Analysis of Paromomycin by HPAE-IPAD

Significance of the Topic

Paromomycin is a broad spectrum aminoglycoside antibiotic used in human and veterinary medicine, notably for treatment of parasitic infections such as leishmaniasis, cryptosporidiosis, and amebiasis. Reliable quantitation of paromomycin is critical to ensure safety, efficacy, and compliance with pharmacopeial standards. Conventional microbial assays lack specificity, are time consuming, and do not provide detailed compositional information. Advanced chromatographic techniques with electrochemical detection offer a fast, sensitive, and specific alternative.

Objectives and Overview

This study presents a high performance anion exchange chromatography method with integrated pulsed amperometric detection (HPAE-IPAD) for assay of paromomycin in bulk and capsule formulations. The aims are to eliminate derivatization, improve specificity and throughput, meet current USP performance requirements, and demonstrate ruggedness across different instruments and consumables.

Methodology and Instrumentation

Instrumentation and conditions:

• Dionex ICS-3000 system with EG II KOH eluent generator and CR-ATC to supply 1.8 mM KOH eluent free of carbonate
• CarboPac PA1 analytical column (4 × 250 mm) with PA1 guard (4 × 50 mm)
• Integrated pulsed amperometric detection using an AAA-Direct waveform on disposable gold working electrodes and pH/Ag/AgCl reference electrode
• Flow rate 0.5 mL/min, temperature 30 °C, injection volume 20 µL

Sample preparation involved dissolving reference standard or capsule contents in water, centrifugation, and dilution to calibration range. Stock solutions were prepared fresh daily and working standards covered 1.25–10 µM.

Main Results and Discussion

Separation and detection:

• Baseline separation of paromomycin isomers I and II achieved within 9 min
• Typical retention times 5.1 min (isomer I) and 8.3 min (isomer II)

Analytical figures of merit:

• Linear dynamic range 1.25–10 µM with R2 ≥ 0.9991
• Limit of quantitation 0.10 µM (S/N = 10), estimated detection limit 0.03 µM (S/N = 3)
• Intra- and interday precision RSD ≤ 2 %
• Recoveries from spiked pharmaceutical formulation 96–107 %
• Method rugged across different columns, eluent cartridges, and analysts

A commercial capsule product labeled 250 mg paromomycin contained 279 ± 10 mg based on this assay, within USP specification of 90–125 % of label claim.

Benefits and Practical Applications

The HPAE-IPAD method allows rapid, specific analysis without derivatization, reducing sample preparation time. Automated KOH generation ensures stable eluent composition and reproducible retention. Disposable electrodes simplify maintenance and minimize variability. This approach can be adopted in QC laboratories for routine batch release and stability testing.

Future Trends and Opportunities

Potential developments include extending electrochemical detection to multi-aminoglycoside panels, coupling with mass spectrometry for structural confirmation, miniaturized or high throughput formats, and real-time online monitoring in manufacturing processes.

Conclusion

The presented HPAE-IPAD assay with automated KOH eluent generation provides a robust, accurate, and precise alternative to traditional microbial methods for paromomycin analysis. It meets USP requirements and demonstrates high sensitivity, reproducibility, and ease of use.

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