Extractability of Incurred Residues using QuEChERS - EURL ...

Extractability of Incurred Residues
M. Anastassiades,
J. Hepperle,
D. Roux,
I. Sigalov
EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
using QuEChERS
1
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

Use of QuEChERS
EUPT
EUPT-FV7 (2005)
EUPT-FV8 (2006)
EUPT-FV9 (2007)
EUPT-FV10 (2008)
EUPT-FV11 (2009)
EUPT-FV12 (2010)
EUPT-SRM5 (2010)
EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
Share of Labs employing QuEChERS
(rough numbers)
1 out of 8 Labs
1 out of 5 Labs
1 out of 4 Labs
1 out of 3 Labs
1 out of 2 Labs
3 out of 5 Labs
Fluazifop: 3 out of 4 labs
Fenbutatin Oxide: 4 out of 5 labs
Abamectin: 4 out of 5 labs
Private sector: 70-80% of labs employ QuEChERS (Source QS)
2
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

QuEChERS Validation
>165 000 recovery figures on SPIKED pesticides
in ~120 different commodities
(Method Validation Database of EURL-DataPool)
but …
EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
How well
do we extract
INCURRED RESIDUES?
3
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

Incurred Residues
not always easily accessible
Can…
be enclosed in cells, vacuoles, starch or wax particles
undergo strong non-covalent interactions with matrix
Various Parameters can influence Extraction-Efficiency:
Solvent Type,
Extraction Temperature,
Extraction Time,
Sample Comminution Grade (particle size)
EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
4
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

QuEChERS
MeCN / Water: 1-phase extraction system
1 min extraction by shaking
MeCN and MeCN-Water mixtures are quite weak in
dissolving lipids and penetrating waxes
Extraction Time and Temperature
expected to have a strong effect
EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
5
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

Question 1:
What are the minimum agitation times required
to extract incurred residues quantitatively ?
EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
6
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

Question 2:
How do longer extraction times impact recoveries of
spiked pesticides ?
Question 3:
What is the impact of Temperature?
EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
7
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

Question 4:
What is the role of particle size?
Does size matter ?
EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
8
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
9
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
10
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

Comparison of Different Approaches
% yields
100
90
80
70
60
50
40
30
20
10
0
By hand 1 min
Geno 10 min
Geno 1 min
Geno 20 min
100%
Geno 60 min
Geno 30 min
EURL-SRM
EU Reference Laboratory for Pesticides
(Geno requiring Grinder Single 60 Residue min setMethods at 100%)
Comparison of Different Extraction Approaches
Matrix: Raisin - Mean of all Pesticide Residues
Average yields of 7 incurred pesticides:
Boscalid, Cyromazine, Carbendazim, Fenhexamide,
Indoxacarb, Methoxyfenozide, Penconazole
Geno 1 min (RT)
Geno 10 min (RT)
Geno 30 min (RT)
Agytax 1 min
Agytax 10 min
Agytax 30 min
Agytax 20 min
Agytax 60 min
Heidolph 10 min
Heidolph 1 min
Heidolph 20 min
Heidolph 30 min
Heidolph 60 min
11
Each condition n=3
Horizontal 60 min
Horizontal 30 min
Orbital 10 min
Orbital 30 min
Ultra Turrax 3 min
Sample: RAISINS
Ultrasonic probe 3 min
Ultrasonic probe 10 min
Frozen Material:
10-15 min extraction
times seem sufficient
for>90% yields
Material at RT:
much shorter
times needed
NOTE:
Times refer to
1 st extraction step
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

Evaluation of Effects
115%
100%
85%
75%
Value of
“Quantitative” Extraction
= “PLATEAU”
EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
(Negative Effects)
Insignificant Effects
Insignificant Effects
Minor Effects
Strong Effects
12
Strong Effects
Minor Effects
Insignificant Effects
Insignificant Effects
(Negative Effects)
Value at
1min shaking by hand
132%
118%
100%
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

Co-extracted Matrix
Extrakt [mg/mL]
120
100
80
60
40
20
0
Residual extract
after evaporation to dryness
1 Min Hand 1 Min 10 Min 20 Min 30 Min
EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
Extraction Time
set at 100%
PLATEAU
Mechanical
shaking
13
Raisins
Extracted with
Geno Grinder
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

