Multivariate analysis for the classification and differentiation of Madeira wines according to main grape varieties

In order to differentiate and characterize Madeira wines according to main grape varieties, the volatile composition (higher alcohols, fatty acids, ethyl esters and carbonyl compounds) was determined for 36 monovarietal Madeira wine samples elaborated from Boal, Malvazia, Sercial and Verdelho white grape varieties. The study was carried out by headspace solid-phase microextraction technique (HS-SPME), in dynamic mode, coupled with gas chromatography–mass spectrometry (GC–MS). Corrected peak area data for 42 analytes from the above mentioned chemical groups was used for statistical purposes. Principal component analysis (PCA) was applied in order to determine the main sources of variability present in the data sets and to establish the relation between samples (objects) and volatile compounds (variables). The data obtained by GC–MS shows that the most important contributions to the differentiation of Boal wines are benzyl alcohol and (E)-hex-3-en-1-ol. Ethyl octadecanoate, (Z)-hex-3-en-1-ol and benzoic acid are the major contributions in Malvazia wines and 2-methylpropan-1-ol is associated to Sercial wines. Verdelho wines are most correlated with 5-(ethoxymethyl)-furfural, nonanone and cis-9-ethyldecenoate. A 96.4% of prediction ability was obtained by the application of stepwise linear discriminant analysis (SLDA) using the 19 variables that maximise the variance of the initial data set.

Varietal flavour compounds of four grape varieties producing Madeira wines

Boal, Malvasia, Sercial and Verdelho are the main white grape varieties used in Madeira wine production. To estimate the free fraction of varietal aroma compounds of these varieties, 39 samples of musts were analysed to determine their content of monoterpenols and C13 norisoprenoids (terpenoids), using dynamic headspace solid-phase microextraction coupled with gas chromatography–mass spectrometry. The r-values for linearity studies of the analytical method used, varied between 0.977 (nerolidol) and 0.999 (linalool). The repeatability for each compound varied between 2.5% (citronellol) and 11.8% (β-ionone). The mean values from three vintages (1998, 1999 and 2000) confirmed that these musts have differentiated contents of terpenoids. In opposition to Verdelho musts, Malvasia showed the highest free terpenoids content. In order to establish relations between the compounds and the varieties under investigation, principal component analysis and linear discriminant analysis were applied to the data, revealing a good separation and classification power between the four groups as a function of varietal origin.

Optimisation of solid-phase microextraction combined with gas chromatography–mass spectrometry based methodology to establish the global volatile signature in pulp and skin of Vitis vinifera L. grape varieties

The volatiles (VOCs) and semi-volatile organic compounds (SVOCs) responsible for aroma are mainly present in skin of grape varieties. Thus, the present investigation is directed towards the optimisation of a solvent free methodology based on headspace-solid-phase microextraction (HS-SPME) combined with gas chromatography–quadrupole mass spectrometry (GC–qMS) in order to establish the global volatile composition in pulp and skin of Bual and Bastardo Vitis vinifera L. varieties. A deep study on the extraction-influencing parameters was performed, and the best results, expressed as GC peak area, number of identified compounds and reproducibility, were obtained using 4 g of sample homogenised in 5 mL of ultra-pure Milli-Q water in a 20 mL glass vial with addition of 2 g of sodium chloride (NaCl). A divinylbenzene/carboxen/polydimethylsiloxane fibre was selected for extraction at 60 °C for 45 min under continuous stirring at 800 rpm. More than 100 VOCs and SVOCs, including 27 monoterpenoids, 27 sesquiterpenoids, 21 carbonyl compounds, 17 alcohols (from which 2 aromatics), 10 C13 norisoprenoids and 5 acids were identified. The results showed that, for both grape varieties, the levels and number of volatiles in skin were considerably higher than those observed in pulp. According to the data obtained by principal component analysis (PCA), the establishment of the global volatile signature of grape and the relationship between different part of grapes—pulp and skin, may be an useful tool to winemaker decision to define the vinification procedures that improves the organoleptic characteristics of the corresponding wines and consequently contributed to an economic valorization and consumer acceptance.

