Analytical

Methods
View Article Online
PAPER View Journal

A simple pre-column derivatization method for the

Cite this: DOI: 10.1039/c7ay00830a
determination of mancozeb technical (fungicide)
by reverse phase HPLC-UV
Published on 02 August 2017. Downloaded by Université Laval on 06/08/2017 10:36:30.

Narendra H. Petha, *a Rama S. Lokhande,b Devender T. Seshadri,c Raju M. Patil,d

Tanaji S. Bhagatc and Jitendra G. Patilc

A simple and fast pre-column derivatization high performance liquid chromatographic method for the
technical analysis of mancozeb was developed and validated in the present study. The mobile phase
consists of a mixture of acetonitrile and 0.1% (v/v) formic acid in the proportion 60 : 40. This was found
to give a sharp peak of the S-ethyl derivative of mancozeb (S-ethyl ethylene bis-dithiocarbamate) at
a retention time of 9.12 minutes. HPLC analysis of mancozeb was carried out at a wavelength of 272 nm
Received 28th March 2017
Accepted 6th July 2017
with a flow rate of 1.0 mL per minute. The linear regression analysis data for the calibration curve
showed a good linear relationship with a regression coefficient of 0.999 in the concentration range of
DOI: 10.1039/c7ay00830a 1 1
8.2 mg L to 32.3 mg L for the derivative of mancozeb technical. The method was validated for
rsc.li/methods specificity, linearity, precision and accuracy.

also developed for HPLC-UV detection of mancozeb in residue

1. Introduction
Mancozeb (a manganese ethylene bis-dithiocarbamate poly-
meric complex with zinc salt) is a non-systemic agricultural
fungicide with multi-site, protective action.1 It falls into
a contact fungicide class that is effective in various fungal
diseases. It controls many fungal diseases in a wide range of
eld crops, fruits, nuts, vegetables, and ornamentals. It is also
used for seed treatment of cotton, potatoes, corn, safflower,
sorghum, peanuts, tomatoes, ax, and cereal grains. Mancozeb
is a complex of ethylene bis-dithiocarbamate (EBDC) with metal
ions in polymeric form [Mn (18–20%, w/w) and Zn (2–2.5%, w/
w)]. Mancozeb in agricultural products is determined by an
indirect method i.e. CS2 liberation by heating under strongly
acidic conditions (typically 4 N H2SO4) and then analysed by
titration with iodine2 but the CS2 liberation method requires
trapping of CS2 and a long acid digestion process which can
lead to CS2 loss and can cause error in analysis. These methods
also cannot differentiate between other dithiocarbamate (DTC)
fungicides. Pre-column derivatization methods were developed
using methylating agents such as methyl iodide3–6 or dimethyl
sulfate (DMS)7 and analysed by HPLC-UV detection. Hazardous
reagents were used in derivatization such as methylating agents
and extraction solvents (CHCl3 and MDC) which are suspected
carcinogens too. Derivatization using 1,2-benzene dithiol8 was
a
Indol Industries Limited, Off S. V. Road, Azad Nagar, Sandoz Baug P.O., Thane-400
069, Maharashtra, India. E-mail: nhp1184@gmail.com; Tel: +91 9930629007
b
Jaipur National University, Jaipur, India. E-mail: rama.lokhande@yahoo.com
c
Indol Industries Limited, India. E-mail: dseshadri-icc@modi.com
d
Institute of Science, Mumbai, India. E-mail: rmpatil_2006@yahoo.com
analysis but it is time consuming because of a long derivatiza-
tion time of about 2 h at 60  C. There are methods also available
which use ion-pairing9–11 reagents for direct analysis of man-
cozeb but ion pairing methods need appropriate HPLC column
cleaning procedures for better column performance and
a mobile phase with ion pair salts is not MS compatible. The
proposed RP-HPLC method is simple, sensitive and reproduc-
ible for the determination of mancozeb. An attempt has been
made to develop simple, safe and fast pre-column derivatization
using a less-hazardous reagent, ethyl iodide, as illustrated in
Fig. 1. This method includes breaking of the complex using
alkaline EDTA and derivatization using ethyl iodide in the same
pot, which eliminates the extraction step in hazardous solvents
reported for methods that use methyl iodide. An advantage of
using ethyl iodide is that it gives better resolution between ethyl
iodide and the S-ethyl derivative as compared to closely eluting
peaks of methyl iodide and the S-methyl derivative as shown in
Fig. 2. The derivative formed was analysed at 272 nm using the
HPLC-UV detection technique. A simple, MS compatible RP-
HPLC mobile phase was used for the determination using
a C8 HPLC column. The developed method was validated
according to SANCO12 and CIPAC13 guidelines to show the
capability of the method.
2. Materials and methods
Ethyl iodide was purchased from S. D. Fine Chemicals. HPLC
grade solvents were used for mobile phase and sample prepa-
ration. Mancozeb standard was purchased from Sigma Aldrich
and mancozeb technical was obtained from Indol Industries

