Determination of aflatoxicol and aflatoxins B1 and M1 in eggs

Procedures from 2 methods, one for aflatoxins B1 and M1 in eggs and one for aflatoxicol in milk, blood, and liver, have been combined to determine the 3 toxins in eggs. The sample is blended with sodium chloride-saturated water and this mixture is then blended with acetone. After separation from the solid residue, the aqueous acetone extract is defatted with petroleum ether. The toxins are next partitioned into chloroform and separated from interferences on a silica gel column. Aflatoxicol is determined by fluorescence measurement after separation on a C18 reverse phase liquid chromatographic column, and aflatoxins B1 and M1 are determined by fluorescence densitometry after separation on a silica gel thin layer chromatographic plate. In a recovery study with eggs, mean recoveries of aflatoxicol added at levels of 0.1, 0.05, and 0.025 ng/g were 87, 77, and 78%, respectively. Mean recoveries of aflatoxins B1 and M1 added at a level of 0.1 ng/g were 75 and 87%, respectively, and at an added level of 0.05 ng/g were 86 and 75%. The within-laboratory precision (repeatability) ranged from 2 to 13%.     

Improved cleanup for liquid chromatographic analysis and fluorescence detection of aflatoxins M1 and M2 in fluid milk products

A rapid method is described for extraction and cleanup of raw and processed milk for determination of aflatoxins M1 and M2 by using a C18 Sep-Pak/silica gel cleanup column combination. Aflatoxins are separated by normal phase liquid chromatography and their concentrations are determined by fluorescence detection in a silica gel-packed flow cell. Recoveries ranged from 99 to 103% with coefficients of variation less than 2% for M1 levels of 0.117-1.17 ng/mL added to raw milk. Similar recoveries were obtained for M2. The coefficient of variation for analysis of 5 subsamples of naturally contaminated milk was less than 1%. Agreement with the official method is satisfactory. Each sample requires less than 25 mL solvent and 10 min actual handling time. Sample chromatograms show no interferences in the M1-M2 elution region and no late-eluting peaks, which permits spacing injections at 13-20 min intervals. Aflatoxin levels as low as 0.03 ppb may be determined by this procedure. Extracts have also been analyzed by thin layer chromatography.     

Distribution of aflatoxins B1 and M1 in contaminated calf and pig livers

The determination of aflatoxins B1 and M1 in multiple sections of livers from 4 calves and 1 pig exposed to high levels of aflatoxins showed a uniform distribution of the aflatoxins in each liver, within the precision of the analytical method used. The thin layer chromatographic method has an expected within-laboratory coefficient of variation of 15%.     

Aflatoxicol and aflatoxins B1 and M1 in the tissues of pigs receiving aflatoxin

Aflatoxicol (AFL) and aflatoxins B1 and M1 were found in tissues (kidney, liver, and muscle) of feeder pigs given an estimated LD50 oral dose of B1 (1.0 mg/kg body weight) provided as a rice culture of Aspergillus flavus and of market-weight pigs fed a naturally contaminated feed, containing aflatoxin B1 at a level of 400 ng/g from corn, for 14 days. The residues in all tissues decreased with time after treatment in both groups, with no detectable residues (approximate detection limits, ng/g, B1 0.03, M1 0.05, AFL 0.01) in pig tissues from the feeding experiment 24 h after withdrawal of aflatoxin-contaminated feed. B1 and M1, when found in the feeding experiment, were at about the same levels in all tissues except the kidney, in which M1 was the dominant aflatoxin. The level of AFL, when detected, was about 10% of the B1 level.     

Extraction and cleanup procedures for determination of diarylphosphates in fish, sediment, and water samples

Methods for determination of triaryl/alkylphosphates (TAPs) in water, fish, and sediment have been extended to determination of the diarylphosphate (DAP) degradation products. DAPs were extracted from water (adjusted to pH 0.5) by use of XAD-2 resin and determined by gas-liquid chromatography as butyl esters. Recovery of diphenylphosphate (DPP) and o-, m-, p-dicresylphosphates (DoCP, DmCP, DpCP) were greater than 95% in water samples fortified at 1, 10, and 50 micrograms/L. DAPs were extracted from fish with methanol and the extracts were cleaned up on reverse phase (C18) silica cartridges. Recoveries were greater than 87% for DPP, DoCP, DmCP, and DpCP in fish muscle fortified at 50, 100, and 500 ng/g. Sediments were refluxed with aqueous methanol and DAPs were recovered by use of XAD-2 resin. Recoveries of DAPs from sediments fortified at 50 and 100 ng/g were greater than 76%. Interferences (1-10 ng/g) from phosphorus or nitrogen-containing GLC peaks prevented sub- ng/g level analysis for DAPs in sediment and fish extracts.     

