Wong Pak Ki. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 86-88). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.iv / Dedication --- p.v / Table of Contents --- p.vi / List of Abbreviations --- p.ix / List of Appendices --- p.xi / List of Figures --- p.xiv / List of Tables --- p.xx / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1 --- Ginseng and Ginsenosides --- p.1 / Chapter 1.2 --- Instrumental Analysis of Ginsenosides --- p.6 / Chapter 1.2.1 --- Thin Layer Chromatography --- p.6 / Chapter 1.2.2 --- Infrared Spectroscopy --- p.7 / Chapter 1.2.3 --- Colorimetry --- p.7 / Chapter 1.2.4 --- Gas Chromatography --- p.7 / Chapter 1.2.5 --- High Performance Liquid Chromatography --- p.8 / Chapter 1.3 --- Objective of the Study --- p.9 / Chapter Chapter 2: --- Experimental --- p.13 / Chapter 2.1 --- History of Electrophoresis and Capillary Electrophoresis --- p.13 / Chapter 2.1.1 --- Electroosmotic Flow (EOF) --- p.14 / Chapter 2.1.2 --- Electrophoretic Migration --- p.18 / Chapter 2.2 --- Reagents and Materials --- p.20 / Chapter 2.2.1 --- Reagents and Glassware --- p.20 / Chapter 2.2.2 --- Instrumentation --- p.20 / Chapter 2.2.3 --- Preparation of Solutions and Wavelength Selection --- p.22 / Chapter 2.2 --- Procedures --- p.23 / Chapter Chapter 3: --- Results and Discussions --- p.24 / Chapter 3.1 --- Initial Selection of the Running Electrolyte --- p.24 / Chapter 3.2 --- Inclusion Additives in the Aqueous Buffer Solution --- p.29 / Chapter 3.2.1 --- Reasons for Addition of Buffer Additives --- p.29 / Chapter 3.2.1.1 --- Cyclodextrin --- p.29 / Chapter 3.3 --- Addition of Surfactants --- p.33 / Chapter 3.3.1 --- Sodium Dodecyl Sulfate (SDS) --- p.35 / Chapter 3.3.2 --- Sodium Cholate --- p.41 / Chapter 3.4 --- Addition of Organic Modifier --- p.43 / Chapter 3.5 --- Effect of pH --- p.46 / Chapter 3.6 --- Effect of the Concentration of the Borate/Phosphate Solution --- p.51 / Chapter 3.7 --- Effect of Capillaries with Different Inner Diameters (I.D.) --- p.54 / Chapter 3.7.1 --- Effect of pH --- p.54 / Chapter 3.7.2 --- Effect of the Buffer Concentration --- p.60 / Chapter 3.7.3 --- Comparison of Migration Time between Capillaries of 50μm and 75μm Inner Diameter --- p.62 / Chapter 3.8 --- Optimization of Other Experimental Parameters --- p.66 / Chapter 3.8.1 --- Applied Voltage --- p.66 / Chapter 3.8.2 --- The Time of Injection --- p.68 / Chapter 3.8.3 --- The Operating Temperature --- p.70 / Chapter 3.9 --- Intra-day and Inter-day Reproducibility --- p.72 / Chapter 3.10 --- Quantitative Analysis of the Ginsenosides --- p.74 / Chapter 3.11 --- Application of the Developed Methodology --- p.78 / Chapter 3.11.1 --- Experimental Procedures --- p.79 / Chapter Chapter 4: --- Conclusion --- p.83 / References --- p.86 / Appendices --- p.89

Ip Yee-man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 85-89). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abstract --- p.iii / Table of Contents --- p.vi / Abbreviations --- p.xi / List of Figures --- p.xiii / List of Tables --- p.xvii / Chapter / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Background --- p.1 / Chapter 1.2 --- Quality control of Chinese herbal medicine --- p.2 / Chapter 1.3 --- Rhizoma Chuanxiong --- p.4 / Chapter 1.3.1. --- General description --- p.4 / Chapter 1.3.2. --- Chemical constituents --- p.4 / Chapter 1.3.3. --- Pharmacology --- p.7 / Chapter 1.3.4 --- Instrumental analysis --- p.9 / Chapter 1.3.4.1 --- Thin Layer Chromatography (TLC) --- p.9 / Chapter 1.3.4.2 --- Gas Chromatography (GC) --- p.10 / Chapter 1.3.4.3 --- High Performance Liquid Chromatography (HPLC) --- p.10 / Chapter 1.3.4.4 --- Capillary Electrophoresis (CE) --- p.10 / Chapter 1.4 --- Objectives of the study --- p.11 / Chapter 