Abstract: A new and sensitive method was developed for the determination of casein phosphopeptides (CPPs) in infant formulas by isotope dilution ultra-high performance liquid chromatography-triple-quadrupole tandem mass spectrometry (UPLC-TQ-MS/MS) under the multiple reaction monitoring (MRM) mode. The sample pretreatment procedure involved the addition of isotope-labeled signature peptide as an internal standard, followed by acid precipitation to remove casein from the sample. The signature peptide was chosen and identified from CPPs with Q Exactive Orbitrap based on Proteome Discoverer protein software searching. CPPs were separated on an ACQUITY UPLC BEH 300 C18 column. The content of CPPs in infant formulas was calculated based on the content of the signature peptide in CPPs and in sample. The results of method validation showed that a good linearity with R2 > 0.999 was achieved within 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 spiked levels in blank infant formula matrices. The limit of quantification (LOQ) of the reported method was 1 mg/100 g for infant formula. This method can be suitable for the determination of the content of CPPs in infant formula.

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