Development of a stable isotope dilution assay for vitamers of the B12 group after biosynthesis of stable isotope-labelled isotopologues

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Vitamin B12 or cobalamin is the vitamin with the most complex chemical structure composed of a cobalt central atom coordinated with a tetracyclic corrin system , an upper ligand (cyanide, methyl, hydroxyl or adenosyl) and 5,6-dimethylbenzimidazole as the lower ligand. From the nutritional point of view, vitamin B12 is one of the critical vitamins, as its natural production is restricted to certain bacteria. Due to natural food-chain enrichment animal-derived foods (i.e., meat, milk, egg, fish) as well as fermented foods are considered to be the dietary sources of vitamin B12 but not plant-derived foods.Therefore, B12 deficiencies in humans are prevalent globally and particularly for vegans and the elderly. To prevent these deficiencies, the knowledge about B12 contents in foods, therefore, is essential. However, analysis of B12 is challenging due to (i) the low concentrations in non-fortified foods with contents of individual vitamers being even lower and (ii) the chemical instability of cobalamins leading to degradation and inter-conversion between forms. Current B12 analysis is carried out either by microbiological assays or HPLC-UV after conversion of all cobalamins into cyano cobalamin. However, these assays lack either specificity or sensitivity, respectively, and they also lack information about the naturally occuring B12 vitamers, which show different stabilities, and, in consequence, possibly different bioavailabilities. Therefore, the application presented here aims at developing an LC-MS/MS based assay involving stable istopologues of natural B12 vitamers as internal standards. As we could already demonstrate for the vitamin groups of folates, pantothenic acid and pyridoxin, these stable isotope dilution assays show superior accuracy compared to other techniques. The stable isotope-labelled compounds will be biosythesized by feeding Propionibacterium freudenreichii with N-15 labelled ammonium sulfate as the sole nitrogen source. The biosynthesized compounds will be purified by chromatographic methods and fully characterized by different spectrometric methods. In a similar way, pseudovitamins of B12 with different lower ligands (i.e. cobamides) will be prepared by Acidipropionibacterium acidipropionici. These compounds will be applied to optimize and validate an LC-MS/MS method for natural forms of cobalamin (native B12 SIDA) and pseudoforms and to perform a method comparison with the standard cyano cobalamin method. Finally, the developed method will be applied to foods and supplements rich in vitamin B 12 and other cobamides.