Simultaneous screening and quantitation of pesticides residue in milk at trace level using high resolution Orbitrap GC-MS

Importance of the topic

Milk is a critical component of human diets worldwide, yet its high water, protein and fat content poses challenges for trace-level pesticide analysis. Regulatory bodies such as the EU and FSSAI set strict maximum residue limits (MRLs) to protect public health. Achieving sensitive, reliable detection of multiple pesticide classes in complex matrices like milk is therefore essential for food safety monitoring and compliance.

Objectives and Study Overview

The study aimed to develop and validate a single analytical workflow capable of simultaneously screening and quantifying 162 pesticide residues in milk. The method was designed to comply with EU SANTE/11813/2017 identification and quantification criteria, as well as FSSAI and EC MRL requirements. Key goals included minimizing false negatives, achieving low limits of detection, and enabling full-scan data acquisition for retrospective analysis.

Methodology and Instrumentation

Sample Preparation

• EN 15662 QuEChERS extraction using 10 g homogenized milk with water and acetonitrile.
• Citrate buffering salts followed by cleanup with 150 mg MgSO₄, 25 mg PSA and 50 mg C18 per mL extract.
• Vortex mixing and centrifugation yielded extracts suitable for GC injection.

Instrument Used

• Thermo Scientific TRACE 1310 GC with TriPlus RSH autosampler and TG-5SIL-MS column (30 m × 0.25 mm × 0.25 µm).
• Programmable temperature vaporizing injector (PTV) in cold splitless mode, 1 µL injection.
• Thermo Scientific Exactive GC Orbitrap GC-MS with EI ionization, resolving power 60 000 (FWHM @ m/z 200), mass range 50–500 Da.
• Data processing in TraceFinder 4.1 software with user-defined filters (±5 ppm mass error, ±0.10 min RT, linearity >0.99, recovery 70–120%, precision <20%).

Main Results and Discussion

• Cleanup optimization highlighted that 50 mg C18 plus 25 mg PSA reduced fat interferences while maintaining sensitivity for most compounds.
• Screening detection limits (SDLs) ranged from 0.001–0.002 mg/kg for 123–137 compounds; full identification and confirmation achieved for up to 162 analytes at 0.05 mg/kg.
• Quantitation limits (LOQs) were between 0.005–0.010 mg/kg, meeting or exceeding EU and FSSAI MRLs.
• Linearity was excellent (R²>0.99) in matrix-matched standards over 0.001–0.100 mg/kg, with residuals <20%.
• Recovery experiments at 0.005, 0.010 and 0.025 mg/kg yielded 72–117% recoveries with RSDs below 15%.
• Full-scan acquisition provided >12 scans per 6 s peak (e.g., fipronil >40 scans), with mass accuracy <5 ppm without lock mass and consistent ion ratios within ±30%.

Benefits and Practical Applications

• High-resolution full-scan GC-Orbitrap MS enables simultaneous targeted and untargeted screening, reducing method development time versus triple quadrupole MS/MS.
• Retrospective data mining supports discovery of additional residues without reanalysis.
• Validated method satisfies stringent regulatory criteria for milk safety testing.
• QuEChERS-based extraction and small-volume PTV injection streamline laboratory workflows and increase throughput.

Future Trends and Opportunities

• Integration of GC-HRMS with LC-HRMS platforms to cover wider pesticide chemistries.
• Advances in automated sample preparation and data processing, including AI-driven peak flagging.
• Development of spectral libraries and in-silico fragmentation tools for non-target screening.
• Application of isotope dilution and high-throughput multiplexing for enhanced quantitation accuracy.

Conclusion

The optimized QuEChERS–GC-Orbitrap HRMS method provides a robust, sensitive and comprehensive approach for screening and quantifying 162 pesticide residues in milk. It achieves low SDLs and LOQs, meets EU and FSSAI regulatory requirements, and offers full-scan capabilities for future retrospective analyses.

References

• Guidance document on analytical quality control and method validation procedures for pesticide residues. SANTE/11813/2017.
• EN 15662:2008 QuEChERS method for pesticide residue analysis.
• Food Chemistry 133 (2012) 473–481: pesticide residues in milk using QuEChERS.
• Thermo Scientific Pesticide Residue Analyzer Reference, Rev. 2.
• FSSAI regulatory requirements: Insecticides Act registrations.
• EC pesticide MRL database for milk.