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QUANTIFICATION OF PESTICIDE LEVELS IN HORSE CHESTNUT
SEEDS
Tursunov Khurshid Obitovich
Yokubova Nilufar Jamshidovna
Tashkent Pharmaceutical Institute
SI “The center for pharmaceutical products safety”
*e-mail: nilufaryokubovapharmi21@gmail.com
https://doi.org/10.5281/zenodo.12684971
Abstract
In the middle of the 20th century, the use of synthetic pesticides became
increasingly prevalent as a means to combat pests such as insects and weeds.
Pesticides, which are substances used to eliminate or control pests, have played
a pivotal role in enhancing food production to meet the rising global demand [1].
These chemicals are vital for safeguarding crops from a wide array of diseases
and pests. Moreover, pesticides find extensive application in non-agricultural
domains, encompassing industrial plant management, pest control in buildings,
and pet care. Despite the escalating reliance on pesticides, pests have shown
growing adaptability to these chemicals, prompting persistent research efforts
aimed at developing effective pest control solutions [2]. Nevertheless, due to the
widespread use of pesticides, complete prevention of their impact on the
environment is unattainable [3]. Hence, it is imperative to regulate the quantity
of pesticides present in various products. In a recent study, we analyzed
pesticides, including atrazine, simazine, hydroxyatrazine, flumethrin, mepiquat,
methylsulfuron, chlorimuron, and chlorsulfuron, in horse chestnut seeds using
high-performance liquid chromatography. The findings from this study revealed
that the levels of pesticides detected in horse chestnut seeds obtained from both
Tashkent City and Qibray district adhered to the requirements stipulated in the
Sanitary Rules and Regulations (SanPiN №0321-15).
Keywords:
pesticide, horse chestnut, high-performance liquid
chromatography, atrazine, simazine, hydroxyatrazine, flumethrin, mepiquat,
methylsulfuron, chlorimuron, chlorsulfuron.
1. Introduction.
Pesticides, also known as plant protection products (PPP), are substances
utilized to control pests, weeds, and diseases [4]. In the 1940s, the development
of the first synthetic pesticides marked a significant milestone in agriculture, as
it substantially contributed to the growth of food production. However, as early
as the 1960s, increasing attention was given to the potential negative effects of
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pesticides on both the environment and human health. This growing concern led
to a reevaluation of the impacts of pesticide use and spurred efforts to develop
more sustainable agricultural practices [5]. It is essential to note that pesticides
have the potential to persist in the environment for prolonged periods, posing
risks to both the environment and human health. The isolation of the Escin
substance from the horse chestnut seeds necessitates adherence to established
pharmacopeia standards, including the limitation of pesticide residue in plant
raw materials. Even though pesticides are not typically directly applied to horse
chestnuts, their presence in the environment can lead to their transfer, even in
small amounts, to the horse chestnut. This research aims to analyze the
pesticides commonly used in Uzbekistan in horse chestnut seeds grown in
Tashkent City and Qibray district and evaluate them according to the
requirements of Sanitary Rules and Regulations (SanPiN №0321-15).
2. Materials and methods.
. The study utilized acetonitrile, deionized water, sodium acetate
trihydrate, Millipore filters (0.45 microns), and working standards of pesticides.
Analyses were performed using the SHIMADZU Nexera 20AR chromatograph
(SHIMADZU, Japan).
2.1. Conditions for determining the amount of atrazine, simazine, and
hydroxyatrazine:
Chromatographic column - Zorbach Eclipse C-18, 4.6x150 mm, 5 mkm;
column temperature - 40°C; detector – UF-detector, 220 nm; flow rate – 1.0
ml/min; injection volume – 10 mkl; mobile phase A – 0.01 M monopotassium
phosphate (pH=7.0); mobile phase B – methanol; solvent – methanol; analysis
duration - 20 minutes; concentration of substances - 0.11 mg/ml. The HPLC
gradient mode is used for mobile phases A and B
(Fig.1).
