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 fo ods was performed by solid-phase extraction and high-performance liqui d chromatography (HPLC) with visible detection at 550 nm using a membr ane filter for the precolumn separation of particulate material. It wa s found that a membrane filter (HLC-Disk 25, 0.45 mu m) was most suita ble for the separation of oily particulates. A sample solution was app lied to a solid-phase extraction cartridge and then cyanocobalamin was eluted using a 50% aqueous acetonitrile solution followed by HPLC. Th is method was suitable for the determination of trace amounts of cyano cobalamin in nutrient samples. The proposed method was simple, rapid ( extraction time: ca. 12 min, analysis time: ca. 12 min), sensitive (de tection limit: ca. 0.15 ng at a signal-to-noise ratio of 3:1), highly selective and reproducible (relative standard deviation: 2.67%) for cy anocobalamin. The calibration graph for cyanocobalamin was linear in t he range of 0.1 to 30 ng. Recovery of cyanocobalamin was over 90% by t he standard addition method.