ABSTRACT: The objective of this project was to establish an advanced analytical method leveraging high-resolution mass spectrometry LC-HRMS to comprehensively profile the human milk lipidome. Mass spectrometry resolves and quantifies compounds based on a fundamental physical metric: the mass-to-charge ratio. This yields an analytical framework marked by high sensitivity and selectivity, enabling robust detection of low quantity analytes within complex isobaric mixtures. The optimized workflow was applied to analyse 189 human milk specimens collected from donor cohorts across three European countries: the Netherlands, Poland, and Italy. Sampling covered three distinct lactational stages: early, transitional, and mature. Ultimately, over 2,000 unique lipid features were identified, with triacylglycerols constituting more than 50% of the pool. These high-dimensional lipidomic profiles were subsequently integrated with comprehensive maternal dietary questionnaires. Our findings reveal profound remodelling within the human milk lipidome across 6 months of lactation. The most substantial adaptations occur between the first three months and the fourth month, a transitional phase that proves pivotal for the biochemical stabilization of mature milk. We identified a directional chain-length shift from shorter saturated chains toward elongated polyunsaturated fatty acids, including DHA, DPA, and ARA – which are responsible for neurodevelopment. In parallel, a continuous redistribution of lipid classes occurred over time: triacylglycerol proportions partially contracted in favor of bioactive polar lipids, especially species implicated in neonatal gastrointestinal maturation, immunomodulation, and neuronal myelination, including phosphatidylethanolamines, sphingomyelins, ceramides, and complex gangliosides. Maternal dietary intake differed substantially among national cohorts. Polish mothers exhibited the highest intakes of dietary cholesterol, total fat, and animal protein, which directly correlated with increased incorporation of shorter, saturated fatty acyl chains into milk lipids; elevated dietary cholesterol was inversely correlated with health-promoting polar phospholipids. The Dutch cohort presented the highest relative energy contribution from dietary protein and fiber, displaying positive associations with phosphatidylcholines and plasmalogens alongside an inverse relationship with phosphatidylinositols. Conversely, Italian participants reported the lowest caloric intake and saturated fat consumption. Despite this, their milk showed no compensatory enrichment in polyunsaturated acyl chains. Crucially, Italian participants demonstrated a significantly lower intake of polyunsaturated fatty acids, including bioactive DHA, compared to Polish mothers. This deficit directly translated into reduced polyunsaturated fatty acid levels in their lipidomic profile, rendering their milk comparatively depleted in critical plasmalogens, phosphatidylethanolamines, and phosphatidylinositols. Collectively, this study maps a precise developmental timeline in which human milk lipid remodeling mirrors the physiological maturation of the maternal mammary gland, continuously adapting to the metabolic demands and rapid development of the infant. The development of this dedicated LC-HRMS method permitted the profiling of over 2000 lipid entities and enabled their semi-quantitative monitoring across lactational stages and dietary choices. We provide clear evidence demonstrating how maternal nutrition and supplementation modify human milk composition, highlighting their downstream implications for neonatal physiology. Providing a unique multi-cohort dataset within Europe, these outcomes establish an essential baseline for future metabolomic and lipidomic investigations. Ultimately, elucidating how maternal diet and external exposures shape human milk chemistry will provide a blueprint for formulating advanced, biologically aligned infant formulas to support infants who cannot be breastfed.
