Integrated multienzyme electrochemical biosensors for the determination of glycerol in wines

The construction and performance of integrated amperometric biosensors for the determination of glycerol are reported. Two different biosensor configurations have been evaluated: one based on the glycerol dehydrogenase/diaphorase (GDH/DP) bienzyme system, and another using glycerol kinase/glycerol-3-phosphate oxidase/peroxidase (GK/GPOx/HRP). Both enzyme systems were immobilized together with the mediator tetrathiafulvalene (TTF) on a 3-mercaptopropionic acid (MPA) self-assembled monolayer (SAM)-modified gold electrode by using a dialysis membrane. The electrochemical oxidation of TTF at +150 mV (vs. Ag/AgCl), and the reduction of TTF⁺ at 0 mV were used for the monitoring of the enzyme reactions for the bienzyme and trienzyme configurations, respectively. Experimental variables concerning both the biosensors composition and the working conditions were optimized for each configuration. A good repeatability of the measurements with no need of cleaning or pretreatment of the biosensors was obtained in both cases. After 51 days of use, the GDH/DP biosensor still exhibited 87% of the original sensitivity, while the GK/GPOx/HRP biosensor yielded a 46% of the original response after 8 days. Calibration graphs for glycerol with linear ranges of 1.0 × 10⁻⁶ to 2.0 × 10⁻⁵ or 1.0 × 10⁻⁶ to 1.0 × 10⁻⁵ M glycerol and sensitivities of 1214 ± 21 or 1460 ± 34 μA M⁻¹ were obtained with GDH/DP and GK/GPOx/HRP biosensors, respectively. The calculated detection limits were 4.0 × 10⁻⁷ and 3.1 × 10⁻⁷ M, respectively. The biosensors exhibited a great sensitivity with no significant interferences in the analysis of wines. The biosensors were applied to the determination of glycerol in 12 different wines and the results advantageously compared with those provided by a commercial enzyme kit.     

Optimisation of a dispersive liquid-liquid microextraction method for the simultaneous determination of halophenols and haloanisoles in wines

A dispersive liquid-liquid microextraction (DLLME) method has been optimised for simultaneously extracting 2,4,6-trichloranisole (TCA), 2,3,4,6-tetrachloroanisole (TeCA), 2,4,6-tribromoanisole (TBA), pentachloroanisole (PCA), 2,4,6-trichlorophenol (TCP), 2,3,4,6-tetrachlorophenol (TeCP), 2,4,6-tribromophenol (TBP) and pentachlorophenol (PCP) from wine. The haloanisoles and halophenols were automatically determined using a gas chromatography-electron-capture detection (GC-ECD) system. Derivatisation of halophenols was performed at the same time as DLLME. Firstly, disperser and extraction solvents, salt addition and temperature conditions were selected. Then, the volume of disperser solvent, extraction solvent and derivatisation agent, and the percentage of base were optimised by means of a central composite design combined with desirability functions. The optimal extraction-derivatisation conditions found were 1.3 mL of acetone, 150 μL of carbon tetrachloride, 75 μL of acetic anhydride and a percentage of base of 0.7%; with no salt addition and at room temperature. Under these conditions, the proposed method showed satisfactory linearity (with correlation coefficients over 0.994), repeatability (below 9.7%) and reproducibility (below 9.9%). Moreover, detection limits were lower than the olfactory threshold of the compounds. The developed method was successfully applied to the analysis of red wine samples. To our knowledge, this is the first time that DLLME has been applied to determine cork taint responsible compounds in wine.     

