K. Karunambigai et al.

/ JPBMS, 2012, 22 (20)

1 Journal of Pharmaceutical and Biomedical Sciences (JPBMS), Vol. 22, Issue 22
Available online at www.jpbms.info

JPBMS

JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL SCIENCES



Reverse phase High Performance Liquid Chromatographic determination of residual
Carbendazim (fungicide) in raw and cooked Indian cereals and pulses

K. Karunambigai*, C. Saravanan, C. A. Sureshkumar, K. Kaveri, M. Thamizhmozhi

Aadhibhagawan College of Pharmacy, Rantham, Cheyyar-604407, Thiruvannamalai, Tamil Nadu, India.
Abstract:
The present work describe a reverse phase HPLC method for line quantification of carbandazim in raw and cooked Indian
Cereals as pulses the retention time of carbandazim is 2.85minutes in RP HPLC using 10%V/ V phosphate butter in
acetonitrile water in ratio 17.37:46 as mobile phase and c18 (5u 250X4.6mm)by hypersil column as stationary phase .
The effluent was monitored by UV detection at 230nm the method was validated for accuracy, precision, specificity in this
study 15 samples of cereals by pulses of different varieties were analyzed for residues of carbandazim in raw as well as
cooked forms. The maximum residual limit for cerbandazim residues in cereals and pulses is 0.5 mg/ kg. The analyzed
amount of carbandazim residues in raw sample of cereals and pulses was 0.0320-18.8171 mg/ kg by collected samples
0.003-0.6776 mg/ kg. The carbandazin plots were within range of 0.1 to 100 mg / ml (R.0.9995), the observed percentage
recovery of carbandazim was found to be 90.09-102-82% which indicated good accuracy and reproducibility of the
method.

Key words: RP-HPLC, Carbendazim, Cereals, Pulses, Determination
Introduction:
The most obvious and rational concern has been for the
presence of pesticides in human food and feed. In order to
limit the contamination of food with chemical residues, it
has been customary to fix administrative action levels to
gauge whether chemicals have been used in accordance
with registered directions and good agricultural practices.
Maximum Residue Limits (MRL) shows the maximum
residue that could result when the chemical is used
according to approved directions, when the crop is
harvested, on the stored grain or when the cereal product
is processed
[1,2]
. Residues greater than the MRL shows that
the chemical has been misused or good agricultural
practice has not been followed.
Carbendazim or MBC [methyl-2-benzimidazole carbamate]
is a widely used systemic fungicide that controls a wide
range of pathogens on a broad range of field crops like
paddy, cereals, fruits, vegetables
[3-14]
, etc. In India, its
consumption is 600-700 tonnes/ year and it is marketed
under more than fifteen brand names. It is also a most
common degradation product of benomyl [methyl-1-
butylcarbomoyl-2-benzimidazole carbamate]. Several
analytical techniques like spectroscopy and gas liquid
chromatographic methods were developed for
determination of carbendazim in various substrates. HPLC
methods were also reported for analysis of carbendazim[
15-
20]
in various substrates like cow milk, urine, faeces, etc.
Carbendazim usage specifies rates from 0.2 to 2 kg ai/ ha
and application is once in year by spraying with intervals
of 7 to 14 days. It is formulated as an aqueous dispersion
or suspension, wettable powder or dispersible powder.
The maximum residue limits for carbendazim are given by
Food & Agricultural organization and World Health
Organization.

Materials and Methods:
Collection of Cereals and Pulses:
About 15 varieties of cereals and pulses were collected
from different parts of Tamilnadu. The collected samples
were stored in well closed container without any addition
of preservatives. The sample collection was done during
JuneSeptember (2002). The varieties of cereals and pulses
analysed for carbendazim are presented in table 1.

Reagents and Chemicals:
All the chemicals used were of analytical grade and
procured from E. Merck, India. The chemicals used for the
study were Ethyl acetate, Chlorofrom, Sulphuric acid,
Sodium hydroxide, Dichloromethane, Anhydrous sodium
sulphate, Acetonitrile HPLC grade and Triethyl amine.
Triple glass distilled water was used. Carbendazim
(reference standard) was generously gifted by
international Institute of Biotechnology and Toxicology,
Padappai 601301 India.

Instrumentation:
The instruments employed for residual analysis of
carbendazim were High performance liquid
chromatography Shimadzu, LC6A, SPD-10ATVP with UV
detector, Injection: Rheodyne needle with hamiltone
loonadzu schweinge syringewith 20ul loop, Column: C18
hypersil, 250x 4.6mm, 5u particle size, Electronic single
pan balance - (shimadzu Libror AEG, 220), Digital PH
meter Model IIIE, Rotary vacuum evaporator.