2. --- "Isolation, Characterization and Identification of Reference Compounds" --- p.13 / Chapter 2.1 --- General experiment procedures --- p.13 / Chapter 2.1.1. --- Solvents for chromatographic separation --- p.13 / Chapter 2.1.2 --- Chromatographic methods --- p.13 / Chapter 2.1.2.1 --- Adsorption column chromatography --- p.13 / Chapter 2.1.2.2 --- Thin layer chromatography --- p.13 / Chapter 2.1.2.3 --- Preparative layer chromatography --- p.14 / Chapter 2.1.3 --- Determination of physical data --- p.14 / Chapter 2.1.3.1 --- Infrared (IR) absorption spectra --- p.14 / Chapter 2.1.3.2 --- Nuclear Magnetic Resonance (NMR) spectra --- p.14 / Chapter 2.1.3.3 --- Mass spectra (MS) --- p.15 / Chapter 2.1.3.4 --- X-ray crystallography --- p.15 / Chapter 2.1.4 --- Authentic reference compounds --- p.15 / Chapter 2.2 --- "Procurement, extraction and initial fractionation of Rhizoma Chuanxiong" --- p.15 / Chapter 2.3 --- Chromatographic separation of the chloroform extract --- p.16 / Chapter 2.3.1 --- Chromatographic separation of fraction F1002 --- p.16 / Chapter 2.3.1.1 --- Spectral data for the characterization of compound 1 [5-(hydroxymethyl)- 2- furancarboxaldehyde] --- p.17 / Chapter 2.3.2 --- Column chromatographic separation of fraction F1003A --- p.17 / Chapter 2.3.2.1 --- Spectral data for the characterization of compound 2 (oleic acid) --- p.18 / Chapter 2.3.2.2 --- Physical data for the characterization of compound 3 (ferulic acid) --- p.18 / Chapter 2.3.3 --- Preparative layer chromatographic separation of Fraction F1010 --- p.19 / Chapter 2.3.3.1 --- Spectral data for the characterization of compound 4 (daucosterol) --- p.19 / Chapter 2.4 --- Column chromatographic separation of the hexane extract --- p.19 / Chapter 2.4.1 --- Removal of fatty acids in fraction F2005 and F2006 by partition --- p.20 / Chapter 2.4.2 --- Column chromatographic separation of fraction F2005M --- p.20 / Chapter 2.4.2.1 --- Spectral data for the characterization of compound 5 (butylidenephthalide) --- p.20 / Chapter 2.4.2.2 --- Spectral data for the characterization of compound 6 (butylphthalide) --- p.21 / Chapter 2.4.3 --- Column chromatographic separation of fraction F2006M --- p.21 / Chapter 2.4.3.1 --- "Spectral data for the characterization of compound 7 (Z, Z'-6.6', 7.3'a- diligustilide)" --- p.21 / Chapter 2.4.4 --- Colum chromatographic separation of fraction --- p.22 / Chapter 2.4.4.1 --- Spectral data for the characterization of compound 8 (pregnenolone) --- p.22 / Chapter 2.4.4.2 --- "Spectral data for the characterization of compound 9 [5,5- oxydimethylenebis(2-furaldehyde)]" --- p.23 / Chapter 2.5 --- Results and Discussion --- p.24 / Chapter 2.5.1 --- Identification of compound 1 [5-(hydroxymethyl)-2- furancarboxaldehyde] --- p.24 / Chapter 2.5.2 --- Identification of compound 2 (oleic acid) --- p.25 / Chapter 2.5.3 --- Identification of compound 3 (ferulic acid) --- p.26 / Chapter 2.5.4 --- Identification of compound 4 (daucosterol) --- p.26 / Chapter 2.5.5 --- Identification of compound 5 (butylidenephthalide) --- p.27 / Chapter 2.5.6 --- Identification of compound 6 (butylphthalide) --- p.28 / Chapter 2.5.7 --- "Identification of compound 7 (Z, Z'-6.6', 7.3'a-diligustilide)" --- p.30 / Chapter 2.5.8 --- Identification of compound 8 (pregnenolone) --- p.31 / Chapter 2.5.9 --- "Identification of compound 9 [5,5'-oxydimethylenebis(2-furaldehyde)]" --- p.32 / Chapter 2.6 --- Conclusions --- p.34 / Chapter 3. --- Analysis of Rhizoma Chuanxiong by Capillary Electrophoresis --- p.35 / Chapter 3.1 --- Introduction --- p.35 / Chapter 3.1.1 --- Capillary electrophoreis system --- p.35 / Chapter 3.1.2 --- Principles of separation --- p.36 / Chapter 3.1.3 --- Considerations on development of analysis method --- p.41 / Chapter 3.2 --- Experimental --- p.43 / Chapter 3.2.1 --- Reagents and materials --- p.43 / Chapter 3.2.2 --- Reference compounds --- p.43 / Chapter 3.2.3 --- Instrumentation and apparatus --- p.44 / Chapter 3.2.4 --- Experimental procedures --- p.45 / Chapter 3.2.4.1 --- Preparation of running buffer solution --- p.45 / Chapter 3.2.4.2 --- Preparation of standard solutions --- p.46 / Chapter 3.2.4.3 --- Preparation of Rhizoma Chuanxiong extracts --- p.47 / Chapter 3.2.4.4 --- Flushing of capillaries --- p.47 / Chapter 3.2.4.5 --- Conditions of separation --- p.48 / Chapter 3.3 --- Results and Discussion --- p.48 / Chapter 3.3.1 --- Preliminary experiments --- p.48 / Chapter 3.3.1.1 --- Addition of surfactants --- p.51 / Chapter 3.3.2 --- Effect of buffer concentration --- p.54 / Chapter 3.3.3 --- Effect of SDS concentration --- p.59 / Chapter 3.3.4 --- Addition of organic modifier --- p.63 / Chapter 3.3.5 --- Reproducibility of the proposed method --- p.68 / Chapter 3.3.6 --- Quantitative analysis of seven standard compounds --- p.70 / Chapter 3.3.7 --- Application of the developed methodology --- p.74 / Chapter 3.3.8 --- Conclusions --- p.83 / References --- p.85 / Appendices / Appendix 1.1.1 1H-NMR spectrum of 5-(hydroxymethyl)-2-furancarboxaldehyde --- p.90 / Appendix 1.1.2 13C-NMR spectrum of 5-(hydroxyinethyl)-2-furancarboxaldehyde --- p.90 / Appendix 1.2 X-ray crystallographic data of ferulic acid --- p.91 / Appendix 1.3 13C-NMR spectrum of butylidenephthalide --- p.96 / Appendix 1.4.1 1 H-NMR spectrum of butylphthalide --- p.97 / Appendix 1.4.2 13C-NMR spectrum of butylphthalide --- p.97 / "Appendix 1.5 X-ray crystallographic data of z, z', 6.6'， 7.3'a-diligustilide" --- p.98 / "Appendix 1.6 X-ray crystallographic data of 5,5'-oxydimethylenebis(2-furaldehyde)" --- p.105 / Appendix 2.1 Details of quantitative analysis of 5-(hydroxymethyl)-2-furancarboxaldehyde --- p.112 / Appendix 2.2 Details of quantitative analysis of ligustrazin hydrochloride --- p.112 / "Appendix 2.3 Details of quantitative analysis of 5,5'-oxydimethylenebis(2-furaldehyde)" --- p.113 / Appendix 2.4 Details of quantitative anlaysis of ferulic acid --- p.113 / Appendix 2.5 Details of quantitative analysis of butylphthalide --- p.114 / Appendix 2.6 Details of quantitative analysis of butylidenephthalide --- p.114 / "Appendix 2.7 Details of quantitative anlaysis of z,z', 6.6', 7.3'a-diligustilide" --- p.115 / Appendix 3.1 Quantitative analysis of Chuanxiong sample from Hong Kong (HK1) --- p.115 / Appendix 3.2 Quantitaive analysis of Chuanxiong sample from Hong Kong (HK2) --- p.116 / Appendix 3.3 Quantitative analysis of Chuanxiong sample from Sichuan (SC1) --- p.116 / Appendix 3.4 Quantitative analysis of Chuanxiong sample from Sichuan (SC2) --- p.117 / Appendix 3.5 Quantitative anlaysis of Chuanxiong samplefrom Fujian (FJ) --- p.117

Analysis of M-components is an important part of the diagnosis of monoclonal gammopathies and for the evaluation of disease response during treatment. In this project, two widely used electrophoresis methods and their corresponding immunotyping method were compared to evaluate the sensitivity of each method for the detection of small M-components. The project included 30 plasma samples from patients with identified M-components; 10 samples containing each IgG, IgA and IgM, respectively. All samples were diluted with normal EDTA plasma to achieve M-components of 5,00g/L. The samples were then serially diluted to achieve M-component concentrations of; 5,00, 2,50, 1,25, 0,63, 0,31 and 0,16g/L. All 180 samples were analysed with agarose gel electrophoresis and capillary electrophoresis. The dilutions above and below the detection level of each method were then analysed with immunofixation and immunosubtraction. The results showed good agreement between agarose gel electrophoresis and capillary electrophoresis in the highest concentrations of IgG and IgM. With agarose gel electrophoresis, IgA was detected in the same location as transferrin and the lowest concentration detected were therefore 1,25g/L. Besides the samples containing IgG, immunofixation was the most sensitive method.

