Sample preparation and analytical method development for monitoring of carbamate insecticides Jitlada Vichapong Department of Chemistry Faculty of Science Khon Kaen University

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

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Carbamate Insecticides “Maximum residue limits (MRLs), mg kg -1 ” Crop MRLs, mg kg -1 EUCodexASEANKFDATACFS* Raw fruit Vegetable *TACFS: Thai Agricultural Commodity and Food Standard 4

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

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

KHONKAEN UNIVERSITY Reduce Alternative solvents  7

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

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

KHONKAEN UNIVERSITY 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

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

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KHONKAEN UNIVERSITY (Potassium ferricyanide) 13

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

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 Isoprocarb APCarbaryl0.04 Carbofuran0.08 LODs for the determination of carbamates 15

SampleRecovery (%) Spiked level (µg mL -1 ) Water I Water II Water III Spiked level (µg g -1 ) Chinese white cabbage Cabbage Cauliflower Cucumber Baby corn Green grape Grapefruit Guava Apple Pear Recovery: 85 – 112% 16

A solvent for micro-extraction of carbamates 2

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

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

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 PPX CBF CBR IPC MTC PMC Linear range: µg mL -1 LOD: – µg mL -1 LOQ: – µg mL -1 RSD: <10.17 EF:

Without RTIL-LLME RTIL-LLME 21

KHONKAEN UNIVERSITY SampleMarket Amount found, μg g -1 PPXCBFCBRIPCMTCPMC WatermelonLocal Market Super Market I Super Market II Cantaloupe1Local Market Super Market I Super Market II Cantaloupe2Local Market StrawberryLocal Market Super Market I OrangeLocal Market Super Market I Super Market II AppleSuper Market I Super Market II Application to sample Range: 0.01– μg g -1 22

KHONKAEN UNIVERSITY SampleRecovery (%) PPXCBFCBRIPCMTCPMC Water I Water II Water III Watermelon Cantaloupe Cantaloupe Strawberry Orange Apple Recovery: 82 – 117% 23

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

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

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

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 CBR IPC MTC PMC Linear range: – 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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KHONKAEN UNIVERSITY Sample % Recovery at different spiked levels (  g mL -1 ) CarbofuranCarbarylIsoprocarbMethiocarbPromecarb Surface water Water Water Commercial juice Guava Orange Grape Apple Fresh juice Guava Orange Grape Sugar apple Longan Watermelon Recovery: 81 – 115%

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 CBR IPC MTC PMC Linear range: – 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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KHONKAEN UNIVERSITY Sample % Recovery at different spiked levels (µg mL -1 ) CarbofuranCarbarylIsoprocarbMethiocarbPromecarb Surface water Water Water Commercial juice Guava Orange Grape Apple Fresh juice Guava Orange Grape Sugar apple Longan Watermelon Recovery: 80 – 112%

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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

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

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 MTM PPX CBF CBR IPC MTC PMC Preconcentration factors = 20 – Linear range: – µg mL -1 LOD: – µg mL -1 LOQ: – µg mL -1 RSD: <9.56 EF: 20 – 125

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

KHONKAEN UNIVERSITY Sample Amount found (  g L -1,  g g -1 ) MTMPPXCBFCBRIPCMTCPMC Water Water Water watermelon guava cowpea cabbage Range: 0.01– 0.73 μg g -1 39

KHONKAEN UNIVERSITY Sample Spiked level %recovery MethomylPropoxurCarbofuranCarbarylIsoprocarbMethiocarbPromecarb Water I 1  g mL  g mL Water II 1  g mL  g mL Water III 1  g mL  g mL Watermelon 1  g g -1   g g -1  Guava 1  g g -1   g g -1  Cowpea 1  g g -1   g g -1  Cabbage 1  g g -1   g g -1  Recovery: 88 – 108% 40

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.

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

Thank you for your kind attention