Analysis of Aminoglycoside Antibiotics by Reversed-Phase HPLC

Significance of the Topic

Aminoglycoside antibiotics remain vital in treating severe bacterial infections, particularly when resistance to other drug classes emerges. Precise analysis of these compounds ensures correct dosage, quality control in pharmaceutical preparations, and monitoring of related impurities. Advanced chromatographic methods improve sensitivity and reliability, supporting both regulatory compliance and patient safety.

Objectives and Study Overview

This application note aims to present optimized reversed-phase HPLC protocols for individual and simultaneous separation of key aminoglycosides, as defined by the Chinese Pharmacopoeia. It also demonstrates quantitative determination of etimicin in a commercial injectable formulation to verify label claims.

Methodology and Instrumentation

Reversed-phase high-performance liquid chromatography was carried out on an Agilent 1200 Series SL system equipped with an evaporative light scattering detector (ELSD). A ZORBAX StableBond SB-C18 column (4.6×150 mm, 3.5 μm) was selected for its low-pH stability and high resolution. Mobile phases comprised trifluoroacetic acid-acidified water and methanol in varying ratios. Key parameters:

• Flow rates: 0.8–1.0 mL/min
• Column temperature: 25 °C
• ELSD settings: 60 °C, 3.5 bar, gain 5, filter 5 s

Main Results and Discussion

Individual methods for gentamicin, kanamycin, and etimicin achieved sharp peaks and baseline separation from related impurities. An isocratic protocol separating six aminoglycosides demonstrated effective resolution within 18 minutes, with minor adjustments to organic composition further improving critical pairs. Analysis of etimicin injection yielded 99.5 % of labeled content. The study also compared calibration strategies for ELSD:

1. Linear fits showed apparent correlation (R2≈0.996) but risked bias at extremes.
2. Polynomial regressions provided exact alignment (R2≈0.9999).
3. Log-log plots combined accuracy and broad dynamic range (R2≈0.9995).

Benefits and Practical Applications

This approach offers:

• High selectivity for structurally similar aminoglycosides.
• Enhanced detection without UV-active chromophores.
• Robustness under acidic conditions, suitable for pharmacopoeial compliance.
• Quantitative reliability for potency assays and impurity profiling.

Future Trends and Potential Applications

Emerging directions include coupling HPLC with mass spectrometry for confirmatory analysis, development of shorter columns with sub-2 μm particles to reduce run times, and automation of calibration curve selection for ELSD detectors. Expanding methods to biodegradable or microfluidic platforms could support point-of-care antibiotic monitoring.

Conclusion

The described reversed-phase HPLC protocols deliver efficient separation and accurate quantitation of aminoglycoside antibiotics using ELSD detection. The methods align with pharmacopeial standards and offer flexibility for both routine quality control and advanced research applications.

Reference

1. Pharmacopoeia of the People's Republic of China, 2005, p. 740
2. Harris D., Quantitative Chemical Analysis, 6th Ed., Macmillan, 2002, p. 626
3. Parriott D., A Practical Guide to HPLC Detection, Academic Press, 1993, p. 158