NUEVA REGLAMENTACION Y GUIAS DE CONTROL DE CALIDAD EUROPEAS

Transcription

1 NUEVA REGLAMENTACION Y GUIAS DE CONTROL DE CALIDAD EUROPEAS Amadeo R. Fernández-Alba EURL-FV European Union Reference Laboratory Pesticide Residues in Fruits and Vegetables

2 World Trade Results Harmonization EU Regulation Food Residue Control New/Upgrade analytical Techniques Social Perception Pesticide Manufacturers

3 1.EU REGULATIONS 2.SOCIAL PERCEPTION 3.PESTICIDE MANUFACTURERS 4.WORLD TRADE 5.RESULTS HARMONIZATION 6. NEW/UPGRADE ANAL. TECH.

4 I. EU PESTICIDE RESIDUE REGULATION Regulation Directive

5 The EU approach and legislation on pesticides residues What is a MRL? Maximum residue level means the upper legal level of a concentration for a pesticide residue in or on food and feed based on good agricultural practice and the lowest consumer exposure necessary to protect vulnerable consumers. What is the main principle in setting a MRL? Maximum residue level should be set at the lowest achievable level consistent with good agricultural practice for each pesticide with a view to protecting vulnerable groups such as children and the unborn.

6 Community Legislation Regulation (EC) No 396/2005 and amendments: Regulation (EC) No 299/2008 of the European Parliament and of the Council of 11 March 2008 amending Regulation (EC) No 396/2005 on maximum residue levels of pesticides in or on food and feed of plant and animal origin, as regards the implementing powers conferred on the Commission Regulation (EC) No 396/2005 of the European Parliament and the Council of 23 February 2005 on maximum residue levels of pesticides in or on food and feed of plant and animal origin and amending Council Directive 91/414/EEC. Available at:

7 Legislation Reg. (EC) No.178/2002 Reg. (EC) No.16/2011 Reg. (EC) No.882/2004 Reg. (EC) No.669/2009 Reg. (EC) No.1213/2008 Reg. (EC) No.901/2009 Reg. (EC) No.915/2010 Harmonised Legislation Comments Food Law, EFSA and Food Safety Official Controls on imports Coordinated Multiannual Community Control Programme, planned within 3 years and including Baby Food (2006/125/EC and 2006/141/EC) and Organic farming samples to be analysed Annex I List of Commodities Reg. (EC) No. 178/2006 Annex II Annexes III A III B Annex IV Existing EU MRLS EU temporary MRLs Pesticides with no MRL (low risk) Reg. (EC) No. 149/2008 Reg. (EC) No. 839/2008 Reg. (EC) No. 822/2009 Reg. (EC) No. 459/2010 Reg. (EC) No. 256/2009 Reg. (EC) No. 1050/2009 Reg. (EC) No. 750/2010 Reg. (EC) No. 765/2010 Reg. (EC) No. 893/2010 Reg. (EC) No. 1097/2009 Reg. (EC) No. 304/2010 Reg. (EC) No. 396/2005 Reg. (EC) No. 299/2008 Annex V Pesticides with default limit other than 0.01mg/Kg Not yet published Annex VI Conversion Factor of MRLs Not yet published Annex VII Fumigants with special derogations Reg. (EC) No. 260/2008 Directive 91/414/EEC Regulation (EC) No. 1107/2009 Regulation (EC) No. 1141/2010 Placing PPP in the market

8 Mechanisms of Control specially through the legislation in place - Inspections: Reg. 882/2004 & Reg. 669/2009 FVO DG SANCO - Pesticide Legislation: Reg. 299/ Coordinated Programs: Reg. 915/ RASFF: Reg. 16/ MRL Database EFSA - Risk Assessment: Reg.178/2002 & Reg.16/ Data Collection of EU Monitoring: Reg. 82/2004

9 The EU approach and legislation on pesticides residue Harmonisation of pesticide MRLs Before Reg. No 396/2005 EU-MRLs National MRLs Default MRLs 250 pesticides 850 pesticides Only in 2 EU Member States 530 pesticides None, except for feed/fish 0,01 mg/kg

10 The EU approach and legislation on pesticides residue Which products are covered by legislation? 315 commodities 10 main groups 190 existing entries from past Directives 125 new entries Mainly minor crops (e.g. cassava, thyme, lupins), but also cocoa, coffee, sugar plants, honey Fish and plants used in animal feed listed, but for the time being exempted from EU-MRL setting