Extractability [%]
110
100
90
80
70
60
50
40
30
20
10
0
1 min by hand
EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
PT-Grape w. incurred residues
> 90%
Geno Grinder Spex Sample Prep
Azoxystrobin
Cymoxanil
Boscalid
Cymoxanil
Azoxystrobi
n
Boscalid
Further compounds of this sample showing NO extractabilility effects:
- Famoxadone
- Spinosad
0 5 10 15 20 25 30 35 Time [min]
14
Set at 100%
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

Yields in [%] ,
(1 min by hand/Frozen matrix = 100 %)
180
160
140
120
100
80
60
40
20
EURL-SRM
0
Frozen 6°C RT Frozen 6°C RT
frozen 6°C RT
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
116 % 122 % 139 % 135 % 132 %
1 min by Hand
15 min Mechan.
For each condition
n=3
Pesticide (logKow):
Metalaxyl (1,75)
Triadimenol (3,08)
Penconazole (3,72)
Myclobutanil (2,94)
Methoxyfenozide (3,72)
Chlorpyrifos-ethyl (4,7)
Imazalil (2,56)
Propargite (3,73)
Indoxacarb (4,65)
Flusilazole (3,87)
Boscalid (2,96)
Metrafenone (4,3)
Raisin
15
3rd Joint EURL-Workshop
frozen 6°C RT
Freiburg, 27 - 28 Sep 2011

Temperature development while thawing
Temperature [°C]
25
20
15
10
5
0
-5
-10
0
Leek
8
14
20
26
EURL-SRM
30 min
32
38
44
50
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
56
62
68
74
80
Raisin
Waiting times:
- 30 min for Thawing
- 2h for RT (would be faster in water bath)
86
92
98
104
16
110
116
Time [min]
15 min extraction of frozen sample overall much faster
2h
Pineapple
122
128
134
140
146
152
158
164
170
176
182
188
194
200
206
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

600
500
400
300
200
100
0
1 min by Hand 15 min mech.
EURL-SRM
Rice
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
114 % 181 % 204 % 376 % 386 %
1 min by Hand 15 min mech. 1 min by Hand 15 min mech.
17
Pesticide (logKow):
Tricyclazole (1,4)
Thiamethoxam (-0,13)
Clothianidin (0,9)
Malathion (2,75)
Kresoxim-methyl (3,4)
Difenoconazole (4,4)
Carbendazim (1,5)
Azoxystrobin (2,5)
Trifloxystrobin (4,5)
Epoxiconazole (3,3)
Tebuconazole (3,7)
Pirimiphos-methyl (4,2)
Cyhalothrin-lambda (7)
Diflubenzuron (3,89)
Propiconazole (3,72)
Chlorpyrifos-ethyl (4,7)
Deltamethrin (4,6)
3rd For each condition
Joint EURL-Workshop
n=3
Freiburg, 27 - 28 Sep 2011

Different fractions – different plant parts?
Rice homogenate
(Real sample)
No increased
values for
incurred
Tricyclazole and
Tebuconazole
EURL-SRM
EU Reference Laboratory for Pesticides
requiring 500 - 1000 Single µm, Residue milledMethods
Sieving
Further Milling
Fractions with different particle sizes
18
> 1000 µm
500 - 1000 µm
250 - 500 µm
125 - 250 µm
63 - 125 µm
Fractions with
small particle size
visibly contained
more peel
particles
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
19
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

Impact of particle size (EUPT2-Wheat further milled)
EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
20
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

Impact of particle size (EUPT2-Wheat further milled)
EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
21
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

Impact of particle size (EUPT2-Wheat further milled)
EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
22
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

Impact of further Comminution (PT-sample)
EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
23
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

Beeswax with incorporated Pesticides - Preparation
1) Spiking of the beeswax
EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
2) Heating beeswax (+ 80 °C) until melting
3) Cooling down to room temperature 4) Overnight storage at – 80 °C
24
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

Beeswax with incorporated Pesticides - Preparation
5) Mincing procedure
4.0 x 1000 rpm
t = 2 min
6) Sieving procedure
3 fractions; particle sizes:
- 500 – 1000 µm
- 250 – 500 µm
- 125 – 250 µm
EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
25
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

