Theses and Dissertations (Chemistry)

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    CpMetal N-heterocyclic carbene complexes : synthesis and applications
    (University of Pretoria, 2017) Landman, Marile; frikkie.malan@up.ac.za; Malan, Frederick Pieter
    The synthesis and catalytic application of new half-sandwich Ni(II), Cr(III), and Ru(II) complexes, all stabilised by either symmetric or asymmetric N-heterocyclic carbene (NHC) ligands, were investigated. The study entailed the synthesis of novel imidazolium halide salts as ligand precursors to the metal-NHC complexes. A range of nine (three symmetric, six asymmetric) imidazolium bromide salts were synthesised and characterized, with N-substituents including either electron-donating or electron-withdrawing groups. The ligands were reacted with nickelocene under inert conditions to form nine [CpNiBr(NHC)] complexes. These complexes were evaluated as catalysts in the Suzuki-Miyaura cross-coupling reaction for which moderate to good activity (53-79% conversion, maximum TOF = 320 h-1) was observed. The catalytic activity of these complexes in the anaerobic oxidation of secondary alcohols was also assessed. Moderate activity (52-84% conversion, TOF = 14-86 h-1) was observed using conventional heating, and excellent activity (90-98% conversion) using microwave heating. DFT studies provided insight into the better performance of the complexes featuring electron-donating NHCs as opposed to those with electron-withdrawing NHC ligands. In addition, DFT-supported energy profile studies explained the lack of formation of α-arylation byproducts, found for similar studies in the literature. By-product formation of the type [NiBr2(NHC)2] during synthesis of the above mentioned nickel NHC complexes led to the subsequent investigation of the reactivity and properties of the biscarbene complexes. Reaction of thiophenol with [CpNiBr(NHC)] or the biscarbene complexes, yielded complexes of the type [CpNi(SPh)(NHC)] and [Ni(SPh)2(NHC)2]. A detailed DFT study revealed structural preferences that were correlated with the experimentally observed structures. The electronic differences among the [CpNiBr(NHC)] complexes were further evaluated through a DFT-supported electrochemistry study, which showed some electrochemical variances due to the different N-alkyl substituents present in the nickel complexes. Reaction of chromocene with the novel non-NO2-containing imidazolium bromide salts, followed by chromium oxidation using CCl4, allowed for the formation, purification and characterisation of five [CpCrBrCl(NHC)] complexes. Exposure of these heterohalo complexes to excess CHCl3 or CCl4 leads to the formation of complexes [CpCrCl2(NHC)]. Upon reaction of chromocene with the novel NO2-containing imidazolium salts, a ligand decomposition reaction occurs. C-N bond cleavage and imidazole protonation result in the formation of [C4H7N2][CpCrBrCl2]. The catalytic activity of the six Cr(III)-NHC complexes was evaluated in the base-free glucose dehydration reaction for which moderate activity (52-81% conversion) was observed. Supplementary DFT studies added insight into the electrochemical properties of the Cr(III) complexes. A range of six new bidentate (benz)imidazolium chloride salts (one symmetric, five asymmetric), each bearing an N-2-methylallyl substituent were synthesised and complexed to ruthenium(II) (three different Ru(II) precursors) via silver transmetallation. Eight new cationic complexes [(C5H4R)Ru(EPh3)(NHC)]PF6 (R = H, Me; E = P, As; NHC = Im(R')(2-methylallyl), BIm(Bn)(2-methylallyl); R' = alkyl) were isolated, purified and characterised. DFT studies illustrated the electronic properties of the complexes, while a catalytic study (tandem transfer hydrogenation-epoxidation of ketone substrates) showed moderate activity (39-62% conversion, TOF = 4-12 h-1) for all of the complexes. Finally, a range of four new C(2)-protected bidentate imidazolium chloride salts (one symmetric, three asymmetric), each containing either an N-2-methylallyl or N-picolyl substituent, were synthesised and characterised. These ligands were found suitable as ligand precursors to abnormal NHCs, and hence were reacted with [(p-cym)RuCl2]2 or [CpRuCl(PPh3)2] to form ten new Ru(II)-NHC complexes (eight Ru-aNHC and two Ru-NHC complexes). The Ru-NHC complexes formed as by-products due to Ag-mediated C(2)-dealkylation in selected cases. The strength of the Ru-aNHC bond was probed in an 1H-NMR titration study with DCl, which showed its superior bond strength when compared to analogous normally bound Ru-NHC complexes. The catalytic activity of the latter ten complexes was screened in both the transfer hydrogenation and alcohol oxidation reactions. The complexes are therefore more suitable as transfer hydrogenation catalysts (50-93% conversion, TOF = 22-60 h-1) than for application in alcohol oxidation catalysis (56-77% conversion, TOF = 2-11 h-1).
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    Development and characterization of flow systems for voltammetric analysis
    (University of Pretoria, 1998) Van Staden, J.F.; Matoetoe, 'M'angaka C.
    Flow-through systems are progressively becoming more important in all branches of analytical chemistry. The increased demand for analysis of more complex environmental samples has resulted in development of reliable, versatile and sensitive techniques. A combination of voltammetric analysis such as differential pulse anodic stripping voltammetry (DP ASV) with flow systems satisfy all the above requirements. Although this combination has great potential, practically it has not been realised yet. This may be partly due to the use of traditional mercury electrodes which has many complications. In order to improve the use of this technique, a glassy carbon electrode (GC) which is more suitable to flow systems compared to mercury-based electrodes was studied. This study describes the electrochemical behaviour of chemicals on the GC electrode in flow systems. The use of a GC with flow systems as a means of detection proved to be very effective and can make electrochemistry more accessible to unskilled scientists. Having characterised and optimised all instrumental parameters of the system, practical evaluation of the technique as a trace metal analyser was explored. Future improvements necessary for optimum development of the technique are also mentioned.
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    Electrochemical reduction of Formic acid using an earth abundant catalyst
    (University of Pretoria, 2019-11) Radhakrishnan, Shankara Gayathri; u13181867@tuks.co.za; Ndlangamandla, Simphiwe
    Copper is one of the most useful electrocatalysts for electrochemically converting CO2 to hydrocarbons and alcohols. Unfortunately, copper suffers from a lack of selectivity and efficiency. Earth-abundant electrocatalysts such as metal porphyrins have been shown to be highly stable and highly selective for products such as carbon monoxide and formic acid. Formic acid is formed with high efficiency on a wide range of materials and can be further reduced to other useful products such as hydrocarbons and alcohols. The aim of this project is to conduct electrochemical formic acid reduction to hydrocarbons or alcohols using copper (II) tetraphenyl porphyrin. Electrochemical Formic acid reduction was conducted using a Proton Exchange Membrane (PEM) cell electrolyser. Water electrolysis was conducted at the anode using a 70:30 IrO2: TaC electrocatalyst. A control experiment was first conducted with a freebase tetraphenyl porphyrin cathode. Thereafter all experiments were repeated with Cu (II) tetraphenyl porphyrin. Products were characterized using liquid injection gas chromatography. Formic acid reduction with freebase tetraphenyl porphyrin did not yield any products at both -1.8 V and -2.1 V. Copper (II) tetraphenyl porphyrin yielded isopropanol and the most conductive copper (II) tetraphenyl porphyrin electrode produced isopropanol with a faradic efficiency of 4.5 % at -2.1 V with current density of -1.71 mA/cm2. The least conductive Cu (II) tetraphenyl porphyrin electrode exhibited a current density of -0.055 mA/cm2 at -2.1 V but produced isopropanol with a faradaic efficiency of 30.4 %. This shows that a high current density does not necessarily equate to an enhanced faradaic efficiency of formic acid reduction to isopropanol. No isopropanol was detected from formic acid reduction using freebase tetraphenyl porphyrin. This indicates that the mechanism of formic acid reduction to isopropanol requires the presence of the copper central metal as the active site.
