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High Sensitivity Quantification of g y QChlormequat and Mepiquat in Seed by

ZIC-HILIC Separation and Triple Quadrupole Detection

Peter AbrahamssonNordic LC/MS SpecialistAgilent Technologies

Quantification of Chlormequat and Mepiquat in Seed by LC/MS/MS

ESAC April 15, 2008

Background

Chlormequat and Mepiquat are two pesticides widely used as plant growth regulators. They act by inhibition of vegetative p g g y y ggrowth and promotion of flowering in a wide range of fruits, vegetables, cereals and cotton. It eliminates in soil through microbiological processes and the end-product is carbonmicrobiological processes and the end-product is carbon dioxide. In animals and humans it accumulates.

EPA (Environmental Protection Agency) has listed theEPA (Environmental Protection Agency) has listed the compounds and hence it requires to be measured.

Quantification of Chlormequat and Mepiquat in Seed by LC/MS/MS

Book by Colborn, Dumanoski, and Myers Raised Public Awareness (1996)Public Awareness (1996)

- reduced immunityreduced immunity- diminished fertility- decreased intelligence……

Quantification of Chlormequat and Mepiquat in Seed by LC/MS/MS

Stability Data on Chlormequat

(Elzner, 1980)

Quantification of Chlormequat and Mepiquat in Seed by LC/MS/MS

Methods for Analysis

Modern methods based on LC/MS/MS (Triple Quadrupole Systems)

Sample preparation based on simple liquid extraction followed by direct injection or alternatively further clean-up by SPE

Separation based on ion-exchange or RP chromatography

Quantification by ESI-LC/MS/MS (MRM)

Best reported LOQ: 8.3 pg on column (corresponds to a 10 µL injection of a sample containing 0.005 mg/kg)

Quantification of Chlormequat and Mepiquat in Seed by LC/MS/MS

Requirement for New Method

Improve Robustness (reduce ion suppression effects)

Improve Sensitivity

Shorten the Analysis time (old method 12 min between i j ti )injections)

Easy To Use – Results Less Dependant on Operator Skills

Quantification of Chlormequat and Mepiquat in Seed by LC/MS/MS

Agilent 1200 RRLC and 6410 QQQ System

Quantification of Chlormequat and Mepiquat in Seed by LC/MS/MS

Analytical Challenge

ChlormequatMepiquat

Very, Very, Very Polar

Quantification of Chlormequat and Mepiquat in Seed by LC/MS/MS

Choice of Column was the Key

ZIC-HILIC column (Merck Sequant AB) is a column with a permanent Zwitter ionic interaction (independant of pH) that owns both ion-exchange properties as well as hydrophilic g p p y pinteractions. Best described as straight phase chromatography

The more polar the longer retention!

Consequence: Higher % organic resulted in higher sensitivity!

Quantification of Chlormequat and Mepiquat in Seed by LC/MS/MS

q g g g y

Sample Preparation Method

Liquid Phase Extraction by Seed• Liquid Phase Extraction by Seed

• Isotopic Labelled Internal Standards (D4 and D3)

• Injection on ZIC-HILIC Column

• Separation in Isocratic Mode

• Detection by Electrospray LC/MS/MS (MRM mode)

Quantification of Chlormequat and Mepiquat in Seed by LC/MS/MS

Sample preparation - detailed

• Weigh 10 g of seed

• Grind and add 20 mL of MilliQ plus 40 mL Methanol with ISTD• Grind and add 20 mL of MilliQ plus 40 mL Methanol with ISTD

• Take an aliqout of 5 mL of extraction solvent

• Add the Apropriate Amount of Standard to the Standard• Add the Apropriate Amount of Standard to the Standard Samples

• Filter

•Inject 1µl Extract on ZIC-HILIC Column (3.5um, 2.1*100mm, 80/19.5/0.5, v/v/v, AcN/MilliQ/FA 25mM NH4Ac overall

t ti @ 0 4 L/ i )concentration @ 0.4mL/min)

