N-Methyl carbamate pesticides

analysis system
 N-Methyl carbamate pesticides
analysis system

1. Three(3) types of measurement conditions

1. Official methods
2. 11 kinds of components simultaneous analysis
3. 18 kinds of components simultaneous analysis
2. High sensitivity fluorescence detection
3. Tools for stable and high sensitivity analysis
Line filter, Clean up column
 Structural formula of carbamate pesticides
H3C H3C H3C
O O O O O O O
(CH3 ) 2NC C NOCNHCH3 CH3 S O C NHCH3 CH3 O S O C NHCH3 CH3 S O C NHCH3
SCH3
H3C H3C H3C
Oxamyl Methiocarb Sulfone Methiocarb
Sulfoxide

CH3 O O O
H3C CH3
CH3NH C O CH3NH C O CH3 O
H3C S C CH N O C NH CH3
O CH CHCH2CH3 (CH3 ) 2N C O N
3
CH3 N
N(CH3 ) 2
O CH3
Aldicarb Bendiocarb Fenobucarb Pirimicarb

O O O O O
O C NHCH3 CH3NH C O CH3NH C O CH3 CH3NH C O CH3NH C O
CH2SCH2CH3 CH CH3

CH3 CH3
CH3
Carbaryl(NAC) Ethiofencarb Isoprocarb Metholcarb(MTMC) Xylylcarb(MPMC)
O O O
CH3 CH3
CH3N C O N C CH3NH C O CH3NH C O O
O SCH3 SCH3
S OCH(CH3 ) 2 O CH3 CH3NH C O
CH3NH C O N C SCH3
CH3 CH3N C O N C CH3 CH3
CH3
Methomyl O Propoxur(PHC) Carbofuran Macbal(XMC)
Thiodicarb
Principle of carbamate pesticides analysis

Step 1: Alkali hydrolysis

O
0.05N NaOH
R O C NHCH3 CH3NH2 + R OH + H2CO3
Heating
N-Methyl carbamete Methylamine

Step 2: Luminescence reaction with o-phtahalaldehyde (OPA)

O SCH2CH2OH
C H Borate buffer
CH3NH2 HSCH2CH2OH N CH3
+ + （pH 9.2）
C H
O 2-mercaptoethanol iso-indole derivatives
OPA
Flow diagram of N-Methyl carbamate
analysis system
Reaction coil
Clean up column
0. 5 I.D. x 2m

Precolumn Carbamatepak CO
PU AS FP
Column

Cooling
Backpressure
coil
LG 0.25I.D. x 1m
coil
0.25 I.D. x 1m
RO
DG 80℃

Reaction coil
0.5 I.D. x 2m
Backpressure Backpressure
Linefilter
capillary capillary

PU PU
 Measurement conditions specified in
official method
Column: Octadecylsilyl silica gel (3.9 mm I.D. x 150 mmL)
Column temperature: 40 degree C
Mobile phase: A: H2O, B: CH3OH, C: THF
Flow rate: 0.6 ml/min
Reagents 1: 50 mMNaOH aq.
Reagent flow rate 1: 0.5 ml/min
Reagents 2: 50 mM Na2B4O7・10H2O / 0.4 mg
OPA in 5 ml CH3OH /2-mercaptoethanol (1000/5/1)
Reagent flow rate 2: 0.5 ml/min
Gradient conditions
Reaction temperature: 80 to 100 °C
Detection : Ex; 340 nm, Em; 455 nm, Gain; x100 A B C
0min 88% 12% 0%
0.1min 90% 0% 10%
20.0min 70% 0% 30%
29.9min 70% 0% 30%
30.0min 88% 12% 0%
1cycle 40min
Chromatogram of carbamete pesticides
(official method)
Concentration of each additive component : 10 ppb
Injection volume : 50 µl
Extraction pre-treatment of carbamate
pesticides in food (official method)
Sample 20g
（H2O 30mL, 2.0h）
Acetone 200mL
Homogenize 5 mL
Filtration (Diatomaceous earth)

Filtrate Residue
Acetone 50mL
Stirring, Filtration
Filtrate

evaporate
0.3mL fractionation Dichloromethane 100 mL
5% NaCl aq. 200 mL
5% NaCl aq. 200 mL
Dichloromethane layer Water
Dichloromethane 100 mL
Boiling
Dichloromethane layer

Dehydration with Na2SO4

Low pressure dry n-Hexane 25mL
CH3CN (saturating with n-Hex)
MeOH 2.0mL 30mL x 3
0.3mL fractionation
dil HCl(pH 3) 3.0mL CH3CN layer
Dichloromethane 100 mL
Filtration CH3CN layer
Inject filtrate into HPLC
Chromatogram of carbamate pesticides added
to extractions of rice (official method)
Concentration of each additive component : 10 ppb
Injection volume : 50 µl
 JASCO Measurement conditions
for the separation of 11 kinds of components

