Peripheral steroids and oxycholesterols are important lipid compounds controlling various functions in the human body. Steroid analysis of biological samples is routinely employed in the clinical environment as an essential source of information on endocrine and metabolic disorders. It has been reported that stress related neurosteroids have been implicated in the development and prognoses of neurodegenerative disorders such as Alzheimer’s disease (AD). These compounds have been identified as possible biomarkers in the diagnosis of AD and other neurodegenerative disorders. Therefore, methods for the simultaneous analysis of steroids from the four major classes (estrogens, androgens, progestogens and corticosteroids) are vital in providing useful and more comprehensive data. Homeostasis of cholesterol in the brain is maintained primarily by metabolism to oxysterols, including oxycholesterols. These oxycholesterols act as a transport form of cholesterol as it readily navigates the blood-brain barrier. Oxycholesterols are generally more bioactive than cholesterol and is of interest in pathophysiology. Moreover, if their production in cells and tissues and/or their introduction with dietary animal fat are excessive, oxycholesterols could indeed contribute to the pathogenesis of various disease processes. The first study in this thesis focuses on a novel supercritical fluid chromatography–tandem mass spectrometry method for targeted analysis of eighteen peripheral steroids. The method is simple and fast. It has sufficient sensitivity for quantification of 18 different steroids in small volume human plasma. Therefore, this novel method can be applied for screening many steroids within 5 minutes providing the possibility to use for routine healthcare practice. The second study involves the quantification of three adrenal steroids in plasma from domesticated White Leghorn (WL) chickens and Red Junglefowl (RJF) birds. The domestication effects on stress induced steroid secretion and adrenal gene expression in chickens are evaluated. The third study focuses on determination of more than ten oxycholesterols in biological samples with a gas chromatography–mass spectrometry method and a supercritical fluid–tandem mass spectrometry method.

<p>LC-MS of protein drugs requires new ideas in bonded phase design rather than adapting bonded phases from the realm of small-molecule drugs. The polymer-shell bonded phase is designed to interact with larger molecules and to shield proteins from the silica substrate. The particles consist of a core of solid silica and a shell of dense polymer brush. The polymer layer is thick enough to protect the protein from interactions with silanols to reduce peak tailing. The polymer contains multiple functional groups that introduce more selectivity. This design gives unprecedented LC resolution and MS sensitivity. Our group has developed polymer shell bonded phases for hydrophobic interaction chromatography (HIC-MS) of antibody-drug conjugates (ADCs), hydrophilic interaction liquid chromatography (HILIC-MS) of glycoproteins, and reversed-phase liquid chromatography (RPLC-MS) of monoclonal antibodies. Since HIC is not in-line compatible with MS due to the high salt levels, it is laborious to identify the constituents of HIC peaks. An MS-compatible alternative to HIC is reported here: native reversed phase liquid chromatography (nRPLC). This employs a mobile phase 50 mM ammonium acetate for high sensitivity in MS, and elution with a gradient of water/isopropanol. The nRPLC-MS data show that all ADC species, ranging from drug-to-antibody ratios of 1 to 8, remained intact and native on the column. As we adapt this concept to intact proteins, we find that lysozyme and α-chymotrypsinogen A are both eluted in their native conformations. We also use the polymer-shell concept to resolve IgG1 free thiol variants by RPLC-MS with 0.5% formic acid. Since there are always other variants besides the intended ones, the need for high MS sensitivity is desired to distinguish subtle mass change between disulfide bond and free thiols. Overall, MS sensitivity increases 10X relative while all of the thiol variants are well resolved by the polymethylmethacrylate bonded phase.</p>

