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Simultaneous Determination of Imidacloprid, Thiacloprid, and

Thiamethoxam in Soil and Water by High-performance Liquid
Chromatography with Diode-array Detection
Guang-Guo Yinga; Rai S. Kookanaa
a
Adelaide Laboratory, CSIRO Land and Water, Glen Osmond, SA, Australia

Online publication date: 10 June 2004

To cite this Article Ying, Guang-Guo and Kookana, Rai S.(2005) 'Simultaneous Determination of Imidacloprid, Thiacloprid,
and Thiamethoxam in Soil and Water by High-performance Liquid Chromatography with Diode-array Detection',
Journal of Environmental Science and Health, Part B, 39: 5, 737 — 746
To link to this Article: DOI: 10.1081/LESB-200030808
URL: http://dx.doi.org/10.1081/LESB-200030808

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 JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH
Part B—Pesticides, Food Contaminants, and Agricultural Wastes
Vol. B39, Nos. 5–6, pp. 737–746, 2004

Simultaneous Determination of Imidacloprid,

Thiacloprid, and Thiamethoxam in Soil and Water by
High-performance Liquid Chromatography with
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Diode-array Detection

Guang-Guo Ying* and Rai S. Kookana

CSIRO Land and Water, Adelaide Laboratory,

Glen Osmond, SA, Australia

ABSTRACT

A high-performance liquid chromatography method with diode-array detection

(HPLC-DAD) is described for the determination of three neonicotinoid
insecticides imidacloprid, thiacloprid, and thiamethoxam in soil and water. The
soil samples were extracted with acetonitrile, while the water samples were
extracted using C18 cartridges. The mean recoveries plus standard deviations for
spiked soil samples were 82  4.2% for thiamethoxam, 99  4.2% for imidaclo-
prid and 94  1.4% for thiacloprid. The recoveries for water samples ranged from
87  3.4% for thiamethoxam to 97  3.9% for imidacloprid and 97  2.6% for
thiacloprid. The limits of quantitation (LOQ) were 0.1, 0.1, 0.01 mg/kg in soil
(5 g), and 2, 2, 0.5 mg/L in water (50 mL) for thiamethoxam, imidacloprid, and
thiacloprid, respectively.

Key Words: Soil; Water; Imidacloprid; Thiacloprid; Thiamethoxam; Analysis;

HPLC.

*Correspondence: Guang-Guo Ying, CSIRO Land and Water, Adelaide Laboratory, PMB 2,
Glen Osmond, SA 5064, Australia; E-mail: guang-guo.ying@ csiro.au.

737

DOI: 10.1081/LESB-200030808 0360-1234 (Print); 1532-4109 (Online)

Copyright & 2004 by Marcel Dekker, Inc. www.dekker.com
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738 Ying and Kookana

INTRODUCTION

Imidacloprid, thiacloprid, and thiamethoxam (TMX) all belong to the

neonicotinoid insecticide class.[1] Imidacloprid, 1-[6-chloro-3-pyridinyl)methyl]-N-
nitro-2-imidazolidinimine, and thiacloprid, (Z)-[3-[(6-chloro-3-pyridinyl)-2-thiazoli-
dinylidene]cyanamide, were developed by Bayer, whereas thiamethoxam, 3-[(2-
chloro-5-thiazolyl)methyl]tetrahydro-5-methyl-N-nitro-4H-1,3,5-oxadiazin-4-imine,
was developed by Syngenta (Fig. 1). They act as an antagonist by binding to
nicotinic acetylcholine receptors in the insect central nervous system. These three
chemicals have a broad-spectrum insecticidal activity and provide an excellent
control of a wide range of chewing and sucking pests, such as aphids, jassids and
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termites.[1–4]

imidacloprid

thiacloprid

thiamethoxam
Figure 1. Chemical structures of imidacloprid, thiacloprid, and thiamethoxam.
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Determination of Insecticides in Soil and Water 739

