Determination of ochratoxin A in currants, raisins, sultanas, mixed dried fruit, and dried figs by immunoaffinity column cleanup with liquid chromatography: interlaboratory study

An interlaboratory study was performed on behalf of the Food Standards Agency to evaluate the effectiveness of an affinity column cleanup liquid chromatographic (LC) method for the determination of ochratoxin A in a variety of dried fruit at European regulatory limits. To ensure homogeneity before analysis, laboratory samples are normally slurried with water in the ratio of 5 parts fruit to 4 parts water, and test materials in this form were used in the study. The test portion was extracted with acidified methanol. The extract was filtered, diluted with phosphate-buffered saline, and applied to an affinity column. The column was washed and ochratoxin A was eluted with methanol. Ochratoxin A was quantified by reversed-phase LC. The use of post-column pH shift to enhance the fluorescence of ochratoxin A by the addition of 1.1 M ammonia solution to the column eluant is optional. Determination was by fluorescence. Currants, sultanas, raisins, figs, and mixed fruit (comprising dried pineapple, papaya, sultanas, prunes, dates, and banana chips), both naturally contaminated and blank (very low level), were sent to 24 collaborators in 7 European countries. Participants were asked to spike test portions of all test samples at a level equivalent to 5 ng/g ochratoxin A. Average recoveries ranged from 69 to 74%. Based on results for 5 naturally contaminated test samples (blind duplicates) the relative standard deviation for repeatability (RSDr) ranged from 4.9 to 8.7%, and the relative standard deviation for reproducibility (RSDR) ranged from 14 to 28%. The method showed acceptable within- and between-laboratory precision for all 5 matrixes, as evidenced by HORRAT values <1.3.     

Method for analysis dried vine fruits contaminated with ochratoxin A

The EU maximum limit of 10 microg kg(-1) of OTA for dried vine fruits has been established since 2002 (European Commission, 2005). The presented work explore the capability of using Fourier infrared spectroscopy attenuated total reflection (FTIR-ATR) for the detection of ochratoxin A (OTA) in dried vine fruits in a range of concentration between 2 and 50 microg kg(-1) OTA. The method developed included a sample pretreatment using a C18 cartridge which was efficient for the isolation of the mycotoxin. The PLS1 analysis of the spectrum of sultanas spiked with different OTA concentrations showed a good correlation between the spectral data and reference concentration for OTA (R(2)=0.85).     

Radiation processing as a post-harvest quarantine control for raisins,dried figs and dried apricots

The commercially packed samples of raisins, dried figs and dried apricots were irradiated using doses in the range of 0.5–1.0 kGy for disinfestation and 0.5–5.0 kGy for sensory analysis with the dose rate ranging from 1.44 to 1.92 kGy/h. Pests on dried fruits were evaluated after 0, 1, 2 and 3 months of storage for irradiated dried figs and 1, 3, 6 and 12 months of storage for raisins and dried apricots. Sensory analysis of dried figs, dried apricots and raisins were carried out after 0, 1, 3, 6 and 12 months of storage. The results indicated that radiation processing at low doses, (∼1.0 kGy) is an effective post-harvest treatment and quarantine control for these products with no adverse effects on sensory (marketing) attributes.     

Rapid analysis of fungal cultures and dried figs for secondary metabolites by LC/TOF-MS

A liquid chromatography-time-of-flight mass spectrometry (LC/TOF-MS) method has been developed for profiling fungal metabolites. The performance of the procedure in terms of mass accuracy, selectivity (specificity) and repeatability was established by spiking aflatoxins, ochratoxins, trichothecenes and other metabolites into blank growth media. After extracting, and carrying out LC/TOF-MS analysis, the standards were correctly identified by searching a specially constructed database of 465 secondary metabolites. To demonstrate the viability of this approach 11 toxigenic and four non-toxigenic fungi from reference collections were grown on various media, for 7-14 days. The method was also applied to two toxigenic fungi, A. flavus (200-138) and A. parasiticus (2999-465) grown on gamma radiation sterilised dried figs, for 7-14 days. The fungal hyphae plus a portion of growth media or portions of dried figs were solvent extracted and analysed by LC/TOF-MS using a rapid resolution microbore LC column. Data processing based on cluster analysis, showed that electrospray ionization (ESI)-TOF-MS could be used to unequivocally identify metabolites in crude extracts. Using the elemental metabolite database, it was demonstrated that from culture collection isolates, anticipated metabolites. The speed and simplicity of the method has meant that levels of these metabolites could be monitored daily in sterilised figs. Over a 14-day period, levels of aflatoxins and kojic acid maximised at 5-6 days, whilst levels of 5-methoxysterigmatocystin remained relatively constant. In addition to the known metabolites expected to be produced by these fungi, roquefortine A, fumagillin, fumigaclavine B, malformins (peptides), aspergillic acid, nigragillin, terrein, terrestric acid and penicillic acid were also identified.     

