An Elevated Resolution GC-TOF-MS Method for the Analysis of Pesticide Residues in Food

Transcription

1 An Elevated Resolution GC-TOF-MS Method for the Analysis of Pesticide Residues in Food

2 Overview Pesticide residues in food EU and worldwide legislation Analytical challenges Targeted screening v. untargeted screening Introduction to exact mass TOF using GCT Premier Targeted screening in food Pear, lettuce and fruit-based baby food TargetLynx application manager Untargeted screening in food ChromaLynx Exact mass scoring of library searches

3 Food Safety Alerts in EU Alert and Information Notifications per Year

4 EU Legislation Regulation 396/05/EC amending 91/414/EC Harmonized for all pesticide/product combinations Range from not detectable to approximately 50mg/kg Not detectable defaults to 0.01 mg/kg if no other level is assigned Recommendation 2006/26/EC of 18 th January 2006 Coordinated monitoring program to ensure compliance with maximum levels of pesticide residues in or on cereals and certain other products of plant origin

5 Worldwide Legislation US Food Quality Protection Act (FQPA) of 1996 o 40 CFR 180 o USDA, Foreign Agricultural Service Japan o Positive List System for Agricultural Chemical Residues in Foods Enforced from 29 th May 2006 Uniform limit set to 0.01 mg/kg o o

6 Analytical Challenges Chemically and structurally diverse analytes Approximately 1000 pesticides registered worldwide Wide variety of matrices (fruits, vegetables, herbs, spices) Method must be Sensitive, low default reporting limit of 0.01mg/kg Selective, reduce or eliminate matrix interferences Multiresidue, multiple targets in a single run Rugged, complex samples with reduced sample clean-up But also generic Food scares and changes in legislation require updating of methods and re-analysis of samples Non target components can be detected post acquisition or retrospectively

7 Trend in Analytical Method Development Sample Preparation Compound Specific Generic Generic Generic Separation / Detection Compound Specific Compound Specific Generic Generic Data Processing Compound Specific Compound Specific Compound Specific Untargeted Results GC-ECD GC-MS (SIR) GC-MS (SIR) GC-MS/MS GC-TOF GC-TOF?

8 System Solution GCT Premier Orthogonal acceleration GC-TOF-MS high resolution, exact mass TargetLynx Advanced quantification with a full range of automatic quality control checks ChromaLynx Peak detection, deconvolution, library searching with exact mass scoring

9 GCT Premier Orthogonal Acceleration TOF Rapid full spectral sampling and acquisition rates Non skewed spectra Narrow chromatographic peaks Accurate chromatographic peak profiling enabling deconvolution Non scanning instrument with a high duty cycle High full scan sensitivity Predictable scan law Routine exact mass calibration with a single lock mass Elevated Resolution Enhanced selectivity

10 GCT Premier Plug in EI/CI ion volumes for rapid change over Resolution Greater than 7000 (Full width half maximum) Exact mass Less than 5 ppm RMS Dynamic range 4 orders linear dynamic range Spectral acquisition rate 20 spectra per second

11 GCT Premier Parameters Electron Impact (EI+) with a mass range of m/z Acquisition Speed = 4 spectra/s Tris(trifluoromethyl)triazine, single point lock mass Dynamic Range Enhancement (DRE) On Low Mass Cut Off = 45Da Electron Energy = 70eV Trap Current = 200µA Source Temperature = 200 C Interface Temperature = 280 C Detector Voltage = 2900V

12 Pesticide Residues Pesticide Residues Azoxystrobin Bifenthrin Biphenyl Bromopropylate Bupirimate Buprofezin Captan Carbaryl Chlorfenvinphos Chlorothalonil Chlorpyrifos Chlorpyrifos-methyl Chlozolinate Cyfluthrin Cyhalothrin-lambda Cypermethrin Cyprodinil DDD DDE o,p-ddt p,p-ddt Deltamethrin Diazinon Dichlofluanid Dicloran Dicofol Difenoconazole Diphenylamine Endosulfan Endosulfan-sulfate Ethion Ethoprophos Fenarimol Fenazaquin Fenbuconazole Fenhexamid Fenitrothion Fenpropathrin Fenvalerate Fludioxonil Folpet Fonofos Furalaxyl HCH-gamma Heptenophos Iprodione Isofenphos Kresoxim-methyl Malathion Mecarbam Metalaxyl Methidathion Myclobutanil Ofurace Oxadixyl Paclobutrazol Parathion-ethyl Parathion-methyl Pendimethalin Pentachloroaniline Permethrin Phenthoate Phosalone Phosmet Phosphamidon Pirimicarb Pirimiphos-ethyl Pirimiphos-methyl Procymidone Profenofos Propargite Propiconazole Propyzamide Prothiofos Pyrazophos Pyridaphenthion Pyrifenox Pyrimethanil Quinalphos Quintozene Simazine Tebuconazole Tebufenpyrad Tecnazene Tefluthrin Tetrachlorvinphos Tetradifon Tolclofos-methyl Tolylfluanid Triazophos Trifloxystrobin Trifluralin Vinclozolin

