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