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.     

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.     

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.     

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.     

A high-performance liquid-chromatographic method for the determination of ochratoxin a in human plasma

Summary A high-performance liquid-chromatographic method is described for the quantitative determination of the mycotoxin ochratoxin A (OTA) in human plasma. The assay involves extraction with chloroform and sodium bicarbonate then HPLC with fluorescence detection. The method was validated in terms of selectivity, recovery, linearity, precision (within-day and between-day variability), accuracy, detection and quantification limits, and the stability of OTA in plasma and treated samples. The limit of detection was 0.4 ng mL⁻¹ of OTA in methanol, corresponding to 0.52 ng ml⁻¹ OTA in plasma. This assay was successfully applied for the determination of OTA levels in human plasma.     

Analysis of ochratoxins from buffered rumen fluid by reversed-phase high-performance liquid chromatography (HPLC)

Summary An efficient procedure for the extraction and analysis of ochratoxins A, B, C and α from buffered rumen fluid has been developed. The samples have been cleaned up byliquid-liquid extraction and the separation of chratoxins was by isocratic elution on a 5μm C₁₈ ODS-column which 0.083 M phosphoric acid/acetonitrile/isopropanol (55/35/10).     

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.     

Simultaneous determination of aflatoxins and ochratoxin A in baby foods and paprika by HPLC with fluorescence detection: a single-laboratory validation study

Mycotoxins are toxic secondary metabolites of fungal origin, the major mycotoxins of food concern are aflatoxins and ochratoxin A. Due to the wide range of matrices susceptible to mycotoxin contamination, the possible co-occurrence, and the very wide range of concentration, validated versatile multi-mycotoxin and multi-matrix methods are strongly requested. A reversed phase HPLC method for the simultaneous determination of aflatoxins and ochratoxin A in baby foods and paprika was set up. Three bulk samples were prepared according to commercial availability, one for paprika and for baby foods, two different bulks were set, a corn based and a multi-cereal based baby food. A single-laboratory validation was performed, for each investigated level ten analyses were performed, relative standard deviations of repeatability (RSD_r) and recovery factors were calculated; RSD_r values ranged from 2% to 10% for AFB₁ and from 3% to 10% for OTA, while the recovery factors ranged from 86% to 96% for AFB₁ and from 77% to 96% for OTA. The checked compliance of the RSD_r and recovery with the values reported in the current EU Regulations confirmed the fitting for purpose of the method. Limit of detection and LoQ values of the method were respectively 0.002 and 0.020 μg/kg for AFB₁ and 0.012 and 0.080 μg/kg for OTA in baby foods; and 0.002 and 0.200 μg/kg for AFB₁ and 0.012 and 0.660 μg/kg for OTA in paprika. The current method represents a good example of the possibility of a multi-mycotoxin and/or a multi-matrix analysis depending on the laboratory research or official control purposes.     

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.     

Simultaneous determination of four aflatoxins and ochratoxin A in ginger and related products by HPLC with fluorescence detection after immunoaffinity column clean‐up and postcolumn photochemical derivatization

Ginger, a widely used spice and traditional Chinese medicine, is prone to be contaminated by mycotoxins. A simple, sensitive, and reproducible method based on immunoaffinity column clean‐up coupled with HPLC and on‐line postcolumn photochemical derivatization with fluorescence detection was developed for the simultaneous determination of aflatoxins (AFs) B₁, B₂, G₁, G₂, and ochratoxin A (OTA) in 25 batches of gingers and related products marketed in China for the first time. The samples were first extracted by ultrasonication with methanol/water (80:20, v/v) and then cleaned up with immunoaffinity columns for analysis. Under the optimized conditions, the LODs and LOQs for the five mycotoxins were 0.03–0.3 and 0.1–0.9 μg/kg, respectively. The average recoveries ranged from 81.3–100.8% for AFs and from 88.6–99.5% for OTA at three spiking levels. Good linearity was observed for the analytes with correlation coefficients all >0.9995. All moldy gingers were contaminated with at least one kind of the five investigated mycotoxins, while none of them were found in normal gingers. Ginger powder samples were contaminated slightly with the contamination levels below the LOQs, while ginger tea bags were mainly contaminated by OTA at 1.05–1.19 μg/kg and ginger black tea bags were mainly contaminated by AFs at 3.37–5.76 μg/kg. All the contamination levels were below the legally allowable limits.     

