High pressure liquid chromatographic determination of ochratoxin A and zearalenone in cereals.

A high pressure liquid chromatographic (HPLC) method has been developed for determining ochratoxin A and zearalenone in cereals. The sample is extracted with phosphoric acid and chloroform. The extract is cleaned by washing on a silica gel column with cyclohexane-ethylene dichloride-ethyl ether. After eluting zearalenone with chloroform, ochratoxin A is eluted with chloroform-formic acid. Zearalenone is extracted into alkaline solution, washed with chloroform, the pH is adjusted, and the zearalenone is extracted back into chloroform. Ochratoxin A is purified by chromatography on aqueous sodium biarbonate-Celite. The mycotoxins are determined by using a liquid chromatograph with 2 columns in series packed with Spherisorb ODS 10 micrometer and 5 micrometers, respectively. Ochratoxin A is detected with a speftrophotofluorometer, coupled in series with an ultra-violet detector for estimation of zearalenone. Detection limits are 1-5 micrograms/kg for ochratoxin A and 2 micrograms/kg for zearalenone.     

Aflatoxins and ochratoxin A in red paprika for retail sale in Spain: occurrence and evaluation of a simultaneous analytical method

Aflatoxins are the only mycotoxins with legal limits for spices in the European Union. A further limit for ochratoxin A is expected to be adopted soon. Thus, rapid simultaneous methods for quantifying the five mycotoxins are sought. Liquid extraction, immunoaffinity column cleanup, and HPLC-FD with a wavelength program were optimized for the analysis of the five mycotoxins in paprika, a complex fatty matrix. The limits of detection ranged from 0.3 to 0.6 microg/kg. Repeatability (RSDr) ranged from 7.9 to 13.4%, and recoveries were between 61.4 and 77.8%, in both cases at the lower spike level. Aflatoxins, when found, were far below the two legal limits of 5 microg/kg for aflatoxin B 1 and 10 microg/kg for total aflatoxins. Ochratoxin A was more frequently found, with a mean of 11.8 microg/kg, and in a more varied range (SD = 18.9 microg/kg). When an automation of the precolumn derivatization step was attempted, the procedure proved to be unfeasible, but experience derived from this trial and from the general employment of this reaction enables some comments on the possibilities and limitations of this procedure and on research for an alternative one to be made.     

Screening method for the detection of aflatoxin, ochratoxin, zearalenone, penicillic acid, and citrinin.

A modification of the official method for ochratoxins and a screening method for zearalenone, aflatoxin, and ochratoxin is described and expanded to include citrinin and penicillic acid. The method uses 0.5N phosphoric acidchloroform (1+10) in the initial extraction; the extract is divided and eluted from 2 columns to provide a quantitative thin layer chromatographic (TLC) method for aflatoxin and ochratoxin in corn and dried beans. Aflatoxin and zearalenone are eluted from one column and ochratoxin, penicillic acid, and citrinin from the other. Ochratoxin A recoveries are low (50%) in peanuts. Zearalenone, penicillic acid, and citrinin were qualitatively recovered from corn and beans; zearalenone and penicillic acid were recovered from peanuts but citrinin was not. Several TLC solvents were used to separate interferences.     

Determination of T-2 and HT-2 toxins in cereals including oats after immunoaffinity cleanup by liquid chromatography and fluorescence detection

A reliable method for the determination of T-2 toxin and HT-2 toxin in different cereals, including oats, as well as in cereal products was developed. After extraction with methanol/water (90/10, v/v) and dilution with a 4% NaCl solution, the toxins were purified with immunoaffinity columns, derivatized with 1-anthroylnitrile, separated by HPLC, and determined using fluorescence detection. Due to the unspecific derivatization reagents, validation parameters were matrix dependent: in the range 10-200 microg/kg, recovery rates of 74-120% with relative standard deviations between 0.5 and 20.3% were obtained. On average, the limit of quantitation was shown to be 8 microg/kg for each toxin. For naturally contaminated samples, comparable results were obtained when analysis was performed according to this method without derivatization as well as according to a method based on a SPE cleanup utilizing tandem mass spectrometric detection in both cases. Using aqueous acetonitrile as extractant resulted in incorrectly high toxin concentrations due to water absorption of dry samples and toxin accumulation in the organic phase in the subsequent phase separation of the extractant. Furthermore, when comparing the commercially available immunoaffinity columns for T-2 and HT-2 toxins, significant differences regarding capacity and cleanup performance were observed.     

Rapid, low cost thin-layer chromatographic screening method for the detection of ochratoxin A in green coffee at a control level of 10 microg/kg.

