Analysis Of Ochratoxin Research Articles

Combination of homogeneous liquid–liquid extraction and vortex assisted dispersive liquid–liquid microextraction for the extraction and analysis of ochratoxin A in dried fruit samples: Central composite design optimization

This paper presents a new analytical procedure based on combination of homogeneous liquid–liquid extraction (HLLE) and vortex-assisted dispersive liquid–liquid microextraction (VA-DLLME) for the accurate and reliable determination of ochratoxin A (OTA) in dried fruit samples. To enable selective extraction of the OTA, six hydrophobic deep eutectic solvents (hDESs) were prepared and tested as extraction solvents. Optimization of DES volume, pH, NaCl amount, and mixing time affecting the efficiency of VA-DES-DLLME step was achieved by central composite design (CCD). Using optimized conditions, the working range was obtained in the range 0.4–350 ng mL−1 with an enrichment factor of 138. The limit of detection was 0.12 ng mL−1. To evaluate the accuracy of the method, the samples were analyzed with both the HLLE-VA-DES-DLLME procedure and reference method. The precision of the method was investigated by intraday/interday studies. The robustness of the method was also evaluated by making minor changes to the optimized conditions. The HLLE-VA-DES-DLLME procedure was successfully applied to dried fruit samples and quantitative recoveries were obtained (92.1–99.2%) confirming its usefulness for implementation in routine analysis of food samples.

Analysis of ochratoxin A, aflatoxin B1 and its biosynthetic precursors in cheese – Method development and market sample screening

The presence of Penicillium and Aspergillus fungi during cheese production may lead to the formation of ochratoxin A, aflatoxins, as well as their partly mutagenic biosynthetic precursors. However, data about the presence of these toxins in final cheese is scarce. In the present study, a quantification method for the toxicologically highly relevant compounds ochratoxin A (OTA), aflatoxin B1 (AFB1), sterigmatocystin (STC), and versicolorin A (VerA) was developed and applied for a market screening of hard, semi-hard, and soft cheese samples. The developed method was based on a QuEChERs approach with dimethylsulfoxide as keeper reagent. This stable isotope dilution assay for OTA, AFB1 and STC achieves high sensitivity (limits of detection: 0.02 μg/kg for AFB1, 0.01 μg/kg for STC and 0.37 μg/kg for OTA). However, in certain hard cheese types, the toxins showed matrix binding effects, leading to reduced recovery rates. When applying the method to market samples, detectable amount of STC in eight out of ten grated hard cheese samples up to 4.99 μg/kg and OTA in three of these samples up to 26.56 μg/kg were revealed. Only three out of the 53 further samples contained detectable amounts of mycotoxin. Thus, it can be assumed that the state of maturation and the process of grating favours the presence of OTA and STC in cheese, based on the market screening of 62 market samples. More comprehensive data are required to draw more detailed conclusions of the parameters determining the susceptibility of certain cheese types for mycotoxin contamination.

Ochratoxin a in breast milk in Morocco: the affecting dietary habits of the lactating mothers and the degree of exposure of newborns “CONTAMILK study”

The aim of the present study is to evaluate the level of contamination of breast milk (BM) by ochratoxin A, among Moroccan lactating mothers in the city of Rabat, and to identify the associated factors of exposure, also to estimate the degree of exposure of the breastfeed infant. The analysis of ochratoxin A (OTA) was accomplished by ELISA method on 82 colostrum samples. OTA was detectable (>0.08 ng/mL) in 55% of samples with a maximum concentration of 10.04 ng/mL, and the levels exceeded 0.5 ng /mL in 50 % of the samples. In addition, several factors and dietary habits affect significantly the level of OTA in the analyzed samples of breast milk including, the consumption of industrial dairy products, the frequency of consumption of canned foods, dried fruits and legumes, also the period of breast milk collection. Besides, OTA was higher than the tolerable daily intake for 49% newborns. However, these results need to be confirmed by multicenter studies to more broadly estimate the levels of exposition of Moroccan population to OTA. Furthermore, awareness campaigns are recommended to inform the public, especially pregnant women and lactating women about appropriate preventive measures to limit exposure to this mycotoxin.

