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Characterization of Romanian Wines byGas Chromatography–Mass SpectrometryVeronica Avrama, Călin G. Floarea, Anamaria Hosub, Claudia

Cimpoiub, Constantin Măruţoiuc & Zaharie Moldovana

a Mass Spectrometry, Chromatography and Applied PhysicsDepartment, National Institute for Research and Development ofIsotopic and Molecular Technologies, Cluj-Napoca, Romaniab Faculty of Chemistry and Chemical Engineering, Babes-BolyaiUniversity, Cluj-Napoca, Romaniac Faculty of Orthodox Theology, Babes-Bolyai University, Cluj-Napoca, RomaniaAccepted author version posted online: 31 Dec 2014.Publishedonline: 09 Feb 2015.

To cite this article: Veronica Avram, Călin G. Floare, Anamaria Hosu, Claudia Cimpoiu,Constantin Măruţoiu & Zaharie Moldovan (2015) Characterization of Romanian Winesby Gas Chromatography–Mass Spectrometry, Analytical Letters, 48:7, 1099-1116, DOI:10.1080/00032719.2014.974054

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Analytical Letters, 48: 1099–1116, 2015Copyright © Taylor & Francis Group, LLCISSN: 0003-2719 print/1532-236X onlineDOI: 10.1080/00032719.2014.974054

Gas Chromatography

CHARACTERIZATION OF ROMANIAN WINES BY GASCHROMATOGRAPHY–MASS SPECTROMETRY

Veronica Avram,1 Călin G. Floare,1 Anamaria Hosu,2

Claudia Cimpoiu,2 Constantin Măruţoiu,3 andZaharie Moldovan11Mass Spectrometry, Chromatography and Applied Physics Department,National Institute for Research and Development of Isotopic and MolecularTechnologies, Cluj-Napoca, Romania2Faculty of Chemistry and Chemical Engineering, Babes-Bolyai University,Cluj-Napoca, Romania3Faculty of Orthodox Theology, Babes-Bolyai University, Cluj-Napoca,Romania

This article reports the determination of the volatile composition of commercial Romanianwines with the goal of identifying characteristic markers. For this purpose, 27 samples fromfour wine-producing areas from three consecutive years were investigated. Analysis wasperformed by gas chromatography–mass spectrometry with liquid–liquid extraction.Forty-eight volatile compounds were identified and characterized as alcohols, ethylesters, fatty acids, phenyl compounds, aldehydes, ketones, lactones, and terpenes as apercentage of the total area of volatile compounds. The results are discussed inrelationship to the grape variety, region, and year of production.

Keywords Gas chromatography; GC–MS; Volatile compounds; White wine

INTRODUCTION

The volatile composition of wine is one of the most important factors that deter-mine its aroma and therefore its quality (Selli et al. 2004; Gil et al. 2006; Câmara,Alves, and Marques 2006a; Coelho et al. 2008). The aromatic profile of wine resultsfrom the combined effects of several different natural compounds (Alves, Nascimento,and Nogueira 2005) following the interaction of the original components of the grapeand those produced during the process of wine making, fermentation, and aging(Gómez-Míguez et al. 2007). Consequently, the wine aroma has been classified intofour groups: aroma variety, characteristic to grape variety; prefermentative aroma

Received 8 July 2014; accepted 28 September 2014.Address correspondence to Zaharie Moldovan, National Institute for Research and Development

of Isotopic and Molecular Technologies, 65-103 Donath Street, 400293 Cluj-Napoca, Romania, E-mail:zaha@itim-cj.ro

Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lanl.

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derived during processing of grapes; fermentative aroma produced by yeast and bac-teria during alcoholic and malo-lactic fermentation; and postfermentative aromaresulting from the changes that occur during storage and aging of wine (Vilanova etal. 2010). The wine aroma also depends on the climate, region, viticulture practices,and physiological ripeness (Alves et al. 2005; Gómez García-Carpintero et al.2012a). The most abundant compounds responsible for the aroma of wine are thoseformed during alcoholic fermentation, i.e., alcohols, esters, acids, phenyl compounds,aldehydes, and ketones (Alves et al. 2005; Sánchez-Palomo et al. 2010). Identificationof these compounds and their relative concentrations can be a useful tool and providean isotopic fingerprint (Avram et al. 2014) for the characterization of wines with dif-ferent geographical origins based on a volatile fraction relationship established withgrape variety, origin, and processing technology used (Gil et al. 2006; Câmara et al.2006a; Welke et al. 2012). Due to their complexity and sometimes low concentrations(in some cases < microgram per liter), the use of preconcentration techniques areessential prior to analysis, performed usually by capillary gas chromatography coupledto mass spectrometry (GC–MS). Some of the most commonly used methods in thepreconcentration step include liquid–liquid extraction (LLE), solid phase extraction(SPE), and solid phase microextraction (SPME).

