Correlation between different clean-up methods and analytical techniques performances to detect Ochratoxin A in wine

Three different clean-up methods and two analytical techniques were compared to determine Ochratoxin A (OTA) in wines. The first clean-up used a MycoSep column, the second an immunoaffinity column (IAC) and the third consisted in a liquid-liquid extraction (LLE) using dichloromethane in acid conditions. Meanwhile, two different OTA determination techniques were also evaluated: a HPLC analysis using a fluorescence detector and an enzyme-linked immunosorbent assays (ELISA) method.Correlations between clean-up methods and analytical techniques to determine OTA in wine were made evaluating linearity, accuracy and precision.Both the two first clean-up methods (solid-phase extraction, SPE) showed a good linear fit (r² = about 0.9999), followed by LLE. The use of immunoaffinity columns showed the best recoveries, even if also the SPE with MycoSep showed good recoveries while the LLE recoveries were the worst ones. The HPLC analysis showed good precision and accuracy, while ELISA method, even with a sufficient linearity, generally underestimated OTA content in wines.     

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.     

Concentration of ochratoxin A in wines from supermarkets and stores of Valencian Community (Spain)

Ochratoxin A (OTA) is a mycotoxin produced by fungi species belonging to the genera Aspergillus and Penicillium being isolated in alcoholic beverages. The aim of this work is developed and applied a procedure for the analysis of OTA in wines. An analytical method based on immunoaffinity column (IAC) for clean-up, liquid chromatography with fluorescence detection (LC-FD), and LC-FD after of OTA methylation was used to determine the occurrence of OTA in wines. Recoveries of this mycotoxin spiked to red wines at 0.5 ng/ml level were >90% with an average of relative standards deviations of 4%. Furthermore, 116 wine samples from designation of origin (DO) and three samples from food stores of Valencian Community (Spain) were examined for the occurrence of OTA being the levels of this mycotoxin ranged from <0.01 to 0.76 ng/ml. Finally, the estimated daily intake of OTA in this study was 0.15 ng/kg bw per day.     

Fluorescence polarization immunoassay for rapid screening of ochratoxin A in red wine

A fluorescence polarization (FP) immunoassay, based on a monoclonal antibody and an ochratoxin A (OTA)-fluorescein tracer, has been developed for rapid screening of OTA in red wine. Wine samples were diluted with methanol and passed through aminopropyl solid-phase extraction columns prior to the FP assay. Average recoveries from samples spiked with OTA at levels of 2.0 and 5.0 ng/mL were 79% with RDS of 11% (n = 6). The limit of detection of the FP immunoassay was 0.7 ng/mL OTA, and the whole analysis was performed in less than 10 min. The assay was tested on 154 red wine samples (naturally contaminated or spiked at level ranging from 0.1 to 5.0 ng/mL) and compared with an high-performance liquid chromatography/immunoaffinity column clean-up method, showing a good correlation (r = 0.9222). Their compliance with the European regulation (2.0 ng/mL OTA maximum permitted level) was correctly assessed for 70% of the analyzed samples of red wine, whereas confirmatory analyses were required for the remaining ones with OTA levels close to the regulatory limit. No false-negative or positive results were observed using the FP immunoassay. The proposed FP assay is a useful screening method for OTA in red wines, when high throughput is required, that could also be used for white and rosé wines, which are known to contain less interfering compounds such as polyphenols.     

An electrochemical immunosensor for ochratoxin A determination in wines based on a monoclonal antibody and paramagnetic microbeads

We report a direct competitive immunosensor for the rapid determination of ochratoxin A (OTA) in wine samples. Magnetic beads (1 ± 0.5 μm diameter) covered with streptavidin were functionalized with a monoclonal antibody against OTA, and then left to incubate in a solution of tracer (ochratoxin conjugated to the enzyme peroxidase) and a range of OTA concentrations (10(-4) to 1,000 ng mL(-1)). After washing and separation steps helped with a magnetic field, a volume of the dispersion was put on screen-printed electrodes under a magnet, and after adding the substrate the p-benzoquinone generated enzymatically was detected by differential-pulse voltammetry. Wine samples (2 mL) were easily prepared simply by adjusting to pH = 7.5 with diluted NaOH and by adding polyvinylpyrrolidone for complexing polyphenols, without any other clean-up or preconcentration steps. The limit of detection for detecting OTA in wines was of 0.11 ± 0.01 ng L(-1), well below the permitted content of the mycotoxin by the European Union (<2 ng mL(-1)). Spiked wines were subjected to immunosensor calibrations to study the matrix effects. OTA concentrations measured with the immunosensor were compared with those obtained by high-performance liquid chromatography coupled to fluorescence detection (AOAC official method 2001.01). The OTA levels from two red wines of "Campo de Borja", Spain, ranged from about 0.027 to 0.033 ng mL(-1) of OTA.     

