E.A. Vargas

Co-occurrence of aflatoxins B1, B2, G1, G2, zearalenone and fumonisin B1 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 B1, B2, G1, G2, zearalenone and fumoni1sin B1 using in-house validated methods. The occurrence of aflatoxin B1, zearalenone and fumonisin B1 was found in 38.3, 30.4 and 99.1% of the samples, respectively. Aflatoxin B1, zearalenone and fumonisin B1 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 B1 and fumonisin B1 was observed in 100% of the aflatoxin-contaminated samples (82 samples). Cooccurrences of aflatoxin B1: zearalenone: fumonisin B1 and aflatoxin B1: aflatoxin B 2: fumonisin B1 were found in 18 and 43 samples, respectively.

Co-occurrence of aflatoxins B1, B2, G1 and G2, ochratoxin A, zearalenone, deoxynivalenol, and citreoviridin in rice in Brazil

A total of 230 samples of processed rice and its sub-products or derived products were analysed to establish the co-occurrence of several mycotoxins. Samples were analysed in the period 2007–2009 due to the outbreak of beriberi associated with the consumption of rice stored in inappropriate conditions in Brazil. According to data from the Ministry of Health, 323 cases of disease were registered in 2006, of which at least 47 cases resulted in death. The occurrence of total aflatoxin (AFT) (aflatoxin B1 + B2 + G1 + G2), ochratoxin A (OTA), zearalenone (ZON), deoxynivalenol (DON), and citreoviridin (CTV) was 58.7%, 40.0%, 45.2%, 8.3% and 22.5%, respectively. From 166 rice samples analysed, 55% had levels <0.11 µg kg−1 for AFT. For OTA and ZON, of 165 rice samples analysed, 28% and 29% were contaminated with levels from 0.20 to 0.24 µg kg−1 and from 3.6 to 290.0 µg kg−1, respectively. One sample (0.6%) was contaminated with 4872.0 µg kg−1 of ZON. A total of 91% of rice samples (n = 165) did not contain detectable DON (<30.00 µg kg−1), although the highest level of contamination was found to be 244 µg kg−1. From the total of 65 samples analysed, 94% had no detectable CTV (<0.9 µg kg−1), with a range from 0.9 to 31.1 µg kg−1 in 6% of the samples. The highest levels of contamination were found in rice sub-products or derived products from the husk and rice bran. Co-occurrence was observed for AFT and ZON in 17.0%, AFT and OTA in 24.2%, AFT and CTV in 6.2%, OTA and CTV in 4.6%, and ZON and CTV in 3.1%. These fractions were also the major contributors for the co-occurrence. The results found show the necessity of monitoring rice production.

Immunoaffinity column clean-up and thin layer chromatography for determination of ochratoxin A in green coffee.

An immunoaffinity clean-up-based method for determining ochratoxin A (OTA) in green coffee aiming at one-dimensional thin layer chromatography (TLC) analysis was established. OTA was extracted with a mixture of methanol and aqueous sodium hydrogen carbonate solution, purified through an immunoaffinity column, separated on normal or reversed-phase (RP) TLC plates and detected and quantified by visual and densitometric analysis. The linear equation of the standard calibration curve by densitometric analysis gave R2 > 0.999 (0.04–84 ng). The mean recovery (R) of OTA from spiked samples (1.8–109 µg kg−1) by densitometric and visual analyses were 98.4 and 103.8%, respectively. The relative standard deviations (RSD) for densitometric and visual analysis varied from 1.1 to 24.9% and from 0.0 to 18.8%, respectively. The RSD for naturally contaminated samples by densitometry (three levels of contamination, n = 3) varied from 11.1 to 18.1%. The correlation (R2) between high-performance liquid chromatography (HPLC) and densitometry, and between visual and densitometric analysis for spiked samples were > 0.99. The limit of detection (LOD) of the method was 0.5 µg kg−1 for normal TLC. Toluene-ethyl acetate-88% formic acid (6:3:1 v/v/v) and acetonitrile-methanol-water-glacial acetic acid (35:35:29:10 v/v/v/v) were regarded as the suitable TLC solvents for eluting both standards and samples on normal and RP TLC plates, respectively. Toluene-acetic acid (99:1 v/v) was chosen as the spotting solvent among several others for giving the best sensitivity and resolution of OTA on TLC plates as well as the best recovery of OTA from standard and sample extract residues. Preliminary studies were carried out to investigate the reuse of the immunoaffinity column and the interference of caffeine in the OTA recovery.

A method for determining fumonisin B1 in corn using immunoaffinity column clean-up and thin layer chromatography/densitometry.

