Optimization and validation of a HPLC method for simultaneous determination of aflatoxin B1, B2, G1, G2, ochratoxin A and zearalenone using an experimental design

A reversed-phase HPLC optimization strategy is presented for investigating the separation and retention behavior of aflatoxin B₁, B₂, G₁, G_2, ochratoxin A and zearalenone, simultaneously. A fractional factorial design (FFD) was used to screen the significance effect of seven independent variables on chromatographic responses. The independent variables used were: (X1) column oven temperature (20–40°C), (X2) flow rate (0.8–1.2?ml/min), (X3) acid concentration in aqueous phase (0–2%), (X4) organic solvent percentage at the beginning (40–50%), and (X5) at the end (50–60%) of the gradient mobile phase, as well as (X6) ratio of methanol/acetonitrile at the beginning (1–4) and (X7) at the end (0–1) of gradient mobile phase. Responses of chromatographic analysis were resolution of mycotoxin peaks and HPLC run time. A central composite design (CCD) using response surface methodology (RSM) was then carried out for optimization of the most significant factors by multiple regression models for response variables. The proposed optimal method using 40°C oven temperature, 1?ml/min flow rate, 0.1% acetic acid concentration in aqueous phase, 41% organic phase (beginning), 60% organic phase (end), 1.92 ratio of methanol to acetonitrile (beginning) and 0.2 ratio (end) for X1–X7, respectively, showed good prediction ability between the experimental data and predictive values throughout the studied parameter space. Finally, the optimized method was validated by measuring the linearity, sensitivity, accuracy and precision parameters, and has been applied successfully to the analysis of spiked cereal samples.     

Aflatoxin B1, ochratoxin A and zearalenone in sorghum grains marketed in Tunisia

A total of 64 samples of sorghum (37 Tunisian sorghum samples and 27 Egyptian sorghum samples) were collected during 2011–2012 from markets in Tunisia. Samples were analysed for contamination with aflatoxin B1, ochratoxin A and zearalenone by High-Performance Liquid Chromatography Coupled with Fluorescence Detection (HPLC-FLD). Aflatoxin B1 was found in 38 samples in the range 0.03–31.7 µg kg^?1. Ochratoxin A was detected in 24 samples with concentrations ranging from 1.04 to 27.8 µg kg^?1. Zearalenone was detected in 21 samples and the concentration varied between 3.7 and 64.5 µg kg^?1. ANOVA analysis of the influence of the country of origin on the incidence and concentration of mycotoxins in the samples studied showed no significant difference (P > 0.05) between the two batches of samples for each of the three mycotoxins studied. The studied mycotoxins contaminate sorghum and may also co-exist because of the diversity of the mycobiota in this cereal.     

Aflatoxins,ochratoxin A and zearalenone in Brazilian corn cultivars

Past surveys indicated that the occurrence of aflatoxins, zearalenone and ochratoxin A was not a problem in corn and corn products in the state of São Paulo, Brazil. However, according to recent studies, a change in pattern has been detected. To obtain a better overview, these toxins were searched for in 110 samples of freshly harvested corn, corresponding to 48 commercial cultivars planted at three different locations in the state. Aflatoxin contamination was found in 60 (54.5%) of the samples, in levels ranging from 6 to 1600 µg kg^?1 aflatoxin B₁. Insect control was exercised, so this was not the main route of corn infection. Endosperm type, germplasm type, number of days to flowering, and length of time the mature corn remained in the field had no effect on aflatoxin contamination. Ochratoxin A was found in two samples (206 and 128 µg kg^?1) and zearalenone in one sample (4640 µg kg^?1). Possible causes of the increase in aflatoxin levels may lie in the changing nature of the commercial cultivars employed, associated with the forsaking of the original landraces, and in a change in the toxigenicity pattern of the corn mycoflora Aspergillus flavus/Aspergillus parasiticus prevailing strains. © 2001 Society of Chemical Industry     

Occurrence of fumonisin B1 and B2 in beer

A total of 29 nationally distributed brands of beer, representing 25 domestic US and four imported brands, were purchased in retail outlets in Lincoln, Nebraska and analysed for concentrations of fumonisin B₁ (FB₁) and B₂(FB₂). Immunoaffinity column extraction and cleanup of fumonisins from the beer samples, coupled with detection and analysis by gradient high performance liquid chromatography (HPLC), provided a limit of quantitation for each toxin of 0.3ng/ml. Of the brands of beer sampled, 86% were positive for FB and 41% were positive for FB2. No beer contained a detectable quantity of FB without a detectable quantity of FB1. The total fumonisin (FB₁ + FB₂) content of positive samples ranged from 0.3 to 12.7ng/ml, with a mean concentration for all positive samples of 4.0 ± 3.4ng/ml (n = 25). Considering that the level of fumonisin contamination of corn in recent harvest years has been minimal, the results of this limited survey could represent levels associated with current agricultural and brewing practices.     

