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This thesis describes the design of a convenient format of solid phase microextraction (SPME) for bioanalysis in pharmaceutical industry and the validation of the approach to the application. An automated in-tip SPME technique coupled with liquid chromatography (LC) and tandem mass spectrometry (MS/MS) for high throughout drug analysis has been developed and applied to the quantitative determination of various drug compounds in different biological fluids from drug discovery to clinical development. The initial research in this thesis focused on a proof-of-concept study using manual multi-fiber approach to determine a drug compound in human plasma from a clinical trial. The proof-of-concept was achieved based on the validation data and a head-to-head comparison with conventional liquid-liquid extraction (LLE) method. An in-tip SPME technique was then proposed to explore the feasibility of SPME automation and two approaches of preparing in-tip SPME fibers were developed including fiber-packed and sorbent-packed fiber preparation. A simple and high throughput in-tip SPME fiber fabricating procedure based on polymer monoliths using photo-polymerization was introduced to prepare 96 fibers simultaneously. The biggest advantage of the in-tip SPME technique is that it is simple and easy to use for automation without introducing any additional devices and in the meantime, the simplicity of SPME is maintained. Automated in-tip SPME was applied to routine drug analysis in drug discovery and development environment. One case study involved the determination of vitamin D3 in human serum with derivatization and the in-tip SPME approach was compared with traditional LLE method using either tubes or 96-well plate extraction. Another study was to use hydrophilic interaction chromatography (HILIC) –MS/MS to determine three polar compounds, imipenem (IMP), cliastatin (CIL) and -lactamase inhibitor (BLI) simultaneously in different biological fluids including rat plasma and mouse blood. The results from both studies clearly demonstrated that in-tip SPME could be used as an alternative sample preparation method in bioanalytical analysis. Matrix effects in bioanalysis using automated in-tip SPME and LC-MS/MS were then thoroughly evaluated for the first time. Our study indicated that the assumption that SPME should provide sample clean up as effective as or better than solid phase extraction (SPE) with no or minimal matrix effects might not be always true, and matrix effects should be investigated in any SPME assays in bioanalysis. Comparisons between in-tip SPME and other automated SPME approaches such as blade/thin film geometries were performed, and the advantages and limitations of using SPME versus conventional sample preparation methods including protein precipitation (PPT), LLE and SPE were summarized. Strategies for in-tip SPME method development and validation and the potential applications and future directions of in-tip SPME in bioanalysis were discussed. Finally, kinetic models were established to describe SPME extraction and desorption processes in a complex matrix with both liquid and solid fiber coatings. The models were successfully applied to different scenarios to estimate the boundary layer (BL) thickness, extraction equilibrium time and total amount of analytes extracted at a given time. The excellent agreements between the model prediction results and experimental data indicated that the SPME modeling approach had great potentials to speed up SPME method development and fiber selection.

