Determination of Tobramycin and Impurities Using HPAE-PAD

Importance of the Topic

Quantitative determination of tobramycin and its impurities is critical in pharmaceutical quality control to ensure clinical safety and efficacy. Aminoglycosides lack chromophores, making UV detection challenging and prone to interference by formulation excipients. High-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) addresses these issues, but reproducible preparation of dilute hydroxide eluents and stable working electrodes are required for reliable operation.

Objectives and Study Overview

This work develops and validates an HPAE-PAD method for tobramycin and its major impurities (kanamycin B, nebramine, neamine), employing:

• An automated eluent generator (EG50) to produce precise, carbonate-free KOH mobile phase.
• A CarboPac™ PA1 column set with a Continuously Regenerated Anion Trap Column (CR-ATC).
• Disposable gold working electrodes with two detection waveforms (Carbohydrate and AAA-Direct).

The method is evaluated for separation performance, sensitivity, linearity, precision, robustness, and long-term reproducibility.

Methodology and Instrumentation

The system consists of a Dionex BioLC platform including GP50 pump with degas, EG50 eluent generator (2 mM KOH isocratic), CR-ATC trap, CarboPac PA1 guard (4 × 50 mm) and analytical (4 × 250 mm) columns, ED50 electrochemical detector, and disposable Au electrodes. Detection employs pulsed amperometry with Carbohydrate and AAA-Direct waveforms. Samples and standards are prepared in high-purity water, injected with a 20 µL loop at 0.5 mL/min, column temperature 30 °C, run time 15 min.

Main Results and Discussion

Separation:

• Complete resolution of tobramycin (RT 5.7 min) from five impurities (RT 3.0–4.7 min) and void peak (RT 2.7 min).
• Peak resolution (tobramycin vs. kanamycin B) averaged 6.00 ± 0.07 (n = 572), exceeding pharmacopeial requirement of 3.0.

Sensitivity and Linearity:

• Carbohydrate waveform linear for tobramycin 0.7–700 pmol (r2 0.9946) and kanamycin B 0.3–500 pmol (r2 0.9874).
• AAA-Direct waveform linear for tobramycin 0.2–750 pmol (r2 0.9935) and kanamycin B 0.12–425 pmol (r2 0.9917).
• Limits of detection: 0.55–2.26 pmol (0.03–0.11 µM) tobramycin (carbohydrate), 0.22–0.36 pmol (0.01–0.02 µM) (AAA-Direct).

Precision and Robustness:

• Intra-day retention time RSD ≤0.4%, peak area RSD ≤4% over 7 days (572 injections).
• Long-term retention time RSD 0.3% over 50 days (2368 injections) using automated eluent generation.
• Robust to 10% variation in eluent concentration, flow rate; disposable electrode lot-to-lot variability <10% RSD.

Benefits and Practical Applications of the Method

• Automated KOH eluent generation eliminates manual preparation errors and carbonate contamination, improving retention time precision.
• Disposable gold electrodes ensure consistent electrochemical response and simplify maintenance.
• Fast isocratic run (15 min), minimal sample preparation, and high sensitivity support routine QC of tobramycin batches.

Future Trends and Opportunities

• Integration of microfluidic HPAE-PAD platforms for high-throughput impurity profiling.
• Extension to other aminoglycoside antibiotics and carbohydrate analytes.
• Coupling with mass spectrometry for structural identification of trace impurities.
• Further waveform optimization for enhanced sensitivity and electrode longevity.

Conclusion

The combination of automated eluent generation, CarboPac PA1 columns, CR-ATC trapping, and disposable gold electrodes delivers a robust, sensitive, and reproducible HPAE-PAD method for quantifying tobramycin and its impurities. This approach meets stringent QC requirements and offers simplified operation for pharmaceutical laboratories.

Reference

1. Physicians’ Desk Reference, 44th Ed., Medical Economics Co., 1990.
2. European Pharmacopoeia, 5th Ed., Council of Europe, 2004.
3. Szunyog J. et al., J. Pharm. Biomed. Anal. 23, 891–896 (2000).
4. Polta J.A. et al., J. Chromatogr. 324, 407–414 (1985).
5. Dionex Application Note 61, 1989.
6. Statler J.A., J. Chromatogr. 92, 244–246 (1990).
7. Cheng J. et al., Anal. Chem. 75, 572–579 (2003).
8. Cheng J. et al., J. Chromatogr. A, 997, 73–78 (2003).
9. ICH Q2A, Validation of Analytical Procedures, 1994.
10. ICH Q2B, Validation of Analytical Procedures: Methodology, 1996.
11. FDA Guidance for Industry, Analytical Procedures and Method Validation, 2000.
12. FDA Reviewer Guidance, Validation of Chromatographic Methods, 1994.
13. FDA Guidance for Industry, Bioanalytical Method Validation, 2001.
14. FDA Guidance for Industry, Submitting Samples and Data for Methods Validation, 1987.
15. USP <1225> Validation of Compendial Methods, USP 27/NF 22 (2004).
16. USP <621> Chromatography, USP 27/NF 22 (2004).
17. Dionex Technical Note 21, Optimal Settings for Pulsed Amperometric Detection, 2004.
18. Dionex AAA-Direct Product Manual, 2004.