Occurrence of ochratoxin A in Turkish wines

A total of 95 wine samples including 34 white, 10 rosé and 51 red wines originating from four different Turkish areas were analysed for ochratoxin A (OTA). An analytical method based on immunoaffinity column (IAC) for clean-up and high performance liquid chromatography with fluorescence detection (HPLC-FD) was used to determine OTA in wines. The limit of detection (LOD) was estimated as 0.006 ng ml^− 1 for white wine and 0.010 ng ml^− 1 for rosé and red wines. The limit of quantification (LOQ) was estimated as 0.020 ng ml^− 1 in white wine and 0.030 ng ml^− 1 in rosé and red wines. Recovery experiments were carried out with spiked samples in the range 0.1–1 ng ml^− 1 of OTA. The average OTA recoveries from spiked white wine samples varied from 79.43% to 85.07%; while the mean recoveries for rosé and red wine samples were in the range of 77.48–83.96% and 76.61–83.55%, respectively. OTA was detected in 82 (86%) wine samples at levels of < 0.006–0.815 ng ml^− 1, which were below the maximum allowable limit established by the European Community. The mean OTA concentration in red wines was slightly higher than in white and rosé wines. Furthermore, our data indicate that the geographic region of origin has strong influence on OTA level for white, rosé and red wines: wines originating from Thrace (n = 44, mean = 0.158 ng ml^− 1) and Aegean (n = 28, mean = 0.060 ng ml^− 1) regions of Turkey were more contaminated with OTA compared with wines originating from central (n = 15, mean = 0.027 ng ml⁻¹) and east Anatolia (n = 8, mean = 0.027 ng ml^− 1) areas. This study showed that the occurrence of OTA in Turkish wines is high, but at levels that probably leads to a non-significant human exposure to OTA by consumption of wines.     

Comparison of methods for the determination of ochratoxin A in wine

Different analytical methods for the determination of ochratoxin A (OTA) in wine have been compared. Sample clean-up was based on solid-phase extraction (SPE) with (i) immunoaffinity or (ii) RP-18 sorbent materials applying different experimental protocols. The detection of OTA was accomplished with high-performance liquid chromatography (HPLC) combined either with electrospray ionisation (ESI) tandem mass spectrometry (MS-MS) or fluorescence detection (FL). Comparative method evaluation was based on the investigation of 18 naturally contaminated red wine samples originating from different European countries. The analytical results are discussed in view of the respective method validation data and the corresponding experimental protocols. In general, analytical data obtained with RP-18 SPE combined with LC-MS-MS detection and immunoaffinity extraction combined with FL offered comparable good results in the sub-ppb concentration level indicating that high selectivity of either the sample clean-up or, alternatively the detection system are equally well-suited to guarantee an accurate OTA analysis in wine.     

Validation of two analytical methods for the determination of ochratoxin A by reversed-phased high-performance liquid chromatography coupled to fluorescence detection in musts and sweet wines from Andalusia

