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 b %. 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 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 which controls wide variety of pests. It is chemically known
as O, O-diethyl O-(3, 5, 6-trichloro-2-pyridinyl-phosphorothioate) (Fig. 1).
Chlorpyrifos kills insects by affecting nervous system which inhibit the
breakdown of acetylcholine and resulting accumulation of acetylcholine in the
synaptic cleft causes overstimulation of neuronal cell. It produces neurotoxicity
and death. Prophenofos is also a broad spectrum organophosphorus pesticide. It
is chemically known as O-4-bromo-2-chlorophenyl O-ethyl S-propyl
phosphorothioate (Fig. 2). 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

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meals in day to day life. To reach the need of vegetables, it is necessary to stop
the wastage of crops carried out by pests. Further, to improve the quantity of
crops sometimes farmers uses high amount of pesticides. Regular or frequent
use of organophosphorus pesticides increases the chances of these pesticides
being found in vegetables which may enter in to the body and damage the
human health.

From the literature review, it was revealed that, no method is available for
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
endeavoured 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 system consisting of binary HPLC pumps
equipped with a photodiode array (PDA) detector and anodyne loop 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 transfer 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 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 6 for UV and
HPLC methods, respectively. From this stock solution, 10 mL aliquot was

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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 mixture was homogenized using a mixer.
Mixture was then transferred to a conical flask, 5 g of sodium bicarbonate was
added and shaken for 5 min. Magnesium sulphate (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 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 homogenised.
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. 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

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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
mm membrane filter and degassed before use. 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.
Correlation coefficient, intercept and slope was determined for both the
pesticides. 8 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
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.

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 UV spectrophotometric method: Known amounts of standard solutions of
chlorpyrifos and prophenofos (4.8, 6 and 7.2 µg/mL) were added to preanalysed
synthetic mixture of chlorpyrifos and prophenofos (6 µg/mL). All samples were
analysed as described in section 2.5. 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 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

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 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.

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 11 order

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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, first-order derivative UV spectrophotometric method was
performed (Fig.3). From spectral observation, 277 nm (zero crossing point for
prophenofos) 289 nm (zero crossing point for chlorpyrifos) were selected as
analytical wavelength for measuring the amplitude of chlorpyrifos and
prophenofos, respectively, as shown in Fig. 3. HPLC method: 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. 4.

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
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

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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 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).

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 4. Conclusion
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 vegetables 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.

Table 1: Validation parameter for UV- spectrophotometric method

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

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Table 2 : Validation parameter for RP-HPLC method
Parameter Chlorpyrifos Prophenofos
Linearity range (µg/mL) (n=6) 0.01-1.5 0.01-1.5
Regression equation Y=8141879x -14225 y = 90697x – 7971
Correlation coefficient(r) 0.9990 0.9990
Intra-day precision (%RSD) (n=9) 0.99-1.02 0.4-1.25
Inter-day precision (%RSD) (n=9) 0.89-1.3 0.47-1.25
Repeatability (%RSD) (n=6) 1.66 1.51
Specificity Specific Specific
LOD (µg/mL) 0.00053 0.00094
LOQ (µg/mL) 0.0016 0.0028
Recovery (%)± SD (n=9) 99.99±0.94 to 101.9±0.68 99.86±0.71 to 101.12±1.02
Robustness Robust Robust

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

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