Imidacloprid and thiacloprid have a water solubility of 0.61 mg/L and 0.185 g/L
at 20 C.[1] Thiamethoxam has a high water solubility of 4.1 g/L and a low log Kow
value of 0.13 at 25 C.[2] Since imidacloprid is the first neonicotinoid insecticide
developed among the three chemicals, high-performance liquid chromatography
(HPLC) with diode-array detection (DAD) or mass spectrometric detection (MSD)
has been developed to determine imidacloprid in vegetables,[5,6] fruits,[7] soil, and
water.[8–10] Imidacloprid has low volatility and high polarity, so direct determination
by gas chromatography (GC) is not possible, but Vilchez et al.[11] used GC-MS to
determine imidacloprid in soil and water after its transformation into a volatile
hydrolyzed product. Recently, Singh et al.[12] reported microwave-assisted extraction
and HPLC analysis for thiamethoxam, imadacloprid and carbendazin residues in
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fresh and cooked vegetable samples. However, there has been no report so far on the
analysis of the other two chemicals, thiacloprid and thiamethoxam in soil and water.
This study will report on the simultaneous determination of imidacloprid,
thiacloprid, and thiamethoxam in soil and water samples using high-pressure liquid
chromatography with a diode array detector.

MATERIALS AND METHODS

Chemicals and Solvents

Thiamethoxam (TMX) standard with a purity of 99.5% was provided by

Syngenta Crop Protection Australia, whereas imidacloprid with a purity of 99.9%
and thiacloprid with a purity of 99.6% were supplied by Bayer CropScience
Germany. Acetonitrile and methanol of HPLC grade were purchased from BDH
England. The stock solutions of imidacloprid, thiacloprid, and thiamethoxam were
prepared in acetonitrile at a concentration of 100 mg/L. Working solutions of the
mixture of the three chemicals were also prepared from the stock solutions at a
concentration of 1 and 10 mg/L.

Instrumentation

An Agilent 1100 series liquid chromatograph was used to analyze imidacloprid,

thiacloprid, and thiamethoxam. It consists of a vacuum degasser, an autosampler, a
quaternary pump and a diode array detector. The instrument was equipped with a
reversed phase column of Prevail Select C18 (150 mm  2.1 mm, 5 m) and a C18 guide
cartridge from Alltech. The UV wavelength used in the experiment was 270 nm for
imidacloprid, 242 nm for thiacloprid and 255 nm for thiamethoxam. The mobile
phase for gradient elution were Milli-Q water (mobile phase A) and acetonitrile
(mobile phase B) delivered at a constant flow rate of 0.5 mL/min. The gradient
program of the mobile phase was as follows: 90% A and 10% B at 0 min, 80% A and
20% B at 3 min, 50% A and 50% B at 10 min, and 90% A and 10% B at 15 min. The
total run time for an HPLC analysis was 15 min. The injection volume of standards
and samples was 25 mL.
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740 Ying and Kookana

Sample Preparation and Analysis

Standard Samples

A series of standard working solutions with a concentration ranging from 0.01

to 10 mg/L were prepared in acetonitrile: water (10:90, v/v) from 100 mg/L stock
solution. These standard samples were run on the HPLC to determine instrumental
detection limit and linearity. A calibration curve was prepared at concentrations of
0.1, 0.5, 1, 2, and 5 mg/L in acetonitrile: water (10:90, v/v).
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Soil Samples

A clay soil from Turretfield, South Australia was used in this fortification test.
Five grams of each soil sample in a tube was spiked at a concentration of 0, 0.005,
0.01, 0.02, 0.1, 0.2, 0.4, 1, and 2 mg/kg from the working solutions of imidacloprid,
thiacloprid, and thiamethoxam mixture. Duplicate samples of each concentration
were prepared for spiking test. After spiking, the tubes were mixed and left in the
laboratory for 2 h. The three compounds in soil were extracted by adding into each
tube with 20 mL of acetonitrile. The tubes were mixed by using a Vortex-mixer for
one minute, and shaken in a mechanical shaker for 1 h. After shaking, these tubes
were centrifuged at 2500 rpm for 15 min. 10 mL of supernatant was taken from each
tube and filtered through a PTFE disk filter (0.45 m) into a test tube. The extracts
were blown to dryness under a gentle nitrogen stream, and re-dissolved in 1 mL of
acetonitrile: water (10:90, v/v) and analyzed by HPLC.
A batch of 7 real contaminated soils from Northern Australia was also analyzed
using the same method as above.