Olfactory perception threshold assessment of volatile acidity in Madeira wines

Madeira wine is a fortified wine with impact in the Madeira Island’s economy. Similarly to other wines, its acidity should be well controlled in order to ensure Madeira wine quality, mostly the volatile acidity. Due to Madeira wine complex flavour, it is crucial to get a better knowledge about the volatile acidity impact in its features, namely determine the perception limit of acetic acid and ethyl acetate, as both are the main contributors for volatile acidity. Firstly, the olfactory perception threshold of volatile acidity was assessed by a trained and an untrained panel, using 5 and 10 years-old Sercial and Malvasia wines. Moreover, the current work also presents the evolution of organic acids, acetic acid and ethyl acetate during 540 days of ageing of Madeira wines (Malvasia, Bual, Verdelho and Sercial), comparing the same wines aged by both traditional ageing processes: canteiro and estufagem. Other wine samples, aged in wood casks (canteiro) for at least 5 years, were also evaluated. HS-SPME followed by GC-MS analysis was used to determine ethyl acetate concentration and IEC-HPLC-DAD was used for the organic acids determination, including acetic acid. The results indicated that acetic acid and ethyl acetate olfactory perception threshold depends essentially on wine’s age. Concerning acetic acid, the untrained panel was in average 5.45 g/L (5 years-old) and 6.22 g/L (10 years-old). Training the expert panel to recognize acetic acid odour, the values decreased for 1.44 g/L (5 years-old) and 1.87 g/L (10 years-old), but still remained higher than the established volatile acidity legal limits. Ethyl acetate threshold was similar for both panels (in average 327.97 mg/L). Both compounds tend to increase exponentially with age, being more evident in sweet wines. Organic acids in young Madeira wines depend mostly on the nature of grape varieties, but this difference is minimized with wine ageing.

Changes in volatile composition of Madeira wines during their oxidative ageing

The influence of the age in the volatile composition of Madeira wines made with Boal, Malvazia, Sercial and Verdelho varieties and aged in oak barrel during 1, 11 and 25 years old was been studied. For this purpose, the evolution of volatile compounds: higher alcohols, ethyl esters, fatty acids, furan compounds, enolic compounds, γ-lactones, dioxanes and dioxolanes, of the four most utilised varieties were determined using liquid–liquid extraction with dichloromeihane. Octan-3-ol was used as internal standard. The wines made with these varieties showed great differences in sugar content and small variations on pH and alcoholic degree. The results show that during ageing, the concentration of fatty acids ethyl esters, acetates and fatty acids decrease significantly contrarily to the great increase of ethyl esters of diprotic acids. There is a strong correlation between sotolon, 2-furfural, 5-methyl-2-furfural, 5-hydroxymethyl-2-furfural and 5-ethoxymethyl-2-furfural with wine ageing. These findings indicate that these compounds can be used as ageing wine markers. Among the molecules studied, sotolon [3-hydroxy-4,5-dimethyl-2(5H)-furanone] was one of the few molecules present in concentrations above the perception threshold in Madeira wines. 5-Eihoxymethyl-2-furfural formed from 5-hydroxymethyl-2-furfural and 2-furfural, derived from sugars, are also involved in the aroma of sweet fortified white wines aged in oxidative conditions. The sensory properties change significantly after long periods of conservation.

