INDOXACARB 612

INDOXACARB
612

CF3
O
CH3
O O
Cl O

N N O
N CH3
O O

ISO Common name Indoxacarb

Chemical name methyl (S)-7-chloro-2,3,4a,5-tetrahydro-2-[methoxy-
carbonyl(4-trifluoromethoxy-phenyl)-carbamoyl]-
indeno[1,2-e][1,3,4]oxadiazine-4a-carboxylate
(IUPAC)
methyl (4aS)-7-chloro-2,5-dihydro-2-[[(methoxy-
carbonyl)[4-(trifluoromethoxy) phenyl]amino]-
carbonyl]indeno[1,2-e][1,3,4]oxadiazine-4a(3H)-
carboxylate (CA; 173584-44-6)
Empirical formula C22H17ClF3N3O7
RMM 527.8
m.p. 88.1 °C
v.p 2.5 × 10-8 Pa (25 °C)
Solubility In water 0.20 mg/l (25 °C). In hexane: 1.307 g/l,
methanol: 109.9 g/l; octan-1-ol: 1.31 g/l; acetone:
>250 g/l; acetonitrile: >250 g/l; ethyl acetate: >250 g/l;
dichloromethane: >250 g/l; dimethylformamide:
>250 g/l; o-xylene: >250 g/l (all at 20 °C)
Description Off-white crystalline solid
Stability Aqueous hydrolysis: DT50 >30 d (pH 5), 38 d (pH 7),
1 d (pH 9)
Formulations Suspension concentrates, water dispersible granules,
dry flowables and emulsifiable concentrates

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 INDOXACARB 612

INDOXACARB TECHNICAL
*
612/TC/M/-

1 Sampling. Take at least 100g.

2 Identity tests

2.1 HPLC. Use the HPLC method below. The relative retention time of the
indoxacarb peak in the sample solution should not deviate by more than 5%
from that of the calibration solution and the UV spectrum measured from this
peak should match that obtained from the calibration substance.
2.2 Infrared. Prepare potassium bromide discs for the technical sample and
indoxacarb reference substance. Scan the discs from 4000 to 400 cm-1. The
spectrum obtained from the sample should not differ significantly from that of
the standard.

3 Indoxacarb

OUTLINE OF METHOD Indoxacarb is determined by high performance liquid

chromatography using a chiral column, UV detection at 310 nm and external
standardisation. The active ingredient content is quantified using a calibration
curve.

REAGENTS

Hexane HPLC grade

Heptane HPLC grade
Propan-2-ol HPLC grade
Ethyl acetate HPLC grade
Water deionised, HPLC grade
Indoxacarb analytical standard of known purity. The analytical standard is a
racemic mixture (exactly 50:50 ratio) of indoxacarb and the R enantiomer.
This means that at a label purity of 99.6 % it contains 49.8 % indoxacarb and
49.8 % R enantiomer.
Mobile phase, hexane (or heptane) – propan-2-ol, 75 + 25 % (v/v). To a 1 l
bottle, add hexane or heptane (750 ml) and of propan-2-ol (250 ml). Mixing of
the mobile phase also may be accomplished using a binary solvent HPLC
pump. Degas prior to use.

*
CIPAC method 2009. Based on a method supplied by DuPont de Nemours, USA.

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 INDOXACARB 612

Calibration solutions. Weigh (to the nearest 0.1 mg) 50, 100, and 150 mg (± 5 mg)
of the analytical standard (a racemic mixture!) into separate volumetric flasks
(100 ml). Note that this is equivalent to 25, 50, and 75 mg of indoxacarb. Add
ethyl acetate (approximately 90 ml) and place the flasks in an ultrasonic bath
for 15 min. Remove the flasks from the ultrasonic bath and allow to stand to
attain room temperature (about one hour). Dilute to volume with ethyl acetate
and mix well. Filter a portion of each standard solution through a 0.2 µm filter
prior to HPLC analysis.

APPARATUS

High performance liquid chromatograph equipped with a constant-flow pump,

constant-temperature column compartment, a sample injector capable of
injecting 5-µl aliquots, a UV detector (310 nm analytical wavelength; 450 nm
reference wavelength) and digital integrator or other data-handling capability.
HPLC column, 250 × 4.6 mm (i.d.), packed with Chiralcel OD 10 µm particle size.
This column must only be exposed to hexane (or heptane) and propan-2-ol. Water
or even acetonitrile would ruin the column. It is important to flush all lines of
the instrument with about 10 – 20 ml propan-2-ol, and then with 15 – 20 ml of the
mobile phase before the column is installed in order to remove aqueous or
acetonitrile mobile phases from the mixing chamber and degasser portions of
the instrument.
Filtering apparatus (for sample and standards solutions) disposable plastic 3 ml
syringes fitted with 0.2 µm (or 0.45 µm) Acrodisc-CR (PTFE or Teflon)
filters
Ultrasonic bath

