Measurement of Chloramphenicol in Honey Using Automated Sample Preparation with LC-MS/MS

Importance of the Topic

Chloramphenicol is an antimicrobial agent with potential carcinogenic effects that has been banned in food-producing animals worldwide. Sensitive and reliable detection in complex matrices like honey is essential for food safety compliance and regulatory monitoring.

Goals and Overview

This study aims to develop a rapid, automated LC-MS/MS workflow for quantifying chloramphenicol in honey. The method employs negative ion heated electrospray ionization and a deuterated internal standard (CAP-d5) to achieve low parts-per-billion detection limits.

Methodology and Instrumentation

Automated sample preparation was performed on the Thermo Scientific Aria TLX-1 system equipped with TurboFlow technology. Honey samples were diluted with water and spiked with CAP-d5. Online extraction used a TurboFlow Cyclone polymer column, followed by elution onto a Hypersil GOLD C18 analytical column. Detection was carried out on a Thermo Scientific TSQ Vantage triple-quadrupole mass spectrometer in negative ion H-SRM mode. Key parameters included:

• Mobile phases: 0.02% acetic acid (aqueous) and methanol
• TurboFlow Cyclone column (0.5×50 mm) and Hypersil GOLD C18 (3×50 mm, 3 µm)
• Transitions monitored: CAP m/z 321→257 (quantifier), 152, 194 (confirmatory); CAP-d5 m/z 326→157, 262
• Total run time: 5 minutes

Main Results and Discussion

Chromatography demonstrated minimal carryover and clear peak separation for CAP and CAP-d5 across pre-blank, LLOQ (0.047 µg/kg), ULOQ (1.5 µg/kg), and post-high blank samples. Matrix-matched calibration showed linearity over two orders of magnitude (r2=0.9944) with coefficients of variation below 19% at LLOQ and under 8% at higher levels. Recoveries, normalized to the internal standard, ranged from 80.9% to 96.0%. No chloramphenicol was detected in surveyed commercial honey samples.
Comparison with traditional offline methods (SPE, QuEChERS, liquid–liquid extraction) indicated sample preparation times reduced by 7–24-fold, LC-MS run times shortened by up to fourfold, and detection limits improved by factors of 5.7–20.

Benefits and Practical Applications

The automated online extraction workflow streamlines sample handling, reduces manual error, and increases throughput. The sensitive LC-MS/MS assay enables routine screening of honey for regulatory compliance at levels well below the EU MRPL of 0.3 µg/kg.

Future Trends and Possibilities

Further enhancements may include multiplexed sample preparation (e.g., Aria TLX-4), expanded workflows for additional veterinary drug residues, miniaturized extraction formats, and integration with high-resolution mass spectrometry. Advances in materials science and software algorithms could further improve sensitivity, reduce matrix effects, and enable real-time monitoring of contaminants in apiculture products.

Conclusion

A robust, fully automated LC-MS/MS method using TurboFlow technology has been established for chloramphenicol analysis in honey with an LOD of 0.023 µg/kg and an LLOQ of 0.047 µg/kg. The approach significantly reduces sample preparation time and enhances sensitivity, supporting high-throughput, regulatory-compliant testing.

Reference

• Commission Decision 2003/18/EC. Official Journal of the European Communities, L71 (2003).
• R. Krell. Value-Added Products from Beekeeping. FAO Agricultural Services Bulletin (1996).
• D. Ortelli, P. Edder, C. Corvi. Analysis of Chloramphenicol Residues in Honey by LC-MS/MS. Chromatographia, 59, 2004.
• C. Pan et al. Determination of Chloramphenicol Residues in Honey by Monolithic LC-MS after QuEChERS. Acta Chromatographica 17 (2006).
• L. Rodziewicz, I. Zawadzka. Rapid Determination of Chloramphenicol Residues in Honey by LC-MS/MS. APIACTA 42 (2007), pp.25-30.