An Ultra Performance Liquid Chromatography Mass Spectrometry Method for the Analysis of Non-derivatized Gentamicin Components

Importance of the topic

Gentamicin is a broad-spectrum aminoglycoside antibiotic composed of several closely related components (C1, C1a, C2, C2a) with subtle structural differences yet distinct toxicity profiles. Precise analysis of each substance is crucial for ensuring drug safety, dosage accuracy, and regulatory compliance.

Objectives and study overview

This study aimed to develop a rapid, isocratic ultra-performance liquid chromatography–mass spectrometry (UPLC–MS) method for the direct analysis of non-derivatized gentamicin components, eliminating the need for derivatization or fluorinated pairing agents, and to demonstrate its applicability to ophthalmic formulations.

Methodology and instrumentation

• Sample preparation: Gentamicin sulfate and 0.3% ophthalmic solutions diluted to 100 µg/mL in MS-grade water.
• Chromatography: ACQUITY UPLC H-Class system with Atlantis Premier BEH Z-HILIC column (1.7 µm, 2.1×100 mm); isocratic mobile phase: 48% 80 mM ammonium formate (pH ~6.3) and 52% 0.1% formic acid in ACN; flow rate 0.8 mL/min; column 55 °C; injection 3 µL; run time ~10 min.
• Mass spectrometry: ACQUITY QDa detector in positive mode; capillary 0.8 kV; probe 600 °C; acquisition 300–630 Da.

Key results and discussion

The method achieved baseline separation of the four major gentamicin components with resolutions >1.39, retention time RSD <1%, and peak area RSD <5%. Optimization identified 80 mM ammonium formate and elevated column temperature as critical for sharp, reproducible peaks. Method robustness was confirmed across multiple column batches and maintained performance in complex ophthalmic matrices, with benzalkonium chloride eluting early and not interfering with target analytes.

Benefits and practical applications

• No derivatization or fluorinated pairing agents required, reducing sample preparation time and instrument contamination risk.
• Short isocratic run minimizes equilibration, boosting throughput.
• Method adaptable to separation and quantification of other aminoglycosides using the same UPLC–MS configuration.

Future trends and opportunities

Potential developments include extension to broader aminoglycoside panels, coupling with high-resolution mass spectrometry for enhanced specificity, automation of sample workflows, and exploration of greener mobile-phase systems.

Conclusion

A fast, reliable UPLC–MS protocol employing Atlantis Premier BEH Z-HILIC and QDa detection was established for non-derivatized gentamicin component analysis, offering reproducible, high-resolution separations ideal for pharmaceutical quality control.

References

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• Alkhateeb FL. et al., Waters Application Note, 2022.