Some Spanish sweet wines are made from raisins, grapes dried by direct exposure to the sun after picking. This drying process can encourage ochratoxin A (OTA) formation. OTA is a mycotoxin formed by several fungi. It has been linked to nephropathy in humans, and may have a long half-life in humans. The aim of this study is to develop and to apply two procedures for the analysis of OTA in grape musts (during the raisining process) and sweet wines, respectively. Reversed-phase high-performance liquid chromatography (RP-HPLC) coupled to fluorescence detection (FLD) was employed in both analytical methods. In grape must, the method involves the direct injection of the sample in a HPLC-FLD system without any kind of prior clean-up procedure. The complexity of the sweet wine samples requires a solid-phase extraction (SPE) clean-up on a C18 column which enables the OTA to be isolated from the matrix. The methods used were statistically validated. The validation also included the comparison of the slopes of the curve obtained with standards and the regression curves obtained by the addition of a standard. Two different studies of standard additions were conducted. One method was validated without sample preparation and it was applied to must samples. The other method was validated with SPE extraction and it was applied to sweet wine samples. Recovery was always better than 89.69%. The limit of detection (S/N = 3) and limit of quantification (S/N = 10) were established at 0.22 and 0.77 μg l⁻¹, respectively. In general, the analytical data obtained provided good results at the sub-μg l⁻¹ concentration level.     

Comparison of different sample treatments for the analysis of ochratoxin A in must, wine and beer by liquid chromatography

Ochratoxin A (OTA) is a mycotoxin produced by some species of Aspergillus and Penicillium verrucosum. It has been found in foods and feed all over the world. There is a great concern about OTA because it is nephrotoxic and probably, carcinogenic to humans. Most of analytical methods developed for OTA in wine, beer and other products are based on LC with fluorescence detection (LC-FLD). In the present work, various procedures for extraction and/or clean-up for determination of OTA in musts, wine and beer by LC-FLD were compared: (1) dilution with polyethylen glycol 8000 and NaHCO3 solution and clean-up an on immunoaffinity column (IAC); (2) extraction with chloroform and IAC clean-up; solid-phase extraction (SPE) on (3) reversed-phase (RP) C18; (4) RP phenylsilane and (5) Oasis HLB cartridges. SPE on phenylsilane and Oasis HLB have not been reported for OTA analysis in beverages. The same LC-FLD conditions and concentration ratio were used. The former procedure was simple, rapid and provided flat baselines, free from most impurity peaks, high OTA recoveries and quite repeatable results. RP C18 using methanol-acetic acid (99.5:0.5) as elution solvent provided good recoveries and precision, thus becoming a cheaper but interesting alternative at 0.1-1 ng/ml spiking levels. Oasis HLB cartridges were usually better than phenylsilane. Possible binding of OTA to proteins or other components was tested by acid treatment before extraction but no significant differences with controls appeared.     

Coacervative extraction of Ochratoxin A in wines prior to liquid chromatography/fluorescence determination

Coacervates made up of reverse micelles of decanoic acid were assessed as a new strategy for the simplification of wine sample treatment in the determination of Ochratoxin A (OTA). Simultaneous extraction/concentration of this contaminant was based on both hydrophobic and hydrogen bond OTA:coacervate interactions. Parameters affecting extraction efficiency and concentration factors were studied. Concentrations of decanoic acid and tetrahydrofuran (THF) were the most influential parameters, being 0.5% of acid and 5% of THF the selected ones. The procedure was very robust, so that the extractions were not influenced by the pH and the nature or concentration of matrix components. OTA recoveries from different types of wines (white, rosé and red) ranged between 85 and 100% and the actual concentration factors varied from 105 to 125 for sample volumes of 15 mL. The detection limits for OTA, after liquid chromatography/fluorimetry (LC/FL) analysis of the coacervate (20 microL), were 4.5 ng L(-1) in white and rosé wines and 15 ng L(-1) in red wines, values which were far below the threshold limit established for OTA by EU directives (2.0 microg L(-1)). No clean-up of the extracts was required for any of the samples analysed. The overall sample treatment took about 15-20 min and several samples could be simultaneously treated using conventional lab equipment. The precision of the method, expressed as relative standard deviation, was about 5%. The approach developed was successfully applied to the determination of OTA in different wine samples from the South of Spain. The concentrations found ranged between 0.015 and 0.091 microg L(-1).     

