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

近年來，智慧型手機成長快速，根據全球市場研究機構TRENDFORCE調查顯示，2013年第四季全球智慧型手機出貨季成長6.5%，達2億六千五百萬支，與去年同期相比成長32.2%。其中小米科技在競爭激烈的市場達成了全球市佔率3%的成績，同時被預估2014年手機出貨上看4,000萬支，取得空前成功。基於此，本研究試圖從使用者參與、口碑行銷與傳遞，以及被廣泛稱為「飢餓行銷」的限量銷售的手法等三個層面對小米所採取的創新作法進行探討，試圖瞭解小米如何在創立至今四年多的時間達成其餘競爭廠商望其相背的成就。 研究結果發現，小米科技發展初期，尚未發行手機硬體前，口碑的形成源自消費者於論壇討論區大量參與MIUI開發與改進所引起關於服務品質、滿意度以及忠誠度的提升。在手機產品上市後，小米科技除了以較競爭廠商更具性能價格比優勢的手機產品吸引消費者外，亦持續透過論壇與服務經營消費者關係，增進了消費者對於品牌口碑傳遞的意願。本研究透過問卷與訪談發現，在小米口碑傳遞內容部分多為其產品之高性能價格比。對於性能價格比優勢，小米科技則是透過限量的搶購模式有效延長產品話題熱度與生命週期，利用常態性關鍵零組件價格走勢降低製造成本，並以手機產品普及後所帶來之服務收入作為其主要營運獲利模式。 本研究亦建議後續研究可針對限量銷售模式、使用者參與程度與口碑內容的影響等方向進一步進行探討，進而對行銷實務上有所貢獻。 / In recent years, a rapid growth in smart-phone usage is observed. According to a study by market research agency, Trendforce, global smart-phone shipment grew at a pace of 6.5% in fourth quarter of 2013 and reached 260 million units. Chinese brand, Xiaomi, alone captured 3% of the global market share during the most competitive period of smart-phone business. It is expected that its 2014 shipment volume will reach 40 million units. Due to this remarkable achievement, this study will analyze the brand from the perspective of end user behavior, word of mouth marketing and the so called "hunger marketing" where limited editions are used, to explore the success of Xiaomi's strategy and its position in the global market within a four year span. The research result indicates that in the early stage of Xiaomi's establishment, prior to their involvement in hardware, the awareness were already establishing from forum discussion of service quality, consumer satisfaction and loyalty, and the development of MIUI. When the hardware eventually launched, Xiaomi's selling point came not only from its low cost strategy, but they continued to participate in forum studies in consumer service level and naturally created brand viral. Consumer market research shows that leading awareness focuses on Xiaomi's high performance/cost ratio. To build on this advantage, Xiaomi uses a limited volume strategy to create brand viral and prolongs product life cycle. It takes advantage of the declining price trend of key components to lower manufacturing costs and income from the software services to enhance operating profit. Further study is recommended on the benefits and results of limited volume strategy, user participation and word of mouth marketing.