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  1. INSPECTORATE FOR HEALTH PROTECTION Food Inspection Service AMSTERDAM “ Developments in GC-MS(/MS) and LC-MS(/MS) Methodology for Pesticide Residue Analysis” André de Kok Research & Development Dept. Pesticide Analysis Group Amsterdam, The Netherlands Lecture presented at the 4th “Meeting on Pesticide Residues”, Rome, Italy; 27 November, 2001

  2. OUTLINE • Analytical Methods Selection Strategy • Extraction and Detection Methods • Scope of Multiresidue Methods • GC-MS EI and CI • GC-MS/MS EI and CI • LVI-GC-MS EI • GC-MS/MS Triple Quad NI-CI • Future Trends & Conclusions

  3. “Fit for Purpose” Analysis CRITERIA • AIM of the analyses • TROUGHPUT OF RESULTS required • INSTRUMENTATION available • ANALYTICAL SKILLS OF PERSONNEL • FINANCIAL BUDGET • TIME available • QUALITY OF DATA !!

  4. Analytical Method Performance CRITERIA • Scope • Sensitivity • Selectivity • Recovery • Run time / Analysis time • Sample Troughput • Robustness • Ease of use • Costs Extraction Cleanup Detection

  5. PESTICIDE RESIDUE ANALYSIS IN FRUITS, VEGETABLES AND CEREALS Multi Residue Methods MRMs vs. Single Residue Methods SRMs Sensitive vs. Selective DETECTION ECD, FPD, NPD <----------> MS, MS/MS GC-amenable vs. LC-amenable Pesticides Organic vs. Aqueous EXTRACTION NO vs. General vs. Selective CLEANUP

  6. ANALYSIS SCHEMEMULTIRESIDUE METHODS DTC SAMPLING HOMOGENIZATION EXTRACTION CLEAN-UP GC HPLC SEPARATION MRMs 5 ITD ECD FPD NPD 1 2 3 4 DETECTION

  7. NEWDITHIOCARBAMATES METHOD ISO-OCTANE EXTRACTION OF CS2 PROCEDURE: - Weigh 50 g sample in 250-ml screw-cap glass bottle - Add 25 ml iso-octane - Add 150 ml SnCl2 in HCl close bottle tightly and put in a water bath - Reaction at 80 ºC for 2 hr under shaking DTC --------> CS2 let cool dowm to room temperature ! - Transfer 1 ml of iso-octane extract into GC autosampler vial

  8. “NEW”DITHIOCARBAMATES METHOD GC-ECD method GC CONDITIONS: Injection: 1 µl splitless Injection temp.: 250 ºC Detector temp.: 300 ºC Carrier gass (He) flow: 2.6 ml/min. Capillary column: 50m x 0.32mm i.d. stationary phase: CP-Sil 8 CB, 1.2 µm film Oven temp.program: 50º (10 min) ---- 50º/min ---> 260 ºC (5 min) Retention time CS2: 5.5 min. Limit of detection: 0.01 mg/kg Limit of determination: 0.02 mg/kg Reporting limit: 0.02 or 0.05 mg/kg

  9. CS2

  10. Miniaturised Acetone Extraction Method • weighing 15 g homogenized crop sample • in 200-ml PTFE centrifuge tube • extraction 30 ml acetone ( “ PolytronR ”) 20 sec ____________________________________ • partitioning 30 ml CH2Cl2 +30 ml petroleum-ether ( “ PolytronR ”) 20 sec • centrifuge 4000 rpm, 4 min. • transfer organic extract to Erlenmeyer flask acetone 30 ml 15 g PE 30 30 CH2Cl2 acetone 30 15 83ml

  11. Alternative Miniaturised Acetone Extraction Method for “polar OP - Pesticides” e.g. for duplicate analysis - samples with exceedings of MRL Method P** PE 30 ml CH2Cl2 30 ml acetone 30 ml + 7. 5 g Na2SO4 7. 5g Recoveries >70% for methamidophos, acephate, omethoate, monocrotofos

  12. MULTIPLE GC- and LC-DETECTION SYSTEMS KvW Alkmaar ORGANIC EXTRACT (acetone / CH2Cl2 / PE) 90 ----> ca. 83 ML Aliquots 15 ML 3 ML Evaporate in waterbath at 40-65ºC and redissolve in iso-octane/toluene, 9:1) GC - ITD GC -NPD GC -FPD idem 200 µL GC -ECD 1 ML 1 ML 1.5 ML SPE CH2Cl2 HPLC-PCR-FLD evaporate and redissolve in MeOH SPE 2 ML 3.0 ML HPLC-UV/FLD

  13. SCOPE GC MRMs KvW Alkmaar • GC-ECD • 55 OC • 20 PYR • GC-NPD (N/P mode) • 150 ON • 100 OP • GC-FPD (P mode) • 100 OP • GC-ITD (Varian Saturn) • 350 pesticides ! PAST

  14. GC-ITD (EI-MODE) IN ROUTINE ANALYSIS KvW Alkmaar • EVALUATION AFTER 3 YEARS (1993-1996, 3000 SAMPLES !) • SCOPE: 350 pesticides / metabolites • Fast Automated identification / quantification • Determination levels of 120 pesticides detected in real samples: • 0.01-0.05 mg/kg for ca. 100 pesticides • 0.1-1 mg/kg for ca. 20 “problem” pesticides • acephate, methamidaphos, monocrotophos, omethoate, • imazalil, prochloraz, bitertanol, • thiabendazole, • endosulfan, captan, folpet, dicofol, • propargite, ................

