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A screening method based on high-performance liquid chromatography (HPLC) interfaced to electrospray ionization mass spectrometry (ESI-MS) with detection of negative ions was devised and the conditions for assaying the complex mixture of gallotannins, ellagitannins and flavonoids of aqueous ethanolic Epilobium hirsutum L. whole plant extract were established. Twenty-eight polyphenolic compounds were tentatively identified and one unique ellagitannin with an odd molecular mass was detected in the extract. This compound was taken as the target and isolated by means of consecutive polyamide and Sephadex LH-20 column fractionation; repeatedly, the isolation and purification were monitored by HPLC/ESI-MS. The compound was isolated and subsequently identified as valoneic acid amide bilactone (epilobamide-A). The parent acid, known as valoneic acid bilactone, was also isolated and mass and ¹H and ¹³C NMR spectra were recorded and completely interpreted. Eventually, all relevant structures were elucidated by established methods of structure analysis and confirmed by 1D and 2D NMR spectroscopy. Further, ESI-MS with in-source collision-induced dissociation of the two valoneic acid derivatives and the formation of distonic radical anions is discussed, together with oxidation processes during fragmentation. © 1997 John Wiley & Sons, Ltd.