1

ANALYSIS OF PESTICIDES ON GRAPES BY CHROMATOGRAPHIC METHOD

Dewa Putu Gede Sudiatmika, I Wayan Muderawan, and Suheimi Syaban
Chemistry Education Department, Ganesha University of Education
Jl. Udayana No. 11 Singaraja-Bali
Email: dsudiatmika@yahoo.co.id
ABSTRACT
The aims of the research are to determine the presence of pesticide residues
and analysis the level (in ppm) of pesticide residues on the grapes in Banjar village.
Methods that used in this research are extraction and gas chromatography. The
grapes samples were obtained at three farmer groups in Banjar village such as,
Tunggal Pamukti (A), Sari Amerta (B) and Amerta Nadi (C). The grapes samples were
chopped into small portions and pulverized by a food blender until it reaches
homogeneous mixture. The homogeneous texture was entered into extraction tube
and extracted by 15 ml of 1% acetic acid in acetonitrile. Shake the tube vigorously for
1 minute and centrifuge at 1500 rcf for 1 minute, continued with chromatography
column to cleanup the pesticide residues from the contaminant of color pigment. The
residue of pesticide was analyzed by using Gas Chromatography and Mass
Spectrometer. Research result shows that there are three kinds of residue pesticide
identified on the grapes sample such as chlorpyrifos, hexaconazole, and piperonyl
butoxide. Sample of Tunggal Pamukti contain 0.0165 0.074 ppm of hexaconazole
and there no chlorpyrifos and piperonyl butoxide are identified, sample of Sari Amerta
contain 0.0047 ppm of chlorpyrifos, 0.345 0.646 ppm of hexaconazole and there no
piperonyl butoxide is identified, and sample of Amerta Nadi contain 0.124 0.664 ppm
of hexaconazole, 0.0017 0.00878 ppm of piperonyl butoxide and there no
chlorpyrifos is identified.
Keywords: Grapes, Pesticide, Pesticide residues
ABSTRAK
Tujuan dari penelitian ini adalah untuk menentukan kandungan residu pestisida
pada buah anggur dan menganalisis (dalam ppm) kadar pestisida pada buah anggur
di Desa Banjar. Metode yang digunakan dalam penelitian ini adalah ekstraksi dan
kromatografi gas. Sampel buah anggur diambil di tiga kelompok tani di Desa Banjar
yaitu Tunggal pamukti (A), Sari Amerta (B) dan Amerta Nadi (C). Sampel buah anggur
dipotong menjadi kecil, dan dihaluskan menggunakan blender hingga terbentuk
campuran homogen. Campuran homogen dimasukan kedalam tabung ekstraksi dan
diekstraksi dengan 1% asam asetat dalan asetonitrile 15 mL. Tabung dikocok selama
1 menit dan disentrifugasi pada 1500 rcf selama 1 menit, dilanjutkan dengan kolom
kromatografi untuk membersihkan residu pestisida dari pengotornya yaitu pigmen
warna. Residu pestisida dianalisis menggunakan Gas Chromatography and Mass
Spectrometer. Hasil analisis menunjukkan tiga residu pestisida yang teridentifikasi
pada sampel anggur yaitu chlorpyrifos, hexaconazole, dan piperonyl butoxide. Sampel
Tunggal Pamukti mengandung hexaconazole 0.0165 0.074 ppm dan tidak terdeteksi
adanya chlorpyrifos dan piperonyl butoxide, sampel Sari Amerta mengandung
chlorpyrifos 0.0047 ppm, hexaconazole 0.345 0.646 ppm dan tidak terdeteksi
adanya piperonyl butoxide, dan sampel Amerta Nadi terkandung hexaconazole 0.124
0.664 ppm, piperonyl butoxide 0.0017 0.00878 ppm dan tidak terdeteksi adanya
chlorpyrifos.
Kata-kata kunci: Anggur, Pestisida, Residu pestisida

