Elsevier

Analytica Chimica Acta

Volume 683, Issue 1, 17 December 2010, Pages 126-135
Analytica Chimica Acta

Optimization of ultrasound assisted-emulsification-dispersive liquid–liquid microextraction by experimental design methodologies for the determination of sulfur compounds in wines by gas chromatography–mass spectrometry

https://doi.org/10.1016/j.aca.2010.10.010Get rights and content

Abstract

A new method was developed for analyzing sulfur compounds in the aroma of white wines using ultrasound assisted-emulsification-dispersive liquid–liquid microextraction coupled with gas chromatography–mass spectrometry detection. In the present work, the analytical method for simultaneous determination of seven sulfur compounds (methylmercaptoacetate, methyl(methylthio)acetate, 2-methylthioethanol, 3-methylthiopropanol, 3-methylthiohexanol, 4-methylthio-4-methyl-2-pentanone and hexanethiol) is reported. Parameters that affect the efficiency of the methodology such as extracting and dispersing solvents, sample volume, ion strength, cavitation time and centrifugation time were investigated using a fractionated factorial 26–1 (R = V) screening design. Then, the factors presenting significant positive effects on the analytical response (extracting volume, ion strength, cavitation time and centrifugation time) were considered in a further central composite design to optimize the operational conditions for the ultrasound assisted-emulsification-dispersive liquid–liquid microextraction procedure. Additionally, multiple response simultaneous optimization by using the desirability function was used to find the optimum experimental conditions. The best results were obtained using pH sample 4.25, extractant volume 150 μL, ionic strength 8.75% NaCl, cavitation time 20 s and centrifugation time 50 s. The use of the optimized ultrasound assisted-emulsification-dispersive liquid–liquid microextraction technique allowed to obtain the best extraction results with the minimum interference from other substances from the matrix, and it allowed to quantify the analytes in white wine samples by calibration graphs. Recoveries ranging from 91.99% to 125.87% for all sulfur compounds proved the accuracy of the proposed method in white wine samples. Method detection limits were in the range of 0.36–1.67 ng mL−1 and limits of quantitation were between 0.63 and 3.02 ng mL−1 for sulfur compounds in white wine samples. The proposed methodology was successfully applied for the determination concentrations of sulfur compounds in different commercial Chardonnay wine samples from Mendoza, Argentine.

Introduction

Wine is not only considered as one of the oldest beverages of the world, it may be also the beverage with the most sophisticated diversity. Wine is a hidroalcoholic solution containing hundreds of compounds; this complexity originates from three major sources: the raw material, which originates from thousands of grape varieties growing on a wide array of geological formations in different climates and altitudes, the fermentation process accomplished by a multitude of yeast and malolactic bacteria species and strains, and the ageing process, which varies owing to different storage methods, container size and material, such as oak barrels of varying origin, but also owing to stocking time, which may range from a few weeks to more than several decades [1].

Flavour is one of the most important parameters of wine quality, since the interaction of aromatic substances with the senses of smell and taste leads to consumer acceptance or rejection. Sulfur compounds have a great sensorial impact and play an important role in flavour of wines. Despite their structure, these compounds are classified in different families: thiol, sulphides, polysulphides, thioesters and heterocyclic compounds [2]. A variety of sensory impressions are possible for wines depending on their unique concentrations, their aromatic properties and synergist–antagonist effect [3]. In general, these compounds are associated to off-flavour because these are described in wines like cauliflower, potato, cabbage, onion, rubber, etc. [4], [5]. Nevertheless, there are some sulfur compounds which are typical of some varieties which contribute to varietal aroma of these wines [6], [7], [8], [9], [10], [11].

There is therefore a growing interest in developing more sensitive and selective analytical methods to analyze sulfur compounds in wine. Nevertheless, few analytical methods for quantifying volatile or semivolatile thiols in wine have been developed. Analyzing these compounds in wine is a great challenge due to the following facts: the complexity of the sample matrix, the low concentration levels (below ng mL−1), and the highly reactive nature of these compounds owing to their oxidation tendency during extraction [12], [13], [14].

The most commonly used methods are based on the liquid–liquid extraction (LLE) of sulfur compounds from wine with an azeotropic mixture [9], [10], [15], or extraction followed by the formation of a reversible complex of thiols with p-hydroxymercurybenzoate [11], [15] prior to gas-chromatography. Although these methods result in very clean extracts with high concentration factors and good sensitivity, they require large volumes of samples and organic solvents and, some one of them, use carcinogenic mercuric derivatives. Moreover, they are often complicated and numerous steps are needed thus increasing the risk of compound loss [16].