This journal is © The Royal Society of Chemistry 2017 Anal. Methods

 View Article Online

Analytical Methods Paper

Published on 02 August 2017. Downloaded by Université Laval on 06/08/2017 10:36:30.

Fig. 1 Reaction scheme of the S-ethyl derivative.

Fig. 2 Chromatographic comparison of the S-methyl and S-ethyl derivatives.

Limited. The conditions used for the chromatographic method 2.2.2 Preparation of the standard. A quantity of 50.2 mg of
is tabulated (Table 1). mancozeb standard was weighed and transferred into a 100 mL

2.1 Preparation of the mobile phase Table 1 HPLC method conditions for mancozeb analysis
2.1.1 Solvent A. HPLC grade acetonitrile, degassed for 15
Instrument HPLC, Shimadzu, LC-20AD
minutes.
with a PDA detector
2.1.2 Solvent B. 0.1% (v/v) formic acid in HPLC grade water, Mobile phase Solvent A-acetonitrile
mixed well and degassed for 15 minutes. Solvent B-0.10% (v/v) formic
2.1.3 Diluent. Mixed solvent A and solvent B in 60 : 40 acid in water
proportion, mixed well and degassed for 15 minutes. Elution program Isocratic, 60 : 40 (A : B)
Column Phenomenex C8, 250 mm 
4.6 mm  5.0 mm
Flow rate (mL per minute) 1.0
2.2 Preparation of the reagent Column oven temperature ( C) 30
2.2.1 Alkaline EDTA solution (25 mM). 6.7 g of disodium Injection volume (mL) 20
Wavelength (nm) 272
EDTA was dissolved in 800 mL of water; the pH of the solution
Run time (minutes) 20.0
was adjusted to 9.5 with sodium hydroxide solution and diluted Diluent Mobile phase
up to 1000 mL with water.

Anal. Methods This journal is © The Royal Society of Chemistry 2017


Paper
Published on 02 August 2017. Downloaded by Université Laval on 06/08/2017 10:36:30.
Fig. 3 Mass spectrum of the S-ethyl derivative of mancozeb.
volumetric ask, and 50 mL of acetonitrile and 0.5 mL of ethyl
iodide were added to it; then 30 mL of alkaline EDTA was added
slowly with stirring. Then the solution was kept for 15 minutes
at 40  C, and aer cooling it was diluted up to the volume with
acetonitrile. An aliquot of 1.0 mL from the solution was pipetted
into a 50 mL volumetric ask; the content was diluted up to the
volume with the diluent to produce the working standard
concentration of 0.009 mg mL 1.
2.2.3 Preparation of the sample. A quantity of 50.5 mg of
mancozeb technical was weighed and transferred into a 100 mL
Fig. 4 Derivatization time at 25  C.
Fig. 5 Effect of temperature on the derivatization rate.
This journal is © The Royal Society of Chemistry 2017
View Article Online
Analytical Methods
volumetric ask, and 50 mL of acetonitrile and 0.5 mL of ethyl
iodide were added to it; then 30 mL of alkaline EDTA was added
slowly with stirring. Then the solution was kept for 15 minutes
at 40  C, and aer cooling it was diluted up to the volume with
acetonitrile. An aliquot of 1.0 mL from the solution was pipetted
into a 50 mL volumetric ask; the content was diluted up to the
volume with the mobile phase and injected into the HPLC
system for the analysis.
Fig. 6 Effect of EDTA concentration.
Fig. 7 Optimization of the derivatization rate at 40  C.
Anal. Methods
 View Article Online

