Recent advances in pesticides residue analyses in food and environmental samples

Recent advances in pesticides residue analyses in food and environmental samples Jahresversammlung 2006 SGLUC Assemblée annuelle 2006 SSCAE 14-15 September 2006 - Solothurn Didier RTELLI Patrick EDDER spco@etat.ge.ch 22 quai Ernest Ansermet, 1211 Genève-4 Laboratory (r)evolution 1892 2006 1906 Chromatography Mikhail Tswett (1872-1919) 1950 1960 1970 1980 1990 2000 2006 GC era MS era GC-MS FID ECD Trap TF PLC era UV DAD Fluo MS/MS ESI APCI 2 1

Challenge for residue laboratory Comprehensive residue analysis ow to analyse more analytes in more sample with better sensitivity in less time? Multiresidue methods C C C Me S C C P S Br Br C C Me C C Et C C C S C C C C S SC C S C C C C C C C Multiresidue C C C methods are the required tools F F 2 C F C C for risk assessment and C C monitoring of a great S C C C C P number Me S C C C SC 2 of various chemical compounds. P Br C C C Me Me 2 I I C C Me Me S C Me C CF C Me C S C P Me 4 2

+ + + + + + + + + + Mass spectrometry Ideally suited for multiresidue analysis Universal and selective Screening and confirmation Compatible with GC and LC Quadrupole + - - + Ion Trap Time of Flight and their combinations QqQ QTrap QToF 5 Quadrupole (single MS) + - - + Mass filter Detection The first MS in common use GC-MS as universal technique since 0 years Excellent quantitative capability Too low sensitivity in full scan mode for residue analysis Selective if using at least three ions Difficult to obtain with LC-ESI-MS 6

Ion Trap Filament Lenses Ion Source Ion Trap + + + + + + + + + + + + + + + Conversion Dynode + + + igh sensitivity in scan mode Sensitivity similar than quadrupole in sim mode Possibility of MS n Less effective than QqQ in MRM mode Smaller linear range than quadrupole 7 Triple Quadrupole (QqQ) Collision gas (Argon) V Mass filter Q1 Collision cell Q2 Mass filter Q Detection Excellent performances for quantitative multiresidue methods Monitoring of known and suspected residue only, unexpected compounds not detected Usually, MRM acquisition in multiple acquisition groups! Retention times moves 8 4

Time f Flight (TF) Full scan acquisition (scan speed ~50-00msec) Compatibility with fast analysis igh resolution MS Accuracy <ppm Resolution ~10'000 FWM Analysis of untargeted compounds Data can be reprocessed at any time ot necessary to set up large nb. of MRM transitions Exact mass measurement give structural informations Good quantitative performance with last generation system 9 Gas chromatography Aldrin Allethrin Azinphos-ethyl Azinphos-methyl Bifenthrin Bromophos ethyl Bromophos methyl Bromopropylate Captafol Captan Chinomethionat Chlorfenvinphos rganochlorines Chlorothalonil Chlorpropham Chlorpyrifos Chlorpyrifos methyl Chlozolinate e.g. Coumaphos Dieldrine, DDT, Cyanophos Lindane Cyfluthrin Cyhalothrin-lambda Cypermethrin rganophosphorous Cyprodinil DDD DDE DDT Deltamethrin Diazinon Dichlofenthion Dichlofluanid Dichlorbenzene-1,4 Dicofol Dieldrine e.g. Diazinon, Dichlorvos, Malathion Diphenylamine Endosulfan-alpha Endosulfan-beta Endosulfan-sulfate Endrine Ethion Pyrethroids Etrimfos Fenhexamide Fenitrothion Formothion C- alpha Fenthion Fludioxonil e.g. Cypermethrin, Fluvalinate Folpet Deltamethrin, FonofosFenvalerate C-beta C-delta Cgamma Lindane Aromatic eptachlor fungicides exachlorbenzene Iprodione Kresoxym methyl Malaoxon Malathion exaconazole Iodofenphos methyl Malaoxon Malathion e.g. Chlorothalonil, Quintozene Mecarbam Methidathion Metolachlor Mevinphos rthophenylphenol Parathion-ethyl Dicarboximide Parathion-methyl fungicides Penconazole Permethrin Phenthoat Phosalone Phosmet e.g. Iprodione, Phosphamidon Procymidone, Pirimiphos Folpet Phosmet ethyl Pirimiphos methyl Procymidone Pyridaben Quinalphos Quinoxyfen Quintozene Soufre Tetrachlorvinphos o low response TetradifonTetramethin for Tolylfluanid Triazamate Trifloxystrobin Vinclozolin carbamates, imidazoles, benzoylureas, sulfonylureas Typical pesticides analysed by GC 10 5

