Simultaneous Separation and Quantification of Aminoglycosides Using Liquid Chromatography and Mass Spectrometry

Significance of the topic

Aminoglycoside antibiotics remain critical in treating serious Gram-negative infections, but their physicochemical properties—high polarity, absence of UV-absorbing chromophores and nonvolatility—pose significant analytical challenges. Traditional assays often require time-consuming derivatizations or ion-pairing reagents, which increase method complexity, cost and instrument maintenance. A streamlined, high-throughput approach is therefore highly valuable for both research and routine quality control laboratories.

Objectives and study overview

This work aimed to develop a fast, robust and user-friendly liquid chromatography–mass spectrometry (LC–MS) method for simultaneous separation and quantification of multiple aminoglycosides using a compact single-quadrupole detector. Key goals included eliminating derivatization steps, reducing equilibration times and demonstrating quantitation suitable for QC environments.

Methodology and used instrumentation

The method employed Hydrophilic Interaction Liquid Chromatography (HILIC) on an Atlantis Premier BEH Z-HILIC column (2.1×100 mm, 1.7 µm) maintained at 50 °C. A quaternary UPLC system delivered a gradient of mobile phase A (0.1% ammonium formate in water, pH 3.0) and B (0.1% formic acid in acetonitrile) at 0.5 mL/min. Injection volume was 3 µL, with samples held at 5 °C. Detection was performed using an ACQUITY QDa single-quadrupole mass detector with electrospray ionization in positive mode (capillary voltage 0.8 kV, source 400 °C, cone 15 V), operated in both full scan (300–630 Da) and Selected Ion Recording (SIR) modes. Data acquisition and processing used Empower 3 CDS.

Main results and discussion

An optimized gradient over 10 minutes achieved baseline separation of sixteen aminoglycosides, of which six compounds yielded strong QDa responses: spectinomycin, streptomycin, amikacin, kanamycin, neamine and sisomicin. Transitioning from full scan to SIR dramatically enhanced signal‐to‐noise and specificity by monitoring individual m/z values. Calibration over 0.5–250 µg/mL returned linear regression coefficients (R2) above 0.99 for all six analytes. Limits of detection reached as low as 0.5 µg/mL, with corresponding limits of quantification meeting QC requirements.

Benefits and practical applications

• Single HILIC–MS assay replaces multiple derivatization-based methods.
• No need for fluorinated ion-pairing agents or UV derivatization.
• Rapid 10-minute run time increases throughput.
• Compact QDa detector offers cost-effective, low-maintenance operation suitable for QC labs.

Future trends and applications

Integration of compact mass detectors with advanced stationary phases may expand to other non‐chromophoric or highly polar drug classes. Automation and miniaturization could further streamline routine antibiotic assays in pharmaceutical, food safety and clinical laboratories. Combining high sensitivity SIR with multiplexed UPLC workflows promises high-throughput screening of broad antibiotic panels.

Conclusion

A straightforward UPLC–QDa method using a zwitterionic HILIC phase provides rapid, sensitive and linear quantification of key aminoglycoside antibiotics without derivatization. Its simplicity and robust performance make it well suited for routine QC environments, enabling efficient antibiotic monitoring with minimal instrument upkeep.

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