1. Sample preparation and analytical method development for monitoring of carbamate insecticides Jitlada Vichapong Department of Chemistry Faculty of Science Khon Kaen University e-mail: jitlada_v@hotmail.com

2. Insecticides ♣ Organochlorines ♣ Organophosphates ♣ Carbamates ♣ Synthetic Pyrethroids Insecticides ♣ Organochlorines ♣ Organophosphates ♣ Carbamates ♣ Synthetic Pyrethroids Herbicides Rodenticides Fungicides Herbicides Rodenticides Fungicides 2

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4. Carbamate Insecticides “Maximum residue limits (MRLs), mg kg -1 ” Crop MRLs, mg kg -1 EUCodexASEANKFDATACFS* 9002- 2008 Raw fruit0.02-10.1-10 0.1-3.00.2-15 Vegetable0.02-20.2-3.0 0.1-5.00.1-3 *TACFS: Thai Agricultural Commodity and Food Standard 4

5. KHONKAEN UNIVERSITY Test kit Spectrophotometry Gas chromatography High performance liquid chromatography 5

6. Require sample preparation Extraction Sample clean-up Matrix removal Analyte preconcentration Carbamates in food & environmental matrices 6

7. KHONKAEN UNIVERSITY Reduce Alternative solvents  7

8. Contents 1.Sequential injection with lab-on-valve (SI-LOV) system for carbamates screening 2.Liquid – liquid microextraction of carbamates using room temperature ionic liquid (RTIL) 3. Ultrasound assisted surfactant-enhanced emulsification microextraction procedure (UASEME) using non - ionic surfactant 4.UASEME procedure using mixed anionic – cationic surfactants 5.On-line renewable micro-solid-phase extraction of carbamates using SI-BI- LOV- HPLC Application: Environmental and food samples 8

9. KHONKAEN UNIVERSITY Sequential injection with lab-on-valve (SI-LOV) system for carbamates screening １ １

10. KHONKAEN UNIVERSITY 1 2 3 4 5 6 1 2 3 4 5 6 7 8 9 10 10-port selection valve waste light source: USB2000 detector LOV unit carrier standards/samples holding coil Syringe pump peristaltic pump computer with a control-software 2  Reagents : 2-aminonaphthalene-1-sulfonic acid (ANSA) : 2,4-dimethoxy aniline (DMA) : 4-aminoantipyrene (4-AP) 10

11. KHONKAEN UNIVERSITY 250 µL NaOH 200 µL carbaryl 400 µL ANSA 50 µL NaNO 2 100 µL HCl Holding coil Direction to flow cell 11

12. KHONKAEN UNIVERSITY 12

13. KHONKAEN UNIVERSITY (Potassium ferricyanide) 13

14. SI-grams of carbaryl – ANSA derivative SI-grams of carbaryl – DMA derivative SI-grams of carbaryl – 4-AP derivative 14

15. KHONKAEN UNIVERSITY ReagentCarbamate detectedLOD (µg mL -1 ) ANSACarbaryl0.02 Methiocarb0.07 Carbofuran0.08 DMACarbaryl0.02 Carbofuran0.02 Methiocarb0.02 Promecarb0.07 Methomyl0.08 Propoxur0.08 Isoprocarb0.08 4-APCarbaryl0.04 Carbofuran0.08 LODs for the determination of carbamates 15

16. SampleRecovery (%) Spiked level (µg mL -1 )0.501.003.00 Water I9295110 Water II9399114 Water III9810295 Spiked level (µg g -1 ) 0.250.500.75 Chinese white cabbage 938985 Cabbage 909287 Cauliflower 9597100 Cucumber 8998102 Baby corn 1099597 Green grape 9993110 Grapefruit 1129887 Guava 9511097 Apple 989399 Pear 889097 Recovery: 85 – 112% 16