Solid-phase micro-extraction (SPME) is a solvent-free extraction technique which combines extraction and preconcentration in one step. Different authors have been studied diverse conditions in order to analyze sulfur compounds from wine samples [17], [18], [19]. The SPME technique has been associated with chemical derivatization for determination of volatile and semivolatile sulfur compounds in wines [20], [21], [22]. All the described methods that use SPME are too expensive and require well-trained analysts. Consequently, they have a restrictive application in the routine analyses to determine sulfur compounds in wine in order to control wine quality or winemaking process.

LPME is a solvent-miniaturized procedure of LLE, compatible with capillary gas chromatography, capillary electrophoresis and liquid chromatography [23], [24]. The single drop microextraction (SDME) method of sample preparation was initially proposed by Jeannot and Cantwell [25]. This technique, which uses a liquid microdrop with an appropriate density, surface-tension and vapor pressure, requires high manual dexterity [26]. The reproducibility of this method is often poor due to the instability of the microdrop that can easily be dislodged from the tip of microsyringe needle. The latter generates inconveniences to reach the thermodynamic equilibrium and, in the worst in the cases, it results in a failed experiment [23].

Dispersive liquid–liquid microextraction (DLLME) is a fairly new method of sample preparation, initially proposed by Rezaee et al. [27]. DLLME is a miniaturized LLE that uses microliter volumes of extraction solvent. This method is based on a ternary component solvent system in which the extraction solvent and disperser solvent are injected into aqueous sample by syringe. The mixture is shaken and a cloudy solution is formed in the test tube. After centrifugation, the extract is taken with a micro syringe and analyzed. The advantages of DLLME are simplicity of operation, rapidness, low cost, high recovery and robustness, high enrichment factors, and environmental benignity [27], [28].

On the other hand, the application of ultrasonic radiation is a powerful aid in the acceleration of various steps of the analytical process, therefore ultrasound assisted liquid–liquid extraction (USALLE) has been used as an alternative to conventional LLE [29], [30]. Regarding liquid–liquid extraction, the main effects of ultrasounds can be summarized as follows: an enormously extended contact surface between both phases to form emulsions with submicron droplet size, the homogenization of the external phase by the action of acoustic flows, and the momentary and localized strong increments of pressure and temperature [30], [31]. In this way, ultrasound assisted emulsification–microextraction (USAEME) can be employed as a simple and efficient extraction and preconcentration procedure for organic compounds in aqueous samples [32].

To best of our knowledge, there is no report on the use of USAEME for the analysis of SCs in wine. The aim of the current work was to develop a fast and sensitive method for the extraction by USAEME and determination by gas chromatography–mass spectrometry (GC–MS) with pressure pulse injection of sulfur compounds (SCs) in white wine samples. Taking into account that a considerable number of variables can affect the extraction yield in the USAEME procedure, and they may also be correlated, optimization was carried out through a multivariate approach. Firstly, a fractionated fractional design was employed as a screening step for the main parameters affecting the extraction and preconcentration processes and then, a central composite design (CCD) was used to optimize the values of the significant variables in order to obtain the best responses. The multiple response criterium was successfully used to optimize peak areas of analytes and the analysis time using the desirability function. The optimized procedure was successfully applied to the determination of SCs in commercial white wines from Mendoza, Argentine.

Section snippets

Standards, reagents and samples

The sulfur compounds studied were: 2-mercaptoethanol [60–24–2] supplied by Fluka (Buchs, Switzerland), while methylmercaptoacetate [2365–48–2], 3-methylthiopropanol [505–10–2], 3-methylthiohexanol [51755–66–9], 4-methylthio-4-methyl-2-pentanone [23550–40–5], hexanethiol [111–31–9], 2-methylthioethanol [5271–38–5], and the internal standard (I.S.) methyl(methylthio)acetate [16630–66–3] were purchased from Aldrich (Milwaukee, WIS, USA). Individual methanolic stock solutions of each SCs were made.

Results and discussion

The efficiency in dispersive liquid–liquid microextraction (DLLME) can be influenced by several variables, such as type and volume of disperser and extractant solvents, ionic strength, pH and volume of sample, way and time of emulsification, centrifugation time, etc. [28], [35], [36]. One major challenge in the utilization of microextraction is the selection of experimental conditions that can provide acceptable response at low analyte concentration. The microextraction requires handling small

Conclusions

Ultrasound assisted-emulsification-dispersive liquid–liquid microextraction coupled to gas chromatography–mass spectrometry provides a simple, efficient and selective methodology for the determination of sulfur compounds in white wine samples. Also, this methodology is characterized by its short-time and low cost of the analysis. The multivariate optimization strategy used, i.e. experimental design and response surface methodology enhanced by the application of desirability function, allowed

Acknowledgements

This work was supported by the National Institute of Agropecuary Technology (INTA) through the projects AETA282821 and PNFRU053941.

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