368 Analyst, June, 1968, Vol. 93, $$. 368-370 A Rapid Procedure for the Identification of Organochlorine Pesticides in Blood and Tissues BY C. E. ELIAKIS, A. S. COUTSELINIS* AND E. C. ELIAKIS (Department of Forensic Medicine and Toxicology, University of Athens, 48 Academy Street, Athens 146, Greece) A method is described for the rapid identification of chlorinated insecti- cides, such as aldrin, dieldrin, op’-DDT and pp’-DDT, in tissues and blood, without the necessity for a previous clean-up procedure of the extracts made from them. Two-dimensional thin-layer chromatography was used for this purpose. The plates were developed in hexane for one dimension and in cyclohexane for the other, and were sprayed with a solution of 0-5 per cent. silver nitrate in ethanol before exposure to germicidal ultraviolet light.The sensitivity of this method is 0.5pg, which is suitable for routine analysis. MANY papers have been published giving methods for the trace detection of chlorinated insecticides. These methods include two steps : an extraction and clean-up procedure for the extracts, and chromatography. The existing extraction and clean-up procedures were studied in an effort to reduce the time factor to a minimum and to develop an improved clean-up technique. Of the chromato- graphic procedures available, thin-layer chromatography, as developed by Kovacsl y2y3 and Abbott, Egan and Thom~on,~ has proved most useful for the semi-quantitative determination of the chlorinated insecticides, as it combines the advantages of rapidity and economy.Consequently, thin-layer chromatography is of particular interest to our laboratory, in which numerous toxicological analyses are carried out annually for the detection of traces of chlorinated insecticides in cases of poisoning that are attributable to the wide use made of these substances in agriculture. In these cases, the specimens examined are of varied nature, consisting frequently of viscera (liver) and blood in an already decomposed condition. As mentioned, the purpose of our laboratory was to find a method for the rapid identi- fication of chlorinated insecticides in tissues and blood. We have found that the use of two-dimensional thin-layer chromatography facilitates the clear identification of chlorinated insecticides such as aldrin, dieldrin of-DDT and +p’-DDT, without the necessity for a previous clean-up procedure of the extracts made from liver or blood.The use of two-dimensional thin-layer chromatography was considered valuable, because with viscera and blood in a decomposed condition, one-dimensional thin-layer chromato- graphy, without previous clean-up of the extracts, did not yield satisfactory results; this was because in most instances when chromatograms were developed with silver nitrate solution, in addition to the spots formed with the chlorinated insecticides, the whole length of the chromatogram course became dark, resulting in a decrease in the sensitivity. On the other hand, when examining blood we found that the extraction method described by Folch, Lees and Sloane Stanley,5 who had applied it to the extraction of total lipids, after being slightly modified by us gave satisfactory results with a short extraction time.The technique described below for the examination of blood is simple and rapid, and its sensitivity has been proved to be quite satisfactory in cases of poisoning. * Present address: Department of Pharmacology, School of Medicine, University of New Mexico, Albuquerque, N.M., 87106, U.S.A. 0 SAC and the authorsELIAKIS, COUTSELINIS AND ELIAKIS 369 EXTRACTION PROCEDURE- One millilitre of total blood was extracted with 6 ml of methanol by shaking the mixture for 30 minutes; 12 ml of chloroform were added and the mixture again shaken for another 30 minutes. The extract was filtered on a filter-paper and the filtrate made up to 20 ml with a mixture of chloroform - methanol (2 + 1 v/v).This solution was then shaken with one fifth of its volume, i.e., 4 ml, of water for 5 minutes. The mixture was then centrifuged and the chloroform layer separated and dried over anhydrous sodium sulphate. After evaporating the solution, the residue was dissolved in 1 ml of hexane and centrifuged at 3000 r.p.m. for 3 to 4 minutes. Following centrifugation, 0-2 ml of the hexane layer was used for chromatography. CHROMATOGRAPHY- Neutral plates (0.25mm thick) were prepared according to the method of StahP by using silica gel G. The plates were activated at 110" C for 1 hour. The spot was located at a distance of 1.5 cm from adjacent sides of the plate. The plates were developed in hexane, dried for half an hour at room temperature, and developed again in cyclohexane.In both systems the length of run was 12 cm from the start. During the run in both solvents, the tanks were saturated in the conventional way. A standard mixture of chlorinated insecticides was chromatographed for each dimension on a separate chromatoplate in the same chromatotank, with the same conditions and development time as before. The positions on the chromatoplate of the chlorinated insecti- cides examined had been previously determined by chromatographing each of them separately in two-dimensional co-ordinates with the same solvents. In addition, standard compounds, together with the sample, were chromatographed for each dimension on the same chromato- plate (Figs. 1 and 2). This was carried out because the RB values, which are influenced by various factors, are not constant.y = Cyclohexane y' = Hexane a = Dieldrin b = pp'-DDT c = op'-DDT d = Aldrin -1 a 0- Y I XI = Position of the standard spot for the development XU = Position of the standard spot for the development p-p' = Limits of chromatogram's development in both in the y' direction in the y direction systems Fig. 1. Two-dimensional thin-layer chromatography of standard compounds370 - I u‘ ELIAKIS, COUTSELINIS AND ELIAKIS Iy 1 - Y’ 0 Y = Cvclohexane d = Aldrin i‘ = Hexane a = Dieldrin b =PP’-DDT c =,op’-DDT XI = Position of the standard spot for the development in the y’ direction p-p’ = Limits of chromatogram’s development in both systems Fig. 2. chromatography Separation of organochlorine insecticides from blood by two-dimensional thin-layer The plates were then dried, sprayed with a solution of 0.5 per cent.silver nitrate in ethanol and exposed to germicidal ultraviolet light for 15 minutes. With the above method, 0.5 pg of aldrin, dieldrin and DDT can be identified as dark spots on a white background. The same chromatographic technique was used for liver, for which the extraction procedure described by Taylor, Rea and Kirby7 was used; the hexane layer was evaporated to 10 ml, 0.2 ml of which was chromatographed. The above-mentioned sensitivity was considered quite satisfactory for identifying traces of chlorinated insecticides in cases of acute poisoning. REFERENCES 1. 2. 3. 4. 5. 6. 7. Kovacs, M. F.. jun., J . Ass. Off. Agric. Chem., 1963, 46, 884. - , Ibid., 1964, 47, 1097. - , Ibid., 1966, 48, 1018. Abbott, D. C., Egan, H., and Thomson, J., J . Ckomat., 1964, 16, 481. Folch, S., Lees, M., and Sloane Stanley, G., J . Biol. Chem., 1957, 226, 497. Stahl, E., Editor, “Thin-Layer Chromatography : A Laboratory Handbook, ” Springer-Verlag, Taylor, A., Rea, R. E., and Kirby, D. R., Analyst, 1964, 89, 497. Received September Mth, 1967 Berlin, Heidelberg and New York; Academic Press Inc., New York and London, 1966.