Development and validation of a liquid chromatography-mass spectrometry method for free monosaccharide analysis in milk from 8 species

Free monosaccharides are important carbohydrates in milk, providing both basic and bioactive nutritional benefits; however, the content and composition of free monosaccharides in milk from different species are still not well understood. The aim of this study was to develop a highly sensitive and accurate liquid chromatography (LC)-MS method for the precise quantification of free monosaccharides in milk from 8 species, including human, cow, goat, sheep, yak, camel, horse, and donkey. The chromatographic conditions and MS parameters were systematically optimized to ensure high resolution, minimal matrix effects, and low detection limits for all 8 target monosaccharides. The method was validated with excellent linearity (R² > 0.996), high recovery rates (94.18%-115.02%), and low CV (<10%), demonstrating robustness across various concentrations. The compositional analysis revealed significant interspecies differences in monosaccharide profiles. Human milk was uniquely enriched in glucose (4,262.98 ± 246.49 ng/mL) and fucose (1,024.80 ± 61.82 ng/mL). In contrast, ruminant milk, such as cow and sheep milk, exhibited high levels of galactose (1,803.56 ± 94.63 ng/mL and 1,230.31 ± 52.33 ng/mL, respectively) and mannose (375.24 ± 16.27 ng/mL and 55.81 ± 3.76 ng/mL, respectively). Principal component analysis and a complementary stacked bar chart effectively visualized the clustering and relative distribution of monosaccharides among species, highlighting their metabolic and functional diversity. This study provides novel insights into the biological roles and evolutionary significance of milk monosaccharides. The developed LC-MS method offers a robust tool for advancing our understanding of milk composition and its implications for neonatal nutrition, dairy product innovation, and human health.