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United States Department of Agriculture

Agricultural Research Service

Title: Cobalamin Speciation Using Reversed Phase Micro High-Performance Liquid Chromatography Interfaced to Inductively Coupled Plasma Mass Spectrometry

Authors
item Yanes Santos, Enrique
item Miller-Ihli, Nancy

Submitted to: Spectrochimica Acta
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 18, 2004
Publication Date: June 18, 2004
Citation: Yanes Santos, E.G., Miller-Ihli, N.J. 2004. Cobalamin speciation using reversed phase micro high-performance liquid chromatography interfaced to inductively coupled plasma mass spectrometry. Spectrochimica Acta. 59:891-899.

Interpretive Summary: A recent report from the National Academy of Sciences highlighted the fact that little information is available related to absorption and bioavailability of the various forms of Vitamin B12 (cobalalmins). Methods are needed which can separate and quantify the important cobalt containing species: cyanocobalamin, hydroxocobalamin, methylcobalamin, and 5'-deoxyadenosylcobalamin as well as the harmful corrinoid analogue cobinamide dicyanide. Current official microbiological methods are time consuming and detect only cyanocobalamin. In this work, capillary-based micro high-performance liquid chromatography (microHPLC) was combined with inductively coupled plasma mass spectrometry (ICP-MS). An optimized reversed-phase separation using a C18 column with a 2 micron particle size produced a baseline separation in 52 minutes. Researchers in both the food industry and the dietary supplement industry will benefit from this method. Analytical chemists interested in combining separation techniques with ICP-MS will gain information related to: impact of organic solvents, selection of nebulizer, lot-to-lot reliability of capillary columns, and the optimization of separation conditions.

Technical Abstract: Micro high-performance liquid chromatography interfaced to inductively coupled plasma mass spectrometry was optimized for the determination and separation of a mixture of cobalt containing species. Four cobalamin species (cyanocobalamin, hydroxocobalamin, methylcobalamin, and 5'-deoxyadenosylcobalamin) representing the various forms of Vitamin B12 as well as the harmful corrinoid analogue cobinamide dicyanide were separated using reversed-phase micro capillary chromatography with columns containing C18 packing material with a 2 micron particle size. Selection of organic solvents for the separation took into consideration compatibility with the inductively coupled plasma mass spectrometer being used for element specific detection. Optimized method conditions included use of a methanol gradient and make-up solution for the nebulizer. Some issues associated with dead volume were overcome by the extension of the gradient program. The total analysis time was 52 minutes. The column-to-column variability was evaluated and was found to be very reasonable (9% RSD on average), confirming that this method is rugged and that the technology should be easily transferred to other laboratories.

Last Modified: 8/30/2014
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