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.     

Synthesis of analogues of ochratoxin A

Four analogues of ochratoxin A (OTA) differing for the aminoacidic moiety were synthesised using ochratoxin α (OTα) as the starting material. The condensation reaction between protected amino acids and OTα, carried out in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC???HCl) and N-hydroxybenzotriazole (HOBt) as coupling agents, followed by deprotection and PTLC purification afforded OTA alanine, leucine, serine and tryptophane analogues in satisfactory yields (33-47%, based on OTα).     

Testing green coffee for ochratoxin A, part III: performance of ochratoxin A sampling plan

Green coffee shipments are often inspected for ochratoxin A (OTA) and classified into good or bad categories depending on whether the OTA estimates are above or below a defined regulatory limit. Because of the uncertainty associated with the sampling, sample preparation, and analytical steps of an OTA test procedure, some shipments of green coffee will be misclassified. The misclassification of lots leads to some good lots being rejected (sellers' risk) and some bad lots being accepted (buyers' risk) by an OTA sampling plan. Reducing the uncertainty of an OTA test procedure and using an accept/reject limit less than the regulatory limit can reduce the magnitude of one or both risks. The uncertainty of the OTA test procedure is most effectively reduced by increasing sample size (or increasing the number of samples analyzed), because the sampling step is the largest source of uncertainty in the OTA test procedure. The effects of increasing sample size and changing the sample accept/reject limit relative to the regulatory limit on the performance of OTA sampling plans for green coffee were investigated. For a given accept/reject limit of 5 microg/kg, increasing sample size increased the percentage of lots accepted at concentrations below the regulatory limit and increased the percentage of lots rejected at concentrations above the regulatory limit. As a result, increasing sample size reduced both the number of good lots rejected (sellers' risk) and the number of bad lots accepted (buyers' risk). For a given sample size (1 kg), decreasing the sample accept/reject limit from 5 to 2 microg/kg relative to a fixed regulatory limit of 5 microg/kg decreased the percentage of lots accepted and increased the percentage of lots rejected at all OTA concentrations. As a result, decreasing the accept/reject limit below the regulatory limit increased the number of good lots rejected (sellers' risk), but decreased the number of bad lots accepted (buyers' risk).     

Quantitative determination of ochratoxin A in vegetable foods

Summary A simple method for the quantitative determination of ochratoxin A (OA) in rice and other vegetable foods (oatmeal, coconut flakes and peas) is described. This procedure implies an acetonitrile-4% KCl -6N HCl (88+10+2) extraction of the acidic OA, subsequent twodimensional thin-layer chromatography (TLC) and detection by fluorescence after exposure to ammonia fumes (excitation at 340 nm; emission at 475 nm). The quantitative detection limit for OA in rice or coconut flakes is 2.4–4 g/kg and the recovery is 96%. For oatmeal and peas the detection limit is only 20 g/kg because of the interference by other metabolites.

