Impurity analysis of L-aspartic acid and glycine by HPLC-UV-CAD

Importance of the Topic

The quality control of amino acids such as L-aspartic acid and glycine is essential in pharmaceutical, nutritional, and industrial applications. Conventional compendial assays rely on time-consuming derivatization and separate analyses for amine-containing and organic acid impurities. A unified, robust method for simultaneous quantitation of both impurity classes can greatly enhance laboratory efficiency and data reliability.

Objectives and Study Overview

This work aims to develop and validate high-performance liquid chromatography methods combining ultraviolet and charged aerosol detection (HPLC-UV-CAD) for the impurity profiling of L-aspartic acid and glycine. Specific goals include replacing derivatization-based assays, reducing overall run times, and achieving equivalent or improved limits of quantification compared to European Pharmacopoeia procedures.

Methodology and Instrumentation

Ion-pair reversed-phase chromatography was employed on a C18 Acclaim Polar Advantage II column.

• Mobile phases contained volatile perfluorinated acids (NFPA for Asp, TDFHA for Gly) with TFA and an ACN gradient for glycine.
• Detection was performed inline with a variable wavelength UV detector (210 nm) and a charged aerosol detector with optimized evaporation temperature and filter settings.
• Sample preparation involved aqueous dissolution of amino acids spiked with standardized impurity stock solutions.
• Instrumentation: Thermo Scientific Vanquish Horizon UHPLC (Binary Pump H, Split Sampler HT, Column Compartment H), Variable Wavelength Detector F, Charged Aerosol Detector H, Chromeleon CDS v7.2.6.

Main Results and Discussion

The L-aspartic acid method achieved baseline separation of six known impurities in a 10 min isocratic run, with CAD limits of quantification down to 8 ng on column. The glycine method employed a 35 min gradient run to resolve nine nonvolatile impurities, achieving LOQs as low as 20 ng. Column equilibration challenges with TDFHA were managed via solvent flushing. Validation followed ICH Q2(R1), demonstrating linearity (R2 > 0.996), accuracy (recoveries 90–111 %), precision (%RSD < 7 %), and robustness. Batch analyses confirmed detection of specified impurities at or below 0.03 % reporting threshold. Compared to Ph. Eur. monographs, total analysis times were reduced by 65 % (glycine) and 90 % (aspartic acid).

Benefits and Practical Applications

• Single-run detection of both amine-containing and organic acid impurities without derivatization.
• Substantial reduction in run time and solvent usage, lowering operational costs.
• Enhanced sensitivity and selectivity suitable for QC laboratories in pharmaceutical, food, and biotechnology sectors.

Future Trends and Potential Applications

Advancements in detector technology and novel ion-pair reagents may further improve sensitivity and reduce equilibration times. The UV-CAD approach can be extended to other polar biomolecules, enabling comprehensive impurity profiling in a range of pharmaceuticals, peptides, and metabolites.

Conclusion

The developed HPLC-UV-CAD methods offer a reliable, efficient alternative to compendial assays for L-aspartic acid and glycine impurity analysis. By combining inline UV and aerosol detection, they deliver comparable or superior sensitivity, drastically lower run times, and simplified workflows, meeting regulatory expectations and laboratory throughput demands.

References

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