Determination of cyanocobalamin in foods by high-performance liquid chromatography with visible detection after solid-phase extraction and membrane filtration for the precolumn separation of lipophilic species

Determination of trace amounts of cyanocobalamin (18 ng/g) in fatty foods was performed by solid-phase extraction and high-performance liquid chromatography (HPLC) with visible detection at 550 nm using a membrane filter for the precolumn separation of particulate material. It was found that a membrane filter (HLC-Disk 25, 0.45 mu m) was most suitable for the separation of oily particulates. A sample solution was applied to a solid-phase extraction cartridge and then cyanocobalamin was eluted using a 50% aqueous acetonitrile solution followed by HPLC. This method was suitable for the determination of trace amounts of cyanocobalamin in nutrient samples. The proposed method was simple, rapid (extraction time: ca. 12 min, analysis time: ca. 12 min), sensitive (detection limit: ca. 0.15 ng at a signal-to-noise ratio of 3:1), highly selective and reproducible (relative standard deviation: 2.67%) for cyanocobalamin. The calibration graph for cyanocobalamin was linear in the range of 0.1 to 30 ng. Recovery of cyanocobalamin was over 90% by the standard addition method.