Time/min
Mobile phase A, %
Mobile phase B, %
3,00
80,0
20,0
5,00
70,0
30,0
7,00
70,0
30,0
10,00
50,0
50,0
12,00
50,0
50,0
14,00
30,0
70,0
16,00
30,0
70,0
18,00
80,0
20,0
20,00
Finish
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Figure 1. The HPLC gradient mode of the mobile phase.
Figure 2. HPLC/DMD profile of the standard sample of simazine.
Figure 3. HPLC/DMD profile of the standard sample of hydroxyatrazine.
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Figure 4. HPLC/DMD profile of simazine and hydroxyatrazine in horse
chestnut seeds. (1-Tashkent, 2-Qibray)
2.2. Conditions for determining the amount of flumethrin:
Chromatographic column - Zorbach Eclipse C-18, 3.0x150 mm, 3.5 mkm;
column temperature - 40°C; detector – UF-detector, 220 nm; flow rate – 1.0
ml/min.; injection volume – 20 mkl; mobile phase – a mixture of methanol and
acetonitrile (55:45); solvent – methanol; analysis duration - 22 minutes;
concentration of substances - 0.25 mg/ml.
Figure 5. HPLC/DMD profile of the standard sample of flumethrin.
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Figure 6. HPLC/DMD profile of flumethrin in horse chestnut seeds.
(1-Tashkent, 2-Qibray)
2.3. Conditions for determining the amount of mepiquat:
Chromatographic column - Silasorb C-18, 4,6x150mm, 5 mkm; column
temperature – 40°C; detector - UF-detector, 230 nm; flow rate-0.7 ml/min.;
injection volume – 20 mkl; mobile phase – 0.05 M monopotassium phosphate
(pH=2.8) and methanol mixture (95:5); solvent – water; analysis duration - 22
minutes; concentration of substances - 0.1 mg/ml.
2.4. Conditions for determining the amount of chlorsulfuron:
Chromatographic column - Zorbach Eclipse C-18, 4.6x150 mm, 5 mkm;
column temperature - 40°C; detector – UF-detector, 226 nm; flow rate – 1.0
ml/min; injection volume – 20 mkl; mobile phase - a mixture of 1.4 g of Sodium
1-hexanesulfonate-containing water per liter, acetonitrile and 98.5% acetic acid
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(940: 50: 10); solvent - mobile phase; analysis duration - 22 minutes;
concentration of substances - 0.15 mg/ml.
Figure 7. HPLC/DMD profile of the standard sample of chlorsulfuron.
Figure 8. HPLC/DMD profile of chlorsulfuron in horse chestnut seeds.
(1-Tashkent, 2-Qibray)
2.5. Conditions for determining the amount of methylsulfuron and
chlorimuron:
Chromatographic column - Zorbach Eclipse C-18, 4.6x150 mm, 5 mkm;
column temperature - 40°C; detector – UF-detector, 234 nm; flow rate – 1.0
ml/min; injection volume – 20 mkl; mobile phase A – 0.05 M sodium acetate
(pH=2.5); mobile phase B – a mixture of methanol and acetonitrile (3:2);
analysis duration - 28 minutes; concentration of substances - 0.1 mg/ml. The
HPLC gradient mode is used for mobile phases A and B
(Fig.9).
Time/min
Mobile phase A, %
Mobile phase B, %
0
0%
100%
1,5
1%
99%
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17,0
57%
43%
18,0
98%
2%
23,0
98%
2%
26,0
1%
99%
28,0
Finish
Figure 9. The HPLC gradient mode of the mobile phase.
Working standard preparation:
0.050 grams of methylsulfuron working standard sample and 0.0500
grams of chlorimuron working standard sample is placed into a 100 ml
volumetric flask, dissolved in a small amount of purified water, diluted to
volume with water, and mixed thoroughly. 10 µL of the resulting solution, 100
µL of the saturated sodium tetraborate solution, and 290 µL of the reagent A are
placed in a microtube and shaken for 3 minutes.