Although LLE is being replaced by more manageable and solvent-freetechniques, this type of extraction is still a reference method for the determinationof wine aroma compounds. The main advantages of this technique are its capacityto extract a wide range of compounds of different volatilities (as long as they havean affinity to the solvent), the high repeatability, and the ability to carrying outsimultaneous extractions (Andujar-Ortiz et al. 2009).

The objective of this work was to determine the volatile composition from aseries of commercial Romanian white wines from different regions to identify anduse some of the compounds as markers for geographical origin and processing tech-nology. The analysis was performed by GC–MS following liquid–liquid extraction.The results are discussed in relationship to the grape variety, region, and productionyear based on classes of compounds and lead the most comprehensive characteriza-tion of Romanian wines to date.

EXPERIMENTAL

Samples

Twenty-seven commercial white wines from the four most important wineregions in Romania were characterized: Oltenia (Oprişor and Vânju-MareVineyards), Muntenia (Cramele Halewood and Ceptura Vineyards), Moldova (HuşiVineyard), and Transylvania (Jidvei Vineyard). The wine varieties were SauvignonBlanc, Fetească Albă, and Riesling from 2008, 2009, and 2010 (Table 1).

Liquid–Liquid Extraction

The protocol used in this work for the extraction of volatile compounds fromwine samples was adapted from Andujar-Ortiz et al. (2009). The 25mL of wine and5mL of methylene chloride (Merck, Germany) were stirred at 0°C for 1 h. Sub-sequently, the mixture was kept for 15min in an ultrasonic bath at the same

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temperature to avoid formation of an emulsion. After separation, the organic phasewas collected and centrifuged. In order to remove water, it was filtered throughanhydrous sodium sulfate and concentrated by rotary evaporation to approximately200 µL. Subsequently 1 µL of extract was analyzed by GC–MS using the splitlessmode.

GC–MS

The wine samples were analyzed using a gas chromatograph (Trace GC)coupled with a mass spectrometer (Polaris Q, Thermo-Finnigan). An HP 5-MScapillary column was used with a methyl-phenyl siloxane class (5%) stationary phase(length of 30M, inner diameter of 0.25mM, thickness of stationary phase of0.25 µL). Helium was used as the carrier gas at a constant flow rate of 1.5 µL/min.The column temperature was programmed to be: initially, 50°C, maintained for2min, and then increased at 10°C/min to 300°C where it was maintained for10min. The temperature of the injector was 250°C. The mass spectrometer wasequipped with an electron impact ionization source (EI) at an ionization energyof 70 electronvolt. The temperatures at the interface and at the ion source were300°C and 250°C, respectively. Acquisition was performed in the full scan modefrom 50 to 650 amu.

Table 1. White wine samples

NumberWine

varieties Wine regionProduction

year

1 SauvignonBlanc

Muntenia (Vineyard I) (Cramele Halewood, DealurileMunteniei)

20082 20093 20104 Oltenia (Vineyard I) (Oprişor, Dealurile Olteniei) 20085 20096 20107 Oltenia (Vineyard II) (Mehedinţi,Vânju-Mare) 20088 20099 201010 Fetească

AlbăMoldova (Dealurile Huşilor) 2008

11 200912 201013 Muntenia (Vineyard I) (Cramele Halewood, Dealurile

Munteniei)2008

14 200915 201016 Muntenia (Vineyard II) (Ceptura, Dealurile Munteniei) 200817 200918 201019 Riesling Muntenia (Vineyard II) (Ceptura, Dealurile Munteniei) 200820 200921 201022 Oltenia (Vineyard II) (Mehedinţi, Vânju-Mare) 200823 200924 201025 Transylvania (Jidvei) 200826 200927 2010

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RESULTS AND DISCUSSION

Separation by GC–MS system was performed after optimization of the con-ditions. Figure 1 shows the analysis of Fetească Albă from Muntenia. Characteristicchromatograms of 4-vinylguaiacol with the selected ion m/z¼ 135 (Figure 1b) andof vanillin derivatives (acetosyringone, acetophenone, ethyl vanillin, and methylvanillin) with the selected ion m/z¼ 151 (Figure 1c) are also shown in Figure 1.