Effect of Coffee Cascara Dietary Fiber on the Physicochemical, Nutritional and Sensory Properties of a Gluten-Free Bread Formulation.

This study aimed to assess the physicochemical, nutritional and sensory properties of gluten-free breads containing isolated coffee cascara dietary fiber (ICCDF) as a food ingredient. ICCDF was obtained by aqueous extraction. The oil and water holding capacity and the nutritional profile of the novel ingredient were determined. Its safety was certificated by analysis of ochratoxin A, caffeine and gluten. Gluten-free bread formulations were prepared enriching a commercial bakery premix in rice protein (8%) and ICCDF (3% and 4.5%). Nutritional profile of the novel gluten-free breads (dietary fiber, protein, amino acids, lipids, fatty acid profile and resistant starch), as well as bread volume, crumb density, moisture, firmness, elasticity and color intensity were determined. A sensory quantitative descriptive analysis of the breads was conducted using eight trained panelists. New breads showed significantly higher (p < 0.05) content of dietary fiber and protein than the control bread. The addition of ICCDF allowed increasing dough yield, a less crumb firmness and a higher crumb elasticity. The nutrition claims “source of protein and high in dietary fiber” were assigned to the new formulations. In conclusion, a certificated gluten-free bread with improved nutritional and physicochemical properties and good sensorial profile was obtained.

Analysis of Ochratoxin A in yoghurt samples with a quartz resonator

Analysis of Ochratoxin A in yoghurt samples with a quartz resonator

UPLC-MS/MS analysis of ochratoxin A metabolites produced by Caco-2 and HepG2 cells in a co-culture system

Ochatoxin A (OTA) is one of the most important mycotoxins based on its toxicity. The oral route is the main gateway of entry of OTA into the human body, and specialized epithelial cells constitute the first barrier. The present study investigated the in vitro cytotoxic effect of OTA (5, 15 and 45 μM) and production of OTA metabolities in Caco-2 and HepG2 cells using a co-culture Transwell System to mimic the passage through the intestinal epithelium and hepatic metabolism. The results derived from MTS cell viability assays and transepithelial electrical resistance measurements showed that OTA was slightly cytotoxic at the lowest concentration at 3 h, but significant toxicity was observed at all concentrations at 24 h. OTA metabolites generated in this co-culture were ochratoxin B (OTB), OTA methyl ester, OTA ethyl ester and the OTA glutathione conjugate (OTA-GSH). OTA methyl ester was the major metabolite found in both Caco-2 and HepG2 cells after all treatments. Our results showed that OTA can cause cell damage through several mechanisms and that the OTA exposure time is more important that the dosage in in vitro studies. OTA methyl ester is proposed as an OTA exposure biomarker, although future studies should be conducted.

Implementation of electrochemical elements for an alternative detection of ochratoxin A

Ochratoxin A (OTA) is a nephrotoxic metabolite, hepatotoxic and carcinogenic produced mainly by Aspergillus and Penicillium fungi. Usually, the mycotoxin analysed through the technique of high performance liquid chromatography. This method is expensive and takes a lot of time. Therefore, expected to automate a device of low cost, minimal instrumentation micropolarographic (MIMP) for the analysis. For this purpose, the right programming environment and the manufacturing of the software for the graphical user interface selected automation of the MIMP through the design and simulation of the circuit, imprint and assembled in a container with connection ports. Lastly, validation and analysis of Ochratoxin A by cyclic voltammetry from analogous MIMP, automatic MIMP and a potentiostat, in order to corroborate the registered data. This way, voltammetry analysis of coumarin is obtained and the simulation of the MIMP electric circuit. Therefore, this are the key data during the investigation, because the molecule of OTA can be monitored through an acid hydrolysis of amides, composed by a phenylalanine and coumaric acid group, generating the significant decrease in the costs and time of analysis.

