LC-MS/MS ANALYSIS OF AMINOGLYCOSIDES IN FOODS BY ZWITTERIONIC HYDROPHILIC INTERACTION CHROMATOGRAPHY

Significance of the Topic

Aminoglycoside antibiotics play a critical role in treating Gram-negative bacterial infections and are sometimes applied as growth promoters in food-producing animals. Monitoring their residues in food matrices such as honey, milk, muscle, and liver is essential for food safety and regulatory compliance. The polar nature of these compounds demands specialized analytical approaches to achieve sensitive, accurate detection at low concentration levels.

Aims and Study Overview

This work aims to develop and validate a rapid and robust LC-ESI-MS/MS method for the simultaneous screening and quantification of 15 common aminoglycosides in various food matrices. A novel zwitterionic HILIC stationary phase based on sulfobetaine chemistry (Atlantis Premier BEH Z-HILIC) was evaluated to improve separation efficiency and peak shape compared with conventional HILIC columns. The study also optimized sample extraction and clean-up procedures using solid-phase extraction cartridges.

Methodology

Sample Preparation and Extraction

• Sample types: honey, milk, bovine/swine muscle, poultry liver.
• Extraction solvent: 10 mM ammonium acetate, 0.4 mM EDTA, 0.5% NaCl, 2% trichloroacetic acid at pH 4.0.
• Procedure: Vortex mixing, refrigeration, centrifugation, pH adjustment to 6.5–7.0, followed by SPE clean-up.
• SPE cartridges tested: Oasis HLB, Oasis WCX, Sep-Pak Accell Plus CM; Oasis HLB provided optimal recoveries.

Instrumentation Used

• Liquid Chromatograph: ACQUITY Premier System with Atlantis Premier BEH Z-HILIC column (2.5 µm, 2.1 × 150 mm) at 50 °C.
• Mass Spectrometer: Xevo TQ-S micro with positive electrospray ionization; desolvation temperature 600 °C, source temperature 150 °C.
• Mobile phases: A – 20 mM ammonium formate in water (pH 3.0); B – 0.1% formic acid in acetonitrile.
• Gradient: 10% A to 75% A in 1 min, to 85% A in 4 min, hold for 3 min; flow rate 0.7 mL/min; run time 10 min.

Results and Discussion

The Atlantis Premier BEH Z-HILIC column achieved baseline separation of all 15 aminoglycosides within 10 minutes. Systematic evaluation of mobile phase pH and buffer concentration revealed optimal retention, peak symmetry, and signal intensity at pH 3.0 and 20 mM ammonium formate. SPE recoveries using Oasis HLB cartridges ranged from 80% to 120% across matrices. Method performance demonstrated excellent sensitivity with limits of quantification between 10 and 100 µg/kg, and linearity coefficients (R2) above 0.99 in matrix-matched calibration across muscle, milk, liver, and honey.

Benefits and Practical Applications

• High throughput analysis with a 10-minute run time.
• Low limits of quantification support compliance monitoring below maximum residue limits.
• Robust SPE clean-up minimizes matrix interferences.
• Applicable to a wide range of food matrices in routine QA/QC laboratories.

Future Trends and Applications

• Integration of high-resolution mass spectrometry to further improve specificity.
• Automation of sample preparation workflows to increase laboratory throughput.
• Expansion to detect emerging aminoglycoside derivatives and related polar antibiotics.
• Application of ion mobility separation to resolve isomeric forms such as gentamicin C1A and C2.

Conclusion

The optimized LC-ESI-MS/MS method employing a novel BEH Z-HILIC column and Oasis HLB SPE offers a fast, accurate, and sensitive solution for aminoglycoside analysis in diverse food products. The approach meets stringent regulatory requirements and enhances routine monitoring capabilities.

References

• Yang J and Rainville P. LC-MS/MS Analysis of Aminoglycosides in Foods by Zwitterionic Hydrophilic Interaction Chromatography. Waters Corporation, 2021.