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-µ 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 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 1 ng of parathion; 2ng of carbophenothion; 5ng of azinphos-methyl, diazinon, ethion, malathion and parathion-methyl; 20 ng of carbaryl, Trithion®-methyl and mevinphos; and 100ng 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 1-naphthyl acetate as substrate, and with bovine and sheep sera as sources of esterase.