Many studies have demonstrated that hypovitaminosis D is widespread through the Western world leading to postulations that it is associated with the development and/or progression of a number of conditions ranging from neurological disorders through to hypertension, type 1 diabetes, kidney disease and cystic fibrosis
. In consequence, many supplemental studies have been conducted with very few successfully altering disease outcomes
. Beyond rickets disease, clinical research on the putative roles of vitamin D in health and disease has proven difficult for a number of reasons including: i) the vitamin exists in multiple forms that are not facile to measure accurately, with considerable questions about several methods
, ii) sources are varied with sunlight generating the primary form of vitamin D3 in the skin and both vitamin D3 and vitamin D2 are available through diet, making supplemental studies very difficult to control, and iii) although studies are conducted globally, there is no worldwide consensus on key quantitative measures of deficiency, insufficiency and sufficiency of vitamin D in blood samples
Vitamin D exists in several forms ranging from the dietary derived D2 and photo-activated D3 analogues, although a fraction of vitamin D3 also arises from the diet. Hydroxylation in the liver affords the circulating 25-hydroxyvitamin-D3 (25OHD3) and 25-hydroxyvitamin-D2 (25OHD2) forms, frequently measured collectively as 25OHD. Additional metabolism in the kidney generates the active metabolites 1-alpha,25-dihydroxyvitamin-D3, (1α25(OH)2D3) and 1-alpha,25-dihydroxyvitamin-D2 (1α25(OH)2D2) along with the minor metabolite 24,25(OH)2D3
[4–6]. Recent efforts have resulted in significant improvements in the understanding of limitation and strengths of various assays for vitamin D forms
Further confounding factors arise as recent studies have revealed the presence of the C-epimer form of 25OHD3 in adult blood samples
, which was subsequently quantified in further studies
[3, 12, 13]. The role of vitamin D epimers is yet to be elucidated but their presence is likely to have an associated function
. Given that the epimers can hamper current assays by cross-reacting, and that they may have biological functions, it is timely to develop an accurate method to analyse all forms of vitamin D, including accurate levels of the epimers. Similar efforts to date resulted in assays quantifying up to five analogues in various combinations
[15–18] but none included the primary forms of vitamin D, 3-epi-25OHD2 or 1α25(OH)2D2.
This paucity of advances, despite the existence of numerous reports, led us to develop a new assay for the simultaneous analysis of eight key analogues of vitamin D including two epimers. The aim of this paper is to report the validated assay along with its application to reference samples to quantify the levels of various analogues of vitamin D.