High-performance liquid chromatography-tandem mass spectrometry method for simultaneous detection of ochratoxin A and relative metabolites in Aspergillus species and dried vine fruits

ABSTRACTA simple, sensitive and reliable quantification and identification method was developed for simultaneous analysis of ochratoxin A (OTA) and its related metabolites ochratoxin alpha (OTα), ochratoxin B (OTB) and mellein. The method was assessed by spiking analytes into blank culture media and dried vine fruits. Performance was tested in terms of accuracy, selectivity and repeatability. The method involves an ultrasonic extraction step for culture samples using methanol aqueous solution (7:3, v/v); the mycotoxin is quantified by high-performance liquid chromatography coupled with electrospray ionisation and triple quadrupole mass spectrometry (LC-ESI-MS/MS). The recoveries were 74.5–108.8%, with relative standard deviations (RSDs) of 0.4–8.4% for fungal culture. The limits of detection (LODs) were in the range of 0.03–0.87 μg l–1, and the limits of quantification (LOQs) ranged from 0.07 to 2.90 μg l–1. In addition, the extraction solutions and clean-up columns were optimised specifically for dried vine fruit samples to improve the performance of the method. Methanol–1% sodium bicarbonate extraction solution (6:4, v/v) was determined to be the most efficient. Solid-phase extraction (SPE) was performed as a clean-up step prior to HPLC-MS/MS analysis to reduce matrix effects. Recoveries ranged from 80.1% to 110.8%. RSDs ranged from 0.1% to 3.6%. LODs and LOQs ranged from 0.06 to 0.40 μg kg–1 and from 0.19 to 1.20 μg kg–1, respectively. The analytical method was established and used to identify and quantify OTA and related compounds from Aspergillus carbonarius and Aspergillus ochraceus in cultures and dried vine fruits. The results showed that A. carbonarius produced OTα, OTB and OTA, whereas A. ochraceus produced OTB, OTA and mellein after 7 days of cultivation. Of 30 commercial samples analysed, 10 were contaminated with ochratoxins; OTB, OTα and mellein were also detected in different samples.

Analysis of ochratoxin A in dried blood spots - Correlation between venous and finger-prick blood, the influence of hematocrit and spotted volume.

We report the improvement of a method for the detection of ochratoxin A (OTA) and its thermal degradation product 2'R-ochratoxin A in dried blood spots (DBS) by high performance liquid chromatographic (HPLC) tandem mass spectrometry (MS/MS). The DBS technique was advanced for the analysis of these two compounds in DBS with unknown amounts of blood as well as varying hematocrit values. Furthermore the comparability of venous vs. capillary blood was investigated. Human whole blood samples were spotted, dried, and extracted with a solvent consisting of acetone, acetonitrile and water for analysis by HPLC-MS/MS. Quantification was carried out by stable isotope labelled internal standards. Blood samples of volunteers (n=50) were used to further optimize and simplify the procedure. Ochratoxin A and 2'R-ochratoxin A concentrations found in the entire spots (approx. 100 μL blood) were compared with punched DBS discs of 8.8mm size containing approximately 20 μL blood. As a result the amounts of both toxins in a punched 8.8mm disc correlate well with the entire DBS. Also the use of capillary blood from finger-pricks versus venous blood was evaluated. The analyte levels correlate as well indicating that the less invasive finger-prick sampling gives also reliable results. The influence of hematocrit was investigated in a range of 25-55% according to the hematocrit in the used real blood samples (34-46% hematocrit). However no significant hematocrit effect was observed for the utilized real blood samples. Moreover different blood volumes were spotted and punched as a minimal spot size is usually recommended for accurate analysis. In this experiment finger-prick samples typically consist of about 90 μL blood. Therefore spots of 75, 100 and 125 μL blood were prepared and analyzed. Similar to the hematocrit effect, no considerable influence was observed.

Man-Made Synthetic Receptors for Capture and Analysis of Ochratoxin A.

