Thin Layer Chromatographic Determination of Citrinin

Abstract Clearly defined zones of citrinin can be obtained on thin layer chromatographic (TLC) plates and measured by fluorodensitometry. Silica gel plates were prepared as a slurry with aqueous 0.05M Na2EDTA (ethylenediaminetetraacetic acid), spread at 0.5 mm wet thickness, and activated at 105°C for 1 hr. Plates were developed in acetic acid-benzene (5+95). The limit of detection was 10 ng citrinin/zone. Densitometric analysis (365 nm excitation, 505 nm emission) revealed that a linear relationship exists for levels of 10 ng to at least 100 ng/zone wtih a coefficient of variation of ±5%.</jats:p

Optimum Conditions for Formation of Aflatoxin Mj-Trifiuoroacetic Acid Derivative

Abstract Because thin-layer chromatographic (TLC) confirmation of identity and reverse-phase liquid chromatographic (LC) determination with fluorescence detection of aflatoxin M1 both require the derivative formed in the reaction of M1 and trifluoroacetic acid (TFA), various reaction conditions were studied to obtain complete derivative formation. Of the various organic solvents tested, the reaction between M1 and TFA proceeded best in the nonpolar solvents hexane and isooctane. Other parameters investigated were reaction temperature and time, aflatoxin M1 concentration, and solvent volume. The following procedure is considered optimum: 200 μL each of hexane and trifluoroacetic acid are mixed with M1 standard in a silylated glass vial or with milk residue in a regular glass vial with a Teflon-lined screw cap and heated 10 min at 40°C. The mixture is evaporated to dryness under N2, and the derivative is saved for TLC or LC. No unreacted aflatoxin M1 was detected by reverse-phase LC after this procedure was incorporated for analysis of milk samples.</jats:p

Stability and Molar Absorptivity of Aflatoxin M1 in Acetonitrile-Benzene (1 + 9)

Abstract A solvent mixture of acetonitrile-benzene (1+9) was investigated as a means to eliminate instability of aflatoxin M1 standard solutions prepared with chloroform. Solutions (10.0 μg/mL) were prepared and sealed in glass ampules that were opened as needed for absorbance measurements on the individual solutions over a 13-month period. A coefficient of variation of 0.69&amp; for the absorbance readings indicated that the solutions were stable. In addition, numerous powdered milk extracts containing aflatoxin M1 were dissolved in both chloroform and acetonitrile-benzene (1+9), and the solutions were spotted on TLC plates and developed. Densitometric measurement of the developed zones showed no differences due to the spotting solvent. The molar extinction coefficient was determined in acetonitrilebenzene (1+9) on 4 different M1 solutions from 2 separate crystalline aflatoxin M1 preparations; the value was 18,815 ± 24.</jats:p

Collaborative Study of Methods for the Determination and Chemical Confirmation of Aflatoxin M1 in Dairy Products

Abstract An international collaborative study involving 19 collaborators was conducted to test methods for the determination and chemical confirmation of aflatoxin M1 in dairy products. For the quantitative method, collaborators assayed samples of liquid and powdered milk, cheese, and butter containing low levels of M1. Statistical results indicated that sensitivity and precision of this method were comparable to other AOAC methods for aflatoxin M1. Impurities were present in blue cheese extracts that tended to interfere with thin layer chromatography. Analysis of liquid milk samples from different areas revealed that some milk extracts may require column chromatography. For the chemical confirmatory method, collaborators prepared acetate and hemiacetal derivatives of M1 in extracts of liquid milk and colby cheese. A sensitivity limit of 30 ng M1 was apparent for the method, and most collaborators easily identified the derivatives. As a result of this collaboration, both methods have been adopted as official first action methods.</jats:p

Reverse Phase Analytical and Preparative High Pressure Liquid Chromatography of Aflatoxins

