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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 homogenate 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 a t 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 a t the 1 ng level and carbaryl, a t 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 organophosphorus 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 chromatographic 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-sprayings of substrateindicator 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 r n e t h ~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.

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 chromatographicenzyme inhibition technique. These pesticides, 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.

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Ten different carbons were investigated as adsorbents in a column cleanup procedure for extracts of lettuce and carrots. Organochlorine pesticides were eluted with 175 nil of 1.5% acetonitrile in hexane. The quantitative elution of 13 pesticides as a mixture with and without the presence of carrot or lettuce extract was determined by gas-liquid chromatography (GLC) with electron capture detection (ECD). The mean recovery values for each pesticide from all carbons, excluding Norit A neutral, ranged from 87 to 106% with a coefficient of variation (C) of 3 to 11. In the presence of plant extracts the individual pesticide recoveries for 6 carbons ranged from 75 lo 99% with a C of 6 to 16. All pesticides from 9 individual carbons had mean recovery values ranging from 93 to 108% with a C of 5 to 10. In the presence of plant extracts these values were 81 to 100% with a C of 8 to 12. Four carbons (Darco G60, Darco S51, Nuchar C-190N, Norit FQP) were considered satisfactory in the cleanup procedure. The procedure was applied to quantitative elution of 42 pesticides representing insecticides, fungicides, and herbicides from a Darco G60-Solka Floe cleanup column. The mean per cent recoveries (3 determinations) ranged from 82 to 112 with C from 2 to 10 for 38 compounds. The mean per cent recovery of EPN was 108 with a C of 13. The per cent recoveries of three compounds were considered low: Thimet 72, Daconil 73, and Guthion 77. Twenty of the pesticides as three mixtures containing 2, 4, and 14 compounds were added to plant extracts of carrots, potatoes, wheat, and pineapples. The mean per cent recovery values ranged from 81 to 108 with a C range of 2 to 10 for 17 compounds