Multiresidue Pesticides Analysis in Solvent Extract of Annatto (Natural Color Additive) Using GCMSTQ8050 NX

Multiresidue Pesticide Analysis in Annatto Solvent Extract Using GCMS-TQ8050 NX

Significance of the topic

Annatto oleoresin is a widely used natural colorant and flavoring ingredient produced from Bixa orellana seeds. Because large-scale cultivation commonly involves pesticide use, traces of multiple pesticides can concentrate during solvent extraction and persist in the final color additive. Reliable multiresidue analysis in this highly pigmented, lipid- and pigment-rich matrix is therefore critical for food safety, regulatory compliance and quality control in the food industry.

Objectives and study overview

• Develop and partially validate a single-laboratory method for simultaneous quantitation of a panel of 49 pesticides in annatto solvent extract.
• Combine a modified QuEChERS extraction/cleanup with ultra-fast GC-MS/MS (Shimadzu GCMS-TQ8050 NX) to achieve trace-level limits of quantification (LOQs) that meet SANTE guidance criteria.
• Demonstrate method linearity, recovery and precision across relevant concentration ranges and identify any analytes with problematic recovery.

Methodology and sample preparation

• Reference standards: Custom pesticide mix purchased for the compounds most often detected in annatto.
• Extraction: Modified QuEChERS approach using acetonitrile as extraction solvent with optimized proportions of NaCl and anhydrous MgSO4 to improve partitioning and recovery from the pigmented oleoresin matrix.
• Cleanup: Dispersive SPE using a combination of C18, graphitized carbon black (GCB), primary-secondary amine (PSA) and anhydrous MgSO4 to remove pigments and coextractives, reduce instrument contamination and lower matrix effects.
• Concentration and solvent exchange: Post-cleanup acetonitrile aliquots were evaporated and reconstituted in ethyl acetate; reconstituted extracts were diluted 10-fold prior to analysis to further minimize matrix load on the instrument.
• Calibration and validation design: Matrix-matched calibration standards from 0.5 to 1000 µg/L with weighted (1/C2) linear regression; recovery experiments at three spiking levels (10, 25 and 100 µg/kg) with six replicates per level; validation evaluated according to SANTE guidance (linearity, accuracy/recovery, repeatability and within-lab reproducibility).

Instrumentation used

• Gas chromatograph–triple quadrupole mass spectrometer: Shimadzu GCMS-TQ8050 NX (EI ionization).
• Autosampler: AOC-20i + s.
• Column: SH-I-5Sil MS, 30 m × 0.25 mm i.d., 0.25 µm film thickness.
• Liner: Topaz splitless single taper with wool.
• GC conditions: Splitless injection (2 µL), injector 250 °C, He carrier with constant linear velocity ~40.4 cm/s; oven program: 80 °C (2 min), 20 °C/min to 180 °C, then 5 °C/min to 300 °C (3 min); run time ~34 min.
• MS conditions: Electron ionization, ion source 230 °C, interface 280 °C, solvent cut 5.0 min, loop time 0.3 s. Multiple reaction monitoring (MRM) transitions selected for target quantitation.
• Software and databases: LabSolutions Insight for data processing and validation parameter evaluation; Shimadzu Smart Pesticides Database used for method optimization.

Main results and discussion

• Scope: The method covered 49 target pesticides representative for annatto seed residues.
• Linearity: Matrix-matched calibration across 0.5–1000 µg/L produced acceptable linearity; calibration points met SANTE requirement of 80–120% accuracy and used weighted (1/C2) linear regression. Representative R2 values for shown compounds were >0.98.
• Limits of quantification (LOQs): Method LOQs were established between 10 and 100 µg/kg. Distribution: 24 pesticides achieved 10 µg/kg LOQ, 21 compounds 25 µg/kg, and 4 compounds 100 µg/kg.
• Recovery and accuracy: At LOQ levels (after the 10× dilution step), most analytes showed mean recoveries within 60–120% with reproducibility (RSDR) <30%—in line with SANTE criteria. Two compounds (Captan and Etoxazole) exhibited reduced mean recoveries (approximately 50–60%) but acceptable repeatability (RSDr ≤20%).
• Precision: Repeatability (RSDr) for six replicate injections at each spiking level was generally <20%; within-lab reproducibility (RSDR) for six independent spiked samples was <30% for the majority of targets.
• Practical matrix handling: The modified QuEChERS cleanup with C18/GCB/PSA effectively reduced pigment interference and instrument contamination, enabling sensitive MS/MS detection even after sample dilution.

Benefits and practical applications >

• The combined modified QuEChERS and GC-MS/MS workflow permits robust multiresidue monitoring of pesticides in a challenging, highly pigmented food additive matrix with trace-level sensitivity.
• Tenfold dilution after cleanup reduces matrix load, prolongs column and source lifetime, and supports reproducible quantitation of many analytes without excessive matrix suppression.
• Use of a comprehensive pesticide database and modern software simplifies method optimization and validation reporting for routine food safety laboratories and QC units.

Future trends and potential applications

• Further matrix-matched or isotopically labelled internal standards could be incorporated to improve accuracy for analytes with lower recoveries (e.g., Captan, Etoxazole) and to compensate for residual matrix effects after dilution.
• Expansion of the target list and validation across additional annatto-derived products (powders, oils, finished food items) would broaden applicability and regulatory coverage.
• Automation of sample cleanup (e.g., online SPE or robotic dispersive-SPE workflows) and higher-throughput GC-MS/MS acquisition strategies could increase laboratory throughput for commercial testing.
• Complementary high-resolution mass spectrometry or orthogonal techniques could be used to identify unexpected non-target contaminants or degradants concentrated during extraction.

Conclusion

The study demonstrates that a tailored QuEChERS extraction/cleanup combined with the Shimadzu GCMS-TQ8050 NX MS/MS platform provides a sensitive, reproducible and rugged approach for quantifying multiple pesticide residues in annatto solvent extracts. Method performance met SANTE validation criteria for most targets, achieving LOQs suitable for routine monitoring while mitigating matrix interference through optimized cleanup and dilution.

References

1. Anastassiades M., Lehotay S. J., Štajnbaher D., Schenck F. J., 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, 86 (2003) 412–431.
2. European Commission Health & Food Safety Directorate: Guidance document on analytical quality control and method validation procedures for pesticide residues and analysis in food and feed. SANTE/11312/2021.