Simultaneous determination of dyes in wines by HPLC coupled to quadrupole orbitrap mass spectrometry

A new method combining the QuEChERS (quick, easy, cheap, effective, rugged, and safe) method with ultra high performance liquid chromatography and ESI quadrupole Orbitrap high‐resolution MS was developed for the highly accurate and sensitive screening of 69 dyes in wines. Response surface methodology was employed to optimize the QuEChERS sample preparation method for the determination of 69 different analytes in wines for the first time. After optimization, the maximum predicted recovery was 99.48% rate for canacert indigo carmine under the optimized conditions of 10 mL acetonitrile, 1.45 g sodium acetate, 107 mg primary secondary amine, and 96 mg C₁₈. For the matrices studied, the recovery rates of the other 68 compounds ranged from 87.2–107.4%, with coefficient of variation < 6.4%. The mass accuracy typically obtained is routinely better than 1.6 ppm and only needed to be calibrated once a week. The LODs for the analytes are in the range 1–1000 μg/kg. This method has been successfully applied on screening of dyes in commercial wines, and it is very useful for the fast screening of different food additives.     

Development and validation of a sequential-injection method with chemiluminescence detection for the high throughput assay of the total antioxidant capacity of wines

This work reports a sequential-injection analysis (SIA) method with chemiluminescence (CL) detection for the rapid assay of the total antioxidant capacity (TAC) in wines. The method exploited the Co(II)-catalysed CL reaction of luminol with hydrogen peroxide in alkaline medium. Zones of sample, hydrogen peroxide, catalyst (Co(II) solution) and alkaline luminol were sequentially aspirated into the holding coil of the SIA manifold. Then, the flow was reversed and the stacked zones were directed to the CL detector. As the zones overlapped, antioxidants in the samples scavenged a portion of hydrogen peroxide and the decrease in the CL intensity was monitored and related to the TAC. The chemical and geometric conditions were studied and the method was validated in terms of linearity, accuracy (trueness and precision), matrix effects, signal additivity and robustness. The reproducibility of the method (expressed as the between-days % relative standard deviation) was between 2.5 and 3.4% and the trueness (expressed as the % recovery in wines spiked with gallic acid) was in the range 96.7-97.3%. The sampling frequency was 60 samples h⁻¹. The proposed SIA-CL method was compared with the DPPH method and the Folin-Ciocalteau (FC) method for the analysis of 25 wine samples.     

Multiresidue determination of 11 new fungicides in grapes and wines by liquid–liquid extraction/clean-up and programmable temperature vaporization injection with analyte protectants/gas chromatography/ion trap mass spectrometry

A gas chromatographic ion trap mass spectrometry (GC-ITMS) method was developed for the determination of 11 new generation fungicides (benalaxyl, benalaxyl-M, boscalid, cyazofamid, famoxadone, fenamidone, fluquinconazole, iprovalicarb, pyraclostrobin, trifloxystrobin and zoxamide) in grapes and wines. Samples were extracted with ethyl acetate:hexane (1:1, v/v) and cleaned-up with graphitized carbon black/primary secondary amine (GCB/PSA) solid-phase extraction (SPE) cartridges using acetonitrile:toluene (3:1, v/v) as eluent. The addition of analyte protectants (3-ethoxy-1,2-propanediol, d-sorbitol and l-gulonic acid γ-lactone) in the final extracts allowed to avoid the matrix-induced response enhancement effect on quantitation process with absolute recoveries ca. 100%. Precision (expressed as relative standard deviation) was lower than 16% for all fungicides. Limits of detection and quantitation were lower than 0.01 mg/kg or mg/L, except for cyazofamid, much smaller in all cases than maximum residue levels (MRLs) established by European Union for grapes and by Switzerland and Italy for wines. The proposed method was applied to determine fungicide residues in three different white grapes for vinification produced in Ribeiro area in Galicia (NW Spain), as well as in their corresponding final wines.     

