References about Madeira Vitiviniculture research

Scientific papers

1. Pereira V, Santos M, Cacho J, Marques JC. Assessment of the development of browning, antioxidant activity and volatile organic compounds in thermally processed sugar model wines. LWT – Food Science and Technology. 2017;75:719-26.

Abstract: The study evaluates the contribution of the fructose and glucose’s degradation for the Madeira wine’s features. The browning index, antioxidant activity and volatile organic compounds developed by the glucose and fructose model systems simulating thermally processed sweet Madeira wines were assessed. Sixteen different fructose/glucose model systems were prepared in synthetic wine and stored at 50 °C for 4 months. Then, three model wines were also submitted to 70 °C for 1 month. The browning index and the antioxidant activity ranged between 0.00 and 0.27 AU and 3.0–65.3 mg(GAE)/L, respectively. The development of several volatile organic compounds was demonstrated (up to 47). The identified compounds were mostly furans, with 5-hydroxymethylfurfural as the most abundant. For the first time, it was shown that the origin of sotolon in sweet wine can be associated with the acid-catalyzed fructose degradation mechanism. Other 2(5H)-furanones were also identified. It could be concluded that part of the browning, antioxidant activity and aroma compounds developed in sweet fortified wines is associated with the thermal degradation of fructose in acid medium.

2. Freitas J, Perestrelo R, Cassaca R, Castillo M, Santos M, Pereira J, et al. A fast and environment-friendly MEPSPEP/UHPLC-PDA methodology to assess 3-hydroxy-4,5-dimethyl-2(5H)-furanone in fortified wines. Food Chemistry. 2017;214:686-93.

Abstract: Sotolon is widely associated with the quality of fortified aged wines, and has also been linked to premature oxidative aging (premox). Here we developed a single, fast and environmental-friendly microextraction by packed sorbent ultra-high pressure liquid chromatography analysis (MEPS/UHPLC-PDA) for sotolon quantification in different wines. The best extraction conditions (loading three times 250 μL samples through the MEPSPEP sorbent and elution with 100 μL of 50% MeOH) were combined with a fast UHPLC separation (5 min separation using acidified 10% MeOH isocratic flow in a CORTECS C18 column) to allow unparalleled minimum sample and solvents volumes usage. The validated methodology showed good linearity (r2 > 0.993) and precision (<5.6%); high recovery (>81%) and detection limits (0.45–2.51 μg/L) far below sotolon odor threshold for any type of wine. The methodology was successfully applied to selected white table and Madeira wines, encompassing therefore a wide range of alcohol and sugar contents. Furthermore, as far we may know, this is the first time a single methodology can be used to assess both wine aging or premox according to the type of wine.

3. Pereira AC, Carvalho MJ, Miranda A, Leça JM, Pereira V, Albuquerque F, et al. Modelling the ageing process: A novel strategy to analyze the wine evolution towards the expected features. Chemometrics and Intelligent Laboratory Systems. 2016;154:176-84.

Abstract: In this work we present a new strategy to monitor the wine evolution during the ageing process. More specifically, we validate a procedure for analyzing how wine evolves during the ageing process in relation to the desired and expected quality features and we apply the proposed methodology to the case of a Portuguese fortified wine, the Madeira wine, where we compare the wine evolution under two different ageing processes. The approach developed consists on modeling samples labeled as aged reference wines (5 year old Madeira wines), produced from four different grape varieties, and then analyze how and in which extent young wines (up to 3 years old) come closer to the reference data set. The analysis is based on a comprehensive set of chemical data, including: polyphenolic composition, organic acids, reducing sugars, color and oenological parameters, commonly used as routine quality control information. The study considers several feature extraction methods, such as: Principal Components of Analysis (PCA), Independent Component of Analysis (ICA) and Partial Least Squares (PLS). The classification methodologies tested were: Linear Discriminant Analysis (LDA), nearest neighbor (k-NN) and Soft Independent Modelling by Class Analogy (SIMCA). The different options of preprocessing/feature extraction/classification were evaluated and compared using a Monte Carlo approach. From our analysis, the best combination of feature extraction/classification methodologies was PLS/LDA, which presented a classification performance of approximately 90% for three out of the four classes modeled, and of about 78% for the remaining one. Regarding the wines monitored during the first 3 years, our analysis revealed that they indeed mature in relation to the five year old reference wines. Furthermore, for some wines, it is possible to detect differences between the two ageing processes analyzed. This study is of particular importance for this type of wines, where the ageing process plays a central role for attaining the expected quality levels, implying significant risks and costs for local and industrial producers. Notwithstanding the specific case study presented, the strategy outlined can be extrapolated to other products with similar characteristics in terms of their monitoring and process control.

4. Perestrelo R, Silva CL, Câmara JS. Quantification of furanic derivatives in fortified wines by a highly sensitive and ultrafast analytical strategy based on digitally controlled microextraction by packed sorbent combined with ultrahigh pressure liquid chromatography. Journal of Chromatography A. 2015;1381:54-63.

Abstract: An improved, reliable and powerful analytical strategy based on digitally controlled microextraction by packed sorbent (MEPS) combined with ultrahigh pressure liquid chromatography (UHPLC) was validated for the simultaneous identification and quantification of major furanic derivatives, namely 5-hydroxymethyl-2-furaldehyde (5HMF), 5-methyl-2-furaldehyde (5MF), 2-furaldehyde (2F) and 2-furyl methyl ketone (2FMK), in fortified wines. To enhance the extraction efficiency of the target furanic derivates, several influencing extraction parameters, such as number of loading cycles, nature of elution solvent and elution volume, were evaluated and optimized. In addition the ability of different MEPS sorbent materials, namely C2, C8, C18, SIL, M1, R-AX, R-CX and PGC, were also tested. The optimal analytical conditions involved loading 3 × 200 μL of wine samples through a C8 sorbent in a MEPS syringe placed in the semi-automatic eVolH syringe followed by elution using 200 μL MeOH:H2O (95:5, v/v). The furanic derivates separation was achieved using a CORTECS UPLC® C18 analytical column in an ultrafast chromatographic run (within 4 min). The method performance was assessed for dry/medium dry (D/MD) and sweet/medium sweet (S/MS) model wines in terms of selectivity, linearity, limit of detection (LOD), limit of quantitation (LOQ), accuracy, precision and matrix effect, using model wine matrix-matched calibration. Good linearity was obtained with a regression coefficient (r2) higher than 0.992. A good precision was attained (RSD < 5%) and low LODs were achieved for D/MD (4.5–129.3 ng L−1) and S/MS (6.9–285.2 ng L−1) model wines. The quantification limits (LOQ) for D/MD model wines ranged from 14.9 to 431.0 ng L−1, whereas for S/MS model wines range from 23.1 to 950.5 ng L−1. The method also afforded satisfactory results in terms of accuracy, ranging from 74 to 97% for D/MD wines and between 84 and 99% for S/MS wines. The MEPSC8/UHPLC–PDA analytical strategy was successfully applied to analyze furanic derivates in 26 fortified Madeira wines from different types (D/MD, S/MS) and ages. The obtained results revealed the analytical strategy as a suitable tool which combines sensitivity, effectiveness, reduced analysis time and simple analytical procedure. Principal component analysis (PCA) suggested that fortified wines can be organized based on their age on PC1, which are mainly characterized by 5HMF.

5. Pereira V, Pereira AC, Pérez Trujillo JP, Cacho J, Marques JC. Amino Acids and Biogenic Amines Evolution during the Estufagem of Fortified Wines. Journal of Chemistry. 2015;2015:9.

Abstract: The current study was focused on the impact of accelerated ageing (heating step) on the amino acid and biogenic amine profiles of fortified wines. In this sense, three Madeira wines from two commonly used grape varieties (one red and the other white) were analysed during the heating, at standard (45°C, 3 months) and overheating (70°C, 1 month) conditions, following a precolumn derivatization procedure using iodoacetic acid, o-phthaldialdehyde, and 2-mercaptoethanol, carried out in the injection loop prior to RP-HPLC-FLD detection. Eighteen amino acids were identified, with arginine being the most abundant. An important decrease of the amino acid levels was detected during the standard heating (up to 30%), enhanced up to 61% by the temperature increase. Cysteine, histidine, and asparagine revealed the greatest decreases at 45°C. Conversely, some amino acids, such as asparagine, slightly increased. Four biogenic amines were identified but always in trace amounts. Finally, it was observed that the accelerated ageing did not favour the biogenic amine development. The results also indicate that the heating process promotes the amino acid transformation into new ageing products.

6. Carvalho MJ, Pereira V, Pereira AC, Pinto JL, Marques JC. Evaluation of Wine Colour Under Accelerated and Oak-Cask Ageing Using CIELab and Chemometric Approaches. Food and Bioprocess Technology. 2015;8:2309-18.

Abstract: The present study aims to analyse the evolution of colour and browning of fortified wines submitted to oak and accelerated ageing induced by temperature, both traditional ageing processes of Madeira wines. These wines were followed during the first 2 years of ageing and were simultaneously compared to aged reference samples, in order to disclose if accelerated ageing favours the wine evolution. The data analysis approaches followed two strategies: the first one was based on the monitoring of wine colour parameters based on CIELab methodology, while the second strategy adopted an untargeted approach, taking advantage of all wine information comprised on the UV-vis spectrum. CIELab results indicated that the greatest variation (up to 92 %) occurs during the vinification, since it was observed that wines produced from the same grape variety (Tinta Negra) acquired different characteristics already on the vinification processes. All wines tend to approach the reference samples during ageing, with the lowest ΔE* value obtained for Malvasia submitted to canteiro at 2 years of ageing (3.29 units). Sweet Tinta Negra wines, previously submitted to estufagem, presented the most pronounced colour change associated with ageing. The multivariate principal component analysis (PCA) model was built with aged reference samples to evaluate how young wines acquire the reference wine’s characteristics and revealed that accelerated ageing increases colour evolution and favours the homogeneity of Malvasia wines.

