ADVANCES IN THE ANALYSIS OF PESTICIDES TYPICALLY ANALYSED BY SINGLE-RESIDUE METHODS

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1 ADVANCES IN THE ANALYSIS OF PESTICIDES TYPICALLY ANALYSED BY SINGLE-RESIDUE METHODS Michelangelo Anastassiades Diana Ströher Kolberg Dorothea Mack Irina Sigalova Daniela Roux

2 SRM-Phobia... SRM-compounds are not very popular...

3 SRM-Phobia... SRMs often require... tedious or unfamiliar sample preparation steps e.g.: ph adjustments Derivatization Cleavage Head-space sampling separation/detection techniques different from those typically used in MRMs and often not available in labs, e.g.: Exotic columns for LC- or GC-separation Exotic mobile phases (LC) Exotic Instrumental-configurations

4 SRM-Phobia... SRMs are thus often Costly and Work-Intensive Effort : Benefit-Ratio is less favourable than that of MRMs As labs are typically overloaded with work..most try to avoid SRMs wherever possible using them only when there no other way out ;-) Covered Pesticides Effort

5 SRM-Challenge... If at all, labs employ SRMs very judiciously targeting only specific pesticide/commodity-combinations Examples: Chlormequat (pears, cereals, mushrooms, carrots) Glyphosate (cereals) Maleic hydrazide (onions, potatoes) This targeted approach Improves the Effort to Benefit Ratio However: Our knowledge about the residue situation of SRM-Pesticides is still very limited Question to be answered: Which SRM-Pesticide/Commodity-Combinations are worth-while monitoring?

6 Let s take a closer look at the SRM pesticides

7 SRM-Pesticides What No makes mattera are the compound the name Divasthey among an SRM-compound? remain the Pesticides? Ugly SAM What is the best name? SRM-Therapy Single Residue Method Single WHY WHY DOES DO EVERYBODY ALL PEOPLE Analyte HATE ME? HATE ME? Method What does Google say? THE WORLD S UGLIEST DOG METHOD

8 What makes a compound an SRM-compound? OBSERVATIONS / REASONS I. Poor Chr/phy and/or Detection II. Degradation - Losses III. Adsorption / Partitioning -Losses IV. Evaporation - Losses - Bad peak-shape - Poor detector response - Degradation (mostly GC) SRM-Therapy - Carbamates, dicofol - Compound Degrades - Hydrolysis - Oxidation - Reactions with matrix - Legislative requirements: V. Residue def. includes bound Residues not in residues or requires release of EURL-FV/SRM the form needed WORKSHOP 2010 common moiety WHY WHY DOES DO EVERYBODY ALL HATE PEOPLE ME? HATE ME? - Comp. interacts w. surfaces - Organotins, Paraquat - Modify solvent (e.g. H + ) - Compound too Polar - Compound Acidic/Basic + unfavourable ph - Compound too Lipophilic + high fat commodity + polar extraction solvent - Compound too Volatile EXAMPLES - Organotins - Abamectin - Captan, Folpet - Ethiofencarb - Chlorothalonil - Quats, Glyphosate - Phenoxy acids - DDT, HCB - Phosphine - Ethylene oxide -CS 2 - Phenoxy-acids - Dithiocarbamates, Amitraz, Prochloraz, Vinclozolin, Diuron POSSIBLE SRM- APPROACHES - Diff. Separation cond. - Diff. Detection cond. - Deriv., APs, Temp. adj. - Control ph, work fast, keep temp. low - One-phase extractions - ph-adjustments - Use non-polar solvents for total fat extraction - Cryo-milling, Special GC-conditions (Headspace sampling ) - Cleavage steps to release bound residues or common moieties

9 Classification SRM or MRM? Classification SRM/MRM varies from lab to lab over the course of time (as technology is progressing) It depends on Sample Preparation Technique Employed: - Trend for methods better covering polar compounds, and being LC/MS amenable (QuEChERS, ChemElut, modif. S19/Luke) Instrumental Analysis Technique Available: - Detection technology Sensitivity/selectivity improves over time, - Chromatographic separation technology LC: UPLC, U-HPLC, new stationary phases e.g. HILIC GC: fast GC, GCxGC Many believe: In future virtually all pesticides will be MRM-amenable ( Dilute & Shoot or Quick & Dirty )?

10 I. Poor Chromatography and/or Detection

11 I. Poor Chromatography and/or Detection Example: Organotins

12 I. Poor Chromatography and/or Detection Example: Organotins QuEChERS-Recoveries % Very poor peak shapes using standard LC-MS/MS MS/MS-conditions Acidic conditions essential to obtain sharp peaks!! H + interrupts unwanted interactions of O-Tins with surfaces Column: Zorbax 3,5 µm; Eclipse XDB-C18; 2,1x 50 mm Mobile phase: A: NH 4 formate (5 mmol) in water +1% formic acid B: NH 4 formate (5 mmol) in methanol +1% formic acid

13 I. Poor Chromatography and/or Detection Example: Organotins ozinn Fenbutatinoxid / T1 (Standard) 519.3/350.9 amu - sample 3 of 103 from sc.wiff ozinn Fenbutatinoxid / T2 (Standard) 517.3/195.0 amu - sample 3 of 103 from sc.wiff Area: 3.075e+003 counts Height: 6.70e+002 cps RT: min Area: 3.599e+003 counts Height: 9.19e+002 cps RT: min Organotins at mg/ml 1% FA in mobile phase ozinn Fenbutatinoxid / (Standard) 519.3/197.0 amu - sample 3 of 103 from sc.wiff Area: 5.177e+003 counts Height: 1.32e+003 cps RT: min Time, min Intensity cps Time, min Intensity cps Time, min Fenbutatinoxid / T1 Fenbutatinoxid / T2 Fenbutatinoxid / ozinn Cyhexatin / T1 (Standard) 369.2/286.9 amu - sample 3 of 103 from sc.wiff Area: 6.717e+003 counts Height: 1.43e+003 cps RT: min ozinn Cyhexatin / T2 (Standard) 367.2/202.9 amu - sample 3 of 103 from sc.wiff Area: 4.534e+003 counts Height: 9.59e+002 cps RT: min ozinn Cyhexatin / (Standard) 365.1/283.1 amu - sample 3 of 103 from sc.wiff Area: 3.128e+003 counts Height: 7.17e+002 cps RT: min Fenbutatin oxide Time, min Intensity cps Time, min Intensity cps Time, min Cyhexatin / T1 Cyhexatin / T2 Cyhexatin / ozinn Fentin / T1 (Standard) 351.0/119.9 amu - sample 3 of 103 from sc.wiff Area: 9.537e+003 counts Height: 1.40e+003 cps RT: min ozinn Fentin / T2 (Standard) 349.0/195.0 amu - sample 3 of 103 from sc.wiff Area: 6.179e+003 counts Height: 9.98e+002 cps RT: min ozinn Fentin / (Standard) 347.0/115.9 amu - sample 3 of 103 from sc.wiff Area: 3.162e+003 counts Height: 4.89e+002 cps RT: min Cyhexatin Time, min 7.29 Intensity cps Time, min Intensity cps Time, min Fentin / T1 Fentin / T2 Fentin / Fentin

14 I. Poor Chromatography and/or Detection and II. Degradation - Losses

15 I./II. Poor Chromatography and Degradation losses Example: Base-Labile Pesticides FOLPET Degradation at High ph High temperatures Phthalimide CAPTAN e.g.: During sample prep. In final extract In GC-inlet Tetrahydrophthalimide Similar behaviour: Dicofol, Captafol, Tolylfluanid, Dichlofluanid, Pyridate

16 Example: Base-Labile Pesticides Reducing losses in extracts Problem: Degradation in extracts of original QuEChERS following PSA-cleanup Rec. % Tolylfluanid stability in extract 7 days 13 days ph after cleanup with PSA ph 4 ph 5 ph 6 ph 7 ph 8 ph 9 MeCN Measured ph in extract Solution: Addition of acid e.g.: Formic acid (5% in ACN) 10 µl per ml extract. Brings ph to ~5 QuEChERS-amenable! OK, but what about instrumental analysis? Tolylfluanid, Dichlofluanid, Pyridate (amenable to LC-MS/MS) Captan, Folpet, Dicofol, Captafol (NOT amenable to LC-MS/MS)

17 I. Poor Chromatography and/or Detection Use of Analyte Protectants to improve GC-Analysis Extract acidification also improves GC-behaviour of Folpet, Captan and Dicofol But better results by addition of ANALYTE PROTECTANTS-MIX to extracts Absolute Error [%] Folpet, Captan work perfectly with APs Dicofol still not fully satisfactory Quantified against solvent-based Calibration Std mix Dichlorvos Dichlobenil Acephate o-phenylphenol Phenmedipham Carbofuran HCH, gamma- Errors due to Matrix Effects with and without Analyte Protectants After ADDING APs to Extract + Cal. Std Chlorothalonil Vinclozolin Parathion-m Carbaryl Methiocarb Diclofluanid Parathion-e Chlozolinate Tolyfluanid Procymidone Captan Folpet Against Solvent w/o APs Against solvent with AP Flubenzimine Ditalimfos Bupirimat Binapacryl Trifloxystrobin Overestimations Tetramethrin1 (no peak in solvent) Dicofol Acrinathrin Bitertanol Iprodione Chinomethionat TPP MEAN ALL