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Quantitative Bestimmung von Ochratoxin A in pflanzlichen Nahrungsmitteln

Zusammenfassung Eine einfache Methode zur quantitativen Bestimmung von Ochratoxin A (OA) in Reis und anderen pflanzlichen Nahrungsmitteln (Haferflocken, Kokosflocken, und Erbsen) wird beschrieben. Zur Extraktion wird Acetonitril-4% KCl -6N HCl (88+10+2) benutzt. Nach der Aufbereitung wird eine Auftrennung durch zweidimensionale DC-Trennung (TLC) vorgenommen. Die quantitative Bestimmung erfolgt durch Fluorescenzphotometrie nach Behandlung der Platten mit gasförmigem Ammoniak (Anregung bei 340 nm, Emission bei 475 nm). Die quantitative Bestimmungsgrenze in Reis oder Kokosflocken beträgt 2,4–4 g/kg und die Wiederfindung (Reis) 96%. Für Haferflocken und Erbsen wurde wegen des Störeffektes durch andere Metaboliten nur eine quantitative Bestimmungsgrenze von 20 g/kg erreicht.

     

Structure-activity relationships for the fluorescence of ochratoxin A: insight for detection of ochratoxin A metabolites

Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus and Penicillium that is widely found as a contaminant of food products. The toxin is a renal carcinogen in male rats, the cause of mycotoxicoses in pigs and has been associated with chronic human kidney diseases. Bioactivation has been implicated in OTA-mediated toxicity, although inconsistent results have been reported, due, in part, to the difficulty in detecting OTA metabolites in vivo. Liquid chromatography (LC) coupled with fluorescence detection (FLD) is the most widely used analytical detection method for OTA. Under acidic conditions the toxin generates blue fluorescence (465 nm) that is due to an excited state intramolecular proton transfer (ESIPT) process that generates an emissive keto tautomer. Disruption of this ESIPT process quenches fluorescence intensity and causes a blue shift in emission maxima. The aim of the present study was to determine the impact of the C5-chlorine atom, the lactone moiety and the amide bond on OTA fluorescence and derive optical parameters for OTA metabolites that have been detected in vitro. Our results highlight the limitations of LC/FLD for OTA metabolites that do not undergo ESIPT. For emissive derivatives, our absorption and emission data improves the sensitivity of LC/FLD (3-4-fold increase in the limit of detection (LOD)) for OTA analogues bearing a C5-OH group, such as the hydroquinone (OTHQ) metabolite and the glutathione conjugate of OTA (OTA-GSH). This increased sensitivity may facilitate the detection of OTA metabolites bearing a C5-OH group in biological fluids and enhance our understanding of OTA-mediated toxicity.     

Determination of zearalenone and ochratoxin A in soil

Mycotoxins are secondary metabolites, formed by the action of fungi on agricultural crops in the field or during storage. These metabolites are highly toxic to animals and humans and high levels have been measured in agricultural crops. In order to evaluate human risks due to ingestion of mycotoxin-contaminated food different methods have been developed for analysis of mycotoxins in cereals and maize. In this project the focus was on mycotoxins in agricultural soil and the fate of these toxins in the soil-water-plant system. Two different mycotoxins were selected in the study: zearalenone (ZON) produced by species of Fusariumor Aspergillusand ochratoxin A (OTA) produced by species of Penicillium. We developed a method for analysis of these toxins in soil. Soil samples were extracted with methanol-water (9:1) and purified by solid-phase extraction (SPE, C8-columns). The final extract was analysed using high-pressure liquid chromatography (HPLC) with fluorescence detection. A Phenyl Hexyl column was used to separate the toxins. The detection limits obtained were 0.1 and 1.0 microg kg(-1) dry weight (dw) for OTA and ZON, respectively. The developed method has been used for analysis of different soils in connection with growth chamber experiments. The soil types used in the growth chamber experiments were a sandy soil, a sandy clay soil, and a soil with high content of organic matter. The recovery was determined as 85.8 and 93.4% and the repeatability to 5.1 and 12.8% for OTA and ZON, respectively. The reproducibility obtained was 8.5 and 15.0% for soil samples, representing concentration levels from 0.2-30 microg kg(-1) dw (OTA) and from 1.0-100 microg kg(-1) dw (ZON).     

