Short Communications High-Performance Thin-Layer Chromatographic Detection of Profenofos in Biological Materials

Vitthal R. Chavan and Bhagwat D. Mali*

Key Words

Profenofos Sulphanilic acid Carbamate insecticides Organophosphorus insecticide HPTLC

1 Introduction technical grade profenofos (Sygenta, Mumbai) was prepared in ethanol. An aqueous solution of 10% w/v sodium hydroxide was Organophosphorus insecticide profenofos is widely used in agri- prepared. Diazotized sulfanilic acid reagent was prepared by culture for crop protection [1]. Due to its easy availability in dissolving 0.5 g sulfanilic acid and 1 g sodium nitrite in 100 mL India, it is misused for homicidal or suicidal purposes. In 2012, of 10% v/v hydrochloric acid. Other diazotized spray reagents the Regional Forensic Science Laboratory, Aurangabad, detect- such as dapsone, p-toluidine, m-nitroaniline, and p-amino ben- ed about 463 human poisoning cases involving organophospho- zoic acid were prepared using requisite amount of acid and sodi- rus insecticides. um nitrite. Instrumental methods such as spectrophotometry [2], gas chro- matography [3–6], gas chromatography–mass spectrometry 2.2 Chromatography (GC–MS) [7–9], and surface plasma resonance [10] are reported in the literature for the determination of profenofos in food and Chromatography was performed on HPTLC (20 × 20 cm) pre- biological materials. Although these methods are sensitive, there coated (silica gel 60 F254) plates (Merck, Darmstadt, Germany). are limitations to their use in routine forensic work, owing to Standard solutions of profenofos, profenofos from extract of vis- their complex matrix which may damage the columns. cera, extract of blank viscera, carbaryl, baygon, carbofuran, and Therefore, high-performance thin-layer chromatography carbosulfan were spotted onto the HPTLC plate. Then it was (HPTLC) is the method of choice for screening biological sam- developed in a previously saturated TLC chamber (saturation ples due to its speed, low cost, and versatility. Several chro- time 20 min) using n-hexane–acetone (8:2) as mobile phase to a mogenic reagents – for example, mercurous nitrate [11], potas- height of about 10 cm from the point of application (develop- sium iodate [12], palladium chloride [13], and orcinol [14] – ment time ca. 15 min). The plate was removed from the cham- have been reported in the literature for TLC detection of ber, dried in air, and sprayed uniformly with 10% sodium organophosphorus insecticides, but none of these shows positive hydroxide solution. After waiting for 5–10 min for complete reaction with profenofos. A selective and sensitive reagent is hydrolysis, it was sprayed uniformly with diazotized sulfanilic therefore required for the detection of profenofos in HPTLC acid reagent. Immediately, an orange spot was observed on the TLC plate at R 0.45 for profenofos standard and profenofos analysis. The use of diazotized sulfanilic acid as spray reagent F extract of viscera. A pink spot for carbaryl (R 0.20), orange for detection and identification of profenofos in routine toxico- F spots for baygon (R 0.25), carbofuran (R 0.32), and brick red logical analysis is reported in this paper. F F

(RF 0.60) for carbosulfan were also observed.

2 Experimental 2.3 Recovery of Profenofos from Biological Material For semi-quantitative determination of profenofos, the insecti- cide (10 mg) was added to minced visceral tissue (stomach, 2.1 Chemicals intestine, liver, etc. ca. 50 g). Approximately, 10 g of ammonium All chemicals used were of analytical reagent grade. Distilled sulphate was then added to this mixture with thorough stirring. water was used throughout. Standard solution (1 mg mL–1) of The mixture was left for a day. The insecticide was then extract- ed with diethyl ether (150 mL) in a separating funnel. The ether extract was transferred to an evaporating dish, and aqueous V.R. Chavan and B.D. Mali, Regional Forensic Science Laboratory, State of Maharashtra, Cantonment, Aurangabad – 431 002, India. phase was re-extracted with diethyl ether (2 or 3 × 50 mL). The E-mail: [email protected] extracts were combined, and the solvent was evaporated at room

Journal of Planar Chromatography 27 (2014) 1, 66–68 DOI: 10.1556/JPC.27.2014.1.12 66 0933-4173/$ 20.00 © Akadémiai Kiadó, Budapest Short Communications

Figure 1

Reaction of profenofos with diazotized sulfanilic acid. temperature. The residue was dissolved in 10 mL ethanol. This solution (10 µL) and standard solutions (10 µL) containing 8.0, 8.5, 9.0, 9.5, and 10.0 µg of profenofos were applied to an acti- vated plate, and this was then developed as described above and sprayed with reagent. The intensity of the orange spot developed for the visceral extract was then compared with those of the standards.

3 Results and Discussion Figure 2 Thin-layer chromatograms obtained from (1) profenofos, (2) profeno- Profenofos (I) (Figure 1) is a derivative of phosphorothionic fos from extract of viscera, (3) blank viscera, (4) carbaryl, (5) baygon, acid and hydrolyzed in alkaline medium to give 4-bromo-6- (6) carbofuran, and (7) carbosulfan. chlorophenol (II) and phosphorothionic acid-O-ethyl-S-propyl ester (III). As phenol couple with diazonium salts [15], 4- bromo-6-chlorophenol (II) also couples with diazotized sulfanil- that the color of some carbamate insecticides for a particular ic acid (IV) to give orange-colored compound (V). reagent is similar to the color of profenofos. They can be distin- guished by their distinct R values and mixed spotting. None of The color of the spot was stable for several days. The mobile F the reagents gives color reaction with profenofos or carbamate phase used gives a compact spot. The limit of detection of the insecticides prior to their alkali hydrolysis (Figure 2). reagent was ~1 µg per spot observed after development. Other diazotized reagents such as dapsone, p-toluidine, m-nitroaniline, In the experiments to determine the recovery of profenofos from and p-aminobenzoic acid, like sulfanilic acid, react with alkali biological materials, the intensity of the orange spot developed hydrolysis products of profenofos and carbamate insecticides for the visceral extract was most similar to that of the standard (baygon, carbaryl, carbofuran, and carbosulfan) to give colored corresponding to 9.0 µg profenofos (n = 3). Hence, recovery of spots. A typical chromatogram is shown in Figure 1. It shows profenofos from this type of sample was approximately 90%.

Table 1

Color reactions of profenofos and carbamates (with detection limits [μg] in parentheses) and the RF values.

Diazotized Profenofos Carbaryl Baygon Carbofuran Carbosulfan reagent

Sulfanilic Orange Pink Orange Orange Brick red acid (1.0) (0.1) (0.1) (0.2) (0.2) Dapsone Orange Violet Red Red Brick red (2.0) (0.1) (0.1) (0.1) (0.2) p-Toluidine Yellow Red Yellow Yellow Yellow (5.0) (0.5) (0.5) (0.2) (0.2) m-Nitroaniline Yellow Pink Orange Orange Orange (2.0) (0.1) (0.2) (0.2) (0.5) p-Aminobenzoic Orange Violet Orange Orange Orange acid (2.5) (0.1) (0.2) (0.2) (0.2)

RF values 0.45 0.20 0.25 0.32 0.60

Journal of Planar Chromatography 27 (2014) 1 67