高效液相色谱法测定饮料中维生素B12的方法验证

Method Validation of Determination of Vitamin B12 Content in Beverages by High Performance Liquid Chromatography

目的对高效液相色谱法(High Performance Liquid Chromatography,HPLC)测定饮料中维生素B_(12)含量方法的检测限、定量限、精密度和准确度进行验证。方法样品中的维生素B_(12)经维生素B_(12)免疫亲和柱吸附,再用甲醇洗脱,氮吹仪吹干溶剂后用0.05%磷酸溶液定容。以液相色谱法测定,外标法定量。选用色谱柱Agilent ZORBAX SB-C_(18)(4.6mm×250mm,5μm),流动相以甲醇-0.05%磷酸溶液梯度洗脱,流速为1.0mL/min,柱温30℃,进样体积100μL,于波长361nm处测定样品。结果维生素B_(12)在11.07ng/m~553.52ng/mL范围内线性关系良好,相关系数r为0.999990,加标平均回收率为72.9%(n=6),检出限为0.06ng/g,定量限为0.20ng/g。结论该方法操作简单、检测时间短、结果稳定,是检测饮料中维生素B_(12)简单有效的方法,解决了传统微生物法的操作复杂、检测时间长和重复性差的缺点。经验证该方法的检测限、定量限、精密度和准确度满足质量控制要求,对检测饮料中维生素B_(12)具有较好的参考作用。

Objective:Validation of the detection limit,quantification limit,precision,and accuracy of the High Performance Liquid Chromatography(HPLC)method for determining vitamin B_(12) content in beverages.Methods:Vitamin B_(12) in the sample was adsorbed on a vitamin B_(12) 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-C_(18) 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 B_(12) 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 B_(12) 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 B_(12) in beverages.