This thesis describes the design of a convenient format of solid phase microextraction (SPME) for bioanalysis in pharmaceutical industry and the validation of the approach to the application. An automated in-tip SPME technique coupled with liquid chromatography (LC) and tandem mass spectrometry (MS/MS) for high throughout drug analysis has been developed and applied to the quantitative determination of various drug compounds in different biological fluids from drug discovery to clinical development. The initial research in this thesis focused on a proof-of-concept study using manual multi-fiber approach to determine a drug compound in human plasma from a clinical trial. The proof-of-concept was achieved based on the validation data and a head-to-head comparison with conventional liquid-liquid extraction (LLE) method. An in-tip SPME technique was then proposed to explore the feasibility of SPME automation and two approaches of preparing in-tip SPME fibers were developed including fiber-packed and sorbent-packed fiber preparation. A simple and high throughput in-tip SPME fiber fabricating procedure based on polymer monoliths using photo-polymerization was introduced to prepare 96 fibers simultaneously. The biggest advantage of the in-tip SPME technique is that it is simple and easy to use for automation without introducing any additional devices and in the meantime, the simplicity of SPME is maintained. Automated in-tip SPME was applied to routine drug analysis in drug discovery and development environment. One case study involved the determination of vitamin D3 in human serum with derivatization and the in-tip SPME approach was compared with traditional LLE method using either tubes or 96-well plate extraction. Another study was to use hydrophilic interaction chromatography (HILIC) –MS/MS to determine three polar compounds, imipenem (IMP), cliastatin (CIL) and -lactamase inhibitor (BLI) simultaneously in different biological fluids including rat plasma and mouse blood. The results from both studies clearly demonstrated that in-tip SPME could be used as an alternative sample preparation method in bioanalytical analysis. Matrix effects in bioanalysis using automated in-tip SPME and LC-MS/MS were then thoroughly evaluated for the first time. Our study indicated that the assumption that SPME should provide sample clean up as effective as or better than solid phase extraction (SPE) with no or minimal matrix effects might not be always true, and matrix effects should be investigated in any SPME assays in bioanalysis. Comparisons between in-tip SPME and other automated SPME approaches such as blade/thin film geometries were performed, and the advantages and limitations of using SPME versus conventional sample preparation methods including protein precipitation (PPT), LLE and SPE were summarized. Strategies for in-tip SPME method development and validation and the potential applications and future directions of in-tip SPME in bioanalysis were discussed. Finally, kinetic models were established to describe SPME extraction and desorption processes in a complex matrix with both liquid and solid fiber coatings. The models were successfully applied to different scenarios to estimate the boundary layer (BL) thickness, extraction equilibrium time and total amount of analytes extracted at a given time. The excellent agreements between the model prediction results and experimental data indicated that the SPME modeling approach had great potentials to speed up SPME method development and fiber selection.

No description available.

Four new synthetic methods employing an oxidative dearomatization - rearomatization strategy are presented. In Chapter 2, a new oxidative dearomatization - radical cyclization - rearomatization approach to form fused oxygen-containing heterocycles is presented. Origins, design, reaction, and optimizations are discussed. In Chapter 3, meta-selective alkylation of catechol mono-ethers is described employing an oxidative dearomatization - radical addition - rearomatization approach using trialkylboranes as source of alkyl radicals. In Chapter 4, a metal-free method to synthesize fluorinated indoles from aniline starting materials is described. Chapter 5 lays the groundwork for para-selective functionalization of catechol mono-ethers. Chapters 6 and 7 highlight the work related to pharmaceutical drug analyses. Chapter 6 presents the FDA approved drugs organized in Disease Focused Posters. Chapter 7.1 and 7.2 present the drug analysis of Sulfur- and Fluorine-Containing Drugs, and Nitrogen-Heterocycle Containing Drugs, respectively.

