Analyst, March, 1968, Vol. 93, $$. 173-177 173 Thin-layer Chromatographic = Enzyme Inhibition Procedure to Screen for Organophosphorus Pesticides in Plant Extracts without Elaborate Clean-up BY C. E. MENDOZA, P. J. WALES, H. A. McLEOD AND W. P. McKINLEY Research Laboratories, Food and Drug Directorate, Department of National Health and Welfare, Ottawa 3, Ontario, Canada) A procedure is described for rapidly screening some organophosphorus pesticides in plant extracts without elaborate clean-up. Azinphos-methyl, carbophenothion, diazinon, ethion, malathion, mevinphos and parathion are extracted with acetonitrile and partitioned into hexane before analysis by the thin-layer chromatographic - enzyme inhibition technique. These pesti- cides, in the presence of apple, beet, carrot, lettuce, pea or potato extractives, are detected on thin-layer chromatographic plates coated with a silica-gel layer.The experiment indicates that the equivalent of only a few milligrams of the original sample is required for semi-quantitative analyses of residues present at, or above, their tolerance levels. THE rapid procedure for screening some organophosphorus pesticides by using thin-layer chromatography and total-phosphorus determination by the molybdenum-blue reaction pro- posed by Abbott, Burridge, Thomson and Webb1 was an improvement over the other procedures summarised in their paper. The analysis, although quantitative, is most suitable for plant materials with known pesticide treatment. Because the spots are not visible, the resolution of a mixture of pesticides must be greatly improved to ensure that the correct areas are scraped off for analysis.Ott, Hearth and Gunther2 reported another rapid thin-layer chromatographic screening procedure, but only for parathion in canned peaches without clean-up. In our laboratory, an enzyme-inhibition technique, in which indoxyl and substituted indoxyl acetates were used with steer-liver homogenate, was developed to detect nanograms of organophosphorus pesticide standards resolved by thin-layer chr~matography.~ Because of its high degree of sensitivity, reproducibility and rapidity of detection, we developed this method to screen for organophosphorus pesticides in plant extracts without elaborate clean-up. The loss or degradation of pesticides incurred by storing the plant extracts at room temperature, and in the cold, was determined by thin-layer chromatography. The efficiency of extracting and partitioning the pesticides into hexane was qualitatively evaluated by thin- layer chromatography in which the capacity of the plates to resolve the pesticides in the presence of plant extractives was evaluated by spotting various amounts of extracts.Various aliquots from each extract were concentrated and analysed by thin-layer chromatography to find the most convenient and simple step for screening organophosphorus pesticide residues. EXPERIMENTAL PESTICIDE STANDARDS*- Purity is expressed in percentages and Canadian tolerance levels5 in p.p.m. A standard solution was prepared containing each of the following pesticides : Azinphos-methyl (Guthion)-93-0 per cent., 1.0 p.p.m.Carbophenothion (Trithion)-94.6 per cent., 0.50 p.p.m. Diazinon-95.8 per cent., 0.10 p.p.m. Ethion-95.0 per cent., 0.10 p.p.m. Malathion-99.5 per cent., 8-00 p.p.m. Mevinphos a-isomer (Phosdrin)-60-0 per cent ., 0.25 p.p.m. Parathion-98.8 per cent., 1.00 p.p.m. The mixed pesticide standard solution was prepared with hexane, with co-solvent acetone used for initial dissolution of azinphos-methyl only; 1 ml of this solution when added to a 8 SAC and the authors.174 [Analyst, Vol. 93 50-g plant sub-sample gave the tolerance levels5 of the seven organophosphorus compounds. Concentrations (w/v) were not corrected for impurities* in the standards. A typical chromatogram of the pesticide standard mixture is shown in Fig.1; spots were white on a blue background. GENERAL PROCEDURE- Extraction6-Extract a 50-g sub-sample with 150 ml of acetonitrile in a food blender at about 6000 r.p.m. for 5 minutes. Repeat the extraction, then filter the homogenate through coarse sintered glass under vacuum and rinse the glassware with acetonitrile. Evaporate the acetonitrile in the filtrate and rinsings with a rotary evaporator at 35" C until only the water phase remains. If frothing occurs, vigorously shake the filtrate with 50 to 100 ml of hexane and continue the evaporation. Partition6-Shake the water phase vigorously with 50 ml of hexane in a 250-ml separating funnel for 30 seconds; repeat the partition step twice. If three layers appear, evaporate this 3-layered