Contemporary analytical methods have the sensitivity required for Ochratoxin A detection and quantification, but direct application of these methods on real samples can be rarely performed because of matrix complexity. Thus, efficient sample pre-treatment methods are needed. Recent years have seen the increasing use of artificial recognition systems as a viable alternative to natural receptors, because these materials seem to be particularly suitable for applications where selectivity for Ochratoxin A is essential. In this review, molecularly imprinted polymers, aptamers and tailor-made peptides for Ochratoxin A capture and analysis with particular attention to solid phase extraction applications will be discussed.

Development of a stir bar sorptive extraction method for analysis of ochratoxin A in beer.

A new, simple, and sensitive method was developed for extraction of ochratoxin A (OTA) in beer combined with HPLC-fluorescence detector. Samples were extracted by stir bar sorptive extraction (SBSE) followed by liquid desorption using commercially available Twister EG-Silicone. The main parameters influencing SBSE, including phase ratio, extraction time, stirring speed, ionic strength, organic modifier, pH, temperature, desorption mode, desorption solvent, and desorption time were optimized. Under the optimal conditions, assay was performed on 4 mL of samples acidified at pH 1.5. The samples were extracted for 180 min at a stirring speed of 800 rpm followed by desorption of analyte using 1 mL methanol under sonication for 45 min. The extract was evaporated at 50 degrees C under a gentle nitrogen stream, then redissolved in 200 microL of methanol-water (50 + 50, v/v). After each use, the stir bar was cleaned by sonication in methanol for 30 min three times. The method provided good linearity of the calibration curve with coefficients of determination greater than 0.999. Recoveries of OTA were greater than 83% with RSD lower than 10%, and LOD of OTA in beer was 0.64 ng/L. This method was applied to determine OTA in 19 beer samples. OTA was detected in 12 samples (63%) in the range of 0.01-0.27 ng/mL.

Analysis of ochratoxin A in grapes, musts and wines by LC–MS/MS: First comparison of stable isotope dilution assay and diastereomeric dilution assay methods

Ochratoxin A (OTA) exhibits potent nephrotoxic, carcinogenic and teratogenic effects and its maximum level in wines has been set to 2μgL−1 by regulation. Consequently, the analytical procedures for OTA determination in wines have to be both very sensitive and reliable. In this paper, we compared two quantification methods: the stable isotope dilution assay (SIDA) and the diastereomeric dilution assay (DIDA). For this purpose, non-natural analogues of OTA were synthesized: the labeled OTA (OTA-d4) as a diastereomeric mixture for the SIDA and one non-natural OTA’s diastereomer (OTA-dia) for the DIDA. To quantify OTA in red grapes, musts or wines, the sample preparation was optimized using immunoaffinity column extraction and the analysis was performed by LC–MS/MS in Multiple Reaction Monitoring mode. A validation procedure in agreement with the International Organization of Vine and Wine recommendations was conducted. It appeared that SIDA quantification exhibited excellent sensitivity (LOD<1ngL−1), accuracy (recovery=98%), repeatability (RSD<3%) and intermediate reproducibility (RSD<4%) compared to quantification by DIDA. Indeed, DIDA method did not provide satisfactory results demonstrating that immunoaffinity extraction is exclusively selective for the natural OTA and not for its diastereomer, which therefore cannot be considered as a good internal standard for this particular method.

Analysis of ochratoxin A in malt beverage samples using dispersive liquid-liquid microextraction coupled with liquid chromatography-fluorescence detection

A simple and economic procedure based on dispersive liquid&amp;ndash;liquid microextraction has been applied to extract and pre-concentrate trace levels of ochratoxin A (OTA) in malt beverage prior to analysis using high performance liquid chromatography with fluorescence detection. The method was based on the formation of fine droplets of a water-immiscible extraction solvent in the sample solution using a water-miscible disperser solvent. The influences of various parameters such as the type and volume of extraction and disperser solvents, centrifuging time, sonication time, and salt concentration on the extraction efficiency of ochratoxin A were investigated. Under optimum conditions, the relative standard deviations for five replicates of 2 ng/ml of OTA were 3.4% as within-day and 6.2% as between-day precisions. The detection limit (S/N = 3) was 0.1 ng/ml and the mean recoveries of OTA from malt beverage samples at spiking levels of 0.5, 2, and 4 ng/ml were in the range of 104&amp;ndash;108.2%.

