Analytica Chimica Acta (v.458, #1)

In Vino Analytica Scientia 2001 by C.J Ducauze; D.N Rutledge (1-2).

The first step of the proposed biogenetic pathway in grapes that leads from carotenoids to C13-norisoprenoids involves the enzymatic degradation of carotenoids by regiospecific oxygenases. Chemical, photochemical and oxidase-coupled degradation of carotenoids to norisoprenoids have been studied in vitro and enzymatic systems shown to be involved in mammals. However, no enzymatic system has been shown to be involved in the formation of C13-norisoprenoids in grapes, despite all recent studies carried out on grapevines supporting such a model. These findings include the preponderance of norisoprenoids possessing 13 carbon atoms that thereby indicates the specificity of cleavage, the configuration of the asymmetric centres and axes common to C13-norisoprenoids and the corresponding carotenoids, the negative correlations observed between the levels of norisoprenoids and carotenoids during berry development, and the in vivo transfer of 13C markers from carotenoids to norisoprenoids in berries between véraison and berry maturity. All of these findings are major arguments in favour of the hypothesis that glycosylated C13-norisoprenoids derive from carotenoids in grape berries. Carotenoids are mostly synthesised from the first stage of fruit formation until véraison, and then degrade between véraison and maturity to produce glycosylated C13-norisoprenoids and other compounds.
Keywords: Carotenoids; Norisoprenoids; Glycosides; Aroma; Grape; Vines; Vitis vinifera; Biogenesis;

Effect of oxygenation on polyphenol changes occurring in the course of wine-making by Vessela Atanasova; Hélène Fulcrand; Véronique Cheynier; Michel Moutounet (15-27).
The influence of controlled oxygenation on the colour and phenolic composition of red wine was studied by UV–VIS spectrophotometry, liquid chromatography (LC) coupled to diode array detection (DAD) and electrospray ionisation mass spectrometry, and thiolysis. The comparison between the control and oxygenated wines demonstrated changes in colour characteristics along with a significant increase in concentrations of pyranoanthocyanins, ethyl-bridged compounds and derived pigments both with storage time and with oxidation. Principal component analysis was applied to wine analysis data measured throughout the conservation period. The effect of the storage time and oxygenation was clearly reflected in this analysis.Mass-spectrometric analysis of the wines demonstrated the presence of compounds which are markers of reactions involving acetaldehyde. Two types of mechanisms were observed. The first concerns acetaldehyde condensation reactions and the second, the cycloaddition process between anthocyanins and flavanols mediated by acetaldehyde, generating tannin-pyranoanthocyanins.The presence in wines of trimeric structures resulting from these mechanisms, as well as the results obtained after thiolysis of the fraction containing polymeric species obtained by Fractogel chromatography, confirm that proanthocyanidins react with acetaldehyde in the same way as flavanol monomers.
Keywords: Wine; Oxygenation; Ageing; Pigments; Colour; Liquid chromatography; Liquid chromatography/electrospray ionisation mass spectrometry;

‘Untypical aging off-flavor’ in wine: formation of 2-aminoacetophenone and evaluation of its influencing factors by Katrin Hoenicke; Thomas J Simat; Hans Steinhart; Norbert Christoph; Martin Geßner; Hans-Jürgen Köhler (29-37).
2-Aminoacetophenone (AAP) is known as the character impact compound responsible for the so called untypical ‘aging off-flavor’ (UTA) in Vitis vinifera wines. The formation of AAP is caused by an oxidative degradation of the phytohormone indole-3-acetic acid (IAA), triggered by sulfuration after fermentation. Using different radical generating systems (hydroxyl and/or superoxide radical generating systems) it could be shown especially that superoxide radicals are responsible for the formation of AAP. Hereby, a pyrrole ring cleavage of IAA yields 3-(2-formylaminophenyl)-3-oxo-propionic acid (FAPOP), N-formyl-2-aminoacetophenone (FAP), and AAP. Analysis of 32 grape musts and their corresponding wines revealed that wines with a higher superoxide radical scavenger activity are less prone to UTA formation. However, UTA is not correlated with the IAA content of the must or wine. Differences in the release of IAA during fermentation of musts derived from early and late harvested grapes indicate a correlation between the ripeness of the processed grapes, the IAA content at the time of sulfuration, and the UTA formation.
Keywords: Untypical aging off-flavor (UTA); Wine; 2-Aminoacetophenone; Indole-3-acetic acid; Superoxide radical; Radical scavengers;

