34 Analyst, January, 1968, Vol. 93, $$. 34-38 Enzymatic Detection of Ten Organophosphorus Pesticides and Carbaryl on Thin-layer Chromatograms: An Evaluation of Indoxyl, Substituted Indoxyl and 1-Naphthyl Acetates as Substrates of Esterases BY C. E. MENDOZA*, P. J. WALES, H. A. McLEOD AND W. P. McKINLEY (Research Laboratories, Food and Drug Directorate, Departmest of National Health and Welfare, Ottawa 3 Ontario, Canada) An enzymatic inhibition method sufficiently sensitive and reproducible for detecting ten organophosphorus pesticides and carbaryl resolved by thin-layer chromatography is described. Reproducible detection of nanogram amounts of these pesticides is achieved with a 450-p thick gel layer, steer-liver homo- genate as source of esterase, and indoxyl or substituted indoxyl acetates (5-bromoindoxyl, 5-bromo-4-chloroindoxyl and 5-bromo-6-chloroindoxyl acetates) as substrates, the esterase and substrate spray solutions being used at a pH of about 8.The coloured products of enzymatic hydrolysis of these substrates are stable and intense ; white spots indicated the sites of pesticides that inhibited the enzyme. Spots persist for days when the amounts present are >l ng of parathion; >2ng of carbophenothion ; > 5ng of azinphos-methyl, diazinon, ethion, malathion and parathion-methyl; 2 20 ng of caxbaryl, Trithiona-methyl and mevinphos ; and > 1 OOng of disulfoton. Carbophenothion and disulfoton are also detected at the 1 ng level and carbaryl, at 5 ng; however, the spots produced disappear within a few hours. Unsatisfactory results are obtained with l-naphthyl acetate as substrate, and with bovine and sheep sera as sources of esterase.ENZYME inhibition reactions have been applied in paper and thin-layer chromatography to detect some organophosphorus pesticides1 s2s3s4 and ~arbamates.~ The substrate l-naphthyl acetate was used with bovine-liver esterase in paper chromatography to detect organophos- phorus pesticides,l s2 and indoxyl and 1-naphthyl acetates were tested with re-constituted dried horse-blood serum in thin-layer chromatography. A change in pH was the criterion used in another method4 to detect cholinesterase-inhibiting insecticides on thin-layer chromato- graphic plates coated with cellulose. Acetylcholine chloride was used as the substrate for human plasma esterase, with bromothymol blue as the pH indicator.Several light re-spray- ings of substrate - indicator solution gave maximum definition of spots; nevertheless, the spots remained unstable. Non-enzymatic methods utilising the reaction of specific moieties in pesticides were even less sensitive than the enzymatic methods in detecting carbay15 and organophosphorus This study was initiated because of the problems we encountered with the l-naphthyl acetate reaction, problems reported with the pH rneth~d,~ and because of the insensitivity of the non-enzymatic methods.6s6s7,* s 9 ~ 1 O ,l1 Other investigators used indoxyl acetate in colori- metry12 and fluorimetryls of cholinesterases and substituted indoxyl acetates in histochemistry of esterases.14 Thus we evaluated indoxyl, substituted indoxyl and l-naphthyl acetates as substrates for the enzymatic detection of ten organophosphorus pesticides and carbaryl ; the factors investigated were the amount of pesticides, pH of esterase and chromogenic solutions, thickness of silica-gel layers and sources of esterases.0 SAC and the authors. p&icides.6,7 9 8 ,9 ,10,11 * National Research Council of Canada postdoctorate fellow, 1965-67.MENDOZA, WALES, MCLEOD AND MCKINLEY 35 EXPERIMENTAL APPARATUS- Chromatographic tanks, 28 x 7 x 26 cm. Glass plates, 20.5 cm2. Sfway-bottles, 50-ml round tyee, No. 130.01 or 130.02-Obtainable from Research Special- Vir Tis@ or all-glass tissue homogenisers. Thin-layer applicator and accessories-Obtainable from Desaga, Heidelberg, West Microsyringes with square-tip needles, 25-pLObtainable from The Hamilton Co., Inc., Bromine $ask-A I-litre Erlenmeyer flask fitted with a wash-bottle fl ow-tube was used.ties Co., Berkeley 7, California, U.S.A. Germany. Whittier, California, U.S.A. REAGENTS- Indoxy1 acetate. 