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Item Development and validation of lc-ms/ms method for determination of non-opioid analgesics in adulterated herbal medicines(Sokoine university of agriculture, 2022) Mpanyakavili, Anna LucasBackground: Herbal medicines are increasingly used worldwide despite the increased concern of their adulteration with conventional drugs. Non-opioid analgesics are among of the conventional drugs reported to be used in adulteration of anti-pain herbal medicines. This rise a need for developing reliable analytical method for determination of adulterated conventional drugs in herbal medicines for quality control. This study aimed at developing a high throughput Liquid Chromatography coupled with Tandem Mass Spectrometry for determination of non-opioid analgesics (acetaminophen, caffeine, acetylsalicylic acid, diclofenac, and ibuprofen) in herbal medicines in one run. The study also aimed at optimizing a sample preparation technique to enhance detection of the adulterants in herbal medicine obtained from the Tanzania market. Methodology: Solid Phase Extraction (SPE) and Ultrasonic Assisted Extraction (UAE) methods were compared in respect of recoveries, extraction time, complexity, matrix effects, and expenses for sample preparation. Waters OASIS Hydrophilic-lipophilic balance (HLB) 200 mg 6 mL, a C 18 sorbent was used in Solid Phase Extraction method optimization. The optimized parameters were effects of filters on sample filtration, sample loading pH, selection of suitable solvents for conditioning, washing, and elution. Ultrasonic bath and centrifuge were used for optimization of ultrasonic assisted extraction method parameters including effectives of organic solvent in extraction, extraction solvent content, and sample to extraction solvent volume ratio. Both methods were evaluated by calculating their matrix effects, absolute recoveries, and percentage recovery for sub- steps. The effective sample preparation method was used in development and validation of an advanced hyphenated method using an Agilent Technologies 1100 series Liquid Chromatography coupled with Waters Tandem Mass spectrometry (LC-MS/MS) with electrospray ionization. The LC-MS/MS method was validated for its selectivity, sensitivity, linearity, accuracy, precision, recovery, matrix effects, and stability. It was then used to analyse 132 samples collected from the markets of four regions in Tanzania. Results and Discussion: The Ultrasonic Assisted Extraction method showed to be efficient to Solid Phase Extraction method for extraction of selected non-opioid analgesics. This was clearly evidenced by high recoveries, minimum extraction time, good peak shape, its simplicity in procedures, and use of less expensive consumables. The absolute recoveries for Ultrasonic Assisted Extraction were at acceptable range (> 60%) ranging from 60 % to 107 % while for Solid Phase Extraction method showed poor recovery except for diclofenac and ibuprofen having 83% and 67%, respectively. The matrix effect expressed as signal suppression/enhancement ranged from 70% - 122% for Ultrasonic Assisted Extraction method and 3% to 124% for Solid Phase Extraction method. From the results the Ultrasonic Assisted Extraction method was chosen for sample extraction for analysis of Herbal Samples. The developed LC-MS/MS method was linear with coefficient of determination of R 2 ≥ 0.9931. The Limit of Detection (LOD) and Limit of Quantification (LOQ) for selected non-opioid analgesics were within the range of 0 – 3.7 μg/mL. The intra-precision of the LC-MS/MS method was expressed as Relative Standard deviation and was less than 9.5% with varying matrix effect among analytes. Twenty one percent (21%) of 132 collected herbal medicines were adulterated with caffeine and acetylsalicylic acid. 1,029.22 μg/mL was one of the highest concentration of caffeine detected in one of the analysed herbal medicines. Conclusion and recommendation: This is the first study in Tanzania to report an analytical method for analysis of five (5) non opioid analgesics in herbal medicines in Tanzania. The developed LC-MS/MS method is suitable for the identification and quantification of 5 non opioid analgesics explored adulterants. The method can be adopted by the regulatory authorities for routine analysis of herbal medicines for monitoring its quality and safety. Further studies are recommended for Ultrasonic Assisted Extraction method using additional internal standards. Key words: Herbal medicines, Adulteration, Analgesics, Solid Phase