ANALYST, JANUARY 1992, VOL. 117 75 Thin-layer Chromatographic Detection of Pyrethroid Insecticides Containing a Nitrile Group Vitthal 6. Patil, Murlidhar T. Sevalkar and Sudhakar V. Padalikar Regional Forensic Science Laboratory, State of Maharashtra, Cantonment, Aurangabad-43 I 002, India A spray reagent for the detection of pyrethroid insecticides containing a nitrile group by thin-layer chromatography is described. These insecticides on alkaline hydrolysis yield cyanide ion, which in turn reacts with copper(ii) acetate and o-tolidine in an acetic acid medium t o give a blue colour. Organochlorine, organophosphorus and carbamate insecticides do not interfere. The limit of detection is about 1 yg. Keywords: Thin-layer chromatography; copper&) acetate; o -tolidine; pyrethroid insecticide Pyrethroid insecticides are used to control a number of insect species on economic crops.These pyrethroids are effective pest control chemicals and have low mammalian toxicity. 1.2 Three major pyrethroid insecticides containing a nitrile group, Liz., fenvalerate, cypermethrin and deltamethrin, have been identified as highly effective contact insecticides. Their use is increasing for the control of insects. Owing to their availabil- ity, insecticides are misused in homicidal and suicidal poison- ing cases. Consequently, characterization of these insecticides is necessary in forensic toxicology. A large number of gas-liquid chromatographic methods"-" for residue analysis of synthetic pyrethroids have been reported, as has autoradiographic thin-layer chromatography (TLC), using l-'C-labelled compounds, particularly in meta- bolic studies where the unlabelled compounds were detected by visualization on silica gel 60 F2s4 chromatographic plates under ultraviolet (UV) light.ks Chromogenic reagents have also been reported, for example, phosphomolybdic acid (20% m/v in ethanol) for the detection of permethrin, cypermethrin and deltamethrin," palladium chloride (0.5% m/v in 12 mol dm-3 HCI) for the detection of deltamethrin10 and silver nitrate impregnated alumina G and irradiation with UV light11 for pyrethroid insecticides in general.In this paper the use of a chromogenic reagent, viz., 20% sodium hydroxide solution, 5% copper(i1) acetate solution and 1% phosphomolybdic acid solution followed by 0.1% o-tolidine in acetic acid, is described for the detection of pyrethroid insecticides containing a nitrile group by TLC.This reagent gives intense blue spots and is selective for pyrethroid insecticides containing a nitrile group. Experimental Reagents All reagents were of analytical-reagent grade. Distilled water was uscd throughout. Sodium hydroxide solution, 20%. Dissolve 20 g of sodium hydroxide in distilled water and dilute the solution to 100 ml. Copper(1i) acetaie solution, 1%. Dissolve 1 g of copper(i1) acetate in distilled water and dilute the solution to 100 ml. Phosphomolyhdic acid solution, 1%. Dissolve 1 g of phosphomolybdic acid in distilled water and dilute the solution to 100 mi. o-Tolidine reagent. Dissolve 0.1 g of o-tolidine in 100 ml of 10% v/v acetic acid.[Caution: o-Tolidine is a known carcinogen .] Procedure A standard glass TLC plate was coated with a slurry of silica gel G in water (1 + 2) to a thickness of 0.25 mm. The plate was activated by heating it at 110 "C for about 1 h. Volumes of 10 yl of standard solutions of fenvalerate, cypermethrin and delta- - Table 1 RF values of three pyrethroid insecticides containing a nitrile group. Solvent system: cyclohcxane-toluene (6 + 4) Insecticide RF value Fen val era t c Cypermethrin Deltarnethrin 0.42 0.38 0.31 methrin in ethanol (each 1 mg ml-l) were spotted on to the plate, which was then developed in a previously saturated TLC chamber using cyclohexane-toluene (6 + 4) as the solvent. After the solvent had eluted a distance of 10 cm up the plate, the latter was removed from the chamber, dried in air and sprayed successively with 20% sodium hydroxide solu- tion, 1% copper(i1) acetate solution and 1% phosphomolybdic acid solution; after 5 min it was sprayed with 0.1% o-tolidine reagent.Intense blue spots of the quinoidal oxidation product of o-tolidine developed. The RF values of three pyrethroid insecticides containing a nitrile group are given in Table 1. Discussion Pyrethroid insecticides containing hydrolysable nitrile moi- eties yield cyanide ion on alkaline hydrolysis. The cyanide ion in turn reacts with copper(ri) acetate and o-tolidine to give a blue colour.12 Normally, the blue colour thus formed fades immediately; however, it is stabilized (>1 h) by the use of phosphomolybdic acid prior to application of the o-tolidine reagent.Other pyrethroids not containing a hydrolysable nitrile group (permethrin, resmethrin, allenthrin, etc.) should not interfere; moreover, organochlorine, organophosphorus and carbamate insecticides do not give a coloured spot. The limit of detection of the method is about 1 pg. The reagent described here is sensitive and selective for pyrethroid insecticides containing a nitrile group, and might find application in the detection of these insecticides in biological materials in forensic toxicology. The authors are grateful to Dr. B. N. Mattoo, Director, Forensic Science Laboratories, State of Maharashtra, Bom- bay, for his valuable advice and encouragement during this work. We also extend our gratitude to Searle (India) Ltd., Bombay, for the supply of technical-grade fenvalerate, and to Nocil Agrochemicals Ltd., Bombay, and Hoechst (India) Ltd., Bombay, for supplying the cypermethrin and deltameth- rin samples, respectively.References 1 Elliott, M., Janes. N. F., and Potter, C., Annu. Rev. Entomof., 1978, 23. 443.76 ANALYST, JANUARY 1992, VOL. 117 2 Elliott, M., Pestic. Sci., 1980, 11, 119. Ruzo. L. 0.. Engel, J. L., and Casida. J. E., J. Agric. Food 3 Chapman. R. A., and Simmons, H. S., J. Assoc. Off. Anal. Chem.. 1979, 27. 725. Chem., 1977.60, 977. 11 Sundararajan, R., and Chawla, R. P., J. Assoc. Of& Anal. 4 Talekar, N. S., J. Assoc. Of5 Anal. Chem., 1977, 60, 908. Chem., 1983, 66. 1009. 5 Lee. Y. W.. Westcott, N. D., and Reichle, R. A..J. Assoc. Off. Feigl, F . , Spot Tests in Inorganic Analysis, Elsevier, Amster- Anal. Chem.. 1978, 61, 869. dam, 1972, p. 209. 6 Roberts. T. R.. and Standen, M. E.. festic. Sci., 1977, 8. 30.5. 7 Ruzo, L. 0.. Holmstead. R. L., and Casida, J . E., J . Agric. Food Chem.. 1977, 25, 1385. 8 Gaughan, L. C., Ackerman, M. E., Unai, T., and Casida, J . E., J. Agric. Food Chem., 1978, 26, 613. 9 Shono, T., Ohsawa, K., and Casida, J. €3.. J. Agric. Food Chem.. 1979. 27, 316. 10 12 Paper I I00988 E Received March 4 , 1991 Accepted July 31, I991