Non-instrumental immunochemical tests for rapid ochratoxin A detection in red wine

Gel-based and membrane-based flow-through immunoassay formats were investigated for rapid ochratoxin A (OTA) detection in red wine. The flow-through set-up consisted of an antibody containing gel or membrane placed at the bottom of a standard solid-phase extraction column (i.e. the flow-through column), combined with a clean-up column. Different clean-up methods were studied for red wine clarification and purification. The optimal method consisted of passing wine, diluted with an aqueous solution containing 1% polyethylene glycol (PEG 6000) and 5% sodium hydrogencarbonate, through strong anion exchange (SAX) silica. An immunoassay for OTA detection in red wine was optimized and a cut-off level at 2 μg L⁻¹ according to EU legislation was achieved with both formats. A more significant colour difference between blank and spiked samples was observed for the gel-based assay making this superior to the membrane-based assay. The proposed rapid gel-based test was compared with a standard immunoaffinity column - high-performance liquid chromatography - fluorescent detection (IAC-HPLC-FLD) method and a good correlation of the results was obtained for naturally contaminated wine samples.     

Ochratoxin A survey in Portuguese wine by LC-FD with direct injection

Wine and grape juices were identified as one of the most important sources of ochratoxin A (OTA), a mycotoxin with diverse toxic effects that naturally appears in food and foodstuffs all over the world.The aim of this study was to assess the OTA levels in Portuguese wines through the application of a simple and accurate method based on liquid chromatography (LC) with direct injection, followed by fluorescence detection (FD).Randomly selected wine samples were used to evaluate the performance of direct injection as efficient, fast, inexpensive and safe sample preparation method. The proposed method was successfully validated. The limit of quantification (LOQ) was 1.0 μg/L and OTA recoveries from wine samples, spiked at the three fortification levels, were higher than 85.4%, with RSDs lower than 9.6% for both red and white wines. The presence of OTA was confirmed by methyl ester derivatization followed by LC analysis.Data on OTA levels were obtained for 60 Portuguese red and white wine samples. OTA was found in 12 samples, nine (26%) red wine samples and three (12%) white wine samples. Only one red wine sample and one white wine sample presented a contamination level above the LOQ, with 1.23 and 2.4 μg/L, respectively. It should be pointed out that this white wine sample exceeded the EC maximum permitted level of 2.0 μg/L. The safe dose established as 120 ng/kg body weight/week was not exceeded by the weekly intake estimated for the samples contaminated above the LOQ.     

Evolution of ochratoxin A content from must to wine in Port Wine microvinification

To study the evolution of ochratoxin A (OTA) content from must to wine during the making of Port Wine, grapes from the five most common varieties of Port Wine were harvested and combined in equal percentages in order to perform microvinifications. Three sets of assays were studied: a blank (A), where the most common Port Wine-making process was used; in the second (B), a solution of OTA was added to the initial must; in the third (C), the grapes were aspersed with an inoculating solution of OTA-producing fungi. Samples were collected, in duplicate, on four different occasions throughout the process. The influence of the addition of SO₂ to the must was also assessed in each set. The quantification of OTA was based on the standard reference method for wines (European Standard prEN 14133), which includes clean-up via immunoaffinity columns and HPLC with fluorescence detection. The limits of detection were 0.076 g/l for wine and 0.114 g/l for must. The method was validated by assessing the precision, accuracy and by obtaining an estimate of the global uncertainty. Overall, the levels of OTA observed during the vinifications dropped by up to 92%, and no grapes used in this work were contaminated naturally.     

Development and Validation of an SPE-LC Method for the Simultaneous Determination of trans-Resveratrol and Selected Flavonoids in Wine

A simple LC method has been developed for the simultaneous determination of the flavonols rutin, myricetin, quercetin, kaempferol and the stilbene, trans-resveratrol, in wines. Sample clean-up was performed with polymeric Nexus ABS ELUT cartridges. The polyphenols were separated in less than 25 min using gradient elution and UV detection at 320 nm. Average recoveries of the analytes from spiked wine ranged from 82.2 to 117.6% and the detection limits, based on spiked extracted synthetic wine, ranged from 0.02 to 0.2 mgL^?1. The method was applied to the analysis of Greek wines.     