11 The EU approach and legislation on pesticides residue DATABASE on Active substances

12 EU MRLs AZOXYSTROBIN MRL (mg/kg) IPRODIONE MRL (mg/kg) Lettuce 3.0 Lettuce 10.0 Grapes 2.0 Cucumber 2.0 Tomatos 2.0 Apples 10.0 Brocoli 0.05 Oranges 0.02 Spinach 0.05 Asparagus 0.02

13 Tomatoe Pepper Cucumber Abamectine Benomyl Cyromazin Dimetomorf Ethiofencarb Flufenoxuron Maximum Residue Levels (MRLs) authorized in EU (mg/kg) Hexaflumuron Hexitiazox Imidacloprid Lufenuron Methomyl Oxamyl Teflubenzuron Tiabendazole Acrinathrin Bifenthrin Buprofecin Chlorpyriphos-me Endosulfan Malathion Methalaxyl Methamidophos Pyrimiphos-methyl Procymidone

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15 Comparison of the different MRLs in rice established by the Codex Alimentarius, Brazil, US, Taiwan, Korea and the European Union for the most used herbicides in rice crops and the principal pesticides found in rice samples. Pesticide US Codex EU Brazil Korea Taiwan Bentazone Clomazone Deltamethrin Glyphosate

16 Pesticide Annex I 91/414 Registered (General) In Turkey Pesticide Annex I 91/414 Registered (General) In Turkey Pyrithiobac Not listed Yes Oxolinic Acid Not listed Yes Imazapic Not listed Yes Fluthiacet-Methyl Not listed Yes Cyclosulfamuron Not listed Yes Chloroneb Not listed Yes Brodifacoum Not listed Yes Anilofos Not listed Yes Pyrimidifen Not listed Yes Triosulfuron Not listed Yes EPN Not listed Yes Acephate Out Yes Agrobacterium radiobacter Out Yes Alachlor Out Yes Aldicarb Out Yes Amitraz Out Yes Atrazine Out Yes Imazapic Out Yes Azinphos-methyl Out Yes Benfuracarb Out Yes Benomyl Out Yes Beta-cypermethrin Out Yes Brodifacoum Out Yes Bromacil Out Yes Bromopropylate Out Yes Bronopol Out Yes Cadusafos Out Yes Carbofuran Out Yes Carbosulfan Out Yes Chlorfenapyr Out Yes

17 MONITORING

18 Pesticide monitored in Coordinated Plan ,4-D Carbofuran Dimethoate Fluazifop Lufenuron Phosmet Tebufenpyrad 4,4 -Methoxychlor Carbosulfan Dimethoate Fludioxonil Malathion Phoxim Tecnazene Abamectin Chlordane Omethoate Flufenoxuron Maneb group Pyraclostrobin Teflubenzuron Acephate Chlorfenapyr Dimethomorph Fluquinconazole Mepanipyrim Pirimicarb Tefluthrin Acetamiprid Chlorfenvinphos Dinocap Flusilazole Mepiquat Pirimiphos-methyl Tetraconazole Acrinathrin Chlormequat Diphenylamine Flutriafol Metalaxyl Prochloraz Tetradifon Aldicarb Chlorobenzilate Endosulfan Folpet Metconazole Procymidone Thiabendazole Amitraz Chlorothalonil Endrin Formetanate Methamidophos Profenofos Thiamethoxam Amitrole Chlorpropham Epoxiconazole Fosthiazate Methidathion Propamocarb Thiacloprid Azinphos-ethyl Chlorpyriphos Ethephon Glyphosate Methiocarb Propargite Thiophanate-methyl Azinphos-methyl Chlorpyriphos-methyl Ethion Haloxyfop Methomyl Propiconazole Tolcloflos-methyl Azoxystrobin Clofentezin Etofenprox HCB Methoxyfenozide Propyzamide Tolylfluanid 177 compounds governed by Regulation (EC) No 396/2005 amending Directive 91/414/EEC Benfuracarb Clothianidin Ethoprophos Heptachlor Monocrotophos Prothioconazole Triadimefon Bifenthrin Cyfluthrin Fenamiphos Alpha-HCH Myclobutanil Pyrazophos Triadimenol Bitertanol Cypermethrin Fenarimol Beta-HCH Oxadixyl Pyrethrins Triazophos Boscalid Cyproconazole Fenazaquin Gamma-HCH Oxamyl Pyridaben Trichlorfon Bromide ion Cyprodinil Fenbutatin oxide Hexaconazole Oxydemeton-methyl Pyrimethanil Trifloxystrobin Bromopropylate DDT Fenbuconazole Hexythiazox Paclobutrazole Pyriproxyfen Triflumuron Bromuconazole Deltamethrin Fenhexamid Imazalil Parathion Quinoxyfen Trifluralin Bupirimate Diazinon Fenitrothion Imidacloprid Parathion-Methyl Quintozene Triticonazole Buprofezin Dichlofluanid Fenoxycarb Indoxacarb Pencycuron Resmethrin Vinclozolin Cadusafos Dichlorvos Fenpropathrin Iprodione Penconazole Spinosad Zoxamide Camphechlor Dicloran Fenpropimorph Iprovalicarb Pendimethalin Spiroxamine Captan Dicofol Fenthion Kresoxim-methyl Permethrin Taufluvalinate Carbaryl Dieldrin Fenvalerate/Esfenvalerate Lambda-cyhalothrin Phenthoate Tebuconazole Carbendazim Difenoconazole Fipronil Linuron Phosalone Tebufenozide