Diss. (sammanfattning) Uppsala : Univ., 2002. / Härtill 5 uppsatser.

Submitted by Priscila Nascimento (pnascimento@icict.fiocruz.br) on 2012-12-10T12:32:54Z No. of bitstreams: 1 iaralice-souza.pdf: 1338553 bytes, checksum: 59fa26df56bce23aa1b5861c460f716f (MD5) / Made available in DSpace on 2012-12-10T12:32:54Z (GMT). No. of bitstreams: 1 iaralice-souza.pdf: 1338553 bytes, checksum: 59fa26df56bce23aa1b5861c460f716f (MD5) Previous issue date: 2001 / Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil. / Neisseria meningitidisdo grupo C é uma bactéria encapsulada causadora dediversas doenças, está associada à altas taxas de mortalidade e portanto é de grande importância para a saúde pública. Bio-Manguinhos está desenvolvendo uma vacina conjugada formada pela ligação covalente do polissacarídeo capsular àanatoxina tetânica e esta vacina, atualmente, está sendo avaliada em estudos clínicosde Fase II em crianças de 1 a 9 anos. A quantificação de componentes livres como polissacarídeos e proteínas faz parte do controle de processo de vacinas conjugadas e tem o objetivo de evitar o aparecimento de reações adversas exacerbadas e/ou redução da imunogenicidade do componente vacinal. A Organização Mundial de Saúde preconiza níveis máximos de proteína livre no conjugado vacinal de 5%, mas não estabelece um limite máximo para o polissacarídeo livre para a vacina conjugada contra o grupo C. Desta forma, o objetivo deste estudo foi desenvolver e validar métodos de controle de qualidade adequados para separar e quantificar estes componentes livres presentes na vacina meningocócica C conjugada brasileira, utilizando a técnica de eletroforese capilar (EC). Para a separação da proteína livre foram comparados os modos de eletroforese capilar de zona livre (CZE) e cromatografia eletrocinética micelar (MEKC). Diferentes condições de migração da amostravariando-se parâmetros como pH, temperatura, tensão, concentração do tampão, ciclodextrinas e de surfactante dodecil sulfato de sódio (SDS) foram estudadas. Os resultados demonstraram que a melhor separação do conjugado foi obtida por MEKC utilizando tampão tetraborato de sódio (TBNa) 150 mM, 25 mM de SDS, 60°C, 30 kV e pH 9,3. Entretanto, nos modos de EC estudados não foi possível obter a separação completa dos componentes, sendo necessária a utilização de outro processo. Por outro lado, por CZE foi possível observar a separação da proteína ativada da nativa, demonstrando a necessidade de otimização da reação de ativação da proteína, a fim de aumentar orendimento da reação de conjugação. A separação completa do açúcar livre presente no conjugado foi obtida empregando CZE utilizando tampão TBNa 50 mM, 40°C, 30 kV e pH 10. Com as condições escolhidas foi possível determinar o conteúdo de polissacarídeo livre nos lotes de conjugado e validar o método proposto, que se mostrou linear na faixa de 0,047 a 0,164 mg/mL, apresentou efeito matriz, 0,0154 mg/mL de limite de detecção e 0,0454mg/mL de limite de quantificação. Após as etapas de validação, foram quantificados alguns lotes de conjugado e observou-se um alto teor de açúcar livre nos lotes com longo período de estocagem a 4°C. Desse modo, fez-se a avaliação de um lote recentemente produzido e obteve-se o valor de 19,08% de polissacarídeo livre. A fim de estimar o tempo de estocagem máximo do conjugado foram realizadas análises