American-Eurasian J. Agric. & Environ. Sci.

, 6 (5): 513-519, 2009

ISSN 1818-6769
IDOSI Publications, 2009

Determination of the Organophosphorus Pesticide in Vegetables

by High-Performance Liquid Chromatography
Sajjad Ahmad Baig, 1Niaz Ahmad Akhtera,
Muhammad Ashfaq and 3Muhammad Rafique Asi
1

Institute of Quality and Technology Management, University of the Punjab, Lahore 54590, Pakistan
2
Nuclear Institute for Agriculture and Biology (NIAB), Faisalabad38000, Pakistan
3
Department of Entomology, University of Agriculture, Faisalabad 38000, Pakistan

Abstract: In the existing study, an effort has been made to evaluate and determine residues of three pesticides
in vegetables, belonging to Organophosphate group, which was extensively used in the Southern part of
Punjab province in Pakistan. Triazophos, profenofos and chlorpyrifos Organophosphorus insecticides, were
commonly used on the summer vegetables like: egg plant, Pumpkins and okra cultivated in study area.
Vegetables were analyzed for pesticide residues using multiresidue analysis by High-Performance Liquid
Chromatography (HPLC) equipped with UV detector. It was observed that 33.0% of the samples were
contaminated with any of the above three pesticides. The results showed that 8% of the samples tested
contained residues higher than the MRLs. Based on observations made in these studies; it is proposed that
more extensive monitoring investigations covering all vegetables from different agro-climatic regions of the
state be carried out to find the exact position of pesticide residues.
Key words: HPLC

Organophosphate pesticides

Vegetables

INTRODUCTION

Pesticide Residues

A huge amount of pesticide is used on crops as well

as on vegetable and their irrational and continual use
becomes the reason of accumulation of pesticide residues
in the primary agriculture products. The problem of
residues
accumulation needs more attention in
vegetables because most of time these are consumed
either raw or without much storage time [4]. Currently
Organophosphorus pesticides (OPs) enjoy wide use in
the world for the pest control with Organochlorine
pesticides [5, 6], which leads to increased world food
production. These Organophosphorus pesticides are also
widely used in Pakistan for controlling the agriculture
pests. In spite of the toxicological problem its efficiency
has been demonstrated in fruit flies, aphids and leaf
miners The continued use of organophosphorus
pesticides increases the possibility that residues of these
compounds could be found in some vegetables [7]
affecting the alimentary security; that is the reason for the
commitment between the public health and the sanitary
defense.
For this reason, it is urgent to develop programs
related to the monitoring of pesticide residues in food to

Agriculture is the single largest sector of Pakistanis

economy with 21 percent contribution to GDP and
employing about 44 percent of the workforce. More than
two-thirds of Pakistans population lives in rural areas
and their livelihood continues to revolve around
agriculture and allied activities [1].
Vegetables are important ingredient of our food
having a high nutritional value. Vegetables, like, okra,
egg plant, spinach, cauliflower, tomato, pumpkin, carrots,
turnips etc. are produced in the country for local
consumption as well as for export purposes [1]. For better
production and aesthetic value, farmers are using a large
amount of insecticides during the entire period of growth
of vegetables, even at fruiting stage and sometimes
farmers also ignored the recommended waiting period
between the harvest and last spray. Owing to this and
other injudicious practice related to pesticide usage,
pesticides become the inner part of vegetable, in the
shape of residues, which could be used by consumers
thus creating health hazards [2, 3].

