Determination chlorpyrifos and prophenofos in

vegetables by HPLC and UV spectrophotometric method

1. Introduction

Pesticides are widely used in agriculture and livestock production to

control insect, plant and fungal pests and diseases. During the past 50 years, the
human population and global agricultural production have both more than
doubled, with productive arable areas increasing by some 10 %. The use of
pesticide products is massive and continues to grow, with an estimated more
than 2.5 million metric tons applied every year worldwide, 40% of this being
herbicides.

Organophosphorus like chlorpyrifos and prophenofos compounds are

widely used as pesticides. Organophosphorus pesticides are highly toxic to
human beings and animals. Due to its hazardous effect on human health, it is
essential to control the unwanted use of pesticides. So it is the utmost
importance to check the level of pesticides in different food materials. Specific
sampling techniques and methods are used for determining the level of
pesticides in food samples. Maximum residue limits (MRL) is the quantity of a
given chemical remaining on food samples. The MRL is different for different
pesticides according to the potency of pesticides. From organophosphorus
pesticides, chlorpyrifos and prophenofos are widely used to protect the different
fruits, citrus, maize, tobacco and vegetables in the world.

Chlorpyrifos is a broad-spectrum pesticide that controls wide variety of

pests. It is chemically known as O, O-diethyl O-(3, 5, 6-trichloro-2-pyridinyl-
phosphorothioate) (Fig. 3). Chlorpyrifos kills insects by affecting the nervous
system which inhibits the breakdown of acetylcholine and resulting
accumulation of acetylcholine in the synaptic cleft causes overstimulation of
neuronal cell. It produces neurotoxicity and death.

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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. Materials and methods

2.1 Instrumentation

The UV spectrophotometric method was developed on a double beam

spectrophotometer made by Shimadzu Inc., model 2450, with spectral width of
1 nm, wavelength accuracy of 0.5 nm and a pair of 10 mm matched quartz cells.
The HPLC method was developed on a system consisting of binary HPLC
pumps equipped with a photodiode array (PDA) detector and anodyne loop
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injector with fixed 20 µL capacity. Borwin PDA software was used for
computational purposes. Sonicator D-compact., capacity 2 L (TRANS-O-
SONIC), hot air oven EIE 108 (EIE Instruments Pvt. Ltd.) were used during the
study. For solid-phase extraction (SPE), solid-phase extractor (SPE 24 position
vacuum manifold set, Phenomenex) and cartridge (C18, Celerity deluxe 30 mg)
procured from Orochem Technologies Inc was used (Sharma, Kothari, Sherikar,
& Mehta, 2014).

2.2 Preparation of standard stock solution

UV spectrophotometric method: Chlorpyrifos (10 mg) was accurately

weighed and transferred to 100 mL volumetric flask and dissolved in ethyl
acetate and acetonitrile to get 100 µg/mL stock solution of chlorpyrifos for UV
and HPLC methods, respectively. For prophenofos, 0.68 mL (93-94 % purity)
of the standard was transferred to 100 mL volumetric flask and dissolved in
ethyl acetate and acetonitrile to get 1000 µg/mL of stock solution of
prophenofos for UV and HPLC methods, respectively. From this stock
solution, 10 mL aliquot was transferred to 100 mL volumetric flask and diluted
with respective solvent to get 100 µg/mL working stock solution of prophenofos
.

2.3 Sample preparation for liquid-liquid extraction (LLE)

Finely cut and chopped 20 g quantity of cabbage and cauliflower was

taken and washed with distilled water. The vegetables were allowed to air dry
for 30 min. Ethyl acetate (50 mL) was added and the mixture was homogenized
using a mixer. The mixture was then transferred to a conical flask, 5 g of
sodium bicarbonate was added and shaken for 5 min. Magnesium sulfate (15 g)
was added further and shaken for 1 hr in mechanical flask shaker. After
shaking, the homogenates of both the vegetables were filtered and 10 mL of the
filtrates were transferred to centrifuge tubes and centrifuged at 1500 rpm for 5

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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.

