1. Anna Sannino, Luciana Bolzoni, Mirella Bandini,
DETERMINATION OF NEW PESTICIDE RESIDUES IN PROCESSED FRUIT PRODUCTS BY LC/MS/MS AND GC/MS
Anna Sannino, Luciana Bolzoni, Mirella Bandini,
SSICA Stazione Sperimentale per l’Industria delle Conserve Alimentari
Experimental Station for the Food Preserving Industry
Viale F. Tanara, 31/A – Parma – Italy - Tel

2. SSICA - Experimental Station of Food Preserving industry Parma site

3. SSICA pesticide residue laboratory
3 technicians
1 GC/MS and 1 GC/ITMS
1 GC/ECD/ECD
1 GC/NPD/FPD
1 LC/UV/FLUOR./COUL.
1 LC/MS/MS

4. Activity of SSICA pesticide residue laboratory
Analysis for the food industry
Setting up of analysis methods

5. Strobilurin ( axoxystrobin, trifloxystrobin, kresoxim-methyl)
Insecticides and fungicides belonging to different chemical classes:
Strobilurin ( axoxystrobin, trifloxystrobin, kresoxim-methyl)
Morpholin (dimethomorph)
Triazol ( tebuconazol, tetraconazol, paclobutrazol, flusilazol, bromuconazol,difeconazol, fenbuconazol
Quinozolin( fenazaquin)
Carbamate ( benfuracarb, fenothiocarb, furathiocarb)
Carboxamide (hexythiazox, tebufenpyrad,
Pyrimidine( ciprodinil, pyrimetanil, mepanipyrim, bupirimate)
Chloronicotinin (imidacloprid)
pirazol (fipronil, fenpyroximate)
Pyrrole ( fludioxonil) hydroxyanilid( fenhexamide)
Pyridazinone( pyridaben)
phenoxybenzil ( ethofenprox)
Benzohydrazide( tebufenozide)

6. 20g of homogeneous sample
Flow chart of method
20g of homogeneous sample
Adjust pH to 6.0
NaCl +Acetone/Blending
ethyl acetate-cyclohexane/Blending
Centrifugation – filtration – evaporation
Clean-up by PSA sorbent (dispersive SPE)
LC/MS/MS
GC/MS

7. Liquid Chromatographic conditions

8. LC/MS/MS PARAMETERS
API triple quadrupole mass spectrometer equipped with a TurboIonSpray (ESI) interface
positive ion mode
Source parameters
55 simultaneous MRM transitions of ion precursor ions
( dwell time: 40ms)
2 or 3 transitions for each pesticide

9. Analyte MRM transitions obtained by LC/MS/MS
1st trans nd trans rd trans
Azoxystrobin / / /329
trifloxystrobin 409/ /206
kresoxim-methyl 314/ / /267
fenazaquin 307/ / /57
indoxacarb 528/ / /218
fenothiocarb 254/ /160
imidachloprid 256/ /175
dimethomorph 388/ /303
benfuracarb 411/ / /190
furathiocarb 383/ /252
fenpyroximate 422/ /135
hexythiazox 353/ /168
tebufenpyrad 334/ /145
tebufenozide 353/ /133
difeconazole 406/ /253
flusilazole 316/ /165
fenbuconazole 337/ /125
paclobutrazole 294/ /70
tebuconazole 308/ / /151
tetraconazole 372/ /161
bromuconazole 378/ / /161
etofenprox 359/ /177
fenehamid 302/ /97
pyridaben 365/ /311
1st trans. used for quantitation

10. Gas chromatographic conditions
-Varian Star 3400 CX gas chromatograph coupled with Model Saturn Ion Trap mass spectrometer;
MS parameters EI mode with 20 µA filament current,
transfer line temparature 280°C,.
detector manifold temperature 50°C,
trap temperature: 200°C,
mass range: m/z
temperature programmable injection port:
initial temperature ,68°C; initial time 0.3min ; rate, 300°C/min; final temperature, 250°C;
column :30 m x 0.25 mm x 0.25 µm Rtx5- MS equipped with a 3 m x 0.25 mm uncoated gap;
Temperature programme: 66°C for 1 min, 25°C/min to 195°C, 195°C for 8 min, 10 °C/min to 230°C, 230°C for 7 min, 20°C/min to 250°C, 250°C for 5 min; 20°C/min to 270°C for 15 min min;
column head pressure 20psi;
Injection volume: 2.5 L of sample 2g/mL

