MetaboLights

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identification was achieved using Lipidex and Compound Discoverer software.MetaboLightsPublicLiquid Chromatography MS - negative - reverse-phaseLiquid Chromatography MS - positive - reverse-phaseBEH C18 10 cm column was used for lipid separation. Mobile phase A consisted of 70% acetonitrile in pure MQ-water with 10 mM ammonium acetate, mobile phase B consisted of 90% isoproponol 10% acetonitrile mixture with 10 mM ammonium acetate. Column temperature was 50°C. Mobile phase flow was set to 0.4 mL/min. Initial conditions were 98% MPA and 2% MPB from 0-2 min; 30% MPB to 5 min; 50% MPB to 6 min; 85% MPB to 20 min; 95% MPB to 21-28 min; equilibration to initial conditions started at 30 min till 35 min. Total run time was 35 minHuman milk as infantile Super Food - the effect of dietary choices and supplementation on the shift of lipidomic profile during different stages of lactation.Mariusz RadkiewiczInstitute of Biochemistry and Biophysicsmilkmass spectrometry assay30 µL of sample was subjected to a new polypropylene Eppendorf tube. 5 µL of internal standard UltimateSplash(TM) mix from Avanti Lipids was added to each sample. 600 µL of chloroform-methanol Folch mixture (2:1, v:v) was added to samples using Repetman automatic pipette. Samples were vortexed for 10 mins at 1,500 RPM in 8°C using ThermoMixer C with ThermoTop from Eppendorf. Next, 120 µL of MQ-grade water was added to initiate phase separation. Samples were additionally vortexed for 1 min at 1,500 RPM at 8°C and spun down at 14,000 RPM for 10 mins at 4°C. Lower organic phase was transferred to a new polypropylene tube and dried in water bath under gentle nitrogen stream for at least 10 mins at 40°C. Lipid residue was reconsituted in 200 µL of MeOH/Tol mixture (9:1, v:v)Homo sapienshttps://www.ebi.ac.uk/metabolights/MTBLS15678Mariusz Radkiewicz. Institute of Biochemistry and Biophysics. m.radkiewicz@ibb.waw.pl.All RAW data was acquired using XCalibur (Thermo Scientific). RAW files were converted to mgfs using ProteoWizard software for Lipidex 1 analysis. For Lipidex 2, RAW files were directly subjected to perform Spectrum Search node. All data was organized in form of CSV filesTime pointm.radkiewicz@ibb.waw.plA total of 189 human milk samples were collected from 36 donors in collaboration with the Human Milk Bank Foundation at the Medical University of Warsaw. The samples were categorized into three distinct lactation stages: Period A (2–12 weeks postpartum), Period B (13–16 weeks), and Period C (17–24 