Determination of the Metabolites of Nitrofuran Antibiotics in Animal Tissues and Food Products by UPLC-MS/MS

Importance of the Topic

Nitrofurans are a class of broad-spectrum antimicrobial agents historically used in veterinary practice. Due to residual toxicity and potential health risks to consumers, regulatory bodies worldwide have prohibited their use in food-producing animals. Despite this ban, nitrofurans and their metabolites continue to appear in imported animal products, triggering border rejections and recalls. Sensitive, reliable analytical methods are essential to enforce Maximum Residue Limits (MRLs) and Minimum Required Performance Limits (MRPLs), ensure food safety, and support both official control and commercial pre-export testing.

Objectives and Overview of the Study

This application note evaluates a targeted ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) method for detecting four nitrofuran metabolites—AOZ, AMOZ, AHD, and SCA—in diverse matrices. The aims were to demonstrate the performance of the Waters ACQUITY UPLC H-Class system with the Xevo TQ-S micro MS, confirm compliance with EU Decision 2003/181/EC MRPLs (1 µg/kg), and assess method suitability for routine official control, pre-harvest, and pre-export testing.

Methodology and Instrumentation

Samples (poultry, aquaculture, honey, egg, fish, kidney) underwent removal of extraneous material, acid hydrolysis to release bound metabolites, and derivatization with 2-nitrobenzaldehyde to form nitrophenyl (NP) derivatives. Extracts were cleaned by C18 solid-phase extraction, spiked with stable isotope internal standards, and filtered.

Used Instrumentation

• ACQUITY UPLC H-Class system with FTN autosampler
• ACQUITY UPLC BEH C18 column (1.7 µm, 2.1 × 100 mm)
• Xevo TQ-S micro mass spectrometer, electrospray ionization (ESI+) source
• MassLynx software for data acquisition
• TargetLynx XS Application Manager for data processing

Chromatographic and MS Conditions

A binary gradient of 0.5 mM ammonium formate (aq.) and methanol at 0.45 mL/min over 11 min achieved baseline separation. Optimal cone and collision energies were set to maximize sensitivity for each NP-derivative. Two MRM transitions were monitored per analyte, and one for each internal standard.

Main Results and Discussion

The method achieved detection at 0.5 µg/kg in all matrices with no observed interferences. Precision in solvent (n=8) yielded 1.7–3.5 % RSD; repeatability in incurred honey (n=20) was 2.8–13 % RSD. Linearity (0.2–5.0 µg/kg) demonstrated r2>0.99 and residuals <15 %. Analysis of a FAPAS honey proficiency test produced 1.41 µg/kg AMOZ versus the assigned 1.50 µg/kg, with 2.9 % RSD. Identification criteria (ion ratio and retention time tolerances) complied with EU Decision 2002/657/EC.

Benefits and Practical Applications

• High sensitivity and specificity for MRPL enforcement and surveillance.
• Capability to meet lower commercial screening limits for pre-export testing.
• Applicability across a broad range of food matrices.
• Rapid throughput (11 min run time) suitable for high-volume laboratories.

Future Trends and Potential Applications

Advances in high-resolution and ion-mobility mass spectrometry may enable simultaneous targeted and non-targeted screening of banned substances. Automation of sample preparation and integration of data analytics will support higher throughput and broader analyte coverage. Emerging derivatization chemistries and microflow LC could further improve sensitivity and reduce solvent consumption.

Conclusion

The UPLC-MS/MS method using the ACQUITY UPLC H-Class and Xevo TQ-S micro provides robust, sensitive analysis of nitrofuran metabolites in diverse animal-derived matrices. It fulfills regulatory requirements for MRPL detection, supports pre-export and pre-harvest testing, and offers reliable performance for routine surveillance.

References

1. Vass M, Hruska K, Franek M. Nitrofuran antibiotics: A review on the application, prohibition and residual analysis. Veterinarni Medicina. 2008;53(9):469–500.
2. Points J, Thorburn-Burns D, Walker MJ. Forensic issues in the analysis of trace nitrofuran veterinary residues in food of animal origin. Food Control. 2015;50:92–103.
3. EU Commission Decision 2002/657/EC of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and interpretation of results. Official Journal of the European Communities. 2002;L221:8–36.
4. EU Commission Decision 2003/181/EC of 13 March 2003 amending Decision 2002/657/EC in regards to setting MRPLs for certain residues in food of animal origin. Official Journal of the European Communities. 2003;L71:17–18.
5. Cooper KM, Mulder PPJ, van Rhijn JA, Kovacsics L, McCracken RJ, Young PB, et al. Depletion of four nitrofuran antibiotics and their tissue-bound metabolites in porcine tissues and determination using LC-MS/MS and HPLC-UV. Food Additives & Contaminants A. 2005;25:406–414.
6. Morphet J, Hancock P. Application of ACQUITY TQD for the analysis of nitrofuran veterinary drug residues in shrimp. Waters Application Note no. 720002299en. September 2007.