Method Validation of Determination of Vitamin B12 Content in Beverages by High Performance Liquid Chromatography
Objective:Validation of the detection limit,quantification limit,precision,and accuracy of the High Performance Liquid Chromatography(HPLC)method for determining vitamin B12 content in beverages.Methods:Vitamin B12 in the sample was adsorbed on a vitamin B12 immunoaffinity column,then eluted with methanol.The solvent was blown dry with a nitrogen blower and then diluted with 0.05%phosphoric acid solution.Determined by liquid chromatography and quantified by external standard method.Select Agilent ZORBAX SB-C18 chromatography column(4.6mm×250mm,5µm),was selected,and the mobile phase was a gradient of methanol 0.05%phosphoric acid solution at a flow rate of 1.0mL/min.The column temperature was 30℃,injection volume was 100µL,and the detection wavelength was 361nm.Results:The linear relationship of vitamin B12 was good within the range of 11.07-553.52ng/mL,with a correlation coefficient r of 0.999990.The average recovery rate of the spiked sample was 72.9%(n=6),and the detection limit was 0.06ng/g,with a quantification limit of 0.20ng/g.Conclusion:This method is easy to operate.It has a short detection time,and stable results.It is a simple and effective method for detecting vitamin B12 in beverages,solving the shortcomings of traditional microbiological methods such as complex operation,long detection time,and poor repeatability.After verification,the detection limit,quantification limit,precision,and accuracy of this method meet the quality control requirements and is a good reference for the detection of vitamin B12 in beverages.
high performance liquid chromatography(HPLC)vitamin B12beverage
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