络合提取/肝素亲和柱净化-高效液相色谱法测定含水解蛋白配方食品中的乳铁蛋白

Determination of lactoferrin in hydrolyzed protein formula foods by complexation extraction/heparin affinity column purification-high performance liquid chromatography

中文关键词:  蛋白水解配方食品  乳铁蛋白  肝素亲和柱  高效液相色谱

英文关键词:hydrolyzed protein formula food  lactoferrin  heparin affinity column  high performance liquid chromatography

中文摘要:

      目的 建立一种络合提取/肝素亲和柱净化-高效液相色谱法测定氨基酸配方及蛋白水解配方等特殊配方食品中乳铁蛋白含量的方法。方法 样品加络合磷酸盐缓冲液溶解提取, 肝素亲和柱净化; 采用C4的300 ?蛋白分析色谱柱分离, 流动相为乙腈-三氟乙酸溶液, 梯度洗脱, 高效液相色谱二极管阵列检测器测定, 外标法定量。结果 乳铁蛋白峰型良好, 与杂质基线分离, 在2~200 μg/mL范围内线性关系良好(r2>0.999), 方法检出限和定量限分别为2 mg/100 g、6 mg/100 g; 在低、中、高3个加标浓度水平下, 平均回收率范围为92.0%~96.2%, 相对标准偏差为2.5%~3.7%。结论 该方法准确可靠、重复性和稳定性良好, 适用于氨基酸配方及蛋白水解配方等特殊配方食品的检测, 解决了国家标准方法不适用于检测氨基酸配方及深度水解蛋白乳粉等特殊配方食品中乳铁蛋白的问题。

英文摘要:

      Objective To establish a method for determining of lactoferrin in special formula foods such as amino acid formulas and protein hydrolyzed formulas by a complexation extraction/heparin affinity column purification-high performance liquid chromatography. Methods The sample was dissolved and extracted with complex phosphate buffer solution, and purified using a heparin affinity column. Separation was achieved using a C4 300 ? protein analysis chromatographiccolumn, with themobile phase consisting of acetonitrile-trifluoroacetic acid solution, followed by gradient elution. Detection was performed using a high performance liquid chromatography diode array detector, and quantification was achieved using an external standard method. Results The lactoferrin peak exhibited a good shape and was separated from the impurity baseline. The method demonstrated a good linear relationship within the range of 2–200 μg/mL (r2>0.999). The limit of detection and limit of quantitation of the method were 2 mg/100 g and 6 mg/100 g, respectively. At 3 spiked concentration levels of low, medium and high, the average recovery rates ranged from 92.0% to 96.2%, with relative standard deviations of 2.5% to 3.7%. Conclusion This method is accurate, reliable and exhibits good repeatability and stability. It is suitable for the detection of lactoferrin in special formula foods such as amino acid formulas and protein hydrolyzed formulas. It addresses the issue that the national standard method is not applicable for detecting lactoferrin in special formula foods like amino acid formulas and deeply hydrolyzed protein milk powder.