2.4 Sample preparation for solid-phase extraction (SPE) for HPLC

Finely cut and chopped 20 g quantity of cabbage and cauliflower was

taken and washed with distilled water. Acetonitrile (50 mL) was added and
homogenized. It was transferred to a conical flask, 10 g of sodium chloride was
added and shaken for 10 min. The solution was filtered and transferred to
centrifuge tubes and centrifuged at 1500 rpm for 5 min. The solution was
evaporated and reduced to 5 mL, extracted with 10 mL acetonitrile and toluene
(3:1, v/v). SPE cartridge was pre-treated with 1mL of distilled water followed
by 1 mL of acetonitrile. After pre-treatment, 2 mL of extracted solution (section
2.5) was loaded to SPE cartridge and eluted with 10 mL acetonitrile.
Acetonitrile solution was collected for further analysis.

2.5 UV spectrophotometric conditions

The solutions of chlorpyrifos and prophenofos were scanned in the

spectrum mode from 200 to 400 nm and overlay spectra were recorded. First-
order spectra was used to separate the overlapping peaks of chlorpyrifos and
prophenofos by measuring absorbance at 277 nm and 289 nm, respectively.

2.6 RP-HPLC conditions

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.

2.7 Method validation

The developed methods were validated as per the International

Conference on Harmonization (ICH) Guidelines for Validation of Analytical
Methods. Various parameters were evaluated, including, specificity, linearity,
limit of detection (LOD), limit of quantification (LOQ), accuracy, precision and
robustness.

2.7.1 Preparation of calibration curve

UV spectrophotometric method : Appropriate volume of aliquots (0.6,

0.8, 1.0, 1.2, 1.4 and 1.6 mL) from standard stock solutions of chlorpyrifos and
prophenofos (100 µg/mL) were transferred to different 10 mL volumetric flasks,
the volume was made up to mark with ethyl acetate to obtain the concentration
of 6, 8, 10, 12, 14 and 16 µg/mL for both the pesticides. The absorbance of
solutions was measured at 277 nm (λmax of chlorpyrifos) and 289 nm (λmax of
prophenofos). The calibration curve was constructed by plotting the absorbance
versus concentration. The correlation coefficient, intercept and slope was
determined for both the pesticides.

HPLC method: Suitable aliquots of standard stock solution of

chlorpyrifos and prophenofos (100 µg/mL) were taken in separate 100 mL
volumetric flasks and diluted with acetonitrile to obtain solutions containing
chlorpyrifos and prophenofos having concentration in range of 0.01, 0.05, 0.1,
0.5, 1.0 and 1.5 µg/mL. The samples were injected in HPLC system under
optimized chromatographic conditions and chromatograms were recorded at
219 nm. Calibration curves were constructed by plotting the peak area versus

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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

Accuracy of the method was determined by % recovery study using

standard addition method at three different levels (80%, 100% and 120%) of
assay concentration.

UV spectrophotometric method: Known amounts of standard solutions

of chlorpyrifos and prophenofos (4.8, 6 and 7.2 µg/mL) were added to pre
analysed synthetic mixture of chlorpyrifos and prophenofos (6 µg/mL). All
samples were analysed as described in section 2.5. The concentration of both
the pesticides were determined using calibration curve and regression equation
of the linearity graph.

HPLC method: Known amounts of standard solutions of chlorpyrifos

and prophenofos (0.9. 1.0 and 1.2 µg/mL) were added to preanalysed synthetic
mixture of chlorpyrifos and prophenofos (0.5 µg/mL). All samples were
analysed as described in section 2.6. Concentration of both the pesticides were
determined by using calibration curve and regression equation of the linearity
graph.

2.7.3 Precision

The precision of the method was verified by repeatability, interday and

intraday precision studies. Intra-day and Inter-day precision was determined by
measuring the response of both the pesticides three times within a day and on
three different days, respectively. Repeatability 9 studies was performed by
analyzing six samples of both the pesticides at 100% test concentrations.

