Extraction and Clean-up of Aminoglycoside Antibiotics from Chicken Meat Using EVOLUTE® EXPRESS WCX Solid Phase Extraction Columns with Analysis by LC-MS/MS

Extraction and Clean-up of Aminoglycoside Antibiotics from Chicken Meat

Significance of the Topic

The use of aminoglycoside antibiotics in poultry farming can lead to residual drug levels in meat and contribute to antimicrobial resistance. Reliable methods for monitoring these residues are essential to ensure food safety, comply with regulatory limits and protect public health.

Objectives and Study Overview

This application note describes the development of a mixed-mode weak cation exchange solid-phase extraction (SPE) followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the quantification of gentamicin, streptomycin, spectinomycin and neomycin in chicken meat. The goal was to achieve high recovery, minimal matrix effects and robust performance across the relevant concentration range.

Methodology

• Sample Pretreatment: Homogenize 5 g of chicken meat, spike with internal standards, extract twice with 20 mL of 10 mM NH₄OAc, 0.4 mM EDTA, 0.5% NaCl and 2% trichloroacetic acid (TCA), vortex, shake, centrifuge and combine supernatants. Filter and adjust pH to 6.5 ± 0.25.
• SPE Cleanup: Condition EVOLUTE EXPRESS WCX (500 mg/6 mL) columns with methanol and water. Load the pretreated extracts (40 mL), wash interferences with water (pH 6.5), elute analytes with 25% formic acid in water (6 mL), dilute to 10 mL and filter through a 0.2 µm disc.
• Chromatography: Use a Waters Acquity UPLC BEH Amide column (1.7 µm, 2.1 × 50 mm) with a gradient of mobile phase A (20 mM heptafluorobutyric acid in H₂O/ACN 95/5) and B (20 mM HFBA in ACN), at 0.2 mL/min, 40 °C, injection volume 7.5 µL.
• Detection: Operate a Xevo TQD triple quadrupole mass spectrometer in positive electrospray ionization mode. Monitor analytes using optimized multiple reaction monitoring (MRM) transitions with specific cone voltages and collision energies.

Used Instrumentation

• EVOLUTE EXPRESS WCX SPE columns, 500 mg/6 mL (Biotage)
• Waters Acquity UPLC system with BEH Amide column
• Waters Xevo TQD triple quadrupole mass spectrometer with ESI source
• Centrifuge, vortex mixer, pH meter and filtration apparatus

Main Results and Discussion

The method exhibited excellent linearity (r² > 0.990) over the target range (100–500 ppb, per USDA-FSIS guidelines). Limits of quantification were 33 ppb for gentamicin, 382 ppb for streptomycin, 75 ppb for spectinomycin and 361 ppb for neomycin. Average recoveries ranged from 80% to 120% across multiple fortification levels, and matrix suppression was effectively controlled by the WCX cleanup.

Benefits and Practical Applications

This workflow delivers a rapid and reliable approach for routine monitoring of aminoglycoside residues in poultry. The mixed-mode SPE simplifies sample preparation, while UPLC-MS/MS ensures high sensitivity and specificity, supporting compliance testing in food safety laboratories.

Future Trends and Opportunities

Advances in SPE sorbent design, fully automated high-throughput platforms and coupling to high-resolution mass spectrometers will enhance sensitivity, broaden analyte coverage and streamline workflows. Expanding the method to include additional antibiotic classes and metabolite profiling is also a promising direction.

Conclusion

The presented SPE-LC-MS/MS method provides an effective solution for the extraction and quantification of aminoglycoside antibiotics from chicken meat, meeting performance criteria for accuracy, precision and sensitivity in regulatory and research settings.

Reference

Biotage 2020. Extraction and Clean-up of Aminoglycoside Antibiotics from Chicken Meat. Application Note AN833.V.1.