Rapid determination of aflatoxins in raw peanuts by liquid chromatography with postcolumn iodination and modified minicolumn cleanup

A method is described for rapid cleanup followed by reverse-phase liquid chromatographic (LC) quantitation of aflatoxins in raw peanuts. A modified minicolumn cleanup is used for sample preparation, and a preliminary estimation of aflatoxin content by minicolumn can be made so that highly contaminated samples can be diluted before LC analysis. The use of the simple, quick minicolumn cleanup eliminates the need for further column or cartridge cleanup, thus greatly reducing sample preparation time. Sensitive quantitation is achieved using a phenyl column, a mobile phase of water-tetrahydrofuran (80 + 20, v/v), and postcolumn derivatization with water-saturated iodine followed by fluorescence detection. The recoveries of aflatoxins B1, B2, G1, and G2 from peanut meal spiked at 3 levels ranged from 71.7 to 88.3% (average 80%) with coefficients of variation from 2.7 to 10.4%.     

Rapid liquid chromatographic determination of aflatoxins M1 and M2 in artificially contaminated fluid milks: collaborative study

An international collaborative study involving 14 collaborators from 5 different countries was conducted to test a rapid liquid chromatographic (LC) method for detecting aflatoxins M1 and M2 in fluid milk. Each collaborator prepared artificially contaminated milk samples (0.078-1.31 ng M1/mL and 0.030-0.13 ng M2/mL) by adding solutions containing various concentrations of aflatoxins M1 and M2 to fresh milk. Recoveries ranged from 85.2 to 102.5% (av. 93.7%) for aflatoxin M1 and from 99.5 to 126.7% (av. 109.8%) for aflatoxin M2. Coefficients of variation averaged 21.4% (M1) and 35.9% (M2). An analysis of variance was calculated from combined data to determine variance components. The within-laboratory variations (So) (repeatability) were 27.9% (M1) and 23.9% (M2), and the among-laboratory variations (Sx) (reproducibility) were 44.5% (M1) and 64.7% (M2). No visual differences were determined between normal or reverse phase LC for contaminated samples; however, there were an insufficient number of collaborators using normal phase to give meaningful separate statistical data. For 26 observations of uncontaminated milk, 3 false M1 positives were reported for normal phase LC determinations and 2 false M1 positives were reported for reverse phase LC determinations. Three normal phase and 11 reverse phase false M2 positives were reported for 104 observations in uncontaminated milk. The reverse phase LC method for determination of aflatoxins M1 and M2 in fluid milk has been adopted official first action.     

Liquid chromatographic method for determination of aflatoxins B1, B2, G1, and G2 in corn and peanut products: collaborative study

A collaborative study of a liquid chromatographic method for the determination of aflatoxins B1, B2, G1, and G2 was conducted in laboratories located in the United States, Canada, South Africa, and Switzerland. Twenty-one artificially contaminated raw peanuts, peanut butter, and corn samples containing varying amounts of aflatoxins B1, B2, G1, and G2 were distributed to participating laboratories. The test portion was extracted with methanol-0.1N HCl (4 + 1), filtered, defatted with hexane, and then partitioned with methylene chloride. The concentrated extract was passed through a silica gel column. Aflatoxins B1 and G1 were derivatized with trifluoroacetic acid, and the individual aflatoxins were determined by reverse-phase liquid chromatography with fluorescence detection. Statistical analysis of the data was performed to determine or confirm outliers, and to compute repeatability and reproducibility of the method. For corn, relative standard deviations for repeatability (RSDr) for aflatoxin B1 ranged from 27.2 to 8.3% for contamination levels from 5 through 50 ng/g. For raw peanuts and peanut butter, RSDr values for aflatoxin B1 were 35.0 to 41.2% and 11.2 to 19.1%, respectively, for contamination levels from 5 through 25 ng/g. RSDr values for aflatoxins B2, G1, and G2 were similar. Relative standard deviations for reproducibility (RSDr) for aflatoxin B1 ranged from 15.8 to 38.4%, 24.4 to 33.4%, and 43.9 to 54.0% for corn, peanut butter, and raw peanuts, respectively. The method has been adopted official first action for the determination of aflatoxins B1, B2, G1, and G2 in peanut butter and corn at concentrations greater than or equal to 13 ng total aflatoxins/g.     

