Simultaneous determination of aflatoxin B1 and ochratoxin A and their natural occurrence in Mediterranean virgin olive oil

Olive oil, the most important dietary fat source of the Mediterranean diet, can be contaminated by mycotoxins. An efficient analytical method for the simultaneous determination of aflatoxin B₁ (AFB₁) and ochratoxin A (OTA) in olive oil is reported. Thirty commercial samples of virgin olive oil, purchased in olive-press plants and supermarkets in southern Italy and North Africa, were analysed to verify the analytical procedure and monitor mycotoxin contamination. A simple, rapid and economic method was set up and tested for both the extractive step and the clean-up procedures for simultaneous AFB₁ and OTA determination in olive oil. Data obtained showed that OTA was detected with high frequency (80%) in samples from both geographical areas (up to 17.0 ng g^-1), while AFB₁ was found from three of four samples from North Africa (up to 2.4 ng g^-1). In addition, 'not labelled' oil samples proved to be more contaminated by OTA then 'labelled' samples (mean values of 2.47 and 0.66 ng g^-1, respectively). These findings indicate that olive oil can be significantly contaminated by mycotoxins and confirm that a scrupulous application of European Regulation 1019/2002 (European Commission 2002), which prohibits the sale of non-labelled olive oil, is strongly recommended. Conventional qualitative parameters such as peroxide number, spectrophotometric evaluation and acid values were not correlated with mycotoxin occurrence.     

Effects of Fusarium toxin-contaminated wheat and feed intake level on the biotransformation and carry-over of deoxynivalenol in dairy cows

An experiment was carried out to examine the effects of feeding Fusarium toxin-contaminated wheat (8.21 mg deoxynivalenol (DON) and 0.09 mg zearalenone (ZON) per kg dry matter) at different feed intake levels on the biotransformation and carry-over of DON in dairy cows. For this purpose, 14 ruminal and duodenal fistulated dairy cows were fed a diet containing 60% concentrate with a wheat portion of 55% (Fusarium toxin-contaminated wheat (mycotoxin period) or control wheat (control period)) and the ration was completed with maize- and grass silage (50 : 50) on a dry matter basis. Daily DON intakes ranged from 16.6 to 75.6 mg in the mycotoxin period at dry matter intakes of 5.6-20.5 kg. DON was almost completely biotransformed to de-epoxy DON (94-99%) independent of the DON/feed intake, and the flow of DON and de-epoxy DON at the duodenum related to DON intake ranged from 12 to 77% when the Fusarium toxin-contaminated wheat was fed. In the serum samples, de-epoxy DON was detected in the range of 4-28 ng ml^-1 in the mycotoxin period, while concentrations of DON were all below the detection limit. The daily excretion of DON and de-epoxy DON in the milk of cows fed the contaminated wheat varied between 1 and 10 µg and between 14 and 104 µg, respectively. The total carry-over rates as the ratio between the daily excretion of DON and de-epoxy DON into milk and DON intake were in the ranges of 0.0001-0.0002 and 0.0004-0.0024, respectively. Total carry-over rates of DON as DON and de-epoxy DON into the milk increased significantly with increasing milk yield. In the urine samples, de-epoxy DON was the predominant substance as compared with DON with a portion of the total DON plus de-epoxy DON concentration to 96% when the Fusarium toxin-contaminated wheat was fed, whereas the total residues of DON plus de-epoxy DON in faeces ranged between 2 and 18% of DON intake in the mycotoxin period. The degree of glucuronidation of de-epoxy DON was found to be approximately 100% in serum. From 33 to 80% of DON and from 73 to 92% of de-epoxy DON, and from 21 to 92% of DON and from 86 to 100% of de-epoxy DON were glucuronidated in the milk and urine, respectively. It is concluded that DON is very rapidly biotransformed to de-epoxy DON in the rumen and only negligible amounts of DON and de-epoxy DON were transmitted into the milk within the range of 5.6-20.5 kg day^-1 dry matter intake and milk yields (fat corrected milk) between 10 and 42 kg day^-1.     

Aflatoxins in Turkish dried figs intended for export to the European Union

Dried figs for export from Turkey from crop years 2003 through 2006 were tested for aflatoxin B1 and total aflatoxins. For export to the European Union, consignments of 0.5 to 10 tonnes of dried figs were sampled according to European Commission regulations, and high-pressure liquid chromatography (HPLC) was used to determine concentrations of aflatoxins Bl, B2, G1, and G2. For each consignment of dried figs, a 30-kg sample (comprising 100 subsamples) was divided into three 10-kg subsamples, which were separately blended and analyzed with HPLC. This monitoring effort was conducted for figs from 2003, 2004, 2005, and up to June 2006, for a total of 10,396 30-kg samples (28,489 analyses). The incidence of contamination with aflatoxin B1 at higher than 2 ng/g was on average 0.6, 2.0, 4.0, and 2.4% for 2003, 2004, 2005, and up to June 2006, respectively, whereas contamination with total aflatoxins at higher than 4 ng/g was 2.6, 3.0, 5.1, and 2.7%. There was significant variability in contamination between replicate 1-kg samples, indicating small numbers of individual contaminated figs were probably responsible. There were also substantial differences in the relative proportions of aflatoxins B1, B2, G1, and G2 among samples, suggesting different contributing fungal sources.     