Ochratoxin A in Wine: Its Determination and Photostability

Abstract

Due to the growing public concern regarding food safety, reliable, nondemanding and robust analytical methods are needed for quantitative determination of toxic compounds in complex matrices. Sample preparation is frequently a crucial step in determination of ochratoxin A (OTA) in wine, and a simplified and automated procedure is described, using solid‐phase extraction coupled on‐line to high pressure liquid chromatography (HPLC) with fluorimetric detection (λ_ex=333 nm, λ_em=460 nm). While the limit of quantitation is frequently better compared to off‐line procedures (30 ng/L), the decisive advantages of the new procedure are the absence of all sample manipulation during preconcentration and subsequent analysis, and consequentially no risk of analyte loss or sample contamination. Furthermore, using the standard addition method, matrix interferences can be avoided and the determination of extraction efficiency is unnecessary. These improvements have important consequences for the overall uncertainty of the analytical procedure. The developed method was applied for determination of OTA in 12 selected Slovenian wines. The typical relative standard deviation (RSD) was 10%. In none of the samples, did the OTA amount exceeded 2 µg/kg, the limit regulated by the EC.

The photo‐stability of the mycotoxin in solutions was examined. During irradiation of OTA solutions, its content was quickly reduced, while three fluorescent degradation products were detected. The degradation proceeds faster in water and 12% ethanolic solutions than in organic solvents or wine. Identification of the fluorescent degradation products was attempted using LC‐MS/MS with electrospray ionization.     

Restricted access supramolecular solvents for sample treatment in enzyme-linked immuno-sorbent assay of mycotoxins in food

A restricted access supramolecular solvent (SUPRAS-RAM) made up of tetradecanoic acid reverse micelles is proposed as a wide-scope and low-cost strategy for the treatment of agrifood samples prior to enzyme-linked immunosorbent assays (ELISA). The approach was assessed for the determination of ochratoxin A (OTA) in wines and spices and aflatoxin B1 (AFB1) in cereals, two ubiquitous mycotoxins that were selected as representative contaminants for this study. The samples were selected to cover a variety of matrices in terms diverse composition and high complexity. Macromolecules such as proteins and carbohydrates were not-co-extracted due to the restricted access properties of the SUPRAS that are provided by chemical and physical mechanisms. In this sense, analyte extraction and clean-up were carried out in a single step. Parameters determining the extraction efficiency were studied and optimized. Certified reference materials were used for method validation. Recoveries of OTA ranged between 83% and 96% in wines (with relative standard deviation, RSD, of about 10%) and between 81% and 93% in spices (RSD 7%). Recoveries for AFB1 in wheat ranged from 75% to 85% (RSD 8%). The detection limits were all below the maximum levels established for OTA and for AFB1 by EU directives. This method offers a green and low-cost alternative to the organic solvent-based extraction and/or immunoaffinity columns-based cleanup of complex samples prior to ELISA.     

Ochratoxin A in wines-assessing global uncertainty associated with the results

Ochratoxin A (OTA) is a mycotoxin (potentially carcinogenic secondary metabolite derived from fungal contamination), produced by some Aspergillus and Penicillium strains. Although present and legislated in different food sources in the human diet, the regulation for wine intake is still under discussion. The Office International de la Vigne et du Vin (OIV) recommended maximum levels in wine of 2 μg l⁻¹. Some reports refer to OTA contamination in wines up to 15 μg l⁻¹ and a special incidence in red wines from the southern regions of Europe and the north of Africa, but the majority of the data available are below 1 μg l⁻¹. When working at such low concentrations, the problem of the uncertainty of the results becomes decisive towards the implementation of legal limits. In order to assess the global uncertainty associated with OTA determination in wines and widen the data set and knowledge of the situation in Portugal, 340 wines were analysed (189 Port Wine, 85 Vinho Verde and 66 wines from other regions in the country) by a high performance liquid chromatography (HPLC)-fluorescence detection (FD) method using immunoaffinity columns for clean up. OTA was detected in 69 wines by the method used, but in concentrations below 0.5 μg l⁻¹, except for two which showed levels up to a maximum of 2.1 μg l⁻¹. However, the global uncertainty for OTA is close to 37% for concentrations above 0.5 μg l⁻¹, and therefore, such value can be below or exceed the OIV limit. In the vicinity of the limit of detection, 0.084 μg l⁻¹, the global uncertainty rises exponentially to a maximum of about 70%. This can be an obstacle when discussing safety intake limits. Ethanol and glucose content did not interfere in the clean up of OTA by immunoaffinity columns.     