5-Bromoindoxyl acetate. 5-Bromo-4-chloroindoxyl acetate. 5-Bromo-6-chloroindoxyl acetate. These chemicals are obtainable from Sigma Chemical Co., St. Louis 18, Missouri, U.S.A. PESTICIDE STANDARDS- The names of the pesticides used are those approved by the Committee on Insecticide Terminology of the Entomological Society of America.16 Purity is expressed as a percentage. Azinphos-methyl (Guthion), 93.0 per cent. Carbaryl, 99.7 per cent. Carbophenothion (Trithion), 94.6 per cent. Diazinon, 95.8 per cent.Disulfoton (Disyston), 96.8 per cent. Ethion, 95.0 per cent. All of the pesticides were dissolved in hexane, with co-solvent acetone for azinphos- methyl, carbaryl and parathion-methyl, and all of the solvents were glass-distilled. Weight per volume concentrations of pesticides were prepared so that 10-pl aliquots contained the amounts given in Table I, but were not corrected for impurities in the standards. Malathion, 99-5 per cent. Parathion-methyl, 99.5 per cent. TrithionB-methyl, 99.0 per cent. Mevinphos a-isomer (Phosdrin) , 60.0 Parathion, 98-8 per cent. per cent. PROCEDURES- Prefiaration of liver homogenate-Use livers from 1 to 2-year old steers, and keep the livers and the glassware in an ice-bath while preparing the homogenate. Grind liver tissues devoid of vascular vessels and membranes, and homogenise 3 g of the ground liver with 27 ml of water in an all-glass homogeniser, or 20 g of ground liver with 180 ml of water in a VirTis@ homogeniser. Spin the combined homogenate in a centrifuge at 2000 r.p.m.for 20 minutes at room temperature, and collect and transfer the supernatant liquids into 13 x 100-mm test- tubes. Seal the tubes with plastic sheet and quickly freeze them in an acetone - dry ice bath; store the tubes at -20" C. Before using the homogenate for pesticide detection, test for esterase activity; thin-layer chromatographic plates sprayed with homogenate should turn intense blue when treated with the 5-bromoindoxyl acetate spray solution. Preparation of 1-naphthyl acetate spray solution-Prepare a solution as described by McKinley and Johal,2 or as outlined by McManus and Mowry,lG modifying the latter as follows.Dissolve 1 g of naphthyl acetate in 50 ml of acetone and add 50 ml of water (solution A). This solution keeps indefinitely in a refrigerator. Prepare, shortly before use, a solution of 20 mg of Fast blue RR in 15 ml of phosphate buffer solution and add 15 ml of water (solution €3). Mix 2 ml of solution A with solution B just before spraying. Preparation of indoxyl and substituted indoxyl acetates spray solution-Dissolve the sub- strate (15mg of indoxyl acetate, 15mg of 5-bromoindoxyl acetate, 1Omg of 5-bromo-4- chloroindoxyl acetate or 10 mg of 5-bromo-6-chloroindoxyl acetate) in 5 ml of absolute ethanol before use (solution C).36 MENDOZA, WALES, MCLEOD AND MCKINLEY [Analyst, Vol.93 Mix 4-0 ml of 0-05 M tris(hydroxymethy1)aminomethane buffer solution, 5.0 ml of 2.00 M sodium chloride, 0.2 ml of 1-00 M calcium chloride and 3.8 ml of water to make 13 ml of solution, or proportionately for a large volume (solution D). Prepare a solution of potassium hexacyanoferrate(II1) and hexacyanoferrate(I1) , 0.05 M with respect to each (solution E). Mix, immediately before spraying, 13ml of solution D with 2ml of solution E and thoroughly mix the resulting solution with solution C; this 20-ml spray solution is sufficient for two plates. Thin-layer chromatographic $Zates-Shake thoroughly for 1 minute a mixture of MN- Kieselgel G-HR@ and water (1 + 2 w/v) in a stoppered flask and spread the slurry 250 or 450-p thick on acetone-rinsed glass plates.As soon as the layer gels, place the plates in a vertical position in an oven at 110" C for 1 hour; store the plates in an air-tight box. Before spotting, scrape the gel off the edges of the plate, about 1-5cm from the top, 0.3 cm from the