Corresponding Author: Sajjad Ahmad Baig, Institute of Quality and Technology Management,
University of the Punjab, Lahore 54590, Pakistan
Tel: +92 3004781314

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Am-Euras. J. Agric. & Environ. Sci., 6 (5): 513-519, 2009

secure consumers. These programs could determine the

amount of the contamination problem and recognise a
way to solve the situation. Pesticide (Organophosphorus)
residue monitoring studies have been reported in many
countries of the world on fruits and vegetables [8-13].
Effects of pesticides have been reported in Pakistan in
vegetables, fruits, feed, cottonseed and fish meal at
different intervals [15-19] The above mention studies in
Pakistan showed that most of the samples were
contaminated by chlorinated pesticides; some of them
were exceeding the maximum residue limits, which can be
threatening for the ultimate consumers. Although, a high
variation of pesticide residues was observed in fruits and
vegetables that could be due to change in the climatic
zone (hot, humid and cold) and variation among different
species of plants. No study has been carried out in
Pakistan to analyze the OPs residues in vegetables, also
the study on pesticide residues in vegetables was also
never carried out in the Southern Punjab, Pakistan.

Profenofos is a broad-spectrum Organophosphorus

pesticide. Profenofos (Ensedan1), O-(4-bromo-2chlorophenyl)O-ethyl S-propyl phosphorothioate, was
developed for those insect strains which developed
resistant to other Organophosphorus pesticides It is
selectively toxic to insects compared to mammals due to
different metabolism of the propylthiol group [22].
Chlorpyrifos is an organophosphate pesticide used
throughout the world around the world and Chlorpyrifos
is currently registered as insecticide with the used in a
wide variety of crops and vegetables The primary
degradant of chloropyrifos is 3, 5, 6-trichloro-2-pyridinol,
a base hydrolysis product [23]. As compare to other
organophosphate pesticide, Chlorpyrifos is fairly stable
and persistent member of organophosphate pesticides
MATERIALS AND METHODS
In this section, all the materials used in the study and
the methods of analysis have been discussed. This
section has been divided into three sub-sections, which
have been discussed in following text.

Selection
of
Pesticide:
However,
nowadays
organochlorine (OC) pesticides were not used primarily
due to their persistence in the environment [20]. At this
time, the use of OPs and CMs glided due to their
availability and quick degradation in the environment
Organophosphorus pesticides have been widely
manufactured and used in the world. And they replaced
the chlorinated pesticides. A survey of the study area i.e.
southern part of the Pakistani Punjab, was carried out to
assess the extent of widely used pesticides before
sample collection and analysis. In the survey, 240 farmers
from four districts (Layyah, Muzaffargarh, Multan and
Khanewal) were interviewed to through a well structured
questionnaire to judge the extent of pesticide use,
practices related to the a pplication of the pesticides
and equipments and safety measures. Results of the
survey showed that many Ops pesticides were used
area but three insecticides (Triazophos, Profenofos
and Chlorpyrifos) were widely used in the area on the
vegetable crops.
Triazophos (C12H16N3SPO3) is a common name
for
O,O-diethyl-O-1-phenyl-1H-1,2,4-triazol-3-yl
phosphorothioate). Triazhphos is a moderately toxic
chemical that is a broad spectrum and no systemic
Organophosphorus pesticide that is used in large
quantities throughout the world [21]. It is used to control
aphids, bollworms, red spiders, fruit borers, leaf hoppers
and cutworms on a variety of crops, such as fruits, cotton,
maize, wheat, cereals and vegetables and is particularly
effective in the control of plant nematodes [22].

Chemicals: Ethyl acetate (HPLC grade), Methanol (HPLC

grade), Sodium chloride (HPLC grade), acetonitrile (HPLC
grade), potassium permanganate (GR 99%)were all
obtained from Merck, Germany)grade. Distal water was
obtained with the help of glass-distilled and further
purified with the help of a Millipore Milli-Q water purifier.
Germany. Analytical standard of profenofos, chlorpyrifos
and triazophos was obtained from Dr Ehrenstorfer Ltd
(Augsburg, Germany). Stock standard solution (1mg/mL)
was prepared by dissolving standard in acetonitrile and
stored in the dark at 4C
Sample Collection Procedure: In this portion of the
chapter sample collection and preparation methodologies
has been discussed. This section is further divided into
two parts;
Sample Collection from Farmers Fields: Sampling was
carried out, from July 2008 to the last week of August 2008
from, the farmers fields of four districts in Southern
Punjab., i.e., Layyah, D.G. Khan, Muzafargarh and Multan.
Some parts of the study area, were suffering from waterlogging and salinity, problems. Different depths of watertables were found in the study area. Normally, in the
month of January, the temperature is low and the
evaporation is slow. The month of June and July, are the
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Am-Euras. J. Agric. & Environ. Sci., 6 (5): 513-519, 2009

