Orbencarb

Materials to be Wheat, soybean, potato and soil

analyzed
Instrumentation Gas-chromatographic determination

1 Introduction

Chemical name (S )-2-Chlorobenzyl diethylthiocarbamate

(IUPAC)
O

Structural formula S N

CI

Empirical formula C12 H16 ClNOS

Molar mass 257.8
Melting point 9.0 ◦ C
Boiling point 158 ◦ C at 1 mmHg
Vapor pressure 12.4 mPa (20 ◦ C)
Solubility Water 24 mg L−1 (20–27 ◦ C)
Very soluble in organic solvents, e.g. acetone, xylene,
n-hexane, ethanol and benzene, all >1 kg L−1 (room
temperature)
Stability Stable to hydrolysis for 60 days at pH 5–9 (20 ◦ C)
Use pattern Orbencarb is a systemic pre-emergence herbicide to
control most annual grasses and broad-leaved weeds
Regulatory position The definition of residues includes orbencarb and its
metabolites, methyl 2-chlorobenzylsulfone (I) and 2-
chlorobenzoic acid (II)

2 Outline of method
The determination of orbencarb in crops and soil is conducted by simultaneous
analysis with its metabolites. In crops, orbencarb, I and II are extracted with acidic

Handbook of Residue Analytical Methods for Agrochemicals.


C 2003 John Wiley & Sons Ltd.
520 Individual compounds

acetone by ultrasonication and transferred into an ethyl acetate–hexane mixture.

After separating II with an alkaline aqueous solution, orbencarb and I remain in the
organic solvent layer. Residues are purified by silica gel column chromatography and
quantified by gas chromatography/electron capture detection (GC/ECD). Orbencarb
and I in soil are extracted by refluxing with water containing methanol, transferred
into dichloromethane, purified by silica gel column chromatography and quantified
by gas chromatography/flame photometric detection (GC/FPD).

O O
OH S

CI CI O
(I) (II)

3 Equipment
Crusher (coffee-mill)
Homogenizer (Polytron mixer)
Ultrasonic cleaner: UC-6100, 600 W, 28 kHz (Sharp)
Round-bottom flasks: 200-, 300-, and 500-mL
Conical beaker: 500-mL
Buchner funnel: 10-cm i.d.
Separatory funnels: 200-, 300-, and 500-mL
Glass funnel
Condenser
Glass chromatography column: 400 × 15-mm i.d. with a stopcock
Column preparation: For orbencarb and I, a silica gel column is prepared by packing
a slurry of silica gel (10 g) in dichloromethane–n-hexane (1 : 1, v/v) into a glass
chromatography column. About a 1-cm layer of anhydrous sodium sulfate is
placed above and below the silica gel bed. For II, a silica gel column is prepared
by packing a slurry of silica gel (10 g) in dichloromethane–n-hexane (1 : 2, v/v)
into a glass chromatography column. About a 1-cm layer of anhydrous sodium
sulfate is placed above and below the silica gel bed
Rotary vacuum evaporator, 40 ◦ C bath temperature

4 Reagents
Acetone, acetonitrile, dichloromethane, ethyl acetate, n-hexane and methanol:
pesticide residue analysis grade
Hydrochloric acid, sodium chloride and sodium hydroxide: special grade
Anhydrous sodium sulfate: special grade
Silica gel: Wakogel C-200 (Wako Pure Chemical Industries, Ltd)
pH test paper
 Orbencarb 521

