Elsevier

Analytica Chimica Acta

Volume 701, Issue 1, 2 September 2011, Pages 98-111
Analytica Chimica Acta

Solid phase extraction in combination with comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry for the detailed investigation of volatiles in South African red wines

https://doi.org/10.1016/j.aca.2011.06.006Get rights and content

Abstract

Comprehensive two-dimensional gas chromatography in combination with time-of-flight mass spectrometry (GC × GC–TOFMS) has been applied for the analysis of volatile compounds in three young South African red wines. In spite of the significant benefits offered by GC × GC–TOFMS for the separation and identification of volatiles in such a complex matrix, previous results utilizing headspace solid phase micro extraction (HS-SPME) demonstrated certain limitations. These were primarily associated with the choice of sample preparation technique, which failed to extract some influential semi-volatile wine constituents. Therefore, in the current report, we utilized solid phase extraction (SPE) in combination with GC × GC–TOFMS for the detailed investigation of particularly low-level semi-volatiles in South African wine. 214 compounds previously reported in grapes and related beverages were tentatively identified based on mass spectral data and retention indices, while 62 additional compounds were positively identified using authentic standards. The method proved particularly beneficial for the analysis of terpenes, lactones and volatile phenols, and allowed us to report the presence of numerous volatile compounds for the first time in Pinotage wines.

Highlights

• A new SPE–GC × GC–TOFMS method for the analysis of wine volatiles is described. • A detailed comparison with HS-SPME–GC × GC–TOFMS is presented. • SPE–GC × GC–TOFMS beneficial for terpenes, volatile phenols and lactones. • Identification by TOFMS is improved by removal of most polar volatiles during SPE. • 276 compounds were identified, many for the first time in Pinotage wines.

Introduction

In excess of 800 volatile compounds have been reported in wine [1]. The levels of these compounds depend on many enological and viticultural factors, and their combined effect determines wine aroma, which is an influential factor in quality assessment. Wine volatiles can broadly be sub-divided into three groups: impact odorants, major volatiles and off-flavors. The former class includes compounds with characteristic odor properties such as varietal compounds [2], which are linked to a specific grape cultivar. Impact odorant may also be imparted to the wine medium from external factors like wood [3] or evolve in wine via chemical reactions during winemaking, from crushing of the grape berries through fermentation, maturation and ageing [3], [4]. Major volatiles commonly exist at higher concentrations and do not contribute to the wine aroma individually, but rather collectively. These compounds are responsible for the so-called base aroma of wines. Most esters and alcohols are categorized as major volatiles, and are mainly produced during fermentation [4]. Off-flavors are volatile compounds associated with deterioration of wine aroma. These compounds could originate in various ways including bacterial spoilage and oxidation [4]. The aroma contribution of a specific compound may be positive and negative, depending on its concentration [5]. Considering the diverse factors that determine the quality and perception of wine aroma, analytical methods suitable for the assessment of the volatile compounds associated with wine aroma are essential.

The study of wine volatiles is normally performed using gas chromatography (GC). Conventional capillary GC (cGC) methods are characterized by high separation power, relatively wide applicability, simplicity and durability. Nevertheless, wine analysis represents a severe analytical challenge due to the large number of volatile compounds. Even the high peak capacity of cGC is not sufficient for the separation of all these compounds existing at different levels in a single sample [6].

Almost two decades since its first appearance in chromatographic literature [7], comprehensive two-dimensional gas chromatography (GC × GC) is receiving increasing attention for the analysis of complex samples. GC × GC is theoretically capable of producing improved separation of complex volatile mixtures due to the distribution of analytes over a retention plane created by two independent columns. The enhanced resolution is a result of unrelated (orthogonal) separation mechanisms provided by the two columns, resulting in much higher peak capacity. In addition, GC × GC is expected to provide more reliable identification of analytes, and highly structured patterns are often obtained due to the presence of two retention mechanisms [6], [8].

