Applications of Solid-Phase Microextraction and Gas Chromatography/Mass Spectrometry (SPME-GC/MS) in the Study of Grape and Wine Volatile Compounds

Volatile compounds are responsible for the wine “bouquet”, which is perceived by sniffing the headspace of a glass, and of the aroma component (palate-aroma) of the overall flavor, which is perceived on drinking. Grape aroma compounds are transferred to the wine and undergo minimal alteration during fermentation (e.g., monoterpenes and methoxypyrazines); others are precursors of aroma compounds which form in winemaking and during wine aging (e.g., glycosidically-bound volatile compounds and C13-norisoprenoids). Headspace solid phase microextraction (HS-SPME) is a fast and simple technique which was developed for analysis of volatile compounds. This review describes some SPME methods coupled with gas chromatography/mass spectrometry (GC/MS) used to study the grape and wine volatiles.


Introduction
Wine aroma is formed by more than 800 volatile compounds and is characteristic of each product [1,2]. Often these compounds are present in very low concentration and are characterized by very low sensory thresholds (between ng/L and μg/L). Usually, the wine aroma profiling needs a sample preparation for isolation and concentration of the volatiles before performing gas chromatographic analysis. Several sample preparation methods for the analysis of grapes and wine were proposed: distillation [3,4], liquid-liquid extraction (LLE) [5,6], solid phase extraction (SPE) [7,8], dynamic headspace extraction [9], and headspace-solid phase microextraction (HS-SPME) [10][11][12][13]. SPME was developed in the 1990s by Pawliszyn and co-workers [14] and every year a thousand papers describing different aspects of this approach, and applications in different fields (chemical analysis, bioanalysis, food science, environmental science, and recently, pharmaceutical and medical sciences), are published [15]. This sample extraction technique was demonstrated to be rapid, simple, and reproducible, with no solvent use, and is suitable for the extraction and concentration of a high number of volatile and semi-volatile compounds from aqueous solutions [16,17]. Moreover, SPME needs a small sample volume and the coupling with gas chromatography and mass spectrometry (GC/MS) provides high sensitivity. For these reasons it has been used to study the volatile profile of many fruit varieties, vegetables, and beverages, including grapes and wine [18][19][20].
This paper reviews the main SPME-GC/MS applications developed to study the volatile and aroma compounds of grapes and wine.

Grape and Wine Volatile Compounds
Since early 80's a great number of studies of grape and wine volatiles have been performed and the main compounds identified are listed in Table 1. Principal in grape are monoterpenes, C13-norisoprenoids, benzene compounds, C6 aldehydes, and alcohols [21][22][23]. These compounds are present in berry skin and pulp in both free (volatile) and glycosidically-bound (non-volatile) form. Free volatile compounds directly contribute to grape and wine aroma while glycosides are flavorless compounds which can act as aroma precursors for enzymatic and acid hydrolysis occurring in winemaking and during wine storage [24].
Main wine volatiles are ethyl esters, acetates, and higher alcohols. Esters are produced by the yeasts during fermentation. Principal are ethyl esters characterized by fruity and floral notes, such as ethyl hexanoate, ethyl octanoate, ethyl decanoate, ethyl dodecanoate, isoamyl acetate, hexyl acetate, and 2-phenylethyl acetate [32,33]. Contents of hexanoic, octanoic, and decanoic acid in wine depend on the yeast strain, fermentation conditions and grape must composition [27]. Higher alcohols are formed by the yeast sugar metabolism (anabolic pathway) as well as via the catabolic or Ehrlich pathway of amino acids [27,34,35]. Rapp and Versini reported that a higher alcohols concentration below 300 mg/L is desirable for the aroma complexity of wine whereas a concentration exceeding 400 mg/L can have a detrimental effect [36].

Analysis of PFBOA-Derivatives
In general, carbonyl compounds contribute to the wine aroma even though they are present in low levels. GC/MS analysis of the O-pentafluorobenzyl (PFB) derivatives formed by reaction with O-(2,3,4,5,6-pentafluorobenzyl)-hydroxylamine (PFBOA) by recording in singular-ion-monitoring (SIM) the mass spectrum base peak signal at m/z 181 (characteristic of PFB-oximes) is a selective and sensitive method. On the other hand, derivatization increases the complexity of analyte peaks in the chromatogram due to formation of two oximes for each carbonyl group (isomers E and Z, except for formaldehyde) [38]. By this method several studies of carbonyl compounds in hydro-alcoholic matrices, such as wine, model wine solutions, and spirits, were performed [30,31,[39][40][41][42][43][44][45][46][47].
Malolactic fermentation (MLF) is an important oenological process performed after the alcoholic fermentation for improving the organoleptic characteristics and the microbiological stability of wine [46]. The process is carried out by lactic acid bacteria: it can occur naturally or be induced by the 2 2-methoxy-3-isobutylpyrazine (green pepper) R=CH(CH 3 ) 2 2-methoxy-3-isopropylpyrazine (green pepper, earthy, raw potato, musty) inoculum of commercial bacteria strains. Conversion of L(−)-malic into L(+)-lactic acid decreases wine acidity [30]; usually, the inoculation of selected bacteria strains enables control over the process [48].
With MLF, together with the other fermentative compounds (esters, sulfur and nitrogen compounds, volatile phenols, and volatile fatty acids), also the carbonyl profile changes, increasing the aromatic complexity of wine [42,49].
In making barrels for wine aging, oak (Quercus sessilis, Q. petraea, Q. robur, Q. peduncolata, Q. alba) is the wood more often used but other species, such as acacia (Robinia pseudoacacia), chestnut (Castanea sativa), cherry (Prunus avium), and mulberry (Morus alba and Morus nigra) are also being considered [55]. SPME-GC/MS was used to study the evolution of wine aroma during aging in 225-L barrels (barriques) made with these wood types. Experimental conditions used are reported in Table 3; main compounds identified are reported in Table 4 [55]. Wines aged in acacia, chestnut and oak wood showed higher contents of vanillin and eugenol and the acacia-aged sample showed an increase of 4-ethylguaiacol. Mulberry-aged wine had a significant decrease of 4-ethylguaiacol and increase of 4-ethylphenol; the wine aged in cherry barrel already showed high levels of 4-ethylguaiacol after three months of aging.

