Effect of bromine oxidation on high-performance thin-layer chromatography multi-enzyme inhibition assay detection of organophosphates and carbamate insecticides

Following high-performance thin-layer chromatography, thiophosphate pesticides, which inhibit choline esterases, are detectable using a multi-enzyme inhibition assay (HPTLC-EI) based on rabbit liver esterase (RLE), Bacillus subtilis (BS2) esterase, or cutinase (from Fusarium solani pisi). Because choline esterase inhibition is more effective after conversion of thiophosphate thions into their corresponding oxons, a pre-oxidation step was added to the HPTLC-EI assay. Bromine vapour was found to be more effective than iodine or UV irradiation for oxidation. Following oxidation, the inhibitory strength of parathion, parathion-methyl, chlorpyrifos, chlorpyrifos-methyl, and malathion, expressed as HPTLC enzyme inhibition factors (fi), increased by approximately 2 orders of magnitude. In contrast, bromine oxidation of organophosphate and carbamate insecticides resulted in a slight reduction in their inhibition factors, due to partial bromination and degradation of the parent compounds, while bromine oxidation increased the inhibition factors for demeton-S-methyl and propoxur. Apple juice and water samples spiked with paraoxon (0.001 mg/L), parathion (0.05 mg/L), and chlorpyrifos (0.5 mg/L) were used to test the HPTLC-EI system, resulting in mean recoveries of 95–106% and 91–102% for RLE and cutinase, respectively.