Profiling of natural products by HPLC combined with DAD and accurate in-source ESI-TOF-MS spectra followed by TLC/HPLC/DAD/ESI-TOF-MS bioactivity screening

Natural products play important and increasing role in pharmaceutical market. In many cases, in comparison with synthetic drugs side affects are less prominent. They are also often a source of new lead compounds, used solely, or for semi-synthesis. Due to its sensitivity and efficiency, HPLC hyphenated with DAD and MS detection systems is widely used in searching for potentially new natural compounds present in plant materials. Nowadays, accurate MS analysis plays special role in rapid qualitative research of potentially novel plant metabolites. Because of instrumental developments with double mass calibrations systems used in modern time-of-flight mass spectrometers (TOF-MS), it is possible to determine exact molecular mass within mass errors even less than 2 ppm. Therefore, errors in molecular formula confirmations have been minimized.

In our research we developed HPLC combined with DAD and accurate ESI-TOF-MS for rapid and efficient dereplication of known metabolites and discovery of potentially new natural compounds. Plant materials were extracted using modern pressurized liquid extraction (PLE), followed by solid-phase extraction (SPE) and then HPLC/DAD/ESI-TOF-MS with accurate in-source MS-fragmentation. In this way, variety of alkaloids (eg. Amaryllidaceae alkaloids), coumarins, chromones, flavonoids and phenolic acids were discriminated in plant samples from Amaryllidaceae, Umbelliferae and Compositae families. Several potentially novel compounds could be determined. Fragmentation pathways were also established, and enabled for example determination of different groups among Amaryllidaceae alkaloids. Moreover, TLC with bioauthography utilizing novel instrumental set up: TLC/HPLC/DAD/ESI-TOF-MS was used for screening of acetyl- and butyrylcholinesterasase inhibitors among investigated plant extracts.