Analytical & Biological Chemistry Research Facility - Doctoral Theses

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    Development of a sensor-based rapid microbial testing platform for the Irish meat industry
    (University College Cork, 2023-01-05) Elisseeva, Sophia; Papkovsky, Dmitri B.; Kerry, Joseph; Department of Agriculture, Food and the Marine, Ireland; Dawn Meats
    Microbial spoilage and foodborne diseases cause significant productivity and economic losses for the food industry. There is a need for novel approaches to extend shelf life of products, improve quality and microbial safety, reduce spoilage and waste, and new assessment methods. Traditional methods are time consuming, labour intensive, centralised, have lengthy time to result, and some cannot analyse crude food homogenates. In this project, funded by the Irish Department of Agriculture, Food and the Marine and performed in partnership with a large Irish food company (Dawn Meats), several new optical oxygen sensor based systems were devised to increase efficiency and accuracy of testing. Chapter 1 (Literature review) describes the state of the art in the area, the range of existing approaches and analytical systems, and their capabilities. Experimental methods used in this study are summarised in Chapter 2 (Materials and Methods). The first experimental section (Chapter 3) describes the development of new multi-parametric toxicity testing platform based on the soluble oxygen probe, MitoXpress-Xtra, and 96 well plate format, which was used to investigate the antimicrobial effects of the compound Lauroyl Arginate Ethyl Ester (LAE) on pure cultures and whole meat microbiota. Through the measurement and analysis of the oxygen probe time profiles (phosphorescent lifetime) under different assay settings, we were able to assess and quantify the toxicity of LAE on different bacterial species, generating dose-response curves, and calculating EC50. The assay allowed for the simultaneous assessment of multiple variables and conditions such as bacterial species, temperature, growth media, sample type, and antimicrobial concentrations. The second experimental part (Chapter 4) describes the new portable and autonomous system(s) based on disposable vials integrated with solid state sensors, tailored for the analysis of meat samples, carcass swabs, and environmental swabs. Along with the disposable sensor vials, the system is composed of two additional parts; a handheld, autonomous sensor reader and a portable incubator/heater. Up to 20 samples were prepared using the standard methods (ISO 4833-1:2013; ISO 18593:2018) in sensor vials, incubated at 30C and measured hourly in a non-invasive, contactless manner. Such a simple system with manual measurements also revealed dissolved oxygen time profiles which were used to determine the threshold time of the sensor signal, which in turn, was used to calculate TVC values (CFU/ cm2 or CFU/g) using developed calibration equations. The method was validated using: i) meat samples and carcass swabs obtained from Dawn Meats, ii) brush swabs of artificially contaminated surfaces with E. coli, iii) swabs of surfaces contaminated with meat microbiota and iv) environmental swabs. No statistical difference was found between the sensor based method and reference method, providing the opportunity for the former to potentially replace the latter. Finally, in Chapter 5 the new respirometric sensor based system was applied to a shelf-life study with four different types of MAP mincemeat samples: beef, turkey, lamb and pork, together with the analysis of these samples by 16S rRNA sequencing. Respirometric profiles revealed unusual linear profiles for pork and lamb mince, the origins of which remain to be investigated. In addition to respirometric microbiological assessment, the whole microbiome of each mincemeat type was analysed using 16S rRNA sequencing, which revealed an overall decrease in alpha diversity with some taxa exhibiting statistically significant changes over shelf-life and after exposure to respirometry. Beta diversity was seen to be dictated by mincemeat type. Overall, the new optical oxygen respirometry systems are highly efficient and attractive for the food industry, with both developed systems improving existing methods in time to result, accuracy, user-friendliness, and on-site use. Furthermore, the portable sensor based system can be combined with cutting edge techniques such as next generation sequencing to provide more detailed information on the microbiota of food samples.