bottom, and 0.5 cm from the sides, to ensure an even solvent front and to prevent loss of gel when in contact with the solvent. With a 25-4 syringe apply the pesticides to the plate 2.5 cm from the bottom of the gel. Spot a constant volume (10 p1) of different concentrations to evaluate the substrates, various amounts of pesticides, pH values of spray solutions and two thicknesses of the gel layer. Development o j the plntes-Equilibrate each tank with 100 ml of 15 per cent. acetone in hexane for about 10 minutes.Chromatograph the plates in this tank until the solvent front reaches the line previously drawn 15 cm from the origin. Air-dry the plates for about 5 minutes and expose them to bromine; yellowing of the plates indicates over exposure. Use a gentle flow of nitrogen to push the bromine cloud out of the storage flask. Bromine converts the thiophosphates into active inhibitors and, as it may inhibit the enzyme, allow the excess to dissipate for 20 minutes. Spray the appropriate enzyme solution gradually and evenly over the plates, making the gel just thoroughly wet. Allow the plates to dry for 20 minutes at room temperature. Spray the chromogenic substrate solution in the same manner; the colour develops in 1 to 30 minutes. RESULTS AND DISCUSSION SENSITIVITY TEST BY THIN-LAYER CHROMATOGRAPHY- Table I shows the amounts of carbaryl and ten organophosphorus compounds enzymati- cally detected on a 450-p thick silica-gel layer, with steer-liver homogenate (1 volume) in 0.05 M tris(hydroxymethy1)aminomethane buffer solution (pH 8-32) (2 volumes) and 5-bromoindoxyl acetate spray solution made with the same buffer solution.Although 1 ng of carbaryl, carbophenothion, disulfoton, and parathion were tested, the smallest amount studied for the other pesticides was intentionally limited to 5 ng. Spots persisted when the amounts present TABLE I SENSITIVTTY TEST ON THIN-LAYER CHROMATOCRAMS WITH 5-BROMOINDOXYL ACETATE AND ESTERASES FROM STEER LIVER* Amount spotted, ngt A I 3 Chemicals . . . . 800 400 200 100 80 50 20 10 8 5 2 1 Azinphos-methyl Carbophenothion Diazinon ..Disulfoton . . Ethion . . .. Malathion . . Parathion-methyl Trithion@-meth yl Mevinphos .. Parathion . . Carbaryl .. * (+) = detected: (u) = unstable or spot disappeared after a few minutes; (0) = not detected; t Constant volume (10 pl) was used for each spot. (-) = not tested.Fig. 1. The eHcct of thickness of silica-gel layer on the detection of 10 organophosphorus pesticides (left, 450 p, and right, 250 p), with 5- bromo-4-chloroindoxyl acetate as substrate for steer-liver esterase, and both spray solutions a t pH 8.32. (Phosdrin is mevinphos; Trithion, carbophenothion ; Disyston, disulfoton ; Guthion, azinphos-methyl. Labels on the bottom of the plates: top row = micrograms of pesticide standards multiplied by 1 x l O F , e.g., Spot No.1 is represented by 8 x pg of mevinphos; b , bottom row = spot numbers.) [To face p. 36A B C Fig. 2 . Thin-layer chromatogram with five substrates for steer-liver esterase to detect organophosphorus pesticides, with 450-p thick silica-gel layer. (Labels on the bottom of each thin-layer chromatographic plate : top row = micrograms of pesticide standards multiplied by 1 x e.g. Spot No. 1 (A) is represented by 2 x 10-2pg of Guthion; bottom row = spot numbers. Thin-layer chromatographic plates at right side: A, top plate, was sprayed with 5-bromoindoxyl acetate; B, middle plate, was sprayed with 5-bromo-4-chloroindoxyl acetate, at pH 8.32 ; C, bottom plate, was sprayed with 1-naphthyl acetate solution at pH 7; Spot Xos. 1 , Phosdrin, i.e., mevinphos ; 2, malathion ; 3, diazinon ; 4, Trithionm-methyl; 5, Trithion or carbophenothion; 6, Disyston or disulfoton; 7, ethion; 8, parathion; 9, methyl parathion ; 10, Guthion or azinphos-methyl) To fuce p.371January, 1968j ENZYMATIC DETECTION OF TEN ORGANOPHOSPHORUS PESTICIDES 37 were >l ng of parathion; 2 2 ng of carbophenothion; >5 ng of azinphos-methyl, diazinon, ethion, malathion, and