Extractability of Incurred Residues
using QuEChERS
M. Anastassiades,
J. Hepperle,
D. Roux,
I. Sigalov
EURL-SRM
EURL-Workshop 27-28 October 2010
1
EU Reference Laboratory
for Pesticide Residues
using Single Residue Methods
QuEChERS
>160 000 recov. figures on SPIKED pesticides
(Method Validation Database of CRL-DataPool)
but …
How well
do we extract
INCURRED residues?
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2
EU Reference Laboratory
for Pesticide Residues
using Single Residue Methods
Incurred Residues
not always easily accessible
enclosed in cells, vacuoles or wax particles
strong non-covalent interactions with matrix sites
Small sample particles are helpful in extraction
$ Good Sample Comminution needed
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3
EU Reference Laboratory
for Pesticide Residues
using Single Residue Methods
QuEChERS
ACN / Water: 1-phase extraction system
1 min extraction by shaking
ACN and ACN-Water mixtures are quite weak in
dissolving lipids and penetrating waxes
Extraction Time and Temperature
$ expected to have an effect
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4
EU Reference Laboratory
for Pesticide Residues
using Single Residue Methods
Question:
What are the minimum agitation times required
to extract incurred residues quantitatively ?
+%
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EU Reference Laboratory
for Pesticide Residues
using Single Residue Methods
Extraction/ Homogen. equipment employed in this study:
Geno Grinder
Spex Sample Prep
Biosan Multi RS-60
Rotation shaker
EURL-SRM
EURL-Workshop 27-28 October 2010
Heidolph
Multi Reax
6
Horizontal shaker
AgytaxⓇ
EU Reference Laboratory
for Pesticide Residues
using Single Residue Methods
Ultra Turrax
Ultrasonic probe
By hand
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7
EU Reference Laboratory
for Pesticide Residues
using Single Residue Methods
Sample: RAISINS
Comparison of Different Approaches
Comparisson of Different Extraction Approaches
%
Matrix: Raisin - Mean of all Pesticide Residues
100
90
Frozen Material:
10-15 min
extraction seem
enough to get
>90% yields
80
70
60
50
Incurred Residues:
Boscalid, Cyromazine, Carbendazim, Fenhexamide,
Indoxacarb, Methoxyfenozide, Penconazole
40
30
Material at RT.:
much shorter
times needed
20
10
Ultrasonic probe 10 min
Ultrasonic probe 3 min
Ultra Turrax 3 min
Orbital 30 min
Orbital 10 min
Horizontal 60 min
condition n=3
Horizontal 30 min
Heidolph 60 min
8
Heidolph 30 min
Heidolph 20 min
Heidolph 10 min
Heidolph 1 min
EURL-Workshop 27-28 OctoberEach
2010
(Geno Grinder 60 min set at 100%)
Agytax 60 min
Agytax 30 min
Agytax 20 min
Agytax 10 min
Agytax 1 min
Geno 30 min (RT)
EURL-SRM
Geno 10 min (RT)
Geno 1 min (RT)
Geno 60 min
Geno 30 min
Geno 20 min
Geno 10 min
Geno 1 min
By hand 1 min
0
NOTES:
% Yields: Average
of all listed residues
Refer
to
Times:
EU Reference
Laboratory
for Pesticide step
Residues
1st extraction
using Single Residue Methods
Comparison of extractability using QuEChERS
1 min by hand vs. 20 min by automated shaker
170
165
Indoxacarb
Raisins
160
(Real sample)
Boscalid
150
145
135
130
Carbendazim
125
120
115
110
Fenhexamid
Penconazole
140
Cyromazine
Extractability increase [%]
(1 min by hand = 100%)
155
105
EURL-SRM
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9
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-0
.5
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-0
.5
to
-1
-1
.5
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-1
100
OW
EU Reference logK
Laboratory
for Pesticide Residues
using Single Residue Methods
Co-extracted Matrix
R e s id u a l e x t r a c t
a f t e r e v a p o r a t io n t o d r yn e s s
120
set at 100%
100
Raisins
PLATEAU
Extrakt [mg/mL]
80
60
40
20
0
1 M in Ha n d
1 M in
1 0 M in
2 0 M in
Ex tr a c tio n T im e
EURL-SRM
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10
3 0 M in
Extracted with
Geno Grinder
EU Reference Laboratory
for Pesticide Residues