C18固相萃取柱-高效液相色谱法测定葡萄干中赭曲霉毒素A

采用C18固相萃取柱对葡萄干样品中赭曲霉毒素A(OTA)净化,以高效液相色谱结合荧光检测器对提取样品进行检测,采用乙腈-水-冰乙酸为流动相,荧光检测器激发波长333nm,发射波长460nm,外标法定量。结果表明,在0.098～12.5μg/L范围内有良好的线性关系(R2=0.9998),检测限为0.07μg/kg,加标回收率在94.0%～108.4%之间,相对标准偏差为0.4%～1.0%,并对采集的10个葡萄干样品进行OTA含量检测,证实了该方法的可行性。该方法可满足对大批量葡萄干样品中赭曲霉毒素A检测的需要。     

Occurrence of ochratoxin A in bread consumed in Morocco

One hundred samples of commercial bread purchased from January to October (2006) from retail bakeshops in five different cities (Rabat, Témara, Salé, Casablanca and Meknès) in Morocco were surveyed for the presence of ochratoxin A (OTA) using liquid chromatography coupled to fluorescence detection. The identification of OTA in positive bread samples was confirmed by methyl ester derivatization. Analytical results showed that forty eight (48%) samples were positive with OTA greater than the limit of quantification (LOQ = 0.051 ng/g). Levels of OTA in positive samples ranged between 0.14 and 149 ng/g. The average contamination of bread samples with OTA was 13 ± 1.5 ng/g. The highest frequency of positive samples (61.5%) and the most contaminated sample (149 ng/g) were found in bread commercialized in the Casablanca area. Twenty six of the positive samples exceeded the maximum level of 3 ng/g set by European regulations for OTA in cereals and derivatives. Based in the results presented in this study, the estimated daily intake of OTA from bread was 126 ng/kg bw/day. The present paper is the first ever drafted on the natural occurrence of OTA in bread consumed in Morocco. Data on the daily intake of OTA by the Moroccan population are also estimated for the first time.     

Occurrence of ochratoxin A in bread consumed in Morocco

One hundred samples of commercial bread purchased from January to October (2006) from retail bakeshops in five different cities (Rabat, Témara, Salé, Casablanca and Meknès) in Morocco were surveyed for the presence of ochratoxin A (OTA) using liquid chromatography coupled to fluorescence detection. The identification of OTA in positive bread samples was confirmed by methyl ester derivatization. Analytical results showed that forty eight (48%) samples were positive with OTA greater than the limit of quantification (LOQ = 0.051 ng/g). Levels of OTA in positive samples ranged between 0.14 and 149 ng/g. The average contamination of bread samples with OTA was 13 ± 1.5 ng/g. The highest frequency of positive samples (61.5%) and the most contaminated sample (149 ng/g) were found in bread commercialized in the Casablanca area. Twenty six of the positive samples exceeded the maximum level of 3 ng/g set by European regulations for OTA in cereals and derivatives. Based in the results presented in this study, the estimated daily intake of OTA from bread was 126 ng/kg bw/day. The present paper is the first ever drafted on the natural occurrence of OTA in bread consumed in Morocco. Data on the daily intake of OTA by the Moroccan population are also estimated for the first time.     

Displacement immunoassay for the detection of ochratoxin A using ochratoxin B modified glass beads

We report here the development of a new assay for the detection of ochratoxin A (OTA) based on the use of its dechlorinated analogue, ochratoxin B (OTB), in a displacement immunoassay. OTB was immobilised on controlled-pore glass beads followed by the binding of anti-OTA antibody, with anti-IgG antibody peroxidase conjugate used as a label. In this manner, an original bio-sensing material was obtained. Upon incubation of this material with OTA, the toxin competes with OTB for the binding sites of the anti-OTA antibodies and releases the antibody-tagged peroxidase complex into the solution. Compared to classic competitive immunoassays, this newly developed displacement immunoassay presents a similar detection limit and assay time. Moreover, the detection, based on the activity of the horseradish peroxidase, is performed for the first time in situ using wine samples.     