com 30, 60 e 90 dias de estocagem a 4°C. Os valores encontrados até 60 dias não foram significativamente diferentes dosdeterminados no tempo zero. No entanto, com 90 dias de estocagem ocorreu uma modificação do perfil do conjugado que impossibilitou a sua quantificação. A metodologia desenvolvida e validada será introduzida no controle de qualidade do lote de conjugado que será submetido aos estudos clínicos de Fase III e na rotina da vacina conjugada estudada. Além disto, o conhecimento adquirido poderá ser empregado no controle de qualidade de outras vacinas conjugadas contra bactérias encapsuladas de interesse epidemiológico no país. / Neisseria meningitidisgroup C is an encapsulated bacterium that causes several diseases and is associated with high mortality rates becoming a serious public health problem. Bio-Manguinhos is developing a conjugate vaccine constituted by covalent attachment of capsular polysaccharide to tetanus toxoid, which iscurrently being evaluated in Phase II clinical studies in children between 1-9 years. Free components quantification is a vaccine process control assay and intended to prevent exacerbated adverse reactions occurrence and/or vaccine immunogenicity reduction. The World Health Organization recommends 5% of free protein maximum level in the conjugate vaccine, but does not set a limit for the free polysaccharide contents. Thus, the aim of this study was to develop and validate quality control methods appropriate to separate and quantify free components present in the conjugate vaccine against N. meningitidisgroup C, using capillary electrophoresis (CE) technique. For free protein separation, free capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) were compared and different sample migration conditions were studied by varying parameters such as pH, temperature, voltage, buffer concentration, cyclodextrin and surfactant sodium dodecyl sulfate (SDS). The results showed that the best separation was obtained by MEKC using sodium tetraborate buffer (TBNA) 150 mM, 25 mM SDS, 60°C, 30 kV and pH 9.3. H owever, the CE did not induce a complete separation of the components suggesting that other techniques should be necessary. On the other hand, native and activated protein separation was possible using CZE, demonstrating the necessity of optimize protein activation reaction in order to increase the conjugation reaction yield. The total free sugar conjugate was completely separated from the conjugate by CZE using 50 mM TBNA buffer, 40°C, 30 kV and pH 10. In these conditions it was possible to determine the free polysaccharide content and validate the proposed method, which was linear in 0.047 to 0.164 mg/mL range, showed a matrix effect, 0.0154 mg/mL of detection limit and 0.0454 mg/mL ofquantification limit.After the validation steps, some conjugate batches were quantified and high levels of free sugar were observed in batches storaged at 4°C for long periods. On the other hand a conjugate batch recently produced was evaluated and showed19.08% of free polysaccharide. In order to estimate the maximum storage time a conjugate batch was analyzed30, 60 and 90 days after the production steps. The values found up to 60 days were not significantly different from that one determined at the initial time. However, with 90 days of storage there was a change in the conjugate profile that impaired its quantification. The methodology developed and validated will be used to evaluate the conjugate batch that will be submitted to Phase III clinical studies and in the routine quality controlof the conjugate vaccine. Moreover, the acquiredknowledge could be used in quality control of other conjugate vaccines against encapsulated bacteria of epidemiological importancein the country.

