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.     

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.     

Molecularly imprinted polymer‐based solid phase clean‐up for analysis of ochratoxin A in beer,red wine,and grape juice

A simple, reliable, and low‐cost method based on molecularly imprinted polymer as a selective sorbent of SPE was proposed for the determination of ochratoxin A (OTA) in beer, red wine, and grape juice by HPLC coupled with fluorescence detection (HPLC‐FLD). Samples were diluted with water and cleaned up with an AFFINIMIP® SPE OTA column. After washing and eluting, the analyte was analyzed by HPLC‐FLD. Under the optimized conditions, LOD and LOQ for OTA were 0.025 and 0.08 ng/mL, respectively. The recoveries of OTA from beer, red wine, and grape spiked at 0.1, 2, and 5 ng/mL ranged from 91.6 to 101.7%. Furthermore, after a simple regenerated procedure, the molecularly imprinted polymer based SPE column could be reused at least 14 times to achieve more than 80% recoveries of OTA in real samples. The developed method was applied to the detection of 30 beer, red wine, and grape juice samples and only four samples were contaminated by OTA with levels below the legal limits.     

Determination of ochratoxin A in wine using liquid-phase microextraction combined with liquid chromatography with fluorescence detection

A liquid-liquid microextraction technique (LPME) has been applied to the extraction of ochratoxin A (OTA) from wine prior to its quantification by HPLC-fluorescence detection. OTA was extracted from wine, through 1-octanol immobilized in the pores of a porous hollow fiber, and introduced into 1-octanol inside the fiber. Recovery was 77%. The method was adequate for quantification of OTA in wine at levels within the range 0.25-10 ng/ml with a LOD of 0.2 ng/ml, and can be a simple and inexpensive alternative to the use of inmunoaffinity columns in order to quantify OTA levels in wine.     

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.     

Determination of glutathione content in grape juice and wine by high-performance liquid chromatography with fluorescence detection

A modified preparation of sample was developed for the determination of glutathione content in grape juice and wine by high-performance liquid chromatography with fluorescence detection, using on-line pre-column derivatization. Ice-cold deoxygenated methanol was used to deactivate the oxidation enzymes in juices or wines and keep the glutathione stable. The optimum recovery of glutathione content in grape juice and wine was obtained when either the sample of grape juice or wine was mixed in ice-cold deoxygenated methanol in the ratio 10:90 (v:v) and further diluted in sodium acetate buffer in the ratio 1:1 (v:v). The optimized method was validated for linearity, limit of detection, limit of quantification, precision and uncertainty. According to the validation data the method is appropriate for the determination of glutathione content in grape juice and wine. Glutathione contents in grape juices made from White Muscat grapes and Sauvignon Blanc wines were analysed. The average glutathione content in 28 young Sauvignon Blanc wines was 12.5 mg L⁻¹.     

A modified HPLC method for the determination of ochratoxin A by fluorescence detection

A high-performance liquid chromatographic method (HPLC) with fluorescent detector is described for the determination of ochratoxin A (OTA). A mobile phase consisting of acetonitrile:water:acetic acid (99:99:2, v/v/v) was used for the resolution of the compound on a C(18) Hypersil column. The retention time for OTA and diflunisal which was used as an internal standard (IS) were 11.7 and 12.8 min, respectively. The method is selective, reliable, reproducable with relative standard deviation (RSD) of 1.70 and linear in the range of 2.5 x 10(-9)-1.5 x 10(-8) M OTA. The limit of detection (LOD) and limit of quantification (LOQ) were 2.5 x 10(-10) M corresponding to 0.1 ng mL(-1) and 8.2 x 10(-10) corresponding to 3.3 ng mL(-1), respectively. Recovery studies were 81.2 +/- 1.9 (SD). The method was applied for analysis of OTA in wheat, corn, red pepper, cheese and wine. The proposed method can be used for the routine analysis of OTA in food and animal feed.     

Comparison of volatile constituents extracted from model grape juice and model wine by stir bar sorptive extraction-gas chromatography-mass spectrometry

A stir bar sorptive extraction (SBSE) method coupled with gas chromatography-mass spectrometry was optimised for the analysis of volatile components of a model wine, based on a previously optimised method used for analysis of the same components in model grape juice. The presence of ethanol in the model wine sample matrix resulted in decreased sensitivity of the method toward most of the volatile constituents. Mean percent relative recoveries and reproducibilities (%CV) were 22.8% and 7.1%, respectively, compared with 28.4% and 8.5% for model grape juice. The mean limit of detection (LoD) ratio (juice:wine) was 0.25. Similar sensitivities for the two sample matrices using this method were achieved by changing the split ratio from 20:1 (grape juice) to 5:1 (wine), giving a mean limit of detection ratio (juice:wine) of 1.0, thus allowing direct comparison of chromatograms of volatile components in the two matrices. This enabled direct comparisons of grape juices and the wines derived from them by alcoholic yeast fermentation. The influence of ethanol concentration in the range 9-15% on method sensitivity is discussed, using an overlay of the total ion chromatograms. The use of a gas saver device for the 5:1 split ratio analysis of desorbed model wine aroma compounds is discussed in terms of preventing extraneous reaction of sorbent and stationary phases with air during analysis.     

