Journal of Chromatography A, 1164 (2007) 145–152 Determination of three major triterpenoids in epicuticular wax of cabbage (Brassica oleracea L.) by high-performance liquid chromatography with UV and mass spectrometric detection Mitja Martelanc, Irena Vovk ∗ , Breda Simonovska National Institute of Chemistry, Laboratory for Food Chemistry, Hajdrihova 19, SI-1000 Ljubljana, Slovenia Received 25 April 2007; received in revised form 29 June 2007; accepted 29 June 2007 Available online 5 July 2007 Abstract Lupeol, together with - and -amyrins in smaller quantities, has been found for the first time in the epicuticular wax of white cabbage (Brassica oleracea L. convar. capitata (L.) Alef. var. alba DC) leaf surface extract. The three triterpenoids were identified by a new high-performance liquid chromatographic (HPLC) method with UV and mass spectrometric (MS) detection using atmospheric pressure chemical ionization (APCI). All three isomeric compounds gave a parent ion peak at m/z 409 [M + H-18] + and the relative intensities of some characteristic fragment ion peaks in tandem mass spectrometric (MS–MS) spectra of this parent ion enabled differentiation between the isomers. An additional peak at m/z 439 [M + H] + , which could be oleanonic or ursonic aldehyde, was detected by HPLC–APCI–MS. Saponification of cabbage leaf surface extract with 20% NaOH in methanol at 65 ◦ C for 2 h had no influence on lupeol, or - or -amyrins, but lead to the formation of three additional compounds, which were not identified. © 2007 Elsevier B.V. All rights reserved. Keywords: Brassica oleracea; White cabbage; Epicuticular wax; Triterpenoids; Lupeol; -Amyrin; -Amyrin; Saponification; HPLC-UV; HPLC–APCI–MS–MS; TLC 1. Introduction Triterpenoids are among the most ubiquitous secondary metabolites in terrestrial and marine flora and fauna [1]. Remark- able diversity is known to exist among carbon skeletal types of triterpenoids, but the most abundant triterpenoids in nature are -amyrin (oleanane type), ursolic acid (ursane type) and friedeline (friedelane type) [2]. Plant triterpenoids, which have a common biosynthetic pathway from six isoprene units via squalene [3], are interesting from an evolutionary and func- tional perspective. In spite of the ubiquity of triterpenoids, their functions are not well known. Recent work concerning the medical uses of this class of compounds suggests their great potential as drugs. Investigations of mixtures of - and -amyrins established their gastroprotective [4], antipruritic [5] and hepatoprotective behavior against acetoaminophen-induced hepatotoxicity [6]. -Amyrin also has topical anti-inflammatory ∗ Tel.: +386 1 4760 341; fax: +386 1 4760 300. E-mail address: irena.vovk@ki.si (I. Vovk). properties [7]. Lupeol and its esters protect against lysosomal membrane damage provoked by cyclophosphamide, which is widely used as an alkylating agent in cancer chemotherapy [8]. Lupeol itself exhibits antitumor activity in murine skin and may inhibit tumor promotion [9]. Brassica oleracea, native to coastal southern and western Europe, has been bred into a wide range of cultivars, includ- ing cabbage, broccoli and cauliflower. Its chemical composition in connection with antioxidant activity of Brassica oleracea contributes to its importance in the human daily diet [10]. In addition, - and -amyrins have been recognized as the compo- nents of the wax of white cabbage, which may have an influence on the insect–plant interactions [11,12]. Many papers have been published on the detection and separation of triterpenoids. Some methods include prior derivatization followed by chromatography on sil- ver nitrate impregnated silica gel columns [13], preparative thin-layer chromatography (TLC) on silica gel [14] or gas chromatography–mass spectrometry [15,16]. A derivatization step was not required for HPLC methods in which UV detec- tors [17–20], refractometers [21], evaporative light-scattering 0021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2007.06.062