Sample pretreatment is an essential step in the analysis of wine volatiles, and several techniques have been described in the literature. Liquid–liquid extraction (LLE) has been extensively applied [1], [3], [9], [10], [11]. Yet, due to limited sensitivity, labor intensiveness, high cost and environmental concerns, LLE is gradually being replaced by alternative methods. Solid phase extraction (SPE) is increasingly being used for the extraction of wine volatiles [3], [9], [12], [13]. Advantages include higher selectivity and sensitivity, ease of automation, and reduced environmental risk. Sorptive-based sample preparation techniques such as solid phase micro extraction (SPME) [14] and stir bar sorptive extraction (SBSE) [15] have also been extensively applied for the investigation of wine volatiles [16], [17], [18], [19].

Despite significant advances, there is no universal sample preparation technique for wine volatiles, and methods are selected based on the analytical goals. Robinson et al. [20], [21] and Schmarr et al. [22] recently reported headspace (HS) SPME–GC × GC–TOFMS procedures for the separation and identification of a large number of wine volatiles. We recently used headspace solid phase micro extraction (HS-SPME) in combination with GC × GC–TOFMS to investigate Pinotage volatiles [23]. While this method provided significantly improved resolution and sensitivity and allowed identification of more than 200 compounds, HS-SPME showed limited application for some influential high-boiling aroma compounds. For example, lactones and volatile phenols were not detected in the previous study. In addition, the extraction of compounds present at high levels in wine, such as highly volatile polar compounds, resulted in tailing peaks, which obscured minor compounds. In order to overcome some of these shortcomings, we report a modified experimental procedure for the exploratory analysis of different classes of wine volatiles in the current work. A different column set and instrumental configuration was used, while the utility of SPE as complementary sample pretreatment technique in combination with GC × GC was investigated specifically for the identification of high-boiling, potentially influential wine volatiles. Work is currently in progress on the adaptation of this screening method for quantitative analysis of wine volatiles.

Section snippets

Chemicals and reagents

Two Pinotage and one Cabernet Sauvignon samples of vintage 2006, supplied by three South African producers, were analyzed. Methanol, ethanol and dichloromethane were obtained from Sigma–Aldrich (St. Louis, MO, USA). NaHCO3 was obtained from UNIVAR® (Helsinki, Finland). Water was purified by Milli-Q water purification system (Millipore, Bedford, MA, USA). Volatile standards (see Table 1) were purchased from Sigma–Aldrich, Fluka (Zwijndrecht, Netherlands), Riedel-de Haën (Steinheim, Germany), and

Results and discussion

The combination of comprehensive two-dimensional gas chromatography with high speed time-of-flight mass spectrometry has enabled us to identify (positively or tentatively) 276 compounds in the three young South African wines. Wine samples for the current study were selected in the first instance to allow comparison with previous results obtained by HS-SPME: the two Pinotage wines were the same wines analyzed before using the HS-SPME–GC × GC–TOFMS [23]. A Cabernet Sauvignon sample was also

Conclusions

While GC × GC–TOFMS as analytical technique has previously been shown to provide significant benefits in terms of the high-resolution and sensitive analysis of wine volatiles, the usefulness of the technique does depend on the sample preparation method for specific target analytes. In the present report, we have demonstrated that the use of SPE on reversed phase material offers several advantages. Primarily, major polar volatiles, which often complicate HS-SPME–GC × GC analyses, may easily be

Acknowledgements

The authors gratefully acknowledge LECO Africa (Dr. Peter Gorst-Allman and Alexander Whaley) for providing instrumentation and software. The authors also thank the Department of Chemistry at University of Pretoria for providing laboratory facilities and Ms. Y. Naudé for experimental support.

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    1

    Current address: Laboratory of Entomology, Wageningen University, PO Box 8031, 6700 EH Wageningen, The Netherlands.

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    Current address: The Faculty of Science, University of the Witwatersrand, Private Bag 3, WITS, 2050, South Africa.

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