18 Analyte Protectants Mixture HO Ethylglycerol H O O H Sorbitol OH OH OH O OH OH Shikimic acid HO O HO OH OH δ-gluconolactone OH O O HO OH OH

19 Area Dicofol calibration curves with and w/o APs No peak Linear but not good enough 0 0,1 0,2 0,3 0,4 0,5 0,6 Dicofol R 2 = Dicofol-with AP with APs w/o APs µg/ml

20 Use of isotopically labelled ISTDs Better with APs because w/o APs often no peaks 1,30 1,15 1,00 0,85 0,70 0,55 0,40 0,25 0,10 1,05 Ratio Dicofol / DicofolD8 - with APs 0,91 0,93 0,94 0,95-0,05 0,01 0,025 0,05 0,1 0,5 for Pesticide µg/ml Residues

21 Use of isotopically labelled ISTDs Flächenverhältnis 4,000 3,000 2,000 1,000 0,000 y = 8,1676x + 0,8362 R 2 = 0, ,1 0,2 0,3 0,4 Dicofol using IL-ISTD-ratio µg Calculated via Standard addition : mg/kg Fläche 30000, , , , , ,000 0,000 y = 49538x R 2 = 0, ,1 0,2 0,3 0,4 µg Dicofol using Area Calculated via Standard addition : mg/kg

22 I. Poor Chromatography and/or Detection Isotopically Labelled ISTDs Folpet in Papaya (MRL=0.01 mg/kg) Fläche pur Only via Area mg/kg R 2 = y = 3E+06x R 2 = 0, ,005 0,01 0,015 0,02 0,025 0,03 0,035 Unacceptable R 2 Should be at least Captan in Blueberries (MRL=0.01 mg/kg) Fläche pur Only via Area mg/kg R 2 = y = 6E+06x R 2 = 0, ,01 0,02 0,03 0,04 0,05 0,06 0,07 ISTDs added to extract FLV BS 138 via ISTD (PCB 138) 0,01 0,008 0,006 0,004 0, mg/kg R 2 = y = 0,1576x + 0,003 R 2 = 0, ,005 0,01 0,015 0,02 0,025 0,03 0,035 0,1 0,08 0,06 0,04 0,02 0 FLV BS 138 via ISTD (PCB 138) mg/kg R 2 = y = 1,0722x + 0,0252 R 2 = 0, ,01 0,02 0,03 0,04 0,05 0,06 0,07 Violation 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0 FLV Folpe t D4 via ISTD (Folpet D4) mg/kg R 2 = y = 12,78x + 0,228 R 2 = 0, ,005 0,01 0,015 0,02 0,025 0,03 0,035 No violation FLV Captan D6 via ISTD (Captan D6) mg/kg y = 71,164x + 2,312 R 2 = R 2 = 0, ,01 0,02 0,03 0,04 0,05 0,06 0,07

23 Chlormequat z-scores (FFP 25%); (* NRL-SRM) z-scores (FFP 25%); (* NRL-SRM) not Specified unacceptable: 1 labs 3 % questionable: 0 labs 0 % acceptable: 37 labs 97 % Median = mg/kg MRRL = 0.02 mg/kg *eupt 017 eupt 038 eupt 095 *eupt 003 *eupt 060 eupt 055 *eupt 086 *eupt 061 *eupt 084 eupt 054 *eupt 001 eupt 021 eupt 029 eupt 019 eupt 091 eupt 008 eupt 037 eupt 005 *eupt 007 *eupt 030 eupt 014 eupt 022 *eupt 046 eupt 026 eupt 042 *eupt 048 z-score eupt 033 *eupt 040 eupt 018 eupt 044 eupt 053 *eupt 043 eupt 049 eupt 050 *eupt 063 eupt 117 eupt 034 *eupt 009 Impact of isotopically labelled ISTDs EUPT-C3/SRM4 Using isotope labelled ISTD

24 Impact of isotopically labelled ISTDs EUPT-C3/SRM4 Impact of using isotopically labelled ISTD on Qn-RSDs Qn Qn- RSD Median= labs 25,8 All Median= labs 16,4 Using isotop. labelled Chlormequat Median= labs 38,0 Other

25 III. Partitioning Losses

26 Pesticide overview % of Pesticides Less polar More polar 10 5 ~4% 0 < ,000 1,000-10,000 10, ,000 Solubility in Water in mg/l >100, % of Pesticides ~4% More polar 0 <-1-1 to 1 0 to 1 1 to 2 2 to 3 3 to 4 4 to 5 5 to 6 >6 LogKow of Pesticides

27 OUT OF SCOPE Strepto -mycin Amino acids (ph dependent) Sugars Quats Glyphosate ph-dependent -3.2 Fosetyl-Al ph-dependent -2.7 Alkanoic acids (~40) Maleic Hydr. ph-dependent -1.9 ph dependent Basic Pesticides ph dependent QuEChERS Traditional MRMs Flavonoids/Anthocyanes Polarity range covered by trad. Fatty MRMs Acids Ureas (~ 30) OPs (~95 compounds) Monoterpenes Carbamates (~30) Pyrethroids (~45) OCs (~20) Phytosterols LogP = LogKow Carotenoids Chlorophyll 17.2 TGs

28 EURL-PROJECT METHOD FOR THE ANALYSIS OF HIGHLY POLAR PESTICIDES

29 Name Formulas LogKow H 2 O sol. g /L pka ADI/ARfD mg/ kg bw 91/414 Status Fosetyl Al -2,7 (ph4) 111 (ph6) / - IN Ethephon (ph7) 800 (ph4) / 0.03 IN Glyphosate -3.2 (ph5) 900 (ph?) Glufosinate (ph5) 500 (ph7) Maleic Hydrazide / - IN 0.02 / 0.02 IN (ph7) 144 (ph7) / - IN Chlormequat / 0.05 IN Mepiquat / 0.3 IN Paraquat / (OUT) Diquat / 0.01 IN

30 Recoveries with QuEChERS... Name QuEChERS Recoveries from Cuccumber ORGANIC phase WATER phase LogP Maleic Hydrazide 30 % 74 % (ph7) Ethephon 2 % 87 % (ph7) Daminozide 6 % 87 % -1.5 (ph7) Fosetyl Al < 1 % 83 % -2,7 (ph4) Amitrol 5 % 105 % (ph7) Method to be developed should not involve liquid-liquid-partitioning

31 Goals of Project... Develop an Attractive Method that labs can readily implement Select relevant pesticides Keep procedure simple and efficient - Avoid troublesome steps (derivatisation, SPE-enrichm. etc.) -Useisotopically labelled ISTDs! Use instr. available or shortly available to most labs (LC-MS/MS) Where possible, simultaneous sample prep. & chromatogr....limitations! Test real samples to localize relevant Pesticide/Commodity Combinations

32 Overview LC-Conditions / ISTD-availability Name LC-MS/MS ESI (+) LC-MS/MS ESI (-) Isotopically Labelled ISTDs Ethephon - ++ NEW (D4) Glyphosate + ++ Existed Glufosinate NEW (D3) Chlormequat ++ - Existed Daminozide ++ - Existed HEPA - ++ Donation ETU ++ - Existed N-Acetylglufosinate - ++ NEW (D3) Streptomycin ++ - dyhidrostreptomycin Cyromazine ++ - Existed MPPA - ++ NEW (D3) AMPA - ++ Existed Mepiquat ++ - Existed Paraquat ++ - Existed Diquat ++ - Existed Amitrol ++ - ordered Fosetyl Al + ++ NEW (D15) Maleic Hydrazide + ++ NEW (D2)

33 Polar Pesticides - Short Introduction

34 Quats Difenzoquat Amenable to QuEChERS

35 Chlormequat and Mepiquat Uses: - Growth regulators in many crops (cereals, grapes, pears...) -In cereals as stem shorteners / strengtheners -Illegal use of chlormequat in carrots, pears Mode of Action: - Systemic Toxicity to mammals: - Comparably low

36 Paraquat and Diquat Uses: - Pre- and post-emergency herbicides - Used on potatoes, canola for defoliation to facilitate mechanical harvest - Used in banana, coffee, palm, citrus plantations to kill weeds incl. for snake precaution - Pre-season weed control for many cultivations Mode of Action: - Non-Systemic (contact-herbicides) - Non-selective (kill everything) Dissipation: - Quite stable, strongly bind on clay thus low residues expected on crops Toxicity: - Paraquat (high), Diquat (intermediate) - High Acute Toxicity to farmers upon inhalation (irreversible lung damage)

37 Maleic hydrazide

38 Maleic Hydrazide Use: Plant growth regulator, herbicide - Inhibits sprouting (potatoes, onions, carrots) - Induces dormancy in citrus fruits - Used as herbicide in combination with 2,4-D Mode of Action: - Systemic, absorbed by leaves and roots, inhibits cell division Toxicity: - Comparably low toxicity to mammals.