GC-ECD, PD, FPD Using two columns of different polarity DB5 SPB20 «Confirmatory methods for organic residues or contaminants shall provide information on the chemical structure of the analyte. Consequently methods based only on chromatographic analysis without the use of spectrometric detection are not suitable on their own for use as confirmatory methods. owever, if a single technique lacks sufficient specificity, the desired specificity shall be achieved by analytical procedures consisting of suitable combinations of clean-up, chromatographic separation(s) and spectrometric detection.» 2002/657/EC 11 GC-MS Currently most commonly applied technique in pesticide residue analysis SIM Electronic Impact (EI) as universal ionisation technique Single Ion Monitoring (SIM) or Full scan acquisition (SCA) 12 6

GC-MS Full scan (m/z 50-500) acquisition Mass spectra for identification Universal library of spectra Complex chromatograms interpretation 1 Automated Mass Spectra Deconvolution Peak MS Spectra t R m/z Spectra 1 Unknown Spectra 2 Library search 2 Target identified C S P Fenitrothion C C 14 7

Sensitivity in scan mode? Lemon extract Deconvolution results (AMDIS software) 2769 components, 1 target Limetin Extract ion chromatogram Dicofol 0.02 mg/kg Typical sensitivity (ion trap) 100pg Fruits/Vegetables ~0.01 mg/kg C Dicofol Complex matrices (baby food, spices..) ~0.1-1 mg/kg 15 Today pesticides analyses by GC @ SPCo Screening - Identification Acquisition full scan 50-500 m/z on GC-ion trap (polarisq - Thermo) - AMDIS deconvolution and automated library search (library >450 spectra) and - Specific extract ions and quantification on ~40 pesticides Confirmation - Quantification - Acquisition full scan 50-500 m/z or - GC-MS (sim) or GC-MS 2 (ion trap) or - GC-ECD, GC-PD 16 8

