Simultaneous determination of phenolic compounds and triterpenic acids in oregano growing wild in Greece by 31P NMR spectroscopy.

(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.