Extraction from Beeswax – Impact of Particle Size
140
120
100
80
60
40
20
0
RT
500
-
1000 µm
22%
28%
50°C
Imidacloprid
(logKow = 0.6)
EURL-SRM
250
-
500 µm
39%
RT
50%
1 min shaking
50°C
26
3rd Correlation between Mean Pesticide Recov. and Extract Weight after evaporation (Melted=100%)
RT /1min : 500-1000µm (22% vs. 20%); 250-500µm (39% vs 36%); 125-250µm Joint (62% EURL-Workshop
vs. 59%)
RT/15min : 500-1000µm (30% vs. 28%); 250-500µm (51% vs 52%); 125-250µm Freiburg, (81% vs. 27 80%) - 28 Sep 2011
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
125
-
250 µm
62%
RT
Acetamiprid
(logKow = 0.8)
500
-
1000 µm
30%
RT
62%
50°C
Carbendazim
(logKow = 1.5)
51%
RT
250
-
500 µm
85%
15 min shaking
50°C
Methiocarb
(logKow = 3.2)
RT
125
-
250 µm
81%
Melted
(set at 100%)
100%
80°C
Difenoconazole
(logKow = 4.4)

First Conclusions
1) SHAKERS
Shaking approach (intensity) less important
2) TEMPERATURE AND TIME
Frozen Samples: ca. 10-15 min shaking time sufficient
Shaking at RT accelerates extraction (~2 min sufficient)
3) ULTRA-TURRAX AND ULTRASOUND-PROBE
Accelerate extraction, but…
… impractical for routine work (sequential approach)
4) PARTICLE SIZE
The smaller the faster the extraction and the higher the yields
EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
27
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
STUDY OF
REAL SAMPLES
(with incurred residues)
28
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

Yield at 20 min mechanical shaking [%]
(1 min manual extraction = 100%)
150
145
140
135
130
125
120
115
110
105
100
95
90
EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
- 1.5 to - 1
Comparison of extractability using QuEChERS
1 min by hand vs. 20 min by automated shaker
Propamocarb
Extrakt [mg/mL]
- 1 to - 0.5
5,0
4,5
4,0
3,5
3,0
2,5
2,0
1,5
1,0
0,5
0,0
1 min
Manually
- 0.5 to 0
Residual extract
after evaporation to dryness
0 to 0.5
Mechanical shaking
2 Min
Extraction Time
0.5 to 1
Oxadixyl
1 to 1.5
10 Min 30 Min
1.5 to 2
2 to 2.5
2.5 to 3
29
Dimethomorph
Boscalid
3 to 3.5
Rocket
(Rucola)
3.5 to 4
Pyraclostrobin
4 to 4.5
Cyprodinil
4.5 to 5
Deltamethrin
5 to 5.5
5.5 to 6
6 to 6.5
6.5 to 7
logK OW
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

Yield at 20 min mechanical . shaking [%]
(1 min by Hand = 100%)
170
165
160
155
150
145
140
135
130
125
120
115
110
105
100
Comparison of extractability using QuEChERS
1 min by hand vs. 20 min by automated shaker (Geno Grinder)
EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
- 1.5 to - 1
- 1 to - 0.5
- 0.5 to 0
Grapes
(PT-sample w. incurred residues)
kindly provided by AGES Innsbruck
0 to 0.5
Cymoxanil
0.5 to 1
1 to 1.5
1.5 to 2
2 to 2.5
Azoxystrobin
30
2.5 to 3
Spinosad
3 to 3.5
Extrakt [mg/mL]
Boscalid
4,5
4,0
3,5
3,0
2,5
2,0
1,5
1,0
0,5
0,0
Tebuconazole
3.5 to 4
1 Min
manually
Trifloxystrobin
4 to 4.5
Residual extract
after evaporation to dryness
5 Min 10 Min 20 Min 30 Min
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011
4.5 to 5
Famoxadone
Mechanical shaking
Extraction Time
5 to 5.5
Fenpyroximate
5.5 to 6
6 to 6.5
logKOW
6.5 to 7

