Ultrasound-assisted emulsification–microextraction for the sensitive determination of Brett character responsible compounds in wines

Abstract

In this study, a method based on ultrasound-assisted emulsification–microextraction (USAEME) was optimised for the determination of Brett character responsible compounds (4-ethylguaiacol (EG), 4-ethylphenol (EP), 4-vinylguaiacol (VG) and 4-vinylphenol (VP)) in wines. For this purpose, once the extraction solvent had been selected, the influence on the extraction efficiency of ratio between extraction solvent and sample volumes, temperature, ionic strength and time was studied using experimental design methodology. The optimum conditions found for USAEME of 5 mL of wine were 160 μL of chlorobenzene at 60 °C during 5 min without salt addition. Then, the analytical performance of the optimised USAEME procedure was evaluated. For all the studied compounds, the proposed method showed correlation coefficients over 0.984. Satisfactory repeatability (below 10%) and inter-day repeatability (below 11%) were obtained at different concentration levels and the method achieved detection limits similar or even lower than previously reported for these compounds in wines. Moreover, the applicability of the method was evaluated by the analysis of real samples. To our knowledge, this is the first time that USAEME has been applied to the determination of Brett character responsible compounds in wine.

Introduction

Food product quality is an issue of increasing importance for consumers. One of the key parameters in determining the quality of a wine is its aroma. The appearance of compounds which reduce the sensory quality of wine can cause serious economical losses to the enological industry. Bearing this in mind, studies focused on quality assurance are continually appearing with the development of new analytical techniques focused on the determination of compounds responsible for undesirable aroma in wine. Some of the most common compounds which may reduce wine quality are the volatile phenols, 4-ethylphenol (EP), 4-ethylguaiacol (EG), 4-vinylphenol (VP) and 4-vinylguaiacol (VG). These compounds are usually present in wine positively contributing to the wine aroma complexity. Vinylphenols appear in wines from the descarboxylation reaction of hydroxycinnamic acids produced by yeasts during fermentation [1], [2], [3], [4], [5]. In addition, the yeasts Brettanomyces/Dekkera may develop after fermentation performing the reduction of the vinylphenols to ethylphenols [6], [7], [8], [9]. High concentration of vinylphenols, whose occurrence is higher in white wines, gives the wine medicinal and spicy odours. On the other hand, ethylphenols are usually present in higher concentrations in red wines giving them phenolic and animal odours [8], [9], [10].

The determination of Brett character responsible compounds in wine is mostly accomplished by gas chromatography (GC). Due to the high complexity of the matrix, sample preparation is a key step in the analytical procedures previous to the determination. Traditionally, liquid–liquid extraction methods with organic solvents were employed for sample preparation for the determination of volatile phenols [11], [12], [13], [14]. However, with increasing concerns in miniaturisation and sample, solvent and time savings, new procedures such as solid-phase extraction (SPE) [15], [16], [17], solid-phase microextraction (SPME) [18], [19], [20], [21] and stir bar sorptive extraction (SBSE) [22], [23] have been developed.

In recent years, a new economical, simple and fast microextraction procedure, named dispersive liquid–liquid microextraction (DLLME), has been satisfactorily applied to the determination of off-flavor responsible compounds in wines such as volatile phenols, haloanisoles and halophenols [24], [25], [26]. DLLME method consists in the rapid injection of an appropriate mixture of an extraction solvent and a disperser solvent into an aqueous sample, thus forming a cloudy solution in which the extraction solvent is dispersed throughout the sample [27]. However, the presence of the disperser solvent, necessary for the correct formation of the dispersion, can diminish the partition coefficient of analytes between the sample and the extraction solvent [28]. Taking this into account, a new approach in this technique, which uses ultrasonic radiation to achieve the dispersion, has been recently proposed [29].

The application of ultrasounds in liquids produces cavitation phenomena, that is, the formation of microbubbles that finally collapse [30], [31]. When the liquid is a mixture of two immiscible phases the result of the cavitation and collapse is an emulsion of the minority phase in the bulk. The cavitational collapse also produces strong variations of pressure and temperature in the interface which may affect the exchange of analytes between the phases [29], [30], [32]. Moreover, ultrasonic radiation produces a homogenisation of the sample [31], [33]. Taking this into account, ultrasound assisted emulsification–microextraction (USAEME) method avoids the use of the third component and takes advantage of the positive effects of ultrasounds in the liquid–liquid extraction procedures. This enables the achievement of more sensitive methods reducing the use of organic solvents and using clean energies such as ultrasonic radiation, in line with the growing trend of environmentally friendly chemistry. Since it was proposed, applications of USAEME in the analyses of several compounds such as pesticides, phenolic preservatives or 2,4,6-trichloroanisole in water have been reported [29], [34], [35]. In wine, USAEME has been successfully applied in the determination of 2,4,6-trichloroanisole, geosmin and 2-methylisoborneol [36], [37].

Therefore, in the present work, USAEME method was optimised for the determination of volatile phenols in wine by GC–tandem mass spectrometry (MS/MS). To perform the optimisation, once the extraction solvent had been selected, the influence of the volume ratio (V_extractant/V_sample), temperature, time and salt addition was evaluated using experimental design methodology. The optimised USAEME method was evaluated and its quality parameters were established. Then, its applicability was evaluated by analysing real wine samples. To our knowledge, this is the first time that USAEME has been proposed as a simple, fast and sensitive method for the determination of Brett character responsible compounds in wine.