Forty-eight volatile compounds were identified belonging to eight differentclasses: alcohols, ethyl esters, fatty acids, phenyl compounds, aldehydes, ketones, ter-penes, and lactones (Table 2). In most cases, the identification of compounds wasachieved by comparing registered mass spectra with those in the NIST library spectraand in some cases with those published in the literature (Câmara et al. 2006a; Vichiet al. 2007). Volatile compounds determined by GC–MS were grouped in classes ofchemical structures and are reported as relative mean concentrations (percentages).The identified compounds were consistent with other results reported in the literature(Rocha et al. 2004; González-Marco et al. 2008; Oliveira et al. 2008; Sáenz-Navajaset al. 2010; Robinson et al. 2011; Losada et al. 2012; Gómez García-Carpintero et al.2012b; Welke et al. 2012). Quantitative analysis was performed on the basic ionchromatographic areas of each compound. The identified compounds and mainparameters are shown in Table 2.

Study of Geo-Climatic Influence

To study the geo-climatic influence, on the volatile composition of SauvignonBlanc, Fetească Albă, and Riesling, wines from Oltenia, Muntenia, Moldova, and

Figure 1. GC–MS of Fetească Albă fromMuntenia: (a) total ion chromatogram (TIC); (b) chromatogramof 4-vinylguaiacol at m/z¼ 135; and (c) chromatogram of acetosyringone, acetophenone, ethyl vanillin,and methyl vanillin at m/z¼ 151.

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Table 2. Identified compound, retention time (tr), and ion used for quantification (m/z)

Compounds tr Ion (m/z)

Alcohols3-Methyl-1-butanol 5.89 57Benzyl alcohol 7.66 792-Phenylethanol 9.01 91Tyrosol 13.71 107Tryptophol 17.61 130AcidsButanoic acid 6.84 60Hexanoic acid 7.49 60Octanoic acid 10.54 60Decanoic acid 12.64 60Dodecanoic acid 14.95 73Tetradecanoic acid 17.19 73Hexadecanoic acid 19.27 73Esters2-Furancarboxylic acid, ethyl ester 7.83 95Hexanoic acid, ethyl ester 6.84 88Octanoic acid, ethyl ester 9.97 88Decanoic acid, ethyl ester 12.74 88Succinic acid, diethyl ester 9.79 101Succinic acid, monoethyl ester 10.89 101Butanedioic acid, hydroxyl-, diethyl ester 11.21 71Succinoic acid, 2-hydroxy-3-methyl-, diethyl ester 10.79 131Butanedioic acid, 2,3-dihydroxy-, diethyl ester 12.42 104Butanedioic acid, 2-(1-methoxy-1-methylethoxy)-3-methyl-, diethyl ester 12.69 85Citric acid, triethyl ester 16.01 157Isomer of ethyl citrate 16.37 157Citric acid, tributyl ester, acetate 21.86 185Phenyl compoundsStyrene 10.98 1044-Vinylguaiacol 11.88 135Benzoic acid, 2,6-dihydroxy-, methyl ester 12.03 136Vanilic acid, methyl ester 14.55 151Benzoic acid, 2,4-dihydroxy-, methyl ester 14.74 136Benzeneacetic acid, 4-hydroxy-, ethyl ester 14.95 107Vanilic acid, ethyl ester 15.35 1513,4,5-Trimethoxyphenol 15.60 1693,4,5-Trimethoxybenzyl alcohol 16.26 198p-Hydroxycinnamic acid, ethyl ester 16.97 147Ferulic acid, ethyl ester 17.50 222m-Tolyl ester 17.99 198o and m-hydroxycinamic acid, ethyl ester 18.17 147Isomer of ferulic acid, ethyl ester 19.05 222AldehydesPhenylacetaldehyde 12.43 91Benzaldehyde, 4-hydroxy-2-methoxy- 12.55 151Benzenebutanal 14.58 104Benzaldehyde, 4-hydroxy-3,5-dimethoxy- 16.25 182KetonesAcetophenone, 4′-hydroxy-3′,5′-dimethoxy- 13.49 181Acetophenone, 4′-hydroxy-3′-metoxy- 14.22 151

(Continued )

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Transylvania were analyzed. The behavior of each class of compounds is discussedwith reference to the primary influence affecting the chemical composition.