• Detection and Quantification by ESI/LC/MS/MS

Quantification of Chlormequat and Mepiquat in Seed by LC/MS/MS

Mass Spectrometer Settings

Heated nitrogen drying gas

Nebulizer tip aimed at 90 degree angle to inlet of capillary

Ions

+

drying gas

Solvent spray-3500 V

++

++

+++ + + + + + + + + + + + + ++

Dielectric capillary•Pos ESI mode

•Gas Temperature 300ºC

Dielectric capillary entrance

•Gas Flow 10L/min

•Nebulizer Pressure 40 Psig

•VCap 3500V

Quantification of Chlormequat and Mepiquat in Seed by LC/MS/MS

•VCap 3500V

Mass Spectrometer Settings cont.

Compound Molecular Ion Product Ion Fragmentor CE

Chlormequat-D4 126 58.1 140 35

Chlormequat 122 58.1 140 35

Mepiquat-D3 117.1 98.2 (Quant)

61.1 (Qual)

140

140

30

30Mepiquat 114 1 98 2 (Quant) 140 30Mepiquat 114.1 98.2 (Quant)

58.1 (Qual)

140

140

30

140

Delta EMV = 400VUnit Res both Quad´s

Quantification of Chlormequat and Mepiquat in Seed by LC/MS/MS

Typical Chromatogram. L1=0.005mg/kg. 800fg on column.Elution order. Chlormequat, Mepiquat.

1.9 min

Quantification of Chlormequat and Mepiquat in Seed by LC/MS/MS

Summary of Results for Chlormequat (Standards)

Level Conc. (mg/kg) RSD (%)

n=4

Average Accuracy (%)

n=6L7 0.3 5.5

L6 0 1 3 0L6 0.1 3.0

L5 0.05 3.2

L4 (QC) 0.025 5.1 (n=6) 100.7

L3 (QC) 0.0125 4.8 (n=6) 104.7

L2 0.01 6.0

L1 0.005 6.1

Quantification of Chlormequat and Mepiquat in Seed by LC/MS/MS

Calibration Curve for Chlormequat

Note: Quadratic curve fit, origin ignored, weightning 1/x2. R2=0.999

Quantification of Chlormequat and Mepiquat in Seed by LC/MS/MS

Summary of Results for Mepiquat (Standards)

Level Conc. (mg/kg) RSD (%)

n=4

Average Accuracy (%)

n=6L7 0.3 2.7

L6 0 1 3 5L6 0.1 3.5

L5 0.05 2.7

L4 (QC) 0.025 6.6 (n=6) 98.7

L3 (QC) 0.0125 2.8 (n=6) 102.3

L2 0.01 8.1

L1 0.005 3.9

Quantification of Chlormequat and Mepiquat in Seed by LC/MS/MS

Calibration Curve for Mepiquat

Note: Linear curve fit, origin ignored, weightning 1/x2. R2=0.997

Quantification of Chlormequat and Mepiquat in Seed by LC/MS/MS

Summary

• QC Samples shows excellent Accuracy and Reproducability

LOQ (s/n 10:1 P:P) 150fg on column (compared to 8 3pg)• LOQ (s/n 10:1 P:P) 150fg on column (compared to 8.3pg)

• Analysis Time 4 minutes between injections

• Method has – succesfully - been running in routine for 6 months

Quantification of Chlormequat and Mepiquat in Seed by LC/MS/MS

Acknowledgement

Lone Möller and Susanne Davidsen, Valsemöllen A/S Who kindly provided the sampels standards the lab and the instrumentsWho kindly provided the sampels, standards, the lab and the instruments

Quantification of Chlormequat and Mepiquat in Seed by LC/MS/MS

Thank You for Your attention

Questions?

Quantification of Chlormequat and Mepiquat in Seed by LC/MS/MS