Sensitive method for the determination of Fipronil and its metabolite Fipronil Sulfone in egg using QuEChERS sample pretreatment and LC-MS/MS detection[LCMS-8060]

Importance of the Topic

Fipronil is a widely used phenylpyrazole insecticide applied in veterinary and agricultural settings. Due to illegal applications in poultry facilities, eggs can be contaminated with fipronil and its toxic sulfone metabolite. European regulations set a stringent maximum residue level (MRL) of 0.005 mg/kg for the sum of these compounds in eggs. Reliable analytical methods are therefore essential to ensure food safety and regulatory compliance.

Objectives and Study Overview

This study aimed to develop a rapid, sensitive, and robust protocol for quantifying fipronil and fipronil sulfone in egg matrices. By combining a standardized QuEChERS sample pretreatment with LC-MS/MS detection, the method targets accurate measurement at levels below the regulatory threshold, while minimizing method development time.

Methodology

A QuEChERS (Quick, Easy, Cheap, Effective, Rugged, Safe) extraction was applied to 5 g of homogenized egg (white and yolk). Samples were diluted with water, extracted with acetonitrile, and treated with pre-weighed salt mixtures for drying and buffering. After centrifugation, a dispersive solid-phase extraction (dSPE) cleanup removed residual interferences. The final extract pH was adjusted with formic acid in acetonitrile prior to analysis.

Used Instrumentation

• UHPLC system: Nexera X2 (Shimadzu) equipped with a Raptor Biphenyl column (100 × 2.1 mm, 2.7 μm)
• Mobile phases: Water and methanol, each containing 2 mM ammonium formate and 0.002% formic acid
• Mass spectrometer: LCMS-8060 triple quadrupole (Shimadzu) operated in MRM and MRM spectrum modes
• MRM spectrum mode: acquisition of 6–12 product ions per analyte to enable library search and improve identification specificity

Main Results and Discussion

Matrix-matched calibration curves were established from 0.0005 to 0.05 mg/kg for fipronil, fipronil sulfone, fipronil-desulfinyl, and fipronil-sulfide. All analytes exhibited excellent linearity (R² ≥ 0.9986) with acceptable precision (%RSD < 5.5%). Control samples spiked at 0.001 and 0.01 mg/kg fell within the calibration range, demonstrating reliable quantification below the 0.005 mg/kg MRL. The MRM spectrum approach provided high-confidence verification without sacrificing sensitivity.

Benefits and Practical Applications

The method offers rapid implementation using commercially available QuEChERS kits and an established LC-MS/MS pesticide package. It ensures high throughput monitoring of egg products for fipronil residues, supporting quality control laboratories and regulatory agencies in food safety surveillance.

Future Trends and Potential Applications

Future developments may include:

• Extension to additional food matrices such as milk and honey
• Automation of sample handling for high-throughput screening
• Incorporation of high-resolution mass spectrometry for broader pesticide profiling
• Integration with spectral libraries and AI-driven data analysis for enhanced specificity

Conclusion

The presented QuEChERS-LC-MS/MS method reliably quantifies fipronil and its sulfone metabolite in eggs at sub-MRL levels. With minimal additional optimization, the protocol meets regulatory requirements and supports routine food safety testing.

References

• EU Commission Regulation No 1127/2014 of 20 October 2014 amending Annexes II and III to Regulation (EC) No 396/2005 regarding maximum residue levels for fipronil and related compounds.