Enantioseparation and Detection of Triazole Fungicides in Wheat Grain and Wheat Straw using the ACQUITY UPC2 System and Xevo TQ-S

Importance of the topic

The enantioselective analysis of triazole fungicides is critical because stereoisomers can display distinct biological activities, environmental behaviors, and toxicological profiles. Accurate measurement of each enantiomer at trace levels supports more precise risk assessments and informs safe agricultural applications.

Objectives and study overview

This work aims to develop a rapid, high-throughput method for the separation and quantitation of five triazole fungicide enantiomers in wheat grain and straw. The target compounds include diniconazole, fenbuconazole, flutriafol, tebuconazole, and uniconazole. The study integrates supercritical fluid chromatography on a chiral stationary phase with tandem mass spectrometry detection to achieve parts-per-trillion sensitivity.

Methodology and instrumentation

Sample preparation

• Wheat straw (1 g) or grain (5 g) were hydrated, acidified, and extracted with acetonitrile following a modified QuEChERS protocol.
• DisQuE QuEChERS salts induced phase separation; the extract underwent cleanup via Oasis MCX solid phase extraction.
• Eluates were dried and reconstituted in methanol prior to analysis.

Chromatography and detection

• ACQUITY UPC2 system equipped with Chiralpak IA-3 column (4.6 × 150 mm, 3 μm) was used.
• Supercritical CO2 mobile phase with methanol or isopropanol/ethanol modifier containing 2 water and 0.1 formic acid provided rapid enantioseparation under 5 minutes.
• Xevo TQ-S tandem quadrupole mass spectrometer operated in ESI positive mode with MRM transitions enabled ppt-level detection.
• RADAR full-scan MS and PICs product ion scans were applied concurrently for qualitative confirmation and interference monitoring.

Main results and discussion

All five fungicide enantiomer pairs achieved baseline resolution in under 3.5 minutes with USP resolutions ranging from 1.7 to 6.8. Calibration curves in matrix matched extracts were linear (r2>0.998) over 0.005 to 50 ng/mL. Limits of quantitation corresponded to approximately 0.01 ng/g in sample. Precision studies (n=4) yielded relative standard deviations below 4  at multiple concentration levels. Recoveries exceeded 75  across fortification levels and matrix effects remained within ±10 . RADAR and PICs tools identified coeluting interferences and guided optimization of cleanup and isocratic elution to enhance specificity.

Benefits and practical applications

• Significant reduction in analysis time compared with HPLC chiral separations (from 13-45 minutes to under 5 minutes).
• High throughput with reliable enantiomer ratio measurement for environmental and degradation studies.
• Trace level quantitation in complex matrices supporting regulatory compliance and QA/QC workflows.
• Orthogonal separation technique complementary to reversed-phase LC for challenging chiral analytes.

Future trends and opportunities

• Extension of UPC2-MS methods to a broader range of chiral agrochemicals and contaminants.
• Advances in chiral stationary phase materials and sub-2 μm particle technologies to further shorten run times.
• Integration of high-resolution mass spectrometry for enhanced identification of unknown stereoisomers.
• Automation and miniaturization of sample preparation using green chemistry principles.
• Implementation in routine environmental monitoring, residue analysis in food and soil, and stereoselective degradation studies.

Conclusion

The combination of supercritical fluid chromatography on a chiral stationary phase with tandem MS detection delivers a robust, sensitive, and rapid approach for enantioselective analysis of triazole fungicides in agricultural matrices. This platform meets the demands for trace-level quantitation, high throughput, and reliable identification required in environmental fate studies and regulatory testing.

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