Assay Validation and Stability

To test for assay stability, the assay was run overnight and every 80 min an injection of 5 mM galanthamine was performed. The assay proved to be stable for over 13 h, but was terminated when maximum data file size was reached. Subsequently, IC50 curves of galanthamine were recorded by injection of 10 mL galanthamine at a concentration of 0, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, and 25 mM. IC50 values were calculated using product peak height as a measure of inhibitory activity. Six galanthamine IC50 curves were recorded over a period of 14 days and the interday variation as calculated from the determined IC50 values was 16.2%.

IC50 curves were also recorded for various other inhibitors and the corresponding IC50 values were calculated. The determined IC50 values of 9-aminoacridine, galanthamine, gallamine, (—)-Huperzine A and thioflavin T were 0.12 mM, 0.38 mM, 6.4 mM, 0.46 mM, and 3.2 mM, respectively. It was difficult to compare these values with comparable values in literature since often different types of AChE, or AChE isolated from different organisms were used and different assay conditions were applied. However, the relative activities of the used inhibitors compared well with those reported in literature, except for (—)-huperzine A, which was found to be relatively less active then was expected.

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