Thin layer chromatographic determination of aflatoxins, ochratoxins, sterigmatocystin, zearalenone, citrinin, T-2 toxin, diacetoxyscirpenol, penicillic acid, patulin, and penitrem A.

A general method is described for determining 16 mycotoxins in mixed feeds and other food products used in the manufacture of these feedstuffs. The mycotoxins are extracted and cleaned up by extracting with solvents of different pH. Thin layer chromatography is used to separate the toxins; toxins are then quantitated by the limit detection method. The minimum detectable concentration of mycotoxins in various products is: aflatoxin B1 or G1, 4--5 micrograms/kg; ochratoxin A or ethyl ester A 140--145 micrograms/kg; citrinin 600--750 micrograms/kg; zearalenone, 410--500 micrograms/kg; sterigmatocystin, 140--145 micrograms/kg; diacetoxyscirpenol, 2400--2600 micrograms/kg; T-2 toxin, 800--950 micrograms/kg; patulin, 750--800 micrograms/kg; penitrem A 14,000--14,500 micrograms/kg; penicillic acid 3400--3650 micrograms/kg.     

Quantitative liquid chromatographic method using fluorescence detection for determining zearalenone and its metabolites in blood plasma and urine

The liquid chromatographic (LC) method described, suitable for use with both blood plasma and urine, is applicable for determination of zearalenone and alpha-zearalenol at levels as low as 0.5 ng/mL plasma and 5 ng/mL urine. The sample is incubated overnight with beta-glucuronidase to analyze for both conjugated and unconjugated forms of zearalenone. The next day, the sample is acidified with H3PO4, extracted with chloroform, and evaporated to dryness. The residue is dissolved in toluene and loaded onto a silica gel cartridge which is washed with toluene and eluted with toluene-acetone (88 + 12). The eluate is evaporated, and the residue is dissolved in chloroform, extracted with 0.18M NaOH, neutralized with H3PO4, and re-extracted with chloroform. The chloroform extract is evaporated, dissolved in mobile phase for LC, and injected onto a normal phase column under the following chromatographic conditions: mobile phase of water-saturated dichloromethane containing 2% 1-propanol, and fluorescence detector, excitation wave-length 236 nm, and 418 nm cut-off emission filter. Recoveries of zearalenone and its metabolites from blood plasma and urine are 80-89% in the range 2.0-10 ng standard/mL plasma, and 81-90% in the range 10-30 ng standard/mL urine. This method was used to analyze blood and urine samples from a pig fed zearalenone-contaminated feed (5 mg/kg), corresponding to 80 micrograms/kg body weight. Zearalenone was rapidly metabolized to alpha-zearalenol, which appeared in the blood only 30 min after feeding. Almost all zearalenone and alpha-zearalenol was found conjugated with glucuronic acid in both blood plasma and urine.     

Liquid chromatographic determination of ochratoxin A in coffee beans and coffee products

A liquid chromatographic method for the determination of ochratoxin A in coffee beans (green and roast), instant coffee, and coffee drink is described. The sample is subjected to extraction with methanol-1% aqueous sodium bicarbonate (1 + 1) and C18 cartridge cleanup. The extract is chromatographed on a Nucleosil 5C18 column with a mobile solvent of acetonitrile-water-0.2M phosphate buffer pH 7.5 (50 + 47 + 3) containing 3 mM cetyltrimethylammonium bromide as an ion-pair reagent. Ochratoxin A is detected with a fluorometer (excitation 365 nm, emission 450 nm). The sensitivity was increased 20-fold by using ion-pair resolution. The detection limits corresponded to 2 micrograms/kg for coffee beans, 5 micrograms/kg for instant coffee, and 0.2 microgram/kg for coffee drink. The recoveries from coffee products were generally better than 80.7% and the relative standard deviations were 3.43-5.93%. The peak coinciding with ochratoxin A can be confirmed by treatment using alcohol (methanol, ethanol, or n-propanol) and H2SO4.     

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.     

Application of manual and automated systems for purification of ochratoxin a and zearalenone in cereals with immunoaffinity columns.

A manual vacuum manifold and an automated solid phase extraction (ASPEC) system were applied for purification of ochratoxin A and zearalenone in wheat, rye, barley, and oat samples with immunoaffinity columns followed by separation with a high-performance liquid chromatograph and fluorescence detection. The immunoaffinity columns for manual sample purification were purchased from a different manufacturer than were those for the automated system. The limit of detection (LOD) for the method for ochratoxin A with a vacuum manifold and ASPEC was 0.1 microg/kg. For the method for zearalenone, the LODs were 1.5 microg/kg with a vacuum manifold and 3 microg/kg with ASPEC. For the methods for ochratoxin A at spiking levels of 0.6 and 2.5 microg/kg, mean recoveries for different cereals varied from 68 to 106%. For the methods for zearalenone, mean recoveries varied from 78 to 117% at spiking levels of 9 and 25 microg/kg. The relative standard deviations of repeatability with various cereals employing both methods were 2-15 and 2-19% for ochratoxin A and zearalenone, respectively.     