Analytical Methods Paper

Published on 02 August 2017. Downloaded by Université Laval on 06/08/2017 10:36:30.

Fig. 8 Chromatogram of the blank.

Fig. 9 Peak purity chromatogram of the mancozeb standard.

Anal. Methods This journal is © The Royal Society of Chemistry 2017


Paper
3. Results and discussion
3.1 Methodology
To ensure maximum mass transfer and formation of the S-ethyl
derivative of mancozeb various parameters were optimized such
as the effect of concentration of alkaline EDTA, derivatization
time and effect of temperature. The formation of the derivative
was conrmed by comparing the mass of the S-ethyl derivative
of mancozeb standard and mancozeb technical, which was 269
(m + 1) as shown in Fig. 3.
3.1.1 Derivatization time. The derivatization time was
Published on 02 August 2017. Downloaded by Université Laval on 06/08/2017 10:36:30.
optimized at room temperature i.e. at 25  C at time intervals of
0, 15, 30, 45 and 60 minutes by keeping the concentration of
EDTA constant. From Fig. 4 (plot of area per mg vs. time) it was
found that the minimum time required for derivatization was
30 minutes at 25  C.
3.1.2 Effect of temperature on derivatization. The effect of
increase in temperature on the rate of formation of the deriva-
tive was checked by keeping the time constant (30 minutes). The
rate of formation was checked at temperature intervals as
shown in Fig. 5 and it was observed that from a temperature of
35  C onwards the derivatization rate remains constant.
3.1.3 Concentration of alkaline EDTA. Alkaline EDTA was
used for breaking of mancozeb (a Mn and Zn complex of EBDC)
and converting it into the sodium salt of EBDC. Complete
conversion into the sodium salt was an important step in the
formation of the derivative. The effect of decrease in the
concentration of EDTA was checked and the results showed
maximum conversion at minimum concentration of EDTA from
Fig. 10 Peak purity chromatogram of the mancozeb technical.
This journal is © The Royal Society of Chemistry 2017
View Article Online
Analytical Methods
10 mM to 100 mM by keeping the time and temperature
constant. The effect was checked by plotting a graph of area
per mg vs. concentration of EDTA in mM as shown in Fig. 6 and
it was observed that a minimum of 20 mM alkaline EDTA was
required for the derivatization.
3.1.4 Optimization of time at 40  C temperature. Finally
using all the optimized conditions, the derivatization time was
optimized at 40  C temperature, 30 mL of 25 mM alkaline EDTA
and 0.5 mL of derivatizing agent (ethyl iodide). From Fig. 7 (plot
of area per mg vs. time at 40  C) it was found that from 15
minutes derivative formation was constant up to 60 minutes.
The optimized conditions for derivatization were 30 mL of
25 mM alkaline EDTA used for breaking 50 mg of mancozeb
complex and derivatization by using 0.5 mL of ethyl iodide
sample for 15.0 minutes at 40  C.
3.2 Method validation
3.2.1 System suitability. System suitability was evaluated
for the number of theoretical plates and tailing factor by
injecting six replications of the standard preparation. The
number of theoretical plates was found to be 13 714 (>2000) and
the tailing factor was 1.06 (<2.0) meeting the acceptance criteria.
3.2.2 Specicity. The specicity of the method was checked
for any interference from the reagent blank, solvent blank and
sample blank. It was observed that there is no interference at
the retention time of the mancozeb derivative due to any of the
blanks. All blank solutions were prepared as per the method-
ology. Fig. 8–10 show that there is no interference at the RT of
the S-ethyl derivative of mancozeb. Therefore, the specicity of
Anal. Methods
 View Article Online

