Ultra-Sensitive and Rapid Assay of Neonicotinoids, Fipronil and Some Metabolites in Honey by UHPLC-MS/MS [LCMS-8060]

Significance of the Topic

The intensive use of neonicotinoid insecticides and related compounds has raised concerns about honeybee health and environmental safety. Monitoring trace levels of these pesticides in honey is critical for assessing compliance with regulatory limits and understanding chronic exposure effects on pollinators.

Objectives and Overview of the Study

This work describes the development and validation of an ultra-sensitive, rapid assay for eight neonicotinoids, fipronil and selected metabolites in honey. The methodology aims to achieve quantification at sub-nanogram per kilogram levels, support high sample throughput and ensure long-term system stability.

Sample Preparation and Methodology

The protocol employs a QuEChERS extraction with dispersive solid-phase cleanup. Key steps include:

• Weighing 5 g honey and spiking with isotopically labeled internal standards.
• Hydration, acetonitrile extraction, salt-induced phase separation and dSPE cleanup using MgSO₄, PSA and C18.
• Direct injection of the clarified extract without evaporation or dilution.

Used Instrumentation

The analysis was performed on a Nexera X2 UHPLC system coupled to LCMS-8060 triple quadrupole MS. Chromatographic and MS parameters:

• Column: ACE SuperC18, 100 × 2.1 mm, 2 µm, 30 °C.
• Mobile phases: water and methanol both containing 0.05 % ammonia, 600 µL/min gradient elution (4 min total).
• Ionization: heated ESI in positive and negative modes with optimized probe voltage and source temperatures.
• MRM acquisition with compound-specific transitions, dwell times and unit quadrupole resolution.

Main Results and Discussion

Method performance highlights:

• Limits of quantification (LOQs) ranged from 0.001 to 0.02 µg/kg in honey, meeting EU SANTE criteria.
• Recoveries of all analytes were between 70 % and 120 % with acceptable precision.
• Calibration exhibited excellent linearity (R² > 0.998) across four orders of magnitude.
• Analysis of nine commercial honey samples detected low-level residues, all below maximum residue limits but accurately quantified.
• Signal stability tests (150 consecutive injections) showed relative standard deviations below 20 % for all targets, demonstrating robust ion source performance.

Benefits and Practical Applications

This streamlined approach delivers:

• High sensitivity enabling detection of trace residues relevant to pollutant exposure studies.
• Minimal sample handling and no solvent evaporation steps, reducing analysis time and potential losses.
• Capability for routine screening of honey, pollen or bee matrices in research, QA/QC and regulatory settings.

Future Trends and Applications

Advances in UHPLC-MS/MS sensitivity and automation may extend this workflow to multi-residue panels, environmental water or food commodity testing. Integration with high-throughput robotic sample preparation could further accelerate monitoring programs and support large-scale ecological impact assessments.

Conclusion

The presented UHPLC-MS/MS method offers ultra-sensitive, reliable quantification of neonicotinoids and fipronil derivatives in honey. Its simplicity, speed and robustness make it a valuable tool for environmental research and regulatory compliance monitoring.