Detection, Confirmation, and Quantitation of Chloramphenicol in Honey

Significance of the Topic

Chloramphenicol is a broad-spectrum antibiotic banned in food due to risks of aplastic anemia and bone marrow suppression. Regulatory bodies set a minimum required performance limit (MRPL) at 300 ppt in honey, driving the need for highly sensitive, cost-effective analytical methods in food safety laboratories.

Objectives and Study Overview

The study aimed to develop a simple, liquid–liquid extraction (LLE) method coupled with the compact Agilent 1260 Infinity II LC system and Ultivo LC/TQ triple quadrupole mass spectrometer. The workflow targets routine confirmation and quantitation of chloramphenicol (CAP) in honey with a limit of quantitation (LOQ) of 50 ppt within a complex matrix.

Methodology

Sample Preparation:

• Weigh 1 g of honey and spike with 10 µL of deuterated CAP (CAP-d5) internal standard.
• Add 1 mL water, vortex for 1 min to homogenize.
• Extract with 6 mL ethyl acetate, vortex for 10 min and centrifuge at 10,000 rpm, 5 °C for 10 min.
• Collect the upper organic phase, evaporate under nitrogen at 50 °C, reconstitute in 1 mL of 95:5 water:methanol, and filter through a 0.2 µm PTFE syringe filter.

Chromatographic and Mass Spectrometric Conditions:

• Column: Agilent InfinityLab Poroshell 120 EC-C18, 2.1 × 100 mm, 2.7 µm.
• Mobile phase: A) Water; B) Methanol; flow rate 500 µL/min, 50 °C column temperature.
• Gradient: 95:5 A:B (0–0.0 min), ramp to 2:98 by 2.5 min, hold until 3.0 min, return to 95:5 by 3.5 min, re-equilibrate to 5.0 min.
• Injection volume: 25 µL.
• MS detection using MRM transitions for CAP (321→151.9, 321→257.1, 321→194.0) and CAP-d5 (326→157.0) with unit resolution and optimized collision energies.

Instrumentation Used

• Agilent 1260 Infinity II flexible pump (G7104C)
• Agilent 1260 Infinity II vial sampler (G7129C)
• Agilent 1260 Infinity II multi-column thermostat (G7116A)
• Agilent Ultivo LC/TQ with AJS ion source (G6465A)

Key Results and Discussion

• Determined limit of detection (LOD) at 25 ppt and recommended LOQ at 50 ppt in honey matrix.
• Calibration curve linear between 50 and 600 ppt (R2 > 0.995) with relative response against CAP-d5.
• Precision (%CV) within acceptable limits for six replicates at LOQ.
• Analysis of commercial honey samples showed CAP levels below the EU MRPL of 300 ppt; spiked samples yielded recoveries between 82% and 107%.

Benefits and Practical Applications

The method eliminates solid-phase extraction, reducing time and cost while maintaining sensitivity. Its simple LLE workflow and compact instrumentation make it suitable for routine food safety testing, QC laboratories, and academic research requiring trace-level antibiotic analysis.

Future Trends and Potential Applications

With ongoing advancements in MS miniaturization and high-throughput automation, this approach can expand to multiplex screening of multiple antibiotic residues. Integration with high-resolution MS and digital data processing will further enhance sensitivity, specificity, and regulatory compliance in food safety analytics.

Conclusion

The developed LLE-based LC–MS/MS method on the Agilent 1260 Infinity II/Ultivo LC/TQ delivers reliable CAP quantitation in honey at 50 ppt LOQ, well below the EU MRPL, combining simplicity, speed, and robustness for routine regulatory and quality-control applications.

References

1. EFSA Scientific Opinion on Chloramphenicol in Food and Feed. EFSA Journal 2014, 12(11), 3907.
2. Commission Decision 2003/181/EC.
3. Fang, Y. et al. Agilent application note 5988-9920EN, 2007.
4. Zhao, L. et al. Agilent application note 5990-3615EN, 2009.
5. Jin-Lan, S. et al. Agilent application note 5991-0013EN, 2012.
6. SANTE/11813/2017 guidelines.