19 Overall frequency of samples with and without measurable residues in the 2008 EU coordinated program ( MRL: Samples with measurable residues below or at the MRL; >MRL: Samples with residues above the MRL). Total number of samples:

20 Origin of samples (EU: EU, Iceland and Norway; Imported: third countries); surveillance and enforcement samples of fruit, vegetables, cereals, processed commodities and baby food

21 Number of residues found in individual surveillance samples from the national and EU coordinated pesticide monitoring programmes 2008

22 II. Social Perception and Consumer Trends

23 In Anlehnung an Flachowsky 2002 Changes in the Attitude towards Food Is there anything to eat? What is there to eat? How Safe is Food How was the Production C o n s u m e r Hunger Appetite Concern P r o d u c t i o n Increase Agric.production Increase Product Quality Increase Product Safety Protect the Environment Year

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25 Motives for Buying Organic-Food Support organic farmers Friendliness to Animals Safe the planet for future generations Better Taste Prevent Illnesses Reminds me of former times Absence of chemicals and additives From Michelangelo Annastassiades (CRL Stuttgart) Source: Frankfurter Rundschau 07/10/ %

26 III. Plant Protection Industry

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28 Pesticides (1 st generation) Diazinon Chlorothalonil Vinclozolina Pirimifos-metil Chlorpyrifos Procimidona Endosulfan Endosulfan sulfato Cipermetrina

29 Pesticides (new generation) Methiocarb Methamidophos Azoxystrobin Spinosad Chemical structures for some well-known and new pesticides

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31 Number of target pesticides (in Almería area) Pesticides covered in 1998 Acephate Acrinathrin Bromopropylate Chlorothalonil Chlorpyriphos Chlorpyriphos-methyl Chlozolinate Dichlofluanid Dichlorvos Dicofol Dimethoate Endosulfan Fenpropathrin Fenthion Flufenoxuron Imidacloprid Lindane Malathion Mecarbam Methamidophos Methidathion Methiocarb Omethoate Parathion-methyl Procymidone Pyrazophos Pyridaphenthion Pyrimiphos-methyl Tetradifon Trichlorfon Quinalphos Vinclozoline

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33 Decrease of the number of authorized substances Absence sometimes of and effective authorized substances Residue appearance of misussed or not authorized pesticides

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36 IV. Results Harmonization

37 European Community 27 countries with quite different size, population, culture, economy. Harmonization is in progress. Social perception = Reality?

38 Labs within Network (EU: N=264) COUNTRY Labs Population per Lab (mio) COUNTRY Labs Population per Lab (mio) Italy Poland Germany Spain France Greece Burgaria Belgium Hungary Romania UK Portugal Slovenia Denmark (1.1) Czech Rep. Slovakia Netherlands Finland Estonia Austria Ireland Lithuania Sweden Cyprus Latvia Malta Luxemburg Average EU

39 No of EU Samples done by MRM EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV No of EU Samples done by MRM EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV EUPT-FV NRL No NRL per year TOTAL:

