Sample preparation and analytical method development for monitoring of

carbamate insecticides

Jitlada VichapongDepartment of Chemistry

Faculty of ScienceKhon Kaen University

e-mail: jitlada_v@hotmail.com

PESTICIDES

Insecticides♣ Organochlorines♣ Organophosphates♣ Carbamates♣ Synthetic

Pyrethroids

Herbicides

Rodenticides

Fungicides

2

Contamination of pesticides

3

Carbamate Insecticides“Maximum residue limits (MRLs), mg kg-1”

CropMRLs, mg kg-1

EU Codex ASEAN KFDA TACFS* 9002-2008

Raw fruit 0.02-1 0.1-10 0.1-10 0.1-3.0 0.2-15

Vegetable 0.02-2 0.2-3.0 0.2-3.0 0.1-5.0 0.1-3

*TACFS: Thai Agricultural Commodity and Food Standard 4

KHONKAENUNIVERSITY

ANALYSIS OF PESTICIDES

Test kit

Spectrophotometry

Gas chromatographyHigh performance liquid chromatography

5

Require sample preparation

Extraction

Sample clean-up

Matrix removal

Analyte preconcentration

Carbamates in food & environmental matrices

6

KHONKAENUNIVERSITY

“GREEN ANALYTICAL CHEMISTRY”

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

KHONKAENUNIVERSITY

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

１

KHONKAENUNIVERSITY

SI-LOV FOR CARBAMATE SCREENING

12

34

5

6

1

2

3

45

67

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

KHONKAENUNIVERSITY

250 µL NaOH

200 µL carbaryl

400 µL ANSA

50 µL NaNO2

100 µL HCl

Holding coil

Direction to flow cell

THE REACTION MECHANISM USING ANSA REAGENT

THE SEQUENCE ORDER 11

KHONKAENUNIVERSITY

---> REACTION MECHANISM USING DMA REAGENT

THE SEQUENCE ORDER

12

KHONKAENUNIVERSITY

THE SEQUENCE ORDER USING 4-AP REAGENT

(Potassium ferricyanide)

13

235 240 245 250 255 260

-0.6

-0.4

-0.2

0.0

0.2

0.4

0.6A

bs

(AU

)

Time (sec)

Blank

CBR 0.05 g mL-1

CBR 0.5 g mL-1

CBR 1.0 g mL-1

CBR 3.0 g mL-1

CBR 5.0 g mL-1

SI-grams of carbaryl – ANSA derivative

225 230 235 240 245-0.6

-0.4

-0.2

0.0

0.2

0.4

0.6

Ab

s (A

U)

Time (sec)

Blank

0.05 g mL-1

0.5 g mL-1

1.0 g mL-1

3.0 g mL-1

5.0 g mL-1

SI-grams of carbaryl – DMA derivative

225 230 235 240 245 250-0.6

-0.4

-0.2

0.0

0.2

0.4

0.6

Abs

(AU

)

Time (sec)

Blank

0.05 g mL-1

0.5 g mL-1

1.0 g mL-1

3.0 g mL-1

5.0 g mL-1

SI-grams of carbaryl – 4-AP derivative

14

KHONKAENUNIVERSITY

Reagent Carbamate detected LOD (µg mL-1)

ANSA Carbaryl 0.02

Methiocarb 0.07

Carbofuran 0.08

DMA Carbaryl 0.02

Carbofuran 0.02

Methiocarb 0.02

Promecarb 0.07

Methomyl 0.08

Propoxur 0.08

Isoprocarb 0.08

4-AP Carbaryl 0.04

Carbofuran 0.08

LODs for the determination of carbamates

15

Sample Recovery (%) Spiked level (µg mL-1) 0.50 1.00 3.00

Water I 92 95 110

Water II 93 99 114

Water III 98 102 95

Spiked level (µg g-1) 0.25 0.50 0.75

Chinese white cabbage 93 89 85

Cabbage 90 92 87

Cauliflower 95 97 100

Cucumber 89 98 102

Baby corn 109 95 97

Green grape 99 93 110

Grapefruit 112 98 87

Guava 95 110 97

Apple 98 93 99

Pear 88 90 97

Ｒｅｃｏｖｅｒy of the spiked

samples Recovery: 85 – 112%

16

Room temperature ionic liquid(RTILs)

