Volatile Character

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Wine can be a very complex matrix to analyze. The Column spoke to Alexandre Pons from Seguin-Moreau in Cognac, France, about his work analyzing volatile compounds in wine and why gas chromatography coupled to tandem mass spectrometry (GC–MS/MS) is his method of choice.- Interview by Kate Mosford

Q. What role do volatile compounds play in wine?

A: In my research field it’s important to keep in mind that volatile compounds give wine its unique personal identity and character.

My current research focuses on the identification of volatile markers associated with the organoleptic quality of red and white wines aged in oak barrels. Indeed, wine ageing in oak barrels constitutes a crucial step in the winemaking process of high‑quality wines. These chemical markers were extracted from toasted oak wood or are produced thanks to chemical mechanisms, mainly oxidative, during wine ageing.

Very small quantities of these compounds may contribute to the complexity and subtlety of wine flavours, but may also be associated with “off‑flavours” that adversely affect quality and are likely to have major financial consequences.

A better understanding of the molecular cause and effect of the ageing process can lead to improved winemaking methods that will increase wine stability (taste and flavour) over a longer time and range of storage conditions.

Nowadays, there is a lot of science in a glass of wine!!

Q. What are the factors that need to be considered when selecting an analytical method for wine analysis? Why is gas chromatography coupled to tandem mass spectrometry
(GC–MS/MS) your method of choice?

A: Wine flavour consists of over 900 volatile compounds resulting from grape vines, yeast, and lactic bacteria metabolisms, and the chemical reactions that occur during ageing. A few of these compounds contribute directly to the flavour of wine (supra‑threshold concentrations). Typically, only about 8–15% of wine volatiles are aroma‑active compounds. Active compounds means that they contribute directly to wine flavour. Many others compounds constitute and contribute to the background noise (flavour of the matrix) but others contribute indirectly to the aroma of wine because of additive or synergetic effect. Compounds such as lactone or esters were demonstrated to contribute to the aroma of wines as families and not as individual component. The aroma-active compounds belong to several chemical families (alcohols, esters, aldehydes, ketones, pyrazine, benzene derivatives, or thiols) and many are found in a huge range of concentrations in wines, from sub-ng/L (benzenemethanethiol) to over 100 mg/L (fusel alcohol).

Considering the wine as a complex matrix the parameters are the same as for others analytical methods assaying trace impact compounds (ng/L): specificity, repeatability, sensitivity, accuracy, range, and precision. Of course, additional considerations such as time parameters (the time necessary to produce a value) are also very important. For all these reasons a tandem mass spectrometry (MS/MS) approach is very interesting. An MS/MS approach is a widespread technique when coupled to high performance liquid chromatography (HPLC) for fragmentation of ions of high m/z values. In the context of our studies, that is, the analysis of compounds with low molecular weight, we observed that MS/MS applied on ions with low m/z values (m/z <100) might also be very useful for certain compounds presenting very fragmented mass spectrum.

Q. What challenges are associated with using tandem mass spectrometry?

A: Liquid chromatography (LC) coupled with MS/MS is a well-established technology in the laboratory. Over recent years, methods such as gas chromatography coupled to tandem mass spectrometry (GC–MS/MS) have been adopted by many laboratories as essentials tools for identification and also quantification of trace volatile compounds in wines. In enology, the big challenge concerns the quantification of thiols compounds, that is, 3-sulfanylhexanol, 4-methyl-4-sulfanylpentan-2-one, furfurylthiol, etc. These compounds are very valuable for the flavour of wines but their quantification is very tricky because of their very low concentrations in wine (some as low as ng/L). The need to overcome peak chromatographic resolution issues during GC–MS analysis historically requires specific extraction conditions such as the reversible chelation between SH function and sodium p-hydroxymercuribenzoate. Very recently, we published a paper on the use of GC–MS/MS with a simple and non‑selective liquid–liquid extraction step for quantification of these thiols with a limit of detection (LOD) below 1 ng/L for some of them (1).

Q. You have identified vanillylthiol for the first time in young red and dry white wines. What analytical challenges did you face in this work?

A: When you try to identify a volatile compound by GC–O (olfactometry) and GC–MS based on its mass spectrum, the first and most important step is the interpretation of mass spectra. Indeed, we can use a database (NIST or FFNSC) but in certain cases volatile compounds are not known, or not described in nature; this was the case for vanillylthiol. We therefore developed hypotheses and then performed a synthesis of the chemicals to either accept or reject them. This process is clearly tricky and time consuming. Finally, you have to develop a quantification method to assay this compound in wines. Validation of the contribution of a volatile compound in a complex matrix requires very specific skills in sensorial analysis, chemistry, and analytical chemistry and of course the latest instrument technology for unknown trace compound identification. In this context GC–MS/MS is a very powerful tool for the analyst.

Q. Why has vanillylthiol not been identified in wine until now?

A: In fact, to our knowledge this is the first time that this thiol has been identified in nature and described precisely in the scientific literature (2). The context of the study was to shed light on the spicy aroma of wines stored for a long time in oak barrels. In enology it is well accepted that compounds such as eugenol or isoeugenol, which is reminiscent of clove with a concentration ranging from 10 to 50 µg/L, contribute to this aroma. These compounds are extracted directly from the toasted oak wood during ageing in barrels. Our study revealed that another compound presenting similar descriptors but a very different chemical structure, concentrations (some µg/L), and perception threshold is able to contribute to this flavour. The precursors of this new thiol are still unknown.

Q. Where will your research in wine analysis take you in the future?

A: For this work the next step concerns the identification of precursors of this thiol compound in order to be able to predict the ability of a process to produce this flavour in wine during barrel ageing. Moreover, based on these results I am developing a method using a comprehensive two-dimensional GC coupled to flame photometric detection‑MS (GC×GC–FPD-MS) approach, that is, nontargeted metabolite profiling, to determine the molecular signature of wines aged in oak
barrels.

References

1. C. Thibon et al., Journal of Chromatography A1415, 123–133 (2015).
2. M. Floch et al., J. Agric. Food Chem.64(6), 1318–1325 (2016).

Alexandre Pons studied chemistry and physics and later enology at the University of Bordeaux (UB) in France. In 2000 he graduated as an enologist (DNO) from the UB. In 2006 he obtained his Ph.D. in enology with Prof. D. Dubourdieu. For 13 years he has worked at Seguin‑Moreau cooperage (France) and also detached at the UB, on the identification of volatile compounds associated with the organoleptic quality of wines and oak wood. In 2007, his work concerning the study of the flavour of the premature aged white wines was awarded the great prize of the Amorim Academy. He is author or co-author of about 22 scientific publications.

E-mail: alexandre.pons@u-bordeaux.fr

Website: http://www.seguin-moreau.fr
               http://www.oenoresearch.univbordeauxsegalen.fr