weeks). Alongside sample collection, participants completed detailed dietary questionnaires that recorded nutritional intake—including fat, carbohydrate, and caloric consumption—as well as supplementation with vitamins and omega-3 fatty acids. To ensure ethical compliance and subject privacy, all samples and questionnaire data were fully anonymized prior to analysis.MetabolomicsLipiDexInstitute of Biochemistry and Biophysics, Polish Academy of SciencesThermo Scientific Q Exactiveuntargeted analysisHealthy dietmilkHomo sapiensLipidomicsWaters ACQUITY UPLC systemexperimental sampledata-independent acquisitionData-Dependent AcquisitionLipiDexInstitute of Biochemistry and Biophysics, Polish Academy of SciencesThermo Scientific Q Exactiveuntargeted analysisHealthy dietmilkHomo sapiensLipidomicsWaters ACQUITY UPLC systemexperimental sampledata-independent acquisitionData-Dependent AcquisitionFor mass spectrometric analysis on the Q-Exactive Orbitrap, the heated electrospray ionization (HESI) source parameters were set to a spray voltage of 4.0 kV for both ESI+ and ESI- modes, an S-lens RF level of 85.0, and an auxiliary gas temperature of 380°C to achieve robust signal intensity while safeguarding the probe hardware. Full MS1 spectra were acquired across a mass range of m/z 200–1600 with an Automatic Gain Control (AGC) target of 1E6 and a maximum injection time (maxIT) of 90 ms. Data-Dependent Acquisition (DDA) was conducted using a Top10 (loop count 10) selection mode with a precursor isolation window of 1.6 m/z and a dynamic exclusion duration of 10 seconds to minimize redundant sampling of dominant lipids. Tandem MS2 fragmentation was performed in the Higher-energy Collisional Dissociation (HCD) cell utilizing stepped normalized collision energies (NCE) of 20–30, coupled with an AGC target of 5E5 and a maxIT of 120 ms to obtain clean, high-quality MS/MS spectra for accurate in silico library matching.Cer[NS] d18:2_18:0Cer[NS] d18:0_22:0PC 18:1_20:1LysoPE-d5 17:0SM d38:1TG 50:1Cer[NS] d17:1_22:0Cer[NS] d18:1_26:0Cer[NS] d18:1_26:1LysoPC-d5 19:0PE 30:0DG 18:2_18:2Plasmenyl-PE P-40:4PA 18:1_18:0Plasmenyl-PE P-40:5Plasmenyl-PE P-40:6Plasmenyl-PE P-40:7PE 38:5PE 38:3PE 38:2PI 17:1_18:0LysoPS-d5 19:0PE 17:0_20:4SM d42:0SM d42:1PS 17:0_20:3Cer[NS] d18:2_22:0HexCer[NS] d18:1_20:0Plasmenyl-PC P-18:0_16:0HexCer[NS] d18:2_24:0PE 38:1TG 50:5PE 38:0Cer[NS] d18:0_18:0DG 14:0_18:2DG 16:0_18:1Cer[AP] t18:0_24:0DG 16:0_18:3PE[OH] OH-35:4Plasmenyl-PE P-18:0_16:0PS-d5 17:0_22:4Cer[AS] d19:0_23:0Cer[AS] d18:0_26:0Cer[AS] d18:0_26:1DG 17:1_18:2Plasmenyl-PE