Drug solution preparation:
1.0 grams of accurately weighed sample is placed into a 25 ml volumetric
flask. Then, 15 ml of water is added and treated with ultrasound for 15 minutes
(can be slightly heated). The resulting solution is then cooled, diluted to volume
with water, mixed, and filtered through a membrane filter with a hole size of
0.45 µm.
Test solution preparation:
10 µL of the drug solution, 100 µL of saturated sodium tetraborate
solution, and 290 µL of the reagent A are placed in a microtube and shaken for 3
minutes.
Reagent A preparation.
The first solution:
0.05 g of phthalaldehyde is placed into a 10 ml volumetric flask, dissolved
in a small amount of methanol, and diluted to volume with the same solvent.
The second solution:
50 ml of 2-mercaptoethanol is diluted to 100 ml with methanol. 1.25 ml of
the first solution and 25 ml of the second solution are mixed.
3. Results and discussion.
The study involved analyzing the levels of various pesticides, including
atrazine, simazine, hydroxyatrazine, flumethrin, mepiquat, methylsulfuron,
chlorimuron, and chlorsulfuron, in horse chestnut seeds harvested from
Tashkent City and Qibray district using high-performance liquid
chromatography
(Fig.10).
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№
Name
of
PESTICIDES
Sanitary Rules
and
Regulations
(SanPiN
№0321-15).
Amount
of
pesticides
in
seeds collected
from Tashkent,
mg/kg
Amount
of
pesticides
in
seeds collected
from
Qibray,
mg/kg
1
Atrazine
Less than 30
mg/kg
-
-
2
Simazine
Less than 30
mg/kg
0,15
0,28
3
Hydroxyatrazine Less than 30
mg/kg
-
0,012
4
Flumethrin
Less than 30
mg/kg
0,245
0,29
5
Mepiquat
Less than 30
mg/kg
-
-
6
Methylsulfuron Less than 30
mg/kg
-
-
7
Chlorimuron
Less than 30
mg/kg
-
-
8
Chlorsulfuron
Less than 30
mg/kg
0,084
0,0024
Figure 10. Results of the analysis of pesticides in horse chestnut seeds.
The findings revealed that horse chestnut seeds from Tashkent contained 0.15
mg/kg of simazine, 0.245 mg/kg of flumethrin, and 0.084 mg/kg of
chlorsulfuron, with no trace of atrazine, hydroxyatrazine, mepiquat,
methylsulfuron, and chlorimuron. In contrast, horse chestnut seeds from Qibray
contained 0.28 mg/kg of simazine, 0.012 mg/kg of hydroxyatrazine, 0.29 mg/kg
of flumethrin, and 0.0024 mg/kg of chlorsulfuron, with the same absence of
atrazine, hydroxyatrazine, mepiquat, methylsulfuron, and chlorimuron.
4. Conclusion.
The horse chestnut seeds collected from Tashkent City and Qibray district were
subjected to a comprehensive analysis to identify the presence of pesticides.
Specifically, the pesticides atrazine, simazine, hydroxyatrazine, flumethrin,
mepiquat, methylsulfuron, chlorimuron, and chlorsulfuron were meticulously
examined. Their concentrations were accurately determined using high-
performance liquid chromatography, ensuring precise results. The findings of
the study revealed that the levels of pesticides in the horse chestnut seeds from
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both Tashkent City and Qibray district were found to be within the permissible
limits outlined by the Sanitary Rules and Regulations (SanPiN №0321-15).
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2. Tankiewicz, M., Fenik, J., & Biziuk, M. (2011). Solventless and solvent-
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3. Gupta, D. K., Chatterjee, S., Datta, S., Voronina, A. V., & Walther, C. (2017).
Radionuclides: accumulation and transport in plants. Reviews of Environmental
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4. Cilia, N., & Kandris, I. (2023). Training in the evaluation of pesticides (plant
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