Method for the determination of 346 Residual Pesticides in Milk using LCMS-8045 and GCMS-TQ8040 NX

Significance of the topic

Milk is a staple food for infants and adults, but its complex matrix presents significant challenges for the detection of trace pesticide residues. Regulatory bodies worldwide set stringent maximum residue limits for pesticides in milk, often tighter than those for fruits and vegetables, driving the need for analytical methods that combine robust sample cleanup with high sensitivity and throughput.

Objectives and overview

The study aimed to develop and validate a unified multiclass, multiresidue workflow capable of quantifying 346 pesticides in milk. Utilizing both LC-MS/MS and GC-MS/MS on Shimadzu LCMS-8045 and GCMS-TQ8040NX instruments, the method covers regulatory requirements of FSSAI, EU and APEDA. The goal was to achieve low limits of quantification (LOQs), high reproducibility and rapid analysis times to meet routine laboratory demands.

Methodology and instrumentation

Sample preparation employed a modified QuEChERS protocol:

• Milk samples deproteinized with acetonitrile then split into two fractions (A and B).
• Fraction A extracted with anhydrous MgSO4, yielding sub-fractions A-I (for LC-MS/MS) and A-II (for GC-MS/MS).
• Cleanup using C18 sorbent and solvent exchanges into mobile phase or ethyl acetate to minimize matrix effects.
• Final reconstitution volumes optimized to avoid dilution and maintain sensitivity.

The instrumental setup included:

• LC-MS/MS: Shimadzu LCMS-8045 coupled to Nexera X2 with a Shim-pack XR-ODS II column; mobile phases with 2 mM ammonium formate/0.02% formic acid; gradient elution over 21 min; ESI mode; co-injection with water (5 µL).
• GC-MS/MS: Shimadzu GCMS-TQ8040NX with AOC-20i injector and SH-Rxi-5Sil MS column; splitless injection (1 µL); helium carrier gas; temperature program from 60 °C to 310 °C over 25 min; EI mode.
• Method optimization leveraged Shimadzu’s MethodPackage Ver.3 and SmartPesticidesDatabase Ver.2, with LabSolutions Insight software for data processing.

Main results and discussion

Validation followed SANTE guidelines, assessing specificity, linearity, recovery and precision:

• Specificity: Blank matrix responses remained below 30% of reporting limits.
• Linearity: Calibration in matrix-matched standards from 2.5 to 40 µg/L (up to 80 µg/L for shared compounds) with R² > 0.99 and accuracy between 80–120%.
• Recovery: At LOQ levels (5 µg/kg or 10 µg/kg), 294 compounds achieved 70–120% recovery, 93 compounds 30–70%, and only three exceeded 120%.
• Precision: Repeatability (RSDr) and reproducibility (RSDR) were below 20% across all compounds, demonstrating method robustness.
• LOQs: Achieved 5 µg/kg for 165 LC-amenable and 118 GC-amenable pesticides; remaining compounds quantified at 10 µg/kg.

Benefits and practical applications

The method offers multiple advantages for routine testing:

• Short analysis times enhance sample throughput and laboratory productivity.
• Reduced injection volumes and flow rates extend column life and improve long-term assay stability.
• Comprehensive analyte coverage meets diverse international regulatory standards.
• High sensitivity and reproducibility support reliable QA/QC in dairy and food safety laboratories.

Future trends and opportunities

Advancements may include automation of sample preparation, integration of high-resolution mass spectrometry for non-target screening, and development of eco-friendly extraction solvents. Expanded pesticide databases and machine-learning algorithms for data processing could further streamline multiclass residue analysis in complex food matrices.

Conclusion

A simple, fast, and sensitive workflow combining modified QuEChERS extraction with dual LC-MS/MS and GC-MS/MS analysis successfully quantifies 346 pesticides in milk at low µg/kg levels. The validated method demonstrates excellent linearity, recovery and precision, making it suitable for high-throughput routine monitoring and regulatory compliance.

References

1. GB2763-2019 National Food Safety Standard — Maximum Residue Limits for Pesticides in Food: https://www.codeofchina.com/standard/GB2763-2019.html
2. Anastassiades M, Lehotay SJ, Štajnbaher D, Schenck FJ. Fast and Easy Multiresidue Method Employing Acetonitrile Extraction/Partitioning and Dispersive Solid-Phase Extraction for the Determination of Pesticide Residues in Produce. J AOAC Int. 2003;86(2):412–431.
3. European Commission SANTE/12682/2019. Guidance document on analytical quality control and method validation procedures for pesticide residues analysis in food and feed.