M. Tech. (Biomedical technology, Faculty of Applied and Computer Science), Vaal University of Technology / Approximately 1% of the world’s population has epilepsy, the second most common neurological disorder after stroke. In South Africa almost 1 in every 100 people has epilepsy, affecting all ages. Levetiracetam (LEV), marketed as Keppra® is an anticonvulsant drug used in the treatment of epilepsy. The daily dosage is 500 mg twice daily with a maximum of 3000 mg. The therapeutic range of LEV is between 12-46 μg/ml. Therapeutic drug monitoring (TDM) should be considered for LEV in patients with poor seizure control or long term treatment. TDM depends on accurate drug concentration measurements. In order to provide an accurate measurement, the High performance liquid chromatography (HPLC) method was developed, compared with a commercially available kit, and the stability of the samples was investigated. Ethical approval was obtained from the Human Research Ethics Committee (Medical), VUT (Ethics reference number: 2015024.4). The study was conducted from January to October 2015. This study involved three groups of volunteers who gave written consent. The first group were fifteen healthy MTech students in the Biomedical Technology Department at the Vaal University of Technology (VUT). Their blood samples were used for the analytical validation of the method and for the stability studies over a 4 weeks period. The second group were six patients from Pathcare Laboratories in Potchefstroom, Klerksdorp and Vereeniging who used Levetiracetam. Their blood samples were used to investigate the influence of different collection tubes as well as the handling and storage of samples on the LEV concentration. The third group were forty four patients from Pathcare Laboratories, Cape Town. Their blood samples were transported to Clinical Pharmacokinetic Laboratory (CPL) for routine therapeutic drug monitoring analysis of LEV and used to compare the newly developed HPLC method and the Commercial kit. The HPLC method was successfully developed and validated to determine LEV in human plasma/serum samples. The calibration curves showed good linearity (r2 = 0,999) over the concentration range of 1 – 60 μg/ml. Accuracy, mean extraction recovery, lower limit of detection (LLOD) and lower limit of quantification (LLOQ) were 98-112%, 97,15% (±1,57), 0,5 and 1,0 μg/ml respectively, in plasma standards. The method was shown to be simple and fast, reproducible and effective for routine laboratory analyses in the future. The agreement between the newly developed method and the ClinRep® HPLC complete commercial kit was the same and there was a statistical significant correlation between the two methods (average r=0.999; p-value < 0.0001, F-test with a true value =0). The method was much cheaper than the commercial kit, used less sample (100 μl) and had a longer running time (15 minutes) to ensure no endogenous interference. The costs of the developed method was 71-82% lower than the three commercial kits available in South Africa. Stability experiments were performed to evaluate the stability of LEV in human plasma/serum, simulating the same conditions which occurred during study samples’ analyses. The % RSD was lower than 5% under all the conditions: freeze, fridge, room temperature and auto sampler over the 4 week period. The results showed that both LEV and the I.S (internal standard) were stable in human serum/plasma under all these conditions. The influence of five different collection tubes, Gold (SST Gel), Red, Purple (EDTA)Green (Heparin) and Blue (Sodium Citrate) was investigated. In two patients, decreased levels were observed in tubes containing blue (sodium citrate) and Green (Heparin). The decrease was not statistically significant. This is an important observation and is an indication that anticoagulants may cause some problems due to drug-protein binding and interference in the matrix effect. A cost effective and reliable HPLC-method with minimal sample preparation time for the routine determination of LEV in plasma/serum samples was developed. It was also shown that the plasma/serum samples were stable at different temperatures over a time period. The only collection tubes that may interfere with the concentrations were the Green (Heparin) and Blue (Sodium Citrate) tubes.

High performance liquid chromatography was used to measure ribonucleoside triphosphates in microbial samples. Anion exchange columns in a radial compression module were used to separate and quantify purine and pyrimidine ribonucleotides. Endogenous ribonucleoside triphosphates were extracted from Escherichia coli and pseudomonas aeruginosa using three different solvents, namely trifluorocetic acid (TFA; 0.5M), trichloroacetic acid (TCA; 6 per cent w/v) and formic acid (1.0M) Extracts were assayed for uridine 5'-triphosphate (ATP), and guanosine 5'-triphosphate (GTP) by using anion exchange radial compression high performance (pressure) liquid chromatography. The three extraction produres were compared for yield of triphosphates. E. coli, the TFA extraction procedure was more sensitive and reliable than TCA and formic acid extraction procedures, but , in P. aeruginosa, the best yields of ATP and GTP were obrained following extraction with TFA. Yields of UTP and CTP increased when extraction was performed in TCA. These data illustrate that different extraction produres produce different measures for different triphosphates, a point often overlooked.

