Date ACCURATE-MASS DATABASE FOR SCREENING OF PESTICIDE RESIDUES IN FRUITS AND VEGETABLES BY GAS CHROMATOGRAPHY TIME-OF-FLIGHT MASS SPECTROMETRY

In practice, target analysis approach (analysing 1-4 of GC and LC amenable compounds) MEANS THAT THE MAJORITY OF THE LOW- FREQUENCY OR MISUSED COMPOUNDS ARE NOT LOOKED FOR

Target methods European Reference Laboratory - FV

Target methods Very Expensive

Target methods Very Expensive Time Consuming

Screening methods Practically all applications have been focused on LC (Time of flight) LC-TOF-MS (libraries containing fragments of compounds and retention time). Focused in GC (single quadrupole), commercial libraries.

GC-TOF/MS (full scan) (Ability to record an unlimited number of compounds) GC libraries (accurate masses of molecular ions and fragments and retention times)

Experimental conditions Injection volume: 2 μl Mode: Splitless MM Inlet Inyector temperature: 28ºC Agilent 72 Q-TOF GC/MS OVEN PROGRAM Rate(ºC/min) Value(ºC) Hold Time(min) Run Time(min) 6 1 1 4 12 2.5 5 31 4.5 Post Run Time: 2 min Post Run temperature: 31ºC BACKFLUSHING CAPABILITIES Retention time locked with constant flow (Trifluralin)

BACKFLUSH CONFIGURATION 2 columns: HP5MSI (35ºC) 15mx25µmx.25µm Constant Flow Column 1 = 1.225ml/min Column 2= 1.425ml/min

Internal Reference Mass European Reference Laboratory - FV

GC-Q-TOF DATABASE Many ions in the mass spectra. The molecular ion is not always present in the spectra.

1ppb Tomate TIC European Reference Laboratory - FV

1ppb Tomate EIC (284.9954_Vinclozolin Parent Ion) European Reference Laboratory - FV Molecular ion Vinclozolin

1ppb Tomate EIC (284.9954_Vinclozolin Parent Ion) European Reference Laboratory - FV Molecular ion Vinclozolin 1ppb Tomate Mass Spectrum (tr = 18.186 min)

1ppb Tomate EIC (284.9954_Vinclozolin Parent Ion) European Reference Laboratory - FV Molecular ion Vinclozolin 1ppb Tomate Mass Spectrum (tr = 18.186 min) NIST MASS INTERPRETER

1ppb Tomate EIC (284.9954_Vinclozolin Parent Ion) European Reference Laboratory - FV Molecular ion Vinclozolin 1ppb Tomate Mass Spectrum (tr = 18.186 min) NIST [C6H6NO2] + [C7H3Cl2NO] + [C1H9ClN ] + [C12H9Cl2NO3 ] + [C1H8Cl2N ] + MASS INTERPRETER

1ppb Tomate EIC (348.9257_Chlorpyrifos Parent Ion) European Reference Laboratory - FV

1ppb Tomate EIC (313.9569_Chlorpyrifos Fragment) 313.9569 Chlorpyrifos Fragment Chorpyrifos

1ppb Tomate EIC (313.9569_Chlorpyrifos Fragment) 313.9569 Chlorpyrifos Fragment Chorpyrifos 1ppb Tomate Mass Spectrum (tr = 2.46 min)

1ppb Tomate EIC (313.9569_Chlorpyrifos Fragment) 313.9569 Chlorpyrifos Fragment Chorpyrifos 1ppb Tomate Mass Spectrum (tr = 2.46 min) [C5H2Cl3NO] + [C9H11Cl2NO3PS ] + [C5H3Cl2NO3PS ] +

