The continuous-flow MS enzyme assay format allows the continuous screening of sample components present in a carrier flow for enzyme inhibition activity. This allows the integration of this assay format in a reversed-phase HPLC system, allowing the screening of complex mixtures after chromatographic separation. For this purpose, a reversed-phase C18 column was inserted between the autoinjector and the mixing union for the addition of the enzyme solution (see Fig. 5.2). Furthermore, a 1:3 flow splitter was placed between the HPLC column and the enzyme assay, resulting in a total flow of 50 mL min-1 directed toward the enzyme assay. The remaining 150 mL min-1 was directed toward waste; in other applications, this flow may be used for UV, fluorescence, or ELSD measurements to obtain additional chemical data on bioactive analytes.
To optimize the on-line HPLC enzyme assay setup, a mixture of five flavonoids spiked with two cathepsin B inhibitors, E-64 and leupeptin, was used as a test sample. Figure 5.5 shows the mass chromatograms obtained when analyzing this mixture by ESI-MS. Figure 5.5a shows the total ion current (TIC) chromato-gram of the test mixture, indicating that the TIC does not provide any information about the (bioactive) compounds injected. Figure 5.5b, c depicts the mass chromatograms of the products AMC (m/z 176) and Z-FR (m/z 456), respectively, reflecting the enzyme inhibition activity of compounds eluting from the HPLC column. The bioactive compounds E-64 and leupeptin cause a temporary decrease of the concentration AMC and Z-FR, detected as negative peaks. The peak heights depend on the concentration of the inhibitor and its binding affinity.
The process of compound characterization and identification is illustrated for the bioactive peaks with retention time (tR) 7.5 min. Figure 5.5g shows the mass spectrum recorded at 7.5 min, obtained after applying background subtraction. Figure 5.5i depicts the EIC chromatograms of the three most abundant masses, together with the EICs of AMC (m/z 176) and Z-FR (m/z 456), reflecting the bioactivity signal. The m/z 613 is not plotted as EIC, because this represents the protonated substrate. Since the active compounds enter the mass spectrometer synchronously with AMC and Z-FR, their identification can be performed on the basis of a retention time and peak shape match. On the basis of this matching, the mass chromatograms for m/z 427 and m/z 459 are identified to correspond tn 3,4 7.5
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