extraction, Ultrasonic assisted extraction, LC-MS/MS. Usuli wa tatizo: Ongezeko la matumizi ya dawa zitokanazo na mimea-dawa linazidi kukua ulimwenguni licha ya wasiwasi uliopo wa kuathiriwa kwa dawa hizo kwa kuchanganywa na dawa za kisasa. Dawa za kutuliza maumivu za kisasa ni miongoni mwa dawa zinazoripotiwa kuongezwa katika dawa zitokanazo na mimea dawa za kupunguza maumivu na hivyo kushusha ubora wake. Hivyo, kuna uhitaji mkubwa wa kuunda njia ya kisayansi yenye kuaminika kwa ajili ya uchambuzi na utambuzi wa dawa zitokanazo na mimea dawa zilizochanganywa na dawa za kisasa ili kudhibiti ubora na kuongeza usalama kwa watumiaji. Utafiti huu ulilenga kutengeneza njia ya kisayansi ya uchambuzi inayotumia teknolojia ya LC-MS/MS ili kubaini aina na kiasi cha dawa za kisasa za kutuliza maumivu (asetaminopheni, kafeini, aspirini, diclofenaki, na ibuprofeni) zilizomo ndani ya dawa zitokanazo na mimea dawa. Utafiti huu pia ulilenga kuboresha mbinu ya utayarishaji wa sampuli ili kuboresha ugunduzi wa dawa za kisasa ndani ya dawa zitokanazo na mimea dawa zinazopatikana katika soko la Tanzania. Methodolojia: Utafiti ulilinganisha njia mbili, Solid Phase Extraction (SPE ) na Ultrasonic Assisted Extraction (UAE), kwa kuzingatia uhuishaji, muda ambao ulitumika kuchukua kiziduo, athari za dutu za ziada, na gharama za uandaaji wa sampuli. Teknolojia ya Waters OASIS Hydrophilic-lipophilic balance (HLB) 200 mg 6 mL, sobanti C18 ilitumika katika mchakato wa kuchambua dutu lengwa. Vigezo stahiki vilipatikana kutokana na athari zilizojitokeza katika uandaaji wa sampuli. Pia teknolojia ya ultrasonic assisted extraction ilitumika kuweka sawa mchakato wa kikemikali wa kuchambua dutu lengwa kutoka kwenye sampuli. Njia zote mbili zilitathiminiwa kwa kukokotoa athari zilizojitokeza, kiasi cha dutu zilizopatikana katika mchakato, na kiasi cha dutu kilichopatikana katika hatua zilizofuata. Njia bora ya kuandaa sampuli stahiki ilitumika katika kutengeneza na kuthibitisha njia yenye ufanisi wa kuchambua dutu lengwa. Kukamilisha zoezi hili, teknolojia ya Agilent Technologies 1100 series Liquid Chromatography na ile ya Waters Tandem Mass spectrometry (LC-MS/MS) yenye electrospray ionization vilitumika. Njia ya LC-MS/MS ilihakikiwa uwezo wake katika kubaini dutu husika, usahihi, utoshelevu, na umadhubuti. Aidha, njia hii ilitumika kuchambua sampuli 132 zilizokusanywa kutoka kwenye masoko ya mikoa minne nchini Tanzania. Matokeo na mjadala: Njia ya UAE ilionyesha kuwa na ufanisi katika kuandaa sampuli ya dawa za kupunguza maumivu zilizochaguliwa kwa ajili ya utafiti. Hii ilithibitishwa na uwezo mkubwa katika kuhuisha, kutumia muda mchache, kutoa taswira nyoofu, urahisi katika kutumia pamoja na utumiaji wa malighafi za gharama ndogo. Uhuishaji halisi wa UAE ulikuwa katika kiwango kinachokubalika, yani, kuanzia 60 % hadi 107 %, wakati njia ya SPE ilionesha uhuishaji dhaifu isipokuwa kwa diclofenac (83%) na ibuprofen (67%). Athari za ziada zilizooneshwa kama alama zilizodhibitiwa (fiche) zilianzia 70% - 122% kwa njia ya UAE na 3% hadi 124% kwa njia ya SPE. Kutokana na matokeo, UAE ilichaguliwa kwa ajili ya kuandaa sampuli zitokanazo na mimea dawa kwa ajili ya uchambuzi. Njia ya LC-MS/MS ililandana na kizigeu-tambuzi, R 2 ≥ 0.9931, upeo wa kubaini pamoja na upeo wa kiasi kwa dawa za kuzuia maumivu zilizotafitiwa zilikuwa kati ya 0 – 3.7 μg/mL. Utashelevu wa ndani wa njia ya LC-MS/MS ilijidhihirisha kama achano sanifu-wiano ambalo lilikuwa 9.5% iliyokuwa na athari za ziada mbalimbali katika dutu zilizochambuliwa. Asilimia ishirini na moja (21%) ya dawa 132 za asili zilikuwa zimechanganywa na kafeini na aspirini. Kiasi cha juu cha kafeini kilichogunduliwa katika dawa zilizochunguzwa ni 1,029.22 μg/mL. Hitimisho na mapendekezo: Huu ni utafiti wa kwanza nchini Tanzania uliotumia njia changanuzi kuchunguza uwepo wa dawa tano za kisasa ndani ya dawa zitokanazo na mimea dawa za kutuliza maumivu. Njia iliyobuniwa ya LC-MS/MS ilifaa katika kutambua na kupima dawa tano za kupunguza maumivu zilizotumika kama viharibifu. Hivyo, njia hii inaweza kutumiwa na mamlaka za udhibiti wa ubora na usalama wa dawa za asili. Inapendekezwa kuwa tafiti zaidi zifanyike kutengeneza njia zingine za LC-MS/MS kwa dawa zingine ili kudhibiti ubora wake. Maneno muhimu: Dawa za mimea-dawa, kuchanganywa, dawa za kutuliza maumivu, uziduzi, LC-MS/MS.Item Development and validation of LC-MS/MS method for determination of non-opioid analgesics in adulterated herbal medicines(Sokoine University of Agriculture, 2022-11) Mpanyakavili, Anna LucasBackground: Herbal