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

Significance of the Topic

A highly sensitive assay for neonicotinoids and fipronil residues in honey addresses growing concerns over pollinator health and food safety. Quantifying trace levels of these pesticides supports environmental monitoring and compliance with regulatory limits, aiding research into colony collapse and ensuring consumer protection.

Objectives and Study Overview

The study aimed to develop and validate an ultra-sensitive, rapid UHPLC-MS/MS method to quantify eight neonicotinoids, fipronil and key metabolites in honey. Emphasis was placed on simple sample preparation, high throughput and robust performance capable of detecting residues far below maximum residue limits.

Methodology and Procedures

Sample Preparation:

• Weighed 5 g of honey with internal standards (thiamethoxam-d3, imidacloprid-d4, clothianidin-d3).
• Performed QuEChERS extraction with acetonitrile and water, followed by dispersive SPE cleanup (MgSO4, PSA, C18).
• Centrifugation and direct transfer into LCMS-certified vials; no evaporation or reconstitution steps.

Calibration and Quantification:

• Prepared matrix-matched calibration from 0.5 pg/mL to 5 ng/mL (1 ng/kg to 10 µg/kg in honey).
• Selected lower limits of quantification to meet 80–120 % accuracy criteria.
• Employed quadratic calibration with 1/x weighting, achieving R² > 0.998.

Instrumental Setup

Liquid Chromatography:

• UHPLC System: Nexera X2
• Column: ACE SuperC18, 100 × 2.1 mm, 2 µm, 30 °C
• Mobile phases: A = water + 0.05 % ammonia; B = methanol + 0.05 % ammonia
• Gradient: 5 % B to 100 % B in 3 min, return to 5 % B in 0.1 min; flow 600 µL/min; run time 4 min
• Injection: 2 µL in POISe mode

Mass Spectrometry:

• Triple quadrupole LCMS-8060 with heated ESI in polarity switching
• MRM transitions optimized for each analyte and labeled ISTDs
• Source temperatures: Interface 400 °C, DL 200 °C, heat block 400 °C
• Gas flows: nebulizing 3 L/min, heating 10 L/min, drying 5 L/min

Results and Discussion

Recovery and Accuracy:

• Spike recoveries in honey ranged 70–120 % (EU SANTE criteria).
• LOQs between 0.001 and 0.020 µg/kg allowed detection far below regulatory limits.

Real Sample Analysis:

• Assayed nine commercial and cosmetic honey samples.
• Detected neonicotinoids at trace levels (< 2 µg/kg), all below maximum residue limits.

Method Stability:

• 150 consecutive injections of spiked extract showed RSDs of 1–17 % across analytes.
• Demonstrated ruggedness and consistent sensitivity over long analytical batches.

Benefits and Practical Applications

The method’s simplicity and speed support high-throughput monitoring in food safety and environmental studies. Its low detection limits enable research on sublethal pesticide exposure in bees and contamination tracking in pollination products.

Future Trends and Potential Applications

Extension of this approach may include:

• Adaptation to pollen, nectar and bee tissue matrices.
• Increased multiplexing for broader pesticide panels.
• Integration with automated sample prep for large-scale surveillance.
• Coupling with high-resolution MS for non-target screening.

Conclusion

An UHPLC-MS/MS method was successfully established for ultra-sensitive, rapid quantification of neonicotinoids, fipronil and metabolites in honey. The streamlined QuEChERS procedure and robust instrument performance delivered reliable results at trace levels, offering a valuable tool for pollinator health research and quality control.

References

• European Commission. Guidance Document on Analytical Quality Control and Method Validation Procedures for Pesticide Residues Analysis in Food and Feed (EU SANTE/11945/2015).
• Shimadzu Corporation. Application News C140: Ultra-Sensitive and Rapid Assay of Neonicotinoids, Fipronil and Some Metabolites in Honey by UHPLC-MS/MS, First Edition December 2016.