液相色谱-荧光法测定乳粉中4种母乳低聚糖

Determination of 4 kinds of breast milk oligosaccharides in milk powder by liquid chromatography-fluorescence method

中文关键词:  2'-岩藻糖基乳糖  3'-唾液酸乳糖  6'-唾液酸乳糖  乳糖-N-新四糖  液相色谱-荧光法

英文关键词:2’-fucosyllactose  3’-sialic acid  6’-sialic acid  lactose-N-neotetraose  liquid chromatography- fluorescence method  milk powder

中文摘要:

      目的 建立液相色谱-荧光法同时测定乳粉中4种母乳低聚糖[2’-岩藻糖基乳糖(2’-fucosyllactose, 2’-FL)、3’-唾液酸乳糖(3’-sialyllactose, 3’-SL)、6’-唾液酸乳糖(6’-sialyllactose, 6’-SL)和乳糖-N-新四糖(lacto-N-neo-tetraose, LNnT)]的含量分析方法。方法 样品加水溶解后, 使用淀粉葡萄糖苷酶或和β-半乳糖苷酶酶解, 用2-氨基苯甲酰胺(2-aminobenzamide, 2-AB)和2-甲基吡啶硼烷(2-methylpyridine borane, 2-PB)衍生后, 采用酰胺键合色谱柱分离, 荧光检测器检测和内标法定量。结果 4种母乳低聚糖在10~600 μg/mL质量浓度范围均有良好的线性关系, 相关系数均为r2>0.999; 4种母乳低聚糖的检出限和定量限分别为0.94~2.31 mg/100 g、3.12~7.69 mg/100 g; 4种母乳低聚糖加标回收率为97.7%~101.5%, 相对标准偏差(relative standard deviation, RSD) (n=7)为0.53%~3.09%。结论 该方法对测定乳粉中的4种母乳低聚糖含量的检测人员、设备和环境要求不高、前处理操作简单, 且具有良好的准确度和精密度, 可为乳粉中母乳低聚糖质量控制提供方法参考。

英文摘要:

      Objective To establish a method for simultaneous determination the content of 4 kinds of human milk oligosaccharides [2’--fucosyllactose (2’-FL), 3’-sialic acid (3’-SL), 6’’-sialic acid (6’-SL) and lactose-N-neotetraose (LNnT)] in milk powder by liquid chromatography-fluorescence method. Methods The samples were dissolved in water, enzymolized with amyloglucosidase or β-galactosidase, derived with 2-aminobenzamide (2-AB) and 2-methylpyridine borane (2-PB), and separated by amide bonding column, detected by fluorescence detector, and quantitated by internal standard methods. Results The 4 kinds of milk oligosaccharides had a good linear relationship in the concentration range of 10–600 μg/mL, and the correlation coefficients (r2) were more than 0.999. The limits of detection and quantification of 4 kinds of milk oligosaccharides were 0.94–2.31 mg/100 g and 3.12–7.69 mg/100 g, respectively; the recovery rates of 4 kinds of milk oligosaccharides were 97.7%–101.5%, and the relative standard deviations (RSD) (n=7) were 0.53%–3.09%. Conclusion This method does not have high requirements for people, equipments and environment for determining the content of 4 kinds of milk oligosaccharides in milk powder. The pre-treatment operation is simple, and it has good accuracy and precision. It can provide a reference for the quality control method of human milk oligosaccharides in milk powder.