A solvent for micro-extraction of carbamates

2

1-butyl-3-methylimidazolium hexafluorophosphate[C4mim][PF6] NN

CH3C4H9

PF6-

18

Analytes

RTIL

IL phase

Aqueous phase

RTIL for extraction of carbamate pesticides

Matrix

IL phase

Sample

Acetonitrile

HPLC

KHONKAENUNIVERSITY

CONDITION

Sample volume 3 mL

[C4MIM][PF6] 100 L

Extraction time 7 min

Dissolving solvent 50 L acetonitrile(to decrease the viscosity)

IL phase

ColumnDetectionMobile phase

Injectionflow rate

Atlantis C18 (5 μm, 150 x 4.6 mm)270 nmACN and 0.1% acetic acid (gradient elution)20 L1.0 mL min-1

19

KHONKAENUNIVERSITY

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

tR Area tR Area

MTM 2.0-15.0 1.00 0.32 2.52 - - - - -

PPX 0.2-10.0 0.10 0.18 3.50 0.02-5.00 0.005 0.52 10.17 20.0

CBF 0.2-10.0 0.10 0.16 3.42 0.02-5.00 0.005 0.48 3.96 20.0

CBR 0.1-10.0 0.02 0.28 3.28 0.01-5.00 0.002 0.32 5.89 10.0

IPC 2.0-15.0 0.50 0.40 4.10 0.04-8.00 0.020 0.62 6.56 25.0

MTC 0.2-10.0 0.05 0.23 1.82 0.05-8.00 0.005 0.49 8.40 10.0

PMC 1.0-15.0 0.50 0.14 4.79 0.40-8.00 0.040 0.30 4.90 12.5

Analytical performance

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: <10.17EF: 10 - 25

RTIL-LLME for carbamates

Without RTIL-LLME RTIL-LLME

21

KHONKAENUNIVERSITY

Sample Market Amount found, μg g-1

PPX CBF CBR IPC MTC PMCWatermelon Local Market - - - - 0.10 0.17  Super Market I - - - 1.28 0.60 1.34  Super Market II 0.04 0.53 0.06 - 0.45 2.61               Cantaloupe1 Local Market - - 0.06 - 0.01 -  Super Market I - - - - 0.37 -  Super Market II - - - - 0.45 -               Cantaloupe2 Local Market 0.02 - 0.06 - 0.11 0.55

               Strawberry Local Market - - - - - -  Super Market I - 0.11 0.02 0.54 0.43 1.55               Orange Local Market - - 0.07 0.18 4.50 -  Super Market I 0.72 0.21 0.16 1.50 0.94 1.81  Super Market II 0.22 0.18 - 0.9 0.38 21.25               Apple Super Market I 0.03 - - - 0.45 1.24  Super Market II - - - - 0.38 1.30               

Application to sample

Range: 0.01– 21.25 μg g-1

22

KHONKAENUNIVERSITY

Sample Recovery (%)

PPX CBF CBR IPC MTC PMCWater I 90 99 102 98 98 89Water II 92 96 87 93 93 104Water III 100 95 103 100 116 94             

Watermelon 115 88 81 90 93 85  96 100 106 92 104 101  96 100 103 103 93 88             

Cantaloupe1 98 103 82 118 117 104  95 96 104 107 101 107

  93 95 98 105 93 94             

Cantaloupe2 83 99 93 101 88 86             

Strawberry 93 102 82 92 103 92  107 103 99 101 107 82             

Orange 83 98 85 107 102 110  89 110 106 106 99 98  100 99 99 98 108 97             

Apple 105 103 102 92 94 92  100 104 99 104 97 112

Recovery

Recovery: 82 – 117%

23

Surfactant-based microextractionUltrasound-assisted surfactant-enhanced emulsification microextraction (UASEME)

OS

O Na

OOSodium dodecylsulphate (SDS)

N BrCetyltrimethyl ammonium bromide (CTAB)

O

OO

OOH

wO

OOH

O

xHO

z y

Tween 20

non-ionic surfactant

Mixed anionic-cationic surfactant 24

dissolving solvent+

Add surfactant and extraction solvent

Sonicated

Principle of UASEME

Sample Cloudy

Phase separation

UASEME with non-ionic surfactant

& mixed anionic–cationic surfactant ４

3

Standard/Sample solution

Column

DetectionMobile phaseInjection volumeMobile phase flow rate

ChromolithFastGradient column (RP-18e, 50–2 mm)270 nm37% MeOH20 L0.5 mL min-1

Sedimented phase 25

ConditionsParameter Condition

non-ionic surfactant

mixed anionic–cationic surfactant

Surfactant Tween 20 SDS - CTAB

Salt Na2SO4 7.0% w/v No salt

Conc. of surfactant 0.01 mol L-1 SDS (0.05 mol L-1) - CTAB (0.01 mol L-1)