UV spectrophotometric method: Intraday and interday precision was

performed on 6, 10 and 14 µg/mL concentrations, for chlorpyrifos and

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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.

HPLC method: Intraday and interday precision was performed using

0.05, 0.5 and 1.5 µg/mL concentrations, for chlorpyrifos and prophenofos.
Repeatability was performed on 0.1 µg/mL concentration of both the pesticides.
Samples were injected under the optimized chromatographic conditions and
responses were recorded at 219 nm. The results were reported in terms of RSD.

2.7.4 Limit of detection (LOD) and limit of quantification (LOQ)

For this determination calibration curve for both the pesticides was
repeated three times. The LOD & LOQ were measured by using mathematical
equations given below

LOD = 3.3 x σ/S

LOQ = 10 x σ/S

Where,

σ = Standard deviation of the intercept

S=Slope of the calibration curve

2.7.5 Robustness

The robustness of the methods was studied by analyzing the samples of

chlorpyrifos and prophenofos with deliberate variations in the optimised
experimental conditions. The changes in the responses of chlorpyrifos and
prophenofos were noted. Results were reported in terms of % RSD.

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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.

HPLC method: Robustness of the method was determined by deliberate

change in flow rate (±0.2 mL/min) and change in λmax (±2 nm) using
concentration 0.1 µg/mL for both the pesticides.

2.7.6 Specificity

The well resolved chromatographic peaks of both the pesticides were

analyzed for peak purity (specificity) by scanning in the range of 200–400 nm.
The specificity of the method was determined by analyzing standard pesticides
and test samples. The peak purity of both the pesticides was determined by
comparing the spectrum at three different regions of the peak, i.e., peak start (s),
peak apex (m), and peak-end (e) by PDA detector.

3 Results and discussion

3.1 Method Optimization

UV spectrophotometric method: Several trials were taken using ethyl

acetate, methanol and acetonitrile as a dilution media. Absorbance of
Chlorpyrifos and Prophenofos was found to be highest in ethyl acetate solvent
compared with other solvents, hence, ethyl acetate was taken as a solvent.
Between 250-400 nm, spectra of both the pesticides were noiseless in ethyl
acetate. Zero-order spectra for both the pesticides having concentration 10
µg/mL were scanned in 200-400 nm range. Zero-order spectra showed the
overlapping of band for both of the pesticides. So, the first-order derivative UV
spectrophotometric method was performed (Fig.1). From spectral observation,
277 nm (zero-crossing point for prophenofos) 289 nm (zero-crossing point for

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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.

3.2 Validation of the developed methods

3.2.1 System suitability test

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

For the UV spectrophotometric method, linear correlations were obtained

between absorbance and concentration for chlorpyrifos and prophenofos in the
ranges of 6 - 16 µg/mL as shown in Table 1. For the HPLC method, linear

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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

The percentage recovery values of chlorpyrifos and prophenofos were

obtained in the range of 98 to 102% and the relative standard deviation (RSD)
values for both the method were less than 2%. The % RSD values of the
accuracy studies for the UV spectrophotometric and HPLC methods are shown
in Tables 1 and 2 respectively, which confirms the accuracy of method.

3.2.4 Precision

Inter-day, intra-day and repeatability variation in the quantification of

chlorpyrifos and prophenofos showed that the RSD values were always less
than 2% during the analysis by both the method. The values of the precision
studies for the UV spectrophotometric and HPLC are depicted in Tables 1 and
2, respectively. These low RSD values show that the methods are precise.

3.2.5 LOD and LOQ

For the UV spectrophotometric method, the LOD values of chlorpyrifos

and prophenofos were 0.44 and 0.33 µg/mL, respectively and LOQ values for
chlorpyrifos and prophenofos were 1.33 and 1.0 µg/mL, respectively (Table 1).
For the HPLC method, the LOD values of chlorpyrifos and prophenofos were
0.00053 and 0.00094 µg/mL, respectively and LOQ values for chlorpyrifos and
prophenofos were 0.0016 and 0.0028 µg/mL, respectively (Table 2).