Analytical characterization of the aroma of Tinta Negra Mole red wine: Identification of the main odorants compounds

A method for the simultaneous determination of major and minor volatiles composition in different types (dry, medium dry, sweet and medium sweet) of a young Tinta Negra Mole (TNM) monovarietal red wine from 2003 harvest has been validated. Wine samples preparation includes a dichloromethane liquid–liquid extraction followed by concentration under a nitrogen atmosphere. The extracted fraction was analysed by gas chromatography–mass spectrometry and give quantitative information for more than 86 analytes whose concentration range from few μg l−1 to 259.1 mg l−1. The method enables high recovery of volatile compounds in wine good linearity with (r2) values higher than 0.980 and good sensitivity. The limits of detection range from 0.003 to 0.534 mg l−1 and limits of quantification from 0.009 to 1.170 mg l−1. The method allows satisfactory determination of more than 80 compounds in the TNM red wines. These wines are characterized by a high content of higher alcohols, ethyl esters, fatty acids and lactones. The levels of sulphur compounds in Tinta Negra Mole medium sweet wines are very low, but they have the highest concentration of carbonyl compounds. Quantitative analysis of the main odorants followed by the determination of aroma index allow us elucidate the aroma of these varieties. On the basis of their odour description and odour threshold, the most powerful odorants of Tinta Negra Mole wines were tentatively established.

Classification of Boal, Malvazia, Sercial and Verdelho wines based on terpenoid patterns

Thirty-six Madeira wine samples from Boal, Malvazia, Sercial and Verdelho white grape varieties were analyzed in order to estimate the free fraction of monoterpenols and C13 norisoprenoids (terpenoid compounds) using dynamic headspace solid phase micro-extraction (HS-SPME) technique coupled with gas chromatography–mass spectrometry (GC–MS). The average values from three vintages (1998–2000) show that these wines have characteristic profiles of terpenoid compounds. Malvazia wines exhibits the highest values of total free monoterpenols, contrary to Verdelho wines which had the lowest levels of terpenoids but produced the highest concentration of farnesol. The use of multivariate analysis techniques allows establishing relations between the compounds and the varieties under investigation. Principal component analysis (PCA) and linear discriminant analysis (LDA) were applied to the obtained matrix data. A good separation and classification power between the four groups as a function of their varietal origin was observed.

3-Hydroxy-4, 5-dimethyl-2 (5 H)-furanone levels in fortified Madeira wines: Relationship to sugar content

The maturation of Madeira wines usually involves exposure to relatively high temperatures and humidity levels >70%, which affect the aroma and flavor composition and lead to the formation of the typical and characteristic bouquet of these wines. To estimate the levels of sotolon [3-hydroxy4,5-dimethyl-2(5 H )-furanone] and their behavior over time, 86 aged Madeira wines samples (1-25 years old), with different sugar concentrations, respectively, 90 g L-1 for Boal, 110 g L-1 for Malvazia, 25 g L -1 for Sercial, and 65 g L-1 for Verdelho varieties, were analyzed. Isolation was performed by liquid-liquid extraction with dichloromethane followed by chromatographic analysis by GC-MS. The reproducibility of the method was found to be 4.9%. The detection and quantification limits were 1.2 and 2.0 µgL-1, respectively. The levels of sotolon found ranged from not detected to 2000 µgL-1 for wines between 1 and 25 years old. It was observed that during aging, the concentration of sotolon increased with time in a linear fashion ( r ) 0.917). The highest concentration of sotolon was found in wines with the highest residual sugar contents, considering the same time of storage. The results show that there is a strong correlation between sotolon and sugar derivatives: furfural, 5-methylfurfural, 5-hydroxymethylfurfural, and 5-ethoxymethylfurfural. These compounds are also well correlated with wine aging. These findings indicate that the kinetics of sotolon formation is closely related with residual sugar contents, suggesting that this molecule may come from a component like sugar.

