Identification of biomarkers for organic milk authenticity using combined untargeted and targeted metabolomics

Organic milk, particularly desert organic milk, commands a market premium that drives economically motivated adulteration. Current authentication lacks mechanistically validated biomarkers. Here, UHPLC-Q-Exactive Orbitrap-based untargeted metabolomics combined with chemometrics and Random Forest analysis was used to compare traditional milk with milk produced under traditional organic and desert organic systems. Fifteen differential metabolites were prioritized, and organic milk showed higher abundances of dehydroascorbic acid, ascorbic acid, fumarate, 2-oxoglutarate, adenine, adenosine, pyrimidine, 5'-S-methyl-5'-thioadenosine, and glycolic acid than traditional milk. Metabolic pathway analysis revealed significant enrichment of six metabolic pathways, including purine and one‑carbon metabolism, reflecting the modulation of the core bovine metabolic network by organic feeding practices. This shift manifested as pronounced nucleoside enrichment. Consequently, adenosine was established as a robust biomarker, unambiguously confirmed via mass spectrometry fragmentation patterns. Targeted quantification by UHPLC-Q-Exactive Orbitrap revealed a definitive gradient: traditional milk < desert organic milk < traditional organic milk. Crucially, mixture models demonstrated that a 50% traditional milk addition drops adenosine entirely below all organic baselines, explicitly including desert organic milk. This mechanistic, quantitative framework provides a high-fidelity tool to safeguard premium dairy supply chains.