Volume of surfactant 30 µL SDS (45 µL) - CTAB (30 µL)

Extractant C6H5Cl C6H5Cl - CHCl3

Volume of extractant 100 µL C6H5Cl (150 µL) - CHCl3 (75 µL)

Sonication time 20 min 10 min

Centrifugation time 3 min 10 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

tR Area tR Area

CBF 0.2-10.0 0.05 1.03 2.27 0.006-5.00 0.0015 0.59 2.76 33

CBR 0.1-10.0 0.02 0.86 3.86 0.002-5.00 0.0004 0.58 1.64 50

IPC 2.0-15.0 0.50 1.22 4.85 0.02-5.00 0.0070 0.40 3.29 71

MTC 0.2-10.0 0.05 0.74 3.85 0.002-5.00 0.0004 0.36 2.86 125

PMC 1.0-15.0 0.50 1.75 3.92 0.006-5.00 0.0030 0.33 4.86 166

Method performance UASEME with non-ionic surfactant

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: <6.32EF: 33 – 166

KHONKAENUNIVERSITY

UASEME using Non-ionic surfactant

28

KHONKAENUNIVERSITY

Sample

% Recovery at different spiked levels (g mL-1)

Carbofuran Carbaryl Isoprocarb Methiocarb Promecarb

0.25 0.50 1.00 0.25 0.50 1.00 0.25 0.50 1.00 0.25 0.50 1.00 0.25 0.50 1.00

Surface water

Water 1 99 93 104 95 98 89 87 108 110 93 97 95 96 93 99

Water 2 94 98 99 97 95 93 98 94 93 93 86 98 82 81 105

Commercial juice

Guava 83 85 87 92 98 97 97 91 83 87 86 85 82 83 92

Orange 98 91 90 92 95 96 99 100 89 82 99 100 99 89 98

Grape 96 92 91 98 99 87 99 89 87 98 93 95 97 99 105

Apple 90 100 102 105 107 87 83 89 88 87 82 81 80 89 98

Fresh juice

Guava 91 90 112 115 89 98 87 88 93 95 97 92 91 90 89

Orange 97 96 92 90 91 90 90 100 107 100 98 97 89 87 87

Grape 102 90 98 98 89 87 105 104 89 99 94 95 91 93 91

Sugar apple 89 97 93 93 92 90 97 98 93 95 100 105 98 97 95

Longan 92 90 98 97 95 98 99 93 92 90 98 99 97 93 93

Watermelon 90 98 99 97 96 93 94 95 93 98 98 97 94 99 93

Recovery: 81 – 115%

Recovery

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

tR Area tR Area

CBF 0.2-10.0 0.05 1.03 2.27 0.006-5.00 0.0005 0.52 1.71 100

CBR 0.1-10.0 0.02 0.86 3.86 0.002-5.00 0.0001 0.55 0.84 200

IPC 2.0-15.0 0.50 1.22 4.85 0.02-5.00 0.0030 0.52 2.05 167

MTC 0.2-10.0 0.05 0.74 3.85 0.002-5.00 0.0003 0.36 0.68 167

PMC 1.0-15.0 0.50 1.75 3.92 0.006-5.00 0.0050 0.22 1.92 100

Method performanceUASEME with mixed anionic–cationic surfactant

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: <6.52EF: 100 – 200

KHONKAENUNIVERSITY

UASEME using Mixed anionic–cationic surfactant

31

KHONKAENUNIVERSITY

Sample

% Recovery at different spiked levels (µg mL-1)

Carbofuran Carbaryl Isoprocarb Methiocarb Promecarb

0.25 0.50 1.00 0.25 0.50 1.00 0.25 0.50 1.00 0.25 0.50 1.00 0.25 0.50 1.00

Surface water Water 1 102 105 89 80 102 108 100 90 95 94 92 91 85 83 97Water 2 99 98 87 94 96 102 101 98 99 85 91 83 96 110 103

Commercial juice Guava 104 103 86 95 94 99 106 97 99 93 87 86 83 81 90Orange 84 105 105 110 107 100 90 91 90 89 88 89 92 102 105Grape 87 89 88 96 94 92 97 93 90 89 110 101 112 85 88Apple 84 92 91 90 87 91 90 102 104 89 92 90 99 96 95