Yield at 20 min mechanical shaking [%]
(1 min by hand = 100%)
170
165
160
155
150
145
140
135
130
125
120
115
110
105
100
EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
- 1.5 to - 1
Comparison of extractability using QuEChERS
- 1 to - 0.5
1 min by hand vs. 20 min by automated shaker
- 0.5 to 0
Cyromazine
0 to 0.5
0.5 to 1
1 to 1.5
Carbendazim
Raisins
(Real sample)
31
1.5 to 2
2 to 2.5
Boscalid
2.5 to 3
3 to 3.5
3.5 to 4
Penconazole
Fenhexamide
4 to 4.5
4.5 to 5
Indoxacarb
logK OW
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

Yield at 20 min mechanical shaking [%]
(1 min by hand = 100%)
160
150
140
130
120
110
100
90
Thiacloprid (pKow = 1.26)
EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
Currant (real sample)
1 min by hand vs. 15 min by automated shaker
Boscalid (pKow = 2.96)
32
Pyraclostrobin (pKow = 3.99)
pesticide ( --> pKow)
Cyprodinil (pKow = 4)
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

Yield at 20 min mechanical shaking [%]
(1 min by hand = 100%)
150
140
130
120
110
100
90
80
Acetamiprid (pKow = 0.8)
EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
Cherry (real sample)
1 min by hand vs. 15 min by automated shaker
Thiacloprid (pKow = 1.26)
33
Boscalid (pKow = 2.96)
pesticide ( --> pKow)
Cyprodinil (pKow = 4)
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

No of studied cases
16
14
12
10
8
6
4
2
0
Average Yield
92 %
Thiacloprid
(LogKow=1.26)
EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
Impact of LogKow
on Extraction Yields of Incurred Residues
Average Yield
86 %
Azoxystrobin
(LogKow=2.5)
34
Average Yield
76 %
Triadimenol
(LogKow=3.24)
85 %
Yield at 1 min manual shaking (15 mechanical shaking set at 100%)
Average Yield
71 %
Cyprodinil
(LogKow=4)
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

Tested Commodities
40
35
30
25
20
15
10
5
0
chive
leek
spring onions
Allium
Crops
strawberries
grapes
red currants
gooseberries
Berries
goji-berries
raspberries
pomelos
EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
34 different commodity types
95 samples / 248 studied pesticides
Ø 2.6 pesticides per sample
kumquats
Citrus
Fruit
pineapples
bananas
papayas
Exotic
Fruit
pitahayas
sweet-peppers
tomatoes
35
melons
Fruiting
Veg.
rocket
celery
parsley
lamb's lettuce
iceberg lettuce
Leafy
Vegetables
cherries
nectarines
apricots
apples
nashi-pears
Pome+Stone
Fruit
LogKow>4
LogKow 2.5-4
LogKow

