Analysis of the Aminoglycoside Antibiotics Kanamycin and Amikacin Matches USP Requirements

Importance of the Topic

Aminoglycoside antibiotics such as kanamycin and amikacin are essential therapies for serious gram-negative infections. Due to their structural similarity and potential nephro- and ototoxicity, rigorous quality control and stability monitoring are critical to ensure patient safety and therapeutic efficacy.

Objectives and Study Overview

This study evaluates both the United States Pharmacopeia (USP) monograph HPAE-PAD assay for kanamycin and amikacin and a revised method employing a CarboPac MA1 column with disposable Au-on-PTFE working electrodes. Key performance characteristics—resolution, precision, linearity, and ruggedness—are assessed to confirm compliance with USP requirements and to demonstrate improvements in throughput and reproducibility.

Methodology and Instrumentation

The analytical method is based on high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD).

• Instrument: Dionex ICS-3000/5000 with Gradient/Isocratic Pump, DC Detector/Chromatography Module, AS Autosampler, and Chromeleon CDS software.
• Column: CarboPac MA1 Analytical (4 × 250 mm) and Guard (4 × 50 mm).
• Eluent: 115 mM NaOH, delivered at 0.5 mL/min at 30 °C.
• Detection: Disposable Au-on-PTFE working electrode and Ag/AgCl reference electrode using a carbohydrate waveform.
• Reagents: Kanamycin sulfate, amikacin disulfate, USP reference standards, deionized water (18 MΩ·cm), and NaOH 50%.
• Sample Preparation: Stock solutions (0.16 mg/mL kanamycin, 0.20 mg/mL amikacin) diluted to assay concentrations (0.008 and 0.020 mg/mL), stored at –40 °C until analysis.

Main Results and Discussion

The method achieves baseline separation of kanamycin (RT ~6.0 min) and amikacin (RT ~7.6 min) with resolution >4 and peak asymmetry of 1.1. Linearity over 2–16 µg/mL (kanamycin) and 4–40 µg/mL (amikacin) yielded correlation coefficients >0.999. Intra- and inter-day RSDs for retention times were <0.16%, and peak area RSDs were <1.2%. Ruggedness tests across column lots and alternative waveforms confirmed robustness. Forced-degradation studies under acidic and basic conditions revealed expected degradation profiles, including deacetylation of amikacin to a kanamycin-like species and late-eluting thermal by-products. Recovery in spiked degraded matrices was 80–86%, indicating method accuracy.

Benefits and Practical Applications

The described HPAE-PAD assay offers high sensitivity, short analysis time (10 min), and minimal sample preparation. Disposable gold electrodes reduce equilibration time and enhance reproducibility across instruments and laboratories. The method meets or exceeds all USP criteria, making it suitable for routine quality control of kanamycin and amikacin in active pharmaceutical ingredients and finished dosage forms.

Future Trends and Opportunities

• Application of shorter columns or gradient elution to further reduce run times.
• Coupling with mass spectrometry for structural elucidation of degradation products.
• Extension of the method to other aminoglycoside antibiotics and related impurities.
• Development of automated workflows for high-throughput pharmaceutical QC laboratories.

Conclusion

The revised HPAE-PAD method employing CarboPac MA1 and disposable Au-on-PTFE electrodes provides a rapid, precise, and accurate assay for kanamycin and amikacin that fully complies with USP guidelines. Its robustness and reproducibility make it a valuable tool for pharmaceutical analysis and stability testing.

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