Rapid thin layer chromatographic determination of aflatoxin M1 in powdered milk

A method has been developed for the detection of aflatoxin M1 in milk. The toxin is extracted with chloroform, the extract is evaporated, and the residue is partitioned between carbon tetrachloride and an aqueous saline-methanol solution. The toxin is once again extracted with chloroform from the methanol solution and analyzed by thin layer chromatography. The limit of detection of M1 in powdered milk is 0.5 microgram/kg; recoveries of added M1 are about 83%. The limit of detection can be improved to 0.3 microgram/kg if the plate is sprayed with an aqueous solution of H2SO4 after development.     

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.     

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.     

Thin layer chromatographic determination of aflatoxin in corn dust

Methods adopted by the AOAC and the American Association of Cereal Chemists for determining aflatoxin in corn were modified, and techniques were developed for application to samples of less than 1 to 10 g instead of the specified 50 g samples. Analysis included chloroform extraction of dust samples or dust collected from glass fiber filters, purification of extracts on a silica gel column of appropriate size, and measurement of aflatoxin by either 1- or 2-dimensional thin layer chromatography (TLC). The solvent for 1-dimensional TLC was chloroform-acetone-water (91 + 9 + 1). Solvents for 2-dimensional TLC were, first direction, ether-methanol-water (95 + 4 + 1, lined tank) and second direction, chloroform-acetone-water (91 + 9 + 1, unlined tank), or first direction, chloroform-acetone-water (91 + 9 + 1, unlined tank) and second direction, toluene-ethyl acetate-formic acid (60 + 30 + 10, unlined tank). When samples weighed less than or equal to 0.1 g, the entire concentrated extract was applied to the TLC plate. About 0.5-1.0 ng aflatoxin B1 could be detected on the plate, making the limit of detection about 9 ng/g for 0.1 g samples.     

Rapid reverse phase liquid chromatographic determination of aflatoxin M1 in milk

A rapid, economical, and reliable liquid chromatographic (LC) method is described for determination of aflatoxin M1 in milk. The method includes an improved AOAC extraction procedure, cleanup of the extract on a silica cartridge, and LC quantitation. Alternatively, a rapid column cleanup procedure can be used. Milk artificially spiked with aflatoxin M1 at 0.05, 0.1, and 0.5 ppb was analyzed using both new approaches as well as an AOAC method coupled with LC for quantitation of the toxin. Recovery of aflatoxin M1 by the first approach of the new method ranged between 93.4 and 99.1%, and for the alternative procedure between 92.4 and 96.8%. The AOAC method gave lower recovery (85.6-90.7%) of toxin, but the results from this method had a somewhat smaller standard deviation for replicate analyses than did results of the new method.     

Thin layer chromatographic determination of aflatoxin B1 in eggs: collaborative study

The thin layer chromatographic method of Trucksess et al. for aflatoxin B1 in eggs was collaboratively studied. Each collaborator analyzed 3 known practice samples and 9 unknown samples containing added aflatoxin B1 at 0, 0.05, 0.10, and 0.30 ng/g. For 9 collaborators, recoveries for the 3 positive levels were: 0--0.13 ng/g (average 98%, coefficient of variation (C.V.) 83%), 0.05--0.18 ng/g (average 102%, C.V. 36%, and 0.11--0.42 ng/g (average 93%, C.V. 31%), respectively. The method has been adopted as official first action.     

Rapid high pressure liquid chromatographic determination of aflatoxin M1 in milk and nonfat dry milk

A modification of the current revised AOAC method, 26.A10-26.A15, is described for the rapid analysis of aflatoxin M1 in milk and nonfat dry milk. The method incorporates chloroform extraction and eliminates the need for column chromatography by using liquid-liquid partition for sample extract cleanup. Quantitation is carried out by using fluorescence detection combined with high pressure liquid chromatography (HPLC) of aflatoxin M1 which has been converted to aflatoxin M2a with trifluoroacetic acid. The method has a detection limit of 0.014 micrograms/L (2 X signal/noise) for whole milk. For 6 samples of naturally contaminated nonfat dry and freeze-dried milk, the modified method gave an average result of 0.698 micrograms/L; the AOAC method gave an average result of 0.386 micrograms/L.     

Automated liquid chromatographic determination of aflatoxin M1 in milk using on-line dialysis for sample preparation

A procedure has been developed for the automated isolation of aflatoxin M1 from decreamed milk. The method uses on-line stopped flow dialysis and subsequent trace enrichment on a reverse-phase column. After a back-flush to the analytical liquid chromatography column, aflatoxin M1 is determined with fluorescence detection. Fully automated analysis is possible with reproducible dialysis recoveries above 50% (CV = 7.5%, n = 25 at the 50 ng/kg level) and determination levels of 20 ng/kg within 20 min.     

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.     

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.     

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.     

Formation of aflatoxin derivatives on thin layer chromatographic plates.

Rapid confirmation of the presence of aflatoxins B-1 and G-1 in foods is provided by reaction with trifluoroacetic acid at the origin of a thin layer chromatographic plate. The procedure has been used successfully with various nuts, grains, coffee and cocoa beans, and other foods.     

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.     

Determination of aflatoxin M1 in milk by liquid chromatography with fluorescence detection

A liquid chromatographic (LC) method is proposed for the determination of aflatoxin M1 in milk. The method was successfully applied to both liquid whole and skim milk and also whole and skim milk powder. The samples are initially extracted with acetonitrile-water followed by purification using a silica gel cartridge and a C18 cartridge. Final analysis by LC was achieved using a radial compression module equipped with a 5 micron C18 column and a fluorescence detector. The method was successfully applied to samples at levels of 10 to 0.08 ppb added aflatoxin M1 with recoveries in the range of 70-98%.     

Simultaneous thin layer chromatographic determination of aflatoxin B1 and ochratoxin A in black olives

A screening method has been developed for simultaneous determination of aflatoxin B1 and ochratoxin A in black olives. The technique includes extraction of both mycotoxins with aqueous methanol, cleanup using lead acetate, defatting with hexane, partitioning in chloroform, and thin layer chromatography. Detection limits are 5-7 micrograms aflatoxin B1 and 20 micrograms ochratoxin A/kg.     

Thin layer chromatographic identification of some sympathomimetic amines.

Thin layer chromatographic behavior of some sympathomimetic amines in the presence of acids in neutral and organic solvent systems is reported. The sympathomimetic amines were dissolved in 0.1N HCl or ethanol and treated with bromocresol green or p-nitrobenzoyl chloride reagents on fiber sheets or precoated glass plates. Two-, 3-, and 4-, component solvent systems were tested. Benzene-ethyl acetate gave 2 spots for each amine standard; the more polar spots were satisfactorily separated. Amines in pharmaccuticals were not separated by any solvent system tested.