PROCEDURE

(a) Operating conditions (typical):

Column 250 × 4.6 mm (i.d.), packed with Chiralcel OD,
10 µm particle size
Mobile phase hexane (or heptane) – propan-2-ol 75 + 25 %
(v/v)
Eluant flow rate 1.0 ml/min
Column temperature 40 °C
Injection volume 5 µl
Detection wavelength 310 nm (bandwidth 4 nm)
Reference wavelength 450 nm (bandwidth 100 nm)
Run time 12 minutes

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 INDOXACARB 612

Retention times
with hexane indoxacarb: 9.5 min
R-enantiomer: 6.9 min
with heptane indoxacarb: 10.3 min
R-enantiomer: 7.5 min

(b) Sample preparation. Weigh (to the nearest 0.1 mg) into a volumetric flask
(100 ml) sufficient sample to contain 65 ± 10 mg indoxacarb (w mg). Add ethyl
acetate (about 90 ml), mix well and place the flask in an ultrasonic bath for
15 min. Remove the flask from the bath and allow to stand to attain room
temperature (about one hour). Dilute to volume with ethyl acetate and mix well.
Filter a portion of the sample solution through a 0.2 µm filter prior to HPLC
analysis.

(c) Determination. Equilibrate the column with mobile phase until a stable
baseline is obtained. Inject, in duplicate, 5 µl each of a solvent blank (ethyl
acetate), standards, and samples, bracketing the samples with calibration
standards.

(d) Calculation. Prepare a calibration curve for indoxacarb by plotting the peak
areas versus the mass of the standards, corrected for purity of the standard, and
corrected to 50 % of the purity as indoxacarb in the racemic analytical standard.
Using the method of least squares, calculate the equation for the straight line
which best fits the experimental calibration data. The correlation coefficient
should be 0.999 or better.
Determine the concentration of indoxacarb for each sample injection using the
following equation:

(H - b) × P
Indoxacarb content = g/kg
a× w

where:
H = indoxacarb peak area in the sample solution
a = slope of calibration curve
b = intercept of calibration curve
P = purity of the indoxacarb standard (g/kg)
w = mass of sample taken (mg)

Repeatability r = 24 g/kg at 960 g/kg active ingredient content

Reproducibility R = 24 g/kg at 960 g/kg active ingredient content

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 INDOXACARB 612

INDOXACARB TECHNICAL CONCENTRATE

*
612/TK/M/-

SCOPE The method is suitable for determining indoxacarb in a mixture

containing indoxacarb and the R-enantiomer in a ratio of approximately 75:25.

1 Sampling. Take at least 100 g.

2 Identity tests
2.1 HPLC. As for indoxacarb technical, 612/TC/M/2.1.
2.2 Infrared. Mix thoroughly 100 mg of the formulation with acetonitrile (5-
10 ml) and filter. Evaporate the filtrate to dryness. Prepare potassium bromide
discs from the residue and from indoxacarb reference substance. Scan the discs
from 4000 to 400 cm-1. The spectrum obtained from the sample should not
differ significantly from that of the standard.

3 Indoxacarb. As for indoxacarb technical 612/TC/M/3.

Repeatability r = 17 g/kg at 556 g/kg active ingredient content

Reproducibility R = 22 g/kg at 556 g/kg active ingredient content

INDOXACARB WATER DISPERSIBLE GRANULES

*612/WG/M/-

SCOPE The method is suitable for determining indoxacarb in a mixture

containing indoxacarb and the R-enantiomer in a ratio of approximately 75:25.

1 Sampling. Take at least 1 kg.

2 Identity tests
2.1 HPLC. As for indoxacarb technical, 612/TC/M/2.1.
2.2 Infrared. Mix thoroughly 100 mg of the formulation with acetonitrile (5-
10 ml) and filter. Evaporate the filtrate to dryness. Prepare potassium bromide
discs from the residue and from indoxacarb reference substance. Scan the discs
from 4000 to 400 cm-1. The spectrum obtained from the sample should not
differ significantly from that of the standard.

*
CIPAC method 2009. Based on a method supplied by DuPont de Nemours, USA.

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 INDOXACARB 612

3 Indoxacarb. As for indoxacarb technical, 612/TC/M/3, except:

(b) Sample preparation. Weigh (to the nearest 0.1 mg) into a volumetric flask
(100 ml) sufficient sample to contain 65 ± 10 mg indoxacarb (w mg). Add 4 ml
of water to the flask containing the sample. Take care to keep the sample at the
bottom of the flask. Do not add the water so fast that some of the sample moves
up the glass and sticks to the side of the flask. Swirl gently to disperse the
granules. Add propan-2-ol (about 10 ml), swirl to mix, and add ethyl acetate
(70 ml). Mix well and place the flask in an ultrasonic bath for 15 min. Remove
the flask from the bath and allow to stand to attain room temperature (about one
hour). Dilute to volume with ethyl acetate and mix well. Filter a portion of the
sample solution through a 0.2 µm filter prior to HPLC analysis.

