Determination of aflatoxins B1, B2, G1, and G2 and ochratoxin A in ginseng and ginger by multitoxin immunoaffinity column cleanup and liquid chromatographic quantitation: collaborative study

The accuracy, repeatability, and reproducibility characteristics of a method using multitoxin immunoaffinity column cleanup with liquid chromatography (LC) for determination of aflatoxins (AF; sum of aflatoxins B1, B2, G1, and G2) and ochratoxin A (OTA) in powdered ginseng and ginger have been established in a collaborative study involving 13 laboratories from 7 countries. Blind duplicate samples of blank, spiked (AF and OTA added) at levels ranging from 0.25 to 16.0 microg/kg for AF and 0.25 to 8.0 microg/kg for OTA were analyzed. A naturally contaminated powdered ginger sample was also included. Test samples were extracted with methanol and 0.5% aqueous sodium hydrogen carbonate solution (700 + 300, v/v). The extract was centrifuged, diluted with phosphate buffer (PB), filtered, and applied to an immunoaffinity column containing antibodies specific for AF and OTA. After washing the column with water, the toxins were eluted from the column with methanol, and quantified by high-performance LC with fluorescence detection. Average recoveries of AF from ginseng and ginger ranged from 70 to 87% (at spiking levels ranging from 2 to 16 microg/kg), and of OTA, from 86 to 113% (at spiking levels ranging from 1 to 8 microg/kg). Relative standard deviations for within-laboratory repeatability (RSDr) ranged from 2.6 to 8.3% for AF, and from 2.5 to 10.7% for OTA. Relative standard deviations for between-laboratory reproducibility (RSDR) ranged from 5.7 to 28.6% for AF, and from 5.5 to 10.7% for OTA. HorRat values were < or = 2 for the multi-analytes in the 2 matrixes.     

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.     

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.     

Determination of fluoride in wine by fluoride selective ion electrode, standard addition method: collaborative study

The accuracy, precision, and reproducibility of a rapid method for determination of fluoride in wine, using a fluoride selective ion electrode, were established by a collaborative study involving 12 laboratories, 5 in Europe and 7 in the United States. The laboratories assayed 6 Youden pairs of fluoride-fortified, red and white wine samples with fluoride concentrations ranging from 0.2 to 3.0 mg/L. The relative standard deviations of repeatability ranged from 1.94 to 4.88%; relative standard deviations of reproducibility ranged from 4.15 to 18.40%. HORRAT values ranged from 0.30 to 0.97. The average recovery was 99.97%. Based on the statistical results of this collaborative study, the Study Director recommends that this method be adopted First Action.     

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.     

基于上转换荧光纳米粒子和金纳米粒子间荧光共振能量转移的高灵敏赭曲霉毒素A检测方法研究

制备了水溶性的上转换荧光纳米材料,在其表面修饰赭曲霉毒素A(OTA)适配体作为能量供体探针;在金纳米粒子表面修饰OTA适配体互补链作为能量受体探针,构建了OTA适配体传感器。在最优条件下,OTA的检测范围为0.001~10 ng/mL,检出限可达0.001 ng/mL。将其应用于啤酒样品中OTA的检测,当加标水平为0.01、0.1、1.0 ng/mL时,回收率为100%~119%,相对标准偏差为4.3%~4.9%,表明该方法可用于实际样品检测。该方法具有灵敏度高、特异性好、操作简单、成本较低等优点。     

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).     

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.     

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.     

Solid-phase extraction followed by liquid chromatography quadrupole time-of-flight tandem mass spectrometry for the selective determination of fungicides in wine samples

In this work, a reliable and selective procedure for the determination of thirteen fungicides in red and white wine samples is proposed. Solid-phase extraction (SPE) and liquid chromatography (LC) tandem mass spectrometry (MS/MS), based on a hybrid quadrupole time-of-flight (QTOF) system, were used as sample preparation and determination techniques, respectively. Extraction and purification of target analytes was carried out simultaneously by using a reversed-phase Oasis HLB (200mg) SPE cartridge combined with acetonitrile as elution solvent. Fungicides were determined operating the electrospray source in the positive ionization mode, with MS/MS conditions adjusted to obtain at least two intense product ions per compound, or registering two transitions per species when a single product was noticed. High selective MS/MS chromatograms were extracted using a mass window of 20 ppms for each product ion. Considering external calibration as quantification technique, the overall recoveries (accuracy) of the procedure ranged between 81% and 114% for red and white wine samples (10-20 mL), spiked at different concentrations between 5 and 100 ng mL(-1). Relative standard deviations of the above data stayed below 12% and the limits of quantification (LOQs) of the method, calculated for 10 mL of wine, varied between 0.1 ng mL(-1) for cyprodinil (CYP) and 0.7 ng mL(-1) for myclobutanil (MYC). The optimized method was applied to seventeen commercial wines produced in Spain and obtained from local supermarkets. Nine fungicides were determined, at levels above the LOQs of the method, in the above samples. The maximum concentrations and the highest occurrence frequencies corresponded to metalaxyl (MET) and iprovalicarb (IPR).     