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    The design, synthesis and biological evaluation of novel compounds against biomarkers of Alzheimer’s disease
    (University of Pretoria, 2019-06) Riley, Darren L.; Panayides, Jenny-Lee; Steenkamp, Vanessa; divanggreunen@gmail.com; Van Greunen, Divan Gerald
    Alzheimer’s disease (AD) is the most common neurodegenerative disease accounting for an estimated 60 – 80 % of dementia cases. The disease affected 5.5 million Americans older than 65 years in 2018 alone, and this number is projected to increase to 13.8 million by 2050. The total cost of care in the United States for people with AD was $277 billion in 2018, making it one of the costliest diseases to treat. Statistics on the prevalence of dementia in South Africa are very limited and according to a study performed in the rural areas of the Eastern Cape in 2017, it was estimated that 352 000 individuals older than 60 are living with dementia. Unfortunately, there are currently no approved therapies which target AD pathology directly and therefore current treatments focus on relieving symptomatic and behavioral aspects of AD. Thus, a lot of focus has been placed on the development of new drugs for different biomarkers as potential treatment of AD. This study consists of the design, synthesis and biological evaluation of novel compounds against biomarkers of AD. In this study, four different series of compounds, consisting of ninety-one analogues, were synthesized as novel compounds against biomarkers of AD. These included; (i) series 1: N-benzylpiperidine carboxamide derivatives; (ii) series 2: 1-(5,6-dimethoxy-8H-indeno[1,2-d]thiazole-2-yl)urea derivatives; (iii) series 3: 1-amino-3-(indeno[1,2-b]indol-5(10H)-yl)propan-2-ol derivatives; and (iv) series 4: 6,7-dimethoxy-1-phenyl-1,4-dihydroindeno[1,2-c]pyrazole-3-carboxamide derivatives. All compounds were evaluated for activity against acetylcholinesterase (AChE), the major biomarker of AD. Derivatized compounds of series 1 were assessed for activity against butyrylcholinesterase (BuChE). Derivatized compounds of series 3 were assessed for activity against the amyloid precursor protein cleaving enzyme 1 (BACE1). The first series of fifteen compounds based upon the skeleton of 5,6-dimethoxy-1-oxo-2,3-dihydro-1H-inden-2-yl 1-benzylpiperidine-4-carboxylate, a compound previously synthesized, were synthesized and evaluated for activity against acetylcholinesterase. A second series of thirty-five compounds designed to have dual acetylcholinesterase and glycogen synthase kinase 3 inhibitory activity, were synthesized based upon the skeleton of 1-benzyl-N-(5,6-dimethoxy-8H-indeno[1,2-d]thiazol-2-yl)piperidine-4-carboxamide. A third series of seventeen compounds with potential β-secretase 1 inhibitors were designed and synthesized based upon the skeleton of 1-(3,6-dichloro-9H-carbazol-9-yl)-3-(naphthalen-1-ylamino)propan-2-ol, a compound previously reported in literature by Macchia and co-workers. A fourth series consisting of twenty-four potential N-methyl-D-aspartate (NMDA) receptor antagonists were designed and synthesized based upon scaffolds previously reported in the literature by the Liotta and Kawai research groups. In order to prepare large quantities of donepezil for use as a stock reagent for the development of libraries of potential BACE1 inhibitors, the preparation of donepezil using continuous flow conditions was attempted. The highest yield obtained for the benzylation of ethyl isonipecotate was 83% at 90 °C with a residence time of 45 minutes as compared to the batch process which took 3 hours to complete with a yield of 79%. Reduction of the (E)-2-[(1-benzylpiperidin-4-yl)methylene]-5,6-dimethoxy-2,3-dihydro-1H-inden-1-one to afford the final product donepezil was obtained with a yield of 80% after 4.5 hours, with no observable de-benzylation in flow as compared to the batch process which was completed after 6 hours with a yield of 78%. Due to time constraints, Stage 2 of the flow synthesis of donepezil could not be completed. However, with only two of the three steps completed for the flow process, the yield of the individual steps was already improved, and there was a reduction in the reaction time.
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    Synthesis, characterization and applications of polymer based conjugate materials for infectious diseases of poverty
    (University of Pretoria, 2018-12) Pilcher, Lynne A.; Balogun, Mohammed; tshweull@gmail.com; Tshweu, Lesego Lovius
    The major disease burden for the majority of the world’s population is due to infectious diseases. The most prevalent are HIV/AIDS, tuberculosis, lower respiratory tract infections, diarrhoeal diseases and malaria. In particular, malaria and tuberculosis have not benefited much from new technological developments in disease management. Most of the drugs used are several decades old and have significant toxicity profiles which impact patient compliance. New potential drugs being discovered stumble on the road to the clinic because of solubility issues. Many just end up being shelved. It is estimated that over 70% of new chemical entities have poor solubility issues. On the other hand, HIV/AIDS remains a major health problem worldwide, as currently there is no cure available. Chronic intake of highly active antiretroviral treatment (HAART) is compulsory to control HIV infection and any non-adherence leads to a quick increase in the viral load. Poor targeting ability of antiretrovirals (ARVs) to latent sites of infection is the main reason for the relapse. Nanomedicines have significantly improved the clinical management of deadly diseases like cancer. Conventional drugs show improved pharmacological indices when designed as nanomedicines. It improves solubility, absorption, clinical performance, and reduces the amount of drug needed to achieve the desired therapeutic effect. The resultant effect is improved solubility and reduced toxicity. The most common method to design nanomedicines is through physical entrapment or encapsulation in polymeric carriers. Today, however, modern delivery systems are being designed by chemical synthesis. Either the drugs are chemically linked to the polymeric carriers or the polymers are chemically derivatized to be more efficient at encapsulating the active agents. In this project, we report on our attempts to chemically modify polymers with active drugs to synthesize smart macromolecular pro-drugs or produce more efficient drug encapsulation systems. The work presented in this project is outlined in the following six chapters. Chapter 1 contains the literature review of the major infectious diseases; namely HIV/AIDS, tuberculosis and malaria; what has been done to increase patient compliance or success of available drugs i.e (to reduce the viral load or attempted to cure the virus with the ARVs in the field of HIV/AIDS, or administration of combination therapy in anti-tuberculosis/malarial field to minimize drug resistance and increase therapeutic success). The use of nanomedicine to ameliorate problems associated with treatment regime for infectious diseases is discussed. Methods for nanoencapsulation or inclusion of the existing drugs within the approved materials is described in detail. A concise general introduction to polymer therapeutics is discussed, followed by a review of polymers that are normally used in polymer-drug conjugation; highlighting advantages and disadvantages of each. Finally a clinical perspective on the use of polymer-drug conjugation for infectious diseases is outlined. Chapter 2 introduces the problems associated with the current ARVs used in the treatment of HIV/AIDS and emphasizes the need to target latent sites of infection using aptamer technology. Aptamers were subsequently conjugated to polyethylene glycol (PEG) using carbodiimide chemistry. The TZM-bl neutralization assay and in vitro stability in human breast milk studies showed that aptamers maintained their binding integrity after pegylation and were more stable than the parent aptamers. Finally the conjugated aptamers were nanoencapsulated into poly(epsilon-caprolactone) [PCL] nanoparticles using a double emulsion method. Nanoparticles of less than 150 nm were produced with a higher surface charge, showing that the nanoparticles were stable. The in vitro binding assay using electrochemical methods showed that the nanoparticles coated with PEGylated RNA aptamers had higher affinity and specifity to HIV-1 gp 120. The overall results demonstrated that these nanoparticles could be used in HIV drug delivery applications to help minimize changes associated with ARVs. Chapter 3 describes nanomedicinal formulations of the anti-TB drug moxifloxacin (Mox). Mox is a relatively hydrophilic drug. The target pathological site where the Mycobacterium tuberculosis resides is a lipid dense granuloma in the lungs. Hence, a large dose will have to be administered to deliver an adequate therapeutic dose i.e. 400 mg is required for Mox. Mox was covalently conjugated into PEG via a releasable amide bond. Similarly a hybrid system was formed by nanoencapsulating the PEG-Mox conjugate into PCL nanoparticles using double emulsion method. The system constitutes the PCL, which is envisaged to increase the hydrophobicity of the PEG-Mox conjugate. PEG-Mox conjugates and PCL-Mox nanoparticles were found to be hemocompatible, inducing only minimal hemolysis. Mox was more toxic than the PEG-Mox conjugate and PCL-Mox nanoparticles. In vitro stability in human plasma showed that PCL-Mox nanoparticles were stable for over 72 hrs. Data obtained emphasizes that PCL nanoparticles could be used as a drug delivery system to minimize the high toxicity of TB drugs Chapter 4 establishes the conjugation of the hydrophobic drug, lumefantrine [Lumf] to water soluble polymers. Lumf is insoluble in water with an octanol-water partition coefficient (logp) of 8.34. As a result a series of Lumf prodrug conjugates were synthesized using two different polymers (polyethylene glycol and p-NAM-stat-p-AA). Average particle size below 200 nm was achieved and PDI values were always below 0.2, which is an indication of the relatively homogeneous size distribution achieved with carbodiimide chemistry. We have for the first time, by applying the polymer therapeutics techniques, synthesized a polymer-drug conjugate of Lumf which has increased the solubility of the drug more than 103 times. Chapter 5 gives the conclusions of the experimental chapters. Through the three different experimental chapters, we have demonstrated that polymer based drug conjugates can be used to address different issues: (1) drug delivery through coating nanoparticles with appropriate aptamers, (2) drug toxicity through encapsulation of a toxic drug in a heamocompatible nanoparticle and (3) greatly improved aqueous solubility of a hydrophobic drug. Furthermore, while there has been much excellent work using polymer based drug conjugates in cancer, we have explored the approach to tackle different problems related to three different infectious diseases of poverty, namely HIV, TB and malaria.
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    Systems thinking for sustainability in chemistry education using life cycle inventories of aspirin synthesis
    (University of Pretoria, 2024) Pilcher, Lynne A.; Dikobe, Dorine; cathrinechimude@gmail.com; Chimude, Cathrine
    As part of a chemistry education research project, this study was designed to assess the effectiveness of an intervention using Life Cycle Inventories (LCI) of pharmaceutical production in introducing systems thinking for sustainability in chemistry education to first year engineering students. Aspirin synthesis was used as an exemplar drug and system. To accomplish the research objective, the research followed a case study design and collected qualitative data. All first-year engineering students taking the 2022 CHM 171 module participated in the one-time intervention. The CHM 171 module is offered by chemistry department on behalf of the Faculty of Engineering, Built Environment and Information Technology (EBIT) of the University of Pretoria. The research participants were drawn from a population of students who had given consent to their data being analysed. This gave rise to 130 individual pre-practical exercises, 85 pre- and post-practical quizzes, and 20 group worksheets being analysed for research purposes. The intervention was centred around a single 3-hour practical session. The data were collected at three points in time: pre-, during, and post-practical. At the beginning of the intervention, the participants were assigned a pre-practical exercise and a pre-practical quiz to collect data on their prior systems thinking skills. The participants then worked in groups during the practical session, where they were introduced to the concepts of life cycle inventories followed by systems thinking. They answered various questions in their groups, covering different dimensions of systems thinking for sustainability. After the practical session, the participants then worked individually on a post-practical quiz, similar in nature to the pre-practical quiz. The post-practical quiz was used to gather their systems thinking for sustainability skills post-intervention. Rubrics, designed through inductive thematic analysis, were used to score participants’ responses for the pre-and post-practical quizzes. The Systems Thinking Hierarchical (STH) model was used for the deductive thematic analysis on the pre- and post-practical quizzes, and the group worksheets, to determine the systems thinking skills shown in the participants’ responses. Quantitative analysis was performed on the pre- and post-practical quizzes to determine if there were any changes between the scores of the two quizzes. A paired T-test revealed that the participants’ systems thinking skills had improved moving from the pre- to post-practical quizzes. The deductive analysis using the STH model showed that the quality of answers had also improved in the post-practical quizzes, and the group worksheets, revealing a move from simplistic reasoning to holistic thinking. The results reveal that the LCIs of aspirin synthesis can be used to introduce systems thinking in chemistry education. Implementation into the curriculum is important as it aids understanding of systems thinking and sustainability.