A transferência de oxigênio para o meio, durante o cultivo de Candida kefyr DSM 70106, mostrou ter grandE influência na produção de inulinase extracelular. O KLa (coeficiente volumétrico de transferência de oxigênio) de 43 h-1 foi o que proporcionou a obtenção de atividade enzimática mais elevada (37,5 U.mL-1). Este parâmetro foi utilizado como critério para ampliação de escala do cultivo em biorreator de 15 litros para 300 litros. O volume de meio obtido foi congelado para utilização nos processos seguintes de separação de células e purificação da inulinase. Para isto foram utilizados três processos diferentes, a saber, filtração (microfiltração, diafiltração e ultrafiltração), extração líquido-líquido por micela reversa e cromatografia de troca iônica em leito fluidizado. Durante a separação de células, a membrana de microfiltração tipo HVLP com 0,45 &#181;m de diâmetro de poro foi a que possibilitou a passagem de maior fluxo de filtrado (324 L.h-1.m-2) quando comparada com outras membranas de poros e materiais diferentes. Associado a este fluxo obteve-se ainda a passagem pela membrana de 99% da enzima presente. Foram também estudados diferentes tipos de módulos de filtração. Os filtros rotativos foram os que apresentaram melhores desempenhos, com destaque para o tipo \"Biodruckfilter\". Fluxo de filtrado de 300 L.h-1.m-2 e transmissão de 99% da enzima foram obtidos neste filtro. Os filtros tipo cassete e os tubulares apresentaram desempenhos, em geral, inferior a 50% quando comparados em termos de fluxo de filtrado. O processo de filtração em escala ampliada, realizada no filtro cassete, apresentou fluxo de filtrado 45% inferior ao obtido na pequena escala. Os estudos de extração líquido-líquido por micela reversa foram feitos utilizando um agente tensoativo catiônico (BDBAC- [N-Benzyl-N-Dodecyl-N bis(2-hydroxyethyl) ammonium chloride] e um aniônico (AOT- sodium-di-2ethyl-hexyl-sulphosuccinate). O BDBAC proporcionou recuperação de 91 % da enzima presente inicialmente. Na ampliação de escala do processo foi verificado um rendimento de 77%, e o aumento da atividade enzimática específica, nos dois casos, foi de aproximadamente 2,8 vezes. Em comparação com os dois processos anteriores, o processo de purificação da inulinase com separação simultânea de células por cromatografia de trocaiônica em leito fluidizado foi o que proporcionou maior aumento da atividade enzimática específica (da ordem de 4,3 vezes). Após adsorção e eluição do leito cromatográfico, 93,1% da enzima foi recuperada. / It has been observed that the production of extracellular inulinase by Candida kefyr DSM 70106 was markedly affected by the rate of oxygen transfer to the medium. The highest inulinase activity (37,5 U.Ml-1) was attained at a KLa value (volumetric coefficient of oxygen transfer) equal to 43 h-1. As such, KLa was chosen as a criterion for scaling-up the bioreactor capacity from 15 L to 300 L. Once the fermentation ended, the whole fermented broth was frozen for further cell separation and inulinase purification. For that, three different processes were used, namely filtration (microfiltration, diafiltration and ultrafiltration), liquid-liquid extraction by reversed micelles and ion-exchange chromatography in fluidized bed. Microfiltration membranes differing in the chemical nature and on the pores diameter were used in cell separations. The highest filtrate flow (FF = 324 L.h-1.m-2) occurred when HVLP type membrane (0,45 &#181;m pore diameter) was employed. Moreover, 99% of inulinase passed through the membrane. Several types of filtration modules were also studied. The rotatory filter, such as the \"Biodruckfilter\" (FF=300 L.h-1.m-2 and 99% of inulinase transmission), presented remarkable performance when compared with cassette and tubulartype filters, in which FF was lower than 50%. Furthermore, filtration carried out with a scaled-up cassette module presented a FF 45% lower than that observed on a smaller scale. Studies on liquid-liquid extraction by reverse micelles were made utilizing a cationic surfactant (BDBAC- [N-Benzyl-N-Dodecyl-N-bis(2-hydroxyethyl) ammonium chloride] and an anionic surfactant (AOT-sodium-di-2-ethyl-hexylsulphosuccinate). The use of BDBAC resulted in a recuperation of 91 % of the enzyme initially present, which decreased about 77% when the process was scaled-up. Neverthless, in both cases the increase in specific enzymatic activity was about 2.8 times. In comparison with the aforementioned processes, the ion-exchange chromatography in fluidized bed, in which inulinase purification and cell separation take place simultaneously, provided. an increase in specific enzymatic activity of about 4.3 times. After adsorption and elution of the chromatographic bed about 93% of the inulinase was recovered.

