Sensitive and Fast Measurement of Aminoglycoside Antibiotics in Milk, Meat or Eggs by HILIC-MS/MS and Identification using MRM Spectrum Mode

Significance of the Topic

Aminoglycoside antibiotics are widely used in livestock, raising concerns over residues in milk, meat, and eggs. Regulatory authorities impose strict maximum residue limits (MRLs) to protect human health. Due to their high polarity, these compounds are challenging to analyze by conventional reversed-phase chromatography. A versatile, high-throughput method capable of sensitive, multi-residue detection and reliable confirmation is essential for food safety laboratories.

Objectives and Study Overview

This study aimed to develop a single HILIC-MS/MS workflow for fast quantitative screening and definitive identification of a broad panel of aminoglycosides across diverse matrices. The method targets regulatory MRLs established in Europe, the USA, and Japan and combines a rapid screening protocol with a multi-transition MRM Spectrum mode for unambiguous confirmation.

Methodology and Instrumentation

• Sample preparation involved protein precipitation with trichloroacetic acid, EDTA chelation, centrifugation, and mixed-mode SPE cleanup in a 96-well format. Final extracts were diluted in 1% formic acid.
• Chromatographic separation used hydrophilic interaction LC on an amide column at 50 °C. Two gradients were optimized: a 3.5 min fast screening gradient and an 11 min confirmation gradient.
• Detection was performed by tandem quadrupole MS with a heated ESI source. Fast MRM acquisition (2 transitions per compound) enabled screening, while MRM Spectrum mode (15 transitions per compound) provided spectral confirmation.

Used Instrumentation

• UHPLC system: Nexera X2
• Chromatography column: Inertsil Amide 3 µm, 100×2.1 mm
• MS detector: LCMS-8060 triple quadrupole
• Sample homogenizer: Retsch Grindomix GM200
• SPE workstation: Biotage Extrahera with WCX Express 96-well plates

Main Results and Discussion

• Limits of quantification in the low pg/mL range on-column were achieved for all target analytes, covering MRLs down to 0.01 mg/kg.
• Calibration curves spanned each regulatory MRL range plus 50%, with lowest points at one-tenth of MRLs or practical detection limits, ensuring compliance across jurisdictions.
• Recovery studies in fortified meat, milk, and egg samples yielded mean recoveries above 80% with relative standard deviations below 14%, demonstrating robustness and minimal matrix effects.
• MRM Spectrum mode provided unambiguous compound identification by matching 15 fragmentation transitions against a spectral library without sensitivity loss relative to the fast screening method.

Benefits and Practical Applications

• Single-method solution for quantitative screening and confirmation of a broad aminoglycoside panel across multiple food matrices.
• High throughput with run times under 4 min for routine screening and under 12 min for confirmatory analysis.
• Avoids ion-pairing reagents, simplifying system maintenance and method sharing.
• Compatible with standard MRLs in major markets, facilitating regulatory compliance and efficient laboratory workflows.

Future Trends and Potential Applications

• Integration with high-resolution mass spectrometry may further enhance selectivity and enable non-targeted screening.
• Automation of sample preparation and data processing can increase throughput and reproducibility.
• Expansion to additional antibiotic classes and emerging contaminants will broaden food safety monitoring capabilities.
• Development of portable or miniaturized platforms could support on-site testing in supply chains.

Conclusion

The presented HILIC-MS/MS method offers a sensitive, rapid, and robust approach for multi-residue analysis of aminoglycoside antibiotics in milk, meat, and eggs. Coupling fast MRM screening with library-based MRM Spectrum confirmation streamlines regulatory compliance and ensures reliable detection across diverse food matrices. This workflow enhances laboratory efficiency and supports comprehensive food safety monitoring.