using Single Residue Methods
PT-Grape w. incurred residues
110
> 90%
100
90
Extractability [%]
80
Geno Grinder Spex Sample Prep
70
Set at 100%
60
1 min by hand
50
Cymoxanil
Boscalid
Azoxystrobi
n
40
30
20
Further compounds of this sample showing NO extractabilility effects:
- Famoxadone
- Spinosad
10
0
0
5
10
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15
20
11
25
30
35
Time [min]
EU Reference Laboratory
for Pesticide Residues
using Single Residue Methods
First Conclusions
SHAKERS
Shaking intensity less important
Ca. 10-15 min shaking of frozen samples sufficient
Employing samples at RT accelerates extraction
ULTRA-TURRAX AND ULTRASOUND PROBE
ƒ Accelerate extraction, but…impractical for routine
work (sequential approach)
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12
EU Reference Laboratory
for Pesticide Residues
using Single Residue Methods
Results for further
REAL SAMPLES
(with incurred residues)
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13
EU Reference Laboratory
for Pesticide Residues
using Single Residue Methods
Comparison of extractability using QuEChERS
1 min by hand vs. 20 min by automated shaker
150
Residual extract
after evaporation to dryness
145
5,0
4,5
140
3,5
130
125
3,0
Deltamethrin
Extrakt [mg/mL]
135
2,5
2,0
1,5
0,5
0,0
2 Min Geno
grinder
10 Min Geno
grinder
30 Min Geno
grinder
Boscalid
1 Min
Manually
115
Extraction Tim e
100
95
Oxadixyl
105
Dimethomorph
110
Rucola
(real
Cyprodinil
120
Pyraclostrobin
1,0
Propamocarb
Extractability increase [%]
(1 min by hand = 100%)
4,0
90
-1
.5
to
-1
-1
to
.5
-0
-
5
0.
to
0
0
to
5
0.
5
0.
to
1
1
EURL-SRM
EURL-Workshop 27-28 October 2010
to
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1.
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1.
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5
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3.
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4.
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5
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5
5.
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6
6
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6.
5
6.
to
7
logKOW
EU Reference Laboratory
for Pesticide Residues
using Single Residue Methods
Currant (real sample)
1 min by hand vs. 15 min by automated shaker
160
110
100
90
Cyprodinil (pKow = 4)
120
Pyraclostrobin (pKow = 3.99)
130
Boscalid (pKow = 2.96)
140
Thiacloprid (pKow = 1.26)
Extractability increase [%]
(1 min by hand = 100%)
150
pesticide ( --> pKow)
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15
EU Reference Laboratory
for Pesticide Residues
using Single Residue Methods
120
110
100
Cyprodinil (pKow = 4)
130
Boscalid (pKow = 2.96)
Extractability increase [%]
(1 min by hand = 100%)
140
Acetamiprid (pKow = 0.8)
150
Thiacloprid (pKow = 1.26)
Cherry (real sample)
1 min by hand vs. 15 min by automated shaker
90
80
pesticide ( --> pKow)
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16
EU Reference Laboratory
for Pesticide Residues
using Single Residue Methods
Comparison of extractability using QuEChERS
1 min by hand vs. 20 min by automated shaker (Geno Grinder)
170
Residual extract
after evaporation to dryness
Grapes
165
(PT-sample w. incurred residues)
4,0
kindly provided by AGES Innsbruck
3,5
Extrakt [mg/mL]
160
4,5
150
3,0
2,5
2,0
1,5
145
1,0
0,5
140
0,0
1 Min
manually
135
5 Min
10 Min
20 Min
30 Min
Boscalid
105
Fenpyroximate
110
Trifloxystrobin
115
Spinosad
Azoxystrobin
125
120
Tebuconazole
130
Famoxadone
Extraction Time
Cymoxanil
Extractability increase [%].
(1 min by Hand = 100%)
155
logKOW
17
7
to
6.
5
6.
5
6
to
6
to
5.
5
5
to
5.
5
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to
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3
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2
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2
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to
1
EURL-Workshop 27-28 October 2010
1.
5
1.
5
1
to
0.