Use of the Mycosep multifunctional cleanup column for liquid chromatographic determination of aflatoxins in agricultural products.

A liquid chromatographic (LC) technique has been developed that uses the Mycosep multifunctional cleanup (MFC) column. MFC columns provide a rapid 1-step extract purification. They are designed to retain particular groups of compounds that may create interferences in analytical methods. At the same time, MFC columns allow compounds of interest to pass through. In the method presented, test samples are extracted in a blender with acetonitrile-water (9 + 1). A portion of the extract is forced through an MFC column designed especially for analysis of numerous mycotoxins. Analytical interferences are retained, while aflatoxins pass through the column. Aflatoxins B1 and G1 are converted to their hemiacetals by heating a mixture of purified extract and water-trifluoroacetic acid-acetic acid (7 + 2 + 1) at 65 degrees C for 8.5 min. An aliquot of this mixture is analyzed by isocratic LC with acetonitrile-water mobile phase and fluorescence detection. A detection limit of less than 0.5 ng/g for aflatoxin B1 was obtained. Average recoveries greater than 95% total aflatoxins (B1, B2, G1, and G2) and coefficients of variation of less than 3% were obtained. The method was successfully applied to the following commodities: corn, almonds, pista-chios, walnuts, peanuts, Brazil nuts, milo, rice, cottonseed, corn meal, corn gluten meal, fig paste, and mixed feeds.     

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.     

Improved method for confirmation of identity of aflatoxins B1 and M1 in dairy products and animal tissue extracts

A method is described for confirming the identity of aflatoxins B1 and M1 in dairy products and liver extracts on a thin layer plate. Extracts and standards containing aflatoxins B1 and M1 are spotted on 10 x 10 cm plates, which are developed 2-dimensionally in mixtures of isopropanol-acetone-chloroform. After the first development, trifluoroacetic acid-hexane (1 + 4) is sprayed on that part of the plate containing the separated extract components and the underdeveloped standard spots of B1 and M1, and the plate is heated 6-8 min at 75 degrees C. Then the plate is developed in a second direction, and the reaction products of B1 and M1 with trifluoroacetic acid from the extract are compared with the same derivatives of the respective standards. The method has been used successfully on extracts of milk, cheese, and liver containing 0.1 ng B1 or M1/g and can be completed in 35-45 min.     

Simple, rapid cleanup method for analysis of aflatoxins and comparison with various methods

A method is described for simple and rapid determination of aflatoxins in corn, buckwheat, peanuts, and cheese. Aflatoxins were extracted with chloroform-water and were purified by a Florisil column chromatographic procedure. Column eluates were concentrated and spotted on a high performance thin layer chromatographic (HPTLC) plate, which was then developed in chloroform-acetone (9 + 1) and/or ether-methanol-water (94 + 4.5 + 1.5) or chloroform-isopropanol-acetone (85 + 5 + 10). Each aflatoxin was quantitated by densitometry. The minimum detectable aflatoxin concentrations (micrograms/kg) in various test materials were 0.2, B1; 0.1, B2; 0.2, G1; 0.1, G2; and 0.1, M1. Recoveries of the aflatoxins added to corn, peanut, and cheese samples at 10-30 micrograms/kg were greater than 69% (aflatoxin G2) and averaged 91%, B1; 89%, B2; 91%, G1; 78%, G2; and 92%, M1. The simple method described was compared with the AOAC CB method, AOAC BF method, and AOAC milk and cheese method. These methods were applied to corn, peanut, and cheese composites spiked with known amounts of aflatoxins, and to naturally contaminated buckwheat and cheese. Recoveries were much lower for the BF method compared with our simple method and the CB method.     

Extraction and cleanup of organochlorine, organophosphate, organonitrogen, and hydrocarbon pesticides in produce for determination by gas-liquid chromatography.

