Reverse phase liquid chromatographic determination and confirmation of aflatoxin M1 in cheese

A systematic method is proposed for determination and confirmation of aflatoxin M1 in cheese by liquid chromatography (LC). A sample of cheese is extracted with chloroform, cleaned up on 2 silica gel columns followed by a Sep-Pak C18 cartridge, and chromatographed on a 5 microns octadecyl silica column with fluorometric detection. The sample extract or standard is treated with n-hexane-trifluoroacetic acid (TFA) (4 + 1) for 30 min at 40 degrees C. Analysis by LC with TFA-treatment of the extract provides quantitative data. Multiple assays of 5 samples of Gouda cheese spiked with aflatoxin M1 at levels of 0.5, 0.1, and 0.05 ng/g showed average recoveries of 93.2, 91.6, and 92.4%, with coefficients of variation of 2.63, 3.97, and 4.52%, respectively. Assay of 5 naturally contaminated cheeses resulted in 0.051-0.448 ng/g of aflatoxin M1. Limit of quantitation is about 0.01 ng/g. The identity of aflatoxin M1 is confirmed by treating aflatoxin M1 or the M2a derivative with TFA-methanol (or ethanol) (3 + 1). The TFA-methanol reaction products of M2a could be detected quantitatively.     

Fluorimetric determination of aflatoxin M1 in cheese

A simple and sensitive method is proposed for the determination of aflatoxin M1 in cheese. The ground cheese sample is extracted with acetone-water (3 + 1). Acetone is evaporated under vacuum, and the aqueous phase is passed through a C18 disposable cartridge. After the cartridge is washed with acetonitrile-water (1 + 9), the toxin is eluted with acetonitrile. The extract is then cleaned up on a silica cartridge. Final analysis is performed by 2-dimensional thin layer chromatography (TLC) combined with fluorodensitometry or by liquid chromatography on a reverse phase C18 column with fluorescence detection. Recovery is greater than 90%, and the coefficient of variation is 6% or less. The detection limit is in the range of 10 ng/kg. The identity of aflatoxin M1 is confirmed by formation of the M2a or acetyl-M1 derivative and rechromatography.     

Development and application of solvent-free extraction for the detection of aflatoxin M1 in dairy products by enzyme immunoassay

The official methods for the quantification of aflatoxin M1 in dairy products (cheese and yogurt) include extraction into dichloromethane or chloroform, evaporation of the solvent, partitioning of the reconstituted residue with hexane, and subsequent analysis. To secure a rapid and inexpensive screen for aflatoxin M1 contamination, a sensitive competitive ELISA, using a rabbit polyclonal antibody, was developed for measuring aflatoxin M1 in milk and used in a comparative study for measuring the extraction efficiency of aflatoxin M1 in aqueous or organic solvent buffers using yogurt samples. An aqueous sodium citrate solution was found to be suitable for extracting aflatoxin M1, thus eliminating the need for organic solvents. The citrate extraction proved to be efficient (recovery ranged from 70 to 124%) in fortified samples of very different kinds of dairy products, including yogurt and six types of cheese. Fourteen yogurt and cheese samples were extracted with citrate solution and analyzed by ELISA. A good correlation was observed (y=0.95x-0.59, r2=0.98) when the data were compared with those obtained through the official method, across a wide range of aflatoxin M1 contaminations (10-200 ng/kg).     

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.     

Simultaneous determination of chloramphenicol and aflatoxin M1 residues in milk by triple quadrupole liquid chromatography-tandem mass spectrometry

A reliable, rapid, and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of chloramphenicol and aflatoxin M(1) in milk has been developed. This method includes simple extraction of sample with acetonitrile, separation on a MGIII-C(18) column using 5 mM ammonium acetate aqueous solution/methanol (60:40, v/v) as mobile phase, and MS/MS detection using multiple reaction monitoring mode. The method was validated according to Commission Decision 2002/657/EC. The limits of detection (LODs) were 0.05 μg/kg for chloramphenicol and 0.005 μg/kg for aflatoxin M(1.) The limits of quantification (LOQs) were 0.2 μg/kg for chloramphenicol and 0.02 μg/kg for aflatoxin M(1). The recovery values ranged from 88.8% to 100.6%, with relative standard deviation lower than 15% in all cases, when samples were fortified at three different concentrations. The decision limits (CCα) and detection capability (CCβ) of the method were also reported. This method has been successfully applied for simultaneous analysis of chloramphenicol and aflatoxin M(1) residues in milk from local supermarkets in China.     