5
0
-0
to
-1
to
0
.5
to
-0
.5
-1
to
.5
EURL-SRM
-1
0.
5
100
EU Reference Laboratory
for Pesticide Residues
using Single Residue Methods
OBSERVATIONS:
(typically reached
after 10-15 min)
Extraction yields
at 1 min by hand vs. Plateau
~60% of tested PESTICIDES
showed yields <85%
in at least one case
Rec. <75%
40 %
Rec. >85%
45 %
of cases
40 %
of cases
Rec. 75-85%
> 80% of tested SAMPLES (fruits, veg. and cereals) showed
yields <85% for at least one of the pesticides contained!!
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18
EU Reference Laboratory
for Pesticide Residues
using Single Residue Methods
FURTHER CONCLUSIONS
Pesticides
The lower the logP the less frequent the effects
LogP < 2.5: ~20% of residues showed yields <85%
LogP > 2.5: ~65% of residues showed yields <85%, but no clear link
between logP and extraction yields
Matrix
Extraction retardation seems more pronounced in certain commodities
Natural surface-wax may play a role (slow penetration by extractant)
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19
EU Reference Laboratory
for Pesticide Residues
using Single Residue Methods
Examples
Lamb’s
lettuce
Auber- Grape Raisin
gine
-
+
Boscalid
(logP = 2,96)
Cyprodinyl
(logP = 4)
Strawberry
+
-
+
Ruccola
Red
Currant
Cherry
++
+
++
-
++
++
- : Minor or no effect : > 85% yield
+ : Intermediate effect : 75-85% yield
++ : Great Effect : <75% yield
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EU Reference Laboratory
for Pesticide Residues
using Single Residue Methods
Ethylacetate
Boscalid
ylacetat-Methode
Extraktionsversuch Ethyla
Fenpyroxymate
Famoxadon
160
Tebuconazole
Spinosyn A
150
(Gehalt bei 1 Min. = 100%)
Gehalt (%)
Azoxystrobin
140
Cymoxanil - sauer-
130
120
110
100
90
80
30
15Min.
Min.
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EU Reference Laboratory
for Pesticide Residues
using Single Residue Methods
Results of
EU-PTs samples
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22
EU Reference Laboratory
for Pesticide Residues
using Single Residue Methods
120
110
130
EURL-SRM
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100
23
140
Bifenthrin (pkow 7.3)
Cyhalothrin-lambda (pkow 7)
Chlorpyrifos-ethyl (pkow 4.7)
Cyprodinil (pkow 4)
Parathion (pkow 3.83)
Dichlofluanid (pkow 3.7)
Diazinon (pkow 3.69)
Myclobutanil (pkow 2.94)
Imazalil (pkow 2.56)
Azoxystrobin (pkow 2.5)
Carbaryl (pkow 2.36)
Pirimicarb (pkow 1.7)
Carbendazim (pkow 1.5)
Acetamiprid (pkow 0.8)
Extractability increase [%]..
(1 min by hand = 100%)
Aubergine (EUPT FV 8)
1 min by hand vs. 15 min by automated shaker
150
90
Pesticides (pkow -->)
EU Reference Laboratory
for Pesticide Residues
using Single Residue Methods
Extractability increase [%]
(1 min by hand = 100%)
120
110
100
130
140
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24
Quinoxyfen (pkow 4.66)
Cyprodinil (pkow 4)
Tolylfluanid (pkow 3.9)
Bupirimate (pkow 3.9)
Penconazole (pkow 3.72)
Tebuconazole (pkow 3.7)
Diazinon (pkow 3.69)
Fenhexamid (pkow 3.51)
Triadimenol (pkow 3.08)
Iprodione (pkow 3)
Pyrimethanil (pkow 3)
Myclobutanil (pkow 2.94)
90
Hexythiazox (pkow 2.53)
Oxamyl (pkow -0.44)
Strawberry (EUPT FV 9)
1 min by hand vs. 15 min by automated shaker
160
150
80
Pesticides (pkow -->)
EU Reference Laboratory
for Pesticide Residues
using Single Residue Methods
Extractability increase [%]
(1 min by hand = 100%)
150
140
130
120
170
160
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25
180
Pendimethalin (pkow 5.2)
Quinoxyfen (pkow 4.66)
