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 (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.

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

Thesis (MSc. (Hons.))--University of Western Sydney, 2004. / A thesis presented to the University of Western Sydney, College of Science, Technology and Environment, School of Science, Food and Horticulture, in fulfilment of the requirements for the degree of Master of Science (Honours). Includes bibliographies.

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

This study was conducted to set up a systematic approach utilizing advantages of both wet lab and bioinformatic methodologies to study biodegradation abilities and microbial bacterial-functional relationship within bioremediation communities. Firstly, 11pharmaceuticals and personal care products (PPCPs)were selected as target chemicals for establishing an effective determination process in analyzing trace-level concentrations in the environment, and understanding the removal routes during pollutants removal process in wastewater treatment process using activated sludge. Ultra performance liquid chromatography-tandem mass spectrometry was utilized to develop a rapid, sensitive and reliable method without solid phase extraction pre-concentration for trace analysis of 11 PPCPs in influent and effluent from municipal wastewater treatment plants. Shorten the detection time and significant reduction of detection cost were achieved due to the omitting usage of solid phase extraction (SPE)process and avoiding the consumption of hydrophiliclipophilic balancced (HLB)cartridge. Research on removal routes of ten selected PPCPs in activated sludge found activated sludge hardly removed carbamazepine. Biodegradation was the sole route to remove acyclovir, metronidazole, benzylparaben, ethylparaben, methylparaben and propylparaben. Both adsorption and biodegradation were involved in the removal of ranitidine and benzophenone-3, while fluoxetine could be totally removed by adsorption in activated sludge. Secondly, as the target microbial community, activated sludge community was used to set up the global bioinformatic analysis process. Both metagenomic and metatranscriptomic approaches were processed to characterize microbial structure and gene expression of activated sludge community. The taxonomic profile showed thatactivated sludge was dominated by Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes and Verrucomicrobiaphyla. Gene expression annotation of nitrogen removal revealed that denitrification-related genes sequences dominated in both DNA and cDNA datasets while nitrifying genes were also expressed in relative high levels. Specially, ammonia monooxygenase and hydroxylamine oxidase demonstrated the high cDNA/DNA ratios, indicating strong nitrification activity. Ammonia-oxidizing bacteria present mainly belonged to Nitrosomonas and Nitrosospira species. A fast method to construct local sub-databases has been established for the quick similarity search and annotation of huge metagenomic datasets. The conducted tests showed sub-database annotation pipeline achieved a speedup of ~150-385 times, and got exactly the same annotation results with those of the direct NCBI-nr database BLAST-MEGAN method. This approach provides a new time-efficient and convenient annotation similarity search strategy for laboratories without access to high performance computing facilities. Thirdly, bisphenol A(BPA), which has a partially known biodegradation pathway and relevant bioremediating genes, was chosen as a model to establish a pipeline for systematical understanding the pathways and gene/bacteria relationships in an enriched microbial community. 11 new metabolites were detected during BPA degradation. Thereby, a novel pathway of degrading BPA metabolite was proposed. Sphingomonas strains were dominant taxa in initial degradation of BPA, while the other taxa were competing BPA metabolites during degradation. Metagenomic binning results showed a cytochrome P450 monooxygenase system, which was previously reported BPA mediator, was sharing by two Sphingomonas strains, showing the undergoing mechanism of competition of the two strains. The observations suggested bacterial specialization may occur in that community that each taxon was selected to degrade certain metabolite in a community economical way. / published_or_final_version / Civil Engineering / Doctoral / Doctor of Philosophy

