Thermal Decomposition of Caffeic Acid in Model Systems: Identification of Novel Tetraoxygenated Phenylindan Isomers and Their Stability in Aqueous Solution Richard H. Stadler,* Dieter H. Welti, Andreas A. Sta ¨ mpfli, † and Laurent B. Fay Nestle ´ Research Centre, Nestec Ltd., Vers-chez-les-Blanc, P.O. Box 44, CH-1000 Lausanne 26, Switzerland Caffeic acid subjected to mild pyrolysis (225-226 °C) under vacuum resulted in rapid decarboxylation and the formation of simple catechol monomers as well as more complex cyclocondensed dimers and polymers. This reaction yielded the same spectrum of products as did acid-catalyzed cyclization of caffeic acid. The major pyrolysis products were identified by reversed-phase HPLC and LC- tandem mass spectrometry. Two novel compounds, identified by MS, 1 H NMR, and 13 C NMR as 1,3-cis- and 1,3-trans-tetraoxygenated phenylindans, were present as major products in both the caffeic acid pyrolysate and the acid-treated sample. The stability and reactivity of the pyrolysis products in weakly buffered aqueous solutions were determined concomitantly by measuring hydrogen peroxide generation and by monitoring the concentration of the individual components by reversed-phase HPLC. Such model studies may provide information pertaining to reaction mechanisms and the nature of the compounds involved in hydrogen peroxide formation in coffee. Keywords: Pyrolysis; caffeic acid; hydrogen peroxide; phenylindan isomers; model system studies INTRODUCTION The formation of hydrogen peroxide in coffee solution over time has been unequivocally demonstrated by numerous investigators (Nagao et al., 1986a,b; Rinkus and Taylor, 1990; Tsuji et al., 1991; Stadler et al., 1994). Much emphasis has been placed on the relationship between hydrogen peroxide generation in coffee and the weak in vitro genotoxic effects that are observed in bacterial and mammalian mutagenicity test systems (Fujita et al., 1985; Itagaki et al., 1992). This contribu- tion of hydrogen peroxide in coffee-mediated mutage- nicity is undisputed, because in vitro mutagenicity can be effectively abolished by addition of antioxidative enzymes such as catalase or peroxidase (Nagao et al., 1986a; Friederich et al., 1985; Itagaki et al., 1992). Reports in this field have shown that dissolved oxygen and water temperature during coffee preparation are decisive factors in hydrogen peroxide formation. This may explain the large fluctuations in the levels of hydrogen peroxide reported in the literature (Fujita et al., 1985; Rinkus and Taylor, 1990; Stadler et al., 1994). The oxidative effects portrayed by coffee seem primarily attributed to polyphenolics which adventitiously form hydrogen peroxide when exposed to oxygen and metal catalysts. However, coffee and certain polyphenolic constituents can also act as potent antioxidants and antimutagens as shown in in vitro assays (Stich et al., 1982; Obana et al., 1986; Stich, 1991; Graf, 1992; Stadler et al., 1994), and coffee as a whole has also been reported to protect against various carcinogens in animal studies (Abraham, 1989; Aeschbacher and Jaccaud, 1990). However, no detailed work has been done to elucidate the mechanisms and chemicals responsible for hydrogen peroxide formation and the antioxidative effects dis- played by coffee. A report by Tsuji and co-workers showed that green coffee beans do not generate hydro- gen peroxide, whereas roasted beans have this ability depending on the degree and duration of roasting (Tsuji et al., 1991). The same authors also propose the involvement of thermal decomposition products of caffeic acid, in particular the substituted benzenediol p-vinyl- catechol. However, no data were presented that could confirm an active role of p-vinylpyrocatechol in hydrogen peroxide production either in model pyrolysis systems or in coffee itself. This dioxystyrene is apparently formed in trace amounts by decarboxylation of caffeic acid during pyrolysis or roasting (Clarke and MacRae, 1983; Heinrich and Baltes, 1987) and, as generally known for o- and p-dihydroxybenzene moieties, can produce hydrogen peroxide when exposed to atmo- spheric conditions (Clapp et al., 1990). Furthermore, this molecule is extremely susceptible to oxidation, resulting in rapid polymerization reactions in solution (Tiedke, 1936) and making isolation and identification difficult. Even though caffeic acid has been implicated indi- rectly in the hydrogen peroxide generation process, there is only limited information on the ability of caffeic acid oxidation or pyrolysis products to produce hydrogen peroxide in model systems. Different pathways and products of chemically induced oxidation as well as autoxidation of caffeic acid at ambient temperatures have been described in the literature. The products of such reactions are diverse and include cyclolignans (Nahrstedt et al., 1990; Gumbinger et al., 1993), ben- zodioxane, and naphthalene-1,2-dihydro- (Cilliers and Singleton, 1991) and tetrahydrofuran-type compounds (Fulcrand et al., 1994). On the other hand, subjection of phenolic acids to thermal treatment results in rapid decarboxylation to furnish substituted styrenes (Klaren de Wit et al., 1971; Rizzi and Boekley, 1992) and simple vinylpyrocatechol monomers (Fiddler et al., 1967; Tsuji et al., 1991). The positive correlation between the degree of roast- ing of the coffee beans and hydrogen peroxide formation suggests that the pertinent reductants are formed during roasting. To gain a better insight into the nature * Author to whom correspondence should be ad- dressed (fax +41/21 785 8553). † Present address: Ciba-Geigy Ltd., K-127.5.02, 4002 Basel, Switzerland. 898 J. Agric. Food Chem. 1996, 44, 898-905 0021-8561/96/1444-0898$12.00/0 © 1996 American Chemical Society