Simultaneous Determination of Organophosphate and Phenoxy-based Herbicides in Black Tea using LC-MS/MS and QuPPePretreatment

Importance of the Topic

Black tea is one of the most widely consumed beverages worldwide, and monitoring pesticide residues is critical to ensure consumer safety. Organophosphate and phenoxy herbicides, which differ greatly in polarity and chemical behavior, require sensitive and versatile analytical methods. Regulatory agencies in the EU and Japan have established strict maximum residue limits (MRLs), driving the need for comprehensive multi‐residue screening in food products such as black tea.

Objectives and Study Overview

This study aimed to develop and validate a rapid LC‐MS/MS method on the Shimadzu LCMS‐8060NX platform for simultaneous determination of highly polar organophosphate herbicides (glyphosate, glufosinate, AMPA) and less polar phenoxy herbicides (MCPA, 2,4-D, 4-CPA) in black tea. A modified QuPPe (Quick Polar Pesticides) pretreatment was applied to prepare samples for analysis.

Methodology and Instrumentation

A modified QuPPe protocol involved the following steps: hydration of tea leaves, extraction with methanol containing formic acid, centrifugation at low temperature, acetonitrile‐induced precipitation, filtration, sample dilution, and ultrafiltration. Calibration standards ranged from 0.5 to 100 ng/mL for organophosphates and from 0.05 to 10 ng/mL for phenoxy herbicides. Stainless steel tubing was replaced with PEEK tubing to prevent chelation of organophosphate analytes.

Instrumentation

• Shimadzu LCMS-8060NX triple quadrupole mass spectrometer
• Heated electrospray ionization interface in negative mode
• Acclaim Trinity Q1 mixed-mode column (3 x 100 mm, 3 μm)
• Mobile phase A: water with 50 mM ammonium formate and 1% formic acid (pH 2.8)
• Mobile phase B: acetonitrile
• Gradient elution over 20 minutes, column temperature 40 °C, injection volume 30 μL
• Multiple reaction monitoring (MRM) with optimized collision energies

Main Results and Discussion

The method achieved separation of all six target compounds within 20 minutes. Calibration curves demonstrated excellent linearity (r2 > 0.998) across the tested ranges. Limits of quantitation in solvent ranged from 0.04 to 1.61 ng/mL, and in tea matrix the LOQs were 0.06 to 2.62 ng/mL (0.0024–0.1048 mg/kg), well below the EU and JP MRLs. Significant matrix suppression was observed, particularly for AMPA, and was mitigated by sample dilution. Intra-day and inter-day precision yielded relative standard deviations below 17%, and recoveries ranged from 83% to 131%. Analysis of four commercial black tea brands detected trace residues below regulatory limits.

Benefits and Practical Applications

• The method allows simultaneous multi-class herbicide screening in a single run, reducing analysis time and resource use
• Sensitivity meets or exceeds regulatory requirements for tea quality control
• The workflow is adaptable to routine laboratory settings for compliance testing

Future Trends and Applications

• Incorporation of isotopically labeled internal standards to further improve quantitation accuracy and address residual matrix effects
• Extension of this approach to additional polar pesticide classes and diverse food matrices
• Automation of sample preparation and high-throughput capabilities to increase laboratory productivity

Conclusion

A robust LC-MS/MS method on the Shimadzu LCMS-8060NX was established for the simultaneous quantitation of organophosphate and phenoxy herbicides in black tea. The method demonstrates high sensitivity, reliable precision, and compliance with stringent regulatory LOQs, making it well suited for routine monitoring and quality assurance.

References

• Chamkasem N Harmon T Analysis of highly polar herbicides in food by LC-MS/MS Anal Bioanal Chem 2016 408 4995-5004
• EURL-SRM QuPPe PO Method Version 12 European Union Reference Laboratory 2021
• Sun Z Zhan Z Simultaneous multi residue analysis of herbicides in rice Shimadzu Application News C250 2021
• Chamkasem N Morris C Multi class pesticide analysis in food matrices Journal of Regulatory Science 2016 4 9-18
• Wang J Chow W Leung D Determination of herbicide residues in tea by LC-MS/MS J AOAC Int 2011 94 1685-1714
• Stahnke H Kittlaus S Kempe G Alder L Evaluation of matrix effects in pesticide analysis by LC-MS Anal Chem 2012 84 1474-1482