New method for determination of (E)resveratrol in wine based on microextraction using packed sorbent and ultra-performance liquid chromatography

An ultra-fast and improved analytical methodology based on microextraction by packed sorbent (MEPS) combined with ultra-performance LC (UPLC) was developed and validated for determination of (E)-resveratrol in wines. Important factors affecting the performance of MEPS such as the type of sorbent material (C2, C8, C18, SIL, and M1), number of extraction cycles, and sample volume were studied. The optimal conditions of MEPS extraction were obtained using C8 sorbent and small sample volumes (50–250mL) in one extraction cycle (extract–discard) and in a short time period (about 3 min for the entire sample preparation step). (E)-Resveratrol was eluted by 1 250mL of the mixture containing 95% methanol and 5% water, and the separation was carried out on a highstrength silica HSS T3 analytical column (100 mm 2.1 mm, 1.8mm particle size) using a binary mobile phase composed of aqueous 0.1% formic acid (eluent A) and methanol (eluent B) in the gradient elution mode (10 min of total analysis). The method was fully validated in terms of linearity, detection (LOD) and quantification (LOQ) limits, extraction yield, accuracy, and inter/intra-day precision, using a Madeira wine sample (ET) spiked with (E)-resveratrol at concentration levels ranging from 5 to 60mg/mL. Validation experiments revealed very good recovery rate of 9575.8% RSD, good linearity with r2 values 40.999 within the established concentration range, excellent repeatability (0.52%), and reproducibility (1.67%) values (expressed as RSD), thus demonstrating the robustness and accuracy of the MEPSC8/UPLC-photodiode array (PDA) method. The LOD of the method was 0.21mg/mL, whereas the LOQ was 0.68mg/mL. The validated methodology was applied to 30 commercial wines (24 red wines and six white wines) from different grape varieties, vintages, and regions. On the basis of the analytical validation, the MEPSC8/UPLC-PDA methodology shows to be an improved, sensitive, and ultra-fast approach for determination of (E)-resveratrol in wines with high resolving power within 6 min.

Dynamic headspace solid-phase microextraction combined with one-dimensional gas chromatography–mass spectrometry as a powerful tool to differentiate banana cultivars based on their volatile metabolite profile

In this study the effect of the cultivar on the volatile profile of five different banana varieties was evaluated and determined by dynamic headspace solid-phase microextraction (dHS-SPME) combined with one-dimensional gas chromatography–mass spectrometry (1D-GC–qMS). This approach allowed the definition of a volatile metabolite profile to each banana variety and can be used as pertinent criteria of differentiation. The investigated banana varieties (Dwarf Cavendish, Prata, Maçã, Ouro and Platano) have certified botanical origin and belong to the Musaceae family, the most common genomic group cultivated in Madeira Island (Portugal). The influence of dHS-SPME experimental factors, namely, fibre coating, extraction time and extraction temperature, on the equilibrium headspace analysis was investigated and optimised using univariate optimisation design. A total of 68 volatile organic metabolites (VOMs) were tentatively identified and used to profile the volatile composition in different banana cultivars, thus emphasising the sensitivity and applicability of SPME for establishment of the volatile metabolomic pattern of plant secondary metabolites. Ethyl esters were found to comprise the largest chemical class accounting 80.9%, 86.5%, 51.2%, 90.1% and 6.1% of total peak area for Dwarf Cavendish, Prata, Ouro, Maçã and Platano volatile fraction, respectively. Gas chromatographic peak areas were submitted to multivariate statistical analysis (principal component and stepwise linear discriminant analysis) in order to visualise clusters within samples and to detect the volatile metabolites able to differentiate banana cultivars. The application of the multivariate analysis on the VOMs data set resulted in predictive abilities of 90% as evaluated by the cross-validation procedure.

Phenolic profile of Sercial and Tinta Negra Vitis vinifera L. grape skins by HPLC–DAD–ESI-MSn: novel phenolic compounds in Vitis vinifera L. grape

