Types of vitamin D test

A vitamin D test measures one of the two metabolites to assess serum vitamin D levels:
  • 25-hydroxyvitamin D (25-OHD)
  • 1,25-dihydroxyvitamin D

Clinically, levels of 25-OHD are measured to determine vitamin D status as it is a more reliable metabolite. There are two different assays1,2 for measuring serum levels of 25-OHD:

  • Liquid chromatography-tandem mass spectrometry
    Sensitive and specific method that is referred to as a ‘gold standard’ test
    Can be slow and requires expensive equipment and skilled staff
  • Commercial immunoassays either using radioactive markers or chemical markers
    Less sensitive as they do not distinguish between the two metabolites of vitamin D
    Cheaper and quicker to conduct (about half the cost of liquid chromatography)

Limitations of vitamin D immunoassays

In the past different immunoassays were reported to yield vastly different results, with inter-assay variation reaching up to 25% at low serum 25-OHD levels (15 nmol/L).3 The accuracy of different immunoassays for measurement of 25-OHD may become more consistent with new reference standards. However, the bias and imprecision of many automated methods may be problematic at the lower, clinically and analytically important range (< 50 nmol/L) of the assay.4

Optimal vitamin D levels

There is debate about the optimal level of serum 25-OHD. Values anywhere between 50 and 110 nmol/L are advocated. It is likely that higher serum 25-OHD levels prevent some disease states, but there is insufficient evidence to recommend targets higher than 75 nmol/L.4

Table 1. Recommended serum concentrations of 25-OHD4
Vitamin D status
Serum 25-OHD concentrations (nmol/L)
≥ 50
Mild deficiency
Moderate deficiency
Severe deficiency
< 12.5

For more information

  1. Lai JK, Lucas RM, Clements MS, et al. Assessing vitamin D status: pitfalls for the unwary. Mol Nutr Food Res 2010;54:1062–71. [PubMed]
  2. Farrell CJ, Martin S, McWhinney B, et al. State-of-the-art vitamin D assays: a comparison of automated immunoassays with liquid chromatography-tandem mass spectrometry methods. Clin Chem 2012;58:531–42.[PubMed]
  3. Lips P. Relative value of 25(OH)D and 1,25(OH)2D measurements. J Bone Miner Res 2007;22:1668-71. [PubMed]
  4. Nowson CA, McGrath JJ, Ebeling PR, et al. Vitamin D and health in adults in Australia and New Zealand: a position statement. Med J Aust 2012;196:686–7.[PubMed]