Headspace solid-phase microextraction–gas chromatography–mass spectrometry for profiling free volatile compounds in Cabernet Sauvignon grapes and wines
Introduction
Grape aroma is comprised of a large number of volatile compounds including alcohols, esters, acids, terpenes, norisoprenoids, thiols, and carbonyl compounds. These aroma compounds are predominantly localized in the exocarp (skin) tissue and many are typically stored as sugar or amino acid conjugates in the vacuoles of the exocarp cells [1]. The skins contain more than half of the total volatile compounds present in grape berries [2], [3]. During winemaking, the “free” aroma compounds are released as a result of physical crushing and subsequent chemical and enzymatic hydrolysis of the conjugated volatiles by grape, yeast, and/or industrial enzymes (glycosidases or peptidases) [4].
The volatile composition of grapes is one of the most important factors determining wine character and quality [5]. However, there have been few studies linking volatile composition in grapes to the final volatile composition in the wine [6]. These limitations are due, in part, to the lack of analytical methods that allow for rapid screening or profiling of multiple volatile compounds that are present at a wide range of concentrations in both grapes and wines.
Previously, volatile aroma analysis relied on distillation, solvent extraction (e.g., pentane/diethyl ether, dichloromethane, Freon 11), or concentration on solid-phase supports (i.e., solid-phase extraction) to isolate and concentrate aroma compounds [7], [8], [9], [10]. These methods are time consuming, result in extensive solvent waste and solvent costs, and can result in losses of some important volatiles depending on solvent selectivity and volatility. In addition, liquid–liquid extractions frequently require heating the sample, which can result in degradation and artifact formation.
Headspace analysis (both static and dynamic) has been widely used for analysis of grape and wine volatiles [11], [12], [13]. However, static headspace analysis often suffers from poor sensitivity for trace volatiles and dynamic headspace analysis suffers from interferences from water and ethanol [14], [15], [16].
Solid-phase microextraction (SPME) is now widely used for analysis of aroma volatiles in many food and beverage matrices [7]. SPME is a solventless sampling technique that can be faster and easier than solvent extractions and distillations, as well as being highly reproducible and sensitive. A range of fiber coatings are commercially available, providing specificity for a wide range of polar, nonpolar, volatile, and semivolatile analytes. Typically, SPME applications have involved extraction of the volatiles in the headspace (HS-SPME) to avoid interferences from nonvolatile matrix components.
Numerous SPME applications for volatiles in wines have been reported (e.g., terpenes, 2,4,6-trichloroanisole, sulfur compounds, diacetyl, methoxypyrazines) [17], [18], [19], [20], [21], [22], [23]. A few HS-SPME methods have been developed for analysis of volatiles in grapes although most of these methods have focused on measuring volatiles in grape varieties where terpenes contribute significantly to the varietal character and dominate the headspace composition (e.g., Muscat, Fernao-Pires, Baga) [24], [25], [26]. Neutral grapes, such as Cabernet Sauvignon, present significant analytical challenges due to the fact that the aroma compounds are present in low concentrations with norisoprenoids, esters, alcohols and aldehydes constituting the majority of the volatiles. There have been no applications of HS-SPME for profiling aroma volatiles in Cabernet Sauvignon grapes.
Analytical methods that would allow aroma profiles to be characterized in both grapes and wines would allow for an improved understanding of how both viticultural and winemaking processes impact volatile composition. Coelho et al. [26] used HS-SPME to monitor volatile formation during maturation of Fernao-Pires grapes but did not relate the volatile composition to sensory properties or follow the composition through winemaking. Fang and Qian [27] successfully measured 33 different compounds in Pinot noir wines of different maturity using a stir bar sorptive extraction technique (this extraction is related to HS-SPME, but the extracting polymer is placed in the liquid phase rather than the headspace and requires modifications to the GC inlet for automated sampling). These authors were able to relate changes in wine composition to differing fruit maturities, but they did not measure volatiles in the grapes to determine how varietal aroma composition may have changed during fermentation.
The aim of this study was to develop a procedure using GC–MS combined with HS-SPME for rapidly profiling the free volatile compounds in Cabernet Sauvignon. We show an application of the optimized method for monitoring changes in volatile composition in grapes harvested at different maturity levels. The same method was further applied to Cabernet Sauvignon wines produced from the same grapes harvested at different maturity levels in order to show the applicability of the method for monitoring selected grape volatiles through the winemaking process.
Section snippets
Grapes and wines
All grapes and wines were donated by J. Lohr Vineyards and Wines (Paso Robles, CA, USA) and were from the 2006 vintage. The grapes were harvested at six different maturity levels (H1–H6; Table 1). Six rows were sampled per harvest and from each row every tenth plant was sampled. Two clusters from the western side of the vine were taken: the first sample was taken closest to the trunk and the second sample was taken from the last cluster on the cordon. After harvest, fruit was immediately
Results and discussion
SPME has become a widely used sample preparation method for volatiles analysis because it eliminates the use of organic solvents, substantially shortens the total extraction time compared to solvent extractions (SPME extractions are typically ≤30 min compared to several hours for liquid–liquid extractions), and allows for convenient automation of the sample extraction step. As with traditional extraction techniques, the development of successful SPME methods requires careful consideration of the
Concluding remarks
We developed a headspace GC–MS method that can be used to profile and quantify 27 free volatile compounds in the headspace of Cabernet Sauvignon fruit. The method is reproducible (RSD < 15%) and relatively rapid (15–20 samples/day). Using the method we were able to observe significant changes in free volatile composition during grape maturation. Selected compounds in the grapes could also be identified in the corresponding wines. The method can now be applied to monitor effects of viticultural and
Acknowledgements
This project was partially funded with support from the American Vineyard Foundation, the California Competitive Grant Program for Research in Viticulture and Enology and the Viticulture Consortium. We thank J. Lohr Vineyards and Wines and Jerry Lohr for donation of the grapes and wines and assistance with this project.
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