Method for quantitation of total vitamin B12 in foods using a highly fluorescent vitamin B12 derivative

A highly fluorescent vitamin B-12 derivative was readily prepared with a fluorescent reagent [6,7-dimethoxy-1-methyl-2(1H)-quinoxaline-3-quinoxaline-3-propionylcarboxylic acid hydrazide] specific for carboxylic acids. Hydroxovitamin B-12 was reduced by NaBH4, and then 4-chlorobutyric acid was added to form carboxypropyl vitamin B-12, which was labeled with the fluorescent reagent in the presence of carbodiimide. An assay for vitamin B-12 with the fluorescent vitamin B-12 derivative and hog intrinsic factor was devised. Vitamin B-12 could be assayed over a range of 0.2-10 mu g/L by this method, which was useful for vitamin B-12 assay in foods.