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2 Analysis of pesticides in herbs by GC-QqQ and LC-QqQ Amadeo R. Fernández-Alba

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4 Introduction In some cultures herbs have been used as a popular beverages for centuries. Increased interest in those beverages has resulted from perceived health benefits that may be associated with their consumption. The EFSA annual report of 2008 revealed that: 5.8% and 4.5% of the tea and herbs samples, respectively, contained more than 5 pesticides, one of the highest percentage of the report. these matrices had the highest percentage of MRL exceedance rates (16% herbs and 9% tea). In multiresidue methods one of the main problems is the wide range of chemical properties of the analytes. Tea leaves comprise sugars, pigments, polyphenols and alkaloids (especially caffeine).

5 Objective The aim of the experiments was to compare and choose the best method for the extraction of pesticides residues in tea. Three popular extraction methods were selected for the comparison: modified QuEChERS with CaCl 2 ethyl acetate method mini-luke Validate the selected method in four different matrices and apply it to real samples. Green tea -Black tea Red Tea - Chamomile

6 Without dilution Tomato Orange Leek Tomato Orange Leek

7 Data base of 300 pesticides Mass 912 components (parent molecules, MS fragment ions and Cl isotopes) m/z: Rt: 7-13 min DB: 227 components

8 Co-extracted matrix components of tomato. LC-TOF-MS Mass m/z: Rt: 4-7 min 2408 Matrix compounds Absolute height counts

9 Data base components- tomato matrix compounds Mass m/z: Rt: 4-7 min m/z: Rt: 7-13 min 2408 Matrix compounds Absolute height counts DB: 227 components Tomato: 656 matrix compounds

10 Co-extracted matrix components of green-tea. LC-TOF-MS m/z: Rt: 4-10 min 9073 Matrix compounds Absolute height counts

11 Data base components- green tea matrix compounds m/z: Rt: 4-10 min m/z: Rt: 7-13 min 9073 Matrix compounds Absolute height counts DB: 227 components Green tea: 3533 matrix compounds

12 Co-extracted matrix components of chamomile. LC-TOF-MS m/z: Rt: 5-11 min 6630 Matrix compounds Absolute height counts

13 Data base components- chamomile matrix compounds m/z: Rt: 5-11 min m/z: Rt: 7-13 min 6630 Matrix compounds Absolute height counts DB: 227 components Chamomile: 3490 matrix compounds

14 Selected Compounds 86 LC a - and GC b - amenable pesticides Acaricide Bifenthrin b Buprofezin a Carbaryl a Carbofuran a Chinomethionat b Chlorpyrifos Methyl b Diazinon b Dichlorvos b Dimethoate a Endosulfan I b Endosulfan II b Endosulfan sulfate b Esfenvalerate b Ethion b Fenpropathrin b Fenvalerate b Isocarbophos b Malathion b Methidathion b Methiocarb sulfone a Methiocarb sulfoxide a Methiocarb a Methomyl a Mevinphos b Monocrotophos a Parathion Ethyl b Phorate b Phosmet b Pirimiphos Methyl a Profenofos a Propargite b Pyridaben b Quinalphos b Tetrachlorvinphos b Triazophos b PGR 2,4 D a 2,4 DP a Herbicide Alachlor b Atrazine a MCPP a Metolachlor a Metribuzin a Propyzamide b Simazine a Terbuthylazine a Trifluralin b Insecticide Acetamiprid a Chlorpyrifos b Cypermethrin b Ethoprophos b Etofenprox b Fenitrothion b Flucythrinate b Fonofos b Imidacloprid a Isofenphos Methyl b Lindane b Parathion Methyl b Permethrin b Pirimicarb a Pyriproxyfen b Terbufos b Thiodicarb a Fungicide Azoxystrobin a Benalaxyl b Biphenyl b Boscalid b Carbendazim a Cyproconazole b Cyprodinil b Dichlofluanid b Dicloran b Fenarimol b Fludioxonil b Imazalil a Iprodione b Kresoxim Methyl b Metalaxyl a Myclobutanil a Omethoate a Oxadixyl b Penconazole b Pyrimethanil b Triadimefon a Triadimenol a Vinclozolin b

15 Spiking procedure A key aspect to achieve reliable recovery results is the spiking process. Herbs are dehydrated samples, so they must be fortified with the mix thoroughly soaking all sample, and dried again. Extend the tea on the crystallizer Add the spiking mix Stir to be sure of the homogenizer procedure Evaporate the solvent

16 Modified QuEChERS method Shake automatically in Agitax 2 g sample + 4 ml H 2 O Shake 30 sec, wait 30 min Add 10 ml acetonitrile and 200 µl TPP (25 ppm) Add 4 g MgSO g NaCl + 1 g NaAc 2 H 2 O g NaAc 6 H 2 O Shake automatically in Agitax Centrifugue 5 min r.p.m. Take 3 ml aliquot Shake 30 second in Vortex Acidify with 10 μl formic acid 5% per ml of extract Add 150 mg CaCl 2 anh mg PSA Centrifugue 5 min r.p.m. Analysis Evaporate to dryness and reconstitute