by Tsoi Yeung-pang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 105-106). / Chapter I. --- Acknowledgements --- p.i / Chapter II. --- Abstract --- p.ii / Chapter III. --- Table of contents --- p.iv / Chapter IV. --- List of Tables and Figures --- p.v / Chapter Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Modes of chromatography / Chapter 1.2 --- Objective of the present study / References / Chapter Chapter 2. --- Instrumentation and theory --- p.8 / Chapter 2.1 --- Instrumentation of HPLC / Chapter 2.2 --- Theory of liquid chromatography / References / Chapter Chapter 3. --- Determination of citrate in pharmaceutical preparations by HPLC using indirect photometric detection --- p.21 / Chapter 3.1 --- Introduction / Chapter 3.2 --- Review of the analytical methods / Chapter 3.3 --- Theory of detection / Chapter 3.4 --- Experimental / Chapter 3.5 --- Results and discussion / Chapter 3.6 --- Conclusion / References / Chapter Chapter 4. --- Determination of camphor and menthol by HPLC using indirect conductometric detection --- p.74 / Chapter 4.1 --- Introduction / Chapter 4.2 --- Review of the analytical methods / Chapter 4.3 --- Theory of detection / Chapter 4.4 --- Experimental / Chapter 4.5 --- Results and discussion / Chapter 4.6 --- Conclusion / References

by Lung Man Tung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1991. / Includes bibliographical references. / ACKNOWLEDGMENT --- p.i / ABSTRACT --- p.ii / Chapter CHAPTER 1 --- GENERAL INTRODUCTION --- p.1 / Chapter 1.1 --- Stationary phases and detectors --- p.2 / Chapter 1.2 --- Internal standardization --- p.5 / Chapter 1.3 --- Common pre-treatment technique --- p.6 / Chapter 1.4 --- Pre-treatment techniques --- p.9 / Chapter 1.5 --- Objectives of this work for the determination of styrene --- p.11 / Chapter CHAPTER 2 --- EXPERIMENTAL --- p.13 / Chapter 2.1 --- Instrumentation --- p.13 / Chapter 2.2 --- Reagents and solutions --- p.16 / Chapter 2.3 --- Preparation of stock solutions --- p.18 / Chapter 2.4 --- Sample preparation --- p.19 / Chapter 2.5 --- Extraction method for food samples --- p.20 / Chapter 2.6 --- Procedure for the analytical finish --- p.22 / Chapter 2.7 --- Assessment of the food matrix effect on the recovery of styrene --- p.23 / Chapter 2.8 --- Confirmatory Procedures --- p.23 / Chapter 2.9 --- Treatment of data --- p.24 / Chapter CHAPTER 3 --- DEVELOPMENT OF METHOD --- p.26 / Chapter 3.1 --- Development of GC method --- p.26 / Chapter 3.2 --- Development of the extraction method --- p.29 / Chapter CHAPTER 4 --- DETERMINATION OF RESIDUAL STYRENE IN POLYSTYRENE POLYMER --- p.50 / Chapter 4.1 --- The proposed method --- p.50 / Chapter 4.2 --- Identification of polymer and one cup for the ice-cream cone --- p.51 / Chapter 4.3 --- Determination of residual styrene monomer in polystyrene container and cup --- p.52 / Chapter CHAPTER 5 --- DETERMINATION OF STYRENE MONOMER MIGRATED INTO FOODSTUFFS --- p.58 / Chapter 5.1 --- Methodolgy and scope --- p.58 / Chapter 5.2 --- Determination of styrene migrated into ice-cream --- p.59 / Chapter 5.3 --- Determination of styrene migrated into Yakult and Yogo --- p.60 / Chapter 5.4 --- Determination of styrene migrated into foodstuff kept at elevated temperature in polystyrene containers --- p.60 / Chapter 5.5 --- Conclusion --- p.74 / REFERENCES --- p.76 / APPENDIX --- p.80

Pharmaceuticals are a group of emerging organic compounds of environmental concern used extensively in human and veterinary medicine. They are continually released into the environment as a result of manufacturing operations and excretion from humans and animals. These compounds enter directly into the municipal sewage systems and into wastewater treatment plants. A large number of important and potentially harmful organic contaminants, such as these pharmaceuticals, are not regulated in drinking and other waters. As a result, conventional technologies at most waste water treatment plants (WWTPs) discharge water that meet regulatory standards, yet are not specifically designed to remove these organic contaminants. Therefore, pharmaceutical compounds and their metabolites remain in discharged effluent and enter into the natural aquatic environment. Concentrations of pharmaceutical residues measured in water are typically reported in the ranges of ug/L to ng/L, which are at least three to four orders of magnitude lower than that required to produce a pharmacological effect. The probability of risks to humans arising from such an acute exposure is unlikely, but the possible effects resulting from life-long exposures and synergistic effects from exposure to many chemicals