No of studied cases
35
30
25
20
15
10
5
0
Pesticides (LogKow)
HEPA ( -2,65 )
Ethephon ( -1,89 )
Omethoate ( -0,74 )
Imidacloprid ( 0,57 )
Oxadixyl ( 0,7 )
Dimethoat ( 0,704 )
Acetamiprid ( 0,8 )
Metamitron ( 0,85 )
Thiacloprid ( 1,26 )
Thiophanate-methyl ( 1,45 )
Carbendazim ( 1,5 )
Pirimicarb ( 1,7 )
Metalaxyl ( 1,75 )
Carbofuran ( 1,8 )
Methidathion ( 2,2 )
Flutriafol ( 2,3 )
Thiabendazol ( 2,39 )
Azoxystrobin ( 2,5 )
Hexythiazox ( 2,53 )
Imazalil ( 2,56 )
Dimethomorph ( 2,72 )
Myclobutanil ( 2,94 )
Boscalid ( 2,96 )
Iprodione ( 3 )
Linuron ( 3 )
Spinosad ( 3 )
Pyrimethanil ( 3 )
Spiroxamine ( 3 )
Triadimefon ( 3,11 )
Flutolanil ( 3,17 )
Iprovalicarb ( 3,18 )
Triadimenol ( 3,2 )
Dithianon ( 3,2 )
Cyazofamid ( 3,2 )
Kresoxim-methyl ( 3,4 )
Fenhexamid ( 3,51 )
Methoxyfenozide ( 3,72 )
Chlorpropham ( 3,76 )
Zoxamide ( 3,76 )
Hexaconazole ( 3,9 )
Tolylfluanid ( 3,9 )
EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
36
Cyprodinil ( 4 )
Pyraclostrobin ( 4 )
Fludioxonil ( 4,12 )
Prochloraz ( 4,12 )
Bitertanol ( 4,15 )
Difenoconazole ( 4,4 )
Prosulfocarb ( 4,48 )
Trifloxystrobin ( 4,5 )
Deltamethrin ( 4,6 )
Indoxacarb ( 4,65 )
Chlorpyrifos ( 4,7 )
Piperonylbutoxid ( 4,75 )
Buprofezin ( 4,93 )
Fenpyroximate ( 5,01 )
Spirodiclofen ( 5,8 )
Etofenprox ( 6,9 )
Cyhalothrin-lambda ( 7 )
-2,65-1,89-0,740,570,70,7040,8 0,851,261,45 1,5 1,7 1,75 1,8 2,2 2,3 2,392,5 2,532,562,722,942,96 3 3 3 3 3 3,113,173,18 3,2 3,2 3,2 3,4 3,513,723,763,763,9 3,9 4 4 4,124,124,15 4,4 4,484,5 4,6 4,654,7 4,754,935,015,8 6,9 7
( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( (
HEP Ethepho AOmetho
Imidaclo n ate Oxadixyl Dimetho prid Acetamiprid Metamitro atThiaclo
Carbendazim
Thio nprid
pP
irimicarb Metalaxyl Carbo Methidathio furan Flutriafo Thiabendazo Azo n lxys
Hexythiazo trol Dimetho bin Imazalil Myclo x mo Bo butanil rph sIpro calid dio Linuro ne Spino Pnyrimethanil sSpiro adTriadimefo
xamine Fluto Iprolanil nTriadimeno
valicarb Dithiano Cyazo l nfamid
Fenhexamid
Kres Metho Chlo xyfeno rpro Zo Hexaco zide xamide pham Tonazo lylfluanid Cypro P yraclo le dinil Fludio sPtro roxo bin chlo Bitertano Difeno nil raz P ro coTriflo sl nazo ulfo Deltamethrin
xys carb le tro Indo bin Chlo Pxacarb ipero rpyrifo Bupro nylbuto Fenpyro s Spiro fezin xidximate
Eto diclo fenpro fen Cyhal x
hanate-
o xim-
o thrin-
LogKow < 2.5
17 Pesticides / 62 cases
Increasing LogKow
LogKow 2.5-4
24 diff. Pesticides / 115 cases
Overall
58 different Pesticides
248 studied cases
LogKow >4
17 Pesticides / 71 cases
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

Extractability of incurred residues – Role of LogKow
Yields at 1min shaking by Hand [%]
120
115
110
105
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
1
Overall
58 different Pesticides
248 studied cases
LogKow4
Average Yield: 74 %
201
211
221
231
241
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

Impact of LogKow on Extraction yields
No of
Average Yield
at 1 min shaking
Pesticide LogKow studied cases (15 min set at 100%)
HEPA -2,65 5 93
Ethephon -1,89 3 99
Imidacloprid 0,57 6 95
Acetamiprid 0,8 4 97
Thiacloprid 1,26 15 92
Carbendazim 1,5 4 91
Pirimicarb 1,7 3 91
Metalaxyl 1,75 3 103
Flutriafol 2,3 6 91
Thiabendazol 2,39 5 90
Azoxystrobin 2,5 11 86
Imazalil
2,56
3,82 (pH9)
9 88
Dimethomorph 2,72 4 87
Myclobutanil 2,94 8 80
Boscalid 2,96 19 81
Iprodione 3 7 78
Triadimefon 3,11 8 74
Triadimenol 3,2 18 76
Fenhexamid 3,51 14 83
Methoxyfenozide 3,72 5 86
Cyprodinil 4 23 71
Pyraclostrobin 4 8 63
Fludioxonil 4,12 6 86
Difenoconazole 4,4
Trifloxystrobin EURL-SRM 4,5
6
13
79
38
74
Indoxacarb EU Reference Laboratory 4,65 for Pesticides 4
requiring Single Residue Methods
82
Differences between
commodities
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

