A Sensitive Method for Direct Analysis of Impurities in Apramycin and Other Aminoglycoside Antibiotics Using Charged Aerosol Detection

Significance of the topic

Accurate detection of impurities in veterinary antibiotics such as apramycin is critical for product quality, safety and regulatory compliance.
Traditional derivatization-based UV methods are time-consuming and may miss non-chromophoric degradation products.

Objectives and overview

The study sought to establish a sensitive non-derivatization HILIC-CAD method for apramycin sulfate and evaluate its performance against the British Pharmacopoeia SCX-UV method and ELSD detection.
The scope was extended to analyze impurities in other aminoglycoside antibiotics using the same approach.

Methodology

Solid phase extraction (anion exchange) was employed to remove interfering sulfate ions and replace them with volatile bicarbonate.
A 30 min gradient HILIC separation was performed using acetonitrile and ammonium formate buffer (pH 2.9).
Detection modes included charged aerosol detection (CAD), evaporative light scattering detection (ELSD) and UV at 568 nm (SCX-UV).

Applied instrumentation

• Thermo Scientific Dionex UltiMate 3000 RSLC system with LPG-3400SD pump, WPS-3000TSL autosampler, TCC-3000RS column compartment and DAD-3000RS diode array detector
• Thermo Scientific Corona Veo RS charged aerosol detector
• Varian ELSD 385-LC evaporative light scattering detector
• Chromeleon 7.2 chromatography data system

Key results and discussion

• HILIC-CAD detected 16 impurities of apramycin at S/N ≥ 3 with 49.6 µg on column, compared to 7 detected by SCX-UV and 12 by ELSD.
• At 20 µg load, CAD identified 7 impurities at S/N ≥ 3 while ELSD detected only 3, demonstrating significantly higher sensitivity and broader dynamic range.
• SPE cleanup effectively removed sulfate interference, revealing early eluting impurities and preserving peak shapes (W0.5 ~ 0.77 min) even at high sample loads (48.9 µg).
• Extension of the method to eleven other aminoglycosides showed robust impurity profiling, with gradient adjustments improving separation where needed.

Benefits and practical applications

• Eliminates time-consuming pre- or post-column derivatization.
• Universal, structure-independent CAD response detects non-volatile and semi-volatile impurities.
• Low nanogram quantitation and high sample loading capacity support comprehensive impurity profiling.
• Applicable in quality control laboratories for veterinary and pharmaceutical products.

Future trends and opportunities

• Integration of CAD with high-resolution mass spectrometry for structural elucidation of unknown impurities.
• Automation of SPE workflows to improve throughput and reproducibility.
• Extension to other antibiotic classes and complex formulations.
• Development of greener mobile phases and faster gradient methods for high-throughput analysis.

Conclusion

The HILIC-CAD method provides a highly sensitive, non-derivatization approach for impurity analysis of apramycin and related aminoglycosides. It surpasses traditional SCX-UV and ELSD techniques in sensitivity, sample load tolerance and impurity coverage, offering a streamlined solution for regulatory and quality control requirements.

Reference

1. Perzynski S, Cannon M, Cundliffe E, Chahwala SB, Davies J. Effects of apramycin on bacterial protein synthesis. Eur J Biochem. 1979;99:623–628.
2. British Pharmacopoeia (Veterinary). London: Her Majesty's Stationery Office; 2013:38–40, 122–125.
3. Barbosa EA, Lourenco FR, Pinto T. Determination of apramycin by HPLC with pre-column derivatization and UV detection. Braz J Pharm Sci. 2011;47:261–268.