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

Abstract

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.

Pesticide residues and greedy scale control on kiwifruit

“…(1: 1) as developing solvent, and bovine liver homogenate, with 5-bromo-indoxylacetate substrate as indicator (Mendoza et al 1968).…”

High-performance thin-layer chromatography in combination with an acetylcholinesterase-inhibition bioassay with pre-oxidation of organothiophosphates to determine neurotoxic effects in storm, waste, and surface water

“…After chromatographic development, 10 mL of freshly prepared NBS (100 µg/mL) was sprayed onto the HPTLC plates with a glass reagent sprayer, followed by a 5-min incubation at room temperature. The following AChE-I assay was performed in parts according to Mendoza et al, Stütz et al, and Weins and Jork [ 20 , 22 , 42 ]. AChE was buffered in TRIS/HCl at a pH of 7.8 (2.5 U/mL).…”

“…Two methods, immersion and spraying, for the application of biosensors on HPTLC plates have been used in a study by Azadniya and Morlock [ 39 ]. Mendoza et al [ 20 ] and Stütz et al [ 23 ] used, as in this study, indoxyl acetate as an esterase substrate but each of them employed a different method to apply the esterase solution on thin-layer plates: immersion or spraying. In a previous work, an immersion and a spray method for application of yeast suspension on HPTLC plates were compared whereas a similar sensitivity and a better precision of the spraying approach were observed [ 40 ].…”

Carbaryl: A Literature Review

“…Limits of detectability were in the sub-nanogram level (WINTERLIN et al 1968). MENDOZA et al (1968) utilized enzymatic inhibition to detect carbaryl and organophosphate insecticides. Steer-liver homogenates served as the source of esterase and substituted or nonsubstituted indoxyl acetates served as substrate.…”