mixture with the rotary evaporator; repeat the partition, continuing only when two layers separate.Combine the three 50-ml hexane fractions in a 200-ml volumetric flask; make up to volume with hexane and thoroughly mix the contents. Discard the water layer. Development of the thin-layer chromatographic plates-The previous paper3 outlined the procedures for preparing the liver homogenate and the 5-bromoindoxyl acetate spray solution. Spot the concentrates and standard mixtures on thin-layer chromatographic plates coated with a 450-p thick Kieselgel G-HR(R) layer. Chromatograph the plates with 20 ml of acetone diluted to 100 ml with hexane, Remove the plates when the solvent front reaches the 15-cm height previously marked. Air-dry the plates for 5 minutes, then evenly treat them with bromine vapour.Allow at least 20 minutes for the excess bromine to dissipate, then spray the plates with steer-liver homogenate diluted with 0 . 0 5 ~ Tris buffer, pH 8.32, until the gel is just thoroughly wet; leave them for 20 minutes at room temperature. Similarly, spray the plates with 5-bromoindoxyl acetate spray solution, and within a few minutes the sites of inhibited enzymes appear as white spots on a blue background. OUTLINE OF EXPERIMENT- Apple, beet, carrot, lettuce, pea and potato samples were divided into 50-g sub-samples, some of which were fortified with the seven organophosphorus pesticides at tolerance levels, and the analyses were carried out on sub-sample extracts refrigerated at about 9" C and on those left at room temperature, 23" to 26" C. Aliquots from the 200-ml hexane fractions were concentrated 20 times.The 4 and 10-ml aliquots of the extracts stored at room temperature were concentrated just before analysis. The 100-ml aliquots were concentrated soon after the partition step and were stored in the cold or at room temperature. The experiment on refrigerated, concentrated 100-ml aliquots, which was replicated once, consisted of control sub-samples, C, and sub-samples fortified after the partition step, F. For each plant sample, one C and two F sub-samples were analysed on duplicate thin-layer chromatographic plates 2 days after extraction. The experiment on extracts and on concentrated 100-ml aliquots left at room temperature consisted of sub-samples C, F and BE (fortified before the extraction step).For each plant sample, two of each of the C, F and BE sub-samples were analysed on duplicate plates 2 days after extraction. The two C and two F sub-samples were further analysed, also on duplicate plates, 5 and 8 or 9 days after extraction. Each sub-sample extract, without elaborate clean-up, was analysed with a gas - liquid chromatograph equipped with electron-capture detector, under the conditions used by McCully and McKinley' but with 190" to 195" C column temperature. RE s ULTS DETERMINATION OF THIN-LAYER CHROMATOGRAPHIC PLATE CAPACITY- The chromatograms of the concentrated aliquots equivalent to 100, 150 and 200 mg of samples (Fig. 2, upper plates, and Fig. 3) were slightly different from one another, but * The impurities were detected, even although they were a small percentage of the standard, because they were probably more potent inhibitors than the parent compound.On the other hand, mevinphos gave only one spot although the other insecticidally active related compound was 40 per cent. MENDOZA et al. : THIN-LAYER CHROMATOGRAPHIC - ENZYME-Carbophenothion -Ethion -Diazinon -Parathion -Malathion i m pu rity -Malathion -Carbophenothion impurity -Azin phos-methyl -Parathion impurity -Mevi n phos Fig. 1. A typical chromatogram of the seven organophosphorus pesticides represented by white spots on an intense blue background' [To face p. 174150-mg sample equiv. wt. 5-Bromoindoxyl acetate pH 8.32 20% acetone in hexane I 2 3 4 5 6 7 C I F2 C I F2 C I F2 Mix Carrot Apple Lettuce 1 2 3 4 5 6 7 C I F2 C I F2 C I F2 Mix Pea Potato Beet 500-mg sample equiv.wt. 5-bromoindoxyl acetate pH 8.32 20% acetone in hexane I 2 3 4 5 6 7 I 2 3 4 5 6 7 C I F2 CI F2 C I F2 Mix C I F2 C I F2 CI F2 Mix Carrot Apple Lettuce Pea Potato Beet Fig. 2. Top, thin-layer chromatograms of six plant extracts (fortified and control), each spot equivalent to 150 mg of sample; extracts stored in a refrigerator 4 to 12 days before. Bottom, thin-layer chromato- grams of the same six plant extracts, each spot equivalent to 500 mg of sample. Silica-gel layer, 450 p thick; steer-liver homogenate and 5-bromoindoxyl acetate spray solutions, pH 8.32 ; solvent system, 20 ml of acetone diluted to 100 ml with hexane. (“Mix” means pesticide standard mixture)Carrot Lettuce 