On-Line Coupling of High-Performance Liquid Chromatography to a Continuous-Flow Enzyme Assay Based on Electrospray Ionization Mass Spectrometry

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Abstract
Liquid chromatography (LC) was coupled on-line to a continuous-flow enzymatic assay using electrospray ionization mass spectrometry (ESI-MS) as readout for the screening of enzyme inhibitors in complex samples. Inhibitors were detected by changes in the concentration of the enzymatic reaction products, indicating the inhibition of enzymatic activity. The molecular masses of the inhibitors were determined with high certainty by using retention time matching and peak shape comparison. Due to the high matching accuracy, baseline separation of coeluting analytes was not necessary in order to identify the correct masses of the bioactive compounds. The continuous-flow system was successfully applied for the screening of complex samples, such as natural extracts. For a red clover extract, detection limits of 0.3−0.8 μmol/L were obtained. System validation was performed by determining the IC50 values of four inhibitors in the flow-injection mode. The IC50 values were in the 0.11−5.6 μmol/L range and correspond closely to data obtained by microtiter plate assays. Detection limits were in the range of 0.018−0.35 μmol/L in the flow-injection mode, and 0.075−0.75 μmol/L in the LC mode. These values are well below the typical compound concentrations (1−10 μmol/L) used in high-throughput screening. Together with an interday precision of 12.6%, these results demonstrate the applicability of the system for bioactivity screening of complex mixtures, generating both chemical and biological information on bioactive compounds in a single run.