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PubMed1980

Rapid method for analysis of aflatoxin M1 in dairy products.

Four methods for determining aflatoxin M1 levels in fluid milk were compared in a search for a simple, routine analytical procedure.. Each method was tested with samples spiked at levels of 0, 0.1, 0.5, and 1.0 ppb. The 3 quantitative methods were comparable in sensitivity (0.1 ng/mL) and recoveries (80-90%). A new method, using a commercially available extraction column that contains an inert hydrophilic matrix to permit extraction of dairy products, is described. The milk is adsorbed on the column and aflatoxin M1 is eluted with chloroform-acetone (9 + 1). The extract is further purified on a small silica gel column and aflatoxin M1 is determined by TLC. This method was simpler and shorter, and was equally sensitive and reproducible in determining aflatoxin M1 in spiked and naturally contaminated fluid milk samples and in spiked nonfat powdered, condensed, and evaporated milk.

Chromatography, Thin Layer↗
PubMedDate not supplied

Robotic analysis of aflatoxin M1 in milk.

An automated aflatoxin M1 assay system capable of performing multiple unattended extractions and chromatographic analyses has been developed. A six-axis anthropomorphic laboratory robot and a flexible computer system are combined to operate a sample turntable, a multisolvent dispensing facility, a solid-phase extraction station, a vacuum manifold, an automatic HPLC sample preconcentration unit, an HPLC, a fluorescence detector and a computing integrator. The system is capable of handling bulk milk samples and can determine aflatoxins at the sub-micrograms/kg level with an accuracy and precision comparable to those of the manual methods of analysis. Time of analysis is reduced. The system can be run in an unattended mode of operation.

Aflatoxin M1↗
PubMedDate not supplied

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.

Aflatoxin M1↗
PubMed2001

Distribution and stability of aflatoxin M1 during processing, ripening and storage of Telemes cheese.

Telemes cheeses were produced using milk that was artificially-contaminated with aflatoxin M1 at the levels of 0.050 and 0.100 microg/l. The cheeses produced in the two cheese-making trials were allowed to ripen for 2 months and stored for an additional 4 months to simulate commercial production of Telemes cheese. Concentrations of aflatoxin M1 in whey, curd, brine, and the produced cheeses were determined at intervals by liquid chromatography and fluorometric detection coupled with immunoaffinity column extraction. Concentrations of aflatoxin M1 in the produced curds were found to be 3.9 and 4.4 times higher than those in milk, whereas concentrations in whey were lower than those in curd and milk. Aflatoxin M1 was present in cheese at higher concentrations at the beginning than at the end of the ripening/storage period, and it declined to concentrations 2.7 and 3.4 times higher than those initially present in milk by the end of the sixth month of storage. Concentrations of aflatoxin M1 in brine started low and increased by the end of the ripening/storage period but only a portion of the amounts of aflatoxin M1 lost from cheese was found in the brine. Results showed that Telemes cheeses produced from milk containing aflatoxin M1 at a concentration close to either the maximum acceptable level of 0.05 microg/l set by the European union (EU) or at double this value, will contain the toxin at a level that is much lower or slightly higher, respectively, than the maximum acceptable level of 0.250 microg of aflatoxin M1/kg cheese set by some countries.

Aflatoxin M1↗
PubMed1986

Use of ultraviolet energy to degrade aflatoxin M1 in raw or heated milk with and without added peroxide.

Raw whole milk was artificially contaminated to contain 1 ppb aflatoxin M1. A thin layer of milk (.1 cm) was irradiated with ultraviolet energy. In the first experiment, milk was held at 90 degrees C for 10 min, cooled to 20 degrees C, and irradiated for 30 min. Amount of aflatoxin M1 decreased equally (56.2 vs. 53.9%) in raw or preheated milk, suggesting no involvement of milk enzymes in degrading aflatoxin M1 by ultraviolet energy. Data obtained when raw milk containing aflatoxin M1 was exposed to ultraviolet energy for 15 to 60 min suggest first order kinetics for the degradation reaction. In another experiment, milk was held at 5, 25, or 65 degrees C while it was being irradiated. Aflatoxin M1 was degraded at all temperatures. Amount of toxin decreased nonlinearly when temperature at which milk was held was increased. Presence in milk of benzoyl peroxide at .002% did not change the extent to which aflatoxin M1 was degraded by irradiation. Amount of toxin, however, decreased by 89.1% in milk containing .05% H2O2 as compared with 60.7% for H2O2-free milk when both were exposed to ultraviolet irradiation for 20 min at 25 degrees C.