Impact of Commodity Type
on Extraction yields of incurred residues
Residue retardation seems more pronounced in certain commodities
But …available Data not sufficient to draw safe conclusions
Possible factors influencing extractability:
Presence of natural surface-wax (slow penetration by extractant)
Time between pesticide application and residue extraction (unknown)
pH (in case of acids and bases)
In any case:
78% of SAMPLES showed yields

EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
40
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
41
3rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011
Pesticides (pkow -->)
90
100
Extractability increase [%]..
(1 min by hand = 100%)
110
120
Acetamiprid (pkow 0.8)
Carbendazim (pkow 1.5)
Pirimicarb (pkow 1.7)
Carbaryl (pkow 2.36)
Azoxystrobin (pkow 2.5)
Imazalil (pkow 2.56)
Myclobutanil (pkow 2.94)
130
Diazinon (pkow 3.69)
Dichlofluanid (pkow 3.7)
Cyprodinil (pkow 4)
Parathion (pkow 3.83)
Chlorpyrifos-ethyl (pkow 4.7)
Cyhalothrin-lambda (pkow 7)
Bifenthrin (pkow 7.3)
140
150
Aubergine (EUPT FV 8)
1 min by hand vs. 15 min by automated shaker

EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
42
3rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011
Pesticides (pkow -->)
80
90
Extractability increase [%]
100
(1 min by hand = 100%)
110
120
Oxamyl (pkow -0.44)
Hexythiazox (pkow 2.53)
Myclobutanil (pkow 2.94)
130
Pyrimethanil (pkow 3)
Iprodione (pkow 3)
Triadimenol (pkow 3.08)
Fenhexamid (pkow 3.51)
Diazinon (pkow 3.69)
Tebuconazole (pkow 3.7)
Penconazole (pkow 3.72)
Bupirimate (pkow 3.9)
Tolylfluanid (pkow 3.9)
Cyprodinil (pkow 4)
140
Quinoxyfen (pkow 4.66)
150
160
Strawberry (EUPT FV 9)
1 min by hand vs. 15 min by automated shaker

EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
43
3rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011
Pesticides (pkow -->)
90
100
110
Extractability increase [%]
(1 min by hand = 100%)
120
130
140
Oxamyl (pkow -0.44)
Methomyl (pkow 0.09)
Acetamiprid (pkow 0.8)
Malaoxon (pkow 2.074)
Boscalid (pkow 2.96)
150
Malathion (pkow 2.75)
Phosmet (pkow 2.96)
Hexythiazox (pkow 2.53)
Triadimenol (pkow 3.08)
Methiocarb (pkow 3.18)
Kresoxim-methyl (pkow 3.4)
Diazinon (pkow 3.69)
160
170
Chlorpyrifos-methyl (pkow 4)
Quinoxyfen (pkow 4.66)
Pendimethalin (pkow 5.2)
180
190
Carrot (EUPT FV 10)
1 min by hand vs. 15 min by automated shaker

EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
44
3rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011
Pesticides (--> pKow)
90
95
Extractability increase [%]
(1min by hand = 100%)
100
105
110
Aldicarb-sulfoxid (pKow = -1.125)
M etham idophos (pKow = -0.8)
Oxam yl (pKow = -0.44)
Aldicarb-sulfon (pKow = -0.57)
M onocrotophos (pKow = - 0.22)
M ethom yl (pKow = 0.09)
3-Hydroxycarbofuran (pKow = 0.21)
Paraoxon-m ethyl (pKow = 1.247)
Thiacloprid (pKow = 1.26)
Aldicarb (pKow = 1.359)
115
120
M etalaxyl (pKow = 1.75)
Carbofuran (pKow = 1.8)
M ethidathion (pKow = 2.2)
Azinphos-m ethyl (pKow = 2.96)
Boscalid (pKow = -2.96)
Triazophos (pKow = 3.34)
Isofenphos-m ethyl (pKow = 3.589)
Diazinon (pKow = 3.69)
Cadusafos (pKow = 3.85)
Phosalon (pKow = 4.01)
Deltam ethrin (pKow = 4.6)
Buprofezin (pKow = 4.93)
125
130
135
Apple (EUPT-FV 11)

EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
45
3rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011
pesticide
90
95
Extractability increase [%]
(1 min by hand = 100%)
100
105
110
115
120
Oxamyl (pKow = - 0.44)
Omethoat (pKow = - 0.74)
Thiacloprid (pKow = 1.26)
Azinphos-methyl (pKow = 2.96)
Kresoxim-methyl (pKow = 3.4)
Ethion (pKow = 4.28)
Triflumuron (pKow = 4.9)
Amitraz (pKow = 5.5)
125
130
Chlorpyrifos (pKow = 4.6)
EPN (pKow = 5.02)
Prothiofos (pKow = 5.67)
Fenpropathrin (pKow = 6)
135
140
Leek (EUPT FV 12)
1 min by hand vs. 15 min by automated shaker

Extractability increase [%]
(1 min by hand = 100%)
120
115
110
105
100
95
90
EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
Apple puree (EUPT SRM 5)
1 min by hand vs. 15 min by automated shaker
Abamectin
46
Fluazifop
FBO
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

Extractability increase [%]
(1 min by hand = 100%)
150
145
140
135
130
125
120
115
110
105
100
EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
- 1.5 to - 1
Comparison of extractability using QuEChERS
1 min by hand vs. 20 min by automated shaker
- 1 to - 0.5
- 0.5 to 0
(EUPT-C4, sample with incurred residues)
Rye
0 to 0.5
0.5 to 1
1 to 1.5
Carbendazim
47
1.5 to 2
2 to 2.5
Carbaryl
2.5 to 3
Spiroxamine
3 to 3.5
Fluquinconazole
3.5 to 4
Kresoxym-methyl
4 to 4.5
Pirimiphos-methyl
4.5 to 5
Deltamethrin
logK OW
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

Extractability increase [%]
(1 min by hand = 100%)
150
145
140
135
130
125
120
115
110
105
100
95
90
85
80
EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
- 1.5 to - 1
Comparison of extractability using QuEChERS
- 1 to - 0.5
- 0.5 to 0
1 min by hand vs. 20 min by automated shaker
0 to 0.5
0.5 to 1
Carbendazim
1 to 1.5
Wheat
(EUPT-C1/SRM2, sample with incurred residues)
1.5 to 2
Azoxystrobin
2 to 2.5
48
2.5 to 3
3 to 3.5
Diazinon
3.5 to 4
Pirimiphos-methyl
4 to 4.5
Deltamethrin
4.5 to 5
5 to 5.5
5.5 to 6
6 to 6.5
6.5 to 7
logK OW
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

How was Data-Reliability ensured?
We have studied…
IMPACT OF EXTENDED EXTRACTION TIMES ON…
a) RECOVERIES
b) MATRIX EFFECTS
Also:
Only sample-compound combinations with good
repeatability included in the study (RSD

IMPACT OF EXTENDED EXTRACTION TIMES
a) Check for Matrix Effects…
Extended extraction times
resulted in :
a) deeper coloured extracts
b) higher dry residue
However…
Impact on matrix-effects
was insignificant
(compounds responsible for
matrix effects are obviously
rather quickly extracted)
EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
50
Extrakt [mg/mL]
5,0
4,5
4,0
3,5
3,0
2,5
2,0
1,5
1,0
0,5
0,0
1 Min
Manual
Raisins
Residual extract
after evaporation to dryness
2 Min
10 Min
Mechanical Mechanical
Extraction Time
30 Min
Mechanical
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

IMPACT OF EXTENDED EXTRACTION TIMES
b) on Recoveries …
430 pesticides tested whether they degrade during
prolonged extraction times (30 min.)
(125+ Pesticides tested on 15 min.) …ongoing…
Commodities used represent lower and upper end of
the physiological pH range
Only very few pesticides showed a clear degradation
effect e.g. captan, folpet, dichlofluanid, chlorothalonil in
the higher pH-range.
EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
51
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
52
More Recovery Data in
EURL-DataPool
Soon!
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