P-16:0_16:0Plasmanyl-PC O-36:2PI 12:0_18:0PI 14:0_18:1PI 16:0_18:1PI 16:0_18:0PI 18:0_18:0Plasmenyl-PE P-18:1_18:1PE-d5 17:0_16:1PG 16:0_18:1PS 14:1_17:0PG 16:0_18:0PG-d5 17:0_16:1Plasmenyl-PE P-18:1_18:2PE 18:1_20:4PE 18:1_20:2SM d31:1TG 46:3PC 38:2PC 38:1PE-NMe2 14:1_20:1PC 16:1_16:1SM d39:1PC 38:4PC 16:1_16:0Plasmenyl-PE P-18:0_20:4SM-d9 18:1_24:1PE 18:2_24:0Plasmenyl-PE P-18:0_20:0PC 14:0_14:0PC 15:0_16:0Plasmenyl-PE P-18:0_20:1TG 10:0_12:0_17:1Plasmenyl-PE P-18:0_20:3PI 18:0_20:1PI 18:0_20:2HexCer[NS] d42:2LysoPE 22:6Cer-d7 18:1_24:1PI 18:0_20:3LysoPE 22:5PI 18:0_20:4PI 18:0_20:5HexCer[NS] d42:1PC 17:0_18:2PS 18:2_18:0Alkenyl-TG P-54:1Plasmenyl-PE P-36:3Plasmenyl-PE P-36:2PS 18:2_18:2Plasmenyl-PE P-36:1PE 18:0_22:4DG-d5 17:0_22:4PE 18:0_22:6Cer[AS] d18:1_22:0PE 18:0_22:5PG-d5 17:0_20:3PE-d5 17:0_20:3Plasmenyl-PE P-18:1_22:2Plasmenyl-PE P-18:1_22:1PE 37:2PE 37:1PE-NMe2 18:2_18:0Plasmenyl-PE P-18:1_22:6SM d28:1Plasmenyl-PE P-18:1_22:5Cer[NS] d18:1_16:0TG-d5 16:0_15:1_16:0Cer[AS] d19:0_24:0Cer[AS] d19:0_24:1PE 18:0_22:0LysoPE 18:0PE 18:0_22:1Plasmenyl-PE P-36:0PI-d5 37:3LysoPI-d5 19:0PE 16:0_18:1Cer[NS] d18:2_16:0Cer[NS] d18:0_24:1PE 16:0_18:2Cer[NS] d18:0_24:0PC 37:2PE 16:0_18:3SM d36:2SM d36:1LysoPE-d5 19:0Cer[NS] d18:1_24:0Cer[NS] d18:1_24:1LysoPC-d5 17:0TG 10:0_10:0_18:2Plasmenyl-PE P-20:0_20:4Cer[NS] d17:1_24:1SM d40:1PE 18:2_18:2PE 32:0PE 18:2_18:0PE 18:2_18:1Cer[NS] d18:2_20:0Cer[AS] d19:0_21:0PC 10:0_22:4PS 18:2_20:0Plasmanyl-PE O-16:0_20:0PS-d5 17:0_20:3Plasmenyl-PE P-18:0_18:2Cer-d7 18:1_16:1PC-d5 17:0_18:1PE 28:1PC 36:0PE 18:1_22:0TG 48:1PC 36:5PC 36:2PS 16:1_17:0PE-d5 17:0_14:1SM d37:1PG 16:0_16:0PG-d5 17:0_18:1Cer[NS] d18:1_25:0Plasmenyl-PE P-18:0_22:5PC 14:0_16:0PE 18:2_22:1Plasmenyl-PE P-18:0_22:4PE 18:2_22:0Plasmenyl-PE P-18:0_22:1PE-NMe2 31:0Plasmenyl-PE P-18:0_22:0PS-d5 17:0_18:1PE 18:1_22:6PE 18:1_22:5LysoPC 18:0PI 18:0_22:4Plasmenyl-PE P-18:0_22:6PI 18:0_22:5LysoPC 18:2PC 12:0_18:2Plasmanyl-PC O-16:0_16:0LysoPC 18:1PC 40:4PC 40:1PE 39:1Cer[AP] t20:0_22:0SM d41:1PE 18:0_20:5TG 18:3_18:3_18:2SM d41:0PC-d5 17:0_22:4PE 16:0_22:6PE 18:2_19:0PA 16:0_16:0PS 17:0_18:1Cer[NS] d18:1_14:0PI-d5 35:1PE 18:0_20:1PE 18:0_20:4PE 18:1_18:0PE 18:0_20:3PE 18:1_18:1Plasmenyl-PE P-34:1PE 18:0_20:0LysoPI-d5 17:0PE-NMe2 16:0_16:0SM d34:1PC 35:1PC 35:2LysoPC 20:3PE 16:0_16:0PC 35:0TG 17:1_18:2_18:2LysoPC-d5 15:0Cer[NS] d18:1_22:2PC 18:2_20:0PC 16:0_17:0PC 16:0_17:1PI-d5 17:0_20:3PC 18:2_20:4Cer[NS] d18:1_22:0LysoPC 16:0DG 18:1_22:2PA 18:2_18:1PG 18:1_18:1PA 18:2_18:0PC 18:0_20:5PG 18:1_18:0LysoPS-d5 15:0PC 16:0_20:4PC 18:0_20:4Alkenyl-DG