Analysis of Residual Pesticides in Solvent Extract of Annatto Using GC-MS/MS

Significance of the topic

Annatto oleoresin is a widely used natural food colorant and flavoring ingredient derived from Bixa orellana seeds. Commercial production commonly involves pesticide use during cultivation, and the dye extraction process can concentrate residues into the oleoresin. Reliable multi-residue pesticide analysis in this highly pigmented, complex matrix is therefore important for food safety, regulatory compliance and consumer protection.

Goals and study overview

This study aimed to develop and validate a rugged, sensitive multi-residue method for quantifying pesticide residues in annatto solvent extract. The approach combined a modified QuEChERS extraction/cleanup with triple-quadrupole gas chromatography tandem mass spectrometry (GC-MS/MS). Validation followed SANTE guidance and targeted 49 pesticides commonly observed in annatto seed/oleoresin.

Methodology and sample preparation

The analytical workflow comprised:

• Extraction: Modified QuEChERS using acetonitrile as extraction solvent and an optimized salt mix (sodium chloride and anhydrous magnesium sulfate) to maximize analyte recovery from the pigmented oleoresin.
• Cleanup: Dispersive SPE with a combination of C18, graphitized carbon black (GCB), primary secondary amine (PSA), and additional anhydrous MgSO4 to remove pigments and matrix interferences, reduce instrument fouling and lower method LOQs.
• Concentration/reconstitution: Post-cleanup acetonitrile aliquots were evaporated and reconstituted in ethyl acetate, producing a 10-fold diluted final extract prior to GC-MS/MS injection.
• Calibration and spiking: Matrix-matched calibration standards over 0.5–1000 μg/L were used with weighted 1/C2 regression. Spike recovery experiments were performed at 10, 25 and 100 μg/kg with six replicates per level.

Used instrumentation

The analysis employed Shimadzu triple-quadrupole GC-MS/MS instrumentation (GCMS-TQ8xxx NX series referenced; figures and text refer to GCMS-TQ8050 NX and GCMS-TQ8040 NX). Key instrumental settings from the method were:

• GC column: SH-I-5Sil MS, 30 m × 0.25 mm i.d., 0.25 μm film.
• Injection: Splitless (high pressure 250 kPa for 1.5 min), 2 μL injection volume, injector temperature 250 °C.
• Oven program: 80 °C (2 min), ramp 20 °C/min to 180 °C, then 5 °C/min to 330 °C (hold 3 min); total run ~34 min.
• Carrier gas: Helium, linear velocity 40.4 cm/s (constant flow).
• MS conditions: Electron ionization (EI), interface temperature ~280 °C, ion-source temperature 230 °C, loop time 0.3 s, solvent cut 5.0 min. Multiple reaction monitoring (MRM) transitions were set individually per analyte.
• Data handling: LabSolutions Insight software for acquisition and validation assessment.

Main results and discussion

Validation followed SANTE/11312/2021 criteria (linearity, recovery, precision, LOQ). Key findings:

• Sensitivity/LOQs: Method LOQs ranged from 10 to 100 μg/kg across 49 pesticides. Specifically, 24 pesticides achieved 10 μg/kg LOQ, 21 pesticides 25 μg/kg, and 4 pesticides 100 μg/kg.
• Linearity: Matrix-matched calibration (0.5–1000 μg/L) produced calibration points with accuracy within the SANTE 80–120% requirement; weighted 1/C2 regression was applied and correlation coefficients were high for target analytes.
• Recoveries: Most analytes returned mean recoveries between 60–120% at spiking levels, meeting SANTE expectations. Two analytes (captan and etoxazole) showed lower mean recoveries (~50–60%) but maintained acceptable repeatability (RSDr ≤20%).
• Precision: Repeatability (RSDr) for six replicates at relevant LOQs was <20% for all analytes; within-laboratory reproducibility (RSDR) was <30% per SANTE criteria.
• Robustness in pigmented matrix: The modified QuEChERS cleanup (notably use of GCB and C18) effectively minimized pigmentation interferences and allowed trace-level detection even after a 10-fold dilution of the final extract, helping reduce matrix effects and instrument contamination.

Benefits and practical applications

The developed method offers several practical advantages:

• Simultaneous multi-residue quantification of a broad pesticide panel in a challenging, highly pigmented food extract.
• Achieves low µg/kg-level LOQs suitable for regulatory monitoring and quality control in food industry laboratories.
• Modified QuEChERS cleanup reduces matrix effects and helps protect GC-MS/MS instrumentation, increasing throughput and method ruggedness.
• Compatibility with routine laboratory workflows: matrix-matched calibration, standard MRM acquisition and commonly available consumables enable implementation in analytical and contract testing labs.

Future trends and potential uses

Possible directions to expand and further improve the approach include:

• Extending analyte scope to include additional polar/non-volatile pesticides or metabolites using complementary LC-MS/MS methods.
• Incorporating isotopically labeled internal standards to improve quantitative accuracy and compensate for residual matrix effects.
• Exploring high-resolution MS (HRMS) for non-target screening to discover unexpected contaminants or degradation products.
• Method miniaturization and solvent reduction to improve sustainability, and automation of cleanup for higher throughput.
• Inter-laboratory validation and participation in proficiency testing to support regulatory acceptance and harmonization.

Conclusion

The combined modified QuEChERS extraction/cleanup and Shimadzu GC-MS/MS method provides a reliable, sensitive and reproducible solution for multi-residue pesticide analysis in annatto oleoresin. The approach delivered LOQs of 10–100 µg/kg for 49 pesticides, acceptable recoveries and precision according to SANTE guidelines, and demonstrated robustness against strong pigmentation and matrix complexity. The method is well suited for routine monitoring and food safety testing of annatto extracts.

References

1. 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. Journal of AOAC International. 2003;86:412–431.
2. European Commission Directorate-General for Health and Food Safety. Guidance document on analytical quality control and method validation procedures for pesticide residues and analysis in food and feed. SANTE/11312/2021.