Vitamin D plays a vital role in skeletal metabolism, calcium homeostasis, [1
] and also in the functioning of the immune, cardiovascular, and reproductive systems [4
]. Vitamin D deficiency leads to rickets and osteomalacia and is also associated with breast and colorectal cancers, multiple sclerosis, dementia, rheumatoid arthritis, diabetes, Parkinson's and Alzheimer's diseases [6
]. Despite numerous reports, the associations of Vitamin D deficiency with health and diseases are subject to debate, partly owing to inadequacies in current approaches to measurement of serum levels. Depending on the source, Vitamin D is produced in two forms: Vitamin D2 and Vitamin D3, which differ by the presence of a double bond and methyl group on the aliphatic side chain. The issues involved in assessing Vitamin D status arise from the complexities of the metabolic pathways leading to a number of active forms. The complex metabolic pathway for Vitamin D3 is summarized in Figure .
Vitamin D3 is formed from its precursor 7-dehydrocholesterol in the skin by ultraviolet B light (medium wavelength, 290-315 nm) and Vitamin D2 originates from dietary sources together with some fraction of D3. In the liver, Vitamins D3 and D2 undergo hydroxylation reactions catalyzed by 25-hydroxylase, which leads to the formation of pharmacologically active metabolites 25OHD3 and 25OHD2 respectively (collectively termed as 25OHD). Further metabolism (in the presence of 1α,hydroxylase) in the kidney produces the pharmacologically active metabolites 1-alpha,25-dihydroxyvitamin-D3 (1α,25(OH)2
D2) and 1-alpha,25-dihydroxyvitamin-D2 (1α,25(OH)2
D3) along with the minor metabolite 24,25(OH)2
Since 25OHD has significant effects on health and wellbeing, there has been a substantial interest in improving the relevant analytical techniques [11
]. Owing to a long serum half-life, measurement of total 25OHD (25OHD2 and 25OHD3) is the routinely used approach for assessing the total circulating Vitamin D status [10
]. In immunoassay techniques, a measure of total metabolite concentration and equivalent detection of both 25OHD2 and 25OHD3 is challenging, as binding proteins show a higher affinity for 25OHD3 than 25OHD2 [15
]. Reports have shown inter-laboratory and inter-method variations in results for Vitamin D determinations [19
LC-MS/MS is currently the best technique available for the correct quantification of 25OHD3 and 25OHD2 [22
] and it also has the capability to overcome most of the problems associated with protein binding assays. LC-MS/MS is a more favourable technique because sample derivatisation is not required, run time is very short and an internal standard is used which usually compensates for any matrix related and instrumental effects [24
However, the LC-MS/MS approach is also subject to interferences [33
]. Along with matrix related, instrumental and analytical interferences, endogenous 25OHD determinations have also been shown to suffer from epimeric and isobaric interferences [38
]. Epimers are non-super imposable (or non mirror images) that only differ in the configuration at one carbon atom (Figure ). Epimers and isobars are compounds with the same molecular weight as Vitamin D metabolites and form the same mass to charge parent and product ion pairs upon ionisation. Moreover, the separation of interfering epimers and isobars is also essential, because they can overlap chromatographically with Vitamin D metabolites or internal standard peaks and give false estimates of true Vitamin D levels. 25OHD3 is the most abundant Vitamin D metabolite in circulation and 3-epi-25OHD3 is the most prevalent epimer of 25OHD3. There are two compounds known to cause isobaric interferences in 25OHD analysis; 1α-hydroxyvitamin-D3 (1αOHD3), which is an exogenous pharmaceutical compound and 7α-hydroxy-4-cholestene-3-one (7αC4), which is an endogenous bile acid precursor [30
Figure 2 Epimerisation and metabolic pathways for Vitamin D3 metabolites. [adapted from reference ].
The epimerization of 25OHD3 and 1α,25-(OH)2
D3 results in the formation of 3-epi-25OHD3 and 3-epi-1α,25(OH)2
D3 epimers respectively as shown in Figure . The epimers of 25OHD differ in configuration at third carbon atom (C-3) (shown by dashed highlights in Figure ) that is attached to a hydroxyl group. Hydroxylation of 3-epi-25OHD3 forms 3-epi-1α,25(OH)2
The aim of this study was to develop a novel LC-MS/MS method that can accurately identify and quantitate 25OHD3 and 25OHD2 and chromatographically separate epimers and isobars.