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    Molecular modelling and crystallographic investigation of novel synthetic cathinone 2-methyl-4'-(methylthio)-2-morpholinopropiophenone (MMMP)
    (University of Pretoria, 2024-03-28) Slabbert, Cara; Govender, Krishna; u16152582@tuks.co.za; Van Heerden, Lanzo Jacques
    2-Methyl-4'-(methylthio)-2-morpholinopropiophenone (MMMP) is a highly modified synthetic cathinone derived from natural occurring cathinone, the main psychoactive alkaloid present in Catha edulis Forsk. or khat. Synthetic cathinones (SCs) belong to the drug class of new psychoactive substances (NPS) which consists of analogues of commonly known abused drugs designed to mimic the psychoactive properties of illicit drugs to circumvent current drug legislations. From a structural perspective, cathinone is a β-keto analogue of amphetamine and SCs are often referred to as “bk-amphetamines”. SCs are capable of evoking psychoactive effects similar to that of amphetamines and cocaine increasing their popularity amongst users as “legal highs”. MMMP itself is commercially available and readily used in the polymer and printing industry in the fixing of thin films, plastics and inks as a Type 1 fragmenting photoinitiator. It was hypothesised that MMMP might have psychoactive properties due to its cathinone origin and isolation in confiscated drug samples. This hypothesis was investigated through molecular modelling techniques to determine if MMMP was capable of interacting with 25 protein targets consisting of 24 monoamine receptors and a transporter which act as common targets for psychoactive substances. From the initial molecular docking results two protein targets, serotonin-1A (SER1A) and serotonin transporter (SERT), were identified as the most likely to form a complex with MMMP. Further analysis through Molecular Dynamics (MD) simulations to evaluate the stability and movement of these complexes revealed that MMMP has a higher probability of interacting and forming a strong complex with the SERT binding pocket, but a weaker complex is formed with SER1A. The crystal structure of MMMP was successfully elucidated through single crystal X-ray diffraction (SCXRD). Analysis of the crystal structure revealed that MMMP crystallises in the non-centrosymmetric space group, Pca21. The unit cell consist of four crystallographically independent conformers with each conformer interacting with itself, with conformers within the same unit cell as well as with conformers in adjacent unit cells through a series of intra- and intermolecular interactions. Three symmetry elements, two glide planes and a screw axis, were also identified.
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    Development of a novel passive sampler for gaseous mercury monitoring
    (University of Pretoria, 2024-06-04) Forbes, Patricia B.C.; Martin, Lynwill; u16341334@tuks.co.za; Kempkes, Geyan
    Mercury as a pollutant has a longstanding history of environmental and health impacts. The ubiquitous global presence of mercury in the environment in combination with its toxicity has given rise to global efforts to control releases and limit exposure and risks associated with the use of mercury. Despite the global transport and deposition of mercury in the environment, there are some areas which lack monitoring data, mainly the southern hemisphere. South Africa, specifically, requires more robust and consistent monitoring, however, implementation of commercially available methods is costly and alternative means of Hg quantitation are needed. Consequently, passive air sampling provides a promising approach to obtain additional data regarding the spatial distribution of Hg across South Africa. Herein a set of novel sulfur-doped 3-D graphene foams were synthesized, characterized, and then employed in a radial passive sampling setup to capture total gaseous mercury (TGM). A chemical vapour deposition (CVD) method was optimized to generate a pristine graphene foam as a cylindrical cartridge. The pristine material was subsequently doped by means of various methods and sulfur sources, namely dimethyl sulfoxide (DMSO), sodium sulfide (Na2S.9H2O), thiourea (SC(NH2)2) and elemental sulfur (S8). Prior to their use in real-world sampling of atmospheric Hg, these derivatized foams were chemically and physically characterized alongside the pristine precursor (where possible) to provide insights into doping efficiencies, surface area differences between foam variants, mass losses upon analysis, surface functionality introduced during doping, and the influence of the polymethyl methacrylate (PMMA) support layer. These characterizations included scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy dispersive X-ray spectroscopy (EDX), Raman spectroscopy, thermogravimetric analysis (TGA), X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR). It was found that the multiple-layered nature of the material, as well as the inherent thinness of the graphene, contributed significantly to the variance seen in the characterization data sets. In addition to this, the influence of the PMMA support layer was identified and the efficient removal thereof was also catered for as it was found to impact characterization and doping efficiencies. Following characterization, selected sorbents, which included the pristine precursor as a candidate foam and point of comparison, were utilized in passive air samplers (PASs) that were deployed in a total of four different deployments, encompassing two at a chosen impacted site (Witbank) and a two at a less impacted site (University of Pretoria). Despite inherently low atmospheric Hg concentrations (ng/m3), the radial passive sampler employed in this study was expected to facilitate high uptake rates of Hg and be viable for ambient atmospheric Hg capture, especially at the impacted site where there are surrounding coal-fired power station point sources. The PASs were also further evaluated by comparison to a globally calibrated commercial passive sampler, the MerPAS®, which was deployed concurrently in the final deployment set at each site. iii Studies were done to determine the linear uptake of Hg by the DMSO-doped graphene foam sorbent specifically, but although linear Hg uptake was not observed, successive deployments offered better Hg capture from the pool of other derivatized foams, including the pristine graphene foam precursor. For all sorbents there was a difference in the mean Hg concentration between the two chosen sites, confirming Witbank as an impacted site. The two best performing foams were the thiourea-doped variant, followed by the sodium sulfide variant. Direct comparison to the Hg captured by the PASs deployed to the responses for the MerPAS® showed a promising similarity in effective Hg capture when the mass differences between the MerPAS® activated carbon sorbent and the graphene foams were considered. The methods which offered the highest doping efficiencies were the thiourea and sodium sulfide-based methods, respectively, which were expected to directly improve Hg captured on the surface of the resulting foams. The results of the deployment, however, inferred that a direct increase in sulfur content alone did not lead to a corresponding increase in captured Hg. Instead, an interplay between the surface area, sulfur content and Hg sorbed was identified which requires further investigation. Quantification of Hg on the deployed samplers was achieved by thermal desorption from the sorbent, amalgamation with gold and then atomic absorption spectroscopy (AAS) by means of a direct mercury analyser. The method was validated by successful participation in a global interlaboratory proficiency testing scheme, for which a Z score of -1.042 was obtained. The calibration procedure for analysis of deployed foams was also optimized as a final matrix-matched calibration, for which the influence of the graphene foam matrix on calibration accuracy and precision was also assessed. The use of intermittent cleaning steps and their effect on the accuracy of the calibration was found to contribute to achieving better fits and more reproducible responses. With respect to a NIST 2962c bituminous coal reference material (0.5% sulfur mass fraction), a reproducibility of 7 % RSD (N=11) and error of 1 % (N=11) were achieved for the final deployments and a limit of detection (LoD) of 0.083 ng Hg was achieved for the final optimized calibration. Quality control limits were also established with respect to the calibration, wherein threshold limits of ±20% around the expected value for the NIST standard reference material (SRM) and a 30 μg/kg aqueous quality control standard were maintained for up to ~six months. Overall, the sorbents utilized in this study require more extensive characterization to offer further insight into the observed trends in their variable Hg capture, surface areas and sulfur contents. The PAS also requires further optimization prior to widespread applications and use in areas of low ambient airborne concentrations. The PASs based on thiourea and sodium sulfide-doped graphene foams that were developed for the first time in this study offered the best Hg capture and should be further optimized. The novel S doped graphene foam PAS shows good potential to bridge the monitoring gaps prevalent in atmospheric Hg monitoring data in South Africa.