A thin-layer chromatographic (TLC) screening method was developed for the detection of ochratoxin A (OTA) in green coffee at a control level of 10 microg/kg. The method is based on extraction of OTA with a mixture of phosphoric acid and dichloromethane, purification by liquid-liquid partition into sodium hydrogen carbonate, separation by normal-phase TLC, and detection by visual estimation of fluorescence intensity under a UV lamp at 366 nm. The method was validated by performing replicate analyses of uncontaminated green coffee material spiked at 3 different levels of OTA (5, 10, and 20 microg/kg), and also by comparing results obtained on a series of test trial green coffees naturally contaminated with OTA (range 0.2 to 136.7 microg/kg) with those measured by a quantitative immunoaffinity/HPLC method. The agreement between the two methods was excellent, and neither false positive nor false negative results were recorded. This screening method is rapid, simple, robust, and very cheap, which makes it particularly well adapted for implementation in coffee-producing countries.     

Occurrence of ochratoxin A in cocoa products and chocolate

In this work, the occurrence of ochratoxin A (OTA) in 170 samples of cocoa products of different geographical origins was studied. An immunoaffinity column with HPLC separation was developed to quantify low levels of OTA in cocoa bean, cocoa cake, cocoa mass, cocoa nib, cocoa powder, cocoa shell, cocoa butter, chocolate, and chocolate cream with >80% recoveries. The method was validated by performing replicate analyses of uncontaminated cocoa material spiked at three different levels of OTA (1, 2, and 5 microg/kg). The data obtained were related on the acceptable safe daily exposure for OTA. The highest levels of OTA were detected in roasted cocoa shell and cocoa cake (0.1-23.1 microg/kg) and only at minor levels in the other cocoa products. Twenty-six cocoa and chocolate samples were free from detectable OTA (<0.10 microg/kg). In roasted cocoa powder 38.7% of the samples analyzed contained OTA at levels ranging from 0.1 to 2 microg/kg, and 54.8% was contaminated at >2 microg/kg (and 12 samples at >3 microg/kg). Ochratoxin A was detected in cocoa bean at levels from 0.1 to 3.5 microg/kg, the mean concentration being 0.45 microg/kg; only one sample exceeded 2 microg/kg (4.7%). In contrast, 51.2% of cocoa cake samples contained OTA at levels > or =2 microg/kg, among which 16 exceeded 5 microg/kg (range of 5-9 microg/kg). These results indicate that roasted cocoa powder is not a major source of OTA in the diet.     

Natural occurrence of ochratoxin A and antioxidant activities of green and roasted coffees and corresponding byproducts

Ochratoxin A is an important mycotoxin that can enter the human food chain in cereals, wine, coffee, spices, beer, cocoa, dried fruits, and pork meats. Coffee is one of the most common beverages and, consequently, it has a potential risk factor for human health related to ochratoxin A exposure. In this study, coffee and corresponding byproducts from seven different geographic regions were investigated for ochratoxin A natural occurrence by HPLC-FLD, nutritional characterization, and antioxidant activities by spectrophotometric assay. The research focused on composition changes in coffee during the processing step "from field to cup". Costa Rica and Indian green coffees were the most contaminated samples, with 13 and 11 microg/kg, respectively, while the Ethiopian coffee was the least contaminated, with 3.8 microg/kg of ochratoxin A. The reduction of ochratoxin A contamination during the roasting step was comparable for any samples that were considered under the recommended level of 4 microg/kg. Total dietary fibers ranged from 58.7% for Vietnam and 48.6% for Ivory Coast in green coffees and ranged from 58.6% for Costa Rica to 61.2% for India in roasted coffee. Coffee silverskin byproduct obtained from Ivory Coast was the highest, with 69.2 and 64.2% of insoluble dietary fibers, respectively.     

Fungal microflora and ochratoxin a risk in French vineyards

To evaluate the ochratoxin A risk in French vineyards, five winemaking regions were investigated. An exhaustive survey of the fungal microflora of 60 grape samples was carried out at two development stages of the berries: end of veraison and harvest time. Potentially toxinogenic fungi isolated from grapes were assessed in vitro for ochratoxin A production. Ochratoxin A was also quantified in musts by high-performance liquid chromatography after cleanup on immunoaffinity columns. Among the 90 species identified, almost half are listed as mycotoxin producers, but only 2 are potentially ochratoxinogenic: Aspergillus carbonarius and Aspergillus niger. Among these strains, only A. carbonarius, isolated from the Languedoc region at harvest time, was found to produce ochratoxin A. These results were in accordance with the presence of ochratoxin A in French southern region musts (0.01-0.43 microg/L) and confirmed the major implication of A. carbonarius in ochratoxin A contamination.     

