Routine Quantitative Pesticide Analysis

in Various Food Matrices

using a GC/Q-TOF

Klaus Wilmers
Chemical and Veterinary Analytical Institute Muensterland-Emscher-Lippe
Chemisches und Veterinäruntersuchungsamt Münsterland-Emscher-Lippe

CVUA-MEL
Joseph-König-Str. 40
48147 Münster
Germany

klaus.wilmers@cvua-mel.de
 New accurate mass
EI pesticide library
and workflow for GC
QTOF!

Agilent 7200 GC/Q-TOF

Today’s webinar
Acquire Full-Spectrum Data Collaborator evaluation of
Agilent GC/Q-TOF for
Comprehensive Pesticide
Screening

Targeted Quantitative Targeted Qualitative Next month’s presentation

Screening Screening Overview of new product
including Qual and Quant
workflows and software features
Outline
• Pesticide analysis in CVUA-MEL

• Requirements for pesticide control in Europe

• GC/Q-TOF: Instrument configuration, Pesticide method and MassHunter software

• Exact Mass Pesticide Library PCDL

• Screening method: development and results

• Target quantification method: development and results

• Conclusion
Pesticide analysis in CVUA-MEL

• Official food control for the district of Muenster with 2.5 million people

• pesticide analysis of 1000 samples per year

• mainly fruit and vegetable samples
Requirements for pesticide control by European legislation

• REGULATION (EC) No 396 / 2005

on Maximum Residue Levels of Pesticides
in or on Food and Feed of Plant and Animal Origin

• 1352 active substances

• 378 products

• more than 200.000 MRLs for defined pesticide - product combinations

• general default MRL of 0.01 mg/kg

for not specifically regulated pesticide - product combinations

• today: more than 60.000 default MRL combinations

• default MRL = required Limit of Detection LOD for pesticide methods

Requirements for pesticide analysis by European legislation

• Document SANTE / 11945 / 2015

Guidance document on
Analytical Quality Control and Method Validation Procedures for
Pesticide Residues Analysis in Food and Feed

• requirements for identification by Mass Spectrometry

unit mass MS minimum 3 diagnostic ions

unit mass MS/MS precursor and minimum 2 product ions

high resolution MS: minimum 2 diagnostic ions

mass accuracy < 5 ppm
Pesticide analysis in CVUA-MEL 2016 / 2017

• Pesticide analysis of 1000 (→ 3000) samples per year

• mainly fruit and vegetable samples

• 450 pesticide compounds

• GC/MS for 180 pesticide compounds

• QuEChERS method for sample extraction with Acetonitrile AcN

• Agilent GC/MSD SingleQuad GC/MSD

Screening in full scan mode
Quantification in SIM mode

• Improvement by new instruments

GC/MS TripleQuad and GC/Q-TOF
Agilent 7200 Accurate-Mass Q-TOF GC/MS

7890 Gas Chromatograph: PostRun ColumnBackflush

G4513 MMI MultiModeInjector: Solvent Vent Injection

7693 Autosampler: Vial Mixer/Heater

MMI
Agilent 7200 Accurate-Mass Q-TOF GC/MS

Useful additional equipment

• Injector cooling system

Start temperature 40 °C
Equilibration time 4 min

• 2nd Data station

Postrun batch processing

• Data storage and backup

~ 4 GB per sample
~ 100 GB for one batch of 25 samples

Current disk space

10 TB for processing
60 TB for storage and backup
Gas chromatograph with backflush system

• PostRun purging the 2-column-system by extended pressure

• keep high boiling compounds from reaching the detector

• extremely useful for dirty QuEChERS sample extracts with high matrix load
The GC injection mode

• QuEChERS samples come in Acetonitrile with high matrix load

• polar solvent with low boilíng point at 82°C

• Injection of 10 µl into to MMI injector in Solvent Vent Mode

• Agilent Ultra Inert Liner with wool - new Liner for each batch of samples
Hydrocarbon mixture C12 – C33
MMI Solvent vent injection 10 µL

Elution of our method´s first pesticide compound (Dichlobenil 7.7 min)

