Analytical methods for vitamins include microbiological assay and high-performance liquid chromatography (HPLC). Vitamin B12 analysis in foods is usually performed by microbiological assay because it is present at trace levels of a few micrograms /100g, and an HPLC method is not sufficiently sensitive and may also be affected by contaminants. However, microbiological assays require culture procedures and takes longer to obtain quantitative results than HPLC assays.
Here we discuss the analysis of vitamin B12 in powdered milk using the HPLC method with reference to the AOAC analysis method*. Large-volume injections using the HPLC method resolved the sensitivity-related problems, and the method was further refined by size-exclusion chromatography (SEC) and introduction onto the analysis column using a heart-cut method, thereby reducing the impact of contamination and large-volume injections on peak shape.