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.     

A Study on Corrosion Processes of Archaeological Glass from the Valencian Region (Spain) and its Consolidation Treatment

The combined use of scanning electron microscopy/energy dispersive X-ray microanalysis (SEM/EDX) and square wave voltammetry (SQWV), is used for studying the corrosion processes that have taken place in buried glass from different archaeological sites in the Valencian Region (Spain). The procedures permit a parallel investigation of morphology and chemical composition. Determination of the chemical composition of the glasses and their alteration crust and identification of the elements responsible for the colour has been also carried out using energy dispersive X-ray microanalysis. The electrochemical response of samples attached to paraffin-impregnated graphite electrodes reveals the presence of different iron and manganese oxide species in the browning areas of the corroded glass. Image analysis applied to microphotographs obtained by means of SEM led to the determination of morphological parameters concerning the corrosion phenomena occurring on the surface of the fragments such as thickness of the corrosion layer and its laminated structure. Additionally, measurement of the thickness of the film of polymer used as coating in the consolidation treatments has been carried out using cryo-scanning electron microscopy (cryo-SEM) combined with image analysis.     

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.     

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.     

Classification of commercial wines from the Canary Islands (Spain) by chemometric techniques using metallic contents

Eleven elements, K, Na, Ca, Mg, Fe, Cu, Zn, Mn, Sr, Li and Rb, were determined in dry and sweet wines bearing the denominations of origin of El Hierro, La Palma and Lanzarote islands (Canary Islands, Spain). Analyses were performed by flame atomic absorption spectrophotometry, with the exceptions of lithium and rubidium for which flame atomic emission spectrophotometry was used. Sweet wines from La Palma were elaborated as naturally sweet with over-ripe grapes and significant differences were found in all the analysed elements with the exceptions of sodium, iron and rubidium with regard to dry wines from the same island. Contrarily, sweet wines from Lanzarote elaborated with grapes in a similar ripening state to dry wines did not present significant differences between them with the exception of strontium, the content of which was greater in dry wines. Among the three islands, significant differences in mean content were found with the exceptions of iron and copper. Cluster analysis and principal component analysis show differences in wines according to the island of origin and the ripening state of the grapes. Linear discriminant analysis using rubidium, sodium, manganese and strontium, the four most discriminant elements, gave 100% recognition ability and 95.6% prediction ability. The sensitivity and specificity obtained using soft independent modelling of class analogy (SIMCA) as a modelling multivariate technique were both 100% for El Hierro and Lanzarote, and 100 and 95%, respectively, for La Palma. The modelling and discriminant capacities of the different metals were also studied.     

Determination of six metals in galician red wines (in northwestern Spain) by capillary electrophoresis

A simple technique is described for the routine simultaneous capillary electrophoretic determination of 6 cations in wine. Separation was achieved on a fused silica capillary column with a UV-Cat-1, alpha-hydroxysobutyric acid and 18-crown-6-ether buffer at pH 4.5 and indirect UV detection at 214 nm. The content of magnesium, sodium, potassium, calcium, manganese, and lithium was determined. The method is quantitative, with recoveries in the 92-102% range, and linear over more than one order of magnitude. The precision is better than 2.5-3.4%. The method is sensible, with detection limits between 0.01 and 0.06 mg/L. Twenty-five red wines with a Certified Brand of Origin from Galicia (north-western Spain) were analyzed by the proposed method. Various wines showed very similar electrophoretic profiles, but significant quantitative differences were observed.     

Concentration of ochratoxin A in coffee products and probabilistic health risk assessment

Coffee is a beverage that people enjoy a lot in their daily lives and is an integral part of people's social life. In this study, the detection of ochratoxin A (OTA) in coffee was carried out by High-performance liquid chromatography with fluorescence (HPLC-FLD) detectors. Furthermore, the amount of ochratoxin A (OTA), Margin of exposure (MOEs), and Hazard quotient (HQ) in different types of coffees; instant, classic, and roasted coffee were calculated using Monte Carlo simulation (MCS) method. The average OTA concentration was in the rage of 3.6 to 26.6 µg/kg. The content of classic and instant coffee found to have.OTA, is below the maximum limit defined by the European Union legislation. The maximum limit for these two types of coffee is 10 µg/kg. The daily intakes of the OTA through classic and instant coffee were also found to be lower than the Tolerance daily intake proposed by Joint FAO/WHO Expert Committee on Food Additives (JECFA). MOEs (neoplastic effect) in adults was classic coffee (171026) > roasted coffee (15390) > instant coffee (8549) and also MOEs (non-neoplastic effects) in children was classic coffee (55790) > roasted coffee (5020) > instant coffee (2789). Consumers of instant coffee are at cancer risk based on neoplastic effects and also consumers of instant coffee and roasted coffee are at cancer risk based on non-neoplastic effects (MOEs lower than 10,000 value). HQ (nephrotoxic effect) in adults was instant coffee (0.132) > roasted coffee (0.097) > classic coffee (0.012). HQ due to consumption of coffee products was lower than 1, hence consumers are at safe non-cancer risk. Therefore, it is recommended reducing the concentration of mycotoxins in coffee products.     