Varietal compounds have been analyzed in wines prepared in the laboratory from four grape varieties grown in Spain. The possibilities for enhancing their aroma afforded by addition of glycosidase enzymes and steeping with the skin were studied. Both treatments increased substances responsible for varietal aroma in all wines, the effect being particularly significant for benzyl alcohol.
Keywords: Wine; Varietal aroma; Glycosidases; Skin;

Effect of maturation in small oak casks on the volatility of red wine aroma compounds by Hector Escalona; Lorraine Birkmyre; John R. Piggott; Alistair Paterson (45-54).
Wood maturation of red wine produces complex interactions between oak extracted compounds and pre-existing components in wine. Wood contributes with some aroma volatiles; but the whole volatile fraction, including the volatiles extracted from grape or produced during fermentation, could be involved in interactions with the non-volatile fraction. Samples of red wine with increasing time of wood contact, matured in 25 l casks of new American oak, were analysed on their volatile composition both in solution and headspace, and phenolic components. There was an increase of acetic acid and acetate esters as the time of wood contact increased. On the other hand, ethyl esters decreased as the time in wood increased. Some volatiles showed different behaviour when comparing their relationship between headspace and solution from two different casks, even though they were treated as replicates. This different evolution among casks was also consistent with different evolution of phenolic compounds, suggesting that there was an interaction among the volatile fraction and phenolic components.
Keywords: Flavour; Aroma; Ageing; Wood; Phenols;

Aroma profile of Portuguese brandies aged in chestnut and oak woods by Ilda Caldeira; A.P. Belchior; M.C. Clı́maco; R. Bruno de Sousa (55-62).
Much research has been made on the physico-chemical composition of brandies aged in oak wood but few sensorial data are available on brandies aged in oak wood and there is no sensorial data on brandies aged in chestnut wood. In the present work a panel of tasters evaluated the aroma attributes of a Portuguese Lourinhã brandy, after 4 years of ageing. The taster panel generated the 11 aroma descriptors and the brandies were evaluated based on those descriptors, with a structured scale. A two factorial experiment (7  woods×3 toasting levels×3 replicates) was used to compare the brandies aged in barrels made from seven different woods and each wood was submitted to three levels of toasting: light, medium and strong, with three replicates of each essay. The analysis of variance showed significant differences between the brandies aged in the seven different woods, concerning the intensities of vanilla, woody, caramel, burned, green, tails, glue and caoutchouc. The highest intensities of caramel and burned were found in the brandies aged in chestnut wood.The toasting level of the barrels had a significant effect on the majority of the aroma descriptors, namely fruity, vanilla, woody, spicy, caramel, burned, smoke, green, tails and glue. In fact, the brandies aged on strong toasted barrels presented the highest intensities of vanilla, woody, spicy, caramel, burned, smoke and the lowest intensities of fruity, green, tails and glue. According to our previous results, this work pointed out the interesting sensorial properties of the brandies aged in chestnut, and thus shows the possibility of the use of this wood on the brandies’ ageing.
Keywords: Brandy; Aroma profile; Ageing; Chestnut wood; Oak wood; Maturation;

Analytical methods for the characterization of proteins and peptides in wines by M.V Moreno-Arribas; E Pueyo; M.C Polo (63-75).
Although proteins and peptides are minor constituents of wine, they make a significant contribution to its quality. Proteins can cause a number of technological problems during vinification and may be responsible for the appearance of turbidity in bottled wine. Peptides exhibit surfactant and sensory properties that can influence the organoleptic characteristics of wine. These reasons make protein and peptide analysis a necessity. In this paper, some of the applications in sample preparation, electrophoretic and chromatographic analysis, and detection of proteins and peptides in wine are examined. Special attention is paid to the methodologies that the authors have used in previously published studies, in some cases developed by them, and in other cases taken from the literature and used routinely in their laboratory.
Keywords: Wine; Proteins; Peptides; Electrophoresis; Liquid chromatography; Mass spectrometry;

