time {min)

Fig. 5.5 On-line HPLC bioactivity screening of a mixture of five flavonoids spiked with two cathepsin B inhibitors, E-64 and leupeptin using acetylcholinesterase as biological target. MS instrument: Shimadzu LCMS-2010 single-stage quadrupole mass spectrometer. (a) TIC chromatogram of the mixture, scan range m/z 75-750; (b) mass chromatogram ofAMC (m/z 176); (c) mass chromatogram ofZ-FR (m/z 456); (d) mass chromatogram of SMC1 (m/z 245); (e) mass chromatogram of SMC2 (m/z 330); (f) mass spectrum recorded at tR = 3.4 min; (g) mass spectrum recorded at tR = 7.5 min; (h) EICs of the most abundant peaks shown in the mass spectrum recorded at tR = 3.4 min; (i) EIC of the most abundant peaks shown in the mass spectrum recorded at tR = 7.5 min.

with the bioactive compound. The mass chromatogram of m/z 255 shows a mismatch in both retention time and peak profile and can be excluded from further data interpretation. The mass chromatograms of m/z 427 and m/z 459 correspond with [leupeptin + H]+ and [leupeptin + H + methanol]+. A similar scheme allowed the identification of negative peak at tR = 3.4 min, resulting in E-64 (m/z 358).

When using MS-based biochemical assays for the screening of complex mixtures, it is essential to perform control experiments to prove that the apparent reduction in product concentrations is, indeed, caused by inhibition of cathepsin B and not by ionization suppression. First of all, the mass chromatograms of the two SMCs (see Fig. 5.5d, e) show no negative peaks at the retention times of the two bioactive peaks, only at the dead time. In addition, we tested the behavior of the overall system in the absence of active enzyme. For this purpose, a solution identical in composition to the enzyme assay solution was used in the continuous-flow reaction detection system. This solution contained deactivated cathepsin B and the products AMC and ZFR. Injections of E-64 did not result in any decrease of the AMC and Z-FR signals, proving that the negative peaks measured under assay conditions, indeed, were the result of cathepsin B deactivation and not of ionization suppression of the AMC and Z-FR signals. Finally, it is important to mention that the peak shapes of the peaks in the AMC and Z-FR chromatograms are virtually identical. It is highly unlikely that ionization suppression due to the injection of E-64 would be identical for substrate conversion products. In conclusion, these control measurements unambiguously confirm that the decrease of product formation monitored by ESI-MS is, indeed, exclusively caused by the presence of cathepsin-B inhibitors in the HPLC effluent.

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