LC-MS/MS multiresidue method used for pesticides analysis in fruits and vegetables 1st International Symposium on Recent Advances in Food Analysis 5-7 November 2003, Prague Didier ORTELLI, Patrick EDDER, Claude CORVI Service de Protection de la Consommation Geneva, Switzerland

Pesticides . ~2000 known pesticides and ~300 widely used . Very different physico-chemical properties . Agricultural practice are different from one country to another . Large variety of commodities to control Service de Protection de la Consommation

Multiresidue method « Simultaneous analysis of a large number of substances in various matrices » . Goals The method must include as many substances as possible and must be accurate, precise, sensitive, fast, widely applicable, selective, easy and robust. Service de Protection de la Consommation

Gas chromatography For many years, gas chromatographic methods have been used for pesticides monitoring GC-ECD, GC-NPD, GC-MS . Limitation to volatile substances . Poorly detectable substances . Poor chromatography (tailing problem etc…) => Complementary analytical method are required for some substances. Service de Protection de la Consommation

Pesticides monitoring Volatile substances: Polar substances: mainly organochlorinated and Various chemical families organophosphous compounds (carbamates, imidazoles, …) GC-ECD, NPD analysis LC-ESI-MS/MS analysis GC-MS confirmation and confirmation Service de Protection de la Consommation

74 Pesticides Acetamiprid Cyproconazole Furathiocarb Oxamyl Tebuconazole Aldicarb Cyprodinil Hexythiazox Oxine Tebufenpyrad Azametiphos Diethofencarb Imazalil Phenthoate Terbufos Azoxystrobin Difenoconazole Imidacloprid Phosalone Thiabendazole Bendiocarb Dimethoate Indoxacarb Pirimicarb Thiacloprid Benfuracarb Dimethomorph Iprovalicarb Prochloraz Thiobencarb Benodanil Dimetilan Isazofos Propamocarb Thiodicarb Benzoximate Dinocap Mepanipyrim Propetamphos Thiofanox Buprofezin Diphenylamine Metalaxyl Propoxur Thiometon Butocarboxym Ethiophencarb Methiocarb Pymetrozine Thiophanate-ethyl Carbaryl Fenazaquin Methomyl Pyridaben Thiophanate-methyl Carbendazim Fenoxycarb Monocrotophos Pyrifenox Tolclophos-methyl Carbofuran Fenpropathrin Myclobutanil Pyrimethanil Triforine Clofentezine Fludioxonil Omethoate Spinosyn A Vamidothion Cymoxanil Fluquinconazole Oxadixyl Spinosyn D Service de Protection de la Consommation

Pesticides H C O H C S 3 N CH O O CH3 O 3 3 OMe N O S O N H C N H C S 3 3 N O N CH N P OMe H C N S CH3 3 H 3 H O CH SCH3 O 3 Thiodicarb Oxamyl Omethoate O H C 3 N O Cl H H N O S Cl N N N Carbaryl Thiabendazole Imazalil N H C 3 N H C 3 OMe O OMe H C OMe H C O O 3 O OMe 3 H CH H 3 O N OMe N N O H H O Cl N O H C O 3 O OCF3 H H H H Diphenylamine Indoxacarb Spinosyn A Service de Protection de la Consommation

Sample preparation Fast Efficient Easy Not selective! The goal of sample preparation is to obtain a sample compatible with the analytical system Service de Protection de la Consommation

Sample preparation . Homogenisation . Weighing 20g . Add 10 ml of water . pH adjustment between 6-7 . Add 40 ml of ethyl acetate . Shake 15 min . Centrifugation . Evaporation of 5 ml of supernatant . Reconstitution in 1 ml of methanol . Filtration on nylon filter 0.45 µm Productivity: ~20 sample / person and day Service de Protection de la Consommation

LC-ESI-MS/MS system Liquid chromatography coupled to a triple quadrupole mass spectrometer QuattromicroTM (Waters-Micromass) equipped with an electrospray source operated in positive ionization mode. Service de Protection de la Consommation

MRM Collision gas Detection Q1 Q2 Q3 Mass filter Collision cell Mass filter Two MS/MS transitions per substances: MH+ as mother ions (if present) and two specific daughter ions Service de Protection de la Consommation

MRM Cyproconazole, 10 µg/ml, 10 µl/min, cone 25 V; Collision 30V Cyproconazole CYPROCONAZOLE 1 (0.505) Scan ES+ 60 3.05e8 100 MH+ 292 % MS scan 292 294 70 85 158 214 56 117 141 236 0 CYPROCONAZOLE3 1 (0.505) Daughters of 292ES+ 70 1.23e7 100 70 OH 125 Cl 125 CH % Daughter scan 3 of 292 125 N N 70 N 139 0 m/z 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 Service de Protection de la Consommation

MS/MS acquisition 25 MS/MS transitions simultaneously Cycle time: <2sec Acquisition is carried out in 11 acquisition groups 1 MS/MS transition by substances Dwell time of 50ms for each MS/MS transitions More compounds could be added to screening Service de Protection de la Consommation