17. A solvent for micro-extraction of carbamates 2

18. 1-butyl-3-methylimidazolium hexafluorophosphate [C 4 mim][PF 6 ] 18 Analytes RTIL IL phase Aqueous phase RTIL for extraction of carbamate pesticides Matrix IL phase Sample Acetonitrile HPLC

19. KHONKAEN UNIVERSITY Sample volume3 mL [C 4 MIM][PF 6 ]100  L Extraction time7 min Dissolving solvent 50  L acetonitrile (to decrease the viscosity) IL phase Column Detection Mobile phase Injection flow rate Atlantis C18 (5 μm, 150 x 4.6 mm) 270 nm ACN and 0.1% acetic acid (gradient elution) 20  L 1.0 mL min -1 19

20. KHONKAEN UNIVERSITY Pesticide HPLC RTIL-HPLC Linear range (µg/mL) LOD (µg/mL) % RSD (n=5) Linear range (µg/mL) LOD (µg/mL) % RSD (n=5) EF tRtR AreatRtR MTM 2.0-15.01.000.322.52 ----- PPX 0.2-10.00.100.183.50 0.02-5.000.0050.5210.1720.0 CBF 0.2-10.00.100.163.42 0.02-5.000.0050.483.9620.0 CBR 0.1-10.00.020.283.28 0.01-5.000.0020.325.8910.0 IPC 2.0-15.00.500.404.10 0.04-8.000.0200.626.5625.0 MTC 0.2-10.00.050.231.82 0.05-8.000.0050.498.4010.0 PMC 1.0-15.00.500.144.79 0.40-8.000.0400.304.9012.5 20 Linear range: 0.01 -8.00 µg mL -1 LOD: 0.002 – 0.040 µg mL -1 LOQ: 0.005 – 0.120 µg mL -1 RSD: <10.17 EF: 10 - 25

21. Without RTIL-LLME RTIL-LLME 21

22. KHONKAEN UNIVERSITY SampleMarket Amount found, μg g -1 PPXCBFCBRIPCMTCPMC WatermelonLocal Market----0.100.17 Super Market I---1.280.601.34 Super Market II0.040.530.06-0.452.61 Cantaloupe1Local Market--0.06-0.01- Super Market I----0.37- Super Market II----0.45- Cantaloupe2Local Market0.02-0.06-0.110.55 StrawberryLocal Market------ Super Market I-0.110.020.540.431.55 OrangeLocal Market--0.070.184.50- Super Market I0.720.210.161.500.941.81 Super Market II0.220.18-0.90.3821.25 AppleSuper Market I0.03---0.451.24 Super Market II----0.381.30 Application to sample Range: 0.01– 21.25 μg g -1 22

23. KHONKAEN UNIVERSITY SampleRecovery (%) PPXCBFCBRIPCMTCPMC Water I909910298 89 Water II92968793 104 Water III1009510310011694 Watermelon1158881909385 9610010692104101 96100103 9388 Cantaloupe19810382118117104 9596104107101107 9395981059394 Cantaloupe28399931018886 Strawberry93102829210392 1071039910110782 Orange839885107102110 89110106 9998 10099 9810897 Apple105103102929492 1001049910497112 Recovery: 82 – 117% 23

24. Ultrasound-assisted surfactant-enhanced emulsification microextraction (UASEME) Sodium dodecylsulphate (SDS) Cetyltrimethyl ammonium bromide (CTAB) Tween 20 non-ionic surfactant Mixed anionic-cationic surfactant 24

25. dissolving solvent + dissolving solvent + Add surfactant and extraction solvent Sonicated Sample Cloudy Phase separation ４ ４ 3 3 Standard/ Sample solution Column Detection Mobile phase Injection volume Mobile phase flow rate Chromolith FastGradient column (RP-18e, 50–2 mm) 270 nm 37% MeOH 20  L 0.5 mL min -1 Sedimented phase 25

