A simple and sensitive LC-ICP-MS method for the accurate determination of vitamin B12 in fortified breakfast cereals and multivitamin tablets

Abstract

A sensitive liquid chromatographic (LC) method coupled with inductively coupled plasma mass spectrometry (ICP-MS) has been developed for the determination of vitamin B₁₂. The method was based on efficient isocratic separation with a mobile phase consisting of 20 mmol L⁻¹ ethylenediaminetetraacetic acid (EDTA) in 25/75 methanol–water mixture (volume fractions) operating at a flow rate of 0.2 mL min⁻¹. After LC separation, ionic cobalt (Co), cyanocobalamin, methylcobalamin, and hydroxocobalamin were measured as ⁵⁹Co by ICP-MS. For Co as cyanocobalamin, the analyte of interest of this work, the method has shown good repeatability with relative standard deviation (RSD) of 3% for ten measurements and excellent linearity between 0.1 ng g⁻¹ and 100 ng g⁻¹ (linear regression, r² > 0.999). The limit of detection (LOD) for cyanocobalamin was found to be less than 1 ng g⁻¹, which permits the method to be employed for the determination of ultra-trace concentrations of vitamin B₁₂ in various types of dietary supplements and fortified food products. 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. To determine total Co 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 Co was achieved only through a complete acid digestion. The method was applied to the determination of vitamin B₁₂ in Standard Reference Material (SRM) 3233 Fortified Breakfast Cereal. SRM 3280 Multivitamin/Multielement Tablet was used for quality assurance of the cereal sample measurements. The vitamin B₁₂ value of (0.187 ± 0.016) mg kg⁻¹ found in SRM 3233 was comparable to (0.219 ± 0.066) mg kg⁻¹ obtained by Grocery Manufacturers Association's Food Industry Analytical Chemists Committee (FIACC) using microbiological assay. The (4.38 ± 0.05) mg kg⁻¹ of vitamin B₁₂ found in quality assurance samples of SRM 3280 was in good agreement with the certified values of (4.8 ± 1.0) mg kg⁻¹.