A liquid chromatographic method optimization for the assessment of low and high molar mass carbonyl compounds in wines

Carbonyl compounds (CC) play an important role in beverage aroma since they may affect flavor of wines, brandies, and beers, among others. For this reason, it is necessary to identify and quantify CC through adequate analytical techniques. This study is a proposal of both developing and optimization of a new analytical methodology that allows investigate C₁–C₈ CC in wines simultaneously by quantifying even those ones that are predominantly present in the adduct form hydroxylalkylsulfonic acids (HASA). The HASA dissociation is undertaken by specific alkaline media (pH 11). The developed methodology employed the LC with UV/VIS detection (λ = 365 nm) technique under gradient elution in the way to reach both free‐CC and bound‐CC quantification. Results showed that binary gradient system using eluent A (MeOH/ACN/H₂O 74.5:0.5:25% v/v/v) and eluent B (MeOH) reached the best separation condition of both lower and higher molecular mass CC. This proposed method allowed simultaneous quantification of formaldehyde, acetaldehyde, propanone, furfuraldehyde, butyraldehyde, benzaldehyde, hexanaldehyde, 2‐ethyl‐hexanaldehyde, E‐pent‐2‐en‐1‐al, and cyclohexanone – all of them were found in white wine (Moscato Canelli) and red wine (Shiraz) produced in the São Francisco Valley, in the Northeastern Region of Brazil – although this optimized method may probably be suitable for quantification of propionaldehyde, isobutyraldehyde, heptanaldehyde, octanaldehyde, benzaldehyde, and E‐hex‐2‐en‐1‐al as well. We could not prove if this method is also able to determine the latter CC group since we have not found these substances present in detectable levels in our real samples considered in this study.     

Determination of primary amino acids in wines by high performance liquid magneto-chromatography

Eight amino acids (ethanolamine, glycine, alanine, β-aminobutyric acid, leucine, methionine, histidine and asparagine) were identified and quantified in Spanish wines by high performance liquid magneto-chromatography (HPLMC) with UV-V spectrophotometry. For this method, the amino acids are first complexed with mono(1,10-phenanthroline)-Cu(II) to confer them paramagnetic properties, and then separated by application of a low magnetic field intensity (5.5 mT) to the stationary phase contained in the chromatographic column. Principal components analysis of the results obtained grouped together the wine samples according to their denomination of origin: “Ribera del Duero”, “Rueda” or “Rioja” (Spain). Through cluster analysis, a series of correlations was also observed among certain amino acids, and between these groupings and the type of wine. These clusters were found to reflect the role played by the amino acids as primary or secondary nutrients for the bacteria involved in alcoholic and malolactic fermentation.     

固相萃取-高效液相色谱检测葡萄酒中赭曲霉毒素A

使用C18小柱固相萃取, C18反相柱(250 mm×4.6 mm i.d.)分离,V(乙腈):V(水):V(乙酸)＝99:99:2为流动相,荧光检测器(激发波长333 nm,发射波长460 nm)检测,测定葡萄酒中赭曲霉毒素A.其质量浓度在6.25～200 ng/mL范围内呈良好线性,相关系数为0.9997.样品经浓缩60倍后,方法检出限为0.027 ng/mL.对红葡萄酒、干红及白葡萄酒进行了加标回收实验,回收率为80.1%～109.8%.平行7份样品加标回收率相对标准偏差为5.9%.对市售6种葡萄酒进行了赭曲霉毒素A的测定.     

Automatic sequential determination of the hydrogen peroxide scavenging activity and evaluation of the antioxidant potential by the 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation assay in wines by sequential injection analysis

An automatic system that performs two analytical procedures, allowing the evaluation of the relative antioxidant capacity of wine samples, was developed. Automation was carried out using a sequential injection analysis (SIA) system that allowed, thanks to its versatility, the development of two methodologies. One is based on the decolorization assay of the 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical, using a spectrophotometric detector. A second methodology allowed the evaluation of the hydrogen peroxide scavenging activity by measuring the oxidation of homovanylic acid (HVA) to its fluorescent dimer, using a fluorescent detector.The developed automatic methodologies were evaluated using trolox as standard and subsequently using other antioxidant substances as gallic acid, caffeic acid, ascorbic acid, catechin and taxifolin which are abundant in wine and whose antioxidant activities were compared to that shown by trolox. The spectrophotometric and fluorimetric assays showed linearity intervals between 0.001 and 0.01 mM, and 0.001 and 0.008 mM of trolox, respectively.The evaluation of the antioxidant power of 20 white and red wine samples, from different Portuguese wine producing regions, was carried out sequentially, in the automatic system. The results were expressed in trolox equivalent antioxidant capacity (TEAC) and presented, for the ABTS and hydrogen peroxide scavenging activity methodologies, detection limits of 8.4 × 10⁻⁷ and 1.4 × 10⁻⁴ mM and relative standard deviation (R.S.D. (%)) in the range 0.6-2.4 and 1-1.8, respectively.     