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 Sample 0-10 C.15 0.20 0.20 Std.0.10 0.15 0.20 Sample 0.10 0.15 0.20 0.20 Std. 0.10 0.15 0.20 Wt. (d Control wt. (g) Con tro I Apple Beet 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 Sample 0.10 0.15 0.20 0-20 Std. 0.10 0.15 0.20 Sample 0.10 0.15 0.20 0.20 Std. 0.10 0.15 0-20 wt. (g) Control Wt. (4 Control Potato Pea 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 Sample 0.10 0.15 0-20 0.20 Std. 0.10 0.15 0.20 Sample 0.10 0.15 0.20 0-20 Std. 0.10 0.15 0-20 ’Vt. (d Con tro I Wt. (g) Con tro I Fig. 3. Thin-layer chromatograms of six crops fortified a t the tolerance levels with seven organo- phosphorus pesticides, developed plates coated with 450-p thick silica-gel layer and sprayed with steer-liver homogenate and 5-bromoindoxyl acetate solutions at pH 8.32. Solvent system was 20 ml of acetone diluted to lOOml with hexane.(“Std.” means pesticide standard mixture)March, 19681 INHIBITION PROCEDURE TO SCREEN FOR ORGANOPHOSPHORUS PESTICIDES 175 markedly different from the chromatograms of the 500-mg samples (Fig. 2, lower plates). All seven organophosphorus pesticides and the enzyme-inhibiting impurities in the standards produced clearly delineated spots. The mevinphos spot from the 100-mg sample faded a few minutes after development ; otherwise, there was no marked difference among the chroma- tograms of the 100, 150 and 200-mg samples. However, separation of the seven pesticides was seldom as distinct in the 500-mg samples (Fig. 2, lower plates) as in the 100, 150 and 200-mg samples (Fig. 3). Pesticides added to carrot, lettuce, apple, beet and pea samples produced well defined spots on thin-layer chromatographic plates.Only carbophenothion and ethion in potato were clearly separated; spots for compounds below ethion were obscured by the streaking effect of the potato components. In general, the spots caused by the organophosphorus pesticides were readily distinguished from those of the plant components. Some areas of enzyme inhibition in the controls were attributable to components in the plant but most disappeared a few hours after develop- ment. Most plant pigments disappeared when brominated, and the remainder did not interfere with the detection of pesticides. In some chromatograms, the gel layer was weakened along the path travelled by plant components; the gel was blown off when sprayed with the homogenate and substrate solutions.Extracts of beet, carrot and lettuce (Fig. 2) weakened the layer most. However, the weakened area did not extend above the position for malathion, even with the 500-mg samples. EFFECT OF STORAGE- The chromatograms of the 2-day old concentrated aliquots left at room temperature (23" to 26" C) did not differ from those of aliquots stored in the cold (about 9" C) for various lengths of time and from the corresponding standard solutions. Storage periods were 30 and 43 days for apple, 26 and 32 days for beet, 23 and 29 days for carrot, 31 and 46 days for lettuce, 30 and 36 days for pea, and 31 and 52 days for potato. Degradation of some plant pigments occurred but did not affect the detection of the added pesticides. Extracts stored in the original and concentrated states at room temperature gave similar thin-layer chromatograms 2, 5 and 8 or 9 days after extraction.The quality of detection of pesticides remained the same, indicating no apparent change in the concentration of pesticides during the storage, although carrot, lettuce and pea extracts showed some pigment degradation. SELECTION OF ALIQUOT SIZE- The chromatograms of 30p1 from concentrates of 4, 10 and 100-ml aliquots of each of the crop extracts (equivalent to 1, 2.5 and 25 g of the crop) did not show any apparent difference among aliquots; the quality of the spots corresponding to the added pesticides did not vary from that of the standards. The 10-ml aliquot was the most convenient; the volume of the solvent required to re- dissolve the residue of the 4-ml aliquot was small and difficult to handle, and the several evaporating and transferring steps required for the 100-ml aliquot were tedious.EFFICIENCY OF EXTRACTION- The chromatograms of BE and F sub-samples suggested no loss of organophosphorus pesticides during the extraction with acetonitrile, flash-evaporation of acetonitrile and partition into hexane. GAS - LIQUID CHROMATOGRAPHY, WITH ELECTRON-CAPTURE DETECTOR, OF EXTRACTS WITHOUT An equivalent of 0.25mg of each plant sample ( 5 4 taken from the 200-ml hexane extract) gave the chromatogram shown in Fig. 4. The pesticides at concentrations simulating tolerance level residues in plant materials gave peaks too disproportionate for simultaneous screening by gas - liquid chromatography.Malathion, carbophenothion and parathion showed as distinct peaks but the malathion peak was off-scale. The peaks for ethion and azinphos- methyl were too low for proper measurement; mevinphos and diazinon were not detected. In addition, constituents or contaminants in some plant extracts interfered with the detection. FURTHER CLEAN-UP-176 MENDOZA et al. : THIN-LAYER CHROMATOGRAPHIC - ENZYME [AnaZyst, Vol. 93 I...,.,,.. 