In our work, the capacity of 1D and 2D 1 H and 13 C homo and heteronuclear nuclear magnetic resonance (NMR) experiments were examined for the characterization of amino acids and anthocyanins, the “minor” but important compounds of wine. The compositions of amino acids and anthocyanins influence the flavour of wines and their composition could be used to differentiate wines according to the vine variety, geographical origin and year of production. Anthocyanins are also the major components responsible for the red wine color and they have a wide range of biochemical and pharmacological effects such as antimicrobial and antioxidant activities.We use the signal intensities in 1D 1 H NMR spectra in the chemometric analysis of white wines from Slovenia. The use of signals of seven amino acids gives a good separation of wines according to the vine variety, while adding also the signal intensities of glycerol, butylene glycol and succinic acid, the separation of wines between the Coastal and continental part of Slovenia was achieved.By using 1D and 2D NMR experiments the reliable assignment of 1 H and 13 C resonances of anthocyanins, peonidine 3-glucoside, petunidine 3-glucoside, cyanidine 3-glucoside, delphinidine 3-glucoside and malvidine 3-glucoside in CD3OD was established, which will serve as a tool for the identification of a particular fraction in liquid chromatographic analysis.
Keywords: Nuclear magnetic resonance; Wine; Amino acids; Anthocyanins; Chemometrics; Geographical origin; Vine variety;

Aroma composition of wine studied by different extraction methods by M Ortega-Heras; M.L González-SanJosé; S Beltrán (85-93).
Several isolation and concentration methods have been developed for the analysis of volatile components in wine. However, it is generally admitted that none of them fulfill all the requirements for the isolation of aroma compounds and that it is necessary to combine different extraction methods to obtain the complete volatile fraction.Three extraction methods have been studied: liquid–liquid extraction, static headspace and a new method developed in our laboratory, which has the same principle as the dynamic headspace technique. The volatiles were swept along with a carrier gas and later condensed in a cold trap. The advantages and disadvantages of the three methods regarding sample preparation, component losses and artifact formation, have been evaluated. Also, the sensitivity and reproducibility have been compared.With the liquid–liquid extraction method used, a new compound has been described for the first time in wine. This compound is 5,6,7,7a-tetrahydro-4,4,7a-trimethylbenzofuran-2(4H)-one.Liquid–liquid extraction and static headspace show good reproducibility; although static headspace is useful for the analysis of highly volatile compounds, it is not able to detect trace compounds due to its lack of sensitivity. The new method allows to obtain an extract, free of artifacts and foreign substances that were not in the original wine. As no solvent is used, the extracts may be tasted and sensorially analyzed.
Keywords: Liquid–liquid extraction; Static headspace; Stripping with nitrogen; Wine volatiles;

Determination of volatile phenols in fino sherry wines by Cristina Domı́nguez; Dominico A. Guillén; Carmelo G. Barroso (95-102).
An easy, fast and reliable analytical method is proposed to determine the concentration of volatile phenols (ethyl- and vinylphenols) in fino sherry wines. The technique employed is a single stage sample treatment by solid-phase extraction (SPE) following a simple, fast procedure that enables 12 samples to be extracted simultaneously and requires a small volume sample and little time. Subsequently, the extracts are analyzed by gas chromatography (GC) with flame ionization detection. The method proposed has been applied to the study of fino sherry wines affected by microbial contamination with yeasts of the Brettanomyces genus, and the relationship of these yeasts with the concentrations of volatile phenols present in this wine.
Keywords: Wine; Ethylphenols; Vinylphenols; Brettanomyces yeast; Solid-phase extraction;

Direct liquid chromatographic analysis of resveratrol derivatives and flavanonols in wines with absorbance and fluorescence detection by Xavier Vitrac; Jean-Pierre Monti; Joseph Vercauteren; Gérard Deffieux; Jean-Michel Mérillon (103-110).
Wine contains a large number of polyphenols including stilbenes, flavanols and anthocyanins. Of these, stilbenes have been reported to have potential chemopreventive activities. We describe the simultaneous determination of six resveratrol derivatives in wines by liquid chromatography (LC) with fluorescence detection. The levels of pallidol, the symmetrical dimer of resveratrol, are reported for the first time. Quantifications were carried out at optimal wavelengths for each compound during separation. A total of 19 red and 30 white commercial wines from South-Western France were analysed, and the highest stilbene concentrations were found in red wines. In addition, the levels of catechins and two flavanonols recently isolated in red wine are reported.
Keywords: Stilbenes; Pallidol; Astilbin; Dihydromyricetin-3-O-rhamnoside; Wine; Fluorescence; Liquid chromatography;