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    Serial identification and structural analysis of synthetic cannabinoids found in herbal mixtures in South Africa
    (University of Pretoria, 2024-04-12) Slabbert, Cara; Wooding, Madelien; Malan, F.P. (Frederick); u21758868@tuks.co.za; Mudau, Mbavhalelo Wendy
    The primary objective of this study was to identify and characterise SCBs in herbal mixtures from South Africa using a serial identification approach. The analysis of seised herbal mixtures was carried out using ultra-performance liquid chromatography coupled with a high-resolution mass spectrometer (UPLC-HRMS) and gas chromatography coupled with amass selective detector (GCMSD). The study identified three SCBs associated with fatalities: N-[[1-[(4-fluorophenyl)methyl]-1Hindazol- 3-yl]carbonyl]-L-valine, methyl ester (AMB-FUBINACA), N-[[1-(5-fluoropentyl)-1H-indazol-3- yl]carbonyl]-3-methyl-D/L-valine, methyl ester (5F-ADB), N-[[1-(5-fluoropentyl)-1H-indazol-3- yl]carbonyl]-3-methyl-L-valine, ethyl ester (5F-EDMB-PINACA) and fifteen other non-SCBs compounds. The study of MS fragment ions predicted possible fragmentation pathways and found that the identified SCBs and analysed reference standards are susceptible to the amide linker group cleavage, followed by cleavage of the fluorinated side chain. These findings suggest that abundant iv fragment ions can be used to screen uncharacterised SCBs belonging to the same family or sharing similar pharmacores. Additionally, we validated isolated SCBs from herbal mixtures using NMR spectroscopy where a reference standard was not available and obtained crystal data using SC-XRD. 1H-NMR and 13C-NMR spectral fingerprinting allowed for cross-validation of the isolated 5F-ADB. 1H-NMR spectroscopy was also successfully used to cross-validate the reference standard (S)-5F-EMBICA, supplied as (S)-5FABICA. SC-XRD crystal data results of (S)-5F-EMBICA were obtained with low quality and hence crossvalidated by NMR spectroscopy. This research, for the first time, to our knowledge reports the 1HNMR spectroscopy data of (S)-5F-EMBICA and its crystal structure and crystallography data. Finally, we conducted in silico analysis to estimate the pharmacokinetics and physicochemical properties of the SCB compounds. We utilised the SwissADME web tool for this purpose, which revealed several key findings, including the lipophilicity enantiomeric discrimination, side chain fluorogroup type effects, hydrophobic nature, TPSA analysis, GI absorption levels and BBB permeation, P-gp binding, and probability for CYP3A4 inhibition. These findings suggest that the SwissADME web tool can be used as a first line of pharmacokinetic properties prediction for SCBs in the absence of bioassays and expertise in advanced computer modelling. In silico analysis was also carried out to characterise selected SCBs further through docking pose predictions on the CB1 receptor using the Maestro molecular modelling software by Schrödinger. The results showed that the indazole ring of the SCB interacts via π-π stacking with phenylalanine residues, particularly Phe 268, Phe 170, Phe 174, and His 178. The study concludes that the (S)-enantiomer has a higher CB1 affinity and more π-π stacking interactions with phenylalanine residues which are important for CB1 agonism. These results can be used to predict the properties of uncharacterised SCBs before their spread in the illicit drug market.
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    Structures and magnetic properties of ionic hybrids of metal halides and carboxyalkylamines.
    (University of Pretoria, 2024-05-17) Rademeyer, Melanie; Sheppard, Charles; shalenebothma@gmail.com; Bothma, Shalene Natalia
    Organic-inorganic hybrid materials combine an organic- and inorganic component at the molecular scale. The resulting hybrid material retains the properties of each component, allowing for the design of a material with specific properties through the selection of the components. This study investigated the structural characteristics and magnetic properties of ionic hybrid compounds prepared from metal halides and n-aminoalkanoic acids, HOOC(CH2)nNH2. In the presence of an acid, the organic component is protonated to form an n-carboxyalkylammonium cation, HOOC(CH2)nNH3+, while the inorganic component forms a perhalidometallate anion, [MaXb]z-, where M is a metal ion and X a halido ligand. Three families of compounds were investigated, namely compounds obtained through the combination of n-aminoalkanoic acids with CuCl2, CuBr2, or MnCl2, in an acidic medium. The metal ions Cu2+ and Mn2+ were chosen to impart magnetic properties to the materials. n-Aminoalkanoic acids of different chain lengths were selected to investigate the effect cation chain length on the structures and magnetic properties. The cations (HOOC(CH2)nNH3+), with n = 2, 3, 4, 5 and 10, were selected. A family of bis(n-carboxyalkylammonium) tetrachloridocuprates, (HOOC(CH2)nNH3)2[CuCl4], was obtained through the combination of the n-aminoalkanoic acids with CuCl2. These compounds exhibit two-dimensional hybrid halide perovskite structures in which bilayers of organic cations alternate with inorganic layers consisting of corner-sharing [CuCl6]4-¬ polyhedra. The compounds with n = 3 and 5 undergo phase transitions to incommensurate phases. Ferromagnetic intralayer interactions, occurring at temperature TC, are observed in these compounds. The ferromagnetic intralayer exchange interactions, JK¬, fall in the range of 13.3(1) K to 15.1(8) K, while TC ranged from 7.9 K to 13.4 K. Weak, interlayer antiferromagnetic interactions were observed for some of the compounds. The combination of the n-aminoalkanoic acids with CuBr2 gave bis(n-carboxyalkylammonium) tetrabromidocuprates, (HOOC(CH2)nNH3)2[CuBr4], and the compounds exhibit two-dimensional hybrid halide perovskite structures. Ferromagnetic intralayer interactions are present, with JK observed between 20.4(1) K to 23.7(3) K, while TC ranged between 10.5 K to 13.4 K. Weak, interlayer antiferromagnetic interactions were identified in all the members of this family. An interesting odd-even effect is observed for the compounds obtained from the combination of the n-aminoalkanoic acids with MnCl2. When the number of carbon atoms in the cation is even, bis(n-carboxyalkylammonium) tetrachloridomanganates, (HOOC(CH2)nNH3)2[MnCl4], are formed, which display two-dimensional hybrid halide perovskite structures. Antiferromagnetic intralayer interactions are present, occurring at temperature TN, with 2JK¬ ranging from -7.72(4) K to -8.28(5) K, and TN¬ ranging from 43 K to 45 K. When the number of carbon atoms in the cation is odd, bis(n-carboxyalkylammonium) diaquatetrachloridomanganates, (HOOC(CH2)nNH3)2[MnCl4(H2O)2], are formed. These compounds have layered structures containing [MnCl4(H2O)2]2- anions. Weak antiferromagnetic intralayer interactions are present, with 2JK = -0.14(2) K when n = 2 and 4. The study showed that the compounds' magnetic properties can be tailored by selecting different components. Magneto-structural correlations were identified in these families of compounds and in related compounds reported in the literature. Identifying these relationships contributes to the fundamental understanding of the magnetic behaviour of these materials.
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    Synthesis of an 8-membered oxygen-containing benzo-fused heterocycle using flow technologies
    (University of Pretoria, 2024-03) Riley, Darren L.; Panayides, Jenny-Lee; bernicecurrie@gmail.com; Currie, Bernice Mercia
    Flow chemistry has become an appealing alternative synthetic technology globally over the last few decades finding application in both academic and industrial laboratories driven largely by the fact that it commonly provides higher selectivity, yield and purity compared to batch chemistry that has become time-consuming, ineffective, and challenging to scale up. This dissertation describes the application and development of a flow process towards the synthesis of an 8-membered oxygen-containing benzo-fused heterocycle. The synthesis consisted of six stages that included two allylation steps, a Claisen rearrangement, an alcohol protection, an aldehyde reduction and a ring-closing metathesis step. The target molecule, (Z)-7-isopropoxy-8-methoxy-3,6-dihydro-1H-benzo[c]oxocine 25, was selected with an interest in developing an improved approach to accessing the scaffold for future structure-activity relationship screening and demonstrating that modern process technologies like flow can also be used routinely to perform fundamental research in a more sustainable and responsible manner. In summary, all six steps were successfully translated into flow and improved yields were shown with the use of greener solvents and operating at unconventionally high temperature and pressures especially when considering the Claisen rearrangement step. Design of experiment was also used in the last two stages which included i) the second allylation which required a strong base such as sodium hydride and was used as a slurry pumping through peristaltic pumps, and ii) the final ring closing metathesis stage in which two ruthenium-based catalysts were investigated with the use of a greener solvent. It was found that the flow approach yielded an overall percentage yield of 37.7 % in 105 minutes, compared to the batch approach that yielded an overall percentage yield of 0.77 % in 154 hours.