have yet to be determined. It has been widely reported that pharmaceuticals and their metabolites that enter into the aquatic environment can have a potential harmful effect on the aquatic ecosystem and can reach drinking water sources. This research focuses on non-steroid anti-inflammatory drugs (NSAIDs), a group of pharmaceuticals which are widely used as analgesic, antipyretic and anti-inflammatory agents. NSAIDs are frequently used because they are easily accessible as over the counter medication and are a group of drugs that do not produce addiction, respiratory depression, or drowsiness. There is an incentive for removing NSAIDs and other pharmaceuticals from the aquatic environment. Thus, quantitative evaluation of the fate of pharmaceuticals, proper risk assessment and improvement of the efficiency of WWTPs need sensitive and reliable analytical methods. The purpose of this project was to provide a method for detecting three common NSAIDs, IBF, KTF, and NAP, in purified water with LLE-GC-FID. And, an investigation of UV photolysis, UV/H2O2, and UV/TiO2 AOPs was performed to determine their effectiveness in treating IBF, KTF, and NAP in purified water. All treatment methods were successful in degrading target compounds with a total degradation of 86% or greater after 45 minutes. A liquid-liquid extraction technique using methylene chloride and BSTFA + 1%TMCS derivatizing agent was determined for detecting low concentrations of IBF, KTF, and NAP with calibration curves showing good linearity with all R2 values greater than 0.9880.

Microporous walled micro-capillary films (MMCFs) are porous polymer films with embedded capillaries. MMCFs have been found to be suitable low-cost chromatography substrates capable of tolerating high flowrates, which suggested they might be a solution to the growing bottleneck in the downstream purification of biopharmaceuticals. However, MMCFs have been mainly tested for binary separations of model proteins and broader capabilities of this technology remain largely unknown. The experimental work presented in this thesis focused on developing MMCFs functionalised with different ligands and their subsequent testing as chromatography media for bioseparations. MMCFs were functionalised to form weak anion (MMCF-DEAE) and weak cation-exchangers (MMCF-gCM and MMCF-CA). Emphasis was placed on low-cost functionalisation methods amenable to single-use applications. To confirm the addition of functional groups on the membranes, a comprehensive characterisation routine was implemented. This included the use of spectroscopy, electron microscopy, elemental analysis and pH titration. The binding performance of these weak ion-exchangers was further assessed by static and dynamic adsorption of model proteins. Next, the functionalised MMCFs were tested for bioseparations with binary and complex mixtures, the latter being more relevant for industrial applications. It was found that MMCFs could selectively separate similarly charged biomolecules using optimised elution strategies. Further, it was observed that MMCF-gCM could capture lysozyme from chicken egg white at a near twenty-fold purity increase compared to the feed. Similarly, this weak cation-exchanger could recover antibodies from unfiltered mammalian cell lysate. The recovered biomolecules were then injected onto MMCF-DEAE to remove nucleic acid impurities in subtractive chromatography mode. Finally, MMCFs were explored for the first time as affinity chromatography supports. Bovine serum albumin (BSA) and Protein A were covalently coupled to the substrate and tested for the capture of relevant analytes. Indeed, it was found that MMCF-BSA could bind bilirubin and MMCF-ProtA could be used to recover antibodies. Given the high cost of Protein A, a cheaper synthetic ligand was coupled onto MMCFs and observed to successfully bind antibodies. Overall, this work has furthered the applications of MMCFs for bioseparations, demonstrating their great versatility and robustness. Furthermore, it opened the field for affinity-based MMCFs, which could have numerous applications in both research and industry. Extensive characterisation methods presented here will greatly simplify future studies with these membranes. While the binding capacity of the developed ion-exchangers was typically two orders of magnitude higher than non-porous MCFs (NMCFs), the low yield achieved with MMCFs currently precludes them from commercial applications. However, optimisation of MMCFs, as outlined in the future work, could make this support more commercially viable and leverage the numerous advantages offered by its unique geometry.