Aflatoxin M1↗
PubMed1982

Aflatoxin M1: in vitro preparation and comparative in vitro metabolism versus aflatoxin B1 in the rat and mouse.

A rapid, simple and relatively inexpensive biotransformation method using microsomes from 3-methylcholanthrene (3MCA) pretreated rats is described for the production of aflatoxin M1 (AFM1) is sufficient quantities for metabolism studies. A comparison is made of the metabolism of AFM1 and AFB1 by the postmitochondrial (S-10) fraction from mouse and rat hepatocytes. AFM1, in both species, is metabolized more slowly than AFB1, with its major metabolites being found in the aqueous fraction and very little associated with S-10 protein. In contrast, AFB1 is metabolized to numerous chloroform-extractable, protein bound and water-soluble metabolites. The toxicological implications of reduced protein binding and slower metabolism for AFM1 are discussed.

Aflatoxin B1↗
PubMed2004

Aflatoxin M1 in yoghurts in Portugal.

Aflatoxin M1 (AFM1) may occur in milk and milk products, resulting from the ingestion of aflatoxin B1 in feedstuffs by dairy cow. Ninety-six samples of commercial yoghurts (48 natural yoghurts and 48 yoghurt with pieces of strawberries) that are produced in Portugal were analyzed for the presence of AFM1 by immunoaffinity column extraction and high-performance liquid chromatography (HPLC). The limit of detection was 10 ng/kg. The recoveries of AFM1 from the samples spiked at levels of 10.0, 50.0, 100.0 and 150.0 ng/kg were 88.0%, 91.0%, 93.0% and 99.0%, respectively. AFM1 was detected in 18 (18.8%) of yogurt samples ranging from 19 to 98 ng/kg, and 78 samples (81.2%) did not reveal the presence of the toxin. Of the 48 natural yoghurts tested, only two (4.2%) were contaminated with 43 and 45 ng/kg of AFM1. Of the 48 yoghurts with pieces of strawberries tested, 16 samples (33.3%) contained levels ranging from 19 to 98 ng/kg; six samples (12.5%) were contaminated with low levels ranging from 19 to 35 ng/kg; four samples (8.3%) were contaminated with levels ranging from 36 to 50 ng/kg, two samples (4.2%) with levels of 51 and 65 ng/kg and four samples (8.3%) presented high contamination levels, from 90 to 98 ng/kg. This paper reports the data of the first survey on the presence of AFM1 in yoghurt in Portugal.

Aflatoxin M1↗
PubMed1978

Confirmatory test for aflatoxin M1 on a thin layer plate.

The identity of aflatoxin M1 can easily be confirmed directly on a thin layer plate by reacting aflatoxin M1 with trifluoroacetic acid (TFA). This confirmation reaction is carried out on the thin layer plate which has been developed in 2 dimensions and used for the quantitation of aflatoxin M1 in the sample. TFA is superimposed on the separated M1 spot. The plate is kept in the dark 3 min, heated to 75 degrees C for 5 min, and developed with chloroform-methanol-acetic acid-water (92 + 8 + 2 + 0.8). The Rf value of the blue-fluorescent derivative is compared with that for the M1 standard. The method was used successfully on extracts of milk, cheese, and liver. M1 quantities on the plate as low as 0.5 ng can be confirmed by this method. The method is also suitable for simultaneous confirmation of aflatoxin B1.

Aflatoxins↗
PubMed1994

Development and evaluation of a minicolumn assay for the detection of aflatoxin M1 in milk.

A practical field method for the chemiselective immobilization and detection of aflatoxin M1 in milk has been developed and is being marketed. In this new method, aflatoxin M1 is selectively adsorbed at the interface of a layer of neutral sand and a band of magnesium silicate (Florisil) packed in a glass minicolumn. Aflatoxin M1, at > or = .5 ppb in contaminated milk, can be easily visualized as a band of bright blue fluorescence. Briefly, raw or homogenized and pasteurized milk is diluted with water (1: 1, vol/vol) and passed through a C18 cartridge. Aflatoxin M1 is then partitioned by polarity, eluted with acetone-methylene chloride, and added to the minicolumn. The minicolumn is washed and viewed under long wave UV light. The limit of detection for this assay was .2 ppb, which was similar to the .3 ppb obtained using an immunoaffinity column, followed by minicolumn detection. The assay was accurate, rapid, easy to perform, and stable.

