A sensitive and repeatable method for characterization of sulfonamides and trimethoprim in honey using QuEChERS extracts with Liquid-Chromatography-Tandem Mass Spectrometry

Significance of Topic

A reliable and sensitive method for detecting sulfonamide antibiotics and trimethoprim in honey is crucial for food safety and public health. Residues of these veterinary drugs can provoke allergic reactions and drive antimicrobial resistance in consumers. Regulatory bodies aim to control these contaminants at levels as low as 10 µg/kg, making robust analytical approaches essential for routine monitoring.

Goals and Study Overview

This study presents the development of a fast, selective and sensitive HPLC-MS/MS assay combined with QuEChERS extraction to quantify 15 sulfonamides and trimethoprim in honey. The method addresses regulatory requirements by achieving low method quantification limits and high repeatability.

Methodology and Instrumentation

Five grams of drug-free honey were fortified with analytical standards and extracted following a QuEChERS protocol. The final extracts were diluted in methanol and analyzed by ultra-high-performance liquid chromatography on a Kinetex 2.6 µm PFP column (100 × 2.1 mm) at 40 °C, using a binary gradient of aqueous and organic mobile phases. A Shimadzu Nexera UHPLC was coupled to a fast-scanning LCMS-8050 triple quadrupole mass spectrometer with electrospray ionization in positive mode. Quantitation employed optimized multiple reaction monitoring transitions for each analyte.

Main Results and Discussion

Calibration curves constructed in 50 % methanol matrix demonstrated linearity over 1–1000 µg/kg (0.5–500 pg on column) with r2 ≥ 0.990 for all compounds. Limits of quantification were 1 µg/kg for all analytes except succinylsulfathiazole and sulfacetamide (2 µg/kg). Recoveries ranged from 53.9 % to 91.4 % for most compounds, while two analytes showed lower extraction efficiency. Precision expressed as relative standard deviation remained below 15 % across concentration levels, confirming method repeatability.

Benefits and Practical Applications

• Rapid sample throughput facilitates routine quality control in the honey industry
• Detection limits comply with or exceed regulatory minimum performance limits
• Minimal sample preparation reduces solvent and time consumption
• High specificity of MRM transitions minimizes matrix interferences

Future Trends and Opportunities

Further improvements may include the use of isotopically labeled internal standards and matrix-matched calibration to enhance accuracy. Expansion to other food matrices, automation of QuEChERS workflows and coupling with high-resolution mass spectrometry could broaden applicability and sensitivity. Advances in miniaturized extraction techniques may reduce sample and solvent volumes.

Conclusion

The combined QuEChERS extraction and HPLC-MS/MS approach provides a fast, sensitive and reliable tool for monitoring sulfonamides and trimethoprim in honey. Its performance meets current regulatory criteria, making it suitable for implementation in food safety laboratories.

Instrumentation Used

• Shimadzu Nexera ultra-high-performance liquid chromatograph
• Shimadzu LCMS-8050 triple quadrupole mass spectrometer
• Kinetex 2.6 µm pentafluorophenyl column (100 × 2.1 mm)
• Electrospray ionization source in positive mode