Electrochemical Studies of Ochratoxin A Mycotoxin at Gold Electrodes Modified with Cysteamine Self‐Assembled Monolayers. Its Ultrasensitive Quantification in Red Wine Samples

The quantification of ochratoxin A is studied at cysteamine self‐assembled monolayer modified gold electrodes in red wine samples by square wave voltammetry. Detection and quantification limits of 0.004 µg L^?1 and 0.012 µg L^?1, respectively, were determined. The recovery percentages were in the range from 146 % to 94.0 % at spiking levels ranging from 0.02 to 5 µg L^?1. The variation coefficients for within‐laboratory repeatability varied from 31.4 to 11.5 % for spiked level from 0.02 to 2.0 µg L^?1. The developed electrochemical method is efficient, reproducible, and ultrasensitive for the quantification of OTA in red wine samples.     

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.     

New method for determination of ochratoxin A in beer using zinc acetate and solid-phase extraction silica cartridges

A new method for the determination of ochratoxin A (OTA) in beer has been developed. The new method has been compared with a reference method currently accepted as AOAC official first action. The limits of detection and quantification of the proposed method were 0.0008 and 0.0025 ng/ml, respectively, while they were 0.0025 and 0.0075 ng/ml, respectively, in the AOAC method used as reference. The recovery levels in the 0.025-0.40 ng OTA/ml spiking range for the proposed and the reference methods were 80.6-87.6% and 78.2-83.8%, respectively. The relative standard deviations of recoveries were 2.6-7.5% for the proposed method and 0.7-6.1% for the reference method. Passing and Bablok regression analysis of recovery data obtained by the proposed method versus data obtained by the reference method on an OTA-spiked beer sample showed good correlation (r2 = 0.9993), while the slope and intercept were 1.049 and -0.0013, respectively. The advantage of the proposed method is the low cost of the materials used in sample preparation because expensive immunoaffinity columns are not needed to clean-up samples while it maintains or even increases the good performance of the reference method. The proposed method was applied to 69 beer samples from different geographic origins (national and imported) but purchased in the Spanish market. They were found to be contaminated with OTA in the range from 0.008 to 0.498 ng/ml (average: 0.070 ng/ml). Five samples surpassed the limit recommended by the European Union (0.2 ng OTA/g).     

Dispersive liquid-liquid microextraction using non-chlorinated, lighter than water solvents for gas chromatography-mass spectrometry determination of fungicides in wine

A novel and low solvent consumption method for the sensitive determination of fungicide residues in wine samples is proposed. Analytes were extracted by dispersive liquid-liquid microextraction (DLLME) and further determined by gas chromatography-mass spectrometry (GC-MS). Under optimized conditions, a binary mixture of acetone and 1-undecanol (0.5 and 0.05 mL, respectively) was used to extract target compounds from diluted (1:1) wine samples. After centrifugation, the floating phase of 1-undecanol was solidified and separated from the liquid hydro-alcoholic matrix. Thereafter, it was allowed to melt at room temperature and injected in the GC-MS system. The method showed relative standard deviations (RSDs, %) below 13%, limits of quantification (LOQs) between 0.2 and 3.2 ng mL(-1) and linear responses for concentrations up to 300 ng mL(-1). The efficiency of the liquid-phase microextraction process was scarcely affected by the characteristics of wine samples, consequently pseudo-external standard calibration (using matrix matched standards of red and white wine) sufficed to achieve acceptable accuracy values: relative recoveries from 81 to 120%. The applicability of the method was demonstrated with commercial wine samples.     

Electrochemical Aptasensor for Zearalenone Based on DNA Assembly and Exonuclease III as Amplification Strategy

A sensitively electrochemical aptasensor was developed to detect zearalenone, utilizing DNA assembly based on hybridization chain reaction to amplify the signal current and exonuclease III to reduce the background current. The linear range 5.0×10⁻⁵ ng/mL-50 ng/mL, and the limit of detection is 0.013 pg/mL. The fabricated aptasensor showed the high specificity toward aflatoxin B1 (AFB1), fumonisin B1 (FB1) and ochratoxin A (OTA), good repeatability and reproducibility. In addition, the average recoveries of spiked corn and beer samples were in the range of 89 % to 102 %. The established method is of great significance in the field of food safety detection.     

Determination of ochratoxin A in domestic and imported beers in italy by immunoaffinity clean-up and liquid chromatography

A method first developed to quantify ochratoxin A in wine has been applied to the analysis of domestic and imported beers in Italy. The method uses commercial immunoaffinity columns for clean-up and high-performance liquid chromatography for quantification of the toxin. Beer was degassed, then diluted with a polyethylene glycol-sodium hydrogencarbonate solution and applied to an OchraTest immunoaffinity column. Ochratoxin A was eluted from the immunoaffinity column with methanol and quantified by reversed-phase HPLC with fluorometric detector. Average recoveries of ochratoxin A from blank beer spiked at levels from 0.04 to 1.0 ng/ml ranged from 93.8% to 100.4%, with relative standard deviations between 3.3% and 5.7%. The detection limit was 0.01 ng/ml based on a signal-to-noise ratio of 3:1. The analysis of 61 samples of domestic (10) and imported (51) beers showed ochratoxin A levels ranging from <0.01 to 0.135 ng/ml with an incidence of contamination of 50% and no substantial difference between strong and pale beers.     

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.     

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.     

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.