Adsorption↗
PubMed1982

Kinetics of transformation of aflatoxin B1 into aflatoxin M1 in lactating mouse: an ELISA analysis.

A new enzyme-linked immunosorbent assay (ELISA) was used to study the kinetics of transformation of aflatoxin B1 into aflatoxin M1 in lactating mice. Aflatoxin M1 concentration in the milk samples reached a maximum 30 min after injection of aflatoxin B1 and decreased thereafter. At the maximum time, the levels of aflatoxin M1 in the samples were proportional to the dosages administered. Aflatoxin B1 was also detected in the milk samples but at a lower concentration.

Aflatoxin B1↗
PubMedDate not supplied

Immunoaffinity column cleanup with liquid chromatography for determination of aflatoxin M1 in liquid milk: collaborative study.

A collaborative study was conducted to evaluate the effectiveness of an immunoaffinity column cleanup liquid chromatographic method for determination of aflatoxin M1 in milk at proposed European regulatory limits. The test portion of liquid milk was centrifuged, filtered, and applied to an immunoaffinity column. The column was washed with water, and aflatoxin was eluted with pure acetonitrile. Aflatoxin M1 was separated by reversed-phase liquid chromatography (LC) with fluorescence detection. Frozen liquid milk samples both naturally contaminated with aflatoxin M1 and blank samples for spiking, were sent to 12 collaborators in 12 different European countries. Test portions of samples were spiked at 0.05 ng aflatoxin M1 per mL. After removal of 2 noncompliant sets of results, the mean recovery of aflatoxin M1 was 74%. Based on results for spiked samples (blind pairs at 1 level) and naturally contaminated samples (blind pairs at 3 levels) the relative standard deviation for repeatability (RSDr) ranged from 8 to 18%. The relative standard deviation for reproducibility (RSDR) ranged from 21 to 31%. The method showed acceptable within- and between-laboratory precision data for liquid milk, as evidenced by HORRAT values at the low level of aflatoxin M1 contamination.

Aflatoxin M1↗
PubMed1981

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.

Aflatoxin M1↗
PubMed2000

[Establishment of monoclonal antibodies against aflatoxin M1].

The monoclonal antibodies against aflatoxin M1 were established. Spleen cells from Balb/c mice immunized with AFM1-oxime-BSA conjugate were fused with murine Sp2/0 myeloma cells. Three hybridoma cell lines secretine monoclonal antibodies against AFM1 were established after the fusion cells subcloned for 3-4 cycles. These antibodies were disignated as 3G2, 3G3 and 6G8, respectively. All of them belonged to the subtype of IgG1. The titres of the antibody in ascites ranged from 1:3.2 x 10(6)-1:20 x 10(6). There was no cross reaction between 6G8 monoclone antibody and other types of aflatoxin.

Aflatoxin M1↗
PubMed2001

Seasonal variations of aflatoxin M1 in the farm milk in Albania.

This paper presents monitoring data on levels of aflatoxin M1 in the farm-gate milk in Albania. The monitoring included 120 evenly distributed samples collected in winter and summer from various farms all over the country. The levels of aflatoxin M1 were determined using the quantitative thin layer chromatography. On average, the winter milk samples revealed higher concentrations of aflatoxin M1 than the summer samples. Thirteen percent of the winter samples resulted above the 0.5 microgram/kg level, as compared to 3% of the summer samples exceeding that level. Skimmed and semi-skimmed milk from the same source contained comparatively lower levels of aflatoxin M1 than whole milk.

Aflatoxin M1↗
PubMedDate not supplied

Development of milk powder reference materials certified for aflatoxin M1 content (Part I).