ther trends GC-MSMS (QqQ) Acquisition in MRM mode in acquisition group Excellent quantitative performances Targeted analyses only! Retention times moves when cutting the column GC-TF Ideally suited when using fast GC or GCxGC 17 Liquid chromatography Typical pesticides analysed by LC Acephate Acetamiprid Acetochlor Aclonifen Alachlor Aldicarb Aldicarb sulfoxide Aldoxycarb (Aldicarb sulfone) Amidosulfuron Amitrole Anilazine Asulam Atrazine Atrazine-2-hydroxy Atrazine-desethyl Atrazine-desethyl-2-hydroxy Atrazine-desethyl-desisopropyl Carbamates Atrazine-desethyl-desisopropyl-2-hydroxy Atrazine-desisopropyl Atrazine- desisopropyl-2- hydroxy Azaconazole Azamethiphos Aziprotryne Azoxystrobin Benalaxyl Bendiocarb Benfuracarb benodanil Benomyl Benoxacor Bentazon Benthiavalicarb e.g. Carbendazim, isopropyl Diethofencarb, Benzoximate Bifenox Iprovalicarb, Bitertanol Thiophanate Boscalid (icobifen) Bromacil Bromuconazole Bupirimate Buprofezin Butocarboxim Carbaryl Carbendazim Carbofuran Carboxin Chlorbromuron Chlorfenapyr Chlorfluazuron Conazoles Chloridazon Chlorotoluron Chloroxuron Chlorpropham Chlorthal-dimethyl Chlorthiamid ofentezine opyralid Cyanazin Cycloxydim Cymoxanil Cyproconazole Cyprodinil Demeton-S-methyl Diafenthiuron e.g. Imazalil, Fenamidone, Myclobutanil, Tebuconazol Dichlorprop-methyl Dichlorprop-P Diclobutrazol Dicrotophos Diethofencarb Difenoconazol Difenoxuron Diflubenzuron Diflufenican Dimefuron Dimethachlor Dimethenamid Dimethoate Dimethomorph Dimetilan Diniconazole Dinocap Dinoseb Sulfonylureas Dinoterb Dioxacarb Diphenylamine Disulfoton Diuron Dodemorph Epoxiconazole Etaconazole Ethiofencarb Ethoxyquin Ethoxysulfuron Fenamidone e.g. Fenamiphos Amidosulfuron, Fenarimol Foramsulfuron, Fenazaquin Fenbuconazole Triasulfuron Fenhexamide Fenobucarb Fenoxycarb Fenpiclonil Fenpropathrin Fenpropidin Fenpropimorph Fenpyroximat Fenuron Fipronil Fluazifop-butyl Fluazinam Flucycloxuron Fludioxonil Triazines Flufenacet Flufenoxuron Flumetralin Fluquinconazole Fluroxypyr Flurprimidol Flusilazole Flutolanil Flutriafol Foramsulfuron Fuberidazole Furalaxyl Furathiocarb aloxyfop-methyl exaconazole exaflumuron exythiazox Imazalil Imidacloprid e.g. Atrazine, Indoxacarb Simazine, Iodosulfuron-methyl Terbuthylazine Ioxynil Iprovalicarb Isazophos Isoproturon Isoxadifenethyl Lenacil Linuron Lufenuron MCPA MCPB Mecarbam Mecoprop Mepanipyrim Metalaxyl Metamitron Metconazole Methabenzthiazuron Phenylureas Methiocarb Methomyl Methoxyfenozide Metobromuron Metolachlor Metolcarb Metosulam Metoxuron Metribuzin Metsulfuron-methyl e.g. Diuron, Monocrotophos Linuron, Monolinuron Chlorbromuron Monuron Myclobutanil apropamide eburon orflurazon uarimol methoate rbencarb rthosulfamuron ryzalin xadiazon xadixyl xamyl xine-copper Paclobutrazol Pencycuron Pendimethalin Strobilurins Phenmedipham Phenthoat Phosalone Picoxystrobin Pirimicarb Prochloraz Promecarb Prometryn Propachlor Propamocarb Propanil Propaquizafop Propargite Propazine Propetamphos Propham Propiconazole Propoxur Propyzamide Prosulfocarb e.g. Azoxystrobin, Pymetrozine Pyraclostrobin Trifloxystrobin Pyridaben Pyridate Pyrifenox Pyrimethanil Pyriproxyfen Quizalofop-P-Ethyl Simazine Simazine-2-hydroxy Spinosad Spirodiclofen Spiroxamine Tebuconazole Tebufenozide Tebufenpyrad Tebutam icotinoids Teflubenzuron Tepraloxydim Terbacil Terbufos Terbumeton Terbuthylazine Terbuthylazine-2- hydroxy Terbuthylazine-desethyl Terbutryn Tetraconazole Thiabendazole Thiacloprid Thiamethoxam Thifensulfuronmethyl Thiobencarb Thiodicarb e.g. Thiofanox Acetamiprid, Thiometon Imidacloprid, Thiophanate ethyl Thiacloprid, Thiophanate Thiametoxam methyl Tolclofos-methyl Tolylfluanid Triadimefon Triadimenol Triasulfuron Triclopyr Tricyclazole Tridemorph Trifloxystrobin Triflumizole Triflumuron Trifluralin Triforine Trinexapac-ethyl Vamidothion and their metabolites 18 9

LC-MS versus GC-MS GC-MS Limitation to amenable GC compounds 100 000 ESI LC-MS Electrospray (ESI) Chemical ionisation (APCI) o limitation in molecular weight Difficult or impossible to ionize apolar analytes MW 10 000 1 000 APCI GC-MS (EI) Apolar Polarity Polar 19 Analytical approach LC-MS/MS is the method of choice due to its capacity to detect analytes at ng/l range associated with high selectivity Sample preparation Low selectivity : solid phase extraction igh concentration factor LC-MS/MS analysis Efficient chromatography MRM detection Mutiresidue screening: 1 precursor and 1 product ion Confirmation : 1 precursor and at least 2 products ions 20 10

LC-MS/MS Collision gas (Argon) V Mass filter Q1 Collision cell Q2 Mass filter Q Detection LC System : Column : Mobile phase : Ionisation source : MSMS system : Acquisition : LC : Acquity UPLC (Waters) Atlantis dc18 µm 2.1 x 100 mm (Waters) Gradient with water and methanol containing 0.1 % of formic acid Electrospray (positive and negative ionisation modes) QuattroMicro QqQ (Waters) mode MRM 25min, 2 injections per extract (herbicides and fungicides/insecticides) 21 ptimisation of MS/MS conditions Cyproconazole, 10 µg/ml, 10 µl/min, cone 25 V; Collision 0V CYPRCAZLE 1 (0.505) 60 100 % 70 85 158 56 117 141 0 CYPRCAZLE 1 (0.505) 70 100 70 125 125 MS scan 214 26 M+ 292 292 294 Scan ES+ Cyproconazole.05e8 C Daughters of 292ES+ 1.2e7 % 0 19 Daughter scan of 292 60 80 100 120 140 160 180 200 220 240 260 280 00 20 40 60 80 400 m/z 22 11