medicines are increasingly used worldwide despite the increased concern of their adulteration with conventional drugs. Non-opioid analgesics are among of the conventional drugs reported to be used in adulteration of anti-pain herbal medicines. This rise a need for developing reliable analytical method for determination of adulterated conventional drugs in herbal medicines for quality control. This study aimed at developing a high throughput Liquid Chromatography coupled with Tandem Mass Spectrometry for determination of non-opioid analgesics (acetaminophen, caffeine, acetylsalicylic acid, diclofenac, and ibuprofen) in herbal medicines in one run. The study also aimed at optimizing a sample preparation technique to enhance detection of the adulterants in herbal medicine obtained from the Tanzania market. Methodology: Solid Phase Extraction (SPE) and Ultrasonic Assisted Extraction (UAE) methods were compared in respect of recoveries, extraction time, complexity, matrix effects, and expenses for sample preparation. Waters OASIS Hydrophilic-lipophilic balance (HLB) 200 mg 6 mL, a C 18 sorbent was used in Solid Phase Extraction method optimization. The optimized parameters were effects of filters on sample filtration, sample loading pH, selection of suitable solvents for conditioning, washing, and elution. Ultrasonic bath and centrifuge were used for optimization of ultrasonic assisted extraction method parameters including effectives of organic solvent in extraction, extraction solvent content, and sample to extraction solvent volume ratio. Both methods were evaluated by calculating their matrix effects, absolute recoveries, and percentage recovery for sub- steps. The effective sample preparation method was used in development and validation of an advanced hyphenated method using an Agilent Technologies 1100 series Liquid Chromatography coupled with Waters Tandem Mass spectrometry (LC-MS/MS) with electrospray ionization. The LC-MS/MS method was validated for its selectivity, sensitivity, linearity, accuracy, precision, recovery, matrix effects, and stability. It was then used to analyse 132 samples collected from the markets of four regions in Tanzania. Results and Discussion: The Ultrasonic Assisted Extraction method showed to be efficient to Solid Phase Extraction method for extraction of selected non-opioid analgesics. This was clearly evidenced by high recoveries, minimum extraction time, good peak shape, its simplicity in procedures, and use of less expensive consumables. The absolute recoveries for Ultrasonic Assisted Extraction were at acceptable range (> 60%) ranging from 60 % to 107 % while for Solid Phase Extraction method showed poor recovery except for diclofenac and ibuprofen having 83% and 67%, respectively. The matrix effect expressed as signal suppression/enhancement ranged from 70% - 122% for Ultrasonic Assisted Extraction method and 3% to 124% for Solid Phase Extraction method. From the results the Ultrasonic Assisted Extraction method was chosen for sample extraction for analysis of Herbal Samples. The developed LC-MS/MS method was linear with coefficient of determination of R 2 ≥ 0.9931. The Limit of Detection (LOD) and Limit of Quantification (LOQ) for selected non-opioid analgesics were within the range of 0 – 3.7 μg/mL. The intra-precision of the LC-MS/MS method was expressed as Relative Standard deviation and was less than 9.5% with varying matrix effect among analytes. Twenty one percent (21%) of 132 collected herbal medicines were adulterated with caffeine and acetylsalicylic acid. 1,029.22 μg/mL was one of the highest concentration of caffeine detected in one of the analysed herbal medicines. Conclusion and recommendation: This is the first study in Tanzania to report an analytical method for analysis of five (5) non opioid analgesics in herbal medicines in Tanzania. The developed LC-MS/MS method is suitable for the identification and quantification of 5 non opioid analgesics explored adulterants. The method can be adopted by the regulatory authorities for routine analysis of herbal medicines for monitoring its quality and safety. Further studies are recommended for Ultrasonic Assisted Extraction method using additional internal standards.Item Investigation of antibacterial effects of herb-herb and herb-antibiotic combinations(Sokoine University of Agriculture, 2022) Ochollah, Mary GeorgeIntroduction Several studies have reported the failure of single-drug treatment regimens for bacterial infections due to emergence of bacterial resistance. Hence, there has been a