26. ParameterCondition non-ionic surfactant mixed anionic–cationic surfactant SurfactantTween 20SDS - CTAB SaltNa 2 SO 4 7.0% w/vNo salt Conc. of surfactant0.01 mol L -1 SDS (0.05 mol L -1 ) - CTAB (0.01 mol L -1 ) Volume of surfactant30 µLSDS (45 µL) - CTAB (30 µL) ExtractantC 6 H 5 ClC 6 H 5 Cl - CHCl 3 Volume of extractant100 µLC 6 H 5 Cl (150 µL) - CHCl 3 (75 µL) Sonication time20 min10 min Centrifugation time3 min10 min 26

27. Pesticide Without UASEME UASEME using non-ionic surfactant Linear range (µg/mL) LOD (µg/mL) % RSD (n=5) Linear range (µg/mL) LOD (µg/mL) % RSD (n=5) EF tRtR AreatRtR CBF 0.2-10.00.051.032.27 0.006-5.000.00150.592.7633 CBR 0.1-10.00.020.863.86 0.002-5.000.00040.581.6450 IPC 2.0-15.00.501.224.85 0.02-5.000.00700.403.2971 MTC 0.2-10.00.050.743.85 0.002-5.000.00040.362.86125 PMC 1.0-15.00.501.753.92 0.006-5.000.00300.334.86166 27 Linear range: 0.002 – 5.00 µg mL -1 LOD: 0.4 – 7.0 µg L -1 LOQ: 1.2 – 15.0 µg L -1 RSD: <6.32 EF: 33 – 166

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29. KHONKAEN UNIVERSITY Sample % Recovery at different spiked levels (  g mL -1 ) CarbofuranCarbarylIsoprocarbMethiocarbPromecarb 0.250.501.000.250.501.000.250.501.000.250.501.000.250.501.00 Surface water Water 1999310495988987108110939795969399 Water 2949899979593989493 86988281105 Commercial juice Guava838587929897 9183878685828392 Orange98919092959699100898299100998998 Grape9692919899879989879893959799105 Apple9010010210510787838988878281808998 Fresh juice Guava91901121158998878893959792919089 Orange979692909190 10010710098978987 Grape1029098 898710510489999495919391 Sugar apple899793 929097989395100105989795 Longan9290989795989993929098999793 Watermelon90989997969394959398 97949993 Recovery: 81 – 115%

30. Pesticide Without UASEME UASEME using mixed anionic– cationic surfactant Linear range (µg/mL) LOD (µg/mL) % RSD (n=5) Linear range (µg/mL) LOD (µg/mL) % RSD (n=5) EF tRtR AreatRtR CBF 0.2-10.00.051.032.27 0.006-5.000.00050.521.71100 CBR 0.1-10.00.020.863.86 0.002-5.000.00010.550.84200 IPC 2.0-15.00.501.224.85 0.02-5.000.00300.522.05167 MTC 0.2-10.00.050.743.85 0.002-5.000.00030.360.68167 PMC 1.0-15.00.501.753.92 0.006-5.000.00500.221.92100 30 Linear range: 0.002 – 5.00 µg mL -1 LOD: 0.1 – 5.0 µg L -1 LOQ: 0.3 – 15.0 µg L -1 RSD: <6.52 EF: 100 – 200

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32. KHONKAEN UNIVERSITY Sample % Recovery at different spiked levels (µg mL -1 ) CarbofuranCarbarylIsoprocarbMethiocarbPromecarb 0.250.501.000.250.501.000.250.501.000.250.501.000.250.501.00 Surface water Water 1 10210589801021081009095949291858397 Water 2 9998879496102101989985918396110103 Commercial juice Guava 104103869594991069799938786838190 Orange 84105 11010710090919089888992102105 Grape 878988969492979390891101011128588 Apple 84929190879190102104899290999695 Fresh juice Guava 92948788959388859291102101929395 Orange 8992911109295889182889299979698 Grape 102101112988992959799100103102878992 Sugar apple 839295968987889899949391909298 Longan 8994961021081109910589889796958987 Watermelon 98889796959987838498971029910098 Recovery: 80 – 112%