Extraction and Clean up: Vegetable samples were

extracted by following the method of 24 with some
modification made by 25 and 26. Ethyl acetate was used
for the extraction because of extraction efficiency, of ethyl
acetate is to be equally good for the extraction of both
polar and non-polar pesticides in all primary agriculture
products [27] g of vegetable sample was grind and then
well-homogenized 50 g from the grind vegetable was
selected and mixed with 20 g anhydrous sodium sulfate +.
1 moler Nacl and 50 ml of ethyl acetate. The whole mixture
was s haken in GFL shaker (Germany) at high speed for
5 minutes and filtered. And then transferred onto a glass
column having 5 ml layer of anhydrous sodium sulfate
and 10 g activated charcoal for bleaching. The mixture was
vacuum-filtered through a Whatman no. 6 filter paper
After filtering solution was shifted into 250 ml round flask
and then evaporate and concentrate with a rotary
evaporator at 55C. and made a volume up to 10 ml and
then filtered through a 0.45 m filter membrane collected
elute was concentrated just to dryness under a gentle
stream of nitrogen gas and re-dissolved in acetonitrile,
volume made up to 500 l for analyzing with HPLC
HPLC analyses were performed on isocratic system
using a Shimadzu Chromatograph including LC-10 AS
pumps, 20-ll Reodyne injector, SPD-10A UV detector
operating at 208 nm and a Supelco C18 analytical column
(25 cm _ 4.6 mm(i.d)). a mixture of water and acetonitrile
(ACN) was used as mobile phase..For the removal of any
impurity, bubbles and dissolved oxygen, the double distal
water and ACN was firstly passed through 0.45 lm filter
paper with filtration assembly and then sonicated for
10. The flow rate was kept at 1 ml/min.
Before analyzing the samples of vegetables, the
HPLC method was validated using chromatographic
parameters including recovery, reproducibility and limits
of detection (LOD) as proposed by 29 and 30. For
recovery experiments, untreated samples of vegetables
were spiked with selected pesticides at 0.5mg/kg and
1.5 mg/kg.
For each fortification level, five replicates were
analyzed by HPLC using UV detector. The efficiency of
the method was validated statistic with recoveries studies,
fortification samples without pesticides (organic
vegetables) by spiking the three different amounts of
pesticides. And then pesticides were extracted from the
vegetables as described early. the reproducibly of all
pesticides were more than 85%.which was satisfactory.
These values were quite satisfactory and meet the
requirements of the European Commission 31, indicating

hottest months in the study area. Rainfall occurs mostly

in the months of July and August, during the monsoon
period. Rabi and Kharif are the two cropping seasons, in
the area. Since the Kharif season covers most part of the
summer and rainy periods, vegetables of Kharif season,
were selected because of the maximum sprays were carried
out on them due to a favorable atmosphere for pests, to
flourish.
Samples of three vegetables i.e., Okra, (Abelmoschus
esculentus L. Moench), Pumpkins, (Cucurbita Pepo)
and Egg plant (Solenum melongena L.), which were
extensively grown vegetables in the study area, were
collected. Information about farmers aptitude, type of
pesticide used and agronomic practices adopted by the
farmers, were also collected with the help of a well
structured questionnaire.
Samples from 108 farms; comprising 32of egg plant,
32 of okra and 32 of pumpkins were collected. Composite
samples: consisted of 12 kgs of each vegetable were
collected from the field, when it was ready for sale. After
selecting the field location and considering all the factors,
which can affect the pesticide residues, the desired
vegetables, were collected in a defined way, such as, S or
an X in the field. During the sample-collection disposable
gloves were used and changed every time before
collecting the next sample. Dust from the samples was
removed with light brushing, without washing prior to
placing them into, the collection bags. Samples were
double packed, in order to save from any other
contamination and transported from the field to the
laboratory. During the transportation of samples, it was
assured that these were protected against any alterations
in the residue-situation. In the laboratory, samples were
freeze in the refrigerator and analyzed within a weeks time
after collection. Only edible parts were selected for the
analysis of pesticide residues.
Sample Collection from Supervised Fields: Three summer
vegetables i.e. okra (Abelmosc hus esculentus L.),
pumpkin (Cucurbita Pepo) and egg plant (Solanum
melongena L.) were grown in the experimental field of,
selective districts respectively. Each vegetable was grown
in a separate plot, measuring 150 x 30 feet in four blocks.
Each plot was separated by fallow strip of 20 feet. The
vegetables, in each season, received normal agronomic
practices, without the spraying of pesticides along with
the selective spray of pesticides. The samples collected
from supervised field trial were used for the spiking of
vegetable.
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that a method can be considered accurate and precise

