Mass Spectrometry & Spectroscopy

Using UV/VIS Spectroscopy for Different types of Vitamin B12 Analysis

Mar 01 2018 Read 7348 Times

Author: Dr Birgit Schelling on behalf of Mettler-Toledo GmbH Analytical

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Vitamin B12 is a common dietary supplement and pharmaceutical ingredient used in the pharma, food and beverage industries. Analysis of vitamin B12 varies from the identification and purity checks of incoming goods to content determination in the final product. Here we describe how UV/VIS spectroscopy can be used in different types of analysis.

Vitamin B12 (cyanocobalamin) is an important compound containing the rare chemical element cobalt at its centre. It plays a key role in the normal functioning of the brain and nervous system, and in the formation of red blood cells. It is also involved in the metabolism of fatty acids and amino acids and is required for DNA synthesis.
Natural sources of vitamin B12 are mainly of animal origin; plants contain only very small amounts. Consequently, vegetarians particularly can be prone to vitamin B12 deficiency although people suffering from malabsorption, caused by other factors, can also be deficient. Vitamin B12 is available as a supplement in many forms, for example in tablets, either in single-or multivitamin preparations, or in processed foods or beverages, such as energy drinks.
In the pharmaceutical and food industries, the vitamin B12 derivatives cyanocobalamin or hydroxocobalamin are more commonly used in preference to cobalamin as they provide better stability and incur lower production costs. These derivatives are easily converted into cobalamin in the human body.
Vitamin B12 is ideally suited for UV/VIS analysis, producing three distinct peaks at 278, 361 and 550 nm, respectively (Figure 1). The most intense peak is at 361 nm. The 550 nm peak is responsible for the red colour of the substance.

Qualitative UV/VIS Analysis

For the identification of cyanobalomin, all three peaks must be verified. In addition, the ratio of absorbance at the peaks of 361 and 278 nm, and the ratio at the peaks of 361 and 550 nm are calculated and have to be within a certain range (see Table 1).

Quantitative UV/VIS Analysis

Quantitative UV/VIS analysis can be used to determine the concentration of cyanocobalamin by comparing an unknown sample with a standard of known concentration. Based on a reference measurement, the concentration of the unknown sample can be easily calculated. The absorbance maximum at 361 nm is typically used for concentration determination. If a mixture with interfering substances at 361 nm is analysed, the peak at 550 nm can also be used.

Derivative Analysis

As cyanocobalamin can degrade to hydroxocobalamin, it is often interesting to compare the amount of each derivative present in the same mixture. Composition can usually be determined by the ratio of absorbance at 351 and 361 nm. Although the spectrum of hydroxocobalamin is similar to that of cyanocobalamin, it is pH dependent. Therefore, as an alternative method, the absorbance at 361 nm of a solution of pH 8 is measured against a solution of pH 2, reflecting the spectral shift of hydroxocobalamin with pH [1].

Identification of Cyanacobalamin

We now present the qualitative analysis method for vitamin B12 according to USP 35-NF 30, which is a very simple and economic method of identification.
Sample preparation: approx. 300 mg of cyanocobalamin standard containing 1.07% cyanocobalamin and mannitol are dissolved in 100 mL of purified water. A 1:100 dilution is then prepared by stepwise dilution. An aliquot of the cyanocobalamin solution is transferred into a 1 cm quartz cuvette and the full spectrum from 200 to 700 nm is measured with the Mettler Toledo UV7 spectrophotometer.
A blank cuvette filled with purified water is used as a reference. As test acceptance criteria, the presence of the three maxima at 278±1, 361±1, 550±2 nm are checked (Figure 1). The absorbance ratio A361/278 must be in the range of 1.70-1.90 and the ratio A361/A550 in the range of 3.15-3.40. Table 1 shows the acceptance criteria and the results of a measured Vitamin B12 sample. All criteria were fulfilled, confirming the identity and purity of cyanocobalamin in the sample.

Pre-defined Method

For convenient routine analysis, the UV7 spectrophotometer contains a pre-defined method for the identification of vitamin B12, which includes the spectrum scan and the automatic evaluation of the relevant results. The peaks in the spectrum are identified and their locations are compared to the nominal data, taking into account permitted tolerances.
The absorbance ratios are also automatically calculated and compared to the nominal values so that the final result displays all acceptance criteria for the identification of cyanocobalamin. The method can be edited to suit user requirements.
Using the UV7 Excellence spectrophotometer, cyanocobalamin identification is a simple, fast and efficient method, with automatic evaluation reducing the risk of error.


1. Görög, S. Ultraviolet-visible spectrophotometry in pharmaceutical analysis, 1995, CRC Press.

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