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

维生素B12或钴胺素是化学结构最复杂的维生素，由钴中心原子与四环corrin系统配位，上配体（氰化物，甲基，羟基或腺苷基）和5,6-二甲基苯并咪唑作为下配体组成。从营养学的角度来看，维生素B12是一种至关重要的维生素，因为它的自然生产仅限于某些细菌。由于天然食物链的富集，动物源性食品（即肉、奶、蛋、鱼）以及发酵食品被认为是维生素B12的膳食来源，而不是植物源性食品。因此，B12缺乏症在全球范围内普遍存在，尤其是素食主义者和老年人。因此，为了预防这些缺陷，了解食物中B12的含量是必不可少的。然而，对B12的分析具有挑战性，因为(i)非强化食品中的浓度较低，个别维生素的含量甚至更低；（ii）钴胺素的化学不稳定性导致不同形式之间的降解和相互转化。目前的B12分析是在将所有钴胺素转化为氰钴胺素后通过微生物测定或HPLC-UV进行的。然而，这些检测方法分别缺乏特异性或敏感性，而且它们也缺乏有关天然存在的B12维生素的信息，这两种维生素表现出不同的稳定性，因此可能存在不同的生物利用度。因此，本文提出的应用旨在开发一种基于LC-MS/MS的检测方法，该方法涉及天然维生素B12的稳定同位物作为内标。正如我们已经证明的叶酸、泛酸和吡哆毒素的维生素群，这些稳定的同位素稀释试验与其他技术相比具有更高的准确性。以N-15标记的硫酸铵作为唯一的氮源，向弗氏丙酸杆菌投料，可合成稳定的同位素标记化合物。生物合成的化合物将通过色谱方法纯化，并通过不同的光谱方法进行充分的表征。以类似的方式，具有不同下配体（即钴酰）的B12假维生素将由acidipropionici细菌制备。这些化合物将用于优化和验证天然形式的钴胺素（天然B12 SIDA）和假形式的LC-MS/MS方法，并与标准氰钴胺素方法进行方法比较。最后，所开发的方法将应用于富含维生素b12和其他维生素酰胺的食品和补充剂。

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.