INTRODUCTION
Vegetables
and
fruits
are
horticultural commodities that contain
lots of vitamins and minerals. They
also have tremendous potential as a
source of income of farmers even
source
of
foreign
exchange.
Consumption of vegetables and fruits
are starting to increase, due to an
awareness that eating fruits and
vegetables mean a healthy life will
increase.
Buleleng regency is located in the
northern part of Bali, known as the
center of Bali's grape. Buleleng
regency has a potential as a center for
grapes production because grapes are
suitable cultivated in Buleleng territory,
especially in the lowlands (0-400 m
asl). The development center of
grapes are still opportunities in the
District of Banjar, Seririt and Gerokgak
to reach 1.500 Ha grapes regions.
Data of Statistics Central Department
of Buleleng regency from 2006 to
2008 shows the development of
grapes production increase from
15.873,00 ton become 22.125,00 ton
(Statistics Central Department of
Agriculture and Livestock Buleleng
regency, 2010).
Statistic Centrals Department of
Buleleng regency also records from
nine districts in Buleleng, there are
three districts as the center of grapes
production, namely Banjar district,
Seririt district and Gerokgak district.
Banjar district become the largest
supplier of grapes with grapes
production amounted to 6.379 tons in
2010. The grapes production in Banjar
village is highest compared with the
production of the other villages. The
production of grapes in 2008 shows in
Tampekan village produced 334 tons
of grapes, Banjar village produced
2043 tons of grapes, Banjar Tegehe
village produced 134 tons of grapes,
Dencarik village produced 1704 tons

of grapes, and Temukus village

produced 1533 tons of grapes
(Statistics Central Department of
Agriculture and Livestock Buleleng
regency, 2008).
Pesticides are chemicals used to
kill and control various pests in the
broad sense (the body of bullies).
Various kinds of pesticides include
insecticide (insect killers), fungicide
(fungus killers), herbicide (weed
killers), larvacide (killer larvae),
rodenticide (rodent killers), and avicide
(bird killers). Two classes of pesticide
often used on fruit crops are
insecticide and fungicide. Fruits are
the most exposed by pesticides, so
many residues are attached to the
skin of grapes. Levels of pesticides
attached to the skin of the fruit has a
negative effect even before consuming
the fruits be washed first. There are
four negative effects, such as
carcinogenic (can cause cancer),
hormone
disruptor
(disrupt
the
hormonal system), neurotoxin (affects
the nervous system), and interfere the
growth and reproductive function.
Some related research about the
residue pesticide on the fruits have
been research by Jeremy C. Shia,
entitled Multi-residue analysis of
pesticides in fruits using a dispersive
Disque sold-phase extraction kit
(Shia, C. J., Michael S. Y. and Diane
M. D., 2008), research by Dr Walter J.
Krol entitled Removal of trace
pesticide residue from produce (Krol,
J. W., 2011), and research by John
Willy entitled Pesticide residue in food
and drinking water (Willy J., 2003).
Based on the facts and the data
above, it is necessary to study
whether the grapes (Vitis vinifera L.) in
Banjar village contains pesticides and
what is the levels of pesticides in the
grapes (Vitis vinifera L.). This research
was extended according to Multiresidue analysis of pesticides in fruits

using a dispersive disque sold-phase

extraction kit procedure (Shia, C. J.,
Michael S. Y. and Diane M. D., 2008).

residue was finally evaporated to 1

mL, prior to chromatographic analysis.
The grape extracts are subsequently
analyzed by Gas ChromatographyMass Spectroscopy on the pesticides
of interest.

RESEARCH METHOD
The pesticides contain in grape
was extended according to multiresidue analysis of pesticides in fruit
RESULT
using disque, a dispersive solid-phase
Based on the research has been
extraction kit procedure (Shia, J. C.,
conducted 15 gram of grapes are
Michael S. Y. and Diane M. D. 2008).
chopped into small portions, and the
Grapes sample are chopped into small
pulverized by a food blender until it
portions, and the pulverized by a food
reaches
homogeneous
mixture.
blender until it reaches homogeneous
Transfer 15 g of the homogeneous
mixture. Transfer 15 g of the
fruit sample into the extraction tube 1,
homogenized fruit sample into the
and then add 15 mL of 1% acetic acid
extraction tube 1, and then add 15 mL
in acetonitrile.
of 1% acetic acid in
acetonitrile. Shake
the tube vigorously
for 1 minute and
centrifuge at 1500 rcf
for
1
minute.
Transfer 1 mL of the
upper layer extract
from tube 1 to cleanup tube 2. Shake
tube 2 vigorously for
1
minute
and
centrifuge at 1500 rcf
at least 1 minute.
The extract obtained
was collected into
Erlenmeyer flask.
Cleanup process
of the extract was
performed
by
chromatography
Figure 1. Chromatogram of mixture
column using 10
of standard pesticide
grams preactivated
adsorbent silica gel
60 G (E. Merck. Art. 7734). Before
use, the columns were conditioned
with 20 mL of acetonitrile without
allowing them dry out. The column
was loaded with 10 mL grape extract
and eluted with 40 mL of acetonitrile.
Eluent was evaporated to dryness at
700C on the rotary evaporator. The

