免疫亲和柱净化-超高效液相色谱-串联质谱法测定奶粉中的维生素B12总量

DETERMINATION OF TOTAL VITAMIN B12 IN MILK POWDER BY ULTRA-PERFORMANCE LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY AFTER PURIFICATION ON AN IMMUNOAFFINITY COLUMN

目的建立免疫亲和柱净化-超高效液相色谱-串联质谱法(UPLC-MS/MS)测定奶粉中的维生素B12(VB12)总量的方法。方法样品经胃蛋白酶酶解后,VB12从结合态脱离出来,再用1%的氰化钾沸水浴30min,将其转化为氰钴胺素,上清液经免疫亲和柱净化,以Waters HSS T3(2.1 mm×100mm,1.7μm)色谱柱分离,流动相由0.1%甲酸水溶液和0.1%甲酸乙腈溶液以梯度洗脱,配合多反应离子扫描(MRM)定性定量分析。结果VB12检测线性相关性好,r≥0.9988;方法回收率用三个浓度进行添加试验,回收率范围为85.6%~92.7%;定量限为2μg/kg。结论本方法前处理简单,净化效率好,灵敏度高,可作为奶粉中VB12总量的有效检测方法。

Objective To develop a method for determing total vitamin B12 in milk powder using ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)after enrichment and purification on an immunoaffinity column.Methods Vitamin B12 was released from samples by pepsin treatment,and then transferred to cyanocobalamin by the KCN-boiling method.The cyanocobalamin was detected as total vitamin B12 after purification and enrichment on an immunoaffinity column.The analysis was performed by an UPLC-MS/MS system with Waters HSS T3(2.1 mm×100mm,1.7μm)column.The mobile phase consisted of 0.1%(v/v)formic acid aqueous solution and acetonitrile containing 0.1%(v/v)formic acid by gradient elution,and multiple reaction monitoring(MRM)mode with electrospray ionization was used.Results The linear calibration curve was obtained with correlation coefficient r≥0.9988.The recoveries were determinated at three concentrations and ranged from 85.6%-92.7%.The lowest limits of quantification(LOQ)was 2μg/kg.Conclusion This method is suitable for the determination of total vitamin B12 in milk powder.