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Information on the latest vitamin D news and research.

Find out more information on deficiency, supplementation, sun exposure, and how vitamin D relates to your health.

Vitamin D blood test: Past and present debates, difficulties, and developments

Most people do not know of the problems that exist in accurately measuring vitamin D levels. If they had their blood drawn five times on the same day, they may well get back five different 25(OH)D levels, and the differences could be significant.

I first wrote about this many years ago, when I reported Quest was not accurate (a problem they have since fixed). Before we agreed to use the ZRT home testing methods, we had the results verified by an independent laboratory and found that the ZRT tests are indeed accurate.

More recently, I have written about the problem of testing accuracy:

Recently Drs William Fraser and Anna Milan of the University of East Anglia in England wrote about the different methods used to measure vitamin D.

Fraser WD, Milan AM. Vitamin D Assays: Past and Present Debates, Difficulties, and Developments. Calcif Tissue Int. 2013 Jan 13.

Here are some of the things they discussed:

Measurement of 25(OH)D is challenging for a number of reasons. Namely, 25(OH)D is unstable in water; it loves fats (lipophilic) and strongly binds with the vitamin D binding protein and other fats. In addition, other fats in the blood are confused with 25(OH)D; it exists in several molecular forms; sunlight rapidly degrades 25(OH)D; and a suitable standardization material has only recently become available.

There are 16 different types of methods used around the world to measure 25(OH)D, and the authors reported that 1,119 labs in the world measure 25(OH)D. Immunoassay methods are used 86 % of the time. Immunoassays are tests that measure the presence of a molecule in a solution with an antibody.

The next largest group was LC-MS/MS. Liquid chromatography (LC) – Mass spectrometry (MS) is a conjoint analytical technique. First, liquid chromatography is used to separate a mixture of compounds using various constituents of a mixture that travel at different speeds, causing them to separate. Then mass spectrometry measures the mass-to-charge ratio of charged particles, specifically identifying them. When used appropriately, especially if preparation techniques are standardized, LC-MS/MS is currently considered the gold standard method for 25OHD measurement, being able to simultaneously quantitate 25OHD2 and 25OHD3, with summation of the two values resulting in total 25OHD.

One criticism made about LC-MS, however, is that there is a multitude of ‘‘home-brew’’ or ‘‘in-house’’ methods used in different labs that have different sample preparation and extraction methods, varying running conditions, and multiple MS detection systems which utilize different methods to detect each molecule of interest. A review of the International Vitamin D External Quality Assurance Scheme (DEQAS) results for the LC-MS tests show that the results vary due to lack of standardization.

However, a greater problem exists with immunoassays. There is significant variability of immunoassays to detect 25(OH)D2, meaning that when ergocalciferol, such as prescription Drisdol is used, some of the immunoassays can’t detect it.

There is also a problem with a form of 25(OH)D, called 3-epi-25OHD, which cannot be separated from 25(OH)D by the majority of current LC-MS methods. Immunoassays do not cross-react with 3-epi-25OHD3, so their total detected 25(OH)D does not include 3-epi-25(OH)D.

In 2009, the National Institute of Standards and Technology (NIST) produced standard reference materials and certified the reference values for 25(OH)D2 and 25(OH)D3.

The authors conclude,

“Measurement of vitamin D and its metabolites is difficult. Many methods have been developed over the years, with immunoassay becoming the most popular method due to a combination of availability, ease of use, relative cost, high output using a small sample volume, and rapid turnaround.”

They also notice and foresee a trend in testing,

“Many clinical laboratories have moved to LC-MS/MS technology, with potential greater specificity and accuracy of measurement. The recent generation of international standard reference materials and the development and acceptance of reference method procedures allied to the technical innovations in sample processing and analysis should all contribute to an improvement in the accuracy, precision, and harmonization of results generated by all methods in use currently and those developed in the future.”

  About: John Cannell, MD

Dr. John Cannell is founder of the Vitamin D Council. He has written many peer-reviewed papers on vitamin D and speaks frequently across the United States on the subject. Dr. Cannell holds an M.D. and has served the medical field as a general practitioner, emergency physician, and psychiatrist.

4 Responses to Vitamin D blood test: Past and present debates, difficulties, and developments

  1. Lynn says:

    Has anyone looked at the effect of supplementation timing in relation to test results? I have started having my patients hold their supplemental vitamin D the day of a blood draw, just in case the dose might falsely elevate their blood levels.

  2. The half life of 25(OH)D is so long and conversion in the liver is so quick that it does not matter if someone takes D that day of the blood test or not.


  3. JBG says:

    Progress in 25(OH)D testing has larger potential importance. It is hard to imagine anything that would cause interest in vitamin D to grow as dramatically as a 25(OH)D test as straightforward and cheap as the self blood test used by diabetics.

  4. Rita and Misty says:


    I agree with you regarding a self-blood test similar to one used by diabetics…and I think we’ll soon see a 25(OH)D like this…

    However, I think that the 25(OH)D test should be made a standard test, administered as part of one’s yearly physical….IMO–this would certainly cause interest in Vitamin D to increase dramatically.

    Also it would help if the medical community would STOP referring to D as a Vitamin, and rather called what it is: A Pro-Hormone….

    AGAIN: D is a pro-hormone, not a vitamin….

    Just my opinions…

    Rita :)