sweet validation of cereal samples – lc-ms/ms analysis

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

National Food Institute. Technical University of Denmark

AgendaShort summary of the MethodNon-linear standard curvesValidation


SweEtSample Clean up

Weigh

• 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

Mix

• Vortex mix for 30 sec • Add 10 ± 0,5 g Na2SO4• Shake for 10 sec

Extract• Ultra sonication for 30 min• Centrifuge 3 min at 3000 rpm (1500 g)

Filtrate• Filtrate the supernatant into a 0.45 µm filter vial (polypropylene)


LC-method• Analytical column: Genesis C18, 100x3 mm, 4 µm

• Solvent A: 10/90 metanol/10 mM ammonium formate, pH 4• Solvent B: Methanol


Pyrimethanil – linear regression0.01 0.02 0.1

Recovery 78 94 232


Pyrimethanil – quadratic regression0.01 0.02 0.1

Recovery 115 143 125


Pyrimethanil – cubic regression0.01 0.02 0.1

Recovery 111 107 104


Metribuzin – linear regressionCompound name: MetribuzinCorrelation coefficient: r = 0.993562, r^2 = 0.987166Calibration curve: 10.1201 * x + -0.00464258Response type: Internal Std ( Ref 1 ), Area * ( IS Conc. / IS Area )Curve type: Linear, Origin: Include, Weighting: 1/x, Axis trans: None

Conc0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00

Res

pons

e

0.0

2.5

5.0

7.5

10.0

Conc

Res

idua

l

-10.0

0.0

10.0

20.0


Injection volumen - chromatograms Fenhexamid

min18.00 19.00

%

0

100

F8:MRM of 8 channels,ES+302>55.5

3.445e+003Fenhexamid17.72630

284419.92

19.3519.24

18.3518.89

min

%

0

100

F8:MRM of 8 channels,ES+302 > 97.3

2.901e+003Fenhexamid17.72618

2590

19.8919.70

18.4618.35 19.46

min18.00 19.00

%

0

100


4.873e+003Fenhexamid17.71727

3828

19.32

19.16

18.08 18.62

19.67

min

%

0

100


5.789e+003Fenhexamid17.71820

5283

19.3219.1118.38

19.62

5 µl2 µl

min18.00 19.00

%

-2

98


2.488e+006Fenhexamid17.67

3181782527076

min

%

-2

98



5749984798504

min18.00 19.00

%

-2

98



1353291106886

min

%

-3

97



2094541750672

0.001 mg/kg

1.00 mg/kg


Injection volumen - standard curvesEpoconazol

Compound name: EpoxiconazoleCoefficient of Determination: R^2 = 0.999832 Calibration curve: 139.264 * x̂ 2 + 89.2489 * x + 0.04741Response type: Internal Std ( Ref 1 ), Area * ( IS Conc. / IS Area )Curve type: 2nd Order, Origin: Include, Weighting: 1/x, Axis trans: None

Conc0.00 0.20 0.40 0.60 0.80 1.00

Res

pons

e

0

50

100

150

200

Conc

Res

idua

l

0.0

10.0

20.0

5 µl2 µl

Compound name: EpoxiconazoleCoefficient of Determination: R^2 = 0.999931 Calibration curve: 228.583 * x̂ 2 + 83.9971 * x + 0.0442506Response type: Internal Std ( Ref 1 ), Area * ( IS Conc. / IS Area )Curve type: 2nd Order, Origin: Include, Weighting: 1/x, Axis trans: None

Conc0.00 0.20 0.40 0.60 0.80 1.00

Res

pons

e

0

100

200

300

Conc

Res

idua

l

0.0

10.0

20.0


Initial validation – LC-compounds• 28 compounds out of 34 had acceptable recoveries

Recoveries, mg/kg LOQ 0,01 0,02 0,1 3-hydroxy carbofuran 118 117 105 0,006Acephat 95 51 18 0,008Carbaryl 110 102 102 0,003Cyprodinil 113 112 100 0,005Demeton-S-methylsulfon 91 96 95 0,003Desmethyl pirimicarb 100 97 87 0,005Epoxiconazole 105 99 89 0,009Fenhexamid 110 105 104 0,008Flusilazole 107 105 98 0,003Imazalil 104 90 84 0,012Imidacloprid 112 105 98 0,004Isoproturon 114 114 108 0,003Kresoxim-methyl 104 109 90 0,007Linuron 106 102 99 0,003Malaoxon 84 85 85 0,002Methacrifos 106 103 96 0,009Methomyl 169 162 135 0,012

Recoveries, mg/kg LOQ 0,01 0,02 0,1 Metribuzin 112 112 101 0,008Omethoat 0 0 41 0,000Oxydemeton-methyl 57 49 45 0,005Paclobutrazole 98 105 101 0,002Pendimethalin 103 99 89 0,004Pirimicarb 101 109 110 0,002Pirimiphos-methyl 113 110 100 0,005Prothioconazole-desthio 102 102 105 0,006Pyraclostrobin 106 106 96 0,008Pyrimethanil 111 107 104 0,005Spiroxamine 3557 2108 632 0,040Tebufenozide 90 93 87 0,005Thiacloprid 104 105 100 0,004Thiodicarb 10 9 12 0,001Triazophos 91 103 108 0,006Tricyclazole 98 97 96 0,002Triticonazole 106 114 109 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

Compound name: SpiroxamineCoefficient of Determination: R^2 = 0.984179 Calibration curve: 245.305 * x̂ 2 + -3.65684 * x + -0.00631267Response type: Internal Std ( Ref 1 ), Area * ( IS Conc. / IS Area )Curve type: 2nd Order, Origin: Include, Weighting: 1/x, Axis trans: None

Conc0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00

Res

pons

e

0

100

200

Conc

Res

idua

l

-400

-200

0

min16.600 16.800 17.000 17.200 17.400

%

-0

100


7.590e+003

130402_13 Smooth(SG,1x2) Samstik A cal 3 i hvede

Spiroxamine17.26735

7171

Spiroxamine17.26735

7171

16.69

min

%

0

100

F6:MRM of 4 channels,ES+298 > 144.24.752e+004

130402_13 Smooth(SG,1x2) Samstik A cal 3 i hvede

Spiroxamine17.24503244522

Spiroxamine17.24503244522


Compounds with low recoveriesLog P Recovery Structure

Acephate -0.89 18-50%

Omethoate -0.74 17-50%

Oxydemeton-methyl

-0.74 40-60%

Thiodicarb 1.62 0-10%

http://www.trc-canada.com/Structures/T344680.png


Compound with high recoveriesLog P Recovery

Methomyl 0.093 135-170%

Compound name: MethomylCoefficient of Determination: R^2 = 0.994937 Calibration curve: 1.84593 * x̂ 2 + 7.71071 * x + -0.00275524Response type: Internal Std ( Ref 1 ), Area * ( IS Conc. / IS Area )Curve type: 2nd Order, Origin: Exclude, Weighting: 1/x, Axis trans: None

Conc0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00

Res

pons

e

0.0

2.5

5.0

7.5

10.0

Conc

Res

idua

l

0.0

20.0


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


Thank you for your attention

sweet validation of cereal samples – lc-ms/ms analysis

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