11. List of pesticides analyzed by GC/MS and ions used for quantitation
RT Quantitation ions
pyrimethanil
ciprodinil
fipronil
mepanipyrim
fludioxonil
buprofezin
bupirimate
kresoxim-methyl
fenexhamid
piridaben
etofenprox

12. Six determinations at each fortification level
Recovery experiments
Tomato puree
lemon concentrated
Apple juice
Six determinations at each fortification level
Two levels of fortification
1-10ppb ppb LC/MS/MS
5-20ppb ppb GC/MS

13. MATRIX EFFECT
HIGHER RESPONSE OF ANALYTES IN MATRIX WHEN ANALYZED BY GCMS (23-83% IN APPLE JUICE AND % IN CITRUS JUICE CONCENTRATED)
LOWER RESPONSE OF ANALYTES IN MATRIX WHEN ANALYZED BY LCMSMS ( 0-44%) FOR THE MOST OF COMPOUNDS). HIGHER RESPONSE FOR INDOXACARB (>40%)
CALIBRATION WAS ACHIEVED BY PREPARING MATRIX-MATCHED STANDARD FROM THE EXTRACTS OF BLANK SAMPLES

14. Usual calibration calculated with polynomial regression( 2nd order)
(404.0 / 372.0): "Quadratic" Regression: y = x^ e+004 x e+004 (r = ) 20 40 60 80
100
120
140
160
180
200
220
240
Concentration, ng/mL
0.0
1.0e5
3.0e5
5.0e5
7.0e5
9.0e5
4.3e6
4.2e6
3.9e6
Azoxystrobin
Calibration curve
3.7e6
3.5e6
3.3e6
3.1e6
2.9e6
2.7e6
2.5e6
2.3e6
Area, counts
2.1e6
1.9e6
1.7e6
1.5e6
1.3e6
1.2e6
1.1e6
1.0e6

15. Usual calibration calculated with polynomial regression( 2nd order)
(388.0 / 301.0): "Quadratic" Regression: y = x^ e+003 x e+005 (r = )
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
Concentration, ng/mL
1.3e7
1.2e7
Dimethomorph
Calibration curve
1.1e7
1.0e7
9.0e6
8.0e6
7.0e6
Area, counts
6.0e6
5.0e6
4.0e6
3.0e6
2.0e6
1.0e6
0.0

16. LIMITS OF QUANTITATION (LOQ) S/N> 10
GC/MS: ppb
LC/MS/MS: ppb
except for: kresoxim-methyl ( 5ppb)
imidacloprid(10ppb)
etofenprox (10ppb)
fenhexamid( 10ppb)

17. Mean recoveries(%) from all spiked matrices and all levels by LC/MS/MS

18. Mean recoveries(%) from all spiked matrices and all levels by GC/MS

19. RSD< 15% for all matrices and all levels by GC/MS and LC/MS/MS
RECOVERIES HIGHER THAN 70% BY LC/MS/MS
RECOVERIES HIGHER THAN 68% BY GC/MS EXCEPT FOR FLUDIOXONIL AND FENHEXAMID THAT ARE ONLY PARTIALLY RECOVERED FROM CLEAN-UP WITH SORBENT (PSA)
RSD< 15% for all matrices and all levels by GC/MS and LC/MS/MS

