Test Catalog

Vitamin D Testing

The Mayo Clinic Laboratories Difference

Updated: September 2013
Published: February 2009

Vitamin D is vital for strong bones. It also has important, emerging roles in immune function and cancer prevention. Deficiencies at any stage of life can have devastating consequences. Similarly, vitamin D toxicity resulting from overmedication can cause serious hypercalcemia. Vitamin D consists of two bioequivalent forms:

  • Vitamin D2: obtained from vegetable sources (dietary sources, supplements)
  • Vitamin D3: derived from both endogenous (synthesized from cholesterol through sun exposure) and exogenous (animal diet) sources

Until recently, vitamin D replacement in the United States consisted exclusively of 25-hydroxyvitamin D2. During the last one to two years an increasing proportion of 25-hydroxyvitamin D3 replacement is being used, in particular, in high-dose prescription formulations.

Vitamin D deficiency is more common than previously believed, especially among adolescents, women, and the elderly. For example, studies have shown that more than 50% of the institutionalized elderly and an equal proportion of women of any age undergoing treatment for osteoporosis have inadequate levels of vitamin D.1 While treatment with vitamin supplementation is easy and inexpensive, many affected individuals go undiagnosed and untreated.

The total 25-hydroxyvitamin D (25-OH-VitD) level (the sum of 25-OH-vitamin D2 and 25-OH-vitamin D3) is the appropriate indicator of vitamin D body stores. Although there is no universal consensus about a treatment cut point, studies suggest 25 to 35 ng/mL as the minimal concentration of 25-OH-VitD needed to avoid the adverse effects of deficiency.2,3,4 By contrast, population reference ranges do not correspond with healthy ranges. In northern latitude locations in particular, one-third of the population may have vitamin D levels less than 25 ng/mL at the end of winter.

Patient Care

People Who Should Be Tested

The following should be tested for vitamin D deficiency:

  • Individuals who receive therapy to prevent or treat osteoporosis
  • Elderly people, especially those with minimal exposure to sunlight
  • Patients with signs and symptoms of hypocalcemia or hypercalcemia
  • Children and adults with suspected rickets and osteomalacia, respectively
  • Patients receiving vitamin D therapy who do not demonstrate clinical improvement

Patient Impact

Vitamin D deficiency testing results in potential impacts:

  • Establishes the need for replacement therapy and guides appropriate treatment, avoiding undermedicating the patient
  • Detects overtreatment, which is rare—occurring only with long-term replacement at doses of almost 10,000 U/day or more

Benefits of Testing

Vitamin D deficiency testing provides:

  • Reliable assessment of vitamin D stores

Technical Comparison

Ligand-Binding Assays

Technical aspects of ligand-binding assays (that is, radioimmunoassay and competitive protein-binding assays) include:

  • There is fundamental difficulty because such assays work best in an aqueous environment, and 25-OH-VitD2 and 25-OH-VitD3 are poorly soluble in water.
    • Manual extraction assays can overcome solubility problems but suffer from increased imprecision.
  • Lack of result comparability occurs between different assays and often between the same assays performed in different laboratories:5
    • A comparison of six laboratories found that "whether a patient is considered as being vitamin D insufficient depends, in large measure, on the laboratory used."4
  • Some assays significantly overestimate vitamin D levels; others significantly underestimate them:
    • A study conducted by the Vitamin D External Quality Assessment Scheme (DEQAS) showed a 31% overestimation by one immunoassay method.6
    • More typically, underestimation is observed. This is particularly the case in patients on vitamin D2 therapy; some immunoassays cannot detect 25-OH-VitD2.
  • Ligand-binding assays cannot distinguish between 25-OH-VitD2 and 25-OH-VitD3. Therefore, it is difficult to judge whether patients are compliant with therapies or suffer from vitamin D malabsorption.

High-Performance Liquid Chromatography (HPLC)

Technical aspects of HPLC include:

  • Overcomes most of the problems of the ligand-binding assays, and previously seen as the "gold standard"
  • Requires a relatively large sample volume (≥1.0 mL)
  • Labor-intensive and time-consuming

Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)

Technical aspects of LC-MS/MS include:

  • Accurate and precise, due to internal standard and physicochemical methodology
    • Is the "gold standard" method
    • Uses standards of defined concentrations
    • Facilitates lab-to-lab and assay-to-assay comparisons with other MS/MS and HPLC assays
    • Uses results to determine whether target therapeutic levels (25-80 ng/mL) have been obtained
  • Not subject to lot-to-lot variation, reagent availability issues, or licensing fees associated with proprietary kits
  • Is faster than HPLC
  • Separates 25-OH-VitD2 and 25-OH-VitD3. In patients who show no improvement on replacement therapy (VitD2), absence of rise in 25-OH-VitD2 may indicate noncompliance or malabsorption, whereas rise in 25-OH-VitD2 and total vitamin D levels may indicate vitamin D resistance.
  • Uses a smaller specimen volume than HPLC (0.25 mL)

How the Mayo Clinic Test Differs

The Mayo Clinic vitamin D deficiency test offers key differentiators:

  • Mayo Clinic-developed and Mayo Clinic-validated LC-MS/MS assay
  • Requires fewer repeat analyses as a result of a wider reportable range
  • Provides 25-OH-VitD2 and 25-OH-VitD3 levels in addition to the total vitamin D level. This allows assessment of the source of the deficiency and also facilitates treatment monitoring.
  • The total 25-OH-VitD measured with the new assay correlates well with our previous methodology, which in itself has been shown to outperform other laboratories and tests.
  • Improves on accuracy and precision of ligand-binding assays, including our previous assay: approximately 10% CV at low 25-OH-VitD levels (10 ng/mL).7


1. Holick MF, Siris ES, Binkley N, et al: Prevalence of vitamin D inadequacy among postmenopausal North American women receiving osteoporosis therapy. J Clin Endocrinol Metab. 2005;90(6):3215-3224

2. Heaney RP: Vitamin D: how much do we need, and how much is too much? Osteoporos Int. 2000;11:553-555

3. Chapuy MC, Preziosi P, Maamer M, et al: Prevalence of vitamin D insufficiency in an adult normal population. Osteoporos Int. 1997;7:439-443

4. Binkley N, Krueger D, Cowgill CS, et al: Assay variation confounds the diagnosis of hypo-vitaminosis D: a call for standardization. J Clin Endocrinol Metab. 2004;89(7):3152-3157

5. Singh, R: Are clinical laboratories prepared for accurate testing of 25-hydroxy vitamin D? Clin Chem. 2008;54(1):222-223

6. Graham DC, Carter R, Jones J, Berry J: How accurate are assays for 25-hydroxyvitamin D? Data from the International Vitamin D External Quality Assessment Scheme. Clin Chem. 2004;50:2195-2197

7. Mayo unpublished quality control data