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41 OFL OFL OFL NRL OFL OFL OFL NRL OFL OFL NRL OFL OFL OFL OFL NRL OFL CRL OFL OFL NRL OFL NRL OFL EURL OFL OFL OFL OFL OFL NRL CRL OFL OFL OFL NRL OFL OFL OFL NRL OFL OFL NRL OFL OFL NRL OFL NRL OFL OFL OFL NRL OFL CRL Network CRL NRL OFL OFL OFL OFL NRL OFL OFL NRL NRL OFL OFL OFL NRL OFL OFL NRL NRL OFL

42 EURL-Central Homepage:

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44 many laboratories have recently invested in new higher mass accuracy MS systems that allow them to greatly increase the scope of their multiresidue methods Another reason for conducting this screening method proficiency test is to gain information from the laboratories on the type of software that they use. Whether they are using commercial software and databases, or whether they are internally constructed and searched manually. This information will provide an overall view of the purpose of this type of test, and if, in a near future, there is the need to develop new software. The EURL-FV aim is to be able to use mass spectrometry based screening methods routinely, following validation. This is in line with the new Document SANCO/10684/2009 ( Method validation and quality control procedures for pesticide residues analysis in food and feed )

45 General European Commission Proficiency Test Scheme EURL-FV CRL-FV Scientific Group Reports Samples and Protocol INFORMATION EUPT Web Page Data Analysis Lab 1 Lab 2 Lab 3 Lab 4 Lab 5

46 EUPTs performed

47 Photo description of the test sample preparation in different EUPT commodities. A B C D E F G H I J K L

48 4,0 3,0 2,0 1,0 z-scores 5,0 12,753 EUPT z-score Results over the 10 tests 10 EUPTs z-score Results FFP RSD 0,0-1,0 4,0 3,0 2,0 1,0 50% 25% 3% of data 3% of data -2,0-3,0-4,0 65% 86% 0,0 of data of data 25% -1,0 50% -2,0 4% of data -3,0 4% of data -4,0-5, , EUPT1 EUPT2 EUPT3 EUPT4 EUPT5 EUPT6 EUPT7 EUPT8 EUPT9 EUPT10 EUPT1 EUPT2 EUPT3 EUPT4 EUPT5 EUPT6 EUPT7 EUPT8 EUPT9 EUPT10

49 Qn RSD for 10 European Commission Proficiency Tests % FFP - RSD EUPT - 1 EUPT - 2 EUPT - 3 EUPT - 4 EUPT - 5 EUPT - 6 EUPT - 7 EUPT - 8 EUPT - 9 EUPT - 10

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52 V. WORLD TRADE

53 Violative o non Violative? Result = 1,65 0,82 mg/kg (k = 2; 95%) 1,65 mg/kg MRL = 1,0 mg/kg

54 1.6 Valor Sin evaluación de la incertidumbre (solo precisión) Con expresión de la incertidumbre (k = 2; 95%) 1.4 mg kg Lab A Lab B Lab A Lab B Lab A Lab B

55 2. 4 Document N SANCO/2007/3131 Results without Uncertainty Results with Uncertainty (50%) (k = 2; 95%) mg/kg mg kg mg/kg Non violative Violative 1. 0 MRL = 1.00 mg/kg Expanded uncertainty 50%

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57 V. NEW ANALYTICAL/INSTRUMENTAL APPROACHES/CHALLENGES

58 SOLVENT EXTRACTION METHOD LC/GC - MS High Sensitivity (LOQ) Multiclass compounds High identification criteria

59 QuEChERS EtOAc method Weigh 15 g of sample in a 50 ml Falcon tube Weigh 10 g of sample in a 50 ml Falcon tube Add 15 AcN with 1% of acetic acid and 200 µl TPP (25 ppm) Add 10 EtOAc and 200 µl TPP (25 ppm). Shake 3 sec. Add 6 g MgSO4 anhidrous g NaAc 3 H2O and shake vigorously for 4 minutes Add 8 g MgSO4 anhidrous g NaCl and shake vigorously for 4 minutes Centrifugue 5 min r.p.m. Centrifugue 5 min r.p.m. Take a 5 ml aliquot from supernant and add 750 mg MgSO4 anh mg PSA Take the supernant to a vial for analysis Centrifugue 5 min r.p.m. Take the supernant to a vial for analysis

60 20 min ( analysis time) + 10 min (equilibration) = 30 min/analysis Around 50 analysis/day