Comparison of different immunoaffinity clean-up procedures for high-performance liquid chromatographic analysis of ochratoxin A in wines

Three immunoaffinity clean-up procedures to analyse ochratoxin A (OTA) in wines were compared. The direct wine clean-up with Ochraprep and OchraTest columns gave equivalent results in terms of recovery and precision if compared with the reference procedure involving a preliminary extraction of OTA with chloroform. OTA quantification limit in wine ranged from 0.020 to 0.045 microg/l. The 'on-flow' OTA emission spectrum (excitation 333 nm) showed a maximum at 460 nm and could be used to confirm the quantitative results. The analysis of 11 red and white wines gave no significant quantitative differences between the three clean-up techniques.     

Solid-phase extraction applied to the determination of ochratoxin A in wines by reversed-phase high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method is described for the analysis of Ochratoxin A at low microg l(-1) levels in samples of artificially contaminated wines. The method involves solid-phase extraction of samples using octadecylsilane cartridges and an additional preconcentration step prior to chromatography with isocratic elution and fluorimetric detection. The method was evaluated for accuracy and precision with relative standard deviations lower than 10%. Recoveries of ochratoxin A added to commercial wines over the range 0.1-3.0 microg l(-1) were higher than 80% in the assays. The performance of the octadecylsilane cartridge method tested compared very favourably with results of other published studies of ochratoxin A which use immunoaffinity columns or solvent extraction techniques.     

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.     

Validation of a liquid chromatography method for the simultaneous quantification of ochratoxin A and its analogues in red wines

A validated high-performance liquid chromatography (HPLC) method with fluorescence detection for the simultaneous quantification of ochratoxin A (OTA) and its analogues (ochratoxin B (OTB), ochratoxin C (OTC) and methyl ochratoxin A (MeOTA)) in red wine at trace levels is described. Before their analysis by HPLC-FLD, ochratoxins were extracted and purified with immunoaffinity columns from 50 mL of red wine at pH 7.2. Validation of the analytical method was based on the following parameters: selectivity, linearity, robustness, limits of detection and quantification, precision (within-day and between-day variability), recovery and stability. The limits of detection (LOD) in red wine were established at 0.16, 0.32, 0.27 and 0.17 ng L(-1) for OTA, OTB, MeOTA and OTC, respectively. The limit of quantification (LOQ) was established as 0.50 ng L(-1) for all of the ochratoxins. The LOD and LOQ obtained are the lowest found for OTA in the reference literature up to now. Recovery values were 93.5, 81.7, 76.0 and 73.4% for OTA, OTB, MeOTA and OTC, respectively. For the first time, this validated method permits the investigation of the co-occurrence of ochratoxins A, B, C and methyl ochratoxin A in 20 red wine samples from Spain.     

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.     

Metal content in southern Spain wines and their classification according to origin and ageing

Young and aged wines from two viticole zones in the Andalusian province of Córdoba (southern Spain) were analysed for their content in Ca, Mg, Fe, Cu, Mn and Zn by flame atomic absorption spectrophotometry, and Na, K, Al and Sr by flame atomic emission spectrophotometry. Significant differences in mean content were found for Na, Mn, Mg, Fe and Zn between wines from Montilla–Moriles and Villaviciosa. Linear discriminant analysis using those variables gave 97.9% recognition ability and 95.7% prediction ability. Cluster and principal component analysis show some differences in wines according to geographical origin and to the ageing of wines. Significant differences between young and aged wines were found in the mean content for Mg, K, Sr, Zn and Mn, obtaining 93.62% recognition ability and prediction ability by using linear discriminant analysis and leave-one-out cross-validation test, respectively. Finally, linear discriminant analysis could also be able to classify the samples according to their provenance and to their ageing simultaneously, obtaining 93.6% of the wines correctly classified.     

Determination of zearalenone and ochratoxin A in soil

Mycotoxins are secondary metabolites, formed by the action of fungi on agricultural crops in the field or during storage. These metabolites are highly toxic to animals and humans and high levels have been measured in agricultural crops. In order to evaluate human risks due to ingestion of mycotoxin-contaminated food different methods have been developed for analysis of mycotoxins in cereals and maize. In this project the focus was on mycotoxins in agricultural soil and the fate of these toxins in the soil-water-plant system. Two different mycotoxins were selected in the study: zearalenone (ZON) produced by species of Fusariumor Aspergillusand ochratoxin A (OTA) produced by species of Penicillium. We developed a method for analysis of these toxins in soil. Soil samples were extracted with methanol-water (9:1) and purified by solid-phase extraction (SPE, C8-columns). The final extract was analysed using high-pressure liquid chromatography (HPLC) with fluorescence detection. A Phenyl Hexyl column was used to separate the toxins. The detection limits obtained were 0.1 and 1.0 microg kg(-1) dry weight (dw) for OTA and ZON, respectively. The developed method has been used for analysis of different soils in connection with growth chamber experiments. The soil types used in the growth chamber experiments were a sandy soil, a sandy clay soil, and a soil with high content of organic matter. The recovery was determined as 85.8 and 93.4% and the repeatability to 5.1 and 12.8% for OTA and ZON, respectively. The reproducibility obtained was 8.5 and 15.0% for soil samples, representing concentration levels from 0.2-30 microg kg(-1) dw (OTA) and from 1.0-100 microg kg(-1) dw (ZON).     