High Performance Liquid Chromatography (HPLC) was used to investigate the utility of this technique for the analytical and preparative separation of components of aquatic fulvic acids (FA). Three modes of HPLC namely adsorption, anion exchange and reversed phase were evaluated. Aquatic fulvic acids were either extracted from surface water and sediment samples collected from the Southwest of the U.S., or were provided in a high purity form from the USGS. On the adsorption mode, a major fraction of aquatic fulvic acid was isolated on a semipreparative scale and subjected to Carbon-13 NMR and FAB Mass Spectroscopy. Results indicated that (1) The analyzed fraction of fulvic acid contains more aliphatic than aromatic moieties; (2) Methoxy, carboxylic acids, and esters are well-defined moieties of the macromolecule; (3) Phenolic components of the macromolecules were not detected in the Carbon-13 NMR spectrum possibly because of the presence of stable free radicals. Results of the anion exchange mode have shown that at least three types of acidic functionalities in aquatic fulvic acid can be separated. Results also indicated that aquatic fulvic acid can be progressively fractionated by using subsequent modes of HPLC. Results of reversed phase mode have shown that (1) The fractionation of aquatic fulvic acid by RP-HPLC is essentially controlled by the polarity and/or pH of the carrier solvent system; (2) Under different RP-HPLC conditions aquatic fulvic acid from several locations are fractionated into the same major components; (3) Fulvic acid extracted from water and sediment from the same site are more similar than those extracted from different sites; (4) Cationic and anionic ion pair reagents indicated the presence of amphoteric compounds within the polymeric structure of fulvic acid. Each mode of HPLC provided a characteristic profile of fulvic acid. The results of this research provided basic information on the behavior of aquatic fulvic acids under three modes of HPLC. Such informations are prerequisite for further investigation by spectroscopic methods.

High Performance Liquid Chromatography (HPLC) has been employed as an analytical tool for the purification and separation of nucleic acids. A Nucleogen DEAE 4000-10 weak anion exchange column, prepacked with modified silica gels, was used to purify and separate a number of Escherichia coli plasmids. Plasmid DNAs were extracted by the alkaline lysis method. The cleared lysate was injected directly onto the Nucleogen column, and the peaks were collected, desalted and analysed by gel electrophoresis. On the chromatogram, the pBR322 formed a distinctive peak at 27 minutes and partial separation was made for the E. coli V517 plasmids. Plasmid pBR322 showed a clear band without any detectable contamination on agarose gel. This purified plasmid DNA is biologically active for enzymatic reaction commonly used in genetic engineering techniques.

The thesis is focused on application of ion chromatography in pharmaceutical analyses of organic ions. Ion chromatography is increasingly used in the field of pharmaceutical analysis. This includes the analysis of impurities and metabolites. In the first part of this thesis, ion chromatography is compared with common separation techniques used in pharmacy, such as gas chromatography and high performance liquid chromatography. In the second part development and validation of methods of ion chromatography for purity evaluation and quality control of active pharmaceutical substances Rivastigmine hemitartrate and Pramipexole hydrochloride were carried out. Key words: ion chromatography, reversed-phase chromatography, ion-pair chromatography, ion-exclusion chromatography, ion-exchange chromatography, GC, HPLC

A Dissertation Submitted to the Faculty of Medicine, University of the Witwatersrand, Johannesburg, in Partial Fulfilment of the Requirements for the Degree of Master of Pharmacy Johannesburg, March 1994 / Preformulation and formulation study of dexchlor- pheniramine maleate (DCPM) for it's inclusion into a gelforming sustained release dosage form was investigated. A modification of the USP apparatus 2 is proposed as an alternative to currently recommended USP dissolution apparatus for floating, gelforming drug delivery systems. In addition, the role of magnesium stearate and talc as dissolution retardants in controlled release matrix tablets is investigated, through application of a factorial design. / IT2018