The development of 3 full-cream milk powder reference materials, certified for their aflatoxin M1 content, is described. The materials were prepared and certified within the Reference Material Programme of the Community Bureau of Reference (BCR). The 3 reference materials, RMs 282, 284, and 285, contain aflatoxin M1 at concentrations of less than 0.05, 0.31 +/- 0.06, and 0.76 +/- 0.05 micrograms/kg, respectively. The preparation, testing for homogeneity, stability of the reference materials, and the certification exercise, which was preceded by 2 intercomparisons of methods, are discussed. Particular emphasis was placed on the independence of the measurements in the certification exercise and the control of errors associated with extraction efficiency and the aflatoxin M1 calibrant. Finally, some guidance is given on avoiding the principal sources of errors in the determination of aflatoxin M1. Details concerning the supply of the reference materials will be provided by BCR on request.

Aflatoxin M1↗
PubMed1981

Rapid screening method for aflatoxin M1 in milk.

A rapid screening method for detecting aflatoxin M1 in milk has been developed, based on minicolumn chromatography and requiring 8-10 min for each test. The minicolumn is packed with dry Florisil (100-200 mesh) on the bottom, anhydrous Na2SO4 as the next layer, topped with neutral alumina (70-200 mesh) to which 8% water (wet basis) has been added. A blue fluorescent band at the Florisil-Na2SO4 interface indicates the presence of aflatoxin M1. The limit of detection is estimated to be about 0.2 microgram/kb. Because several items are disposable, both the time to maintain glassware and the cost per determination are reduced.

Aflatoxin M1↗
PubMed1998

Automated column-switching high-performance liquid chromatography for the determination of aflatoxin M1.

An extractionless method for determining aflatoxin M1 (AFM1), a major metabolite of aflatoxin B1 (AFB1), in human urine was developed. The biological fluid is injected directly into the chromatographic system after simple dilution and centrifugation. A pre-column, packed with a cation-exchange phase and coupled on-line to a column-switching liquid chromatography (LC) system, is used for sample pre-treatment and concentration. The analytes are non-selectively desorbed with the LC eluent and cleaned by means of a column-switching procedure. Pre-treatment and analysis were performed within 40 min. Average AFMI recovery reached 97% in the 10-100 ng/l range of urine. The detection limit of AFM1 in urine and milk was 2.5 ng/l for 1 ml of injected sample. A comparison with an immunoaffinity column clean-up and LC method was performed. The method was applied to determine AFM1 in the urine of AFB1 gavaged rats, and in the urine of both potentially exposed and supposedly unexposed workers. The method was also extended to milk.

Aflatoxin M1↗
PubMed2004

Aflatoxin binders II: reduction of aflatoxin M1 in milk by sequestering agents of cows consuming aflatoxin in feed.

Sequestering agents bind dietary aflatoxin B1 (AFB1) and reduce absorption from an animal's gastrointestinal tract. As a result, they protect an animal from the toxic effects of AFB1 and reduce transfer of the metabolite, aflatoxin M1 (AFM1), into milk. Three experiments, using late-lactation Holstein cows fed AFB1-contaminated feed, were conducted to evaluate several potential sequestering agents for their abilities to prevent or reduce the transmission of AFM1 into milk. Six agents previously tested in our laboratory for AFB1 binding in vitro were evaluated in these experiments. These were: SA-20, an activated carbon (AC-A); Astra-Ben-20, a sodium bentonite (AB-20); MTB-100, an esterified glucomannan (MTB-100); Red Crown, a calcium bentonite (RC); Flow Guard, a sodium bentonite (FG); and Mycrosorb, a sodium bentonite (MS). Five of the six sequestering agents significantly (P < 0.01) reduced AFM1 contamination of milk (AB-20, 61%; FG, 65%; MS, 50%; MTB-100, 59%; and RC, 31%); whereas, AC-A, activated carbon, had no effect on AFM1 transmission at 0.25% of feed. By the first milking (1 day after cows consumed contaminated feed), AFM1 appeared in milk, then reached maximum levels after three days, and was absent from milk within four days after AFB1 was removed from the feed. Sodium bentonites at 1.2% of feed showed good potential as AFB1 binders; MTB-100, a yeast cell wall product, was equally effective at 0.05% in feed. Potential AFB1 binding agents should be evaluated experimentally to demonstrate efficacy. Our data show that sequestering agents can reduce AFM1 in milk of cows fed AFB1-contaminated feed.

Aflatoxin B1↗