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.     

Rapid quantitation and confirmation of aflatoxins in corn and peanut butter, using a disposable silica gel column, thin layer chromatography, and gas chromatography/mass spectrometry

A simple, rapid, and solvent-efficient method for determining aflatoxins in corn and peanut butter is described. Aflatoxins B1, B2, G1, and G2 were extracted from 50 g sample with 200 mL methanol-water (85 + 15). A portion of the extract was diluted with 10% NaCl solution to a final concentration of 50% methanol, and then defatted with hexane. The aflatoxins were partitioned into chloroform. The chloroform solution was evaporated, and the residue was placed on a 0.5 g disposable silica gel column. The column was washed with 3 mL each of hexane, ethyl ether, and methylene chloride. Aflatoxins were eluted with 6 mL chloroform-acetone (9 + 1). The solvent was removed by evaporation on a steam bath, and the aflatoxins were determined using thin layer chromatography (TLC) with silica gel plates and a chloroform-acetone (9 + 1) developing solvent. Overall average recovery of aflatoxin B1 from corn was 82%, and the limit of determination was 2 ng/g. For mass spectrometric (MS) confirmation, aflatoxin B1 in the extract from 3 g sample (20 ng/g) was purified by TLC and applied by direct on-column injection at 40 degrees C into a 6 m fused silica capillary gas chromatographic column. The column was connected directly to the ion source. After injection, the temperature was rapidly raised to 250 degrees C, and the purified extract was analyzed by negative ion chemical ionization MS.     

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.     

High pressure liquid chromatographic determination of aflatoxins in corn.

A high pressure liquid chromatographic (HPLC) method is proposed for determining aflatoxins in corn. The sample is extracted with methanol-10% NaCl (4 + 1), pigments are precipitated with zinc acetate, and the extract is cleaned up on a small (2 g) silica gel column. Aflatoxins in the purified extract are resolved by normal phase HPLC on a microparticulate (10 micrometer) silica gel column with water-saturated chloroform-cyclohexane, acetonitrile solvent, and detected by fluorescence on a silica gel-packed flowcell. The method was compared with chloroform-water extraction of the official CB method on 15 samples of contaminated corn. In 5 of the 6 samples containing aflatoxins B1, B2, G1, and G2, methanol-10% NaCl extracted more aflatoxin than did cloroform-water, as measured both by HPLC and by thin layer chromatography. In samples containing only B1 and B2, the 2 extraction solvents were virtually equivalent. Agreement was good between HPLC and TLC for each extraction solvent. Average recovery of aflatoxins B1, B2, G1, and G2 added to yellow cornmeal at 3 levels was greater than 90%.     

Evaluation of enzyme-linked immunosorbent assay of cleanup for thin-layer chromatography of aflatoxin B1 in corn, peanuts, and peanut butter

A simple, rapid enzyme-linked immunoassay (ELISA) was used to evaluate the performance of each step (extraction, filtration, solvent partition, and silica gel column chromatography) of a solvent-efficient thin-layer chromatographic (TLC) method which is undergoing interlaboratory collaborative study for the determination of aflatoxin B1 in corn, raw peanuts, and peanut butter. The apparent average recoveries using the ELISA method were about 30 to 50% higher than those using the TLC method if only the amount of B1 added to the samples was used in the calculations. After the cross-reaction of the antibody with other aflatoxins added to the samples was considered, the amounts recovered approached the levels of aflatoxins added in all 3 commodities tested. With no cleanup treatment, ELISA recoveries at aflatoxin B1 levels above 7.5 ng/g were 84, 79, and 103% for corn, raw peanuts, and peanut butter, respectively. The coefficients of variation were between 5.2 and 25.2%. With each cleanup step in the TLC method, ELISA detected a progressive decrease in recovery from 150.5 to 105.3% (before correction for the presence of other aflatoxins) or from 93.5 to 65.4% (after correction for other aflatoxins) of B1 added to the samples. The ELISA data support the conclusion obtained from previous studies that cleanup treatments were not necessary in the ELISA. When large amounts of other aflatoxins are present, an understanding of the cross-reactivity of antibody with other aflatoxins in the ELISA is essential for final interpretation of the data.     

Production and isolation of aflatoxin M1 for toxicological studies

One hundred mg aflatoxin M1 was produced and purified for toxicological studies. Aspergillus flavus NRRL 3251 was cultured on rice to produce aflatoxins B1, B2, M1, and M2, B1 and B2 were separated from M1 and M2 by a normal phase low pressure liquid chromatography (LC) column. M1 was then separated from M2 by a reverse phase low pressure LC column. Recoveries of aflatoxins from the LC columns were about 90%. The purified M1 was confirmed by ultraviolet-visible spectrometry, mass spectrometry, nuclear magnetic resonance spectrometry, optical rotation, and its mutagenicity to Salmonella typhimurium TA98.     

