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.     

Survey of 1975 wheat and soybeans for aflatoxin, zearalenone, and ochratoxin

Wheat samples (102 lots) were collected from Virginia, North Carolina, southeastern Missouri, southern Illinois, and Kentucky. Soybean samples (180 lots) were collected from Virginia, Illinois, Iowa, Minnesota, Nebraska, Alabama, Arkansas, and Texas. Samples of both commodities were analyzed for zearalenone, aflatoxin, and ochratoxin by the Eppley method. None of the 3 mycotoxins was detected in soybeans. Aflatoxins and ochratoxin A were not detected in wheat, but zearalenone was detected in 19 of 42 samples collected in Virginia. Half of the Virginia samples were collected because they were mold-damaged. Zearalenone levels ranged from 0.36 to 11.05 ppm; the identity of the zearalenone was confirmed by gas-liquid chromatography and mass spectroscopy. Gibberella zea infection (6-60%) was detected in all of the zearalenone-positive samples; 6-60% of the kernels in the samples tested contained G. zea.     

Co-occurrence of ochratoxin A and citrinin in cereals from Bulgarian villages with a history of Balkan endemic nephropathy

Cereal samples were collected in 1998 from Bulgarian villages without [control village (C), n = 20] or with [endemic villages (E); E1, n = 21; E2, n = 30; E3, n = 23] a history of Balkan endemic nephropathy (BEN). Sampling included foods (wheat, corn) and feeds (barley, oats, wheat bran). Analysis of ochratoxin A and citrinin was done by enzyme immunoassays (EIA), with detection limits of 0.5 and 5 ng/g, respectively. Ochratoxin A-positive results were confirmed by HPLC after immunoaffinity chromatography. Highest toxin levels were found in wheat, wheat bran, and oats. For ochratoxin A, the percentages of positives were 35% (C), 29% (E1), 30% (E2), and 47% (E3), the mean/median values of positives were 1.5/1.3 ng/g (C), 11/1.6 ng/g (E1), 18/1.6 ng/g (E2), and 3.5/1.5 ng/g (E3). For citrinin, 5.0% (C), 14% (E1), 3.3% (E2), and 13% (E3) were positive, and the mean/median values were 6.1/6.1 ng/g (C), 180/83 ng/g (E1), 10/10 ng/g (E2), and 84/20 ng/g (E3). Highest concentrations of ochratoxin (maximum = 140 ng/g) and citrinin (maximum = 420 ng/g) were found in samples from endemic villages. Co-contamination with ochratoxin A and citrinin was found for one sample (14% of positives) from village C and for six samples (22% of positives) from villages E1-E3. Citrinin levels in these samples were 2-200 times higher than those of ochratoxin A.     

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.     

Browning indicators in model systems and baby cereals.

In many countries, baby cereals are the first solid food given to 3-4-month-old babies after weaning and to infants aged 6-12 months. Various simple technologies are traditionally used in the processing of cereals, including toasting, hydrolysis, and drying. In this study color and hydroxymethylfurfural (HMF) assays have been used to evaluate heat effects induced during the manufacture of these foods. The baby cereals analyzed were wheat, rice, oat, and four mixtures of flours. No HMF was detected in the raw flours. Toasting the flours increased HMF values by between 1.1 and 4.53 mg/kg and color (DeltaE) values by 2.51-9.34. The drying step increased HMF values by between 1.14 and 19.60 mg/kg. High values of HMF coincided with the addition of ingredients containing HMF. Color and HMF contents in sugar-amino acid model systems were much higher than in sugar systems at temperatures >100 degrees C and low moisture content.     

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.     

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.     

Determination of aflatoxins, ochratoxin a, and zearalenone in mixed feeds, with detection by thin layer chromatography or high performance liquid chromatography

A sensitive, reliable, and economical method for the determination of 6 mycotoxins in mixed feeds is described. The feed is extracted with chloroform-water and the extract is cleaned up by using a disposable Sep-Pak silica cartridge. The procedure requires less time (15 min from sample extraction to extract preparation) and less solvent (approximately one-tenth) compared with conventional methods and is suitable for a fast, economical screen. Additional cleanup procedures, involving dialysis or extraction into base, are described for samples containing high levels of interfering compounds. Thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) with fluorescence detection are described for identification and estimation of mycotoxins. The method has been applied to a wide range of mixed feeds, including laboratory animal diets, and raw materials. The limit of detection is 1 microgram/kg for all mycotoxins measured by HPLC.     

