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Determination of Aflatoxins B1 and M1 in Milk
Determination of Aflatoxins
and
in Milk
W. C. JACOBSON, W. ¢. HARMEYER, and H. G. WISEMAN
Division of Veterinary Research, Food and Drug Administration U.S. Department of Health, Education, and Welfare Beltsville, Maryland 20204 Abstract
sufficient to permit analysis slightly below 0.1 /zg p e r liter.
Cows fed contaminated rations containing the mold raetabolite aflatoxin B i secrete low levels of aflatoxin B1 and its metabolite afiatoxin M 1 in milk. To establish the relationship between ingested aflatoxin and milk-secreted aflatoxin, a sensitive method is needed to determine aflatoxins B i and M1 in milk. A method is presented b y which good recoveries, within the limits of visual comparison, were obtained from samples containing 0.5 parts per billion aflatoxin. Deproteinized milk (80% methanolie filtrates) was adjusted to 50% by addition of 4% salt solution and defatted by extraction with hexane. Aflatoxins were extracted from the alcoholic phase with chloroform which was removed in vacuo. The residue was dissolved in benzene :hexane (1:1) and chromatographcd on a partition column of Celite coated with an aqueous solution of calcium chloride to separate aflatoxins B1 and MI. The elated B1 was passed through another partition column of Celite coated with methanol :water (1:1) for additional cleanup. Aflatoxin eluates were evaporated to d~Tness and residues were dissolved in chloroform and spotted on thin-layer chromatographic plates for fluorescence comparisons.
Experimental Procedure
Reagents and materials. All solvents and reagents were A C S grade; the solvents were glass-distilled before use. The acid-washed Celite 545 was extracted with methanol, washed with water, and dried at 150 C for t 2 hours before use. F o r thin layer chromatography (TLC), Kieselguhr-formamide-water plates were prepared by the method of Adye and Mateles (1) except that 10 ml of formamide were included in the aqueous slurry used for coating. The plates were dried at 40 C for 90 minutes, stored at 4 C until used, and, after developmeat, were viewed over a long wave ultraviolet light. 1 l~efere~ce standards preparation. The comparison standard of aflatoxin B1 was obtained from Aspergillus flavus 2 cultured on a ricewheat medium. Crude aflatoxin was extracted from the culture medium with methanol. The alcoholic filtrate was diluted 50% by adding 4% sodium chloride solution and then defatted by extracting with hexane. The crude aflatoxin was extracted from the aqueous methanol with chloroform and the extract was decolorized by swirling with 10 g of copper carbonate (7) for each 400 ml of chloroform. The mixture was then filtered through a 4-mm bed of Celite on a coarse fritted glass Biichner funnel and the filtrate was evaporated to dryness in vacuo. The residue was dissolved in benzene and chromatographed on a column of silicie acid :Celite (2:1) coated with a stationary phase of formamide :water (3:1) in the proportions of 48 nd of liquid phase to 90 g of the adsorbents. Aflatoxin B1 was eluted with benzene saturated with formamide and then B 1 fractions which contained fluorescent impurities as shown by TLC were rechromatographed on silicie acid :Celite. The eluates were evaporated to dryness in vacuo and dissolved in small volumes of chloroform; the aflatoxins were
Introduction
I t has been established that cows which have consumed aflatoxin B 1 will secrete the metabolite aflatoxin Mi in their milk (2, 3, 4, 5). I n the analysis of milk for afiatoxin Mi, a dried powdered sample is commonly used. To eliminate uncertainties of losses by drying or prolonged storage, a method for direct analysis of aflatoxins in fluid milk is desirable. A method has been devised by which afiatoxins M 1 and B1 can be quantitatively recovered from fluid milk at the 0.5 ppb level. Cleanup is
1 Ultra-Violet Products, Inc., San Gabriel, California. 2 Food and Drug Administration, Division of Microbiology Isolate M93.
