Abstract
Beta-casomorphin peptides including beta-casomorphin 5 (β-CM5) and beta-casomorphin 7 (β-CM7) in raw cow milk were analyzed using a liquid chromatography-tandem mass spectrometry (LC-MS/MS). Milk samples were acidified with HCl followed by centrifuged and cleaned-up using solid-phase extraction (SPE). The eluents were evaporated to dryness and subsequently reconstituted with methanol. Limits of detection (LOD) were 0.32 ng/g for β-CM5 and 0.24 ng/g for β-CM7 and limits of quantification (LOQ) were 1.06 ng/g and 0.69 ng/g for β-CM5 and β-CM7, respectively. Recoveries ranged from 89.7% to 99.4% for β-CM5 and from 96.3% to 98.9% for β-CM7. Intra-day precision was ranged between 7.3% and 22.7% for β-CM5; and 4.5% and 7.3% for β-CM7, meanwhile inter-day precision resulted 7.3%–16.5% for β-CM5 and 9.3%–12.8% for β-CM7. The concentration of β-CM5/7 was analyzed in different milk samples. β-CM7 was detected in 21 out of 30 samples and ranged from 0.76 to 8.41 ng/g milk, while β-CM5 was unequivocally identified in 3 samples, its concentration was 0.4–0.64 ng/g. This is the first report to describe the natural occurrence of β-CM5/7 in raw milk using LC-MS/MS. The present study provides evidence of β-CMs, especially β-CM7 that can be released in raw milk from different breeds.