Metabolite fingerprinting of bioactive compounds in Echinacea pallida by high-performance liquid chromatography with diode array and electrospray ionization-mass spectrometry detection

This study was aimed at developing a novel, reliable and fully validated method for the simultaneous analysis of biologically active caffeic acid derivatives and polyacetylenes/polyenes in Echinacea pallida (Nutt.) Nutt. plant material and natural products by HPLC-UV/DAD, HPLC-ESI-MS and MS/MS using ion trap (IT) and triple quadrupole (TQ) mass analyzers. Even if it is more difficult to optimize the separation of such different constituents in one run, a method for the simultaneous determination of both caffeic acid derivatives and polyacetylenes/polyenes in E. pallida is useful because it can reduce both the time and the sample size required for the analysis. In addition, a technique able to provide a complete fingerprint of the secondary metabolites is of great interest in the ambit of phytochemical analysis and quality control of E. pallida raw material and derivatives, widely used in phytotherapy. The HPLC analyses were carried out on an Ascentis C18 column (250 mm × 4.6 mm I.D., 5 um), with a mobile phase composed by H2O and ACN both containing 0.1% formic acid, under gradient elution. A relevant novel aspect of the proposed technique is that the combination of MS and MS/MS data with retention times and UV spectra made the peak identification very reliable. The fragmentation patterns of caffeic acid derivatives and polyacetylenes/polyenes obtained by IT and TQ are discussed in detail for the first time in the present work. The oxidation mechanism of monocarbonylic acetylenes is demonstrated for the first time in this study using MS and MS/MS data. The practical applicability of the technique was demonstrated by the quantitative analysis of E. pallida root extracts and commercially available dietary supplements to provide reliable chromatographic fingerprints of their bioactive secondary metabolites.