growing interest in researching and developing new antibacterial drugs, containing several combined ingredients as one of the means to combat bacterial resistance. Among the successful reported antibacterial combination treatments include herb-herb and herb-antibiotic. This study investigated the antibacterial effects of single and combined extracts from leaves, stems and root barks of Commiphora swynnertonii and Synadenium glaucescens. In addition, the study evaluated antibacterial effects of combined crude extracts of two medicinal plants, also the extracts were combined with selected three antibiotics namely ciprofloxacin, ampicillin, and erythromycin. Methods C. swynnertonii and S. glaucescens were respectively collected from Manyara and Njombe regions of Tanzania. The standard antibiotics used as positive control in this study were gentamicin, ciprofloxacin, ampicillin, and erythromycin. Extraction of plant materials was done by soxhlet and macerations methods using dichloromethane and methanol solvents respectively. The extracts were assessed for their effects on Gram-positive bacteria (Staphylococcus aureus and Enterococcus faecalis) and Gram- negative bacteria (Escherichia coli, Klebsiella pneumonia, and Pseudomonas aeruginosa). Minimum Inhibitory Concentrations (MIC) was determined by broth micro dilution method while Fractional inhibitory concentration (FIC) indices were calculated from MIC values of individual and combined crude extracts to determine combination effects. Findings Strong antibacterial activities were demonstrated by all extracts of S. glaucescens (MIC 0.01-0.37mg/mL) and methanol extracts of C. swynnertonii (MIC 0.04-0.37mg/mL) against Gram-positive bacteria Synergistic effect was observed in combination of methanol extracts of C. swynnertonii stem barks and S. glaucescens leaves against S.aureus (∑FIC 0.5), other synergistic effects were observed against E. faecalis in combination of dichloromethane extracts of C. swynnertonii stem barks and S. glaucescens stem barks (∑FIC 0.5), and C. swynnertonii root barks and S. glaucescens root barks (FIC index 0.3). For the remaining combinations of crude extracts, mainly additive effects were observed. The combinations of crude extracts with antibiotics revealed synergism when ciprofloxacin was combined with all tested crude extracts against E. coli (ΣFIC of 0.02), combination of ciprofloxacin with extract from root barks of C. swynnertonii (ΣFIC of 0.5) against S. aureus, root barks of S. glaucescens (ΣFIC of 0.1) against S. aureus and combination of ampicillin with all tested crude extracts (ΣFIC of 0.03-0.1) against E. faecalis. Nevertheless, antagonism was observed between the combinations of ampicillin and erythromycin with all tested crude extracts against Gram- negative bacteria (ΣFIC of 4-8). Conclusions When two or more plant parts are combined among themselves or with antibiotics, the resulting antibacterial effects may either be synergistic, additive or antagonistic depending on the phytochemical contribution of each plant or part and also the bacteria or diseases intended. Recommendations Synergistic effects demonstrated in this study provide promising good combinations to be considered as alternative antimicrobials. Toxicity studies of the combinations of S. glaucescens stem barks of and C. swynnertonii leaves and S. glaucescens root barks and C. swynnertonii root barks which showed synergistic effects against Gram-positive bacteria are recommended in the future.Item Higher order perturbation analysis of plasma and gravitational waves(Universiteit Antwerpen, 2004) Karugila, Geoffrey KamahangaPlateau [1] initiated experimentally and theoretically the stability of a liquid cylinder with surface tension. Lord Rayleigh [2] improved this work and developed the linearized theory for sound waves (although the calculation of sound velocity goes back to Newton and Laplace) and for the Plateau experiments which was then applied to all kinds of fields: gravitation [3. 