高效液相色谱法测定牛奶及奶粉中免疫球蛋白含量

Determination of Immunoglobulin in Milk and Milk Powder by HPLC

建立高效液相色谱法(HPLC)测定牛奶及奶粉中免疫球蛋白含量。在磷酸盐缓冲液条件下通过HiTrap^(TM) Protein G HP亲和柱结合免疫球蛋白,甘氨酸盐缓冲液洗脱,用CAPCELL PAK C18色谱柱在流动相为乙腈︰0.1%三氟乙酸(5︰5),流速1.0 mL/min,室温条件下进行分离,检测波长280 nm,进样量10μL。Ig G在5~200μg/m L质量浓度范围内相关系数R2为0.999 6,线性关系良好。牛奶基质检出限为10.0 mg/kg,定量限为30.0 mg/kg;奶粉基质检出限为100 mg/kg,定量限为300 mg/kg。在牛奶基质加标10~100 mg/kg、奶粉基质加标100~1 000 mg/kg时,方法回收率为92.4%~98.0%,相对标准偏差(n=6)为0.914%~1.52%,精密度良好,准确度高,可适用于牛奶及奶粉中IgG含量的测定。

A high performance liquid chromatography(HPLC) method was established for the determination of immunoglobulin in milk and milk powder.In the condition of phosphate bufer,immunoglobulin was bound by HiTrap^(TM) Protein G HP affinity column and eluted by glycine bufer.Chromatography was performed on CAPCELL PAK C18 column.Acetonitrile︰0.1%trifluoroacetic acid(5︰5) was used as mobile phas.The flow rate was 1.0 mL/min,and the separation was carried out at room temperature.The detection wavelength was 280 nm.The injection volume was 10 μL.The correlation coefficient R2 of IgG in the concentration range of 5-200 μg/mL was 0.999 6,indicating a good linear relationship.The detection limit of milk matrix was 10.0 mg/kg and the quantification limit was 30.0 mg/kg.The detection limit of milk powder matrix was 100 mg/kg and the quantification limit was 300 mg/kg.The recoveries were 92.4%-98.0%,and the relative standard deviations(n=6) were 0.914%-1.52% when the standards were 10-100 mg/kg for milk matrix and 100-1 000 mg/kg for milk powder matrix.The method had good precision and accuracy,which could be used for the determination of IgG in milk and milk powder.