39 Fosetyl Aluminium Phoshorous acid (Phosphonic acid) Fosetyl (Ethylphosphonic acid) H Current Residue Definition: Fosetyl-Al (sum of fosetyl, phosphorous acid and their salts, expressed as fosetyl)

40 Fosetyl-Aluminium Use: - Fungicide - Control of diseases caused by fungi (e.g. Phythophthora) - On a variety of crops (e.g. berries, fruiting vegetables, vine) Note: Fosetyl-Al not the only source of phosphonic acid - Phosphonic acid + salts used as plant strengtheners (incl. organic farming). - Additional source: foliar P fertilizers. Mode of Action: - Systemic, absorbed by leaves and roots, Phosphonic acid degradant activates plant s defence system against fungal infections, e.g. peronospora in vine cultures Toxicity: - Comparably low toxicity to mammals.

41 Amitrol = 3AT

42 Amitrol Use: - Non-Selective Herbicide (unlike all other triazoles Fungicides) - Control annual weeds mostly in non-crop lands - Also in food cultivations e.g. Kiwi (NZ), Soy, Cereals (pre-planting) Mode of Action: - Systemic, absorbed by leaves and roots, Toxicity: - Proven endocrine disruption activity, - Strongly suspected to induce thyroid cancer Included in a biocide ban approved by European Parliament in Jan. 2009

43 Ethephon/HEPA Currently, no legal relevance in food but toxicological concerns HEPA HO

44 Ethephon Use: - Plant growth regulator - promotes pre-harvest ripening in apples, berries, citrus etc. - facilitates harvesting by loosening the fruits - shortens and strengthens the stem in cereals, maize etc. - accelerates post-harvest ripening in mango, pine apples etc. Mode of Action: - Systemic, decomposes to ethylene, affects growth and ripening Toxicity: Comparably low toxicity to mammals.

45 Daminozide

46 Daminozide Use: - Plant growth regulator - Inhibition of internodial elongation. More compact plants. - Formerly used on apples and peanuts - Not used in Europe any more Mode of Action: - Systemic, absorbed by the leaves, translocated throughout plant Toxicity: - Comparably low toxicity to mammals.

47 Glyphosate, Glufosinate Trimethylsulfonium Trimesium No legal relevance in food Counter-ion of Glyphosate with proper MRL 3-MPPA = MPP NAG = N-acetyl-glufosinate NAG is formed in transgenic crops as metabolite, next to check

48 Glyphosate and Glufosinate Use: - Broad spectrum Herbicides Against weeds, grasses, As desiccants to dry off crops before harvest (potato, canola ) Transgenic crops resistant to glyphosate / glufosinate (Corn, Soy, Canola ) Combination with other herbicides (often double resistant GM-crops) Glyphosate makes 60% of worldwide herbicide sales!!! Mode of Action: - Systemic - Inhibit production of amino acids essential for plant growth Toxicity: - Comparably low toxicity to mammals and insects.

49 pka=0.78 O H HOPCH 2 NHCH 2 COOH + OH pka=2.29 pka=5.96 pka=10.98 HOPCH 2 NHCH 2 COOH O - + O H HOPCH 2 NHCH 2 COO - O OPCH 2 NHCH 2 COO - + O - OPCH2 NHCH 2 COO - O EURL-FV/SRM WORKSHOP O O O - H H O HOPCH 2 NHCH 2 COOH OH H Minor tautomer Glyphosate, ph dependency Ref: Chamberlain et al. (1996). Pestic. Sci., 47.

50 Multiresidue Method for SRM Analytes Weigh in 10g of frozen sample (5 g for dry material) Add ISTD-solution Add 10 ml water (for dry material) Add 10ml of Extraction solvent (MeOH w. 1% Formic Acid) Shake vigorously 1 min Centrifuge 5min, 4000RPM Filter Aliquot (through 0,45µm syringe filter) Fill in vial Perform LC-MS/MS analysis

51 Multiresidue Method for Paraquat Diquat Weigh in 10g of frozen sample (5 g for dry material) Add ISTD-solution Add 10 ml water (for dry material) Add 10ml of Extraction solvent MeOH M HCl in water (1:1) Filter Aliquot (through 0,45µm syringe filter) Centrifuge 5min, 4000RPM Fill in vial Perform LC-MS/MS analysis Shake 1 min, heat 15 min at 80 C and re-shake 1 min Variation for Paraquat + Diquat

52 Polar Pesticides Measurement Validation Analysis of real samples

53 Pesticide Name Amitrol & Co. Quats & Co. Methods Glyphosate & Co. Fosetyl /MH Amitrol x Ordered Isotopically labelled ISTDs ETU x NEW D PTU x NEW D Chlormequat x x Existed D Mepiquat x x Existed D Daminozide x x Existed D6 Cyromazine x x NEW D Paraquat x Existed D Diquat x Existed D Glufosinate/MPPA/NAG x 3x NEW D3/D3/D /++/- Glyphosate/AMPA x Existed G: 13 C2 15 N AMPA: 13 C 15 N Glyphosate& Co. COLUMN: AS11 (Dionex) 250x2mm; Eluent: A: H2O; B: 1mM citric acid adj. to ph11 w. DMA (Gradient: 0%B to 50%B) EURL-FV/SRM WORKSHOP COLUMN: 2010ObeliscR (SIELC) 150x2mm 5μ; Fosetyl / MH Almería/Spain, October Eluent: 2010 A: 50 mm NH4formate + 0,1% FA ; B: ACN (Gradient: 3%A using to Single 70%A) Residue Methods ESI neg ESI pos ++ (+) Ethephon/HEPA x 2x NEW D4/D N-Acetylglufosinate x NEW D Fosetyl-Al Screening x NEW D15 (=3x5) ++ (+) Maleic hydrazid x NEW D2 ++ (+) COLUMN: ObeliscR (SIELC) 150x2mm 5μ; Quats & Co. Eluent: A: 20 mmol NH 4 -formate in H2O (ph 3 w. FA); B: ACN (Gradient: 20%A to 80%A) Amitrol & Co. Eluent: A: 50 mm NH 4 formate in H2O; B: ACN (Gradient: 3%A to 70%A)

54 Quats & co. Chlormequat Mepiquat Diquat Paraquat Require stronger extraction conditions than other compounds Generic conditions OK for screening Problems with simultaneous analysis of this pair Daminozide Trimesium cation Amitrol

55 LC-MS/MS Method: Quats & co. Paraquat/Diquat and Amitrol could NOT be included in same LC-method - Paraquat/Diquat require acidic eluent: A: 20 mmol NH 4 formate in water (ph3 with FA) B: ACN Gradient: 20% A to 90% A - Amitrol hates acidity A: 50 mmol NH 4 formate in H2O; B: ACN Gradient: 3% A to 70% A Chlormequat Mepiquat Daminozide Trimesium work well under both conditions

56 LC-Column used... Column used for Quats : ObeliscR by Sielc R stands for RP-Charakter, but we have used it in NP-mode! LiSC (Liquid Separation Cell) Zwitterionic Ligands Small pores act like living cells Pore-openings act as a membrane Different environment inside and outside the cells