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    Synthesis of novel quorum sensing inhibitors of DSF
    (University College Cork, 2022) Horgan, Conor; O'Sullivan, Tim; Irish Research Council; Higher Education Authority
    Antimicrobial resistance (AMR) has become a growing concern among the medical community with many previously effective antibiotics losing their efficacy. Much of this AMR is thought to stem from biofilm formation controlled by cell-to-cell signalling. Chapter 1 introduces quorum sensing with a particular focus on the Diffusible Signal Factor (DSF) family of autoinducers. Given that these molecules contain a carboxylic acid, this chapter also contains a review of the literature relating to carboxylic acid bioisosteres which have emerged since 2013. Quorum sensing interference is an increasingly attractive target for combatting bacterial infections. Accordingly, the work in this thesis focusses on the synthesis of sulfonamide-based bioisosteric derivatives of Burkholderia DSF (BDSF). In Chapter 2, 15 novel N-acyl sulfonamide analogues of BDSF are prepared and tested for biological activity. Some of these compounds display significant activity against many strains of bacteria both in vitro and in vivo. The cis-α,β-unsaturated double bond, a key factor in the biological activity of BDSF, is susceptible to isomerisation. The preparation and subsequent biological evaluation of 16 potentially more stable aromatic N-acyl sulfonamide analogues of BDSF is detailed in Chapter 3. Many of these compounds significantly inhibited biofilm formation and enhanced the efficacy of last-resort antibiotics against S. maltophilia and B. cenocepacia. In Chapter 4, X. fastidiosa, a bacterium responsible for diseases such as olive quick decline syndrome, is introduced. Some of the aromatic N-acyl sulfonamide analogues inhibited the formation of biofilm biomass and cell growth in the bacterium. The synthesis and subsequent testing of the parent signalling molecules and aromatic analogues containing a longer alkyl chain is also described. A practical synthetic route to a new class of aryl sulfonamide analogues of BDSF is presented in Chapter 5. Using SwissADME and MarvinSketch, important ADME properties of a virtual library containing 16 potential aryl sulfonamides, and the pre-existing olefinic and aromatic N-acyl sulfonamides are analysed to determine their suitability as drug candidates.
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    Applications of Broadband Acoustic Resonance Dissolution Spectroscopy (BARDS) in pharmaceutical analysis
    (University College Cork, 2022-02-08) O'Mahoney, Niamh; Fitzpatrick, Dara; National University of Ireland
    Dissolution testing is one of the most time-consuming, costly, and laborious tasks in the pharmaceutical industry, and yet it is a cornerstone of quality control testing and product release. Dissolution testing is a prerequisite for the quality control and release to market of nearly every prescription and over the counter product. It is one of the few technologies which has not undergone disruption of any kind. Practices have changed little in decades, with liquid samples being taken at regular intervals over several hours and tested using expensive analytical instruments. Most routine testing can take upwards of a day to perform. Speeding up these processes is vital. This thesis highlights a modern complementary approach to existing dissolution testing practices for powder, pellet, tablet and liquid formulations called Broadband Acoustic Resonance Dissolution Spectroscopy (BARDS). BARDS is an innovative technique that can economise production processes for drug formulations. BARDS is based on reproducible changes in a solvent's compressibility as a sample dissolves. It is a rapid and straightforward method that utilises a magnetic stir bar to mix added solute and induce a vessel's acoustic resonance containing a fixed volume of solvent. As a sample is wetted and subsequently dissolved, gas is released from the solvent, altering the resonance frequency. Adding a solute to a solvent reduces the solubility of dissolved gases in solution, leading to gas oversaturation and outgassing of the solvent, changing the solvent system's compressibility, and reducing the velocity of sound in the solvent. In the results section of this thesis, Broadband Acoustic Resonance Dissolution Spectroscopy (BARDS) is used to characterise several pharmaceutical formulations, including enteric-coated microspheres, tablets and multiple-unit pellet systems (MUPS). Effervescent tablets, chewable tablets and liquid formulations were also analysed. A single replicate BARDS measurement can provide data relevant to multiple dissolution processes in a time-efficient manner by tracking the Erosion of the enteric coating, Disintegration, Deaggregation and overall Dissolution of the formulations while assessing the formulation's integrity using an EDDDI Plot. BARDS can determine the thickness of the drug and enteric coatings, characterise various dosage forms and test formulation integrity. Ultra Violet -Visable Spectroscopy (UV-Vis) has been used in the cross-validation of the technique. Tablets, pellets, and multiple-unit pellet system (MUPS) formulations were examined to investigate the effect of polymer coating and