"Foxy Smelling Compounds" and 3-Alkyl-2-Methoxypyrazines in Grape Juice
2'-Aminoacetophenone (o-AAP) is the main compound identified as the cause of the aging note-the so-called "hybrid note", "foxy-smelling" or "American character"-typical of V. labruscana grapes, even though it was also found in some V. vinifera wines such as Müller-Thurgau, Riesling, and Silvaner [56]. This note is variously described as "acacia blossom," "naphthalene note," "furniture polish," "fusel alcohol," and "damp cloth," and causes a considerable number of wine rejections. The formation of o-AAP in grape is promoted by several factors, such as reduced nitrogen fertilization in combination with hot and dry summers, and the risk increases in wines made with grapes harvested early. The phytohormone indole-3-acetic acid (IAA) is the principal precursor of o-AAP through non-enzymatic processes [57,58]. Also, methyl anthranilate (MA) contributes to the typical foxy taint of wines made with American and wild vine grapes, although it was also found in some V. vinifera white wines in concentrations of up to 0.3 μg/L [59].
For analysis of o-AAP in wine a direct-immersion SPME method by using a DVB/CAR/PDMS fiber and GC/MS, was proposed [60]; instead, analysis of MA in grape juice was performed by using a PDMS fiber [61].

Volatile Phenols in Wine
4-Ethylphenol (4-EP) and 4-ethylguaiacol (4-EG) are associated with wine defects that can form during winemaking or, more commonly, wine aging. These compounds are characterized by sensorial characteristics described as "stable", "animal" and "phenolic" and their presence is particularly detrimental for the product [74][75][76]. They are produced by winery contaminants, such as Brettanomyces and Dekkera yeasts, through processes of decarboxylation and reduction of ferulic and p-coumaric acids present in the grape [75]. Sensory thresholds of 4-EP and 4-EG in wine are 440 μg/L and 33 μg/L, respectively [77].
4-EG and 4-EP in wine were also analyzed by a multiple-headspace SPME method. By performing three consecutive extractions of the sample with a CW/DVB fiber, the possible matrix effects were minimized by providing a LOD of 0.06 μg/L for both 4-EG and 4-EP [81].

Higher Alcohols and Esters in Wine
By using a PDMS 100-μm fiber, effective methods for analysis of higher alcohols and aliphatic esters in wine were performed [82,83]. This coating fiber showed high affinity for non-polar compounds such as ethyl esters and acetates [84][85][86], while CW/DVB fiber is suitable for more polar compounds, such as 1-hexanol, hexen-1-ol, 1-octanol, and monoterpenols [84].
Antalick et al., developed a SPME and GC/MS-SIM method which provided simultaneous determination of 32 esters in wine in concentration between ng/L and mg/L [87]. Seven different fibers were tested: DVB/CAR/PDMS 50/30 μm, CAR/PDMS 85 μm, PDMS 100 μm, PDMS/DVB 65 μm, PA 85 μm, CW/DVB 70 μm, and polyethyleneglycol (PEG) 60 μm. PDMS was the most efficient in extracting the less polar and less volatile compounds; for more volatile esters the best coating was CAR/PDMS, and aromatic esters were better recovered by CW/DVB. In general, the PDMS fiber showed high efficiency for all compounds and provided LOQs between 0.4 ng/L and 4.0 μg/L.
Recent applications showed that the tri-phase fiber DVB/CAR/PDMS provides extraction of the highest number of wine volatiles, including ethyl esters (56% of the compounds identified), alcohols, and acids [2,88].
A SPME method for analysis of 13 volatile sulfur compounds in wine (i.e., DMS, EtSH, DES, MTA, ETA, ME, DMDS, DEDS, BT, HMT, MTB, MTP, and MTE) with b.p. ranging from 35 °C to 231 °C by using a CAR/PDMS/DVB 50:30 μm 2 cm length fiber, was developed [90]. By addition of MgSO4 (1.0 M) to increase ionic strength of solution and performing the extraction at 35 °C, the method showed a high sensitivity for all the analytes.

Conclusions
Grape aroma is composed of a hundred compounds and wine's volatile profile also includes a number of fermentative compounds. SPME coupled to GC/MS showed to be effective for studying several classes of these analytes without solvent use. It often resulted in a high-sensitive technique for quantitative analysis of compounds for which the standard is available with high reproducibility. Moreover, the use of a multiphase fiber coupled to MS and multivariate data analysis allows sampling automation and statistical treatment of fragment abundances for the identification of compounds [95,96].
On the other hand, different to most of the sample preparation methods performed by liquid-liquid extraction and SPE, the selectivity of SPME fiber often changes dramatically for the different analytes. As a consequence, it is rarely possible to perform the semi-quantitative profiling of the sample on the internal standard signal, which is particularly useful in the characterization of grape varieties and the monitoring of the winemaking processes.
Logically, by increasing the standards commercially available new SPME-GC/MS applications are developed, and methods for the profiling of specific classes of grape aroma compounds, such as terpenols and norisoprenoids, could be particularly useful.