Column: CarbamatePak (4.0 mm I.D. x 150 mmL)

Column temperature: 40 degree C
Mobile phase: A: H2O, B: CH3OH
Flow rate: 1.0 ml/min
Reagents 1: 50 mMNaOH aq.
Reagent flow rate 1: 0.5 ml/min Gradient conditions
Reagents 2: 50 mM Na2B4O7・10H2O / 0.4 mg OPA A B
in 5 ml CH3OH /2-mercaptoethanol (1000/5/0.5) 0min 85% 15%
Reagent flow rate 2: 0.5 ml/min
20.0min 35% 65%
Reaction temperature: 80 °C
Detection: Ex; 339 nm, Em; 455 nm, Gain; x100 25.0min 35% 65%
25.1min 0% 100%
30.0min 0% 100%
30.1min 85% 15%
1cycle 50min
 Chromatogram of 11 components
of carbamate pesticide (standard samples)
μV 6
2.0E+05
1.0ppm,10µl Injection
1
9

7
1.5E+05
10
5 11

3
2
1.0E+05 4

5.0E+04

8
0.0E+00

5.00 10.00 15.00 20.00 25.00 30.00

[min]

1. Oxamyl 7. Ethiofencar
2. Methiocarb sulfoxide 8. Pirimicarb 　
3. Methiocarb sulfone 9. Isoprocarb(MIPC) 　
4. Aldicarb 10.Fenobcarb(BPMC)
5. Bendiocarb 11.Methiocarb
6. Carbaryl
 JASCO Measurement conditions
for the separation of 18 kinds of components
Column: CarbamatePak (4.0 mm I.D. x 150 mmL)
Column temperature: 40 °C
Mobile phase: A: H2O, B: CH3OH, C: THF
Flow rate: 1.0 ml/min
Reagents 1: 50 mMNaOH aq.
Reagent flow rate 1: 0.5 ml/min Gradient conditions
Reagents 2: 50 mM Na2B4O7・10H2O / 0.4 mg OPA
A B C
in 5 ml CH3OH /2-mercaptoethanol (1000/5/0.5)
Reagent flow rate 2: 0.5 ml/min 0min 88% 12% 0%
Reaction temperature: 80 °C 0.2min 88% 12% 0%
Detection: Ex; 339 nm, Em; 455 nm, Gain; x100 3.0min 90% 0% 10%
20.0min 85% 0% 15%
35.0min 70% 0% 30%
40.0min 70% 0% 30%
40.1min 88% 12% 0%
1cycle 50min
 Chromatogram of 18 kinds of components of
carbamate pesticide (standard samples)
uV
2.0E+04

10 ppb

2
1.6E+04

17
16
13

18
8
3

12

14
10
4

15
6

11
5
1

1.2E+04

7
8.0E+03
2

100 ppb

16
6.0E+04

13

17
12

14
3

18
4

10

15
11
6

9
5

4.0E+04
1

2.0E+04
7

0.0E+00
10.0 20.0 30.0 40.0 [min]

1. Oxamyl 7. Pirimicarb 13.Macbal(XMC)　

2. Methomyl 8. Metholcarb(MTMC)　 14.Carbaryl　　
3. Methiocarb sulfoxide 9. Propoxur(PHC) 15.Ethiofencarb　
4. Aldicarb 10.Carbofuran 16.Isoprocarb(MIPC)
5. Methiocarb sulfone 11.Bendiocarb 17.Fenobcarb(BPMC)
6. Thiodicarb 12.Xylylcarb(MPMC) 18.Methiocarb
 Detection limit of carbamate pesticides
Compornents (A) Detection limit* (B) Minimum standard Ratio between (A) and (B)
detection concentration**

Oxamyl 0.54 20 37
Methiocarb sulfoxide 0.36 50 137
Methiocarb sulfone 0.32 50 152
Aldicarb 0.56 20 35
Bendiocarb 0.36 20 56
Carbaryl 0.26 100 396
Ethiofencarb 0.26 100 374
Isoprocarb 0.22 500 2190
Fenobucarb 0.36 300 836
Methiocarb 0.42 50 118

* Injection volume

** Notice of Ministry of Health and Welfare No. 166

 Reproducibility

（Concentration: 20ppb, Injection volume: 50µl, n=5）

Compoments Retention time (min) Peak height(µV) Peak area (mV sec)

AV. S.D. R.S.D.(%) AV. S.D. R.S.D.(%) AV. S.D. R.S.D.(%)

