Analytica Chimica Acta 635 (2009) 188–195 Contents lists available at ScienceDirect Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca Rapid and novel discrimination and quantification of oleanolic and ursolic acids in complex plant extracts using two-dimensional nuclear magnetic resonance spectroscopy—Comparison with HPLC methods Vassiliki G. Kontogianni a , Vassiliki Exarchou b , Anastassios Troganis c , Ioannis P. Gerothanassis a,∗ a Section of Organic Chemistry & Biochemistry, Department of Chemistry, University of Ioannina, Ioannina GR-45110, Greece b NMR Center, University of Ioannina, Ioannina GR-45110, Greece c Department of Biological Applications & Technology, University of Ioannina, Ioannina GR-45110, Greece article info Article history: Received 4 November 2008 Received in revised form 23 December 2008 Accepted 12 January 2009 Available online 18 January 2009 Keywords: Oleanolic acid Ursolic acid NMR Mixture analysis 1 H– 13 C heteronuclear single-quantum coherence 1 H– 13 C heteronuclear multiple-bond correlation abstract A novel strategy for NMR analysis of mixtures of oleanolic and ursolic acids that occur in natural prod- ucts is described. These important phytochemicals have similar structure and their discrimination and quantification is rather difficult. We report herein the combined use of proton–carbon heteronuclear single-quantum coherence ( 1 H– 13 C HSQC) and proton–carbon heteronuclear multiple-bond correlation ( 1 H– 13 C HMBC) NMR spectroscopy, in the identification and quantitation of oleanolic acid (OA) and urso- lic acid (UA)in plant extracts of the Lamiaceae and Oleaceae family. The combination of 1 H– 13 C HSQC and 1 H– 13 C HMBC techniques allows the connection of the proton and carbon-13 spins across the molecular backbone resulting in the identification and, thus, discrimination of oleanolic and ursolic acid with- out resorting to physicochemical separation of the components. The quantitative results provided by 2D 1 H– 13 C HSQC NMR data were obtained within a short period of time (∼14min) and are in excellent agreement with those obtained by HPLC, which support the efficiency of the suggested methodology. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Natural environment and especially plants continue to provide a dynamic research area in the field of drug discovery [1], since phytochemicals can be possible starting materials of novel bioactive compounds [2]. Triterpenoids is a group of phytochemicals that has been extensively studied for their pharmacological properties, due to their high abundance in common plants and fruits. Especially the triterpenes ursolic and oleanolic acid are present in many fruits and herbs and, thus, they have been safely consumed worldwide for many centuries without apparent ill effects [2,3]. For this reason literature furnishes numerous data on both analysis and biological activity of the two acids. Oleanolic acid (OA) and ursolic acid (UA) are position isomers of the methyl group on the ring E (Scheme 1). These two triterpenes may occur as free acids and as aglycones of saponins. Both OA and UA are of interest as therapeutics and their biological activities are rather similar considering the closeness of their chemical struc- tures. The antioxidant [4,5], anti-inflammatory [6,7], antitumour ∗ Corresponding author. Tel.: +30 2651098389; fax: +30 2651098398. E-mail address: igeroth@cc.uoi.gr (I.P. Gerothanassis). [8], anti-HIV [9], antimicrobial [10,11], gastroprotective [12] and hypoglycemic [13] properties of OA and UA are well documented. A detailed list of data on the biological activity is available herein [14–22] showing the importance of these pentacyclic triterpene acids and their potency to be used in medicine. The simultaneous determination of OA and UA is a difficult task considering their structure similarity. Common procedures such as gas chromatography including the necessary silylation [23–26] or methylation step [27], liquid chromatography coupled with UV [28,29] and MS spectrometry [30–32] have been used for the anal- ysis of OA and UA. These analytes have weak chromophores, low UV absorption and the resolution by LC seems difficult on reversed phase, as these molecules are position isomers. For this reason several methods have been suggested for the simultaneous OA and UA determination, such as high-performance thin layer chro- matography after iodine derivatization [33], micellar electrokinetic capillary chromatography [34], the use of a LC system with porous graphitic carbon and evaporative light scattering detection [35], capillary supercritical fluid chromatography [36], and nonaque- ous capillary electrophoresis [37]. The addition of cyclodextrins to the mobile phase has been also investigated to improve the separation of the isomers [38] and cyclodextrin-modified micel- lar electrokinetic chromatography [39] has been reported. These 0003-2670/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2009.01.021