Abstracts Category : Other

Comparingdiene derivatisation methods of dry blood spot samples for vitaminD metabolites quantification by liquid chromatography-tandem massspectrometry

by Akanyang AnnahFaithful Rapholo

This dissertation describes the elucidation andimplementation of derivatisation in the quantification ofbiologically active vitamin D metabolites in limited volume serumand dry blood spot samples (DBS) using the liquid chromatographytandem-mass spectrometry (LC-MS/MS) analytical technique. Thismanuscript describes in detail the development and validation ofan analytical methodology, highlighting the role derivatisationand mass spectrometry plays in the structural characterisation andquantification of vitamin D metabolites. The first chapter reviewscomprehensively, the history of vitamin D biosynthesis discoveryas an anti-rickets agent, the biochemistry of vitamin D, itsmetabolic pathway, functions in the different biological systemsand the consequences of its deficiency in the body. The secondchapter reviews the current methods and techniques utilised forthe detection and characterization of vitamin D metabolites, withspecific emphasis based on the contribution made by derivatisationand mass spectrometry. A brief introduction to derivatisation isprovided, with specific focus on PTAD and Amplifex diene reagents(Cooksontype reagents) used in this study. The importance ofsensitivity and selectivity of targeted analytes is describedfirst in detail for underivatised analytes, followed by PTAD andAmplifex derivatised samples. Chapter 2 also describes theimportance of vitamin D quantification using liquidchromatography, the strengths and limitations of LC-MS/MS when usedin isolation and after derivatisation. Also discussed, is howcombining these techniques can overcome inherent limitations inLCMS/MS and enhance analytical performance. In Chapter 3 thematerials and methods used and the study design is laid out,describing a brief introduction of the routinely used clinicaldiagnostics assay enzyme-linked immunosorbent assay (ELISA) as areference method and is compared to an LC-MS/MS assay, toascertain discrepancies and agreement between both methodologiesfrom the same volunteer samples. Chapters 3 and 4 describes thecomprehensive development, optimisation and validation of thehighly sensitive PTAD derivatives LC-MS/MS assay for thequantification of active vitamin D metabolites, as well as thedevelopment of method using Amplifex diene derivatisation. Alsodiscussed, is sample preparation optimisation of DBS and Mitramicro-samples. A holistic approach was taken to the development ofthe methodologies to provide data from which the requiredanalytical information can be obtained for method evaluation andstatistical analysis. The validated PTAD derivatives method isapplied to the quantification of vitamin D metabolites in limitedvolume (100 L) clinical human serum samples from 30 volunteerscompared to results obtained using the clinical diagnostics ELISAtechnique. In Chapter 4 data analysis is described and the resultsare further discussed and a conclusion made based on the findingsfrom the study. This study envisaged that combination of limitedsampleAdvisors/Committee Members: Cromarty, Allan Duncan (advisor), Van Tonder, Jacob John (coadvisor).