Analysis of Pesticide Residues in Spinach using Phenomenex roQ™ QuEChERS AOAC Kits by LC/MS/MS and GC/MS

Significance of the Topic

The analysis of pesticide residues in leafy vegetables such as spinach is critical for ensuring food safety and compliance with international maximum residue limits. Spinach’s high pigment content, water content and fibrous matrix pose significant challenges for multi-residue pesticide analysis. The development of robust sample preparation techniques and sensitive instrumental methods enables laboratories to deliver reliable, high-throughput monitoring of diverse pesticide classes in complex food matrices.

Objectives and Study Overview

The study aimed to evaluate the performance of Phenomenex roQ™ QuEChERS AOAC 2007.01 extraction kits combined with PSA/GCB dispersive SPE for cleanup of spinach samples. Eighteen representative pesticides plus two internal standards were extracted and analyzed by both LC–MS/MS and GC–MS to demonstrate method versatility, recovery, precision and sensitivity against AOAC acceptance criteria.

Methodology

• Sample Pretreatment: Fresh spinach was frozen with liquid nitrogen, homogenized to a powder, and stored at –80 °C.
• QuEChERS Extraction: 15 g sample was spiked, mixed with 15 mL acetonitrile containing 1% acetic acid and internal standards. A pre-weighed salt packet (6 g MgSO₄, 1.5 g sodium acetate) induced phase separation.
• dSPE Cleanup: 8 mL supernatant was treated with a PSA/GCB blend (1.2 g MgSO₄, 0.4 g PSA, 0.4 g GCB) to remove moisture, fatty acids, and pigments.
• Solvent Exchange:
  • LC–MS/MS aliquot: 250 µL extract evaporated and reconstituted in aqueous formic acid/methanol.
  • GC–MS aliquot: 4 mL extract evaporated with toluene, re-diluted and dried over MgSO₄.

Instrumentation

• LC–MS/MS: Agilent 1200 HPLC with Luna® C18(2) 150 × 3.0 mm, 3 µm column; AB SCIEX API 4000 triple quadrupole; positive ESI; scheduled MRM.
• GC–MS: Agilent 6890N GC with 5973 MS; Zebron™ ZB-5MSi 30 m × 0.25 mm × 0.25 µm column; splitless injection; helium carrier gas; electron ionization.

Main Results and Discussion

• Pigment Removal: PSA/GCB dSPE produced visually clear extracts with most chlorophyll removed. Highly pigmented matrices were effectively cleaned without extensive matrix interferences.
• Recoveries and Precision: For 18 pesticides, mean recoveries ranged from 70% to 120% and RSDs remained below 15%, meeting AOAC 2007.01 criteria.
• Planar Analyte Adsorption: Chromenetically planar pesticides (e.g., cyprodinil, pymetrozine, thiabendazole) showed reduced recoveries due to GCB adsorption. Adding toluene or adjusting sorbent composition can mitigate loss.
• LC Injection Solvent Optimization: Early-eluting peaks exhibited splitting with 25% acetonitrile; switching to 25% methanol in formic acid eliminated distortion and improved peak shape.
• GC–MS Separation: Baseline resolution achieved for all target compounds. GC–MS provided complementary sensitivity for apolar and semi-polar pesticides.

Benefits and Practical Applications

• High Throughput: Pre-measured kits streamline extraction and cleanup, reducing manual weighing errors and labor time.
• Versatility: Applicable to diverse food matrices by modifying sorbent blends (e.g., adding C18 for high-fat samples).
• Regulatory Compliance: Achieves regulatory limits for routine monitoring of multi-class residues in produce.
• Dual Detection: Combined LC–MS/MS and GC–MS expands compound coverage from polar to non-polar pesticides.

Future Trends and Possibilities for Use

• Advanced Sorbents: Development of novel graphitized carbon derivatives to reduce non-specific adsorption of planar analytes.
• Miniaturization: Micro-QuEChERS formats and automated dSPE on 96-well plates for ultra-high throughput screening.
• High-Resolution Mass Spectrometry: Integration with QTOF or Orbitrap platforms for non-targeted screening and confirmatory analysis.
• Green Chemistry: Exploration of alternative solvents and sorbents to reduce environmental impact.

Conclusion

The Phenomenex roQ™ QuEChERS AOAC kits combined with PSA/GCB dSPE offer a fast, reliable, and reproducible workflow for the multi-residue analysis of pesticides in spinach. The approach delivers clear extracts, robust recoveries, and compatibility with both LC–MS/MS and GC–MS detection. Method adaptations accommodate both highly pigmented matrices and challenging analytes, making this format a valuable tool for modern residue testing laboratories.

References

1. Anastassiades et al., J. AOAC Int., 2003, 86(2), 412–431.
2. Lehotay et al., J. AOAC Int., 2005, 88(2), 595–614.
3. Lehotay, J. AOAC Int., 2007, 90(2), 485–520.
4. Pinto et al., Talanta, 2010, 81(2), 385–391.
5. Brondi et al., Bull. Environ. Contam. Toxicol., 2011, 86, 18–22.