Use of Phosphomolybdic Acid as a Chromogenic Reagent for the Selective Detection of Monocrotophos in Biological Samples.
Harishchandra Jirimali1*, Saiprasad Santre1,2, Vidya Kokne1,2, Bhausaheb More1*
1Regional Forensic Science Laboratory, Nanded (MS) India- 431601.
2Department of Chemistry, Yeshwant Mahavidyalaya, Nanded (MS) India- 431602.
*Corresponding Author E-mail: hdj739@gmail.com, bhaumore1@gmail.com
ABSTRACT:
Organophosphorus pesticides, especially Monocrotophos, [dimethyl (2E)-4-(methylamino)-4-oxobut-2-en-2-yl phosphate] have seen extensively used in agricultural practices in India due to their effectiveness in controlling insect pests due to its systemic nature1. The misuse and accessibility of organophosphorus pesticides in India have led to an increase in cases of both accidental and intentional poisonings. Monocrotophos, marketed under various trade names such as Phoskill, Nuvacron, Monophos and Azodrin is highly toxic and poses rapid symptoms and fatal outcomes, particularly in developing countries where awareness and regulation are limited2-3.
According to forensic casework reports from Maharashtra, India, hundreds of poisoning cases involving Monocrotophos have been reported annually, establishing the urgent need of for efficient detection methods in forensic toxicology laboratories. Rapid, selective and inexpensive detection methods are urgently needed, especially for complex matrices like biological tissues. Toxicologist face significant challenges in identifying such insecticides due to the complex nature of biological matrices like viscera, where pesticides residues may exist in very small amounts amid numerous interfering substances4-8.
While instrumental techniques such as x-Ray Fluroscence HPLC, GC and GC-MS are available and highly sensitive they are often inaccessible in routine labs due to their cost and need for skilled operators. Consequently, thin layer chromatography (TLC) Remains a widely used and reliable method in such settings for preliminary detection due to its simplicity and speed. It is frequently used in forensic science due to its affordability, simplicity and adaptability8-21.
Many chromogenic reagents have been developed and applied to improve the selectivity and visibility of TLC-based pesticide detection. Traditional regions like Vanillin, diazotized sulphanilamide, and methanolic ferric chloride have been used to detect Monocrotophos after alkaline hydrolysis forming colored complexes6-8. However, these often suffer from drawbacks such as poor selectivity, interference from co-extracted matrix compounds, or unstable color formation4.
To address these limitations, this study introduces a novel chromogenic spray reagent based on Phoshophomolybdic Acid (PMA) for specific TLC-based detection of Monocrotophos in biological materials. The reaction between PMA and Monocrotophos produces a stable light purple-pink color due to redox process involving Mo (VI) to Mo(V), With no significant interference from Visceral components or structurally similar insecticides. This research focuses on introducing Phosphomolybdic Acid (PMA) as a chromogenic reagent for the identification of Monocrotophos in Biological samples. The development of such a reagent provides a low-cost, rapid, and reproducible method suitable for use in resource-limited laboratories, thereby improving the accuracy and efficiency of pesticide-related toxicological investigations.
2. MATERIALS AND METHODS:
2.1 Chemicals and Reagents:
All chemicals used were of analytical grade. Monocrotophos (Phoskill 36 % SL) United phosphorous Ltd GIDC Ankaleshwar Gujarat. was obtained from a local vendor. Solvents such as Acetone (HPLC Grade) and Hexane were purchased from Molychem, Mumbai. Hydrochloric acid was sourced form Avantor Performance Materials, Thane. Phosphomolybdic acid and Ethanol were obtained from Changshu Hongsheng Fine Chemicals Co. Ltd., Jiangsu Province. Deionized water was prepared using an Xtrapure Lablink instrument.
2.2 Preparation of Chromogenic Reagent:
1 gm of Phosphomolybdic Acid was dissolved in 30 mL of Ethanol with continuous stirring for 5 minutes. Complete dissolution was indicated by the formation of a yellow solution.
2.3 Extraction of Monocrotophos:
2 mL of Phoskill formulation was dissolved in 5 mL diethyl ether. The solution was used for TLC spotting.
2.4 Extraction of Monocrotophos from visceral sample:
Approximately 50 g of viscera (stomach and intestine) was mixed with 2 mL Monocrotophos and 25 mL diethyl ether. After ultrasonication and stabilization, 100 mL chloroform was added. The organic layer was separated, evaporated to dryness, and redissolved in acetone for TLC analysis. A Blank viscera sample was similarly processed without Monocrotophos to serve as a reference.