Received for publication August 26, 1970. 21
22
J A C O B S O N E T AL.
then crystallized by slow addition of nine volumes of hexane to yield pure aflatoxin B 1. Purity of the preparation was determined by TLC; 50 ng revealed only one spot. The molar absorbaney in ethanol at 361 n m was 23,000. To prepare the standard aflatoxin M1, a cow was given a daily dose of 40 mg of aflatoxin B 1 in 50 ml of ethanol by way of tureen fistula for four days. Total urines before and after dosing were collected in separate fractions. Urines were extracted with chloroform and the extracts were evaporated to dryness in vacuo and dissolved in methanol. A n equal volume of 4% sodium chloride solution was added and the resulting 50% methanol solutions were extracted with hexane for partial cleanup and then with chloroform to remove the aflatoxins. (The aqueous-alcoholic phase retained considerable extraneous matter.) The chloroform extracts were dried in vacuo and the residues ehromatographed on 122 × 2.5-cm columns of Celite coated with 44% aqueous solution of calcium chloride (30 ml/100 g Celite). The residues were transferred to columns by successive washings with the following eluting solvents, in order: hexane; hexane:benzene (1:1) ; benzene ; benzene :acetone (99:1) ; benzene :acetone (98:2) ; benzene :chloroform (70:30) ; and chloroform. Similar fractions from experimental and control urines were spotted on adjacent tracks of TLC plates; in this way, a slow-moving fluorescent spot not present in the control urine was located. The fraction containing the slow-moving compound was streaked on a TLC plate and, after development, the streak was scraped off the plate and eluted into chloroform. When the chloroform eluate was developed on a TLC plate, only one spot appeared. Spectrophotometrie peaks were determined to be 226, 265, and 357 nm; peak ratios were 122, 64.0, and 100, and agreed with values TABLE 1. Recovery of aflatoxin B 1 and 1~1 added to milk at 0.5 parts per billion. Recovered Sample 1 2 3 4 5 6 Control milk Avg recovery
B1 (ppb) 0.5 0.5 0.5 0.5 0.4 0.5 0.0
M1 (%) 100 100 100 100 80 100 0 97
(ppb) 0.5 0.5 0.5 0.5 0.4 0.4 0.0
JOURNAL OF DAIRY SCIENCE "VOL. 54, ~TO. 1
(%) 100 100 100 100 80 80 0 93
of 121.6, 61.1, and 100 reported by ttolzapfel et al. (5). The concentration of solution was calculated by using the value reported by ttolzapfel et al. (5) for the molar absorbancy at 357 nm. Sample extractio~ and purification. Recoveries were determined with both fresh and market milks. A volume of aflatoxin standard solution was added to the milk sample before methanol extraction. A 75-ml sample of milk was blended with 300 ml of methanol for two minutes in a l-liter Waring Blendor j a r ; 25 g of Celite were then added and the mixture was blended for an additional 30 seconds. The mixture was filtered by vacuum through a 1-cm bed of Celite 545 on a coarse fritted glass Biichner funnel. The bed of Celite and the precipitated casein were pressed and washed with 75 mI of methanol. The filtrate and washing (approximately 375 ml) were transferred to a 1-liter separatory funnel and adjusted to a methanol concentration of 50% by adding 4% sodium chloride solution. The alcoholic solution was extracted with four successive 200-ml volumes of hexane and then extracted with four successive ]00-ml volmues of chloroform to remove the aflatoxin. The chloroform extracts were combined and shaken with 300 ml of 4% sodium chloride solution, transferred to a l-liter round bottom flask, and evaporated to dryness for the following column chromatographic procedure. A fine stainless steel screen was placed in the bottom of a 20 X 450-mm chromatographic tube equipped with a stopcock. The column material was prepared by blending 100 g of Celite with 750 ml of hexane in a W a r i n g Blendor j a r ; 4 ml of water were then added, followed by 30 ml of aqueous 44.4% CaC12 while the mixture was blending. A portion of slurry was added to the chromatographic tube and firmly packed with pressure (500 mm of mercury) to a height of 250 mm. The residue was dissolved in 25 ml of benzene, 25 ml of hexane were added, and the dissolved material was added to the column. When the initial charge sank to the level of the top of the column, 150 ml of benzene:hexane (1:1) were added to elute some of the impurities, and additional impurities were eluted with 200 ml of benzene. A 200-ml volume of benzene :acetone (98:2) eluted the aflatoxin B 1. Some of the slow-moving impurities were eluted with 150 ml of benzene:acetone (05:5) and the aflatoxin M 1 was then eluted with 200 ml benzene :chloroform (70:30). :For preparation of a column to reehromatograph the aflatoxin B 1 fraction, 100 g of Celite
23
AFLATOXIN ANALYSIS