Determination of aflatoxins in high-pigment content samples by matrix solid-phase dispersion and high-performance liquid chromatography

A fast, efficient, and cost-effective method was developed for the analysis of aflatoxins in farm commodities with high-pigment content, such as chili powder, green bean, and black sesame. The proposed method involved matrix solid-phase dispersion (MSPD) and high-performance liquid chromatography (HPLC)-fluorescence detection (FLD) with postcolumn electrochemical derivatization in a Kobra cell. The MSPD procedure combined the extraction with neutral alumina and pigment cleanup with graphitic carbon black (GCB) in a single step. The recoveries of aflatoxins ranged from 88% to 95% with the relative standard deviations (RSD) less than 6% (n = 6). The limits of detection (LODs) were 0.25 ng/g aflatoxin B1, G1, and 0.10 ng/g aflatoxin B2, G2, respectively. The analytical results obtained by MSPD were compared to those of the immunoaffinity column (IAC) cleanup method. No significant differences were found between the two methods by t-test at the 95% confidence level.     

Immunoaffinity column coupled with solution fluorometry or liquid chromatography postcolumn derivatization for determination of aflatoxins in corn, peanuts, and peanut butter: collaborative study

An AOAC/IUPAC (International Union of Pure and Applied Chemistry) collaborative study was conducted to evaluate the effectiveness of an immunoaffinity column for the determination of aflatoxin. The test portion is extracted with methanol-water (7 + 3), filtered, diluted to less than 30% methanol with water, and applied to the affinity column. The column is washed with water and the concentrated aflatoxins are eluted with methanol. Total aflatoxins are determined by solution fluorometry with bromine (SFB), and individual toxins are determined by reverse-phase liquid chromatography with postcolumn derivatization with iodine (PCD). Corn naturally contaminated with aflatoxins, and peanuts, peanut butter, and corn containing added aflatoxins (B1:B2:G1:G2 = 7:1:3:1) were sent to 24 collaborators in the United States, France, Canada, and the Republic of South Africa. Twelve collaborators used the SFB method, 9 used the PCD method, and 3 used both SFB and PCD methods. Twenty collaborators completed the study (10 used the SFB method, 7 used the PCD method, and 3 used both SFB and PCD methods). Test portions were spiked at 10, 20, and 30 ng/g. For SFB analyses, recoveries of total aflatoxins were 123, 105, and 107%, respectively; the relative standard deviation for repeatability (RSDr) ranged from 11.75 to 16.57%, and the relative standard deviation for reproducibility (RSDR) ranged from 10.97 to 33.09%. For PCD analyses, recoveries were 81, 81, and 83%, respectively; the RSDr ranged from 5.20 to 17.22%, and the RSDR ranged from 4.68 to 50.77%. The RSDr for aflatoxins B1 and G1 for spiked test portions ranged from 5.45 to 23.55%, and the RSDR ranged from 4.21 to 57.28%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of isotope-labeled aflatoxins for LC-MS/MS stable isotope dilution analysis of foods

Aflatoxins are a group of very carcinogenic mycotoxins that can be found on a wide range of food commodities including nuts, cereals, and spices. In this study, the first LC-MS/MS stable isotope dilution assay (SIDA) for the determination of aflatoxins in foods was developed. The development of this method was enabled by easily accessible isotope-labeled (deuterated) aflatoxins B2 and G2, which were synthesized by catalytic deuteration of aflatoxin B1 and G1, purified, and well-characterized by NMR and MS. All four aflatoxins of interest (B1, B2, G1, and G2) were quantified in food samples by using these two labeled internal standards. The response factors (RF) of the linear calibrations were revealed to be matrix independent for labeled aflatoxin B2/aflatoxin B2 and labeled aflatoxin G2/aflatoxin G2. For labeled aflatoxin B 2/aflatoxin B 1 and labeled aflatoxin B2/aflatoxin G1 matrix-matched calibration was performed for the model matrices almonds and wheat flour, showing significant differences of the RFs. Limits of detection (LOD) were determined by applying a statistical approach in the presence of the two model matrices, yielding 0.31 microg/kg (aflatoxin B1), 0.09 microg/kg (aflatoxin B2), 0.38 microg/kg (aflatoxin G1), and 0.32 microg/kg (aflatoxin G2) for almonds (similar LODs were obtained for wheat flour). Recovery rates were between 90 and 105% for all analytes. Coefficients of variation (CV) of 12% (aflatoxin B1), 3.6% (aflatoxin B2), 14% (aflatoxin G1), and 4.8% (aflatoxin G2) were obtained from interassay studies. For further validation, a NIST standard reference food sample was analyzed for aflatoxins B1 and B2. The method was successfully applied to determine trace levels of aflatoxins in diverse food matrices such as peanuts, nuts, grains, and spices. Aflatoxin contents in these samples ranged from about 0.5 to 6 microg/kg.     

Minicolumn detection method applied to almonds: collaborative study.