Chlorpyrifos-methyl (pkow 4)
Diazinon (pkow 3.69)
Kresoxim-methyl (pkow 3.4)
Methiocarb (pkow 3.18)
Triadimenol (pkow 3.08)
Phosmet (pkow 2.96)
Boscalid (pkow 2.96)
Malathion (pkow 2.75)
Hexythiazox (pkow 2.53)
Malaoxon (pkow 2.074)
Acetamiprid (pkow 0.8)
Methomyl (pkow 0.09)
Oxamyl (pkow -0.44)
Carrot (EUPT FV 10)
1 min by hand vs. 15 min by automated shaker
190
110
100
90
Pesticides (pkow -->)
EU Reference Laboratory
for Pesticide Residues
using Single Residue Methods
Extractability increase [%]
(1min by hand = 100%)
130
125
120
115
110
105
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95
26
135
Buprofezin (pKow = 4.93)
Deltam ethrin (pKow = 4.6)
P hosalon (pKow = 4.01)
Cadusafos (pKow = 3.85)
Diazinon (pKow = 3.69)
Isofenphos-m ethyl (pKow = 3.589)
Triazophos (pKow = 3.34)
Boscalid (pKow = -2.96)
Azinphos-m ethyl (pKow = 2.96)
M ethidathion (pKow = 2.2)
Carbofuran (pKow = 1.8)
M etalaxyl (pKow = 1.75)
Aldicarb (pKow = 1.359)
Thiacloprid (pKow = 1.26)
P araoxon-m ethyl (pKow = 1.247)
3-Hydroxycarbofuran (pKow = 0.21)
M ethom yl (pKow = 0.09)
M onocrotophos (pKow = - 0.22)
Oxam yl (pKow = -0.44)
Aldicarb-sulfon (pKow = -0.57)
M etham idophos (pKow = -0.8)
100
Aldicarb-sulfoxid (pKow = -1.125)
Apple (EUPT-FV 11)
90
Pesticides (--> pKow)
EU Reference Laboratory
for Pesticide Residues
using Single Residue Methods
Leek (EUPT FV 12)
1 min by hand vs. 15 min by automated shaker
105
100
Triflumuron
Oxamyl
Omethoat
Ethion
Azinphos-methyl
110
Thiacloprid
115
Kresoxim-methyl
120
Chlorpyrifos
EPN
125
Prothiofos
130
Amitraz
Extractability increase [%]
(1 min by hand = 100%)
135
Fenpropathrin
140
95
90
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27
EU Reference Laboratory
for Pesticide Residues
using Single Residue Methods
Apple puree (EUPT SRM 5)
1 min by hand vs. 15 min by automated shaker
120
105
100
FBO
Fluazifop
110
Abamectin
Extractability increase [%]
(1 min by hand = 100%)
115
95
90
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EU Reference Laboratory
for Pesticide Residues
using Single Residue Methods
Comparison of extractability using QuEChERS
1 min by hand vs. 20 min by automated shaker
140
(EUPT-C4, sample with incurred residues)
Spiroxamine
130
125
Carbaryl
120
115
110
105
Kresoxym-methyl
Fluquinconazole
135
Deltamethrin
Rye
Pirimiphos-methyl
145
Carbendazim
Extractability increase [%]
(1 min by hand = 100%)
150
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to
4.
5
4.
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to
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1.
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0.
5
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-0
.5
to
0
-0
.5
to
-1
-1
.5
to
-1
100
logKOW
29
EU Reference Laboratory
for Pesticide Residues
using Single Residue Methods
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30
EU Reference Laboratory
for Pesticide Residues
using Single Residue Methods
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 study (RSD <12 % at n=5)
$ good homogeneity,
$ little influence of measurement uncertainty
At least n=3 for each experimental settings
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EU Reference Laboratory
for Pesticide Residues
using Single Residue Methods
IMPACT OF EXTENDED EXTRACTION TIMES
a) on Recoveries …
430 pesticides tested whether they degrade during
prolonged extraction times
using commodities representing the 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.