Headspace solid phase microextraction (HS-SPME) was investigated as a solvent-free alternative method for the extraction and determination of 4-ethylphenol (EP) and 4-ethylguaiacol (EG) in red wine by capillary gas chromatography with flame ionization detection (FID) and compared to liquid–liquid extraction.For HS-SPME, better results were obtained with saturated sodium chloride samples, at 55 °C, using a 85 μm polyacrylate fiber. An absorption time of 40 min was needed to reach the absorption equilibrium for EG. This 40-min duration corresponds to the beginning of EP equilibrium and was selected for the experiments. In these conditions, the calibration graphs were linear in the range 5–5000 μg l−1 and the sensitivity was nearly the same for the two compounds. The detection limits were in the low μg l−1 range. In model wine solutions, result obtained with the liquid–liquid extraction method exhibit a linear calibration between 25 and 10,000 μg l−1 with a detection limit of 1 μg l−1, but, the relative standard deviations of the EP and EG result in the low concentration range (<50 μg l−1) are higher than those obtained by HS-SPME (15% compared to 2% for EP and 12% compared to 5% for EG). Taking into account the numerous volatile compounds in wine, HS-SPME is a rapid and valid alternative technique for use in the determination of ethylphenols at trace levels.
Keywords: Ethylphenols; Red wine; Headspace analysis; Solid phase microextaction; Gas chromatography;

The optimization of an ultrasound-assisted extraction (UAE) method for tartaric and malic acids from grape derived samples is shown. A fractional factorial experimental design allowed for the determination of the effects of seven extraction variables. Relationships between all the variables were examined. By applying graphical analysis, the best extractions conditions were obtained. The most important variables were the extracting liquid and the extraction temperature. Later, a central composite design was applied for optimizing the temperature and the composition of the extracting liquid. The optimized method was applied to grapes and to winemaking by-products. The repeatability of the method was studied and the recovery of tartaric and malic acids was established. Organic acids quantification was done by liquid chromatography (LC) using a post-column buffer and a conductivity detector.
Keywords: Ultrasound-assisted extraction; Tartaric acid; Malic acid; Grapes; Grape seeds;

The performance of sequential injection (SI) systems has often been criticized for its low sampling frequency. The present work describes a SI system where an injection valve and an additional pump were incorporated to enhance sample throughput rate. The proposed system was applied to the enzymatic determination of glycerol and ethanol in wines, using spectrophotometric detection and immobilized glycerol and alcohol dehydrogenases. The method proposed was applied to the determination of ethanol between 0.10 and 0.50% (v/v) and glycerol between 0.03 and 0.30 g l−1. These ranges were appropriate for determination in table and port wines, since samples were diluted 50 times before introduction into the system. The results obtained from 15 wine samples were statistically comparable to those obtained by the reference methods, with good repeatability (R.S.D.<3.4%, n=10). The sampling rate was 22.5 h−1, corresponding to 45 determinations per hour. This way, the time required for each determination was decreased by 30% when compared to a conventional SI system.
Keywords: Glycerol; Ethanol; Wine; Sequential injection; Glycerol dehydrogenase; Alcohol dehydrogenase; Port wine;

Comparison of wine aromas with different tannic content aged in French oak barrels by E.M. Dı́az-Plaza; J.R. Reyero; F. Pardo; M.R. Salinas (139-145).
A method has been developed to determine the most significant volatiles of a wine aged in barrels: trans- and cis-whiskylactone, guaiacol, 4-ethylguaiacol, eugenol, 4-ethylphenol, vanillin, furfural and γ-butyrolactone. This method consists in an extraction with dichloromethane, according to classical methods, but emulsion formation is avoided and the later analysis is carried out by thermal desorption-gas chromatography with mass spectrometric detection. This method is quite rapid and gives calibration graphs with linear regression coefficients between 0.97 and 0.99 for each compound and variation coefficients of variation <10%. This method has been applied to three wines with different tannic content (free-run or base wine and two other from base wine by adding both enological tannin and its own press wine) aged 6 months in French oak barrels (Allier). Sensorial analyses were carried out by expert tasters. All results were used in discriminant analyses, but only the lactones and vanillin were necessary to properly classify all the wines according to tannic content.
Keywords: Wine; Tannin; Aroma; French oak; Volatiles; Thermal desorption-gas; Chromatography–mass spectrometry;