A análise de fármacos e outras moléculas relacionadas em fluidos biológicos é essencial no âmbito farmacêutico. Atualmente, a demanda por análises rápidas e mais complexas impulsiona a química analítica para o desenvolvimento de soluções inovadoras. A cromatografia líquida multidimensional com acoplamento de colunas para injeção direta de fluidos biológicos tem ganhado atenção nos últimos anos. Ao mesmo tempo, o acoplamento entre cromatografia líquida e espectrometria de massas proporcionou marcante desenvolvimento científico na área biomédica e bioquímica. Esta tese apresenta os diversos estágios na redução da escala em sistemas de column switching utilizando colunas RAM, para a análise de fármacos em fluidos biológicos. Na escala convencional, com colunas de 4,6 mm de diâmetro interno, desenvolveu-se um sistema para a análise de fluoxetina em plasma. A metodologia desenvolvida foi adequadamente validada para aplicação na monitorização terapêutica, com tempo de análise de 20 minutos (incluído o preparo de amostras) e consumo de apenas 100 µL de amostra. Avaliou-se a escala microbore (2,1 mm), a qual apresentou excelente potencialidade para o acoplamento com a espectrometria de massas utilizando ionização por electrospray. Na primeira etapa em escala capilar, com colunas de 520 µm de diâmetro interno, desenvolveu-se um sistema para análise de fluoxetina em plasma. Esse sistema proporcionou análises em 25 minutos, também aplicáveis à monitorização terapêutica, consumindo poucos microlitros de amostra. Finalmente, foi desenvolvido um sistema de column switching capilar com colunas na ordem de 200 µm. Esse sistema foi acoplado à espectrometria de massas em tandem proporcionando, inovadoramente, análises altamente sensíveis e simultâneas, com baixo consumo de amostras. Um grupo de cinco antidepressivos e o albendazol, com seus produtos de biotransformação, tiveram suas análises validadas em menos de 8 minutos, consumindo menos de um microlitro de amostra. Esse sistema capilar contrasta com os sistemas convencionais comumente utilizados, os quais consomem entre centenas e milhares de vezes mais amostra para atingir a mesma detectabilidade. / Analysis of drugs and other related molecules in biofluids is essential in the pharmaceutical field. Nowadays, the development of innovative solutions in analytical chemistry has been pushed by the needs for speed and more complex analysis. Lately, multidimensional liquid chromatography using column switching for direct injection of biofluids has gained attention. At the same time, liquid chromatography hyphenated with mass spectrometry provided remarkable scientific development in biomedical and biochemical area. This thesis presents the scale reduction steps in RAM column switching, for drug analysis in biofluids. In the conventional scale, using 4.6 mm i.d. columns, a system was developed, providing fluoxetine analysis in plasma. The developed method resulted in a 20 min long run, including the sample preparation step, which consumed 100 µL of sample. The method was adequately validated, being applicable to therapeutic drug monitoring. The microbore scale (2.1 mm) was evaluated, presenting great potentiality for coupling with electrospray-mass spectrometry. In the first capillary scale step, using 520 µm columns, a system was developed for fluoxetine analysis. Fluoxetine analysis was achieved in 25 min, within the application range for therapeutic drug monitoring, and consuming few microliters of sample. Finally, a RAM capillary column switching system employing columns on the order of 200 µm was developed, in an innovative way. This system was coupled with a tandem mass spectrometer, rendering sensitive and simultaneous analysis with reduced sample volume. The analysis of one group containing five antidepressants, as well as the analysis of albendazol and its metabolites was validated. These analyses took only 8 minutes and consumed less than one microliter of sample. In contrast with conventional systems, this system consumes about hundreds or thousands times less sample, with the same detectability.

Uma metodologia rápida e seletiva foi desenvolvida para a quantificação da piridoxina em medicamentos. O método foi padronizado para aplicação da espectrofotometria derivada no ultravioleta na análise direta da vitamina em preparações multivitamínicas sólidas (cápsulas) e líquidas (solução oral e injetável). As interferências do espectro UV convencional devidas aos excipientes (veículos) e demais fármacos presentes foram eliminados. As retas de calibração foram calculadas, obtendo-se, para a derivada de 1ª ordem, o coeficiente de correlação linear de 0.99997. Os resultados foram estatisticamente estudados e determinaram-se o desvio padrão, coeficiente de variação e intervalo de confiança. O método foi empregado na análise de amostras comerciais e simuladas e os resultados, quando comparados com aqueles provenientes da aplicação do método da Farmacopéia Americana XXII rev., evidenciaram nítidas vantagens quanto à exatidão e precisão, além da facilidade operacional. / A rapid and selecrive method for rhe dererminarion of pyridoxine in pharmaceuticals has been described. The procedure has been developed using direct UV first-derivative spectrofotometry in solid and liquid preparations (tablets, oral solution and injection). Spectral inrerferences from formulation excipienrs and other drugs in simple UV spectrophotometric methods have been eliminated by the application of the proposed method. Calibration curves have been made and the correlation coefficienr for. the first-order derivative was 0,99997. Standard deviation, coefficient of variation and confidence interval were calculated. The method was applied in the analysis of commercial and simulated samples. The results when compared with those obtained by using the USP 22nd. ed. official method shows clear advanrages related to accuracy, precision and practical application.