Determination of ochratoxin A by monoclonal antibody-based enzyme immunoassay

A simple and rapid indirect enzyme-linked immunosorbent assay was developed for the quantitative determination of ochratoxin A in barley after the successful production of a high affinity, specific monoclonal antibody. A rapid sample cleanup was achieved by extracting ochratoxin A from barley with chloroform and partitioning the toxin into bicarbonate buffer; the buffer solution was then added directly to the assay plate and ochratoxin A content was assessed. Recoveries were greater than 85% and detection limits were 5 micrograms ochratoxin A/kg barley.     

Rapid, sensitive liquid chromatographic method for determination of zearalenone and alpha- and beta-zearalenol in wheat

A rapid, sensitive liquid chromatographic (LC) method is described for quantitative determination of zearalenone and alpha- and beta-zearalenol in wheat. The procedure incorporates an internal standard, zearalenone oxime, to facilitate quantitation and automated analysis. A sample, buffered with pH 7.8 phosphate, is extracted with water-ethanol-chloroform (2 + 50 + 75) and cleaned up. The final residue is dissolved in LC mobile phase and injected onto a reverse phase RP-18 column under the following conditions: water-methanol-acetonitrile (5 + 3 + 2) mobile phase; fluorescence (excitation wavelength 236 nm, 418 nm cut-off emission filter) and UV (254 nm, range 0.0025 AU) detectors. The limit of detectability (twice background) is 0.5 ng for zearalenone and alpha-zearalenol standards on the fluorescence detector and 4 ng for beta-zearalenol on the UV detector, which is equivalent to 20 micrograms zearalenone and 20 micrograms alpha-zearalenol/kg, and 160 micrograms beta-zearalenol/kg feed. Standard curves are linear over the range 0-35 ng zearalenone and alpha-zearalenol on the fluorescence detector and 0-50 ng beta-zearalenol on the UV detector. Recoveries of all compounds are 87.5-101% in the range 0.1-3.0 mg/kg (ppm).     

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.     

Simultaneous extraction and fractionation and thin layer chromatographic determination of 14 mycotoxins in grains.

A simple, systematic analytical method for multiple mycotoxins was developed for detecting 14 mycotoxins; aflatoxins B1, B2, G1, and G2, sterigmatocystin, T-2 toxin, diacetoxyscirpenol, neosolaniol, fusarenon X, zearalenone, ochratoxin A, citrinin, luteoskyrin, and rugulosin. These mycotoxins were extracted with 20% H2SO4-4% KCl-acetonitrile (2 + 20 + 178), defatted with isooctane, and transferred to chloroform. The chloroform extract was cleaned up by silica gel column chromatography; the first 10 toxins were eluted with chloroform-methanol (97 + 3) and the remaining 4 toxins with benzene-acetone-acetic acid (75 + 20 + 5). Each fraction was analyzed by thin layer chromatography for the final determination. The method has been applied to polished rice, rough rice, corn, wheat, and peanuts as an analytical screening procedure. The detection limits in these commodities ranged from 10.00 to 800.0 microgram/kg, depending on the mycotoxin, but all limits were superior to those obtained for the individual mycotoxins by using other methods.     

Simultaneous thin layer chromatographic determination of aflatoxin B1 and ochratoxin A in black olives

A screening method has been developed for simultaneous determination of aflatoxin B1 and ochratoxin A in black olives. The technique includes extraction of both mycotoxins with aqueous methanol, cleanup using lead acetate, defatting with hexane, partitioning in chloroform, and thin layer chromatography. Detection limits are 5-7 micrograms aflatoxin B1 and 20 micrograms ochratoxin A/kg.     

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.     

Rapid thin layer chromatographic determination of zearalenone in corn, sorghum, and wheat

A rapid method is described for determining zearalenone in corn, sorghum, and wheat. The mycotoxin is extracted with a mixture of acetonitrile and 4% KCl in HCl. The extract is cleaned up with isooctane, evaporated, and redissolved in chloroform. Zearalenone is separated by thin layer chromatography; identity is confirmed with various developing solvents and spray reagents. Zearalenone is then quantitated by the limit detection method. The minimum detectable concentration is 140-160 micrograms/kg when aluminum chloride solution is used as spray reagent, and 85-110 micrograms/kg when Fast Violet B salt is used as spray reagent.     

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.     