A mini-dissertation in partial compliance with the requirements for a Master's Degree in Technology: Homoeopathy, Durban Institute of Technology, 2002. / The purpose of this study was to compare the amount of caffeine extracted from triturated samples and ethanolic samples of Coffea tosta using high pressure liquid chromatography (HPLC) as a method of analysis. The study wanted to expand on homoeopathic pharmaceutical knowledge, specifically looking at the two methods of remedy preparation of plant materials. From the same batch of ground roasted coffee beans, using the decimal scale of dilution, the mother tincture (bill) and the first triturated (bill) samples were prepared. The subsequent 2xH and 3xH triturated and ethanolic potencies were then made in accordance with homoeopathic methodology. Each group contained three different dilution levels (bill, 2xH and 3xH), 18 samples per group giving a total of36 samples that were analysed using HPLC. Three samples were analysed from the three dilution levels in each Group, in total there were 18 samples from the triturated group and 18 from the ethanolic group. . The samples were analysed quantitatively using the highly accurate and advanced method of high pressure liquid chromatography. This method gives accurate readings of the caffeine concentrations of a sample compared to a caffeine standard. This allowed for quantification of the caffeine concentration of each sample. The percentage caffeine was calculated from each sample. The aim of the study was to evaluate the difference in each method of preparation by measuring the amount of caffeine extracted from the samples. The results obtained from the inter-Group Mann-Whitney and ANOVA tests showed that there was a significant difference between the ethanolic dilutions and triturated dilutions with regards to the 1xH and 2xH dilutions. In the 1xH dilution the ethanolic method retained / M

by Chuen-shing Mok. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 192-193). / Chapter Chapter 1 --- INTRODUCTION --- p.1 / Chapter Chapter 2 --- INSTRUMENTATION AND THEORY --- p.9 / Chapter Chapter 3 --- INDIRECT CONDUCTOMETRIC DETECTION OF AMINO ACIDS AFTER HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC SEPARATION --- p.17 / Chapter Chapter 4 --- DETERMINATION OF MONOSODIUM GLUTAMATE IN FOODS WITH HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY USING INDIRECT CONDUCTOMETRIC DETECTION --- p.52 / Chapter Chapter 5 --- HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC DETERMINATION OF ACTIVE INGREDIENTS IN COUGH-COLD SYRUPS WITH INDIRECT CONDUCTOMETRIC DETECTION --- p.83 / Chapter Chapter 6 --- HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC DETERMINATION OF ATROPINE AND ATROPINE- LIKE ALKALOIDS IN PHARMACEUTICAL PREPARATIONS WITH INDIRECT CONDUCTOMETRIC DETECTION --- p.144 / Chapter Chapter 7 --- CONCLUSION --- p.194