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    Anti-inflammatory and anti-diabetic properties of plant extracts and compounds from Scabiosa columbaria and Sclerocarya birrea
    (University of Pretoria, 2023) Maharaj, Dashnie Naidoo; Maharaj, Vinesh J.; chidinma.ezeofor@unn.edu.ng; Ezeofor, Chidinma Christiana
    Given that South Africa is home to around 10% of all flowering plant species known to humans, the country is blessed with an abundance of natural resources. About 24,000 plant species have yet to be fully uncovered for the benefit of humanity, making this a significant resource. The use of traditional medicine to cure illnesses is still widespread in South Africa despite the country's rapid urban and infrastructure development, more westernization, and accessibility to typical western medical institutions. Intricately woven within South African culture, the use of medicinal plants to heal illnesses is still prevalent. Additionally, due to Africa's failing healthcare system, using medicinal plants for health reasons is a well-accepted alternative that is practiced by all races, classes, and socio-economic classes. Bio-active extracts, fractions, and compounds have been identified as effective treatments for inflammatory and diabetic disorders based mostly on ethnomedicinal and empirical knowledge on traditional applications of plants. South Africa is a country with a high plant diversity of over 30,000 species of higher plants and 3,000 of these plant species have been found to be used in the traditional medicine for anti-inflammatory purposes. Inspired by the traditional uses of plant species, various scientists have studied the anti-inflammatory activities of South African plant species. Current cosmeceutical and drug discoveries rely on the massive screening of natural product libraries against various extracellular and intracellular molecular targets to find novel chemotypes with the desired mode of action. In the pharmaceutical and cosmeceutical industries, there is an increasing interest in the demand for natural ingredients with potential health benefits, such as anti-inflammatory, anti-diabetic, and anti-cancer properties, in an effort to replace or lessen the usage of synthetic products. With 60% of marketed pharmaceuticals and cosmeceuticals, natural ingredients have long been a key source of medicinal and cosmeceutical scaffolds. Nature continues to prove to be a source of new bioactive molecules with high safety profiles, despite the fact that many synthetic chemists are focused on synthesizing potent compounds with high toxicity profiles for pharmaceutical and cosmeceutical goals. Therefore, natural product chemists keep looking for novel leads. As a continuation of these efforts, this study aimed to identify and develop new natural anti-inflammatory ingredients from selected South African plant species for commercial application in different market sectors based on their traditional uses and literature data and to isolate and characterize biologically active compounds using modern hyphenated analytical techniques from biologically active plant species. A literature survey was carried out to identify South African plants based on their traditional uses to include in this study. A scoring system was applied to rank them and 3 plant species (Scabiosa columbaria, Commiphora pyracanthoides and Pelargonium capitatum) belonging to three families were selected. The 3 plant species were collected from the University of Pretoria Experimental farm, KwaZulu Natal and Limpopo and extracted singly using cosmetic acceptable solvents (acetone, ethanol water/ethanol (1:1) and water). The extracts were tested in different assays (anti-inflammatory and skin even tone). After testing different extracts of the plant species, the ethanol extract of S. columbaria roots was selected for further evaluation to identify the compound/s responsible for the anti-inflammatory activity and development as a potential active herbal ingredient based on good anti-inflammatory efficacy data and no anti-inflammatory reports in the literature. UPLC-QTOF-MS analysis of the ethanol extract of S. columbaria roots led to the tentative identification of fifteen compounds which are loganic acid (53) scrophuloside A1 (peak 2), 3,4-dicaffeoylquinic acid (peak 3), cantleyoside (peak 4), sylvestroside III (peak 5), triplostoside A (54), hederagenin (55), maslinic acid (peak 8), 2-isoursolic acid (56), glycyrrhetaldehyde (peak 10), pomaceic acid (57), euscaphic acid (58) and 3-oxoglycyrrhetinic acid (61). The presence of loganic acid (62), cantleyoside -dimethyl-acetal (63), ursolic acid (64), 2-isoursolic acid (65), 24-nor-2α,3β-dihydroxyolean-4(23),12-ene (66) and hederagenin (67) in the ethanol extract of S. columbaria roots was confirmed by isolation and structure elucidation of the compounds using MS and NMR data. Significant reduction of nitric oxide levels in RAW 264.7 macrophages was observed for ursolic acid (64) (12.5, 25 and 50 µg mL-1; 0.0702, 0.0558 and 0.0357 µg/mLrespectively), 24-nor-2α,3β-dihydroxyolean-4(23),12-ene (66) (12.5, 25 and 50 µg mL-1; 0.0543, 0.0327 and 0.0231 µg/mLrespectively) and hederagenin (67) (12.5 and 25 µg mL-1; 0.0735 and 0.0513 µg/mLrespectively) compared to the positive control aminoguanidine (12.5 µg mL-1; 0.0336 µg mL-1). At a concentration of 25 and 50 µg mL-1, 24-nor-2α,3β-dihydroxyolean-4(23),12-ene (66) demonstrated a potent reduction in nitric oxide level in RAW 264.7 macrophages. The compounds identified in the ethanol extract of S. columbaria roots will be used as chemical markers for quality control purposes, for batch-to-batch reproducibility that is required for commercializing the herbal ingredient. The anti-inflammatory activity of 24-nor-2α,3β-dihydroxyolean-4(23),12-ene (66) has been reported for the first time in this work. The active compounds (ursolic acid (64), 24-nor-2α,3β-dihydroxyolean-4(23),12-ene (66) and hederagenin (67)) were structurally similar and contained a β-hydroxy group at C-3. The compound 2- isoursolic acid (65) was inactive and had a hydroxy group at C-2 instead of C-3, which suggests that the position of the β-hydroxy group may play a role in the nitric oxide inhibition activity. A concentrated form of the ethanol extract of S. columbaria roots can be developed to have a higher concentration of the active compounds for commercial application as an anti-inflammatory ingredient. Diabetes is a global health problem and a national economic burden. Although there are many anti-diabetic medications on the market, there is still a need for innovative treatment agents with increased efficacy and less side effects. Because they are more diverse and have minimal side effects than synthetic medications, pharmaceuticals made from natural products are more appealing. In line with this quest, the Department of Science and Innovation (DSI) established the African Traditional Medicines collaboration, where research was carried out to find and create a new natural anti-diabetic ingredient. S. birrea was chosen because its leaves and stem bark have historically been used to treat conditions including diabetic mellitus. Although type-2 diabetes mellitus was reportedly inhibited by S. birrea leaf extract, the compounds responsible for the anti-diabetic activity have not yet been identified, and their discovery will be helpful for commercial use. In order to discover the chemical compounds in S. birrea that are responsible for the anti-diabetic activity and to employ them as chemical markers for quality control purposes, this Ph.D. research was carried out. Of all the extracts of S. birrea leaves tested, aqueous extract 4 showed statistically significant activity including at the lowest test concentration (0.01 µg mL-1) and was selected to isolate and identify the compounds responsible for the anti-diabetic activity (glucose uptake activity). UPLC-QTOF-MS analysis of the spray-dried aqueous leaf extracts of S. birrea (aqueous extracts 1 and 4) led to the tentative identification of sixteen compounds which are quinic acid (peak 1), gallic acid (peak 2), procyanidin B2 (peak 3), gallocatechin (peak 4), Pistafolin A (peak 5), epicatechin (peak 6), myricetin-3-O-β-D-glucuronide (88), gossypin (peak 8), quercetin-3-O-(6''-galloyl)-β-D-glucopyranoside (peak 9), myricetin-3-O-α-L-rhamnopyranoside (peak 10), quercetin-3-O-β-D-glucuronide (89), quercetin-3-O-arabinoside (peak 12), quercetin-3-O-α-L-rhamnopyranoside (peak 13), kaempferol-3-O-α-L-rhamnopyranoside (peak 14), myricetin (90) and quercetin (peak 16). The presence of myricetin (91), myricetin-3-O-β-D-glucuronide (92) and quercetin-3-O-β-D-glucuronide (93) in the aqueous leaf extract of S. birrea was confirmed by isolation and structure elucidation of the compounds using MS and NMR data. Myricetin-3-O-α-L-rhamnopyranoside (peak 10), gallic acid (peak 2), quercetin-3-O-arabinoside (peak 12) and quercetin-3-O-α-L-rhamnopyranoside (peak 13) were previously reported to occur in S. birrea. Quinic acid (peak 1), myricetin (90) and quercetin (peak 16) were previously reported to have anti-diabetic activity. Myricetin-3-O-β-D-glucuronide (92) and quercetin-3-O-β-D-glucuronide (93) have not been previously reported to occur in S. birrea. Myricetin (91), myricetin-3-O-β-D-glucuronide (92) and quercetin-3-O-β-D-glucuronide (93) significantly increased the glucose uptake in differentiated C2C12 myocyte cells at different test concentrations; myricetin (91) (0.1 and 10 µg mL-1; 85.7 and 109.1%, respectively), myricetin-3-O-β-D-glucuronide (92) (0.1 and 10 µg mL-1; 61.6 and 88.8%, respectively) and quercetin-3-O-β-D-glucuronide (93) (0.1 and 10 µg mL-1; 40.9 and 43.9%, respectively) compared to the treatment of insulin (0.1 µM; 100%). At a concentration of 10 µg mL-1, myricetin (91) demonstrated both a potent and concentration-dependent stimulatory action on glucose uptake in the C2C12 myocytes, matching that of insulin, the positive control. Myricetin-3-O-β-D-glucuronide (92) has not been previously reported to have anti-diabetic activity, and the combination of this compound with other known anti-diabetic compounds in S. birrea contributes to the plant’s anti-diabetic efficacy. These anti-diabetic compounds will be used as chemical markers for quality control purposes required for commercializing the herbal ingredient. This study provides scientific data to support the commercial application of the aqueous extract of S. birrea leaves as an anti-diabetic ingredient. By finding novel active components and molecules responsible for the biological efficacy to be exploited as chemical markers for commercial application, the project's ultimate purpose was accomplished. The indigenous knowledge on the use of medicinal plants proves to be useful for identifying ingredients and developing products for the various market sectors, and this research provides scientific evidence on the value of South Africa's plant biodiversity as a continuing source of biologically active ingredients. Secondly the bioassay guided isolation method used in the isolation of active compounds proved to be a useful technique with the bioassay acting as a specific detector at every purification step. Though it can be criticized for being time consuming and resource intensive compared to the conventional method of isolation, the approach compensates for these shortcomings by its effectiveness.
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    The electrochemical reduction of CO2 to value-added products
    (University of Pretoria, 2024-02-23) Radhakrishnan, Shankara Gayathri; u17023000@tuks.co.za; Klopper, Reinard
    Carbon dioxide is the fourth most abundant gas in the atmosphere making up 0.035 %. Carbon dioxide prevents heat from escaping into the outer space and is used by plants to make oxygen. The levels of carbon dioxide have significantly increased since the start of the industrialisation era and the effects thereof is felt on a global scale. The aim of this project is to electrochemically transform the inert CO2 molecule into value added products such as formic acid, carbon monoxide, methanol, methane etc., as an energy storage media and reduce its global footprint. Use of water electrolysis as a medium for production of hydrogen ion (green hydrogen route) has been employed. IrO2:TaC 70:30 wt. % was selected as a suitable anode electrocatalyst and In2O3 enriched with 0.15 wt. % PTFE as the cathode electrocatalyst under investigation. The electrocatalysts synthesized were characterised using scanning electron microscopy and powder X-ray diffraction techniques. The IrO2:TaC 70:30 wt. % proved to be a good system for the water electrolysis in accordance to our previous studies and with literature1,2 Catalyst inks were prepared to deposit the electrocatalysts via spray coating methods thereby making 5 sets of catalyst coated membranes. The anode electrocatalyst IrO2:TaC 70:30 wt % was deposited directly onto the Nafion® membrane while the cathode electrocatalyst PTFE infused In2O3 onto the carbon paper gas diffusion layer. Humidified gaseous CO2 as well as studies were also performed along with the usual aqueous CO2 which eliminates solubility issues and the need of electrolytes. Linear sweep voltammetry for ECR revealed onset potentials ranging from -1.10 V to -1.49 V which corroborated with literature while showing some changes with each membrane, accompanied with high current densities and low ohmic resistance. Tafel slopes revealed mechanistic changes over potentials applied indicating a possible change in product formation with increasing potentials. Chronoamperometry experiments were conducted using applied potentials of -1.6 V, and, -1.8 V, with average current densities reaching up to -99 mA/cm2 in bubbled CO2 and -223.32 mA/cm2 in CO2 humidified in H2SO4. Gas injected gas chromatography was used to analyse gaseous products. Products obtained were carbon monoxide, methane, ethane, and ethylene with the best results yielding a Faradaic efficiency of 4.36 % for ethane formation.
