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

Area of Science:

• Analytical Chemistry
• Biochemistry

Background:

• Effect-directed analysis (EDA) is crucial for identifying toxic compounds in complex samples.
• Biochemical detectors in chromatography can suffer from reduced sensitivity due to band broadening.

Purpose of the Study:

• To develop and validate an on-line acetylcholinesterase inhibition assay for effect-directed analysis using reversed-phase high-performance liquid chromatography (RP-HPLC).
• To investigate methods for minimizing band spreading and enhancing detector sensitivity in flow-injection assays.

Main Methods:

• Coupling RP-HPLC with an on-line colorimetric acetylcholinesterase inhibition assay using Ellman's reagent.
• Utilizing glass capillaries for enzyme inhibition and substrate conversion at a flow rate of 100 µL/min.
• Implementing air-segmentation in knitted reactors to extend reaction times while maintaining chromatographic resolution.

Main Results:

• The system achieved limits of detection of 13 ng for carbofuran (carbamate) and 7.4 ng for paraoxon-ethyl (organophosphate) using isocratic elution.
• Gradient elution successfully separated a mixture of five insecticides, with inhibitory effects detected at high resolution.
• Air-segmentation and knitted reactors minimized band broadening, with the enzyme inhibition detector signal broadening by only a factor of 1.4 after 8 minutes of reaction time.

Conclusions:

• The developed on-line assay is effective for the effect-directed analysis of toxic samples, particularly insecticides.
• Air-segmentation is a viable strategy to improve sensitivity in biochemical flow assays without compromising chromatographic performance.
• This method offers a sensitive and high-resolution approach for detecting enzyme inhibitory compounds in environmental and biological samples.