Anthocyanin-derived pigments and colour of red wines by A.M. Vivar-Quintana; C. Santos-Buelga; J.C. Rivas-Gonzalo (147-155).
The colouring material of a red wine was fractionated by medium pressure liquid chromatography (LC) on a C18 reversed-phase column. Three fractions were collected and studied by liquid chromatography–diode array detection–mass spectrometry (LC–DAD–MS) and spectrophotometry in order to evaluate their pigment composition and colour characteristics. Fraction 1 represented 31% of the dry material (DM) of the wine and contained grape anthocyanins. Fraction 2 (61% DM) also contained anthocyanins together with several vitisin-like pigments, which appeared as very minor peaks in the liquid chromatograms. Fraction 3 (8% DM) exclusively showed anthocyanin-derived pigments. Diverse anthocyanin-derived pigments present in fractions 2 and 3 were identified by their mass spectra. It was shown that an erroneous notion about the components with influence on the colour of the wine is obtained if only the liquid chromatographic profiles are taken into account. At pH 4 the major contribution to the colour was made by the anthocyanin-derived pigments, which were hardly detected in the chromatograms, where the anthocyanins greatly predominated. An explanation is that the chromatograms are obtained in very acidic conditions at which the anthocyanins exist in the highly coloured flavylium form; however, at the usual pH value of the wine (3.2–4.0) they exist mainly as colourless hemiacetal structures. In contrast, the colour of the new pigments is much less affected by the pH and even if they may have lower molar absorptivity at 520 nm at very acidic pH values, their colour expression at the pH of the wine is higher than that of the anthocyanins.
Keywords: Anthocyanin-derived pigments; Red wine; Liquid chromatography–mass spectrometry; Wine colour;

Volatile sulphur compounds in wines related to yeast metabolism and nitrogen composition of grape musts by N. Moreira; F. Mendes; O. Pereira; P. Guedes de Pinho; T. Hogg; I. Vasconcelos (157-167).
The influence of nitrogen compounds in grape musts on the content of sulphur compounds of wines was studied. Different vinifications were performed with the addition of methionine (20 mg l−1) and/or cysteine (40 mg l−1) to grape musts before alcoholic fermentation. Six grape musts, with different nitrogen composition, from cultivars of the ‘Vinhos Verdes’ Region, in Portugal, were used. Addition of methionine to grape musts enhanced the content of wines in 3-(methylthio)-1-propanol, acetic acid-3-(methylthio)propyl ester, 3-(methylthio)propionic acid and some unidentified sulphur compounds. Increase of cysteine concentration in musts led to the production of wines with high levels of hydrogen sulphide and cis-2-methyltetrahydro-thiophene-3-OL and also unidentified sulphur compounds; however, the content of 3-(methylthio)propionic acid in the wines decreased considerately with the addition of cysteine to grape musts. This work showed that cultivars from the Vinho Verde Region show different sulphur compound contents. Avesso wines, elaborated from grape musts with low amino acids level, presented the highest total sulphur compounds content. Wines from Azal branco and Alvarinho were characterised by high contents of 4-(methylthio)-1-butanol and 3-(methylthio)propionic acid, respectively. A high content of N-(3-(methylthio) propyl)-acetamide and dimethylsulphone characterise the Loureiro wines. In contrast, Trajadura wines, produced from a must rich in amino acids, presented a low total sulphur compounds content; however, these wines were also characterised by high concentrations of 4-(methylthio)-1-butanol, acetic acid-3-(methylthio)propyl ester and hydrogen sulphide.
Keywords: Sulphur compounds; Amino acid composition; Fermentation; Wine;