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    Chemical analysis and survey of mycotoxins in South African produced grains with emphasis on those produced by stenocarpella maydis in maize
    (University of Pretoria, 2024-02-20) Maharaj, Vinesh J.; Rohwer, Egmont Richard; hannalien.meyer@sagl.co.za; Meyer, Johanna Christina
    During the latter half of the previous century, new mycotoxins were discovered as secondary metabolites of fungi in major grains before harvest. The prevalence of these toxic compounds and the negative health effects caused when consumed in contaminated food and feed present a significant challenge for producers, traders, processors, and consumers worldwide. Until recently, there was a lack of information on the occurrence of well-known mycotoxins such as aflatoxins, fumonisins, and deoxynivalenol in commercially produced maize and wheat in South Africa (SA). Additionally, there was no available information on whether the toxins produced by the fungus Stenocarpella maydis in maize also occur in maize delivered after harvest for further processing. The main objective of this study was to gain insight into the prevalence of grain-related mycotoxins in South African-produced wheat and maize and to better understand the potential risks associated with toxins produced by Stenocarpella maydis infection in maize. Accurate reporting of the presence and concentration levels of specific mycotoxins is crucial in managing the risk associated with mycotoxins to guarantee the safety of food and feed products for consumers. However, developing analytical methods for the simultaneous analysis of a large number of organic compounds at µg/kg concentration levels is challenging. This is mainly due to the varying physical and chemical properties of the compounds, the availability of chemical standards for quantitative analysis, and the complex nature of the commodities (grains, fruits, nuts etc.) to be tested. In this study, extraction methods were investigated to develop a fit-for-purpose and cost-effective LC-MS/MS analytical method to analyse different mycotoxin groups simultaneously in grain samples. The developed LC-MS/MS method was able to analyse 14 different mycotoxins, including diplodiatoxin, in grains. It was successfully validated according to international method guidelines to ensure reliable results. A comprehensive survey was conducted over four wheat and maize production seasons in South Africa to determine the occurrence and concentration ranges of aflatoxins (AFB1, AFB2, AFG1, AFG2), deoxynivalenol (DON) and 15-acetyl-deoxynivalenol (15-ADON), fumonisins (FB1, FB2, FB3), ochratoxin A (OTA), T-2 toxin (T-2), HT-2 toxin (HT-2) and zearalenone (ZON) in commercially produced wheat and white and yellow maize in all the production provinces. The wheat and maize samples collected after harvest at the storage facilities were analysed using the validated LC-MS/MS method. The mycotoxin results of the first comprehensive maize survey were based on analyses of 1400 maize samples collected over 4 production seasons from 2014 to 2017. A follow-up survey was conducted to determine for the first time the prevalence of diplodiatoxin in commercially produced maize in South Africa, the primary toxin produced by Stenocarpella maydis. A low mycotoxin risk was reported in SA produced wheat. Only deoxynivalenol was present in 12.5% of the 160 wheat samples collected over 4 production seasons (2015–2018). The maximum concentration levels found ranged from 362 µg/kg to 593 µg/kg, far below the regulated maximum allowable levels in wheat. Aflatoxin B1 was seldom present in commercially produced maize in South Africa. The mycotoxins found were DON, 15-ADON, fumonisins, and ZON with at least one of these mycotoxins found in 83% of the maize in the first season, and 80%, 63% and 62% in the successive seasons. Deoxynivalenol and fumonisins were the most prevalent in maize. Sixty-nine percent of the maize samples was contaminated with DON in the first year, and 41%, 23% and 37% of the samples in the following three seasons. Occurrence in white and yellow maize only differed in the 4th season with 54% of white maize and only 19% of yellow maize contaminated with DON. Higher annual mean DON concentrations were reported in white maize in the seven different production provinces. Between 41% and 57% of the maize samples contained fumonisins, with similar prevalence in white and yellow maize in most of the production provinces. The mean FUM concentrations were well below the 4000 µg/kg regulatory value. Overall, less than 10% of the samples were contaminated with ZON. Diplodiatoxin was the major toxin found in maize inoculated with S. maydis isolates. The inclusion of diplodiatoxin in the validated LC-MS/MS analytical method was possible with the availability of pure diplodiatoxin extracted from the inoculated maize. In the maize survey, conducted from 2019 to 2022, diplodiatoxin was for the first time reported in commercial produced white and yellow maize in 5 of the production provinces. A large seasonal variation in prevalence was found with diplodiatoxin present in 13%, 20%, 1% and 51% of the maize samples in the 4 consecutive seasons. The presence of diplodiatoxin in maize delivered to food and feed processors highlighted the importance to further investigating the toxicity of diplodiatoxin. The suitability of the zebrafish embryo toxicity test method was verified with diplodiatoxin assays. This study has successfully reported on the occurrence of mycotoxins related to grains in commercially produced wheat and maize in South Africa. The concentration ranges, regional variations, and seasonal trends confirmed the importance of continuously monitoring mycotoxins in the grain value chain to ensure that the food and feed are safe for consumers.
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    The semiochemistry of fruits as seen through the lens of Tephritid fruit flies and their parasitoids
    (University of Pretoria, 2024-02) Rohwer, Egmont Richard; Ahmed Yusuf, Abdullahi; Mohamed, Samira A; Dekker, Teun; mianorn@gmail.com; Njurai Miano, Raphael
    The tephritid fruit fly is a term well-known in fruit and vegetable production. Several techniques including the use of parasitoids have been deployed for fruit fly control. In Sub-Saharan Africa, Fopius arisanus (Sonan) and Diachasmimorpha longicaudata (Ashmead) (Hymenoptera: Braconidae) have been introduced to supplement the existing native parasitoids. Although the effectiveness of parasitoids is known, there is a knowledge gap in the semiochemical-mediated interactions among tree-attached fruits, fruit flies, and parasitoids. Here, I aimed to compare the attraction of fruit flies and parasitoids to different fruits, evaluate fruit fly performances, in terms of recovered puparia, in these fruits and elucidate the fruits’ headspace volatile compounds. First, the attraction of Bactrocera dorsalis (Hendel), F. arisanus and D. longicaudata to the headspace volatiles of different treatments of three varieties of mangoes were compared. B. dorsalis and the two parasitoids were differentially attracted to the mango headspace volatiles compared to the control, clean air. A higher number of B. dorsalis puparia was recovered in the Apple mango variety (81.7%) but none from the Kent variety. Gas chromatography- mass spectrometry revealed several organic compounds with qualitative and quantitative differences. The majority of tentatively identified compounds were esters (33.8%). Most compounds were produced at higher concentrations by fruit fly-infested mangoes than non-infested ones. A similar approach to B. dorsalis' was followed on Ceratitis cosyra, the two parasitoids and its native Psyttalia cosyrae (Wilkinson). C. cosyra and the parasitoids differentially responded to the treatments of the three mango varieties. Ceratitis cosyra performed better in Kent mango (72.1% of the 287 puparia recovered) compared to Apple and Haden varieties. Esters were the main components of the non-infested ripe and the late post-oviposition larval stages of the three mango varieties. At the same time, monoterpenes and sesquiterpenes were dominant in the other mango treatments. The performances of B. dorsalis, C. cosyra, Zeugodacus cucurbitae and B. latifrons on different species of fruits (mango cv. Haden, banana cv. Fhia-17, and tomato cv. Improved Nouvelle F1) were investigated and the headspace volatiles of different treatments of the three mango varieties, ripe bananas and tomatoes analyzed using GC-MS and GC-electroantennographic detection (EAD). The fruit fly species performed differentially in the different fruits. There were overlapping detections of most EAD-active compounds across the four fruit fly species and parasitoids with esters being the most prevalent class of compounds. This study represents the first report of the interactions of different fruit fly species and their parasitoids to in situ headspace volatiles of different treatments of mangoes and the subsequent changes in the headspace components of these mango treatments. Results obtained not only provide a better understanding and add new knowledge to science on the dynamic interactions of the selected tephritid fruit fly species and their parasitoids to a variety of hosts with different physiological states but also show a convergence of fruit fly and parasitoid antennal-active compounds hence presenting an informed foundation for future reference in developing sound Integrated Pest Management (IPM) strategies for managing fruit flies without harming parasitoids.