Au cours de ce travail, nous avons mis au point une nouvelle interface CE/MALDI-MS automatisée, équipée d’une cellule UV/visible déportée, et d’une distribution automatique de matrice intégrée. Ce nouveau système a été évalué sur des mélanges différents de protéines entières, de digestats de protéines et d’anticorps monoclonaux (mAbs). Les résultats obtenus lors de cette évaluation ont montré la complémentarité de la nouvelle interface avec les systèmes analytiques classiques. De plus, nous avons montré la première séparation et analyse de mAbs entier par CE/MALDI-MS. Dans un second travail, la nouvelle interface a été utilisée pour effectuer la première analyse Top Down de proteine entière et de mAbs par collecte et enrichissement de fraction. Cette stratégie a montré la répétabilité du système permettant de séparer des analytes d’intérêt et d’enrichir les dépôts MALDI jusqu'à de très hautes quantités d’analytes permettant l’obtention de spectre Top Down. Au cours du troisième travail, le nouveau système CE/MALDI-MS a été utilisé dans une stratégie originale à 2 dimensions de séparation et de collecte d’isoformes de mAbs entiers ou partiellement digérés suivi d’infusions et analyses à l’aide du nanosprayer CESI. Pour cela, nous avons mis au point des conditions électrophorétiques dit « asymétriques » séparant les mAbs dans des conditions très salées mais collectés dans un milieu compatible avec l’ESI-MS. Cette stratégie inédite a permis d’effectuer la première séparation et caractérisation de mAbs par CE-MS. Parallèlement, nous avons mis au point le premier dosage plasmatique d’ITPP par MALDI-TOF MS et surtout la création de CEToolbox, une application Androïd gratuite pour smartphone et tablette permettant le calcul des principales grandeurs mathématiques pour la caractérisation et l’optimisation des séparations par électrophorèse capillaire. / In this work, we developed a new interface CE/MALDI-MS automated, equipped with a UV/visible cell remote, and integrated automatic distribution of matrix. This new system has been evaluated on different mixtures of intact protein, digested protein and monoclonal antibodies (mAbs). The results obtained during this evaluation showed the complementarity of the new interface with conventional analytical systems. Furthermore, we have shown the first separation and analysis of mAbs by CE/MALDI-MS. In a second work, the new interface was used to perform the first Top Down analysis for intact protein and mAbs by fraction collection and enrichment. This strategy has shown the repeatability of the system for separating analytes and the enrichissment the MALDI deposits up to very high amounts compatible for Top Down approach. In the third work, the new system CE/MALDI-MS has been used in an original 2-dimensional strategy of separating and collecting intact mAbs isoforms or partially digested followed by infusions and analyzes with CESI nanosprayer. For this, we have developed electrophoretic condition so-called "asymmetric" allowing the separation of mAbs under very salty conditions but collected in a totally compatible solution with ESI-MS. This novel strategy allowed for the first separation and characterization of mAbs by CE-MS. Meanwhile, we have developed the first plasma level of ITPP by MALDI-TOF-MS and particularly the creation of CEToolbox as a Free Android application for smartphone and tablet enabling the calculation of the main mathematical quantities for characterization and optimization of CE separations.