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35. KHONKAEN UNIVERSITY LiChroprep  RP-18 (25 – 40  m)  -column 34  L 6 mL 80% acetonitrile in 0.1% acetic acid (200  L) Column Detection Mobile phase Injection flow rate Atlantis C18 (5 μm, 150 x 4.6 mm) 270 nm ACN and 0.1% acetic acid (gradient elution) 20  L 1.0 mL min -1 35

36. 1. Bead Packing (conditioning) plug Bead analyte HPLC analyte Interference 2. Sample loading 3. Washing 5. Bead discarding Sample analyteInterference 4. Elution 36

37. Pesticide HPLCSI-BI-LOV-HPLC Linear range (µg/mL) LOD (µg/mL) % RSD (n=5)Linear range (µg/mL) LOD (µg/mL) % RSD (n=5) tRtR AreatRtR MTM2.0-15.01.000.322.520.2-10.00.020.683.67 PPX0.2-10.00.100.183.500.01-7.00.0020.263.67 CBF0.2-10.00.100.163.420.01-7.00.0020.663.48 CBR0.1-10.00.020.283.280.005-7.00.0010.303.28 IPC2.0-15.00.500.404.100.04-12.00.0040.725.37 MTC0.2-10.00.050.231.820.01-6.00.0010.252.61 PMC1.0-15.00.500.144.790.1-12.00.010.252.37 Preconcentration factors = 20 – 125 37 Linear range: 0.005 – 12.00 µg mL -1 LOD: 0.001 – 0.020 µg mL -1 LOQ: 0.003 – 0.060 µg mL -1 RSD: <9.56 EF: 20 – 125

38. KHONKAEN UNIVERSITY 1, methomyl 2, propoxur 3, carbofuran 4, carbaryl 5, isoprocarb 6, methiocarb 7, promecarb HPLC SI-BI-LOV-HPLC

39. KHONKAEN UNIVERSITY Sample Amount found (  g L -1,  g g -1 ) MTMPPXCBFCBRIPCMTCPMC Water 1 ------- Water 2 ------- Water 3 ------- watermelon -0.020.010.100.390.260.29 guava ---0.090.19-- cowpea --0.160.10--- cabbage -0.040.060.080.220.020.73 Range: 0.01– 0.73 μg g -1 39

40. KHONKAEN UNIVERSITY Sample Spiked level %recovery MethomylPropoxurCarbofuranCarbarylIsoprocarbMethiocarbPromecarb Water I 1  g mL -1 108105 10010893 2  g mL -1 8893961039810493 Water II 1  g mL -1 108107103 10610295 2  g mL -1 891049310199101102 Water III 1  g mL -1 102 1031059310798 2  g mL -1 88939610310510494 Watermelon 1  g g -1  10310794 10298 2  g g -1  991081029790103 Guava 1  g g -1  105 10096105102 2  g g -1  101 10298104102 Cowpea 1  g g -1  10290100101 2  g g -1  999799102101104 Cabbage 1  g g -1  989710192100103 2  g g -1  105100969210093 Recovery: 88 – 108% 40

41. 1. The novel automated flow-based system with suitable sample preparation was successfully developed for fast screening of carbamate insecticides. 2. The HPLC conditions for simultaneous determination of carbamate insecticides were obtained. 3. The simple microextraction procedures prior to HPLC analysis were obtained. 4. The UASEME procedures using both non-ionic surfactant and mixed anionic – cationic surfactants for preconcentration of carbamate insecticides were obtained. 5. The on-line sample preparation system coupled to HPLC technique was obtained for automated extraction and preconcentration of carbamates prior to HPLC analysis. 6. The developed methodologies were successfully applied for determination for carbamates in various samples.

42. Prof. Dr. Tadao Sakai, AIT (JAPAN) Prof. Dr. Norio Teshima, AIT (JAPAN) Asst. Prof. Dr. Rodjana Burakham, KKU

44. Thank you for your kind attention