Fresh juice Guava 92 94 87 88 95 93 88 85 92 91 102 101 92 93 95Orange 89 92 91 110 92 95 88 91 82 88 92 99 97 96 98Grape 102 101 112 98 89 92 95 97 99 100 103 102 87 89 92Sugar apple 83 92 95 96 89 87 88 98 99 94 93 91 90 92 98Longan 89 94 96 102 108 110 99 105 89 88 97 96 95 89 87Watermelon 98 88 97 96 95 99 87 83 84 98 97 102 99 100 98

Recovery: 80 – 112%

Recovery

UASEME using mixed anionic–cationic surfactant

KHONKAENUNIVERSITY

Online SPE-HPLC５

KHONKAENUNIVERSITY

SI-BI-LOV-HPLC

LiChroprep RP-18 (25 – 40 m)

-column 34 L

6 mL80% acetonitrile in 0.1% acetic acid (200 L)

ColumnDetectionMobile phase

Injectionflow rate

Atlantis C18 (5 μm, 150 x 4.6 mm)270 nmACN and 0.1% acetic acid (gradient elution)20 L1.0 mL min-1

35

1. Bead Packing (conditioning)

plugBead

analyte HPLC

analyteInterference

2. Sample loading

3. Washing

5. Bead discarding

Sample

analyte Interference

4. Elution

SI-BI-LOV-HPLC

36

Pesticide HPLC SI-BI-LOV-HPLC

Linear range

(µg/mL)

LOD (µg/mL)

% RSD (n=5) Linear range

(µg/mL)

LOD (µg/mL)

% RSD (n=5)

tR Area tR Area

MTM 2.0-15.0 1.00 0.32 2.52 0.2-10.0 0.02 0.68 3.67

PPX 0.2-10.0 0.10 0.18 3.50 0.01-7.0 0.002 0.26 3.67

CBF 0.2-10.0 0.10 0.16 3.42 0.01-7.0 0.002 0.66 3.48

CBR 0.1-10.0 0.02 0.28 3.28 0.005-7.0 0.001 0.30 3.28

IPC 2.0-15.0 0.50 0.40 4.10 0.04-12.0 0.004 0.72 5.37

MTC 0.2-10.0 0.05 0.23 1.82 0.01-6.0 0.001 0.25 2.61

PMC 1.0-15.0 0.50 0.14 4.79 0.1-12.0 0.01 0.25 2.37

Method performance

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: <9.56EF: 20 – 125

KHONKAENUNIVERSITY

1, methomyl

2, propoxur

3, carbofuran

4, carbaryl

5, isoprocarb

6, methiocarb

7, promecarb

HPLC

SI-BI-LOV-HPLC

Online preconcentration

KHONKAENUNIVERSITY

Sample Amount found (g L-1, g g-1)

MTM PPX CBF CBR IPC MTC PMCWater 1 - - - - - - -

Water 2 - - - - - - -

Water 3 - - - - - - -

watermelon - 0.02 0.01 0.10 0.39 0.26 0.29

guava - - - 0.09 0.19 - -

cowpea - - 0.16 0.10 - - -

cabbage - 0.04 0.06 0.08 0.22 0.02 0.73

Sample determination

Range: 0.01– 0.73 μg g-1

39

KHONKAENUNIVERSITY

SampleSpiked level

%recovery

Methomyl Propoxur Carbofuran Carbaryl Isoprocarb Methiocarb Promecarb

Water I 1 g mL-1 108 105 105 105 100 108 93

  2 g mL-1 88 93 96 103 98 104 93

Water II 1 g mL-1 108 107 103 103 106 102 95

  2 g mL-1 89 104 93 101 99 101 102

Water III 1 g mL-1 102 102 103 105 93 107 98

  2 g mL-1 88 93 96 103 105 104 94

Watermelon 1 g g-1 103 107 94 94 102 98

  2 g g-1 99 108 102 97 90 103

Guava 1 g g-1 105 105 100 96 105 102

  2 g g-1 101 101 102 98 104 102

Cowpea 1 g g-1 102 90 100 101 101 101

  2 g g-1 99 97 99 102 101 104

Cabbage 1 g g-1 98 97 101 92 100 103

  2 g g-1 105 100 96 92 100 93

RECOVERY

Recovery: 88 – 108%

40

CONCLUSION1. 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.

ACKNOWLEDGEMENTS

Prof. Dr. Tadao Sakai, AIT (JAPAN)

Prof. Dr. Norio Teshima, AIT (JAPAN)

Asst. Prof. Dr. Rodjana Burakham, KKU

ACKNOWLEDGEMENTS

Thank you for your kind attention