1. INTRODUCTION:

Rodents are the main pests that are responsible for the destruction of agricultural crops like stored grains, and they are also their domestic pests that are spreading diseases like plague and destroying household things. Rodenticides are the chemicals that are used to control the rodents like rats, mice, moles, and rabbits^1-2. Generally, phosphide compounds are the inorganic rodenticides that are popularly used for domestic rodent control.

Bromadiolone {3-[3-(4’-Bromo[1,1’-biphenyl]-4-yl)-3-hydroxy-1-phenylpropyl]-4-hydroxy-2H-1-benzo-pyran-2-one} a potent rodenticide, is a warfarin derivative and second-generation dicoumarin rodenticide. Bromadiolone is used in bait forms and can be used for domestic as well as industrial pest control. Due to its attractive bait formulation, rodents are easily attracted towards it. Due to its anticoagulation action, internal haemorrhage the death of the rodents^3-5.

The analytical methods like thin layer chromatography (TLC), UV- Vis spectroscopy, High-performance liquid chromatography (HPLC) and Mass chromatography (MS), are the methods used for the detection and quantification of the pesticides^6-15. The sophisticated instruments like HPLC and GC needed costly chemicals, maintenance and the purification of the sample, so the methods like thin layer chromatography can be preferred for the detection and quantification purpose. For the detection of bromadiolone, thin-layer chromatography is reported, but very few chromogenic reagents are available for the detection of bromadiolone^16-17.

In the present study, o-Tolidine was modified by simple diazotization reaction and used as a chromogenic reagent for the detection of bromadiolone^18,19. The present method the bromadiolone was separated from visceral sample and detected by using prepared chromogenic reagent. The result shows the selective detection of bromadiolone in the presence of other interfering pesticides and the extraction is more than 85%. In this method the reagent preparation process is easy and results are reproducible that’s why this method is useful for the detection of bromadiolone in forensic samples.

2 MATERIALS AND METHODS:

2.1 Chemicals and Reagents

All reagents and chemicals were of analytical grade. Deionized water was used throughout the experiments. A standard sample of Bromadiolone purchased from Sigma Aldrich and its 2mg/mL solution was prepared by dissolving it in acetone. Acetone HPLC grade, sodium nitrite, o-Tolidine, hexane were purchased from Molychem, Mumbai. Hydrochloric acid from Avantor performance Materials India Pvt. Ltd. Thane. Acetic acid from Merk Life Sci Pvt. Ltd., Mumbai. Ethanol from Changshu Hongsheng Fine Chemicals Co. Ltd. Jiangsu Province. All chemicals were used without further purification, and deionized water from Xtrapure Lablink instrument was used in all experiments.

2.2 Preparation of Chromogenic Reagent:

Take 100mL of 0.1M o-Tolidine solution and 10mL of 2 M HCl, mix the together, and place them in an ice bath, maintain the temperature at 0-5°C. After 5 minutes, slowly added 10mL of 0.1M NaNO₂ to the reaction mixture with continuous stirring. Kept the stirring for 10-15 minutes and observe the colour of the reaction mixture. The formation of wine-red colour indicates the completion of the diazotization reaction^18-19.

2.3 Preparation of Standard Bromadiolone Solution:

10mg of bromadiolone accurately weight and dissolved in 2mL of acetone and used throughout the experiments.

2.4 Extraction of Bromadiolone from Visceral Sample:

50g of viscera-containing pieces of stomach and intestine was taken in a glass beaker chopped well, and 20mg of bromadiolone standard was added in it. Stirred the resulting mixture and then ultrasonicated for 10min, then it kept for stabilization. Afterward 50mL of diethyl ether was added in it for the extraction purpose, and kept it for 24 Hrs. The upper ether layer was separated from viscera gently and kept it for evaporation to dryness. The dried mass was redissolved in an appropriate amount of acetone and used for spotting on TLC. The same procedure was used for blank viscera without adding the bromadiolone. A plain viscera sample is taken and diethyl ether was added in it and for extracted it to make a control sample.

2.5 Thin Layer Chromatography:

Thin-layer chromatography plates were prepared on cleaned glass plates by making a slurry of silica powder in water. The prepared plates were air-dried and activated at 100°C for 30min in oven. After activation, the silica plates were used for the spotting of bromadiolone standard and prepared viscera samples. First of all, the extract of viscera that is prepared without bromadiolone (1), extract made by addition of bromadiolone in viscera (2) and standard bromadiolone sample (3). 10microlitres of each sample were spotted on the activated silica plates. The TLC plate was developed in Chloroform: Acetone (7:3) and 1mL of Acetic acid (glacial). The developed plates were dried at room temperature and sprayed by using diazotized o-Tolidine chromogenic reagent.