Alcohols

Higher alcohols are produced during alcoholic fermentation from carbohy-drates and amino acids and play an important role in the flavor of the wine accordingto the type and concentration (Câmara, Alves, and Marques 2006b). Quantitatively,they are the largest group of volatile compounds (more than 50%) in the wines; theseresults are confirmed by other published data (Zamúz and Vilanova 2006). However,their concentrations varies by type and vineyard as shown in Figure 2. The mostimportant alcohols were aromatic: 2-phenylethanol, tyrosol, triptophol, and benzylalcohol. The only aliphatic alcohol present was 3-methylbutanol, but at a low concen-tration. In some samples, the concentration was less than 1% of the alcohols ident-ified; a result also reported in the literature (Welke et al. 2012).

Figures 2a and b are very similar due of the high abundance of 2-phenylethanolrelative to total compounds from alcohol family (more than 90%). The 2-Pheny-lethanol represents 68% of the total volatile fraction. A similar result was reportedby other authors (Vilanova et al. 2010; Sagratini et al. 2012). The compound2-phenylethanol is formed by chemical degradation of phenylalanine followingtransamination, decarboxylation, and reduction reactions (Uzunov et al. 2011) andhas a rose-like odor (Gómez García-Carpintero et al. 2012a).

The concentration of 2-phenylalanine amino acid increased following contactwith the grape skin and yeast. Thus, the wines aging on yeast lees (so called autolysisof wines) or a longer contact with grape skin during maceration may increase theconcentration of 2-phenylethanol (Selli et al. 2006; Losada et al. 2012). The averageconcentrations of the identified alcohols were 64.8% for Sauvignon Blanc, 67.2% forFetească Albă, and 57% for Riesling.

An increase in alcohol percentage was observed with time. Exceptions includeSauvignon Blanc fromMuntenia (vineyard I) and Riesling from Oltenia (vineyard II)and Transylvania (Jidvei). This behavior can be explained by an increasing contri-bution of the esters and other components in wines during maturation. Therefore,the mentioned exceptions may be due to the specific vinification processes.

For Sauvignon Blanc, the average level of alcohols are 59.9% and 60.7% inOltenia I and Muntenia I vineyards but differ significantly in Oltenia II vineyard,73.8%. This behavior can be attributed to the differences in the vinification process;a hypothesis which is also sustained by the relative stability of the values obtainedin the Oltenia II vineyard. For Fetească Albă, the average values of alcohols fromMuntenia are 64.8% in vineyard I and 62.4% in vineyard II and are different from

Table 2.. Continued

Compounds tr Ion (m/z)

Lactones2(3H)-Furanone, dihydro-3-(phenylmethyl)- 13.57 91γ-Decalactone 11.78 85Terpene2-Acetyl-carene 13.05 163

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the value in the Moldova vineyard, 74.3%. This should indicate that in Muntenia,the vinification processes and climate were similar but are different from thosein the Moldova vineyard. For Riesling, the average values obtained in Munteniaand Oltenia vineyards were 53.1% and 50.5%, respectively, but significantly differ-ent from the value of 67.3% obtained in the Transylvania vineyard. This trend maybe caused by vinification techniques or geo-climate.

The average concentration strongly depended upon the geo-climatic conditionsspecific to the grape region. The concentration differences among regions are between11% and 16%. Also, the vinification process contributes 1–3%. Small quantities ofalcohols are produced by the hydrolysis of esters during maturation in wood (Câmaraet al. 2006b).

Ethyl Esters

Ethyl esters represent a class of compounds that also contribute significantly tothe aroma of the wine. They play a positive role in wine flavor being responsible forthe floral and fruity character. Thirteen esters were identified: three fatty acid ethylesters (hexanoic-, octanoic- and decanoic acid), nine esters of dicarboxylic acids (suc-cinic, malic, citric), and an ethyl ester of a heterocyclic carboxylic acid (2-furoic acid).The main ester contributors were monoethyl succinate, diethyl succinate, and diethylmalate with average relative concentrations of 11.3%, 7.0%, and 8.7%, respectively,with small differences based on grape variety. The maximum of succinic acid esterswas present in Riesling wine.

Figure 2. Relative concentrations of alcohols determined in Sauvignon Blanc, Fetească Albă, andRiesling: (a) total alcohols and (b) 2-phenylethanol.