Minimized discrimination of low boiling / early eluting pesticide compounds

The pesticide GC method

• Solvent vent injection of 10 µL sample in AcN at 40°C

Same as
• Oven temperature program from 40 to 310°C in 40 minutes Agilent
GC/MS
• Retention time locking with Chlorpyrifos-methyl at 18.11 minutes Pesticide
Analyser
• Post run backflush for 2 minutes at 310°C method

The pesticide MS method

• 4 GHz TOF aquisition mode for high resolution spectra

• Mass detection from m/z 50 to 550 at 5 scans/second

• MassCal repeated before every sample in batch (2 minutes during GC cooldown)

Total ion chromatogram of pesticide mixture at 0.1 ppm
x10 1 +EI TIC Scan MultiMix_1.D
3.4 1 ISTD 1
3.3
3.2
3.1
3
2.9
2.8
2.7
2.6
2.5
2.4
2.3
2.2
2.1
2
1.9
1.8
1.7
1.6
1.5
1.4
1.3
1.2
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
Counts (%) vs. Acquisition Time (min)
Extracted ion chromatogram for system quality check
x10 5 +EI EIC(163.1117, 170.9637, 285.9256) Scan 15_multi.D
7 1 1

6.8
6.6
6.4
6.2 Etofenprox
6
5.8
5.6 163.1117
5.4
5.2
5
4.8
4.6
4.4
4.2
4
3.8
3.6
3.4 Chlorpyrifos-methyl 18.1 min
3.2
3
2.8
285.9256
2.6
2.4
2.2
2
1.8
1.6
1.4 Dichlobenil
1.2
1
0.8 170.9637
0.6
0.4
0.2
0
5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
Counts vs. Acquisition Time (min)

Daily Check
• Retention time locking
• Mass accuracy
• Sensitivity
• Peak shape
PCDL manager
PCDL = Personal Compound and Database Library
• Manage target compounds

Qualitative analysis
• Target identification based on PCDL
• Extract chromatograms
• Extract spectra

Quantitative analysis
• Target quantification
• Calibrate and quantitate sample batches
The Agilent Exact Mass Pesticide Library

• Identification of pesticide compounds by MS based on 70 eV fragmentation

• Fragmentation of most compounds without ESI-like molecular ion

• Chlopyriphos-methyl: dominant 286 fragment and small 321 molecular ion

Library spectrum
110
285.92557

Abundance
100.00
100

90

80

70

60
124.98206
50 45.37
40
78.99434
30
25.15
20
46.99500 196.91965
10 7.53 6.64

0
25 50 75 100 125 150 175 200 225 250 275 300 325
m/z

• High resolution exact mass pesticide library built in 2014

• Today: 852 pesticide compounds with exact mass spectra and retention times
PCDL manager - edit and update compounds
PCDL manager - add additional compounds
Quan or Qual: data analysis workflow strategy

Acquire Full-Spectrum
Data

Yes Able to No
Calibrate?

Targeted Quantitative Targeted Qualitative Untargeted

Screening Screening Screening
Quan & PCDL Qual & PCDL
Pesticide Screening Solution
Targeted Approach using Untargeted Approach
Agilent GC/Q-TOF Pesticide using NIST GC/MS
PCDL Library
Qual: Method development
QualMethod - European proficiency test 2015

Matrix Broccoli

Pendimethalin at 0.05 mg/kg

and 3 other GC targets:

Permethrin
Diazinon
Bupirimate

were successfully identified.

QualMethod - European proficiency test 2016
+EI EIC(248.0949) Scan 20160210_02.D
x10 5 Noise (PeakToPeak) = 411.78; SNR (21.212min) = 500.8
Matrix Spinach leaves
2.8

Penconazol at 0.02 mg/kg SN >500 2.6

2.4

2.2
21.212
2
1.8

1.6
and 3 other GC targets:
1.4

1.2
Chlorantraniliprol
1
Metalaxyl
0.8
Triadimenol 0.6

0.4
were successfully identified. 0.2

0
21.15 21.2 21.25 21.3
Counts vs. Acquisition Time (min)