7. Perestrelo R, Silva C, Câmara JS. A useful approach for the differentiation of wines according to geographical origin based on global volatile patterns. Journal of Separation Science. 2014;37:1974-81.

Abstract: In this study, the feasibility of solid-phase extraction combined with gas chromatography and mass spectrometry in tandem with partial least squares discriminant analysis was evaluated as a useful strategy to differentiate wines according to geographical origin (Azores, Canary and Madeira Islands) and types (white, red and fortified wine) based on their global volatile patterns. For this purpose, 34 monovarietal wines from these three wine grape-growing regions were investigated, combining the high throughput extraction efficiency of the solid-phase extraction procedure with the separation and identification ability. The partial least squares discriminant analysis results suggested that Madeira wines could be clearly discriminated from Azores and Canary wines. Madeira wines are mainly characterized by 2-ethylhexan-1-ol, 3,5,5-trimethylhexan-1-ol, ethyl 2-methylbutanoate, ethyl dl-2-hydroxycaproate, decanoic acid, 3-methylbutanoic acid, and (E)-whiskey lactone, whereas 3-ethoxypropan-1-ol, 1-octen-3-ol, (Z)-3-hexenyl butanoate, 4-(methylthio)-1-butanol, ethyl 3-hydroxybutanoate, isoamyl lactate, 4-methylphenol, γ-octalactone and 4-(methylthio)-1-butanol, are mainly associated with Azores and Canary wines. The data obtained in this study revealed that solid-phase extraction combined with gas chromatography and quadrupole mass spectrometry data and partial least squares discriminant analysis provides a suitable tool to discriminate wines, both in terms of geographical origin as well as wine type and vintage.

8. Perestrelo R, Barros AS, Rocha SM, Câmara JS. Establishment of the varietal profile of Vitis vinifera L. grape varieties from different geographical regions based on HS-SPME/GC–qMS combined with chemometric tools. Microchemical Journal. 2014;116:107-17.

Abstract: The varietal volatile profiles of pulp and skin fractions from the main red and white Vitis vinifera L. grape varieties used in the production of Madeira wine (Portugal) were established by headspace solid phase microextraction (SPME) combined with gas chromatography–quadrupole mass spectrometry (GC–qMS) tandem with chemometric tools. A total of 60 varietal volatile compounds — 27 in pulp and 42 in skin of V. vinifera L. grapes studied, were identified, including 27 monoterpenic, 26 sesquiterpenic and 7 C13 norisoprenoid compounds. Principal Component Analysis (PCA) and Hierarchical Clusters Analysis (HCA) results demonstrated that, independently of grape fractions, a distribution by grape variety (white and red) as well as by Terroir effect, was attained using the varietal volatiles from skin grape fraction. Bual, Malvasia Cândida and Malvasia Branca (white varieties) exhibited different varietal volatile profiles, independently of grape fractions, skin or pulp. This difference could be explained by the highest sesquiterpenic level found in Bual grape variety, and monoterpenic level found in Malvasia Cândida and Malvasia Branca grape varieties. The obtained data suggests that climatic factors, such as altitude, temperature and humidity influenced the varietal profile from the target varieties, as the varietal profile of grapes cultivated at Estreito de Câmara de Lobos (32°39′50.59″N; 16°58′48.28″W) and Jardim da Serra (32°41′42.36″N; 16°59′37.32″W) showed qualitative and quantitative differences (expressed as GC peak area) when compared to varietal profile of grapes cultivated at Estreito da Calheta (32°44′0.09″N; 17°11′14.80″W), independently of the grape fractions (pulp or skin). Moreover, the cultivation of recommended grapes to produce Madeira wines — Malvasia Branca, Terrantez and Bastardo, should be promoted as their varietal volatile profile is quite similar to that found in noble varieties (Malvasia Cândida and Sercial, respectively).

9. Pereira V, Cacho J, Marques JC. Volatile profile of Madeira wines submitted to traditional accelerated ageing. Food Chemistry. 2014;162:122-34.

Abstract: The evolution of monovarietal fortified Madeira wines forced-aged by traditional thermal processing (estufagem) were studied in terms of volatiles. SPE extracts were analysed by GC–MS before and after heating at 45 °C for 3 months (standard) and at 70 °C for 1 month (overheating). One hundred and ninety volatile compounds were identified, 53 of which were only encountered in baked wines. Most chemical families increased after standard heating, especially furans and esters, up to 61 and 3-fold, respectively. On the contrary, alcohols, acetates and fatty acids decreased after heating. Varietal aromas, such as Malvasia’s monoterpenic alcohols were not detected after baking. The accelerated ageing favoured the development of some volatiles previously reported as typical aromas of finest Madeira wines, particularly phenylacetaldeyde, β-damascenone and 5-ethoxymethylfurfural. Additionally, ethyl butyrate, ethyl 2-methylbutyrate, ethyl caproate, ethyl isovalerate, guaiacol, 5-hydroxymethylfurfural and γ-decalactone were also found as potential contributors to the global aroma of baked wines.

10. Pereira V, Albuquerque F, Cacho J, Marques J. Polyphenols, Antioxidant Potential and Color of Fortified Wines during Accelerated Ageing: The Madeira Wine Case Study. Molecules. 2013;18:2997.

Abstract: Polyphenols, antioxidant potential and color of three types of fortified Madeira wines were evaluated during the accelerated ageing, named as estufagem. The traditional estufagem process was set to 45 °C for 3 months. Overheating conditions, 1 month at 70 °C, were also examined. Total polyphenols (TP), total monomeric anthocyanins (TMA) and total flavonoids (TF) were assessed by spectrophotometric methods, while individual polyphenols and furans were simultaneously determined by HPLC-DAD. Antioxidant potential (AP) was estimated by ABTS, DPPH and FRAP assays, while color was evaluated by Glories and CIELab. Traditional estufagem decreased the TP and AP up to 20% and 26%, respectively, with final values similar to other wines. TMA of the Madeira wines from red grapes decreased during estufagem. Six hydroxybenzoic acids, three hydroxycinnamic acids, one stilbene, three flavonols and three flavan-3-ols were found in these wines. The prominent phenolics were hydroxycinnamates and hydroxybenzoates, even after estufagem. Most polyphenols decreased, with the exception of caffeic, ferulic, p-coumaric, gallic and syringic acids. Finally, both chromatic systems revealed that all wines tended to similar chromatic characteristics after estufagem. The study suggests that estufagem can be applied without high impact on polyphenols and antioxidant potential of these fortified wines.

11. Fernandes PJ, Barros N, Câmara JS. A survey of the occurrence of ochratoxin A in Madeira wines based on a modified QuEChERS extraction procedure combined with liquid chromatography–triple quadrupole tandem mass spectrometry. Food Research International. 2013;54:293-301.

Abstract: Over the past years, to ensure food safety, the European Union (EU) has adopted specific legislation concerning the control of mycotoxins residue levels in different kinds of food. In this study, a fast, selective and sensitive reversed-phased liquid chromatography with tandem mass spectrometry (LC–MS/MS) methodology was developed and validated for quantification of ochratoxin A (OTA) in Madeira wines. Sample extraction and purification were performed with a modified QuEChERS-based (quick, easy, cheap, effective, rugged, and safe) procedure. Firstly, the homogenized samples are extracted and partitioned using an organic solvent and salt solution. Then, the supernatant is further extracted and cleaned using a dispersive solid phase extraction (dSPE) technique. Finally clear wine extracts were concentrated under vacuum to near dryness and taken up into initial mobile phase. Careful optimization of the LC–MS/MS parameters was achieved in order to attain a fast separation with the best sensitivity. The detection was carried out on a triple–quadrupole tandem mass spectrometer (MS/MS) by electrospray ionization in positive ion mode (ESI+) with multiple reaction monitoring (MRM). MS/MS conditions were optimized in order to increase selectivity, selecting the corresponding product ions (precursor-to-fragment m/z 404 → 239; m/z 404 → m/z 358) for quantification and identification. The performance of the method was assessed and compared to the European Commission (EC) Regulations, by studying the selectivity and specificity, limit of detection (LOD), limit of quantification (LOQ), linear dynamic range, matrix effect, accuracy, precision, robustness/ruggedness and uncertainty. The validation was performed by analyzing recovery samples at four different spiked concentrations, 0.5, 1.0, 5.0 and 10.0 μg/kg, with four replicates (n = 4) at each concentration. Recoveries ranged from 87.2% to 102.6%, with relative standard deviations below 9% in all cases. The intra-day precision and inter-day precision, expressed as relative standard deviation, were lower than 7% and 14%, respectively. Matrix effects were observed by comparing the slope of matrix-matched standard calibration with that of solvent. Good linearity was achieved at the concentration levels of 0.50–22.5 μg/L. The LOQ and LOD, 0.4 and 0.1 μg/kg, respectively, at the signal-to-noise ratio (S/N) of 10 and 3, were lower than the concentration usually permitted by legislation in wines. The method was successfully applied to evaluate the occurrence of OTA in 30 Madeira wine samples. None of the analyzed samples exceeded the maximum permissible limit for wines (2.0 μg/kg) set by the EC, which confirms that the risk of OTA occurrence in Madeira wines is, as expected, is very low.

12. Perestrelo R, Caldeira M, Câmara JS. Solid phase microextraction as a reliable alternative to conventional extraction techniques to evaluate the pattern of hydrolytically released components in Vitis vinifera L. grapes. Talanta. 2012;95:1-11.

Abstract: 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.