57 Kartoffel 1%As mit MeOH mit 0,1 ug/is - Trimethylsulfonium-Kation 77.1 / 62.0 (Unknown) 77.1/62.0 amu - sample 10 of 38 from Area: 8.04e+005 counts Height: 1.01e+005 cps RT: min 9.5e4 9.0e4 8.5e4 8.0e4 7.5e4 7.0e4 6.5e4 6.0e4 5.5e4 5.0e4 4.5e4 4.0e4 3.5e4 3.0e4 2.5e4 2.0e4 1.5e4 1.0e Time, min Kartoffel 1%As mit MeOH mit 0,1 ug/is - Trimethylsulfonium-Kation 77.1 / 62.0 (Unknown) 77.1/62.0 amu - sample 10 of 38 from Area: 8.04e+005 counts Height: 1.01e+005 cps RT: min 9.5e4 9.0e4 8.5e4 8.0e4 7.5e4 7.0e4 6.5e4 6.0e4 5.5e4 5.0e4 4.5e4 4.0e4 3.5e4 3.0e4 2.5e4 2.0e4 1.5e4 1.0e Time, min Mehl 1%As mit MeOH mit 0,1 ug/is - Trimethylsulfonium-Kation 77.1 / 62.0 (Unknown) 77.1/62.0 amu - sample 12 of 38 from Area: 1.06e+006 counts Height: 1.03e+005 cps RT: min 1.00e5 9.50e4 9.00e4 8.50e4 8.00e4 7.50e4 7.00e4 6.50e4 6.00e4 5.50e4 5.00e4 4.50e4 4.00e4 3.50e4 3.00e4 2.50e4 2.00e4 1.50e4 1.00e Time, min Kartoffel 1%As mit MeOH mit 0,1 ug/is - Daminozide 161/143 (Unknown) 161.2/143.1 amu - sample 10 of 38 from _Validierun... Area: 3.96e+005 counts Height: 3.20e+004 cps RT: min 3.2e4 3.0e4 2.8e4 2.6e4 2.4e4 2.2e4 2.0e4 1.8e4 1.6e4 1.4e4 1.2e4 1.0e Time, min Kartoffel 1%As mit MeOH mit 0,1 ug/is - Daminozide 161/143 (Unknown) 161.2/143.1 amu - sample 10 of 38 from _Validierun... Area: 3.96e+005 counts Height: 3.20e+004 cps RT: min 3.2e4 3.0e4 2.8e4 2.6e4 2.4e4 2.2e4 2.0e4 1.8e4 1.6e4 1.4e4 1.2e4 1.0e Time, min Mehl 1%As mit MeOH mit 0,1 ug/is - Daminozide 161/143 (Unknown) 161.2/143.1 amu - sample 12 of 38 from _Validierung Qu... Area: 3.62e+005 counts Height: 2.95e+004 cps RT: min 2.8e4 2.6e4 2.4e4 2.2e4 2.0e4 1.8e4 1.6e4 1.4e4 1.2e4 1.0e Time, min Karotte 1%As mit MeOH mit 0,1 ug/is - Chlormequat / 58.2 (Unknown) 122.1/58.2 amu - sample 9 of 38 from _Validieru... Area: 1.95e+006 counts Height: 1.55e+005 cps RT: min 1.5e5 1.4e5 1.3e5 1.2e5 1.1e5 1.0e5 9.0e4 8.0e4 7.0e4 6.0e4 5.0e4 4.0e4 3.0e4 2.0e4 1.0e Time, min Karotte 1%As mit MeOH mit 0,1 ug/is - Chlormequat / 58.2 (Unknown) 122.1/58.2 amu - sample 9 of 38 from _Validieru... Area: 1.95e+006 counts Height: 1.55e+005 cps RT: min 1.5e5 1.4e5 1.3e5 1.2e5 1.1e5 1.0e5 9.0e4 8.0e4 7.0e4 6.0e4 5.0e4 4.0e4 3.0e4 2.0e4 1.0e Time, min Mehl 1%As mit MeOH mit 0,1 ug/is - Chlormequat / 58.2 (Unknown) 122.1/58.2 amu - sample 12 of 38 from _Validierung... Area: 2.33e+006 counts Height: 2.21e+005 cps RT: min 2.2e5 2.0e5 1.8e5 1.6e5 1.4e5 1.2e5 1.0e5 8.0e4 6.0e4 4.0e4 2.0e Time, min Karotte 1%As mit MeOH mit 0,1 ug/is - Mepiquat / 98.1 (Unknown) 114.2/98.1 amu - sample 9 of 38 from _Validierung... Area: 2.35e+006 counts Height: 1.28e+005 cps RT: min 1.20e5 1.10e5 1.00e5 9.00e4 8.00e4 7.00e4 6.00e4 5.00e4 4.00e4 3.00e4 2.00e4 1.00e Time, min Karotte 1%As mit MeOH mit 0,1 ug/is - Mepiquat / 98.1 (Unknown) 114.2/98.1 amu - sample 9 of 38 from _Validierung... Area: 2.35e+006 counts Height: 1.28e+005 cps RT: min 1.20e5 1.10e5 1.00e5 9.00e4 8.00e4 7.00e4 6.00e4 5.00e4 4.00e4 3.00e4 2.00e4 1.00e Time, min Mehl 1%As mit MeOH mit 0,1 ug/is - Mepiquat / 98.1 (Unknown) 114.2/98.1 amu - sample 12 of 38 from _Validierung Q... Area: 1.73e+006 counts Height: 1.14e+005 cps RT: min 1.10e5 1.00e5 9.00e4 8.00e4 7.00e4 6.00e4 5.00e4 4.00e4 3.00e4 2.00e4 1.00e Time, min Karotte 1%As mit MeOH mit 0,1 ug/is - Diquat / (Unknown) 184.2/156.1 amu - sample 9 of 38 from _Validierung Q... Area: 7.99e+004 counts Height: 5.78e+003 cps RT: min Time, min Karotte 1%As mit MeOH mit 0,1 ug/is - Diquat / (Unknown) 184.2/156.1 amu - sample 9 of 38 from _Validierung Q... Area: 7.99e+004 counts Height: 5.78e+003 cps RT: min Time, min Mehl 1%As mit MeOH mit 0,1 ug/is - Diquat / (Unknown) 184.2/156.1 amu - sample 12 of 38 from _Validierung Qu... Area: 1.18e+005 counts Height: 1.04e+004 cps RT: min 1.00e Time, min 6.70 Kartoffel 1%As mit MeOH mit 0,1 ug/is - Paraquat / (Unknown) 186.2/171.1 amu - sample 10 of 38 from _Validieru... Area: 1.57e+005 counts Height: 1.02e+004 cps RT: min 1.00e Time, min Kartoffel 1%As mit MeOH mit 0,1 ug/is - Paraquat / (Unknown) 186.2/171.1 amu - sample 10 of 38 from _Validieru... Area: 1.57e+005 counts Height: 1.02e+004 cps RT: min 1.00e Time, min Mehl 1%As mit MeOH mit 0,1 ug/is - Paraquat / (Unknown) 186.2/171.1 amu - sample 12 of 38 from _Validierung... Area: 8.48e+005 counts Height: 9.37e+004 cps RT: min 9.0e4 8.5e4 8.0e4 7.5e4 7.0e4 6.5e4 6.0e4 5.5e4 5.0e4 4.5e4 4.0e4 3.5e4 3.0e4 2.5e4 2.0e4 1.5e4 1.0e Time, min LC-MS/MS Method: Quats-Co with ObeliscR Trimesium 77/62 Daminozide 161/143 Chlormequat 122/ ppm on potato Mepiquat 114/98 Diquat 184/156 Paraquat 186/171 In ten sity, cp s In te n sity, cp s Inte nsity, cp s In ten sity, cp s In te n s ity, c p s In ten sity, cp s 0.1 ppm on pineapple In te n s ity, c p s Intensity, cps In te n s ity, c p s Intensity, cps Intensity, cps In te n s ity, c p s 0.2 ppm on wheat flour In te n sity, c p s In te n sity, c p s In te n sity, c p s In te n sity, c p s Intensity, cps Intensity, cps

58 LC-MS/MS of Diquat Importance of Extract Composition Peak most probably from [M-H + ] + formed in non-acidified extracts and eluting as such Mix neutral 0,1 ug/ml - Diquat 92.1 / 92.1 (Unknown) 92.1/92.1 amu - sample 2 of 38 from080529_validierung Quats.wiff Area: 2.22e+005 counts Height: 1.03e+004 cps RT: min Mix neutral 0,1 ug/ml - Diquat / (Unknown) 183.2/157.1 amu - sample 2 of 38 from080529_validierung Quats.wiff Area: 5.85e+006 counts Height: 4.91e+005 cps RT: min Mix neutral 0,1 ug/ml - Diquat184.2 / (Unknown) 184.2/128.0 amu - sample 2 of 38 from080529_validierung Quats.wiff Area: 1.12e+005 counts Height: 5.86e+003 cps RT: min Intensity, cps 1.00e [M] 2+ Intensity, cps 4.5e5 4.0e5 3.5e5 3.0e5 2.5e5 2.0e5 1.5e5 1.0e5 5.0e Most abundant MRM Very small In te n s ity, cp s Std in MeOH Time, min Mix sauer 0,1 ug/ml verd in 1%As mit MeOH - Diquat 92.1 / 92.1 (Unknown) 92.1/92.1 amu - sample 4 of 38 from080529_validierung... Area: 2.28e+005 counts Height: 1.42e+004 cps RT: min Time, min Mix sauer 0,1 ug/ml verd in 1%As mit MeOH - Diquat / (Unknown) 183.2/157.1 amu - sample 4 of 38 from080529_validie... Area: 4.21e+004 counts Height: 3.04e+003 cps RT: min Time, min Mix sauer 0,1 ug/ml verd in 1%As mit MeOH - Diquat184.2 / (Unknown) 184.2/128.0 amu - sample 4 of 38 from _Validier... Area: 2.83e+005 counts Height: 1.76e+004 cps RT: min Intensity, cps 1.4e4 1.3e4 1.2e4 1.1e4 1.0e In te n sity, c ps small Intensity, cps 1.7e4 1.6e4 1.5e4 1.4e4 1.3e4 1.2e4 1.1e4 1.0e Std in MeOH + 1% FA Time, min Time, min Time, min m/z 92/92 m/z 183/157 M 2+ [M-H + ] + Deprotonated D. m/z 184/128 M + Reduced D. (Radical)

59 Recoveries - Quats & Co. -method Matrix: Wheat flour 0.1 mg/kg Name Matrix mached cal. (with ISTD) matrix mached cal.) (w/o ISTD) Matrix Effects cal. in solv.= 100% Rec. RSD Rec. RSD Compound ISTD Chlormequat Mepiquat Diquat Paraquat Daminozide Trimesium no ISTD no ISTD Recoveries using the generic extraction method Column: ObeliscR (SIELC) 150x2mm 5µ; Eluent: A: 20 mmol NH4formate at ph 3.0; B: acetonitrile (gradient: 20% A to 80% A)