Rapid analysis of aflatoxins B1, B2, and ochratoxin A in rice samples using dispersive liquid–liquid microextraction combined with HPLC

A novel, simple, and rapid method is presented for the analysis of aflatoxin B₁, aflatoxin B₂, and ochratoxin A in rice samples by dispersive liquid–liquid microextraction combined with LC and fluorescence detection. After extraction of the rice samples with a mixture of acetonitrile/water/acetic acid, mycotoxins were rapidly partitioned into a small volume of organic solvent (chloroform) by dispersive liquid–liquid microextraction. The three mycotoxins were simultaneously determined by LC with fluorescence detection after precolumn derivatization for aflatoxin B₁ and B₂. Parameters affecting both extraction and dispersive liquid–liquid microextraction procedures, including the extraction solvent, the type and volume of extractant, the volume of dispersive solvent, the addition of salt, the pH and the extraction time, were optimized. The optimized protocol provided an enrichment factor of approximately 1.25 and with detection of limits (0.06–0.5 μg/kg) below the maximum levels imposed by current regulations for aflatoxins and ochratoxin A. The mean recovery of three mycotoxins ranged from 82.9–112%, with a RSD less than 7.9% in all cases. The method was successfully applied to measure mycotoxins in commercial rice samples collected from local supermarkets in China.     

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.     

Determination of ochratoxin A and T-2 toxin in alcoholic beverages by hollow fiber liquid phase microextraction and ultra high-pressure liquid chromatography coupled to tandem mass spectrometry

A new method for the determination of ochratoxin A and T-2 toxin in alcoholic beverages (wine and beer) by hollow fiber liquid microextraction was optimized. The extraction step was followed by ultra high-pressure liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). The extraction procedure was based on the extraction of mycotoxins from the sample to the organic solvent (1-octanol) immobilized in the fiber, and afterwards, they were desorbed in a mixture of acetonitrile/water (80:20, v/v) at pH 7 prior to chromatographic determination. Different variables affecting the extraction process such as organic solvent, salt content, extraction time and desorption solution were studied. The developed method was validated in wine and beer, using white wine and alcoholic beer as representative matrices for both types of samples. Relative recoveries higher than 70% were obtained for the selected mycotoxins. Good linearity (R² > 0.993) was obtained and quantification limits (0.02-0.09 μg L⁻¹) below European regulatory levels were achieved. Repeatability, expressed as relative standard deviation, was always lower than 12%, whereas interday precision was lower than 21%. The proposed method was applied to the analysis of several types of wines and beers and ochratoxin A was detected in a rosé wine at 1.1 μg L⁻¹.     

Supramolecular solvent-based microextraction of ochratoxin A in raw wheat prior to liquid chromatography-fluorescence determination

A supramolecular solvent made up of reverse micelles of decanoic acid, dispersed in a continuous phase of THF:water, was proposed for the simple, fast and efficient microextraction of OTA in wheat prior to liquid chromatography-fluorescence determination. The method involved the stirring of 300 mg-wheat subsamples (particle size 50 μm) and 350 μL of supramolecular solvent for 15 min, subsequent centrifugation for 15 min and the direct quantitation of OTA in the extract, previous 5.7-fold dilution with ethanol/water/acetic acid (49.5/49.5/1), against solvent-based calibration curves. No clean-up of the extracts or solvent evaporation was needed. Interactions between the supramolecular solvent and major matrix components in the wheat (i.e. carbohydrates, lipids and proteins) were investigated. The reverse micelles in the extractant induced gluten flocculation but only in the coacervation region of lower analytical interest (i.e. at percentages of THF above 11%). The quantitation of OTA was interference-free. Representativity of the 300 mg-wheat subsamples was proved by analysing a reference material. OTA recoveries in wheat ranged between 84% and 95% and the precision of the method, expressed as relative standard deviation, was 2%. The quantitation limit of the method was 1.5 μg kg⁻¹ and was below the threshold limit established for OTA in raw cereals by EU directives (5.0 μg kg⁻¹). The method developed was validated by using a certified reference material and it was successfully applied to the determination of OTA in different wheat varieties from crops harvested in the South of Spain. OTA was not detected in any of the analysed samples. This method allows quick and simple microextraction of OTA with minimal solvent consumption, while delivering accurate and precise data.     