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The following ethyl esters of fatty acids were identified: ethyl hexanoate, ethyloctanoate, and ethyl decanoate. These compounds are primarily produced in the firststage of alcoholic fermentation (Gil et al. 2006) and contribute the aromas of greenapples, pears, and pineapple (Noguerol-Pato et al. 2009; Gómez García-Carpinteroet al. 2012b). The levels of these esters were approximately 0.1% in most samples.Ethyl hexanoate was slightly higher than for the others, consistent with the literature(Câmara et al. 2006b). Riesling from the Oltenia region (vineyard II) contained thehighest percentages in all years of production, reaching 0.15% in 2010. Becausethe content and composition of the ester fraction is influenced by fermentationconditions, a possible explanation for the low level may be a higher temperatureand low pH during fermentation (Belitz, Grosch, and Schieberle 2009).

In Figure 3, the total concentrations of ethyl esters for three consecutive yearsare shown. An increasing trend of their contribution from year to year that corre-lated with the downward trend observed for alcohols was present. This correlationoccurred because esters are the second class of compounds in the order ofabundance. Their relative average values were approximately 30%.

The averages were 28.6% for Sauvignon Blanc, 26.5% for Fetească Albă, and35.6% for Riesling. The values of ethyl esters obtained for each vineyard forSauvignon Blanc and Fetească Albă were inversely correlated with alcohol abun-dances as can be seen in Figures 2a and b. An average of 22% was obtained forSauvignon Blanc in vineyard II of Oltenia that was significantly smaller comparedwith 31.2% in vineyard I of Oltenia and 32.5% in vineyard I of Muntenia. ForFeteascăAlbă fromMoldova region, an average of 19.1% was obtained that was alsosmaller compared with values of 30.5% and 29.9% obtained from vineyards I and IIof Muntenia, respectively. These observations are consistent with the differencesbetween vinification techniques or geo-climatic conditions that led to this behavior.Riesling contained the highest values of esters. The average values were 33.3% inMuntenia (vineyard II), 43.2% in Oltenia (vineyard II), and 27.2% in Transylvania.Although a similar correlation was expected with the corresponding alcohols levels, amore pronounced contribution of esters appeared in vineyard II of Oltenia.

The ethyl ester concentration increased with age except for Transylvaniavarieties. In addition to the climate contribution, ester concentration may alsoincrease during aging due to esterification reactions (Câmara et al. 2006b; Coetzeeand du Toit 2012). We assume, therefore, that the levels depend substantially on

Figure 3. Concentrations of ethyl esters in wine.

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grape variety and on the fermentation conditions in each vineyard. The type ofenzymes and the factors influencing their action, such as temperature, oxygen, pH,and nitrogen sources, are essential.

Fatty Acids

Wine fatty acids are formed in the first two stages of alcoholic fermentation,but can be found also in low concentrations in the original composition of the mustbefore fermentation. The formation of volatile fatty alcohol during fermentation isrelatively low in terms of quantity, but are very important in terms of flavor (Câmaraet al. 2006b) which may be described as having fruity, cheese, fatty, and rancid notes(Gil et al. 2006; Tufariello, Capone, and Siciliano 2012; Gómez García-Carpinteroet al. 2012a).

Although the presence of C6–C10 fatty acids is linked with the presence of anunpleasant aroma, they are important to the aromatic equilibrium of wine as theyprevent the hydrolysis of corresponding esters. It was found that concentrationsof 4–10mg/L of C6 fatty acids provide a pleasant aroma, while levels above 20mg/L have a negative effect (Gil et al. 2006; Muñoz-González et al. 2011; Tufarielloet al. 2012).

Seven fatty acids were identified: butanoic, hexanoic, octanoic, decanoic, dode-canoic, tetradecanoic, and hexadecanoic. Their levels were low in all types of wine inconcordance with previous studies (Gómez García-Carpintero et al. 2012a). Forbutanoic acid, the variation was small: the mean values were approximately 0.15%for Sauvignon Blanc and Fetească Albă and 0.28% for Riesling. The concentrationsof C10, C12, C14, and C16 acids were approximately 0.01% in all samples. Hexanoicand octanoic acids accounted for the largest contribution with average values of0.9% and 1.3%. The higher level of the octanoic acid relative to hexanoic acid iscommon as reported in literature (Vilanova et al. 2009; Losada et al. 2012; GómezGarcía-Carpintero et al. 2012a). The distribution of fatty acids is shown in Figure 4.

The average acid concentrations were 2.9% for Sauvignon Blanc, 2.1% forFetească Albă, and 3% for Riesling. The range of the individual values is quite wideshowing a minimum value of 0.74% and a maximum of 5.01%. The acid levels wereaffected by the type of yeast, temperature, oxygen, pH, and nitrogen sources. Also,some technological processes such as skin maceration or clarification of the must

Figure 4. Concentrations of fatty acids in wine.

