The Analytical Science Behind Winemaking

LCGC spoke to Rosa Perestrelo of Centro de Química da Madeira located in Madeira University (Portugal) about wine analysis. She reveals the science behind winemaking and offers her tips for selecting the best analytical method for analysis.

Q. What are your main research interests? How did you become involved in these areas?A: My main research interests are related to Madeira wine, because this product has a significant impact on the Madeira economy. Over the last few years, I have focused on the characterization of the volatile profile of Madeira wine obtained from noble grape varieties (for example, Malvasia, Bual, Verdelho, and Sercial), as well as Tinta Negra, which is not considered a noble variety but represents about 80% of total production. My recent research in this field has been related to the development of rapid, simple, and inexpensive analytical methods to identify volatile compounds that might be potential wine ageing markers (such as furan compounds and lactones), as well as Madeira wine quality markers to ensure its authenticity and to avoid potential fraud.

My involvement in the oenology field came out of the proposal for my bachelor thesis, which involved collaborating with local businesses, such as Instituto do Vinho do Bordado e Artesanato da Madeira (IVBAM). This collaboration allowed me to have direct contact with producers, increasing my knowledge in viticulture and thus combining the analytical part with oenology.

Q. How has the field of wine analysis developed over the years?A: Wine analysis has evolved immensely over recent years, as I will demonstrate with Madeira wine. The qualification of wines is normally assessed by physicochemical and sensorial parameters. As far as we know, Nogueira and Nascimento (1) were the first authors to characterize these parameters for a large set of Madeira wine samples from different Vitis vinifera L. grapes according to standard methodology and in agreement with the European Union regulation for commercialization (EU Nº 4252, 1988). After this, several works have been developed to obtain the aroma profile of Madeira wine produced from Vitis vinifera L. grape varieties, since the aroma is the most important organoleptic characteristic and a key attribute to consumers. This resulted in identifying about 300 chemically different volatile compounds belonging to a variety of different chemical groups. Câmara et al. (2,3) then focused on the development of analytical methods to establish potential wine ageing markers, such as heterocyclic acetals and furanic compounds. More recently, Pereira et al. (4) used statistical tools to obtain models to ascertain more robust information and ensure the authenticity of Madeira wines. Using different methodologies, Perestrelo et al. (5) obtained more complete information in this field and in a shorter analysis time.

The first study on Madeira wines in the sensorial field was performed by Campo et al. (6), and these authors concluded that Madeira wine sensorial proprieties are highly dependent on the winemaking process, particularly the Estufagem process. In Estufagem, also called the baking process, the wine is placed in large coated vats, and the temperature is slowly increased (about 5 ºC per day) and maintained at 45–50 ºC for at least for 3 months. Finally, some wines are submitted to an ageing process in oak casks, from a minimum of 3 years to 20 years, or even longer. Pereira et al. (7) have performed several studies focused on storage time and temperature to achieve the optimal Estufagem conditions to improve Madeira wine sensorial properties.

The health benefits of wine explain the growth of interest in the characterization and evaluation of phenolics and the antioxidant capacity. Regarding Madeira wines, some studies have been performed to quantify the phenolics using different analytical methods (8,9). The latest studies on Madeira wine have focused on developing fast analytical tools to detect potential undesirable compounds, such as biogenic amines and ethyl carbamate (10,11).

It can be verified that there has been a remarkable evolution in wine analysis, as demonstrated by Madeira wine, going from empiric information to a more profound knowledge of this matrix. This evolution in wine analysis has identified some issues that will be addressed going forwards. For example, the grape varietal profile can be taken into account in the winemaking process to maximize the aroma profile that may result in a greater aromatic complexity. Another area that might be improved is the control of the Estufagem process conditions to avoid the formation of undesirable compounds such as ethyl carbamate.

Q. What are the factors that need to be considered when selecting an analytical method for wine analysis? Do you have a preferred method of analysis?A: The main factor in choosing an analytical method for wine analysis resides in the analytical target. Criteria such as accuracy, precision, sensitivity, selectivity, robustness, ruggedness, operation scale, analysis time, sample preparation, equipment availability, and cost should be considered. These parameters have to be taken into account since the outcome can be influenced, for example by the presence of some interference compounds. Moreover, nowadays the time of analysis is an important factor in choosing an analytical method because it is crucial to analyze the highest number of samples in the shortest time with suitable analytical performance. Moreover, it is important to select extraction techniques that involve less organic solvents, as well as extraction steps to make a “greener” method.

Relative to the preferred method of analysis, I opted to use easy, simple, automated, inexpensive techniques such as solid-phase microextraction (SPME) and microextraction by packed sorbent (MEPS). As for the analytical tools, I currently use ultrahigh-performance liquid chromatography (UHPLC), liquid chromatography coupled to mass spectrometry (LC–MS) and comprehensive two-dimensional gas chromatography coupled to a time of flight mass spectrometer (GC×GC–ToF-MS) because these techniques are in consonance with the previously reported criteria.

Q. Do different varieties of grape present different analytical challenges?A: The different grape varieties, in my experience, do not present analytical challenges. Nevertheless, it could depend on the kind of target compounds. For example, if we are focused on the glycosidically bound volatiles, this could present an analytical challenge because it is related to the sugar content.