Chromatography Column: Nucleosil 100-5 C18 HD, 70 x 2 mm I.D. Gradient: [A] 0.1% of formic acid in water [B] 0.1% of formic acid in MeOH Flow rate: 300 µl/min Injection: 10 µL %B%B%B Spiked sample at 100 µg/kg 100100100 505050 0000 000000 222222 444444 666666 888888 101010101010 121212121212 141414141414 161616161616 181818181818 202020202020 TimeTimeTimeTimeTime Service de Protection de la Consommation

Validation . Difficult to validate for all combination of substances and matrices. Validation was carried out for: 1. All substances 2. Representative matrices Service de Protection de la Consommation

Method validation 1. Response fonction Linearity verified in the analytical domain 10-1000 µg/kg 2. Detection and quantification limits LOD <10 µg/kg for almost all compounds 3. Extraction recovery Majority of compounds with recovery >80% 4. Accuracy and repeatability 80% 162 13.52 314 > 162 100 100 627728 3.27e6 76507 3.67e5 Area Area 314 -> 162 % % 12.35 2866 -5 -5 131102-02 Sm (Mn, 1×2) MRM of 2 Channels ES+ 131102-01 MRM of 2 Channels ES+ 314 > 120 314 > 120 100 100 3.27e6 3.67e5 13.60 Area 526587 314 -> 120 % % ? 12.35 2910 -5 Time -5 Time 10.00 11.00 12.00 13.00 14.00 15.00 10.00 11.00 12.00 13.00 14.00 15.00 Service de Protection de la Consommation

Non-compliant sample . Confirmatory method is needed to identified without ambiguity the incriminated substance and to quantified without error at the level of interest. External matrix matched calibration for quantification Test non-compliant sample using a second MS/MS transition for the incriminated substance Service de Protection de la Consommation

Pesticides monitoring program Analytical strategy in our laboratory today: ~2000 samples/year Acetonitrile-Hexane extract Ethyl acetate extract GC-ECD, NPD, FPD analysis LC-MS/MS analysis and GC-MS confirmation confirmation Volatile substances: Polar substances: mainly organochlorinated and Various chemical families: organophosphous compounds carbamates, imidazoles, conazoles… Service de Protection de la Consommation

Pesticides monitoring program 1265 fruits samples from the Swiss market Results from january 2002 to september 2003 Nb of Without With residue With residue FRUITS sample residue MRL Pome fruits 246 80 (32%) 164 (67 %) 2 (2 %) Stone fruits 217 84 (39 %) 130 (60 %) 3 (1 %) Berries and small fruits 480 143 (30 %) 292 (61 %) 45 (9 %) Citrus fruits 172 31 (18 %) 119 (69 %) 22 (13 %) Exotic fruits 85 57 (67 %) 25 (29 %) 3 (4 %) Miscellaneous fruits 65 56 (86 %) 9 (14 %) 0 (0 %) 451 739 75 TOTAL 1265 (35.6%) (58.4%) (5.9%) Service de Protection de la Consommation

Pesticides monitoring program 1306 vegetables samples from the Swiss market Results from january 2002 to september 2003 Nb of Without With residue With residue VEGETABLES sample residue MRL Root vegetbales 40 38 (95 %) 2 (5 %) 0 (0 %) Leafy vegetables 445 182 (41 %) 205 (46 %) 58 (13 %) Fruiting vegetables 616 350 (57 %) 245 (40 %) 21 (3 %) Legume vegetables 42 34 (81 %) 8 (19 %) 0 (0 %) Miscellaneous vegetables 163 116 (71 %) 34 (21 %) 13 (8 %) 720 494 92 TOTAL 1306 (55.2%) (37.8%) (7.0%) Service de Protection de la Consommation

Pesticides monitoring program 50% 43.3% 45% 40% Vegetables 35% 30% 25% 19.3% 20% 13.8% 15% 8.9% Majority of samples 10% 5.9% 4.0% 3.0% 1.8% 5% contained more 0% 0 1 2 3 4 5 6 >6 than one residues! 50% Number of residues 45% 40% Fruits 35% 30% 26% 25% 19% 20% 20% 17% 15% 9% Statistical data from 10% 4.3% 2.7% 2.0% 5% january to september 0% 0 1 2 3 4 5 6 >6 2003 Number of residues Service de Protection de la Consommation

Multiresidue sample! Swiss Grape sample MRL . Chlorpyrifos (I) 1.08 mg/kg 0.5 . Folpet (F) 0.78 mg/kg 3 . Fenitrothion (I) 0.65 mg/kg 0.5 . Bromopropylate (A) 0.62 mg/kg 2 . Fludioxonil (F) 0.52 mg/kg 3 . Dicofol (A) 0.32 mg/kg 2 . Cyprodinil (F) 0.30 mg/kg 3 . 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) 0.03 mg/kg 1 Service de Protection de la Consommation

Acknowledgement . Dr Patrick EDDER, Dr Claude CORVI . Maria BLANCO, Corinne BERGER, Eric PITTET . Waters-Micromass Service de protection de la consommation Geneva – Switzerland www.geneve.ch/consommation Service de Protection de la Consommation

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