Rapid thin layer chromatographic method for determining aflatoxin B1, ochratoxin A, and zearalenone in corn

A multimycotoxin thin layer chromatographic method is described for the analysis of corn. Aflatoxins are extracted from the samples with acetonitrile-water, and sodium bicarbonate is added to separate the acidic ochratoxin from zearalenone and aflatoxin B1. After chloroform extraction, 1N NaOH is added to separate zearalenone and aflatoxin B1. The separated mycotoxins are spotted on TLC plates, which are then examined under ultraviolet light. The following recoveries (%) were obtained for corn samples: aflatoxin B1 71, ochratoxin A 87, and zearalenone 85. The limits of detection for the respective mycotoxins were 2, 40, and 200 ppb.     

Comparative study of three different methods for the determination of aflatoxins in tahini

Aflatoxins spiked at three different levels (6.5, 13.0, and 19.5 microg/kg) in tahini, a sesame butter, were analyzed by using three different methods: high-performance liquid chromatography (HPLC), fluorometry, and enzyme-linked immunosorbent assay (ELISA). An immunoaffinity column was used for cleanup and purification of extracts prior to detection by HPLC and fluorometry. All methods were statistically evaluated for accuracy, precision, and simple correlations. Additionally, 14 tahini samples randomly obtained from Turkish retail markets were analyzed using an immunoaffinity column cleanup procedure coupled with the HPLC detection method. The fluorometric determination method involving an immunoaffinity column cleanup step was found to be highly correlated with the HPLC method (r = 0.978). Both methods were found to be effective due to their high recoveries and low variance for the prediction of total aflatoxin contamination in tahini samples. The ELISA method, due to its high variation in replicates, was found to be applicable only as a screening method. The survey study demonstrated the need for control of aflatoxin contamination of foodstuffs involving sesame seeds as an ingredient.     

Survey of aflatoxins, ochratoxin A, zearalenone, and sterigmatocystin in some Brazilian foods by using multi-toxin thin-layer chromatographic method

A previously published method for ochratoxin A was evaluated and proved appropriate for simultaneous determination of aflatoxins, ochratoxin A, sterigmatocystin, and zearalenone, with considerable savings in time and reagent costs. The detection limits were 2, 5, 15, and 55 micrograms/kg, respectively. The recoveries and coefficients of variation obtained with artificially contaminated samples were 91-101% and 0-16% for aflatoxin B1, 98-117% and 0-17% for sterigmatocystin, and 96-107% and 0-17% for zearalenone, respectively. The coefficients of variation for naturally contaminated samples (aflatoxins in rice and ochratoxin A in beans) ranged from 0 to 8%. The method was used to survey 296 samples that included 10 cultivars of dried beans, 8 types of corn products, 3 types of cassava flour, and both polished and parboiled rice between May 1985 and June 1986 in Campinas, Brazil. Only aflatoxin B1 (9 samples, 20-52 micrograms/kg), aflatoxin G1 (4 samples, 18-31 micrograms/kg), and ochratoxin A (5 samples, 32-160 micrograms/kg) were found. The average contamination percentage was 4.7%; beans showed the highest (6.6%) and rice showed the lowest (3.3%) incidence rates. Zearalenone and sterigmatocystin were not detected. Positive samples were confirmed by chemical derivatization, corroborated by development in 3 solvent systems.     

Production of polyclonal antibodies against ochratoxin A and its detection in chilies by ELISA

Polyclonal antibodies were produced for Ochratoxin A (OA) by injecting OA-bovine serum albumin (BSA) conjugate subcutaneously at multiple sites into a New Zealand White inbred rabbit. Antiserum could be used at a dilution exceeding 1:100 000 in an indirect competitive enzyme-linked immunosorbent assay (ELISA), and detected OA concentrations up to 0.1 ng/mL. The 50% inhibition binding (I(50)) of OA was 5 ng/mL. Antibodies did not react with ochratoxin B, coumarin, 4-hydroxycoumarin, L-phenylalanine, and aflatoxin B1. OA contamination in chilies (Capsicum annum L.) collected from commercial markets and cold storage units was determined. The mean recoveries from OA-free chilies spiked with 1 to100 microg of OA per kg of chili sample were 90-110% with a standard deviation of <10%. Of 100 chili samples tested, 26 were found to contain over 10 microg/kg of OA. In 12 samples the OA concentration varied from 10 to 30 microg/kg, in 10 samples from 30 to 50 microg/kg, in 3 samples from 50 to100 microg/kg, and in one sample it was 120 microg/kg. This is the first record in India of OA in chilies, a major component of cooked foods in this country, and it is noteworthy that OA contamination exceeded the permissible limit for human consumption of less than 20 microg/kg in over 26% of the market samples tested.     

