Nuclear magnetic resonance (NMR) spectroscopy is emerging as a promising technique for the analysis of bovine milk, primarily due to its non-destructive nature, minimal sample preparation requirements, and comprehensive approach to untargeted milk analysis. These inherent strengths of NMR make it a formidable complementary tool to mass spectrometry-based techniques in milk metabolomic studies. This review aims to provide a comprehensive overview of the applications of NMR techniques in the quality assessment and authentication of bovine milk. It will focus on the experimental setup and data processing techniques that contribute to achieving accurate and highly reproducible results. The review will also highlight key studies that have utilized commonly used NMR methodologies in milk analysis, covering a wide range of application fields. These applications include determining milk animal species and feeding regimes, as well as assessing milk nutritional quality and authenticity. By providing an overview of the diverse applications of NMR in milk analysis, this review aims to demonstrate the versatility and significance of NMR spectroscopy as an invaluable tool for milk and dairy metabolomics research and hence, for assessing the quality and authenticity of bovine milk.

Over the years, NMR spectroscopy has become a powerful analytical tool for the identification and quantification of a variety of natural compounds in a broad range of food matrices. Furthermore, NMR can be useful for characterizing food matrices in terms of quality and authenticity, also allowing for the identification of counterfeits. Although NMR requires minimal sample preparation, this technique suffers from low intrinsic sensitivity relative to complementary techniques; thus, the detection of adulterants or markers for authenticity at low concentrations remains challenging. Here, we present a strategy to overcome this limitation by the introduction of a simple band-selective homonuclear decoupling sequence that consists of double irradiation on ¹H during NMR signal acquisition. The utility of the proposed method is tested on dihydrosterculic acid (DHSA), one of the cyclopropane fatty acids (CPFAs) shown to be a powerful molecular marker for authentication of milk products. A quantitative description of how the proposed NMR scheme allows sensitivity enhancement yet accurate quantification of DHSA is provided.

Misfolding and aggregation of intrinsically disordered proteins into amyloid fibrils are central to neurodegenerative diseases such as Parkinson’s, Alzheimer’s, and Huntington’s. Increasing evidence suggests that transient, low-populated oligomeric intermediates, rather than mature fibrils, are key cytotoxic species. Natural polyphenols have shown promise as amyloid inhibitors, though their mechanisms of action remain unclear due to the complexity of early aggregation. This perspective explores how solution-state NMR can quantitatively assess inhibitor mechanisms. Building on recent literature elucidating the aggregation mechanisms of the huntingtin exon 1 protein (htt^ex1), responsible for Huntington’s disease, we propose a kinetic framework that integrates early reversible oligomerization with downstream fibril formation and models the impact of small-molecule binding at distinct stages of the pathway. We show that monomer sequestration and inhibition of elongation-competent nuclei produce distinct aggregation profiles, resolvable through global fitting of NMR and kinetic data. This mechanistic insight enables classification of inhibitors by target stage—monomeric, oligomeric, or fibrillar—and demonstrates how polyphenols serve as a biologically relevant case study for applying this general NMR-driven framework to the design of small-molecule amyloid inhibitors.

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.