61 Acefato Metalaxil Metamidofos/ Dimetoato Diclorvos Clorpirifos Metil Vinclozolina Paration Metil Metiocarb Clorpirifos Fention Triadimefon Dietofencarb Procimidona Triadimenol Quinometionato Endosulfan I Hexaconazol Buprofezin Bupirimato Endosulfan II Miclobutanil Benalaxil Endosulfan Sulfato Dicofol / Nuarimol Iprodiona Fenpropatrin Tebufenpirad Tetradifon Fosalone Piridaben Cipermetrinas Deltametrin Permetrin Difenoconazol Etoprofos Pirimifos Metil Penconazol Acrinatrin Fenamifos Bifentrin Ciprodinil Bromopropilato Pirimetanil GC-IT-MS (Internal Ionization) EI/PCI - MS/MS kcounts 600 Spiked pepper extract (0.50 mg/kg) PCI Lindano Propizamida Diazinon Mecarbam Quinalfos Oxadixil Ofurace Triazofos Fenarimol Pirazofos minutos

62 Diclorvos Metamidofos GC-QqQ-MS/MS DICLORVOS METAMIDOFOS m/z m/z 10 5 Ion: 145 Ion: Analyst, (2001) min

63 LC-QqQ-MS Intensity, cps 1,3e5 1,2e5 1,1e5 1,0e5 9,0e4 8,0e4 7,0e4 6,0e4 5,0e4 4,0e4 3,0e4 2,0e4 1,0e4 0,0 2 5,16 4,80 1 4,12 6,12 3 5,78 4 7, , ,11 22, , Dinocap 2 Thiocyclam 3 Thiabendazole, Parathion 4 Methomyl 5 Fenuron 6 Acetamiprid 7 Thiacloprid 8 Imazalil, Aldicarb 9 Monuron 10 Metolcarb 11 Carbofuran 12 Prometryn 13 Diuron, Ethiofencarb 14 Difenoxuron 15 Dimetomorph 16 Methiocarb 17 Terbuthylazine, Spinosyn A 18 Methidation 19 Promecarb 20 Azoxystrobin, Prochloraz, Spinosyn D 21 Diflubenzuron 22 Chlorfenvinphos, Triflumizole 23 Benalaxyl 24 Parathion, Difenoconazole 25 Buprofezin 26 Spiromesifen Time, min 36,51 16,82 29,21 19,59 39,80 9,94 15,83 26,40 14,62 35,25 9,65 25,47 37, ,04

64 CHALLENGES

65 Matrix Effects In Pesticide Residue Analysis: Is The Battle Over?

66 Problems Technical Identification Quantification

67 LC-QqQ-MS/MS Skimer After 20 injections GC-Q-MS Liner after 40 injections

68 XIC of +MRM (297 pairs): 226.2/170.0 amu from Sample 1 (MRMs 100) of Data MRM pesticides_02.wiff (Turbo Spray) Max. 1.1e6 cps. Intensity, cps 1.05e6 1.00e6 9.50e5 9.00e5 8.50e5 8.00e5 7.50e5 7.00e5 6.50e5 6.00e5 5.50e5 5.00e5 4.50e5 4.00e5 3.50e5 3.00e5 2.50e5 2.00e5 1.50e5 1.00e5 5.00e Time, min

69 Number of compounds Results x TIC MRM 50 ppb std chromatogram 0.1%HCOOH % formic acid 100 ppb Distribution of retention times take into account the peak width; HPLC application using 0.1% formic acid as modifier I. II. III. IV. V. Time windows ,1 18,1 20,9 23,4 Start time (min) of 33 time windows Retention time (min) Counts vs. Acquisition Time (min)

70 Results Examples for common transitions: butoxycarboxin aldicarb sulfone fluometuron - diuron x MRM ( > ) 50 ppb std chromatogram 0.1%HCOOH x MRM ( > ) 50 ppb std chromatogram 0.1%HCOOH / /148.0 specific specific Aldicarb sulfone=aldoxycarb x MRM ( > ) 50 ppb std chromatogram0.1%hcooh / x MRM ( > ) 50 ppb std chromatogram 0.1%HCOOH / x MRM ( > ) 50 ppb std chromatogram 0.1%HCOOH specific 223.0/ Counts vs. Acquisition Time (min) x MRM ( > ) 50 ppb std chromatogram0.1%hcooh 223.0/ Counts vs. Acquisition Time (min) both scanned transitions are common scanned unscanned transitions are common