Validation of the procedure for the simultaneous determination of aflatoxins ochratoxin A and zearalenone in cereals using HPLC-FLD

Method validation for quantitative analysis of aflatoxins (AFs), ochratoxin A (OTA) and zearalenone (ZEA) in cereals using HPLC with fluorescence detector (FLD) is described. Mycotoxins were extracted with methanol?:?water (80?:?20) and purified with a multifunctional AOZ immunoaffinity column before HPLC analysis. The validation of the analytical method was performed to establish the following parameters: specificity, selectivity, linearity, limits of detection (LOD) and quantification (LOQ), accuracy, precision (within- and between-day variability), stability, robustness, measurement of performance, and measurement of uncertainty. Calibration curves were linear (r?>?0.999) over the concentration range, from the LOQ to 26, 40 and 400?ng/g for AFs, OTA and ZEA, respectively. LOD and LOQ were 0.0125 and 0.05?ng/g for aflatoxin B1 (AFB1) and G1 (AFG1), 0.0037 and 0.015?ng/g for aflatoxin B2 (AFB2) and G2 (AFG2), as well as 0.05 and 0.2?ng/g for OTA and 0.5 and 2?ng/g for ZEA, respectively. The mean recovery values were 77–104% for different concentrations of AFs, OTA and ZEA in spiked cereal samples. Both intra- and inter-day accuracy and precision were within acceptable limits. This method was successfully applied for the simultaneous determination of mycotoxins for 60 cereal samples collected from Malaysian markets. Fifty per cent of the cereal samples were contaminated with at least one of these mycotoxins, at a level greater than the LOD. Only one wheat sample and two rice samples were contaminated with levels greater than the European Union regulatory limits for AFs and OTA (4 and 5?ng/g). The means and ranges of mycotoxins obtained for the cereal samples were 0.4?ng/g and 0.01–5.9?ng/g for total AFs; 0.18?ng/g and 0.03–5.3?ng/g for OTA; and 2.8?ng/g and 2.4–73.1?ng/g for ZEA, respectively. The results indicate that the method is suitable for the simultaneous determination of AFs, OTA and ZEA in cereals and is suitable for routine analysis.     

Co-occurrence of aflatoxins B 1 , B 2 , G 1 , G 2 , zearalenone and fumonisin B 1 in Brazilian corn

Two hundred and fourteen unprocessed corn samples (1997-98 harvest), collected at wholesale markets in different regions in Brazil, were surveyed for the occurrence of mycotoxins. The samples were analysed for aflatoxins B 1 , B 2 , G 1 , G 2 , zearalenone and fumoni1sin B 1 using in-house validated methods. The occurrence of aflatoxin B 1 , zearalenone and fumonisin B 1 was found in 38.3, 30.4 and 99.1% of the samples, respectively. Aflatoxin B 1 , zearalenone and fumonisin B 1 contamination levels varied from 0.2 to 129, 36.8 to 719, and 200 to 6100 μg/kg, respectively. The cooccurrence of the two carcinogenic mycotoxins aflatoxin B 1 and fumonisin B 1 was observed in 100% of the aflatoxin-contaminated samples (82 samples). Cooccurrences of aflatoxin B 1 : zearalenone: fumonisin B 1 and aflatoxin B 1 : aflatoxin B 2 : fumonisin B 1 were found in 18 and 43 samples, respectively.     

