Screening and Low-Level Quantitation of Chloramphenicol in Commercial Honey Samples Using Miniaturized LC/MS System

Significance of the Topic

Chloramphenicol is a broad-spectrum antibiotic known to cause severe hematological disorders at trace levels. Regulatory bodies worldwide enforce a maximum residue limit of 0.3 ng/mL in foodstuffs, including honey. Routine monitoring of chloramphenicol in commercial honey is essential to protect consumer health and ensure compliance with international standards.

Objectives and Study Overview

This study presents a miniaturized LC/MS workflow for the sensitive screening and quantification of chloramphenicol in honey. The method aims to achieve a limit of quantitation (LOQ) at 50 pg/mL (ppt), well below the European MRPL, while offering a streamlined sample preparation protocol suitable for high-throughput routine analysis in food testing laboratories.

Methodology and Instrumentation

Sample preparation relies exclusively on liquid–liquid extraction (LLE) to minimize time and cost, using ethyl acetate as the extraction solvent. A deuterated analogue (CAP-D5) serves as an internal standard to correct for matrix variability.

• LC system: Agilent 1260 Infinity II Quaternary Pump, Vialsampler and Multicolumn Thermostat
• Mass spectrometer: Agilent Ultivo LC/TQ with electrospray ionization in negative mode
• Column: Poroshell EC-C18, 2.1 × 100 mm, 2.7 µm
• Mobile phase gradient: water/methanol (initial 95:5, ramp to 2:98 over 2.5 min, re-equilibration)
• MRM transitions: 321→151.9, 321→257.1, 321→194 for CAP; 326→157 for CAP-D5

Key Results and Discussion

The method exhibits a limit of detection (LOD) of 25 ppt and a practical LOQ of 50 ppt to account for matrix complexity. Calibration from 50 to 600 ppt yielded excellent linearity (R2 = 0.995). Recovery tests at the LOQ level (50 ppt spike) across five honey brands demonstrated recoveries between 86 % and 120 %. All commercial samples tested below the 0.3 ng/mL regulatory threshold. These results affirm the method’s sensitivity, specificity, and robustness for routine honey screening.

Benefits and Practical Applications

This protocol offers multiple advantages for food safety laboratories:

• Ultra-low LOQ well below regulatory limits
• Rapid sample preparation without SPE cartridges
• High throughput capability with miniaturized LC/TQ instrumentation
• Reliable quantitation using a deuterated internal standard

Future Trends and Applications

The approach can be extended to other prohibited veterinary drugs or biological matrices by adjusting extraction solvents and MRM parameters. Integration with automated liquid handlers and multiplexed assays will further increase throughput. Emerging high-resolution MS platforms may provide additional confirmation capabilities and reduced false positives.

Conclusion

A streamlined LC/TQ method using miniaturized instrumentation and simple LLE has been developed for chloramphenicol quantitation in honey at 50 ppt LOQ. The workflow meets international regulatory requirements, delivers robust performance, and is suitable for routine food safety analysis.

References

1. EFSA Journal 2014;12(11):3907. Scientific Opinion on Chloramphenicol in Food and Feed.
2. European Commission Decision 2003/181/EC.
3. SANTE/11813/2017. Guidance Document.
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