Carbamates cholinesterase inhibition detection

L. Pogacnik and M. Franko, Optimisation of FIA system for detection of organophosphorus and carbamate pesticides based on cholinesterase inhibition. Talanta 54, 631-641 (2001). 

Tests for specific classes of chemicals include the measurement of transmitter stimulated adenyl cyclase and Na/K-ATPase for chemicals that affect receptor function or cholinesterase inhibition for organophosphates or carbamates. Electrophysiological techniques may detect chemicals such as DDT or pyrethroids, which affect impulse transmission. 

Certain specialized reagents are used to visualize compounds based on their biological activity. Cholinesterase-inhibiting pesticides (e.g., organophos-phates, carbamates) are detected sensitively by treating the layer with the enzyme and a suitable suljstrate, which react to produce a colored product over the entire layer except where colorless pesticide zones are located due to their inhibition of the enzyme-substrate reaction. 

Organophosphates, thiocarbamates, carbamates, carbamoyloximes, dithiocarbamates, and ureas were included among 100 pesticides and metabolites detected on TLC plates by their cholinesterase inhibiting properties. After developing the plates in ether, xylene, di-n-butyl ether, n-butyl acetate or methyl-isobutyl ketone, the plates were exposed to bromine vapor. The compounds were oxidized to their oxo derivatives, which exhibited more effective cholinesterase inhibitory activity. Bovine liver suspension served as enzyme source and the substrates 2-naphthyl acetate and Fast Blue B salt as the chromogenic agents (163a). 

Enzymatic techniques have also been employed in the analysis of these compounds. The toxicity of carbamate insecticides is due to the inhibition of the enzyme acetylcholine esterase, so the determination of these compounds can be achieved by enzyme inhibition (2,83,119), bioassay (118,167), or enzyme-linked immunosorbent assay (ELISA) (168-171). In the detection of carbamates by fluorimetric enzyme inhibition, the effluent from a reversed-phase chromatographic column was incubated with cholinesterase, which was introduced via a postcolumn reagent delivery pump. Then, the resulting partially inhibited cholinesterase was reacted with N-methyl indoyl acetate to produce a fluorophore and a reduction in the baseline fluorescence (172). 

Method. A diagram of the apparatus is shown in Fig.4.29. Any suitable liquid chromatograph may be used. The AutoAnalyzer is modified such that the liquid sampler is fitted to the end of the chromatographic column. The proportioning pump is by-passed. The set-up of the AutoAnalyzer is the same as that for normal measurements of cholinesterase. The application of this technique to the determination of CGA 18809 in plum-leaf extract is shown in Fig.4.30. A comparison is made with UV analysis of the same extract. The limit of detection for CGA 18809 is c . 20 ng at a 3 1 signal to noise ratio. The relative inhibitions of several organophosphates and carbamates are compared in Table 4.9. Diazoxon may be detected in low picogram quantities. 

The determination of pesticides has become increasingly important in recent years because of the widespread use of these compounds, which is due to their large range of biological activity and a relatively low persistence. The development of biosensors for pesticides is the subject of considerable interest, particularly in the areas of food and environmental monitoring. Several enzymes such as cholinesterase enzymes (AChE, BChE) and urease have been used in the design of direct electrochemical biosensors for the detection of pesticides (Larsen et al., 2007). Analytical devices based on the inhibition of cholinesterase have been widely used for the detection of organic phosphate compounds and carbamate pesticides. 

The cholinesterase electrode was constructed on this basis for the total determination of organophosphates and carbamates [32]. This electrode is also sensitive to other toxic compounds with very low detection limits (paraquat (3 ppm) trichlorophenol (20 ppm) methylazinphos (150 ppb) lindane (15 ppm) and mercury (4 ppm)) [135]. This type of biosensor uses enzymatic inhibition and can be incorporated in an automated system for the quality control of water, and, notably, waste water [291]. 

Basanta R, Nunez A, Lopez E, Fmiandez M, Diaz-Fierros F (1995) Measurement of cholinesterase activity inhibition for the detection of organophosphorus and carbamate pesticides in water. Int J Envirrai Stu 48 211—219... 

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