BENEFITS OF USING HYBRID SURFACE TECHNOLOGY IN LC ANALYSIS OF OLIGOSACCHARIDES

Importance of the Topic

The reliable analysis of oligosaccharides is essential in food science, biotechnology and pharmaceutical quality control. However, metal surfaces in liquid chromatography systems can adsorb polar analytes like sugars, reducing sensitivity and reproducibility. Hybrid surface technology (MaxPeak HPS) offers a barrier to metal interactions, promising more robust, high-performance LC analysis of complex carbohydrates.

Objectives and Study Overview

This study evaluates the impact of MaxPeak HPS on the liquid chromatography–fluorescence detection (LC-FLR) analysis of galactooligosaccharides (GOS) following AOAC Official Method 2021.01. Key aims include:

• Comparing peak responses and reproducibility for 2-AB labeled dextran (DP ≥ 6) on systems with and without MaxPeak HPS.
• Investigating carry-over behavior across repeated injections.
• Assessing whether quantification of GOS is affected by hybrid surface treatment.

Methodology and Instrumentation

Sample Preparation:

• Dextran calibration ladder and GOS samples derivatized with 2-aminobenzamide following AOAC 2021.01.
• Internal standards included for relative peak-area calculations.

LC Conditions:

• Column temperature: 25 °C; injection volume: 2 µL; run time: 60 min.
• Mobile phase A: acetonitrile; B: 100 mM ammonium formate (pH 4.4); gradient elution over 60 min.
• Detection: fluorescence (Ex 330 nm, Em 420 nm) with Empower 3 CDS software.

System Configurations:

• Without MaxPeak HPS: ACQUITY H-Class Plus with ACQUITY FLR detector and Glycan BEH Amide Column (1.7 µm, 2.1×150 mm).
• With MaxPeak HPS: Arc Premier System (BSM) with ACQUITY FLR detector and XBridge Premier Glycan BEH Amide Column (2.5 µm, 2.1×150 mm).

Main Results and Discussion

1) Peak Response and Consistency

• Systems with MaxPeak HPS achieved higher relative peak areas for dextran DP ≥ 6 and maintained consistency injection-to-injection.
• Non-HPS systems produced lower responses that increased over successive injections but never matched HPS performance.

2) Carry-Over Analysis

• MaxPeak HPS effectively eliminated carry-over, even for high-DP oligomers.
• Systems without HPS showed measurable residual peaks after repeated injections.

3) Impact on GOS Quantification

• Quantitative results for GOS remained equivalent with or without MaxPeak HPS, likely due to predominance of oligosaccharide chains smaller than DP 6.
• Dextran ladder served as molecular weight marker without influencing final GOS assay accuracy.

Benefits and Practical Applications

• Enhanced sensitivity and reproducibility in LC analysis of oligosaccharides, supporting tighter quality control.
• Elimination of carry-over reduces sample cross-contamination and simplifies method validation.
• Broad applicability to peptides, phosphoglycans and other polar analytes prone to metal adsorption.

Future Trends and Opportunities

The adoption of hybrid surface treatments is expected to expand across chromatographic and mass spectrometric platforms. Ongoing developments may include:

• Further optimization of surface chemistries for targeted analyte classes.
• Integration into micro- and nano-LC systems for proteomics and glycomics.
• Extension to high-throughput industrial assays and real-time monitoring in bioprocessing.

Conclusion

Incorporating MaxPeak HPS into LC systems and columns significantly improves the analysis of high-DP oligosaccharides by increasing signal intensity, ensuring injection-to-injection consistency and eliminating carry-over. While quantitation of smaller GOS chains remains unaffected, the overall method robustness is enhanced, benefiting diverse carbohydrate analysis workflows.

Reference

• DeLano M, Walter TH, Lauber M, et al. Anal Chem. 2021;93(14):5773-5781.
• Yang J, Wilson I, Rainville P. J Chromatogr B. 2022;1204:123336.
• Lauber M, Walter TH, Gilar M, et al. Low Adsorption HPLC Columns Based on MaxPeak High Performance Surfaces. Waters White Paper 720006930EN. 2020.
• Walter TH, Trudeau M, Simeone J. Low Adsorption UPLC Systems and Columns Based on MaxPeak HPS: The ACQUITY Premier Solution. Waters White Paper 720007128EN. 2021.