高效液相色谱法检测奶及奶制品中未变性乳铁蛋白

Detection of native lactoferrin in milk and dairy products using high performance liquid chromatography

中文关键词:  奶  奶制品  未变性乳铁蛋白  高效液相色谱  检测

英文关键词:milk  dairy  non-denatured lactoferrin  high performance liquid chromatography

中文摘要:

      目的 建立一种基于高效液相色谱法(high performance liquid chromatography, HPLC)测定奶及奶制品中未变性乳铁蛋白的分析方法。方法 本研究探索了奶及奶制品前处理方法、分析未变性乳铁蛋白的方法的液相色谱条件、检出限、定量性、线性范围、重复性、再现性等关键参数。结果 利用磷酸盐缓冲液对样品进行稀释, 用1 mL规格的HiTrapTM Heparin HP肝素亲和柱进行富集和纯化为简单有效的前处理方法, 分析未变性乳铁蛋白的液相色谱条件为: XBridge Protein BEH C4色谱柱(4.6 mm250 mm, 3.5 μm), 柱温60℃。针对液态奶样品, 本方法的检出限为2.5 mg/kg、定量限为5.0 mg/kg, 回收率为106.0%~107.5%; 针对乳粉样品, 本方法的检出限为5.0 mg/kg、定量限为10.0 mg/kg, 回收率为103.5%~107.7%。奶及奶制品中未变性乳铁蛋白的定量上限为100 mg/kg, 在线性范围内具有良好的线性关系(r2>0.999)。平行性实验、日间实验、日内实验和实验室间比对实验表明, 本方法具有良好的重复性和再现性。结论 本方法前处理简单、检测速度快, 可以实现对不同类型奶及奶制品中未变性乳铁蛋白的定量分析, 可为企业和质检机构评价奶及奶制品品质提供标准。

英文摘要:

      Objective To establish an analytical method for the determination of native lactoferrin in milk and dairy products based on high performance liquid chromatography (HPLC). Methods Key parameters such as the pretreatment methods of milk and dairy products, the liquid chromatography conditions for the analysis of native lactoferrin, limit of detection, limit of quantification, linear range, repeatability, and reproducibility were explored. Results Phosphate buffer was used to dilute the samples, and a 1 mL HiTrapTM Heparin HP affinity column was used for enrichment and purification, which was a simple and effective pretreatment method. The liquid chromatography conditions for the analysis of native lactoferrin were: XBridge Protein BEH C4 column (4.6 mm× 250 mm, 3.5 μm), with a column temperature of 60℃. For liquid milk samples, the detection limit of this method was 2.5 mg/kg, the quantitative limit was 5.0 mg/kg, and the recovery rate ranged from 106.0% to 107.5%. For milk powder samples, the detection limit of this method was 5.0 mg/kg, the quantitative limit was 10.0 mg/kg, and the recovery rate ranged from 103.5% to 107.7%. The quantitative upper limit of native lactoferrin in milk and dairy products was 100 mg/kg, and it showed a good linear relationship (r2>0.999) within the linear range. Parallel tests, inter-day experiments, intra-day experiments, and comparisons between laboratories indicated that this method had good repeatability and reproducibility. Conclusion This method features simple pretreatment and fast detection speed, enabling quantitative analysis of native lactoferrin in different types of milk and dairy products. It can provide standards for enterprises and quality inspection agencies to evaluate the quality of milk and dairy products.