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before fermentation may lead to an increase in fatty acids (Pérez Olivero and PérezTrujillo 2011). A decrease in acid concentration by variety as a function of age wasobserved (Figure 3). This trend is in contrast with the results for ethyl esters (asshown in Figure 2), confirming that esterification of acids is a continuously processin presence of ethyl alcohol (Losada et al. 2012).

Phenyl Compounds

The primary phenyl compounds responsible for wine aroma originate fromgrapes or can be generated during alcoholic fermentation by degradation of phenolicacids. Volatile phenols are considered to be characteristic components of wine flavor.Their influence to the final product can be positive or negative, depending on theirconcentration (Tufariello et al. 2012; Gómez García-Carpintero et al. 2012a).

The main phenols in white wines are 4-ethylguaicol, 4-vinylguaicol, and 4-vinyl-phenol (Tufariello et al. 2012). The presence of these compounds in wines is associatedwith Brettanomyces (Dekker) yeast but also may originate from wood barrels inwhich maturation occurs (Ortega-Heras, González-Sanjosé, and González-Huerta2007). In white wines and at high concentrations, vinylphenols may be responsiblefor heavy pharmaceutical odors, but at moderate or low concentrations may be asso-ciated with a pleasant aroma of spice (Gil et al. 2006; Sánchez-Palomo et al. 2010).

Fourteen phenolics were identified including 4-vinylguaiacol, styrene, vanillicacid methyl ester, ethyl esters of vanilic, ferulic-, p-hydroxycinnamic-, and o- andm-hydroxycinnamic acids. According to literature, a higher concentration of ethylp-coumarate or ethyl ferulate may indicate that the wine was aged in barrels (Hixsonet al. 2012). Also, 4-vinylguaiacol is found only in white wines and its presence in thered and rosé wines serve as an indicator for recognizing a mixture with white wines(Gil et al. 2006).

A higher concentration of ethyl p-coumarate was observed in Sauvignon Blancfrom Oltenia (vineyard I) in 2008 (1.02%). The 4-Vinylguaiacol level was low, withaverage values below 0.1%. The Fetească Albă variety was an exception; in Moldovaand Muntenia (vineyard II) the values were close to 0.2%. Figure 5 shows the totalphenyl profile in the three types of wine.

The average values were 1.9% for Sauvignon Blanc, 1.6% for Fetească Albă,and 2% for Riesling. The maximum value was obtained for Riesling wine in vineyard

Figure 5. Concentrations of phenyl compounds in wine.

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II of Muntenia, which was primarily due to the high values of ethyl o- or m-couma-rate (1.28%) and of m-tolyl ester (1.6%). A significant difference also appearedbetween averages at the vineyards: 4.2% for vineyard II of Muntenia and 0.9% invineyard II of Oltenia and Transylvania. Similar results, but not so pronounced,were observed in Sauvignon Blanc where the vineyard averages were 3.4% forOltenia I, 1.3% for Oltenia II, and 1.0% for Muntenia I. For Fetească Albă, theaverage values were: 1.5% in Moldova, 1.0% in Muntenia I, and 2.2% in MunteniaII, showing more homogeneous distributions.

No dependence of phenyl compounds concentration was observed for any winevariety based on year. As the concentrations of phenyl compounds are not dependentsignificantly on the type of grape or geo-climatic conditions (variation from a year toother), the significant differences may be explained by longer times of skin contact usedto produce a floral and fruity wine (Selli et al. 2006; Gómez-Míguez et al. 2007).

Aldehydes

Four aromatic aldehydes were detected: phenylacetaldehyde; benzaldehyde;4-hydroxy-2-methoxy-, benzenebutanal, and benzaldehyde; and 4-hydroxy-3,5-dimethoxy- (syringic aldehyde). The most important for wine aroma are phenylacet-aldehyde and syringic aldehyde. Phenylacetaldehyde is formed during alcoholicfermentation by decarboxylation of 2-oxo-3-phenylpropanoic acid (Sarrazin,Dubourdieu, and Darriet 2007) and contributes the note of aged wood (Campoet al. 2006). Syringic aldehyde is a phenolic aldehyde and together with vanillinand its derivatives is formed by thermal degradation of lignin during burning oakwood (Liberatore et al. 2010; Gómez García-Carpintero et al. 2012b). As its concen-tration is low in the majority of wines, not exceeding 0.01% and absent in some Sau-vignon Blanc samples, its contribution to total aldehydes is low. Phenylacetaldehydewas present in all samples at between 0.11% and 0.50%. A large value for FeteascăAlbă in 2009 from Moldova was observed due mostly to a higher concentration ofbenzenebutanal. This compound was not reported in previous papers. It is likely thatthis compound was produced from the container in which the wine was produced.The average concentration of aldehydes was only 0.49% of the total volatile com-pounds identified excluding benzenebutanal.

