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

维生素B12或钴胺素是具有最复杂化学结构的维生素，其由与四环咕啉系统配位的钴中心原子、上部配体（氰化物、甲基、羟基或腺苷）和作为下部配体的5，6-二甲基苯并咪唑组成。从营养学的角度来看，维生素B12是重要的维生素之一，因为它的天然生产仅限于某些细菌。由于天然食物链富集动物源性食品（即，肉类、奶类、蛋类、鱼类）和发酵食物被认为是维生素B12的膳食来源，而非植物性食物。因此，维生素B12缺乏症在全球范围内普遍存在，尤其是素食者和老年人。因此，要预防这些缺陷，对食品中B12含量的了解至关重要。然而，B12的分析具有挑战性，因为（i）非强化食品中的浓度较低，单个维生素的含量甚至更低，以及（ii）钴的化学不稳定性导致降解和形式之间的相互转化。目前的B12分析是在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，这些测定分别缺乏特异性或灵敏度，并且它们也缺乏关于天然存在的B12维生素的信息，其显示不同的稳定性，并且因此可能具有不同的生物利用度。因此，本文提出的应用旨在开发一种基于LC-MS/MS的测定方法，该方法涉及天然B12维生素的稳定异源物作为内标。正如我们已经证明的维生素组叶酸，泛酸和吡啶，这些稳定的同位素稀释测定显示出上级的准确性相比，其他技术。稳定的同位素标记的化合物将通过用N-15标记的硫酸铵作为唯一氮源喂养费氏丙酸杆菌来生物合成。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表征。以类似的方式，将通过Acidipropionibacterium acidipropionici制备具有不同低级配体（即钴酰胺）的B12的假维生素。这些化合物将用于优化和验证钴胺素天然形式（天然B12 SIDA）和假形式的LC-MS/MS方法，并与标准氰基钴胺素方法进行方法比较。最后，开发的方法将应用于富含维生素B 12和其他钴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.