Method for the determination of 431 Residual Pesticides in Honey using LCMS-8050 and GCMS-TQ8040 NX

Importance of the Topic

Honey is a widely consumed product valued for its nutritional and medicinal properties, but it can accumulate trace levels of pesticide residues from agricultural sources. Monitoring multiple pesticide classes in honey is critical to protect consumer health and ensure compliance with regulatory maximum residue limits (MRLs) set by authorities such as the European Union.

Objectives and Study Overview

The study aimed to develop and validate a rapid, sensitive, and reproducible multi-residue method for determination of 431 pesticides in honey. Two analytical platforms were used: LC-MS/MS for 244 pesticides and GC-MS/MS for 214 pesticides, with 27 analytes overlapping between both techniques. Key validation parameters included limit of quantification (LOQ), linearity, specificity, recovery, repeatability, and within-laboratory reproducibility according to SANTE guidelines.

Used Instrumentation

• Shimadzu LCMS-8050 triple quadrupole mass spectrometer coupled with Nexera X2 UHPLC system
• Shim-pack XR-ODS column (100 × 3.0 mm I.D., 2.2 µm)
• Shimadzu GCMS-TQ8040 NX tandem quadrupole GC-MS/MS with AOC-20i + s autosampler
• SH-Rxi-5Sil MS capillary column (30 m × 0.25 mm I.D., 0.25 µm) with Topaz splitless liner
• LabSolutions Insight software for data processing and validation evaluation

Methodology and Sample Preparation

A single liquid–liquid extraction protocol was applied to 10 g honey samples spiked with target pesticides. Sodium sulfate was added to aid phase separation, and ethyl acetate served as the extraction solvent. The extract was divided for parallel analysis:

• For GC-MS/MS: cleanup using primary-secondary amine (PSA) sorbent and anhydrous MgSO₄ to remove co-extractives
• For LC-MS/MS: reconstitution in methanol–water (60:40, v/v)

Final extracts were filtered (0.22 µm nylon) and injected: 1 µL in splitless mode for GC and 20 µL co-injection with water for LC. Mobile phases, gradients, temperatures, and MS/MS transitions were optimized to achieve rapid 20 min LC runs and 34 min GC runs.

Main Results and Discussion

Linearity: Matrix-matched calibration curves showed R² ≥ 0.98 over 0.2–20 µg/L (LC) and 1–50 µg/L (GC) with weighted 1/C² regression. Accuracy of calibration standards fell within 80–120 %.

LOQ Performance:

• LC-MS/MS: 123 compounds at 1 µg/kg, 18 at 2 µg/kg, 80 at 5 µg/kg, 21 at 10 µg/kg, 2 at 25 µg/kg
• GC-MS/MS: 158 compounds at 5 µg/kg, 50 at 10 µg/kg, 1 at 20 µg/kg, 5 at 25 µg/kg

Recovery and Precision:

• Mean recoveries at LOQ were 70–120% for 190 LC analytes and for 207 GC analytes; 17 LC compounds and 7 GC compounds fell below 70%, while 37 LC compounds exceeded 120%.
• Repeatability (RSDr) and within-lab reproducibility (RSDR) at LOQ were <20% in all cases, meeting SANTE criteria.

Specificity: No interfering peaks exceeded 30% of the reporting limit in blank matrix.

Benefits and Practical Applications

• The method offers comprehensive coverage of over 400 regulated pesticides in a single honey matrix.
• Simple extraction and cleanup streamline sample preparation and reduce analysis time.
• Low LOQs allow reliable detection below common MRLs, enabling food safety monitoring and compliance testing.
• Integration with Shimadzu LCMS and GCMS method libraries simplifies parameter optimization.

Future Trends and Opportunities

• Expansion of compound databases and inclusion of emerging pesticide chemistries.
• Automation and high-throughput workflows using online SPE or robotic sample handling.
• Coupling with high-resolution mass spectrometry for non-target screening and retrospective data mining.
• Application of miniaturized extraction and ambient ionization techniques to further accelerate analysis.

Conclusion

A robust, fast, and sensitive LC-MS/MS and GC-MS/MS method was developed and validated for 431 pesticide residues in honey. The procedure meets stringent regulatory criteria for LOQ, recovery, and precision, supporting routine food safety laboratories in multi-residue compliance testing.

References

• Guidance document on analytical quality control and method validation procedures for pesticide residues and analysis in food and feed. SANTE/11312/2021.