Transcript SweEt validation of Cereal samples – LC-MS/MS analysis Anne Kruse Lykkeberg; Gitte Andersen & Mette Erecius Poulsen; Nordic Pesticide Workshop, 2013, Porvoo/Borgå.

SweEt validation of Cereal
samples – LC-MS/MS analysis
Anne Kruse Lykkeberg; Gitte Andersen
& Mette Erecius Poulsen;
Nordic Pesticide Workshop, 2013, Porvoo/Borgå
Agenda
Short summary of the Method
Non-linear standard curves
Validation
National Food Institute. Technical University of Denmark
SweEtSample Clean up
Weigh
Mix
Extract
Filtrate
•5 ± 0,05 g grinded dry sample (1 mm sieves)
•Add 10 ± 1,0 ml H2O
•And 10 ± 0,05 ml ethyl acetat med 1 % acetic acid
•Vortex mix for 30 sec
•Add 10 ± 0,5 g Na2SO4
•Shake for 10 sec
•Ultra sonication for 30 min
•Centrifuge 3 min at 3000 rpm (1500 g)
•Filtrate the supernatant into a 0.45 µm filter vial (polypropylene)
National Food Institute. Technical University of Denmark
LC-method
• Analytical column: Genesis C18, 100x3 mm, 4 µm
• Solvent A: 10/90 metanol/10 mM ammonium formate, pH 4
• Solvent B: Methanol
National Food Institute. Technical University of Denmark
Pyrimethanil – linear regression
Recovery
National Food Institute. Technical University of Denmark
0.01
0.02
0.1
78
94
232
Pyrimethanil – quadratic regression
Recovery
National Food Institute. Technical University of Denmark
0.01
0.02
0.1
115
143
125
Pyrimethanil – cubic regression
Recovery
National Food Institute. Technical University of Denmark
0.01
0.02
0.1
111
107
104
Metribuzin – linear regression
Compound name: Metribuzin
Correlation coefficient: r = 0.993562, r^2 = 0.987166
Calibration curve: 10.1201 * x + -0.00464258
Response type: Internal Std ( Ref 1 ), Area * ( IS Conc. / IS Area )
Curve type: Linear, Origin: Include, Weighting: 1/x, Axis trans: None
Residual
20.0
10.0
0.0
-10.0
Conc
Response
10.0
7.5
5.0
2.5
0.0
0.00
0.10
0.20
0.30
0.40
National Food Institute. Technical University of Denmark
0.50
0.60
0.70
0.80
0.90
Conc
1.00
Injection volumen - chromatograms
Fenhexamid
5 µl
2 µl
97
100
0.001
mg/kg
F8:MRM of 8 channels,ES+
302 > 97.3
F8:MRM of 8 channels,ES+
1.720e+006
302
> 97.3
Fenhexamid
2.901e+003
Fenhexamid
17.70
17.72
209454
618
1750672
98
100
2590
%
F8:MRM of 8 channels,ES+
F8:MRM of 8 channels,ES+
302 > 97.3
302 > 97.3
4.727e+006
Fenhexamid
5.789e+003
Fenhexamid
17.67
17.71
574998
820
4798504
5283
%
%
%
19.70
18.35 18.46
19.46
19.89
18.38
-3
0
100
98
1.00
mg/kg
%
%
minmin
F8:MRM
8 channels,ES+
F8:MRM
of of
8 channels,ES+
302>55.5
302>55.5
1.091e+006
3.445e+003
Fenhexamid
Fenhexamid
17.70
17.72
135329
630
2844
1106886
19.92
19.35
19.24
18.35
18.89
-2
0
min
min
100
98
F8:MRM
F8:MRMofof8 8channels,ES+
channels,ES+
302>55.5
302>55.5
4.873e+003
2.488e+006
Fenhexamid
Fenhexamid
17.71
17.67
727
318178
3828
2527076
19.32
%
%
19.16
18.08
0
-2
min
18.00
18.00
19.00
min
19.00
National Food Institute. Technical University of Denmark
19.11 19.32 19.62
0
-2
18.00
18.00
19.67
18.62
19.00
19.00
min
min
Injection volumen - standard curves
Epoconazol
Compound name: Epoxiconazole
Coefficient of Determination: R^2 = 0.999832
Calibration curve: 139.264 * x^2 + 89.2489 * x + 0.04741
Response type: Internal Std ( Ref 1 ), Area * ( IS Conc. / IS Area )
Curve type: 2nd Order, Origin: Include, Weighting: 1/x, Axis trans: None
Residual
20.0
5 µl
2 µl
10.0
0.0
Conc
Response
200
150
100
50
0
0.00
0.20
0.40
0.60
0.80
Conc
1.00