545 were blended with 750 ml of hexane in a W a r i n g Blendor; 4 ml of water were added and followed by 30 ml of methanol:water (1:1) while the mixture was blending. A portion of slurry was added to the chromatographic tube and firmly packed with pressure (500 mm of mercury) to a height of 250 ram. The fraction containing the aflatoxin B 1 was evaporated to dryness and the residue dissolved in 100 ml of hexane :chloroform (95:5). After the sample had been added to the column, an additional 100 ml volume of hexane :chloroform was used to wash the flask and to wash the impurities through the column. To elute the aflatoxin B1, 200 ml of hexane :chloroform (90:10) were passed through the column. The eluate was evaporated to dryness, and the residue was dissolved in 2 ml of chloroform and transferred to a 12-ml centrifuge tube with a bulb type syringe. The flask was rinsed with three 2-ml portions of chIoroform and the rinsings were transferred to the centrifuge tube. The tube was placed in a heating block at 60 C and the chloroform removed with a jet of water-pumped nitrogen. A 1-ml volume of chloroform was used to wash down the sides of the tube and evaporated to dryness. The tube was cooled and the residue dissolved in 0.1 ml of chloroform. A volume sufficient to contain 1-7 ng of aflatoxin B 1 was spotted on a TLC plate with the apparatus shown in Figure :l. The aflatoxin B 1 standard was spotted on the plate in varying quantities and the spots were dried with cool air from a heat gun. The plate was developed with benzene saturated with formamide and viewed with an ultraviolet light provided with a 365-nm filter. The concentration was measured by the fluorescence of the spots relative to that of the standard spots. The aflatoxin IV[1 fraction from the Celite:
CaCI2 column was similarly evaporated to dryness, dissolved in chloroform, reduced to a 0.l-rot volume, and spotted on TLC plates along with the aflation M 1 standard in varying quantities. The plates were developed with benzene:chloroform (25:75). The concentration was estimated by visual comparison of the fluorescence of standard and test spots under ultraviolet light. Results and Discussion
Roberts and Allcroft (6) recently described a method for determining M1 in fluid milk at 0.3 ppb with aflatoxin B1 as the comparison standard. F o r more accurate comparisons of R~ values, it is desirable to use aflatoxin MI as a standard. Recoveries of afiatoxins B I and M1 added to milk at the 0.50 ppb level were satisfactory, subject to the errors of visual comparison; values are shown in Table 1. The equivalent of 13.4 ml of milk was spotted on the plate for the aflatoxin )/[1 recoveries, and 12.5 ml of milk for the B1 recoveries. The relatively little chromatographic background produced at these plate loadings permitted measurement of 1.25 ng of aflatoxin without difficulty. We consider that milks containing aflatoxins at 1.25 ng/12.5 ml (0.1 ppb) can be measured with confidence by this method. Although Roberts and Allcroft (6) have reported a change in the brightness of the fluorescence of the aflatoxin M 1 spots during or after short exposure to ultraviolet light, on these TLC plates a ]0-ng spot of aflatoxin M1 irradiated for five minutes showed only a minor residual spot after re-development; the major amount was advanced by the solvent. Afiatoxin B1 under similar circumstances is almost totally immobilized by irradiation. References
(1) Adye, J., and 12. I. Mateles. 1964. Incorporation of labelled compounds into ariatoxins. Biochim. Biophys. Acta, 86: 418. (2) Allcroft, R., and R. B. A. Carnaghan. 1963. Groundnut toxicity: An examination for toxin in human food products from animals fed toxic groundnut meal. Vet. Record. 75 : 259.
Fief. 1. Thin-layer chromatographic plate spotter. Screw-fed metering syringe on adjustable tilting stage.
(3) Allcroft, R., and B. A. Roberts. 1968. Toxic groundnut meal: The relationship between ariatoxin 131 intake by cows and excretion of aflatoxin Mx in milk. Vet. Record, 82: 116. (4) De Iongh, H., R. O. Vies, and 3". G. van Pelt. 1964. Milk of mammals fed an ariatoxin-containing diet. Nature, 202: 466. JOURNAL OF DAIRY SCIRI~CR ~OL. 54, 1~0. 1
24
JACOBSON ET AL.
(5) Holzapfel, C. W., P. S. Steyn, and I. F. H. Purchase. 1966. Isolation and structure of aflatoxins M 1 and M 2. Tetrahedron Letters No. 25, 2799. (6) Roberts, B. A., and R. Allcroft. 1968. A note on the semi-quantitative estimatioa of aflatoxin M 1 in liquid milk by thln-layer
JOURNAL OF DAIRY SCIENCE YOL. 54, ~S~O. 1
chromatography. Food Cosmet. Toxicol., 6: 339. (7) Wiseman, H. G., W. O. Jaeobson, and W. C. Harmeyer. 1967. Note on removal of pigments from chloroform extracts of aflatoxin cultures with copper carbonate. J . Ass. Offici. Anal. Chem., 50 : 982.
and
in Milk
W. C. JACOBSON, W. ¢. HARMEYER, and H. G. WISEMAN
Division of Veterinary Research, Food and Drug Administration U.S. Department of Health, Education, and Welfare Beltsville, Maryland 20204 Abstract
sufficient to permit analysis slightly below 0.1 /zg p e r liter.