Ochratoxin exposure and risk assessment to human health.

The consumption of foods contaminated with mycotoxins (particularly ochratoxin A) causes various hepatic and renal disorders, severe immunodeficiency, many other disorders and the presence of these mycotoxins or their metabolites in various fluids (such as serum, urine). In regard to the mycotoxicological analysis of ochratoxin A in urine we have established the specific work protocol and have used an ELISA Kit to determine ochratoxin A. The mycotoxicological analysis of ochratoxin A in urine have shown the presence of OTA in samples of urine with values between 51.88 ppt - 85.15 ppt (0.05 - 0.08 ppb). The values obtained in the sample analysis are relatively high, the case studies presented highlighting a correspondence between the disease's specific at the investigated patients, the results of the biochemical and hematological analysis, the nutritional analysis and the presence of OTA in the biological liquid analysed.

Analysis of Ochratoxin a Blood Levels in Bladder Cancer Cases and Healthy Persons from Pakistan

The mycotoxin ochratoxin A (OTA), a well-known human nephrotoxic and carcinogenic agent, is a public health concern in many countries. Exposure is assessed by means of mycotoxin analysis in food commodities and by human biomonitoring of OTA in blood samples. Data available from several European countries and some studies in Africa, Asia, and the Americas indicate frequent detection of OTA. Thus far, data from developing countries that compare blood levels in healthy and diseased individuals are scarce. Thus, the aim of this investigation was to determine OTA levels in blood samples of bladder cancer patients (n = 96) and healthy controls (n = 31) from Pakistan. OTA in blood plasma was analyzed after extraction by high-performance liquid chromatography (HPLC) with fluorescence detection. Among samples of 87 cancer patients and 30 controls, 92% in total contained quantifiable amounts of OTA. In bladder cancer cases the median OTA concentration was 0.19 ng/ml (mean 0.296; range: 0.03 to 3.41 ng/ml), and in healthy controls the median OTA was 0.19 ng/ml (mean 0.3; range: 0.04 to 1.24 ng/ml). The OTA levels found in the Pakistanian cohorts were comparable to those reported previously for the general population in the European Union. In conclusion, OTA is not likely to play a major role in the etiology of bladder cancer in the Karachi cohort, at least as the sole risk factor.

Analysis of Ochratoxin A in Wine by High-Resolution UHPLC-MS

A method for determination of ochratoxin A (OTA) in wines using a new-solid phase extraction clean-up procedure followed with ultra performance liquid chromatography (UHPLC)-Orbitrap MS based on two scan events (full-scan Fourier transform mass spectrometer [FTMS] and higher energy-induced collision dissociation[HCD] data-dependent MS/MS) in positive ionization mode has been developed. The limit of detection (LOD) was estimated at 0.46 μg l−1 for white wine, 0.53 and 0.54 μg l−1 for rose and red wines, respectively. The limit of quantification (LOQ) was estimated at 1.57 μg l−1 in white wine, 1.77 and 1.81 μg l−1 in rose and red wines. Recovery experiments were carried out with spiked samples at three concentration levels (2, 5 and 10 μg l−1). The OTA recoveries in spiked white wine samples varied from 69.6 % to 99.8 %, while the recoveries for rose and red wine samples were in the range of 63.0–110.2 % and 63.6–103.2 %, respectively. Finally, based on the results, it is concluded that the combination of C18 cartridge with conventional particle packed columns and UHPLC LTQ-Orbitrap XL is an appropriate procedure for OTA analysis in wines.

