Multiresidue Analysis of Pesticides in Bovine Milk by GC/MS/MS with Bond Elut QuEChERS EN Kits

Significance of the Topic

Monitoring pesticide residues in bovine milk is critical for ensuring food safety, protecting consumer health, and complying with regulatory standards. Milk is a widely consumed nutrient source, especially by infants and children, who are more vulnerable to toxic effects. Pesticides used in animal husbandry or introduced through contaminated feed can partition into milk fat and persist through processing steps such as homogenization and ultra‐high‐temperature treatment. A robust, multi‐residue analytical method enables laboratories to detect trace levels of a broad spectrum of pesticides and metabolites, supporting national control programs and international trade requirements.

Objectives and Study Overview

This work aimed to develop and validate a quantitative method for simultaneous determination of 60 pesticides and metabolites in ultra‐high‐temperature (UHT) whole bovine milk. The targets include compounds listed in Brazil’s National Plan for Control of Residues and Contaminants (PNCRC) and those with Codex Alimentarius maximum residue limits. Key goals were to establish method performance in terms of sensitivity, accuracy, precision, and applicability to real commercial milk samples.

Methodology and Used Instrumentation

The sample preparation employed a two-step QuEChERS workflow:

• Primary extraction: 10 g milk with 10 mL acetonitrile and a ceramic homogenizer, using an Agilent BondElut QuEChERS EN kit (method 15662EN) containing MgSO₄, NaCl, and citrate buffers.
• Dispersive cleanup: 6 mL extract treated with a dispersive SPE kit containing PSA, C18EC, and MgSO₄ to remove fats and matrix interferents.
• Final extract transferred to a glass vial for analysis.

GC/MS/MS analysis was performed on an Agilent 7890A gas chromatograph coupled to a 7000B triple quadrupole MS in electron-impact mode with constant flow/midcolumn backflush configuration (Pesticide Analyzer 412):

• Column: two Agilent J&W HP-5ms UI (15 m × 0.25 mm, 0.25 µm) with Purged Ultimate Union for backflush.
• Injection: 1 µL splitless, inlet at 280 °C; helium carrier in constant flow at 1 mL/min.
• Oven program: 60 °C (1 min) → 170 °C at 40 °C/min → 310 °C at 10 °C/min → 280 °C at 16 °C/min, total run 20.75 min.
• MS settings: transfer line 280 °C, source 300 °C, Q1/Q2 at 180 °C, solvent delay 2.3 min, multiple reaction monitoring (MRM) transitions per Agilent Pesticide Analyzer database.

Main Results and Discussion

Method validation demonstrated:

• Limits of detection (LODs) between 0.0005 and 0.0369 mg/kg and limits of quantitation (LOQs) from 0.004 to 0.1 mg/kg.
• Recoveries of 70–120% for all compounds except dichlorvos (137%), with relative standard deviations (RSDs) below 20% at LOQ levels, meeting SANCO/12571/2013 criteria.
• Calibration curves over six levels showed linearity with R² values above 0.96 for all analytes.

The method was applied to 28 UHT whole‐milk samples (14 brands, two production batches). Chlorpyrifos was detected in four samples at 0.0022–0.0033 mg/kg and cypermethrin I in one sample at 0.037 mg/kg, all below respective LOQs but above LODs.

Benefits and Practical Applications of the Method

This QuEChERS-GC/MS/MS approach offers:

• High throughput with simple, fast sample preparation suitable for routine monitoring.
• Broad multi-residue coverage, enabling simultaneous analysis of herbicides, insecticides, acaricides, organochlorines, and polychlorinated biphenyls in a single run.
• Excellent sensitivity and selectivity through MRM detection, reducing false positives from complex milk matrix.
• Compliance with national and international MRL standards, supporting regulatory control, food industry quality assurance, and public health surveillance.

Future Trends and Potential Applications

Emerging directions include:

• Integration of high-resolution mass spectrometry for non-target screening and identification of novel contaminants.
• Miniaturized extraction techniques and automation to further increase laboratory throughput and reduce solvent consumption.
• Extension of the methodology to other dairy products (cheese, yogurt) and biological fluids.
• Data analytics and machine-learning tools for pattern recognition and risk assessment based on large-scale monitoring datasets.

Conclusion

A validated QuEChERS-based GC/MS/MS method provides reliable quantitation of 60 pesticide residues in UHT bovine milk at sub-MRL levels. The procedure combines efficient fat removal, robust chromatographic separation with midcolumn backflush, and sensitive MRM detection. Application to commercial samples confirmed the method’s suitability for routine surveillance, ensuring consumer safety and regulatory compliance.

Reference

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10. Agilent Technologies. GC/MS/MS Pesticide Residue Analysis Reference Guide, Publication 5991-2389EN, 2013.