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. / Dissertation (MSc)--University of Pretoria, 2017. / Pharmacology / MSc / Unrestricted
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:up/oai:repository.up.ac.za:2263/63038 |
| Date | January 2017 |
| Creators | Rapholo, Akanyang Annah Faithful |
| Contributors | Cromarty, Allan Duncan, akanyangrapholo@yahoo.com, Van Tonder, Jacob John |
| Publisher | University of Pretoria |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Dissertation |
| Rights | © 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. |
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