ACCURATE MASS DATABASE ### Formula Retention Time Mass Compound name # Formula RT Mass Cpd C17H16Br2O3 28,12 425,9466 Bromopropylate C13H9Br2O 28,12 338,92 Bromopropylate F1 C7H6BrO 28,12 184,962 Bromopropylate F2 C6H4Br 28,12 154,9496 Bromopropylate F3 C13H12BrCl2N3O 27,92 374,9541 Bromuconazole C1H8BrCl2O 27,92 292,9136 Bromuconazole F1 C13H12Cl2N3O 27,92 296,357 Bromuconazole F2 C1H9Cl2O 27,92 215,3 Bromuconazole F3 C7H3Cl2O 27,92 172,9561 Bromuconazole F4 C8H7Cl2 27,92 172,9925 Bromuconazole F5 C13H24N4O3S 24,1 316,1569 Bupirimate C11H18N3O3S 24,1 272,169 Bupirimate F1 C11H18N3O 24,1 28,145 Bupirimate F2 C5H4N2O 24,1 18,324 Bupirimate F3 C16H23N3OS 23,85 35,1562 Buprofezin C9H9N2S 23,85 177,486 Buprofezin F1 C7H5NO 23,85 119,371 Buprofezin F2 C7H8N 23,85 16,657 Buprofezin F3 C7H11N2OS 23,85 171,592 Buprofezin F4 C8H16N2S 23,85 172,134 Buprofezin F5 Continue...

ACCURATE MASS DATABASE ### Formula Retention Time Mass Compound name # Formula RT Mass Cpd C17H16Br2O3 28,12 425,9466 Bromopropylate C13H9Br2O 28,12 338,92 Bromopropylate F1 C7H6BrO 28,12 184,962 Bromopropylate F2 C6H4Br 28,12 154,9496 Bromopropylate F3 C13H12BrCl2N3O 27,92 374,9541 Bromuconazole C1H8BrCl2O 27,92 292,9136 Bromuconazole F1 C13H12Cl2N3O 87 PESTICIDES 27,92 296,357 Bromuconazole F2 C1H9Cl2O 27,92 215,3 Bromuconazole F3 C7H3Cl2O 2 FRAGMENTS 27,92 AT LEAST FOR EACH 172,9561 PESTICIDE Bromuconazole F4 C8H7Cl2 RETENTION TIMES 27,92 OF EACH PESTICIDE 172,9925 Bromuconazole F5 C13H24N4O3S 24,1 316,1569 Bupirimate C11H18N3O3S 24,1 272,169 Bupirimate F1 C11H18N3O 24,1 28,145 Bupirimate F2 C5H4N2O 24,1 18,324 Bupirimate F3 C16H23N3OS 23,85 35,1562 Buprofezin C9H9N2S 23,85 177,486 Buprofezin F1 C7H5NO 23,85 119,371 Buprofezin F2 C7H8N 23,85 16,657 Buprofezin F3 C7H11N2OS 23,85 171,592 Buprofezin F4 C8H16N2S 23,85 172,134 Buprofezin F5 Continue...

Data base in Excel file (csv format)

Data base in Excel file (csv format) Retention time tolerance:,5 min Mass error tolerance 1 ppm

% compounds % compounds 1µg/Kg_ 1µg/Kg_Spring onion 2µg/Kg_ 2µg/Kg_Spring onion 5µg/Kg_ 5µg/Kg_Spring onion 1µg/Kg_Spring onion 1µg/Kg_ % compounds % compounds 1µg/Kg_ 1µg/Kg_ European Reference Laboratory - FV 2µg/Kg_ 2µg/Kg_ 5µg/Kg_ 5µg/Kg_ 1µg/Kg_ 1µg/Kg_ TOMATO AUTOMATIC IDENTIFICATION POTATO 1 9 8 7 6 5 4 3 2 1 1 9 8 7 6 5 4 3 2 1 SPRING ONION ORANGE 1 9 8 7 6 5 4 3 2 1 1 9 8 7 6 5 4 3 2 1 % DETECTED % NOT DETECTED % ONE FRAGMENT DETECTED