Determination of ochratoxin A in grains by immuno-ultrafiltration and HPLC-fluorescence detection after postcolumn derivatisation in an electrochemical cell

The paper presents a new sample clean-up method based on immuno-ultrafiltration for the analysis of ochratoxin A in cereals. In contrast to immunoaffinity chromatography, in immuno-ultrafiltration, the antibodies are used in non-immobilised form. Ochratoxin A was extracted with ACN/water (60/40, v/v), and the extract was loaded onto the ultrafiltration device. After a washing step with phosphate-buffered saline, containing 0.05% Tween 20, ochratoxin A was eluted with MeOH/acetic acid (99/1, v/v). The detection of ochratoxin A was carried out with high-performance liquid chromatography and a fluorescence detector coupled to an electrochemical cell (Coring cell). The electrochemical cell was used to eliminate matrix interferences by oxidising matrix compounds. The method was validated by repeatedly analysing spiked barley and rye samples as well as a certified wheat reference material. Recoveries and standard deviations (1 SD) were found to be 71 ± 9%, 77 ± 12% and 77 ± 8% in wheat, barley and rye, respectively. The limit of detection (S/N = 3) and limit of quantitation (S/N = 10) were determined to be 0.4 μg kg(-1) and 1 μg kg(-1). The analysis of the certified reference material resulted in ochratoxin A concentrations which were in the range assigned by the producer. Additionally, the effect of the electrochemical cell on other widely used clean-up techniques, namely the immunoaffinity clean-up and multifunctional columns (Mycosep #229), was evaluated. In all clean-up methods, an improvement of the chromatogram quality was registered.

Molecularly Imprinted Polymers for Ochratoxin A Extraction and Analysis

Molecularly imprinted polymers (MIPs) are considered as polymeric materials that mimic the functionality of antibodies. MIPs have been utilized for a wide variety of applications in chromatography, solid phase extraction, immunoassays, and sensor recognition. In this article, recent advances of MIPs for the extraction and analysis of ochratoxins are discussed. Selection of functional monomers to bind ochratoxin A (OTA) with high affinities, optimization of extraction procedures, and limitations of MIPs are compared from different reports. The most relevant examples in the literature are described to clearly show how useful these materials are. Strategies on MIP preparation and schemes of analytical methods are also reviewed in order to suggest the next step that would make better use of MIPs in the field of ochratoxin research. The review ends by outlining the remaining issues and impediments.

Influencing factors on bread-derived exposure to ochratoxin A: Type, origin and composition

The nearly ubiquitous consumption of cereals all over the world renders them an important position in international nutrition, but concurrently allocates exposure to possible contained contaminants. Mycotoxins are natural food contaminants, difficult to predict, evade, and reduce, so it is important to establish the real contribution of each contaminated food product, with the aim to evaluate mycotoxin exposure. This was the key objective of this survey and analysis for ochratoxin A content on 274 samples of commercialized bread in the Portuguese market, during the winter 2007. Different bread products were analyzed through an HPLC-FD method, including traditional types, novel segments, and different grain based bread products. A wide-ranging low level contamination was observed in all regions and types of bread products analyzed, especially in the Porto and Coimbra regions, and in the maize and whole-grain or fiber-enriched bread. Nevertheless, the exposure through contaminated wheat bread continues to be the most significant, given its high consumption and dominance in relation to the other types of bread.

Analysis of ochratoxin A and ochratoxin B in traditional Chinese medicines by ultra-high-performance liquid chromatography–tandem mass spectrometry using [ 13C 20]-ochratoxin A as an internal standard

The paper reported a reliable analytical method for simultaneous determination of ochratoxin A (OTA) and ochratoxin B (OTB) in traditional Chinese medicines (TCMs) by ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS). The development of the method and investigations on the matrix influence were described in particular. The matrix effects were thereby minimized by using a reliable internal standard and a simple sample pretreatment. The established method was further validated by determining the linearity ( R 2 ≥ 0.9990), average recovery (86.3–114.2%), sensitivity (limit of quantitation 0.03–0.19 ng mL −1) and precision (relative standard deviation ≤ 13.1%). It was shown to be a suitable method for simultaneous determination of OTA and OTB in various TCMs. Finally, a total of 51 TCMs widely used in China were screened for OTA and OTB with the proposed method. The results showed that only 4 samples were contaminated with ochratoxins at low levels, indicating that it was low risk of ochratoxins to consumers who occasionally used TCMs.