Typescript (photocopy). / Digitized by Kansas Correctional Industries

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 1979. / MICROFICHE COPY AVAILABLE IN ARCHIVES AND SCIENCE. / Vita. / Includes bibliographical references. / by John Milan Lavoie, Jr. / Ph.D.

Siu Yuen Yu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 122-139). / Abstracts in English and Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iii / Acknowledgments --- p.v / Table of Contents --- p.vi / List of Figures --- p.x / List of Tables --- p.xvi / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter CHAPTER 2 --- LITERATURE REVIEW --- p.7 / Chapter 2.1 --- Traditional methods for studying zooplankton feeding selectivity --- p.7 / Chapter 2.1.1 --- Cell counting after laboratory feeding experiments --- p.7 / Chapter 2.1.2 --- Direct examination of gut contents --- p.8 / Chapter 2.1.3 --- Use of radioactive tracers --- p.9 / Chapter 2.1.4 --- Gut fluorescence method --- p.9 / Chapter 2.2 --- High Performance Liquid Chromatography analysis of phytoplankton pigments --- p.11 / Chapter 2.2.1 --- Principle --- p.11 / Chapter 2.2.2 --- Pigments as signature markers of phytoplankton --- p.11 / Chapter 2.2.3 --- Development of HPLC analysis of phytoplankton pigments --- p.16 / Chapter 2.2.4 --- Advantages of HPLC analysis of phytoplankton pigments --- p.17 / Chapter 2.2.5 --- Limitation of HPLC analysis of phytoplankton pigments --- p.18 / Chapter 2.3 --- Zooplankton feeding selectivity --- p.19 / Chapter 2.3.1 --- Ecological importance of zooplankton feeding selectivity --- p.19 / Chapter 2.3.2 --- Factors affecting zooplankton feeding selectivity --- p.19 / Chapter 2.3.3 --- Feeding selectivity of zooplankton studied in this study --- p.21 / Chapter 2.3.3.1 --- p. avirostirs --- p.21 / Chapter 2.3.3.2 --- Paracalanus spp --- p.22 / Chapter 2.3.3.3 --- Pseudevadne tergestina --- p.23 / Chapter 2.4 --- Pigment degradation in guts of zooplankton --- p.24 / Chapter 2.4.1 --- Experimental design --- p.24 / Chapter 2.4.2 --- Pigment degradation --- p.24 / Chapter 2.5 --- "Tolo Harbour, Hong Kong" --- p.26 / Chapter 2.5.1 --- Site description --- p.26 / Chapter 2.5.2 --- Phytoplankton and zooplankton in Tolo Harbour --- p.27 / Chapter CHAPTER 3 --- MATERIALS AND METHODS --- p.28 / Chapter 3.1 --- Field sampling --- p.28 / Chapter 3.1.1 --- Study of seasonal patterns in zooplankton feeding selectivity --- p.28 / Chapter 3.1.1.1 --- Collection of phytoplankton and zooplankton for pigment analysis --- p.28 / Chapter 3.1.1.2 --- Collection of phytoplankton and zooplankton for plankton enumeration --- p.30 / Chapter 3.1.2 --- Collection of phytoplankton and zooplankton for laboratory feeding experiments --- p.32 / Chapter 3.2 --- Laboratory experiments and data analysis --- p.33 / Chapter 3.2.1 --- Study of seasonal patterns in zooplankton feeding selectivity --- p.33 / Chapter 3.2.1.1 --- HPLC of