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

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.