60 Recoveries - Amitrol & Co. -method Matrix: Cucumber 0.01 mg/kg Name Matrix-Mached cal. (with ISTD) Solvent-Based Cal. (with ISTD) Matrix Effects cal. in solv.= 100% Amitrol 107 (no ISTD) Rec. RSD Rec. RSD Compound (no ISTD) Mepiquat Chlormequat ETU PTU 99 (no ISTD) (no ISTD) Cyromazine Daminozide

61 Recoveries Matrix: Orange 0.01 mg/kg Name Matrix-Mached cal. (with ISTD) Solvent-Based Cal. (with ISTD) Matrix Effects cal. in solv.= 100% Amitrol 109 (no ISTD) Rec. RSD Rec. RSD Compound (no ISTD) Mepiquat Chlormequat Daminozide Column: ObeliscR (SIELC) 150x2mm 5µ; Eluent: A: 50 mmol NH4formate; B: acetonitrile

62 Recoveries - Amitrol & Co. -method Matrix: Cucumber 0.01 mg/kg Amitrol 85 /43 Mepiquat 114/98 Chlormequat 122/58 Daminozide 161 /143 Cyromazine 167/68 ETU 103/44 PTU 117/100

63 Glyphosate & Co. Glyphosate Glufosinate AMPA 3-MPPA Ethephon HEPA

64 LC-Column used Anion-Exchange Column Active group: e.g. quarternary amine (permanently cationic) -N(CH 3 ) 3 + OH - Column used for Glyphosate & Co.: IonPac AS11 by Dionex Quarternary Ammonium bound on a DVB/Ethyl-DVB co-polymer

65 LC-MS/MS Glyphosate AMPA 168/63 110/63 Glufosinate 180/63 MPPA 150/63 problems with RT-shift / peak shape can occur depending on matrix! (ISTD fortunately matches for this) Ethephon 143/107 Spargel mit 0.05 ug/is bw. ml in MeOH+1%AS - Glyphosat / 62.9 (Unknown) 167.9/62.9 amu - sample 25 of 34 from Validierun... Area: 2.12e+004 counts Height: 3.43e+003 cps RT: min Spargel mit 0.05 ug/is bw. ml in MeOH+1%AS - AMPA / 62.8 (Unknown) 110.0/62.8 amu - sample 25 of 34 from Validierung Et... Area: 3.99e+004 counts Height: 3.78e+003 cps RT: min Spargel mit 0.05 ug/is bw. ml in MeOH+1%AS - Glufosinate / 62.9 (Unknown) 180.0/62.9 amu - sample 25 of 34 from Validieru... Area: 4.42e+004 counts Height: 7.09e+003 cps RT: min Spargel mit 0.05 ug/is bw. ml in MeOH+1%AS - MPPA / 62.8 (Unknown) 150.9/62.8 amu - sample 25 of 34 from Validierung Et... Area: 1.27e+005 counts Height: 2.01e+004 cps RT: min Spargel mit 0.05 ug/is bw. ml in MeOH+1%AS - Ethephon / (Unknown) 142.9/106.9 amu - sample 25 of 35 from Validieru... Area: 4.77e+004 counts Height: 6.63e+003 cps RT: min e4 1.9e4 1.8e4 1.7e4 1.6e4 1.5e4 1.4e4 1.3e ,1 ppm In ten s ity, c ps Inten sity, c ps Inte nsity, cp s Inte nsity, cps 1.2e4 1.1e4 1.0e Intensity, cps asparagus Time, min Time, min Time, min Time, min Time, min Kartoffel mit 0.05 ug/is bw. ml in MeOH+1%AS - Glyphosat / 62.9 (Unknown) 167.9/62.9 amu - sample 27 of 34 from Validieru... Area: 2.06e+004 counts Height: 3.38e+003 cps RT: min Kartoffel mit 0.05 ug/is bw. ml in MeOH+1%AS - AMPA / 62.8 (Unknown) 110.0/62.8 amu - sample 27 of 34 from Validierung E... Area: 4.11e+004 counts Height: 5.63e+003 cps RT: min Kartoffel mit 0.05 ug/isbw. ml in MeOH+1%AS - Glufosinate / 62.9 (Unknown) 180.0/62.9 amu - sample 27 of 34 fromvalidier... Area: 4.74e+004 counts Height: 7.01e+003 cps RT: min Kartoffel mit 0.05 ug/is bw. ml in MeOH+1%AS - MPPA / 62.8 (Unknown) 150.9/62.8 amu - sample 27 of 34 from Validierung E... Area: 2.00e+005 counts Height: 2.62e+004 cps RT: min Kartoffel mit 0.05 ug/is bw. ml in MeOH+1%AS - Ethephon / (Unknown) 142.9/106.9 amu - sample 27 of 35 from Validier... Area: 4.78e+004 counts Height: 7.27e+003 cps RT: min In ten s ity, c p s Time, min Intensity, cps Time, min Inte nsity, cps Time, min Inten sity, cp s 2.6e4 2.4e4 2.2e4 2.0e4 1.8e4 1.6e4 1.4e4 1.2e4 1.0e Time, min Intensity, cps Time, min 0,1 ppm potato Erdbeere mit 0.05 ug/is bw. ml in MeOH+1%AS- Glyphosat / 62.9 (Unknown) 167.9/62.9 amu - sample 28 of 34 fromvalidieru... Area: 2.37e+004 counts Height: 3.90e+003 cps RT: min Erdbeere mit 0.05 ug/is bw. ml in MeOH+1%AS - AMPA / 62.8 (Unknown) 110.0/62.8 amu - sample 28 of 34 from Validierung E... Area: 8.87e+004 counts Height: 9.32e+003 cps RT: min Erdbeere mit 0.05 ug/is bw. ml in MeOH+1%AS - Glufosinate / 62.9 (Unknown) 180.0/62.9 amu - sample 28 of 34 fromvalidier... Area: 8.30e+004 counts Height: 1.25e+004 cps RT: min Erdbeere mit 0.05 ug/is bw. ml in MeOH+1%AS - MPPA / 62.8 (Unknown) 150.9/62.8 amu - sample 28 of 34 fromvalidierung E... Area: 1.25e+005 counts Height: 1.96e+004 cps RT: min Erdbeere mit 0.05 ug/is bw. ml in MeOH+1%AS - Ethephon / (Unknown) 142.9/106.9 amu - sample 28 of 35 from Validier... Area: 5.16e+004 counts Height: 7.93e+003 cps RT: min Intensity, cps Time, min 9.60 Intensity, cps Time, min Intensity, cps 1.20e4 1.10e4 1.00e Time, min 7.03 Intensity, cps 1.9e4 1.8e4 1.7e4 1.6e4 1.5e4 1.4e4 1.3e4 1.2e4 1.1e4 1.0e Time, min 7.39 Intensity, cps Time, min ,1 ppm strawberry

66 HEPA in real samples Note RT shift, but ISTD behaves the same Oats mg/kg Figs 0.12 mg/kg Pineapple mg/kg HEPA D4 (IS) 129/78.8 HEPA 125/94.8 HEPA 125/78.9 T HEPA 125/62.8

67 Chromatographic experiments - Ethephon Ethephon 0,1 ug/ml MeOH Ethephon / (Unknown) 142.9/106.9 amu - sample 69 of 112 from Area: 1.03e+005 counts Height: 1.29e+004 cps RT: min Ethephon ,1 ug/ml in MeOH - Ethephon / (Unknown) 142.9/106.9 amu - sample 4 of 75 from Area: 5.66e+005 counts Height: 1.11e+004 cps RT: min 1.20e4 1.10e4 1.00e Time, min Ethephon 0,1 µg/ml In SOLVENT 1.10e4 1.00e Time, min Kalib Apfel 0,05 ug/ml Ethephon - Ethephon / (Unknown) 142.9/106.9 amu - sample 12 of 93 from Area: 2.71e+004 counts Height: 3.62e+003 cps RT: min nearly no suppression 1.64 Ethephon 0,05 µg/ml in APPLE Kalib Apfel 0,05 ug/ml Ethephon - Ethephon / (Unknown) 142.9/106.9 amu - sample 7 of 75 from Area: 1.16e+004 counts Height: 7.16e+002 cps RT: min extreme suppression Time, min Dionex AS11 250x2mm A: water B: 1 mmol citric acid in water, adjusted to ph 11 with Dimethylamin Time, min Atlantis HILIC 100x2mm A: 100 mmol NH4formate in water B: acetonitrile

68 Recoveries - Glyphosate & Co. - Method Matrix: Wheat flour 0.1 mg/kg Name Matrix mached cal. (with ISTD) Matrix mached cal. (w/o ISTD) Matrix Effects cal. in solv.= 100% Rec. RSD Rec. RSD Compound ISTD Glyphosate Glufosinate no ISTD no ISTD AMPA MPPA no ISTD no ISTD Ethephon

69 HEPA-Recoveries Matrix: Orange 0.02 mg/kg Name Rec. Matrix mached cal. (with ISTD) RSD (n=5) HEPA Isotope-labelled ISTD provided by Bayer CropScience

70 Fosetyl, MH - Method Fosetyl-Aluminium Maleic Hydrazide

71 LC-Column used... Column used for Fosetyl andmh : ObeliscR by Sielc R stands for RP-Charakter, but we have used it in NP-mode! LiSC (Liquid Separation Cell) Zwitterionic Ligands Small pores act like living cells Pore-openings act as a membrane Different environment inside and outside the cells