A method for determining fumonisin B1(FB1) in corn was developed and the clean-up optimized in order to give an extract suitable for one-dimensional thin layer chromatographic (TLC) analysis. FB1 was extracted with a solution of methanol:water (80:20,v/v), purified through an immunoaffinity column and separated on a C18 reversed phase TLC plate. The FB1 was visualized with 0.1mol/l sodium tetraborate, 0.40mg/ml fluorescamine in acetonitrile and 0.01mol/l boric acid:acetonitrile (2:3,v/v) for fluorescence detection, and quantified by densitometric analysis. Water, acetonitrile:water (1:1v/v) and acetonitrile:water (4:1v/v) were evaluated as TLC solvents for running both standards and samples together with derivatization procedures aimed at improving separation, resolution, sensitivity and linearity. The mean recovery for FB1 for spiked samples was found to be 85% and the linear equation of standard calibration curve by densitometric analysis gave an r2 value higher than 0.99. The maximum coefficient of variation for replicate analysis of spiked samples was 19%. The absolute amount of FB1 standard detectable on a TLC plate was 2 ng, giving a detection limit for the method of 0.1mg/kg. The method has been shown to be robust in the application of FB1 monitoring in corn (214 samples) collected in different regions of the country. FB1 was detected in 99% of these samples in the range of 0.2 to 6 mg/kg.

Determination of aflatoxin risk components for in-shell Brazil nuts.

A study was conducted on the risk from aflatoxins associated with the kernels and shells of Brazil nuts. Samples were collected from processing plants in Amazonia, Brazil. A total of 54 test samples (40 kg) were taken from 13 in-shell Brazil nut lots ready for market. Each in-shell sample was shelled and the kernels and shells were sorted in five fractions: good kernels, rotten kernels, good shells with kernel residue, good shells without kernel residue, and rotten shells, and analysed for aflatoxins. The kernel : shell ratio mass (w/w) was 50.2/49.8%. The Brazil nut shell was found to be contaminated with aflatoxin. Rotten nuts were found to be a high-risk fraction for aflatoxin in in-shell Brazil nut lots. Rotten nuts contributed only 4.2% of the sample mass (kg), but contributed 76.6% of the total aflatoxin mass (µg) in the in-shell test sample. The highest correlations were found between the aflatoxin concentration in in-shell Brazil nuts samples and the aflatoxin concentration in all defective fractions (R 2 = 0.97). The aflatoxin mass of all defective fractions (R 2 = 0.90) as well as that of the rotten nut (R 2 = 0.88) were also strongly correlated with the aflatoxin concentration of the in-shell test samples. Process factors of 0.17, 0.16 and 0.24 were respectively calculated to estimate the aflatoxin concentration in the good kernels (edible) and good nuts by measuring the aflatoxin concentration in the in-shell test sample and in all kernels, respectively.

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).

Modelling uncertainty estimation for the determination of aflatoxin M1 in milk by visual and densitometric thin-layer chromatography with immunoaffinity column clean-up

The uncertainty of aflatoxin M1 concentration in milk, determined by thin-layer chromatography (TLC) with visual and densitometric quantification of the fluorescence intensities of the spots, was estimated using the cause-and-effect approach proposed by ISO GUM (Guide to the expression of uncertainty in measurement) following its main four steps. The sources of uncertainties due to volume measurements, visual and densitometric TLC calibration curve, allowed range for recovery variation and intermediary precision to be taken into account in the uncertainty budget. For volume measurements the sources of uncertainties due to calibration, resolution, laboratory temperature variation and repeatability were considered. For the quantification by visual readings of the intensity of the aflatoxin M1 in the TLC the uncertainty arising from resolution calibration curves was modelled based on the intervals of concentrations between pairs of the calibration standard solutions. The uncertainty of the densitometric TLC quantification arising from the calibration curve was obtained by weighted least square (WLS) regression. Finally, the repeatability uncertainty of the densitometric peak areas or of the visual readings for the test sample solutions was considered. For the test samples with aflatoxin M1 concentration between 0.02 and 0.5 µg l−1, the relative expanded uncertainties, with approximately 95% of coverage probability, obtained for visual TLC readings were between 60% and 130% of the values predicted by the Horwitz model. For the densitometric TLC determination they were about 20% lower. The main sources of uncertainties in both visual and densitometric TLC quantification were the intermediary precision, calibration curve and recovery. The main source of uncertainty in the calibration curve in the visual TLC analysis was due to the resolution of the visual readings, whereas in the densitometric analysis it was due to the peak areas of test sample solutions followed by the intercept and slope uncertainties of the calibration line.

Producing a sulfamethazine quality control material under the framework of ISO/CD Guide 80

The increasing use of antimicrobial agents such as sulfonamides by the pig industry is of concern, since residues in both pork and its by-products, when derived from animals treated improperly, can endanger human health. The aim of this study was to establish the production conditions and to evaluate the homogeneity and the stability of sulfamethazine in porcine liver quality control material, produced ‘in-house’ for use in ring tests of the laboratory network of residues and contaminants of the Ministry of Agriculture, Livestock and Food Supply, Brazil. In the process of preparing the material, a FOSS blender was used, where the samples were ground to obtain a homogeneous mass, which was packed in polypropylene bottles. The material resulting from this process of homogenisation was sampled and analysed by LC/MS/MS. The analytical results were statistically evaluated by one-way ANOVA. According to statistical evaluation, the material produced was considered homogeneous, with 95% confidence. Stability tests were performed with the bottles stored under the specified storage conditions. They were randomly selected and analysed in duplicate by the same analytical method as the homogeneity study. The analytical results were statistically evaluated by the procedures for a stability check described in ISO 13528:2005, indicating that the material was unstable under the conditions of storage.