Simultaneous determination of phenolic compounds and triterpenic acids in oregano growing wild in Greece by 31 P NMR spectroscopy Alexia Agiomyrgianaki a and Photis Dais a * 31 P nuclear magnetic resonance (NMR) spectroscopy was used to detect and quantify simultaneously a large number of phenolic compounds and the two triterpenic acids, ursolic acid and oleanolic acid, extracted from two oregano species Origanum onites and Origanum vulgare ssp. Hirtum using two different organic solvents ethanol and ethyl acetate. This analytical method is based on the derivatization of the hydroxyl and carboxyl groups of these compounds with the phosphorous reagent 2-chloro-4,4,5,5- tetramethyl-1,3,2-dioxa phospholane and the identification of the phosphitylated compounds on the basis of the 31 P chemical shifts. Unambiguous assignment of the 31 P NMR chemical shifts of the dihydroxy- and polyhydroxy-phenols in oregano species as well as those of the triterpenic acids was achieved upon comparison with the chemical shifts of model compounds assigned by using two-dimensional NMR techniques. Furthermore, the integration of the appropriate signals of the hydroxyl derivatives in the corresponding 31 P NMR spectra and the use of the phosphitylated cyclohexanol as an internal standard allowed the quantification of these compounds. The validity of this technique for quantitative measurements was thoroughly examined. Copyright © 2012 John Wiley & Sons, Ltd. Supporting Information may be found on the online version of this article. Keywords: oregano species; phenolic compounds; triterpenic acids; 31 P NMR Introduction Origanum species is an annual herb widely distributed in Eurasia, North Africa and North America. [1,2] It is commonly used in countries located in the Mediterranean basin and in Latin American cuisines as a spice under the commercial name ‘oregano’. Moreover, this well known aromatic herb is endowed by medicinal properties due to its high content of carvacrol, thymol and other phenolic compounds. In this respect, it finds interesting applications in pharmacology. [3] In addition, oregano contains notable quantities of two triterpenic acids, ursolic acid and oleanolic acid, in their free forms. Triterpenic acids have been used for centuries in folk medicine as antiflammatory, antidiabetes and hepatoprotective agents, and recently they attracted attention in the scientific community because of their anticarcinogenic activity. [4,5] Several analytical methods have been used in the past for the detection and quantification of the oregano constituents including gas and liquid chromatography, [1–3,6] mass spectrometry [2,3,6,7] and NMR spectroscopy. [8–10] The application of two-dimensional (2D) NMR spectroscopy to oregano extracts [8] succeeded in detecting the phenolic compounds o-coumaric acid, ferulic acid, caffeic acid, p-hydroxybenzoic acid and vanillic acid. The flavonols taxifolin, aromadendrin, eriodictyol, naringerin and apigenin, the phenolic acid rosmarinic acid and the monoterpene carvacrol were identified after their separation by HPLC-UV, subsequent purifica- tion with solid-phase extraction and finally detection with NMR and MS (LC-UV-SPE-NMR-MS). [9] No quantification of these oregano constituents was reported in the aforementioned studies. The determination of triterpenic acids in oregano was achieved by using 2D 1 H- 13 C heteronuclear experiments, namely, heteronuclear single quantum coherence (HSQC) and heteronuclear multiple bond correlation (HMBC). [10] Calibration curves with standard com- pounds were required for the quantification of oleanolic and ursolic acids. In this work, an alternative methodology based on 31 P NMR spectroscopy is evaluated for the detection and quantification of minor constituents in two oregano species (Origanum onites and Origanum vulgare ssp. Hirtum) extracted by two different solvents. This method, introduced in an earlier publication, [11] is based on the derivatization of the labile hydrogens of the hydroxyl and car- boxyl groups by the phosphitylating reagent 2-chloro-4,4,5,5-tetra- methyl-1,3,2-dioxaphospholane (compound I), according to the re- action shown in Fig. 1, and the use of the 31 P chemical shifts to identify the labile centres (compound II). Compound I reacts rapidly (<15 min) and quantitatively under mild conditions (within the NMR tube) with the hydroxyl and carboxyl groups. This method has been already applied to determine polyphenols, diacylglycer- ols, total free sterols and free acidity in vegetable oils. [11–14] The pros and cons of this methodology against earlier analytical methods and the possibility of its application to other plant extracts will be discussed as well. * Correspondence to: Photis Dais, NMR Laboratory, Department of Chemistry, University of Crete, Voutes Campus, 71003 Hraklion, Crete, Greece. E-mail: dais@chemistry.uoc.gr a NMR Laboratory, Department of Chemistry, University of Crete, Voutes Campus, 71003 Hraklion, Crete, Greece Magn. Reson. Chem. (2012) Copyright © 2012 John Wiley & Sons, Ltd. Research Article Received: 21 January 2012 Revised: 24 March 2012 Accepted: 27 March 2012 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/mrc.3877