Diazomethane: In a distillation flask equipped with an distillation funnel and a cooler,

place a solution of 5 g of potassium hydroxide in 8 mL of water and 25 mL
of ethanol. Warm the distillation flask to 65 ◦ C in a water-bath. Add a solution
of 21.5 g (0.1 mol) of N -methyl-N -nitroso- p-toluenesulfamide in 130 mL of di-
ethyl ether through the instillation funnel in 5 min. If the distillation funnel be-
comes empty, pour 20 mL of diethyl ether into the funnel, and distill it gradually.
Continue distillation until the distilled ether solution becomes colorless. About 3 g
of diazomethane is contained in the whole resultant ether distillate. Caution: these
procedures should be conducted in a laboratory hood
Orbencarb, methyl 2-chlorobenzylsulfone (I), 2-chlorobenzoic acid (II), methyl 2-
chlorobenzoate: analytical standard materials (Ihara Chemical Industries Co., Ltd)
Orbencarb and I standard solution for gas chromatography: 1.0 µg mL−1 in acetone
Methyl 2-chlorobenzoate standard solution for gas chromatography: 0.1 µg mL−1 in
n-hexane

5 Sample preparation
Wheat grains and soybeans are ground in a coffee mill. Potato is chopped finely with
a cutter.

6 Procedure
6.1 Extraction
6.1.1 Plant material
Weigh 50 g of the sample into a 500-mL conical beaker, add 150 mL of ace-
tone, 50 mL of water and 2.5 mL of concentrated HCl and sonicate the mixture
for 30 min. For soybean and potato, add 150 mL of acetone, 50 mL of water and
4 mL of concentrated HCl, homogenize the mixture with a Polytron and sonicate for
30 min.
Filter the mixture through a filter paper by suction and collect the filtrate in a
500-mL round-bottom flask. Wash the residue and the beaker with 100 mL of acetone
and filter and collect the washings in the same manner. Concentrate the combined
acetone extracts in the round-bottom flask to about 60 mL, using a rotary evaporator
under reduced pressure at 40 ◦ C.

6.1.2 Soil
Weigh 50 g (dry soil base) of the sample into a 500-mL round-bottom flask and add
120 mL of methanol and 40 mL of water. Attach a condenser to the flask and reflux
at 75 ◦ C for 1 h. Filter the mixture through a filter paper by suction and collect the
filtrate in a 500-mL round-bottom flask. Wash the residue and the flask with 80 mL
of methanol and filter and collect the washings in the same manner. Combine the
filtrates in a 500-mL separatory funnel.
522 Individual compounds

6.2 Cleanup
6.2.1 Plant material
Transfer the concentrated sample extract (as described in Section 6.1.1) into a 200-mL
separatory funnel and shake twice with 100 mL of ethyl acetate–n-hexane (1 : 1, v/v)
solvent mixture.
Combine the ethyl acetate–n-hexane layer in a 500-mL separatory funnel, add
70 mL of 0.9 M sodium hydroxide solution and 10 mL of saturated sodium chloride
aqueous solution (pH 10 or higher), shake the mixture and collect the organic layer.
Wash the residual alkaline aqueous layer with 30 mL of n-hexane and combine the
n-hexane layer with the organic layer. Using this partitioning procedure, Orbencarb
and I are partitioned into the organic layer and II is partitioned into the alkaline
aqueous layer.
Dry the organic solvent layer through 80 g of anhydrous sodium sulfate on a glass
funnel and collect the dried solution in a 300-mL round-bottom flask. Evaporate
the solvent under reduced pressure. Dissolve the residue in 150 mL of n-hexane and
transfer the solution into a 300-mL separatory funnel. Extract twice with 100 mL of
acetonitrile. Combine the acetonitrile extracts in a 500-mL round-bottom flask and
evaporate the solvent under reduced pressure. Dissolve the residue in a small amount
of column-eluting solvent (dichloromethane–n-hexane, 1 : 1, v/v) and transfer the
solution to the top of the silica gel column. After eluting the column with 60 mL
of solvent of the same composition (discard), elute orbencarb and I with 150 mL
of dichloromethane. Collect the eluate in a 300-mL flask and evaporate the solvent
under reduced pressure. Dissolve the residue in an appropriate volume of acetone for
analysis.
The analytical procedure for the alkaline aqueous layer containing II is as follows.
After acidifying the solution (about pH 2) with 4 mL of concentrated HCl, extract
twice with 100 mL of dichloromethane. Dry the dichloromethane extract with anhy-
drous sodium sulfate and collect the dried solution in a 300-mL round-bottom flask.
Evaporate the solvent under reduced pressure. Dissolve the residue in a mixed solvent
consisting of 4 mL of ethyl acetate, 0.5 mL of methanol and 30 µL of concentrated
HCl. To this mixture, add 7 mL of diazomethane–diethyl ether solution and allow
the mixture to stand at room temperature for 1 h. Concentrate the reaction mixture
to 0.5 mL under reduced pressure and evaporate the solvent in a gentle stream of
nitrogen. Dissolve the residue in a small volume of dichloromethane–n-hexane (1 : 2,
v/v) and transfer the solution to the top of column. Elute with solvent of the same
composition, discard 60 mL of the initial eluate and collect 100 mL of the subsequent
eluate in a 200-mL round-bottom flask. Concentrate the eluate to 0.5 mL under re-
duced pressure, evaporate the solvent in a gentle stream of nitrogen and dissolve the
residue in an appropriate volume of acetone for analysis.