Testing green coffee for ochratoxin A, part II: Observed distribution of ochratoxin A test results

The suitability of 4 theoretical distributions (normal, lognormal, negative binomial, and gamma) to predict the observed distribution of ochratoxin A (OTA) in green coffee was investigated. One symmetrical and 3 positively skewed theoretical distributions were each fitted to 25 empirical distributions of OTA test results for green coffee beans. Parameters of each theoretical distribution were calculated by using Methods of Moments. The 3 skewed theoretical distributions provided acceptable fits to each of the 25 observed distributions. Because of its simplicity, the lognormal distribution was selected to model OTA test results for green coffee. Using variance equations determined in previous studies, mathematical expressions were developed to calculate the parameters of the log normal distribution for a given OTA lot concentration and test procedure. Observed acceptance probabilities were compared to an operating characteristic curve predicted from the lognormal distribution, and all 25 observed acceptance probabilities were found to lie within the 95% confidence band associated with the predicted operating characteristic curve. The parameters of compound gamma distribution were used to calculate the fraction of OTA contamination beans within a contaminated lot. The percent-contaminated beans were a function of the lot concentration and increased with lot concentration. At a lot concentration of 5 microg/kg, approximately 6 beans per 10,000 beans are contaminated.     

Immunochromatographic assay for the detection of ochratoxin A

The detection of mycotoxins—toxic contaminants of fungal origin—is an important problem in the food and feed quality control. An immunochromatographic system was developed for the detection of ochratoxin A (OTA), which is one of the priority contaminants in grain. Monoclonal antibodies against OTA and their conjugates with colloidal gold nanoparticles were prepared. The detection is based on the competition of OTA in a sample and an OTA-protein conjugate immobilized on a test strip for the binding to anti-bodies on the colloidal particle surface. The method was tested in the analysis of plant extracts (maize and barley extracts). It was shown that OTA can be detected in a medium with a high content of an organic solvent (up to 35% of methanol). The disappearance of the line in the test zone is visually detected at OTA concentrations starting from 50 ng/mL. In the case of the video-digital detection of changes in the color intensity of the test zone, the limit of detection of OTA is 5 ng/mL. The duration of the assay is 10 min.     

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.     

Natural occurrence of deoxynivalenol (DON) in wheat based noodles consumed in Malaysia

In this first study performed in noodle samples consumed in Malaysia for the presence of deoxynivalenol (DON), a total of 135 sample of noodles, comprised of instant noodle, yellow alkaline noodle and white salted noodle, were randomly collected from food stores and analyzed for DON using high performance liquid chromatography (HPLC) with a PDA (photodiode array) detector at 218 nm. The objective of this study was to investigate the DON contamination levels in different types of wheat-based noodle consumed in Peninsular Malaysia. An acetonitrile:water (17:83 v/v) mixture was used as a mobile phase and clean-up was accomplished with a Mycosep 225 column. There was a high variation in the DON concentrations in all types of noodle group as well as between brands. Only one sample of instant and yellow alkaline noodle each were contaminated with DON, at concentrations of 1.003 and 1.243 ng/g, respectively. The minimum detectable concentration for the DON was 0.627 ng/g in instant noodle. In the case of white salted noodle, none of the samples contained any detectable amount of DON. The results indicate a low occurrence of DON mycotoxins in commercial noodle products in Malaysia.     

A comparative study of extraction apparatus in HPLC analysis of ochratoxin A in muscle

Ochratoxin A (OTA) is a secondary fungal metabolite produced by several moulds, mainly by Aspergillus ochraceus and by Penicillium verrucosum, that occurs in meat products. The aim of this work was to optimize an efficient extraction procedure for the determination of OTA in muscle tissue in order to assess its occurrence in meat samples. Three different apparatus, a Waring blender, a switching apparatus, and an ultrasonic processor, were evaluated to verify the efficiency of extraction. The analytical methods proposed involve the extraction with chloroform-orthophosphoric acid, cleanup through an immunoaffinity column, high-performance liquid chromatography/fluorescence detection for separation and identification of OTA, and confirmation with liquid chromatography/FD after methylation of OTA in muscle tissue. The limit of quantification of the proposed method was 0.04 μg kg⁻¹. Recoveries of OTA, using switching apparatus, ranged from 90.3 to 103.2% for chicken muscle spiked at 2.4 and 0.48 μg kg⁻¹, respectively, with a within-day relative standard deviation of 17 and 15.3%. The proposed method was applied to 38 chicken, swine, and turkey muscle samples and the presence of OTA was confirmed in five samples. Finally, the estimated daily intake of OTA in this study was between 23 pg kg⁻¹ body weight per day for swine samples and 18 pg kg⁻¹ body weight per day for turkey samples.