17 Modified QuEChERS method Shake automatically in Agitax 2 g sample + 4 ml H 2 O Shake 30 sec, wait 30 min Add 10 ml acetonitrile and 200 µl TPP (25 ppm) Shake automatically in Agitax Add 4 g MgSO g NaCl + 1 g NaAc 2 H 2 O g NaAc 6 H 2 O Centrifugue 5 min r.p.m. 5 times dilution Take 3 ml aliquot Shake 30 second in Vortex Acidify with 10 μl formic acid 5% per ml of extract Add 150 mg CaCl 2 anh mg PSA Centrifugue 5 min r.p.m. Analysis Evaporate to dryness and reconstitute

18 Ethyl acetate method 2 g sample + 4 ml H 2 O Shake 30 sec, wait 30 min Shake 3 s by hand Shake automatically in Agitax 15 min Add 210 mg GCB g MgSO 4 anhidrous g NaCl Centrifugue 5 min r.p.m. Analysis Add 10 ml ethyl acetate and 200 µl TPP (25 ppm) Evaporate to dryness and reconstitute 5 times dilution

19 miniluke extraction method Shake vigorously 30s by Ultra-Turrax Shake vigorously 30s by Ultra-Turrax 2 g sample + 4 ml H 2 O Shake 30 sec, wait 30 min Add 20mL Petroleum ether: Dichloromethane (1:1) Add 10mL of Acetonitrile and 200 µl TPP (25 ppm) Centrifugue 5 min. at 4000 r.p.m. 5 times dilution Evaporate to dryness 3 ml portion of the upper layer Reconstitute with 1 ml of adecuate solvent Analysis

20 LC and GC analysis LC analysis: Mobile phases: 0.1% FA in H 2 O and AcN Reversed-phase C8 analytical column of 4.6 mm x 150 mm and 5 µm particle size (Agilent Zorbax SB) 6410 LC-QQQ-MS/MS (Agilent Technologies) Dynamic MRM software features GC analysis: Ultra Inert GC column, HP-5MS UI 15 m 0.25 mm i.d μm (Agilent) 7000 GC-QQQ-MS/MS (Agilent Technologies) Retention Time Locking (RTL) and Blackflushing 39 segment SRM method

21 # Compounds European Reference Laboratory - FV Recoveries QuEChERS EtOAc 10 5 miniluke Concentration level: 100 µg/kg (n=5) Recovery (%) n.d.

22 Recoveries Number of compounds QuEChERS Ethyl Acetate method miniluke % % < 60% Not detected 3 7 4

23 Lowest recoveries with modified QuEChERS method P vap pkow modified QuEChERS Compound (Pa, 20ºC) (ph 7, 20ºC) Rec (%) RSD(%) Ometoathe a 3, ,74 19,8 15,3 Monocrotophos a 2, ,22 34,0 12,6 MCPA a 4, ,81 45,5 19,3 2,4-D a < 1, ,83 46,6 22,8 Dichlofluanid b* 1, ,70 49,4 8,3 Dicamba a 1, ,88 51,7 15,9 Carbendazim a 1, ,48 56,9 17,2 a Ansalysed by LC b Ansalysed by GC * Thermical degradation into the GC system CaCl 2

24 Lowest recoveries with ethyl acetate method P vap pkow EtOAc method Compound (Pa, 20ºC) (ph 7, 20ºC) Rec (%) RSD(%) Cyprodinil b 5, ,00 0,0 0,0 Pirimiphos Methyl a 2, ,90 31,1 9,9 Imazalil a 1, ,56 31,9 7,1 Chinomethionat b 2, ,78 33,1 8,7 Ometoathe a 3, ,74 37,4 6,3 Terbufos b 3, ,51 37,7 16,4 Boscalid b 7, ,96 39,3 5,3 Terbuthylazine a 1, ,40 44,4 6,7 Diazinon b 1, ,69 46,3 7,6 Azoxystrobin a 1, ,50 47,1 6,7 Pyrimethanil a 1, ,84 48,4 10,3 Myclobutanil a 2, ,89 48,4 6,1 Phorate b 8, ,86 48,7 3,8 a Ansalysed by LC b Ansalysed by GC GCB P vap pkow EtOAc method Compound (Pa, 20ºC) (ph 7, 20ºC) Rec (%) RSD(%) Thiodicarb a 2, ,62 51,6 6,6 Metolachlor a 4, ,40 51,9 6,8 Kresoxim Methyl b 2, ,40 52,7 3,5 Fenarimol b 6, ,69 53,1 2,0 Penconazole b 3, ,72 53,1 2,6 Iprodione b 5, ,10 54,8 4,2 Alachlor b 2, ,09 55,5 6,8 Isofenphos Methyl b 4,04 55,9 4,1 Malathion b 4, ,75 56,8 3,5 Fludioxonil b 3, ,12 56,9 3,3 Quinalphos b 3, ,44 57,4 4,0 Fonofos b 2, ,94 57,7 14,5 Benalaxyl b 6, ,54 58,2 4,8 Vinclozolin b 1, ,02 59,0 6,4

