Development of an assay for vitamin D in biological samples

Abstract

The role of vitamin D in bone health has been well established. Recent studies have provided a greater focus on the ways in which vitamin D and its metabolites have additional functions in the body that subsequently influence conditions such as common cancers, autoimmune diseases, cardiovascular diseases, depression and schizophrenia. Vitamin D is known to have more than 50 metabolites. The major circulating form in man is 25(OH)D3; having a long half-life, this compound is considered the principal biomarker therefore the best indicator of vitamin D status. Measuring 25(OH)D3 will, however, only provide an incomplete picture of vitamin D status that may or may not correlate with functions in disease. The 1,25dihydroxyvitamin D metabolites (1,25(OH)2D3 and 1,25(OH)2D2) are the most active forms that are responsible for bone health; evidence has now accumulated indicating its role in brain development. The 24,25-dihydroxyvitamin D metabolites (24,25(OH)2D3 and 24,25(OH)2D2) are responsible for bone fracture repair. The biological roles of vitamin D-sulfate compounds (D3-S, D2-S, 25(OH)D3-S and 25(OH)D2-S) are unclear to date, probably due to the lack of sufficiently sensitive assay methods to study them. It has been suggested that sulfated compounds are the storage forms of the non-conjugated metabolites,and may have similar potencies. Considering the above information, and given the diverse biological roles of vitamin D, it is important to accurately quantify as many forms as possible. Although widely used, immunoassay-based methods are not suitable for this purpose because of their inability to differentiate between some of these compounds. Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) has been the method of choice in recent years because of its high selectivity and sensitivity. This thesis describes the development and validation of an LC-MS/MS method for the quantitative analysis of 12 vitamin D compounds, specifically: D2, D3, 25(OH)D2, 25(OH)D3, 24,25(OH)2D2, 24,25(OH)2D3, 1,25(OH)2D2, 1,25(OH)2D3, D3-S, D2-S, 25(OH)D3-S and 25(OH)D2-S in biological fluids. During the initial method development, samples of human plasma and serum were compared. It was found that serum yielded higher concentrations of vitamin D compounds. Protein precipitation was compared with saponification method for extraction of vitamin D from biological fluids and found to be more effective. Vitamin D metabolites are known to have low ionisation efficiencies therefore produce weak signals in iii MS detector. Pre-column derivatisation procedure using 4-phenyl-1,2,4-triazoline-3,5-dione, (PTAD) was attempted to enhance the ionisation efficiencies of 12 vitamin D compounds. However, this method failed to detect the sulfated compounds, except for traces of 25(OH)D3-S and the derivatisation procedure proved unsuitable for sulfated compounds. The principal analytical challenge was that of extracting and chromatographing vitamin D compounds with very different lipophilicities in a single analysis procedure. The aim was therefore to o...