Water Samples

Tap water (50 mL each) was spiked at a concentration of 0, 0.5, 1, 2, 10, 20, 40,
100, and 200 mg/L. Water samples were extracted using C18 solid phase extraction
cartridges (Alltech Hi-Flow C18 cartridge, 3 mL, 500 mg sorbent). Each cartridge was
first conditioned using 2 mL of methanol and 2 mL of Milli-Q water. Then 50 mL of
water sample was passed through the cartridges at a flow rate of about 5 mL/min.
The cartridges were washed with Milli-Q water and dried by passing through air for
one hour. The three compounds was eluted from each cartridge using 2  2 mL of
methanol. The extracts were blown to dryness under a gentle nitrogen gas and
re-dissolved in 1 mL of acetonitrile:water (10:90, v/v) for HPLC analysis.

RESULTS AND DISCUSSION

Three insecticides (imidacloprid, thiacloprid, and thiamethoxam) were separated

on a reversed phase HPLC C18 column with a retention time of 5.0 min for
thiamethoxam, 6.7 min for imidacloprid, and 8.9 min for thiacloprid (Fig. 2), despite
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DAD1 A, Sig=242,16 Ref=400,100 (YING\SPIKE012.D)

mAU
50
thiacloprid

40

30

20

10

0 2 4 6 8 10 12 14 min
DAD1 B, Sig=270,4 Ref=400,100 (YING\SPIKE012.D)
mAU
imidacloprid
ORDER

50

40

30

20

10
Determination of Insecticides in Soil and Water

0
REPRINTS

0 2 4 6 8 10 12 14 min
DAD1 C, Sig=255,4 Ref=550,100 (YING\SPIKE012.D)
mAU
50
thiamethoxam
40

30

20

10

0 2 4 6 8 10 12 14 min

Figure 2. Liquid chromatograms of the three insecticides at a concentration of 2 mg/L. Conditions: Prevail
Select C18 column (150 mm  2.1 mm, 5 m). UV wavelength: 270 nm for imidacloprid, 242 nm for thiacloprid, and
255 nm for thiamethoxam. (View image in color online.)
741
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742 Ying and Kookana

A.
*DAD1, 6.694 (127 mAU,Apx) Ref=4.600 & 7.107 of INSEC010.D
mAU
120

100

80

60
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40

20

220 240 260 280 300 320 340 360 380 nm

B.
*DAD1, 4.402 (89.0 mAU,Apx) Ref=4.188 & 4.775 of SPIKE003.D
mAU

80

70

60

50

40

30

20

10

220 240 260 280 300 320 340 360 380 nm

Figure 3. UV spectra of thiacloprid (A), thiamethoxam, (B) and imidacloprid (C).

(continued) (View image in color online.)

their similarity in chemical structure and properties. The highest UV absorbance is at

242, 255, and 270 nm for thiacloprid, thiamethoxam, and imidacloprid, respectively
(Fig. 3). For convenience, we could also use single UV absorbance 255 nm for all
three insecticides. Within the concentrations of 0.025 to 10 mg/L for each compound,
this analytical method gave a very good linearity with the correlation coefficients
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Determination of Insecticides in Soil and Water 743

C.
*DAD1, 3.793 (16.9 mAU, - ) Ref=2.126 & 5.666 of INSEC002.D
mAU

14

12

10

8
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220 240 260 280 300 320 340 360 380 nm

Figure 3. Continued.

(R2) more than 0.999. Under the conditions used, the instrumental limit of detection
(LOD) for the three compounds on the HPLC was determined to be 0.01 mg/L.

Soil and Water Extraction

Analyses of the soil and water extracts showed no interferences with compound
peaks (Fig. 4). The LOD of each compound was determined as the level for which
the signal to noise ratio was greater than 3. It was 0.01 mg/kg for imidacloprid and
thiamethoxam and 0.005 mg/kg for thiacloprid in soil (5 g). However, the LOQ was
defined as the lowest quantified quantity with repeatability in agreement with all the
quality criteria, and recoveries more than 70%.[9] The LOQ was 0.1 mg/kg for
imidacloprid and thiamethoxam and 0.01 mg/kg for thiacloprid in soils (5 g). Since
only half of the extract from each soil sample was used to concentrate into the final
1 mL for analysis, the limits of detection and quantitation could be further lowered if
the whole extract was used. The extraction recoveries were found to be 82  4.2% for
thiamethoxam, 99  4.2% for imidacloprid, and 94  1.4% for thiacloprid for the
soils spiked at concentrations above the LOQ.
The LOD and LOQ were also determined based on the same principle used for
the spiked soil samples. The LOD in water (50 mL) was 0.5 mg/L for both
thiamethoxam and imidacloprid, and 0.25 mg/L for thiacloprid. And the LOQ
in water was 2, 2, and 0.5 mg/L for thiamethoxam, imidacloprid, and thiacloprid,
respectively. Thiacloprid had lowest LOQ for both soil and water samples. Water
extraction using C18 cartridges gave recoveries of 87  3.4% for thiamethoxam,
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744 Ying and Kookana

