Study On The VB₁₂ In Animal Food By Pre-column Derivatization

Objective:An analytical method for Vitamin B12 in animal food was built in this research.Methods:The sample preparation methods were set up as follows:Animal food was extracted under the hydrolysis of pepsin under the condition of pH=4.0,0.05mol acetate buffer,every 20 grams of liver need 10mg KCN and every 20 grams of fish and meat need 5mg KCN to transform the vitamin B12 into a CN-VB12 form in pre-column derivatization. With the certification in room temperature at 5000 rpm for 10min,then separete the fat of solution by LLE with 1:1 of ether: petroleum ether. The water layer was finally applied to the SPE column, which is AlltechTM C8 SPE column is under the gradient operation. The chromatographic conditions was set up as follows:The separetion column was CAPCELL PAK MG C18 column, the eluent was operated by a gradient elution procedures with both water and acetonitrile, the flow rate was 1mL/min, the column temperature was kept at 25℃, and the wavelength was set at 361nm.A comparison is also done among the data from this study,USDA databaseSR23 and microbiology method.Results:The detection concentration limit of assay was 0.01μg,the linear range of method was 0.01μg/mL-10μg/mL, the detection concentration limit was 0.997μg/100g, limit of quantitation was 1.33μg/100g,the average recovery is 88.7%-103.2%.Conclusions:The method was applied to detect the concentration of Vitamin B12 in some kinds of animal foods. It was enough to wipe out impurity of samples. The results was proved to be simply, easily to extend and reliable.