H

Fig. 5.8 Analysis of a Narcissus extract by HPLC coupled to the MS-based AChE assay. MS instrument: Q-T0F2 (Waters) equipped with a Waters Z-spray electrospray (ESI) source. (a) Total ion current (TIC); (b) product trace (choline) m/z 104; (c) substrate trace (acetylcholine) m/z 146; (d)

system monitoring compound (SMC) detected at m/z 113; (e-j) MS traces, MS and MS/MS spectra for bioactive compound detected at an elution time of 33.5 min; (k) MS spectrum of active peak at t = 33 min; (l) MS/MS spectrum of galanthamine.

peak in the SMC trace, excluding ion suppression and thus indicating that these peak was caused by AChE inhibitory activity. A mass spectrum of the peak area was recorded (Fig. 5.8k), and the extracted ion chromatograms of the most prominent m/z values were constructed (Fig. 5.8e-h). Of these extracted ion chromatograms only the extracted ion chromatogram of m/z of 288 showed a peak matching with the activity peak. Although other compounds having an m/z of 288 eluted from the column, they did not show any sign of activity in the acetylcho-line and choline traces. The m/z of 288 corresponded with the calculated M+H+ of galanthamine. The MS/MS spectrum of galanthamine is presented in Fig. 5.8l. From the TOF-MS/MS data from the extract, extracted ion chromatograms of the major m/z values, 198 and 213, present in the MS/MS spectrum of galanthamine were constructed (Fig. 5.8i, j). Both extracted chromatograms of these m/z values showed a peak matching the activity peak indicating that indeed galanthamine is responsible for the AChE inhibitory activity present in the Narcissus extract. Albeit to a lesser extent, these daughter ions also showed peaks at the same retention time as the peaks present in the extracted ion chromatogram of m/z 288. This indicates that the other masses may be derivatives of galanthamine that fragment during ionization. The amount of galanthamine was determined by connecting the HPLC system to the MS. Performing an MS/MS experiment using the height of the daughter peak m/z 288 ! 198 to quantify galanthamine, it was established that 1 mM galanthamine was present in the 50 x diluted crude extract that was injected in the screening assay.

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