P-18:1_20:5PC 18:0_20:3DG 18:3_18:2Cer[NS] d18:0_14:0PE 34:2Cer[NS] d18:1_19:0PA 18:2_18:2Cer-d7 18:1_18:1Cer[AP] t18:1_25:0PI 34:0PE 18:3_18:0PC-d5 17:0_16:1PE 16:0_17:1TG 10:0_18:2_18:2TG 42:2Cer[NP] t20:0_26:0Cer[NP] t20:0_26:1PS-d5 17:0_16:1TG 18:2_18:2_20:4Cer-d7 18:1_20:1Plasmenyl-PE P-16:0_22:1Plasmanyl-PC O-16:0_18:2Plasmenyl-PE P-16:0_22:6Plasmenyl-PE P-16:0_22:4Plasmenyl-PE P-16:0_22:5PC 16:0_16:0PE-NMe 32:0PC 14:0_18:2Cer[NS] d18:1_23:0Cer[AS] d18:1_26:0PE 16:0_20:4PI 18:1_18:1Cer[AP] t20:0_24:1Cer[AP] t20:0_24:0TG 14:0_16:0_18:1PI 18:1_18:0PA 16:0_18:2PC-d5 17:0_20:3PA 16:0_18:1TG 12:0_16:0_18:1PI-d5 33:1HexCer[NS] d34:1HexCer[NS] d34:2PI 17:0_18:1PI 17:0_18:2Plasmenyl-PE P-32:1TG 16:0_16:0_18:1Cer[NS] d18:1_12:0PE 17:0_18:2PE-NMe2 16:0_18:0PE 17:0_18:1PE-NMe2 16:0_18:1LysoPE 18:1Cer[NS] d18:0_20:0PE-NMe2 16:0_18:2LysoPE 18:2LysoPG-d5 17:0PS 18:1_18:1PS 18:1_18:0LysoPE-d5 15:0PE 14:0_18:2PE 40:3PE 40:5PE 12:0_18:2Cer[NS] d18:1_20:0Cer[AS] d18:2_24:0HexCer[NS] d41:1SM d44:2SM d44:1PG 17:1_17:1LysoPS-d5 17:0Cer[AP] t20:0_25:0Cer[NS] d18:2_24:1Plasmenyl-PC P-16:0_18:2DG 18:1_20:0HexCer[NS] d18:2_22:0PE 36:2SM-d9 18:1_20:1PE 36:1PE 36:0Cer[NS] d18:0_16:0Plasmanyl-PE O-38:1PC 18:1_18:0PC 18:1_18:1PE 16:1_17:1PI 18:3_18:0Cer[NS] d18:1_17:0Cer[AS] d18:0_24:0Cer[AS] d18:0_24:1Cer[AS] d19:0_25:0LysoPE 17:1Cer[NS] d17:1_16:0Plasmenyl-PE P-16:0_18:3PI 16:0_16:0Plasmanyl-PE O-18:0_20:0Plasmenyl-PE P-16:0_18:1Plasmenyl-PE P-16:0_18:2PE-d5 17:0_18:1SM d33:1PG-d5 17:0_14:1PC-d5 17:0_14:1PI 18:2_18:0Plasmenyl-PC P-16:0_20:4PE 14:0_17:1PI 16:0_20:4Plasmenyl-PE P-16:0_20:0LysoPE 20:1PS-d5 17:0_14:1SM-d9 18:1_22:1Plasmenyl-PE P-16:0_20:4Plasmenyl-PE P-16:0_20:5LysoPS 18:0Cer-d7 18:1_22:1PC 16:0_18:1LysoPE 20:4PC 16:0_18:0Cer[NS] d18:1_21:0LysoPE 20:3PC 16:0_18:3HexCer[NS] d40:2LysoPE 20:2PC 16:0_18:2Plasmenyl-PE P-38:2SM d45:1Cer[AS] d18:1_24:0Plasmenyl-PE P-38:1Plasmenyl-PE P-38:0Plasmanyl-PE O-18:0_18:1TG 10:0_18:3_18:2Cer[AS] d18:1_24:1PC 18:2_18:1Plasmenyl-PE P-38:5PC 18:2_18:0Cer[AP] t20:0_26:1PC 18:2_18:2Cer[AP] t20:0_26:0PE 35:0Plasmenyl-PE P-18:1_20:2Plasmenyl-PE P-18:1_20:1Plasmenyl-PE P-18:1_20:4Cer[NS] d18:2_23:0PE-d5 17:0_22:4PE 35:1PE 16:1_16:1PE 16:1_16:0Cer[NS] d18:1_18:0PI-d5 31:1Cer[AS] d19:0_26:0LysoPG-d5 19:0Cer[AS] d19:0_26:1Plasmenyl-PE P-30:0LysoPE 16:0PI-d5 39:4falseHuman milk as infantile Super Food - the effect of dietary choices and supplementation on the shift of lipidomic profile during different stages of lactationThe 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.2026-09-222026-09-15MTBLS15678