71 Without dilution Tomato Orange Leek Tomato Orange Leek

72 Distribution of mass diferences between ions in database and matrix Da Da Da Da Da Da Da Garlic Coliflower Rice Pepper R=15000=300/0.02 R=7500=300/0.04

73 Leek Dil10 Standard 100ppb Triflumuron? > Ratio 0.6 Triflumuron > Ratio 0.7 Triflumuron? > Triflumuron > 138.9

74 TRIFLUMURON in LC-TOF-FULL SCAN TRIFLUMURON 11,22 359,0405 [M+H] + C15H11ClF3N2O3 TRIFLUMURON Cl37 11,22 361,0375 [M+H+2] + C15H11 37 ClF3N2O3 TRIFLUMURON F1 11,22 156,0211 C7H7ClNO x10 6 +ESI EIC( ) Scan Frag=90.0V EUPT_SC_1PPB_PUERRO.d Smooth 1.6 TRIFLUMURON F1 Cl37 11,22 158,0181 C7H7 37 ClNO EIC 359, Counts vs. Acquisition Time (min) x ESI Scan (9.994 min) Frag=90.0V EUPT_SC_1PPB_PUERRO.d Subtract * * * The fragment and the isotopic profile of the triflumuron doesn t appear Counts vs. Mass-to-Charge (m/z)

75 Problems Quantification

76 Without Clean-up With Clean-up

77 Matrix Matched Calibration Istopically Labeled Internal Standards

78 Practical design of a batch of samples QC PROCEDURES FOR PEST. RES. ANAL. Guidelines for Residues Monitoring Control in the European Union Document 7826/VI/97 ISO 17025

79 STANDARDS A) 100 Standards x 40 /Standard = 4000 B) 200 Standards x 40 /Standard = 8000 C) 600 Standards x 40 /Standard = SOLUTIONS 3-6 months 1-2 YEARS QC Double Check (every day) Stability?

80 How to overcome the problems? I. Easy and very practical solutions II. Specific strategies III. Most powerful MS technologies

81 HIGH SENSITIVE LC-MS

82 Preparation of the samples QuEChERS No dilution Dilution 2x Dilution 5x Dilution 8x Dilution 10x Dilution 15x Dilution 25x Dilution 50x Dilution 100x LC-MS/MS

83 Matrix effect Standard 1 μg/l 3.000/ amu Expected RT: 9.6 ID: Acetamiprid2 from Sample 59 (1 ppb zumo mix naranja dil5x) of QuECh... Max. 4.9e4 cps XIC of +MRM (114 pairs): / amu Expected RT: 9.9 ID: Imazalil2 from Sample 50 (1 ppb zumo mix pera ) of QuEChERS efecto e5 9.5e5 9.0e5 Max. 9.8e5 cps. Matrix Dil.5x Matrix no dilution 8.5e5 XIC of +MRM (114 pairs): / amu Expected RT: 12.1 ID: Carbofuran2 from Sample 8 (1 ppb puerro dil5x) of.wiff (Turbo Spray... Max. 1.8e5 cps. In te n s ity, c p s 8.0e5 7.5e5 7.0e5 6.5e5 6.0e5 5.5e5 5.0e5 4.5e5 x2 1.8e5 1.7e5 1.6e5 1.5e5 1.4e5 1.3e5 1.2e5 x e5 3.5e5 3.0e5 2.5e5 In te n s ity, c p s 1.1e5 1.0e5 9.0e4 8.0e4 x2 2.0e5 7.0e4 1.5e e e Time, min 5.0e4 4.0e4 0.0 Acetamiprid 6.0e e Time, min 2.0e4 1.0e4 0.0 Imazalil Carbofuran Time, min

84 % Signal Supression Matrix effect Acetamiprid 70,0 60,0 50,0 40,0 30,0 20,0 10,0 0,0 0 No Dil Dil2x 2 Dil5x Dil8x 4 Dil10x Dil15x 6 8 Dil25x Dil50x Leek Orange Tomato

85 Matrix effect Acetamiprid Leek 1,40E+07 1,20E+07 1,00E+07 8,00E+06 6,00E+06 4,00E+06 2,00E+06 0,00E Solvent Dil 15x Dil 5x No Dil

