Highly sensitive simultaneous analysis of tetracyclines and β-lactams antibiotics in edible meat by LC/MS/MS

Importance of the Topic

Veterinary antibiotics such as tetracyclines and β-lactams are extensively used in livestock to treat and prevent infections. Excessive use can lead to antibiotic resistance and pose risks to human health due to residue in edible meat. Regulatory agencies worldwide have established maximum residue limits for these compounds, driving the need for sensitive, selective, and high-throughput analytical methods to ensure food safety.

Objectives and Study Overview

This study aimed to develop a streamlined sample pretreatment and simultaneous LC-MS/MS method for quantifying 20 veterinary drugs—including tetracyclines, penicillins, and cephems—in chicken breast and thigh. The protocol targets high sensitivity, robust linearity, and reliable recovery to support routine monitoring in food quality control.

Methodology

Sample Pretreatment:

• Homogenize 2.5 g meat and spike with standards (final 2 ng/mL).
• Add acetonitrile for protein precipitation, vortex, then hexane for lipid removal.
• Extract analytes with McIlvaine buffer (0.05 mol/L EDTA, pH 6.5).
• Centrifuge and load supernatant onto InertSep PLS-2 SPE cartridge.
• Wash cartridge with water, elute with 50% acetonitrile, dilute to volume.

Instrumentation

The analysis employed a Shimadzu Nexera X3 UHPLC coupled to an LCMS-8060NX triple quadrupole.

• Column: Shim-pack Scepter C18-120 (100 × 2.1 mm, 1.9 µm).
• Mobile phases: A) 2 mM ammonium formate + 0.1% formic acid in water; B) methanol.
• Gradient: B from 5% to 70% (0–8 min), 95% at 10–15 min, return to 5% by 20 min.
• Flow rate: 0.4–0.6 mL/min, column temperature 40 °C.
• MS: ESI positive in MRM mode; DL 200 °C, heat block 450 °C, interface 400 °C.

Main Results and Discussion

All 20 compounds were baseline-separated within a 10-minute run. Matrix-matched calibration curves exhibited excellent linearity (R2≥0.996). Accuracy ranged from 80.7% to 114.8%, and repeatability at 2 ng/mL showed RSD ≤7.3%. Recovery tests in chicken breast achieved 70–120% for 80% of analytes; in thigh, 75% of compounds fell within this range. Amoxicillin showed lower recovery due to its high polarity and limited SPE retention.

Benefits and Practical Applications

The method offers:

• Simultaneous quantification of multiple antibiotic classes.
• High throughput suitable for routine screening in QA/QC labs.
• Robust performance under regulatory residue limits.

Future Trends and Applications

Advancements may include:

• Extension to other food matrices (milk, eggs, fish).
• Integration of high-resolution mass spectrometry for non-targeted screening.
• Automation of sample preparation and data processing.
• Adoption of microflow LC and novel SPE sorbents for enhanced sensitivity.

Conclusion

The presented LC-MS/MS protocol combining efficient pretreatment with a rapid UHPLC run delivers sensitive, linear, and reproducible quantification of tetracyclines and β-lactams in edible meat. It fulfills regulatory requirements and supports streamlined monitoring in food safety laboratories.

Reference

• BUNSEKI KAGAKU, Vol. 66, No. 5, pp. 369–374 (2017)
• Food Hygiene and Safety Science, Vol. 61, No. 4, pp. 109–118 (2020)