Determination of ochratoxin A in beer marketed in Spain by liquid chromatography with fluorescence detection using lead hydroxyacetate as a clean-up agent

A new sample treatment for liquid chromatographic analysis of ochratoxin A (OTA) in beer is proposed. Degassed beer is mixed with lead hydroxyacetate, which precipitates some bulk components but does not remove OTA. The precipitate is separated and the acidified liquid is extracted with chloroform. The solvent is evaporated and the residue is dissolved in mobile phase (acetonitrile-water, 40:60, v/v; acidified at pH 3.0 with phosphoric acid) and separated by liquid chromatography using fluorescence detection. The limit of detection was 0.005 ng/ml. The average recovery rate and the average RSD of recovery in the spiking level range 0.01-0.5 ng/ml were 95.5% and about 5%, respectively. The method is cheaper that other alternative ones using immunoaffinity columns or other solid-phase extraction cleanup:The separation was optimised with regard to composition and flow of the mobile phase and no interference from the matrix was found. The method was applied to 88 samples of beer (domestic and imported) marketed in Spain. OTA was detected in 82.9% of them. The range for positive samples was 0.007-0.204 ng of OTA/ml.     

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.     

Particle size distribution of major inorganic species in atmospheric aerosols from Majorca (Spain)

Atmospheric aerosols collected by means of a cascade impaction system at the campus of the University of the Balearic Islands (Majorca, Spain) from November 1993 to February 1994 were analysed for chloride, nitrate, sulphate, ammonium, calcium, magnesium, sodium and potassium. Based on particle size distribution, the species studied were classified into three groups: (a) concentration decrease with particle size (sulphate and ammonium), (b) concentration increase with particle size (chloride, calcium, magnesium and sodium), and (c) independent of particle size (nitrate and potassium). A principal component analysis (PCA) revealed a clear relationship between particle size and analyte origin. Also, the origin of sulphate and potassium fine and coarse particles was found to be different.     

Photobiological characteristics and photosynthetic UV responses in two Ulva species (Chlorophyta) from southern Spain

The effect of different wavebands of artificial UV (UVB and UVA) and photosynthetically active radiation (PAR) was assessed in two species of the genus Ulva, U. olivascens and U. rotundata, from southern Spain in order to test for possible differences in acclimation of photosynthesis. Both species share similar morphology but are subject to different light environments: U. rotundata is an estuarine alga, inhabiting subtidal locations, while U. olivascens is an intertidal, sun-adapted organism. Algae were exposed to three different UV conditions, PAR+UVA+UVB, PAR+UVA and PAR for 7 d. Short-term exposure (6 h) was also carried out, using two PAR levels, 150 and 700 micromolm(-2)s(-1). Pigment contents and photosynthesis vs. irradiance curves from oxygen evolution were used to contrast sun- and shade adaptation between these species. O2-based net photosynthesis (Pmax) and PAM-chlorophyll fluorescence (optimal quantum yield, Fv/Fm) were used as parameters to evaluate photoinhibition of photosynthesis in the experiments. The results underline different photobiological characteristics among species: the subtidal U. rotundata had higher contents of pigments (Chl a, Chl b and carotenoids) than the sun-adapted U. olivascens, which resulted in higher thallus absorptance and P-I parameters characterized by higher photosynthetic efficiency at limiting irradiances (alpha) and lower saturating points for photosynthesis (Ek). After 7 d exposure, photoinhibition of Fv/Fm was close to 40-45% in both species. Differences between UV treatments were seen in U. rotundata after 5 d and after 7 d in U. olivascens, in which PAR+UVA impaired strongly photosynthesis (80%). Such patterns were correlated with a progressive decrease in pigment contents, specially chlorophylls. In short-term (6 h) exposures, combinations of UVA+UVB and high PAR level resulted in high rates of photoinhibition of chlorophyll fluorescence (68-92%) in U. rotundata, whereas in U. olivascens photoinhibition ranged between 42% and 53%. Photoinhibition under low PAR combined to UV radiation was lower than observed under high PAR. Net O2-Pmax revealed similar response among the species, with maximal photoinhibition rates close to 60% in algae incubated under high PAR+UVA+UVB. In the case of UV exposure in combination with low PAR, the highest photoinhibition rates were measured in U. rotundata.