The influence of local factors on the prediction of fumonisin contamination in maize

BACKGROUND: Contamination by mycotoxins is a major concern to the maize industry in north‐east Italy where maize grain is often spoiled by Fusarium spp. In this work, fumonisins, deoxynivalenol and zearalenone were determined and an artificial neural network (ANN) model suitable for predicting mycotoxin contamination of maize at harvest time was developed. RESULTS: The occurrence of deoxynivalenol and zearalenone was very limited, while fumonisins concentration ranged from 163 and to 3663 µg kg^?1 in 2007, and from 333 to 11473 µg kg^?1 in 2008. Statistical data analysis of factors affecting fumonisins concentration revealed that irrigation, chemical treatment against the European corn borer and harvest date significantly affected the level of contamination (P < 0.05), although the relevance of the factors was different in 2007 and 2008. The neural network approach showed a significant correlation between ascertained values and predictions based on agronomic data. CONCLUSION: This is the first time that an artificial neural network has been used to predict fumonisin accumulation in maize: the prediction has been shown to have the potential for the development of a new approach for the rapid cataloging of grain lots. Copyright © 2012 Society of Chemical Industry     

Natural co-occurrence of aflatoxin B1, fumonisin B1 and ochratoxin A in barley and corn foods from Korea

A survey for aflatoxin B₁ (AFB₁), fumonisin B₁ and ochratoxin A (OTA) was conducted on 127 samples that included 30 food-grade barley, 32 barley foods, 18 food-grade corn and 47 corn foods, randomly collected during 1998-99 in Seoul, Korea. The presence of mycotoxins was analysed by direct competitive enzyme-linked immunosorbent assay (ELISA), and most of the positive samples from ELISA were confirmed using high-performance liquid chromatography (HPLC). Recoveries of AFB₁ and OTA spiked at 10 ng g -1 and FB₁ spiked at 50 ng g^-1 were 106, 87 and 105% by ELISA, whereas those by HPLC were 80, 79 and 84%, respectively. Detection limits by ELISA for AFB₁, FB₁ and OTA were 1, 5 and 5 ng g^-1, and those by HPLC were 0.6, 35 and 1 ng g^-1. Naturally occurring AFB₁, FB₁ and OTA were found in 4/32 (12%), 2/32(6%) and 4/32 (12%) samples of barley foods with an average of 26, 16 and 9 ng g^-1, respectively. AFB₁ and FB₁ in corn foods were detected in 4/47 (8%) and 9/47 (19%) samples with the average being 20 and 74 ng g^-1, while no OTA was found in any corn foods samples. No AFB₁, FB₁ or OTA was detected in any of food-grade barley and corn samples. This is the first report on the natural co-occurrence of AFB₁ and FB₁ in barley and corn foods as well as on surveillance of OTA in Korea.     

干燥方式、储存温度和品种对玉米中伏马毒素B1的影响

伏马毒素B_1广泛存在于玉米中,是玉米及其制品质量安全的重要危害因子。选取23个玉米品种为研究对象,对不同干燥方式(烘干、晒干和晾干)和不同储藏温度(4、15、25℃、常温)对其中伏马毒素B_1的影响进行研究,结果表明:玉米进行烘干、晒干和晾干后放置3个月后,伏马毒素B_1含量为烘干晒干晾干,玉米水分含量与伏马毒素B_1含量显著相关(r=0.678);不同温度中放置3个月后,伏马毒素B_1含量由小到大为4℃15℃25℃室温;烘干、4℃储存玉米中伏马毒素B_1含量分别为0～1.39 mg/kg(平均值0.40 mg/kg)、0.08～1.60 mg/kg(平均值0.78 mg/kg),均低于美国FDA规定的最高限量(2 mg/kg);品种对玉米中伏马毒素B_1含量影响显著。烘干、4℃储存、选取高抗镰刀菌玉米品种可有效控制在储藏过程中伏马毒素B_1对玉米的污染。     

Detoxification of zearalenone and ochratoxin A by ozone and quality evaluation of ozonised corn