A.
DAD1 C, Sig=255,4 Ref=550,100 (YING\REP00005.D)

8.920
mAU

5.095

6.772
80

60

40

20
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0 2 4 6 8 10 12 14 min

B.
DAD1 C, Sig=255,4 Ref=550,100 (YING\REP00010.D)

8.896
mAU

200

175
6.751
5.083

150

125

100

75

50

25

0 2 4 6 8 10 12 14 min

Figure 4. Liquid chromatograms of the three insecticides in the extracts of a soil sample (A)
and a water sample (B) at the wavelength of 255 nm. (View image in color online.)

97  3.9% for imidacloprid, and 97  2.6% for thiacloprid. Although these three
insecticides have high water solubilities, this experimental study demonstrated that
they could be easily extracted from water by using C18 cartridges.
This HPLC method gave a very good analytical repeatability in retention time
and amount for the three compounds. Repeated analysis of one soil extract (2 mg/
kg) and one water extract (0.2 mg/L) within one day showed an excellent
repeatability with the relative standard deviation (R.S.D.) of less than 0.7% for
retention time and less than 0.1% for amount.

Application of the Method

This HPLC method was used to analyze soil samples collected from a pesticide
trial site. First batch of 7 samples including 2 untreated controls were collected and
extracted with acetonitrile as described in the method. Thiamethoxam was found in
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Determination of Insecticides in Soil and Water 745

the five treated soil samples at concentrations ranging from 2.072 to 8.621 mg/kg
with an average of 5.844  2.496 mg/kg (mean  SD); but the other two chemicals
were not detected in all soil samples. After one year, another 7 soil samples were
collected and analyzed. The concentration of thiamethoxam in the treated soils
ranged from 0.236 to 1.852 mg/kg with an average of 1.017  0.769 mg/kg
(mean  SD). This monitoring study demonstrated that this method was simple,
robust and could be used for the routine analysis of the three neonicotinoids in soils.

CONCLUSIONS
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A rapid and reliable analytical method was described for the simultaneous
determination of three neonicotinoid insecticides in soil and water using HPLC with
diode-array detection at the wavelength of 242, 255, and 270 nm. Soil samples were
extracted with acetonitrile without further cleanup steps. Water samples were
extracted using C18 cartridges and eluted with methanol. The present study showed
that this method is adequate for routine measurement of imidacloprid, thiacloprid,
and thiamethoxam in soil and water samples.

ACKNOWLEDGMENTS

The authors would like to thank Syngenta Crop Protection Australia and Bayer
CropScience for providing technical grade standards.

REFERENCES

1. Tomlin, C.D.S., Ed. The Pesticide Manual, 12th Ed.; British Crop Protection
Council: UK, 2000; 537 pp.
2. Maienfisch, P.; Angst, M.; Brandl, F.; Fischer, W.; Hofer, D.; Kayser, H.;
Kobel, W.; Rindlisbacher, A.; Senn, R.; Steinemann, A.; Widmer, H. Chemistry
and biology of thiamethoxam: a second generation neonicotinoid. Pest. Manag.
Sci. 2001, 57, 906–913.
3. Maienfisch, P.; Huerlimann, H.; Rindlisbacher, A.; Gsell, L.; Dettwiler, H.;
Haettenschwiler, J.; Sieger, E.; Walti, M. The discovery of thiamethoxam:
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6. Mukherjee, I.; Gopal, M. Environmental behaviour and translocation of

imidacloprid in eggplant, cabbage and mustard. Pest. Manag. Sci. 2000, 56,
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8. Baskaran, S.; Kookana, R.S.; Naidu, R. Determination of the insecticide
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E.R. A LC/APCI-MS/MS method for analysis of imidacloprid in soils, in

plants, and in pollens. Anal. Chem. 2003, 75, 2027–2033.
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Received May 14, 2004

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