After centrifuge the samples

2. Determination of Pesticides in
cleanup by 10 grams of silica gel G60
Grapes
and use 40 mL acetonitrile as eluent.
The entire sample in sampling
After
cleanup,
place of A was
sample
was
analyzed using GCevaporated by using
MS.
The
rotary evaporator at
chromatogram
temperature of 700C
shows there are 72
up to 1 mL of
different
peaks
sample. Based on
detected by GC-MS
the procedures that
that assumed as
have
been
pesticide residues.
conducted
were
Compared
the
found 9 different
chromatogram with
samples
to
be
standard
analyzed by using
chromatogram used
GC-MS.
only one peak that
1. Analysis
of
have same peak,
Standard
which is pesticide
Pesticide
residue
of
The mixture of
hexaconazole.
standard
pesticide
Respectively,
in
was analyzed using
sample A1 contain of
(a)
(b)
GC-MS. Figure 01
hexaconazole with
shows
the
Rt detected at 22.45,
chromatogram
of
area detected of
mixture of standard
1908 and the levels
pesticide. Based on
of
pesticide
the
chromatogram
contained is 0.0165
can be shown there
ppm, sample A2 with
(a)
(b)
are 3 peaks that
Rt detected at 22.46,
Figure 3. Chromatogram of (a)
detected by GC-MS.
area detected of
sample
B1
and
(b)
sample
B2
The first peak is
8557 and the levels
chlorpyrifos with Rt
of
pesticide
at 20.74 and area
contained is 0.074
detected of 15308,
ppm, sample A3 with
second
peak
is
Rt detected at 22.46,
hexaconazole with
area detected of
Rt at 22.45 and area
6559 and the levels
detected of 7669,
of
pesticide
and third peak is
contained is 0.057
piperonyl
butoxide
ppm. The Figure 2
(c)
with Rt at 24.44 and
shows
the
area detected of
chromatogram
of
all
Figure 2. Chromatogram of (a)
70505. The Figure 1
samples in sampling
sample A1, (b) sample A2, and (c)
shows
the
places of A.
sample A3
chromatogram
of
The entire sample
mixture pesticide standard.
in sampling place of B was analyzed
using GC-MS. The chromatogram

shows there are 89 peaks detected by

GC-MS that assumed as pesticide
residues.
Compared
the
chromatogram
with
standard
chromatogram used there are two
peaks that have same peaks, which
are chlorpyrifos and hexaconazole.
Respectively, in sample B1 contain of
hexaconazole with Rt detected at
22.46, area detected of 39695 and the
levels of pesticide contained is 0.345
ppm and sample B2 contains of
chlorpyrifos with Rt detected at 20.70,
area detected of 1096 and the levels
of
pesticide
contained is 0.0047
ppm, and contain of
hexaconazole with
Rt detected at 22.45,
area detected of
74362 and the levels
of
pesticide
contained is 0.646
ppm. The Figure 3
shows
the
chromatogram of all
samples in sampling
places of B.
The entire sample
in sampling place of
C was analyzed
using GC-MS. The
chromatogram
(a)
shows there are
many
peaks
detected by GC-MS
that assumed as
pesticide residues.
Compared
the
chromatogram with
standard
chromatogram used
there are two peaks
that
have
same
peaks, which are
hexaconazole
and
piperonyl butoxide.
Respectively,
in
sample C1 contains
(c)