ptimisation of MS/MS conditions More than 250 compounds have been tested 2 Chromatography Column: Atlantis dc18 µm 2.1 x 100 mm (Waters) Mobile [A] 0.1% of formic acid in water phase [B] 0.1% of formic acid in Me Flow rate: 00 µl/min Injection: 10 µl 100 80 Gradient % B 40 0 0 1 4 15 21 25 Time [min] 24 12

MS/MS Acquisition 4 MS/MS transitions simultaneously Cycle time: ~2 sec Acquisition is carried out in 1 acquisition groups 1 MS/MS transition per substances Dwell time of 40ms for each MS/MS transitions 25 Chromatography Abundance Abundance 88 erbicides Time 0 5 10 15 20 25 min. 146 Fungicides Insecticides Acaricides 26 1

Today pesticides analyses by LC-MSMS @ SPCo Screening Quantification Acquisition in MRM mode with one transition per substances First run (25 min) for 146 fongicides, insecticides and acaricides Second run (25 min) for 88 herbicides 27 Today pesticides analyses by LC-MSMS @ SPCo Confirmation Quantification 2 new extractions are made from the intact foodstuff QC sample prepared with an equivalent blank matrices Acquisition in MRM mode with all transitions (at least two) for the substance(s) to confirm over all time range Use of fresh standard solution 1 st transition 2 nd transition Standards Blank QC Samples 28 14

ther trends LC-TF igh resolution spectra Untargeted analysis Fast analysis (UPLC) LC-QTrap (QqLIT) igher sensitivity of linear ion trap than quadrupole Combining quantitative performance of triple quadrupole with sensitive ion trap scans 29 Applications 0 15

Pesticides residues analyses @ SPCo Food control ~1500 samples/year Fruits and vegetables Cereals Animal foodstuff (meat, fish, honey, ) Processed products (oil, wine, babyfood, ) Water analysis ~100 samples/year Drinking water (tap water, mineral water, ) Geneva lake survey River pollution 1 Generic sample preparation for foodstuffs Adapted for a large number of substances Fast, efficient, easy, robust and not selective omogenisation Weighing Ethyl acetate extraction Purification Matrix solid phase dispersion (MSPD) and/or Gel permeation chromatography (GPC) Evaporation - Concentration Filtration Injections GC-MS and LC-MSMS 2 16

Routine analysis LC-MSMS screening Target compounds 146 Fongicides, Insecticides 88 erbicides GC-MS full scan screening Deconvolution and library search Extract ions on target pesticides Search for >00 pesticides in routine at ppb level in 24hours Pesticides monitoring program 2004 FRUITS Analysés Sans résidus Avec résidus conformes Avec résidus non conformes Fruits à pépins 0 9 (0 %) 20 (67 %) 1 ( %) Fruits à noyau 101 16 (16 %) 82 (81 %) ( %) Baies 281 5 (12 %) 199 (71 %) 47 (17 %) Agrumes 128 16 (1 %) 10 (80 %) 9 (7 %) Fruits exotiques 5 2 (66 %) 11 (1 %) 1 ( %) LEGUMES Analysés Sans résidus Avec résidus conformes Avec résidus non conformes Légumes tubercules 16 12 (75 %) 4 (25 %) --- Légumes à tiges 25 2 (92 %) 2 (8 %) --- Légumes à feuilles 204 66 ( %) 12 (60 %) 15 (7 %) Légumes fruits 65 99 (27 %) 226 (62 %) 40 (11 %) Légumineuses 12 9 (75 %) (25 %) --- Fines herbes 146 5 (6 %) (2 %) 60 (41 %) AUTRES Analysés Sans résidus Avec résidus conformes Avec résidus non conformes Aliments pour bébés 58 48 (8 %) 8 (14 %) 2 ( %) uiles, margarines 4 2 (94 %) 1 ( %) 1 ( %) Vins 265 41 (15 %) 206 (78 %) 18 (7 %) Eaux de boisson 1 1 (42 %) 18 (58 %) --- Autres 104 7 (6 %) 65 (62 %) 2 (2 %) TTAL 185 52 (29,0 %) Service de 1104 Protection (60,1 %) de la Consommation 199 (10,9%) 4 17