when the accuracy of data is between 70 and 110%, with
relative standard deviations (RSDs) not higher than 20%.

RESULTS AND DISCUSSION

P(OCH2CH3)2
O

N
N

Fig 1: Structure of Triazophos

This research work was completed in two phases.

In the first phase, a survey was conducted, for
determining the nature and frequency of pesticide
currently being used. Only those farmers were
interviewed who have at least 3 acres for cultivation. As
such structured questionnaire will developed to collect
information from the farmers about their, knowledge of the
adverse health effects of pesticides, work practices related
to the pesticide use, use of protective measures and also
about an inventory of pesticides used on the farm On
the bases of the questionnaire, the Organophosphorus
pesticide for further study was selected.
It was found that 35 samples, out of 108, were found
to be contaminated with some of the organophosphate
pesticides, studied. Profenofos was found to be the
maximum in the analyzed samples of okra (25%), egg plant
(17%) and pumpkins (5%), where as, Chlorpyrifos was
found to be the second most common op pesticide in the
vegetable. It was observed that frequency of pesticide
residues detection in the okra was directly related to their
usage in the area.

P(OCH 2CH3)2
O

N
N

Fig 2: Structure of Profenofos

S
Cl

O P(O CH 2 C H 3 ) 2

Cl
Cl
Fig 3: Structure of Chlorpyrifos

Egg Plant: Out of a total of 36 samples of egg plant,

analyzed, 17 were found to be contaminated with the
residues of pesticides; Chlorpyrifos was detected in a
maximum number of egg plant samples, as compared to
the other two OP pesticides. It was determined that
8 samples, out of 36 samples, were found to be polluted
with the chlorpyrifos -residues and out of these 8
samples, three samples contained pesticide-residues, more
than the MRLs The results of organophosphate pesticide
residues, detected in vegetables, samples collected from
the farm gate, are, presented with the Fig 4 and Table 1.
The results indicated that at a raw stage, the highest
amount of chlorpyrifos residues found in egg plant was
1.45 mg/kg (ND 1.45 mg /kg) and 17% of the egg plant
samples were seen contaminated with profenofos.
One sample of the egg plant contained profenofos
residues more than the MRLs. Triazophos was detected
in a minimum number of egg plant samples, as in
comparison to the other OPs. The initial data collected in
the survey showed that chlorpyrifos, was used more on
egg plant, in the area, as in comparison to other
organophosphates.
516