Distribution of aflatoxin in pistachios. 7. Sequential sampling

Sequential sampling for aflatoxin testing in pistachios is evaluated using the aflatoxin distribution and Monte Carlo results previously obtained (J. Agric. Food Chem. 1999, 47, 3771-3775). The sequential protocol is modeled on the current EU test protocol by applying a three-step sampling, using 10, 20, and 30 kg sample averages. An acceptance level of 15 ng/g of total aflatoxin, under consideration for U.S. standards, is applied. Optimization leads to indifference regions of 2-30 ng/g for the first two steps. The resulting OC curve approximates that for a single 50 kg sample. The sequential protocol is applied to the results for a set of 1293 lots of the 1998 crop year, each tested with a single 10 kg sample. Ninety-five percent of the lots would have been accepted on the basis of the single test and 1.5% would have been rejected, whereas 3.5% of the lots would have required retesting.     

Liquid chromatographic determination of aflatoxin M1 in milk

The official AOAC method for aflatoxin M1 in milk was modified by replacing cellulose column chromatography with cartridge chromatographic cleanup and replacing thin layer chromatographic (TLC) determination with liquid chromatographic (LC) quantitation to yield a new method for bovine and porcine milk. An acetone extract of milk is treated with lead acetate and defatted with hexane, and M1 is partitioned into chloroform as in the AOAC method. Chloroform is removed by evaporation under a stream of nitrogen at 50 degrees C. The residue is dissolved in chloroform, the vessel is rinsed with hexane, and the 2 solutions are applied in sequence to a hexane-activated silica Sep-Pak cartridge. Less polar impurities are removed with hexane-ethyl ether, and M1 is eluted with chloroform-methanol, and determined by C18 reverse phase LC using fluorescence detection. Recoveries of M1 added to bovine milk at 0.25, 0.50, and 1.0 ng/mL were 90.8, 93.4, and 94.1%, respectively. The limit of detection was less than 0.1 ng M1/mL for both bovine and porcine milk.     

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 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, 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.     

Effects of injection solvent and mobile phase on efficiency in reverse-phase liquid chromatographic determination of aflatoxin M1

The effects of injection solvent and mobile phase composition on the reverse-phase liquid chromatographic determination of aflatoxin M1 (M1) were examined. M1 was converted to the more highly fluorescent derivative aflatoxin M2a (M2a). Using a C-18 column and a mobile phase of H2O-MeCN-MeOH (60 + 20 + 20) (MP-A), M2a was dissolved in various ratios of MeCN-H2O prior to injection. Chromatographic efficiency for the M2a peak varied from ca 2000 theoretical plates when injected in 30% aqueous MeCN to ca 9000 plates when injected in water alone. However, using the same C-18 column but with a mobile phase of H2O-IPA-MeCN (80 + 12 + 8) (MP-B), the M2a peak exhibited 25,000 plates when injected in 30% aqueous MeCN and 10,000 plates when injected in water alone.     

Comparison of radioimmunoassay and enzyme-linked immunosorbent assay for determining aflatoxin M1 in milk

Using a highly specific antibody against aflatoxin M1, a radioimmunoassay (RIA) and an enzyme-linked immunosorbent assay (ELISA) were developed for the quantitation of M1 in milk. RIA was sensitive in the range of 5-50 ng per assay but was subject to interference by whole milk. Extraction and cleanup were therefore necessary for the detection of M1 in milk at 0.5 ng/mL. An ELISA procedure was developed by using an aflatoxin M1-carboxymethyl-horseradish peroxidase conjugate as the ligand. Competitive assays revealed that this system was relatively more sensitive for M1 than for B1, and had a much lower degree of cross-reactivity for aflatoxins B2, G1, G2, B2a, and aflatoxicol. As low as 0.25 ng M1/mL in artificially contaminated milk (raw, whole, skim) could be detected by ELISA in 3 h without extraction or cleanup. Because of its simplicity, sensitivity, and specificity, ELISA is the preferred method for monitoring aflatoxin M1 in milk.     

Direct enzyme-linked immunosorbent assay for determining aflatoxin M1 at picogram levels in dairy products

Protocols for detecting picogram quantities of aflatoxin M1 in dairy products were established. Milk samples were subjected to a reverse phase Sep-Pak C18 cartridge treatment before analysis by an enzyme-linked immunosorbent assay (ELISA) according to previously published procedures. M1 in yogurt, brick cheddar, and ripened Brie cheese was extracted by a modified Pons method, subjected to a normal phase silica cartridge treatment, and analyzed by ELISA. The detection limits for M1 in milk, yogurt, cheddar, and Brie were 10, 10, 50, and 25 ppt (ng/kg), respectively. Recovery for M1 added to these products was in the range 70-110%. Good agreement was found for M1 levels in several naturally contaminated milk samples analyzed by both ELISA and liquid chromatography.     