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EU Reference Laboratory
for Pesticide Residues
using Single Residue Methods
IMPACT OF EXTENDED EXTRACTION TIMES
b) On Matrix Effects…
Raisins
Extended extraction times
resulted in :
a) deeper coloured extracts
b) higher dry residue
R e s id u a l e x tra c t
a fte r e v a p o ra tio n to d ry n e s s
However…
5 ,0
4 ,5
(compounds responsible for
matrix effects are obviously
rather quickly extracted)
Extrakt [mg/mL]
$ Impact on matrix-effects
was insignificant
4 ,0
3 ,5
3 ,0
2 ,5
2 ,0
1 ,5
1 ,0
0 ,5
0 ,0
1 M in M a n u a lly
2 M in G e n o
g r in d e r
1 0 M in G e n o
g r in d e r
3 0 M in G e n o
g r in d e r
Ex tra c ti o n T i m e
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EU Reference Laboratory
for Pesticide Residues
using Single Residue Methods
QuEChERS - Citrate buffered
No cleanup, always n=2,
matrix = cucumber
1 min
Extraction
by Hand
Compound
Rec. %
30 min
Extraction with GenoGrinder
Notes
BEFORE adding
partitioning/buffering salts
Deviation
Rec. %
%
103
2
102
-6
100
-2
102
-4
98
0
107
-3
103
2
96
-7
104
0
88
-8
91
11
97
-5
102
2
98
-4
106
5
101
-10
AFTER adding
partitioning/buffering salts
Deviation
Rec. %
%
98
-2
88
-18
104
1
99
-7
96
-2
91
-17
91
-10
97
-6
95
-8
103
8
81
-2
95
-7
101
1
90
-12
99
-2
105
-6
1-Naphthalene acetamide
1-Naphthylessigsaeure
2,4,5-T
2,4,5-TP
2,4-D
2,4-DB
2,4-DP
2-Naphtoxy acetic acid
3-Hydroxycarbofuran
4-CPA
Acephat
Acetamiprid
Acibenzolar-S-methyl
Aciflourfen
Acrinathrin
a-Endosulfan
101
108
103
106
98
110
101
103
104
95
82
102
100
102
101
112
Aldicarb
108
129
19
70
-35
Aldicarb-sulfon
Aldicarb-sulfoxid
Aminocarb
94
86
89
86
100
92
-9
15
3
89
94
95
-6
9
6
Amitraz
27
25
-8
28
5
Ancymidol
Anilophos
Aspon
Azaconazole
Azinphos-methyl
Azoxystrobin
91
101
104
101
108
99
103
102
106
98
83
102
13
2
2
-4
-23
3
99
101
95
103
80
97
8
0
-8
2
-26
-3
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34
Oxidation prone, plus other problems
with measurement ??
Always low recovery if extract not
stabilized (requires D-SPE w. PSA
w/o acidification)
Oxidation to the oxon?
EU Reference Laboratory
for Pesticide Residues
using Single Residue Methods
EURL-SRM
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35
EU Reference Laboratory
for Pesticide Residues
using Single Residue Methods
EURL-SRM
EURL-Workshop 27-28 October 2010
36
EU Reference Laboratory
for Pesticide Residues
using Single Residue Methods
EURL-SRM
EURL-Workshop 27-28 October 2010
37
EU Reference Laboratory
for Pesticide Residues
using Single Residue Methods
EURL-SRM
EURL-Workshop 27-28 October 2010
38
EU Reference Laboratory
for Pesticide Residues
using Single Residue Methods
EURL-SRM
EURL-Workshop 27-28 October 2010
39
EU Reference Laboratory
for Pesticide Residues
using Single Residue Methods
EURL-SRM
EURL-Workshop 27-28 October 2010
40
EU Reference Laboratory
for Pesticide Residues
using Single Residue Methods
EURL-SRM
EURL-Workshop 27-28 October 2010
41
EU Reference Laboratory
for Pesticide Residues
using Single Residue Methods
EURL-SRM
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42
EU Reference Laboratory
for Pesticide Residues
using Single Residue Methods
EURL-SRM
EURL-Workshop 27-28 October 2010
43
EU Reference Laboratory
for Pesticide Residues
using Single Residue Methods
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
EURL-SRM
EURL-Workshop 27-28 October 2010
44
EU Reference Laboratory
for Pesticide Residues
using Single Residue Methods
Thank you very much…
EURL-SRM
EURL-Workshop 27-28 October 2010
45
EU Reference Laboratory
for Pesticide Residues
using Single Residue Methods