parathion-methyl ; >20 ng of carbaryl, TrithionB-methyl and mevinphos ; and >lo0 ng disulfoton. Carbophenothion and disulfoton were also detected at the 1 ng level and carbaryl, at 5 ng; however, the spots disappeared within a few hours. The levels of organophosphorus pesticides used in the subsequent experiments were based on this test and on the possibility of applying this method in pesticide residue analyses.EFFECT OF GEL THICKNESS- Intense backgrounds and well defined spots were consistently obtained when the gel layer was 450-p thick, but not when 250-p thick (Fig. 1). The substrates 5-bromoindoxyl and 5-bromo-4-chloroindoxyl acetates were used in this test. Each substrate solution was prepared with 0-05 ivi tris(hydroxymethy1)aminomethane buffer (pH 8-32), and liver homo- genate (1 volume) was diluted with the same buffer solution (2 volumes). CONPARISON OF INDOXYL AND SUBSTITUTED INDOXYL ACETATES- To compare the sensitivity of detection with indoxyl, 5-bromoindoxyl, 5-bromo-4- chloroindoxyl and 5-bromo-6-chloroindoxyl acetates, the same amounts of each organophos- phorus pesticide were chromatographed on 450-p thick silica-gel layers.Liver homogenate (1 volume) diluted with 0.05 M tris(hydroxymethy1)aminomethane buffer (pH 8-32) (2 volumes) and substrate solution, made with the same buffer solution, were sprayed on to thin-layer chromatographic plates. Indoxyl and substituted indoxyl acetates detected nanogram amounts of ten organo- phosphorus compounds and carbaryl resolved on thin-layer chromatograms (Fig. 2, top and middle plates). Indigo compounds produced by the hydrolysis of the substrates gave coloured backgrounds, leaving the sites of inhibition by pesticides as white spots; the colours produced were blue for indoxyl and 5-bromoindoxyl acetates, turquoise for 5-bromo-4-chloroindoxyl acetate and pink for 5-bromo-6-chloroindoxyl acetate.These backgrounds were stable and the spots lasted for months. 5-Bromo-4-ch1oroindoxy1, 5-bromoindoxyl and indoxyl acetates produced more intensely coloured backgrounds than 5-bromo-6-chloroindoxyl acetate ; an intense colour is preferred because it delineates more distinctly the sites of enzyme inhibition. Both indoxyl and Ei-bromo- indoxyl acetates produced a blue centre in the disulfoton spot; although this peculiarity is not understood, it may be used as a criterion for the presence of disulfoton. Unlike 5-bromo-4- chloroindoxyl acetate, 5-bromo-6-chloroindoxyl acetate produced well defined spots for mevinphos and diazinon, and it consistently gave stable spots for disulfoton. COMPARISON OF TWO PREPARATIONS OF ~-NAPHTHYL ACETATE SPRAY SOLUTION- The preparation of I-naphthyl acetate spray described here gave the same background intensity as that of McKinley and Johal.2 Both gave unsatisfactory analyses of ten organo- phosphorus pesticides at the levels detected by the indoxyl acetates.Spots corresponding to the organophosphorus compounds at nanogram levels were not clearly defined (Fig. 2, bottom plates) ; therefore, confirmation of pesticides based on these spots was not reliable. Unsatisfactory results were obtained when 1-naphthyl acetate was tested with two gel thicknesses, phosphate buffer solutions (pH 5.3 and 7.19), B.D.H. buffer solution (pH 7.0) and two homogenate dilutions (1 volume of homogenatej5hs 2 or 6 volumes of buffer solution2). 1-Naphthyl acetate was not tested at pH 8.32 because of its rapid hydrolysis to naphthol, which readily coupled with Fast blue RR.EFFECT OF PH- The pH values were chosen to cover the range 5 to 9 and the same tris(hydroxymethy1)- aminomethane buffer solution was used to make each pair of homogenate and substrate sprays. Solutions of 5-bromoindoxyl acetate made with 0-05 M tris(hydroxymethy1)amino- methane buffer were used with liver homogenate (1 volume) in the same buffer solution (2 volumes) on 450-p thick silica-gel layer. A t pH 8.32 and 9.10, 5-bromoindoxyl acetate gave intense and uniform blue backgrounds; the sites of