The sample for the proficiency test 2017 has just arrived from Spain on Tuesday…
Unknown Screening - using Qual and NIST
x10 8 +EI TIC Scan 20_probe_10.D
Sample: poisoned(?) Owl (liver and kidney) 1.4 1 1

1.2

• find 251 compounds by deconvolution 1

0.8

0.6

• NIST library search: 0.4

0.2
Barbituric acid derivative at 14.2 min
0
x10 7 +EI TIC Scan 20_probe_06.D
5 1 1
4.5
4
3.5

• repeated after dilution of sample 1:100 3

2.5
2
1.5
• NIST library search: Pentobarbituric acid 1
0.5
0

x10 3 +EI EIC(226.1313) Scan 20_probe_06.D Smooth

1 1
1.2

1
• extract high resolution trace 0.8

for molpeak(!) at m/z 226.1313 ± 10 ppm 0.6

0.4

• confirmed by RT and spectra of standard 0.2

0
6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
Counts vs. Acquisition Time (min)
Quan method setup: inject TargetMix - run QUAL & PCDL
Quan method - parameters for pesticide analysis

Everyday strategy for automated processing: allow false positives and

prevent false negatives

“use bigger windows“

• Retention time window ± 0.25 minutes (exclude)

• Mass extraction range ± 50 ppm (exclude)

• Qualifier ratio tolerance 30 % (warning)

• Quantification method by area

• Integration Cutoff automated (Agile2)

• Smoothing none
Quan method - Results: Endosulfan sulfate 0.025 mg/kg in spiked zucchini
Quan method - Results: European proficiency test 2016 Spinach leaves

Metalaxyl

Triadimenol

Chlorantraniliprol
SureMass - optimize calibration curve

Problem: limited dynamic range - saturated calibration curves

0.01 - 0.25 ppm

chlorobenzilate
in cucumber

R2=0.92

Solution: new QuanMethod feature SureMass

Using SureMass
R2=0.99
Difficult compounds - 2016´s dirty dozen

Targets not automatically detected

for a matrix calibrated batch in the
Quality control sample spiked at 0.025 ppm

• Buprofezin Various matrix effects

• Chlorothalonil depending on
• Fenvalerat water content
• Lenacil sugar content
• Metamitron starch content
• Oxadixyl protein content
• Paraoxon fat content
• Phthalimid acid content of the sample
• Pyrethrin and
• Quinomethionat amount of sample extracted
• Triticonazole
• Zoxamid
Optimizing for difficult compounds and matrices

Adapt QuEChERS sample preparation

Compound not stable optimize

or matrix overload: sample extraction and clean-up

Adapt Quan method

Compound response too low: use more intense fragments

Compound response too high: use less intense fragments

Isomeric compounds mismatched: decrease retention time window

Fragment ratio irreproducible: use higher mass fragments

Bad calibration curve: check for matrix compounds

decrease mass extraction window
decrease retention time window
Quan method - Results: compound recovery in various matrices
Combine Quan and Qual
Analyzed Sample: Strawberry

Quan analysis of 180 calibrated targets: Cyprodinil 0,647 mg/kg

Anything else?

Qual analysis
covers now
852 pesticide compounds
in the PCDL library

• no additional injection
• no calibration

Qual result: Fluopyram

(not in GC scope)
Combine Quan and Qual
Analyzed Sample: Prunes

Quan analysis of 180 calibrated targets: Iprodion 0,118 mg/kg

Retrospective View
on
finished ‘old‘ samples

• no additional injection
• no calibration

Qual result: Fenhexamid

(not in GC scope)
GC/Q-TOF and PCDL in routine pesticide analysis

GC method OK for retention times > 7 minutes

MS method OK with 1 quantifier and 2 qualifier fragments

Screening
and Quantification OK for > 95% of the target compounds
at LOD of 0.01 ppm

Dynamic range
for Quantification OK with SureMass
above 0.1 ppm

Conclusion The Agilent GC/Q-TOF is suitable for routine pesticide

analysis according to the European requirements
and
offers efficient screening possibilities for additional
targets and unknown compounds
Thank you for your attention !

Klaus Wilmers

CVUA-MEL

Joseph-König-Str. 40
48147 Münster
Germany

klaus.wilmers@cvua-mel.de