Profiling galactosyloligosaccharide-containing samples by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD)

Significance of the Topic

Galactosyloligosaccharides (GOS) are prebiotic carbohydrates that escape digestion in the upper gastrointestinal tract and selectively stimulate beneficial bacteria in the colon. They are widely used in functional foods and infant formulas to enhance gut health. Reliable analytical methods for profiling GOS composition are essential for product development, quality control, and regulatory compliance.

Objectives and Study Overview

The primary aim was to develop and demonstrate a robust high-performance anion-exchange chromatography method with pulsed amperometric detection (HPAE-PAD) for detailed profiling of GOS in commercial Bimuno GOS powder and syrup. The study also evaluated the impact of maltodextrin additives and confirmed peak identities using enzymatic treatment.

Methodology and Instrumentation

The method employed a Thermo Scientific Dionex ICS-5000+ system equipped with a CarboPac PA200 analytical column (3 × 250 mm) and PA200 guard column (3 × 50 mm). A binary eluent of 100 mM NaOH (Eluent A) and 1 M sodium acetate in 100 mM NaOH (Eluent B) was applied in a linear gradient from 50 to 150 mM sodium acetate over 45 minutes at 0.5 mL/min. Detection was performed by pulsed amperometry on a gold on PTFE disposable electrode. Key consumables included 0.2 µm syringe filters, Amicon Ultra-15 centrifugal devices, and amyloglucosidase for enzymatic treatment. Sample preparation protocols covered dissolution, filtration, and optional enzyme digestion to distinguish maltodextrin interferences.

Main Results and Discussion

Initial chromatograms of Bimuno GOS powder suggested oligosaccharide peaks from DP1 to DP13. However, enzymatic hydrolysis with amyloglucosidase removed peaks corresponding to α-1,4-linked glucose chains, confirming these as maltodextrins introduced during spray drying. Bimuno GOS syrup exhibited complex clusters of GOS peaks that remained after enzyme treatment, indicating genuine galactosyloligosaccharide structures. Adjusting the acetate gradient (20–100 mM) improved resolution of cluster components. An infant formula spiked with Bimuno GOS powder showed both maltodextrin and GOS patterns; enzyme treatment again eliminated maltodextrin peaks, enabling accurate GOS profiling.

Benefits and Practical Applications

• The HPAE-PAD method permits direct detection of underivatized oligosaccharides with high resolution.
• Enzymatic treatment efficiently removes maltodextrin interferences, improving specificity for GOS.
• Using the actual GOS product as calibration standard ensures accurate quantitation in fortified foods and formulas.
• The approach supports quality control of prebiotic ingredients, product development, and regulatory testing.

Future Trends and Potential Applications

The integration of HPAE-PAD with mass spectrometry or NMR can provide deeper structural characterization of GOS clusters. Automated sample preparation and advanced data analysis will streamline profiling in complex matrices. Expanding the method to other prebiotic oligosaccharides and functional food components will enhance its utility in nutraceutical research and personalized nutrition.

Conclusion

A reliable HPAE-PAD protocol for profiling galactosyloligosaccharides in powder and syrup matrices was established. Enzymatic removal of maltodextrin interferences and use of appropriate calibration standards deliver accurate GOS profiles suitable for quality control and product development in the food and nutraceutical industries.

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