86 Leek m/z (Matrix) Thiacloprid (m/z )

87 % Signal Supression Matrix effect Ethiofencarb 90,0 80,0 70,0 60,0 50,0 40,0 30,0 20,0 10,0 0, No No Dil Dil Dil2x Dil2x Dil5x Dil5x Dil8x Dil8x Dil10x Dil10x Dil15x Dil15x Dil25x Dil25x Dil50x Dil50x Dil100x Leek Orange Tomato

88 Matrix effect Ethiofencarb Naranja 8,00E+06 7,00E+06 6,00E+06 5,00E+06 4,00E+06 3,00E+06 2,00E+06 1,00E+06 0,00E Solvent Dil 15x Dil 5x Sin dil

89 Tomato Tomato 17% No matrix effect 0% 35% Improves No matrix before effect or with Dil.15x 6% 0% 26% Improves at before high dilutions or with Dil.15x (50x-100x) 48% Doesn t Improves improve at high with dilutions dilution 68% (50x-100x) Doesn t improve with dilution Orange Orange Leek 17% Leek 0% 35% 4% 13% 7% No matrix effect Improves before or with Dil.15x 76% 48% Improves at high dilutions (50x-100x) Doesn t improve with dilution

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92 FULL SCAN High resolution Mass accuracy

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96 Intensity, cps 4.2e7 3.8e7 3.4e7 3.0e7 2.6e7 2.2e7 1.8e7 1.4e7 1.0e7 6.0e6 2.0e Time, min (a) XIC [ m/z ] (b) XIC [ m/z ] (c) XIC [ m/z ] Azoxystrobin Time, min (a) XIC [ m/z ] Time, min (b) XIC [ m/z ] Time, min (c) XIC [ m/z ] ppm Thiacloprid Time, min Time, min Time, min

97 Sulfadimethoxine OCH 3 OCH 3 N RESOLUTION O S O N H 2 N N OCH 3 H 3 N N OCH 3 Exact Mass: O O H 2 N S C 12 H 15 N 4 O 4 S + Exact Mass: H 2 N Exact Mass: GHz 4 GHz

98

99 DATABASE SEARCH PESTICIDES in solvent (20-500ppb)

100 DATABASE SEARCH PESTICIDES in solvent (20-500ppb)

101 Intensity, cps Intensity, counts 1.9e5 1.7e5 1.5e5 1.3e5 1.1e5 9.0e4 7.0e4 5.0e4 3.0e4 1.0e4 6.8e4 6.4e4 6.0e4 5.6e4 5.2e4 4.8e4 4.4e4 4.0e4 3.6e4 3.2e4 2.8e4 2.4e4 2.0e4 1.6e4 1.2e Time, min ACETAMIPRID C 10 H 11 H 3 C CH 3 N 11 N Cl [M+H] + m/z Chlorine isotopic profile N N Cl N XIC m/z, amu FIGURE 1

102 PROBLEMS sensitivity quantification ident. Param.

103 RETROSPECTIVE ANALYSIS EASY TO IMPLEMENT

104 Additional Advantages

105 Accurate Mass of Diagnostic Frag. Ions Pesticide Family Compound Diagnostic Ion Accurate Mass of Diagnostic Ion Phenylureas Diuron Fenuron Isoproturon Monuron N O Organophosphates Triazines Chlorpyrifos methyl Dimethoate Malathion Deethylatrazine Deethylterbuthylazine Deisopropylatrazine N Cl S P N O O Cl Atrazine Terbuthylazine H 2 N N NH 3 N N N H N NH 3 Fluorobenzylureas Diflubenzuron Lufenuron Flufenoxuron Teflubenzuron Hexaflumuron F O NH Neonicotinoids Acetamiprid Thiacloprid F Cl N Conazole Fungicides Bromuconazole Propiconazole Imazalil Cl Cl

106 Searching for UNKNOWN pesticides in food by LC/TOF-MS (Using the chlorine isotope signature) Intensity, cps 6.0e7 5.8e7 1.6e5 5.6e7 1.5e5 5.4e7 1.5e5 5.2e7 1.4e5 1.4e5 5.0e7 1.3e5 4.8e7 1.3e5 4.6e7 1.2e5 4.4e7 1.2e5 4.2e7 1.1e5 4.0e7 1.1e5 3.8e7 1.0e5 3.6e7 9.5e4 3.4e7 9.0e4 3.2e7 8.5e4 3.0e7 8.0e4 2.8e7 7.5e4 2.6e7 7.0e4 Intensity, counts 2.4e7 6.5e4 N N -CH 3 O Cl Imazalil methyl-derivate Imazalil DBE Cl NON TARGET 2.2e7 6.0e4 2.0e7 5.5e e7 5.0e e7 4.5e e7 4.0e e7 3.5e e e e e e e6 1.5e4 4.0e e e m/z, amu Time [min] CH An impurity from commercial Methyl formulations substituted containing Imazalil imazalil DBE 7.5 Ion Trap MS/MS m/z m/z