Abstract Reverse phase high pressure liquid chromatography (HPLC) was investigated and found applicable for determining 6 aflatoxins. Aflatoxins M2, M1, G2, G1, and B1 were completely resolved and B2 was satisfactorily separated on a C18 (10 μm, 4 mm id × 30 cm) column with an eluting solvent of acetonitrile-water (35 + 65) and flow rate of 1.5 ml/min. Compounds were detected by ultraviolet absorbance at 350 nm. Peak height and retention time reproducibility of multiple injections was excellent with coefficients of variation of 1.0% (M1) and 1.9% (B1) when laboratory temperatures were relatively constant throughout a day ; however, coefficients of variation increased significantly when temperatures varied by 10°F. HPLC determinations of aflatoxin B1 added to uncontaminated corn extracts at 20 and 50 ppb levels were 83±14 and 92.5±8%, respectively, of those expected. Comparison of com extract assays (6–98 ppb B1) by thin layer chromatography (TLC) and HPLC revealed that HPLC values averaged 25% less than TLC values. Numerous peaks and background interferences were present in corn extracts which made interpreting chromatograms difficult. None of the cleanup procedures tried was successful in removing these interferences. Preparative HPLC was used to isolate and purify quantities of aflatoxins M1, B1, and G1 from silicic acid column mixtures of M1-M2, B1-B2, and G1-G2. Separations were achieved on C18/Porasil B (35–75 μm) columns (⅜″ od × 8′) developed with acetonitrile-water mixtures (M1, 20+80; B1, 35+65; G1, 25+75) at 9.0 ml/min. These columns permitted isolation of 40 mg aflatoxin B1 or G1 in less than 3 hr. Aflatoxin M1 required 2 HPLC steps.</jats:p

Determination of Aflatoxins in Animal Tissues

Abstract A method for the determination of aflatoxins in animal tissues has been developed, and applied successfully to beef, swine, chicken, and human livers, and to beef kidney, heart, spleen, muscle, and blood. Blended tissue is denatured with citric acid and extracted with dichloromethane on a wrist-action shaker. After filtration, the extract is partially purified on a silica gel column, and aflatoxins B1 and M1 are determined by 2-dimensional thin layer chromatography and densitometry. Recoveries of Bi and Mi added to meat tissues and blood were approximately 90 and 80%, respectively. The method gave results for a contaminated freeze-dried liver comparable to analyses by 3 other published meat tissue methods. The method is rapid and has a determination limit ≤0.1 ng/g. In addition, the method uses less toxic and smaller quantities of solvents and chemicals.</jats:p

Collaborative Study of Three Screening Methods for Aflatoxin in Corn

Abstract The 3 rapid screening methods studied all involved chromatography of partially purified extracts on minicolumns. In one method acetonitrile-water is used to extract aflatoxin from corn and a lead acetate precipitation removes impurities from the extracts. The second method specifies acetone-water as the extracting solvent and ammonium sulfate as the precipitant. The third method is based on acetone-water as the extracting solvent and ferric gel as the precipitant. The most sensitive method was the ferric gel precipitation, which could detect 5 ppb aflatoxin, but it was very time-consuming. The most satisfactory method (timewise) used the ammonium sulfate and had a sensitivity of 10 ppb. This method also led to the best differentiation between levels of aflatoxin. These 2 methods have been adopted as official first action.</jats:p

Aflatoxin M1: Analysis in Dairy Products and Distribution in Dairy Foods Made from Artificially Contaminated Milk

Abstract A published method to determine aflatoxin M1 in fluid and concentrated milks was refined to permit assays of a wider range of dairy products. Milk products that were spiked with aflatoxin Mx were processed with commercial dairy cultures, rennet, and organic acids to make several cheeses and butter. Cheeses that were representative of various curd-precipitation methods or high cooking temperatures were included. When milk was set with lactic acid cultures and small quantities of rennet to produce short-set cottage cheese or heated with lactic acid starter to prepare ricotta cheese, about 71–74% of the aflatoxin M1 was recovered in the whey fractions. Nearly equal quantities of M1 were detected in curds and whey from long-set, acidprecipitated cottage cheese and from rennetprecipitated cheeses, such as colby, cheddar, and swiss. Results were similar with queso bianco cheese products that were produced by direct addition of organic acids. According to analysis, only 16% of the available M1 was in the butter while the remainder was in the buttermilk.</jats:p