13. Gonçalves J, Mendes B, Silva CL, Câmara JS. Development of a novel microextraction by packed sorbent-based approach followed by ultrahigh pressure liquid chromatography as a powerful technique for quantification phenolic constituents of biological interest in wines. Journal of Chromatography A. 2012;1229:13-23.

Abstract: A novel analytical approach, based on a miniaturized extraction technique, the microextraction by packed sorbent (MEPS), followed by ultrahigh pressure liquid chromatography (UHPLC) separation combined with a photodiode array (PDA) detection, has been developed and validated for the quantitative determination of sixteen biologically active phenolic constituents of wine. In addition to performing routine experiments to establish the validity of the assay to internationally accepted criteria (linearity, sensitivity, selectivity, precision, accuracy), experiments are included to assess the effect of the important experimental parameters on the MEPS performance such as the type of sorbent material (C2, C8, C18, SIL, and M1), number of extraction cycles (extract-discard), elution volume, sample volume, and ethanol content, were studied. The optimal conditions of MEPS extraction were obtained using C8 sorbent and small sample volumes (250 μL) in five extraction cycle and in a short time period (about 5 min for the entire sample preparation step). The wine bioactive phenolics were eluted by 250 μL of the mixture containing 95% methanol and 5% water, and the separation was carried out on a HSS T3 analytical column (100 mm × 2.1 mm, 1.8 μm 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 gave satisfactory results in terms of linearity with r2-values > 0.9986 within the established concentration range. The LOD varied from 85 ng mL−1 (ferulic acid) to 0.32 μg mL−1 ((+)-catechin), whereas the LOQ values from 0.028 μg mL−1 (ferulic acid) to 1.08 μg mL−1 ((+)-catechin). Typical recoveries ranged between 81.1 and 99.6% for red wines and between 77.1 and 99.3% for white wines, with relative standard deviations (RSD) no larger than 10%. The extraction yields of the MEPSC8/UHPLC–PDA methodology were found between 78.1 (syringic acid) and 99.6% (o-coumaric acid) for red wines and between 76.2 and 99.1% for white wines. The inter-day precision, expressed as the relative standard deviation (RSD%), varied between 0.2% (p-coumaric and o-coumaric acids) and 7.5% (gentisic acid) while the intra-day precision between 0.2% (o-coumaric and cinnamic acids) and 4.7% (gallic acid and (−)-epicatechin). On the basis of analytical validation, it is shown that the MEPSC8/UHPLC–PDA methodology proves to be an improved, reliable, and ultra-fast approach for wine bioactive phenolics analysis, because of its capability for determining simultaneously in a single chromatographic run several bioactive metabolites with high sensitivity, selectivity and resolving power within only 10 min. Preliminary studies have been carried out on 34 real whole wine samples, in order to assess the performance of the described procedure. The new approach offers decreased sample preparation and analysis time, and moreover is cheaper, more environmentally friendly and easier to perform as compared to traditional methodologies.

14. Mendes B, Gonçalves J, Câmara JS. Effectiveness of high-throughput miniaturized sorbent- and solid phase microextraction techniques combined with gas chromatography–mass spectrometry analysis for a rapid screening of volatile and semi-volatile composition of wines—A comparative study. Talanta. 2012;88:79-94.

Abstract: In this study the feasibility of different extraction procedures was evaluated in order to test their potential for the extraction of the volatile (VOCs) and semi-volatile constituents (SVOCs) from wines. In this sense, and before they could be analysed by gas chromatography–quadrupole first stage masss spectrometry (GC–qMS), three different high-throughput miniaturized (ad)sorptive extraction techniques, based on solid phase extraction (SPE), microextraction by packed sorbents (MEPS) and solid phase microextraction (SPME), were studied for the first time together, for the extraction step. To achieve the most complete volatile and semi-volatile signature, distinct SPE (LiChrolut EN, Poropak Q, Styrene-Divinylbenzene and Amberlite XAD-2) and MEPS (C2, C8, C18, Silica and M1 (mixed C8-SCX)) sorbent materials, and different SPME fibre coatings (PA, PDMS, PEG, DVB/CAR/PDMS, PDMS/DVB, and CAR/PDMS), were tested and compared. All the extraction techniques were followed by GC–qMS analysis, which allowed the identification of up to 103 VOCs and SVOCs, distributed by distinct chemical families: higher alcohols, esters, fatty acids, carbonyl compounds and furan compounds. Mass spectra, standard compounds and retention index were used for identification purposes. SPE technique, using LiChrolut EN as sorbent (SPELiChrolut EN), was the most efficient method allowing for the identification of 78 VOCs and SVOCs, 63 and 19 more than MEPS and SPME techniques, respectively. In MEPS technique the best results in terms of number of extractable/identified compounds and total peak areas of volatile and semi-volatile fraction, were obtained by using C8 resin whereas DVB/CAR/PDMS was revealed the most efficient SPME coating to extract VOCs and SVOCs from Bual wine. Diethyl malate (18.8 ± 3.2%) was the main component found in wine SPELiChrolut EN extracts followed by ethyl succinate (13.5 ± 5.3%), 3-methyl-1-butanol (13.2 ± 1.7%), and 2-phenylethanol (11.2 ± 9.9%), while in SPMEDVB/CAR/PDMS technique 3-methyl-1-butanol (43.3 ± 0.6%) followed by diethyl succinate (18.9 ± 1.6%), and 2-furfural (10.4 ± 0.4%), are the major compounds. The major VOCs and SVOCs isolated by MEPSC8 were 3-methyl-1-butanol (26.8 ± 0.6%, from wine total volatile fraction), diethyl succinate (24.9 ± 0.8%), and diethyl malate (16.3 ± 0.9%). Regardless of the extraction technique, the highest extraction efficiency corresponds to esters and higher alcohols and the lowest to fatty acids. Despite some drawbacks associated with the SPE procedure such as the use of organic solvents, the time-consuming and tedious sampling procedure, it was observed that SPELiChrolut EN, revealed to be the most effective technique allowing the extraction of a higher number of compounds (78) rather than the other extraction techniques studied.

15. Silva CL, Gonçalves JL, Câmara JS. A sensitive microextraction by packed sorbent-based methodology combined with ultra-high pressure liquid chromatography as a powerful technique for analysis of biologically active flavonols in wines. Analytica Chimica Acta. 2012;739:89-98.

Abstract: A new approach based on microextraction by packed sorbent (MEPS) and reversed-phase high-throughput ultra high pressure liquid chromatography (UHPLC) method that uses a gradient elution and diode array detection to quantitate three biologically active flavonols in wines, myricetin, quercetin, and kaempferol, is described. In addition to performing routine experiments to establish the validity of the assay to internationally accepted criteria (selectivity, linearity, sensitivity, precision, accuracy), experiments are included to assess the effect of the important experimental parameters such as the type of sorbent material (C2, C8, C18, SIL, and C8/SCX), number of extraction cycles (extract-discard), elution volume, sample volume, and ethanol content, on the MEPS performance. The optimal conditions of MEPS extraction were obtained using C8 sorbent and small sample volumes (250 μL) in five extraction cycle and in a short time period (about 5 min for the entire sample preparation step). Under optimized conditions, excellent linearity View the MathML source, limits of detection of 0.006 μg mL−1 (quercetin) to 0.013 μg mL−1 (myricetin) and precision within 0.5–3.1% were observed for the target flavonols. The average recoveries of myricetin, quercetin and kaempferol for real samples were 83.0–97.7% with relative standard deviation (RSD, %) lower than 1.6%. The results obtained showed that the most abundant flavonol in the analyzed samples was myricetin (5.8 ± 3.7 μg mL−1). Quercetin (0.97 ± 0.41 μg mL−1) and kaempferol (0.66 ± 0.24 μg mL−1) were found in a lower concentration.

The optimized MEPSC8 method was compared with a reverse-phase solid-phase extraction (SPE) procedure using as sorbent a macroporous copolymer made from a balanced ratio of two monomers, the lipophilic divinylbenzene and the hydrophilic N-vinylpyrrolidone (Oasis HLB) were used as reference. MEPSC8 approach offers an attractive alternative for analysis of flavonols in wines, providing a number of advantages including highest extraction efficiency (from 85.9 ± 0.9% to 92.1 ± 0.5%) in the shortest extraction time with low solvent consumption, fast sample throughput, more environmentally friendly and easy to perform.

16. Silva CL, Pereira J, Wouter VG, Giró C, Câmara JS. A fast method using a new hydrophilic–lipophilic balanced sorbent in combination with ultra-high performance liquid chromatography for quantification of significant bioactive metabolites in wines. Talanta. 2011;86:82-90.

Abstract: This manuscript describes the development and validation of an ultra-fast, efficient, and high throughput analytical method based on ultra-high performance liquid chromatography (UHPLC) equipped with a photodiode array (PDA) detection system, for the simultaneous analysis of fifteen bioactive metabolites: gallic acid, protocatechuic acid, (−)-catechin, gentisic acid, (−)-epicatechin, syringic acid, p-coumaric acid, ferulic acid, m-coumaric acid, rutin, trans-resveratrol, myricetin, quercetin, cinnamic acid and kaempferol, in wines. A 50-mm column packed with 1.7-μm particles operating at elevated pressure (UHPLC strategy) was selected to attain ultra-fast analysis and highly efficient separations. In order to reduce the complexity of wine extract and improve the recovery efficiency, a reverse-phase solid-phase extraction (SPE) procedure using as sorbent a new macroporous copolymer made from a balanced ratio of two monomers, the lipophilic divinylbenzene and the hydrophilic N-vinylpyrrolidone (Oasis™ HLB), was performed prior to UHPLC–PDA analysis. The calibration curves of bioactive metabolites showed good linearity within the established range. Limits of detection (LOD) and quantification (LOQ) ranged from 0.006 μg mL−1 to 0.58 μg mL−1, and from 0.019 μg mL−1 to 1.94 μg mL−1, for gallic and gentisic acids, respectively. The average recoveries ± SD for the three levels of concentration tested (n = 9) in red and white wines were, respectively, 89 ± 3% and 90 ± 2%. The repeatability expressed as relative standard deviation (RSD) was below 10% for all the metabolites assayed. The validated method was then applied to red and white wines from different geographical origins (Azores, Canary and Madeira Islands). The most abundant component in the analysed red wines was (−)-epicatechin followed by (−)-catechin and rutin, whereas in white wines syringic and p-coumaric acids were found the major phenolic metabolites. The method was completely validated, providing a sensitive analysis for bioactive phenolic metabolites detection and showing satisfactory data for all the parameters tested. Moreover, was revealed as an ultra-fast approach allowing the separation of the fifteen bioactive metabolites investigated with high resolution power within 5 min.

