Assay Methods for Vitamins B12 and D


Vitamin B12 refers to active vitamers, known as Cobalamins and they contain cobalt. The structure of Vitamin B12 consists a Corrin (macrocyclic ring), which is formed through linking four-reduced pyrole rings. Crystalline Vitamin B12 can be heated for upto 100 degrees centigrades without loosing its stableness. More to this the Aqeous solutions of the vitamins at 4 to 7 pH levels are easily acutoclaved without much loss. Handling of colabins is very sensitive to lights, alkaline conditions and reducing agents.

According to Hurst (2008) plant based Vitamin B12 cannot be detected unless when it occurs in microorganisms. As such, the most potent sources of Vitamin B12 are seafood and animal products such as meat and milk.

Assay methods to determine physiochemical components

Spectrophotometric determination: This is the most common assay in Vitamin B12 especially for use in pharmaceutical products (Hurst, 2008). However, it is unsuitable for use in components that have a complex-sample matrix. In Food analysis, this assay is expensive and has specificity problems associated with radio-ligand analysis. According to Eitenmiller et al (2008), this shortcoming led to the development of fluorescent derivatives assays for use in food analysis. The latter when bind with the hog IF yields a fluorescence-dilution assay. This is based the competitive binding of B12-fluorescenent derivative and Vitamin B12 for sites on the IF (Eitenmiller et al, 2008). The results obtained from this assay compare favorably to results obtained from Microbiological assays and chemiluminescence as discussed hereunder.

Protein-binding assays: The hog Intrinsic Factor (IF) and the “Radioisotope dilution Assay” (RIDA) are used for vitamin B12 determination in food and are so far the most specific vitamin B12 binding protein (Sato et al, 2002). The use of protein-binding assays is especially common because RIDA kits are available for commercial use. The RiDA method and the microbiological methods have a close correlation co-efficient, except that the RIDA method requires the use of a radioisotope, and the applicable facilities and equipments for it to be successful. According to Hurst (2008) the Elisa enzyme immune assay falls in the RIDA category, and is among the recent developments which are attained in a micro plate. Elisa Enzyme Immunoassay contains a specific antibody for use in Vitamin B12 quantification in pharmaceuticals and foods. While using Elisa Enzyme immunoassay with food samples, one need to remove the fat samples first, and depending on the food being analyzed, one may need to have samples of enzymatic treatments.

High performance liquid Chromatography determination: This assay is less sensitive to light, alkalinity and reducing agents. As such, it is widely used in B12 analysis. Reversed-phase HPLC methods can be used in analyzing the different vitamin B12 isomers. Other analytical methods viable under this include solid extraction phase, column switching and post column labeling (Hurst, 2008).According to Eitenmiller et al (2008), most procedures in this assay observe concentration and clean up steps in order to increase the assay’s potent in low-concentration products.

Chemiluminescence assays: Several methods have been developed for analyzing Vitamin B12. In the recent past, attention has shifted to fluorescent based derivatives for the development of assays, which compared well with chemiluminescence based assays for vitamin B12. Chemiluminescence analyzers need to be fully automated, with IF and “acridinium ester-labeled B12” (Hurst, 2008). In this kind of assay, the Vitamin B12 extraction is done using distilled water. Microbiological techniques of vitamin extraction must be applied and filter papers used in the extraction. The Vitamin needs to be extracted after 1 day at 4degrees centigrade temperatures and in a dark room (Husrt, 2008). The standard set for this extraction include determining compounds spectrophotometrically through gauging its absorbance at 361 nanomolar (Sato et al, 2002). When this is done, the resulting B12 compounds are then applied to the chemiluminescence analyzer. According to Eitenmiller et al (2008), Chemiluminescence assays are most used in Flow Injection Analysis (FIA) since they are rapid procedures. The simplicity and speed of the procedure makes it viable for use in foods, serum and pharmaceuticals.

Microbiological Assays: this involves the extraction of B12 using classical methods from homogenates through boiling the same with potassium Cyanide (KCN) at acidic levels. After centrifuging, the upper layer of the liquid is then used as the assay. For the Microbiological method to be effective, samples of the supernatant should be diluted. A vitamin B12 assay medium is also necessary for use in this method. The use of l.leiscmanii (ATCC7830) is common, and in this case the test culture should be measured at 600 nanomolars. Microbiological assay methods are not only sensitive, but also more tedious. They also consume more time during sample incubation and preservation of the strain (altekrusej, 2005).

Optical Bio-sensor (protein-binding) assay: this assay uses R-protein (haptocorrin) as the main element in the protein-binding process. The R-protein is used with the bio-sensor due to its stability in solutions when compared to IF and its attraction to corrinoids. The results of this assay can be compared well with isotope-labeled protein binding assays and the microbiological assays. However, Eitenmiller et al (2008) notes that hapten-specific antibodies for the Vitamin B12 were difficult to form in this assay.


Altekrusej, A. (2005). Vitamin B12 Assay Medium. Web.

Eitenmiller, R., Ye, L., and Landen, W. (2008). Vitamin analysis for the health and food sciences. Ed. New York: CRC press.

Hurst, W.J. (2008). Methods of analysis for functional foods and nutraceuticals. Ed. New York: CRC press.

Sato, K., Muramatsu, K., & Amano, S. (2002). Application of Vitamin B12-targeting site of lactobacillus helveticus B-1 to Vitamin B12 Assay by Chemiluminescence method. Analytical biochemistry. Vol. 1(1).

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