Okra: Out of a total, 36 samples of okra analyzed, 15 were

found to be contaminated with the residues of selected
OP pesticides. The results of residues detected in
vegetable samples collected, from the farm gate are
presented through the Fig 4.2 and Table 4.3. The results
indicated that at raw stage the highest Profenofos residue
was found in egg plant were 1.6 mg/kg (ND 1.6mg /kg);
whereas the four samples were contained pesticide
residues of Profenofos more than the MRLs and 7(19%)
samples of okra were observed to be contaminated with
the residues of chlorpyrifos, that the one sample was
contained residues of chlorpyrifos exceeded from the
MRLs. Five samples of okra found to be contaminated
with the residues of triazophos. The initial data collected
in the survey showed that the usage of profenofos was
more on the okra crops as compared to that on others.
During the survey it was also observed that, in many
cases, okra was grown near the cotton field and the
farmers used same treatment of insecticide, without
differentiating the two crops.
Pumpkins: A total of 36 samples of pumpkins, were
analyzed, out of which only 5 samples were found
to be contaminated with pesticide residues. The
residues of triazophos were not found, for the main reason
of this that no spray of triazophos was reported to be
carried out on pumpkins. In the area, most of the
pumpkins were produced without the application of any
pesticide sprays, which, if at all carried out, pesticide were
carried out on early stage. No sample of the pumpkins was
found to contain more than one type of pesticideresidues.

Am-Euras. J. Agric. & Environ. Sci., 6 (5): 513-519, 2009

Table 1: Pesticide residues (mg kg_1) in fresh vegetables, collected from study area
Sr.No

Vegetable

Insecticide Analyses

Okra

36

Profenofos

9(25%)

Nd-.1.6

Triazophos

5(14%)

Nd-.9

.5*

Egg plant

Pumpkin

36

36

Contamination

MRLs

Chlorpyrifos

7(19%)

Nd-1.7

1.5**

Profenofos

6(17%)

Nd-1.45

Triazophos

4(11%)

Nd.-.8

Chlorpyrifos

8(22%)

Nd-1.34

1 **

Profenofos

2(5%)

Nd-.45

0.05*

Triazophos

1(2.5 %)

Nd-.32

Nd

.2**

Chlorpyrifos
*Japanese MRLs

Range of Residues mg/kg

** FAOMRLs

Fig. 4: Occurrence of pyrethroid pesticide residues in fresh vegetables samples

Fig. 5: Organophosphate Residues in Vegetables

On the whole, 35 samples out of 108 were found to be
contaminated with selected type of (organophosphate)
pesticide-residues. From the results, it was clear that
although all selected vegetables were found to be
contaminated with the pesticide-residues, yet okra was
seen to be affected the most from the organophosphate
pesticides. Mostly, only one type of residues was found
in the vegetable samples. But in some sample more than
one type of residues were detected. Out of 35 pesticidecontaminated samples, 9 were contained residue more
than their MRLs
On a further investigation, from the farmers through
an open discussion, it was found that most vegetable
growers, who were using toxic pesticides, on a high

residues level, did not care for the quality of their produce
as they, were neither interested in ensuring the vegetable
safety, nor followed the good agricultural practices,
because of their ignorance or lack of such a training.
It was further noticed that most of farmers did not fallow
the concept of Good Agriculture Practices. And they
did not follow the with holding periods. A withholding
period (WHP) is the minimum time you must wait between
applying an agrochemical and final harvesting or grazing
of the crop. Such a period allows enough time to decay to
an acceptable level [32]. Most of the vegetable samples
were brought to the market, after first day of spraying,
contained residues, which exceeded their MRLs. Result of
this study, were verified from the study of 33. Although,
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Am-Euras. J. Agric. & Environ. Sci., 6 (5): 513-519, 2009

it was cleared that level of pesticides in the primary

products were less as compared to the previous studies
in Pakistan. Yet these results generally agree with the
other studies already carried out, locally [34] and
international with 35 This was mainly due to the using of
organophosphorus pesticides and new chemistry
products, instead of organochlorines pesticide which are
more persistent and have more chances of accumulation
in food [36] As an outcome of this study, it is suggested
that a wide-range of studies, on the monitoring of all
summer vegetables and other crops grown in different
agro-climatic regions of Pakistan should be undertaken for
determining the level of pesticide of pesticide residues
and developing awareness about Good Agriculture
Practices

7.

8.

9.

10.

ACKNOWLEDGEMENT
The authors thank to university of Punjab, Pakistan
and also to HEC (Higher Education Commission) Pakistan
for providing financial aids for this project.
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