Chromatography Forum

I am looking for a good, rugged method for the analysis of cyanocobalamin in multivitamin/mineral formulations. Any help would be greatly appreciated.

Given the lack of replies from our usually vocal participants, I'll explain why it is hard.

First, I have been able to separate B12 nicely. 0-20% acetonitrile, pH 3.4 buffer, polar-embedded column. It comes off in a group with biotin, folate and riboflavin.

The catch is that the normal levels of B12 are very low, even in vitamin supplements and fortified foods. Unfortunately, plant pigments and some dyes also elute in the same region, and cause a lot of false positives with UV detection. Furthermore, there are related substances that are poorly resolved, and are biologically inactive. B12 in solution is unstable in the presence of ascorbic acid, so even if it is an ingredient, it is not really there in some products.

Bioassay is a pain, but still is the most reliable for complex samples. If I had no choice but to use HPLC, I would go for LC/MS/MS and hope for the best.

Thanks for the information. We are seeing results all over the place in multivitamin capsules, even in the same batch. Thinking that it is problem with separating the b12 from other components, physically, not chromatography. Currently using acetate buffer. We may look at solvent extractions.

I had the same experience with multivitamins; too many interfering peaks. I was also using aqueous extraction (pH 3.4) because I wanted to get all the water-soluble vitamins in one step. Struck out on B12 and biotin, but got the rest.