The recent introduction of flavored wine and malt beverages

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1 EBELER ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 2, FOOD COMPOSITION AND ADDITIVES Solid-Phase Microextraction for the Enantiomeric Analysis of Flavors in Beverages SUSAN E. EBELER University of California, Davis, Department of Viticulture and Enology, One Shields Ave, Davis, CA GAY M. SUN Aerojet Fine Chemicals, Rancho Cordova, CA MEERA DATTA, PHIL STREMPLE, and ALLEN K. VICKERS J&W Scientific, Inc., Folsom, CA Solid-phase microextraction combined with gas chromatographic/mass spectrometric analysis and separation on a chiral cyclodextrin stationary phase was a rapid, reliable technique for profiling chiral aroma compounds in flavored alcoholic beverages. Several enantiomeric terpenes, esters, alcohols, norisoprenoids, and lactones were identified in berry-, peach-, strawberry-, and citrus-flavored wine and malt beverages (wine coolers). Using this technique, we were able to confirm the addition of synthetic flavoring to several beverages, consistent with label designations. The recent introduction of flavored wine and malt beverages has created a need for methods that can be used to rapidly profile and authenticate the volatile composition of these beverages. Constant and Collier (1) used gas chromatography with flame ionization detection (GC/FID) combined with solid-phase microextraction (SPME) to profile volatile components in fruit-flavored malt beverages, including lagers and ales. Over 40 volatiles were identified, and all of the products had distinguishable GC/FID profiles. Two raspberry-flavored products contained i-methyl- -ionone and n-methyl- -ionone, compounds that are not found in nature; however, their presence was consistent with label indications of flavor addition. In addition, propylene glycol, a flavor carrier, and benzyl benzoate, a flavor stabilizer and enhancer, were identified in several of the products, again indicating probable flavor addition to these products. Although measurements of characteristic flavor constituents and their quantity and distribution in aroma extracts can be precise and reproducible, they can give only limited information on the source and origins of the flavoring (2). However, as reviewed by Marchelli et al. (3), the enantiomeric composition of flavorings is often specific to the natural source or origin. For example, enantiomeric distribution of (E)- -ionone may be used as a marker for the authenticity of raspberry flavor because natural sources contain the Received March 13, Accepted by JL May 31, enantiopure (R)-form (4). Therefore, the enantiomeric ratio of key flavor compounds has been used as an important criterion for differentiating natural flavor compounds from their synthetic counterparts (2, 3, 5). Methods for analysis of chiral flavor compounds typically involve quantitative and qualitative measurements by gas chromatography (GC) with cyclodextrin-based stationary phases. However, routine analysis of volatile composition by GC is often limited by sample preparation techniques that are time consuming, require expensive equipment and solvents, and/or lack sensitivity and precision. Care must also be taken to avoid racemization during sample preparation steps. SPME offers a rapid, solvent-free alternative to traditional sample preparation techniques (e.g., liquid/liquid extractions and static and dynamic headspace sampling) for extracting flavors from food and beverages (6 12). A number of SPME fiber coatings are available to provide optimal sensitivity and selectivity for a wide range of analytes. In addition, SPME requires no modification of the GC procedure and is readily automated. The objective of this study was to demonstrate the application of SPME combined with enantioselective analysis by gas chromatography/mass spectrometry (GC/MS) to profile important chiral aroma compounds in flavored alcoholic beverages. Experimental Chemicals and Samples All chemicals were purchased commercially (Aldrich Chemical Co., Milwaukee, WI, or Fluka Chemical Corp., Milwaukee, WI) and were of the highest grade available. All beverage samples were purchased locally (Folsom, CA). Gas Chromatographic Conditions All analyses were performed by using a Hewlett-Packard, Willmington, DE 6890 gas chromatograph interfaced to a 5973 mass-selective detector and ChemStation, Hwelett-Packard, Willmington, DE software. The column was CycloSil-B (heptakis[2,3-di-o-methyl-6-o-t-butyldimethyl silyl]- -cyclodextrin), 30 m 0.25 mm 0.25 µm (J&W Scientific, Inc., Folsom, CA). Helium was used as the carrier gas at 37 cm/s (linear velocity measured at 40 C). The inlet of the

2 480 EBELER ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 2, 2001 Figure 1. Chromatogram obtained by gas chromatography with mass-selective detection (GC/MSD) for a berry-flavored wine cooler with natural and artificial flavors. Figure 2. Chromatogram obtained by GC/MSD for a raspberry Merlot wine flavored with natural and artificial flavors.

3 EBELER ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 2, Figure 3. flavors. Chromatogram obtained by GC/MSD for a raspberry-flavored grape wine with raspberry and other natural Figure 4. Chromatogram obtained by GC/MSD for a blackberry-flavored wine beverage with natural flavors.

