同位素内标加长肽段稀释UHPLC-MS/MS检测特殊医学用途婴儿配方食品中过敏原β-酪蛋白

Determination of β-Casein in Infant Formula for Special Medical Purposes by Extended Specific Peptide-Isotope-labeled Internal Standard Dilution-UPLC-MS/MS

关键词:  特殊医学用途婴儿配方食品  β-酪蛋白  同位素内标加长肽段  四级杆/轨道阱高分辨质谱  超高效液相色谱串联质谱

Key words:  infant formula for special medical purposes  β-casein  extended specific peptide -isotope-labeled internal standard  Q-Orbitrap MS  UPLC-MS/MS

摘要:

目的 根据筛选得到的β-酪蛋白征肽段，建立特殊医学用途婴儿配方食品中过敏原β-酪蛋白的稳定同位素加长肽段稀释-UPLC-MS/MS定量检测技术。方法 首先利用胰蛋白酶的特异性对过敏原β-酪蛋白进行酶解，经四级杆串联轨道阱高分辨质谱（Q-Orbitrap MS）分析，筛选得到β-酪蛋白征肽段VLPVPQK。然后将添加同位素内标加长肽段SQSKVL*（13C6，15N）PVPQKAVPY的样品进行酶解，在ESI正离子、反应监测（MRM）模式下经UPLC-MS/MS分析，采用稳定同位素内标肽段进行定量。结果 VLPVPQK在0.0025~1.5 μmol·L-1浓度范围内线性良好，相关系数大于0.999。经换算，β-酪蛋白的方法检出限为1 μmol·kg-1，定量限均为3 μmol·kg-1，阴性基质加标平均回收率为56.0%~63.0%，相对标准偏差为1.8%~4.7%，采用建立的方法对不同类型特殊医学用途婴儿配方食品中β-酪蛋白的含量分析。结论 该方法灵敏度高、特异性强，不仅适用于特殊医学用途婴儿配方食品中过敏原β-酪蛋白的含量测定，还可以判断部份水解配方生产工艺中蛋白质的来源，解决目前行业所面临的产品质量评价的难题。

Abstract:

ABSTRACT: OBJECTIVE The objective of this study was to obtain special peptides from β-casein by orbitrap high resolution mass spectrometry (Orbitrap MS), then establish an analytical method for the qualitative and quantitative determination of β-casein based on extended specific peptide-isotope-labeled internal standard dilution-UPLC-MS/MS. METHODS Bovine β-casein in infant formula for special medical purposes was enzymolized by trypsin, and the specific peptide (VLPVPQK) of β-casein was screened by Q-Orbitrap MS analysis. The samples diluents which had been mixed with extended specific peptide-isotope-labeled internal standard (SQSKVL*(13C6，15N)PVPQKAVPY) was enzymolized by trypsin, and detected by UPLC-MS/MS with ESI positive ion and multi-reaction monitoring (MRM) mode. RESULTS The specific peptide of β-casein was linear in the concentration range of 0.0025~1.5 μmol·L-1, and the correlation coefficients was above 0.999. The detection limit of β-casein was 1 μmol·kg-1, with quantification limit of 3 μmol·kg-1, respectively. The average recovery were 56~63%, with RSD from 1.8%~4.7%. CONCLUSION Our results demonstrated that this method is capable of analyzing the content of β-casein in infant formula for special medical purposes with excellent sensitivity and specificity, and also can judge the production process of part of the hydrolyzed formula and the source of the hydrolyzed protein to solve the problem of product quality evaluation faced by the industry.