Analysis of zearalenone in cereal and Swine feed samples using an automated flow-through immunosensor

The development of a sensitive flow-though immunosensor for the analysis of the mycotoxin zearalenone in cereal samples is described. The sensor was completely automated and was based on a direct competitive immunosorbent assay and fluorescence detection. The mycotoxin competes with a horseradish-peroxidase-labeled derivative for the binding sites of a rabbit polyclonal antibody. Control pore glass covalently bound to Prot A was used for the oriented immobilization of the antibody-antigen immunocomplexes. The immunosensor shows an IC(50) value of 0.087 ng mL(-1) (RSD = 2.8%, n = 6) and a dynamic range from 0.019 to 0.422 ng mL(-1). The limit of detection (90% of blank signal) of 0.007 ng mL(-1) (RSD = 3.9%, n = 3) is lower than previously published methods. Corn, wheat, and swine feed samples have been analyzed with the device after extraction of the analyte using accelerated solvent extraction (ASE). The immunosensor has been validated using a corn certificate reference material and HPLC with fluorescence detection.     

Rapid purification of fumonisins and their hydrolysis products with solid-phase extraction columns

Fumonisins B(3) and B(4) (FB(3) and FB(4)) were recovered from the 50:50 acetonitrile/water extract of corn cultures of a strain of Fusarium moniliforme that does not make FB(1) or FB(2) by stirring the extract with IRA-68, a weak anion-exchange resin. The fumonisins were desorbed with 5% acetic acid in the same solvent. After dilution with water, the desorbed fumonisins were separated into FB(3) (FB(3) and FA(3)) and FB(4) (FB(4), FC(4), and FA(4)) fractions with a tC(18) solid-phase extraction (SPE) cartridge. The FB(3) fraction was then separated into FB(3) and FA(3) by using an NH(2) SPE cartridge and eluting with 5% acetic acid and increasing amounts of acetonitrile in water. Finally, FB(1) and FA(3) were hydrolyzed with calcium hydroxide. After recovery from the reaction mixture using a tC(18) cartridge, the hydrolyzed and partially hydrolyzed analogues were separated and the unreacted fumonisins recovered by using an NH(2) cartridge, initially in the normal-phase mode with increasing amounts of water in acetonitrile and then in the reversed-phase mode after the addition of 5% acetic acid to the solvent and eluting in the reverse order.     

Mycotoxins in grain dust: method for analysis of aflatoxins, ochratoxin A, zearalenone, vomitoxin, and secalonic acid D

A multimycotoxin method is presented to quantitate aflatoxins, ochratoxin A, zearalenone, secalonic acid D, and vomitoxin in grain dust. Dust spiked with these mycotoxins was extracted sequentially with methylene chloride followed by acetonitrile-water (86 + 14). Vomitoxin was recovered in the latter extract and all other mycotoxins were recovered in the methylene chloride. Aflatoxins and ochratoxin were quantitated by fluorescence measurement on silica thin layer chromatographic plates. The other mycotoxins were quantitated after cleanup by reverse phase liquid chromatography and ultraviolet detection. Recoveries from dust spiked in the parts per billion (ng/g) range were approximately 80% (SD = 15-29%) for all mycotoxins. Minimum detectable amounts ranged from less than 0.5 ng/g for aflatoxins to 20 ng/g for zearalenone.     

Rapid screening method for aflatoxins and zearalenone in corn.

The methanol-water extraction system used in AOAC Method II for aflatoxins extracts both the aflatoxins and zearalenone from corn. Using this methanol-water extraction system as a base, a rapid screening procedure has been developed for these mycotoxins. The methanol-water extract is defatted with hexane and the pigments are precipitated with copper carbonate. The aflatoxins and zearalenone are subsequently extracted into chloroform and are then detected by half-plate TLC. An elapsed time of about 1 hr is required to analyze 1 sample. The sensitivity of the method is about 2 mu-g/kg for aflatoxin B-1 and 100 mu-g/kg for zearalenone.     

