High-performance liquid chromatography paired with UV photodiode-array and electrospray mass spectrometry was investigated as a method for the analysis of alkamides in the roots and achenes of the popular herb Echinacea purpurea (L.) Moench. All alkamides showed very abundant peaks in the reconstructed total ion current chromatogram. Nine alkamides were identified in the root of E. purpurea, similar to the fingerprint reported by Bauer and Remiger [Planta Med., 55 (1989) 367]. Several minor alkamides, not identifiable by previously published techniques, have been also tentatively identified. Two major peaks were identified in E. purpurea achenes as undeca-2E,4Z-diene-8, 10-diynoic acid isobutylamide (1) and dodeca-2E,4E,8E,10E/Z tetraenoic acid isobutylamide (8/9). The isomer pair, tetraene 8/9, was purified as a standard for quantification of alkamide content in E. purpurea achenes and roots, as well as for the achenes of E. angustifolia DC and E. pallida (Nutt.) Nutt.