13 Sample Preparation Extraction of the baby food, lettuce and pear samples Weigh 10g homogenized sample into a centrifuge tube Add 10ml acetonitrile with 1% acetic acid, 4g anhydrous MgSO 4 and 1.66g sodium acetate and shake immediately Centrifuge at 4300g for 5min Transfer 1ml aliquot of acetonitrile layer to a vial and then blow down gently with nitrogen to add 0.2ml toluene Transfer 1ml extract to a micro-centrifuge vial Add 150mg anhydrous MgSO mg PSA + 50mg carbon sorbent and vortex mix for 30s Centrifuge at 5000g for 1min Transfer into a GC vial and submit for GC-TOF-MS analysis

14 % Recovery Recovery at 0.01mg/kg from fruit-based baby food (n = 5) Mean % Recovery and % RSD (in parenthesis) Target is % with < 30% RSD Biphenyl 119 (7) Heptenophos 114 (6) Tecnazene 90 (4) Diphenylamine 104 (5) Ethoprophos 119 (9) Trifluralin 112 (5) Dichloran 89 (5) Simazine 102 (4) Gamma-HCH 106 (9) Quintozene 64 (5) Propyzamide 106 (2) Fonofos 115 (8) Pyrimethanil 59 (5) Diazinon 114 (12) Phosphamidon-e 93 (12) Tefluthrin 115 (9) Chlorothalonil 51 (9) Pirimicarb 88 (2) Pentachloroaniline 40 (9) Phosphamidon-z 100 (4) Vinclozolin 107 (4) Chlorpyrifos-methyl 97 (8) Parathion-methyl 103 (5) Carbaryl 106 (5) Tolclofos-methyl 107 (8) Metalaxyl 108 (3) Fenitrothion 98 (5)

15 % Recovery Pirimiphos-methyl 102 (5) Dichlofluanid 111 (7) Malathion 105 (4) Chlorpyrifos 85 (5) Parathion-ethyl 98 (3) Dicofol BD 105 (9) Pirimiphos-ethyl 97 (6) Cyprodinil 52 (2) Chlorfenvinphos-e 82 (6) Pendimethalin 85 (4) Pyrifenox (I) 101 (3) Chlozolinate 105 (4) Tolylfluanid 106 (6) Chlorfenvinphos-z 114 (2) Isofenphos 104 (4) Captan 98 (8) Mecarbam 104 (6) Quinalophos 89 (4) Phenthoate 98 (3) Furalaxyl 106 (1) Folpet 70 (13) Procymidone 104 (3) Methidathion 99 (3) Pyrifenox (II) 99 (9) Paclobutrazol 103 (9) Tetrachlorvinphos 97 (6) Endosulfan (I) 105 (7) Prothiofos 81 (7) Fludioxonil 95 (3) Profenofos 90 (4) p,p'-dde 102 (4) Myclobutanil 101 (5) Kresoxim-methyl 101 (5) Buprofezin 100 (9) Bupirimate 98 (4) Endosulfan (II) 99 (5) o,p-ddt 104 (3) Oxadixyl 98 (5) p,p-ddd 102 (7) Ethion 97 (4) Triazophos 94 (10) Ofurace 100 (4)

16 % Recovery Ofurace 100 (4) Propiconazole (I) 94 (5) Endosulfan-sulfate 103 (5) Trifloxystrobin 100 (6) Fenhexamid 94 (4) p,p-ddt 97 (3) Propiconazole (II) 78 (15) Tebuconazole 92 (7) Propargite 100 (9) Iprodione 99 (7) Pyridaphenthion 94 (4) Phosmet 84 (3) Bromopropylate 99 (4) Bifenthrin 95 (3) Fenpropathrin 102 (6) Tebufenpyrad 91 (3) Fenazaquin 46 (10) Tetradifon 89 (6) Phosalone 73 (4) Cyhalothrin-λ 99 (4) Fenarimol 99 (6) Dicofol 99 (2) Pyrazophos 51 (9) Permethrin cis 88 (6) Permethrin trans 95 (11) Fenbuconazole 78 (7) Cyfluthrin (I) 89 (29) Cyfluthrin (II) 119 (12) Cyfluthrin (III-IV) 165 (8) Cypermethrin (I) 130 (11) Cypermethrin (II) 103 (22) Cypermethrin (III- IV) 114 (11) Fenvalerate (I) 99 (6) Fenvalerate (II) 90 (17) Difenoconazole (I) 87 (10) Difenoconazole (II) 87 (6) Deltamethrin 90 (7) Azoxystrobin 103 (5)