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    Intermolecular bonding in water clusters from the molecular-wide and electron density-based (MOWED) perspective : a theoretical study
    (University of Pretoria, 2023-12-14) Cukrowski, Ignacy; De Lange, Jurgens; stefanzaaiman@gmail.com; Zaaiman, Stéfan
    Cooperativity is a strange phenomenon in water clusters, characterized by a non-linear decrease in the average electronic energy of a water molecule or hydrogen bond with an increase in cluster size. The main aim of this theoretical study was to determine the effect cooperativity has on a water cluster and how it manifests, using novel theoretical tools and methodologies. Specifically, the MOlecular Wide Electron Density (MOWED) approach was used in this study to explore water clusters. Modelled water clusters (which included 2D cyclic and various 3D conformers) displayed the expected non-linear decrease in electronic energy. A novel equation was developed and fitted to the clusters in order to predict maximum cooperativity effects and their relative rates of changes. The equation was then adapted to be able to accommodate any property for the applicable water clusters. The equation predicted a maximum stability for cyclic water clusters of –8.316 kcal/mol per water molecule. This is the limit in stability relative to the dimer that a cyclic water cluster can reach due to cooperativity. The relationship between electron delocalization and cooperative stabilization was also explored extensively. It was found that intermolecular electron delocalization increases non-linearly with increasing cluster size and can be used to explain the origins of cooperativity. Intermolecular delocalization and interaction energies were also further decomposed into atomic and fragment contributions, and notably the 3-atom oxygen fragments contributed the most to the stability of the water clusters. Visualization of electron delocalization revealed ‘highways’ that electrons travel through within the water clusters, and 1D cross-section of an H-bond showed that a substantial amount of delocalized electron density is contributed by atoms other than the three present. The investigation of electron delocalization reveals that cooperativity is truly a molecular-wide event that is driven primarily by O-atoms and directed by H-atoms. The mechanistic limits to the number of electrons that can be delocalized were also investigated and found to be primarily O-localized density. Various cooperativity–induced effects – effects that result from cooperativity – were investigated. The atomic charge for oxygen showed a contribution from delocalized electrons resulting in the increased negative charge for the oxygen atom. The total interaction energy decreased while the exchange-correlation increased and had a positive sign. However, the total classic electrostatic interactions decreased and had a larger magnitude than total interaction energy. The increase and positive sign for exchange-correlation resulted from intramolecular interactions. Overall, the total intermolecular interaction energy and both its components contributed to the stability of the water cluster. The topological properties showed a stability increase at the critical point and increased covalency with the incremental increase of water molecules. Geometrical descriptors resulted in the same conclusions as found above. Finally, similar cooperativity effects were revealed in a series of 3D hexamer clusters, where the number of water molecules remain constant but the number and nature of H-bonds increases. The same mechanism of intermolecular electron delocalization along ‘highways’ connecting neighbouring O-atoms were revealed to be the primary driver of cooperative stabilization. Unlike the cyclic structures, the primary source of delocalized electrons was shown to be intramolecular delocalization (such as O–H covalent bonds).
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    Thermal and Structural Characterization of Fipronil
    (University of Pretoria, 2023-06-13) Focke, Walter Wilhelm; Van der Merwe, E.M. (Elizabet Margaretha); Rademeyer, Melanie; u11327414@tuks.co.za; Moyo, Dennis Simbarashe
    Fipronil is a widely used insecticide which exerts selective toxicity towards the GABA receptors of insects and has no known pesticide resistance in target insects. This, in turn, has led to the increase in the usage of fipronil as an alternative to commonly used pesticides in residential, industrial, commercial and agricultural settings. In this study, fipronil was considered for use as the chemical active in a controlled-release polymer system applied as alternative to indoor residual spraying in the fight against malaria. However, before incorporating the insecticide in a polymer, the vaporization and polymorphic behaviour of fipronil had to be studied. This information is important in providing guidance for the implementation of suitable safety measures during polymer processing to prevent exposure or contact to substances which could potentially harm one's health. On the other hand, polymorphism affects various physicochemical properties like the melting point, solubility, stability and manufacturability of a compound, which are extremely important for quality control and assurance. Differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), powder X-ray diffraction (PXRD) and single crystal X-ray diffraction (SCXRD) were used to study the vaporisation and polymorphic behaviour of fipronil. Sublimation and evaporation rates were determined using isothermal TGA. From these results, vapour pressures were deduced on the assumption that the fumes behaved like ideal gases and that mass loss was controlled by diffusion through the gas present in the partially-filled crucible. For the latter process, the diffusion coefficient was estimated using the Fuller correlation. Results obtained using benzoic acid as the calibration standard suggested that it is possible to estimate vapour pressures to within 12% with this TGA method. The enthalpies of sublimation and of evaporation were determined as 120 ± 4 and 72 ± 5 kJ mol-1, respectively. Polymorphic behaviour was studied through a systematic comparison of the thermal and structural properties of different crystal forms, including those obtained in this study and in literature. DSC was particularly useful in differentiating between two different crystal forms found in the as-received neat fipronil. Two polymorphs were successfully separated through sublimation of neat fipronil. A metastable, lower melting polymorph and a thermodynamically stable, higher melting form were obtained in the sublimate and residue, respectively. The lower melting polymorph was found to be unstable at high heating rates, with evidence suggesting a solid-solid phase transition to the stable form at low heating rates. Solvent recrystallization of neat fipronil in acetonitrile, acetone, ethyl acetate and methanol yielded five different crystal forms of fipronil. TGA curves revealed that all five crystal forms, except for the acetone-derived sample, were solvate pseudo-polymorphs exhibiting solvent loss between 60 and 100 °C. The acetone-derived sample was found to be a hemihydrate exhibiting mass loss at 120 °C. SCXRD studies revealed that three of the five forms had similar structural characteristics, while the other two forms differed notably from each other and the rest of the structures. Despite these differences, all five forms exhibited near-identical intra- and intermolecular hydrogen bond networks. The sublimation and evaporation enthalpies were successfully determined and the data indicates that fipronil will likely sublime at polymer processing conditions above 150 °C. The study also demonstrated, by DSC analysis, that the lower melting form in the neat, as-received fipronil would be converted to the high melting, thermodynamically stable form at the polymer processing conditions.
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    Porphyrin-based porous organic polymers enriched with Fe3O4  nanoparticles as electrosensors for the detection of endocrine disrupting chemicals in water
    (University of Pretoria, 2023-11) Nombona, Nolwazi; u15179941@tuks.co.za; Berrange, Lu-Nita
    Endocrine disrupting chemicals (EDCs) are commonly found in products such as pesticides, plastics, and pharmaceuticals. These chemicals are of significant concern due to their potential to disrupt the endocrine system, which plays essential roles in growth and reproduction. The detection of EDCs in water requires extensive equipment training and is expensive; therefore, it remains a significant challenge. Various catalysts such as multiwalled carbon nanotubes and graphene has been used as sensors to detect EDCs but porous organic polymers (POPs) and Porphyrin-based POPs (Py-POPs) have gained interest in electrochemical research owing to their porosity, stability, and tuneable structure. The structure of POPs depends on the synthetic method, the monomer used, and reaction conditions. Porphyrins are cyclic tetrapyrroles that consist of a conjugated π-electron system. Due to this conjugation, Py-POPs are stable structures that can coordinate metal ions. Metalated porphyrin-based POPs (MPy-POPs) are redox-active and chemically stable with improved electrochemical performance. In light of this, metalated porphyrin-based POPs (MPy-POPs) were used as sensing platforms for the detection of 2-phenylphenol, an endocrine disrupting chemical. Iron and zinc Py-POPs were synthesized and enriched with Fe3O4 nanoparticles to form nanocomposite materials. The morphological and structural characteristics of the composites were analysed using a variety of techniques, which includes ultraviolet-visible (UV-Vis) spectroscopy, Fourier transform infrared spectroscopy (FTIR), powder X-ray diffraction (PXRD), scanning electron microscopy (SEM), tunnel electron microscopy (TEM) and energy dispersive X-ray (EDX) analysis. The synthesized composites had spherical morphology. The Fe3O4@MPy-POP catalysts were used for the electrocatalytic detection of 2-phenylphenol. Catalytic currents ranged between 24.7 µA and 41.5 µA, with peak potentials between 0.61 V and 0.65 V. The limit of detection ranged from 0.75 mM to 2.6 mM for the redox active catalysts. Real sample analysis using sunblock and apple peels were analyzed for the detection abilities of the synthesized catalysts. This study shows that these composites are promising materials for the detection endocrine disrupting chemicals in water.