Semiautomatic flow-injection method for determination of volatile acidity in wines.

A flow-injection (FI) method based on analytical pervaporation was assessed for its routine use in the determination of volatile acidity in winery laboratories. The new method was compared with both the official method and the Mathieu method, which is most often used in Spanish wineries, by testing 30 different wines, including young and aged, and sweet and dry wines from Montilla-Moriles appellation d'origine. The robustness of the new method was established, and then all 3 methods were studied in terms of range of linearity and regression of the calibration curve, repeatability, reproducibility, sensitivity, detection and quantitation limits (LOD and LOQ, respectively), and time of analysis. The FI method surpassed the Mathieu method in reproducibility and both the Mathieu and official methods in LOD and LOQ and sensitivity; it also required less personnel involvement and shorter analysis time. The statistical criteria established by the Office International de la Vigne et du Vin were applied to the data and the results obtained indicated that the differences between the analytical parameters of the 3 methods are not significant and can be applied indistinctly. The correlation of the methods was studied by taking them 2 by 2, and the corresponding equations, coefficients, and deviations confirmed the statistical results. Thus, the new method can be used in winery laboratories with clear advantages over its 2 counterparts (the routine and official methods).     

New method for the determination of saturation concentration of Dichlorvos

Summary Saturation concenrtation of DDVP at room temperature (28°) has been determined by a colorimetric method. The saturation concentration at 28° are 9.39 and 8.55 mg/l for AR and technical grade samples respectively. The method consists of coupling DDVP vapours in gas-tight tubes with alkaline resorcinol at a water bath of 60–65°, when a yellow chromophore with an absorption maximum at 490 nm is formed. The linear relationship between the concentration of DDVP and absorbance at 490 nm holds good in the range of 1 to 20g DDVP. The method has been checked with a gas Chromatographie method for accuracy. These values are also verified by a physical method based on Dalton's Law of partial pressures in a gaseous mixture.

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Eine neue Methode zur Bestimmung der Sättigungskonzentration von Dichlorvos

Zusammenfassung Die Sättigungskonzentration von Dichlorvos (DDVP) bei Raumtemperatur (28° C) wurde kolorimetrisch bestimmt. Sie beträgt bei 28° C für das analytisch reine Präparat 9,39 mg/l bzw. für das technisch reine Präparat 8,55 mg/l. Das Verfahren besteht in der Kupplung von DDVP-Dampf in gasdichten Röhrchen mit alkalischem Resorcin bei Wasserbadtemperatur (60–65°), wobei eine gelbe Verbindung mit einem Absorptionsmaximum bei 490 nm gebildet wird. Die Absorption bei dieser Wellenlänge ist zwischen 1 und 20g DDVP linear. Das Verfahren wurde gaschromatographisch überprüft. Außerdem wurden die Ergebnisse auch physikalisch mittels des Gesetzes von Dalton für Partialdrucke in gasförmigen Gemischen verifiziert.

     

Quantification of polyphenols with potential antioxidant properties in wines using reverse phase HPLC

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 (r(2) >0.994) within test ranges. Detection limits ranged between 0.03 and 11.5 microg/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.     

