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Advancements in Pesticide Residue Analysis: GC-MS(/MS) and LC-MS(/MS) Methodology

This lecture discusses the methodology for pesticide residue analysis, covering analytical methods, extraction, detection, and multiresidue approaches using GC-MS and LC-MS. It also outlines criteria for "fit-for-purpose" analysis and performance evaluation. Various extraction and detection methods are explored, focusing on multiresidue methods in fruits, vegetables, and cereals. New developments like miniaturized acetone extraction methods are presented for improved sensitivity and selectivity in pesticide detection. The application of multiple GC and LC detection systems is detailed, emphasizing the scope and efficiency of pesticide analysis.

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Advancements in Pesticide Residue Analysis: GC-MS(/MS) and LC-MS(/MS) Methodology

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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

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