Highly Selective Detection and Identification of Nitrofuran Metabolites in Honey Using LC-MS/MS

Importance of the Topic

Use of nitrofurans in food-producing animals has been banned globally due to their potential carcinogenic and genotoxic effects. Residues of their metabolites in honey pose a public health concern and challenge regulatory agencies to enforce strict limits, such as the EU’s 0.3 ppb threshold. Sensitive, accurate methods for detecting these residues are essential for ensuring food safety and maintaining export market access.

Objectives and Study Overview

This application study demonstrates a quantitative LC-MS/MS method capable of detecting four nitrofuran metabolites (AOZ, AMOZ, SEM, AHD) in honey at levels down to 0.3 ppb. Conducted by Food Science Laboratories (Argentina) in collaboration with Thermo Fisher Scientific, the work aims to validate performance metrics (sensitivity, linearity, recovery, precision) against EU requirements.

Methodology and Instrumentation

Sample Preparation:

• Honey (2 g) spiked with AOZ, AMOZ, SEM, AHD.
• Acid hydrolysis with 125 mM HCl and derivatization by 2-nitrobenzaldehyde at 37 °C for 17 h.
• pH neutralization with potassium phosphate to pH 7.0.
• Liquid–liquid extraction with ethyl acetate, centrifugation; evaporation under nitrogen; reconstitution and cartridge filtration; hexane elution.

Chromatography:

• HPLC system: Thermo Scientific Surveyor MS Pump and Autosampler.
• Column: 100 × 2.1 mm, 3 µm.
• Mobile phases: (A) water + 0.05% acetic acid; (B) methanol + 0.05% acetic acid.
• Gradient: 0–3 min (90%A/10%B) to 21–25 min (90%A/10%B).

Mass Spectrometry:

• TSQ Quantum Discovery triple quadrupole.
• Positive electrospray ionization (ESI), SRM mode.
• Scan width 0.002 m/z; scan time 0.1 s; peak width 0.7 FWHM in Q1 and Q3.
• Argon collision gas at 1.5 mTorr.
• Analyte transitions optimized for each metabolite and corresponding deuterated internal standard.

Key Results and Discussion

Calibration and Linearity:

• Five calibration levels (0.63–8.34 ppb) yielded R² ≥ 0.995 for all metabolites.
• Linearity supports accurate quantitation across the target range.

Limits of Detection and Quantification:

• LOD: 0.04 ppb (AMOZ) to 0.08 ppb (SEM).
• LOQ: 0.09 ppb (AMOZ) to 0.18 ppb (SEM).

Precision and Recovery:

• Intra-day RSD ≤ 9.0% at LOQ and ≤ 3.6% at higher levels for most analytes.
• Mean recoveries ranged from 88.5% (AHD) to 101.2% (AOZ).
• Analytical range validated up to 4 ppb with recoveries within 70–120% and CV ≤ 8.3%.

Chromatographic Separation:
The gradient effectively resolved all four metabolites and deuterated standards within a 25 min run, with clear SRM peaks at 0.6 ppb (see identification of AOZ at LOQ level).

Benefits and Practical Applications

This LC-MS/MS assay delivers:

• High sensitivity exceeding EU MRPL requirements.
• Robust sample preparation adaptable to honey and similar matrices.
• Reliable quantitation for routine quality control in food safety laboratories.
• Rapid throughput with a single 25 min analytical run.

Future Trends and Opportunities

Advances in high-resolution MS and automated sample cleanup promise further improvements in throughput and sensitivity. Implementation of multiplexed workflows may enable simultaneous screening of broader antibiotic classes. Integration with data-driven compliance platforms can streamline regulatory reporting and global trade inspections.

Conclusion

The described LC-MS/MS method using the TSQ Quantum Discovery system offers a validated, sensitive, and precise approach for detecting nitrofuran metabolites in honey at sub-ppb levels. It meets stringent FDA and EC regulations, ensuring reliable food safety monitoring and protection of consumer health.

Used Instrumentation

• Thermo Scientific Surveyor MS Pump and Surveyor Autosampler.
• 100 × 2.1 mm, 3 µm HPLC column.
• Thermo Fisher Scientific TSQ Quantum Discovery triple quadrupole mass spectrometer.

References

1. Herrlich P., Schweiger M. Nitrofurans: discrimination of specific mRNA classes. Proc. Natl. Acad. Sci. U.S.A. 73 (10): 3386–3390.
2. NTP. Toxicology and carcinogenesis studies of nitrofurazone in F344/N rats and B6C3F1 mice. Technical Report Series No. 337.
3. McCracken R.J., Kennedy D.G. Determination of 3-amino-2-oxazolidinone in porcine tissues by LC-thermospray MS. J. Chromatogr. 691: 87–94.
4. Food Standards Agency. Reporting limits for nitrofuran and chloramphenicol residues. June 30, 2003.
5. Food Science Laboratories. Determination of nitrofurans residues in honey by LC-MS/MS. Analysis Method, 2005.