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Comparing diene derivatisation methods of dry blood spot samples for vitamin D metabolites quantification by liquid chromatography-tandem mass spectrometry
Dissertation (MSc)--University of Pretoria, 2017.
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| Format: | Thesis |
| Language: | English |
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University of Pretoria
2017
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| _version_ | 1867613442414739456 |
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| access_status_str | Open Access |
| author2 | Cromarty, Allan Duncan |
| author_browse | Cromarty, Allan Duncan |
| author_facet | Cromarty, Allan Duncan |
| collection | Thesis |
| dc_rights_str_mv | © 2017 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. |
| description | Dissertation (MSc)--University of Pretoria, 2017. |
| format | Thesis |
| id | oai:repository.up.ac.za:2263/63038 |
| institution | University of Pretoria (South Africa) |
| language | English |
| last_indexed | 2026-06-10T12:36:12.984Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UPSpace — University of Pretoria Institutional Repository |
| publishDate | 2017 |
| publishDateRange | 2017 |
| publishDateSort | 2017 |
| publisher | University of Pretoria |
| publisherStr | University of Pretoria |
| record_format | dspace |
| source_str | UPSpace — University of Pretoria Institutional Repository |
| spelling | oai:repository.up.ac.za:2263/63038 Comparing diene derivatisation methods of dry blood spot samples for vitamin D metabolites quantification by liquid chromatography-tandem mass spectrometry Cromarty, Allan Duncan akanyangrapholo@yahoo.com Van Tonder, Jacob John Rapholo, Akanyang Annah Faithful Amplifex diene Clinical diagnostics Derivatisation DBS samples ELISA LC-MS/MS Limited volume samples Mass spectrometry PTAD Vitamin D metabolites Health sciences theses SDG-03 Health sciences theses SDG-17 Dissertation (MSc)--University of Pretoria, 2017. This dissertation describes the elucidation and implementation of derivatisation in the quantification of biologically active vitamin D metabolites in limited volume serum and dry blood spot samples (DBS) using the liquid chromatography tandem-mass spectrometry (LC-MS/MS) analytical technique. This manuscript describes in detail the development and validation of an analytical methodology, highlighting the role derivatisation and mass spectrometry plays in the structural characterisation and quantification of vitamin D metabolites. The first chapter reviews comprehensively, the history of vitamin D biosynthesis discovery as an anti-rickets agent, the biochemistry of vitamin D, its metabolic pathway, functions in the different biological systems and the consequences of its deficiency in the body. The second chapter reviews the current methods and techniques utilised for the detection and characterization of vitamin D metabolites, with specific emphasis based on the contribution made by derivatisation and mass spectrometry. A brief introduction to derivatisation is provided, with specific focus on PTAD and Amplifex diene reagents (Cooksontype reagents) used in this study. The importance of sensitivity and selectivity of targeted analytes is described first in detail for underivatised analytes, followed by PTAD and Amplifex derivatised samples. Chapter 2 also describes the importance of vitamin D quantification using liquid chromatography, the strengths and limitations of LC-MS/MS when used in isolation and after derivatisation. Also discussed, is how combining these techniques can overcome inherent limitations in LCMS/MS and enhance analytical performance. In Chapter 3 the materials and methods used and the study design is laid out, describing a brief introduction of the routinely used clinical diagnostics assay enzyme-linked immunosorbent assay (ELISA) as a reference method and is compared to an LC-MS/MS assay, to ascertain discrepancies and agreement between both methodologies from the same volunteer samples. Chapters 3 and 4 describes the comprehensive development, optimisation and validation of the highly sensitive PTAD derivatives LC-MS/MS assay for the quantification of active vitamin D metabolites, as well as the development of method using Amplifex diene derivatisation. Also discussed, is sample preparation optimisation of DBS and Mitra micro-samples. A holistic approach was taken to the development of the methodologies to provide data from which the required analytical information can be obtained for method evaluation and statistical analysis. The validated PTAD derivatives method is applied to the quantification of vitamin D metabolites in limited volume (100 μL) clinical human serum samples from 30 volunteers compared to results obtained using the clinical diagnostics ELISA technique. In Chapter 4 data analysis is described and the results are further discussed and a conclusion made based on the findings from the study. This study envisaged that combination of limited sample volume and DBS, derivatisation and LCMS/ MS is a powerful tool in vitamin D metabolite analysis and provided evidence of a positive increase in sensitivity and selectivity between derivatised compared to underivatised samples. A 10-fold increase in signal-to-noise-ratio (S/N) was observed when comparing PTAD derivatised, and Amplifex diene derivatised versus underivatised samples. Chapter 5 presents suggested future directions and considerations in the areas of vitamin D metabolite derivatisation and DBS sampling technique analysis using LC-MS/MS research based on the results presented in this dissertation. em2026 Pharmacology MSc Unrestricted SDG-03: Good health and well-being SDG-17: Partnerships for the goals 2017-11-07T07:37:57Z 2017-11-07T07:37:57Z 2017 2017 Dissertation Rapholo, AAF 2017, Comparing diene derivatisation methods of dry blood spot samples for vitamin D metabolites quantification by liquid chromatography-tandem mass spectrometry, MSc Dissertation, University of Pretoria, Pretoria, viewed yymmdd <http://hdl.handle.net/2263/63038> http://hdl.handle.net/2263/63038 en © 2017 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. application/pdf University of Pretoria |
| spellingShingle | Amplifex diene Clinical diagnostics Derivatisation DBS samples ELISA LC-MS/MS Limited volume samples Mass spectrometry PTAD Vitamin D metabolites Health sciences theses SDG-03 Health sciences theses SDG-17 Comparing diene derivatisation methods of dry blood spot samples for vitamin D metabolites quantification by liquid chromatography-tandem mass spectrometry |
| title | Comparing diene derivatisation methods of dry blood spot samples for vitamin D metabolites quantification by liquid chromatography-tandem mass spectrometry |
| title_full | Comparing diene derivatisation methods of dry blood spot samples for vitamin D metabolites quantification by liquid chromatography-tandem mass spectrometry |
| title_fullStr | Comparing diene derivatisation methods of dry blood spot samples for vitamin D metabolites quantification by liquid chromatography-tandem mass spectrometry |
| title_full_unstemmed | Comparing diene derivatisation methods of dry blood spot samples for vitamin D metabolites quantification by liquid chromatography-tandem mass spectrometry |
| title_short | Comparing diene derivatisation methods of dry blood spot samples for vitamin D metabolites quantification by liquid chromatography-tandem mass spectrometry |
| title_sort | comparing diene derivatisation methods of dry blood spot samples for vitamin d metabolites quantification by liquid chromatography tandem mass spectrometry |
| topic | Amplifex diene Clinical diagnostics Derivatisation DBS samples ELISA LC-MS/MS Limited volume samples Mass spectrometry PTAD Vitamin D metabolites Health sciences theses SDG-03 Health sciences theses SDG-17 |
| url | http://hdl.handle.net/2263/63038 |