CRL for Cereals and Feeding stuff

National Food Institute

Danish Technical University

Method validation report

Determination of pesticides in cereals using
the QuEChERS method and GC-ITD

Susan Strange Herrmann and

Mette Erecius Poulsen
March 15, 2007
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Appendix 6, Technical Report 2006

Contents
1. Introduction.....................................................................................................................................3
2. Principle of analysis........................................................................................................................3
3. Validation design.............................................................................................................................3
4. Calibration curves ...........................................................................................................................4
5. Precision - repeatability and reproducibility..................................................................................4
6. Accuracy - Recovery........................................................................................................................5
7. Criteria for the acceptance of validation results............................................................................5
8. Detection limit, LOD .......................................................................................................................5
9. Results..............................................................................................................................................6
10. Conclusion.....................................................................................................................................6
11. References .....................................................................................................................................6
Appendix 1 - Summary of statistical values….………………………………...…………………...9
Appendix 2 - Ions used for MS quantification……………………..………...…………...……….15
Appendix 3 - Chromatograms of rice and rye…………………………………………...…..…….19

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1. Introduction

This report describes the validation of the QuEChERS method combined with GC-ITD for
determination of pesticide residues in cereals. The QuEChERS method has an extraction and clean-
up step, which has been developed to be Quick, Easy, Cheap, Efficient, Rugged and Safe. The
method has already been validated on fruits and vegetables1, but the data available on cereals is
limited. The method validated here was based on the procedure for dry matrixes (<30% water
content) according to the document CEN/TC 275/WG 4 N 0204 (CEN document)(available as a
draft). Even though cereals have a fat content of about 2%2 no attempt has been made to remove the
fat from the extract, e.g. freezing out as proposed in the CEN document, since no problems caused
by fat has been observed.

2. Principle of analysis
Cold water/ice water, acetonitril and an internal standard are added to the milled sample. The
sample was shaken and a salt and buffer mixture was added and the sample was shaken again. After
centrifugation the supernatant was transferred to a tube with PSA and MgSO4. After shaking and an
additional centrifugation step the extract was analysed by GC-ITD and large volume injection. The
injection volume was 8 µl. Instrument specifications as setting are presented in details in Poulsen
and Granby 20003.

3. Validation design
The method was validated for 83 pesticides, isomers or degradation products in four types of flour,
oat, rice, rye and wheat.

The validation was performed at three concentration levels as double determinations. The
concentration levels were 0.01, 0.02 and 0.2 mg/kg. Thus a total of 6 samples per flour type were
spiked and analysed. A blank sample was included for each matrix. The experiments were carried
out once on oat, rice and rye and twice on wheat, in total 5 experiments (See Table 1). The
experiments were performed by two different technicians and on different days.

Table 1. Validation design, spike levels and matrices

Experiment 0 mg/kg 0.01 mg/kg 0.02 mg/kg 0.2 mg/kg

1 – wheat x x x x x x x
2- rye x x x x x x x
3- rice x x x x x x x
4 -maize x x x x x x x
5- wheat x x x x x x x

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4. Calibration curves
The calibration curve was determined by the analysis of each of the 83 pesticides at 5 calibration
levels, i.e. 0.00289, 0.0087, 0.0289, 0.0868 and 0.289 µg/ml. The calibration curves were best fitted
to a linear curve. The majority of the correlation coefficients (R) were higher or equal to 0.98.
Examples of calibration curves are presented in Figure 1.
Pyrimethanil Vinclozolin
Y = 1,27328e+007*X R^2 = 0,9951 W: Equal Y = -3287,46+4,14883e+006*X R^2 = 0,9958 W: Equal
4000000
1200000
3500000
3000000 1000000

2500000 800000
Area

Area
2000000
600000
1500000
400000
1000000
500000 200000

0 0
0,0 0,1 0,2 0,3 0,0 0,1 0,2 0,3

Penconazole Tebuconazole
Y = 7,43815e+006*X R^2 = 0,9969 W: Equal Y = 5,52891e+006*X R^2 = 0,9942 W: Equal

2000000 1500000

1500000
1000000
Area

Area

1000000

500000
500000

0 0
0,0 0,1 0,2 0,3 0,0 0,1 0,2 0,3

Figure 1: Calibration curves for pyrimethanil, vinclozolin, penconazole and tebuconazole.