Isolation and determination of paspalitrem-type tremorgenic mycotoxins using liquid chromatography with diode-array detection

A liquid chromatographic (LC) method is described for the isolation and determination of the tremorgenic mycotoxins paxilline (Penicillium paxilli NRRL 6110), paspaline, paspalinine and paspalicine (Claviceps paspali). Following a Soxhlet extraction of a mould-contaminated matrix using chloroform, the crude extract was partitioned between hexane and 80% aqueous methanol. The latter fraction, containing the desired toxin(s), was evaporated to dryness, the residue dissolved in methylene chloride and the solution analysed by liquid chromatography using a Supelcosil LC-Si column eluted with methylene chloride-diethyl ether (9 + 1, v/v). A mixture containing standards of these compounds was similarly analysed. All toxins were detected using a UV diode-array detector. The generated UV spectra and chromatographic data of the standard toxins were stored in a computer as a library and used to identify these toxins in a crude mixture. The purity of the separated peaks and the amount of toxin in the crude mixture were also determined. The toxins were isolated by selectively collecting the eluted peaks using a programmable fraction collector equipped with a peak level sensor. Further confirmation of compound identity was achieved by mass spectrometry using the direct inlet probe method. In comparison with methods used previously to isolate these toxins, the present technique is fast and allows the acquisition of complete UV spectral information and chromatographic data and the isolation of multiple toxins in a single chromatographic operation.     

Development of the custom polymeric materials specific for aflatoxin B1 and ochratoxin A for application with the ToxiQuant T1 sensor tool

Two polymers were computationally designed with affinity to two of the most abundant mycotoxins aflatoxin B1 (AFB1) and ochratoxin A (OTA) for application in the ToxiQuant T1 System. The principle of quantification of AFB1 and OTA using the ToxiQuant T1 instrument comprised of a fluorimetric analysis of mycotoxins adsorbed on the polymer upon exposure to UV light. High affinity of the developed resins allowed the adsorption of both toxins as discrete bands on the top of the cartridge with detection limit as low as 1 ng quantity of mycotoxins.     

Optimization of Matrix Solid-Phase Dispersion method for simultaneous extraction of aflatoxins and OTA in cereals and its application to commercial samples

A method based on Matrix Solid-Phase Dispersion (MSPD) has been developed for the determination of 5 mycotoxins (ochratoxin A and aflatoxins B and G) in different cereals. Several dispersants, eluents and ratios were tested during the optimization of the process in order to obtain the best results. Finally, samples were blended with C₁₈ and the mycotoxins were extracted with acetonitrile. Regarding to matrix effects, the results clearly demonstrated the necessity to use a matrix-matched calibration to validate the method. Analyses were performed by liquid chromatography-triple quadrupole-tandem mass spectrometry (LC-QqQ-MS/MS). The recoveries of the extraction process ranged from 64% to 91% with relative standard deviation lower than 19% in all cases, when samples were fortified at two different concentrations levels. Limits of detection ranged from 0.3 ng g⁻¹ for aflatoxins to 0.8 ng g⁻¹ for OTA and the limits of quantification ranged from 1 ng g⁻¹ for aflatoxins to 2 ng g⁻¹ for OTA, which were below the limits of mycotoxins set by European Union in the matrices evaluated. Application of the method to the analysis of several samples purchased in local supermarkets revealed aflatoxins and OTA levels.     

Determination of ochratoxin A in wine by liquid chromatography tandem mass spectrometry after combined anion-exchange/reversed-phase clean-up

Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus ochraceus and Penicillium verrucosum. It has been found and analysed in several foods and feeds. Owing to its toxicity and occurrence in food and feed, the European Community has issued directives and some countries have their own regulations for OTA contents in food, feed and beverages. This work describes a method for the determination of OTA in mulled and red wine. It is based on combined anion exchange/reversed-phase clean-up and was analysed by liquid chromatography coupled with tandem mass spectrometry (multiple reaction monitoring). The method was validated with natural contaminated and spiked wine samples with OTA contents from 1.34 to 3.48 g kg^–1. Owing to its accuracy, good reproducibility and repeatability, this easy method is a good alternative to liquid chromatography–fluorescence detection methods.     

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%.