Immunosorbents coupled on-line with liquid chromatography for the determination of fluoroquinolones in chicken liver.

Four fluoroquinolones were analyzed in fortified chicken liver using an automated, on-line immunoaffinity extraction method. The fluoroquinolones were extracted from the liver matrix using an immunoaffinity capture column containing anti-sarafloxacin antibodies covalently cross-linked to protein G. After interfering liver matrix components had been washed away, the captured fluoroquinolones were automatically eluted directly onto a reversed phase column. Liquid chromatographic analyses were performed by isocratic elution using 2% acetic acid/acetonitrile (85:15) as the mobile phase and an Inertsil phenyl column with fluorescence detection at excitation and emission wavelengths of 280 and 444 nm, respectively. No significant interferences from the sample matrix were observed, indicating good selectivity with the immunoaffinity column. Overall recoveries from fortified liver samples (20, 50, and 100 ng/g) ranged between 85.7 and 93.5% with standard deviations of <5%. The limit of quantification for each fluoroquinolone was 1 ng/mL. The limits of detection, based on a signal-to-noise ratio of 5:1, were 0.47, 0.32, 0.87, and 0.53 ng/mL for ciprofloxacin, enrofloxacin, sarafloxacin, and difloxacin, respectively.     

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.     

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.     

Distribution of aflatoxin in pistachios. 6. Seller's and buyer's risk.

The seller's risk-the probability of a set of samples exceeding an agreed upon aflatoxin level when the lot mean does not-and the buyer's risk-the probability of a lot exceeding this level when a set of samples do not-have been computed using a parametrized experimental aflatoxin distribution and Monte Carlo simulation. The calculations are exemplified using the proposed EC standards (three 10 kg samples, 4 ng/g of total aflatoxin, basis kernels only) as well as for samples up to 250 kg and for varied lot aflatoxin levels. It is found that within this sample size range the seller's risk is as high as 42% at 10 kg and increases with increasing sample size to 80% at 250 kg. Only by reducing lot levels to 0.2 ng/g of total aflatoxin, basis kernels, can the risk be brought down to 2.5%, independent of sample size. The buyer's risk is as high as 58% at 10 kg but falls to 11% at 250 kg samples. The implications for both seller and buyer strategies are discussed.     

Aflatoxins and fumonisins in corn from the high-incidence area for human hepatocellular carcinoma in Guangxi, China

A comparative study on the natural occurrence of aflatoxins and Fusarium toxins was conducted with corn samples from high- and low-incidence areas for human primary hepatocellular carcinoma (PHC) in Guangxi, China. In samples from the high-risk area, aflatoxin B(1) was the predominant toxin detected in terms of quantity and frequency, with its concentration ranging between 9 and 2496 microg/kg and an 85% incidence of contamination. Among the samples, 13 (76%) exceeded the Chinese regulation of 20 microg/kg for aflatoxin B(1) in corn and corn-based products intended for human consumption. Significant differences in aflatoxin B(1), B(2), and G(1) and total aflatoxin concentrations in corn between the areas were found (P < 0.05). The average daily intake of aflatoxin B(1) from corn in the high-risk area was 184.1 microg, and the probable daily intake is estimated to be 3.68 microg/kg of body weight/day, 3.20 times the TD(50) in rats. Corn samples from both areas were simultaneously contaminated with fumonisins B(1), B(2), and B(3). Aflatoxin B(1) may play an important role in the development of PHC in Guangxi.     

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.     

Determination of aflatoxins in vegetable oils

A simple method is proposed for determination of aflatoxins in vegetable oils. The method was successfully applied to both crude and degummed oils. The oil sample, dissolved in hexane, was applied to a silica column and washed with ether, toluene, and chloroform; aflatoxins were eluted from the column with chloroform-methanol (97 + 3). As quantitated by thin layer chromatography and liquid chromatography, the oils analyzed contained aflatoxin B1 at levels of 5-200 micrograms/kg. Recoveries of aflatoxin B1 standards added to aflatoxin-free oils were between 89.5 and 93.5%, with coefficients of variation of 6.3-8.0%.