National Food Institute. Technical University of Denmark
Initial validation – LC-compounds
• 28 compounds out of 34 had acceptable recoveries
Recoveries, mg/kg
0,01
0,02
0,1
3-hydroxy carbofuran
118
117
105
Acephat
95
51
18
Carbaryl
110
102
102
Cyprodinil
113
112
100
Demeton-S-methylsulfon 91
96
95
Desmethyl pirimicarb
100
97
87
Epoxiconazole
105
99
89
Fenhexamid
110
105
104
Flusilazole
107
105
98
Imazalil
104
90
84
Imidacloprid
112
105
98
Isoproturon
114
114
108
Kresoxim-methyl
104
109
90
Linuron
106
102
99
Malaoxon
84
85
85
Methacrifos
106
103
96
Methomyl
169
162
135
LOQ
0,006
0,008
0,003
0,005
0,003
0,005
0,009
0,008
0,003
0,012
0,004
0,003
0,007
0,003
0,002
0,009
0,012
National Food Institute. Technical University of Denmark
Recoveries, mg/kg
0,01
0,02
0,1
Metribuzin
112
112
101
Omethoat
0
0
41
Oxydemeton-methyl
57
49
45
Paclobutrazole
98
105
101
Pendimethalin
103
99
89
Pirimicarb
101
109
110
Pirimiphos-methyl
113
110
100
Prothioconazole-desthio 102
102
105
Pyraclostrobin
106
106
96
Pyrimethanil
111
107
104
Spiroxamine
3557
2108 632
Tebufenozide
90
93
87
Thiacloprid
104
105
100
Thiodicarb
10
9
12
Triazophos
91
103
108
Tricyclazole
98
97
96
Triticonazole
106
114
109
LOQ
0,008
0,000
0,005
0,002
0,004
0,002
0,005
0,006
0,008
0,005
0,040
0,005
0,004
0,001
0,006
0,002
0,009
Spiroxamin
• LogP = 2.89
• Low sensitivity LOD = 0.04 ng/ml
– Only i SWEET – not QuEChERS
• Recovery is good at the highest level
130402_13 Smooth(SG,1x2)
Samstik A cal 3 i hvede
Spiroxamine
17.24
5032
44522
100
F6:MRM of 4 channels,ES+Compound name: Spiroxamine
298 > 144.2Coefficient of Determination: R^2 = 0.984179
4.752e+004Calibration curve: 245.305 * x^2 + -3.65684 * x + -0.00631267
Response type: Internal Std ( Ref 1 ), Area * ( IS Conc. / IS Area )
Curve type: 2nd Order, Origin: Include, Weighting: 1/x, Axis trans: None
Spiroxamine
17.24
5032
44522
0
Residual
%
0
-200
min
130402_13 Smooth(SG,1x2)
Samstik A cal 3 i hvede
F6:MRM of 4 channels,ES+
298>100.2
7.590e+003
Spiroxamine
17.26
735
7171
100
Response
Spiroxamine
17.26
735
7171
%
-400
Conc
200
100
16.69
-0
min
16.600
16.800
17.000
17.200
17.400
National Food Institute. Technical University of Denmark
0
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
Conc
1.00
Compounds with low recoveries
Log P
Recovery
Acephate
-0.89
18-50%
Omethoate
-0.74
17-50%
Oxydemetonmethyl
-0.74
40-60%
Thiodicarb
1.62
0-10%
National Food Institute. Technical University of Denmark
Structure
Compound with high recoveries
Methomyl
Log P
Recovery
0.093
135-170%
Residual
Compound name: Methomyl
Coefficient of Determination: R^2 = 0.994937
Calibration curve: 1.84593 * x^2 + 7.71071 * x + -0.00275524
Response type: Internal Std ( Ref 1 ), Area * ( IS Conc. / IS Area )
Curve type: 2nd Order, Origin: Exclude, Weighting: 1/x, Axis trans: None
20.0
0.0
Conc
10.0
Response
7.5
5.0
2.5
0.0
0.00
National Food Institute. Technical University of Denmark
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
Conc
1.00
Conclusions
• Samples in ethyl acetate gives non linear standard curves
for many pesticides
• Using Quadratic fit and i few cases Cubic fit gives
– Acceptable residuals with random distributions
– Acceptable recoveries at three levels
• Compounds with Log P below 0 gives a poor recovery
using the SweEt method
National Food Institute. Technical University of Denmark
National Food Institute. Technical University of Denmark