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.     

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.     

Rapid determination of ochratoxin A in cereals and cereal products by liquid chromatography

A new method based on extraction with octylsilica (C8) followed by liquid chromatography coupled with fluorescence detection (LC-FLD) was studied to determine ochratoxin A (OTA) from cereals and cereal products. Optimization of different parameters, such as type and amount of solid phase, type and volume of eluent and amount of sample were carried out. Recovery of OTA from rice samples spiked at 10 ng/g level was of 86% with relative standard deviation of 5%. The limits of detection and quantification of the proposed method were 0.25 and 0.75 ng/g, respectively. Furthermore, LC-FLD after of OTA methylation and liquid chromatography coupled to mass spectrometry with an electrospray interface were used for confirmation of OTA in all studied samples. The proposed method was applied to 62 samples of cereals and cereal products and the presence of OTA was found in seven samples.     

Comparative study of screening methodologies for ochratoxin A detection in winery by-products

The worldwide contamination of winery by-products by mycotoxins may present a serious hazard to human and animal health. Mycotoxins are secondary metabolites of fungi with possible adverse effects on humans, animals, and crops that result in illnesses and economic losses. Mycotoxins are under continuous survey in Europe, but the regulatory aspects still need to be set up for winery by-products, which may be used in animal feed. The aim of this study was to implement a simple but reliable analytical methodology for ochratoxin A (OTA) quantification in grape pomaces in order to perform a survey of samples from the Douro Demarcated Region, Portugal. The method involved a unique preparation step, solvent extraction, followed by high-performance liquid chromatography (HPLC) with fluorescence (FL) detection. A comparative study was performed with two extraction solvents (ethyl acetate and methanol) as well as using extraction on an immunoaffinity column. The linearity range for OTA analysis was 0.05–23.5 μg L⁻¹ with a detection limit of 0.05 μg L⁻¹ and a precision (expressed by the coefficient of variation under repeatability conditions) of 0.4–14.7%. The percentage of recovery was on average 23.5 ± 3.6% (extraction with ethyl acetate) or 70.1 ± 2.5% (extraction with 70% methanol). Accounting for the recovery factor and the chromatographic detection limit, as well as the preconcentration factor, the limit of detection in grape pomaces is 0.04 μg kg⁻¹ (ethyl acetate extraction) and 0.33 μg kg⁻¹ (methanol extraction). Samples from 12 out of 13 sites in the Douro Demarcated Region showed OTA presence with concentrations not exceeding 0.4 μg kg⁻¹. Both developed methods for evaluation of OTA in grape pomace are simple but efficient. Figure Extraction of ochratoxin A (OTA) from grape pomaces allows simple but efficient quantification of OTA in winery by-products by HPLC-FL     

Solid-phase extraction of ochratoxin A from wine based on a binding hexapeptide prepared by combinatorial synthesis

In this paper we describe the preparation of a hexapeptide library by combinatorial synthesis and the identification of a peptide with sequence Ser-Asn-Leu-His-Pro-Lys, which showed good affinity (K(eq)=3.4 x 10(4) M(-1)) towards the mycotoxin ochratoxin A (OTA). An immunoaffinity-like stationary phase supporting such a hexapeptide was used to develop a solid-phase extraction method for the quantification of OTA in wine samples at concentration levels down to 0.10 microg l(-1). Several different wine samples fortified with OTA at 2 and 4 microg l(-1) levels showed recovery of 94.7% and 98.4% at 2.0 and 4.0 microg l(-1), respectively, without any effect on the extraction efficiency of the matrix. The efficacy of this approach was successfully tested by comparison with an immunoaffinity extraction performed on commercial cartridges.     

超高效液相色谱-电喷雾串联质谱法直接测定黄酒和葡萄酒中氨基甲酸乙酯

建立了超高效液相色谱-电喷雾串联质谱(UPLC-ESI-MS/MS)直接测定黄酒和葡萄酒中氨基甲酸乙酯含量的方法。黄酒和葡萄酒样品经蒸馏水简单稀释后,过0.22 μm微孔滤膜,直接进行UPLC-MS/MS分析检测。以Waters Acquity UPLCTMBEH C18色谱柱为分析柱,乙腈和0.1%(v/v)乙酸水溶液为流动相,采用电喷雾正离子(ESI+)模式电离,多反应监测(MRM)模式检测,以氨基甲酸丁酯(BC)作为内标进行定量。结果表明: 方法在2～500 μg/L的范围内线性关系良好(相关系数大于0.995),其对黄酒和葡萄酒的检出限为1.7 μg/L,定量限为5.0 μg/L,可达到黄酒和葡萄酒中氨基甲酸乙酯的检测要求。当添加水平为10、20和100 μg/L时,黄酒和葡萄酒中待测组分的回收率为90%～102%,日内精密度(n=6)为0.8%～4.5%,日间精密度(n=6)为1.4%～5.6%。该方法样品处理简单,前处理过程不使用有机溶剂,测定快速、准确,灵敏度高,非常适合黄酒和葡萄酒中氨基甲酸乙酯的快速检测和定量分析。     

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.     