4 482 EBELER ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 2, 2001 Table 1. Peak areas and ratios for the 2 isomers of -ionone in berry-flavored beverages Sample Fragment ion Peak area of first-eluting isomer Fragment ion Peak area of second-eluting isomer Ratio a Berry-flavored wine cooler (natural and artificial flavoring) Raspberry wine (Merlot; natural and artificial flavors) Raspberry-flavored grape wine (raspberry & other natural flavors) m/z m/z m/z m/z m/z m/z Blackberry-flavored wine beverage (natural flavors) m/z m/z m/z m/z m/z m/z a Ratio: peak area of first-eluting isomer: peak area of second-eluting isomer. gas chromatograph and the transfer line of the mass-selective detector were maintained at 250 and 280 C, respectively. The oven temperature was programmed to increase at 2 C/min from an initial temperature of 40 C to a final temperature of 190 C. All injections were performed in the split mode with the split ratio set at 1:5. Headspace SPME Headspace SPME sampling was done manually by using a polyacrylate fiber (84 µm; Supelco, Inc., Bellefonte, PA). Samples (10 ml) were placed in 15 ml glass headspace vials fitted with Teflon crimp-seal closures. Sodium chloride (1 g/10 ml sample) was added before the vial was sealed and before sampling. After conditioning according to the manufacturer s directions, the SPME fiber was inserted into the headspace of the vial, allowed to equilibrate at room temperature (27 C) for 1 h without stirring, and then injected into the inlet of the gas chromatograph. Peak identifications were determined by comparison of retention times with those of authentic standards purchased Figure 5. Chromatogram obtained by GC/MSD for a peach-flavored wine cooler with natural flavors.

5 EBELER ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 2, Figure 6. Chromatogram obtained by GC/MSD for a strawberry-flavored white Zinfandel with natural flavors. commercially. In addition, GC/MS spectra were obtained for both samples and standards and the spectra were compared for final confirmation of peak identity. Results and Discussion Headspace SPME Analysis Several studies have evaluated the variables that can influence the extraction sensitivity and selectivity of the SPME technique (6 9, 13 16). These variables can include the choice of SPME fiber, salt additions, sampling temperature, and sampling time. Our purpose for this study was to develop a general procedure that could be used to profile volatile compounds with a range of boiling points and polarities. A polyacrylate fiber and the addition of salt to the beverage before sampling were shown to give optimum results for this purpose. Sampling times of 60 min were used for all samples. Shorter analysis times (20 30 min) were also evaluated; however, the longer extraction times provided better overall sensitivity for the relatively low concentrations of flavor found for these products. The sampling time also coincided with the GC analysis time, so that the fiber was always in either the injector or the headspace of a vial to minimize analyte loss and contamination of the fiber by ambient air. Although manual sampling was used for this study, the method could be readily automated. Sample Profiles Raspberry-flavored malt beverage and wines. Four different berry-flavored wines and malt beverages were analyzed (Figures 1 4). Labels indicated that both natural and artificial flavors were used, depending on the product. GC aroma profiles varied among the samples; however, α-ionone, a key aroma component of raspberry flavor, was identified in all samples. Other positively identified chiral compounds included ethyl 2-methyl butanoate, limonene, linalool, α-terpineol, and 2-ethyl-1-hexanol. α-ionone is present as the enantiopure (R)-form in raspberries and other natural sources and can be used as a marker of the authenticity of natural products (4). However, a comparison of peak areas for the 2 isomers of α-ionone indicated that both isomers were present in approximately equal quantities in all samples (Table 1; the absolute configuration of each peak was not determined). An unidentified coeluting component interfered with analysis of the first-eluting isomer in 2 of the beverage samples. However, a comparison of ion ratios for the predominant fragment ions of α-ionone (m/z 93, 121, and 136) also indicated that similar amounts of both isomers were present in these products (Table 1). These results are consistent with the labeling for the use of artificial flavoring in 2 of the products. In the products labeled as containing only natural flavors, it is unclear whether the observed α-ionone composition is due to the addition of synthetic flavoring or to the addition of other natural flavorings with different isomer com-

