Impurity Analysis of Aminoglycoside Antibiotic Using the Agilent InfinityLab Poroshell 120 HILIC-Z Column with ELSD Detection

Significance of the Topic

Aminoglycoside antibiotics are essential agents in treating severe bacterial infections, but their high polarity and the presence of structurally similar impurities pose analytical challenges. Efficient impurity profiling ensures drug safety, quality control and regulatory compliance.

Aims and Overview of the Study

This study aimed to develop a robust liquid chromatographic method to separate the impurity neamine from the aminoglycoside antibiotic ribostamycin. The focus was on using a hydrophilic interaction chromatography (HILIC) approach with minimal system or solvent modifications compared to conventional reversed-phase methods.

Methodology and Instrumentation

The separation employed an Agilent InfinityLab Poroshell 120 HILIC-Z column (2.1 × 100 mm, 2.7 µm) with a mobile phase gradient of 100 mM ammonium acetate (A) and acetonitrile (B). The gradient ran from 65 % B to 55 % B over 5 minutes, followed by a 5 minute hold and re-equilibration. Evaporative light scattering detection (ELSD) was chosen to address low UV absorbance of analytes.

• Agilent 1260 Infinity II binary pump
• Agilent 1260 Infinity II vialsampler
• Agilent 1260 Infinity II multicolumn thermostat
• Agilent 1290 Infinity II ELSD detector
• OpenLAB chromatography data system

Sample preparation involved dissolving ribostamycin sulfate and neamine in water, diluting with acetonitrile to 50 % organic content, and spiking to represent both native and 2 % impurity levels.

Main Results and Discussion

The method achieved clear baseline separation of neamine and ribostamycin within a 9 minute run. Peak shapes were sharp and symmetric, and the zwitterionic HILIC-Z stationary phase provided excellent resolution. The ELSD parameters (nebulizer and evaporator at 40 °C, gas flow 1.6 SLM) delivered stable response for quantitation.

Benefits and Practical Applications

This HILIC-Z approach offers rapid, reproducible analysis of highly polar aminoglycosides and their impurities without the need for ion-pairing reagents. It streamlines QC workflows in pharmaceutical development and manufacturing, enabling reliable impurity detection and quantification.

Future Trends and Opportunities

Advancements may include coupling HILIC-Z with mass spectrometry for structural elucidation, miniaturized flow systems for higher throughput, and automated sample handling. Expanding the method to other polar drug classes and impurity types will further enhance its utility.

Conclusion

The developed HILIC-Z/ELSD method on the Agilent InfinityLab Poroshell 120 column enables effective separation and quantification of aminoglycoside impurities. Its simplicity, speed and robustness make it well suited for routine pharmaceutical impurity profiling.