17 % Recovery % Recovery

18 GC Conditions Agilent 6890N GC CTC Analytics CombiPal Autosampler Cryo cooled PTV in solvent vent mode 5μl acetonitrile injected at 50 C Multiple baffle liner used to give a large surface area Solvent is vented using helium at a pressure of 5kPa and flow of 20ml/min Vent closed after 0.5min Injector ballistically heated to 300 C Remaining material in the liner transferred to column

19 GC Conditions Column Restek, Rxi-5ms, 30m x 0.25mm i.d., 0.25μm Constant flow of 1.0 ml/min helium Oven temperature program Ramp rate, C/min Hold, min 50 C C/min C/min 7 Run time = 34min

20 Total Ion Chromatogram (TIC) Biphenyl Azoxystrobin

21 Targeted Screening TOF Compound list is known Exact mass chromatograms, 0.02 or 0.05 Da window Full spectrum technique o Any ion can be chosen for quantification Number of residues/ions can be increased without loss in sensitivity Screening is based on one or more exact mass chromatograms TargetLynx o Highlight samples that have concentrations greater than the reporting level do not pass quality control criteria

22 Selectivity of Exact Mass Chromatograms Chlorothalonil, 0.01mg/kg in baby food, m/z = Da 0.02 Da

23 Selectivity of Exact Mass Chromatograms Methidathion, 0.01mg/kg in baby food, m/z = Da 0.02 Da

24 Selectivity of Exact Mass Chromatograms Endosulfan sulfate, 0.01 mg/kg, m/z = Da 0.02 Da Pear Lettuce Baby food

25 Propyzamide, 0.01 mg/kg in Pear Sensitivity Increasing the Number of Ions

26 Propyzamide Spectrum

27 TargetLynx Browser

28 Application to Real Samples Real samples analyzed Pear, 11 samples Lettuce, 12 samples Matrix-matched standards used for calibration TargetLynx used for automatic quantification with a full range of quality control checks Two or three exact mass chromatograms per residue were extracted For illustration purposes Reporting Level = 0.005mg/kg Maximum Residue Limit = 0.010mg/kg

29 Lettuce Fenitrothion 0.023mg/kg

30 Pears Chlorpyrifos 0.023mg/kg

31 Untargeted Screening TOF Compound list is unknown Full spectrum technique with no spectral skew Fast acquisition speed enables deconvolution, 5-10 spectra/s Exact mass facilitates identification, < 5 ppm RMS High resolution allowing enhanced selectivity, > 7000 FWHM ChromaLynx o Automatic peak detection and deconvolution o Automatic production of clean component spectra o Automatic identification via library searching o Exact mass scoring

32 Deconvolution of Lettuce 5 Components?

33 Deconvolution of Lettuce with ChromaLynx ChromaLynx found 8 Components!

34 Extracted Mass Chromatograms 3 Components in Lettuce Mecarbam Isofenphos Chlorfenvinphos-z

35 Pear ChromaLynx ChromaLynx found ~ 500 Components

36 Untargeted Screening Exact Mass Scoring Library searching can be scored by exact mass in ChromaLynx

37 Untargeted Screening Thiabendazole in Pear

38 Untargeted Screening DCPA in Lettuce

39 Untargeted Screening DCPA in Lettuce

40 Targeted Confirmation DCPA in Lettuce Lettuce sample was re-extracted and quantified using the established single quadrupole method Validation recoveries (n=5) % at 0.01mg/kg Confirmation based on 3 ions. literature ion ratios in parenthesis 301 (100%) 299 (80%) 332 (30%) Concentration of DCPA = 0.07mg/kg (measured ratios) 301 (100%) 299 (78%) 332 (28%)

41 Summary An exact mass GC-TOF-MS method has been developed for the screening of approximately 100 pesticide residues to concentration levels of 0.01 mg/kg or less in pear, lettuce and fruit-based baby food Good linearity and recoveries for the majority of pesticides were obtained A targeted approach using TargetLynx and an untargeted approach using ChromaLynx were used for screening Joint approach led to the successful identification of incurred target and non-target residues in pear and lettuce The targeted approach correlated well with previous results obtained from established single quadrupole method

42 Acknowledgements Waters Corporation, Manchester, UK Peter Hancock Central Science Laboratory, York, UK Richard J. Fussell Cristiana Leandro University of York, York, UK Cristiana Leandro Brendan J. Keely