% of pesticides detected European Reference Laboratory - FV AUTOMATIC IDENTIFICATION VS MANUAL IDENTIFICATION TOMATO ORANGE SPRING ONION POTATO 1 9 8 7 6 5 4 3 2 1 1µg/Kg 2µg/Kg 5µg/Kg 1µg/Kg 1µg/Kg 2µg/Kg 5µg/Kg 1µg/Kg 1µg/Kg 2µg/Kg 5µg/Kg 1µg/Kg 1µg/Kg 2µg/Kg 5µg/Kg 1µg/Kg Automatic identification Manual identification

NOT AUTOMATICALLY IDENTIFIED One ion is automatically detected Cpd Label Name Formula Score Mass Mass (DB) Diff (Tgt, ppm) Diff (Tgt, mda) 16 Cpd 16: Napropamide Napropamide C17 H21 N O2 81,36 271,1563 271,1572-3,6 -,98 C17H21NO2 22,97 271,1572 Napropamide C12H11O 22,97 171,81 Napropamide F1 C7H14NO 22,97 128,175 Napropamide F3 12,2 ppm Napropamide in tomato at 5 µg/kg -81,31 ppm,84 ppm Napropamide (RT 23,4 min) coeluting with this peak

QUANTIFICATION European Reference Laboratory - FV

MATRIX EFFECT European Reference Laboratory - FV

Area Area Area Area European Reference Laboratory - FV GC-QqQ/MS GC-QTOF/MS Chlozolinate Chlozolinate 35 3 25 2 15 1 5 1 2 3 C(µg/Kg) 6 5 4 3 2 1 5 1 15 2 25 C(µg/Kg) 5-1-5-1-25µg/Kg 1-2-5-1-2µg/Kg Endosulphan sulfate Endosulphan sulfate 9 75 6 45 3 15 5 1 15 2 25 3 C(µg/Kg) 6 5 4 3 2 1 5 1 15 2 25 C(µg/Kg)

Area Area Area Area European Reference Laboratory - FV GC-QqQ/MS GC-QTOF/MS Metalaxyl Metalaxyl 6 5 4 3 2 1 1 2 3 C(µg/Kg) 6 5 4 3 2 1 5 1 15 2 25 C(µg/Kg) 5-1-5-1-25µg/Kg 1-2-5-1-2µg/Kg Vinclozolin Vinclozolin 6 5 4 3 2 1 5 1 15 2 25 3 6 5 4 3 2 1 5 1 15 2 25 C(µg/Kg) C(µg/Kg)

Area Area Area Area European Reference Laboratory - FV GC-QqQ/MS GC-QTOF/MS Ametryn Ametryn 15 1 125 1 75 5 25 5 1 15 2 25 8 6 4 2 5 1 15 2 25 C µg/kg) C(µg/Kg) 5-1-5-1-25µg/Kg 1-2-5-1-2µg/Kg Cadusafos Cadusafos 25 125 2 15 1 75 1 5 5 5 1 15 2 25 25 5 1 15 2 25 C (µg/kg) C(µg/Kg)

Area Area Area Area European Reference Laboratory - FV GC-QqQ/MS GC-QTOF/MS Chlorpyrifos-Methyl Chlorpyrifos_Methyl 8 6 4 2 5 1 15 2 25 C(µg/Kg) 12 1 8 6 4 2 5 1 15 2 25 C(µg/Kg) 5-1-5-1-25µg/Kg 1-2-5-1-2µg/Kg Ethofumesate Ethofumesate 2 15 1 5 5 1 15 2 25 C (µg/kg) 12 1 8 6 4 2 5 1 15 2 25 C(µg/Kg)

Area Area Area Area European Reference Laboratory - FV GC-QqQ/MS GC-QTOF/MS Malathion Malathion 25 1 2 8 15 6 1 4 5 5 1 15 2 25 C (µg/kg) 2 5 1 15 2 25 C(µg/Kg) 5-1-5-1-25µg/Kg 1-2-5-1-2µg/Kg 2 15 1 5 Nuarimol 1 2 C (µg/kg) 8 6 4 2 Nuarimol 5 1 15 2 25 C(µg/Kg)

