A Sensitive LC-MS/MS Method for Quantifying Beta-Casomorphin 5 in Pasteurized Milk

This study aimed to apply a highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify beta-casomorphin 5 (β-CM5) content in pasteurized milk (PM) and to evaluate the effect of heat treatment on this peptide. Commercial PM samples were collected from local supermarkets, and heat treatment of industrial milk (IP) samples was performed at 62°C and 72°C for 5 min. The milk samples were purified using solid-phase extraction (SPE) and analyzed for β-CM5 using the LC-MS/MS combined with stable isotope-labeled internal standards. The method achieved high sensitivity, with a limit of detection of 0.03 ng/g. The concentration of β-CM5 ranged from 0.10 to 0.55 ng/g for commercial PM samples and from 0.10 to 0.13 ng/g for IP samples. These results suggest that industrial heat treatment had a very slight effect on β-CM5, and variation in this peptide content in PM samples is primarily driven by differences in raw milk origin, such as endogenous enzyme activity and beta-casein content, rather than pasteurization conditions.