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高效液相色谱串联质谱法测定婴幼儿配方奶粉中酪蛋白磷酸肽含量

胡蓓1,张京顺2,柯星3,蒋易蓉4,周健5,果基施锑6,任一平7   

  1. 1. 浙江工业大学 化学工程学院
    2. 浙江省疾病预防控制中心
    3. 浙江警察学院
    4. 浙江大学生物系统工程与食品科学学院
    5. 宁波市疾病预防控制中心
    6. 阿尔拉食品原料贸易(北京)有限公司
    7. 清华大学长三角研究院
  • 收稿日期:2017-08-14 修回日期:2018-07-13 出版日期:2018-09-25 发布日期:2018-09-18
  • 通讯作者: 任一平 E-mail:renyiping@263.net

Determination of casein phosphopeptides in infant formula by LC-MS/MS

Bei HU 2, 2, 2,Jian Zhou3, 2,Ping YiRen   

  • Received:2017-08-14 Revised:2018-07-13 Online:2018-09-25 Published:2018-09-18
  • Contact: Ping YiRen E-mail:renyiping@263.net

摘要: 本文建立了一种同位素稀释-高效液相色谱串联质谱法定量检测婴幼儿配方奶粉中酪蛋白磷酸肽(Casein Phosphopeptides,CPPs)含量的方法。CPPs原料经Q Exactive Orbitrap 扫描,Uniprot蛋白质数据库初步确定原料中所含肽段,根据质谱响应以及加标回收率等方法学验证实验进一步筛选确定CPPs的特征肽段。样品经过前处理后,采用ACQUITY UPLC BEH 300 C18色谱柱分离,多反应离子监测模式定量测定其中的CPPs特征肽。根据CPPs原料与CPPs特征肽含量的折算系数,从而确定出样品中CPPs的含量。方法学验证结果表明,本文所建立的方法在10-150 mg/100 g范围内线性关系良好,相关系数0.999。在高、中、低三水平加标实验中,回收率在96.2%-100.2%之间,日间重现性在5.3%-7.8%之间。方法定量限为1 mg/100 g,可以满足婴幼儿配方奶粉中不同CPPs含量水平的定量要求。

关键词: 酪蛋白磷酸肽, 特征肽, 高效液相色谱串联质谱, 婴幼儿配方奶粉

Abstract: A new and sensitive determination method was developed for casein phosphopeptides (CPPs) in infant formulas, based on the signature peptide by ultra high-performance liquid chromatography and triple-quadrupole tandem mass spectrometry under the multiple reaction monitoring mode. The simple pretreatment procedures included the addition of the isotope-labeled signature peptide as internal standard, followed by acid precipitation to remove casein in the sample. The signature peptide was chosen and identified from CPPs ingredients by ultra high-performance liquid chromatography and Q Exactive Orbitrap based on Proteome Discoverer protein software search. CPPs were separated on an ACQUITY UPLC BEH 300 C18 column.The content of CPPs in infant formulas was calculated by the content of the signature peptide in ingredients and the content of the signature peptide in sample. The results of method validation showed that good linearity with R2 > 0.999 were achieved in the range from 10 to 150 mg/100 g. Satisfactory recovery (96.2%-100.2%) and inter-day reproducibility (5.3%-7.8%) were obtained at three-level spiking experiments in blank infant formula matrices. The limit of quantification of the reported method was 1 mg/100 g for infant formula. It was suitable for the determination of CPPs content in infant formula.

Key words: casein phosphopeptides, signature peptide, Ultra high-performance liquid chromatography–tandem mass spectrometry, infant formula

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