Pesticides findings 2005 Fruits Vegetables b of sample 609 581 Excluding organically grown samples Positive samples 84% 60% % of positive samples 65% 60% 55% 50% 45% 40% 5% 0% Introduction of LC-MSMS in the laboratory 1998 1999 2000 2001 2002 Year 200 2004 2005 2006* 5 More than 20 years of pesticides residue analysis @ SPCo 16% 14% Introduction of LC-MSMS in the laboratory % of non compliant samples 12% 10% 8% 6% 4% 2% 0% 1980 1982 1985 1987 1990 1991 1992 199 1994 6 1995 Year 1996 1997 1998 1999 2000 2001 2002 200 2004 2005 2006* 18

Multiresidue samples 40% 5% Data 2005 1668 samples Frequency 0% 25% 20% 15% 10% 5% 0% 0 1 2 4 5 6 7 8 9 10 >10 b. of pesticides residue per sample 7 Multiresidue samples Grape sample Swiss MRL Chlorpyrifos (I) 1.08 mg/kg 0.5 Folpet (F) 0.78 mg/kg Fenitrothion (I) 0.65 mg/kg 0.5 Bromopropylate (A) 0.62 mg/kg 2 Fludioxonil (F) 0.52 mg/kg Dicofol (A) 0.2 mg/kg 2 Cyprodinil (F) 0.0 mg/kg Fenazaquin (A) 0.21 mg/kg 0.20 Fenpropathrin (I) 0.09 mg/kg 0.02 Cymoxanil (F) 0.04 mg/kg 0.05 Dimethomorph (F) 0.04 mg/kg 2 Tebuconazole (F) 0.0 mg/kg 1 8 19

Water analysis 9 Generic sample preparation for water Adapted for a large number of substances Fast, efficient, easy, robust and not selective Concentration factor : 5000x Sample Extraction Elution Evaporation Reconstitution Me Elution ITRGE 40 C 100 µl Me/2 500 ml 40 20

Pesticides residues in drinking waters Sum of pesticides [ng/l] 100 90 80 70 60 50 40 0 20 10 0 Sum of pesticides residues in 26 commercial drinking waters MRL Individual 100 ng/l Sum 500 ng/l A B C D E F G I J K L M P Q R S T U V W X Y Z Samples ~50% of samples contained residues 19 different pesticides were identified 41 River pollution Pollution of the river Allondon occurring after an important rain in July 2005 42 21

Pesticides monitoring in Geneva lake Commission International pour la Protection des Eaux du Léman Basin of Geneva lake ~8'000km 2 ~1'500'000 inhabitants Results Foramsulfuron () 81 ng/l Metalaxyl (F) 52 ng/l Atrazine () 27 ng/l Amidosulfuron () 19 ng/l Atrazine-deséthyl () 21 ng/l Metolachlor () 1 ng/l Atrazine-deisopropyl () 17 ng/l Fenarimol (F) 15 ng/l Terbutylazine () 11 ng/l Simazine () 17 ng/l Propiconazole 11 ng/l Pymetrozine 10 ng/l Iodosulfuron-methyl () 10 ng/l Sampling Depth: 0 m 10 ct. 2005 + 2 other substances < 10 ng/l 4 Pesticides monitoring in Geneva lake 5 ng/l 27 ng/l 19 ng/l 1.6 ng/l 17 ng/l 4 ng/l 9 ng/l 1 ng/l 21 ng/l 6 ng/l 10 ng/l 0.5 ng/l 81 ng/l 1.8 ng/l 2.6 ng/l 52 ng/l 44 22

Water processing 0.5 0.0 0.0 Station Le Prieuré In collaboration with Service Industriels de Genève Sum of pesticides residues Total concentration [µg/l] 0.25 0.20 0.15 0.10 0.05 0.00 Preoxydation Acidification Floculation Sand filtration 0.22 zonation 0.10 Charcoal filtration Tap water 0.002 45 Conclusions A revolution occurred these last 10 years in residue analysis. Mass spectrometry has opened many doors for multiresidue analysis. ew technologies from the single quad to last generation MS are available for a wide range of applications, performances and cost. Comprehensive residue analysis is now in routine 46 2

Acknowledgements Dr Emmanuelle CGARD Maria BLAC Corinne BERGER Gaëlle RAVI Philippe JA Joëlle AÇZ www.geneve.ch/consommation 47 Thank you for your attention! 48 24