3.2.6 Comparison of LLE and SPE for pesticides from sample

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

The robustness of the methods was studied by carrying out deliberate

variations in optimized method parameters for chlorpyrifos and prophenofos.
The assay values were calculated in the changed parameters. The assay values
in the changed parameters were within the accepted range. The % RSD value
was found to be 1.53 in UV method by measuring absorbance at 277 ± 2 nm
and 289 ± 2 nm for chlorpyrifos and prophenofos respectively. In HPLC
method, flow rate of mobile phase was changed to 1.0 ± 0.2 mL/min and %
RSD was found to be 1.17. In HPLC, the detection wavelength was varied by
219 ±2 nm and % RSD was found to be less than 0.845 which indicates that the
method proved to be robust (Table 1 and 2).

3.3 Effect of washing solvent

Cleaning effect of washing solutions were estimated by HPLC method.

Chlorpyriphos was found to be 0.018 mg/kg without washing, which is above
MRL value (0.01 mg/kg). After washing with tap water it was found to be less
than 0.015 mg/kg. After washing with warm water and soda-salt solution
chlorpyrifos was found to be 0.012 and 0.008 respectively. Which indicates
that, for washing solution, soda-salt solution (5%) was most effective as
compared to other cleaning agents. Prophenofos was not found in any collected
sample.

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

Parameter Chlorpyrifos Prophenofos

Estimating 277 nm 289 nm
wavelength
Linearity range 6-16 6-16
(µg/mL) (n=6)
Regression equation Y=0.0009x - 0.001 y=0.003x - 0.0017
Correlation 0.9985 0.9995
coefficient (r)
Intra-day precision 0.15-1.26 0.68-1.38
RSD (%) (n=9)
Inter-day precision 1.05-1.69 0.62-1.63
RSD (%) (n=9)
Repeatability RSD 0.25-1.38 0.49-1.58
(%) (n=6)
LOD (µg/mL) 0.44 0.33
LOQ (µg/mL) 1.33 1.0
Recovery (%) ± SD 100-100.16 ± 0.43 100-100.16 ± 0.43
(n=9)
Robustness Robust Robust

Table 2 : Validation parameter for RP-HPLC method

Parameter Chlorpyrifos Prophenofos

Linearity range 0.01-1.5 0.01-1.5
(µg/mL) (n=6)
Regression equation Y=8141879x y = 90697x –

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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

Fig. 1. First-order overlay UV spectra of chlorpyrifos (10 µg/mL) at 277 nm in

ethyl acetate and prophenofos (10 µg/mL) at 289 nm in ethyl acetate

Fig. 2. HPLC chromatogram of chlorpyrifos and prophenofos (0.05 µg/mL) at

219 nm in acetonitrile

Fig. 3. Chemical structure of chlorpyrifos

Fig. 4. Chemical structure of prophenofos

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 Reference

Paranthaman, R., Sudha, A., & Kumaravel, S. (2012). Determination of

pesticide residues in banana by using high-performance liquid chromatography
and gas chromatography mass spectrometry. American Journal of Biochemistry
and Biotechnology, 8(1)

Dasika, R., Tangirala, S., & Naishadham, P. (2012). Pesticide residue

analysis of fruits and vegetables. J Environ Chem Ecotoxicol, 4(2), 19–28

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.

European Food Safety Authority. (2010). Conclusion on the peer review

of the pesticide risk assessment of the active substance bitertanol. EFSA
Journal, 8(10), 1850. doi:doi:10.2903/j.efsa.2010.1850

Tang, H. P. (2013). Recent development in analysis of persistent organic

pollutants under the Stockholm Convention. TrAC Trends in Analytical
Chemistry, 45, 48–66.

Topuz, S., Özhan, G., & Alpertunga, B. (2005). Simultaneous

determination of various pesticides in fruit juices by HPLC-DAD. Food
Control, 16(1), 87–92.

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.

19
 Keehner, D. M., & Shamim, M. (1999). Environmental Risk Assessment
for Prophenofos. Washington: United States Environmental Protection Agency,
Washington.

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