On-line coupling of high-performance liquid chromatography to a continuous-flow enzyme assay based on electrospray ionization mass spectrometry.

摘要: 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 concentration reaction products, indicating inhibition activity. The molecular masses determined with high certainty retention time matching and peak shape comparison. Due accuracy, baseline separation coeluting analytes not necessary order identify correct bioactive compounds. system successfully applied samples, such natural extracts. For red clover extract, detection limits 0.3-0.8 micromol/L obtained. System validation performed determining IC(50) values four flow-injection mode. 0.11-5.6 range correspond closely data obtained microtiter plate assays. Detection 0.018-0.35 mode, 0.075-0.75 LC These are well below typical compound concentrations (1-10 micromol/L) used high-throughput screening. Together an interday precision 12.6%, these results demonstrate applicability bioactivity mixtures, generating both chemical biological information on compounds single run.