Reversed-phase liquid chromatographic method for the determination of ochratoxin A in wine

In this paper, we propose a new, rapid, highly sensitive and reproducible RP-HPLC-FLD method for the detection of ochratoxin A (OTA) in wine, by directly injecting the liquid in the chromatographic system without any extraction or clean-up. An alkaline mobile phase (NH4Cl:CH-CN 85:15 (v/v), 20 mM, pH 9.8) was used to obtain a distinct fluorescence enhancement. This improvement allows to reach, without an immunoaffinity clean-up or concentration, a detection limit of 0.05 ng/ml, which is similar to those commonly obtained after immunoaffinity purification and acidic elution. The method was statistically validated and directly applied to a series of wine samples.     

Worldwide interlaboratory study on the determination of ochratoxin A in different wine type samples

Interlaboratory studies are decisive tools to help the validation of a specific analytical methodology or to assess the reproducibility of the use of different methods to analyze a given compound or compounds in certain sample matrices. In this work, homogeneous samples of two white wines (“White Wine” and “White Liqueur Wine”) and one red wine (“Red Fortified Wine”) from Portugal with different production techniques and characteristics, namely in alcohol strength (10.5%, 16.0% and 19.0% ethanolic content, respectively), were analyzed for their contents in ochratoxin A (OTA), a mycotoxin generated from fungal contamination. White Liqueur Wine was naturally contaminated, whereas the other two wine type were spiked with ethanolic OTA solutions. The participation of 24 laboratories from 17 countries of five continents was ensured for this study. Although with no restrictions in terms of analytical methodology to employ, 75% of the laboratories resorted to immunoaffinity columns clean-up followed by high performance liquid chromatography with fluorescence detection (HPLC-FD), most of them in accordance with the European Standard EN 14133. For White Wine samples, the general mean OTA concentration was 1.96 μg/l (two outliers) with interlaboratorial standard deviation (s_L) of 0.53 μg/l; for White Liqueur Wine, mean of 1.59 μg/l (one outlier), with s_L = 0.59 μg/l; and for Red Fortified Wine, mean of 2.73 μg/l (no outliers), with s_L = 0.96 μg/l. Outliers were determined by Cochran and Grubbs tests. The Horrat index, recommended by the Association of Official Analytical Chemists (AOAC) for the quality assurance of the collaborative study was, on average, 1.7. This study proved that OTA determination in wines is reproducible, regardless of the methodology employed.     

Determination of ochratoxin A in wine by means of immunoaffinity column clean-up and high-performance liquid chromatography

A new and accurate method to quantify ochratoxin A (OA) in table wine has been developed. The method uses commercial immunoaffinity columns for clean-up and high-performance liquid chromatography (HPLC) with fluorescence detection for quantification of the toxin. Wine was diluted with a solution containing 1% polyethylene glycol (PEG 8000) and 5% sodium hydrogencarbonate, filtered and applied to an OchraTest immunoaffinity column. The column was washed with a solution containing sodium chloride (2.5%) and sodium hydrogencarbonate (0.5%) followed by water. OA was eluted with methanol and quantified by reversed-phase HPLC with fluorometric detection (excitation wavelength 333 nm, emission wavelength 460 nm) using acetonitrile-water-acetic acid (99:99:2) as mobile phase. Average recoveries of OA from white, rosé and red wine samples spiked at levels from 0.04 to 10 ng/ml ranged from 88% to 103%, with relative standard deviations (RSDs) between 0.2 and 9.7%. Detection limit was 0.01 ng/ml based on a signal-to-noise ratio of 3:1. The method was applied successfully to 56 samples of red (38), rosé (8), white (9) and dessert (1) wine. The levels of OA ranged from <0.01 to 7.6 ng/ml with red wines more contaminated than rosé and white wines. A good correlation (r=0.987) was found by comparative analysis of 20 naturally contaminated samples using this method and the method of Zimmerli and Dick with better recoveries of OA and better performances for the new method. Several advantages of this method with respect to the actually available methods have been pointed out, with particular reference to red wine which appears to be the most difficult to analyze.