Analysis of the Fusarium mycotoxins fusaproliferin and trichothecenes in grains using gas chromatography-mass spectrometry

A method is described using gas chromatography-mass spectrometry (GC-MS) for the simultaneous detection of the Fusarium mycotoxins fusaproliferin and seven trichothecenes from grains. Sample purification of the raw extract was carried out with commercial solid phase extraction columns, and the recovery of the more polar analytes was increased by rinsing the column with acetonitrile. A significant matrix effect was found for the analysis of fusaproliferin and trichothecenes; thus, the calibrants should be prepared in a blank matrix. The response was linear in the range used. The mean recovery for fusaproliferin was 60.4 or 62.9%, depending on the spiking level. With respect to the trichothecenes, the recovery was generally higher (70.2-125.3%). The method proved to be repeatable for the analysis of fusaproliferin and trichothecenes. The limit of detection for fusaproliferin in the blank matrix mixture was 50 microg/kg, and that for trichothecenes was 5-15 microg/kg. Thirty-eight Finnish grain samples were analyzed for fusaproliferin and trichothecenes with the method developed. Fusaproliferin was not detected in any of the samples. The mean levels of deoxynivalenol, 3-acetyldeoxynivalenol, nivalenol, HT-2 toxin, and T-2 toxin in Finnish grain samples were 272, 17, 150, 40, and <20 microg/kg, respectively.     

Automated fluorometric determination of thiamine and riboflavin in infant formulas.

Commercial infant formulas were analyzed simultaneously for thiamine and riboflavin by an automated fluorometric method and by the AOAC manual fluorometric methods. For 10 products, the mean thiamine and riboflavin results determined using the automated method ranged from 104 to 113% and 90 to 112%, respectively, of those by the AOAC manual methods. The coefficients of variation for thiamine and riboflavin ranged from 1.05 to 3.90% and 0.60 to 2.48%, respectively, for the automated methods, and 1.48 to 3.86% and 0.69 to 10.9%, respectively, for the manual methods. Using the automated method, mean recoveries of thiamine and riboflavin added to samples were 103 and 104%, respectively. The automated method used a common sample preparation to determine both thiamine and riboflavin, and gave results equivalent to, or better than, those obtained by the manual methods.     

Stable isotope dilution analysis of the fusarium mycotoxin zearalenone

Zearalenone is a secondary metabolite produced by molds of the Fusarium genus. Beside its nonsteroidal molecular structure, zearalenone has estrogenic activity and can disrupt the function of the endogenous hormone 17beta-estradiol in animals and possibly in humans. It can frequently be found in all major cereal grains as well as in processed food. Because of the estrogenic properties of zearalenone and its metabolites, legal regulations are installed in the European Union setting maximum levels in cereals and cereal products. Routine analysis of zearalenone in various commodities is carried out by HPLC with fluorescence detection, but due to the development of multi-mycotoxin methods and the reduced sample cleanup, HPLC-MS/MS has become a fast and efficient alternative. However, to achieve a reliable quantitation with this technique suitable internal standards are required. This paper reports the synthesis of stable isotope labeled 3,5- d 2-zearalenone (ZON) as internal standard for stable isotope dilution analysis. Furthermore, a method for the analysis of zearalenone by HPLC-MS/MS using 3,5- d 2-zearalenone as IS has been developed. Fifteen cereal products from the German retail markets were analyzed, of which seven contained ZON in levels from 4.9 to 45.0 microg/kg.     

Occurrence of zearalenone-4-beta-D-glucopyranoside in wheat

An LC-MS method was developed for the analysis of zearalenone-4-beta-D-glucopyranoside and zearalenone in wheat (Triticum aestivum). The limit of determination for zearalenone-4-beta-D-glucopyranoside and zearalenone was 10 microg/kg. The recovery rates were calculated to be 69% and 89% at a concentration of 100 microg/kg for zearalenone-4-beta-D-glucopyranoside and zearalenone, respectively. Twenty-four Bavarian wheat samples from a 1999 harvest were analyzed. Zearalenone was present in 22 of 24 field samples, the levels ranged from 11 to 860 microg/kg. Zearalenone-4-beta-D-glucopyranoside was found in 10 of the zearalenone positive samples (42%) at levels ranging from 17 to 104 microg/kg. The amounts of zearalenone-4-beta-D-glucopyranoside were correlated to those of zearalenone (r2 = 0.86, b = 0.10). After gastrointestinal hydrolyzation, zearalenone-4-beta-glucopyranoside might be implicated in the development of a zearalenone-syndrome. Therefore, more attention should be focused on conjugated mycotoxins in food and feed.     