enzyme inhibited by pesticides were white and well defined. However, at pH 7.19, this substrate produced a speckled background and indistinct spots. At pH 5-30, it exhibited very pale and uneven backgrounds; the sites of enzyme inhibition were either invisible or barely discernible.This enhancement of indigo dye production at higher pH agrees with previous findings.l5J7 Characteristic responses were observed for each substrate.38 MENDOZA, WALES, MCLEOD AND MCKINLEY SHEEP AND BOVINE SERA AS SOURCES OF ESTERASES- The sera (1 volume) were diluted with 0.05 M tris(hydroxymethy1)aminomethane buffer (pH 8-32) (1, 3, 4 or 6 volumes) ; 5-bromoindoxyl acetate solution was made with the same buffer solution. These sera were unsatisfactory for detecting the resolved pesticides because only minute amounts of indigo compound were hydrolysed from 5-bromoindoxyl acetate sprayed on thin-layer chromatographic layers, 450-p thick. Sera diluted with B.D.H. buffer solution (pH 7.0) likewise produced only faint backgrounds when used with the l-naphthyl acetate spray solution of McKinley and Johal.2 CAUSES OF, AND REMEDIES FOR, FAILURES IN THE DEVELOPMENT OF THE BACKGROUND Unreactive oxidising solution-Re-spray the plate with 2 ml of the oxidising solution diluted with 13 ml of 0.05 M tris(hydroxymethy1)aminomethane buffer (pH 8-32).Insuficient amount of enzyme on plate-Re-spray with homogenate solution, diluted as before. Two tubes of homogenate diluted as specified are sufficient to spray two plates. Insu$cient amount of szcbstrate on plate-Re-spray with the same substrate; 20 ml are usually adequate for two plates. Inadequate dissi$ation of excess of bromi.pze-This cannot be remedied. Thin gel Zayer-The gel layer should be about 450-p thick.Inadequate spray-bottle-That listed under Apparatus has a fine orifice and is recom- mended; sprayers that work solely on the creation of a vacuum around the orifice are un- satisfactory. CONCLUSION Ten organophosphorus pesticides and carbaryl can be detected with precision in nano- gram amounts by using indoxyl or substituted indoxyl acetates as substrate for steer-liver esterase. Spray solutions, with pH values not lower than 8, produced intensely coloured backgrounds and well defined spots; the intensity of the colours and the definition of the spots were enhanced by using a 450-p instead of a 250-p thick silica-gel layer. The technique used in this experiment is being developed as a method for the analysis of pesticide residues in plant materials. We are indebted to Mrs. C. R. Sherwood for technical assistance in certain phases of the work, to Mr. B. Korda and Mr. H. Baird for photography, and to Dr. K. A. McCully, Dr. J. B. Rogers and Dr. K. Murray for their critical review of the manuscript. COLOURS- 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. REFERENCES McKinley, W. P., and Read, S. L., J . Ass. Ofl. Agric. Chem., 1962,45, 467. McKinley, W. P., and Johal, P. S., Ibid., 1963,46, 840. Ortloff, R., and Franz, P., 2. Chemie, Lpz, 1965, 5, 388. Menn, J. J., and McBain, J. B., Nature, 1966,209, 1351. Chiba, M., and Morley, H. V., J . Ass. Ofl. Agric. Chem., 1964,47, 667. MacRae, H. F., and McKinley, W. P., Ibid., 1961, 44, 207. Kovacs, M. F., jun., Ibid., 1964, 47, 1097. Watts, R. R., Ibid., 1965,48, 1161. Stanley, C. W., J . Chromat., 1964, 16, 467. Barney 11, J. E., Ibid., 1965,20, 334. Irudayasamy, A., and Natarajan, A. R., Analyst, 1966, 90, 503. Gunther, J., and Ruff-Sondermeier, D., Nuturwisseraschajtera, 1965, 52, 540. Guilbault, G. G., and Kramer, D. N., Analyt. Chem., 1965,37, 120. Holt, S. J., irt Danielli, J. F., Editor, “General Cytochemical Methods,” Academic Press, Inc., New York, 1968, Volume 1, p. 376. Kenaga, E. E., Bull. Ent. SOC. Amer., 1966, 12, 161. McManus, J. F. A., and Mowry, B. W., “Staining Methods, Histologic and Histochemical,” Harper and Row, Publishers, New York, 1960, p. 172. Gehauf, B., and Goldenson, J., Analyt. Chem., 1957,29, 276. Received May 19th 1967