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<i>β</i>-酪蛋白

刘柱; 阮昊; 陈万勤; 徐潇颖; 陈爽; 朱价; 罗英; 石云峰; 华颖

doi:10.13748/j.cnki.issn1007-7693.20241226

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-UHPLC-MS/MS

摘要

摘要:
目的根据筛选得到的β-酪蛋白特征肽段，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-UHPLC-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-酪蛋白特征肽段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、多反应监测模式下经UHPLC-MS/MS分析，采用稳定同位素内标肽段进行定量。
结果 VLPVPQK浓度在0.0025~1.5 μmol·L⁻¹内线性良好，相关系数>0.999。经换算，β-酪蛋白的方法检出限为1 μmol·kg⁻¹，定量限为3 μmol·kg⁻¹，阴性基质加标平均回收率为56.2%~63.0%，相对标准偏差为1.8%~4.7%。采用所建方法可成功对不同类型特殊医学用途婴儿配方食品中β-酪蛋白的含量进行检测分析。
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，还可以判断部分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:
OBJECTIVE To develop a stable isotope-labeled extended peptide Dilution-UHPLC-MS/MS quantitative method for allergenic β-casein in infant formula for special medical purposes based on the screened signature peptides.
METHODS Based on the specific digestion of the allergen β-casein with trypsin, the specific peptide VLPVPQK was identified through quadrupole-Orbitrap mass spectrometry (Q-Orbitrap MS) analysis. Subsequently, samples spiked with the stable isotope-labeled extended internal standard peptide SQSKVL*(¹³C₆, ¹⁵N)PVPQKAVPY were enzymatically digested and analyzed by UHPLC-MS/MS under electrospray ionization positive mode with multiple reaction monitoring, enabling accurate quantification using the isotope-labeled internal standard.
RESULTS The concentration of the signature peptide VLPVPQK showed good linearity within the range of 0.0025–1.5 μmol·L⁻¹, with a correlation coefficient greater than 0.999. The limit of detection for β-casein was 1 μmol·kg⁻¹, and the limit of quantification was 3 μmol·kg⁻¹. The average recoveries in negative matrix ranged from 56.2% to 63.0%, with relative standard deviations between 1.8% and 4.7%. The developed method was successfully applied for the detection and analysis of β-casein content in different types of infant formula for special medical purposes.
CONCLUSION The developed method demonstrates high sensitivity and specificity, making it not only suitable for quantifying the allergenic β-casein in infant formula for special medical purposes, but also applicable to identifying the protein source in partially hydrolyzed formula production processes, thereby addressing current industry challenges in product quality assessment.

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