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    Development of analytical methods for N-nitrosamine disinfection byproducts in drinking water
    (University of Pretoria, 2023-10-23) Forbes, Patricia B.C.; u21832227@tuks.co.za; Tyhali, Akhona
    N-nitrosamines (NAs) are widely recognized as cancer-causing and genotoxic substances that emerge in water primarily as a result of chlorination or chloramination disinfection processes when nitrogen-containing compounds are present. N-nitrosodimethylamine (NDMA) is an NA that has garnered considerable attention due to its highly carcinogenic properties. Solid-phase extraction (SPE) is the preferred method for isolating NAs from water samples, with analysis primarily relying on gas chromatography-mass spectrometry (GC−MS) or liquid chromatography-mass spectrometry (LC−MS). Despite this, comprehensive research into the distribution and potential consequences of NAs in water remains largely unexplored on the African continent. There is a pressing need for extensive research geared towards the development of sensitive yet user-friendly analytical techniques for detecting NAs and monitoring their presence in African water sources, especially in drinking water, to better assess potential health risks for the human population. Molecularly imprinted polymers (MIPs) are widely acknowledged for their durability, adaptability, and ability to effectively emulate antibody-antigen systems. These polymers were thus synthesized with microcavities designed with the intent to exclusively recognize and bind to NDMA for utilization in the extraction of NDMA from water and methanol samples using SPE and dispersive solid-phase extraction (dSPE) techniques. Computational simulations were undertaken to identify an appropriate template for the synthesis of MIPs with selectivity towards NDMA. The difference in binding energies between the dimethylformamide and methacrylic acid complex (DMF−MAA) and N-nitrosodimethylamine and methacrylic acid complex (NDMA−MAA) in both acetonitrile and water was found to be -1.081 kcal.mol-1 and -1.012 kcal.mol-1, respectively. This observation suggested that DMF could serve as a suitable template for the synthesis of MIPs targeting NDMA, as the binding energies of these complexes exhibited relatively close values. Additionally, it is of paramount importance that the template is complementary in shape and size to the target molecule to allow the creation of microcavities that would be able to bind to it. In this study, DMF was the template of choice for NDMA due to similarities in their structures. The choice of water and acetonitrile for the simulations was deliberate, as these solvents mirror those employed in the actual MIP synthesis process. The DMF template was efficiently removed from the synthesized polymer by heat (158 oC for one hour) after an initial washing step to remove unreacted reagents. This approach is more environmentally friendly than typical methods that employ large solvent volumes for removing templates from MIPs. The optimal synthetic method for MIPs and non-imprinted polymers (NIPs) employed 1,1’-azobis(cyclohexanecarbonitrile) as an initiator and temperatures around 75-76 oC for 24 hr. The characterization of the resulting polymers was done using scanning electron microscopy, thermogravimetric analysis, Fourier-transform infrared spectroscopy, and the Brunauer-Emmett-Teller (BET) method. Thermal analysis showed that the backbone of all the polymers collapsed around 290 oC and that there was close to 100% thermal decomposition for all polymers around 470 oC which was comparable to temperatures that were found in the literature for similar MIPs. The FTIR spectrum of DMF had a completely different fingerprint region when compared to that of MIP−DMF. In addition to the results of the analysis of the polymer washings by GC−MS, this proved that DMF was efficiently removed from the MIP−DMF polymer. Despite the large particles that were observed for the non-imprinted polymers (NIPs) which may indicate a smaller specific surface area, the BET method showed that the NIP contained more pores and thus the additional spaces and interconnected pores in it rendered a larger overall surface area, which resulted in a higher specific surface area than that for the MIPs which had smaller particles. LC−MS and GC−MS methods were developed for NAs analysis. For the LC−MS, atmospheric pressure chemical ionization (APCI) outperformed electrospray ionization (ESI) and hence was chosen as the ionization mode of choice for further analyses. The LC−MS method was used for the analysis of eluates from the SPE and dSPE experiments. The GC−MS method was used in the stability analysis of NAs, for the analysis of anhydrous extracts from the SPE and dSPE experiments, and to provide further evidence that the DMF removal from the MIPs was successful. Despite the extensive precautions taken to preserve the nitrosamine mix standard in securely sealed amber bottles and vials stored under refrigeration, along with conducting experiments under low-light conditions, a stability assessment revealed that NAs exhibit significant instability, particularly those with lower molecular masses, such as NDMA and N-nitrosomethylethylamine (NMEA). These compounds were observed to degrade progressively over time, with eight of the nine NAs in a nitrosamine mix standard displaying a decline in response (in terms of peak areas) of 60% or more by the fifth day of the stability analysis. Consequently, the detection of NAs at trace levels becomes even more challenging due to their inherent instability. The optimal conditions for the SPE of NAs by LC−MS were found to be: one hour sorbent drying time, 10 mL elution volume, 1 M ionic strength, and a basic pH of 11.60 using HC-C18 cartridges. 500 mg MIP and NIP cartridges were found to block during the sample loading step when SPE experiments were conducted, but this challenge was overcome by reducing the sorbent mass to 90 mg. Upon loading the MIP−DMF cartridges with 500 mL water samples spiked with 1 mL of 10 µg/mL of NDMA to provide a final concentration of 0.02 µg/mL, it was found that the sorbents were either unable to retain NDMA or interacted very strongly with it, making it difficult to elute NDMA by dichloromethane (DCM), as NDMA was not detected in the eluates by GC−MS. To provide further evidence of whether the synthesized MIPs could retain NDMA and whether they are selective, dSPE experiments were attempted as they are relatively easy and can be completed in a short time as opposed to the cartridge based SPE method, which makes dSPE potentially attractive for commercial laboratory applications. 5 mg of MIPs and NIP were determined to be a suitable sorbent mass for the dSPE procedure, but 20 mg was used for the HC C18 as it was difficult to weigh 5 mg of it accurately due to its high density. The amount of NDMA that was detected by the GC−ToFMS in the supernatant of the MIP−NDMA was 1.4 greater than that detected in the HC-C18 supernatant as determined by the ratio of the NDMA peak areas. The amount of NDMA detected in the supernatants of the MIP−DMF and NIP was 9.2 and 5.7 greater, respectively, than that detected in the HC-C18 supernatant. The MIP−NDMA performed more similarly to HC-C18, whilst the MIP−DMF and the NIP did not perform as well. There was no NDMA detected in the extracts from either the loaded synthesized polymers or the HC-C18. It is possible that the extraction solvents employed (DCM and methanol) may not have been sufficiently strong to release any extracted NDMA, making it present at concentrations that were not detectable by the GC−ToFMS. Alternatively, NDMA might have been strongly bound to the materials, hindering its release, or the bound NDMA underwent degradation during the extraction procedure. Further dSPE experiments were conducted using a nitrosamine mix standard as a spiking solution to determine the selectivity of the MIPs. Percentages of how much was retained and eluted, degraded or irreversibly retained, or not retained by the sorbents for each N-nitrosamine were determined after analysis by UPLC−QToFMS. Of the three NAs that were retained and eluted from the sorbents, MIP−DMF extracted 14% of the NDBA whilst MIP−NDMA and HC C18 extracted 10% and 20% of the NDBA, respectively. The MIP−DMF extracted more of the N-nitrosopiperidine (NPip) (8%), followed by the MIP−NDMA (7%) and the HC-C18 (3%). The MIP−DMF also extracted more of the N-nitrosodiphenylamine (NDPhA) (15%), followed by the MIP−NDMA (14%) and the HC-C18 (12%). The remaining analytes or portions of analytes that were not retained and eluted were either irreversibly retained by the sorbent material or degraded, whilst some portions were not retained at all. Of the two NAs extracted by the NIP, 7% and 6% were extracted for N-nitrosopyrrolidine (NPyr) and NPip, respectively. Most of the NAs were either irreversibly retained by the sorbent material or were degraded. The dSPE method utilized did not prove effective in fully assessing the potential sorption of NDMA by the MIPs synthesized in this study. In comparison to the cartridge SPE experiments, the dSPE results for the HC-C18 were notably poorer (although it is acknowledged that a lower mass of sorbent was used). This suggests that the dSPE method employed may not be well-suited for extracting the nine target NAs in this study. Nonetheless, it is worth noting that the dSPE method offers convenience and rapidity in contrast to the cartridge based SPE method. Overall, this study contributes to the understanding of N-nitrosamine extraction and detection, specifically NDMA, from water matrices. It highlights the development of MIPs as a potential solution for improved extraction and the challenges associated with the stability and selective sorption of N-nitrosamines, providing valuable insights into the field of water quality assessment and the need for more effective analytical techniques.
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    Evaluation of labelling mechanisms and analytical procedures for Ga-68 labelled peptides used in positron emission tomography
    (University of Pretoria, 2023) Rohwer, Egmont Richard; Zeevaart, Jan Rijn; Ebenhan, Thomas; biljana.marjanovic-painter@necsa.co.za; Marjanovic-Painter, Biljana
    Nuclear Medicine imaging involves the monitoring of disease processes by using radioactive signals produced by radiopharmaceuticals (injected intravenously) to monitor the pharmacokinetic behaviour of compounds non-invasively in the human body by special cameras. The most common imaging technique using radioactivity is Positron Emission Tomography (PET). The radiopharmaceuticals are compounds made of two main components, firstly the radionuclide which provides a signal for detection and secondly, a molecular vehicle which is responsible for biochemical functions, binding, metabolism, and clearance. If the radiopharmaceutical is peptide based, these two structural components are made to exist as functional units and their performance is enabled, enhanced, and controlled through the use of different chelators, spacers, and modifications. Receptor expressing tissues will be targeted with these receptor binding peptides and are used for imaging in different applications, for example in specific targeting of tumors. This thesis highlights the importance of validation of analytical methods in Quality control according to Good Manufacturing Practices, current Good Radiopharmacy Practice and Good Laboratory Practices with a specific application in the field of Radiochemistry and Radiopharmaceutical development. It is important to include these aspects in the design process, as early as the stage of product conception. The aim was to evaluate the gallium-68 radiopharmaceutical development pipeline and provide constructive guidance on best practices. The development of ubiquicidin-derived radiopharmaceuticals, as an exemplary application, was critically investigated using a systematic reviewing strategy (Chapter 4). It was found that the systematization and unification of criteria for preclinical imaging and larger clinical trials are still required to ensure the translation of this new radiopharmaceutical into the clinic. It was observed that there was not enough testing done across different sizes of animals, preventing better and easier translation to humans. Kits are gaining popularity as a known convenient labelling procedure for gallium-68 based radiopharmaceuticals in the Nuclear Medicine clinic. Cold kits are a benefit to small radiopharmaceutical sites, but their quality should be checked in the same way as if they were produced by big pharmaceutical producers. The purpose of this thesis was to describe analytical methods and step-by-step validation thereof, (according to European Pharmacopoeia requirements) using readily available instrumentation in laboratories. The validation of the analytical methods that can be used during cold kit production of different gallium-68 radiopharmaceuticals is described in Chapter 3. Validated methods were applied to the PSMA-11 kit as an example and it was found that this product complied with all set requirements.