JPC – Journal of Planar Chromatography – Modern TLC

https://doi.org/10.1007/s00764-020-00015-2

SHORT COMMUNICATION

Development method of high-performance thin-layer

chromatographic detection of synthetic organophosphate insecticide
profenofos in visceral samples
Umakant D. Pawar 1 & Chandrakant D. Pawar 2 & Ulka K. Kulkarni 3 & Rajendra K. Pardeshi 4

Received: 9 November 2019 / Accepted: 28 January 2020

# Akadémiai Kiadó, Budapest, Hungary 2020

Abstract
The synthetic pesticide profenofos is a member of organophosphate insecticide compounds. It is an important synthetic insec-
ticide and has a diversified role in agriculture. During the last decade, profenofos was frequently misused in homicidal, acci-
dental, and suicidal poisoning cases. Since then, a large number of visceral samples have been received for toxicological chemical
analysis. Thin-layer chromatography (TLC) and high-performance thin-layer chromatography (HPTLC) were the preferred
methods of choice. By observing the results of toxicological analyses, the conclusion can be drawn that profenofos plays a
major role in poisioning. So we developed a new reagent which was found to be a selective spray reagent for profenofos in routine
analysis by HPTLC.

Keywords High-performance thin-layer chromatography . Synthetic organophosphate insecticide . Toxicology . Forensic

science . Post-mortem visceral samples

1 Introduction ability to inhibit the enzyme acetylcholinesterase receptors

Profenofos is chemically 4-bromo-2-chloro-
1-[ethoxy(propylsulfanyl)phosphoryl]oxybenzene, and it
belongs to the family of organophosphate insecticides. It
is mostly used as an insecticide for a variety of crops
including cotton, maize, potato, sugar beet, vegetables,
etc. throughout the world [1].
It is a highly effective insecticide which causes adverse
effect for humans, animals and the environment. The profeno-
fos poisoning cases to humans primarily involve endocrine
system disrupter causing hormonal imbalance and it has the
* Rajendra K. Pardeshi
rkpardeshiudp@gmail.com
1
Regional Forensic Science Laboratories, Aurangabad, Maharashtra,
India
2
Department of Chemical Technology, Dr. Babasahab Ambadker
Marathwada University, Aurangabad, Maharashtra, India
3
Directorate Forensic Science Laboratories Kalina,
Mumbai, Maharashtra, India
4
Sant Ramdas College Ghansawangi, Jalna, Maharashtra, India
[2].
Profenofos is easily available for use and frequently en-
countered in forensic casework, since it is frequently misused
in homicidal, accidental and suicidal poisoning cases. In the
year 2018, the regional forensic laboratory, Aurangabad,
India, detected several cases of human poisoning by use of
profenofos. In routine forensic toxicology department, the
poisoning samples analyses are generally performed by thin-
layer chromatography (TLC) and high-performance thin-layer
chromatography (HPTLC), ultraviolet (UV), high-
performance liquid chromatography (HPLC), gas chromatog-
raphy (GC), gas chromatography–mass spectrometry (GC–
MS), etc. [3]; although these methods are very sensitive and
accurate, they are highly expensive and also time-consuming.
HPTLC and TLC methods are the versatile techniques for the
analysis of a large number of chemical substances, drugs, and
pesticides. These techniques can be conveniently and easily
used for routine toxicological work. The chemical structure of
profenofos consists of easily hydrolyzed groups.
For the detection of profenofos poisoning, some instrumen-
tal methods were used. The instruments like spectrophotome-
try [4], GC [5–7], GC–MS [8–11] and surface plasma reso-
nance [12], etc. were used for the detection of profenofos. All
those instruments were used for the identification of poisoning
 JPC-J Planar Chromat

cases, but due to limitations like cost, complex matrix which Visualization: spray reagent A, then wait for 15 min, then
results in the damage of column and time, these methods were spray reagent B.
not used for routine forensic works. HPTLC is the method of
choice for the screenings of biological samples due to its
2.2 Extraction of profenofos from biological materials
speed, cost and versatility. There are several chromogenic re-
agents reported in the literature such as mercuric nitrate
About an amount of 100 g of viscera was taken including
followed by diphenyl cabazone [13], mercuric nitrate follow-
pieces of stomach, intestine, along with pieces of liver, kidney,
ed by potassium ferrocyanide [14], Nessler’s or Tollen’s re-
spleen and lungs containing profenofos poison from post-
agents [15], alkaline resorcinol and aqueous phenylhydrazine
mortem visceral sample. This material was cut into fine pieces
hydrochloride reagent [16], sodium hydroxide, potassium fer-
and minced carefully. To this minced visceral sample, diethyl
rocyanide and o-toludine [17], bromophenol blue [18], diazo-
ether (50 mL) was added and it was kept at room temperature
tized sulfanilic acid reagent [19] and cupric ferrocyanide re-
for 24 h. After 24 h, the diethyl ether layer was transferred to a
agent [20], etc. These all reagents are reported for the detec-
round-bottom flask and evaporated under reduced pressure.
tion of organophosphorus insecticide by TLC. In the present
The final residue was transferred into a steel capsule and evap-
communication we have reported a new spraying reagent for
orated to dryness. The residue was re-dissolved in methanol
the identification of poisonous visceral sample by profenofos
(2 mL) and was used for HPTLC.
by simple HPTLC method. We identified the profenofos poi-
son from the visceral samples by using HPTLC method and
by using chromogenic spray reagents. We screened a series of 2.3 Thin-layer chromatography
spraying reagents for the identification of profenofos poisons
from biological (visceral) samples. Profenofos reacts with cu- For identification of profenofos residue, pre-coated TLC
pric acetate spraying reagent to produce an intense brown- plates (silica gel 60 F254, Merck Ltd. Darmstadt, Germany)
colored compound in the presence of sodium hydroxide, by were used. The samples were spotted on TLC plate with fine
undergoing hydrolysis reaction. The chromogenic reagent capillary tube along with standard profenofos. The TLC plate
does not react with the organochlorine insecticides, carbamate was run in hexane:acetone (7:3, v/v) developing solvent. The
insecticides, and pyrethroids. Visceral constituents including
amino acids, peptides, proteins, fats, etc. do not interfere with
the spraying reagent.
In continuation of our research work for the identification
or detection of different poisons by using different spraying
reagents, we developed cupric acetate for the identification of
organophosphorous poisons with the hope it gives quick re-
sults in routine sample analysis ultimately helping in the fo-
rensic field [21–23].

