超高效液相色谱-串联质谱法测定乳与乳制品中牛乳铁蛋白

建立了超高效液相色谱-串联质谱技术定量检测乳制品中牛乳铁蛋白含量的方法。样品经脱脂、酶解后，利用超高效液相色谱-四极杆/静电场轨道阱高分辨质谱（UPLC-Q/Exactive-HRMS）和Protein Pilot软件分析，实现了乳铁蛋白及其肽段的鉴定；通过初级局部序列搜索工具（basic local alignment search tool，BLAST）与Uniprot数据库对比分析筛选出牛乳铁蛋白的8个特征肽段；选择其中3个响应强度高、稳定性好的特征肽段进一步通过超高效液相色谱-三重四极杆质谱（UPLC-QqQ-MS）进行验证和多反应监测（MRM）定量研究，并考察了空白基质匹配的外标法和内标法对乳铁蛋白检测结果的差异。采用外标法定量时，3个肽段在各自范围内线性关系良好，检出限为0.023~0.041 mg/kg，定量限为0.077~0.137 mg/kg，平均回收率为93.8%~103.9%，日内精密度≤8.8%，日间精密度≤9.5%。该方法抗干扰能力强，灵敏度高，重复性好，适用于乳及乳制品中牛乳铁蛋白的含量测定。

Determination of bovine lactoferrin in milk and dairy products by ultra performance liquid chromatography-tandem mass spectrometry

An ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was established for the quantitative detection of bovine lactoferrin in dairy products. The samples were treated by degreasing and tryptic hydrolysis. Proteins of bovine lactoferrin and peptides were identified using ultra performance liquid chromatography-quadrupole/electrostatic orbitrap-high resolution mass spectrometry (UPLC-Q/Exactive-HRMS) and analyzed by Protein Pilot software. Eight species-specific marker peptides of bovine lactoferrin were identified by comparison of the basic local alignment search tool (BLAST) with the Uniprot database. Three markers with high response strength and stability were chosen for further quantitative research by UPLC-triple quadrupole mass spectrometry (QqQ-MS). The method showed a good linear relationship within its own range. The limits of detection and limits of quantification were 0.023-0.041 and 0.077-0.137 mg/kg, respectively. The observed recoveries were in the range of 93.8%-103.9%. The intra-day and inter-day RSDs were lower than 8.8% and 9.5%, respectively. This method presents various advantages such as strong anti-interference, high sensitivity and reproducibility, and it is suitable for the quantitative analysis of bovine lactoferrin in dairy products.