Analysis of Paromomycin by HPAE-IPAD

Importance of the Topic

Paromomycin is an aminoglycoside antibiotic widely used in human and veterinary medicine to treat bacterial and parasitic infections, including leishmaniasis and amebiasis.
Accurate quantitation and identification of its active components are critical for drug safety, efficacy, and regulatory compliance.
Conventional microbial assays lack specificity, accuracy, and speed, while traditional HPLC methods often require time-consuming derivatization.

Objectives and Study Overview

The study aimed to develop a robust high-performance anion-exchange chromatography method with integrated pulsed amperometric detection (HPAE-IPAD) for precise determination of paromomycin in pharmaceutical formulations.
Key goals included eliminating derivatization, improving sensitivity and throughput, meeting USP compendial requirements, and evaluating performance in a commercial capsule product.

Methodology and Instrumentation

An electrolytically generated potassium hydroxide eluent (1.8 mM) was delivered at 0.5 mL/min using an EGC II KOH cartridge with a continuously regenerated anion trap to eliminate carbonate interference.
Separation was achieved on CarboPac PA1 analytical and guard columns at 30 °C with a 20 µL injection.
Detection employed a disposable AAA Au working electrode under a six-potential AAA-Direct waveform in integrated pulsed amperometric mode.
Calibration standards ranged from 1.25 µM to 10 µM; samples were prepared by dilution of bulk material and capsule suspensions.

Instrumentation

• Dionex ICS-3000 HPAE system with SP/DP pump and DC detector compartment
• Eluent generator EGC II KOH cartridge with CR-ATC trap and vacuum degas kit
• CarboPac PA1 analytical (4×250 mm) and guard (4×50 mm) columns
• AAA Au disposable working electrodes and pH/Ag/AgCl reference electrode
• Chromeleon chromatography workstation

Main Results and Discussion

• Baseline separation of paromomycin isomers I and II at ~5.1 min and ~8.3 min in a 16 min run, with minimal interference from baseline dips and oxygen peaks.
• Linearity over 1.25–10 µM (r² ≥ 0.9991); LOQ of 0.10 µM (S/N=10) and LOD of 0.03 µM (S/N=3).
• Intraday precision (n=3) RSD <2% for USP standard and ≤1% for drug substance and capsule; interday precision (n=9) RSD <2%.
• Spike recovery in commercial capsule at 0.5, 1.0, and 2.5 µM was 96–107%, confirming accuracy.
• Ruggedness across analysts, columns, and eluent cartridges yielded overall RSD <2% for standards and <3.6% for capsule assay.
• Assay of Humatin capsules yielded 279±10 mg paromomycin (3.7% RSD), within USP 90–125% specification.

Benefits and Practical Applications

• High specificity and sensitivity without chemical derivatization streamline analytical workflow.
• Automated eluent generation ensures reproducible carbonate-free hydroxide levels and stable retention times.
• Disposable electrodes simplify maintenance and enhance inter-laboratory consistency.
• Method complies with USP performance requirements for routine pharmaceutical quality control.

Future Trends and Opportunities

Potential developments include on-line sample preparation integration, coupling with mass spectrometry for structural confirmation, expansion to related aminoglycosides, and portable HPAE-IPAD systems for field analysis.

Conclusion

The described HPAE-IPAD method provides a rapid, accurate, and reproducible assay for paromomycin in pharmaceutical formulations, leveraging automated eluent generation and disposable electrodes to meet regulatory standards and support routine quality control.

References

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