Analysis of Aminoglycosides in Foods Using a Zwitterionic Stationary Phase and Liquid Chromatography-Tandem Mass Spectrometry

Significance of the Topic

Ensuring the safety of food products requires sensitive and accurate detection of aminoglycoside antibiotics in complex matrices. Residues of these highly polar and toxic compounds can pose health risks, contribute to allergic reactions, and promote antimicrobial resistance. Regulatory bodies worldwide have established maximum residue limits (MRLs) for aminoglycosides in animal-derived foods. A robust analytical method that meets these standards is essential for routine monitoring and consumer protection.

Objectives and Overview of the Study

This work aimed to develop and validate a hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC-MS/MS) method for simultaneous analysis of 17 aminoglycosides in milk, muscle (beef and pork), liver, and honey. Key goals included: optimizing chromatographic conditions on an Atlantis Premier BEH Z-HILIC column; establishing a simple extraction and cleanup using trichloroacetic acid (TCA) and Oasis HLB solid‐phase extraction (SPE); and demonstrating method performance against regulatory criteria.

Methodology and Instrumentation

• Sample preparation: Homogenized food samples were extracted with 2 % TCA, 10 mM ammonium acetate (pH 4.0) containing EDTA and NaCl. Extracts were adjusted to pH 6.75 and cleaned up on Oasis HLB cartridges without reconstitution.
• Chromatography: Atlantis Premier BEH Z-HILIC column (2.1 × 150 mm, 2.5 µm) at 50 °C. Gradient elution from 20 % to 95 % aqueous mobile phase (20 mM ammonium formate, pH 3.0) over 5 min, with acetonitrile/0.1 % formic acid as organic phase. Flow rate 0.7 mL/min, injection volume 6 µL, run time 10 min.
• Mass spectrometry: Xevo TQ-S micro in positive electrospray mode. Multiple reaction monitoring (MRM) transitions optimized to avoid interferences (e.g., alternative transitions for spectinomycin and dihydrostreptomycin). Source at 150 °C, desolvation at 600 °C, cone gas 50 L/h, desolvation gas 1000 L/h.

Main Results and Discussion

• Chromatographic optimization: Higher aqueous content reduced retention times, while pH 3.0 and 20 mM ammonium formate delivered optimal peak shape, resolution, and signal intensity for all 17 analytes in 10 min.
• Matrix effects: Ranged from strong ion suppression (<20 %) to enhancement (>170 %), necessitating matrix-matched calibration.
• SPE recovery: Most aminoglycosides showed 40–100 % recoveries at 1000 µg/kg; kasugamycin had low recovery due to its low pKa.
• Method performance: Limits of quantification were below established MRLs. Linearity (R²>0.99) was achieved over 4–6 calibration levels. Intra-day retention time precision was <1 % RSD. Accuracy in spiked samples (200 µg/kg) ranged from 86 % to 122 % with SD 4–23 %.

Benefits and Practical Applications of the Method

• Reliable separation of 17 aminoglycosides with no ion-pairing reagents or high buffer concentrations.
• High sensitivity and specificity meeting global regulatory requirements for milk, meat, liver, and honey.
• Streamlined sample preparation with minimal handling and no reconstitution step.
• Compatibility with routine high-throughput analysis in quality assurance, veterinary control, and research laboratories.

Future Trends and Possibilities for Application

• Extension to additional food matrices and environmental samples of veterinary drug residues.
• Development of automated, high-throughput SPE workflows and on-line sample cleanup.
• Integration with high-resolution mass spectrometry for broader screening and identification of novel aminoglycoside derivatives.
• Application of green chemistry principles to reduce solvent consumption and waste.
• Implementation of data-driven quality control and AI-based prediction of matrix effects.

Conclusion

A rapid 10 min HILIC-MS/MS method using Atlantis Premier BEH Z-HILIC columns and Oasis HLB SPE effectively separates and quantifies 17 aminoglycosides in diverse food samples. The protocol offers robust sensitivity, accuracy, and precision well below regulatory MRLs, providing a powerful tool for food safety laboratories.

Reference

1. U.S. Food and Drug Administration. Tolerances for Residues of New Animal Drugs in Food. 21 CFR § 556 (2020).
2. European Commission. Commission Regulation (EU) No 37/2010 on Pharmacologically Active Substances and Their Classification Regarding MRLs in Foodstuffs. Official Journal of the European Union L15 (2010).
3. Ministry of Agriculture and Rural Affairs of China. National Food Safety Standard GB 31650-2019 on Maximum Residue Limits for Veterinary Drugs in Foods, effective April 1, 2020.
4. The Japan Food Chemical Research Foundation. Positive List System – Antibiotics.
5. Codex Alimentarius. CX/MRL 2-2018. MRLs and Risk Management Recommendations for Veterinary Drug Residues. FAO/WHO.
6. USDA FSIS. Screening for Aminoglycosides by LC/MS-MS, CLG-AMG4.03 (2020).
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