Simple SummaryBovine milk is important for human nutrition, especially for infants. However, per-fluoroalkyl substances (PFASs) are harmful chemicals that can contaminate milk and pose a threat to both humans and animals. This study evaluated how 14 different PFASs were distributed within milk by analyzing the content of whole milk, skim milk, and cream. Samples were taken from 23 cows in Northern Italy that were not exposed to known sources of PFASs. The most common PFAS found in all three fractions was perfluorobutanoic acid (PFBA), followed by perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA). PFOS was found in higher concentrations in cream compared to raw and skimmed milk. Multiparous cows had higher levels of PFASs in their milk. To fully understand the risks of consuming dairy products contaminated with PFASs and their potential impact on cattle health status, further investigation is necessary. Additionally, more studies are needed to uncover the mechanisms behind the excretion of these substances in milk.AbstractBovine milk is a pillar of the human diet and plays a key role in the nutrition of infants. Perfluoroalkyl substances (PFASs) are well-recognized highly stable organic compounds that are able to pollute ecosystems persistently and threaten both human and animal health. The study aimed to analyze the distribution of 14 PFASs within the milk matrix by comparing their content in whole milk, and its skimmed and creamed fractions. Raw milk samples were individually collected from 23 healthy cows (10 primiparous and 13 multiparous) reared on a farm in Northern Italy not surrounded by known point sources of PFASs. Each sample was fractioned in whole, skim, and cream components to undergo PFAS analysis using liquid chromatography–high-resolution mass spectrometry. All samples contained at least one PFAS, with perfluorobutanoic acid (PFBA) being the primary contaminant in all three fractions, followed by perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA). PFOS was shown to be significantly (p< 0.001) more concentrated in cream than in raw and skimmed milk. Multiparous cows showed a higher frequency of positive samples in all analyzed fractions. Further research is necessary to assess the risk of dairy diets and high-fat dairy products and to investigate the toxicological effects of PFASs on cattle, even in environments without known PFAS sources.