Analytical Methods Paper

the method was conrmed by the absence of interfering peaks
(ghost peaks) at the analyte elution time from blank
chromatograms.
3.2.3 Linearity. The linearity of the detector response for
the S-ethyl derivative of mancozeb was demonstrated in the
range of 8.2 to 32.3 mg L 1. The regression coefficient was
found to be 0.999 which also shows that along with the linear
detector response the formation of the derivative (S-ethyl) was
also linear with the weight of mancozeb (Fig. 11).
3.2.4 Precision (repeatability). The precision under the
conditions of repeatability (for seven sample preparations) was
Published on 02 August 2017. Downloaded by Université Laval on 06/08/2017 10:36:30.
mancozeb at three concentration levels, 0.5%, 1.0% and
1.5%, of the target concentration to the sample examined. The
mean percentage recoveries (98 to 102%) were 98.95, 98.65 and
99.10 at each concentration level respectively. From the
percentage recoveries the method was found to be accurate.
3.3 Sample results
Experimental results of the amount of mancozeb in mancozeb
technical expressed as a percentage of label claim were in good
agreement with the label claims14 thereby suggesting that there
is no interference from any of the excipients which are normally

determined separately for the determination of mancozeb (the
S-ethyl derivative of EBDC) in each replicate of the test item. The
% RSD for the mancozeb content (85% w/w) was found to be 0.4
which is well within the expected % RSD (#2.0%) calculated as
per the Horwitz equation and hence the method was found to
be precise.
3.2.5 Accuracy. To conrm the accuracy of the proposed
method, recovery experiments were carried out by the standard
addition technique. The accuracy of the method was deter-
mined by adding known amounts of reference standard
present. The S-ethyl derivative of mancozeb standard was used
as the standard for analyzing three different batches of man-
cozeb technical using the proposed procedure and the results
were compared with those of the conventional CS2 liberation
method. The purity of mancozeb standard known from the CS2
liberation method was considered as the purity of the S-ethyl
derivative of mancozeb standard. It was found that the active
ingredient content results by HPLC and by the CS2 method were
comparable as shown in Table 2 in mancozeb technical and
Table 3 in the mancozeb WP formulation.

4. Conclusion
The specicity, precision and accuracy results show that this
method is suitable for the quantication of mancozeb. A
simple, safe and fast pre-column derivatization reverse phase
HPLC method was successfully developed for the estimation of
mancozeb in mancozeb technical and its WP formulations.

Abbreviations

RP-HPLC Reverse phase-high performance liquid

chromatograph
Fig. 11 Linearity curve of mancozeb.
UV Ultraviolet detector
DTCs Dithiocarbamates
CHCl3 Chloroform
Table 2 Active ingredient content in mancozeb technical MDC Methylene dichloride
EDTA Ethylene diamine tetra acetic acid
Active content
(%, w/w)
EBDC Ethylene bis-dithiocarbamate
Mean STD. MS Mass spectrometer
Batch no. W1 W2 W3 content DEV. % RSD By CS2 PDA Photodiode array detector
RT Retention time
1 85.5 84.9 85.8 85.4 0.458 0.537 85.5 STD. Standard deviation
2 85.2 85.1 85.7 85.3 0.321 0.377 85.2
DEV.
3 86.1 85.1 85.6 85.6 0.500 0.584 85.4
% RSD Percentage relative standard deviation
W Number of weights

Table 3 Active ingredient content in the mancozeb WP formulation

Active content Acknowledgements

(%, w/w)
Mean STD. The authors would like to thank Indol Industries Ltd. (Thane-
Batch no. W1 W2 W3 content DEV. % RSD By CS2 MH, India) for providing the sample of mancozeb technical.
The authors would like to thank Dr A. G. Pawar, Exec. Vice
1 80.2 79.8 80.7 80.2 0.45 0.56 80.5
2 80.8 80.1 79.9 80.3 0.47 0.59 80.6 President R&D Indol Industries Ltd., Thane, India for
providing necessary facilities to carry out the work.