17. Pérez Trujillo JP, Conde JE, Pérez Pont ML, Câmara J, Marques JC. Content in metallic ions of wines from the Madeira and Azores archipelagos. Food Chemistry. 2011;124:533-7.

Abstract: This study determines for the first time Na, K, Ca, Mg, Fe, Cu, Zn, Mn, Sr, Li and Rb contents in wines from the archipelagos of Madeira and Azores (Portugal). The greater part of the mean content for the different parameters fell within the ranges described in the literature, except for sodium whose higher content may be due to the effect of marine spray. ANOVA was used to establish the metals with significant differences in mean content between the wines from both archipelagos, between table and liquor wines of Madeira, and between wines of Pico and Terceira Islands from the Azores archipelago. Principal component analysis shows differences in the wines according to the wine-making process and/or the equipment employed. Stepwise linear discriminant analysis achieves a good classification and validation of wines according to the archipelago of origin, and the island in the case of Azores wines.

18. Perestrelo R, Barros AS, Câmara JS, Rocha SM. In-Depth Search Focused on Furans, Lactones, Volatile Phenols, and Acetals As Potential Age Markers of Madeira Wines by Comprehensive Two-Dimensional Gas Chromatography with Time-of-Flight Mass Spectrometry Combined with Solid Phase Microextraction. Journal of Agricultural and Food Chemistry. 2011;59:3186-204.

Abstract: The establishment of potential age markers of Madeira wine is of paramount significance as it may contribute to detect frauds and to ensure the authenticity of wine. Considering the chemical groups of furans, lactones, volatile phenols, and acetals, 103 volatile compounds were tentatively identified; among these, 71 have been reported for the first time in Madeira wines. The chemical groups that could be used as potential age markers were predominantly acetals, namely, diethoxymethane, 1,1-diethoxyethane, 1,1-diethoxy-2-methyl-propane, 1-(1-ethoxyethoxy)-pentane, trans-dioxane and 2-propyl-1,3-dioxolane, and from the other chemical groups, 5-methylfurfural and cis-oak-lactone, independently of the variety and the type of wine. GC × GC-ToFMS system offers a more useful approach to identify these compounds compared to previous studies using GC−qMS, due to the orthogonal systems, that reduce coelution, increase peak capacity and mass selectivity, contributing to the establishment of new potential Madeira wine age markers. Remarkable results were also obtained in terms of compound identification based on the organized structure of the peaks of structurally related compounds in the GC × GC peak apex plots. This information represents a valuable approach for future studies, as the ordered-structure principle can considerably help the establishment of the composition of samples. This new approach provides data that can be extended to determine age markers of other types of wines.

19. Pereira V, Albuquerque FM, Ferreira AC, Cacho J, Marques JC. Evolution of 5-hydroxymethylfurfural (HMF) and furfural (F) in fortified wines submitted to overheating conditions. Food Research International. 2011;44:71-6.

Abstract: As furfural (F) and 5-hydroxymethylfurfural (HMF) are essentially formed from sugar dehydration, especially in food submitted to heat, they can be found in beverages, as well as fortified sweet wines. In order to assess the impact of temperature on Madeira winemaking, three traditional varieties of Madeira wines (Malvasia, Sercial and Tinta Negra Mole) were studied to evaluate the F and HMF contents. The wines were produced by two vinification processes, following traditional and modern methodologies, heated at standard conditions (30 °C and 45 °C, for 4 months) and compared with the same wines submitted to overheating conditions (55 °C, for 4 months). The RP-HPLC-DAD methodology used for the control of F and HMF during the process showed no significant changes in the wines maintained at 30 °C (canteiro) and a noticeable but controlled increase in the wines heated at 45 °C (estufagem) where values up to about 150 mg/L of HMF could be found in sweet wines. The strong relation of this compound with the sugar content and baking temperature stood out in the wines submitted to overheating conditions where values higher than 1 g/L could be found for sweeter wines, with HMF level being in general higher than F. The results clearly suggest that the amount of HMF in these fortified wines can be easily controlled when submitted to adequate conditions of heating during estufagem and storage. Furthermore, different temperatures for the baking of sweet and dry wines may be considered.

20. Pereira AC, Reis MS, Saraiva PM, Marques JC. Madeira wine ageing prediction based on different analytical techniques: UV–vis, GC-MS, HPLC-DAD. Chemometrics and Intelligent Laboratory Systems. 2011;105:43-55.

Abstract: The present work aims to analyze the feasibility of different analytical measurement procedures for Madeira wine ageing prediction. In order to properly identify and quantify the chemical compounds qualified for characterizing wine evolution during the ageing period, chromatographic and spectroscopic analyses were carried out. Twenty-six samples, representative of ten harvest years and covering an ageing period of 20 years, were analyzed in terms of their volatile and phenolic composition, as well as characterized in terms of absorbance measurements in the UV and Visible region. Then, multivariate prediction models were established by applying PLS regression to each chemical data set, after which they were compared in terms of their ageing prediction ability. The optimum number of PLS dimensions to consider in each estimated model was obtained based on the minimization of the root mean squared error of Monte Carlo validation. With such estimated models, the prediction interval estimates based on the bootstrap percentile approach were also computed for the available samples, in order to test model’s prediction ability, once each sample is successively removed from the data set. Our analysis shows that Madeira wine age, produced from a known grape variety, can be predicted with good accuracy from its volatile and phenolic composition, as well as from UV–vis absorbance measurements. The PLS models estimated are able to predict wine age with a root mean square error of 0.9, 1.1, and 1.4 years, respectively. The sample-specific prediction intervals computed also allowed for the analysis of differences between observed and predicted values, and confirmed the interesting wine age prediction abilities of the proposed methodologies. A compromise between model accuracy and cost of analysis can be established in order to decide which methodology to use, according to the particular application scenario, as the more time-consuming and complex techniques (GC-MS and HPLC-DAD) are also those leading to more accurate results, but UV–vis also enabled us to come up with acceptable age predictions.

21. Pereira AC, Reis MS, Saraiva PM, Marques JC. Development of a fast and reliable method for long- and short-term wine age prediction. Talanta. 2011;86:293-304.

Abstract: Wine age prediction based on its intrinsic characteristics can provide significant assistance to oenologists’ quality evaluations, concerning wine ageing process control and wine quality assurance. Simpler, faster, cheaper and affordable analytical procedures would be greatly welcome to establish such a practice. In this study, we present a new and reliable strategy to predict wine age, in the long and short-term, centered on the use of wine UV–vis absorbance data, coupled with proper chemometric techniques. The strategy followed consists essentially in first pre-processing the UV–vis data, secondly to carry out variable selection over such pre-processed data sets, and finally to use the set of selected variables for developing a PLS model focused on wine age prediction. We tested different data pre-processing methodologies, namely first and second derivatives, multiplicative scatter correction, standard normal variate and orthogonal signal correction, as well as different variable selection approaches, specifically interval partial least squares, VIPS, genetic algorithms and the wavelet transformation combined with a genetic algorithm. In both case studies, regarding long and short-term ageing periods, we have found out that it is indeed possible to predict wine ages, in our case Madeira wine ages, with an accuracy of 1.4 years for longer ageing periods, and of 3 months for wines of an age comprised in the first two years of ageing. The genetic algorithm revealed to be very useful for proper wavelet coefficients selection, leading to the most parsimonious model among all those analyzed, which also presents the best predictive performance found.

22. Gonçalves J, Câmara JS. New method for determination of (E)-resveratrol in wine based on microextraction using packed sorbent and ultra-performance liquid chromatography. Journal of Separation Science. 2011;34:2376-84.

Abstract: 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 (50–250 μL) 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 1×250 μL of the mixture containing 95% methanol and 5% water, and the separation was carried out on a high-strength silica HSS T3 analytical column (100 mm×2.1 mm, 1.8 μm 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 60 μg/mL. Validation experiments revealed very good recovery rate of 95±5.8% RSD, good linearity with r2 values >0.999 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 0.21 μg/mL, whereas the LOQ was 0.68 μg/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.

23. Rudnitskaya A, Rocha SM, Legin A, Pereira V, Marques JC. Evaluation of the feasibility of the electronic tongue as a rapid analytical tool for wine age prediction and quantification of the organic acids and phenolic compounds. The case-study of Madeira wine. Analytica Chimica Acta. 2010;662:82-9.