Role of melanoidins in sweet wines by M.D. Rivero-Pérez; S. Pérez-Magariño; M.L. González-San José (169-175).
Melanoidins from several Spanish sweet wines were isolated by dialysis. Browning indexes (absorbances at 420 and 345 nm) and CIELab parameters of the wines and their dialysed fractions were measured. The antioxidant activity, which was expressed as Trolox equivalent antioxidant activity (TEAC), was determined using the N,N-dimethylphenylene diamine dihydrochloride (DMPD) method.Results showed a good correlation of colour parameters L , h and absorbances at 345 and 420 nm between the dialysed fractions and the original sweet wines. However, the TEAC of dialysed fractions did not show any significant correlation with TEAC of the original wines. So brown polymers of high molecular weight (>12000 Da) isolated by dialysis, which could be mainly melanoidins, play an important role on the colour of these sweet wines, but not in their antioxidant activity.
Keywords: Sweet wines; Melanoidins; Colour; Antioxidant activity;

Chemical characterization of Italian red wines from different geographical locations in the Apulia, region of southern Italy, have been performed by means of chromatographic, analyses routine analyses (density, alcohol content, acidity, dry extract and ash content), inductively coupled plasma-atomic emission spectrometric measurements and nuclear magnetic resonance (1H NMR) spectrometric determinations. Multivariate statistical methods were applied separately to the analytical and NMR data. The results showed that Apulian red wines are divided in three groups according to their geographical origin.
Keywords: Italian red wines; NMR; Multivariate statistical analysis; Geographical origin; ICP-AES;

Multivariate classification of rosé wines from different Spanish protected designations of origin by S Pérez-Magariño; M Ortega-Heras; M.L González-San José (187-190).
Wine composition depends on many factors which are especially important for quality wines from specific regions, such as protected designation of origin (PDO) wines. Nineteen analytical parameters were analysed in commercial rosé wines from different Spanish PDOs (Ribera del Duero (RD), Rioja (Rj), Valdepeñas and La Mancha (M-V)), and from two consecutive vintages. Stepwise linear discriminant analysis (SLDA) was used to differentiate and classify these wines by their geographical origin. All the wines were made from “similar” varieties of grapes. The final model selected 12 variables, being ethanol and calcium the most important variables for the differentiation of the three PDOs. The evaluation of the model was done by crossvalidation, obtaining a global percentage of correct classification of 98.8% and of global prediction of 97.3%. These results can be considered satisfactory and acceptable, and the selected variables can be useful to differentiate these wines by their origin.
Keywords: Rosé wines; PDO; Classification; Multivariate analysis;

Characterisation of young Cognac spirit aromatic quality by O. Lablanquie; G. Snakkers; R. Cantagrel; G. Ferrari (191-196).
In order to determine the quality of young Cognac spirits, mainly appreciated by the absence of defects, a new method has been developed, based on sniffing and olfactometry (GC–O) and associated with sensory and chemical analysis. During 3 years, good quality spirits selected first by Cognac merchant were analysed and selected by an original panel of tasters used to judge those spirits. The aromatic profile were established and compared. Then, the most different spirits were analysed by sniffing coupled with gas chromatography. The olfactometric profile obtained were compared. We noticed that they have all the same aromatic base constituted with 18 specific areas. Six of them were unknown and we are trying now to identify them.
Keywords: Spirits; Quality; Aroma; Olfactometry;

Ion exchange techniques have been used to reduce the content of iron, copper and manganese in white wines. Two exchanger resins have been compared, a chelating resin, the active group of which is iminodiacetate and a Dowex, acidic cation exchange resin.The results obtained show that the technique of using exchanger resins is extremely effective in lowering the metal content of wines, although on occasions, their use alters the organoleptic characteristics of the wine. Treated wines present lower polyphenolic and aromatic profiles than the untreated wines. Polyphenolic and metallic reductions would explain why treated wines present a notably reduced susceptibility to browning.
Keywords: Metals; White wine; Browning; Ion exchange;