2.5 Thin Layer Chromatography:
TLC plates were prepared by applying a silica-water slurry to clean glass plate. Plates were air-dried and activated at 100 oC. Samples were spotted: (1) viscera extract without Monocrotophos, (2) Spiked viscera with Monocrotophos, (3) Monocrotophos extract from formulation. Plates were developed using Hexane: Acetone (8:2), dried, the sprayed with PMA reagent followed by dil. HCl. Monocrotophos spots appeared as light purple-pink coloration. (Fig. 1)
Fig. 1: Thin Layer Chromatography from: (a) Blank viscera, (b) Monocrotophos- spiked viscera extract, (c) Monocrotophos extract from formulation
3. RESULTS AND DISCUSSION:
The prepared chromogenic spray reagent based on Phosphomolybdic Acid (PMA) Exhibited highly specific and visually distinct results for the identification of Monocrotophos on thin layer chromatography (TLC) plates. When the PMA reagent was applied on TLC plates containing Monocrotophos extracts from various sources (standard formulation, spiked viscera and blank samples) a stable purple-pink color developed which was absent in samples without Monocrotophos. This visual distinction confirms the specificity of the reagent.(Fig. 2)
Fig. 2: Simultaneous detection of various pesticides for Monocrotophos selectivity: (a) Monocrotophos (b) Profenophos (c) Triazophos (d) Quinalphos (e) Cypermethrin (f) Chlorpyriphos (g) 2,4- D
Upon application of the Hexane: Acetone (8:2) Solvent system, the Rf value of Monocrotophos was consistent and reproducible across all experiments, providing a reliable point of comparison. Among all tested substances, Only Monocrotophos Produced the distinct pink chromogenic spot while other co-extracted pesticides and matrix constituents like amino acids and proteins did not interfere with the detection. In a acid catalyzed reaction the cleavage of the Monocrotophos occurs and it release the phosphate monoester ions forming complexes with the phosphomolybdic acid resulting in the appearance of colour spot. The mechanism behind the color development is a redox reaction where in Molybdenum (VI) in PMA is the reduced to Molybdenum(V) in the presence of Monocrotophos. This reaction leads to the formation of light purple-pink complex that remains stable over time, enabling confident visual identification without the need for instrumental quantification2-3. (Fig. 3).
Fig. 3: Proposed reaction mechanism of Monocrotophos with Phosphomolybdic Acid
A comparative evaluation of Monocrotophos From the biological matrix was calculated to be approximately 90% which alliance well with findings from earlier studies V. B. Patil et al. and Pawar er al., Demonstrating excellent extraction efficiency and analytical reproducibility2,7. Furthermore, these newly developed method was validated by performing TLC analysis alongside older chromogenic reagents like vanillin and diazotized sulphanilamide. Unlike this traditional reagent, which often produces weaker or less stable color formations and can react with other insecticides or matrix components6-7, The PMA region showed higher selectivity and a distinct advantage in color stability and sensitivity. The spots located on TLC are stabled for more than 96 hours at room temperature, suggesting reliability and stability of the reagent this method is also reproducible and can be used for the detection of Monocrotophos in forensically important samples like soil, water and foodstuff.
This enhanced specificity and reliability make the PMA-based chromogenic reagent a powerful tool in the preliminary screening of Organophosphate poisoning cases in forensic settings. The technique is cost-effective, easy to implement, and does not require advanced instrumentation or highly trained personnel, making it highly suitable for widespread forensic and toxicological application in resource-constrained environments.
4. CONCLUSIONS:
Phosphomolybdic Acid is a promising new chromogenic reagent for TLC-based identification of Monocrotophos. It proven to be a reliable and specific tool for the detection of Monocrotophos in forensic biological samples . With its ability to produce a stable, distinctive purple pink color through a simple redox mechanism, these reagent offers a practical solution for routing forensic analysis, particularly in resource-limited settings. High recovery rate of method its selectivity against non target compounds and its use make it a valuable addition to the forensic toxicologist’s tool kit. It serves as a complementary.
5. CONFLICT OF INTEREST:
The authors have no conflicts of interest regarding this investigation.
6. ACKNOWLEDGEMENT:
The authors are grateful to the Director General of Legal and Technical, and the Director, Directorate of Forensic Sciences, Department of Home, Government of Maharashtra, for their support and encouragement.
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Received on 24.05.2026 Revised on 12.06.2026 Accepted on 27.06.2026 Published on 04.07.2026 Available online from July 30, 2026 Asian J. Research Chem.2026; 19(4):391-394. DOI: 10.52711/0974-4150.2026.00058 ©A and V Publications All Right Reserved
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