Zearalenone (ZEN) and ochratoxin A (OTA) are secondary toxic metabolites of fungi that can contaminate a wide range of food and feedstuff. In this study, the effects of ozone treatment on ZEN and OTA and the quality of ozonised corn are investigated. Ozone significantly affects ZEN and OTA solutions. ZEN was undetectable 5 s after being treated with 10 mg l^–1 ozone. However, OTA was resistant to ozonation with a degradation rate of 65.4% after 120 s of treatment. Moreover, ZEN and OTA solutions were difficult to degrade after being dried by a nitrogen stream. Results showed that ozone effectively degraded ZEN and OTA in corn. The degradation rates of ZEN and OTA in corn increased with ozone concentration and treatment time. The degradation of ZEN and OTA at different ozone concentrations appropriately conformed to first-order kinetics with an R² value > 0.8749. Furthermore, under the same conditions, corn with increased moisture content (MC) (19.6%) was more sensitive to ozone than corn with a low MC (14.1%). When treated with 100 mg l^–1 ozone for 180 min, ZEN and OTA in corn with 19.6% MC decreased by 90.7% and 70.7%, respectively. To evaluate the quality of ozonised corn, subsequent quality experiments were conducted using corn samples treated at different times with 100 mg l^–1 ozone. The MC of corn decreased after ozone treatment. The whiteness and yellowness of the corn increased and decreased with increasing time, respectively. The fatty acid value of the corn increased significantly (p ≤ 0.05) after 180 min of treatment. This study verified that ozone can effectively degrade ZEN and OTA in corn, but slightly affected corn quality.     

Occurrence of ochratoxin A and citrinin in Czech cereals and comparison of two HPLC methods for ochratoxin A detection

The aims of the study were to obtain information about the occurrence of ochratoxin A (OTA) and citrinin (CIT) in cereals harvested in the Czech Republic and to compare two analytical procedures for detecting OTA. A total of 34 cereal samples, including two matrix reference materials (R-Biopharm, Germany), were analysed. The results were compared with the limit for raw cereal grains used as a foodstuff according to Commission Regulation No. 1881/2006, which allows a maximum OTA level of 5 µg kg^?1. Compared were two methods based on the high-performance liquid chromatography principle, one using the immunoaffinity columns OchraTest? (VICAM) and the second based on solvent partition (PART), both followed by fluorescence detection. The highest OTA contents were found in two barley samples. According to the method employed, the results for the first sample (malting barley) were VICAM = 31.43 µg kg^?1 and PART = 44.74 µg kg^?1. For the second sample (feeding barley) they were VICAM = 48.63 µg kg^?1 and PART = 34.40 µg kg^?1. Two samples of bread wheat had an OTA content approaching the legal limit (VICAM = 4.71 µg kg^?1 and PART = 6.03 µg kg^?1; VICAM = 4.12 µg kg^?1 and PART = 3.95 µg kg^?1). CIT was analysed using the PART method only, and its highest content (93.64 µg kg^?1) was found for the malting barley sample with high OTA content (44.74 µg kg^?1 as analysed using PART).     

A survey of the fungal contamination and presence of ochratoxin A and zearalenone on Spanish feed and raw materials

The incidence of fungal contamination in 91 samples of feed and raw materials used for animal feeding in Spain has been studied. Sample analysis was accomplished in a new culture medium to which a beta‐cyclodextrin had been added, and a comparison with other more usual culture media was performed. Ochratoxin A (OTA) and zearalenone (ZEA) contamination of all the samples was evaluated by RP‐HPLC with fluorescence detection. The fungal genera found, such as Penicillium and Fusarium, included mycotoxigenic strains as OTA and ZEA (33.3 and 26.4% incidence respectively). One sample of corn and another of cotton seed were contaminated with levels of OTA above the 5 and/or 10 µg kg^?1 recommended by the legislation of several European countries, whereas none of the samples contaminated with ZEA surpassed the legislation limits suggested by the official agencies. Copyright © 2004 Society of Chemical Industry     

Fumonisin B1, zearalenone and deoxynivalenol production by Fusarium moniliforme,F proliferatum and F graminearum in mixed cultures on irradiated maize kernels

The impact on fungal growth and mycotoxin formation of interactions between fumonisin‐producing isolates of Fusarium moniliforme and F proliferatum and a zearalenone (ZEA)‐ and deoxynivalenol (DON)‐producing isolate of F graminearum inoculated together on irradiated maize at 15 and 25 °C and at 0.98, 0.95 and 0.93 a_w was studied. The presence of F graminearum decreased the fungal populations (CFU g⁻¹ grain) of F moniliforme and F proliferatum under almost all conditions tested. In the presence of F moniliforme, CFUs of F graminearum increased significantly at 25 °C, especially at 0.93 and 0.95 a_w, while the presence of F proliferatum caused them to increase at 15 °C. The presence of F graminearum always inhibited FB₁ production, except at 25 °C and 0.98 a_w where it increased. However, the observed differences were not statistically significant. There was no effect of fungal interaction on ZEA production by F graminearum; however, when paired with F moniliforme and F proliferatum, DON production by F graminearum was significantly stimulated, especially at 0.98 a_w. © 2000 Society of Chemical Industry     

