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).

Calculate the price
Make an order in advance and get the best price
Pages (550 words)
*Price with a welcome 15% discount applied.
Pro tip: If you want to save more money and pay the lowest price, you need to set a more extended deadline.
We know how difficult it is to be a student these days. That's why our prices are one of the most affordable on the market, and there are no hidden fees.

Instead, we offer bonuses, discounts, and free services to make your experience outstanding.
How it works
Receive a 100% original paper that will pass Turnitin from a top essay writing service
step 1
Upload your instructions
Fill out the order form and provide paper details. You can even attach screenshots or add additional instructions later. If something is not clear or missing, the writer will contact you for clarification.
Pro service tips
How to get the most out of your experience with Online Academic Experts
One writer throughout the entire course
If you like the writer, you can hire them again. Just copy & paste their ID on the order form ("Preferred Writer's ID" field). This way, your vocabulary will be uniform, and the writer will be aware of your needs.
The same paper from different writers
You can order essay or any other work from two different writers to choose the best one or give another version to a friend. This can be done through the add-on "Same paper from another writer."
Copy of sources used by the writer
Our college essay writers work with ScienceDirect and other databases. They can send you articles or materials used in PDF or through screenshots. Just tick the "Copy of sources" field on the order form.
See why 20k+ students have chosen us as their sole writing assistance provider
Check out the latest reviews and opinions submitted by real customers worldwide and make an informed decision.
IT, Web
Awesome paper in such a short amount of time. Was in a jam with another service who didn't deliver, but Theresa did the job correctly. I will use her again to do my essays for sure!
Customer 452967, November 27th, 2021
English 101
Thank you for this excellent analysis of the Alchemist.
Customer 453213, June 15th, 2022
Emergency Management
Overall, I think it's a great service. I have yet to submit the assignment, so I don't know what kind of grade I'll get. But customer service was excellent. Reviews were made and the assignment proofed for satisfaction.
Customer 453015, December 15th, 2021
thank you so much
Customer 452749, June 10th, 2021
Looks amazing!
Customer 453283, September 2nd, 2022
Don't really see any of sources I provided, but elsewise its great, thank you!
Customer 452697, May 8th, 2021
Social Work and Human Services
Great work I would love to continue working with this writer thought out the 11 week course.
Customer 452667, May 30th, 2021
It's the second time I use this service and it does not let me down. Work quality is so good for its price!
Customer 452985, December 3rd, 2021
Thank you so much for your attention to this assignment and timeliness.
Customer 453111, November 12th, 2022
English 101
very good paper thank you.
Customer 453083, February 18th, 2022
quick and before due date/time.
Customer 453027, January 29th, 2022
Social Sciences
Very fast report with excellent information on the topic. Thank you for your speedy service in such a short time.
Customer 453213, June 15th, 2022
Customer reviews in total
Current satisfaction rate
3 pages
Average paper length
Customers referred by a friend
15% OFF your first order
Use a coupon FIRST15 and enjoy expert help with any task at the most affordable price.
Claim my 15% OFF Order in Chat
Live ChatWhatsApp