Detection of Chloramphenicol in Honey Using Automated Solid-Phase-Extraction and HPLC-MS/MS-Detection

Importance of the Topic

Honey is widely consumed worldwide and strict control of antibiotic residues is essential to protect public health and ensure compliance with EU regulations. Chloramphenicol, a potent broad-spectrum antibiotic banned in food-producing animals due to severe side effects and potential genotoxicity, has been repeatedly detected in imported honey. Reliable and high-throughput analytical workflows are therefore needed for routine monitoring.

Objectives and Study Overview

This study presents a fully automated solid-phase extraction (SPE) workflow using the FREESTYLE robotic system coupled to HPLC-MS/MS for the quantification of chloramphenicol in honey. The method aims to compare automated and manual SPE in terms of recovery, reproducibility, and sample throughput.

Methodology and Instrumentation

Samples: 5 g blossom honey spiked with chloramphenicol and isotopic internal standard.

Sample Preparation:

• Dilution in water, addition of internal standard.
• Liquid–liquid extraction with ethyl acetate.
• Evaporation under nitrogen, reconstitution in water.

Automated SPE (FREESTYLE SPE):

• Conditioning: methanol, then water.
• Loading: 9 mL sample.
• Washing and drying steps.
• Elution with ethyl acetate/methanol (80:20, v/v).
• Final evaporation and reconstitution.

HPLC-MS/MS Analysis:

• Column: EC 150/2 NUCLEODUR π2, 5 µm; gradient 5–95 % acetonitrile in water.
• Flow rate: 0.3 mL/min; injection 5 µL; column at 35 °C.
• Detector: AB Sciex QTRAP 5500, ESI negative, MRM transitions m/z 320.9→152.0/256.0 for chloramphenicol.

Main Results and Discussion

Recovery and Precision:

• Automated SPE recoveries: 86.8 ± 4.4 % (chloramphenicol) and 95.6 ± 4.5 % (d5-IS).
• Manual SPE recoveries: 74.6 ± 2.7 % and 80.3 ± 4.7 %, respectively.

Automated processing showed higher recoveries and comparable reproducibility (< 5 % RSD). Chromatograms demonstrated clear resolution of chloramphenicol peaks from matrix interferences. Calibration curves were linear from 0.5 to 100 ng/mL (R² > 0.99).

Benefits and Practical Applications

• High sample throughput with unattended automated SPE.
• Improved recovery and consistency versus manual extraction.
• Reduced hands-on time and risk of operator error.
• Suitable for routine monitoring laboratories complying with EU regulations.

Future Trends and Applications

Automation platforms may be extended to multi-residue analysis of other banned antibiotics and contaminants. Integration with laboratory information management systems (LIMS) and further miniaturization of SPE formats will streamline workflows. Advances in high-resolution mass spectrometry and data analytics could enhance sensitivity, specificity, and regulatory compliance in food safety testing.

Conclusion

The fully automated SPE method coupled with HPLC-MS/MS provides a robust, reproducible, and high-throughput approach for the detection of chloramphenicol in honey. Automation yields higher recoveries than manual protocols and supports reliable compliance monitoring under stringent regulatory limits.

References

• Regulation (EEC) 2377/90, Annex IV – banned pharmacologically active substances.
• Regulation (EU) 37/2010 – maximum residue limits for veterinary medicinal products in foodstuffs of animal origin.