A survey of ochratoxin A occurence in Greek wines

A total of 150 wines, including 123?dry wines (64 red, 49 white and 10 rosé) and 27 dessert wines (14 red and 13 white), were obtained from various viticulture and oenological practices across Greece during the period 1999–2006 and analyzed for ochratoxin a (OTA) using immunoaffinity clean-up and HPLC with fluorescence detection. There was a high frequency of OTA in commercially available wines (69% positive samples). However, the level of contamination was relatively low, with only one sample marginally reaching the EU permitted maximum level (2.0?µg?l^?1). A total of 91% of the samples had OTA concentrations <1.0?µg?l^?1. The higher concentrations were found in wines from the southern regions, especially in dessert-type wines. There were no significant differences based on wine color or production years. Furthermore, there was no difference between conventional or organic cropping systems in terms of OTA presence.     

Relative severity of fumonisin contamination of cereal crops in West Africa

Traditional and improved varieties of maize, pearl millet and sorghum were planted by small-scale farmers under the direction of the International Institute for Tropical Agriculture in two Nigerian agro-ecological zones: the Sudan Savanna and the Northern Guinea Savanna. Samples were collected for the determination of Fusarium infection and fumonisin (B₁, B₂ and B₃) contamination. A previous paper reported Aspergillus infection and aflatoxin contamination of these samples. Fusarium infection levels, measured by per cent kernels infected, were modest with mean levels for the above cereals of 16% ± 11% (SD), 12% ± 7% and 13% ± 16%, respectively. However, the Fusarium species recovered from maize were predominantly the fumonisin producers F. verticillioides and F. proliferatum, together making an infection rate of 15% ± 10%, whereas these species were present to a limited extent only in the other two cereals, 1% ± 1% for pearl millet and 2% ± 6% for sorghum. Fumonisin contamination was variable but reflected the diversity of Fusarium producers in these three cereals. Mean levels were 228 ± 579 µg kg^–1 (range < 5–2860 µg kg^–1) for maize, 18 ± 7 µg kg^–1 (range = 6–29 µg kg^–1) for pearl millet and 131 ± 270 µg kg^–1 (range < 5–1340 µg kg^–1) for sorghum. Together with previous results on aflatoxin, this study confirmed the co-occurrence of aflatoxins and fumonisins in maize as well as in the traditional African cereals, millet and sorghum (89% co-occurrence across all three cereals). The low fumonisin levels may be ascribed to the use of good agricultural practices. Of the Fusarium species present, those in maize consisted mainly of fumonisin producers, the opposite of what was observed in pearl millet and sorghum. It is concluded that replacement of maize by pearl millet and sorghum could improve food safety with regards to aflatoxin B and fumonisin B exposure.     

Effect of processing on ochratoxin A content in dried beans

Dried pink beans naturally contaminated with ochratoxin A (OTA) and dried carioca beans artificially contaminated with OTA by inoculation with Aspergillus ochraceus (ATCC 22947) were tested for ochratoxin A levels as follows: dried beans were washed with water for 2, 60 or 120 min, soaked in water for 60, 120 min or 10 h, and cooked for 60 or 120 min. At each step, test water and beans were separated. Test water, raw beans and cooked beans were analyzed for OTA. The amount of OTA partitioned into water and in residual beans was determined by methanol–sodium bicarbonate extraction, buffer dilution, immunoaffinity column cleanup, liquid chromatographic separation and fluorescence detection. The results demonstrated that the distribution of OTA in processing water and beans depends on the method of preparation. All treatments (washing, soaking and cooking) when applied individually reduced the amounts of OTA retained in bean flour and whole beans. Higher amounts of OTA remained in whole beans than in bean flour after removing the processing water. The combination of the three treatments eliminated about 50% of the toxin from whole beans. This study provides evidence that discarding the washing, soaking and cooking water leads to a significant reduction in OTA contamination in dried beans.