Determination of Streptomycin and Impurities Using HPAE-PAD

Significance of the Topic

Streptomycin is a critical aminoglycoside antibiotic used in clinical settings. Ensuring its purity and quantifying trace impurities are essential for patient safety and regulatory compliance. Traditional UV and refractive index detectors often fail to detect non-chromophoric aminoglycosides and their degradation products at low levels. High-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAE-PAD) offers sensitive, selective analysis without derivatization, filling a key gap in pharmaceutical quality control.

Objectives and Study Overview

This application note demonstrates a validated HPAE-PAD method, aligned with the United States Pharmacopeia (USP) monograph for streptomycin sulfate, to:

• Separate streptomycin A from its known impurities and late-eluting degradation products;
• Assess system suitability, precision, linearity, detection limits, and ruggedness;
• Evaluate purity in fermentation broth and commercial drug substances;
• Demonstrate feasibility for routine QC and compliance with ICH guidelines.

Instrumentation Used

• Thermo Scientific Dionex ICS-3000 Ion Chromatography system with dual-pump gradient and vacuum degassing
• Dionex CarboPac PA1 analytical column (4 × 250 mm) with guard column
• Electrochemical detector (PAD) with Ag/AgCl reference and disposable gold working electrodes
• Autosampler with diverter valve and 20-µL injection loop
• Eluent organizer, eluent bottles, 0.2-µm nylon filters, vacuum pump

Methodology

Analyses employed an isocratic 70 mM NaOH mobile phase at 0.5 mL/min, 30 °C column temperature, and pulsed amperometric detection using a carbohydrate waveform. Calibration covered 0–4000 pmol injections, with a target 41 µM concentration. System suitability was confirmed by resolution between streptomycin A and its thermal degradation peak (>5.8). Detection limits were determined at 1.7 pmol (LOD) and 5.6 pmol (LOQ). Spike recovery in diluted YPD fermentation broth (1000×) yielded 82.6% and 92.9% for 10 µM and 41 µM spikes.

Main Results and Discussion

Chromatograms show baseline separation of up to 20 impurities, including a late-eluting degradation product at 160 min. Streptomycin A exhibited excellent linearity (r2 > 0.999 over 80–1600 pmol), peak asymmetry <1.4, and efficiency >2200 plates. Precision (n = 34) gave <0.6% RSD for retention time and <2% for peak area. Method ruggedness was confirmed under ±10% variations in NaOH concentration, flow rate, and temperature, with minimal impact on key parameters. Comparative analysis of USP and commercial drug lots revealed total impurity levels of 9–15% before and after thermal treatment.

Benefits and Practical Applications

• High sensitivity and specificity for aminoglycosides without derivatization;
• Robust quantitation of major and minor impurities to meet pharmacopeial criteria;
• Rapid throughput with single-step isocratic elution;
• Direct analysis of fermentation samples, supporting process monitoring and QC.

Future Trends and Opportunities

Advances in microfabricated disposable electrodes may further improve reproducibility. Integration with parallel IC conductivity detection can profile counter-ions and enhance potency determination. Adapting gradient PAD methods could shorten run times and resolve even more complex impurity profiles.

Conclusion

This HPAE-PAD approach provides a validated, sensitive, and reliable assay for streptomycin A and its impurities. It meets USP and ICH guidelines, supports both drug substance and fermentation-derived sample analysis, and enhances pharmaceutical QC for aminoglycoside antibiotics.

References

1. United States Pharmacopeia, USP 30–NF 25, Monograph Streptomycin Sulfate.
2. Thermo Scientific Dionex Application Note 19033, Disposable Gold Electrodes for Aminoglycoside Analysis, 2003.
3. ICH Q2(R1) Validation of Analytical Procedures: Text and Methodology.
4. Dionex CarboPac PA1 Column Product Manual.
5. Hanko V.; Rohrer J. Determination of Streptomycin Using HPAE-PAD. Thermo Fisher Scientific Application Note 181, 2007.