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A simple and sensitive HPLC-ICP-MS method for the accurate determination of Vitamin B12 in Fortified Breakfast Cereals and Multivitamin Tablet Reference Materials



Siva K. Chinthalapati, Lee L. Yu, John E. Schiel, Stephen E. Long


A sensitive high performance liquid chromatography (HPLC) coupled with inductively coupled plasma mass spectrometry (ICP-MS) method has been developed for the determination of vitamin B12. The method was based on efficient isocratic separation with a mobile phase consisted of 20 mM ethylenediaminetetraacetic acid (EDTA) in 25/75 methanol/water mixture operating at a flow rate of 0.2 mL/min. After HPLC separation, ionic cobalt, cyanocobalamin, methylcobalamin, and cyanocobalamin were measured as 59Co by ICP-MS. For Co as cyanocobalamin, the method has shown good repeatability relative standard deviation (RSD) of around 3 % for ten measurements and excellent linearity between 0.1 ng/g and 100 ng/g (linear regression, R2 > 0.99). The limit of detection (LOD) value for cyanocobalamin was found to be less than 1 ng/g, which permits the method to be employed for the determination of ultra-trace concentrations of vitamin B12 in various types of dietary supplements. Cyanocobalamin in aqueous solution was found to decompose under the ambient light of the laboratory; therefore, dark room conditions are required for the determination of vitamin B12 in the form of cyanocobalamin to minimize the photon-induced decomposition. In an effort to determine total cobalt in a commercial high purity cyanocobalamin using direct ICP-MS measurement as part of an effort to characterize the chemical for use as a calibrant, it was observed that quantitative measurement of cyanocobalamin was achieved only through a complete acid digestion. The method was applied to the determination of vitamin B12 in candidate Standard Reference Material (SRM) 3233 Fortified Breakfast Cereal. SRM 3280 Multivitamin/Multielement Tablet was used for quality assurance of the cereal sample measurements. The amount of vitamin B12 found at 0.187 ± 0.016 mg/kg and 4.38 ± 0.05 mg/kg were in good agreement with the certified values of 0.210 ± 0.040 mg/kg and 4.8± 1.0 mg/kg for SRM 3233 and 3280, respectively.
Journal of Analytical Atomic Spectrometry


Vitamin B12, cyanocobalamin, HPLC, ICP-MS, Standard reference material, dietary supplement.


Chinthalapati, S. , Yu, L. , Schiel, J. and Long, S. (2013), A simple and sensitive HPLC-ICP-MS method for the accurate determination of Vitamin B12 in Fortified Breakfast Cereals and Multivitamin Tablet Reference Materials, Journal of Analytical Atomic Spectrometry, [online], (Accessed July 18, 2024)


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Created April 10, 2013, Updated November 10, 2018