Optimization of the derivatization method for the liquid-chromatographic determination of carboxylic acids in wines

Several reagents and experimental conditions were tested to optimize the yield of the derivatization procedure for determining carboxylic acids in wines. Two techniques were applied for optimization: a modified sequential simplex method and a simultaneous modelling method. The results obtained were similar; advantages of the latter method are discussed. The optimized conditions (phenacyl bromide as reagent with 18-crown-6 as catalyst with a reaction time of 90 min at 90 ° C) were used to determine the main carboxylic acids in several varietal wines of the Tarragona region.     

Exposure assessment to ochratoxin A from the consumption of Italian and Hungarian wines

A total of 267 wine samples including 19 dessert, 186 red, 11 rosé and 51 white produced mostly in the years 1997–2002 in Italian and Hungarian regions were analyzed for ochratoxin A (OTA) using inmunoaffinity column (IAC) clean-up and HPLC with fluorimetric detection. None of Hungarian wine samples were contaminated with this mycotoxin. For Italian red wines, 84% of the samples were positive for OTA ranged from 0.01 to 4.00 ng/mL. Furthermore, OTA was detected in 63% of dessert, in 56% of rosé and in 19% of white wine samples ranged from 0.01 to 1.64, from 0.01 to 1.04 and from 0.01 to 0.21 ng/mL, respectively. A study of OTA daily exposure assessment in Italian wines was also carried out outlining a quite low contribution to the overall daily intake.     

Influence of different bentonites on the rare earth element concentrations of clarified Romanian wines

The rare earth element (REE) concentrations of 19 Romanian young wine samples originating from the Dealurile Moldovei viticulture area were determined by double focusing inductively coupled plasma mass spectrometry (DF-ICP-MS) after microwave-assisted digestion with nitric acid. The determination of Eu was hampered by the BaO molecular interference. Generally, the red wine samples were more concentrated for REEs than the white wine samples studied. The REE concentrations of the four bentonites (Gelbenton, Evergel, BW200, Tükrös) determined after their fusion were higher by three orders of magnitude than those of the wine samples. After a simulated wine purification process performed with these bentonite samples and a red and white pool samples, the REE concentrations of the clarified wine samples increased by 1.2–1.5 times for red, and 1.3–3 times for white wines in case of the fibrous bentonite sample (Gelbenton), by about 2–5 times in case of the bentonite containing ovalbumin, caseine and gelatine (Evergel), meanwhile this factor was about 20–25 for Na bentonite powder samples (BW200, Tükrös). On basis of the chemometric evaluation using the REE concentrations as input data, the majority of the Feteasca wines belonged to the same cluster as well as the two Cabernet Sauvignon to another subcluster. The adequate choice of the bentonite may allow the use of REEs as fingerprints for determining the wine provenance.     

Determination of multi-class pesticides in wines by solid-phase extraction and liquid chromatography-tandem mass spectrometry

This work reports a new sensitive multi-residue liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for detection, confirmation and quantification of forty-six pesticides and transformation products belonging to different chemical classes in wines. The proposed method makes use of a solid-phase extraction (SPE) procedure with Oasis HLB cartridges that combines isolation of the pesticides and sample clean-up in a single step. Analysis is performed by liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-MS/MS) operated in the selected reaction monitoring (SRM) mode, acquiring two specific precursor-product ion transitions per target compound. An investigation of matrix effects has been performed during method validation showing medium to low effects for the majority of the compounds. Limits of detection (LODs) were in the range 0.0003–0.003 mg L⁻¹ and limits of quantification (LOQs) were in the range 0.001–0.01 mg L⁻¹. The average recoveries, measured at two concentration levels (0.010 and 0.050 mg L⁻¹), were in the range 70–110% for most of the compounds tested with % relative standard deviations below 20%, while a value of 0.010 mg L⁻¹ has been established as the method limit of quantification (MLOQ) for all target species. Expanded uncertainty values were in the range 10–40% while the Horrat ratios were below 1. The method has been successfully applied to the analysis of 60 wine samples in the course of an annual monitoring study with carbendazim-benomyl, thiophanate-methyl and carbaryl being the most frequently determined pesticides.     