Effects with Ethyl acetate Method
Yield at 20 min mechanical shaking [%]
(1 min by hand = 100%)
160
150
140
130
120
110
100
90
80
Cymoxanil (pKow = 0.6)
EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
Azoxystrobin (pKow = 2.5)
Spinosyn A (pKow = 3.0
Extraction Time: 30minutes
53
Grape sample
Boscalid (pKow = 3.0)
Tebuconazole (pKow = 3.7)
Famoxadone (pKow = 4.8)
Fenpyroxymate (pKow = 5.0)
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

Effects with Acetone Method
Yield at 20 min mechanical shaking [%]
( 1 min by hand = 100 % )
160
150
140
130
120
110
100
90
80
Metalxyl
Imazalil
Myclobutanil
Boscalid
Triadimenol
EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
Raisin sample
Methoxyfenozide
Penconazole
Propargite
Flusilazole
(pKow -->)
54
Flufenoxuron
Metrafenone
Deltamethrin
Indoxacarb
Chlorpyrifos-ethyl
Bifentrin
Pesticide pKow
Metalxyl 1.75
Imazalil 2.56
Myclobutanil 2.94
Boscalid 2.96
Triadimenol 3.20
Methoxyfenozide 3.72
Penconazole 3.72
Propargite 3.73
Flusilazole 3.74
Flufenoxuron 4.00
Metrafenone 4.30
Deltamethrin 4.60
Indoxacarb 4.65
Chlorpyrifos-ethyl 4.70
Bifentrin 6.00
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

Comparison of Methods
EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
QuEChERS (ACN:Water 1:1)
55
Triadimefon Triadimenol
1 min by Hand (Frozen) 80 70
1 min by Hand (RT) 95 110
1 min Ultra Turrax (Frozen) 94 93
5 min Mechanical (Frozen) 95 90
15 min Mechanical (Frozen) 100 100
15 min Ultrasound (Frozen)
Ethylacetate (EtAc:Water 1:1)
Triadimefon Triadimenol
1 min by Hand (Frozen) 86 94
1 min by Hand (RT) 103 124
1 min Ultra Turrax (Frozen) 122 143
5 min Mechanical (Frozen) 96 101
15 min Mechanical (Frozen) 100 100
15 min Ultrasound (Frozen)
Acetone (Acetone:Water 3:1)
% (15 min = 100%)
% (15 min = 100%)
Triadimefon Triadimenol
1 min by Hand (Frozen) 90 84
1 min by Hand (RT) 89 87
1 min Ultra Turrax (Frozen) 98 94
5 min Mechanical (Frozen) 94 91
15 min Mechanical (Frozen) 100 100
15 min Ultrasound (Frozen)
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

Interlaboratory Comparison
All labs using QuEChERS
Pineapple with
Incurred residues
EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
1 min by hand vs. 15 min automatic shaking
(1 min by Hand = 100%)
Lab 1
Lab 2
Lab 3
Lab 4
Lab 5 (thawed)
Lab 6 (thawed)
56
Triadimefon
134
132
126
138
112
102
Triadimenol
137
125
131
144
102
103
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

OUTLOOK
• Perform additional recovery experiments and collect
recovery data from other labs (datapool)
• Study additional commodities w. incurred residues to
elucidate the role of commodity type
• Organize an inter-laboratory comparison with real samples
• Study impact of pH on extractability of ionizable compounds
• Further study impact of particle size (dry commodities)
• Further study effects in the case of ethylacetate- and
acetone-based procedures
• Study extractability of microencapsulated pesticides
EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
57
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

SUMMARY
Experiments with real samples have shown the need for
PROLONGED EXTRACTION TIMES (e.g. 10-15 min)
to increase the recoveries of incurred residues.
The use of MECHANICAL SHAKERS is indicated
GOOD COMMINUTION (SMALL PARTICLE SIZES) is
essential to improve accessibility of incurred residues
and achieve high extraction yields.
EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
58
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011

EURL-SRM
EU Reference Laboratory for Pesticides
requiring Single Residue Methods
Thank you very much…
59
3 rd Joint EURL-Workshop
Freiburg, 27 - 28 Sep 2011