UPLC/MS/MS Determination of Aminoglycoside Antibiotics in Meat and Milk

Significance of the topic

Aminoglycoside antibiotics are essential in veterinary medicine for treating livestock but pose analytical challenges due to their poor solubility in organic solvents and strong protein binding. Reliable detection in meat and milk is critical to ensure food safety and comply with regulatory maximum residue limits.

Study objectives and overview

The goal of this work was to develop and validate an ultraperformance liquid chromatography tandem mass spectrometry method for quantifying aminoglycoside residues in bovine milk and muscle at parts per billion levels. Key steps include aqueous extraction, solid phase cleanup, ion-pairing reversed-phase chromatography, and sensitive MS/MS detection.

Methodology and instrumentation

• Sample preparation – Homogenize 2 g meat or 10 mL milk; extract with 20 mL aqueous buffer containing trichloroacetic acid to precipitate proteins; adjust pH to 6.5.
• SPE cleanup – Use Oasis HLB 96-well plate; condition with methanol and water; load sample; wash with water; elute with acidified isopropanol mixture; add ion-pairing reagent.
• UPLC conditions – ACQUITY UPLC with HSS PFP column; gradient from 20 to 80 percent acetonitrile with heptafluorobutyric acid as ion-pairing agent; 0.5 mL/min flow; 35 °C.
• MS/MS detection – ACQUITY TQD in positive electrospray; multiple reaction monitoring transitions optimized for each analyte; cone and collision energies set for best sensitivity.

Results and discussion

Recovery in milk at 10 ppb ranged from 75 to 93 percent (RSD ≤ 13 percent) and at 200 ppb from 70 to 87 percent (RSD ≤ 14 percent). In muscle, recoveries at 50 ppb were 84 to 103 percent (RSD ≤ 12 percent) and at 1600 ppb 83 to 97 percent (RSD ≤ 14 percent). Matrix effects remained below 12 percent in milk and 30 percent in meat. Use of an aqueous eluent improved cleanup compared with methanol, and heptafluorobutyric acid provided optimal peak shape and sensitivity. Chromatograms demonstrated clear resolution of isobaric isomers and signal to noise above 100:1 at LOQ.

Practical benefits and applications

• Rapid extraction and cleanup enabling analysis of 20 samples per operator per day.
• Low detection limits suitable for regulatory monitoring of milk and meat.
• Straightforward SPE protocol with high reproducibility.
• Robust UPLC/MS/MS workflow adaptable to quality control and research laboratories.

Future trends and potential applications

• Extension to other food matrices and additional antibiotic classes.
• Integration of high resolution mass spectrometry for non targeted screening.
• Automation of sample preparation for higher throughput.
• Investigation of alternative chromatographic modes such as HILIC with improved peak performance.

Conclusion

The presented method combines aqueous extraction with TCA, SPE cleanup on Oasis HLB, ion-pairing UPLC on HSS PFP column, and sensitive MS/MS detection to achieve reliable quantitation of aminoglycosides in bovine milk and meat at low ppb levels. The protocol meets regulatory requirements and offers a practical solution for routine monitoring.

Reference

1. Screening and Confirmation for Aminoglycosides by UHPLC-MS-MS United States Department of Agriculture Food Safety and Inspection Service
2. Plozza T Trenerry VC Zeglinski P Nguyen H Johnstone P The Confirmation and Quantification of Selected Aminoglycoside Residues in Animal Tissue and Bovine Milk by Liquid Chromatography Tandem Mass Spectrometry International Food Research Journal 2011 18(3) 1077-1084