This study represents the first phytochemical research of phenolic components of Sercial and Tinta Negra Vitis vinifera L. The phenolic profiles of Sercial and Tinta Negra V. vinifera L. grape skins (white and red varieties, respectively) were established using high performance liquid chromatography–diode array detection–electrospray ionisation tandem mass spectrometry (HPLC–DAD–ESI-MSn), at different ripening stages (véraison and maturity). A total of 40 phenolic compounds were identified, which included 3 hydroxybenzoic acids, 8 hydroxycinnamic acids, 4 flavanols, 5 flavanones, 8 flavonols, 4 stilbenes, and 8 anthocyanins. For the white variety, in both ripening stages, hydroxycinnamic acids and flavonols were the main phenolic classes, representing about 80% of the phenolic composition. For red variety, at véraison, hydroxycinnamic acids and flavonols were also the predominant classes (71%), but at maturity, anthocyanins represented 84% of the phenolic composition. As far as we know, 10 compounds were reported for the first time in V. vinifera L. grapes, namely protocatechuic acid-glucoside, p-hydroxybenzoyl glucoside, caftaric acid vanilloyl pentoside, p-coumaric acid-erythroside, naringenin hexose derivate, eriodictyol-glucoside, taxifolin-pentoside, quercetin-glucuronide-glucoside, malylated kaempferol-glucoside, and resveratrol dimer. These novel V. vinifera L. grape components were identified based on their MSn fragmentation profile. This data represents valuable information that may be useful to oenological management and to valorise these varieties as sources of bioactive compounds.

Solid phase microextraction as a reliable alternative to conventional extraction techniques to evaluate the pattern of hydrolytically released components in Vitis vinifera L. grapes

In present research, headspace solid-phase microextraction (HS-SPME) followed by gas chromatography–mass spectrometry (GC–qMS), was evaluated as a reliable and improved alternative to the commonly used liquid–liquid extraction (LLE) technique for the establishment of the pattern of hydrolytically released components of 7 Vitis vinifera L. grape varieties, commonly used to produce the world-famous Madeira wine. Since there is no data available on their glycosidic fractions, at a first step, two hydrolyse procedures, acid and enzymatic, were carried out using Boal grapes as matrix. Several parameters susceptible of influencing the hydrolytic process were studied. The best results, expressed as GC peak area, number of identified components and reproducibility, were obtained using ProZym M with b-glucosidase activity at 35 °C for 42 h. For the extraction of hydrolytically released components, HS-SPME technique was evaluated as a reliable and improved alternative to the conventional extraction technique, LLE (ethyl acetate). HS-SPME using DVB/CAR/PDMS as coating fiber displayed an extraction capacity two fold higher than LLE (ethyl acetate). The hydrolyzed fraction was mainly characterized by the occurrence of aliphatic and aromatic alcohols, followed by acids, esters, carbonyl compounds, terpenoids, and volatile phenols. Concerning to terpenoids its contribution to the total hydrolyzed fraction is highest for Malvasia Cândida (23%) and Malvasia Roxa (13%), and their presence according previous studies, even at low concentration, is important from a sensorial point of view (can impart floral notes to the wines), due to their low odor threshold (μg/L). According to the obtained data by principal component analysis (PCA), the sensorial properties of Madeira wines produced by Malvasia Cândida and Malvasia Roxa could be improved by hydrolysis procedure, since their hydrolyzed fraction is mainly characterized by terpenoids (e.g. linalool, geraniol) which are responsible for floral notes. Bual and Sercial grapes are characterized by aromatic alcohols (e.g. benzyl alcohol, 2-phenylethyl alcohol), so an improvement in sensorial characteristics (citrus, sweet and floral odors) of the corresponding wines, as result of hydrolytic process, is expected.

Characterization of volatile substances in apples from rosaceae family by headspace solid‐phase microextraction followed by GC‐qMS