Abstract: A set of fourteen Madeira wines comprising wines produced from four Vitis vinifera L. varieties (Bual, Malvasia, Verdelho and Tinta Negra Mole) that were 3, 6, 10 and 17 years old was analysed using HPLC and an electronic tongue (ET) multisensor system. Concentrations of 24 organic acids, phenolic and furanic compounds were determined by HPLC. The ET consisting of 26 potentiometric chemical sensors with plasticized PVC and chalcogenide glass membranes was used. Significance of the effects of age and variety on the ET response and wine composition with respect to the organic acids, phenolics and furanic derivatives were evaluated using ANOVA—Simultaneous Component Analysis (ASCA). Significance of the effects was estimated using a permutation test (1000 permutations). It was found that effects of age, grape variety and their interaction were significant for the HPLC data set and only the effect of age was significant for the ET data. Calibration models of the HPLC and ET data with respect to the wine age and of the ET data with respect to the concentration of the organic acids and phenolics were calculated using PLS1 regression. Models were validated using cross-validation. It was possible to predict wine age from HPLC and ET data with the accuracy in cross-validation of 2.6 and 1.8 years respectively. The ET was capable of detecting the following components (mean relative error in cross-validation is shown in the parentheses): tartaric (8%), citric (5%), formic (12%), protocatehuic (5%), vanillic (18%) and sinapic (14%) acids, catechin (6%), vanillin (12%) and trans-resveratrol (5%). The ET capability of predicting Madeira wine age with good accuracy (1.8 years) as well as quantify of some organic acids and phenolic compounds was demonstrated.

24. Perestrelo R, Petronilho S, Câmara JS, Rocha SM. Comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry combined with solid phase microextraction as a powerful tool for quantification of ethyl carbamate in fortified wines. The case study of Madeira wine. Journal of Chromatography A. 2010;1217:3441-5.

Abstract: An analytical methodology based on headspace solid phase microextraction (HS-SPME) combined with comprehensive two-dimensional gas chromatography—time-of-flight mass spectrometry (GC × GC–ToFMS) was developed for the identification and quantification of the toxic contaminant ethyl carbamate (EC) directly in fortified wines. The method performance was assessed for dry/medium dry and sweet/medium sweet model wines, and for quantification purposes, calibration plots were performed for both matrices using the ion extraction chromatography (IEC) mode (m/z 62). Good linearity was obtained with a regression coefficient (r2) higher than 0.981. A good precision was attained (R.S.D. <20%) and low detection limits (LOD) were achieved for dry (4.31 μg/L) and sweet (2.75 μg/L) model wines. The quantification limits (LOQ) and recovery for dry wines were 14.38 μg/L and 88.6%, whereas for sweet wines were 9.16 μg/L and 99.4%, respectively. The higher performance was attainted with sweet model wine, as increasing of glucose content improves the volatile compound in headspace, and a better linearity, recovery and precision were achieved. The analytical methodology was applied to analyse 20 fortified Madeira wines including different types of wine (dry, medium dry, sweet, and medium sweet) obtained from several harvests in Madeira Island (Portugal). The EC levels ranged from 54.1 μg/L (medium dry) to 162.5 μg/L (medium sweet).

25. Pereira V, Câmara JS, Cacho J, Marques JC. HPLC-DAD methodology for the quantification of organic acids, furans and polyphenols by direct injection of wine samples. Journal of Separation Science. 2010;33:1204-15.

Abstract: This article proposes a simple and sensitive HPLC method with photo-diode array detection for the analysis of organic acids, monomeric polyphenols and furanic compounds in wine samples by direct injection. The chromatographic separation of 8 organic acids, 2 furans and 22 phenolic compounds was carried out with a buffered solution (pH 2.70) and acetonitrile as mobile phases and a difunctionally bonded C18 stationary phase, Atlantis dC18 (250×4.6 mm, 5 μm) column. The elution was performed in 12 min for the organic acids and in 60 min for the phenolic compounds, including phenolic acids, stilbenes and flavonoids. Target compounds were detected at 210 nm (organic acids, flavan-3-ols and benzoic acids), 254 nm (ellagic acid), 280 nm (furans and cinnamic acid), 315 nm (hydroxycinnamic acids and trans-resveratrol) and 360 nm (flavonoids). The RSD for the repeatability test (n=5) of peak area and retention times were below 3.1 and 0.3%, respectively, for phenolics and below 1.0 and 0.2% for organic acids. The RSDs expressing the reproducibility of the method were higher than for the repeatability results but all below 9.0%. Method accuracy was evaluated by the recovery results, with averaged values between 80 and 104% for polyphenols and 97–105% for organic acids. The calibration curves, obtained by triplicate injection of standard solutions, showed good linearity with regression coefficients higher than 0.9982 for polyphenols and 0.9997 for organic acids. The LOD was in the range of 0.07–0.49 mg/L for polyphenols (cinnamic and gallic acids, respectively) and 0.001–0.046 g/L for organic acids (oxalic and lactic acids, respectively). The method was successfully used to measure and assess the polyphenolic fingerprint and organic acids profile of red, white, rosé and fortified wines.

26. Pereira AC, Reis MS, Saraiva PM, Marques JC. Aroma ageing trends in GC/MS profiles of liqueur wines. Analytica Chimica Acta. 2010;659:93-101.

Abstract: Madeira wine has been studied with the main goal of acquiring a better understanding about the evolution of its properties over time. For that purpose, flexible and reliable data analysis tools were employed to characterize wines at different ageing stages, using flavour chromatography measurements. In this paper we present the results from such a study, where the main differences in the aroma profiles and their development in different types of aged Madeira wines are analyzed and evaluated according to their discriminating power. An exploratory multivariate data analysis was conducted using two different tools, namely biplots and contributions plots obtained through principal component analysis (PCA). In order to take advantage of the maximum amount of information provided by the chromatography data sets, a new approach that incorporates samples variability in the analysis of the statistical significance of contributions estimates, was developed and tested. In this way, it was possible to analyze which volatile compounds have statistically significant and/or similar contributions regarding the observed separation of wine samples from different groups. Furthermore, since several chemical compounds are expected to change together as a result of the ageing-related chemical reactions, they were clustered according to a similarity criterion relative to their importance in the trends observed in the scores space. Results obtained provide a sound basis for the differentiation and characterization of the ageing process followed by Madeira wines.

27. Pereira AC, Reis MS, Saraiva PM, Marques JC. Analysis and assessment of Madeira wine ageing over an extended time period through GC–MS and chemometric analysis. Analytica Chimica Acta. 2010;660:8-21.

Abstract: Wine is one of the world’s higher value agricultural products. The present work is centred on Madeira wine, a fine and prestigious example among Portuguese liqueur wines,with the main goal to deepen our understanding of relevant phenomena going on during the winemaking process, in particular during ageing of “Malmsey” Madeira wine. In this paper we present the results obtained from the chemical characterization of how its aroma composition evolves during ageing, and the development of a robust framework for analyzing the identity of aged Madeira wines. An extended ageing period was considered, covering a time frame of twenty years, from which several samples were analyzed in terms of their aromatic composition. The multivariate structure of this chemical information was then processed through multivariate statistical feature extraction techniques such as principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA), in order to identify the relevant patterns corresponding to trends associated with wine ageing. Classification methodologies for age prediction were developed, using data from the lower dimensional sub-spaces obtained after projecting the original data to the latent variable spaces provided by PCA or PLS-DA. Finally, the performance for each classification methodology developed was evaluated according to their error rates using cross-validation methodologies (Leave-One-Out and k-fold Monte Carlo). Results obtained so far show that quite interesting classification performances can indeed be achieved, despite the natural variability present in wine products. These results also provide solid bases which can be used to build up available frameworks which assist quality monitoring and identity assurance tasks.

28. Perestrelo R, Nogueira JMF, Câmara JS. Potentialities of two solventless extraction approaches—Stir bar sorptive extraction and headspace solid-phase microextraction for determination of higher alcohol acetates, isoamyl esters and ethyl esters in wines. Talanta. 2009;80:622-30.

Abstract: A stir bar sorptive extraction with liquid desorption followed by large volume injection coupled to gas chromatography–quadrupole mass spectrometry (SBSE-LD/LVI-GC–qMS) was evaluated for the simultaneous determination of higher alcohol acetates (HAA), isoamyl esters (IsoE) and ethyl esters (EE) of fatty acids. The method performance was assessed and compared with other solventless technique, the solid-phase microextraction (SPME) in headspace mode (HS). For both techniques, influential experimental parameters were optimised to provide sensitive and robust methods. The SBSE-LD/LVI methodology was previously optimised in terms of extraction time, influence of ethanol in the matrix, liquid desorption (LD) conditions and instrumental settings. Higher extraction efficiency was obtained using 60 min of extraction time, 10% ethanol content, n-pentane as desorption solvent, 15 min for the back-extraction period, 10 mL min−1 for the solvent vent flow rate and 10 °C for the inlet temperature. For HS-SPME, the fibre coated with 50/30 μm divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) afforded highest extraction efficiency, providing the best sensitivity for the target volatiles, particularly when the samples were extracted at 25 °C for 60 min under continuous stirring in the presence of sodium chloride (10% (w/v)). Both methodologies showed good linearity over the concentration range tested, with correlation coefficients higher than 0.984 for HS-SPME and 0.982 for SBES-LD approach, for all analytes. A good reproducibility was attained and low detection limits were achieved using both SBSE-LD (0.03–28.96 μg L−1) and HS-SPME (0.02–20.29 μg L−1) methodologies. The quantification limits for SBSE-LD approach ranging from 0.11 to 96.56 μg L−and from 0.06 to 67.63 μg L−1 for HS-SPME. Using the HS-SPME approach an average recovery of about 70% was obtained whilst by using SBSE-LD obtained average recovery were close to 80%. The analytical and procedural advantages and disadvantages of these two methods have been compared. Both analytical methods were used to determine the HAA, IsoE and EE fatty acids content in “Terras Madeirenses” table wines. A total of 16 esters were identified and quantified from the wine extracts by HS-SPME whereas by SBSE-LD technique were found 25 esters which include 2 higher alcohol acetates, 4 isoamyl esters and 19 ethyl esters of fatty acids. Generally SBSE-LD provided higher sensitivity with decreased analysis time.