of hexaconazole with Rt detected at

22.45, area detected of 14306 and the
levels of pesticide contained is 0.124
ppm and contain of piperonyl butoxide
with Rt detected at 24.39, area
detected of 6154 and the levels of
pesticide contained is 0.0058 ppm;
sample C3 contains of hexaconazole
with Rt detected at 22.40, area
detected of 76425 and the levels of
pesticide contained is 0.664 ppm and
contain of piperonyl butoxide with Rt
detected at 24.46, area detected of
9287 and the levels of pesticide
contained is 0.00878
ppm; sample C4
contains
of
hexaconazole with
Rt detected at 22.39,
area detected of
64978 and the levels
of
pesticide
contained is 0.564
ppm and contain of
piperonyl
butoxide
with Rt detected at
24.43, area detected
of 1849 and the
levels of pesticide
contained is 0.0017
ppm.
Except,
in
sample C2 there are
no peaks that same
(b)
compared
by
chromatogram
standard used. The
Figure 4 shows the
chromatogram of all
samples in sampling
places of C.
DISCUSSION
The
extraction
was used 1% acetic
acid in acetonitrile. In
this process the aim
of adding 1% acetic
acid in acetonitrile is
as
a
solvent.
(d)

Figure 4. Chromatogram of (a)

sample C1, (b) sample C2, (c)
Sample C3, and (d) sample C4

Acetonitrile is a good solvent to

analysis pesticide residue. Mastovska
K. and Lehotay SJ. in their research
showed by addition of acetic acid in
acetronile will improve the stability of
these analytes. For several reasons,
acetonitrile was found to the most
suitable solvent for extraction of a
wide polarity range of pesticide
residues from produce (Mastovska K.
and Lehotay SJ. 2004).
Cleanup process of pesticides on
the grapes extract carried out by
chromatography
column.
In
chromatography column was occurred
color degradation, where the color of
grapes extract from red become
colorless solution. Color degradation
happened as a function of silanol
groups in silica gel. Normal-phase
silica gel which ends with the-OH
(silanol groups) will be polar and will
hold the polar component in the
stationary phase.
The grapes extract have red color
because still consist of anthocyanins
and tannins on it. Anthocyanins and
tannins
are
a
phytochemical
compound that gives red color in the
grapes which can be found on the skin
of grapes. From their structure
anthocyanins and tannins have polar
properties. Anthocyanins and tannins
will combine to form polymeric
pigments. Its compounds will hold by
silanol groups in the chromatography
column. So the grapes extract can
separate from the contaminant of color
pigment.
From the result analysis of
chromatogram in sample A were taken
from farmer group of Tunggal Pamukti,
can be known from three places
sampling all gift the same result for the
pesticide residue in grapes was
hexaconazole. It can be known by
compared the chromatogram of
standard with chromatogram of
sample, where hexaconazole has

retention time at 22.45. Hexaconazole

as a pesticide residue in sample A is
under maximum levels that allowed
about 0.1 ppm (BSN, 2008), where in
sample A1 contained 0.0165 ppm,
sample A2 contained 0.074 ppm, and
sample A3 contained 0.057 ppm. So,
the average of pesticide residue levels
in sample A is 0.0492 ppm.
Hexaconazole can be found in Anvil
fungicide and Cypermetrin insecticide.
Sample B were taken from Sari
Amerta farmer group are detected two
pesticide residues known based on
standard
chromatogram,
namely
chlorpyrifos and hexaconazole. In two
sampling places such as sample B1 is
containing hexaconazole 0.345 ppm
and sample B2 are contains
chlorpyrifos
0.0047
ppm
and
hexaconazole 0.646 ppm.