107 Applications

108 Applications *Out of Annex I **Pending ***Never notified N.A. Not analyzed Code Matrix Origin Screening by LC-TOF-MS Confirmation by LC-QQQ- MS 6 Pomegrana India Below TOF LOD Acefate (3ppb) te Carbendazim (155 ppb) Carbendazime (315ppb) Difenoconazole (14.7 ppb) Below TOF LOD Below TOF LOD Difenoconazole (41ppb) Methiocarbsulfoxide (4ppb) Omethoate * (1.8ppb) 7 Pear Sudáfrica Below TOF LOD Metoxifenozide (0.2ppb) Thiacloprid (13 ppb) Thiacloprid (52ppb) 8 Pear Chile Iprodione (214 ppb) Iprodione (415ppb) 10 Lime Brasil Imazalil (9 ppb) Imazalil (44ppb) Prochloraz (242 ppb) Prochloraz (490ppb) 11 White Grape Argentina Myclobutanil* (8 ppb) Myclobutanil * (8ppb) 12 Red Plum Sudáfrica Iprodione (81.69 ppb) Iprodione (116ppb) 13 White Plum Sudáfrica FREE OF PESTICIDES FREE OF PESTICIDES 14 Kiwi Italia Fenhexamid (2399 ppb) Fenhexamide (5884 ppb) 15 Bean Marruecos FREE OF PESTICIDES FREE OF PESTICIDES 16 Bean Marruecos FREE OF PESTICIDES FREE OF PESTICIDES 17 Pineapple Costa Rica Triadimefon * ( 54 ppb) Triadimefon * (74ppb) Triadimenol * (120 ppb) Triadimenol * (219ppb) 18 Mango Perú Thiabendazole (188 ppb) Thiabendazole (210ppb)

109 x Cpd 1: 4.096: +ESI ECC Scan Frag=190.0V garlic_2.d INTERFERENCE OF CARBENDAZIM IN GARLIC Cpd1: Cpd 1: Counts vs. Acquisition Time (min) x Cpd 1: 4.096: +ESI MFE Spectrum ( min) Frag=190.0V garlic_2.d (M+H) [M+H] + MASS SPECTRUM OF THE INTERFERENCE ? (2M+H)+ [2M+H] Response vs. Mass-to-Charge (m/ z) x ESI Scan (3.195 min) Frag=190.0V mix160pest_100ppb_29abr.d Subtract MASS SPECTRUM OF CARBENDAZIM [C 10 H 9 O] [M+H] + [C 9 H 10 N 3 O 3 ] Counts vs. Mass-to-Charge (m/ z)

110 7 x ESI EIC( ) Scan Frag=90.0V Sens1mix160pestFS20ppbPeppers_1.d Noise (PeakToPeak) = ; SNR (9.166min) = Bupirimate LC/Q/TOF TOF MODE C 13 H 24 N 4 O 3 S Counts vs. Acquisition Time (min) x10 6 +ESI Scan (9.166 min) Frag=90.0V Sens1mix160pestFS20ppbPeppers_1.d FULL SCAN SPECTRUM * [M+H]+ Calculated Mass: Counts vs. Mass-to-Charge (m/z)

111 5 x ESI EIC Product Ion Frag=90.0V (** -> , , , ) Sens1mix160pestMSMS20ppbPeppers_1.d Smooth Noise (PeakToPeak) = ; SNR (9.176min) = EIC Bupirimate LC/Q/TOF Q/TOF MODE C 13 H 24 N 4 O 3 S x Counts vs. Acquisition Time (min) +ESI Product Ion (9.190 min) Frag=90.0V CID@20.9 ( [z=1] -> **) Sens1mix160pestMSMS20ppbPeppers_1.d MS/MS SPECTRUM (P.I) [M+H]+ Calculated Mass: Counts vs. Mass-to-Charge (m/z)

112

113 SENSITIVITY Target analysis

114 Non target analysis SENSITIVITY + SEARCHING CAP. & SOFTWARE