29. Pereira V, Pontes M, Câmara JS, Marques JC. Simultaneous analysis of free amino acids and biogenic amines in honey and wine samples using in loop orthophthalaldeyde derivatization procedure. Journal of Chromatography A. 2008;1189:435-43.

Abstract: This work presents a RP-HPLC method for the simultaneous quantification of free amino acids and biogenic amines in liquid food matrices and the results of the application to honey and wine samples obtained from different production processes and geographic origins. The developed methodology is based on a pre-column derivatization with o-phthaldialdehyde carried out in the sample injection loop. The compounds were separated in a Nova-Pack RP-C18 column (150 mm × 3.9 mm, 4 μm) at 35 °C. The mobile phase used was a mixture of phase A: 10 mM sodium phosphate buffer (pH 7.3), methanol and tetrahydrofuran (91:8:1); and phase B: methanol and phosphate buffer (80:20), with a flow rate of 1.0 ml/min. Fluorescence detection was used at an excitation wavelength of 335 nm and an emission wavelength of 440 nm. The separation and quantification of 19 amino acids and 6 amines was carried out in a single run as their OPA/MCE derivatives elute within 80 min, ensuring a reproducible quantification. The method showed to be adequate for the purpose, with an average RSD of 2% for the different amino acids; detection limits varying between 0.71 mg/l (Asn) and 8.26 mg/l (Lys) and recovery rates between 63.0% (Cad) and 98.0% (Asp). The amino acids present at the highest concentration in honey and wine samples were phenylalanine and arginine, respectively. Only residual levels of biogenic amines were detected in the analysed samples.

30. Paixão N, Pereira V, Marques JC, Câmara JS. Quantification of polyphenols with potential antioxidant properties in wines using reverse phase HPLC. Journal of Separation Science. 2008;31:2189-98.

Abstract: A RP-HPLC method with photodiode array detection (DAD) was developed to separate, identify and quantify simultaneously the most representative phenolic compounds present in Madeira and Canary Islands wines. The optimized chromatographic method was carefully validated in terms of linearity, precision, accuracy and sensitivity. A high repeatability and a good stability of phenolics retention times (< 3%) were obtained, as well as relative peak area. Also high recoveries were achieved, over 80.3%. Polyphenols calibration curves showed a good linearity (r2 >0.994) within test ranges. Detection limits ranged between 0.03 and 11.5 μg/mL for the different polyphenols. A good repeatability was obtained, with intra-day variations less than 7.9%. The described method was successfully applied to quantify several polyphenols in 26 samples of different kinds of wine (red, rosé and white wines) from Madeira and Canary Islands. Gallic acid was by far the most predominant acid. It represents more than 65% of all phenolics, followed by p-coumaric and caffeic acids. The major flavonoid found in Madeira wines was trans-resveratrol. In some wines, (–)-epicatechin was also found in highest amount. Canary wines were shown to be rich in gallic, caffeic and p-coumaric acids and quercetin.

31. Oliveira e Silva H, Guedes de Pinho P, Machado BP, Hogg T, Marques JC, Câmara JS, et al. Impact of Forced-Aging Process on Madeira Wine Flavor. Journal of Agricultural and Food Chemistry. 2008;56:11989-96.

Abstract: The aim of this study was to determine the optimal temperature and baking time to obtain a Madeira wine considered typical by an expert panel. For this purpose simultaneous descriptive analyses of typical Madeira wines were performed, and seven descriptors were selected: “dried fruit”, “nutty”, “musty”, “baked”, “oak”, “mushroom”, and “brown sugar”. Up to 10 odor-active zones were the most frequently cited by the members of the GC−olfactometry panel as corresponding to the panel’s descriptors. The odor importance of each of the zones reported by the GC-O analysis was ranked by AEDA. Three odor zones were identified as common to both Malvasia and Sercial wines and had retention indices (RI) of 1993 (“brown sugar” and “toasted”), 2151 (“brown sugar”), and 2174 (“nutty”, “dried fruits”); sotolon was identified as responsible for this last aroma. Several molecules were selected to be quantified on baked wines on the basis of AEDA results and expected Maillard volatiles, such as sotolon, furfural, 5-methylfurfural, 5-ethoximethylfurfural, methional, and phenylacetaldehyde. It was observed that typicity scores were positively correlated with the concentrations of sotolon and sugar and baking time and negatively with the fermentation length.

32. Perestrelo R, Caldeira M, Rodrigues F, Câmara JS. Volatile flavour constituent patterns of Terras Madeirenses red wines extracted by dynamic headspace solid-phase microextraction. Journal of Separation Science. 2008;31:1841-50.

Abstract: A suitable analytical procedure based on static headspace solid-phase microextraction (SPME) followed by thermal desorption gas chromatography–ion trap mass spectrometry detection (GC–ITDMS), was developed and applied for the qualitative and semi-quantitative analysis of volatile components of Portuguese Terras Madeirenses red wines. The headspace SPME method was optimised in terms of fibre coating, extraction time, and extraction temperature. The performance of three commercially available SPME fibres, viz. 100 μm polydimethylsiloxane; 85 μm polyacrylate, PA; and 50/30 μm divinylbenzene/carboxen on polydimethylsiloxane, was evaluated and compared. The highest amounts extracted, in terms of the maximum signal recorded for the total volatile composition, were obtained with a PA coating fibre at 30°C during an extraction time of 60 min with a constant stirring at 750 rpm, after saturation of the sample with NaCl (30%, w/v). More than sixty volatile compounds, belonging to different biosynthetic pathways, have been identified, including fatty acid ethyl esters, higher alcohols, fatty acids, higher alcohol acetates, isoamyl esters, carbonyl compounds, and monoterpenols/C13-norisoprenoids.

33. Paixão N, Perestrelo R, Marques JC, Câmara JS. Relationship between antioxidant capacity and total phenolic content of red, rosé and white wines. Food Chemistry. 2007;105:204-14.

Abstract: Phenolic compounds are one of the most important quality parameters of wines, since they contribute to wine organoleptic characteristics such as colour, astringency, and bitterness. Furthermore, several studies have pointed out that many show biological properties of interest, related to their antioxidant capacity. This antioxidant activity has been thoroughly studied and a wide variety of methods have been developed to evaluate it. In this study, the antioxidant activity of commercial Terras Madeirenses Portuguese wines (Madeira Island) was measured by three different analytical methods: [1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay, 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS+)) radical cation decolourisation, and ferric reducing/antioxidant power (FRAP) for the evaluation of reducing power (PR) and correlate them with the total phenolic content determined with the Folin–Ciocalteu’s reagent using gallic acid as a standard. The total polyphenol concentration was found to vary from 252 to 1936 mg/l gallic acid equivalents (GAE). The antiradical activity varied from 0.042 to 0.715 mM Trolox equivalents and the antioxidant capacity varied from 344 to 1105 mg/l gallic acid equivalents (GAE). For the reduction power we obtained 3.45–3.86 mM quercetin equivalents.

34. Câmara JS, Alves MA, Marques JC. Classification of Boal, Malvazia, Sercial and Verdelho wines based on terpenoid patterns. Food Chemistry. 2007;101:475-84.

Abstract: 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.

35. Perestrelo R, Fernandes A, Albuquerque FF, Marques JC, Câmara JS. Analytical characterization of the aroma of Tinta Negra Mole red wine: Identification of the main odorants compounds. Analytica Chimica Acta. 2006;563:154-64.

Abstract: 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.

36. Campo E, Ferreira V, Escudero A, Marqués JC, Cacho J. Quantitative gas chromatography–olfactometry and chemical quantitative study of the aroma of four Madeira wines. Analytica Chimica Acta. 2006;563:180-7.

Abstract: The aroma profile of four Madeira wines from the most emblematic grape varieties, Malvazia, Boal, Verdelho and Sercial, has been studied by sensory analysis, quantitative gas chromatography–olfactometry (GC–O) and gas chromatography–mass spectrometry (GC–MS). The aroma of the wines was characterized as candy, nutty, maderized, toasty, lacquer and dried fruit. The GC–O was carried out on extracts obtained by a dynamic headspace technique. The GC–O profiles of Madeira wines were compared to the GC–O profiles obtained from three young white monovarietal wines made with the same varieties so that the identification of odorants related to the particular process of elaboration of Madeira wines was possible. The aroma profile of Madeira wines was extremely complex, and was rich in sotolon, phenylacetaldehyde, wood extractable aromas, and lacked of the most important varietal aromas, such as linalool, 3-mercaptohexyl acetate and methoxypyrazines. A large number of potentially important and unknown odorants, most of them specific to Madeira wines, was also detected by GC–O. The GC–MS results confirmed most of the results of the GC–O study, which suggests that the proposed GC–O strategy is a useful tool for screening the presence of active odorants in wine.

37. Câmara JS, Alves MA, Marques JC. Development of headspace solid-phase microextraction-gas chromatography–mass spectrometry methodology for analysis of terpenoids in Madeira wines. Analytica Chimica Acta. 2006;555:191-200.

Abstract: A dynamic headspace solid-phase microextraction methodology was developed for analysis of varietal aroma compounds in must and Madeira wine samples, a spirit wine with an ethanol content of 18% (v/v). The factors with influence in the headspace solid-phase microextraction efficiency such as: fibre coating, extraction time and temperature, pH, ionic strength, ethanol content, desorption time and temperature, were optimised and the method validated. The best results were obtained for a 85 μm polyacrylate fibre, with a 60 min headspace for must and 120 min for wine samples, in a 2.4 ml sample at 40 °C with 30% of NaCl. The extract is injected in the splitless mode in a GC–MS Varian system, Saturn III, and separated on a Stabilwax capillary column. The linear dynamic range of the method covers the normal range of occurrence of analytes in wine with typical r2 between 0.985 (β-ionone) and 0.998 (linalool) for musts and between 0.980 (α-terpineol) and 0.999 (linalool) for must and wine samples, respectively. For must samples the reproducibility ranges from 2.5% (citronellol) to 14.4% (nerolidol) (as R.S.D.), and from 4.8% (citronellol) to 14.2% (nerolidol) for wine samples. The analysis of spiked samples has shown that matrix effects do not significantly affect method performance. Limits of detection obtained are in low μg l−1 range for all compounds analysed in this study.

