A New Chromogenic Spray Reagent for Detection and

Identification of Monocrotophos
Vijay R. Chandegaonkar, Jaiprakash N. Sangshetti, Devanand B. Shinde, and Dhananjay V. Mane*

Key Words
Biological materials
Extraction
Monocrotophos
TLC
Benzil
Chromogenic reagent

1 Introduction
Monocrotophos (nuvacron, azodrin) is widely used in agriculture to protect crops from insects, similar to other organophosphorus insecticides, for example malathion, parathion, methyl
parathion, fenthion, and methyl demeton. Owing to their ready
availability they are often misused for homicidal or suicidal purposes. The forensic laboratories of Maharashtra detected
approximately 1500 cases of monocrotophos poisoning during
2007 [1].
For routine forensic toxicological work detection of organophosphorus insecticides is achieved by thin-layer chromatography (TLC) because of its simplicity and rapidity. Other methods
(for example GC, HPLC) [2] have been reported in the literature
but there are limitation to their use in routine forensic work
owing to the complex matrix which may damage the columns.
TLC can moreover be used to screen many samples in the time
taken by these instrumental methods to screen one sample only.
TLC is therefore the method of choice for screening biological
samples.
Several chromogenic reagents have been used for detection and
identification of monocrotophos insecticides including mercurous nitrate [3], potassium iodate starch [4], sodium carbonate
chloranil acetone [5], mercuric nitrate-diphenyl carbazone [6],
palladium chloride [7], and vanillin acetone [8]. These reagents
are either of low sensitivity or are not specific, and there is very
limited literature available on the detection and identification of
monocrotophos by TLC and HPTLC. This paper reports use of
benzil as a specific and selective spray reagent for detection and
identification of monocrotophos on thin-layer chromatography
plates.

V.R. Chandegaonkar, Forensic Science Laboratory, Aurangabad, (MS)-431002,

India; J.N. Sangshetti and D.B. Shinde, Department of Chemical Technology, Dr
B.A.M. University, Aurangabad, (MS)-431004, India; and D.V. Mane, Department of Chemistry, Chhatrapati Shivaji College, Omerga (MS)-413606, India.
E-mail: manedv.2007@rediffmail.com

Journal of Planar Chromatography 22 (2009) 6, 457458

0933-4173/$ 20.00 Akadmiai Kiad, Budapest

2 Experimental
2.1 Chemicals and Reagents

All reagents and chemicals were of analytical grade. Glass-distilled water was used throughout the work. A standard solution
of monocrotophos (1 mg mL1) was prepared by dissolving
13.5 mg 74% technical grade monocrotophos (Hindustan
CibaGeigy, Mumbai, India) in ethanol (10 mL). An aqueous
solution of sodium hydroxide (5% w/v) was prepared by dissolving 5 g sodium hydroxide pellets in distilled water and diluting to 100 mL. Benzil reagent (5% w/v) was prepared by dissolving benzil (5 g) in acetone (100 mL).
2.2 Chromatography

Glass TLC plates (10 cm 15 cm) were coated with slurry of silica gel G (Acne chemicals, Mumbai, India) in water (1:2) to produce uniform 0.25-mm layers. These were left to dry at room
temperature. Before use the plates were activated by heating in
an oven at 100C for ca 1 h then stored in a desiccator. Standard
monocrotophos solution and standard solutions of other
organophosphorus insecticides (malathion, parathion, fenthion,
dimethoate, and phorate), organochloro insecticides (endosulfan
and BHC), carbamate insecticides (baygon and carbaryl), and
pyrethroids (cypermethrin, deltamethrin), and extracts from visceral tissue spiked with a standard solution of monocrotophos
were applied to the plates and these were then developed to a
distance of 10 cm with chloroformacetone 7:3 as a mobile
phase in a TLC chamber previously saturated with mobile phase
vapor. After development the plate was removed from the chamber, dried in air, and sprayed with 5% sodium hydroxide solution
then 5% benzil reagent. The plate was then heated in oven at
100C for 10 min and then cooled at room temperature. A typical chromatogram is shown in Figure 1. A pink spot was
observed (RF 0.48).
2.3 Recovery of Monocrotophos from Biological Material

Monocrotophos (1.35 mg) was added to 100 g minced visceral

tissue, mixed well and left for a day. The insecticide was then
DOI: 10.1556/JPC.22.2009.6.14

457

Short Communications

3 Results and Discussion

Monocrotophos on alkaline hydrolysis yields dimethylphosphoric acid and N-methylacetoacetamide. The methylene group
of N-methylacetoacetamide is located alpha to two carbonyl
groups, which increases the reactivity of the two alpha hydrogen
atoms. This active methyl group reacts with the chromogenic
reagent benzil to give a pink compound (Figure 2).
It was observed that pink spots of RF 0.48 were obtained from
standard technical grade monocrotophos and from monocrotophos extracted from visceral tissue, whereas no spots were
observed for other organochloro, organophosphorus, carbamate,
and pyrethroid insecticides (Figure 1).
Figure 1
Thin-layer chromatogram obtained from: (a) standard monocrotophos, (b) monocrotophos from visceral extract, (c) blank viscera,
(d) dimethoate, (e) endosulfan, (f) carbaryl, (g) cypermethrin.

From the recovery experiment it was observed that the intensity

of the pink spot obtained from the visceral extract was comparable with that of the spot obtained from 8.5 g technical grade
monocrotophos and hence recovery was 85% (n = 3).The intensity of the spot was determined by visual comparison of spot
from visceral extract with that from the standard. The reagent
reported here is selective and sensitive (ca 0.5 g) for monocrotophos and hence can be routinely used for the detection and
identification of residual monocrotophos in biological materials
related to forensic toxicology.
Acknowledgments

dimethylphosphoric
acid

N-methylacetoacetamide

N-methylacetoacetamide

Pink compound

Figure 2
Reaction of monocrotophos with benzil.

The authors are grateful to Dr R. Krushnmurti, Director, Forensic Science Laboratories, State of Maharashtra, Mumbai, India,
and to Dr M. S. Shingare, Professor and Head, Department of
chemistry, Dr B.A.M.U., Aurangabad, India, for their keen interest and valuable guidance in this work.

References
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State of Maharashtra, Mumbai, 2007.
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[3] G.B. Kawale, V.D. Joglekar, V.P. Barve, H.S. Mahal, Sci. Cult. 38
(1972) 373374.

extracted with ethyl acetate (150 mL), the solvent was evaporated at room temperature, and the residue was dissolved in 1 mL
ethanol. This solution (10 L) and standard solutions (10 L)
containing 8.0, 8.5, 9.0, and 9.5 g monocrotophos were spotted
on an activated plate that was then developed as described
above. The intensity of the pink spot obtained from the visceral
extract was compared with those obtained from the known standards and found to be most similar to the spot obtained from
8.5 g monocrotophos (n = 3), hence the recovery was ca 85%

[4] V.B. Patil, S.V. Padlikar, G.B. Kawale, Analyst 112 (1987)
17651766.
[5] V.B. Patil, M.V.Garad, J. Planar Chromatogr. 14 (2001) 210212.
[6] V.D. Joglekar, H.S. Mahal, Arch. Kriminol. 142 (1968) 170171.
[7] J. Baumler, U.S. Rippstein, Helv. Chim. Acta 44 (1961)
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[8] V.R. Chandegaonkar, D.B. Shinde, D.V. Mane, J. Planar Chromatogr. 21 (2008) 199200.
Ms received: April 26, 2008
Accepted: July 3, 2009

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