6.2.2 Soil material

Add 250 mL of water and 10 mL of saturated sodium chloride solution to the
sample extract (as described in Section 6.1.2) and extract twice with 150 mL of
dichloromethane. Dry the dichloromethane extract with anhydrous sodium sulfate,
 Orbencarb 523

collect the dried solution in a 500-mL round-bottom flask and evaporate the solvent
under reduced pressure. Dissolve the residue in 150 mL of n-hexane and transfer the
solution into a 300-mL separatory funnel. Extract twice with 100 mL of acetonitrile.
Collect the acetonitrile layer in a 300-mL round-bottom flask and evaporate the solvent
under reduced pressure. Dissolve the residue in a small volume of dichloromethane–
n-hexane (1 : 1, v/v). Thereafter, conduct the cleanup of the soil sample by silica
gel column chromatography as described in Section 6.2.1 to prepare the sample for
determination of orbencarb and I.

6.3 Gas-chromatographic determination

Inject an aliquot (Vi ) of the solution derived from Section 6.2 (VEnd ) into the gas
chromatograph.

Operating conditions for orbencarb and I

Gas chromatograph
Column
Column temperature
Injection port temperature
Detector
Detector temperature
Gas flow rates
Attenuation
Injection volume
Retention time
Minimum detectable amount
Operating conditions for methylated II
Gas chromatograph
Column
Column temperature
Injection port temperature
Detector
Detector temperature
Gas flow rate
Attenuation
Injection volume
Retention time
Minimum detectable amount
Hitachi model 163
Glass, 3-mm i.d. × 1.0 m, packed with 3% NPGS
on Chromosorb W HP, 100–120 mesh
190 ◦ C
230 ◦ C
Flame photometric detector fitted with a 394-nm
sulfur-specific filter
240 ◦ C
Nitrogen carrier gas, 40 mL min−1
Hydrogen, 35 mL min−1
Oxygen, 20 mL min−1
128 × 1000
0.5–6 µL
Orbencarb 3.2 min, I 3.8 min
0.5 ng
Hitachi Model 163
Glass, 3-mm i.d. × 1.0 m, packed with 3% PEGA
on Chromosorb W HP, 100–120 mesh
200 ◦ C
240 ◦ C
Electron capture detector (63 Ni, 10 mCi, pulse in-
terval 100 µs)
240 ◦ C
Nitrogen carrier gas, 50 mL min−1
32 × 10
1–5 µL
3.9 min
0.1 ng
524 Individual compounds

7 Evaluation
7.1 Method
Quantitation is performed by the calibration technique. Construct a fresh calibration
curve with orbencarb standard solutions for each set of analyses. Using log–log paper,
plot the peak heights in millimeters against the injected amount of orbencarb in
nanograms. Peak heights of orbencarb on the chromatogram of a sample extract
were measured. Orbencarb was quantified by comparing the peak height with the
calibration curve. If I and II are determined simultaneously, the respective calibration
curves should be prepared.