4, 5], plasma [6], (magneto)hydrodynamics [7, 8, 9], energy principles [10]. The linear theory flourished tremendously in the past century to a large extent due to the goal of fusion. Soon the need for nonlinear theories was manifest e.g. Callebaut [11, 12]. We therefore see in the previous decades a lot of work on nonlinear theory of plasma waves and instabilities being done. These yield either exact solutions or approximate ones. Often exact solutions are obtained after that the equations have been approximated. We may mention the papers with the exact solutions by Malfliet et al. [13, 14, 15], Hereman et al. [16, 17, 18], Verheest et al. [19, 20, 21] on solitary waves and those of Khater et al. on Backlund transformations and Painleve analysis [22, 23, 24, 25]. Amiranashvili et al. [26] gave some exact solutions for standing waves in bounded plasmas without using the solitary wave theory but with some boundary conditions. Callebaut and Tsintsadze [27, 28] e.g., neglecting some higher order terms, dealt with the nonlinear bunching of Alfven waves and the filamentation and modulation of weakly ionized magnetized plasmas. In fact, except for the approaches leading to solitary wave solutions, the nonlinear methods usually yield approximations and usually one has barely an idea how long these are valid in the behavior of the plasma. The approach used in this thesis exploits the Fourier analysis for nonlinear systems. It is rather different from the approaches just mentioned as it allows some insight in the convergence. Moreover, it gives useful results for the many cases where one can not find a closed form for the solitary waves. Indeed the solitary waves are an exceptional and rare case, comparable with a polynomial (as is clear e.g. from Malfliet’s work, see references cited) while the general solution is an infinite series. The polynomial may use a function (e.g. tanh) instead of the (combined) variable itself. Similarly the series may use any function although the customary ones are exponentials and (co)sines. Infinite Fourier series may in principle be considered as an exact solution, but in practice it often is an approximation, which, however, allows clear insight on its validity. The set of (partial differential) equations (e.g. equations (2.1) - (2.4)), together with some initial and/or boundary conditions, defines a set of func tions (which are, of course, interrelated). From the Fourier theory it is known that if a periodic function is continuous from — oo to +oo and has a deriva tive which is piecewise monotonous and continuous, then the function may be developed in a Fourier series which is absolutely and uniformly convergent in any interval. In the thesis we deal with the single variable x (= co t + k • r), which is the combination of the four independent variables i.e. the angular frequency ( cj = 2 tt z /, v = frequency), the time (Z), the wave vector (fc) and space (r). Thus the conditions have to be satisfied for the function(s) of this combined variable. In particular the function should be periodic in co t and in k r. However, an exponential growth is easily accommodated just like the periodic situations as was the case in a hydrodynamical problem [12]. Hence under rather general conditions it is possible to expand the functions defined by the set of equations. When will this breakdown? E.g. when the series diverges, i.e., physically speaking, when instability leading to dis- rupture develops meaning that a (large) amount of energy has been made available (either injected externally or freed by the system itself from e.g. its potential energy or, more generally, from its free energy). The convergence of the series puts conditions on the linear theory, mainly on its amplitude. In fact a linearized theory can never determine its own limitations: that has to be done by the nonlinear analysis. In the previous works [12] it turned out that some experimental situa tions, in particular the oscillations and instabilities of a liquid jet, could be explained very well by this method. Moreover some cases appeared where the nonlinear theory showed that the linear theory was good even up to de struction of the configuration, while for wavelengths much larger than the diameter of the jet the nonlinear terms became dominant. Another breakdown of the method may occur e.g. when the function(s) is (are) not periodic. However, in the linear perturbation theory one works usually with a periodic perturbation and this generates naturally higher order terms which are periodic too as is obvious e.g. in our present work and in the works of Callebaut [12, 29, 30]. For a non-periodic solution one has to take a wholly different start in the linear theory, e.g. by using a series in t and/or re, ?