5. Precision - repeatability and reproducibility

As precision often varies with analyte concentration, repeatability and in-house reproducibility were
calculated for all matrices and all pesticides and degradations products at all three spiking levels.

The repeatability is given as the relative standard deviation on the results from two or more analysis
of identical samples, by the same operator, on the same instrument and within a short period of
time. Repeatability is calculated from the double determinations.

In-house reproducibility is relative standard deviation on results obtained under reproducibility

conditions, with the same method on the same sample by different operators within a larger period
of time. The In-house reproducibility is a combination of the repeatability variance and the in-house
reproducibility.

In appendix 1 are the calculated values for repeatability and In-house reproducibility presented for
the validated compounds.
The repeatability and reproducibility has been calculated in accordance to ISO 5725-24.

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6. Accuracy - Recovery
Certified reference material was not available for all pesticides in all matrices. In the absence of
reference materials, trueness has been calculated as the recovery of the validated compounds from
the four cereal matrices at the three spiking levels.

The recoveries for each of the validated compounds are presented in Appendix 1.

7. Criteria for the acceptance of validation results

For the pesticides to be accepted as validated the following criteria for precision and accuracy must
to be fulfilled:
1. The standard deviation of the relative repeatability and reproducibility must be less than or
equal to the standard deviation proposed by Horwitz5.
2. The average relative recovery must be between 70 and 110%6.

If the above mentioned criteria have been met, the detection limits have been calculated.
An example of accepted results (repeatability, reproducibility and recovery) is shown in Table 2.

Table 2: Example of accepted results for repeatability, with-in laboratory reproducibility and
Horwitz standard deviations

Tebufenpyrad
Spiking level (mg/kg) 0.011 0.022 0.217
Number of results 10 10 10
Repetitions 5 5 5

Recovery (mg/kg) 0.012 0.024 0.218

Recovery (%) 108 109 100

Sr (mg/kg) 0.0005 0.0015 0.0065

RSDr (%) 4.1 6.2 3.0
SR (mg/kg) 0.0012 0.0030 0.0165
RSDR (%) 9.9 12.4 7.2

RSDHorwitz 31.5 28.4 20.1

Recovery (mg/kg): mean absolute recovery in mg/kg. Recovery (%): Mean relative recovery in %. Sr (mg/kg): The
standard deviation on the absolute repeatability in mg/kg. RSDr, (%): The standard deviation on the relative
repeatability in mg/kg. SR (mg/kg): The standard deviation on the absolute reproducibility in mg/kg. RSDR (%): The
standard deviation on the relative reproducibility in mg/kg. RSDHorwitz: the Horwitz value at the relevant concentration.

8. Detection limit, LOD

The calculation of the detection limit (LOD) has been based on the results of the lowest spiking
level for which the results met the acceptance criteria, as three times the standard deviation of the
absolute recoveries.

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The limits of determination for the pesticides included in the validation are presented in Appendix
1. The ions used for quantification are presented in Appendix 2.

9. Results
The QuEChERS method, in accordance to CEN/TC 275/WG 4 N 0204, has been tested for 83
pesticides, isomers and degradation products in cereal flour, represented by oat, rice, rye and wheat.

The criteria for acceptance were met for 62 out of 83 pesticides, isomers and degradation products.
The LODs ranged from 0.003 mg/kg to 0.11 mg/kg with a median at 0.009 mg/kg. Some of the
compounds could only be validated at the highest fortification level (0.217 mg/kg) or at the second
highest fortification levels (0.022 mg/kg), and in several cases this was due to high recovery at the
lower levels.