A rapid, multiresidue procedure utilizing the minimal cleanup necessary for gas-liquid chromatographic (GLC) analysis is presented. The samples are extraced with acetone and partitioned with methylene chloride-petroleum either to remove water. The organophosphorus and organonitrogen compounds are then quantitated by GLC, using a KCl thermionic detector. A Florisil cleanup of the extract is performed prior to the determination of organochlorine compounds by a GLC electron capture detector. Carbon-hydrogen compounds such as biphenyl and o-phenylphenol undergo the Florisil cleanup and may also be quantitated by GLC. Quantitative recoveries for 15 organophosphorus, 9 organochlorine, 5 organonitrogen, and 2 hydrocarbon pesticides show the range in polarities of pesticides recovered, from Monitor to biphenyl. The method is simple and fast with a great potential for the analysis of many more compounds.     

Resolution of aflatoxins B1, B2, G1, and G2 by high-pressure liquid chromatography.

Aflatoxins were completely resolved as sharp peaks in the order BU-B2-G1-G2 by high-pressure liquid chromatography on a small particle (10 mum) porous silica gel column in 7-13 min (B1 through G2) by a water-saturated chloroform-cyclohexane-acetonitrile elution solvent (25+7.5+1.0), with detection by ultraviolet absorbance at 360 nm. The relationship between peak height and amount injected was linear over a 5-400 ng range for each aflatoxin. Both retention times and peak heights were highly reproducible, multiple injections of mixed standards giving coefficients of variation of 1.0-1.4% (retention time) and 1.6-2.8% (peak height) for the 4 aflatoxins. Detection was highly sensitive, with mean peak height, mm/ng, of 7.1 (B1), 6.4 (B2), 4.5 (G1), and 4.1(G2), allowing detection of 1-2 ng of each aflatoxin.     

New confirmatory test for aflatoxins B1 and B2.

A new confirmatory test for aflatoxins B1 and B2 is described. The test involves treatment of aflatoxins with excess sodium borohydride for 10 min at room temperature, to yield a fluorescent trihydroxy derivative of each aflatoxin. The test is sensitive and simple and gives no side reactions. The test is also applicable to aflatoxins G1 and G2.     

Simple, rapid, and inexpensive cleanup method for quantitation of aflatoxins in important agricultural products by HPLC

A chemical cleanup procedure for low-level quantitative determination of aflatoxins in major economically important agricultural commodities using HPLC has been developed. Aflatoxins were extracted from a ground sample with MeOH/H2O (80:20, v/v), and after a cleanup step on a minicolumn packed with Florisil, aflatoxins were quantified by HPLC equipped with a C18 column, a photochemical reactor, and a fluorescence detector. Water/MeOH (63:37, v/v) served as the mobile phase. Recoveries of aflatoxins B1, B2, G1, and G2 from peanuts spiked at 5, 1.7, 5, and 1.7 ng/g were 89.5+/-2.2, 94.7+/-2.5, 90.4+/-1.0, and 98.2+/-1.1, respectively (mean+/-SD, %, n=3). Similar recoveries, precision, and accuracy were achieved for corn, brown and white rice, cottonseed, almonds, Brazil nuts, pistachios, walnuts, and hazelnuts. The quantitation limits for aflatoxins in peanuts were 50 pg/g for aflatoxin B1 and 17 pg/g for aflatoxin B2. The minimal cost of the minicolumn allows for substantial savings compared with available commercial aflatoxin cleanup devices.     

Reduction of aflatoxins B1 and B2 with sodium borohydride.

Reduction of aflatoxin B1 and aflatoxin B2 with sodium borohydride quantitatively yielded new fluorescent derivatives, designated as aflatoxin RB1 and aflatoxin RB2. Mass spectrometric data showed that RB1 and RB2 were trihydroxy derivatives of B1 and B2, respectively. Nuclear magnetic resonance analysis revealed that new chemical shifts were present in aflatoxins RB1 and RB2 in addition to those of the parent aflatoxins. The new compounds had lower melting points and different ultraviolet and infrared spectra compared to aflatoxins B1 and B2 and the monohydroxy derivative aflatoxicol. They were lses toxic to chick embryos than the parent toxins. Since the reduction yields were quantitative and since the reduction products could be detected at low levels comparable to those for B1 and B2, the reduction reaction could be used as a confirmatory test for both aflatoxins B1 and B2. Preliminary results obtained from gas-liquid chromatography (GLC) analysis of the trimethylsilyl derivatives of aflatoxins RB1 and RB2 indicated that these compounds could furnish the basis for developing an analytical GLC method for aflatoxins B1 and B2.     

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.