38. Câmara JS, Alves MA, Marques JC. Multivariate analysis for the classification and differentiation of Madeira wines according to main grape varieties. Talanta. 2006;68:1512-21.

Abstract: 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.

39. Câmara JS, Alves MA, Marques JC. Changes in volatile composition of Madeira wines during their oxidative ageing. Analytica Chimica Acta. 2006;563:188-97.

Abstract: 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.

40. Alves RF, Nascimento AMD, Nogueira JMF. Characterization of the aroma profile of Madeira wine by sorptive extraction techniques. Analytica Chimica Acta. 2005;546:11-21.

Abstract: The characterization of the aroma profile of 33 samples of Madeira wine from five monovarieties (Sercial, Verdelho, Boal, Malvasia and Tinta Negra Mole) having different type and categories is presented, using solid phase microextraction and stir bar sorptive extraction techniques (SPME and SBSE) followed by capillary gas chromatography and mass spectrometry detection (GC–MS). Headspace SPME/GC–MS provided effectiveness to identify the major constituents of the aroma profile of Madeira wine, where no remarkable differences occur among the samples studied. The volatile compounds are mainly constituted by ethyl octanoate (11.3–256.9 μg L−1), ethyl decanoate (21.5–210.5 μg L−1), ethyl decenoate (0.1–112.8 μg L−1), diethyl succinate (0.9–65.6 μg L−1), ethyl dodecanoate (1.2–6.5 μg L−1), ethyl nonanoate (0.6–5.2 μg L−1), ethyl hexanoate (0.2–3.7 μg L−1) and isoamyl octanoate (0–2.2 μg L−1). C13 norisoprenoids such as vitispirane (0.9–7.0 μg L−1) and 1,1,6-trimethyl 1,2-dihydro naphthalene (0.7–12.5 μg L−1), as well as phenyl ethanol (0–8.1 μg L−1), were also found in Madeira wine samples. The powerful capabilities of SBSE followed thermal desorption and GC–MS analysis allowed higher ability for profiling traces and ultra traces of compounds in Madeira wine samples, including esters (80.7–89.7%), carboxylic acids (1.6–4.2%), alcohols (3.5–8.2%), aldehydes (0.9–3.7%), pyrans (0.2–1.7%), lactones (&lt;3%), monoterpenes (0.1–1.4%), sesquiterpenes (0.1–0.8%) and C13 norisoprenoids (1.7–6.5%), which some of them play a remarkable impact on the aroma complexity. C13 norisoprenoids in particular, seem to play an important role on Madeira wine bouquet since presenting very low sensorial threshold limits. Excellent correlation between Madeira wine ageing and the abundance of cis-oak lactone was attained showing to be an important chemical descriptor to characterize reserves and Vintages as well as a contributor to wine flavour. The differentiation between reserves, dry/medium dry and sweet/medium sweet young wines could be well established by means of chemometric analysis, using particular aroma compounds such as diethyl succinate, cis-oak lactone and ethyl octanoate as discriminating variables.

41. Câmara JS, Marques JC, Alves MA, Silva Ferreira AC. 3-Hydroxy-4,5-dimethyl-2(5H)-furanone Levels in Fortified Madeira Wines: Relationship to Sugar Content. Journal of Agricultural and Food Chemistry. 2004;52:6765-9.

Abstract: 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-hydroxy-4,5-dimethyl-2(5H)-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 μg L-1, respectively. The levels of sotolon found ranged from not detected to 2000 μg L-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.

42. Câmara JS, Herbert P, Marques JC, Alves MA. Varietal flavour compounds of four grape varieties producing Madeira wines. Analytica Chimica Acta. 2004;513:203-7.

Abstract: 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.

43. Câmara JS, Marques JC, Alves A, Ferreira ACS. Heterocyclic acetals in Madeira wines. Analytical and Bioanalytical Chemistry. 2003;375:1221-4.

Abstract: The maturation of Madeira wines usually involves exposure to relatively high temperatures which affect the aroma and flavour composition leading to the formation of the typical and characteristic bouquet of these wines. The formation of heterocyclic acetals (1,3-dioxanes and 1,3-dioxolanes) was investigated in order to determine levels and for possible use as indicators of wine age. The results show a linear correlation of the investigated acetals with wine age but suggest that the acetalization reaction is not particularly affected by the drastic oxidative conditions observed during maturation.

44. Nogueira JMF, Nascimento AMD. Analytical Characterization of Madeira Wine. Journal of Agricultural and Food Chemistry. 1999;47:566-75.

Abstract: For a correct characterization of Madeira wine commercially available in the market, 52 samples having different types and ages, representative of the delimited region of Madeira (liquor wine of quality produced in delimited region), were analyzed in relation to physicochemical and sensorial parameters. Standard methodology for spirits and alcoholic beverages as well as a council of tasters were adopted respectively to quantify such parameters, according to European Union regulations. The main physicochemical parameters analyzed demonstrated that Madeira wine represent a high quality beverage without toxicological risks, as all constituents have in general contents clearly below the maximum concentration admissible by the national and/or international rules. A reasonable differentiation of properties could be achieved between samples having different types and ages, when principal component, discriminant and cluster analyses were applied to the analytical data, especially for physicochemical parameters. Keywords: Madeira wine; physicochemical analysis; sensorial analysis; quality control; differentiation; multivariate analysis; principal component analysis; discriminant analysis; cluster analysis

45. Carvalho ML, Barreiros MA, Costa MM, Ramos MT, Marques MI. Study of heavy metals in Madeira wine by total reflection X-ray fluorescence analysis. X-Ray Spectrometry. 1996;25:29-32.

Abstract: The suitability and high sensitivity of total-reflection x-ray fluorescence analysis (TXRF) were used for multi-element analysis of several species of Madeira wine taken directly from the usual glass bottles. In addition, the uptake of lead by Madeira wine kept in a crystal vessel (30% lead) as a function of time was also studied. The detection limits were in the range 0.001–0.10 mg 1−1 and the precision was in the range 5–10%. The organic matrix of the wine was removed by wet digestion in nitric acid.

46. Ferreira MA, Fernandes JO. The Application of an Improved GC-MS Procedure to Investigate Ethyl Carbamate Behavior During the Production of Madeira Wines. American Journal of Enology and Viticulture. 1992;43:339-43.

Abstract: A gas chromatography-mass spectrometry (GC-MS) method for ethyl carbamate quantification in Madeira wines is presented. The method is applied to follow the evolution of ethyl carbamate levels during the fermentation and baking processes. Three wine samples were specifically prepared from the same grape must and fortified with the same wine spirit, at different °Baumé, after which the wines were submitted to the same baking conditions: 45°C for 90 days. The samples were analyzed before baking and after 30, 60, and 90 days baking. An increase in the ethyl carbamate yields was established at different fermentation levels and baking stages, respectively. The wine spirit used revealed no ethyl carbamate

Doctoral Thesis

1. Perestrelo RMdS. Evaluation of the potencial of Vitis vinifera L. grapes used to produce Madeira wine: impact of winemaking process [Doctoral]. Funchal: Universidade da Madeira; 2013.
2. Pereira ACR. New methodologies for Madeira wine ageing characterization and monitoring [Doctoral ]. Coimbra: Universidade de Coimbra; 2012.

Abstract: Wine quality assessment has commonly been carried out by producers who evaluate its sensory qualities and routine physico-chemical parameters to make a final verdict over the wine’s market value. However, additional tools are currently required in order to quantify and guarantee quality standards, to ensure the authenticity of the wine’s declared attributes, as well as to protect brands.

In the particular case of fortified wines, monitoring the ageing process is crucial for the winemaker. During the ageing process, significant quantities of wine develop their final features which will grant the wine its final value on the market. It is within this context that the motivation of this thesis arose: to provide an innovative contribution to the analysis assessment and monitoring of fortified wine’s ageing phenomena. The case study is Portuguese Madeira wine, a fortified wine which has great potential for ageing and developing its well-known unique characteristics.

The strategy outlined to follow the Madeira wine ageing process is based on the development of reliable tools to predict wine age, as well as on the inverse problem of studying the influence of wine age in the evolution of wine composition and properties. The strategy followed relies on the fact that wine age can be predicted in a rigorous and reliable way, based on its intrinsic features. By comparing the estimated wine age with the known wine age (according to the harvest year of production) the producer’s evaluation can be reinforced by a quantitative assessment that may help him to determine wine quality, the potential for further ageing or to back up decisions regarding a possible correction by blending wines.

The ageing process analysis was carried out in two different temporal scales: long and short ageing periods. First, the work was oriented towards the characterization of an extended period of ageing, covering the wine ages in which Madeira wine is commonly sold, and then directed to the exploration of the first ageing years, during which key and distinguishable features are expected to start developing.

The analysis of the long-term ageing period started with a study of an exploratory nature, in which different styles of Madeira wines, of two different ageing times, were analyzed based on their volatile composition. Different ageing trends were identified for different styles of Madeira wines. A new statistical method was developed to assess the contribution of volatile compounds to the ageing process and, according to this information, establish similarities and differences between the ageing processes of the different wines styles. Built upon these results, the second stage of the analysis of the long-term ageing period continued with the analysis of the ageing process for a single grape variety, covering an ageing period of twenty years, in order to develop a rigorous framework for age prediction and monitoring. The data for volatile and phenolic composition, as well as absorbance values of the electromagnetic UV-Vis spectrum were compared in terms of their potential to support the development of wine age prediction models. A compromise between model accuracy and cost of analysis was established in order to decide which methodology to use, according to a particular application scenario. Monte Carlo and bootstrap residuals approaches were used to validate the models and to establish sample-specific prediction intervals for all estimates presented, respectively. Also, a rapid and reliable strategy to predict wine age was proposed in the long and short-term, centered on the use of wine UV-Vis absorbance data, coupled with proper chemometric techniques.