A new spectrophotometric method for the determination of promethazine-HCl from pure and pharmaceutical preparations

Promethazine-HCl reacts with potassium persulphate to give a pinkish red color complex exhibiting maximum absorbance at 515 nm. The reaction is selective for promethazine-HCl, with 1 μg/mL as visual limit of identification and provides a basis for a new spectrophotometric determination method. The color forming reaction obeys Beer's Law from 0.001 to 0.125 mg/mL of promethazine-HCl. The relative standard deviation does not exceed 0.005 mg/mL. The method is successfully applied to pure and pharmaceutical formulations of promethazine-HCl. The quantitative assessment of tolerable amounts of possible interferants was also studied. The results are reproducible within ±1% and in good agreement with those obtained by the standard procedure.     

Optimisation of stir bar sorptive extraction for the analysis of volatile phenols in wines

An easy, fast and reliable analytical method is proposed for the determination of the concentration of volatile phenols (ethyl- and vinylphenols) in wines. The novel stir bar sorptive extraction (SBSE) technique is employed, following a simple and fast procedure that allows 15 samples to be extracted simultaneously using very small sample volume. Extracts are desorbed in a thermodesorption system (TDS) coupled on-line to a gas chromatograph-mass spectrometry system. The SBSE offers better recovery and linear regression coefficient (r2) for the four volatile phenols than solid-phase extraction (SPE). The mass spectrometric detection in selected ion monitoring mode contributes to the lower detection limit and good sensibility obtained with this method.     

Field-amplified sample injection and in-capillary derivatization for capillary electrophoretic analysis of metal ions in local wines

In-capillary derivatization and field-amplified sample injection (FASI) coupled to capillary zone electrophoresis (CZE) was evaluated for the analysis of metals (Co(II), Cu(II), Ni(II), and Fe(II)) using 2-(5-Nitro-2-Pyridylazo)-5-(N-Propyl-N-Sulfopropylamino)Phenol (Nitro-PAPS) as the derivatizing agent. For FASI, the optimum conditions were water as sample solvent, 1 s hydrodynamic injection (0.1 psi) of a water plug, 5 s of electrokinetic introduction (10 kV) of the sample. The in-capillary derivatization was successfully achieved with zone-passing strategy in order tandem injection of Nitro-PAPS reagent (0.5 psi, 7 s), a small water plug (0.1 psi, 1 s), and metal ion introduction (10 kV, 5 s). The solution of 45 mmol L^− 1 borate pH 9.7 and 1.0 × 10^− 5 mol L^− 1 Nitro-PAPS containing 20% acetonitrile was used as the running buffer. The limit of detection obtained by the proposed method was lower than those from pre-capillary derivatization about 3–28 times. The recovery of the method was comparable to pre-capillary derivatization method. In-capillary derivatization-FASI-CZE was applied to analysis of metals in wine samples. The results were compared with those obtained by CZE with pre-capillary derivatization method and atomic absorption spectrometry (AAS).     

Optimisation of a liquid chromatography-tandem mass spectrometric method for the determination of acrylamide in foods

A robust and fitted routine method resulting from an analytical optimisation has been applied for the determination of acrylamide in several foods including mainly potato and cereal products. For the sample treatment, different materials were evaluated for filtration and purification of the extract. To increase the performances in terms of sensitivity, a preconcentration to small volume was introduced before liquid chromatography coupled to tandem mass spectrometry analysis on a μ-Bondapak C₁₈ column using d₃-acrylamide as internal standard. For identification, relative retention time and two diagnostic ions were monitored. A limit of detection of 10 μg kg⁻¹, a limit of quantitation of 20 μg kg⁻¹, mean recoveries ranging from 100 to 115%, coefficients of variation from 1.36 to 8.06% for repeatability and from 3.3 to 18.2% for reproducibility within the laboratory and a measurement uncertainty of 42% were obtained during an in-house validation procedure. Results of tests, validation data and Z-score obtained during participation to proficiency studies are presented.