0 4 8 12 16 3 w O --I 0 4 8 I 6 -'-26 30 34 Time, minutes A = Malathion B = Parathion C = Carbophenothion D = Ethion E = Azinphos-methyl Fig. 4. Chromatograms (gas - liquid chromato- graph equipped with electron-capture detector) of pesticide standard mixture and 6 fortified plant extracts. (Mevinphos and diazinon peaks are not showing and others are very disproportionate) DISCUSSION AND CONCLUSION A sensitive and reproducible detection of the seven organophosphorus pesticides in samples equivalent to 150 mg was obtained by using thin-layer chromatographic plates with a 450-p thick silica-gel layer.The plates were sprayed with steer-liver homogenate, followed by 5-bromoindoxyl acetate spray solution, both at about pH 8-32. The sample, equivalent to 150 mg, used for thin-layer chromatographic analysis was most conveniently obtained from a 10-ml aliquot of the extract in hexane concentrated to 0.5 ml. From the chromatograms of the seven organophosphorus pesticides in the presence of plant extractives, gas - liquid chromatography with electron-capture detector was shown, under the conditions used, to be unreliable for screening purposes.GENERAL COMMENTS- To a certain extent, the application of this method to the screening for pesticides in crops of unknown history is perhaps based on whether the enzyme inhibitor is present or not (see pea control), whether the amount of the inhibition may be attributed to the pesticides or not (see apple and potato controls), and whether the intensity of the inhibition may be interpreted as caused by the presence of pesticides at questionable levels or not (see carrot, lettuce and beet controls; the quality of inhibition will not correspond to malathion tolerance level, if it is malathion). In this experiment, some expediency for semi-quantitative analysis was demonstrated by spotting a fraction rather than the entire aliquot.Based on the lowest limit of detection of individual pesticides by this procedure, a fraction of an aliquot may be selected to detect only that pesticide present at, or above, the tolerance level. Thus, a pesticide tolerated atMarch, 19681 INHIBITION PROCEDURE TO SCREEN FOR ORGANOPHOSPHORUS PESTICIDES 177 relatively high levels in foods (e.g., malathion) or a strong inhibitor (e.g., parathion) can be detected by thin-layer chromatography of aliquots that are too small for the detection of other pesticides present at their tolerance levels. This procedure is readily adaptable to routine analysis of the compounds and crops studied. As solutions can be stored for at least 8 days at room temperature, or at least 23 days in the cold, several samples may be extracted before simultaneous analysis by thin-layer chromatography; solutions showing questionable residues may then be re-analysed for confirmation. We propose that this procedure be used to screen for any of the seven organophosphorus pesticides studied in apple, beet, carrot, lettuce, pea or potato samples. Reduction of sub- sample size to reduce operating time further and extension of the scope to cover more pesticides, crops and developing systems will be investigated. Acknowledgement is made to Dr. K. A. McCully and Dr. T. K. Murray for review and criticism, and to Mr. H. Baird and Mr. B. Korda for photography. REFERENCES 1. 2. 3. 4. 6. Abbott, D. C., Burridge, A. S., Thomson, J., and Webb, K. S., Analyst, 1967, 92, 170. Ott, D. E., Hearth, F. E., and Gunther, F. A., Bull. Envir. Contam. Toxicol., 1966, 1, 175. Mendoza, C. E., Wales, P. J., McLeod, H. A., and McKinley, W. P., Analyst, 1968, 93, 34. Kenags, E. E., Bull. Ent. SOC. Amer., 1966, 12, 161. “1954 Office Consolidation of the Food and Drug Act and of the Food and Drug Regulations with Amendments t o July 14, 1966,” issued by Department of National Health and Welfare, Queen’s Printer and Controller of Stationery, Ottawa, Canada. 6. McLeod, H. 1967, 50, A., Mendoza, C. E., 1216. wale‘s; P. J., and McKinley, W. P., J . Ass. Off, Analyt. Chem., 7. McCully, K. A., and McKinley, W. P., J . Ass. Off. Agric. Chem., 1964, 47, 662. Received July Sth, 1967