刘春丽，陈汉峰，杨 静，卢迪勋，谢颖君，东方宁
（捷通（广州）检测技术服务有限公司，广东 广州 510730）
摘要：目的对高效液相色谱法（High Performance Liquid Chromatography，HPLC）测定饮料中维生素B12 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B12 经维生素B12 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 ZORBAXSB-C18（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B12 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B12 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B12 具有较好的参考作用。
关键词：高效液相色谱法（HPLC）；维生素B12；饮料
Method Validation of Determination of Vitamin B12 Content in Beverages by High PerformanceLiquid Chromatography
LIU Chun-li，CHEN Han-feng，YANG Jing，LU Di-xun，XIE Ying-jun，DONG Fang-ning
（Access（Guangzhou）Testing Technology Service Co.，Ltd，Guangzhou 510730，China）
Abstract: Objective:Validation of the detection limit，quantification limit，precision，and accuracy of the HighPerformance 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. Thesolvent was blown dry with a nitrogen blower and then diluted with 0.05% phosphoric acid solution. Determined byliquid chromatography and quantified by external standard method. Select Agilent ZORBAX SB-C18 chromatographycolumn（4.6mm×250mm，5μm），was selected，and the mobile phase was a gradient of methanol 0.05% phosphoricacid solution at a flow rate of 1.0mL/min. The column temperature was 30℃，injection volume was 100μL，and thedetection 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 was72.9%（n=6），and the detection limit was 0.06ng/g，with a quantification limit of 0.20ng/g. Conclusion: This methodis easy to operate. It has a short detection time，and stable results. It is a simple and effective method for detectingvitamin 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 ofvitamin B12 in beverages.
Key words: high performance liquid chromatography（HPLC）；vitamin B12；beverage
中图分类号：O657.7+2 文献标志码：A 文章编号：1007-7871（2024）04-0019-05
2024，Vol.27，No.4