20. LC/MS/MS chromatograms of 9 pesticides in fortified apple juice
Sample Name: "mela tq +m.q" Sample ID: "" File: "Datamixnewmela.wiff"
Peak Name: "404.0 / 372.0" Mass(es): "404.0/372.0 amu"
Comment: "" Annotation: ""
Sample Index:
Sample Type: Unknown
Concentration: N/A
Acq. Date: /20/03
Acq. Time: :40:15 PM
Modified: No
Bunching Factor: 1
Noise Threshold:
Area Threshold:
Num. Smooths:
RT Window: sec
Expected RT: min
Sep. Width:
Sep. Height:
Exp. Peak Ratio:
Exp. Adj. Ratio:
Exp. Val. Ratio:
Use Relative RT: No
Int. Type: Base To Base
Retention Time: min
Area: e counts
Height: e cps
Start Time: min
End Time: min 6 8 10
Time, min
0.0
2000.0
4000.0
6000.0
8000.0
1.0e4
1.2e4
1.4e4
1.6e4
Intensity, cps
8.13
Peak Name: "314.0 / 206.0" Mass(es): "314.0/206.0 amu"
Noise Threshold:
Area Threshold:
Expected RT: min
Retention Time: min
Area: e counts
Height: e cps
Start Time: min
End Time: min
500
1000
1500
2000
2500
3000
3500
8.64
8.77
Comment: "" Annotation: ""
Peak Name: "409.0 / 186.0" Mass(es): "409.0/186.0 amu"
Sample Name: "mela tq +m.q" Sample ID: "" File: "Datamixnewmela.wiff"
Modified: No
Acq. Date: /20/03
Acq. Time: :40:15 PM
Concentration: N/A
Sample Type: Unknown
Sample Index:
Retention Time: min
Use Relative RT: No
Int. Type: Base To Base
Area: e counts
Start Time: min
Exp. Adj. Ratio:
End Time: min
Height: e cps
Exp. Val. Ratio:
Num. Smooths:
Area Threshold:
Noise Threshold:
Bunching Factor: 1
Exp. Peak Ratio:
RT Window: sec
Expected RT: min
Sep. Height:
Sep. Width:
1.3e4
8.94
1.2e4
1.0e4
azoxystrobin ppb
kresoxim-methyl 10ppb
8000.0
trifloxystrobin 2ppb
6000.0
4000.0
2000.0
0.0 6 8 10
Time, min
Height: e cps
Start Time: min
End Time: min 6 8 10
Time, min
1000
2000
3000
4000
5000
6000
7000
8000
8700
9.02
Sample Name: "mela tq +m.q" Sample ID: "" File: "Datamixnewmela.wiff"
Peak Name: "254.0 / 72.0" Mass(es): "254.0/72.0 amu"
Comment: "" Annotation: ""
Sample Index:
Sample Type: Unknown
Concentration: N/A
Acq. Date: /20/03
Acq. Time: :40:15 PM
Modified: No
Bunching Factor: 1
Noise Threshold:
Area Threshold:
Num. Smooths:
RT Window: sec
Expected RT: min
Sep. Width:
Sep. Height:
Exp. Peak Ratio:
Exp. Adj. Ratio:
Exp. Val. Ratio:
Use Relative RT: No
Int. Type: Base To Base
Retention Time: min
Area: e counts
Height: e cps
Start Time: min
End Time: min
9000
8.25
Peak Name: "256.0 / 209.0" Mass(es): "256.0/209.0 amu"
Noise Threshold:
Area Threshold:
Expected RT: min
Retention Time: min
Area: e counts
Height: e cps
Start Time: min
End Time: min 2
Comment: "" Annotation: ""
Sample Name: "mela tq +m.q" Sample ID: "" File: "Datamixnewmela.wiff"
Sample Type: Unknown
Sample Index:
Acq. Date: /20/03
Concentration: N/A
Peak Name: "307.0 / 57.0" Mass(es): "307.0/57.0 amu"
Acq. Time: :40:15 PM
Peak Name: "528.0 / 249.0" Mass(es): "528.0/249.0 amu"
Sample Name: "mela tq +m.q" Sample ID: "" File: "Datamixnewmela.wiff"
Comment: "" Annotation: ""
Acq. Time: :40:15 PM
Acq. Date: /20/03
Sample Index:
Concentration: N/A
Sample Type: Unknown
End Time: min
Start Time: min
Sep. Height:
Sep. Width:
Expected RT: min
Exp. Peak Ratio:
Exp. Adj. Ratio:
Exp. Val. Ratio:
RT Window: sec
Num. Smooths:
Modified: No
Bunching Factor: 1
Noise Threshold:
Area Threshold:
Use Relative RT: No
6.6e4
9.62
Exp. Val. Ratio:
Use Relative RT: No
Area Threshold:
Exp. Adj. Ratio:
Num. Smooths:
Sep. Height:
RT Window: sec
Noise Threshold:
Sep. Width:
Expected RT: min
Exp. Peak Ratio:
Bunching Factor: 1
Modified: No
Area: e counts
Height: e cps
Int. Type: Base To Base
Retention Time: min
Int. Type: Base To Base
Area: e counts
Retention Time: min
6.0e4
5.0e4
indoxacarb ppb
Intensity, cps
fenazaquin ppb
fenothiocarb ppb
4.0e4
3.0e4
2.0e4
1.0e4
0.0 6 8 10
Time, min 4
Time, min
200
400
600
800
1000
1200
1400
1550
Intensity, cps
3.04
Sample Name: "mela tq +m.q" Sample ID: "" File: "Datamixnewmela.wiff"
Peak Name: "388.0 / 301.0" Mass(es): "388.0/301.0 amu"
Comment: "" Annotation: ""
Sample Index:
Sample Type: Unknown
Concentration: N/A
Acq. Date: /20/03
Acq. Time: :40:15 PM
Modified: No
Bunching Factor: 2
Noise Threshold:
Area Threshold:
Num. Smooths:
RT Window: sec
Expected RT: min
Sep. Width:
Sep. Height:
Exp. Peak Ratio:
Exp. Adj. Ratio:
Exp. Val. Ratio:
Use Relative RT: No
Int. Type: Base To Base
Retention Time: min
Area: e counts
Height: e cps
Start Time: min
End Time: min 6 8 10
500
1500
2000
2500
3000
3500
4000
4500
4975
7.23
7.10
Peak Name: "411.0 / 195.0" Mass(es): "411.0/195.0 amu"
Bunching Factor: 1
Noise Threshold:
Area Threshold:
Expected RT: min
Retention Time: min
9.11
Start Time: min
Height: e cps
End Time: min
Area: e counts
1.20e5
1.00e5
imidacloprid ppb
8.00e4
benfuracarb ppb
dimetomorph ppb
6.00e4
4.00e4
2.00e4
0.00 6 8 10
Time, min