The criteria for acceptance were not met for 21 of the compounds. Results for binapacryl,
fenamiphos, fludioxonil, flutolanil, hexaconazole and iodofenphos did not meet the acceptance
criteria due to interfering matrix peaks in all four types of flour. Besides these six pesticides it was
not possible to quantify diethofencarb, flusilazole and kresoxim-methyl in rice samples because of
interfering matrix peaks. A large matrix peak was observed in rice samples at a retention time of
about 14 minutes to about 16 minutes indicating the clean up was not sufficient for rice. A
chromatogram of a spiked rice sample is shown in Appendix 3.
Another fifteen compounds did not elute in one of the large matrix peaks but still could not meet the
acceptance criteria. For some of these compounds the ion ratios were low compared to the noise
ratio resulting in high repeatability and reproducibility. For other compounds the repeatability was
acceptable whereas the reproducibility was considerately higher than the relevant Horwitz value.

The results for the different pesticides, which were accepted, are listed in Appendix 1.

It is expected that the problems with interfering matrix could partly be eliminated if the extracts
were analysed on a MS quadropol instrument or at GC/MS/MS. Further analysis will be performed
to eliminate the problems and meet the acceptance criteria for the remaining 21 pesticides.

10. Conclusion
The method was validated for 62 pesticides, isomers or degradation products. The detection limits
ranged from 0.003-0.11 mg/kg, with a median at 0.009 mg/kg. Work on the method will continue,
particularly detection the pesticide on quadropol instrument and MS/MS.

11. References
1
http://www.quechers.com/
2
The Composition of Foods – fourth edition by Erling Saxholt, Gyldendals, 1996.
3
Poulsen, M.E., Granby, K. (2000): Validation of a multiresidue method for analysis of pesticides
in fruit, vegetables and cereals by GC/MS iontrap system. In Principle and Practices of Method
Validation, edited by A. Fajgelj and A Ambrus. Special Publication No 256 from The Royal
Society of Chemistry. ISBN 0-85404-783-2.

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4
ISO 5725-2:1994. Accuracy (trueness and precision) of measurement methods and results – Part 2.
Basic method for the determination of repeatability and reproducibility of standard measurement
method. First edition. December 1994.
5
W. Horwitz, Anal. Chem., 1982; 54, 76A.
6
Quality Control Procedures for Pesticide Residue Analysis- Guidelines for Residues Monitoring in
the European Union, SANCO/10232/2006, 24/March/2006, European Commission, Brussels, 2006.

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Appendix 1 - Summary of statistical values

Summary of statistical data based on data obtained in connection to the validation of 83 pesticides,
isomers and degradation products in cereals using the QuEChERS method in accordance to
CEN/TC 275/WG 4 N 0204. Data in italics have not met the acceptance criteria.

Fortification level
(mg/kg) 0.011 0.022 0.217 LOD
Aclonifen RSDr, % 15 8 3 0.007
RSDR, % 32 14 8
Recovery,% 74 82 98
Acrinathrin RSDr, % 5 14 5 0.008
RSDR, % 26 23 9
Recovery,% 95 93 84
Benalaxyl RSDr, % 12 4 5 0.046
RSDR, % 20 9 7
Recovery,% 153 131 99
Bifenthrin RSDr, % 46 12 1 0.012
RSDR, % 33 17 11
Recovery,% 125 108 94
Bitertanol RSDr, % 14 24 7 0.006
RSDR, % 22 18 10
Recovery,% 90 95 102
Bromophos-ethyl RSDr, % 10 9 4 0.011
RSDR, % 40 20 11
Recovery,% 94 92 93
Bromopropylate RSDr, % 12 4 6 0.009
RSDR, % 26 19 11
Recovery,% 111 104 94
Carbofenthion RSDr, % 16 15 5 0.008
RSDR, % 21 29 12
Recovery,% 122 102 103
Carbofuran RSDr, % 17 3 0.013
RSDR, % 17 5
Recovery,% 114 109
Chlorfenvinphos RSDr, % 9 5 2 0.009
RSDR, % 27 13 7
Recovery,% 118 113 105