72 LC-MS/MS Maleic Hydrazide Onion with 0.06 mg/kg maleic hydrazide Kalib 0,03 ug/is auf Zwiebel 1%AS in MeOH - Maleic hydrazid / 81.8 T (Unknown) 111.0/81.8 amu - sample 28 of 38 from Vali... Area: 5.29e+004 counts Height: 5.99e+003 cps RT: min Kalib 0,03 ug/is auf Zwiebel 1%AS in MeOH - Maleic hydrazid / 41.9 (Unknown) 111.0/41.9 amu - sample 28 of 38 from Validie... Area: 9.09e+003 counts Height: 1.32e+003 cps RT: min In te n s ity, cp s In te n s ity, cp s Time, min Time, min Maleic hydrazid 111/82 Maleic hydrazid 111/42 Kalib 0,03 ug/is auf Zwiebel 1%AS in MeOH - Maleic hydrazid D2 113/84(IS) (Unknown) 113.0/83.8 amu - sample 28 of 38 from Vali... Kalib 0,03 ug/is auf Zwiebel 1%AS in MeOH / 41.9 (Unknown) 113.0/41.9 amu - sample 28 of 38 from Validierung_Malein_Fo... Area: 1.83e+006 counts Height: 2.02e+005 cps RT: min Area: 4.36e+005 counts Height: 6.45e+004 cps RT: min 2.2e e5 2.0e5 6.0e4 1.9e5 1.8e5 5.5e4 1.7e5 5.0e4 1.6e5 1.5e5 4.5e4 In te n sity, cp s 1.4e5 1.3e5 1.2e5 1.1e5 1.0e5 9.0e4 8.0e4 In te n s ity, cp s 4.0e4 3.5e4 3.0e4 2.5e e4 6.0e4 2.0e4 5.0e4 1.5e4 4.0e4 3.0e e4 2.0e4 1.0e Time, min Time, min ISTD Maleichydrazid H. D2 113/84 D2 111/84 ISTD Maleichydrazid H. D2 113/42 D2 111/42

73 LC-MS/MS of Fosetyl Grapes 0,5 g/ml with 0,03 mg/kg Atlantis HILIC - late RT Longer RT, less suppression with neutral extracts Dionex-AS11 - early RT Traube 0,5 g/ml - Fosetyl-Al / 81.0 (Unknown) 109.0/81.0 amu - sample 55 of 86 from Mc_Malein... Area: 2.81e+004 counts Height: 5.69e+003 cps RT: min Traube 0,5 g/ml - Fosetyl-Al / 63.0 (Unknown) 109.0/63.0 amu - sample 55 of 86 from Mc_Malein... Area: 8.38e+003 counts Height: 1.60e+003 cps RT: min Traube 0,5 g/ml - Fosetyl-Al / 81.0 (Unknown) 109.0/81.0 amu - sample 37 of 67 from Mc_Eth Area: 2.80e+004 counts Height: 3.30e+003 cps RT: min Traube 0,5 g/ml - Fosetyl-Al / 63.0 (Unknown) 109.0/63.0 amu - sample 37 of 67 from Mc_Ethep Area: 8.95e+003 counts Height: 1.02e+003 cps RT: min Intensity, cps Intensity, cps Time, min Fosetyl-Al / Time, min Fosetyl-Al / 63.0 Moderate matrix effects (20% suppression) Time, min Time, min Fosetyl-Al / 81.0 Fosetyl-Al / 63.0 Strong matrix effects (80% suppression!) HILIC also works well for Fosetyl Glyphosate & Co.-Method not optimal but suitable for Fosetyl SCREENING

74 LC-MS/MS of Phosphorous acid 81/ ppm w. Glyphosate+co method Problem: no isotopically labelled ISTD Phoshorous acid (Phosphonic acid) 81/35 Shifts of H between O and P Exchange of OH in aqueous media

75 Recoveries - Fosetyl & MH - method Fosetyl: Strawberry 0.1 mg/kg Maleic Hydrazide: Onion 0.1 mg/kg Compound Matrix mached cal. (with ISTD) Matrix mached cal. (No ISTD) Matrix Effects cal. in solv.= 100% Rec. RSD Rec. RSD Compound ISTD Fosetyl-Al Maleic Hydrazide

76 LODs Limits of detection in fruits, vegetables and cereals Method Pesticide Cucumber Orange Barley M1 Amitrol M1 ETU M1 PTU M1/M2 Chlormequat 0.005/ / /0.01 M1/M2 Mepiquat 0.005/ / /0.02 M1/M2 Cyromazine 0.01/ / /0.02 M1/M2 Daminozide 0.01/ / /0.04 M1/M2 Trimethylsulfonium-Cation 0.01/ / /0.01 M1/M2 Morpholine n.a. M2 N,N-Dimethylhydrazine M2 Diquat M2 Paraquat M3 Ethephon M3 HEPA M3 Glyphosate M3 AMPA M3 Glufosinate M3 MPPA M3 N-AG M3/M4 Fosetyl 0.005/? 0.005/? 0.005/? M4 Maleic Hydrazide M5 Streptomycin 0.01 n.a. n.a. M5 Kasugamycin 0.01 n.a. n.a.

77 Use of ISTDs Approx. cost of ISTDs Name of ISTD If added to sample (2µg) -cent per portion (exemplary) If added to 1 ml sample extract aliquot (0.1µg) Chlormequat D4 6 c 0.3 c Mepiquat D3 136 c 7 c Diquat D4 8 c 2 c Paraquat D6 146 c 7 c Glyphosate 1,2-13 C2 15 N 660 c 33 c Ethephon D4 13 c 0.6 c Fosetyl-Al D15 8 c 0.4 c Maleic hydrazide D2 12 c 0.6 c Notes: If detections are rather seldom ISTD can be added in a 2 nd analysis in case of positives If RTs are shifting, adding ISTD is highly recommended, at least to the 1 ml aliquot (vial) - RT of ISTD gives additional certainty to identification

78 Residue findings of polar pesticides in real samples

79 Chlormequat and Mepiquat - Residues Data from CVUA Stuttgart Commodities Chlormequat Mepiquat Mushrooms* (champignons, oyster) +++ (also in organic*) Pears ++ (decreasing) Cereals +++ (also in organic**) Fruiting vegetables (tomato, sweet pepper) +++ (also in organic*) ++ (decreasing) +++ (also in organic**) + + * From the cereals used as a substrate for fungi growth ** In organic often due to cross-contamination in mills

80 Glyphosate / Glufosinate - Residues Glyphosate: Analyzed: 261 samples (28 different commodities) from 16 countries Positive: 5 samples (asparagus, shallots, oats, bran) from 3 countries MRL-violations: none Samples analyzed with residues Min (mg/kg) Max (mg/kg) Asparagus ,01 0,04 Onions, shallots 4 1 0,02 Cereals ,52 0,61 Glufosinate: 1x in potatoes AMPA, 3-MPPA: No detections

81 Ethephon - Residues Analyzed: 1162 samples (79 different commodities) from 45 countries Positive: 74 samples (12 commodities) from 18 countries, MRL-violations: 11 Matrix Country of origin No. of samples No. of findings Ethephon mg/kg Organic growth >MRL Pineapple Africa Brazil Costa Rica to 1.0 Ghana Honduras Kamerun to x yes Unknown Panama to 0.4 Pineapple Juice Unknown to x yes Apple Germany to 0.1 Pear Italy Durian Thailand to Figgs Brazil to

82 Ethephon Residues cont. No. >MRL Country of samples No. of Ethephon Organic Matrix of origin findings mg/kg growth Sweet Pepper Spain to 1.2 Kaki Italy Spain to Pomelo China to Cherry Germany Grapes Argentina Italy to 0.78 Namibia Spain South Africa to x yes Tomato Belgium and The Netherlands to 0.68 Total (positiv matrices)

83 Ethephon Residues newer data (1890 samples) Pineapples Grapes Tomatoe s Sweet peppers Figs Apples Pineapple juices Persimmons Pomelos Oranges Cherries Durians Plums Peaches Mangoes Grapefruits Pears with residues residues > MRL ARfD exceedance number of samples 47 6 ARfD exceedances!!