As copaíferas fazem parte de um importante gênero de plantas que produzem um exsudato denominado como oleorresina, que é constituído por uma mistura de sesquiterpenos e diterpenos ácidos. A medicina popular utiliza a oleorresina de diferentes espécies de Copaifera com diversas indicações, tais como: ação anti-inflamatória, antimicrobiana, cicatrizante, antitumoral, entre outras. Desta forma, numerosos pesquisadores têm investigado a composição química e as atividades biológicas e toxicológicas deste produto natural, a fim de reconhecer os benefícios e a segurança deste produto. Todavia, estudos da composição química e das atividades biológicas dos constituintes das folhas são escassos. Assim, neste projeto buscou-se investigar a composição química de diferentes extratos de folhas e de oleorresinas de copaíferas coletadas na flora brasileira, bem como produtos comerciais (óleo de copaíba) propondo e aplicando métodos analíticos em cromatografia para determinação dos principais constituintes das amostras. Dez amostras de folhas e oleorresinas autênticas de diferentes espécies foram coletadas no norte e sudeste do Brasil (C. langsdorffi Desf., C. duckei Dwyer, C. reticulata Ducke, C. multijuga Hayne, C. paupera (Herzog) Dwyer, C. pubiflora Benth, C. lucens Dwyer, C. oblogifolia Mart, C. trapezifolia Hayne, e Copaifera sp.) e mais seis amostras comerciais foram adquiridas. Os compostos voláteis foram separados da oleorresina bruta utilizando-se o aparelho de Clevenger. Os padrões cromatográficos foram isolados utilizando-se diferemntes modalidades cromatográficas. As oleorresinas de C. multijulga e C. langsdorffii apresentaram os maiores teores de óleo volátil das amostras com 92 e 72%, respectivamente. Três diterpenos foram isolados da oleorresina de C. duckei: os ácidos ent-poliáltico, ent-diidroagático e ent-agático-15-metil éster. Uma análise qualitativa foi otimizada usando microextração em fase sólida seguido de análise em CG-EM, a qual revelou a presença em comum do ?-copaeno, ?-elemeno, ?-cariofileno e ?-bergamoteno em todas as amostras de oleorresina analisadas. Dois métodos analíticos utilizando CLUE-EM/EM e CG-DIC foram desenvolvidos, otimizados e validados para análise de nove diterpenos ácidos e quatro sesquiterpenos, os quais apresentaram adequada seletividade/especificidade, faixa de trabalho, limite de detecção, limite de quantificação, precisão e exatidão. Todavia, em ambos os métodos há necessidade de controle rigoroso das condições analíticas dos sistemas cromatográficos. Os resultados obtidos aplicando os dois métodos indicaram a presença frequente do ?-cariofileno e dos ácidos ent-copálico e ent-caurenoico como as substâncias mais encontradas em oleorresinas de copaíferas autênticas e comerciais. A análise qualitativa empregando CLUE-EM indicou a presença dos ácidos di-O-metil-3,5-di-O-galoilquínico (AGQ-8), 5\',5\'\'-di-O-metil-4,5-di-O-galoilquínico (AGQ-9), 5\',5\'\',5\'\'\'-tri-O-metil-3,4,5-tri-O-galoilquínico (AGQ-16), afzelina e quercitrina nos extratos das folhas de nove amostras de copaíferas. Os métodos analíticos desenvolvidos são confiáveis para as análises dos componentes fixos e voláteis e os resultados obtidos neste trabalho confirmam a variabilidade da composição química das oleorresinas desse gênero vegetal. / Copaifera plants are part of an important genus that produces an exsudate called oleoresin, which consists of a mixture of sesquiterpenes and acid diterpenes. Folk medicine uses the oleoresin of different Copaifera species for different health problems, such as: anti-inflammatory, antimicrobial, wound healing and antitumor, among others. Thus, numerous researchers have investigated the chemical composition, biological and toxicological activities of this natural product in order to recognize its benefits and safety. However, investigation of chemical composition and biological activities of leaf contituents are scarce. In this project we aimed to investigate the chemical composition of different leaf extracts and Copaifera oleoresins collected in the Brazilian flora and acquired in the market (copaíba oil) by proposing and applying chromatographic analytical methods to determine their main constituents. Ten samples of leaves and authentic oleoresins of different species were collected in northern and southeastern parts of Brazil (C. langsdorffi Desf., C. duckei Dwyer, C. reticulata Ducke, C. multijuga Hayne, C. paupera (Herzog) Dwyer, C. pubiflora Benth, C. lucens Dwyer, C. oblogifolia Mart, C. trapezifolia Hayne, e Copaifera sp.) and six commercial samples were purchased. The volatile fraction of the crude oleoresin was obtained by using Clevenger apparatus, and the chromatographic standards were isolated by using different chromatographic techniques. The oleoresins of C. langsdorffii and C. multijuga displayed the highest volatile oil contents. Three diterpenes were isolated from C. duckei oleoresin: ent-polialthic, ent-dihydroagathic and ent-agathic-15-metil ester acids. A qualitative analysis was optimized using solid phase micro-extraction followed by GC-MS analysis, which revealed the presence of ?-copaene, ?-elemene, ?-caryophyllene and ?-bergamotene in all investigated oleoresins. Two analytical methods using UPLC-MS/MS and GC-FID were developed, optimized and validated for the analysis of nine acid diterpenes and four sesquiterpenes, which gave adequate selectivity/specificity, range, limit of detection, limit of quantification, precision and accuracy. However, in both methods there is a need for rigorous control of the analytical conditions. The results generated by the two methods indicated the frequent presence of ?-caryophyllene, ent-copalic and ent-kaurenoic acids as the compounds frequently found in authentic and commercial Copaifera oleoresins. Qualitative analysis of leaf extracts using UPLC-MS/MS indicated the presence of 5\',5\'\'-di-O-methyl-3,5-di-O-galloylquinic acid (GQA-8), 5\',5\'\'-di-O-methyl-4,5-di-O-galloylquinic acid (GQA-9), 5\',5\'\',5\'\'\'-tri-O-methyl-3,4,5-tri-O-galloylquinic acid (GQA-16), afzelin and quercetrin in all studied species. The analytical developed methods are reliable for the analyses of both fixed and volatile compounds of the oleoresins. The obtained results confirm the variability among the composition of the Copaifera oleoresins.