The minimum measured value for aldehydes was 0.16% and the maximum was0.78%. The variety averages were 0.43% for Sauvignon Blanc, 0.42% for FeteascăAlbă (value calculated excepting the mentioned outlier value), and 0.60% for Riesl-ing. The average level of aldehydes from each vineyard did not vary significantly(0.43%). A slightly higher level was observed for the Rieslings, especially in the Trans-ylvania vineyard, where the average for three years was 0.89%. An explanation isbased on the significant contribution of the benzenebutanal and phenylacetaldehyde.Additionally, the concentration of phenylacetaldehyde may increase after alcoholicfermentation by botrytized grapes (Sarrazin et al. 2007).

Ketones

The ketones found were acetovanillone (acetophenone, 4′-hydroxy-3′-methoxy-) and acetosyringone (Acetophenone, 4′-hydroxy-3′,5′-dimetoxy-)

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(Figure 6). These, together with syringic aldehyde and 4-vinylguaiacol, are producedduring wine aging in oak barrels to enhance the sensory quality of wines (Ortega-Heras et al. 2007; Liberatore et al. 2010; Gómez García-Carpintero et al. 2012b).

The averages were 0.14% for Sauvignon Blanc, 0.29% for Fetească Albă, and0.23% for Riesling from the total volatile compounds identified. Higher levels forFetească Albă and Riesling were observed compared to Sauvignon Blanc. However,this behavior was not associated with the wine type; rather, the values depended to agreater extent on the vineyard and production year. Thus, for FeteascăAlbă, a signifi-cant difference between the levels of ketones obtained in the two vineyards in theregion of Muntenia was observed. The averages for the three years were 0.13% invineyard I and 0.47% in vineyard II and confirm that the vinification process has amajor influence. Longer contact with wood may have caused the higher values fromvineyard II. Regarding the temporal evolution, although in some cases, there was anincrease in ketones as wine was aged; in other cases, it was almost constant ordecreased in support of this hypothesis.

Lactones

Two lactones were identified: dihydro-3-(phenylmethyl)-2(3H)-furanone andγ-decalactone (Figure 7). The latter was more abundant. The γ-Decalactons areamong the most important components contributing to sensory characteristics ofwines aged in oak barrels (Perestrelo et al. 2006; Câmara et al. 2006b; Losada

Figure 6. Concentrations of ketones in wine.

Figure 7. Concentrations of total lactones in wine.

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et al. 2011). The aroma of lactones depends on their chemical structure (functionalgroup and the side chain length) and are described as “fruity” (γ-hexalactone),“coconut” (γ-octalactone), “peach, milky” (γ-decalactone), or “sweet floral”(γ-dodecalactone) (Perestrelo et al. 2006; Câmara et al. 2006b).

The averages were 1.17% for Sauvignon Blanc, 1.23% for Fetească Albă, and1.44% for Riesling. Lactones are the third most important compound class (averageconcentration around of 1.25%), following fatty acids and phenolic compounds.Despite the relatively small differences between the mean values obtained on the typesof wine, a more pronounced difference was present for average lactones from the vine-yards. Thus, for Fetească Albă in the Muntenia region, there was a significant differ-ence between the levels obtained in the two vineyards. The mean values for the threeyears were 0.89% in vineyard I and 1.53% in vineyard II. This is similar to the resultsobserved for ketones. These results may be due to the different conditions of fermen-tation or aging. If the wine is fermented on lees or aged in oak barrels, the concentra-tions of phenols, ketones, phenolic aldehydes, and lactones may increase.

Terpenes

Terpenes are heterogeneous chemical compounds from the structural point ofview and are widely found in nature. In spite of their structural variety, terpenes arebiosynthesized from structural unit of isoprene (2-methyl-1,3-butadiene). The basicformula of terpenes is a multiple of this structural unit, (C5H8)n, where n is the num-ber of isoprene units linked. Although developed primarily in conifers, terpenesappear also in grapes in free form or linked to sugar molecules, when they form areserve of odorless aroma. Subsequently, terpenes can be released from sugarsthrough the action of several enzymes, contributing to the characteristic floral flavorof wine (Liberatore et al. 2010).

