Simultaneous separation, identification and activity evaluation of three butyrylcholinesterase inhibitors from Plumula nelumbinis using on-line HPLC-UV coupled with ESI-IT-TOF-MS and BChE biochemical detection.

We have firstly established a method of high-performance liquid chromatography-ultraviolet analysis coupled with electrospray ionization-ion trap-time-of-flight mass spectrometry and butyrylcholinesterase biochemical detection (HPLC-UV-ESI-IT-TOF-MS-BChEBCD). Applying this on-line method to the identification of BChE inhibitors in a Plumula nelumbinis sample, three alkaloids, namely liensinine, isoliensinine, and neferine, have been detected as having a strong BChE inhibition activity for the first time; in addition, norisoliensinine and 6-hydroxynorisoliensinine were proposed as two new compounds identified by their UV and MS data. The HPLC fingerprint, the MS fragments of the components, and the BChE activity profile could be simultaneously recorded during real-time analysis of complex samples using this on-line approach. Tacrine, a BChE inhibitor, was used as a positive reference compound, and its detection limit in the biochemical detection system was 1 nmol. The BChE activity of 1g of P. nelumbinis sample was equal to that of 127.88 μmol tacrine. The proposed on-line method has been validated as having good precision and reproducibility, and could be used to rapidly identify BChE inhibitors and to screen potential drugs for the treatment of Alzheimer's disease in complicated samples.