Area Area Area Area European Reference Laboratory - FV GC-QqQ/MS GC-QTOF/MS 6 5 4 3 2 1 Pirimiphos-Methyl 5 1 15 2 25 C (µg/kg) 1 8 6 4 2 Pirimiphos-Methyl 5 1 15 2 25 C(µg/Kg) 5-1-5-1-25µg/Kg 1-2-5-1-2µg/Kg 15 Triadimefon 8 Triadimefon 1 5 5 1 15 2 25 C (µg/kg) 6 4 2 5 1 15 2 25 C(µg/Kg)

Area Area Area Area European Reference Laboratory - FV GC-QqQ/MS GC-QTOF/MS Prothiophos Prothiofos 15 12 9 6 3 5 1 15 2 25 C (µg/kg) 1 8 6 4 2 5 1 15 2 25 C(µg/Kg) 5-1-5-1-25µg/Kg 1-2-5-1-2µg/Kg Terbumeton Terbumeton 3 25 2 15 1 5 5 1 15 2 25 C (µg/kg) 15 1 5 5 1 15 2 25 C(µg/Kg)

RELATIVE ABUNDANCES AT DIFFERENT CONCENTRATIONS 1µg/Kg FLUTOLANIL 2µg/Kg 1µg/Kg 2µg/Kg 5µg/Kg 1µg/Kg Absolute Abundances 173.214 6513.68 137994.63 19672.92 342524.94 145.265 19536.64 42949.29 739.21 149.59 281.664 7659.46 25692.66 39432.82 11548.5 5µg/Kg Relative Abundances to base peak 173.214 145.265 3% 31% 37% 41% 281.664 12% 19% 21% 3% 1µg/Kg

111.9954 139.993 111.9954 1µg/Kg 2µg/Kg 5µg/Kg BOSCALID 1µg/Kg 2µg/Kg 5µg/Kg 1µg/Kg Absolute Abundances 139.993 15757.57 43265.9 1434 2121 342.327 7855.58 8177.92 3216 6335.3 111.9954 11613.48 219.98 52541 12291 Relative Abundances to base peak 139.993 342.327 44% 19% 3% 32% 111.9954 74% 46% 5% 51% 111.9954 1µg/Kg

154.855 1µg/Kg TERBUMETON 169.964 21.1355 154.855 154.855 2µg/Kg 5µg/Kg 1µg/Kg 2µg/Kg 5µg/Kg 1µg/Kg Absolute Abundances 21.1355 7423.91 17674.94 54219.45 92597.9 169.964 8499.35 19325.86 6249.8 91847.98 154.855 5352.16 1216.7 38487.3 6385.87 Relative Abundances to base peak 21.1355 169.964 114% 19% 114% 99% 154.855 72% 58% 71% 69% 169.964 154.855 1µg/Kg

RELATIVE ABUNDANCES IN DIFFERENT MATRICES European Reference Laboratory - FV 152.925 1µg/Kg PROPAZINE 2µg/Kg 5µg/Kg 1µg/Kg 2µg/Kg 5µg/Kg 1µg/Kg Absolute Abundances 214.859 1443.78 29185.33 71157.2 144882.28 172.516 7969.87 24995.55 6173.32 128452.48 152.925 2947.45 7185.62 19546.38 34594.88 Relative Abundances to base peak 214.859 172.516 76% 86% 87% 89% 152.925 28% 25% 27% 24% 1µg/Kg

152.925 1µg/Kg PROPAZINE 2µg/Kg 5µg/Kg 1µg/Kg 2µg/Kg 5µg/Kg 1µg/Kg Absolute Abundances 214.859 2689.61 54278.41 125928.74 213349.14 172.516 27217.56 4579.22 11175.51 26968.92 152.925 7313.1 12299.85 2829.23 54131.23 Relative Abundances to base peak 214.859 172.516 12% 83% 88% 97% 152.925 27% 23% 22% 25% 1µg/Kg

