免疫亲和柱净化/高效液相色谱法测定乳粉中维生素B_(12)含量

Determination of Vitamin B_(12) Content in Milk Powder by Immunoaffinity Column Purification-HPLC Method

建立了免疫亲和柱净化/高效液相色谱法测定乳粉中维生素B12的方法。采用磷酸盐缓冲液溶解样品后,通过维生素B12免疫亲和柱、甲醇洗脱、浓缩富集后直接测定。采用Symmstry C18色谱柱为分析柱,柱温为35℃,流动相为0.03 mol/L磷酸二氢钾(磷酸调至pH 3.0)-乙腈(85∶15),流速为1.0 mL/min,紫外检测器波长为361 nm。在优化条件下,维生素B12在20～300μg/L范围内呈良好线性关系,相关系数(r)为0.999 7,检出限为0.6μg/kg;样品的加标回收率为90%～96%;相对标准偏差(RSD)为1.1%～2.4%。该法具有样品处理简单方便、灵敏度高、重现性好、分析时间短等优点,可以满足调制乳粉及婴幼儿配方乳粉中微量维生素B12含量的测定要求。

An immunoaffinity column purification -high performance liquid chromatographic method was developed for the determination of vitamin B_ 12 in milk powder. The sample was dissolved with phosphate buffer, and purified through the vitamin B_12 immunity affinity column. After eluted with methanol, and concentrated, the target compound was directly measured on a Symmstry C_18 column. The optimal conditions were as follows： column temperature： 35 ℃, mobile phase： 0. 03 mol/L potassium dihydrogen phosphate （ phosphate adjustable pH 3.0） - acetonitrile （ 85 ： 15 ）, flow rate ： 1.0 mL/min, detection wavelength： 361 nm. Under the optimal conditions, the calibration curve was linear in the range of 20 -300 μg/L , with correlation coefficient（r） of 0. 999 7 and a detection limit of 0. 6 μg/kg. The spiked recoveries were in the range of 90% -96% with RSD of 1. 1% -2.4%. Compared with the national standard method, the proposed method has the advantages of easy sample handling, high sensitivity, good reproducibility and short analysis time, and could meet the require- ment for the determination of trace amounts of vitamin B_12 in milk powder and infant formula.