Repeatability r = 8.1 g/kg at 300 g/kg active ingredient content

Reproducibility R = 11 g/kg at 300 g/kg active ingredient content

4 Suspensibility

REAGENTS and APPARATUS. As for indoxacarb technical, 612/TC/M/3 and

MT 184, except:
add at:
Calibration solutions
Prepare in the same way standard solutions at other concentrations, if needed.

PROCEDURE

(a) Preparation of suspension and determination of sedimentation. MT 184.

(b) Determination of indoxacarb in the bottom 25 ml of suspension. After removal

of the top 225 ml of suspension, add to the remaining 25 ml propan-2-ol (75 ml).
Place the cylinder in an ultrasonic bath for 5 min. Allow to cool to room
temperature, mix well by inverting the cylinder several times, then take a
suitable aliquot of the solution for the determination of the mass of indoxacarb
(Q g).

(c) Calculation
111 ( c - Q )
Suspensibility = %
c
where:
c = mass of indoxacarb in the sample taken for the preparation of
the suspension (g)
Q = mass of indoxacarb in the bottom 25 ml of suspension (g)

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 INDOXACARB 612

INDOXACARB EMULSIFIABLE CONCENTRATES

*
612/EC/M/-

1 Sampling. Take at least 1 l.

2 Identity tests
2.1 HPLC. As for indoxacarb technical, 612/TC/M/2.1.
2.2 Infrared. Mix thoroughly 100 mg of the formulation with acetonitrile (5-
10 ml) and filter. Evaporate the filtrate to dryness. Prepare potassium bromide
discs from the residue and from indoxacarb reference substance. Scan the discs
from 4000 to 400 cm-1. The spectrum obtained from the sample should not
differ significantly from that of the standard.

3 Indoxacarb. As for indoxacarb technical, 612/TC/M/3.

Repeatability r = 5.9 g/kg at 161 g/kg active ingredient content

Reproducibility R = 9.0 g/kg at 161 g/kg active ingredient content

INDOXACARB SUSPENSION CONCENTRATES

*
612/SC/M/-

SCOPE The method is suitable for determining indoxacarb in a mixture

containing indoxacarb and the R-enantiomer in a ratio of approximately 75:25.

1 Sampling. Take at least 1 l.

2 Identity tests
2.1 HPLC. As for indoxacarb technical, 612/TC/M/2.1.
2.2 Infrared. Mix thoroughly 100 mg of the formulation with 5-10 ml of
acetonitrile and filter. Evaporate the filtrate to dryness. Prepare potassium
bromide discs from the residue and from indoxacarb reference substance. Scan
the discs from 4000 to 400 cm-1. The spectrum obtained from the sample should
not differ significantly from that of the standard.

*
CIPAC method 2009. Based on a method supplied by DuPont de Nemours, USA.

79
 INDOXACARB 612

3 Indoxacarb. As for indoxacarb technical, 612/TC/M)/3.

Repeatability r = 3.3 g/kg at 141 g/kg active ingredient content

Reproducibility R = 6.4 g/kg at 141 g/kg active ingredient content
4 Suspensibility. As for indoxacarb water dispersible concentrates,
612/WG/M/4.

Fig. 37 UV spectrum of indoxacarb technical

Fig. 38 Infrared spectrum of indoxacarb technical

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 INDOXACARB 612

DAD1 A, Sig=310,4 Ref=360,100 (080807\041-0102.D)

mAU

400

300

200 9.519 - KN128

100

0
0 2 4 6 8 10 min

Fig. 39 Chromatogram of indoxacarb standard run with mobile phase made

with hexane

DAD1 A, Sig=310,4 Ref=360,100 (061908\003-0302.D)

mAU

250

10.322 - KN128
200

150

100

50

0 2 4 6 8 10 12 min

Fig. 40 Chromatogram of indoxacarb standard run with mobile phase made

with heptane

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 INDOXACARB 612

DAD1 A, Sig=310,4 Ref=360,100 (080807\042-0202.D)

mAU

400

300

200 9.504 - KN128

100
6.977 - KN127

0
0 2 4 6 8 10 min

Fig. 41 Chromatogram of a mixture of indoxacarb and the R-enantiomer run

with mobile phase made with hexane

DAD1 A, Sig=310,4 Ref=360,100 (061908\002-0202.D)

mAU

250

200 10.331 - KN128

150

100
7.542 - KN127

50

0 2 4 6 8 10 12 min

Fig. 42 Chromatogram of a mixture of indoxacarb and the R-enantiomer run

with mobile phase made with heptane

82