Determination of Tobramycin and Impurities Using HPAE-PAD

Importance of the Topic

This application details a high‐performance anion‐exchange chromatography with pulsed amperometric detection (HPAE-PAD) method for quantifying tobramycin and its related aminoglycoside impurities. Because tobramycin lacks a UV chromophore and is often contaminated with structurally similar antibiotics (kanamycin B, nebramine, neamine), sensitive and selective detection is essential in pharmaceutical quality control to ensure patient safety and regulatory compliance.

Objectives and Overview

The study demonstrates how an automated eluent generator and disposable gold electrodes improve reproducibility and sensitivity of tobramycin impurity analysis. It evaluates method performance against key validation parameters—linearity, limits of detection (LOD/LOQ), precision, robustness, and long‐term retention time stability—aligned with pharmacopeial and regulatory guidelines.

Methodology and Instrumentation

Eluent generation produced a stable, carbonate-free 2 mM KOH mobile phase, eliminating manual preparation errors. Separation employed a CarboPac PA1 analytical column with guard, 0.5 mL/min isocratic flow, 30 °C, and 20 µL injections. Detection used pulsed amperometry with two waveforms: a carbohydrate waveform for extended electrode life, and the AAA-Direct waveform for enhanced sensitivity.

Instrumentation

• Dionex BioLC system (GP50/IP25 pump, vacuum degas, GM-4 mixer)
• ED50 electrochemical detector with pH/Ag/AgCl reference
• Disposable Au working electrodes (CarboPac and AAA-Direct certified)
• EG50 eluent generator with EGC II KOH cartridge and CR-ATC trap column
• CarboPac PA1 analytical (4×250 mm) and guard (4×50 mm) columns
• AS50 autosampler with thermal compartment, EO1 eluent organizer
• Chromeleon chromatography workstation

Main Results and Discussion

The method achieved baseline separation of tobramycin from five impurities within 15 min, with resolution >6.0. Using the AAA-Direct waveform, linear response extended from 0.22 to 750 pmol for tobramycin (r2 = 0.9935) and 0.12 to 425 pmol for kanamycin B (r2 = 0.9917). LODs ranged 0.22–2.3 pmol (tobramycin) and 0.12–1.4 pmol (kanamycin B). Retention time RSD was 0.3% over 7 days and 0.3% over 50 days; peak area RSD was <4%. Method robustness was confirmed under ±10% eluent concentration and flow variations and electrode or column replacements.

Benefits and Practical Applications

• Automated eluent generation ensures precise, carbonate-free KOH and consistent retention times.
• Disposable Au electrodes deliver low noise, high sensitivity, and eliminate polishing.
• High reproducibility supports routine QC and regulatory compliance.
• Rapid isocratic run enables 15-min throughput per sample.
• Reduced pump wear and improved laboratory safety by avoiding manual caustic preparation.

Future Trends and Opportunities

Advances may include miniaturized eluent generation modules, integrated microfluidic PAD detectors, on-line impurity profiling, and AI-driven chromatography optimization. Further development of high-capacity anion-exchange media and novel waveforms could expand detection to emerging aminoglycoside derivatives.

Conclusion

This HPAE-PAD method with eluent generation and disposable electrodes provides a rugged, high-sensitivity, and highly reproducible platform for tobramycin and impurity analysis, meeting stringent pharmaceutical quality requirements with minimal manual intervention.

Reference

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