  15. LC Multiresidue Methods (MRMs) • N-methylcarbamates (NMC) • 20 parents + 12 metabolites • Phenylurea Herbicides (PUH) • 15 pesticides • Benzoylphenylurea Insecticides(BPU) • 8 pesticides • Benzimidazole fungicides • Thiabendazole + Carbendazim (incl. benomyl) • Conazole fungicides • Prochloraz + Imazalil 1 HPLC-PCR-FLD 2 HPLC-PCR-FLD 3 HPLC-DAD or LC-MS/MS 4 HPLC-UV/FLD 5

  16. USE OF MULTIRIDUE METHODSGC KvW Amsterdam PRESENT All GC-amenable pesticides with LODITD < = LODsel. det. and accurate quantitation 1 ITD If LODITD > LODsel. det. (GC) and If LODITD > “ 0-tolerance “ (0.01 - 0.05 ppm) : use GC-FPD, -ECD or -NPD and/or CI-MS (/MS) If pesticides are not detectable by GC-ITD : use HPLC or LC-MS/MS

  17. GC Multiresidue Methods (MRMs) GC-ITD: 1 mixture of reference pesticides (76x) 6 mixtures of other pesticides ------------------------------------------------- Total: ca. 400x GC-FPD: 1 mixture of reference pesticides (7x) - dichlorvos, methamidofos, acephate - omethoate, monocrotophos, - mecarbam, azinphos-methyl GC-ECD: 1 mixture of reference pesticides (13x) - dichloran, lindane, chlorothalonil, - dichlofluanide, aldrin (I.S.), - tolylfluanide, captan, folpet, - endosulfan-a, b, & sulphate, - cypermethrin, deltamethrin [ GC-NPD: - prochloraz + imazalil ] optional

  18. GC-ITD TOTAL ION CHROMATOGRAM Mixture ITD-1, Reference Pesticides (76x)

  19. VALIDATION OF MULTIRESIDUE METHODS GC-MRM’s ITD: Mixture 1,2,3,............ ECD: Mixture 1 FPD: Mixture 1 HPLC-MRM’s NMC: Mixture 1 (polar parents and metabolites) Fungicides: - Thiabendazole + Carbendazim - Prochloraz + Imazalil ( 1 ASPEC/SPE system + 2 parallel HPLC-UV systems) Detection/Determination limits + Linearity calibration curve (7 levels, n=6) (solvent + matrix) Recoveries: 6 matrices, 3 levels, n=6)

  20. LIMITS definition/calculation Validation vs. QC (check) • Limit of Detection LODc • 3 x SD of estimated LOD ~S/N = 3/1 • Limit of Determination LODm 6 x SD of estimated LOD ~S/N = 6/1 • Limit of Quantitation LOQ • 10 x SD of estimated LOD ~S/N = 10/1 • Lowest Calibration Level LCL S/N = variable Reporting LimitRL~ LCL ~LODm Check: x Response LCL Example : GC-ITD x x o 0.05 0.2 0.8 Conc. (~mg/kg) RL 0.025

  21. TRENDS Related to Pesticides Legislation, Enforcement and Monitoring, and Pesticides Analysis EU QA/QC Requirements - quantitation and identification - MS confirmation (GC-MS criteria) EU “Babyfood Directive” - 0.01 mg/kg (10 ppb) limit for all pesticides EU MRL-setting Directives - more “0-tolerances” (0.01-0.05 mg/kg) EU border control (3rd-countries = non-EU import) - more 72-hr respons analyses - shorter analysis time / high throughput

  22. TRENDS and SOLUTIONS in pesticide residue analytical methods GC Shorter analysis times: “ Fast GC” (short, narrow-bore columns) “ Rapid MS” (short, wide-bore columns) GC-MS with higher scan rates (Time of Flight, TOF, 5000 scans/sec) Higher selectivity and Lower detection limits: GC-MS/MS (EI/CI mode) Ion Traps (ITD) or Triple-Quads (pos./neg. mode) Lower detection limits: on-line or off-line cleanup (SPE, GPC, LC) + concentration large-volume injection (LVI)

  23. Grape Sample: 0.01 mg/kg of PIRIMICARB GC-ITD EI-MS mode m/z 166 TIC m/z 166

  24. Grape Sample: spiked with 0.1 mg/kg Chlorfenvinphos CI-MS Acetonitrile liquid- Chemical Ionisation Fit 978 m/z 359 (M+1) S/N: 60/1

