PHYTOCHEMICAL ANALYSIS, VOL. zyxwvuts A 7, 237-244 (1996) zyxwvut BA Mass Spectrometry of Labelled Triterpenoids: Thermospray and Electron Impact Ionization Analysis Horacio HeinZen*??:, Jan X. de Vries,O Patrick Moyna,?: Gerd Remberg,? Ruben Martinez5 and Lutz F. Tietzet zyxwvutsr CBA $ Citedra de Farmacognosia y Productos Naturales, Facultad de Quimica, Universidad de la Repliblica, Avenida General Flores 21 24, Montevideo, Uraguay zyxwvutsr BA Q Medizinische Klinik der Universitat, Abteilung Klinische Pharrnakologie, Bergheimerstrasse 58, D-69 115 Heidelberg, Germany Institut fur Organische Chernie der Universitat, Tarnmanstrasse, D-37077 Gottingen, Germany The mass spectra of unlabelled and specifically labelled triterpenes and derivatives were recorded using thermospray liquid chromatography (TSP-LC) and electron impact (EI) ionization. These mass spectra were used for the development of assays to characterize the products from the in zyxw DCBA vitro oxidative biodegradation of triterpenoids and the identification of compounds in plant extracts. Analysis using gas chromatography-mass spectrometry (GC-MS) with EI ionization of the free products, acetates and trimethylsilyl derivatives, sho lower limits of detection and higher selectivity compared with those with TSP-LC ionization. TSP-L may be useful for the determination of the molecular ions of highly oxygenated tritepenoids. Accur the EI fragment ions were recorded for triterpenes of the lupane series and fragmentation routes ar Based on these characteristic fragmentations, 0-methyl-P-lupeol has been identified in plant e Orbygnia species. Keywords: Triterpenoids; thermospray liquid chromatography-mass spectrometry; gas chromatography-mass spectro- metry; electron impact ionization; lupanes; 0-methyl-P-lupeol. INTRODUCTION Triterpenes are a widespread class of compounds in the plant kingdom where they occur free or as glycoconjugates (Harborne, 1989) but their biological significance has not been fully clarified as yet. Although closely related to sterols from a biosynthetic and structural point of view, they cannot efficiently replace them in biological systems such as membrane constituents or as precusors, for example, of insect hormones (Nes and Heftmann, 1981). Unconjugated triterpenes are often found in the epicu- ticular waxes of plants (Tulloch, 1976; Garcia et zyxwvu GFEDC al., 1995) where their main fuction is to prevent water loss and to constitute the first defensive barrier to predators (i.e. bacteria, fungi, insects) (Tulloch, 1976). Information about the metabolism and enzymatic biodegradation pathways of the pentacyclic triterpenes is, however, limited and for this purpose a series of triterpenes labelled with stable isotopes (I3C and *H) has been synthesized (Tietze et al., 1991). Mass spectra of unlabelled and labelled triterpenes, together with some sterols and derivatives, were recorded with electron impact ionization (EI) using a direct introduction probe (DIP) or after capillary gas chromatographic (GC) separa- tion. Ionization using thermospray liquid chromato- graphy-mass spectrometry (TSP-LC-MS) was also per- formed. MS has been used for the development of quantitative assays and for the identification of triterpenoids from biodegradation products and from plant extracts. Although the EI ion fragmentations of triterpenes of the lupane series have been investigated (Budzikiewicz et al., * Author to whom correspondence should be addressed. 1963), new fragmentation routes are proposed in the pr study based on the MS of labelled compounds and on the measurement of exact masses. EXPERIMENTAL Materials. Compounds 5,9,10,14,15 and 16 (Table 1) were synthesized according to the general scheme previo described (Tietze et al., 1991) and characterized by mass and protonnuclear magnetic resonance spectrometry (NMR). According to this scheme, the vinylic double bon was ozonized and rebuilt employing the Wittig or Lomba methodologies with the appropriate label in the newly introduced methylene at C-29. The deuterium label introduced in the C-2 of the triterpene skeleton by exchang of 9 with CH30D/CH30Na. Compounds 1,6,11,17 and 19 were commercially available (Carl Roth GmbH; Heid berg, Germany); compound 22 was isolated from Orbygniu phaleraru (St. Hill) (Garcia et al., 1995); compound 8 was obtained by oxidation of 11 with chromic acid on a polymer support (Aldrich Chemie; Steinheim, Germany) (Cainelli et al., 1976). For trimethylsilylation, bis(trimethylsily1) tr fuoroacetamide (BSTFA; Merck, Darmstadt, Germany) or a 3: 1 mixture of trimethylsilyl imidazol and BSTFA (Pierc Rockford, IL, USA) were used. Thermospray liquid chromatography-positive ion mass trometry (TSP-LC-MS). TSP-LC-MS positive ion spectra wereobtained with a model5986A quadrupole mass spectrometer (Hewlett Packard, Bad Homburg, Germa coupled to a Vestec model 721A thermospray LC-MS CCC 0958-0344/96/050237-08 zyxwvutsrq 0 1996 by John Wiley & Sons, Ltd. Received 9 December I994 Accepted (revised) 2 January 1996