formulation core degradation over time. BARDS can enable the rapid development of solid drug formulation dissolution and disintegration testing as an In-Process and In-Line Control test and drug stability analysis. In combination with minimal Ultra Violet - Visible Spectroscopy usage, BARDS can effectively track these changes, therefore assessing a formulation's stability. BARDS data also indicates which aspect of a formulation may be unstable, whether a coating, sub-coating or core. Paediatric and geriatric formulations were studied in detail in this thesis. Chewable, effervescent and liquid dosage forms were characterised for formulation attributes using BARDS. Nutraceutical and pharmaceutical chewable and effervescent formulations were investigated in tablet form to examine how the formulation disintegrates and dissolves. The data show that a solid oral dose formulation has an intrinsic acoustic signature specific to the method of manufacture and excipient composition. Effervescent tablets disintegrate rapidly due to a chemical reaction. The reaction causes carbon dioxide gas production and subsequent release, resulting in the characteristic effervescent fizz, which can be tracked acoustically using BARDS. This thesis will highlight BARDS as a rapid characterisation technique to track the chemical reaction associated with effervescent dosage forms. BARDS can be used as an analytical tool to quantify the dissolution of liquid formulations. This has been demonstrated by a test model using two different liquid formulation types, suspension formulations and syrup formulations. Similarities between different brands were apparent when tested. BARDS can qualitatively discriminate between Active Pharmaceutical Ingredient (API) dosage, API type, and discriminate whether an API has been partially dissolved in the suspension media before addition to the BARDS instrument. In conclusion, various applications with the tremendous novelty of this platform technology have been proven. This project can potentially impact the methodology for dissolution testing with a high capability to influence regulatory policies and practices worldwide. BARDS can enable the rapid development of solid drug formulation dissolution and disintegration testing as an in-process control test and drug stability analysis. The data shows that a dosage formulation has an intrinsic acoustic signature specific to the method of manufacture, excipient composition and elapsed time since the production of a product. In addition, BARDS is a time-efficient, cost-effective and green approach to formulation characterisation.
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    Studies in the synthesis and analysis of novel heterocyclic synthetic cannabinoid receptor agonists
    (University College Cork, 2022-03-08) Alam, Ryan; Keating, J J; Eli Lilly and Company
    Over the past decade, synthetic cannabinoid receptor agonists (SCRA) have come to represent a diverse category of new psychoactive substance (NPS). However, from a chemical perspective, little is known about the clandestine chemical syntheses of SCRAs. To contextualise the latter paucity of information, this thesis introduces the pharmacological significance of the endocannabinoid system and describes the subsequent advances that have been made to develop synthetic cannabinoid-based therapeutics. The repurposing of cannabinoids that were developed as a part of legitimate drug discovery programmes, for recreational use as illicit NPS, is then discussed with a particular focus on the structural diversity, toxicological adverse effects, legislative control, and chemical characterisation of newly emerging SCRA ligands. Following the assembly of the key heterocyclic building block in the synthesis of indazole-type SCRA ligands, 1H-indazole-3-carboxylic acid, this work describes the development and application of a protocol for the regioselective (>99%) N-1 alkylation of the indazole scaffold to generate a wide range of structurally diverse N-1 substituted indazole derivatives in excellent yield (up to 99%). Additionally, the latter optimised N-alkylation protocol is also shown to regioselectively (> 99%) afford N-2 substituted indazoles. Through the variation of “tail” and “head” group motifs inspired by structural trends observed in newly emerging cannabimimetic SCRA NPS, we have generated a prophetic library of novel 1,3-disubstituted indazole-3-carboxamide derivatives. The subsequent discussion of notable spectroscopic features of these heterocyclic SCRA analogues provides further information of forensic interest. Finally, the development and optimisation of a methodology for an expedient approach to analogous, novel, 1,3-disubstituted pyrazolo[3,4-b]pyridine-3- carboxamide SCRA derivatives in high yield (up to 99%), via palladium-catalysed aminocarbonylation is described. This aminocarbonylation protocol is applied to the synthesis of isomeric 4-, 5-, 6-, and 7-azaindazole analogues of the known illicit indazole SCRA ligand, MDMB-PINACA, to facilitate their unambiguous spectroscopic differentiation and structural confirmation. The latter work provides a robust and convenient methodology for the synthesis of pyrazolopyridine-3-carboxamides and presents the first reported spectroscopic discussion of azaindazole-type SCRA NPS.
