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 1,25-dihydroxyvitamin 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 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 overcome this challenge, and to simplify the method to be suitable for routine analysis. The compounds were extracted using online solid phase extraction (SPE) following serum protein precipitation with acetonitrile (without derivatisation). Pentafluorophenyl (PFP) stationary phase, used as the guard column / SPE trapping device, and as the analytical column enabled the extraction and separation of all 12 compounds in a single procedure. Both the extraction step and the separation step were optimised including the chromatographic separation of 25(OH)D3 from the inactive isomer 3-epi-25(OH)D3. The effects of mobile phase pH on separation and the effects of precipitant solvent on the protein precipitation efficiency were also investigated.Detection limits for all 12 compounds were in the picomole range when using a 500 µL sample volume. Recovery percentages ranged from 92% to 99%. A stable isotope labelled-internal standard (SIL-IS) for each of the 12 vitamin D compounds was used as the co-eluting internal standard to correct for matrix effects in the MS detector as well as to correct for the procedural errors during sample extraction and clean up. In order to demonstrate the applicability of the proposed method, the validated method was then applied to assay for vitamin D compounds in mouse brain samples and donations of human serum samples collected from volunteers.Although the method developed and validated was simple, sensitive and accurate and amenable for the routine assay of 12 vitamin D compounds in serum, it was expensive to develop and implement because of the SIL-IS compounds required for each of the 12 vitamin D compounds. A standard addition method of calibration, instead of using SIL-IS, was proposed to overcome the matrix effects in MS detection. Since the conventional standard addition procedure does not address procedural errors, an additional internal standard (not co-eluting) was included in the proposed method. The accuracy of this improved standard addition method was validated through the determination of recovery, and by comparison with existing method SIL-IS method. Recoveries determined on human serum samples showed that the proposed method of standard addition yielded more accurate results than internal standardisation using SIL-IS. The precision of the proposed method of standard addition also proved superior to the conventional method of standard addition.