The volatile composition of different apple varieties of Malus domestica Borkh. species from different geographic regions at Madeira Islands, namely Ponta do Pargo (PP), Porto Santo (PS), and Santo da Serra (SS) was established by headspace solid-phase microextraction (HS-SPME) procedure followed by GC-MS (GC-qMS) analysis. Significant parameters affecting sorption process such as fiber coating, extraction temperature,extractiontime,sampleamount,dilutionfactor,ionicstrength,anddesorption time,wereoptimizedanddiscussed.TheSPMEfibercoatedwith50/30 lmdivinylbenzene/carboxen/PDMS (DVB/CAR/PDMS) afforded highest extraction efficiency of volatile compounds, providing the best sensitivity for the target volatiles, particularly whenthesampleswereextractedat508Cfor30 minwithconstantmagneticstirring. A qualitative and semi-quantitative analysis between the investigated apple species has been established. It was possible to identify about 100 of volatile compounds amongpulp(46,45,and39),peel(64,60,and64),andentirefruit(65,43,and50)inPP, PS,andSSapples,respectively.Ethylesters,terpenes,andhigheralcoholswerefound tobethemostrepresentativevolatiles. a-Farnesene,hexan-1-olandhexyl2-methylbutyratewerethecompoundsfoundinthevolatileprofileofstudiedappleswiththelargestGCarea,representing,onaverage,24.71,14.06,and10.80%ofthetotalvolatilefractionfromPP,PS,andSSapples.InPPentireapple,themostabundantcompoundsidentified were a-farnesene (30.49%), the unknown compound m/z (69, 101, 157) (21.82%) andhexylacetate(6.57%).RegardingPSentireapplethemajorcompoundswere a-farnesene(16.87%),estragole(15.43%),hexan-1-ol(10.94),andE-2-hexenal(10.67).a-Farnesene(30.3%),hexan-1-ol(18.90%),2-methylbutanoicacid(4.7%),andpentan-1-ol(4.6%) werealsofoundasSSentireapplevolatilespresentinahigherrelativecontent.Principal component analysis (PCA) of the results clustered the apples into three groups according to geographic origin. Linear discriminant analysis (LDA) was performed in order to detect the volatile compounds able to differentiate the three kinds of apples investigated. The most important contributions to the differentiation of the PP, PS, and SS apples were ethyl hexanoate, hexyl 2-methylbutyrate, E,E-2,4-heptadienal, pethylstyrene,andE-2-hexenal.

Determination of lipophilic and hydrophilic antioxidants in Solanum Lycopersicum L. tomato from Gordal variety by LL-USAE combined with UHPLC-PDA/FLR

Tomato (Lycopersicon esculentum L.) is one of the main constituents of the Mediterranean diet. Its consumption has been proposed to reduce the risk of cardiovascular diseases and certain types of cancer. It is therefore one of the most popular and extensively consumed vegetable crop worldwide. To gain insights on the potential of Lycopersicon esculentum L. as bioactive food, two analytical methodologies were developed to determine the levels of the lipophilic -tocopherol, α-tocopherol, β-carotene, lycopene; and hydrophilic antioxidants ascorbic acid. The quantification of total carotenoids (β-carotene and lycopene) was assessed through a liquid–liquid ultrasound assisted extraction (LL-USAE) in combination with ultraviolet-visible spectroscopy (UV-Vis), according to method of mean, for total carotenoids (λmáx = 450 nm. The ultra-high performance liquid chromatographic using both photodiode array and fluorescence detection (UHPLC-PDA/FLR), allows the identification and quantification of the target lipophilic and hydrophilic antioxidants. This methodology UHPLC-PDA/FLR is fast, simple and revealed a high sensitivity for the compounds under study. The limits of detection (LODs) and quantification (LOQs) obtained were much lower (about 10 times) than the reported in literature. The method LL-USAE/UV-Vis was validated and applied to different tomato foodstuffs. The results reveal a small increase of carotenoids content during maturation, reaching the maximum level when ripe. These results complement those obtained by the ORAC and TBARS assays that show an increase of antioxidant capacity during maturation. The LODs ans LOQs obtained were also about 10 times lower than reported in literature. The carotenoid content was also evaluated by LL-USAE/UV-Vis in different tomatoes varieties. Regional variety present the high carotenoid level, followed by campari and gordal, and at last grape. This methodology was also applied to different processed food samples containing tomatoes derivatives. Highest carotenoids content were obtained in concentrated tomato foodstuffs.