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    Hydrophilic interaction liquid chromatography for biologically important metal complex speciation and low molecular weight ligand analysis
    (University College Cork, 2022-07-14) Alsaeedi, Majidah; Glennon, Jeremy; Ministry of Higher Education of Saudi Arabia
    Metal ions play crucial roles both from a biological and an environmental perspective. They circulate around the human body by coordinating with selected biological molecules to generate stable and strong bonding to reach other organs in the body, such as the brain, liver, tissues and kidney. Therefore, understanding their forms distribution and concentration fluctuations in a biological fluids and tissues is of great interest. This research provides an overview of the roles of selected metal ions as well as their toxicity and their transportation within the human body. The significance of the labile metal pools concept and the role of low molecular weight compounds as metal chelators are discussed. Analytical techniques used for metal speciation and metallomics are reviewed, with a particular emphasis on the use and potential of hydrophilic interaction liquid chromatography (HILIC). Post chromatographic detection methods are compared including inductively coupled plasma-mass spectrometry (ICP-MS) and electrospray ionization-mass spectrometry (ESI-MS). The potential of the zwitterionic HILIC stationary phases for the fast and efficient separation of labile metal is investigated. Specifically the aqueous metal complex species distribution analysis for the low molecular weight Ni(II)-histidine was investigated at various metal-ligand ratios and as a function of pH. While selective species separation and detection was not achievable using silica bonded reversed phase or on non-bonded silica particle HILIC stationary phases, the two stepwise species, Ni(II)-His1 and Ni(II)-His2, as well as free His, were rapidly separated within 120 seconds on the Z-cHILIC phosphocholine bonded. The identities of Ni(II)-His1 and Ni(II)-His2 species were confirmed using HILIC electrospray ionization- mass spectrometry (HILIC-ESI-MS) at negative mode supported with offline analysis of collected fractions by inductively coupled plasma-mass spectrometry (ICP-MS). In addition, sensitive, rapid and efficient separation of three selected catecholamines (CAs), namely dopamine (DA), epinephrine (EPI), and norepinephrine (NE), known to be aluminium (III) and iron (III) chelators was achieved in a time of 40 seconds using a poroshell Z-HILIC column coupled to electrochemical detection based on a boron-doped diamond electrode. The applicability of the proposed method was investigated in the determination of the urinary CAs in a real urine sample following phenylboronic acid (PBA) solid-phase extraction (SPE). Al(III) and Fe(III)-CA speciation was also investigated using poroshell Z-HILIC and core-shell RP-amide columns. The chromatographic behaviours of both metal complexes are discussed. Also, the production of aminochrome forms of each CAs during the chromatographic separation was observed and carefully monitored. Attention was then placed on the separation and detection of urinary selected monoamine neurotransmitters (MNTs) including serotonin, dopamine, norepinephrine, homovanillic acid , 5-hydroxyindoleacetic acid, and vanillylmandelic acid that are of importance as biomarkers for neuroblastoma and carcinoid tumors. The chromatographic separation was carried out using the Z-cHILIC column, with isocratic elution is followed by amperometric detection at a BDD electrode. LODs between 40 to 150 nM are obtained for the MNTs. The method was applied for the analysis of the selected MNTs in urine samples following phenylboronic acid (PBA) solid-phase extraction. The research outcomes presented represent an advancement in our knowledge and application of HILIC for the separation of biologically important biological ligands and selected metal complexes of importance in metal-related health conditions such as in neurodegenerative diseases and allergy.