2 Experimental

2.1 Chemicals and reagents

All the reagents were of analytical reagent scale. Standard

profenofos was purchased from Coromandel Fertilizer Ltd.
(Ankleshwar, Gujarat, India). Cupric acetate and sodium hy-
droxide reagent ware purchased from S. D. Fine Chem. Ltd.
(Mumbai, India).
Preparation of standard solutions:
Profenofos solution was prepared in ethanol (2 mg mL−1).
[A] A mixture of 10% of sodium hydroxide solution:10 g
of sodium hydroxide was dissolved in 100 mL of distilled
water. Fig. 1 TLC showing profenofos residues using chromogenic spray
[B] Cupric acetate solution: 4 g of cupric acetate was dis- reagent: b) blank viscera, b) viscera with profenofos poisoning, and c)
solved in 100 mL distilled water. standard profenofos
JPC-J Planar Chromat
Scheme 1 Reaction of
profenofos with copper acetate.
TLC plate bearing standard profenofos and visceral sample
and blank viscera (non-poisonous history cases) spots was
dried and the chromatogram was developed in a pre-
saturated tank containing the developing solvent mentioned
above. After developing the plate, the solvent front (distance
travelled by the solvent) was immediately marked and the
extra solvent was evaporated (dried) in a fume hood. The
plates were then sprayed with the above-mentioned cupric
acetate reagent and then the plate was kept in an oven at
120 °C for 10 min. A brown-colored spot with white back-
ground was clearly visible at Rf 0.44, as shown in Fig. 1. Rf
values and color of visceral sample and standard profenofos
were compared and they were matching with each other.
3 Results and discussion
Profenofos is an organic compound which reacts with cupric
acetate reagent to give an intense brown-colored compound.
The color species formed in the coordination complex of the
profenofos reacts with the reagent. The color of the spot re-
mains stable. The limit of detection with this reagent is ap-
proximately 4 μg. Profenofos and spray reagent copper ace-
tate react with each other in the presence of sodium hydroxide.
The reaction was done under basic condition to hydrolyze the
profenofos in the first part and by elimination of sodium phen-
oxide and later when treated with copper acetate to eliminate
and give copper sulfate and some parts of sodium acetate due
to the presence of hydroxide from base. The detailed reaction
is mentioned in Scheme 1. The copper reagent does not react
with organochlorine insecticides like endosulfan, BHC, DDT.
With organophosphorus insecticides dimethoate, phorate,
monocrotophos, etc. it does not show any reaction. The syn-
thetic pyrethroids like fenvalerate, cypermethrin and delta-
methrin the reagent does not react. The visceral constituents
like amino acids, peptides, proteins and fats do not interfere
with the chromogenic reagents. Although mercuric nitrate
followed by diphenylcarbazone, mercuric nitrate followed
by potassium ferrocyanide, Nessler’s or Tollen’s reagent, al-
kaline resorcinol, sodium hydroxide, potassium ferricyanide
and o-toluidine, palladous chloride reagent, diazotized
sulfanilic acid reagent, etc. are used for the identification of
profenofos poisons. It has been reported for the detection of
organophosphorus insecticides, the chromogenic reagent uti-
lized in the proposed method is cheap and does not involve
any critical reaction condition or tedious sample preparation.
Hence, this can be used routinely for the detection of profeno-
fos in post-mortem visceral materials.
4 Conclusion
To the best of our knowledge, cupric acetate reagent was used
the first time for the identification of profenofos in post-
mortem visceral samples (in fatal poisoning cases of profeno-
fos). The proposed reagent is simple and can be used routinely
for the identification of organophosphate insecticide profeno-
fos. On the basis of TLC, we can easily identify the profenofos
poisons. Further work on new spray reagents is in progress
and will be communicated in due course.
Acknowledgments Authors are grateful to Dr. K. V. Kulkarni, Director,
Forensic Science Laboratories, Home Department State of Maharashtra,
Mumbai, India, and Head of the Department of Chemistry, Dr. Babasahab
Ambedkar Marathwada University, Aurangabad, MS, India, for helpful
discussion and suggestions.
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