ISSN NO- 2230 7885
CODEN JPBSCT
NLM Title: J Pharm Biomed Sci.


Research article
K. Karunambigai et al. / JPBMS, 2012, 22 (20)

2 Journal of Pharmaceutical and Biomedical Sciences (JPBMS), Vol. 22, Issue 22
Instrumentation and operational conditions for RP- HPLC
Mode of operation : Isocratic
Temperature : Ambient
Flow rate : 1ml/ min
Load : 20ul
UV detection : 282 nm
Injection interval : 10 min

Mobile phase:
The mobile phase used was 10% w/ v PH 7 Phosphate
buffer in acetonitrile water in the ratio of 17:37:46.

Preparation of buffer:
The phosphate buffer was prepared by mixing solutions of
0.067 M disodium hydrogen phosphate (Na2 HPO4) and
0.67M potassium dihydrogen orthophosphate (KH2 PO4)
AT the ratio of 3: (v/ v), so that the ph of the prepared
buffer was 6.98 =0.05.

Preparation of standard solution:
An accurately weighed quantity of 100 mg of carbendazim
was dissolved in minimum quantity of o.1 N hydrochloric
acid and made up 100 ml with acetonitrile to obtain 1000
g/ ml of carbendazim solution. The stock solutions were
further diluted to get different concentration and a
calibration curve was plotted.

Quantitati Ve Analysis of Carbendazim:
Linearity and calibration:
Calibration of instruments is essential in obtaining
accurate analysis. The linearity of an analytical procedure
is its ability to obtain test results within a given rage which
are directly proportional to the concentration of analyte in
the sample. A minimum of 5 concentrations are required
for linearity. It is evaluated by linear regression analysis of
the plot of signals as a function of analyte concentration.
The correlation coefficient and slope of regression line
should be satisfactory.
Varying quantities of stock solution of carbendazim was
diluted with acetonitrile to give different concentration
and the injections were made at an interval of 10 minutes.
The peak areas were measured and tabulated. The detector
response (UV AT 282 nm) of the developed RP-HPLC
protocol by standard solutions of carbandazim (0.1to 100
g/ ml in mobile phase) were measured.

Quantitative Analysis of Carbendazim in Raw
Cereals and Pulses:
About 20 g of sample was weighed followed by extraction
and clean up process.

Extraction:
About 20 g of the sample was weighed and homogenized in
a heavy duty waring blender and then extracted with 100
ml of ethylactate and filtered. The solid residue was re-
extracted with 50 ml of ethylacetate and again filtered. The
extracts were combined and evaporated to dryness on a
rotary vaccum evaporator. The residues were dissolved in
3x 20 ml of 0.5 N sulphuric acid and passed through a
funnel with a glass wool plug into a separating tunnel for
further clean up.
Clean up:
The acid extract was washed with 3x40 ml of chloroform
and the chloroform layers were discarded after phase
separation. The PH of the acidic aqueous layer was
adjusted between 8.5 to 9 with 6% V/ V aqueous sodium
hydroxide. Then the solution was extracted with 2x25 ml
of dichloromethane. The extracts were combined and dried
by passing it through anhydrous sodium sulphate and
evaporated to dryness on a rotary vacuum evaporator. The
residues were immediately dissolved in 5 ml of water:
acetonitrile (55:45) solution filtered through 0.2m
membrane filter and analysed by RP-HPLC.

Quantitative Analysis of Carbendazim in
Cooked Cereals and Pulses:
About 20 g of sample was weighed and cooked under
steam. The extraction and clean up procedure was done as
per that of the raw samples. the sample solutions were
then filtered through 0.2 m membrane filter and analysed
by RP-HPLC. The sample were fortified with 1 g of
carbendazim and injected. Each solution was run thrice at
an interval of 10 min to ensure elution of earlier injection.
The amount of carbendazim present in both raw and
cooked cereals was calculated by comparing the sample
peak area with the standard peak area.

Amount of Carbendazimin in each sample per kg=Sample peak
area Concentration of standardDilutiion factor 1000
Standard peak area weight taken


Results and Discussion:
The UV spectrum of carbendazim is presented in Fig.1. The
chromatogram of carbendazim standard, in raw cereals
and pulses and in cooked cereals and pulses (RP-HPLC)
under the experimental condition were presented in Fig .2,
3 & 4 respectively.