Cows fed contaminated rations containing the mold raetabolite aflatoxin B i secrete low levels of aflatoxin B1 and its metabolite afiatoxin M 1 in milk. To establish the relationship between ingested aflatoxin and milk-secreted aflatoxin, a sensitive method is needed to determine aflatoxins B i and M1 in milk. A method is presented b y which good recoveries, within the limits of visual comparison, were obtained from samples containing 0.5 parts per billion aflatoxin. Deproteinized milk (80% methanolie filtrates) was adjusted to 50% by addition of 4% salt solution and defatted by extraction with hexane. Aflatoxins were extracted from the alcoholic phase with chloroform which was removed in vacuo. The residue was dissolved in benzene :hexane (1:1) and chromatographcd on a partition column of Celite coated with an aqueous solution of calcium chloride to separate aflatoxins B1 and MI. The elated B1 was passed through another partition column of Celite coated with methanol :water (1:1) for additional cleanup. Aflatoxin eluates were evaporated to d~Tness and residues were dissolved in chloroform and spotted on thin-layer chromatographic plates for fluorescence comparisons.
Experimental Procedure
Reagents and materials. All solvents and reagents were A C S grade; the solvents were glass-distilled before use. The acid-washed Celite 545 was extracted with methanol, washed with water, and dried at 150 C for t 2 hours before use. F o r thin layer chromatography (TLC), Kieselguhr-formamide-water plates were prepared by the method of Adye and Mateles (1) except that 10 ml of formamide were included in the aqueous slurry used for coating. The plates were dried at 40 C for 90 minutes, stored at 4 C until used, and, after developmeat, were viewed over a long wave ultraviolet light. 1 l~efere~ce standards preparation. The comparison standard of aflatoxin B1 was obtained from Aspergillus flavus 2 cultured on a ricewheat medium. Crude aflatoxin was extracted from the culture medium with methanol. The alcoholic filtrate was diluted 50% by adding 4% sodium chloride solution and then defatted by extracting with hexane. The crude aflatoxin was extracted from the aqueous methanol with chloroform and the extract was decolorized by swirling with 10 g of copper carbonate (7) for each 400 ml of chloroform. The mixture was then filtered through a 4-mm bed of Celite on a coarse fritted glass Biichner funnel and the filtrate was evaporated to dryness in vacuo. The residue was dissolved in benzene and chromatographed on a column of silicie acid :Celite (2:1) coated with a stationary phase of formamide :water (3:1) in the proportions of 48 nd of liquid phase to 90 g of the adsorbents. Aflatoxin B1 was eluted with benzene saturated with formamide and then B 1 fractions which contained fluorescent impurities as shown by TLC were rechromatographed on silicie acid :Celite. The eluates were evaporated to dryness in vacuo and dissolved in small volumes of chloroform; the aflatoxins were
Introduction
I t has been established that cows which have consumed aflatoxin B 1 will secrete the metabolite aflatoxin Mi in their milk (2, 3, 4, 5). I n the analysis of milk for afiatoxin Mi, a dried powdered sample is commonly used. To eliminate uncertainties of losses by drying or prolonged storage, a method for direct analysis of aflatoxins in fluid milk is desirable. A method has been devised by which afiatoxins M 1 and B1 can be quantitatively recovered from fluid milk at the 0.5 ppb level. Cleanup is
1 Ultra-Violet Products, Inc., San Gabriel, California. 2 Food and Drug Administration, Division of Microbiology Isolate M93.
