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Prophenofos is also a broad spectrum of organophosphorus pesticide. It is
chemically known as O-4-bromo-2-chlorophenyl O-ethyl S-propyl
phosphorothioate (Fig. 4). It is mainly used for control pests in vegetables and
cotton (European Food Safety Authority, 2010). Prophenofos pesticide inhibits
acetylcholine esterase and causes neurotoxicity in insects. Its mode of action is
similar to that of chlorpyrifos for non-targeting organisms.
From the literature review and ground level survey, it was found that
chlorpyrifos and prophenofos are frequently used to control the pests in
cauliflower and cabbage. These vegetables are commonly used in salads and
meals in the day to day life. To reach the need for vegetables, it is necessary to
stop the wastage of crops carried out by pests. Further, to improve the quantity
of crops sometimes farmers use high amount of pesticides. Regular or frequent
use of organophosphorus pesticides increases the chances of these pesticides
being found in vegetables which may enter into the body and damage the
human health.
From the literature review, it was revealed that no method is available for
the simultaneous estimation of chlorpyriphos and prophenofos in vegetable
samples. Hence, it is essential to develop a simple, accurate, and easy method to
monitor the level of pesticides in vegetable samples. Therefore it was
endeavored to develop and validate UV spectrophotometric and HPLC methods
2.1 Instrumentation
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min. Supernatant from the centrifuges tubes were collected, evaporated at 80ºC
and reduced up to 2 mL. Further, 20 mL mixture of ethyl acetate and
cyclohexane (1:1, v/v) was added. Then, 1mL of final solution was transferred
to 10 mL volumetric flask and diluted up to the mark with ethyl acetate and
acetonitrile for UV and HPLC methods, respectively.
Hibar C18 analytical column (250 mm × 4.6 mm, 5.0 µm) was used as a
stationary phase and column temperature was maintained at 24°C. The injected
sample volume was 20 µL. The Mobile phase composition was acetonitrile:
water (90:10, v/v). The mobile phase was filtered through a nylon 0.45 µ, 47
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mm membrane filter and degassed before use. The flow rate of mobile phase
was 1 mL/min. The detection wavelength for both the pesticides was selected as
219 nm using PDA detector.
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concentration and the regression equations were calculated for both the analyte.
Each response was the average of six determinations.
2.7.2 Accuracy
2.7.3 Precision
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prophenofos. Repeatability was performed on 10 µg/mL concentration of both
the pesticides. A first order spectrum was generated and absorbance was
measured at 277 nm and 289 nm for chlorpyrifos and prophenofos respectively.
The results were reported in terms of RSD.
For this determination calibration curve for both the pesticides was
repeated three times. The LOD & LOQ were measured by using mathematical
equations given below
LOQ = 10 x σ/S
Where,
2.7.5 Robustness
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UV spectrophotometric method: Robustness of the method was
determined by measuring bsorbance at wavelength ± 2 nm of λmax for both the
pesticides using 10 µg/mL solutions.
2.7.6 Specificity
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chlorpyrifos) were selected as analytical wavelengths for measuring the
amplitude of chlorpyrifos and prophenofos, respectively, as shown in Fig. 1.
HPLC method: The Mobile phase was optimized by taking several trials.
Finally, acetonitrile: water (90:1, v/v) was selected as optimized mobile phase
as it showed very well resolved peaks of both the analytes with good peak shape
and symmetry. Hibar C18 (250 mm × 4.6mm, 5 µ) column was selected as
stationary phase. The flow rate of mobile phase was set as 1 mL/min, and total
runtime was 8 min. The estimation for both the pesticides was made at 219 nm.
A complete resolution of peaks, with clear baseline separation, was obtained
with retention times of 3.2 and 5.52 min for chlorpyrifos and prophenofos
respectively, as shown in Fig. 2.
A system suitability test for HPLC was performed before each validation
run. Five replicate of standard solutions of both the pesticides were injected in
HPLC. Different parameters were monitored for HPLC. Asymmetry of the
chromatographic peak was found to be 1.2 for chlorpyrifos and 1.12 for
prophenofos. Resolution between the peaks was 12.34. Number of theoretical
plates were found to be 7009 for chlorpyrifos and 9688 for prophenofos with
capacity factor 0.28 and 1.1, respectively. The chromatogram showed the
retention time of 3.2 and 5.52 for the chlorpyrifos and prophenofos,
respectively.
3.2.2 Linearity
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correlations were obtained between peak area and concentration for chlorpyrifos
and prophenofos in the ranges of 0.01–1.5 µg/mL as shown in Table 2.
3.2.3 Accuracy
3.2.4 Precision
Two extraction methods, LLE and SPE were compared by adding known
amount of chlorpyrifos (0.1 µg/mL) and prophenofos (0.1 µg/mL) in the
sample. The % amount recovered from the sample by LLE was found to be
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90.88 % for chlorpyrifos and 89.15 % for prophenofos. The % amount
recovered from the sample by SPE were found to be 97 % for chlorpyrifos and
94.5 % for prophenofos. From the results it was found that SPE is more efficient
extraction technique as compare to LLE.
3.2.7 Robustness
4. Conclusion
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Developed and validated UV spectrophotometric and HPLC methods are
efficient for the determination of chlorpyrifos and prophenofos pesticides in
vegetables. It was found that the methods were accurate, precise, sensitive,
robust and easy to apply. Samples collected from different regions, chlorpyrifos
were found to be of the concentration of 0.018mg/kg, which is above MRL
value (0.01 mg/kg), while, prophenofos was not found in any of the analysed
samples. Both the methods were compared statistically , which shows, there is
no significant difference among both the method. Among the different cleaning
solutions tested for pesticide removal from vegetable soda-salt (5%) solution
was found to be the most effective. It showed the highest relative cleaning
capacity as compared to the other cleaning agents tested. The pesticide residues
are present in higher amount in the vegetables that we are consuming. Hence, it
is of utmost importance to keep an eye over the use of pesticides to protect the
crops and the present methods can be helpful to monitor the over usage of
pesticides.
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Table 1: Validation parameter for UV- spectrophotometric method
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-14225 7971
Correlation 0.9990 0.9990
coefficient(r)
Intra-day precision 0.99-1.02 0.4-1.25
(%RSD) (n=9)
Inter-day precision 0.89-1.3 0.47-1.25
(%RSD) (n=9)
Repeatability 1.66 1.51
(%RSD) (n=6)
Specificity Specific Specific
LOD (µg/mL) 0.00053 0.00094
LOQ (µg/mL) 0.0016 0.0028
Recovery (%)± SD 99.99±0.94 to 99.86±0.71 to
(n=9) 101.9±0.68 101.12±1.02
Robustness Robust Robust
Figure captions
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Reference
Eaton, D. L., Daroff, R. B., Autrup, H., Bridges, J., Buffler, P., Costa, L.
G., … Nadel, L. (2008). Review of the toxicology of chlorpyrifos with an
emphasis on human exposure and neurodevelopment. Critical Reviews in
Toxicology, 38(sup2), 1–125.
Zhang, Y., Li, X., Liu, H., Zhang, Y., Zhao, F., & Yu, Q. (2013). Study
on universal cleaning solution in removing blended pesticide residues in
Chinese cabbage. Journal of Environmental Chemistry and Ecotoxicology, 5(8),
202–207.
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Keehner, D. M., & Shamim, M. (1999). Environmental Risk Assessment
for Prophenofos. Washington: United States Environmental Protection Agency,
Washington.
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