Figure 1. UV Spectrum of Carbendazim
Figure 2. Chromatogram of Carbendazim standard
K. Karunambigai et al. / JPBMS, 2012, 22 (20)

3 Journal of Pharmaceutical and Biomedical Sciences (JPBMS), Vol. 22, Issue 22

Figure 3. Chromatogram of Carbendazim in raw cereals and pulses

Figure 4. Chromatogram of Carbendazim in Cooked cereals and pulses
The linearity data of carbendazim is presented in Table 2.
The system sutability data is presented in table 3. The LOD
and LOQ of carbendazim are presented in Table 4. Table 5
represents quantitative data and statistical analysis for raw
cereals and pulses after extraction and cleanup for
determination of carbendazim. Table 6 represents the
quantitative data and statistical analysis for cooked cereals
and pulses samples after extraction and cleanup for the
determination of carbendazim after fortification with 1 ug
of carbendazim. Table 7 presents the recovery data of
carbendazim in the developed protocol. The comparison of
carbendazim content is raw and cooked cereals and
pulsesis presented in Table 8.
Table 1. Varieties of Cereals and Pulses samples analysed for
Carbendazim
Sample no Common name
I Horse gram
II Millet
III Green gram
IV Red gram
V Groundnut
VI Bengal gram
VII Barley
VIII Oats
IX Maize
X Black gram
XI Ragi
XII Sorghum
XIII Chickpea
(Black)
XIV Wheat
XV Chickpea
(White )

Table 2. Linearity data of Carbendazim
S.no Concentration ( g/ ml) Peak area
1 0.1 133796
2 0.2 266180
3 0.3 452383
4 0.4 593819
5 0.5 762128
6 1 1272409
7 2 2625585
8 3 4357796
9 4 5230370
10 5 7004253
11 10 9390596
12 20 21527424
13 30 32554304
14 40 44767305
15 50 56555117
16 100 114672844

Table 3. System suitability data of the developed method
Parameter Carbendazim
Nuber of Theoritical Plates 1357
Tailing factor 1.12

Table 4. Limit of detection and Limit of quantification data of
Carbendazim in present study

Table 5. Quantitative analysis of Carbendazim in raw cereals and pulses
Sample
Peak
Areas
Amount
Mg/ kg
Standard
Deviation
X10-3
Relative
Standard
Deviation
Standard
Error of
Mean x 10-3

I 40343224 18.8171 0.252 0.134 0.145
II 9995829 2.0276 0.289 0.014 0.231
III 2072117 0.9521 0.4 0.042 0.176
IV 6363779 2.9612 0.306 0.013 0.176
Parameter Carbendazim ug/ ml
Limit of Detection 0.0825
Limit of Quantification 0.2751
K. Karunambigai et al. / JPBMS, 2012, 22 (20)

4 Journal of Pharmaceutical and Biomedical Sciences (JPBMS), Vol. 22, Issue 22
V 588189 0.2736 0.252 0.0092 0.145
VI 669525 0.3101 0.252 0.081 0.145
VII 69257 0.0320 0.153 0.477 0.882
VIII 2072810 0.9732 0.351 0.036 0.203
IX 232377 1.0956 0.212 0.019 0.122
X 318709 0.1488 0.321 0.202 0.173
XI 2642259 1.2277 0.208 0.017 0.120
XII 773906 0.3635 0.3 0.083 0.173
XIII 253633 0.1171 0.2 0.171 0.115
XIV 289658 0.1350 0.154 .0113 0.088
XV 259208 0.1219 0.361 0.296 0.208

Table 6. Quantitative analysis of Carbendazim in cooked cereals and pulses
Sample
Peak
Areas
Amount
Mg/ kg
Standard
Deviation
X10-3
Relative
Standard
Deviation
Standard
Error of
Mean x 10-3

I 176416 0.0484 0.257 0.535 0.148
II 547796 0.2579 0.222 0.086 0.128
III 731513 0.3621 0.252 0.069 0.145
IV 121482 0.0174 0.265 1.521 0.153
V 317552 0.1289 .0252 0.195 0.145
VI 308340 0.1211 0.268 0.219 0.154
VII 100329 0.0054 0.264 0.049 0.153
VIII 664291 0.3034 0.400 .0132 0.231
IX 757172 0.3759 0.202 0.053 0.115
X 126578 0.0201 0.158 0.787 0.091
XI 251739 0.6776 0.252 0.037 0.176
XII 251736 0.0896 0.306 0.341 0.176
XIII 93613 0.0016 0.233 14.446 0.134
XIV 154470 0.0358 0.252 0.707 0.145
XV 91465 0.0003 0.153 41.659 0.0882
*Each value is mean of three readings
Table 7.Recovery studies of Carbendazim
Sample
Area of plain
Cooked samples
Area of spiked
Samples
Area of plain
And standard
%Recovery
I 513426 1058610 1057823 100.07
II 2764829 3284630 3309226 99.26
III 3882468 4386588 4426865 90.09
IV 174832 728382 719229 101.27
V 1434978 1907790 1979375 96.38
VI 1267853 1848196 1812250 101.98
VII 55982 599436 600379 99.84
VIII 3452620 3925698 3997017 98.21
IX 3894321 4542678 4438718 102.34
X 7025261 7749564 7569658 95.18
XI 7025261 7749564 7569658 95.18
XII 979423 1507320 1523820 98.92
XIII 17632 559044 562029 99.46
XIV 354382 546978 547339 99.93
Area of standard Carbendazim (1g/ ml) =544397.