The analysis of the short-term ageing period took into account samples produced from different grape varieties. A single model for the four types of wine analyzed provided an accurate evaluation of wine age, when UV-Vis absorbance data was used.

Concerning the polyphenolic composition information, an exploratory data analysis was conducted and important patterns were found regarding the different evolution trends of different styles of wines during the first two years of ageing.

In summary, original approaches were developed and applied in this work for analyzing the analytical information currently available for Madeira wines. The main challenge was to extract and model the relevant information in order to obtain a comprehensive and systematic description of the wine trends across the ageing period which can further be used to establish powerful strategies to assess, control and optimize wine quality.

3. Pereira VNG. Effect of the estufagem process on the chemical constituents of Madeira wines [Doctoral ]. Funchal: Universidade da Madeira; 2011.

Abstract: Madeira wine is a product of well-established reputation, whose aroma and flavour is the result of unique combinations. Particularly, its maturation may include estufagem, wherein wine is usually heated at 45 °C for three months. During this period, several chemical changes may occur, so it is essential to assess its impact on the wine. In this sense, the main objective of the thesis was to evaluate the effect estufagem on the chemical constituents of Madeira wine, specifically on those molecules potentially important in the development of its typical features.

Firstly, analytical methodologies capable of determining the target compounds, combining precision and reproducibility to execution effectiveness, were developed. Then various monovarietal Madeira wines were analysed during estufagem under standard and overheating conditions in order to assess its effect. The following compounds were evaluated: furans, amino acids, biogenic amines, polyphenols, organic acids and volatile compounds. In addition, the total polyphenolic composition, the antioxidant potential and the colour of these wines were also evaluated.

The results show that most constituents change due to the heating process. Particularly, the heating promotes the development of 5-hydroxymethylfurfural (HMF) in sweet wines submitted to estufagem at higher temperatures. Moreover, estufagem provides the decrease of most amino acids, suggesting their involvement in the formation of the bouquet of these wines. Regarding the total polyphenol content and antioxidant potential of these wines they do not seem to be greatly affected by the heating step, however most monomeric polyphenols decrease during this process. The thermal processing of Madeira wines favours the development of the volatile composition, especially of volatiles usually reported as typical aromas of Madeira wines. Finally, it was demonstrated that the thermal degradation of sugars, especially of fructose, promotes the emergence of volatile compounds identified in baked wines.

4. Câmara, JS. Caracterização Aromática de Castas Produtoras de Vinho Madeira – Boal, Malvasia, Sercial e Verdelho [Doctoral]. Funchal: Universidade da Madeira; 2004

 

Master Thesis

1. Rodrigues PMS. Madeira wine vinification process: analysis and control [Master]. Funchal: Universidade da Madeira; 2016.

Abstract: The present study was done in collaboration with J. Faria e Filhos company, a Madeira wine producer, and its main goal was to fully characterize three wines produced during 2014 harvest and identify possible improving points in the winemaking process. The winemaking process was followed during 4 weeks, being registered the amounts of grapes received, the fermentation temperatures, the time at which fermentation was stopped and evolution of must densities until the fortification time. The characterization of musts and wines was done in terms of density, total and volatile acidity, alcohol content, pH, total of polyphenol, organic acids composition, sugars concentration and the volatile profile. Also, it was developed and validated an analytical methodology to quantify the volatile fatty acids, namely using SPME-GC-MS. Briefly, the following key features were obtained for the latter methodology: linearity (R2=0.999) e high sensitivity (LOD =0.026-0.068 mg/L), suitable precision (repeatability and reproducibility lower than 8,5%) and good recoveries (103,11-119,46%). The results reveal that fermentation temperatures should be controlled in a more strictly manner, in order to ensure a better balance in proportion of some volatile compounds, namely the esters and higher alcohols and to minimize the concentration of some volatiles, namely hexanoic, octanoic and decanoic acids, that when above their odours threshold are not positive for the wine aroma. Also, regarding the moment to stop the fermentation, it was verified that it can be introduced changes which can also be benefit to guarantee the tipicity of Madeira wine bouquet.

2. Miranda AFS. Olfactory perception threshold assessment of volatile acidity in Madeira wines [Master]. Funchal: Universidade da Madeira; 2015.

Abstract: 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.

3. Silva JCVd. Color measurement using a smartphone applied to Madeira wines [Master]. Funchal: Universidade da Madeira; 2015.

Abstract: Computer vision is a field that uses techniques to acquire, process, analyze and understand images from the real world in order to produce numeric or symbolic information in the form of decisions .

This project aims to use computer vision to prepare an app to analyze a Madeira Wine and characterize it (identify its variety) by its color. Dry or sweet wines, young or old wines have a specific color.

It uses techniques to compare histograms in order to analyze the images taken from a test sample inside a special container designed for this purpose.

The color analysis from a wine sample using an image captured by a smartphone can be difficult. Many factors affect the captured image such as, light conditions, the background of the sample container due to the many positions the photo can be taken (different to capture facing a white wall or facing the floor for example).

Using new technologies such as 3D printing it was possible to create a prototype that aims to control the effect of those external factors on the captured image.

The results for this experiment are good indicators for future works. Although it’s necessary to do more tests, the first tests had a success rate of 80% to 90% of correct results.

This report documents the development of this project and all the techniques and steps required to execute the tests.

4. Leça JMdS. Evolution of ethyl carbamate during Madeira wine ageing by GC-MS: a new methodology [Master]. Funchal: Universidade da Madeira; 2014.

Abstract: Recently, ethyl carbamate (EC) was reclassified by the International Agency for Research on Cancer (IARC) as “probably carcinogenic to humans” and occurs mainly in fermented beverages. Nowadays many countries have set limit values for EC in alcoholic beverages. In this sense and taking into account the low concentrations found in alcoholic beverages, the scientific community has shown interest for the development of new analytical methods, whereby its simplification plays an important role in the EC control and prevention.

Firstly, a simple, rapid and sensitive methodology was developed for the EC quantification in fortified wines by microextraction by packed sorbent (MEPS) with gas chromatography coupled with a mass spectrometer detector (GC-MS). This method showed good linearity (R2 = 0.999) and sensitivity (LOD = 1.5 μg/L). The accuracy of the method was assessed by means of repeatability and reproducibility (RSD < 7%). Moreover, a good recovery has been demonstrated (97 – 106%) as well as its applicability (16 fortified wines). Thus, the developed methodology has proven to be an excellent approach for routine quantification of EC in fortified wines. The EC evolution was also evaluated during a year and half of Madeira wine ageing submitted to two traditional ageing methods, estufagem and canteiro, in order to evaluate the formation kinetic. The results revealed that estufagem process increased the formation kinetic and promoted a linear increase of the EC concentration (R2 ≥ 0.977), proportionally to the ageing time (4 months). However, when the wines are firstly submitted to estufagem and then undergo canteiro ageing, the EC values remain almost constant during the following 14 months. The results suggest that estufagem does not seem to be the critical factor in the EC formation, but instead the amount of precursors in the medium.

5. Martins CSF. Estratégias para a redução de carbamato de etilo em vinhos Madeira [Master]. Aveiro: Universidade de Aveiro; 2011.

Abstract: Ethyl carbamate (EC) is a potentially toxic compound, it was re-classified in 2007 as probably human carcinogen compound (Group 2A) by the International Agency for Research on Cancer (IARC). This compound has been detected on several fermented foods and alcoholic beverages, namely Madeira wine. Thus, they were studied some intrinsic (vine variety, type of wine and process) and extrinsic factors (light, temperature and storage time) to the wine that condition the formation of EC. In parallel, it was established the profile of volatile organic acids in order to explain the observed increase in acidity during the aging of Madeira wines. Headspace solid phase microextraction combined with comprehensive multidimensional gas chromatography time-of-flight mass spectrometry (HS-SPME/GC × GC – ToFMS) was the methodology applied in these studies. The several assays were performed with synthetic model wines and Madeira wines. This study allowed concluding that the vine variety and the type of wine (two intrinsic factors), the time and temperature of storage showed influence on the EC levels. The comparative study envolving 2 sugars, glucose and sorbitol (with and without a carbonyl group) showed that glucose catalyze the reaction of EC formation. The raise of temperature that occurs during the “estufagem” and storage gives the external energy needed to promote the reaction of EC formation. An absence/presence of the light didn’t demonstrate significant effects in the formation of EC. The profile of volatile organic acids includes 18 compounds, from which 4 were reported for the first time in Madeira wines. For the wines under this study, it was illustrated that the total acidity increases with ageing, which may be explained by the raise of short-chain (C3 – C5) and long-chain organic acids (C14 – C18). These acids may have been originated by hydrolysis reactions that occur during ageing and in oak casks where the wine is stored. In short, this work shows that the EC formation is conditioned by multiple factors; it is not possible to specify a single parameter to change in the winemaking process or storage to reduce its formation. There was clearly an increase in knowledge about the factors involved that allows us to suggest some strategies: i) the process of winemaking and storage should be adapted to the vine variety, type of wine and aging time expected, ii) vine varieties which originate sweet wines or medium-sweet, more likely to result in wines with higher content of EC, should preferably be processed at low temperatures (optimize the binomial time/temperature) and iii) whenever is possible the wines should be stored at 4 ° C, considering the financial constraints, especially the sweet and medium-sweet wines.