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

Importance of the topic

Aminoglycoside antibiotics are frequently administered to livestock, poultry and dairy animals to treat bacterial infections. Due to their strong binding to animal tissues, residues in meat, milk or eggs can pose risks to human health. Regulatory bodies worldwide impose maximum residue limits (MRLs) for these compounds in different commodities. Analytical methods that can sensitively, quickly and reliably screen a broad spectrum of aminoglycosides across various matrices are essential for food safety laboratories and regulatory control.

Objectives and study overview

The study aimed to develop a single, high-throughput HILIC-MS/MS method for simultaneous quantification of multiple aminoglycosides in meat, milk and eggs without ion-pairing reagents. Key goals included:

• Reaching low limits of quantification (LOQs) covering relevant MRLs from Europe, Japan and the USA.
• Ensuring robust extraction recoveries and minimal matrix effects to avoid matrix-matched calibration.
• Implementing a rapid quantitative screening workflow and an identification protocol based on multi-MRM spectrum mode for confirmatory analysis.

Materials and methodology

Sample preparation:

• Five grams of homogenized meat or mixed eggs (or 10 mL milk) spiked with internal standard (ribostamycin).
• Extraction with ammonium acetate/EDTA/NaCl/trichloroacetic acid solution, followed by centrifugation and repeat extraction.
• pH adjustment to 6.5 ± 0.25 and cleanup by mixed-mode SPE in 96-well plates; elution with 10 % formic acid.
• Final dilution (1:5) in mobile phase A before injection.

Analytical conditions:

• Chromatography: HILIC on an amide column (100 × 2.1 mm, 3 µm) at 50 °C with water/ammonium formate/acid (A) and acetonitrile (B), ultra-fast gradient for screening (4.5 min) and extended gradient for confirmation (13 min).
• Mass spectrometry: Triple quadrupole with heated ESI in positive mode; two MRM transitions per compound for quantification, and up to 15 MRM transitions for spectrum generation in confirmatory mode.

Instrumentation used

• Liquid chromatograph: Nexera X2 (Shimadzu).
• HILIC column: Inertsil Amide 3 µm, 100 × 2.1 mm (GL Sciences).
• Mass spectrometer: LCMS-8060 triple quadrupole (Shimadzu) with heated ESI source.
• Automation: Extrahera SPE workstation (Biotage) with WCX Express 96-well plates (30 mg sorbent).

Main results and discussion

Calibration and sensitivity:

• Calibration ranges combined all relevant MRLs; LOQs set at 1/10 of the lowest MRL or the practical S/N>10 limit, and upper levels at MRL+50 %. Range spanned from 1.3 to 245 µg/kg depending on compound.
• Typical chromatograms at LOQs showed clear, well-resolved peaks for all target analytes.

Recovery and matrix effect:

• Spiking experiments at 100 ng/g in meat, egg and milk matrices yielded mean recoveries above 90 % for all analytes.
• No detectable matrix interference in blank samples and consistent recoveries across matrices, confirming efficient extraction and cleanup.

Identification using multi-MRM spectrum mode:

• Fifteen MRM transitions per compound were acquired to build an optimized fragment spectrum.
• Software-driven merging against a reference library provided unambiguous compound identification.
• Ultrafast MRM acquisition ensured no sensitivity loss when increasing transition number.

Benefits and practical applications of the method

• One method covers all major food matrices (meat, milk, eggs) and a wide panel of aminoglycosides, aligning with global regulatory requirements.
• High-throughput sample preparation and rapid chromatography enable large-scale screening.
• Excellent recoveries and low matrix effects remove the need for matrix-matched calibration.
• Multi-MRM spectrum mode allows confident confirmation without additional reagents or columns.

Future trends and potential applications

Expected developments include:

• Extension of the HILIC-MS/MS workflow to emerging polar veterinary drugs and transformation products.
• Integration with high-resolution mass spectrometry for non-target screening and retrospective data mining.
• Automation enhancements in sample preparation to further increase throughput and reproducibility.
• Expansion of spectral libraries and machine-learning based identification to improve specificity in complex matrices.

Conclusion

The presented HILIC-MS/MS method with multi-MRM spectrum mode delivers a sensitive, robust and high-throughput solution for both quantitative screening and confirmatory identification of aminoglycoside residues in animal-derived foods. Its broad applicability, streamlined workflow and compatibility with regulatory standards make it a valuable tool for food safety monitoring and quality control.