3 RESULTS AND DISCUSSION:

Bromadiolone is a hydroxycoumarin derivative, a potential rodenticide used for domestic applications to control the rodents. The toxicity of bromadiolone is very high, so sometimes it is found in accidental poisoning and suicidal poisoning cases. In the present study, the diazotized chromogenic reagent is prepared for the detection of bromadiolone. Here, diazotized o-Tolidine chromogenic reagent is prepared by reacting HCl, NaNO₂, and o-Tolidine as mentioned in the experimental section. The bromadiolone standard and visceral extract samples were spotted on TLC as mentioned in the experimental section. The chromogenic reagent was sprayed after developing the TLC, and it showed reddish-pink coloured spot. The formation of reddish-pink spots might be due to the reaction of diazotised compound present in the chromogenic spray and the bromadiolone. Fig.1 shows the TLC spotted with extract of blank viscera (a), extract of viscera containing bromadiolone (b), and standard bromadiolone sample (c). Spot (a) doesn’t show any reddish colour due to the absence of bromadiolone in control viscera extract. Spot (b) and (c) shows reddish colour due to the presence of bromadiolone. The Reddish-pink coloured spot of bromadiolone located at the R_F value of 0.80. The colour of the spot remains stable for more than 12 hrs. Possibly, the diazotized o-Tolidine forms an azo compound with the para position of hydroxycoumarin ring.

Fig. 1. Thin layer chromatogram obtained from:(a) without bromadiolone viscera (control) (b) Viscera containing Bromadiolone (c) Standard Bromadiolone

Selective detection of pesticides is important due to the interference of other pesticides and biological compounds. In the present method for the selective detection of bromadiolone in the presence of other pesticides, the TLC was spotted and sprayed with newly prepared diazotized o-Tolidine reagent as shown in Fig. 2. Here the presence of bromadiolone confirmed by the formation of reddish-pink colour as seen in Fig. 2 (a), whereas the TLC doesn’t show formation of spot in presence of other pesticides as, Thimet (b), Monocrotophos (c), Triazophos (d), Chloropyrifos (e), 2,4-D (f), Glyphosate (g), Imidachloprid (h), Profenophos (i), Rogar (j).

Fig. 2. Simultaneous detection of various pesticides for selectivity of Bromadiolone (a)bromadiolone (b)Thimet (c)Monocrotophs (d) Triazophos (e) Chloropyrifos (f) 2-4 D (g) Glyphosate (h) Imidachloprid (i) Profenophos (j) Rogar

The possible mechanism of reaction of chromogenic reagent with bromadiolone and the formation of reddish-pink coloured azo adduct, is shown in Fig. 3.

Though o-tolidine and its derivatives were widely used as chromogenic reagent, here diazotized o-Tolidine was prepared and first time used for the detection and identification of bromadiolone in commercial and biological samples. This is a simple method of detection and recovery of the bromadiolone found to be around 85%. The spots located on TLC are stabled for more than 12 hours at room temperature, suggesting reliability and the stability of the reagent. This method is also reproducible and can be used for the detection of bromadiolone in forensically important samples like soil, water, and foodstuff.

Fig. 3. Reaction mechanism of Diazotized o-Tolidine with Bromadiolone

4 CONCLUSIONS:

Diazotized o-Tolidine reagent was prepared by the simple reaction of hydrochloric acid, sodium nitrite, and o-Tolidine. The prepared reagent was stable for more than five days at 0 to 5^oC used for the detection of bromadiolone by using thin-layer chromatography. The reddish-pink spot formed by the reaction of reagent and bromadiolone was stable for more than 12 hours. This method is used for the detection of bromadiolone in visceral samples and also rodenticide bait samples. This method showed more than 85% of bromadiolone in the visceral sample and results are reproducible. All the experimental results suggest that this method can be applied for the detection of the bromadiolone in forensically important samples.

5. CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

6.ACKNOWLEDGEMENT:

Authors are grateful to Director General of legal and technical, and Director, Directorate of Forensic sciences, Department of Home, Government of Maharashtra.

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Received on 20.12.2025 Revised on 09.01.2026

Accepted on 27.01.2026 Published on 10.04.2026

Available online from April 13, 2026

Asian J. Research Chem.2026; 19(2):109-112.

DOI: 10.52711/0974-4150.2026.00018

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