Carbohydrates are important biopolymers, but they have been less studied compared to proteins and nucleic acids, for example. The initial focus was to develop Capillary Zone Electrophoresis (CZE)-based techniques for the analysis of monosaccharides, oligosaccharides, polysaccharides and glycosaminoglycans (GAGs). As the project progressed and in part due to the Covid pandemic and through an external calibration with LEO Pharma (Denmark) the focus was directed more towards novel methods (such as CZE, High Performance Anion Exchange Chromatography (HPAEC), viscometry, Thermogravimetric Analysis (TGA) and Fourier-Transform Infrared Spectroscopy (FT-IR)) for the quantification of heparin in porcine mucosa and methods to follow changes in heparin structure when exposed to low and high pH at elevated temperatures. Several novel analytical methods were attempted, and findings include, a lower limit of detection (0.2 ppm) than previously published for the analysis heparin using Azure A. The effect of salt, buffer or pH on this interaction was assessed and intermediate pH values and low salt concentrations were optimal, this is important from a processing point of view. CZE was used for the separation of monosaccharides (glucose, galactose, mannose, arabinose, and xylose with linear calibration curves in each case and mixtures of up to five monosaccharides could be separated. GAGs (heparin, tinzaparin, hyaluronic acid and chondroitin sulphate) were also analysed by CZE, and the linearity of their calibration curves were assessed. When heparin was stored at pH 1 for extended periods of time (up to 800 hours), it appears that two degradation mechanisms occur. During the early stages (up to 100 hours) there is desulphation followed by depolymerisation (decrease in molecular weight) when incubated for longer periods of time. Results were similar at pH 12, although the results from TGA may suggest that changes in heparin structure are different depending on the pH of incubation. Finaly, and to the best of our knowledge, this for the first time that Azure A has been used as a simple method to determine the amount of heparin in raw mucosa (up to 140 mg/kg) and that different extraction conditions (amount of enzyme, temperature, and time of extraction) have an influence on the amount of heparin extracted, although not significantly (a = 0.05). This is a significant improvement, as heparin is usually measured in terms of activity and not in terms of mass. Overall, it is clear that simple analytical techniques, such as UV-vis with Azure A dye could be useful in the determination of heparin in raw mucosa and it could also be used as part of a regime which could be used in the estimation of any changes in heparin composition when stored/ incubated under different conditions.
| Date of Award | 6 Dec 2024 |
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| Original language | English |
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| Supervisor | Gordon Morris (Main Supervisor) & Alan Smith (Co-Supervisor) |
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