84 Ethephon and HEPA in real samples Sample Ethephon mg/kg HEPA mg/kg Pomelo Figs Grapes Pomelo Figs Figs Figs Figs Figs Orange Pineapple Pineapple 0.65 From straw Oat - used to grow Champignons - on mushrooms 0.17 Champignons Champignons

85 Maleic Hydrazide - Residues Analyzed: 177 samples (15 commodities) from 13 countries Positive: 15 samples (3 commodities garlic, onion and shallots) from 4 countries. MRL-violations: 1 MRL-violation in shallots from France with 17.2 mg/kg. 1 organic onion sample from Italy with 1.7 mg/kg. Commodity Country of Origin No. of findings Minimum Value (mg/kg) Maximum Value (mg/kg) Garlic France 1 14,3 France Remarks Germany Illegal use Onions Italy organic The Netherlands Unknown 1 0,2 Shallots France > MRL

86 Fosetyl Aluminium Residue Findings Analyzed: 1241 samples (85 commodities) from 44 countries Positive: 37 samples (in 9 dif. commodities) from 9 countries MRL-violations: None Matrix Country of origin No. of samples No. of findings Fosetyl mg/kg Organic growth Lettuce, ice Spain to Lettuce, cabbage Germany Lettuce, lollo France Strawberry Germany to Greece Spain to 1.1 Cucumber Germany to 2.6 Italy to x yes Maroc x yes Spain and x yes Litchi Thailand Maracuja Columbia Melon Italy Rucola Germany Italy Tomato Italy Spain The Netherlands Grape EURL-FV/SRM Total WORKSHOP for Pesticide 6 Residues

87 Intermediate Conclusion

88 Ethephon Fosetyl-Al Chlormequat Glyphosate Maleic hydrazide

89 Do SRM! Do SRM! Do SRM! Do SRM! Don t do SRM Don t do SRM Don t do SRM Don t do SRM

90 III. Partitioning Losses (due to unfavorable ph)

91 Nicotine in Mushrooms

92 Nicotine in Mushrooms - Background Information: In 2008 high levels of Nicotine detected in mushrooms at CVUA Sigmaringen CVUA Stuttgart consulted for confirmation (LC-TOF, -MS/MS GC-MS ) Since then many findings by various labs in mushrooms mainly from China Most affected dried Porcini (Boletus edulis) but also Truffles and Chanterelles Porcini are reported to be not cultivable China (Yunnan Region) largest producer (80% of EU-imports from CN) Chinese authorities say : tobacco is also widely cultivated in Yunnan region Nicotine is a naturally occurring alkaloid in tobacco (Nicotiana tabacum) where it occurs at concentrations ranging from 2% to 8% Cross-contamination in drying/packing sites may be an issue Intentional use of nicotine as pesticides is also speculated

93 Nicotine in Mushrooms Risk assessment/management EFSA ARfD : mg/kg body weight; ADI: mg/kg bw per day 99% of dried mushroom samples contained Nicotine (conc. often above 1 ppm) Provisional MRL-Proposal: Dried ceps with a nicotine > 2,3 mg/kg should be withdrawn from the market and safely disposed of. Monitoring program for European wild mushrooms initiated

94 NICOTINE PROPERTIES: (K. Chamberlain et al., Pestic. Sci., 47, 265 (1996) Basic: pka 1 = 3.1; pka 2 = 8.2 (i.e. predominantly protonated at ph<8.2 and double protonated at ph<3.1) Polar: logp = 0.93 (25 C/unionised), the lower the ph the lower the logp Volatile: Pvap = 5.6 Pa (25 C). Evaporation losses reduced at low ph (ionized) N CH 3 N H

95 Nicotine by QuEChERS Optimisation of ph at extraction/partitioning-step Recovery (%) Recovery of Nicotine from fresh mushrooms at different ph (spiked residues) Recovery for Nicotine with QuEChERS - ph-dependant Natural ph LC-QToF, ISTD TPP ph-value before extraction Using Nicotine D3 as ISTD losses were compensated irrespective of ph (Rec %)

96 Extraction of incurred residues from Dried Porcini QuEChERS -impactofph area/area ISTD 2,5 E xtraction rates of N icotine from P orcini using QuE C he R S (incurred residues) 2 1,5 1 0,5 0 natural ph-value

97 Extraction of incurred residues from Dried Porcini QuEChERS ph 10, Variation of Temperature and Time area/area ISTD 2,0 Extraction rates of Nicotine from Porcini using QuEChERS (incurred residues) 1,5 1,0 0,5 0,0 1 min (RT) 10 min (RT) 30 min (RT) 1 min Ultra- Turrax (RT) 20 min (60 C) 20 min (90 C) extraction time

98 QuEChERS modification for Nicotine 10 g Fresh mushrooms or 2 g dried sample + 10 ml water Bring ph to by addition of NaOH 5N Add 4 g MgSO 4 + 1g NaCl Shake 1 min by hand Shake for 30s Centrifugue 5 min. at 3500 r.p.m. Add 150 mg MgSO 4 anh mg PSA per ml extract Centrifugue 5 min. at 3500 r.p.m. Take aliquot and acidify w. 5% Formic Acid to ph~5 Analysis via GC or LC Take alicuot Validation (n=5) using Champignon 10 g Mean Recovery: 106% RSD: 4,1%

99 Ad-hoc PT on Nicotine in dried mushrooms in collaboration with COOP-Switzerland 2 Boletus (CN, BiH) + 1 Mu-Err (CN) w. incurred Nicotine (~1.0/0.7/2.5 mg/kg) 4,0 3,0 2,0 1,0 Preliminary Data Possibly systematically underestimating z-scores 38 Acceptable (= 90%) 2 Questionable (= 5%) 2 Unacceptable (= 5%) z-score 0,0-1,0-2,0-3,0-4,0 = Methanol employed for extraction = Petrol ether employed for extraction ND All other labs variations of QuEChERS z-scores for Boletus mushroom (Bosnia and Herzegovina) z-score for Boletus mushroom (China) z-score for Mu-Err mushroom (China) Lab-Code

100 V. Pesticides requiring inclusion of metabolites or reaction products

101 Acidic Pesticides - Bound Residues

102 Acidic pesticides cleanup issue Example: Extract of red currant Losses of acidic components after cleanup 12 mg/ml 10 8 Citrate-Buffered Sample ph 5.1 ph=7.8 ph=8.3 9 ph Acidic compounds interact with PSA. Thus skip PSA cleanup ,4 ph=3.5 1,0 0, Raw Extract PSA 25 mg/ml PSA 50 mg/ml mg co-extractives/ml Extract ph 0

103 QuEChERS alkaline hydrolysis Example 2,4-D Selective systemic herbicide Control of broad leaved weed Plant growth regulator used to prevent premature fruit drop Formulations include free acid, salts, esters May form conjugates Method designed to convert all possible residues to free acid Acids are often covalently bound to matrix components and thus their concentration underestimated!

104 Acidic pesticides ph-issue: Ionization of pesticides at low or high ph-values Acids: HX H + + X - Bases: B+H + BH + Ionic form prefers to stay in the water phase ph-range of agricultural samples: ~2.5 7

105 Acidic pesticides influence of ph on recovery LC-MS/MS, ESI (-), No PSA Cleanup 120 Recovery % max. ph Ioxynil ph 6 ph 5 ph 4 ph 3 Imazethapyr Imazaquin Naphthylacetic acid Fluoxypyr Mecoprop Triclopyr 2,4,5-TP Propyzamid Bentazon Bromoxynil Fluazifop Bromacil 2,4-DB MCPB Picloram Clopyralid Imazapyr Benazolin Dicamba MCPA 2,4-D 2,4,5-T 2,4-DP 4-CPA lower pka general trend higher pka 0

106 QuEChERS schematic description with AH Citrate-Buffered QuEChERS Weigh 10 g of Frozen Sample Shake Add 10 ml Acetonitrile Add ISTD-Solution Shake Add 4 g MgSO 4 / 1 g NaCl / Citrate Buffer (ph 5-5.5) Shake and Centrifuge Analysis of acidic pesticides by LC-MS/MS alkaline hydrolysis: Add NaOH and store for 30 min at RT, then neutralize w. H 2 SO 4 optionally: Freeze-out of extracted fat over night

107 Alkaline cleavage or the release of phenoxy-acids Alkaline Hydrolysis for the release of phenoxy-acid pesticides wheat sample Relativ concentration At 70 C ph min 715 At Room Temperature ph min 336 ph min 275 ph 9 30 min Wheat flour with incurred residues ph 12/30 min ph 10/30 min ph 12/30min ph 10/60 min ph 10/30 min ph 9/30 min QuEChERS w/o hydrol.