25 Green Tea Blank LC-QToF-MS Full Scan Total Ion Chromatogram Mini Luke method EtOAc method QuEChERS method

26 Green Tea Blank GC-Q-MS Full Scan Total Ion Chromatogram Mini Luke method EtOAc method QuEChERS method

27 Set-up Green tea Modified QuEChERS Ethyl Acetate method miniluke method Validation in 4 different matrices with the best method Chamomile Green tea Red Tea Black tea

28 Set-up Green tea Modified QuEChERS Ethyl Acetate method miniluke method Validation in 4 different matrices with the best method Chamomile Green tea Red Tea Black tea

29 Compounds (%) European Reference Laboratory - FV Recoveries 50 Concentration level: 25 μg/kg (n=5) Green Tea Red Tea Black Tea Camomile Recovery (%)

30 Compounds (%) European Reference Laboratory - FV Recoveries 50 Concentration level: 25 μg/kg (n=5) Green Tea Red Tea Black Tea Camomile Recovery (%)

31 Compounds (%) European Reference Laboratory - FV Recoveries Concentration level: 100 μg/kg (n=5) Green Tea Red Tea Black Tea Chamomile Recovery (%)

32 Compounds (%) European Reference Laboratory - FV Recoveries Concentration level: 100 μg/kg (n=5) Green Tea Red Tea Black Tea Chamomile Recovery (%)

33 Compounds (%) European Reference Laboratory - FV LODs Benalaxyl Cypermethrin Fonofos Quinalphos Profenofos ,1-0,25 0,25-0,5 0, > 100 Fonofos Mevinphos Green tea Red Tea Black Tea Chamomile LOD (μg/kg) Quinalphos Propargite Mevinphos

34 Linearity and repetitivity Linearity 7 calibration points, 0,5-200 μg/l All the compounds had a R 2 >0.99 Repetitivity Two concentration levels: 5 and 20 μg/l RSD<17%

35 Compounds (%) European Reference Laboratory - FV Matrix effects Green tea Red Tea Black Tea 4 Chamomile 2 0 < -50% -50% - (-20%) -20% % 20% - 50% 50% - 100% 100% - 200% 200% - 300% 300% - 400% 400% - 500% > 500% Slope Variation (%)

36 Blank matrices extracted with modified QuEChERS method LC-QToF-MS Full Scan Total Ion Chromatogram

37 Blank matrices extracted with modified QuEChERS method LC-QToF-MS Full Scan Total Ion Chromatogram

38 Real samples 75 real samples of different kind of herbs bought in: 26 samples 26 samples 9 samples 6 samples 3 samples 1 sample 1 sample 1 sample 1 sample 1 sample

39 Real samples Results 8.0% 24.0% 68.0% Under MRL Above MRL Blank Uncertainty of 50% is considered to check the MRL exceedance.

40 # Samples European Reference Laboratory - FV Real samples Distribution of the pesticides in the samples # Pesticides

41 # Samples European Reference Laboratory - FV Real samples Distribution of the pesticides in the samples # Pesticides Most found: Biphenyl (37), Acetamiprid (36), Carbendazim (36), Chlorpyrifos (34), Imidacloprid, (31), Bifenthrin (30) and Endosulfan (Sum) (22).

42 APPLICATION TO REAL SAMPLES SAMPLE PESTICIDE DETECTED CONCENTRATION (μg/kg) Red tea_1 Chamomille_3 Green tea_5 Black tea_10 Acetamiprid Bifenthrin Biphenyl Carbendazim Imidacloprid Carbendazim Carbofuran Chlorpyrifos Endosulphan I Metalaxyl Etofenprox Penconazole Acetamiprid Bifenthrin Carbendazim Endosulphan I Endosulphan II Endosulphan sulphate Imidacloprid Methomyl Acetamiprid Bifenthrin Carbendazim Endosulphan I Endosulphan II Endosulphan sulphate Imidacloprid In red, values above MRL