21. Apple juice unfortified
Overlaid GC/MS Chromatogram Plots
Plot 1: h:\fungi840.sms Ions: all
Plot 2: h:\fungi837.sms Ions: all
8.75
9.00
9.25
9.50
9.75
minutes
2.5
5.0
7.5
10.0
12.5
15.0
kCounts
Ions: all fungi840.sms
S/N. 55
S:14650
N: 265
Ions:
Apple juice fortified
with 5 ppb Pyrimethanil
Apple juice unfortified

22. 102 samples supplied from market.
Include: nectars, juices, purees of
stone apricot, peach, plum
Pome apple, pear
Citrus orange, lemon
( organic and conventional products, baby foods)

23. Highest residue found ppb
Results of 102 analyzed samples
Samples N° P
PHL
Pesticide found
Highest residue found ppb
Organic 33 2
Cyprodinil
6.7
Baby food 19 3
tebuconazole
6.1
conventional 50
Tebuconazole
Tetraconazole
fenbuconazole
3.3
N° number of samples; P positive samples,with residues higher than quantitation limits; PHLpositive samples with residues higher than MRL

24. LC/MS/MS analysis of a positive sample
LC/MS/MS chromatogram of a positive sample

25. LC/MS/MS analysis of a positive sample
6.0
6.2
6.4
6.6
6.8
7.0
7.2
7.4
7.6
7.8
8.0
8.2
8.4
8.6
8.8
9.0
9.2
9.4
9.6
9.8
Time, min
500
1000
1500
2000
2500
3000
LC/MS/MS analysis of a positive sample
7.88
8500
8000
7500
Apricot nectar
7000
6500
6000
5500
Tetraconazole
2.2 ppb
5000
4500
Intensity, cps
4000
3500

26. Positive sample (cyprodinil 6.1 ppb)
GC/MS CHROMATOGRAMS OF CYPRODINIL
IN 2 SAMPLES OF BABY HOMOGENIZED PLUM
Plot 1: d:\prova\prova219.sms Ions: all
Plot 2: d:\prova\prova216.sms Ions: all
kCounts
S/N:99
Ions: all prova219.sms
12.5
s:11586
N:117
Positive sample (cyprodinil 6.1 ppb)
10.0
Ions:
7.5
5.0
Negative sample
2.5
0.0
13.00
13.25
13.50
13.75
14.00
minutes

27. Conclusions
Extraction-partition method is capable of quantitatively extracting all pesticides
Fenhexamide and fludioxonyl are partially recovered from dispersive SPE clean-up
LC/MS/MS provide to be a technique with high selectivity and sensitivity (0.5-2ppb) for the 24 of 30 pesticides
None of analyzed samples contained residues > 10 ppb. Few samples (8%) found to be contaminated at levels > LOQs