7.2 Recoveries and limits of detection

The recoveries from untreated control samples fortified with orbencarb at 0.2 mg kg−1
for crops and 0.5 mg kg−1 for soils were 85–98 and 89–101%, respectively. The limit
of detection was 0.005 mg kg−1 for crops and 0.01 mg kg−1 for soils. The recovery
of I was 92–102% from 0.2 mg kg−1-fortified crops and 95–98% from 0.5 mg kg−1 -
fortified soils. The recovery of II was 68–79% from 0.2 mg kg−1 -fortified crops and
73–77% from 0.5 mg kg−1 -fortified soils. The limits of detection of I and II were the
same as those of orbencarb.

7.3 Calculation of residues

The residue R, expressed in mg kg−1 orbencarb, I or II, is calculated from the fol-
lowing equation:

R = (WA × VEnd )/(Vi × G) × F

where
G = sample weight (g)
VEnd = terminal volume of sample solution from Section 6.2 (mL)
Vi = portion of volume of VEnd injected into the gas chromatograph (µL)
WA = amount of orbencarb for Vi read from the calibration curve (ng)
F = 1 for orbencarb and I and 0.92 for II (factor for conversion of methylated
II to II)

8 Important points

8.1 Liquid–liquid partition

The partition rates of orbencarb and I in aqueous solutions (pH 2–12) into ethyl
acetate–n-hexane (1 : 1, v/v) were as high as 87–90%, and the partition rate did not
differ depending on the pH of the liquid. On the other hand, the recovery of II from
aqueous solutions into ethyl acetate–n-hexane (1 : 1, v/v) was 85% at pH 2, 34% at
 Orbencarb 525

pH 7 and <3% at pH 12. Therefore, when orbencarb, I and II are separated by the
partition procedures under alkaline conditions, the recovery of II will decrease unless
the liquid is sufficiently alkaline. Since the property of the liquid extract of the sample
is slightly different according to the type of analytical sample, it should be confirmed
with a pH test paper that the pH of the aqueous layer is more than 10. Since the
partition rate from n-hexane into acetonitrile is 99% for I and 75% for orbencarb, it is
necessary to carry out the extraction twice to achieve a higher recovery of orbencarb.

8.2 Cleanup

Activated carbon column chromatography (1 g of Dalco G60 and 5 g of Avicel cellu-

lose mixture): I and orbencarb elute into 0–50 mL and 100–300 mL of acetone eluate,
respectively; a recovery of 95% or higher for both analytes is achieved for column
cleanup, but it takes 2–3 h to complete.

8.3 Evaporation
Since methylated II is highly volatile, evaporation of the solvent to dryness should
be avoided.

8.4 Detection
For gas-chromatographic analysis, orbencarb and I are detected with good peak shapes
with a column using as liquid phases silicone SE-30, Thermon-3000, FFAP and
PEGA, but NPGS is superior for separation from impurities.

8.5 Determination of Metabolite II in soil

The recovery is low on extracting II from soil by refluxing with water containing
methanol. Metabolite II in soil can be extracted by sonication with alkaline methanol.
Using this alkaline extraction procedure, another metabolite, 2-chlorobenzylsulfonic
acid, is also extracted simultaneously.1

Reference
1. M. Ikeda, Y. Asano, and K. Ishikawa, ‘Analytical methods for chlorobenzylsulfonic acid in crops,
soils and water,’ in “Abstracts of the 13th Annual Meeting of the Pesticide Science Society of
Japan,” p. 145 (1988).

Further reading

M. Ikeda, T. Unai, and C. Tomizawa, J. Pestic. Sci., 11, 85 (1986).

M. Ikeda, T. Unai, and C. Tomizawa, J. Pestic. Sci., 11, 97 (1986).

Mitsumasa Ikeda, Yoshihiro Saito, and Akira Yagi

Kumiai Chemical Industry Co., Ltd, Shizuoka, Japan