Multi-Enzyme Inhibition Assay for Detection of Insecticidal Organophosphates and Carbamates by High-Performance Thin-Layer Chromatography. 1. Basics of Method Development

A recently introduced microtiter-plate multienzyme-inhibition assay using rabbit liver esterase (RLE), Bacillus subtilis (BS2) esterase, and cutinase from Fusarium solani pizi has been successfully transferred to high-performance thin-layer chromatography. Paraoxon, malaoxon. and carbofuran as esterase inhibitors with high. medium. and low inhibitory activity, respectively, were used to optimize method performance with regard to enzyme concentration, incubation time, and time of immersion in alpha-naphthyl acetate-fast blue salt B substrate. For paraoxon as strongest inhibitor, limits of detection (LOD) of 1.3, 1.2, and 540 pg per band were determined using RLE, BS2, and cutinase, respectively. Respective LODs were 7.9, 7.4, and 760 ng per band for malaoxon, and 33.54, and 1420 ng per band for carbofuran. With regard to the LODs of strong, medium, and weak inhibitors, the detectability range is favorably reduced for the low-sensitivity cutinase (0.54-1420 ng per band) whereas it was approximately 3 x 10(4) and 5 x 10(4) for RLE and BS2, respectively.