phytoplankton pigments --- p.33 / Chapter 3.2.1.2 --- Fluorometric measurement of chlorophyll-α --- p.35 / Chapter 3.2.1.3 --- Plankton identification and enumeration --- p.36 / Chapter 3.2.2 --- Laboratory feeding experiments for investigation of pigment degradation in zooplankton gut --- p.37 / Chapter CHAPTER 4 --- RESULTS --- p.41 / Chapter 4.1 --- Information on Tolo Harbour --- p.41 / Chapter 4.1.1 --- Temperature and salinity in Tolo Harbour --- p.41 / Chapter 4.1.2 --- Plankton composition and community in Tolo Harbour --- p.43 / Chapter 4.1.2.1 --- Phytoplankton --- p.43 / Chapter 4.1.2.2 --- Zooplankton --- p.50 / Chapter 4.2 --- Seasonal zooplankton feeding selectivity investigated by HPLC phytoplankton pigment analysis --- p.53 / Chapter 4.2.1 --- Verification of HPLC pigment analysis by fluorometric analysis --- p.53 / Chapter 4.2.2 --- Correlations between phytoplankton cell densities and pigment concentrations in water samples --- p.55 / Chapter 4.2.3 --- Feeding selectivity of zooplankton on different phytoplankton groups --- p.73 / Chapter 4.2.4 --- Feeding selectivity of zooplankton on dinoflagellates --- p.87 / Chapter 4.2.5 --- Feeding selectivity of zooplankton on diatoms --- p.87 / Chapter 4.3 --- Feeding selectivity on phytoplankton by other cladoceran - Pseudevadne tergestina --- p.89 / Chapter 4.4 --- Pigment degradation in zooplankton guts after ingestion of phytoplankton --- p.90 / Chapter 4.5 --- Clearance rates of P. avirostris and Paracalanus spp. in feeding experiments --- p.101 / Chapter CHAPTER 5 --- DISCUSSIONS --- p.105 / Chapter 5.1 --- Experiment design --- p.105 / Chapter 5.2 --- Seasonal zooplankton feeding selectivity investigated by HPLC phytoplankton pigment analysis --- p.108 / Chapter 5.2.1 --- Correlations between phytoplankton cell densities and pigment concentrations in water samples --- p.108 / Chapter 5.2.2 --- Feeding selectivity of zooplankton on different phytoplankton groups --- p.108 / Chapter 5.2.3 --- Feeding selectivity of Pseudevadne tergestina --- p.111 / Chapter 5.3 --- Feeding experiments for investigating pigment degradation in guts of zooplankton --- p.112 / Chapter 5.3.1 --- Principle --- p.112 / Chapter 5.3.2 --- Degradation for different pigments in guts of P. avirostris and Paracalanus spp. --- p.112 / Chapter 5.4 --- Clearance rates of P. avirostris and Paracalanus spp. --- p.114 / Chapter 5.4.1 --- p. avirostris --- p.114 / Chapter 5.4.2 --- Paracalanus spp. --- p.115 / Chapter 5.5 --- Limitations of HPLC analysis of phytoplankton pigments --- p.116 / Chapter 5.6 --- Environmental events related to feeding selectivity of zooplankton in Tolo Harbour --- p.118 / Chapter 5.6.1 --- Energy transfer in trophic level --- p.118 / Chapter 5.6.2 --- Abilities of p. avirostris and Paracalanus spp. to control red tides in Tolo Harbour --- p.118 / Chapter CHAPTER 6 --- CONCLUSION --- p.120 / REFERENCES --- p.122 / APPENDIX