Oxamyl 7.053 0.0049 0.069 30266 209 0.69 334369 2703 0.78

Methiocarb sulfoxide 12.030 0.0078 0.065 13909 162 1.17 167168 1797 1.08

Methiocarb sulfone 13.481 0.0103 0.076 6063 272 4.49 70661 2618 3.71

Aldicar 15.691 0.0124 0.079 20345 134 0.66 259049 4301 1.66

Bendiocarb 18.667 0.0122 0.065 15682 172 1.10 204956 4937 2.41

Carbaryl 19.892 0.0121 0.061 18425 131 0.71 226471 5722 2.53

Ethiofencarb 20.524 0.0120 0.059 14743 212 1.44 182936 6307 3.45

Isoprocarb 21.795 0.0161 0.074 18081 317 1.76 226641 6834 3.02

Fenobucarb 24.356 0.0192 0.079 13120 215 1.64 172654 2573 1.49

Methiocarb 25.003 0.0237 0.095 6712 154 2.30 90581 1729 1.91

AV.: Average S.D.: Standard deviation R.S.D. : Relative standard deviation

 GPC Pre-treatment system

Procedure
Sample
Comparing with conventional pre-treatment
method based on solvent extraction, using GPC
for pre-treatment of residual pesticides makes Solvent extraction
possible to save a lot of time.
It is used for removing fat (Triglyceride etc.) and GPC Clean-up
pigments (chlorophyl etc.)
Preparative

HPLC, GC
GPC Chromatogram of standard samples and
brown rice uV
2
3.0E+5
1
3
2.0E+5
4 Calibration solution 1
1.0E+5 5

0.0

6
6.0E+5
Calibration solution
4.0E+5 7 of target components 2
2.0E+5

0.0

4.0E+5

Brown rice + STD

2.0E+5

0.0
10.0 20.0 30.0 40.0
[min]

Preparatives (Eluent position of target components)

Preparative column: Shodex CLNpak EV-2000 (20mm I.D. x 300mm L)，

Shodex CLNpak EV-G (20mm I.D. x 100mm L)
Column temperature: room temperature
Solvent: Cyclohexane/Ethyl Acetate(70/30)
Flow rate: 5.0 mL/min
Injection volume: 5.0 mL
Detection wavelength: 254 nm
Standard samples: 1. Cone oil , 2. Bis(2-ethyl hexyl) phthalete, 3. Methoxychlor,
4. Perylene, 5. Sulfer, 6. Quinomethionate, 7. Fulvalinate
Chromatogram of brown rice added carbamate
uV
pesticides
14 Standards 1000ppb
2.0E+5 8
3 13
16 18
12
2 9
17
10
11 15
1 5
1.0E+5 4

7
0.0

STD + Brown rice

1.2E+5
14
8 13
16 18
3 9 12
8.0E+4 10
2 17
1 11

4.0E+4 5

4
6 7
0.0

10.0 20.0 30.0 40.0 50.0 60.0

[min]

18 components of standards ware added to be 1 ppm

 N-methyl carbamate analysis with semi-micro
HPLC
μV
［Conditions］
Column: CarbamatePak
(2.1mm I.D. x 150mmL)
Column temp.: 40 °C
1.0E+04 Eluent: A; H2O / B; CH3OH / C; THF
13 15
Gradient eluent 1cycle 55min
8 17
3 4 9 16 18 Flow rate: 0.2ml/min
1 2 10 11 12 14
5 6 Reaction Eluent-1: 50mM NaOH
0.0E+00 0.1ml/min 80°C
Reaction Eluent-2: OPA* 0.1ml/min 40°C
13 16 17
Wave length: W.L.Ex 339nm,
4 8 12
4.0E+04
1
3
9 14 18
W.L.Em 455nm
11 15
2 10 Injection volume: 5µL
6
2.0E+04 5 *) Sodium Tetraborate buffer/OPA/Mercaptoethanol
(1000/5/0.05)
7
0.0E+00
10.0 20.0 30.0 40.0 50.0 [min]

1. Oxamyl 7. Pirimicarb 13.Macbal(XMC)

2. Methomyl 8. Metholcarb(MTMC) 14.Carbaryl
3. Methiocarb sulfoxide 9. Propoxur(PHC) 15.Ethiofencarb
4. Aldicarb 10.Carbofuran 16.Isoprocarb(MIPC)
5. Methiocarb sulfone 11.Bendiocarb 17.Fenobcarb(BPMC)
6. Mthiodicarb 12.Xylylcarb(MPMC) 18.Methiocarb
 Summary

The best method can be selected from 3 types of methods
for the target components of pesticides

The system offers a good reproducibility with high
sensitivity over 35 times of standard

Stable and high sensitivity analysis