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Fortification level
(mg/kg) 0.011 0.022 0.217 LOD
Chlorobenzilate RSDr, % 6 3 2 0.005
RSDR, % 16 14 6
Recovery,% 104 104 99
Chloropropylate RSDr, % 9 3 2 0.006
RSDR, % 18 14 6
Recovery,% 102 104 99
Chlorpyriphos RSDr, % 5 0.11
RSDR, % 19
Recovery,% 92
Chlorpyriphos-methyl RSDr, % 7 6 5 0.012
RSDR, % 31 17 6
Recovery,% 142 117 101
Chlorthal-dimethyl RSDr, % 14 16 2 0.007
RSDR, % 27 24 31
Recovery,% 89 89 89
Cyprodinil RSDr, % 9 6 2 0.003
RSDR, % 9 9 7
Recovery,% 93 96 97
Dialifos RSDr, % 9 9 9 0.007
RSDR, % 20 8 10
Recovery,% 110 111 104
Diazinon RSDr, % 14 5 0.042
RSDR, % 18 7
Recovery,% 130 101
Diclofenthion RSDr, % 11 12 6 0.003
RSDR, % 11 12 6
Recovery,% 95 92 96
Diethofencarb RSDr, % 7 6 23 0.003
RSDR, % 8 8 34
Recovery,% 103 102 92
Dioxathion RSDr, % 27 7 3 0.013
RSDR, % 29 20 9
Recovery,% 85 103 100

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Fortification level
(mg/kg) 0.011 0.022 0.217 LOD
Ethion RSDr, % 14 9 5 0.004
RSDR, % 13 10 6
Recovery,% 94 100 102
Etrimfos RSDr, % 8 12 5 0.078
RSDR, % 29 23 12
Recovery,% 192 143 102
Fenarimol RSDr, % 27 11 8 0.012
RSDR, % 29 16 9
Recovery,% 128 113 100
Fenchlorphos RSDr, % 7 7 3 0.014
RSDR, % 38 19 5
Recovery,% 141 116 98
Fenitrothion RSDr, % 6 5 5 0.007
RSDR, % 17 9 6
Recovery,% 142 118 104
Fenoxaprop-p-ethyl RSDr, % 14 8 2 0.004
RSDR, % 12 12 5
Recovery,% 109 98 98
Fenpropathrin RSDr, % 10 19 4 0.007
RSDR, % 19 24 8
Recovery,% 109 100 100
Fenpropimorph RSDr, % 8 6 3 0.012
RSDR, % 20 17 11
Recovery,% 139 112 104
Flusilazole RSDr, % 8 7 5 0.004
RSDR, % 11 9 8
Recovery,% 105 102 103
Fonofos RSDr, % 24 14 9 0.007
RSDR, % 22 21 11
Recovery,% 91 94 96
Furathiocarb RSDr, % 5 21 5 0.005
RSDR, % 14 15 9
Recovery,% 112 111 115

