensional separations that have been investigated to show its aspects, drawbacks and potential ability to separate highly complex mixtures. Measurements for the performance of multidimensional chromatography, the effects of the first and subsequent dimensions and the approaches to pairing dimensions are shown with experimental examples. Fundamental and practical features of multidimensional chromatography are explained as well as theoretical discussions on current and future multidimensional chromatography performance. Experimentally, very high peak capacities were obtained (ca. 7000) and an algorithm to predict how to best optimize a two-dimensional separation based on the time used and performance was created for designing experiments.

Oxidative protein surface mapping has gained popularity over recent years within the mass spectrometry (MS) community for gleaning information about the solvent accessibility of folded protein structures. The hydroxyl radical targets a wide breadth of reactive amino acids with a stable mass tag that withstands subsequent MS analysis. A variety of techniques exist for generating hydroxyl radicals, with most requiring sources of radiation or caustic oxidizing reagents. The purpose of this research was to evaluate the novel use of electrochemistry for accomplishing a comparable probe of protein structure with a more accessible tool. Two different working electrode types were tested across a range of experimental parameters, including voltage, flow rate, and solution electrolyte composition, to affect the extent of oxidation on intact proteins. Results indicated that the boron-doped diamond electrode was most valuable for protein research due to its capacity to produce hydroxyl radicals and its relatively low adsorption profile. Oxidized proteins were collected from the electrochemical cell for intact protein and peptide level MS analysis. Peptide mass spectral data were searched by two different “hybrid” software packages that incorporate de novo elements into a database search to accommodate the challenge of searching for more than forty possible oxidative mass shifts. Preliminary data showed reasonable agreement between amino acid solvent accessibility and the resulting oxidation status of these residues in aqueous solution, while more buried residues were found to be oxidized in “non-native” solution. Later experiments utilized higher flow rates to reduce protein residence time inside the electrochemical flow chamber, along with a different cell activation approach to improve controllability of the intact protein oxidation yield. A multidimensional chromatographic strategy was employed to improve dynamic range for detecting oxidation of lower reactivity residues. Along with increased levels of oxidation around “reactive hotspot” sites, the enhanced sensitivity of these measurements uncovered a significant level of background oxidation in control proteins. While further work is needed to determine the full utility that BDD electrochemistry can lend protein structural studies, the experimental refinements reported here pave the way for improvements that could lead to a high-throughput structural pipeline complementary to predictive modeling efforts.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (p. 161-186).

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.

Submitted by Cláudia Bueno (claudiamoura18@gmail.com) on 2015-11-03T16:46:05Z No. of bitstreams: 2 Dissertação - Lilian Ribeiro Batista - 2015.pdf: 1964612 bytes, checksum: b9bd7ed726cf48d3b1c896f2d3efb42a (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-11-04T09:38:10Z (GMT) No. of bitstreams: 2 Dissertação - Lilian Ribeiro Batista - 2015.pdf: 1964612 bytes, checksum: b9bd7ed726cf48d3b1c896f2d3efb42a (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-11-04T09:38:10Z (GMT). No. of bitstreams: 2 Dissertação - Lilian Ribeiro Batista - 2015.pdf: 1964612 bytes, checksum: b9bd7ed726cf48d3b1c896f2d3efb42a (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-03-06 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Despite the efforts of many pharmaceutical companies to replace or reduce the content, many liquid medications still contains ethanol, which needs to have its contents regulated and monitored by quality control. Ethanol analyzes are generally carried out using one-dimensional gas chromatograph, however this technique does not always have adequate separation for the analysis of complex samples. The multidimensional chromatography has emerged as an innovative alternative because it allows the combination of two or more independent separation steps, significantly increasing the power of separation of the corresponding one-dimensional techniques. In this way, towards doing ethanol analysis in syrups for child and adult audience, a method by multidimensional gas chromatography-mass spectrometry was developed and validated for quality control purposes in the pharmaceutical industry. The analytical method developed led to a gain in selectivity by using the headspace extraction phase of ethanol in miniaturized and automated sample preparation. The use of HS-MDGC / GCMS provided a better separation of syrup components allowing to evidence that some commercial syrup have ethanol content above the maximum levels recommended by ANVISA. The method was validated according to standards established by ANVISA. The method was validated according ANVISA standards, presenting LOD 0.03% (v / v) and the LOQ of 0.06% (v / v) ethanol. The recovery values were 96.71% to 101.38%. Commercial samples of syrup in selected pharmacies in Goiânia/GO were analyzed by the proposed method and the content of ethanol in some samples was higher than maximum value allowed for this medicines. / Apesar dos esforços de muitas empresas farmacêuticas em substituir ou reduzir o teor alcoólico, muitos medicamentos líquidos ainda contém etanol, o qual precisa ter seu teor regulado e monitorado por técnicas específicas e precisas de controle da qualidade. Análises de etanol são geralmente realizadas utilizando cromatografia gasosa unidimensional, porém esta técnica nem sempre apresenta adequada separação para a análise de amostras complexas. A cromatografia multidimensional surgiu como uma alternativa inovadora, pois permite a combinação de duas ou mais etapas de separação independentes, aumentando significativamente o poder de separação das técnicas unidimensionais correspondentes. Assim, visando efetuar a análise de etanol em xaropes destinados ao público infantil e adulto, um método via cromatografia gasosa multidimensional acoplada à espectrometria de massas foi desenvolvido e validado para fins de controle de qualidade na indústria farmacêutica. O método analítico desenvolvido proporcionou ganho de seletividade pela utilização do headspace como fase extratora de etanol em sistema miniaturizado e automatizado de preparo de amostra. O uso de HS-MDGC-MS proporcionou a separação mais adequada dos componentes do xarope, permitindo evidenciar que alguns xaropes comerciais apresentam teor de etanol acima dos teores máximos preconizados pela Anvisa. O método foi validado segundo as normas estabelecidas pela Anvisa apresentando LD de 0,03% (v/v) e o LQ de 0,06% (v/v) de etanol. Os valores de recuperação foram de 96,71% a 101,38%. Amostras comerciais de xarope selecionados em farmácias de Goiânia/GO foram analisadas pelo método proposto, sendo que em algumas dessas amostras a concentração de etanol obtida foi superior aos limites máximos permitidos nestes medicamentos.