N
o
.
1
2
3
4
5
6
7
8
9

This pesticide residue can be found in

Anvil
fungicide
and
Cypermax
insecticide. Based on BSN the levels
of chlorpyrifos in farmer group of Sari
Amerta are under maximum levels
that allowed about 0.5 ppm and for the
levels
of
hexaconazole
were
exceeded
the
allowable
limit
Table 1. Residue pesticide
maximum 0.1 ppm.
contained on the sample
The biggest levels of
Farm S Chlo Hexa Pipero hexaconazole
that
er
a ropy cona
nyl
detected because of
grou m rifos zole
butoxi
the farmer used Anvil
ps
pl
de
fungicide
and
e
Cypermax
insecticide,
this
A
0.01
pesticide residue of
1
65
hexaconazole
can
ppm
Tung
be
found
in
both
of
A
- 0,07
gal
pesticides. So, by
2
4
Pam
using
both
of
ppm
ukti
pesticides make the
A
0.05
residue
of
3
7
hexaconazole
is
ppm
increasing.
B
0.34
Sample C were
1
5
Sari
taken from Amerta
ppm
Ame
Nadi was detected
B 0.00 0.64
rta
two
kinds
of
2
47
6
pesticide
residue,
ppm ppm
C
0.12 0.0058 namely
hexaconazole
and
1
4
ppm
piperonyl butoxide.
ppm
In four sampling
C
places in Amerta
Ame 2
rta
C
0.66 0.0087 Nadi groups gives a
different levels of
Nadi 3
4
8 ppm
pesticide contained,
ppm
C
0.56 0.0017 such as in sample
C1
contains
of
4
4
ppm
hexaconazole
0.124
ppm
ppm and piperonyl
butoxide
0.0058
ppm, sample C2 there is no pesticide
residue detected based on standard
chromatogram that used, sample C3
contains of hexaconazole 0.664 ppm
and piperonyl butoxide 0.00878 ppm,

and
sample
C4
contains
of
hexaconazole
0.564
ppm
and
piperonyl butoxide 0.0017 ppm. Based
on BSN the levels of hexaconazole in
farmer group Amerta Nadi was
exceeded the allowable limit maximum
about 0.1 ppm and for the levels of
piperonyl
butoxide
was
under
maximum limit allowed about 2 ppm.
The pesticide residue that detected in
Amerta Nadi groups can be found in
pesticide trade mark such as in Anvil
and Cypermax that contain of
hexaconazole pesticide residue and
Bamex
that
contain
piperonyl
butoxide.
The calculation result of residue
pesticide contained in all grapes
samples in Banjar Village gives in
Table 1. From the Table 1 can be
known that hexaconazole as a residue
pesticide that can found in all
samples, it can be found in Anvil
fungicide. Anvil is systemic fungicide
with suspension shaped and brownish
white color used to controlling the
disease in rubber plant, soybeans,
coffee, watermelon, and grapes.
Excessive use of this pesticide can
cause poisoning through the eyes,
mouth, and skin. Symptoms of
poisoning
this
pesticide
are
characterized by loss of body balance,
dizziness, and nausea.
Chloropyrifos residue only found in
sample B (Sari Amerta), this
insecticide
found
in
Cypermax
insecticide. Cypermax is an insecticide
as a contact poison and stomach
poison with emulsified concentrates
shaped. This insectide used when
infestations occur by insect. Research
by the U.S. EPA in 1993 chlorpyrifos
was classified in Group E, evidence of
non-carcinogenicity for humans (U.S.
Environmental Protection Agency,
2005).
Piperonyl butoxide residue that only
found in sample C (Amerta Nadi), this

insecticide found in Bamex insecticide.

Piperonyl butoxide inhibits breakdown
of pesticides by insects. Without
piperonyl butoxide, an insect may
degrade a pesticide before an effect
can occur. The addition of piperonyl
butoxide to a pesticide reduces the
amount of pesticide required to be
effective. The U.S. EPA classifies
piperonyl butoxide as a group C
carcinogen (Tracking Report U.S.
Environmental Protection Agency,
1997). This means that piperonyl
butoxide is considered a possible
human carcinogen based on limited
evidence of cancer in laboratory
animals.
CONCLUSION
The analysis of chromatogram
results of GC-MS shown form three
standards used, the grapes from
Banjar village contain pesticide
residue of chlorpyrifos, hexaconazole
and piperonyl butoxide.
Sample of Tunggal Pamukti contain
0.0165 0.074 ppm of hexaconazole
and there no chlorpyrifos and
piperonyl butoxide are identified,
sample of Sari Amerta contain 0.0047
ppm of chlorpyrifos, 0.345 0.646
ppm of hexaconazole and there no
piperonyl butoxide is identified, and
sample of Amerta Nadi contain 0.124
0.664 ppm of hexaconazole, 0.0017
0.00878 ppm of piperonyl butoxide
and there no chlorpyrifos is identified.

ACKNOWLEDGEMENT
We would like to deliver respectfully
thank for Forensik Laboratory Polresta
Denpasar for the helps in analysis
data of sample by using Gas
Chromatography-Mass Spectroscopy.
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