Liquid chromatographic determination of aflatoxins, ochratoxin A, and zearalenone in grains, oilseeds, and animal feeds by post-column derivatization and on-line sample cleanup

A liquid chromatographic method using on-line sample cleanup, reverse flow analytical column loading, gradient elution, and postcolumn derivatization with iodine permits direct, rapid determination of aflatoxins B1, B2, G1, and G2, as well as ochratoxin A and zearalenone. Limits of quantitation are 5 ppb for the aflatoxins and ochratoxin A and 30 ppb for zearalenone. This procedure performs well as a multimycotoxin screen for cereal grains and oilseeds, with more limited success in complete animal feeds.     

Chemical confirmatory tests for ochratoxin A, citrinin, penicillic acid, sterigmatocystin, and zearalenone performed directly on thin layer chromatographic plates

Published tests have been improved and a new procedure is described for chemical confirmation of mycotoxins directly on thin layer plates. After extraction and preliminary cleanup chromatography with n-hexane or chloroform, the mycotoxins ochratoxin A, citrinin, penicillic acid, sterigmatocystin, and zearalenone were easily separated by thin layer chromatography (TLC) using toluene-ethyl acetate-90% formic acid (6 + 3 + 1) developing solvent. In chemical confirmatory methods, the developed chromatogram was exposed to vapors of pyridine, acetic anhydride, or a mixture, or the mycotoxins were over-spotted. With this treatment, ochratoxin A, citrinin, penicillic acid, and zearalenone were converted to new fluorescent compounds, and observed under 365 nm light after re-chromatography with the same developing solvent. Sterigmatocystin was confirmed chemically using TLC plates impregnated with 0.6N H2SO4 or 10% oxalic acid in methanol. The described procedures are satisfactory for confirming mycotoxins present in standards, artificially contaminated grain samples (barley, corn, oat, rye, and wheat), and extracts from both fungal cultures and naturally contaminated grain samples.     

Screening and quantitation of ochratoxin A in corn, peanuts, beans, rice, and cassava

To answer the need for simple, economical, rapid methods for mycotoxins, a procedure for screening and quantitation of ochratoxin A was developed. A methanol-aqueous KCl extraction is used, followed by cleanup with clarifying agents and partition into chloroform. Part of the chloroform extract is used for screening and the other part for quantitation by thin layer chromatography (TLC). The screening procedure takes 40 min, using a silica gel/aluminum oxide minicolumn developed for this purpose. The limits of detection are 80 and 10 micrograms/kg, respectively, for minicolumn screening and TLC quantitation. Ammonium sulfate is efficient in cleaning samples of corn and cassava; cupric sulfate is better with peanuts, beans, and rice. Tests were conducted on triplicate spiked samples of yellow corn meal, raw peanuts, dried black beans, polished rice, and cassava flour at different levels (400, 200, 80, 40, and 10 micrograms/kg). Recoveries ranged from 86 to 160% and the coefficients of variation ranged from 0 to 26%.     

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.     

Degradation of ochratoxin a by Brevibacterium species

The ability to degrade ochratoxin A was studied in different bacteria with a well-known capacity to transform aromatic compounds. Strains belonging to Rhodococcus, Pseudomonas, and Brevibacterium genera were grown in liquid synthetic culture medium containing ochratoxin A. Brevibacterium spp. strains showed 100% degradation of ochratoxin A. Ochratoxin α was detected and identified by high-performance liquid chromatography-mass spectrometry (HPLC-MS) as a degradation product in the cell-free supernatants. The degradation of ochratoxin A is of public concern for food and environmental safety, because it could contribute to the development of new biological ochratoxin A detoxification systems in foodstuffs. In this study, the degradation of ochratoxin A by bacteria belonging to the food chain was demonstrated for the first time.     

Liquid chromatographic determination of aflatoxins in feedstuffs containing citrus pulp

A liquid chromatographic (LC) method was developed for the determination of aflatoxins in feedstuffs containing citrus pulp. The feed-stuff sample is extracted with chloroform, followed by Sep-Pak Florisil cartridge cleanup and Sep-Pak C18 cartridge cleanup. The final eluate (water-acetone, 85 + 15, v/v) is submitted to reverse-phase liquid chromatography with water-methanol-acetonitrile (130 + 70 + 40, v/v/v) as mobile phase and postcolumn derivatization with iodine. Citrus components are removed from the extract efficiently. The limit of detection for aflatoxin B1 is less than 1 microgram/kg. Other aflatoxins can also be detected and measured. Recoveries of aflatoxins B1, B2, G1, and G2 for dairy rations spiked at 13, 5, 10, and 4 micrograms/kg were 87, 86, 81, and 82%, respectively. Corresponding coefficients of variation were 3.1, 3.6, 5.2, and 3.8%, respectively.