Determination of tobramycin using HPAE-IPAD on a compact ion chromatography system

Importance of the Topic

Tobramycin is a key aminoglycoside antibiotic often accompanied by kanamycin A and B impurities from fermentation or degradation. Accurate measurement of these compounds is essential for product quality, patient safety, and regulatory compliance.

Objectives and Study Overview

This work aimed to develop and validate a high-pressure anion exchange chromatography method with integrated pulsed amperometric detection (HPAE-IPAD) for tobramycin, kanamycin A, and B using the Thermo Scientific Dionex Integrion HPIC system.

Methodology and Instrumentation Used

Chromatographic separation employed a CarboPac PA1 guard (4×50 mm) and analytical (4×250 mm) column at 30°C with 2 mM KOH eluent generated by the EGC 500 KOH cartridge and purified through a CR-ATC 600 trap column and high-pressure degasser. The flow rate was 0.5 mL/min and 20 µL injections were performed via an AS-AP autosampler. Detection was by IPAD using an AAA-Direct gold working electrode and pH/AgCl reference electrode in Chromeleon CDS software.

Main Results and Discussion

Tobramycin and its impurities were resolved in a 16-minute run with excellent baseline stability. Reproducibility (RSD) was <1.7% for tobramycin and <0.5% for impurities. Calibration showed linearity (r2>0.999) for tobramycin (0.5–100 µM) with quadratic fitting and for kanamycins A and B (0.1–10 µM). The limit of detection for tobramycin was 1.3 nM on column. Recovery studies yielded 98.6% accuracy. The AAA-Direct waveform outperformed the conventional carbohydrate waveform.

Benefits and Practical Applications of the Method

• Automated and precise eluent generation reduces operator error and CO₂ interference.
• High-pressure capability ensures robust performance and shorter runtimes.
• Consumable monitoring streamlines maintenance and traceability.
• Applicability for routine QA/QC of antibiotic drug substances.

Future Trends and Potential Applications

• Integration with fully automated labs and real-time process monitoring.
• Coupling HPAE-IPAD with mass spectrometry for structural confirmation.
• Miniaturization and accelerated gradient methods for high-throughput screening.
• Advanced data analytics and AI-driven method optimization.

Conclusion

The updated HPAE-IPAD method on the Dionex Integrion HPIC system provides a sensitive, reproducible, and efficient approach for quantifying tobramycin and related impurities, supporting stringent quality control in pharmaceutical analysis.

Reference

1. Hanko VP et al. J Pharm Biomed Anal. 2008;47(4–5):828–833.
2. Thermo Scientific Application Note 61. 2004.
3. Thermo Scientific Technical Note 176. 2016.
4. USP General Chapter <621> Chromatography, USP39–NF34. 2016.