The minicolumn screening method for aflatoxins was collaboratively tested on naturally contaminated almonds. The nuts were extracted, and the extract was cleaned up and applied to a Velasco-type minicolumn. This permits the detection of total aflatoxins (B1, B2, G1, G2) as a fluorescent band on the Florisil layer of the column. The results of 20 collaborators are presented. Samples containing 0, 2, 5, 10, and 25 ng aflatoxin/g were analyzed. Ninety-six per cent of the samples containing 5--25 ng total aflatoxins/g and 83% of the negative samples were correctly identified. The method has been adopted as official first action for detection of total aflatoxin levels of greater than or equal to 5 ng/g.     

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.     

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.     

Beta-cyclodextrin post-column fluorescence enhancement of aflatoxins for reverse-phase liquid chromatographic determination in corn

beta-Cyclodextrin enhances the fluorescence of aflatoxins B1 and G1 in aqueous systems. This effect was utilized in developing a unique reverse-phase liquid chromatographic (LC) method for determination of aflatoxins B1, B2, G1, and G2 (B1 detection limit 1 ppb), without preparing derivatives of B1 and G1. The aflatoxins are dissolved in methanol or the mobile phase for injection onto the LC system. Using a mobile phase of methanol-beta-cyclodextrin (1 + 1), the aflatoxins are resolved on a C18 column. Fluorescence of the aflatoxins is enhanced by post-column introduction of an aqueous concentrated beta-cyclodextrin solution. All 4 aflatoxins elute within 10 min in the order G2, G1, B2, B1. Fluorescence responses for B1 and G1 standards were linear over the concentration range 0.5-10 ng, yielding correlation coefficients (r) of 0.9989 and 1.000, respectively. The average peak response ratio for G1:B1 for the mobile phase-enhancement solution described was 0.765 with a coefficient of variation (CV) of 0.98%. CVs were 6.2, 9.0, and 7.5% for multiple assays of aflatoxin B1 in 3 samples of naturally contaminated corn. For samples of corn spiked to a total B1 content of 8.3 ng/g, average B1 recovery was 90% (CV 11.7%).     

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.     

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.     

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.     

High pressure liquid chromatographic determination of aflatoxins in peanut butter using a silica gel-packed flowcell for fluorescence detection

A high pressure liquid chromatographic method has been developed for determining aflatoxins B1, B2, G1, and G2 in peanut butter. The method is based on extraction with acidified aqueous methanol, partition of the aflatoxin into methylene chloride, and purification of the extract on a 2 g silica gel column. The extracted aflatoxins are resolved on a microparticulate (10 micrometer) porous silica gel column in ca 10 min with a water-washed chloroform-cyclohexane-acetonitrile solvent that contains 2% isopropanol. The fluorescence detection system determines aflatoxins B1, B2, G1, and G2 at low levels, i.e., 0.25 ppb B1, 0.5 ppb G1, and 0.2 ppb B2 and G2. Multiple assays of 5 samples of naturally contaminated peanut butters containing total aflatoxins (B1 + B2 + G1 + G2) at levels of 1, 2, 3, 9, and 17 ppb gave intralaboratory coefficients of variation of 7, 4, 4, 11, and 3%, respectively. Samples spiked at levels of 5, 9, and 17 ppb total aflatoxins showed recoveries of 79, 81, and 81%, respectively.     

Liquid chromatographic determination of aflatoxin levels in peanut butters using an immunoaffinity column cleanup method: international collaborative trial

Thirteen laboratories in 7 different countries participated in a collaborative trial to evaluate the immunoaffinity column cleanup procedure with quantitation by fluorescence liquid chromatography (post-column derivatization) for the determination of aflatoxins in peanut butters. Participants were sent 10 randomly numbered samples of roasted peanut butter for analysis (5 pairs of undisclosed duplicates). Two of the pairs were "blank" peanut butters to which aflatoxin standards had been added; these "spiked" samples were used for recovery purposes. The other 3 pairs of samples were a nominal "blank" and 2 naturally contaminated peanut butters. A full statistical presentation of the results is given. Coefficients of variation (CVs) for the total aflatoxin determinations for mean levels of 4, 15, and 38 microns/kg were between 32 and 44% for the blank and 2 trial samples. Recovery levels for the 2 spiked samples were 51-67%, with aflatoxin B1 recovery of 60%. Relative standard deviations for method repeatability (RSDr) and reproductibility (RSDR) for the 3 trial samples were 15-26% and 33-45%, respectively.     

Determination of aflatoxin M1 in milk and milk products contaminated at low levels

A new method is described for the determination of aflatoxin M1 in milk and dairy products by thin layer chromatography. The main characteristic is the extraction system using an alkaline solution. Lipids are removed by centrifuging at low temperatures, and the aflatoxins are then extracted with CHCl3. The method has 2 options: Technique II (detection limit 0.02 ppb) requires cleanup on a chromatographic column; this is not necessary in Technique I (detection limit 0.1 ppb). The recovery rate in both techniques is over 92.8% in milk and yoghurt. This method may also be used for other aflatoxins. Because of the advantages of the method, Technique II is recommended for aflatoxin M1 control in milk, where a low detection limit is necessary. Technique I is proposed for experimental aflatoxin production studies in dairy products, which require analysis of a large number of samples but which do not require a very low detection limit.     

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.