Chow Ka-man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 52-55). / Abstracts in English and Chinese. / Chapter 1 --- Introduction / Chapter 1.1 --- Chemical sensors --- p.1 / Chapter 1.2 --- Quartz crystal microbalance --- p.4 / Chapter 1.3 --- Concept of affinity mass sensor --- p.8 / Chapter 1.4 --- Film immobilization technologies --- p.9 / Chapter 1.5 --- Research outlines --- p.13 / Chapter 2 --- Experimental / Chapter 2.1 --- Sensor fabrication --- p.14 / Chapter 2.2 --- Flow-through cell --- p.16 / Chapter 2.3 --- Analysis procedures --- p.19 / Chapter 2.4 --- Response curve --- p.19 / Chapter 2.5 --- Experimental setup --- p.21 / Chapter 3 --- Detection of ascorbic acid by affinity mass sensor based on 3-aminophenylboronic acid / Chapter 3.1 --- Conventional analytical methods --- p.23 / Chapter 3.2 --- Research method - affinity mass sensor based on APBA --- p.24 / Chapter 3.3 --- To locate the binding site in ascorbic acid --- p.25 / Chapter 3.3.1 --- Steric energy calculated by molecular modeling --- p.26 / Chapter 3.4 --- Optimization of experimental variables --- p.29 / Chapter 3.4.1 --- Effect of pH --- p.29 / Chapter 3.4.2 --- Effect of sample volume --- p.30 / Chapter 3.4.3 --- Effect of flow velocity --- p.30 / Chapter 3.5 --- Calibration and Reproducibility --- p.32 / Chapter 3.6 --- Kinetic analysis --- p.33 / Chapter 3.7 --- Stability of sensor --- p.37 / Chapter 3.8 --- Interference studies --- p.37 / Chapter 3.9 --- Determination of ascorbic acid in real samples --- p.39 / Chapter 3.9.1 --- Results and Discussion --- p.39 / Chapter 3.10 --- Comparison with conventional ascorbic acid sensors --- p.42 / Chapter 3.11 --- Summary --- p.42 / Chapter 4 --- Boronic acid derivatives for the detection of sugars / Chapter 4.1 --- Scope of this work --- p.43 / Chapter 4.2 --- Results and Discussion --- p.44 / Chapter 4.3 --- Summary --- p.49 / Conclusion --- p.50 / References --- p.52 / List for tables --- p.56 / List for figures --- p.57 / Appendix I --- p.59 / Appendix II --- p.61

An analytical method was developed for the determination of the â-agonists clenbuterol, cimaterol and salbutamol in bovine retina. The method involved extraction into a pH 8.5 tris-HCl buffer, followed by protease enzyme digestion and immunoaffinity column cleanup before analysis by liquid chromatography with tandem mass spectrometry detection LC/MS/MS. The LOD for clenbuterol, salbutamol and cimaterol were 0.64, 1.20 and 1.92 ng/g respectively. The identities of the analytes were able to be confirmed to an acceptable standard. An analytical method was also developed for the analysis of the â-agonists clenbuterol, salbutamol, cimaterol, ractopamine, and mabuterol in bovine urine and emu muscle. The urine and muscle samples were digested with â-glcuronidase enzyme and cleaned up using a Bond Elute Certify SPE. The extracts were analysed by LC/MS/MS. Deuterated internal standards were used for quantitation. The LOD for urine [less than] 1ng g and for emu muscle it was [less than] 0.3 ng/g. The last part of the work describes the simultaneous gas chromatography-mass spectrometric analysis of diethylstilbestrol DES, hexestrol HEX, dienestrol DIEN, zeranol ZER, taleranol TAL and zeralenone ZON in fresh full cream and fresh skim milk. The analytes were analysed as their trimethyl silane (TMS) derivatives. A three phase solvent system was used for extraction and the extract was cleaned up using a combination of the anion exchange and hydrophobic properties of an anion exchange SPE. The detection limits for DES, DIEN, HEX, ZER, TAL and ZON were 9.6, 9.6, 16.8 , 7.2 , 13.5 and 34.8 ng/L respectively. / Doctor of Philosophy(PhD)

Thesis (Ph. D.)--Pennsylvania State University, 1982. / Includes bibliographical references.