Screen-printed biosensors for the control of wine quality based on lactate and acetaldehyde determination by Alina Avramescu; Thierry Noguer; Maria Avramescu; Jean-Louis Marty (203-213).
Biosensors for d-lactate and acetaldehyde were developed, based on screen-printed electrodes and NAD+-dependent dehydrogenases. Modification of screen-printed electrodes with the mediator Meldola Blue or with Meldola Blue-Reinecke salt resulted in sensitive, low cost and reliable NADH detectors. The biosensors were realised in two configurations, as disposable and reusable devices. Single-use sensors were obtained by simple deposition of enzyme and cofactor on the surface of mediator-modified electrodes. Chronoamperometry was used for the detection of substrates in small volumes of samples (25 μl). Immobilisation of dehydrogenases by entrapment in poly(vinyl alcohol) bearing styrylpyridinium groups (PVA-SbQ) allowed sensors to be obtained with sufficient operational stability. Amperometry in stirred solutions was the detection technique with biosensors for multiple use. The 3σ detection limits for acetaldehyde were 1 μM by amperometry and 6 μM by chronoamperometry and for d-lactate-0.03 μM and 0.05 μM for reusable and disposable biosensors respectively. The biosensors were applied in the analysis of some French and Romanian wines.
Keywords: NADH; Meldola Blue; Screen-printed electrodes; d-Lactate; Acetaldehyde; Wine;

Yeast cells sucrose biosensor based on a potentiometric oxygen electrode by Lucian Rotariu; Camelia Bala; Vasile Magearu (215-222).
A new microbial biosensor based on an immobilised microorganisms (Saccharomyces cerevisiae) and a potentiometric oxygen electrode is described. Determination is based on the respiratory activity of the microorganism in presence of different sugars (sucrose and glucose). A response time of ca. 4 min for the steady-state method and 2 min for the initial slope method was obtained. Potentiometric detection has the advantage of an extended calibration range and a low detection limit. The calibration curve for sucrose was linear in the range 1×10−5 to 3×10−2  M. This biosensor was used for selective monitoring of sucrose in the presence of glucose, using a second anti-interference enzymatic layer with glucose oxidase (GOD) and catalase (CAT). Interference of glucose in the determination of sucrose decreases from 15% for a microbial biosensor to a maximum 3.5% for the hybrid biosensor. The hybrid biosensor was used to determine sucrose in soft drinks. A good correlation between the results for the biosensor and a spectrophotometric method with dinitrosalicylic acid was achieved.
Keywords: Sucrose; Biosensor; Potentiometry; Yeast; Food analysis;

Trace metal studies of selected white wines: an alternative approach by L. Sauvage; D. Frank; J. Stearne; M.B. Millikan (223-230).
A number of white wines from vineyards outside the metropolitan region of Melbourne were analyzed for the trace metals copper, iron, potassium, sodium, magnesium and calcium by atomic absorption spectrometry (AAS). The 24 wines analyzed included Chardonnay, Sauvignon Blanc, Riesling, Gewürztraminer and Pinot Gris. In addition, preliminary transmission near infrared (NIR) spectrometric analyses for these metals in the above wines indicated, using both multiple linear regression (MLR) and partial least squares (PLS), that promising results may be obtained for some metals. The squared correlation coefficients (R 2) and the ratios of the standard error of cross validation (SECV) and the standard deviation (S.D.) for the calibration models for potassium, sodium, magnesium and calcium were acceptable, however those for iron and copper were not. Although, only a small number of wines were used in this study, the preliminary data indicated that NIR spectrometry may provide a suitable, rapid method for analysis for some metals in white wines, and warrants a further investigation using a larger number of white wines over a wider range of locations.
Keywords: Australian white wine; Trace metals; Atomic absorption spectrometry; Near infrared;

