Effect-directed analysis by high-performance liquid chromatography with gas-segmented enzyme inhibition

Abstract

A reversed-phase high-performance liquid chromatography system with UV-detector was equipped with an on-line acetylcholinesterase inhibition assay to achieve effect-directed analysis of potentially toxic samples. The enzyme activity was detected colorimetrically using Ellman's reagent. The inhibition and substrate conversion took place in glass capillaries at a 100 μL/min flow rate. Extra-column band spreading in the reaction coils reduces the sensitivity and separation power of biochemical detectors severely. Knitted reactors exhibited no reduction of longitudinal dispersion in the tested flow range. The implementation of air-segmentation allowed an extended inhibition and substrate conversion time without a significant loss of chromatographic resolution. The limit of detection of two model compounds carbofuran (carbamate) and paraoxon-ethyl (organophosphate) was determined to be 13 ng (injected mass) and 7.4 ng, respectively, applying an isocratic chromatography method. A mixture of five insecticides was separated by a gradient elution and the inhibitory effect on the enzyme activity could be detected with high resolution. The band width at half height of the enzyme inhibition detector signal after a reaction time of about 8 min or 4.2 m of capillary, respectively, increased only by a factor of 1.4 compared to the UV-detector signal.