Sin Wai-Chu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references. / Abstracts in English and Chinese. / ABSTRACT --- p.i / 論文摘要 --- p.ii / ACKNOWLEDGEMENT --- p.iii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Electroplating history --- p.1 / Chapter 1.2 --- Electroplating bath --- p.7 / Chapter 1.3 --- Electroplating analytical methods --- p.8 / Chapter 1.3.1 --- Metal content and elemental impurities analysis --- p.10 / Chapter 1.3.2 --- "Metal complex, inorganic anion and cation analysis" --- p.11 / Chapter 1.3.3 --- Organic brighteners and levelers analysis --- p.12 / Chapter 1.4 --- HPLC literature review --- p.15 / Chapter 1.5 --- My research work --- p.16 / Chapter 1.6 --- References for Chapter 1 --- p.19 / Chapter Chapter 2 --- General Experimental --- p.23 / Chapter 2.1 --- The HPLC System --- p.23 / Chapter 2.2 --- The factors that affect the separation --- p.26 / Chapter 2.2.1 --- The composition of the solvent system --- p.27 / Chapter 2.2.2 --- The selection of column --- p.30 / Chapter 2.2.3 --- The most suitable analytical wavelength for UV detection --- p.34 / Chapter 2.3 --- Challenges in analyzing electroplating baths solution --- p.35 / Chapter 2.3.1 --- High metal content --- p.36 / Chapter 2.3.2 --- Strong ligand or complexing agent --- p.36 / Chapter 2.3.3 --- Interference --- p.37 / Chapter 2.3.4 --- Extreme pH --- p.37 / Chapter 2.3.5 --- Other difficulties --- p.38 / Chapter 2.3.6 --- Maintenance of HPLC instrument --- p.38 / Chapter 2.4 --- References for Chapter 2 --- p.38 / Chapter Chapter 3 --- Palladure 200 bath HPLC analysis --- p.41 / Chapter 3.1 --- Introduction --- p.41 / Chapter 3.2 --- Experimental --- p.43 / Chapter 3.3 --- Problems in the existing UV analysis for monitoring Palladure200 process --- p.45 / Chapter 3.4 --- HPLC method development for monitoring Palladure 200 process --- p.49 / Chapter 3.5 --- Analysis of aged Palladure 200 plating bath from production line --- p.55 / Chapter 3.6 --- Conclusion --- p.57 / Chapter 3.7 --- References for Chapter 3 --- p.58 / Chapter Chapter 4 --- Nickel PC3 bath HPLC analysis --- p.59 / Chapter 4.1 --- Introduction --- p.59 / Chapter 4.2 --- Experimental --- p.60 / Chapter 4.3 --- Problems in the existing Titration method for monitoring Nickel PC3 process --- p.62 / Chapter 4.4 --- HPLC method development for monitoring Nickel PC3 process --- p.63 / Chapter 4.4.1 --- Identify individual component of Nickel PC3 process --- p.63 / Chapter 4.4.2 --- Set up a calibration curve for the Nickel PC3 Additive --- p.67 / Chapter 4.4.3 --- Analysis of aged Nickel PC3 plating bath from production line --- p.68 / Chapter 4.5 --- Conclusion --- p.71 / Chapter 4.6 --- References for Chapter 4 --- p.72 / Chapter Chapter 5 --- Solderon SC bath HPLC analysis --- p.73 / Chapter 5.1 --- Introduction --- p.73 / Chapter 5.2 --- Experimental --- p.74 / Chapter 5.3 --- Instability in the existing Cyclic Voltammetric Stripping (CVS) method for monitoring Solderon SC process --- p.76 / Chapter 5.4 --- HPLC method development for monitoring Solderon SC process --- p.77 / Chapter 5.4.1 --- Identify the individual components --- p.77 / Chapter 5.4.2 --- Set up a calibration curve for the Solderon SC Primary --- p.82 / Chapter 5.4.3 --- Analysis of aged Solderon SC plating bath from production line --- p.84 / Chapter 5.5 --- Conclusion --- p.86 / Chapter 5.6 --- References for Chapter 5 --- p.86 / Chapter Chapter 6 --- Copper Gleam PPR bath HPLC analysis --- p.87 / Chapter 6.1 --- Introduction --- p.87 / Chapter 6.2 --- Experimental --- p.89 / Chapter 6.3 --- Problems in the existing Cyclic Voltammetric Stripping (CVS) method for monitoring Copper Gleam PPR process --- p.91 / Chapter 6.4 --- HPLC method development for monitoring Copper Gleam PPR process --- p.92 / Chapter 6.4.1 --- Identify Individual components and copper PPR additivein standard bath --- p.92 / Chapter 6.4.2 --- Set up a calibration curve for the Copper Gleam PPR Additive --- p.95 / Chapter 6.4.3 --- Analysis of aged Copper Gleam PPR plating bath from production line --- p.96 / Chapter 6.4.5 --- Study of H202 effect --- p.101 / Chapter 6.4.6 --- Study of air agitation effect --- p.104 / Chapter 6.4.7 --- Study of Copper anode effect --- p.105 / Chapter 6.5 --- Conclusion --- p.107 / Chapter 6.6 --- References for Chapter 6 --- p.107 / Chapter Chapter 7 --- Silverjet220 bath HPLC analysis --- p.109 / Chapter 7.1 --- Introduction --- p.109 / Chapter 7.2 --- Experimental --- p.110 / Chapter 7.3 --- HPLC method development for monitoring Silverjet 220 process --- p.112 / Chapter 7.3.1 --- Identify individual components and Silverjet 220 Additive in the plating bath --- p.112 / Chapter 7.3.2 --- Optimize the condition for HPLC analysis --- p.117 / Chapter 7.3.3 --- Analysis of aged Silverjet 220 plating bath from production line --- p.119 / Chapter 7.4 --- Conclusion --- p.122 / Chapter 7.5 --- References for Chapter 7 --- p.123 / Chapter Chapter 8 --- Conclusions and Further Studies --- p.124 / Chapter 8.1 --- Conclusions --- p.124 / Chapter 8.2 --- Further Studies --- p.126 / APPENDIX --- p.128 / The User guide for HPLC --- p.128 / HPLC System Calibration Maintenance --- p.135 / HPLC System Preventive Maintenance --- p.145

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