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.     

Wet digestion,manual and automated analysis of total sulphur in plant material

Abstract

Accurate and reproducible determinations of total sulphur can be made following digestion of plant samples with a nitric/ perchloric acid mixture containing dichromate and metavanadate. Care should be taken to control digestion temperatures. The occurrence of dense fumes of perchloric acid and the deposition of red chromium trioxide are not satisfactory indicators of the digestion endpoint. Digestion for lh. beyond the change in colour of the digest from green to olive‐brown was necessary, in our work, for consistent results.

Methods for reliable manual and automated determination of sulphate sulphur are described; the sulphur values obtained with our samples, were the same by both methods.     

Simple method for simultaneous detection of aflatoxin and zearalenone in corn.

A method is described for the simultaneous detection of alfatoxin and zearalenone in corn at 5 and 200 ppb, respectively. No evaporation of solvent is required and the procedure is simple enough to be considered for use at marketing locations. The presence of absence of these myocotoxins can be determined in 10-20 min/sample. The procedure involves an initial blender extraction with methanol, partitioning of fat and pigments into 1-1,2-trichlorotrifluoroethane (Freon-113) from an aqueous ammonium sulfate layer, followed by extraction of aflatoxin from the aqueous layer with chlorobenzene. The chlorobenzene extract can be spotted directly onto a thin layer chromatographic plate which requires only 4 min development. Concentrations of aflatoxin and zearalenone can be estimated by visual comparison of sample spots with standards.     

Survey of U.S. wheat for ochratoxin and aflatoxin.

A total of 291 hard red winter wheat samples, 286 hard red spring wheat samples, and 271 soft red winter wheat samples were analyzed for the presecne of ochratoxin and aflatoxin. Samples in all grades came from those collected during crop years 1970-1973 for grade determinations by the Agricultural Marketing Service, U.S. Department of Agriculture. Sensitivity limits of the analytical method as carried out were 1-3 ppb aflatoxin B1 and 15-30 ppb ochratoxin A. No aflatoxin was detected in any sample. Three samples of hard red winter wheat (Grades U.S. No. 4 and 5 and Sample Grade) contained ochratoxin A (trace, 35, and 25 ppb, respectively). Eight of the hard red spring wheats contained ochratoxin A (15-115 PPB); these were in Grades U.S. No. 4 and 5 and Sample Grade.     

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.     

Collaborative study of a method for the determination of ochratoxin A in green coffee.

A method for the semiquantitative determination of ochratoxin A in green coffee has been studied collaboratively by 11 laboratories. The average recovery for the 7 samples spiked at 3 levels of ochratoxin A was 69.1%, ranging from 60.5 to 85.6%. This is comparable to other visual thin layer chromatographic methods of mycotoxin detection. The method has been adopted as official first action for the determination of ochratoxin A in green coffee beans.     

Analysis of bisphenol A and alkylphenols in cereals by automated on-line solid-phase extraction and liquid chromatography tandem mass spectrometry

An on-line solid-phase extraction (SPE) following a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method was established for the simultaneous analysis of bisphenol A (BPA), nonylphenol (NP), and octylphenol (OP) in cereals (including rice, maize, and wheat). The target compounds were extracted by acetonitrile, purified by an automated on-line SPE cartridge, and analyzed by LC-MS/MS under the negative-ion mode. Mean recoveries fortified at three concentration levels ranged from 81.6 to 115.7%, and the coefficient of variation ranged from 4.6 to 19.9% (n = 6). The limits of quantification (LOQs) of the method were 0.5, 0.5, and 0.25 μg/kg for BPA, NP, and OP, respectively, in both rice and maize, while the LOQs in wheat were 0.5, 1.25, and 0.5 μg/kg for BPA, NP, and OP, respectively. This method was applied in the analysis of rice, maize, and wheat from a local market. As a result, NP occurred in all cereal samples at the concentration range of 9.4-1683.6 μg/kg and BPA was detected in a few samples.