An improved method for galactosyl oligosaccharide characterization

Importance of the Topic

Galactosyl oligosaccharides GOS serve as important prebiotics that selectively stimulate beneficial gut bacteria and support digestive and immune health. Detailed profiling of their degree of polymerization DP composition and linkage patterns is critical for understanding structure activity relationships and ensuring quality control in food and pharmaceutical industries.

Objectives and Study Overview

The main goal of this work was to develop an improved high resolution method for GOS characterization by coupling high performance anion exchange chromatography with pulsed amperometric detection HPAE PAD to high resolution accurate mass MS HRA MS. The study aimed to separate individual GOS oligomers DP2 to DP10 without derivatization assign DP values accurately and assess the impact of post column desalting on chromatographic performance.

Methodology and Instrumentation

An HPAE system using a Dionex CarboPac PA300 4 µm analytical column operated at 30 °C provided high efficiency separation with a 70 minute gradient of sodium hydroxide and sodium acetate. Detection was performed in parallel by pulsed amperometry and by a Q Exactive Orbitrap mass spectrometer in negative electrospray mode. A Dionex ERD 500 electrolytically regenerated desalter removed sodium from the eluent replacing it with hydronium ions to enable sensitive MS analysis. Key components included:

• Dionex ICS-5000+ or ICS-6000 reagent-free IC system with DP pump and PAD detector
• Dionex CarboPac PA300-4µm 2×250 mm column and guard
• Dionex ERD 500 desalter operating at 250 mA regenerant current
• Q Exactive Orbitrap mass spectrometer with high energy collisional dissociation for MS2

Results and Discussion

The method achieved baseline separation of 28 GOS peaks corresponding to DP2 through DP6. Mass measurement errors were below 1.7 ppm confirming high accuracy. Desalting caused an average resolution loss of only 14.5 %, demonstrating minimal impact on peak shape. MS2 fragmentation provided characteristic glycosidic and cross-ring ions for unambiguous DP assignment, though linkage stereochemistry remained undetermined.

Benefits and Practical Applications

• No derivatization is required enabling direct analysis of complex matrices
• Parallel PAD and high resolution MS delivers both quantitative and structural information
• Reduced need for fraction collection accelerates workflow
• Applicable to routine quality control and research into prebiotic mixtures

Future Trends and Opportunities

Advances may include integration of ion mobility for linkage differentiation automation of MS2 interpretation using machine learning and further miniaturization of HPAE columns for higher throughput. Expansion of this approach to other food oligosaccharides and glycomics applications will broaden its impact.

Conclusion

The combination of HPAE-PAD with high resolution Orbitrap MS and efficient desalting delivers a powerful platform for detailed GOS profiling. This workflow achieves high sensitivity resolution and accuracy without derivatization and supports both research and quality assurance in prebiotic analysis.

Reference

1. Macfarlane G Steed H Macfarlane S Bacterial metabolism and health related effects of galacto oligosaccharides and other prebiotics J Appl Microbiol 2008 104 305 344
2. Gibson G R Roberfroid M B Dietary modulation of human colonic microbiota introducing the concept of prebiotics J Nutr 1995 125 1401 1412
3. Ganzle M G Enzymatic synthesis of galactooligosaccharides and other lactose derivatives hetero oligosaccharides from lactose Int Dairy J 2012 22 116 122
4. Cardelle Cobas A Villamiel M Olano A Corzo N Study of galactooligosaccharide formation from lactose using pectinex Ultra SP L J Sci Food Agr 2008 88 954 961
5. Hernández Hernández O Calvillo I Lebrón Aguilar R Moreno F J Sanz M L Hydrophilic interaction liquid chromatography coupled to mass spectrometry for the characterization of prebiotic galactooligosaccharides J Chromatogr A 2012 1220 57 67
6. Rohrer J S Basumallick L Hurum D High performance anion exchange chromatography with pulsed amperometric detection for carbohydrate analysis of glycoproteins Biochemistry Moscow 2013 78 697 709
7. Rohrer J S High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection HPAE PAD for the Determination of Oligosaccharides in Food and Agricultural Products ACS Symposium Series 2003 849 16 31
8. Thermo Fisher Scientific Application Note 71993 Profiling galactosyloligosaccharide containing samples by HPAE PAD AN71993 EN
9. Coulier L Timmermans J Bas R Van Den Dool R Haaksman I Klarenbeek B Slaghek T Van Dongen W In depth characterization of prebiotic galacto oligosaccharides by a combination of analytical techniques J Agr Food Chem 2009 57 8488 8495
10. Thermo Fisher Scientific Technical Note 71 TN71 Eluent Preparation for HPAE PAD TN70669 EN
11. Thermo Fisher Scientific Technical Note 72478 TN72478 Instrument configuration for native N linked oligosaccharide characterization by HPAE PAD MS EN
12. Thermo Scientific Dionex ERD 500 Product Manual 031761 05