Analysis of the Aminoglycoside Antibiotics Kanamycin and Amikacin Matches USP Requirements

Significance of the topic

The aminoglycoside antibiotics kanamycin and amikacin are critical broad-spectrum agents against Gram-negative infections. Their narrow therapeutic window and potential ototoxicity and nephrotoxicity necessitate robust quality control to ensure safety and efficacy.

Objectives and overview of the study

The study aims to evaluate and refine the USP HPAE-PAD assay for kanamycin and amikacin using a CarboPac MA1 column and disposable Au-on-PTFE electrodes. Key goals include assessing precision, linearity, resolution, and robustness of the method.

Methodology and instrumentation

The method employs high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) using a Dionex ICS-3000/5000 system. A CarboPac MA1 analytical column (4×250 mm) and guard column (4×50 mm) are eluted with 115 mM NaOH at 0.5 mL/min and 30 °C. Detection uses disposable gold-on-PTFE working electrodes with an Ag/AgCl reference. Standards are prepared in high-purity water to 0.008 mg/mL (kanamycin) and 0.02 mg/mL (amikacin).

Main results and discussion

• The retention times are 6.0 min for kanamycin and 7.6 min for amikacin, with resolution >4 (USP requires >3) and asymmetry of 1.1 (<2).
• Linearity was demonstrated over 2–16 µg/mL for kanamycin (r²=0.9993) and 4–40 µg/mL for amikacin (r²=0.9991).
• Precision studies showed retention time RSD <0.16% and peak area RSD <1.2% (n=9), meeting USP criteria. Between-day precision remained <3% RSD.
• Ruggedness was confirmed across column lots and waveforms, with consistent performance. The disposable electrodes improved reproducibility and reduced equilibration time.
• Forced degradation under acidic (0.5 M HCl, 100 °C, 1 h) and basic (0.5 M NaOH, 120 °C, 2 h) conditions generated degradation products eluting before 10 min and late-eluting peaks (~21–22 min). Amikacin base hydrolysis produced kanamycin-like species at 6 min.

Benefits and practical applications

• The method meets USP monograph requirements for assay of kanamycin and amikacin in API and drug products.
• Disposable electrodes ensure high lab-to-lab reproducibility and shorter equilibration.
• A 10-minute run time supports high throughput.
• No chemical derivatization is needed, simplifying sample preparation.

Future trends and opportunities

• Adaptation to other aminoglycosides and comprehensive impurity profiling.
• Integration into stability-indicating methods during formulation development.
• Automation and high-throughput screening for quality control labs.
• Coupling with mass spectrometry to identify unknown degradants.

Conclusion

The refined HPAE-PAD method using CarboPac MA1 and disposable Au-on-PTFE electrodes provides accurate, precise, and robust analysis of kanamycin and amikacin. It fulfills USP requirements and offers efficient, high-throughput quality control without derivatization.

References

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• Stead DA. Current Methodologies for the Analysis of Aminoglycosides. J Chromatogr B. 2000;747:69–93.
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• Dionex Corp. Determination of Streptomycin and Impurities Using HPAE-PAD. Application Note 181, LPN 1887, 2007.