Butyltin compounds are widespread contaminants which have also been found in some wines, determined by liquid–liquid extraction followed by alkylation with a Grignard reagent and gas chromatography–mass spectrometric (GC–MS) analysis. A promising alternative to this extraction/derivatization method is the one-step tetraethylborate in situ ethylation/solid-phase micro-extraction (SPME) method. In this work, a SPME–GC–MS method for the determination of butyltin compounds in wine was optimised. The optimised parameters concerned the pre-treatment with tetramethylammonium hydroxide, matrix modification with sodium chloride, tetraethylborate concentration, extraction time and temperature, and the GC separation program. The analytical figures of merit of the optimised method (range, limit of detection (LOD) and reproducibility) were evaluated. The sensitivity (range 20–1421 kcounts μg−1  l−1 as Sn) and LOD (range, 0.01–0.2 μg l−1 as Sn) depended greatly on the butyltin species to be measured and on the type of wine. For the tested species (monobutyltin, dibutyltin and tributyltin) the highest sensitivities were achieved for Port wine samples, followed by red wine>white wine>white Verde wine. The method allowed acceptable repeatability (relative standard deviation (R.S.D.), 6–8%; n=4) and reproducibility (R.S.D., 8–9%; n=3).
Keywords: Tetraethylborate in situ ethylation; Headspace solid-phase micro-extraction; Butyltin compounds; Wine; Gas chromatography–mass spectrometry;

Micelle mediated methodology for the determination of free and bound iron in wines by flame atomic absorption spectrometry by E.K. Paleologos; D.L. Giokas; S.M. Tzouwara-Karayanni; M.I. Karayannis (241-248).
A methodology utilizing the cloud point phenomenon for the determination of free and tannin-bound iron in wines is presented. The method employs precipitation of the tannins and other phenolic and insoluble compounds in the micelles of a non-ionic surfactant mixture (TX-100 and TX-45) upon increase of the solution temperature, which are subsequently separated from the initial solution by centrifugation. The surfactant-rich-phase containing the tannins and the insoluble iron fraction is directly aspirated into the nebulizer of a flame atomic absorption spectrometer after its uptake with a methanolic solution of HNO3. The supernatant is submitted to the same cloud point extraction procedure for the determination of free iron species in the presence of a chelating agent, ammonium pyrrolidine dithiocarbamate (APDC), in order to form water-insoluble complexes with free iron. The complexes are extracted in the micelles and directly analyzed after they are diluted in a methanolic solution as described above. The total content of iron was also determined by conventional methods for comparison, yielding satisfactory results.The calibration graph was rectilinear up to 0.35 mg l−1 Fe, with detection limits of 0.02 mg l−1 with a relative standard deviation of 2.4%. The method was successfully applied to red and white wines.
Keywords: Wine; Iron species; Cloud point extraction; Atomic absorption spectrometry;

We have introduced a non-toxic electrode material similar to dental amalgam for use in voltammetry. Its electrochemical properties are like a silver electrode. However, it possesses a higher overvoltage towards hydrogen than silver, and therefore enables detection of metals like zinc, nickel and cobalt. As such solid electrodes are found to give stable results over several weeks, without any maintenance, and because this method greatly facilitates monitoring of heavy metals, attempts to apply such methods to various samples have been are carried out. The present paper deals with the determination of zinc and lead at nanogram per milliliter levels in wines and spirits with only minor treatment of the samples. The procedure may easily be adapted to continuous monitoring.We have previously found that audible sound may greatly increase the voltammetric signal using liquid mercury as well as silver as electrode material. This is also applied to the actual systems.Finally, model determinations of thallium in brandy with the dental amalgam electrode are compared with atomic absorption spectrometric (AAS) measurements. It was found that the electrode could be used repeatedly, without fouling, and with results close to those found by the AAS method.
Keywords: Voltammetry; Dental amalgam electrode; Wine; Online; Medium exchange; Zinc; Cadmium; Thallium; Lead;

Optimisation of the method for determination of the temperature of saturation in wines by Paulo Cameira dos Santos; F Gonçalves; M.N De Pinho (257-261).
The temperature of saturation of potassium hydrogentartrate (KHT) in wines was studied as an analytical parameter for wine tartaric stability evaluation. Two types of wines were used: “Vinho Verde” and “Port wine”. The temperature of saturation was determined by the intersection of the plots of conductivity versus temperature for a wine sample and the same wine sample with added KHT, by raising the temperature. The heating rate used was optimised in terms of accuracy and time necessary for the determination. An optimal value of 0.5 °C min−1 was found for both types of wine. The saturation temperature was determined for eight untreated wines and after two different tartaric stabilisation treatments, cold stabilisation and electrodialysis. The temperature of saturation values allowed the evaluation of the tartaric stability achieved and the comparison of the two treatments.
Keywords: Temperature of saturation; Wines tartaric stability; Potassium hydrogentartrate;