Q. You have also researched the establishment of furanic derivatives fingerprint of fortified wines using MEPS combined with reversed-phase UHPLC. Could you talk a little about this research? What analytical challenges did you face in this work, and how did you overcome them?A: Furanic derivatives are a group of compounds that are formed during nonenzymatic browning reactions, such as caramelization, which involves the degradation of sugars, and the Maillard reaction, involving Amadori rearrangement (7), and could be used as potential wine ageing markers (5). The work represents a new, reliable, and powerful analytical method based on MEPS combined with reversed-phase UHPLC as a strategy for the simultaneous determination of furanic derivatives. According to the validation parameters, this is a suitable tool which combines sensitivity, effectiveness, reduces analysis time (4 min), and is a simple analytical procedure. However, before validation, several influencing extraction parameters, such as MEPS sorbent, number of cycles, and elution solvent and volume were evaluated and optimized to improve the extraction efficiency of the target furanic derivatives. Moreover, this analytical method combined with principal component analysis (PCA) allows the studied wines from different vintages and varieties to be organized by age.

The main difficulty in this study concerned the validation step because different sugar contents are observed in different types of Madeira wine. In sweet/medium sweet wines the sugar content range from 65 to 150 g/L, while the dry/medium dry from 49 to 96 g/L. To solve this issue two calibration curves were built using model synthetic wine with different glucose content to represent these wine types. This work is already completed and it could be verified that the sugar content affects the detection and quantification limit as well as the recoveries. This was also verified in the ethyl carbamate quantification (10).

Q. Where will your research in wine analysis take you in the future? A: The research I have performed in wine analysis has resulted in a deep knowledge on this matrix. This background has allowed me to find the strengths and weaknesses from the harvest process to the final product. For example, the work developed in the establishment of the varietal profile of Vitis Vinifera L. grape varieties offers the winemaker the possibility to improve the organoleptic characteristics of Madeira wines. The vinification and ageing process is an important stage for the organoleptic and sensorial properties of Madeira wine, which make this a renowned product. Nevertheless, in this process undesirable compounds, such as ethyl carbamate, can be formed. According to the legislation, the limit for this compound is 100 μg/L. I envision that in the future we can maximize the extraction of varietal compounds from the grapes and control the conditions used in the winemaking process to overcome the potential issues that result in undesirable compounds in Madeira wine.

Acknowledgements
I would like to thankLCGC for this wonderful opportunity to share the work developed over the last years in wine and winemaking process. I also wish to acknowledge Centro de Química da Madeira and Madeira University for providing the conditions to perform the experimental work and the scientific environment necessary to develop and expand my scientific knowledge.

References

(1) J.M.F. Nogueira and A.M.D. Nascimento, J. Agric. Food Chem. 47, 566–575 (1999).
(2) J.S. Câmara, M.A. Alves, and J.C. Marques, Anal. Chim. Acta 563, 188–197 (2006).
(3) J.S. Câmara, J.C. Marques, A. Alves, abd A.C.S. Ferreira, Anal. Bioanal. Chem. 375, 1221–1224 (2003).
(4) A.C. Pereira, M.S. Reis, P.M. Saraiva, and J.C. Marques, in Computer Aided Chemical Engineering, S. Pierucci and G.B. Ferraris, Eds. (Elsevier, 2010) pp. 247–252.
(5) R. Perestrelo, A.S. Barros, J.S. Câmara, and S.M. Rocha, J. Agric. Food Chem. 59, 3186–3204 (2011).
(6) E. Campo, V. Ferreira, A. Escudero, J.C. Marqués, and J. Cacho, Analytica Chimica Acta 563, 180–187 (2006).
(7) V. Pereira, F.M. Albuquerque, A.C. Ferreira, J. Cacho, and J.C. Marques, Food Res. Int. 44, 71–76 (2011).
(8) C.L. Silva, J.L. Gonçalves, and J.S. Câmara, Anal. Chim. Acta 739, 89–98 (2012).
(9) V. Pereira, J.S. Câmara, J. Cacho,and J.C. Marques, J. Sep. Sci. 33, 1204–1215 (2010).
(10) R. Perestrelo, S. Petronilho, J.S. Câmara, and S.M. Rocha, J. Chromatogr. A 1217, 3441–3445 (2010).
(11) V. Pereira, M. Pontes, J.S. Câmara, and J.C. Marques, J. Chromatogr. A 1189, 435–443 (2008).

Rosa Perestrelo

was born in 1982 in Madeira Island (Portugal). She studied chemistry at Madeira University and completed a master in green chemistry at the Faculty of Sciences of Lisbon University (both in Portugal). She completed her PhD in 2013, where she evaluated the potential of

Vitis vinifera

L. grapes used to produce Madeira wine. Her research focuses on the detailed characterization of several food matrices (for example, fruits, wine). Another recent area of interest is the establishment of the metabolic profile of cancer patients using several extraction techniques (SPME, MEPS) combined with different chromatographic technique, such as liquid chromatography tandem mass spectrometry (LC–MSn), gas chromatography coupled to mass spectrometry (GC–MS), and comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC×GC–ToF-MS). Currently, she works as liquid chromatography–mass spectrometry technician at Madeira University.