Assay Methods for Vitamin D

Vitamin D is a combination of compounds, which consists of 9 -10-secosteroids. The 10-secosteroids only differ in the structure of the side chains and are thus identified as a single entity since they are structurally related to four-ringed compounds (cyclopentanoperhydrophenahthrenes). Vitamin D derives its photochemical reactions from this compound. D vitamins are unstable in acid solutions and stable in alkaline solutions. Further, they are reasonably soluble in ethanol, oil, and fats and highly soluble in solvents such as ether, methanol, and chloroform. All the D vitamers are highly sensitive to light, especially in the solution form. This however can be stabilized through the addition of vitamins A and E into the solutions (McDowell, 2000). Since only a few foods contain Vitamin D, the Assay methods develop over the years strive to be sensitive, accurate, and reliable.

Vitamin D assays are divided into biological and chemical assays. Biological assays are sensitive methods of Vitamin D analysis capable of detecting 120 ng of the vitamin, while chemical assays are less sensitive and hence able to detect almost 9 times more vitamin components than the biological assays. Gas chromatography is an exception to these two processes and uses an electronic-capture detector, which can detect as little as 50 pg of the D vitamin. This technique is especially important to detect the vitamins, because Vitamin D, just like vitamin B12 is a potent vitamin, with only a small amount needed in the tissues.

Biological assays: According to Remington: the science and practice of Pharmacy (2005), the biological essay is the most accurate and effective Vitamin D assay.

  • Rat-line tests: Here young rats have drained off their vitamin D reserves in the body, to the point where they develop rickets. They are then fed with vitamin D for seven days after becoming rachitic. The shortcoming with this is that its time consuming (McDowell, 2000).
  • 25-hydroxy-vitamin D assay: This is a fairly new assay patented in 2003. The assay consists of processes where the 25-hydroxy-vitamin D is assayed from a blood sample. The pH is then lowered to 5.5 in order to disassociate the 25-hydroxy-vitamin D from the binding proteins in the Vitamin. The next step involves determining the 25-hydroxy-vitamin D concentration in the sample. This assay method is distinct from others because it does not remove the vitamin D binding protein. As such, this assay provides a viable way of measuring both ergocalciferol (25-OH Vitamin D2) and cholecalciferol (25-OH Vitamin D3) (Sackrison et al, 2003)

Colorimetric Assays: these are easy to execute but require sufficient vitamins in the test sample in order for the test to react (Remington: the science and practice of Pharmacy, 2005). Insufficient amounts lack the sensitivity necessary to bring up colorimetric changes. The most important factor in this assay is the color reaction, which is made possible by the ring structure, which brings about the vitamin activity. This assay however has several drawbacks, which include inadequate specifics, which makes vitamin quantification hard. More this, the requirement of the sufficient vitamin can pose hindrances, since, without them, a measurable color change may fail to occur.

Protein-binding essay: the most common is the 1, 25-dihydroxy vitamin D, which involves partially purifying acetonitrile serum extract through SEP-PAK C18. The process is followed by liquid chromatography, which is done under high pressure. The results attained are then subjected to a protein binding assay. A receptor specifically drawn from healthy chicken’s duodenal mucosa is used in the assay. Two milliliters of the sample is necessary in order to give the assay sensitivity of 17pmol/l. According to France & Labor (2008), this assay is rapid, technically simple, and uses a binding protein (duodenal mucosa), which is readily available from a healthy chicken. In addition to the binding protein being readily available, it is a stable protein for use as a reagent.

Others: throughout the years, researchers have developed several assays for Vitamin D analysis. Colorimetric is one such assay that uses aniline –HCI and antimony chloride. This assay is primarily used to assess the vitamin D presence in pharmaceuticals and has a working range of 3.2 to 6.5 miles. The Trifluoroacetic acid (ultraviolent absorption) is another assay that uses a vitamin D solution with 5.7 nmol and expects it to absorb 0.10 nmol at 264nm. The advantage of using this is that the conversion of pro-vitamin D to either D3 or D2 can be easily observed. Ultraviolet fluorescence assay is based on acetic anhydride-sulfuric acid properties, which act by inducing fluorescence to the vitamin.

Bioassay techniques used in Vitamin D analysis have the benefit of being specific and sensitive. However, they consume more time, are expensive, and even the slightest form of contamination could alter the results completely. Regardless of this, however, McDowell (2000) states that “the physiological effects of bioassays are quantifiable, while the effects of vitamins as observed in the assays are directly proportionate to the same in theory.” Chemical assays on the other hand are less costly, fast, but have a higher probability of giving non-specific results when compared with the bioassays.


France, M.W. & Labor, B. (2008). A competitive protein binding assay for 1, 25-dihydroxy vitamin D in the blood. Irish Journal of Medical Science. Vol. 150(1).

Mcdowell, L. R. (2000). Vitamins in animal and human nutrition. Ed. London: Wiley-Blackwell

Remington: the science and practice of Pharmacy. (2005). Remington: the science and practice of Pharmacy. New York: Lippincott Williams & Wilkins

Sackrison, J., Miller, A.., Kamerud, J., Ersfeld, D., Olson, G., & Macfarlane, G. (2003). Vitamin D. United States patent application 20040132104. Web.

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