1µg/Kg 19.45 CHLORPYRIFOS METHYL 19.45 19.45 2µg/Kg 5µg/Kg 1µg/Kg 2µg/Kg 5µg/Kg 1µg/Kg Absolute Abundances 285.9261 2466.83 56468.81 142328 2147.22 124.9826 1655.15 2412.67 6614.67 145653.33 19.45 5369.61 16857.85 3913.32 8463.63 Relative Abundances to base peak 285.9261 124.9826 43% 43% 46% 68% 19.45 22% 3% 27% 39% 19.45 1µg/Kg

19.45 124.9826 1µg/Kg CHLORPYRIFOS METHYL 19.45 19.45 2µg/Kg 5µg/Kg 1µg/Kg 2µg/Kg 5µg/Kg 1µg/Kg Absolute Abundances 285.9261 7434.42 22382.88 72778.96 1456.23 124.9826 5132.83 12894.29 2858.71 66677.6 19.45 266.15 693.35 1963.65 44127.69 Relative Abundances to base peak 285.9261 124.9826 69% 58% 39% 47% 19.45 35% 31% 26% 31% 19.45 1µg/Kg

248.28 1µg/Kg TRIFLURALIN 248.28 248.28 2µg/Kg 5µg/Kg 1µg/Kg 2µg/Kg 5µg/Kg 1µg/Kg Absolute Abundances 264.232 1297.47 2915.49 76583.45 134799.16 36.72 789.28 15394.74 46812.6 8595.51 248.28 282.65 421.54 1336 2492.55 Relative Abundances to base peak 264.232 36.72 59% 53% 61% 64% 248.28 23% 15% 17% 18% 248.28 1µg/Kg

186.46 1µg/Kg TRIFLURALIN 186.46 2µg/Kg 5µg/Kg 1µg/Kg 2µg/Kg 5µg/Kg 1µg/Kg Absolute Abundances 264.232 26643.72 4981.53 124512.23 196694.5 36.72 15638.56 28918.62 87336.68 14629.5 186.46 5121.2 8316.86 23355.94 4866.36 Relative Abundances to base peak 264.232 36.72 59% 58% 7% 74% 186.46 19% 17% 19% 24% 1µg/Kg

% Pesticides European Reference Laboratory - FV ACCURACY (%RSD, n =1) 1 9 8 7 6 5 4 % RSD < 5 % RSD > 5 < 1 % RSD >1 3 2 1 TOMATO POTATO ORANGE SPRING ONION

% pesticides European Reference Laboratory - FV 1 9 8 7 LIMITS OF DETECTION Retention time and at least two fragments with error lower than 5 ppm (correct relative abundance) 6 5 4 1 2-5 1 3 2 1 TOMATO POTATO SPRING ONION ORANGE

% compounds European Reference Laboratory - FV LIMITS OF DETECTION Retention time and at least one fragment with error lower than 5 ppm (other fragments with error higher than 5 ppm but with the correct relative abundance) 1 9 8 7 6 5 4 1µg/Kg 2µg/Kg 5µg/Kg 1µg/Kg 3 2 1 Spring onion

