Isolation and analysis of vitamin B12 from plant samples

• A new assay procedure was developed and evaluated for determination of vitamin B12.
• Vitamin B12 was quantified in a range of matrices.
• Hippophae rhamnoides was found to be a significant source of vitamin B12.
• Above 98% of active vitamin B12 was found in Hippophae rhamnoides.

Based on increased demands of strict vegetarians, an investigation of vitamin B12 content in plant sources, was carried out. The vitamin B12 concentration was determined by RP-HPLC with UV detection, after prior matrix isolation by immunoaffinity chromatography (IAC). Vitamin B12 was extracted in the presence of sodium cyanide, to transform all forms of cobalamin into cyanocobalamin. Diode array detector was used to monitor vitamin B12, after its chromatographic separation under gradient elution with a mobile phase consisting of acetonitrile and trifluoroacetic acid 0.025% (w/v). The method demonstrated excellent linearity with a limit of detection 0.004 μg/ml. The method precision was evaluated for plant samples and it was below 0.7% (n = 6). Significant amounts of vitamin B12 in plants were detected in Hippophae rhamnoides (37 μg/100 g dry weight), in Elymus (26 μg/100 g dry weight) and in Inula helenium (11 μg/100 g dry weight).