Comparing diene derivatisation methods of dry blood spot samples for vitamin D metabolites quantification by liquid chromatography-tandem mass spectrometry
Year: 2017
Type: dissertation
Abstract: This dissertation describes the elucidation and implementation of derivatisation \nin the quantification of biologically active vitamin D metabolites in limited volume \nserum and dry blood spot samples (DBS) using the liquid chromatography \ntandem-mass spectrometry (LC-MS/MS) analytical technique. This manuscript \ndescribes in detail the development and validation of an analytical methodology, \nhighlighting the role derivatisation and mass spectrometry plays in the structural \ncharacterisation and quantification of vitamin D metabolites. \nThe first chapter reviews comprehensively, the history of vitamin D biosynthesis \ndiscovery as an anti-rickets agent, the biochemistry of vitamin D, its metabolic \npathway, functions in the different biological systems and the consequences of \nits deficiency in the body. The second chapter reviews the current methods and \ntechniques utilised for the detection and characterization of vitamin D \nmetabolites, with specific emphasis based on the contribution made by \nderivatisation and mass spectrometry. A brief introduction to derivatisation is \nprovided, with specific focus on PTAD and Amplifex diene reagents (Cooksontype \nreagents) used in this study. The importance of sensitivity and selectivity of \ntargeted analytes is described first in detail for underivatised analytes, followed \nby PTAD and Amplifex derivatised samples. Chapter 2 also describes the importance of vitamin D quantification using liquid \nchromatography, the strengths and limitations of LC-MS/MS when used in \nisolation and after derivatisation. Also discussed, is how combining these \ntechniques can overcome inherent limitations in LCMS/MS and enhance \nanalytical performance. In Chapter 3 the materials and methods used and the \nstudy design is laid out, describing a brief introduction of the routinely used clinical \ndiagnostics assay enzyme-linked immunosorbent assay (ELISA) as a reference \nmethod and is compared to an LC-MS/MS assay, to ascertain discrepancies and \nagreement between both methodologies from the same volunteer samples. Chapters 3 and 4 describes the comprehensive development, optimisation and \nvalidation of the highly sensitive PTAD derivatives LC-MS/MS assay for the \nquantification of active vitamin D metabolites, as well as the development of \nmethod using Amplifex diene derivatisation. Also discussed, is sample preparation optimisation of DBS and Mitra micro-samples. A holistic approach \nwas taken to the development of the methodologies to provide data from which \nthe required analytical information can be obtained for method evaluation and \nstatistical analysis. The validated PTAD derivatives method is applied to the \nquantification of vitamin D metabolites in limited volume (100 μL) clinical human \nserum samples from 30 volunteers compared to results obtained using the clinical \ndiagnostics ELISA technique. \nIn Chapter 4 data analysis is described and the results are further discussed and \na conclusion made based on the findings from the study. This study envisaged \nthat combination of limited sample volume and DBS, derivatisation and LCMS/ \nMS is a powerful tool in vitamin D metabolite analysis and provided evidence \nof a positive increase in sensitivity and selectivity between derivatised compared \nto underivatised samples. A 10-fold increase in signal-to-noise-ratio (S/N) was \nobserved when comparing PTAD derivatised, and Amplifex diene derivatised \nversus underivatised samples. \nChapter 5 presents suggested future directions and considerations in the areas \nof vitamin D metabolite derivatisation and DBS sampling technique analysis using \nLC-MS/MS research based on the results presented in this dissertation.
Author: Akanyang Annah Faithful Rapholo
Language: English
Subfield: Analytical Chemistry
Field: Chemistry
Domain: Physical Sciences
Open Access status: green
OpenAlex ID: W2767521567