©2015 Waters Corporation 1

Enantioseparation and Trace Level Detection of Fungicides by UPC2 and Xevo TQ-S micro

Peter Hancock Marion Twohig

©2015 Waters Corporation 2

Introduction

Crop protection chemicals provide benefits in terms of an increase in crop yields however, they must also be managed responsibly in order to minimize any adverse effects

Extensive studies and trials are carried out in support of agrochemical product registration Study data are submitted to regulatory bodies, e.g. EPA, along with the registration application. Studies ensure that any risks associated with using the product are characterised and properly understood so that it can be safely applied in the field

©2015 Waters Corporation 3

Chiral Pesticides

When a crop protection active ingredient (AI) contains one of more stereogenic centers the enantioselective behaviour must be studied as it is known that enantiomers can exhibit different bioactivities

Analytical methods used to evaluate the influence of

stereochemistry on the degradation dynamics, environmental fate and final residue levels help to establish a more accurate risk assessment of crop protection products

Detection sensitivity and specificity offered by tandem quad

MS/MS is advantageous for determining trace levels in complex matrices like field crops or soil

©2015 Waters Corporation 4

Experimental Description

ACQUITY UPC2 and Xevo TQ-S micro are used for the trace level enantioanalysis of five triazole fungicides in wheat grain and wheat straw

DisQue extraction modified for dry commodities was performed followed by SPE using Oasis MCX

Chiral separations using the new Trefoil AMY1, 2.5 µm column

followed by MRM detection allowed concentrations of part per trillion levels to be reproducibly detected and quantified

©2015 Waters Corporation 5

Triazole Fungicides

(R)-Flutriafol (S)-Flutriafol

(R)-Tebuconazole (S)-Tebuconazole

(R)-Fenbuconazole (S)-Fenbuconazole

(R)-Uniconazole (S)-Uniconazole

(R)-Diniconazole (S)-Diniconazole

* * * *

* * * *

* *

©2015 Waters Corporation 6

UPC2 Conditions

Column: ACQUITY UPC2 AMY1, 3.0 x 150 mm, 2.5 µm Column temp: 40 0C ABPR: 3000 psi Flow rate: 2.0 mL/min Make up solvent: 90:10 methanol/water + 0.1% formic acid Injection volume: 4 µL Co-solvent (B): Methanol Gradient: 88% CO2 (hold 0.1 min) → 65% CO2

(hold 2.0 min) in 1.5 min

Learn more at Waters.com : Click Here

©2015 Waters Corporation 7

Xevo TQ-S micro Conditions

Ionisation: ESI+ Capillary: 1.5 kV Cone voltage: 30 V Source temp: 150 °C Desolvation temp: 500 °C Desolvation gas: Nitrogen @ 1000 L/hr Collision gas: Argon @ 3.2 x 10-3 mBar Uniconazole: 292.3 → 70.0 @ 23 eV Flutriafol: 302.2 → 70.0 @ 15 eV Tebuconazole: 308.3 → 70.0 @ 20 eV Diniconazole: 326.3 → 70.0 @ 24 eV Fenbuconazole: 337.3 → 70.0 @ 18 eV

Learn more at Waters.com Click Here

©2015 Waters Corporation 8

Triazoles Enantioseparation

Fenbuconazole

Uniconazole

Tebuconazole

Flutriafol

Diniconazole 10 ng/g in

wheat grain

©2015 Waters Corporation 9

Enantiomer 1 R2 = 0.9983

Enantiomer 2 R2 = 0.9985

Linear Calibration

Uniconazole 0.05 - 50 ng/mL

Wheat Straw

Compound name: Uniconazole 1Correlation coefficient: r = 0.999172, r^2 = 0.998345Calibration curve: 13622.2 * x + 305.816Response type: External Std, AreaCurve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

ng/mL-0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 42.5 45.0 47.5 50.0

Res

pons

e

-0

200000

400000

600000

ng/mL

Res

idua

l

-5.0

0.0

5.0

Compound name: Uniconazole 2Correlation coefficient: r = 0.999270, r^2 = 0.998540Calibration curve: 13765.2 * x + 328.733Response type: External Std, AreaCurve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

ng/mL-0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 42.5 45.0 47.5 50.0

Res

pons

e

-0

200000

400000

600000

ng/mL

Res

idua

l

-5.0

0.0

5.0

10.0

©2015 Waters Corporation 10

Trace Level Detection Limits Xevo TQ-S micro

0.05 ng/mL level for all compounds spiked into extracted wheat grain

matrix

Fenbuconazole

Uniconazole

Tebuconazole

Flutriafol

Diniconazole

©2015 Waters Corporation 11

Summary

Enantioseparation of five triazole fungicides performed in less than 3.5 minutes using Trefoil AMY1 column

Xevo TQ-S micro provided linear calibrations of the triazole fungicides in wheat grain and wheat straw at trace levels successfully enveloping regulatory concentrations

Xevo TQ-S micro provided excellent sensitivity to relevant trace levels in wheat grain matrix

Combination of Trefoil columns, ACQUITY UPC2 and Xevo TQ-S micro successfully met expectations for this application