Table 8. Comparison of Carbendazim content in raw and cooked cereals
and pulses
Sample
Amount in raw
Samples (mg/ kg )
Amount in cooked
Samples (mg/ kg )
I 18.8171 0.484*
II 2.0276 0.2579*
III 0.9521 0.3621*
IV 2.9612 0.0174*
V 0.2736 0.1289*
VI 0.3101 0.1211*
VII 0.0320 0.0054*
VIII 0.9732 0.3034*
IX 1.0956 0.3759*
X 0.1488 0.201*
XI 1.2277 0.6776*
XII 0.3635 0.0896*
XIII 0.1171 0.0016*
XIV 0.1350 0.0358*
XV 0.1219 0.003*

The wavelength for quantification of carbendazim was
selected as 282 nm. The linearity lies in the range of 0.1-
100 ug/ ml (r=0.9995). The retention time of carbendazim
is 2.85 minutes in RP-HPLC using 10%v/ v phosphate
butter in acetonitrile water in the ratio of 17:37:46 as
K. Karunambigai et al. / JPBMS, 2012, 22 (20)

5 Journal of Pharmaceutical and Biomedical Sciences (JPBMS), Vol. 22, Issue 22
mobile phase and C18 (5u , 250x 4.6mm) hypersil column
as stationary phase.
In the present study, 15 samples of cereals and pulses of
different varieties were analysed for residues of
carbendazim in raw as well as cooked forms. The
maximum residual limit for carbendzim residues in cereals
and pulses was 0.5 mg / kg. The amount of analysed
carbendazim residues in raw samples of cereals and pulses
was 0.0320-18.8171mg/ kg, and in cooked samples of
cereals and pulses is 0.003-0.6776 mg/ kg,
Out of the 15 raw cereals and pulses samples taken for
study only 8 samples are found to have their residues
within the maximum residual limit. The remaining 7
samples have their residues beyond the maximum residual
limit specified by Food & Agricultural Organisation and
World Health Organization.
It was found that cooking reduces the residues of
carbendazim significantly (p<0.001). From the 15 samples
analysed, only one lies outside the limits. Though in raw
samples 7 samples exceeded their limits, cooking reduced
the amount and only on exceeded the limit.
The accuracy, precision, specificity, suitability and validity
of the developed RP=HPLC method was further verified by
recovery studies. The observed percentage recovery of
carbendazim was found to be 90.09-102.82 % which
indicated good accuracy and reproductibility of the
method.

Summary and Conclusion:
In the present study, RP-HPLC method was developed for
the analysis of residual carbendazim in raw and cooked
samples of cereals and pulses. The developed RP-HPLC
method utilises 10 % v/ v phosphate buffer in acetonitrile
and water in the ratio of 17:37:46 as mobile phase and
hypersil C 18 (5u, 250 x 4.6mm) colum as a stationary
phase. The eluted carbendazim was detected at 282 nm.
It was observed that in raw samples, 7 samples exceeded
their maximum residual limits specified by Food
&Agricultural Organisation and World Health Organization.
The process of cooking reduced the carbendazim content
significantly (p<0.001) and only one sample exceeded the
maximum uses of the fungicide carbendazim. The recovery
studies showed that the percentage recovery was found to
be 90.09 -102.82 %.
From the above analytical data and recovery studies it was
found that the developed RP-HPLC method for the
quantification of residual carbendazim was accurate and
precise.

Acknowledgements:
The authors are thankful to our chairman and directors,
Aadhibhagawan College of pharmacy, Rantham, Cheyyar,
Thiruvannamalai district, Tamilnadu for providing
necessary facilities to carry out this research work.

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Corresponding Author:-
Mr.C.Saravanan,
Asst. Professor, Aadhi Bhagawan College of Pharmacy,
Rantham, Cheyyar-604407,
Thiruvannamalai District, Tamil Nadu





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