Characterization of the Anticoagulant Nadroparin by 2D-LC With High‑Resolution MS

Significance of the Topic

The structural complexity of low molecular weight heparins demands advanced analytical methods to guarantee drug safety by detecting impurities and verifying batch consistency.

Objectives and Study Overview

To establish a two-dimensional liquid chromatography method coupled with high-resolution mass spectrometry for comprehensive profiling of the anticoagulant nadroparin, enabling resolution of oligosaccharide chain lengths and compositions.

Methodology and Instrumentation

A two-dimensional workflow was developed combining:

• First dimension: Size exclusion chromatography to separate degrees of polymerization (DP6–DP16).
• Second dimension: Ion-pair reversed-phase chromatography using pentylamine and hexafluoroisopropanol buffers to resolve isomeric oligosaccharide compositions.
• Detection: Agilent 6546 LC/Q-TOF with dual Jet Stream ESI for high-resolution mass analysis.

Instrument control and data processing were performed using Agilent MassHunter software suite.

Main Results and Discussion

High retention time precision (<0.1% RSD) permitted reliable time-based heart-cutting into the second dimension.
Twenty-eight distinct nadroparin compositions across DP6 to DP16 were identified, revealing diverse sulfation patterns.
Pentylamine adduct formation enhanced negative ion detection and preserved sulfated structures in the MS spectra.
The method demonstrated the ability to resolve multiple isobaric and isomeric oligosaccharides within the same chain length.

Benefits and Practical Applications

• Robust quality control assay for commercial and generic heparins.
• Automated desalting step reduces sample preparation and instrument contamination.
• Detailed top-down characterization supports biosimilar equivalence studies.
• Enhanced safety monitoring by detecting known and unknown heparin contaminants.

Future Trends and Potential Applications

Advances in stationary phases and multidimensional separation techniques will further improve resolution of complex glycans.
Integration with machine learning algorithms may accelerate data interpretation and pattern recognition in polysaccharide analysis.
The workflow could be extended to other biopharmaceuticals, such as glycoproteins and complex oligosaccharides.

Conclusion

The developed 2D-LC/HRMS platform provides a powerful, high-resolution approach to dissect the structural heterogeneity of nadroparin, ensuring product quality and safety through detailed molecular profiling.

Used Instrumentation

• Agilent 1290 Infinity II Bio two-dimensional LC system
• Agilent 6546 LC/Q-TOF mass spectrometer
• Agilent MassHunter Acquisition and Qualitative Analysis software

References

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