Automated fluorometric determination of vitamin C in foods.

Two automated fluorometric methods were compared with the official AOAC methods for determining vitamin C in fortified ready-to-eat cereals, fruits, vegetables, baby foods, flour products, pet foods, meats, frozen dinners, juices, and nutritional health bars. Each sample was analyzed in duplicate on 2 days and included a recovery study on all products. Egberg's semiautomated method (Method 1) and Roy's automated method (Method 2) were compared with the manual AOAC tritrimetric and fluorometric methods. The correlation factor for Methods 1 and 2 were 0.999 and 0.979, respectively, when compared with the AOAC methods. The recovery study showed average recoveries of 97.8% for Method 1, 99.3% for Method 2, and 100.6% for the AOAC methods. The results suggest that Method 1 is the method of choice for the majority of the products analyzed.     

Simultaneous immunoaffinity column cleanup and HPLC analysis of aflatoxins and ochratoxin A in Spanish bee pollen

Bee pollen is a major substrate for mycotoxins growth when no prompt and adequate drying is performed by the beekeeper after collection by bees. Regulatory limits for aflatoxins and ochratoxin A are currently in force in the European Union for a rising list of foodstuffs, but not for this. An immunoaffinity column cleanup process has been applied prior to the analysis of aflatoxins B(1), B(2), G(1), and G(2) and ochratoxin A (OTA). Optimization of the HPLC conditions has involved both a gradient elution and a wavelength program for the separation and fluorimetric quantitation of all five mycotoxins at their maximum excitation and emission values of wavelength in a single run. The higher limit of detection (mug/kg) was 0.49 for OTA and 0.20 for aflatoxin B(1). Repeatability (RSDr) at the lower limit tested ranged from 9.85% for OTA to 6.23% for aflatoxin G(2), and recoveries also at the lower spiked level were 73% for OTA and 81% for aflatoxin B(1). None of the 20 samples assayed showed quantifiable values for the five mycotoxins.     

Quantification of zearalenone in various solid agroenvironmental samples using D6-zearalenone as the internal standard

Because of its pronounced estrogenicity, zearalenone may be of concern not only in the aqueous but also in the terrestrial environment. Therefore, we developed several analytical methods to quantify zearalenone in different solid matrices of agroenvironmental relevance (i.e., plant organs, soil, manure, and sewage sludge). The use of D(6)-zearalenone as the internal standard (IS) was essential to render the analytical method largely matrix-independent because it compensated for target analyte losses during extract treatment and ion suppression during ionization. Soil and sewage sludge samples were extracted with Soxhlet, whereas plant material and manure samples were extracted by liquid solvent extraction at room temperature. Absolute recoveries for zearalenone were 70-104% for plant materials, 105% for soil, 76% for manure, and 30% for sewage sludge. Relative recoveries ranged from 86 to 113% for all matrices, indicating that the IS was capable to largely compensate for losses during analysis. Ion suppression, between 8 and 74%, was in all cases compensated by the IS but influenced the method quantification levels. These were 3.2-26.2 ng/g(dryweightdw) for plant materials, 0.7 ng/g(dw) for soil, 12.3 ng/g(dw) for manure, and 6.8 ng/g(dw) for sewage sludge. Plant material concentrations varied from 86 ng/g(dw) to more than 16.7 microg/g(dw), depending on the organ and crop. Soil concentrations were between not detectable and 7.5 ng/g(dw), depending on the sampling depth. Zearalenone could be quantified in all manure samples in concentrations between 8 and 333 ng/g(dw). Except for two of the 85 investigated sewage sludge samples, zearalenone concentrations were below quantification limit.     

Mycotoxins in animal feedstuffs: sensitive thin layer chromatographic detection of aflatoxin, ochratoxin A, sterigmatocystin, zearalenone, and T-2 toxin

Improvements have been made to a previously described multi-mycotoxin method that involved a membrane cleanup step. Using 2-dimensional thin layer chromatography and appropriate solvent systems, aflatoxin B1 can be detected in mixed feedstuffs and various ingredients at levels ranging from 0.1 to 0.3 microgram/kg. Corresponding detection limits for ochratoxin A and sterigmatocystin are 5 to 20 microgram/kg and for T-2 toxin and zearalenone 20 to 200 microgram/kg.