Sol-gel immunoaffinity chromatography for the clean up of ochratoxin A contaminated grains

This paper describes the application of sol-gel immunoaffinity columns for clean up of ochratoxin A contaminated cereal crops. Monoclonal antibodies selective for OTA have been entrapped into the pores of a sol-gel matrix in order to prepare immunoaffinity columns. Different parameters such as amount of entrapped antibodies and loading conditions were optimized to obtain highest possible recoveries of OTA. The method has been found to be a suitable tool in sample preparation prior to HPLC-FLD determination and as selective as conventional commercially available immunoaffinity columns. In the clean up of different cereals mean recoveries of 82±5%, 90±6% and 91±3%, were obtained for wheat, barley and rye, respectively, with sol-gel columns containing 1mg of anti-OTA antibodies. The detection limit (signal-to-noise ratio, 3) was 0.5 μg/kg and the limit of quantification (signal-to-noise ratio, 10) determined to be 1 μg/kg. Sol-gel columns can be reused 7 times without significant loss of recovery. After 10 applications the recovery decreased to approx. 50%.     

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).     

Modified magnetic nanoparticles in an electrochemical method for the ochratoxin A determination in Vitis vinifera red grapes tissues

This work described the development and characterization of an electrochemical method using square wave voltammetry (SWV) combined with the use of modified magnetic nanoparticles (MNPs), which had shown a rapid and sensitive determination of ochratoxin A (OTA) in wine grapes (Cabernet Sauvignon, Malbec and Syrah) post-harvest tissues. The wine grapes were inoculated with Aspergillus ochraceus to obtain OTA in artificially infected samples. The OTA was directly determined using square-wave voltammetry. The current obtained is directly proportional to the concentration of OTA present in the samples. This method has been used for OTA determination in wine grapes and it was validated against a commercial ELISA test kit. The limits of detection calculated for electrochemical detection and the ELISA were 0.02 and 1.9 μg kg⁻¹, respectively and the coefficients of variation for accuracy and precision dates were below 5.5%. This method promises to be suitable for the detection and quantification of OTA in apparently healthy fruits post-harvest for assuring safety and quality of food as well as consumer's health.     

Determination of Ochratoxin A in wine at sub ng/mL levels by solid-phase microextraction coupled to liquid chromatography with fluorescence detection

Solid-phase microextraction (SPME), using a polydimethylsiloxane/divinylbenzene (PDMS/DVB) fiber, interfaced with liquid chromatography-fluorescence detection (LC-FD) has been applied to the determination of Ochratoxin A (OTA) in wine samples. Compared to the most widely adopted extraction/clean-up procedure based on immunoaffinity columns (IAC), the solventless extraction is simpler and cost-effective, requiring the simple immersion of the fiber in diluted wine samples. Furthermore, a fast LC separation is achieved under isocratic conditions. The linear range investigated in wine was 0.25-8 ng/mL; at fortification levels of 0.5 and 2 ng/mL, within-day intra-laboratory precision (repeatability) values, expressed as RSD%, were 5.9 and 5.1, respectively, whereas between days (n = 4) precision was 8.5 and 7.1%, respectively. The limit of detection (LOD) at a signal-to-noise (S/N) ratio of 3 was 0.07 ng/mL; the limit of quantification (LOQ) calculated at S/N = 10 was 0.22 ng/mL, well below the European regulatory level of 2 ng/mL. The potential of the method has been demonstrated by the analysis of a number of different wine samples.     

Simple and Fast Picomolar Detection of Ochratoxin A Using a Reusable Label Free Aptasensor Built with a Layer‐by‐layer Procedure

A label free impedimetric aptasensor for simple, fast and reusable picomolar detections of Ochratoxin A (OTA) in grape juices was designed. Two main factors were observed to affect the accurate detections of the toxin: i‐lateral interactions between self‐assembled aptamers ii‐ adsorption of large molecules present in complex matrixes like grape juices. Lateral interactions between aptamers were minimized by constructing the aptasensor in a Layer‐by‐Layer procedure. The interferences associated to the unspecific and irreversible adsorption of large molecules present in grape juice, were reduced by submitting samples to ultrafiltration prior to analysis. With this protocol, a 0.12 pM limit of detection and 0.24 pM limit of quantification in spiked grape juices were achieved after only 5–7 mins of interaction with the samples. The presented aptasensor can be recovered after a simple immersion in hot water (90 °C) for ten minutes.