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.     

Thin layer chromatographic determination of citrinin.

A thin layer chromatographic (TLC) method is described for the determination of citrinin in feeds. Citrinin is extracted from feed with methanol and water, the mixture is made alkaline with 10% sodium carbonate, and the aqueous solution is filtered and extracted with chloroform to remove most of the interfering materials. The aqueous layer is acidified with 2N HCl and extracted with chloroform. The chloroform extract is concentrated and spotted on a thin layer chromatographic (TLC) plate which is developed in chloroform-acetone-ethanol-water (60 + 40 + 10 + 1). The citrinin is viewed under ultraviolet light after TLC. Either visual or fluorodensitometric quantitation is used. Recoveries of citrinin from various feed samples spiked at levels of 2.0--5 micrograms/g were 75--92%. The proposed method can detect 0.5 micrograms/g feed, including corn, silage, ready mixed feeds, and feed pellets.     

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.     

Thin layer chromatographic determination of sterigmatocystin in cheese

A one-dimensional thin layer chromatographic method has been developed for determining sterigmatocystin in cheese. Cheese is extracted with acetonitrile-4% KCl (85 + 15). A simplified liquid-liquid partition cleanup is used, and the sample extract is passed through a cupric carbonate column for final purification. Sterigmatocystin is visualized by spraying the plate with aluminum chloride. The fluorescence of the spot is enhanced 10-fold by additional plate spraying with a silicone-ether mixture, enabling sterigmatocystin detection and quantitation at 2 and 5 micrograms/kg, respectively. Average recoveries were 88.3 and 86.4% at the 10 and 25 micrograms/kg levels, respectively.     

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.     

Gas chromatographic screening method for T-2 toxin, diacetoxyscirpenol, deoxynivalenol, and related trichothecenes in feeds

A gas chromatographic method for screening trichothecene mycotoxins in feeds is described. Feed is extracted with acetonitrile-water, and the toxins are purified with charcoal-alumina-Celite, Florisil, and silica mini-columns. Deoxynivalenol (DON), nivalenol (NIV), diacetoxyscirpenol (DAS), T-2 toxin, and their fungal metabolites are hydrolyzed to their corresponding parent alcohols (DON, NIV, scirpentriol, or T-2 tetraol) by alkaline hydrolysis. After derivatization to their pentafluoropropionyl analogs, they are quantitated by capillary gas chromatography with electron capture detection. Identity can be confirmed and sensitivity can be increased by using negative chemical ionization mass spectrometry with no additional sample workup. Recoveries of DAS, DON, and T-2 toxin averaged, respectively, 80, 65, and 85% in corn; 84, 65, and 88% in soybeans; and 70, 57, and 96% in mixed feeds at concentrations ranging from 0.1 to 2.0 ppm. Recoveries of 15-monoacetoxyscirpenol (MAS), HT-2, NIV, and T-2 tetraol were 97, 97, 86, and 56%, respectively, in corn at a concentration of 0.25 ppm: A detection limit of 0.02 ppm in corn, soybeans, and mixed feeds, and 0.05 ppm in silages is estimated.     

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.     

Thin layer chromatographic detection and indirect gas chromatographic determination of three carbamate pesticides.

Carbamate pesticide residues are extracted from vegetables and fruits with methylene chloride. The extracts are spotted on silica gel plates and the pesticides are detected by an enzymatic inhibition technique. For quantitative determination, aliquots of the methylene chloride extracts are evaporated to dryness in a rotary evaporator. After the residues are dissolved in ethanol, 0.5N NaOH is added in the hydrolysis step. To remove a number of possible interferences the hydrolyzed phenols are steam-distilled and treated with 1-fluoro-2,4-dinitrobenzene and/or 4-chloro-alpha,alpha,alpha-trifluoro-3,5-dinitrotoluene to form the ether derivatives. Efficiency in the conversion of the phenolic moieties to the phenyl ethers is about 100%. The resulting electron-capturing derivatives enable the carbamate pesticides to be detected in vegetables and fruits at the 0.05 ppm level. Recoveries of 90-94% were obtained from vegetables and fruits fortified with 0.5-2.0 ppm carbaryl, Mesurol, and propoxur.     

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.     

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.     

Thin layer chromatography and densitometric determination of aflatoxins in mixed feeds containing citrus pulp

A method for the accurate one-dimensional thin layer chromatographic (TLC) determination of aflatoxins B1, B2, G1, and G2 in mixed feeds is presented. The aflatoxins are extracted from the sample with chloroform and purified by solvent partitioning. Each aflatoxin is separated from pulp interference by thin layer chromatography on aluminum-backed silica plates. The separated aflatoxins are detected by fluorescence densitometry. Average recoveries for samples spiked from 10 to 100 ppb B1 and G1 and from 3 to 30 ppb B2 and G2 are 82, 84, 95, and 94% for B1, B2, G1, and G2, respectively. The above recovery data, when analyzed for overall method repeatability, produced relative standard deviations of 6.8, 4.3, 6.9, and 7.6% for B1, B2, G1, and G2, respectively. Minimum detection level is less than 1 ppb for each aflatoxin. B1 is confirmed by trifluoroacetic acid derivative formation on a silica TLC plate.     

Substances interfering with the gas-liquid chromatographic determination of T-2 mycotoxin.

Two substances interfering with the gas-liquid chromatographic (GLC) detection of T-2 mycotoxin were identified as 1-glyceryl-monooleate and 1-glycerylmonolinoleate. These monoglycerides are natural products formed by species of Fusarium growing on cereal grains and are also additives contained in liquid vegetable and animal fats added to the feed mixture. The monoglycerides can be removed from the analytical sample by resolution by thin layer chromatography prior to separation by GLC. Trimethylsilyl ether derivatives of the monoglycerides and T-2 toxin have almost identical retention times on 3% OV-1 columns, whereas the trifluoroacetyl and pentafluoropropionyl derivatives give baseline separation on the same column. The monoglycerides can be misidentified as the T-2 toxin in analyses involbing GLC.     

Thin layer chromatographic detection and spectrophotometric determination of the trisodium salt of 1,3,6-pyrenetrisulfonic acid in D&C Green No. 8

A thin layer chromatographic method is presented for separating the reaction by-product 1,3,6-pyrenetrisulfonic acid (trisodium salt) (PTS) from D&C Green No. 8 (8-hydroxy-1,3,6-pyrenetrisulfonic acid). PTS is detected visually, extracted from the adsorbent, and determined spectrophotometrically. Recoveries of PTS added at 0.75-6.73% levels to 8-hydroxy-1,3,6-pyrenetrisulfonic acid ranged from 80.0 to 94.8%.     

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.     

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.     

Thin layer chromatographic separation and spectrodensitometric determination of higher and lower sulfonated subsidiary dyes in FD&C Yellow No. 6.

A thin layer chromatographic method is presented by which higher and lower sulfonated subsidiary dyes are simultaneously separated from FD&C Yellow No. 6. After separation, the colors are quantitated directly, using a spectrodensitometer. Recoveries of 1, 2, and 5% of added 1-(4-sulfophenylazo)-2-naphthol-3,6-disulfonic acid ranged from 92 to 111%. Recoveries of 1, 2, and 5% of added 1-(4-sulfophenylazo)-2-naphthol ranged from 90 to 110%.