/, 2, or some adequate combination of those, or if nevertheless a periodic or exponential start is used as first order term, to adapt profoundly the nonlinear terms. Such nonlinear approaches have been elaborated in various ways in the literature see e.g. the cited references of Malfliet et al.; Hereman et al.; Verheest et al.; Khater et al.; Callebaut and Tsintsadze.Item Targeting the removal of Pyridine Herbicides from an Aqueous environment using polymer inclusion membrane(La Trobe University, 2019) Mwakalesi, Alinanuswe JoelThe beneficial use of herbicides on pastures to increase crop yields is offset by associated risks from chemical runoff contaminating environmental waters. Most commonly used methods to remove residues from environmental waters are ineffective, while some produce degradation by-products which can also affect the environment. The successful use of polymer inclusion membranes (PIMs) as a simple remediation method to remove pyridine herbicides, such as picloram (4-amino-3,5,6-trichloro-2-pyridinecarboxyIic acid), and related herbicides from an aqueous environment was reported. An optimum PIM composition of 25 wt%CTA, 30 wt% Aliquat 336 and 45 wt% NPOE was used to transport picloram with an initial flux of 294 (± 14) * 10 8 mol.m 2.s 1 and transport efficiency of 95 ± 1%. The PIM was reused in five consecutive transport cycles with a negligible change in flux during later cycles. The results indicate that PIMs provide a potential alternate method for the removal of troublesome herbicides fr om environmental waters. Degradation products from herbicides are considered emerging contaminants and studies on effective removal methods from environmental waters are limited. The successful tiansport of 4-amino-2-chloropyridine (ACP) as a model degradation product of pyridine- based herbicides using PIMs was demonstrated. An optimal membrane composition of 20wt%CTA, 40 wt% Aliquat and 40wt%NPOE producing an initial flux of 413 (± 7) 10 8 mol.m“2.s 1 and 98 ± 1% transport efficiency was identified. Preferential transport of ACP, involving the formation of aggregates with the carrier, over picloram was noted. The results indicate the opportunity for future investigations of the extraction and transport of degradation products of pyridine-based herbicides using PIMs. The use of natural compounds from plants as active chemicals for various applications is regarded as a “green chemistry approach” because of die reduced risk of contaminating the environment. However, there are limited investigations on the use of iplant-based compounds as components in PIMs. Anacardic acid (AA) derived from cashew nut shell liquid was investigated as a “green cationic carrier’’ for some representative organic compounds. An 20 wt% dodecanol optimal and membrane composition 40 wt% NPOE of producing 30 wt% CTA. an initial 10 wt% A A. flux of 364 (± 16) x to x mol.m 2.s 1 and transport efficiency of 98 ± 1% for the transport of ACP as a model compound was demonstrated. The results were comparable with using a commercial carrier, bis-(2-ethylhexyl) phosphoric acid. The membrane also displayed effective competitive transport of ACP, paraquat and diquat with an average transport efficiency of 97±l%. Therefore, the potential use of plant derived natural compounds as “green chemicals” in membranes is an exciting novel development and worthy of further investigations. PIMs have emerged as a powerful tool for the pre-treatment of samples because of their versatility and easy fabrication. The optimal membranes were tested for the pre concentration of picloram, ACP. paraquat and diquat as representative compounds from a low initial concentration of 500 pg/L in samples of natural water. The effective pre concentration of the compounds was indicated by the significant increase in HPLC signal intensities of the post-concentrated samples. The results indicate the suitability of PIMs as an alternate method of sample pre-treatment and the potential for fabrication and application in passive sampling devices. The results from this thesis indicate the potential of PIMs as a viable method to remove and measure problematic herbicides and degradation products from natural waters using commercial and “green” carriers. However, more investigations simulating real-world conditions, such as long-term use of similar membrane chemistry in hollow fibre extractors and passive sampling devices, are needed to fully demonstrate this exciting potential. Statement of Authorship Except where reference is made in the text of the thesis, this thesis contains no material published elsewhere or extracted in whole or in part from a thesis accepted for the award of any other degree or diploma. No other person’s work has been used without acknowledgment in the main text of the thesis. This thesis has not been submitted for the award of any degree or diploma in any other tertiary institution.