2023 FDA Science Forum
Determination of Vitamin B12 in Multivitamins using HPLC-ICP-MS
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Contributing OfficeCenter for Food Safety and Applied Nutrition
Abstract
Vitamin B12, also known as cobalamin, is one of eight B vitamins with a key role in the normal functioning of the brain and nervous system. Naturally occurring cobalamins are only found in animal products such as meat, eggs, and dairy. Dietary supplements such as multivitamins can be used to increase nutrient intake. Traditional methods for vitamin B12 determination include microbiological assays, spectrophotometric, and chromatographic techniques, many of which are limited in sensitivity, specificity, and efficiency.
Inductively coupled plasma-mass spectrometry (ICP-MS) is a sensitive, selective, and robust technique capable of quantifying vitamin B12 by measuring the complexed cobalt in cobalamins. In the present study, vitamin B12 was determined in multivitamins using high performance liquid chromatography (HPLC) coupled with ICP-MS. Homogenized samples were mixed with a sodium acetate buffer and heated in a water bath in the presence of enzymes to break down starch and release protein-bound cobalamins. After centrifugation, the supernatant was filtered and analyzed by HPLC-ICP-MS. Active forms of vitamin B12 (i.e., adenosyl-, cyano-, hydroxo- and methyl-cobalamin) were separated under gradient elution on a reversed-phase column, with mobile phases consisting of water and methanol with formic acid.
Multivitamins in the forms of tablets, capsules, liquids, soft gels, and powders with declared levels and forms of cobalamins were analyzed to verify method performance. Standard reference materials and fortified analytical portions were used for validation. Experiments were carried out with subdued lighting and amber vials to avoid photolytic degradation of the analytes.
