Determination of ochratoxin A in Portuguese rice samples by high performance liquid chromatography with fluorescence detection

A sensitive and accurate analytical method for the determination of ochratoxin A (OTA) in rice, based on extraction with phosphate-buffered saline/methanol, an immunoaffinity column (IAC) for clean-up, and high performance liquid chromatography with fluorescence detection (HPLC-FD), is described. The limit of quantification of the proposed method was 0.05 g kg^–1. Recovery of OTA from rice samples spiked at 0.05 g kg^–1 was 92%, with a within-day RSD of 5.4%. The proposed method was applied to 42 rice samples from Portugal and the presence of OTA was found in six samples at concentrations ranging from 0.09 to 3.52 g kg^–1. The identification of OTA was confirmed by methyl ester derivatization and then HPLC analysis. The daily intake of OTA by the Portuguese population was also estimated.     

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.     

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.     

核酸适配体识别-荧光法检测赭曲霉素A

建立了核酸适配体识别-荧光探针技术检测赭曲霉素A(OTA)的新方法.基于微孔板上固定的核酸适配子与目标物质OTA结合时构象发生变化,导致预先与其互补杂交的FAM标记短链DNA解离,引起荧光信号发生变化,据此可实现对OTA的定量检测.当微孔板包被亲和素浓度为25 mg/L、适配子浓度为50 nmol/L,FAM标记互补短...     

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.     

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.     

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.     

Quantification of ochratoxin A in foods by a stable isotope dilution assay using high-performance liquid chromatography-tandem mass spectrometry

A stable isotope dilution assay (SIDA) was developed for quantification of the mycotoxin ochratoxin A (OTA) by using [2H5]-OTA as internal standard. The synthesis of labelled OTA was accomplished by acid hydrolysis of unlabelled OTA and subsequent coupling one of the products, ochratoxin alpha, to [2H5]-L-phenylalanine. The mycotoxin was quantified in foods by LC-tandem MS after extraction with buffers containing [2H5]-OTA and clean-up by immuno affinity chromatography or by solid phase extraction on silica. The method showed a sufficient sensitivity with a low detection and quantification limit of 0.5 and 1.4 microg/kg, respectively, and good precision in inter-assay studies showing a CV (n = 3) of 3.6%. The analysis of certified reference materials resulted in a low bias of 2.1% from the certified values and revealed excellent accuracy of the new method. To prove the suitability of SIDA. OTA was quantified in a number of food samples and resulted mainly in not detectable OTA contents. However, three samples of raisins exceeded the legal limit of 10 microg/kg and highlighted the need for further controlling the contamination with the mycotoxin.     

液相色谱-串联质谱法测定宠物食品中赭曲霉毒素A和B

建立了同时测定宠物食品中赭曲霉毒素A和B的液相色谱-串联质谱分析方法。样品经乙腈/水(1∶1,V/V)提取,HLB固相萃取柱净化。采用Agilent ZOBRAX C_(18)柱(150×2.1mm,5μm)分离,以0.1%甲酸水溶液-乙腈作为流动相,梯度洗脱。目标化合物在多反应监测模式(MRM)下进行检测,外标法定量。在优化的条件下,赭曲霉毒素A和B在0.1~10.0ng·mL~(-1)范围内呈良好的线性关系,相关系数均不低于0.9993,方法定量限分别为0.1μg·kg~(-1)和0.05μg·kg~(-1)。方法平均回收率为78.3%~107.5%,相对标准偏差不大于9.5%。该方法前处理简单、选择性好、灵敏度高,可用于宠物食品中赭曲霉毒素A和B的测定。     

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.