Monoterpenes are particularly abundant in aromatic grape varieties such as:Muscat, Riesling, and Gewürztraminer (Masa and Vilanova 2008; Dziadas and Jeleń2010). As most terpenoids occur in micro-concentrations in grapes, must, and wine,their quantification is often difficult (Câmara, Alves, and Marques 2006c). Thus, inthese samples, only one terpene was determined, 2-acetyl-carene, a bicyclic monoter-pene. Its percentage in the three types of wine was small (under 0.3%) but consistentwith the literature (Welke et al. 2012). The variation of 2-acetyl-carene in wine isshown in Figure 8.

Figure 8. Concentrations of 2-acetyl-carene in wine.

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The averages based on variety are close: 0.11% for Sauvignon Blanc, 0.11% forFetească Albă, and 0.16% for Riesling. The maximum level of 2-acetyl-carene wasobtained for Fetească Albă in 2008 in Muntenia region. Wine from 2008 had thehighest concentrations of 2-acetyl-carene for many wine samples. This observationis consistent with climatic conditions. Because one of the most important factors thatinfluence the characteristic aroma of a particular variety of grape is ripening stage, apossible explanation for the higher concentration of 2-acetyl-carene in 2008 is that inthis year the weather conditions allowed a delayed harvest and the grapes reachedfull maturity. Terpenoids are higher in wine produced from ripe grapes (SánchezPalomo et al. 2007).

Effect of the Variety on the Volatile Fraction

To determine the influence of grape variety on the volatile composition,Sauvignon Blanc, Riesling, and Fetească Albă produced in the Muntenia region wereanalyzed. Thus, the behavior of the major volatile compounds (higher alcohols andethyl esters) was studied in these assortments. The values by compound familyshowed a downward trend of alcohol contribution to the total volatile compoundsas the wines were aged. This behavior is, as mentioned, correlated with the increasedcontribution of ethyl esters. As alcohols and esters are major components of wine(the totality of minor compounds are about 10%), their correlation is evident andan increase of ethyl esters content in older wines was observed. This correlationsupports the idea that ethyl esters are formed from acids and alcohols present in wineduring aging.

The alcohol averages over the three years were 53.1% for Riesling, 60.7% forSauvignon Blanc, and 64.8% for Fetească Albă in vineyard I and 62.4% in vineyardII. The averages over the three years for ethyl esters were 36.3% for Riesling, 32.5%for Sauvignon, and 30.5% for Fetească Albă in vineyard I and 29.9% in vineyard II.The concentrations of alcohols were close for Sauvignon Blanc and Fetească Albăbut significantly lower for Riesling. A similar situation was observed for ethyl estersbut the quantities were higher for Riesling.

CONCLUSIONS

LLE GC–MS was robust and rapid for the determination of volatiles in wine.Forty-eight compounds were determined from 0.001% to 75%. Detailed analyses ofthe compounds allow correlation with the technology used at a vineyard, region, andproduction year. Average concentration of alcohols strongly depended upon thegeo-climatic conditions specific to the cultivation region. The range of total alcoholswas between 53.10% and 67.33%. A decrease in the alcohol percentage was observedwith time due to a higher contribution by esters during maturation. Ester concen-tration showed a strong dependence on grape variety. For example, the averagevalue for Fetească Albă was 26.5% but for Riesling the average was 35.6%. Anincrease was observed from year to year and the values correlated with the down-ward trend observed for alcohols.

The concentrations of acids depended primarily on vineyard processing (typeof yeast and specific technology). In the same region (Oltenia) for the same type

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of grape (Sauvignon Blanc) but in different vineyards, the average of the acids was of1.33% in vineyard Oltenia II but 3.7% in vineyard Oltenia I. The main factor influ-encing phenolic levels was vinification technology (longer time of skin contact duringthe preparation of the must). The maximum level was found to be 6.61% (Rieslingsort, Muntenia II).

The concentrations of ketones and lactones depended upon the contact timewith wood during the aging in oak barrels. Thus, for Fetească Albă, significantdifferences were observed between the levels of ketones obtained in two vineyardsfrom Muntenia: 0.13% at vineyard I and 0.47% at vineyard II. A similar resultwas obtained for lactones: the concentrations were 0.89% in vineyard I and1.53% in vineyard II from the same region. The levels of terpenes were characteristicof climatic condition (ripening stage of the grape). The highest level was observed forall varieties in 2008 because weather conditions allowed a delayed harvest.

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