ANALYSIS OF OLIGOSACCHARIDES BY HILIC: POTENTIAL ADSORPTION ISSUE AND SOLUTION

Significance of the Topic

Oligosaccharides such as β-galactooligosaccharides (GOS) are widely used as prebiotics in food and beverage formulations. Precise measurement of these compounds in infant formula is critical for product quality, regulatory compliance, and consumer safety. Hydrophilic interaction liquid chromatography (HILIC), as defined by AOAC Official Method 2021.01, offers a standardized route for saccharide analysis, yet unexpected adsorption phenomena can undermine analytical reliability for higher-molecular-weight dextran standards.

Study Objectives and Overview

This work explores the adsorption and carry-over tendencies of dextran oligosaccharides with degree of polymerization (DP) ≥6 under AOAC-specified HILIC conditions. It compares analytical performance between conventional metal-oxide surfaces and systems employing hybrid organic–inorganic coatings (MaxPeak HPS), and assesses the effect on GOS quantification in infant formula samples.

Experimental Methodology

Sample Preparation Procedures:

• Derivatized dextran standards and samples with 2-aminobenzamide (2-AB) according to AOAC 2021.01 protocols.
• Constructed a dextran calibration ladder to serve as molecular weight markers.

Chromatographic Conditions:

• Column temperature held at 25 °C.
• Injection volume set to 2 µL.
• Mobile phase A: acetonitrile; mobile phase B: 100 mM ammonium formate, pH 4.4.
• Gradient elution over a 60-minute run.
• Fluorescence detection: excitation at 330 nm, emission at 420 nm.

Used Instrumentation

• Without MaxPeak HPS: ACQUITY H-Class Plus system with FLR detector and XBridge Glycan BEH Amide column (2.5 µm, 2.1 × 150 mm).
• With MaxPeak HPS: Arc Premier (BSM) system with FLR detector and XBridge Premier Glycan BEH Amide column (2.5 µm, 2.1 × 150 mm) featuring a hybrid organic–inorganic surface barrier.
• Data management via Empower CDS software.

Main Results and Discussion

• On the standard HILIC setup, dextran DP ≥ 6 experienced significant adsorption losses and variable signal intensities over successive injections, indicating analyte–surface interactions.
• The MaxPeak HPS configuration effectively eliminated such adsorption, delivering consistently higher and stable fluorescence responses.
• GOS quantification in infant formula remained unaffected by system choice, as treated samples mainly contained lower-DP oligosaccharides (DP < 6).

The observations suggest a novel adsorption mechanism for higher-DP saccharides on metal oxide surfaces, which is mitigated by hybrid surface technology.

Benefits and Practical Applications

• Improved assay robustness and reproducibility for high-DP dextran standards.
• Removal of carry-over enhances data integrity and analytical throughput.
• Maintains AOAC 2021.01 compliance while boosting performance for routine quality control.

Future Trends and Potential Applications

• Broader adoption of hybrid surface coatings to prevent analyte adsorption across diverse compound classes.
• Design of specialized low-adsorption stationary phases for complex matrix analyses.
• Implementation in high-throughput QA/QC laboratories for reliable polysaccharide profiling.

Conclusion

Incorporating MaxPeak HPS technology into HILIC systems significantly enhances the analysis of dextran oligosaccharides (DP ≥ 6) by preventing adsorption and carry-over. This advancement underpins accurate prebiotic quantification in infant formulas and paves the way for broader applications of hybrid surface solutions in liquid chromatography.

References

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• Lauber M., Walter T.H., Gilar M., et al. Waters White Paper, 720006930EN, October 2020.
• Walter T.H., Trudeau M., Simeone J. Waters White Paper, 720007128EN, May 2021.