A novel chromatographic method was devised that makes use of the superb group separation power of normal phase supercritical fluid chromatography (SFC) combined with a fast second separation by a resistively heated gas chromatograph (GC). The SFC was operated isothermally with stopped flow to provide the time required for the GC analysis. The GC analysis had a typical cycle time of 1 minute. During this time the GC column was independently heated at a rate of 450°C/min to 250°C and actively cooled down again to -50°C before the next GC injection takes place. This was achieved with an in-house designed, resistively heated, temperature programmable gas chromatograph. Various temperature measurement circuits were also evaluated. An interface was developed that allows transfer between the SFC and the GC in such a way that the entire eluent from the first separation is analysed by the second separator. Chromatographic resolution was not lost during the transfer process from the first to the second separation stages. The interface also allows for the exchange of the carrier gas used in the second gas chromatographic separation to provide for the maximum separation speed. In the first separation, a silica gel packed column and the novel application of a silica gel porous layer open tubular capillary column was used for SFC group separation. The SFCxGCftp was applied to petrochemical samples and essential oils and the results were compared to that obtained with a commercially available GCxGC system. / Thesis (PhD)--University of Pretoria, 2003. / Chemistry / unrestricted

Separation technologies have occupied a central role in the current practices of analytical methods used for drug analysis today. As the emphasis in contemporary drug analysis shifts towards ultra-trace concentrations, the contribution from unwanted matrix interferences takes on greater significance. In order to single out a trace substance with confidence from a rapidly expanding list of drug compounds (and their metabolites) in real complex specimens, analytical technologies must evolve to keep up with such trends. Today, the task of unambiguous identification in forensic toxicology still relies heavily upon chromatographic methods based on mass spectrometric detection, in particular GC-MS in electron ionisation (EI) mode. Although the combined informing power of (EI) GC-MS has served faithfully in a myriad of drug application studies to date, we may ask if (EI) GC-MS will remain competitive in meeting the impending needs of ultra-trace drug analysis in the fut ure? To what extent of reliability can sample clean-up strategies be used in ultra-trace analysis without risking the loss of important analytes of interest? The increasing use of tandem mass spectrometry with one-dimensional (1D) chromatographic techniques (e.g. GC-MS/MS) at its simplest, considers that single-column chromatographic analysis with mass spectrometry alone is not sufficient in providing unambiguous confirmation of the identity of any given peak, particularly when there are peak-overlap. Where the mass spectra of the individual overlapping peaks are highly similar, confounding interpretation of their identities may arise. By introducing an additional resolution element in the chromatographic domain of a 1D chromatographic system, the informing power of the analytical system can also be effectively raised by the boost in resolving power from two chromatographic elements. Thus this thesis sets out to address the analytical challenges of modern drug analysis through the application of high resolut ion comprehensive two-dimensional gas chromatography (GC„eGC) to a series of representative drug studies of relevance to forensic sciences.