Using UHPLC MS MS to analyze neonicotinoid compounds and fipronil and its metabolites

Importance of the Topic

Neonicotinoid insecticides and fipronil remain under scrutiny for their impact on pollinators and food safety. Their widespread use in agriculture has led to trace residues in honey, raising concerns about environmental exposure and human consumption. A robust analytical method is critical to ensure compliance with strict EU regulations and to safeguard bee populations and honey quality.

Objectives and Study Overview

This study aimed to develop a rapid, sensitive, and reproducible ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method to quantify multiple neonicotinoids, fipronil and its metabolites in honey.

• Evaluate QuEChERS extraction combined with dispersive solid-phase extraction (dSPE) cleanup for high recovery.
• Optimize multiple reaction monitoring (MRM) transitions on a triple quadrupole mass spectrometer.
• Determine limits of quantitation (LOQs), method recovery, precision and accuracy according to EU guidance.
• Apply the method to nine commercial honey varieties as real-world samples.

Used Instrumentation

• Nexera X2 UHPLC system equipped with an ACE SuperC18 column (100 mm × 2.1 mm, 2 µm).
• Shimadzu LCMS-8060 triple quadrupole mass spectrometer with electrospray ionization (ESI).
• QuEChERS extraction kits and dSPE cleanup tubes (MgSO4, PSA, C18 sorbents).

Methodology

The sample preparation and analysis workflow included:

• Weighing ~5 g honey and spiking with isotopically labeled internal standards (thiamethoxam-D3, imidacloprid-D4, clothianidin-D3).
• Addition of acetonitrile and water, vortex mixing, and shaking.
• Salting out with MgSO4 and citrate salts, centrifugation, followed by dSPE cleanup (MgSO4, PSA, C18).
• UHPLC separation using a 4-minute gradient (5–100% methanol with 0.05% ammonia) at 0.6 mL/min and column temperature of 30 °C.
• MRM detection in both positive and negative modes, optimized for each analyte and internal standard.
• Calibration from 0.5 pg/mL to 5 ng/mL in acetonitrile, corresponding to 1 ng/kg to 10 µg/kg in honey.

Main Results and Discussion

The method achieved recoveries between 70% and 93% for all target compounds, meeting EU SNATE criteria (70–120%). LOQs ranged from 0.001 µg/kg for fipronil and its sulfone to 0.02 µg/kg for clothianidin, imidacloprid and nitenpyram. Precision studies on 150 replicate injections of spiked honey yielded relative standard deviations (RSD) below 18%.
The analysis of nine commercial honey samples revealed detectable levels of certain neonicotinoids and fipronil metabolites, but none exceeded regulatory limits. The high sensitivity allowed quantification of residues down to sub-µg/kg levels.

Benefits and Practical Applications

This UHPLC-MS/MS approach offers several advantages:

• Rapid 4-minute analysis per sample, increasing laboratory throughput.
• High sensitivity and selectivity for simultaneous monitoring of multiple compounds.
• Simplified sample preparation with QuEChERS and dSPE, reducing solvent use and cost.
• Applicability to routine quality control, environmental monitoring and regulatory compliance.

Future Trends and Applications

Emerging needs include extending the method to other bee-related matrices such as pollen and wax, as well as adapting workflows for high-throughput screening of new pesticide classes. Integration with automated sample preparation and data processing will further enhance efficiency. Research into residue degradation products and isotopic tracers could improve source tracking and risk assessment.

Conclusion

A sensitive, fast, and robust UHPLC-MS/MS method was established for quantifying neonicotinoids, fipronil and its metabolites in honey. The protocol demonstrated excellent recoveries, low detection limits and high reproducibility. It provides a valuable tool for laboratories engaged in pesticide monitoring, regulatory testing and pollinator health studies.

Reference