REAL SAMPLES Muestra GC-TOF automatico GCQQQMS GC-TOF manual Pepper Boscalid (.25mg/Kg) Boscalid(.23mg/Kg) Iprodione (.2mg/Kg) Iprodione(.27mg/Kg) _1 Pyriproxifen (.54mg/Kg) Pyriproxifen (.62mg/Kg) _2 Bifenthrin 1F(,12mg/Kg) Bifenthrin (.13mg/Kg) Ion 166.99 error higher than1 ppm Melon_1 Bupirimate (.15mg/Kg) Bupirimate(.13mg/Kg) Buprofezin(.49mg/Kg) Buprofezin(.16mg/Kg) _3 Metalaxyl(.6mg/Kg) Metalaxyl(.68mg/Kg) Pirimicarb(.33mg/Kg) Pirimicarb(.24mg/Kg) Bupirimate(.11mg/Kg) Bupirimate(.48mg/Kg) Zucchini Penconazole(.91mg/Kg) Penconazole(.74mg/Kg) Brussels sprouts Boscalid(.21mg/Kg) Boscalid(.12mg/Kg) _4 Bifenthrin (.1mg/Kg) Bifenthrin (.125mg/Kg) Ion 166.99 error higher than1 ppm Bean_1* Iprodione 1F(.83mg/Kg) Iprodione(1.2mg/Kg) Saturation* Azoxystrobin ND Azoxystrobin(.337mg/Kg) Coelution Bean_2 Iprodione(.36mg/Kg) Iprodione(.25mg/Kg) Azoxystrobin ND Azoxystrobin(.11mg/Kg) Coelution Bean_3 Iprodione(.19mg/Kg) Iprodione(.78mg/Kg) Azoxystrobin ND Azoxystrobin(.16mg/Kg) Coelution Melon_2 Bupirimate(.11mg/Kg) Bupirimate(.8mg/Kg) Bean_4* Iprodione ND(1.82mg/Kg) Iprodione(1.7mg/Kg) Saturation* Bean_5* Iprodione ND(1.27mg/Kg) Iprodione(1.7mg/Kg) Saturation* Iprodione(.15mg/Kg) Iprodione(.11mg/Kg) Grape Myclobutanil.41mg/Kg Myclobutanil(.41mg/Kg) *These samples were injecting diluted 1:2 to obtain a correct identification and quantification and the mass spetrum show at least 2 fragments ions.

Real Sample Bean C=,8 ppm IPRODIONE NOT DETECTED AUTOMATICALLY Rt= 27.8974 min Real Sample C=.276ppm ### Formula Retention Time Mass Compound name Description # Formula RT Mass Cpd Comments C13H13Cl2N3O3 27,81 329,334 Iprodione C12H1Cl2N3O3 27,81 314,99 Iprodione F1 C7H3Cl2NO 27,81 186,9592 Iprodione F3 DETECTED AUTOMATICALLY Rt= 27.8974 min

The system has 2 modes of operation : 2GHz Extended Dynamic Range (EDR) mode 4Ghz High Resolution (HR) mode

Benalaxyl 2GHz Fragment Benalaxyl F1 Molecular formula Experimental mass Theorethic al mass Error (ppm) C18 H2 N O 266,1542 266,1539,83 4GHz Fragment Benalaxyl F1 Benalaxyl F2 Benalaxyl F4 Benalaxyl F5 Molecular formula Experimental mass Theorethical mass Error (ppm) C18 H2 N O 266,152 266,1539,83 C13 H16 N O3 234,112 234,1125-2,19 C12 H16 N 26,1174 26,1176 -,81 O2 C11 H14 N O 176,173 176,17 1,67

Benalaxyl 2GHz 4GHz

-34.7 ppm -23,7 ppm Benalaxyl 2GHz 2,1 ppm 5 ppm 1,13ppm ppm 6,4 ppm 4GHz 1,8ppm

% Pesticides European Reference Laboratory - FV 2 ppb in Automatic identification 1 9 8 7 6 5 4 3 2 1 Detected no detected 1 fragment detected 2GHz 4GHz

CONCLUSIONS European Reference Laboratory - FV

15 real samples were processed with the developed method, results were compared with those obtained by GC-QQQ-MS, from a total of 22 positives 14 were correctly identified automatically, and three pesticides were not identified correctly by GCQTOF because the high concentration saturate the detector, five pesticide were not correctly identified because the some of the ions showed errors higher than 1 ppm. This work is a first approach to the accurate mass database for automatic identification and quantification in GC-TOF-MS. It can be considered as a starting point for a large database and for an improved methodology for automatic identification. High Resolution (HR) mode can be considered as a alternative to improve the effectiveness of the automatic identification.