108 Alkaline Hydrolysis Level of MCPA in berries Goosebeery Strawberry Relative Response of MCPA [%] no alkaline hydrolysis with alkaline hydrolysis

109 Alkaline Hydrolysis Level of 2,4-D in different citrus samples 0,7 no alkaline hydrolysis with alkaline hydrolysis 0,6 x 2.5 0,5 x 2.2 mg/kg 0,4 0,3 x 1.8 x 3.4 factor: x 3.3 0,2 x 2.6 x 1.8 x 5.0 x 3.0 0,1 x sample 01 sample 02 sample 03 sample 04 sample 05 sample 06 sample 07 sample 08 sample 09 sample 10

110 Matrix: Method: Cucumber QuEChERS Acid Ester Acid Esters Conjug. Residue Definition 2,4,5-T 2,4-D 2-butyl butoxyethyl ethylhexyl isooctyl isopropyl methyl octyl methyl butyl ethyl isobutyl isooctyl isopropyl 2,4-DP methyl x Dichlorprop, incl. Dichlorprop-p Carfentrazone ethyl x x Chlorthal dimethyl x Chlorthal-dimethyl Cinidon ethyl x x x Redidue Definition 2,4,5-T (F) Carfentrazone-ethyl (determined as carfentrazone and expr. as carfentrazoneethyl) Cinidon-ethyl (sum of cinidon ethyl and its E-isomer) Clodinafop propargyl x Clodinafop and its S-isomers, expr. as clodinafop (F) Cyhalofop butyl x x Cyhalofop-butyl (sum of cyhalofop butyl and its free acids) Dicamba methyl x Dicamba Dichlorprop 2-ethylhexyl x Dichlorprop, incl. Dichlorprop-p Diclofop methyl x x Diclofop (sum diclofop-methyl and diclofop acid expr. as diclofop-methyl) Diethatyl ethyl Dinoseb acetate x Dinoseb isooctyl Fenoprop methyl Fenoxaprop P-ethyl Flamprop isopropyl butyl Fluazifop methyl Flumiclorac pentyl x 2,4-D (sum of 2,4-D and its esters exp. as 2,4-D) Experiments performed in advance to QuEChERS I II III IV V VI VII VIII AH AH AH EH (1 mg) EH (2 mg) EH (5 mg) EH (2 mg) VII + II RT 30 min 80 C 30 min RT 16 h RT 3 h RT 3 h RT 3 h RT 16 h remaining esters 5% AH = alkaline hydrolysis, ph 12, re-neutralized with H 2 SO 4 remaining esters 5-20% EH = incubation of buffered samples with esterase from porcine liver (buffering at ph 6-9 with EURL-FV/SRM remaining esters WORKSHOP > 20% 2010 for phosphate Pesticide buffer Residues

111 Matrix: Method: Cucumber QuEChERS Acid Ester Acid Esters Conjug. Residue Definition Haloxyfop ethoxyethyl methyl Ioxynil octanoat (x) x Ioxynil, incl. its esters expr. as ioxynil (F) MCPA 1-butyl butoxyethyl ethyl ethylhexyl thioethyl MCPB ethyl x x x Mecoprop methyl 1-octylester 2,4,4-trimethylpentyl diethyl di-isopropyl Redidue Definition Mefenpyr Nitrothal Picloram isooctyl x Picloram Triclopyr 2-butoxyethyl x Triclopyr Trinexapac ethyl x Trinexapac x x x x x x x Haloxyfop incl. haloxyfop-r (Haloxyfop-R methyl ester, haloxyfop-r and conjugates of haloxyfop-r expr. as haloxyfop-r) (F) (R) MCPA and MCPB (MCPA, MCPB incl. their salts, esters and conjugates expr. as MCPA) (F) (R) MCPA and MCPB (MCPA, MCPB incl. their salts, esters and conjugates expr. as MCPA) (F) (R) Mecoprop (sum of mecoprop-p and mecoprop expressed as mecoprop) Experiments performed in advance to QuEChERS I II III IV V VI VII VIII AH AH AH EH (1 mg) EH (2 mg) EH (5 mg) EH (2 mg) VII + II RT 30 min 80 C 30 min RT 16 h RT 3 h RT 3 h RT 3 h RT 16 h remaining esters 5% remaining esters 5-20% remaining esters > 20% AH = alkaline hydrolysis, ph 12, re-neutralized with H 2 SO 4 EH = incubation of buffered samples with esterase from porcine liver (buffering at ph 6-9 with phosphate buffer

112 Amitraz

113 Amitraz Use: - Acaricide and Insecticide mite control in many different crops pome fruit, citrus, stone fruit, strawberries, cucurbits, solanaceous etc. To control varroa mite in Honey-production Mode of Action: - Non-systemic - Interacts with the nervous system of mites and ticks Toxicity: Relatively high toxicity to mammals

114 Amitraz Recoveries with QuEChERS quite good Better stability of Amitraz in extracts at high ph PSA cleanup (w/o acidification) increases ph and thus stability Amitraz 100,0 90,0 80,0 70,0 60,0 50,0 40,0 30,0 20,0 10,0 0,0 ph3 ph4 ph5 ph6 ph7 ph8 ph9 ph10 0 Tage 1 Tag 2 Tage 4 Tage 9 Tage Measurement of acidified extract should be performed within one day. Better don t acidify QuEChERS extract!

115 Amitraz Degradation of amitraz and formation of DMPF (amidine( amidine) during storage of QuEChERS extracts at diff. ph Residual Amitraz in % 120 DMPF formation expressed in amitraz equivalents d 1 d 2 d 3 d Extract Storage time 4 d 9 d 17d ph d 1 d 2 d 3 d Extract Storage time 4 d 9 d 17d ph

116 Amitraz Residue Definition: Amitraz including all metabolites containing 2,4-dimethylaniline expressed as Amitraz Traditional methods involve: Conversion of Amitraz and degrad.. products to 2,4-dimethylanilin Different approaches: - Alkaline hydrolysis, - Acidic hydrolysis, - Combination of both

117 Degradation paths of Amitraz CH 3 CH 3 CH 3 N N N 1:1 H 3 C N N CH 3 H Amitraz (minor residue) H 3 C CH 3 H + 1:2 In presence of matrix nonquantitative 1:1 1:2 OH - H 3 C Amidine (main residue) OH - 1:1 In presence of matrix nonquantitative H 3 C CH 3 NH 2 Aniline or DMA (very minor/ n.d.) H + 1:1 H 3 C CH 3 O NH H Amid (minor residue)

118 Amitraz Acidic hydrolyses were quantitative: Amitraz Aniline Amid Aniline Amidin remains stable Aniline remains stable Alkaline hydrolyses were not quantitative (especially in presence of matrix) Amitraz Amid Amid Aniline Amidin Aniline Aniline remains stable (closed vessel to avoid losses!)

119 Amitraz Amitraz and its 3 main metabolites well detectable by LC-MS/MS So, total DMA can be calculated (virtual( hydrolysis) Virtual vs. chemical Hydrolysis: Virtual hydr.: only covers the detected compounds. Chemical hydr.: covers also other DMA-containing metab./conj. Question: Are there any significant levels of other metabolites/conjugates releasing DMA upon hydrolysis? Observation: Alkaline hydrolysis did not increase determined residue level in real samples In any case: Acidic hydrolysis does not transform amidine to aniline Risk of losses alkaline hydrolysis is very high.

120 Amitraz - Calculating the Total Residue Important: Mind stechiometries of degradation pathways - Amidin - Amid - DMA Amitraz 1:1 Amitraz 2:1 or 1:1 (depending of pathway) Amitraz 2:1 CASE 1: 1 Amidin is the only metabolite detected (as in pears) 1:1 stechiometry (conversion factor based on MW-difference) CASE 2: 2 Amidin + at least one of other metabolites present 1) Expression of all metabolites as DMA (1:1 stechiometry), and calculation of Amitraz from DMA (2:1 stechiometry) 2) Expression of Amidin as Amitraz (1:1), Amid + DMA are ignored as they can be formed as by-products during the degradation of Amitraz to Amidin. Take the highest of both

121 Amitraz-Findings in pears (2008): CVUA Stuttgart* Origin Amitraz (parent) Metabolite: N-2,4-Dimethylphenyl-N-methylformamidine Amitraz (sum) MRL- Exceedance ARfD- Exceedance [%] Turkey ND 1,6 2,9 Yes 2647 Turkey ND 1,31 2,4 Yes 2191 Turkey 0,001 1,2 2,2 Yes 2008 Turkey <0,001 1,21 2,2 Yes 2008 Turkey 0,003 0,96 1,7 Yes 1552 Turkey ND 0,84 1,52 Yes 1387 Turkey ND Amitraz parent 0,81 1,5 Yes 1369 Turkey ND 0,79 1,4 Yes 1278 Turkey ND not detected or at 0,69 1,2 Yes 1095 Turkey ND very low levels in 0,59 1,1 Yes 1004 Turkey ND ,59 1,1 Yes 1004 Turkey ND 0,49 0,89 Yes 812 Turkey ND 0,49 0,89 Yes 812 Turkey ND Residue situation 0,48 0,87 Yes 794 Turkey ND 0,47 0,85 Yes 776 is clearly Turkey ND 0,42 0,76 Yes 694 Turkey ND underestimated 0,28 0,51 Yes 465 Turkey ND if only parent is 0,27 0,49 Yes 447 Spain ND 0,013 0,024 No 22 targeted Italy ND 0,32 0,58 Yes 529 Unknown ND 0,9 1,6 Yes 1460 Risk Assessment: German Child 2-5 y. old, 97.5 percentile (232 g/portion); Variability Factor: 7 (JMPR model); ARfD 0.01 (EFSA) * Amitraz-article:

122 Amitraz-Findings in Pears (2008): CVUA Stuttgart Turkish Pear-samples: 18 analyzed; 18 >ARfD Other countries: Italy (11/1 >ARfD), Argentina (10 samples/0 findings), Germany (7/0), Spain (6/1 <ARfD), South Africa (5/0), China (4/0) Chile (1/0), France (1/0),

123 Amitraz-Findings in Pears (2009): CVUA Stuttgart 8 Turkish Pear-samples analyzed, 8 >ARfD Total Amitraz Amitraz parent % ARfD (~140 x) (~100 x) nd nd 183 Turkey has withdrawn amitraz registration. Farmers have 2-year period of grace to get rid of stock. This had obviously a negative effect in terms of agricultural practice