Validation of a high-performance liquid chromatography analytical method for ochratoxin A quantification in cocoa beans

A validated high-performance liquid chromatography (HPLC) method with fluorescence detection for the quantitative analysis of ochratoxin A (OTA) in cocoa beans is described. OTA was extracted with methanol-3% sodium hydrogen carbonate solution and then purified with immunoaffinity columns before its analysis by HPLC. The validation of the analytical method was based on the following criteria: selectivity, linearity, limit of detection and quantification, precision (within- and between-day variability) and recovery, robustness and uncertainty. Detection and quantification limits were 0.04 and 0.1 μg kg^-1, respectively. Recovery was 88.9% (relative standard deviation = 4.0%). This method was successfully applied to the measurement of 46 cocoa bean samples of different origins. A total of 63% of cocoa bean samples was contaminated with a level greater than the limit of detection. The means and medians obtained for cocoa bean were 1.71 and 1.12 μg kg^-1, respectively. Surveillance controls should be set up in both crops and factories involved in transformation processes to avoid this mycotoxin in final products.     

Determination of zearalenone in corn flour and a cheese snack product using high-performance liquid chromatography with fluorescence detection

The presence of the mycotoxin zearalenone in corn flour and a cheese snack derived from this was determined. Thirty-eight samples (corn flour and cheese snacks) of different brands were analysed for zearalenone using high-performance liquid chromatography with fluorescence detection. Zearalenone was detected in corn flour and cheese snack samples with average content of 0.377 ppm (maximum, 0.889 ppm) and 0.832 ppm (maximum, 1.471 ppm) respectively. The recovery from spiked corn flour and cheese snack samples ranged from 70-87%. The method had a limit of detection of 0.01 µg ml^-1. The linearity of method was determined (y = 5.88 x + 0.25, r² = 0.9999), and optimum assay range was 0.05-30 µg ml^-1. The occurrence of zearalenone in the maize product confirms the need to assess the exposure of the Iranian population to this mycotoxin.     

Design of a sampling plan to detect ochratoxin A in green coffee

The establishment of maximum limits for ochratoxin A (OTA) in coffee by importing countries requires that coffee-producing countries develop scientifically based sampling plans to assess OTA contents in lots of green coffee before coffee enters the market thus reducing consumer exposure to OTA, minimizing the number of lots rejected, and reducing financial loss for producing countries. A study was carried out to design an official sampling plan to determine OTA in green coffee produced in Brazil. Twenty-five lots of green coffee (type 7 - approximately 160 defects) were sampled according to an experimental protocol where 16 test samples were taken from each lot (total of 16 kg) resulting in a total of 800 OTA analyses. The total, sampling, sample preparation, and analytical variances were 10.75 (CV = 65.6%), 7.80 (CV = 55.8%), 2.84 (CV = 33.7%), and 0.11 (CV = 6.6%), respectively, assuming a regulatory limit of 5 µg kg^-1 OTA and using a 1 kg sample, Romer RAS mill, 25 g sub-samples, and high performance liquid chromatography. The observed OTA distribution among the 16 OTA sample results was compared to several theoretical distributions. The 2 parameter-log normal distribution was selected to model OTA test results for green coffee as it gave the best fit across all 25 lot distributions. Specific computer software was developed using the variance and distribution information to predict the probability of accepting or rejecting coffee lots at specific OTA concentrations. The acceptation probability was used to compute an operating characteristic (OC) curve specific to a sampling plan design. The OC curve was used to predict the rejection of good lots (sellers' or exporters' risk) and the acceptance of bad lots (buyers' or importers' risk).     

Occurrence of mycotoxins and ergosterol in maize harvested over 5 years in Northern Italy

Maize samples collected from storage bins and feed mills in Northern Italy between 1995 and 1999 were surveyed for the occurrence of aflatoxin B₁ (AFB₁), zearalenone (ZEA), deoxynivalenol (DON) and fumonisin (FB₁); further, ergosterol was analysed as a fungal growth marker. The incidence and mean content of AFB₁ were generally low; nevertheless, a remarkable contamination was found in two samples (109 and 158 μg kg^-1), while five others exceeded 20 μg kg^-1. DON and ZEA mean levels were significantly higher in 1996 (2716 and 453 μg kg^-1) with respect to the other years, when mean contents ranged from 7 to 30% and from 3 to 17%, respectively, expressed in per cent of 1996 contents. FB₁ was present in all samples and was by far the most remarkable mycotoxin in Northern Italian maize, with the exception of samples from 1996. The average level was 3064 μg kg^-1, 69.6% of samples resulted over 1000 μg kg^-1 and 16.9% over 5000 μg kg^-1. Significant correlations were found between ergosterol and the major mycotoxin(s) in each year (FB₁ in 1995 and 1997-99; ZEA + DON in 1996). Consequently, ergosterol seems to be a good index of the toxicological quality of maize. Climatic conditions influenced the growth of different fungal species. In 1996, the first 20 days of October were extremely rainy; these weather conditions delayed the harvest until the first week of November and favoured the growth of DON and ZEA producing fungi and the synthesis of mycotoxins. On the contrary, the temperate and dry climate of the other years supported the growth of FB₁-producing fungi.     

