Vitamin D testing for medical laboratories

Vitamin D is a common deficiency test performed in a clinical lab setting due to its importance to bone and mineral metabolism and the belief that vitamin D is effective in the prevention and treatment of rickets and osteomalacia.1 Studies have also revealed that higher vitamin D levels are associated with a decreased incidence of malignancies and cancers.2 Vitamin D deficiency has been linked in studies to an increased risk for diseases including autoimmune diseases, type 1 and type 2 diabetes, rheumatoid arthritis, and more.3 There has also been controversy where studies have determined that it isn’t very helpful for most people to know their vitamin D levels and scant evidence that taking a vitamin D supplement would be of any benefit to the patient. The Endocrine Society still recommends regular vitamin D screening for individuals at risk for deficiency. The development of updated testing protocols enables laboratorians to give the providers the most up-to-date information for their patients.

Vitamin D2 and vitamin D3

Vitamin D represents a family of molecules including the two main isoforms vitamin D3 (cholecalciferol) and vitamin D2 (ergocalciferol). Vitamin D3 is the more common form of vitamin D and is produced by the body with sunlight exposed skin causing the conversion of 7-dehydrocholesterol to cholecalciferol. Vitamin D2 is derived from fungal and plant sources and is in most vitamin supplement preparations. Testing for vitamin D isoforms have developed over recent years.

Old testing methods were not able to distinguish between the D2 and D3 forms of the vitamin and were only able to report the total result. Newer methods such as liquid chromatography-tandem mass spectrometry (LC-MS/MS), however, can report levels of both D2 and D3 and then add them together for a total level as a panel. Both vitamin D2 and vitamin D3 are converted to 25-hydroxyvitamin D in the liver, which is one of the active forms on which testing is performed.

Testing for vitamin D

There are two forms of activated vitamin D for testing performed:

  • 25-hydroxyvitamin D [25(OH)D]/calcidiol: The most abundant circulating form of vitamin D and the most common measure of serum levels.4
  • 1,25-dihydroxyvitamin D [1,25(OH)2D]/calcitriol: Although the most metabolically active form, circulating 1,25(OH)2D is generally not considered to be a reliable measurement of vitamin D due to its very short half-life.4

Vitamin D status is usually assessed by measuring the serum 25-hydroxyvitamin D (25(OH)D) concentration. There has been a dramatic increase in 25-OHD requests over recent years prompting many labs to consider the use of automated immunoassays.

Labs have also developed their own methods, such as liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), to measure vitamin D.5 Clinical lab professionals observed that some vitamin D assays from commercial sources and platforms have occasionally produced inconsistent results from the identical specimens. In some instances, these differences were large enough to affect whether a patient would be classified as having sufficient or deficient vitamin D levels.5

Vitamin D Standardization Program

To correct these disparities, the Vitamin D Standardization Program (VDSP), an initiative of the National Institutes of Health Office of Dietary Supplements (NIH ODS), was launched in 2010 in collaboration with the National Institutes of Health (NIH), the Centers for Disease Control and Prevention (CDC), the National Institute for Standards and Technology (NIST), and Ghent University in Belgium. The VDSP goals are to standardize vitamin D measurements in national health surveys worldwide, promote standardized 25- hydroxyvitamin measurements for assay manufacturers, and conduct an international research program devoted to 25(OH)D and its lab measurement. Critical factors in establishing successful standardization programs include minimizing matrix effects in the standard reference material that may lead to spurious results and maintaining commutability of standards across all manufacturers and all methods.6 Current testing in clinical labs include multiple different methodologies. 

Vitamin D assays

Assays currently available in the market (U.S. and EU) can be classified as binding assays and chemical assays. Methodologies for 25(OH)D measurements include high performance liquid chromatography (HPLC), radioimmunoassay (RIA), automated immunoassays, and LC-MS/MS.7

Chemiluminescence immunoassays (CLIA), radioimmunoassay (RIA), and binding protein assays belong to the binding assays group, while chemical assays include high-performance liquid chromatography (HPLC) and LC-MS/MS. The specificity and accuracy of these methods can be somewhat variable. Both RIA and CLIA are immunoassays, in which the accuracy of the method will depend on the specificity of the antibody used (how well the antibody recognizes D2 and D3). The binding assays are affected by the matrix effects due to the tight binding of the vitamin D-binding protein to vitamin D. Automated immunoassays are the most popular due to the relative low cost, availability, using small sample  volume, rapid turnaround, and ease of use. 