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Fortification level
(mg/kg) 0.011 0.022 0.217 LOD
Heptachlor RSDr, % 8 17 10 0.073
RSDR, % 28 25 11
Recovery,% 195 150 104
Isofenphos RSDr, % 12 6 3 0.011
RSDR, % 27 15 5
Recovery,% 119 118 109
Kresoxim-methyl RSDr, % 15 3 3 0.005
RSDR, % 13 8 5
Recovery,% 109 103 105
Methidathion RSDr, % 19 6 0.057
RSDR, % 41 8
Recovery,% 132 110
Molinate RSDr, % 19 11 15 0.11
RSDR, % 34 16 19
Recovery,% 106 121 90
Myclobutanil RSDr, % 11 56 34 0.021
RSDR, % 14 50 57
Recovery,% 124 114 90
Oxadixyl RSDr, % 23 4 2 0.009
RSDR, % 29 27 55
Recovery,% 95 99 90
Parathion-methyl RSDr, % 10 10 4 0.008
RSDR, % 15 9 5
Recovery,% 171 132 106
Penconazole RSDr, % 9 8 3 0.003
RSDR, % 9 11 7
Recovery,% 102 100 100
Pendimethalin RSDr, % 17 6 2 0.004
RSDR, % 16 9 7
Recovery,% 83 86 97
Phenthoat RSDr, % 8 8 4 0.009
RSDR, % 22 13 5
Recovery,% 132 114 105

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Fortification level
(mg/kg) 0.011 0.022 0.217 LOD
Phorat RSDr, % 7 0.051
RSDR, % 8
Recovery,% 96
Phosalone RSDr, % 16 6 0.047
RSDR, % 27 7
Recovery,% 146 107
Phosmet RSDr, % 20 4 0.12
RSDR, % 22 17
Recovery,% 147 110
Pirimiphos-ethyl RSDr, % 8 5 3 0.012
RSDR, % 29 17 5
Recovery,% 136 115 103
Pirimiphos-methyl RSDr, % 7 6 0.038
RSDR, % 21 5
Recovery,% 113 107
Profenophos RSDr, % 27 7 7 0.013
RSDR, % 34 19 8
Recovery,% 102 109 105
Propham RSDr, % 26 15 0.098
RSDR, % 63 13
Recovery,% 180 111
Propyzamide RSDr, % 9 12 3 0.005
RSDR, % 15 13 4
Recovery,% 105 105 101
Prothiofos RSDr, % 51 7 9 0.005
RSDR, % 51 7 13
Recovery,% 122 102 100
Pyrimethanil RSDr, % 12 9 3 0.004
RSDR, % 12 11 5
Recovery,% 102 96 102
Quinalphos RSDr, % 7 5 4 0.006
RSDR, % 14 13 6
Recovery,% 124 118 104

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Fortification level
(mg/kg) 0.011 0.022 0.217 LOD
Sulfotep RSDr, % 8 12 9 0.094
RSDR, % 43 22 14
Recovery,% 169 135 105
Tebuconazole RSDr, % 38 11 4 0.056
RSDR, % 54 63 9
Recovery,% 133 169 104
Tebufenpyrad RSDr, % 4 6 3 0.003
RSDR, % 10 12 7
Recovery,% 108 109 100
Tetradifon RSDr, % 12 43 1 0.065
RSDR, % 66 52 10
Recovery,% 245 139 107
Tetrasul RSDr, % 13 11 6 0.006
RSDR, % 22 13 11
Recovery,% 84 82 80
Trichloronat RSDr, % 6 8 4 0.013
RSDR, % 36 18 10
Recovery,% 127 111 97
Trifloxystrobin RSDr, % 14 17 4 0.006
RSDR, % 17 13 9
Recovery,% 116 110 108
Trifluralin RSDr, % 13 14 12 0.004
RSDR, % 16 16 13
Recovery,% 86 92 95
Vinclozolin RSDr, % 15 10 1 0.014
RSDR, % 37 19 4
Recovery,% 130 117 103

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Appendix 2 – List of ions used for MS quantification

