Study of a complex secondary metabolites with potent anti-radical activity by two dimensional TLC/HPLC coupled to electrospray ionization time-of-flight mass spectrometry and bioautography.

The high performance liquid chromatography (HPLC) coupled to electrospray ionization (ESI) octopole orthogonal acceleration time-of-flight (TOF) mass spectrometry (MS) was developed to separate secondary metabolites belonging to different groups of compounds with a wide range of polarity, i.e. phenolic acids, flavonoids, sesquiterpenes and others present in minute amounts, in a single run. Moreover, application of ESI-TOF-MS with a simple in source type of fragmentation and high mass accuracy measurements (around 2 ppm) in both positive and negative ionization modes with different and optimized fragmentor voltages, enabled the identification of each single compound based on proposed fragmentation patterns. In this way a total number of 14 compounds in methanolic and water-methanolic (1:1, v/v) extract from fruits of Eryngium amethystinum and 6 compounds in extract from fruits of E. planum have been identified for the first time. Additionally, two dimensional thin layer chromatography/high performance liquid chromatography/electrospray-ionization-time-of-flight mass spectrometry (TLC/HPLC/ESI-TOF-MS) system was developed for identification of compounds with the highest anti-radical activity revealed in TLC-DPPH bioauthographic test. This on-line procedure enables significant improvement in terms of efficiency and faster structural analysis of the potential antioxidant natural compounds directly in the plant extracts screened for bioactivity tests. This effect - directed analysis (EDA) method enables separation, identification and preliminary screening of plant extracts for anti-radical properties and can be easily adopted for other bioactivities.