43 APPLICATION TO REAL SAMPLES SAMPLE DETECTED PESTICIDE CONCENTRATION (μg/kg) SAMPLE DETECTED PESTICIDE CONCENTRATION (μg/kg) Green tea_34 Green tea_35 Acetamiprid Bifenthrin Biphenyl Carbendazim Carbofuran Clorpyrifos Endosulphan I Endosulphan II Endosulphan sulphate Fenpropathrin Imidacloprid Methomyl Pyridaben Triazophos Acetamiprid Bifenthrin Biphenyl Carbaril Carbendazim Clorpyrifos Endosulphan I Endosulphan II Endosulphan sulphate Fenpropathrin Imidacloprid Methomyl Triazophos Black tea_41 Black tea_57 Black tea_10 Black tea_21 Acetamiprid Biphenyl Carbendazim Methiocarb sulfone Pyridaben Acetamiprid Bifenthrin Buprofezin Chlorpyrifos Imidacloprid Methiocarb Sulfone Acetamiprid Bifenthrin Carbendazim Endosulphan I Endosulphan II Endosulphan sulphate Imidacloprid Acetamiprid Biphenyl Carbendazim Chlorpyrifos Imidacloprid Methiocarb sulfone Pyridaben In red, values above MRL

44 APPLICATION TO REAL SAMPLES SAMPLE PESTICIDE DETECTED CONCENTRATION (μg/kg) Green tea_9 Black tea_10 Green tea_75 Acetamiprid Bifenthrin Carbaril Carbendazim Endosulphan I Endosulphan sulphate Imidacloprid Methomyl Propargite Acetamiprid Bifenthrin Carbendazim Endosulphan I Endosulphan II Endosulphan sulphate Imidacloprid Acetamiprid Bifenthrin Buprofezin Carbendazim Carbofuran Chlorpyrifos Endosulfan I Endosulfan II Endosulfan suphate Imidacloprid Fenvalerate/Esfenvalerate RR/SS Fenvalerate/Esfenvalerate RS/SR Methomyl Pyridaben Triazophos In red, values above MRL

45 APPLICATION TO REAL SAMPLES SAMPLE PESTICIDE DETECTED CONCENTRATION (μg/kg) Rooibos tea_19 Carbendazim Chlorpyrifos Etofenprox Imazalil Iprodione Methomyl Herbs and fruits_23 Biphenyl Boscalid Carbendazim Carbofuran Chlorpyrifos Endosulphan sulphate Fludioxonil Iprodione Pirimetanil Pirimicarb Propargite Pyrimethanil Pyrimiphos methyl Black-green tea_30 Acetamiprid Bifenthrin Biphenyl Carbendazim Chlorpyrifos Endosulphan I Endosulphan II Endosulphan sulphate Imidacloprid Methomyl Monocrothophos Triazophos In red, values above MRL

46 Conclusions From the three MRM the preferred method for the analysis of pesticides residues in green tea was QuEChERS with calcium chloride. This method was the best in terms of recoveries and the full scan chromatograms were the least influenced with coextracted matrix compounds. The method was validated studying recoveries, limits of detection, linearity, repeatability and matrix effects, with good results. It was applied to real samples from different countries, where 92% of them were positives, as average they contained 5 pesticides per sample and 24% exceeded the EU MRL.

47 Tea infusion European Reference Laboratory - FV

48 Infusion for LC analysis European Reference Laboratory - FV 2 g sample 50mL of boiled milliq water Stir during 15 min Take 900µL of infusion + 100µL of acetonitrile Filter 27.8 times dilution Analysis by LC-MS/MS

49 Transfer (%) of the pesticides from the leaves to the infusion Water solubility

50 Ring Test Tea Results European Reference Laboratory - FV

51 Participating labs Number of participating labs in each country

52 Preparation of test material

53 Results Compound Lab 1 (France) Lab 3 (Sweden) Lab 4 (Germany) Lab 5 (Austria) EURL- FV EU Average (mg/kg) EU RSD (%) Lab 2 (Sri Lanka) Lab 6 (Thailand) Lab 7 (Thailand) EU and 3 rd Countries RSD (%) Acetamiprid Bifenthrin NA Buprofezin Carbofuran Detected 26.3 Chlorpyrifos Detected NA 20.8 Cypermethrine NA Detected NA 33.2 Endosulfan Alpha NA Endosulfan Beta NA Endosulfan Sulfate NA NA Fenpropathrin NA NA Fenvalerate NA Detected Imidacloprid NA 22.2 Methomyl NA NA Pyridaben NA Triazophos Concentration expressed as mg/kg NA: Not Analised

54 Concentration (mg/kg) Acetamiprid Bifenthrin Buprofezin Carbofuran Chlorpyrifos Cypermethrine Endosulfan Alpha Endosulfan Beta Endosulfan Sulfate European Reference Laboratory - FV Fenpropathrin Imidacloprid Methomyl Pyridaben Triazophos Fenvalerate European labs results Lab 1 (France) Lab 3 (Sweden) Lab 4 (Germany) Lab 5 (Austria) EURL-FV

55 Thank You for Your Attention

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