The first automated vitamin D assay developed was based on Competitive-Protein Binding Assays (CPBA) for the Nichols Advantage analyzer. It has the advantages of being inexpensive, can be performed on small sample size, and is co-specific for 25-OH-D2 and 25-OH-D3.7 This assay underestimated 25-OH-D at low levels and overestimated it at high levels. Immunoassay methods were first reported in the 1980s with RIA. This assay formed the basis for a subsequent chemiluminescent detection-based system. The RIA requires a small sample size and the incorporation of iodine-125 as a tracer. It is not subjected to nonspecific interference, and in addition to being rapid it is inexpensive and accurate.7  

However, it still requires the use of radionuclides, and some RIA assays discriminate between 25-OH-D2 and 25-OH-D3. The challenges with vitamin D immunoassays include demonstrated accuracy and specificity and the recognition of fluctuations of assay performance.

Chemical assays have traditionally been more technically involved but are now able to accommodate a higher number of tests per workday. Chemical methods (HPLC and LC-MS/MS) can report vitamin D2 and D3 independently.8 Ultraviolet quantitation following HPLC is a very stable, repeatable assay, and provides separate quantitation of 25-OH-D2 and 25-OH-D3.

Nevertheless, it requires a larger sample size, needs a preparation step before chromatography and sometimes is subject to interferences with other compounds measured in the ultraviolet spectrum. This assay also requires a high level of technical expertise.8

Isotope dilution LC-MS/MS is currently considered the most desirable method for 25OHD measurement, being able to simultaneously quantitate 25OHD2 and 25OHD3, with summation of the two values resulting in total 25OHD.9 A criticism of LC-MS/MS is that there is a multitude of ‘‘home-brew’’ or ‘‘in-house’’ methods available using different sample preparation and extraction methods, varying running conditions and buffers, different HPLC systems, and multiple MS detection systems which utilize different transitions for each  molecule of interest.9 

Blood vs. hair testing

Currently, the best method to measure the presence of vitamin D is blood. However, there is a recent study regarding extraction and determination of vitamin D concentration from human hair.10 This method has the capability to account for the high variability of 25(OH)D3 concentration and could capture a large seasonal difference, since hair only grows approximately one cm per month. Whereas blood can only account for a snapshot of vitamin D and is not able to provide information of vitamin D year-round. These findings potentially present a new approach to epidemiological studies relating vitamin D to bone and non-bone related medical conditions which have been associated with its deficiency.10


In conclusion, vitamin D is a compound with some controversial methods and studies on both sides of the spectrum touting the benefits of routine testing. Coupled with the disparity and consistency between methodologies, what remains a concern is how to introduce well-standardized assays in clinical labs in the coming years. More labs are moving to the LC-MS/MS technology with potential greater specificity and accuracy of measurement. However, it is clear that a considerable bias still exists between methods even though the re-standardized assays are now traceable to the National Institute of Standards and Technology Standard Reference Material 2972 (NIST SRM 2972).


  1. Pilz S, Zittermann A, Trummer C, et al. Vitamin D testing and treatment: a narrative review of current evidence. Endocr Connect. 2019;8(2):R27-R43.
  2. Souberbielle JC, Body JJ, Lappe JM, et al. Vitamin D and musculoskeletal health, cardiovascular disease, autoimmunity and cancer: Recommendations for clinical practice. Autoimmune Rev. 2010 Sep;9(11):709-15.
  3. Holick MF, Binkley NC, Bischoff-Ferrari HA, et al. Evaluation, treatment, and prevention of vitamin D deficiency; an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab. 2011 Jul;96(7):1-20. 
  4. Regence (2019, January 1). Medical Policy Manual, Laboratory Policy no. 52. Vitamin D Testing. Retrieved from
  5. Freeman J, Wilson, K. (2013, August 1). Vitamin D Progress Towards Standardization. Retrieved from
  6. National Institutes of Health. Vitamin D Standardization Program (VDSP). Retrieved from
  7. Galior K, Ketha H, Grebe S, Singh RJ. 10 years of 25-hydroxyvitamin-D testing by LC-MS/MS-trends in vitamin-D deficiency and sufficiency. Bone Rep. 2018;8:268-273. Published 2018 May 23. doi:10.1016/j.bonr.2018.05.003.
  8. Atef S. ( 2017 , January 16) Insight into Vitamin D Assays in Clinical Laboratory. Retrieved from
  9. Fraser W, Milan A. (2013). Vitamin D Assays: Past and Present Debates, Difficulties, and Developments. Calcified tissue international. 92. 10.1007/s00223-012-9693-3.
  10. Lina Zgaga, et al. 25-Hydroxyvitamin D Measurement in Human Hair: Results from a Proof-of-Concept study, Nutrients (2019). DOI: 10.3390/nu11020423.