Trace Level estimation of Glyphosate and Glufosinatein Red Chilli Pepper using FMOC-derivatization

Importance of the topic

Glyphosate and glufosinate are widely used herbicides whose residues in food commodities are tightly regulated because of potential human-health risks. Red chilli powder is a chemically complex, highly pigmented plant matrix that poses analytical challenges (poor chromatographic behavior, ion suppression, low ionization efficiency) for these very polar analytes. Reliable trace-level measurement methods that satisfy regulatory limits (down to 0.001 mg/kg in this study) are essential for food safety monitoring, trade compliance and consumer protection.

Objectives and study overview

This study demonstrates an LC–MS/MS workflow based on FMOC-Cl derivatization to enable sensitive, robust quantitation of glyphosate and glufosinate in red chilli powder at trace levels. Key aims were to: achieve an LOQ of 0.001 mg/kg, demonstrate acceptable recoveries in a difficult matrix using pre-spike (matrix-matched) samples, compare performance in electrospray ionization (ESI) positive and negative modes, and show compliance with current regulatory criteria (including SANTE ion-ratio requirements).

Methodology

Sample preparation and derivatization (paraphrased):

• Weighed 2.5 g of red chilli powder and soaked in 10 mL of 1% formic acid, then extracted with 10 mL dichloromethane.
• After centrifugation, 1 mL of the supernatant was taken; 0.7 mL of 5% borate buffer and 0.3 mL of 40 mM FMOC-Cl were added to effect FMOC derivatization.
• Derivatization was performed at 40 °C for 45 minutes in the dark with agitation.
• Samples were cooled, acidified with 0.05 mL formic acid, centrifuged (5,000 rpm, 5 min) and analyzed.

Chromatography and MS conditions (summary):

• Reversed-phase UHPLC gradient using mobile phase A = 5 mM ammonium acetate and B = methanol. Initial conditions are 99% A / 1% B, ramping to 1% A / 99% B by 7.0 min, returning to initial composition around 9.1 min; total run ~13 min. Flow = 0.4 mL/min.
• Column thermostatted at 40 °C; autosampler at 5 °C; injection volume 20 µL.
• ESI with Agilent AJS source operated in both positive and negative polarities; capillary voltages ~+2700 V and −3500 V, nozzle 500 V. Gas settings: drying gas ~9 L/min at 300 °C, sheath gas ~10 L/min at 300 °C, nebulizer ~35 psi.

Instrumentation used

The experiments were performed on an Agilent 1290 UHPLC coupled to an Agilent 6475 triple-quadrupole LC/TQ mass spectrometer. Column referenced as Agilent P/N 693975-302 (operation at 40 °C). Standard instrument settings summarized above were used for both ESI(+) and ESI(−) acquisitions.

Results and discussion

Sensitivity and linearity:

• The method achieved quantitation at 0.001 mg/kg (1 ppb) in red chilli powder for both glyphosate and glufosinate.
• Calibration was linear from 0.001–0.1 mg/kg (1–100 ppb). Correlation coefficients were very high: r2 > 0.9996 (ESI negative) and r2 > 0.995 (ESI positive), demonstrating excellent linear response across the target range.
• Matrix blanks showed signal well below the LOQ, indicating limited background interference at the targeted levels.

Mode comparison and matrix effects:

• ESI negative mode delivered superior signal-to-noise and peak quality compared with positive mode for the FMOC derivatives; area counts and S/N were higher in negative polarity.
• ESI positive spectra exhibited greater baseline disturbance consistent with stronger matrix effects, yet linearity and ion-ratio requirements were still met in positive mode.

Regulatory compliance and quality criteria:

• The method met SANTE (2026) criteria including ion-ratio acceptability for both analytes in the challenging red chilli matrix.
• Performance aligns with regulatory sensitivity expectations from agencies such as FDA, EU (MRLs), and FSSAI for commodity monitoring.

Additional observations:

• Pre-spike (matrix-matched) sample preparation produced satisfactory recoveries, indicating the derivatization and cleanup strategy is effective for this matrix.
• Derivatization with FMOC-Cl mitigates issues related to poor chromatography and low ionization efficiency of underivatized polar herbicides, facilitating routine LC–MS/MS measurement.

Benefits and practical applications

The method offers several practical advantages for commercial and regulatory laboratories:

• Sensitivity down to 0.001 mg/kg allows compliance with the most stringent MRL/LOQ expectations for spices and other plant matrices.
• FMOC derivatization improves chromatographic retention and MS response for highly polar analytes, reducing false negatives and increasing method robustness.
• Dual-polarity acquisition (ESI− and ESI+) provides flexibility: ESI negative offers better sensitivity for these derivatives, while positive mode remains a viable option when required by laboratory workflows or instrumentation constraints.
• Relatively short UHPLC run (~13 min) supports reasonable sample throughput for routine monitoring campaigns.

Future trends and potential applications

Opportunities to expand and modernize this approach include:

• Automation of derivatization and sample handling to improve throughput and reproducibility, particularly for high-volume testing laboratories.
• Extension to multi-residue methods that combine FMOC-based detection of phosphonate/polars with conventional pesticide panels, or migration to high-resolution MS for additional confirmatory power.
• Use of isotopically labeled internal standards to further correct matrix effects and improve quantitative accuracy.
• Optimization toward greener solvents and minimizing solvent volumes to reduce environmental impact and cost.
• Adaptation for other challenging matrices (spices, dried herbs, fermented products) and incorporation into surveillance programs for imported/exported goods.

Conclusion

An FMOC-Cl derivatization workflow combined with targeted UHPLC–MS/MS detection provides a robust solution for trace-level determination of glyphosate and glufosinate in red chilli powder. The method achieves the targeted LOQ of 0.001 mg/kg, demonstrates excellent linearity and acceptable recoveries in a difficult matrix, meets current ion-ratio and regulatory criteria, and offers operational flexibility through dual-polarity acquisition. This makes it a practical approach for commercial testing laboratories and regulatory monitoring.

References

1. Code of Federal Regulations, Title 40, Part 180 — pesticide tolerances (US EPA regulatory framework for tolerances).
2. European Commission — EU Pesticides Database, maximum residue limits for glyphosate and glufosinate (EU regulatory MRL listings).
3. Food Safety and Standards Authority of India — Contaminants Regulations, 2026 (national limits and guidance for contaminants).