  25. COMPARISONGC-ITD (EI/CI) vs. GC-ECD/NPD/FPD KvW Alkmaar PRODUCT: LETTUCE PESTICIDE FPD NPD ECD ITD ITD -EI -CI metalaxyl _ _ 0.008 endosulfan(a-) 0.29 0.29 _ endosulfan(b-) 0.23 0.23 0.22 endosulfan-SO4 matrix 0.54 0.44 iprodion 0.08 0.06 0.07 0.06 deltamethrin 0.10 + + procymidon _ _ _ 0.01 oxadixyl 0.58 + +________________________________________ metalaxyl 0.06 0.07 0.08 iprodion 0.25 0.26 0.22 0.21 tolclophos -methyl 0.42 0.42 0.40 0.40

  26. COMPARISONGC-ITD (EI/CI) vs. GC-ECD/NPD/FPD KvW Alkmaar PRODUCT: ORANGE PESTICIDE FPD NPD ECD ITD ITD -EI -CI azinphos -methyl 0.50 + 0.59 0.45 imazalil 0.68 + 3.9 4.0 dicofol + 0.10 + o-phenyl-phenol 0.48 0.65 dimethoate 0.04 - - 0.05 chlorpyrifos 0.02 0.02 - 0.02 PRODUCT: SWEET PEPPER dicloran + 0.17 + + procymidon 0.13 0.11 0.14 0.14 vinchlozolin 0.15 0.10 0.12 0.14 methamidophos 0.03 - - 0.03 ________________________________________ metalaxyl 0.06 0.07 0.08 iprodion 0.25 0.26 0.22 0.21 tolclophos -methyl 0.42 0.42 0.40 0.40

  27. GC-ITD: Acetonitrile CI-mode • Automated method, routinely used • Acetonitrile easy-to-use reagent gas • More sensitive than EI-mode • Less interference from matrix peaks • Over 70% show molecular ion • Less sensitive for some pesticides • Not enough specific ions for unambiguous identification • Less good repeatability • CI can not replace EI, but is supplementary

  28. Grape Sample: spiked with 0.01 mg/kg Chlorfenvinphos EI-MS/MS Full-scan EI-MS/MS Fit 729 S/N: 6/1 m/z 267 -----> 159

  29. Grape Sample: spiked with 0.005 mg/kg PROCYMIDON LARGE_VOLUME INJECTION GC-EI-MS 5-µL INJECTION m/z 283 S/N: 7/1

  30. PDA chromatogram (260 nm, 3D) of a paprika extract fortified with 0.05 mg/kg of BENZOYLPHENYLUREAS

  31. IonChromatograms (full scan MS) of a paprika extract, fortified with 0.05 mg/kg of BENZOYLPHENYLUREAS diflubenzuron teflubenzuron HPLC conditions: Superspher 100 RP-18 (150 x 3 mm ID); 75% ACN in water; 0,50 ml.min-1 MS conditions: ESI-; source voltage: 4,5 kV; capillary. voltage -30 V; capillary. temp: 175°C

  32. Ion Chromatograms (full scan MS/MS) of a pepper extract, fortified with 0.05 mg/kg of BENZOYLPHENYLUREAS LC-MS/MS Diflubenzuron MS/MS Teflubenzuron MS/MS HPLC condities: Superspher 100 RP-18 (150 x 3 mm ID); 75% ACN in water; 0,50 ml.min-1 MS condities: ESI-; source voltage: 4,5 kV; capillary. voltage -30 V; capillary. temp: 175°C

  33. Ion Chromatograms of a pear extract, with incurred residue ofCHLORMEQUAT LC-MS MS spectrum m/z 122 m/z124 UV spectrum DAD: 205nm HPLC conditions: Shodex RSpak DE-613 (150 x 6 mm); 50% MeOH in 25 mM ammonium acetate; 0,75 ml.min-1 MS conditions: ESI+; source voltage: 4,5 kV; capillary voltage: 25 V; capillary temp: 270°C DAD conditions: scan range 200-400 nm; scanrate 5 Hz

  34. Ion Chromatograms (full scan MS/MS) of a lettuce extract, with incurred residue of PROPAMOCARB LC-MS/MS 189 144 m/z 189 m/z 189 144 102 m/z 189 102 HPLC conditions: Shodex RSpak DE-613 (150 x 6 mm); 50% MeOH in 25 mM ammonium acetate; 0,75 ml.min-1 MS conditions: ESI+ ; source voltage: 4,5 kV; capillary voltage 25 V; capillary temp: 270°C

  35. Pepper Sample with incurred residue of IMIDACLOPRID ( 0.23 mg/kg) LC-MS/MS m/z 212  128 degradation m/z 256  210 HPLC conditions: Superspher 100 RP-18 (150 x 3 mm ID); 25% ACN in 0.1% acetic acid; 0.50 ml.min-1 MS conditions: APCI+ ; vaporizer temp: 400°C; discharge needle: 4 kV; cap. voltage 25 V; cap. temp: 150°C

  36. FUTURE TRENDS and CONCLUSIONS • GC-MS (MS/MS) and LC-MS/MS • Expansion of scope of Multiresidue methods • More Single-Residue Methods (SRMs) • Decrease of detection limits