Anal. Methods This journal is © The Royal Society of Chemistry 2017


Paper
References
1 EXTOXNET Pesticide information prole http://extoxnet.
orst.edu/pips/mancozeb.html.
2 CIPAC, 34/TC/M-3, method for Active Content in Mancozeb
technical.
3 L. Chi-Chu, H. Ming-Hsun and H. Ming-Der, Use of HPLC
and atomic absorption methods to distinguish propineb,
zineb, maneb, and mancozeb fungicides, J. Agric. Food
Chem., 1996, 44(9), 2720–2723.
4 K. Nobuyuki, H. Noriko and N. Munetomo, Rapid analysis
Published on 02 August 2017. Downloaded by Université Laval on 06/08/2017 10:36:30.
method of dithiocarbamate pesticides in agricultural
products by high performance liquid chromatography,
Shokuhin Eiseigaku Zasshi, 1995, 36(2), 244–251.
5 X. Hui, HPLC determination of mancozeb 80% WP aer
methylation, Nongyao, 2007, 46(8), 540–541.
6 K. Gustafsson, Haakan and T. Richard, High-pressure liquid
chromatographic determination of fungicidal
dithiocarbamate, J. Agric. Food Chem., 1981, 29(4), 729–732.
7 C. Dong, F. Xin-Gang, Q. Dong-Mei, L. Xiao-Lan, C. Li,
W. Chen-Rui and Z. Yong-Quan, Simplied method for
determination of mancozeb residues in apple using ultra
performance liquid chromatography mass spectrometry,
Fenxi Huaxue, 2010, 38(4), 508–512.
8 D. Ikram, T. Karine, D. Evelyne and M. Nicholas,
Identication and Quantitation by High-Performance
Liquid Chromatography of Mancozeb Following
This journal is © The Royal Society of Chemistry 2017
View Article Online
Analytical Methods
Derivatization by 1,2-Benzenedithiol, J. Anal. Toxicol., 2004,
28(1), 41–45.
9 X. Mei, S. Jingquan, W. Xinran and H. Qing, Mancozeb
analysis method using high performance liquid
chromatography, Faming Zhuanli Shenqing, patent CN
2013-10305749, 2013.
10 V. Lishaut, H. Schwack and Wolfgang, Selective trace
determination of dithiocarbamate fungicides in fruits and
vegetables by reversed-phase ion-pair liquid chromatography
with ultraviolet and electrochemical detection, J. AOAC Int.,
2000, 83(3), 720–727.
11 K. W. Weissmahr, C. L. Houghton and D. L. Sedlak, Analysis
of the Dithiocarbamate Fungicides Ziram, Maneb, and Zineb
and the Flotation Agent Ethylxanthogenate by Ion-Pair
Reversed-Phase HPLC, Anal. Chem., 1998, 70(22), 4800–4804.
12 EUROPEAN COMMISSION Directorate General Health and
Consumer Protection, Guidance for generating and
reporting methods of analysis of technical material and
preparations, SANCO/3030/99 rev.4 11/07/00.
13 CIPAC Guideline for analytical methods for the
determination of relevant impurities referred to in FAO
and/or WHO specications for pesticide technical grade
active ingredients and formulations, CIPAC rev.7, June
2009.
14 Food and Agriculture Organization of the United Nations
Rome, 1988 (FAO) Specication 101/TC/S (1989).
Anal. Methods