<jats:title>Abstract</jats:title><jats:p><jats:bold>Introduction – </jats:bold><jats:italic>Callistemon lanceolatus</jats:italic> DC. (Myrtaceae) is a plant rich in polyphenols, and is used as anticough, antibronchitis and insecticide in folk medicine. Because of the biological importance of plant polyphenols, particularly tannins, a phytochemical study was of interest to investigate the constitutive poyphenols in the extracts of flowers and leaves.<jats:p><jats:bold>Objective – </jats:bold>To avoid time‐consuming methodology for isolation of a complex mixture of known metabolites, HPLC‐ESI/MS was employed for fast picking up of the new compounds followed by identification of the structures with UV and one‐ and two‐dimensional NMR.<jats:p><jats:bold>Methodology – </jats:bold>Flowers and leaves were separately extracted with hot aqueous methanol under reflux (70°C). Pre‐isolation of the total extracts was achieved through column chromatographic fractionation on polyamide with water–methanol for gradient elution. The main fractions were purified using repeated column chromatography on cellulose and/or Sephadex LH‐20 with suitable eluents. HPLC‐ESI/MS analyses were carried out in the single ion monitoring (SIM) and negative ion modes. The pure compounds in methanol–water (1:1) were analysed by direct infusion ESI/MS. Final structure elucidation was obtained by one‐ and two‐dimensional NMR.<jats:p><jats:bold>Results – </jats:bold>Two new metabolites namely quercetin 3‐<jats:italic>O‐β</jats:italic>‐D‐glucuronopyranoside <jats:italic>n</jats:italic>‐butyl ester (<jats:bold>1</jats:bold>) and <jats:italic>n</jats:italic>‐butylgallate 4‐<jats:italic>O</jats:italic>‐(2′,6′‐di‐<jats:italic>O</jats:italic>‐galloyl)‐<jats:italic>β</jats:italic>‐<jats:sc>d</jats:sc>‐glucopyranoside (<jats:bold>4</jats:bold>) along with nine known ones were identified from the aqueous methanol extracts of flowers and leaves.<jats:p><jats:bold>Conclusion – </jats:bold>The study has shown that <jats:italic>Callistemon lanceolatus </jats:italic>is rich in polyphenols. HPLC‐ESI/MS may be used, in negative ion mode, as an efficient and rapid analytical tool for investigating complex plant extracts. Copyright © 2008 John Wiley & Sons, Ltd.