Compound Ions for quantification by MS

Aclonifen 194 212 264
Acrinathrin 181 208 289
Amitraz 132 147 162 293
Benalaxyl 148 266 325
Bifenthrin 165 166 181
Binapacryl 83
Biphenyl 152 153 154
Bitertanol 170 171
Bromophos-ethyl 303 331 359
Bromopropylate 183 339 341
Carbofenthion 157 199 342
Carbofuran 149 164
Chlorfenvinphos 267 269 323
Chlorobenzilate 139 251 253
Chloropropylate 139 251 253
Chlorothalonil 264 266 268
Chlorpyriphos 197 314
Chlorpyriphos-methyl 286 288
Chlorthal-dimethyl 303 332
Cyprodinil 224 225
Dialifos 208 210 357
Diazinon 179 199 304
Diclofenthion 223 251 279
Dicofol 139 251
Diethofencarb 196 225 267
Dioxathion 197 270
Ditalimfos 130 243 299
Ethion 231 233 384
Ethoxyquin 145 147 202

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Compound Ions for quantification by MS

Etridiazole 140 183 211 246
Etrimfos 181 277 292
Fenamiphos 195 260 303
Fenarimol 251 330
Fenchlorphos 285 286 287
Fenitrothion 260 277
Fenoxaprop-p-ethyl 288 289
Fenpropathrin 181 265
Fenpropimorph 128
Fludioxonil 127 154 182 248
Flusilazole 206 233 315
Flutolanil 173 281 323
Fonofos 246
Furathiocarb 135 163 194 325
Heptachlor 272 274 337
Hexachlorbenzen 249 282 284 286
Hexaconazole 175 214 231
Iodofenphos 125 377 379
Isofenphos 121 185 213
ISTD-triphenylphosphate 325 326
Kresoxim-methyl 116 131 206
Methidathion 85 145
Molinate 98 126 154
Myclobutanil 152 179 181
Oxadixyl 132 163 233
Parathion-ethyl 109 139 291
Parathion-methyl 125 246 263
Penconazole 248 250
Pendimethalin 162 191 252
Phenthoat 246 274

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Compound Ions for quantification by MS

Phenylphenol-2 141 169 170
Phorat 75 231 260
Phosalone 182 184 367
Phosmet 160 161
Pirimiphos-ethyl 168 318 333
Pirimiphos-methyl 276 290 305
Profenophos 337 339
Propargite 173 201 350
Propham 93 137 179
Propyzamide 173 175 255
Prothiofos 239 267 309
Pyridaben 147 309 311 364
Pyrimethanil 198 199
Quinalphos 146 156 157 298
Spiroxamine 100 126 144 198 282
Sulfotep 266 294 322
Tebuconazole 125 250 252
Tebufenpyrad 171 276 318 333
Tetradifon 159 229 356
Tetrasul 252 254 324
Tolylfluanid 137 181 238
Trichloronat 269 297 299
Trifloxystrobin 116 131 190
Trifluralin 264 306 335
Vinclozolin 198 212 214 285

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Appendix 3 – Examples of chromatograms obtained by GC-MS analysis

RT: 5.10 - 37.28

15.66 NL:
36 1.99E8
TIC F: MS
070219pr1
34
1
12.33
32

30

28
24.45
26

24

22
Relative Abundance

20 18.08

18

16

14
32.31
12
15.86
10 23.95 33.41
29.59

8 6.60 9.66 13.84 28.91 31.20 34.41

20.70
6
24.74
27.72
4 8.03 18.47 26.75 35.03
11.81 19.31 22.62
2

10 15 20 25 30 35
Time (min)

Figure 2. Cromatogram of a rice sample fortified with 0.022 mg/kg.

RT: 5.06 - 37.02

27.34 31.30 NL:
50 1.16E8
TIC F: MS
48
061221pr1
46 4
14.75
44
42 32.26
40 22.81 34.75
30.50
38 34.80

36
34
32
30 26.79
Relative Abundance

28 24.27

26
34.16
24
22
20
12.09
18 17.89 29.22
16
14
12 24.39

10 20.12
35.14
8
14.96
6 9.54
4 6.60 15.25 18.33 26.45
7.65 13.83 17.11 21.32
11.36
2
0
10 15 20 25 30 35
Time (min)

Figure 3. Cromatogram of a rye sample fortified with 0.022 mg/kg

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