Structural insights on oligosaccharides in commercial infant formula products using ion chromatography-mass spectrometry (IC-MS)

Significance of the Topic

Functional oligosaccharides such as human milk oligosaccharides (HMOs), fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS) and malto-oligosaccharides (MOS) play a vital role in gut health and development of the infant microbiome. Their structural diversity directly influences prebiotic activity, immune modulation and nutrient absorption. Detailed characterization of these molecules is essential for quality control and formulation of infant nutrition products.

Objectives and Study Overview

The study aimed to develop a derivatization-free ion chromatography–mass spectrometry (IC-MS) workflow for qualitative assessment of functional oligosaccharides in commercial infant formula. Three products—two bovine-based (liquid and powder) and one soy-based—were evaluated for a range of oligosaccharide types and polymerization degrees. Structural isomer resolution and linkage characterization were key focus areas.

Methodology

Sample preparation involved dissolution (for powder), defatting by centrifugation, protein precipitation with cold ethanol, drying and cleanup using porous graphitic carbon filter plates. Purified oligosaccharides were then directly injected into the IC-MS system without chemical derivatization.

Instrumental Setup

• Dionex ICS-6000 HPIC system with pulsed amperometric detection (PAD) for simultaneous conductivity and MS detection
• Thermo Scientific CarboPac PA300-4 µm analytical and guard columns
• Electrolytically regenerated desalter (ERD500-2 mm) for salt removal
• Thermo Scientific Q Exactive HF-X Hybrid Quadrupole-Orbitrap mass spectrometer in negative ESI mode
• HESI-II source: spray voltage 3.2 kV, capillary 320 °C, sheath/auxiliary gas flows 40/20 units
• Full MS (m/z 400–2000) at 60 000 resolution; MS2 at 15 000 resolution, HCD energy 28

Main Results and Discussion

• Separation of isomeric MOS standards: Maltohexaose (α-1,4 linkages) vs 6-α-D-maltotriosyl-maltotriose (α-1,4/α-1,6 mix). Unique cross-ring fragments (0,2A4, 0,3A4, 0,4A4) enabled linkage assignment.
• Oligosaccharide profiles in products A–C spanned DP3 to DP17, covering FOS, GOS, MOS and HMOs. High-performance anion exchange chromatography resolved matrix components from target analytes.
• Product A (liquid bovine formula) contained 2′-fucosyllactose (2′-FL) and low levels of 3′-sialyllactose (3′-SL), likely from bovine milk. Products B (powder bovine) and C (soy liquid) lacked HMOs, showing mainly GOS and short-chain FOS.
• Orbitrap MS2 data provided high-accuracy fragment masses (<5 ppm) for confident structural annotation without the need for reference standards.

Benefits and Practical Applications

• Derivatization-free workflow reduces sample prep time and artifact formation.
• High-resolution isomer separation supports regulatory compliance and labeling accuracy.
• Structural insights guide formulation of prebiotic blends for infant nutrition and functional foods.

Future Trends and Applications

Emerging trends include quantitative IC-MS methods for absolute concentration profiling, integration with automation and artificial intelligence for high-throughput screening, expanded HMO libraries for deeper structural coverage, and application to clinical studies of microbiome interactions and personalized nutrition solutions.

Conclusion

The developed IC-MS approach offers a robust, high-resolution platform for qualitative structural characterization of diverse oligosaccharides in complex matrices. It enables differentiation of isomeric linkages and comprehensive profiling of prebiotic components in infant formula, supporting quality control and research into functional benefits.

References

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