6 484 EBELER ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 2, 2001 Figure 7. Chromatogram obtained by GC/MSD for a citrus-flavored wine cooler with natural flavors (21; reprinted with permission from the American Chemical Society). positions, or whether α-ionone has undergone racemization by exposure to UV light during storage (17). The chiral ester, ethyl 2-methylbutanoate, was identified in 3 of the samples, with the 2 isomers present in approximately equal concentrations. The S(+)-form predominates in apples and pineapple (18). The enantiomeric composition in raspberries is not known. Peach-flavored malt beverage. Natural peach flavors are dominated by lactones, particularly -decalactone (4-decanolide; 19). Only one isomer of -decalactone was identified in the peach-flavored malt beverage (Figure 5), and it was tentatively identified as the (R)-configuration, which is known to predominate in natural peach flavors (3). Other chiral compounds positively identified in this sample were the 2 isomers of 2-ethyl-1-hexanol, present in approximately equal amounts. Strawberry-flavored malt beverage (wine cooler). (R)- -Decalactone (4-decanolide), an important odor-active compound in strawberries (20), was tentatively identified in the strawberry-flavored wine (Figure 6). Ethyl 2-methylbutanoate, was also identified. Consistent with reports by Takeoka et al. (18), the second-eluting isomer of this ester, tentatively identified as the S(+)-configuration, predominated. These results are consistent with the use of a natural strawberry flavor in this beverage. Other positively identified chiral compounds were (R)-limonene and 2-ethyl-1-hexanol. Citrus-flavored wine cooler. Terpenes predominate in the headspace of a naturally flavored citrus-flavored malt beverage (Figure 7). We previously showed that ratios of 4 enantiomeric terpenes, limonene, linalool, terpinen-4-ol, and α-terpineol, were comparable to those of a naturally flavored carbonated soda beverage (21). In particular, the R(+)-isomer of limonene predominanted in both the wine cooler and in the soda, in agreement with published data for natural citrus flavors (17). Care must be taken when information on enantiomeric ratios of acidic beverages is interpreted, however, because racemization of some compounds readily occurs (e.g., linalool and α-terpineol; 3, 22). Summary Information about enantiomeric composition is frequently used as a reliable parameter for determining the naturalness of flavorings (2, 3, 5). Our results show that headspace SPME combined with GC/MS analysis and separation on chiral stationary phases provides a simple and rapid method for profiling chiral lactones, norisoprenoids (α-ionone), esters, alcohols, and terpenes in flavored alcoholic beverages. The method is readily automated and the addition of an internal standard to the samples before analysis could be used to provide additional quantitative information about the flavor composition. Using this technique, we were able to confirm the addition of synthetic flavorings to several products, consistent with

7 EBELER ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 2, designations on the labels. Enantiomer profiles of 2 products were inconsistent with label designations, which indicated the use of flavorings from natural sources only. However, additional analyses are required to unambiguously verify the source of flavorings for these samples. Acknowledgments Partial financial support for Susan E. Ebeler was provided by a Faculty Development Award from the University of California, Davis. Special thanks are given to Jeanee Tollefson for technical assistance. References (1) Constant, M., & Collier, J. (1997) J. Am. Soc. Brew. Chem. 55, (2) Mosandl, A. (1995) Food Rev. Int. 11, (3) Marchelli, R., Dossena, A., & Palla, G. (1996) Trends Food Sci. Technol. 7, (4) Lehman, D., Dietrich, A., Schmidt, S., Dietrich, H., & Mosandl, A. (1993) Z. Lebensm. Unters. Forsch. 196, (5) Mussinan, C.J., & Hoffman, P.G. (1999) Food Technol. 53, (6) Yang, X., & Peppard, J. (1994) J. Agric. Food Chem. 42, (7) Yang, X., & Peppard, J. (1995) LC GC 13, (8) De la Calle Garcia, D., Magnaghi, S., Reichenbacher, M., & Danzer, K. (1996) J. High Resolut. Chromatogr. 19, (9) De la Calle Garcia, D., Reichenbacher, M., & Danzer, K. (1998) J. High Reso. Chromatogr. Chromatogr. Commun. 21, (10) Evans, T.J., Butzke, C.E., & Ebeler, S.E. (1997) J. Chromatogr. A. 786, (11) Scarlata, C.S., & Ebeler, S.E. (1999) J. Agric. Food Chem. 47, (12) Hayasaka, E., & Bartowsky, E.J. (1999) J. Agric. Food Chem. 47, (13) Louch, D., Motlagh, S., & Pawliszyn, J. (1992) Anal. Chem. 64, (14) Bucholz, K.D., & Pawliszyn, J. (1994) Anal. Chem. 66, (15) Field, J.A., Nickerson, G., James, D.D., & Heider, C. (1996) J. Agric. Food Chem. 44, (16) Song, J., Gardner, B., Holland, J., & Beaudry, R. (1997) J. Agric. Food Chem. 45, (17) Bauer, K., Garbe, D., & Surburg, H. (1990) in Common Fragrance and Flavor Materials, VCH, Weinheim, Germany, 218 pp (18) Takeoka, G., Flath, R.A., Mon, T.R., Buttery, R.G., Teranishi, R., Güntert, M., Lautamo, R., & Szejtli, J. (1990) J. High Resol. Chromatogr. 13, (19) Berger, R.G. (1991) in Volatile Compounds in Foods and Beverages, H. Maarse (Ed.), Marcel Dekker, Inc., New York, NY, pp (20) Latrasse, A. (1991) in Volatile Compounds in Foods and Beverages, H. Maarse (Ed.), Marcel Dekker, Inc., New York, NY, pp (21) Ebeler, S.E., Sun, G.M., Vickers, A.K., & Stremple, P. (2000) in Aroma Active Compounds in Foods and Beverages G.R. Takeoka, M. Güntert, & K.-H. Engel (Eds), American Chemical Society, Washington, DC, in press (22) Rapp, A. (1988) in Wine Analysis, Modern Methods of Plant Analysis, H.F. Linskens & J.F. Jackson (Eds), Springer-Verlag, Berlin, Germany, pp 29 66