ANALYSIS OF AMINOGLYCOSIDES IN FOODS BY LC-MS/MS USING A ZWITTERIONIC STATIONARY PHASE

Significance of the topic

Aminoglycoside antibiotics are critical for treating Gram-negative bacterial infections and have been used as growth promoters in food-producing animals. Their polar nature and widespread use in food animals necessitate reliable analytical methods to monitor residues in complex matrices such as milk, honey, eggs, muscle, liver, and other tissues. Accurate detection at low concentrations ensures consumer safety and regulatory compliance.

Objectives and overview

The study aimed to develop a robust LC-ESI-MS/MS method for quantifying 15 aminoglycosides in various food matrices using a zwitterionic hydrophilic interaction liquid chromatography (Z-HILIC) stationary phase and a mass spectrometry-friendly mobile phase. Key goals included:

• Minimizing buffer concentration while maintaining chromatographic performance.
• Optimizing sample extraction and clean-up to achieve high recoveries across diverse foods.
• Establishing method sensitivity, linearity, and quantitation limits suitable for monitoring at or below maximum residue limits.

Methodology and instrumentation used

• Chromatography system: Arc™ Premier LC System equipped with Waters Atlantis Premier BEH Z-HILIC Column (2.5 µm, 2.1 × 150 mm) maintained at 50 °C.
• Mobile phases: A = water with 20 mM ammonium formate, adjusted to pH 3.0; B = acetonitrile with 0.1% formic acid; gradient elution over 10 min at 0.7 mL/min flow rate.
• Mass spectrometry: Xevo™ TQ-S micro triple quadrupole in positive electrospray mode; source temperature 150 °C, desolvation temperature 600 °C, capillary voltage 1.5 kV.
• Data acquisition: MassLynx™ V4.2 software for instrument control and quantitation.
• Sample preparation: Solid-phase extraction using Oasis™ HLB cartridges (6 mL, 500 mg) for cleanup of spiked food samples (1 µg/g).

Main results and discussion

The study investigated the effects of mobile phase pH and buffer concentration on aminoglycoside retention, peak shape, and signal intensity. Key findings included:

• Optimal chromatographic behavior at pH 3.0, yielding sharp peaks and reproducible retention times for all analytes.
• A buffer concentration of 20 mM ammonium formate provided a balance between ionization efficiency and chromatographic resolution.
• Oasis HLB cartridges achieved recoveries above 80% for 14 of the 15 aminoglycosides across milk, honey, beef, pork, and liver matrices.
• Limits of quantitation ranged from 10 to 100 µg/kg depending on analyte and matrix; all calibration curves demonstrated excellent linearity (R2 > 0.98) over 10–2500 µg/kg.

Benefits and practical applications

The developed method offers several advantages for routine food safety monitoring:

• High throughput: 10-minute runtime and simple gradient program enable rapid analysis of multiple samples.
• Wide applicability: Effective cleanup and detection across diverse animal-derived matrices.
• Regulatory relevance: Sensitive quantitation below typical maximum residue limits for aminoglycosides.
• MS-friendly mobile phase: Reduced salt content minimizes ion source contamination and extends instrument uptime.

Future trends and application possibilities

Potential developments and extensions of this work include:

• Automation of sample preparation to further increase laboratory throughput.
• Expansion of the target analyte list to include newer aminoglycoside derivatives or related polar veterinary drugs.
• Integration with high-resolution mass spectrometry for non-target screening and confirmation of unexpected residues.
• Miniaturization of sample prep using micro-SPE or in-tube solid-phase extraction to reduce solvent use and waste.

Conclusion

The LC-ESI-MS/MS method employing a zwitterionic HILIC column and a low-buffer mobile phase demonstrates robust performance for the simultaneous analysis of 15 aminoglycosides in food matrices. Excellent recoveries, sensitivity, and linearity make this approach suitable for routine screening and regulatory compliance.

References

• Yang J, Alkhateeb F, Rainville P. Analysis of aminoglycosides in foods by LC-MS/MS using a zwitterionic stationary phase. Waters Corporation; 2022.