Human milk oligosaccharides are pivotal for shaping the infant gut microbiome and immune development, yet their structural diversity hampers routine identification and quantification. We report an optimized capillary electrophoresis-mass spectrometry workflow that enables sensitive, isomer‑selective profiling of 10 biologically relevant human milk oligosaccharides in colostrum and early‑lactation breast milk. Human milk oligosaccharides were first neutralized to stabilize sialic acids and derivatized with Girard's reagent P, introducing a permanent positive charge to enhance electrophoretic resolution and electrospray ionization efficiency. Separation in a linear‑polyacrylamide‑coated capillary (0.25 M formic acid, 30 kV) and mass spectrometry detection with a nanoCEasy interface achieved baseline resolution of all targets except positional isomers lacto-N-difucohexaose I/II. Incorporation of Girard's reagent P‑labeled maltoheptaose as an internal standard improved migration time precision to < 0.5% RSD and reduced peak‑area repeatability to 9%-25% RSD. Limits of detection were 0.8-290 ng/mL, corresponding to fg-pg on‑column amounts and outperforming precedent APTS-based CE/LIF methodologies. Application to colostrum and milk samples from a single donor (1-3 months postpartum) revealed pronounced variation. Colostrum was dominated by 2'‑fucosyllactose and fucosylated lacto-N-fucopentaose isomers, whereas sialylated human milk oligosaccharides were present in smaller amounts. Longitudinally, 2'‑fucosyllactose remained the most abundant species, while lacto-N-fucopentaose and lacto-N-neotetraose/lacto-N-tetraose diminished markedly by Month 3. The presented capillary electrophoresis-mass spectrometry platform delivers reasonably fast (< 70 min), high‑sensitivity human milk oligosaccharide fingerprinting from minimal sample volumes and is readily adaptable to large‑cohort studies, offering new opportunities to elucidate the nutritional dynamics of the maternal milk glycome during lactation.

Department of Analytical Chemistry Faculty of Chemistry Jagiellonian University Kraków Poland

Department of Food Chemistry and Nutrition Faculty of Pharmacy Jagiellonian University Medical College Kraków Poland

Doctoral School of Exact and Natural Sciences Jagiellonian University Kraków Poland

Institute of Analytical Chemistry of the Czech Academy of Sciences Brno Czech Republic

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