Fusarium mycotoxins in milling streams from the commercial milling of maize imported to the UK,and relevance to current legislation

A study in three large commercial UK maize mills showed that Fusarium mycotoxins, such as deoxynivalenol, zearalenone and fumonisins present at mill intake, are distributed in milling streams approximately according to their occurrence in the maize seed structure. Fractions derived from the endosperm tended to contain the lowest levels of mycotoxins. Concentrations of mycotoxins within the endosperm are also related to the particle size. However, the products derived from the embryo or outer seed layers contained the highest mycotoxin levels being concentrated up to five times or more, although these components are normally used for animal feed or industrial use. The general pattern of mycotoxin distribution found when milling French and Argentinean maize was similar, although very variable, and it is concluded that this variability stems from different milling strategies used at each mill and from the nature and condition of each consignment of maize. Mycotoxins in maize grits (particle sizes >500 µm) were usually reduced by the greatest amount when compared with the whole maize, while flour (≤500 µm) could be both reduced or increased depending on the mill and consignment. Thus, in most situations mycotoxin concentrations in whole maize that meet European Commission legislation on intake should give rise to levels in milled ingredients that should also do so. However, this was not always true in some ingredients, especially for fumonisins in those fractions with particle size ≤500 µm.     

Effect of grain storage and processing on chlorpyrifos-methyl and pirimiphos-methyl residues in post-harvest-treated wheat with regard to baby food safety requirements

A study was undertaken to assess the effects of storage intervals and of milling procedures on the dissipation of chlorpyrifos-methyl and pirimiphos-methyl residues in post-harvest-treated wheat grain and to obtain scientific data on the compliance of the processed products with safety requirements concerning baby foods. The insecticide formulations were applied on stored wheat at recommended rates (20 ml t^-1). The initial concentration levels in whole grain were determined in samples taken 1 h after treatment. The dissipation of residues and their distribution in different fractions of the milled grain were studied after various storage intervals, from 7 to 270 days after treatment. Samples of treated grain were milled in a fractionating laboratory mill and eight fractions — bran, semolina, three types of groats and three types of flour — were collected and analysed for pesticide residues. The residues were determined by an analytical method based on acetone extraction, graphitized carbon clean-up and GC-ECD, respectively, and GC-NPD determination of residues. The limits of determination of both pesticides were 0.005 mg kg^-1, which is high enough for enforcement of the European Commission Directive that established a maximum residue level of 0.01 mg kg^-1 for any pesticide in cereal-based baby food. The results showed that the pesticides chlorpyrifos-methyl and pirimiphos-methyl applied post-harvest on wheat as grain protectants were distinguished by relatively low rates of degradation in the grain under practical storage conditions. Milling did not significantly reduce the bulk of the chemicals but resulted in the distribution of residues in various processed products. The main part of the insecticides deposited on the grain remained in the bran and partly in semolina fractions. After 270 days of treatment, the residues of chlorpyrifos-methyl were within the range 0.8-2.1 mg kg^-1 and of pirimiphos-methyl — between 0.6 and 3.7 mg kg^-1 in the various types of flour.     

Determination of ochratoxin A in grapes of Greek origin by immunoaffinity and high-performance liquid chromatography

A simple analytical method for ochratoxin A (OTA) determination in grapes is described, using aqueous methanolic extraction, an immunoaffinity column clean-up step and high-performance liquid chromatography with fluorescence detection. Mean recovery was 94% (RSD = 4.0%) with a detection limit of 0.4 ng g^-1 and quantification limit of 1.20 ng g^-1. Repeatability (r) and reproducibility (R) were 1.17 and 1.34, respectively. OTA determinations were applied to 50 grape samples (23 different varieties) originating from representative regions of Greece. Results showed the presence of OTA in 86% of samples tested (n = 50). Traces were found in 56% of samples but OTA was not detectable in 14% of samples. Traces were also found in 4% of red, organically grown samples. The most contaminated were three samples of red grapes, two from Central Greece (2.69 and 1.41 ng g^-1), both table and wine-making grapes. The third sample (1.46 ng g^-1), originating from the island of Samos, was used only in wine-making. Mean (1.06 ng g^-1) and median (0.76 ng g^-1) OTA concentrations in red grapes were slightly higher compared to the mean (0.82 ng g^-1) and median (0.65 ng g^-1) concentrations in white grape samples. The study shows that the potential risk for a person of 60 kg ranged from 0.9 to 9 ng kg^-1 bw day^-1 and is dependent on the quantity of grapes consumed daily.