Differentiating cyclopropane fatty acids to support milk authenticity through GC-MS and NMR spectroscopy

Cyclopropane fatty acids (CPFAs), are emerging markers for food authentication in premium dairy products like Parmigiano Reggiano and Haymilk, where silage feeding is prohibited. This study focuses on two CPFAs-dihydrosterculic acid (DHSA) and lactobacillic acid (LBA)-in milk from cows fed diets with or without silage. Using both NMR spectroscopy and a refined GC-MS method, we quantified CPFAs and assessed their potential as dietary markers. NMR enabled rapid, non-destructive screening via cis-methylene proton signals, whereas GC-MS provided accurate quantification and separation of DHSA and LBA. DHSA levels were ∼ 3-fold higher in maize-silage-fed cows compared to non-silage-fed cows, making DHSA, or the DHSA/LBA ratio, robust markers of feeding regime. In contrast, LBA remained stable across diets and was unexpectedly detected in Haymilk, suggesting alternative metabolic origins. These results demonstrate the value of combining NMR and GC-MS for accurate CPFA quantification and provide new insights into their role in dairy authentication.