Received for publication August 26, 1970. 21
22
J A C O B S O N E T AL.
then crystallized by slow addition of nine volumes of hexane to yield pure aflatoxin B 1. Purity of the preparation was determined by TLC; 50 ng revealed only one spot. The molar absorbaney in ethanol at 361 n m was 23,000. To prepare the standard aflatoxin M1, a cow was given a daily dose of 40 mg of aflatoxin B 1 in 50 ml of ethanol by way of tureen fistula for four days. Total urines before and after dosing were collected in separate fractions. Urines were extracted with chloroform and the extracts were evaporated to dryness in vacuo and dissolved in methanol. A n equal volume of 4% sodium chloride solution was added and the resulting 50% methanol solutions were extracted with hexane for partial cleanup and then with chloroform to remove the aflatoxins. (The aqueous-alcoholic phase retained considerable extraneous matter.) The chloroform extracts were dried in vacuo and the residues ehromatographed on 122 × 2.5-cm columns of Celite coated with 44% aqueous solution of calcium chloride (30 ml/100 g Celite). The residues were transferred to columns by successive washings with the following eluting solvents, in order: hexane; hexane:benzene (1:1) ; benzene ; benzene :acetone (99:1) ; benzene :acetone (98:2) ; benzene :chloroform (70:30) ; and chloroform. Similar fractions from experimental and control urines were spotted on adjacent tracks of TLC plates; in this way, a slow-moving fluorescent spot not present in the control urine was located. The fraction containing the slow-moving compound was streaked on a TLC plate and, after development, the streak was scraped off the plate and eluted into chloroform. When the chloroform eluate was developed on a TLC plate, only one spot appeared. Spectrophotometrie peaks were determined to be 226, 265, and 357 nm; peak ratios were 122, 64.0, and 100, and agreed with values TABLE 1. Recovery of aflatoxin B 1 and 1~1 added to milk at 0.5 parts per billion. Recovered Sample 1 2 3 4 5 6 Control milk Avg recovery
B1 (ppb) 0.5 0.5 0.5 0.5 0.4 0.5 0.0
M1 (%) 100 100 100 100 80 100 0 97
(ppb) 0.5 0.5 0.5 0.5 0.4 0.4 0.0
JOURNAL OF DAIRY SCIENCE "VOL. 54, ~TO. 1
(%) 100 100 100 100 80 80 0 93
of 121.6, 61.1, and 100 reported by ttolzapfel et al. (5). The concentration of solution was calculated by using the value reported by ttolzapfel et al. (5) for the molar absorbancy at 357 nm. Sample extractio~ and purification. Recoveries were determined with both fresh and market milks. A volume of aflatoxin standard solution was added to the milk sample before methanol extraction. A 75-ml sample of milk was blended with 300 ml of methanol for two minutes in a l-liter Waring Blendor j a r ; 25 g of Celite were then added and the mixture was blended for an additional 30 seconds. The mixture was filtered by vacuum through a 1-cm bed of Celite 545 on a coarse fritted glass Biichner funnel. The bed of Celite and the precipitated casein were pressed and washed with 75 mI of methanol. The filtrate and washing (approximately 375 ml) were transferred to a 1-liter separatory funnel and adjusted to a methanol concentration of 50% by adding 4% sodium chloride solution. The alcoholic solution was extracted with four successive 200-ml volumes of hexane and then extracted with four successive ]00-ml volmues of chloroform to remove the aflatoxin. The chloroform extracts were combined and shaken with 300 ml of 4% sodium chloride solution, transferred to a l-liter round bottom flask, and evaporated to dryness for the following column chromatographic procedure. A fine stainless steel screen was placed in the bottom of a 20 X 450-mm chromatographic tube equipped with a stopcock. The column material was prepared by blending 100 g of Celite with 750 ml of hexane in a W a r i n g Blendor j a r ; 4 ml of water were then added, followed by 30 ml of aqueous 44.4% CaC12 while the mixture was blending. A portion of slurry was added to the chromatographic tube and firmly packed with pressure (500 mm of mercury) to a height of 250 mm. The residue was dissolved in 25 ml of benzene, 25 ml of hexane were added, and the dissolved material was added to the column. When the initial charge sank to the level of the top of the column, 150 ml of benzene:hexane (1:1) were added to elute some of the impurities, and additional impurities were eluted with 200 ml of benzene. A 200-ml volume of benzene :acetone (98:2) eluted the aflatoxin B 1. Some of the slow-moving impurities were eluted with 150 ml of benzene:acetone (05:5) and the aflatoxin M 1 was then eluted with 200 ml benzene :chloroform (70:30). :For preparation of a column to reehromatograph the aflatoxin B 1 fraction, 100 g of Celite
23
AFLATOXIN ANALYSIS
545 were blended with 750 ml of hexane in a W a r i n g Blendor; 4 ml of water were added and followed by 30 ml of methanol:water (1:1) while the mixture was blending. A portion of slurry was added to the chromatographic tube and firmly packed with pressure (500 mm of mercury) to a height of 250 ram. The fraction containing the aflatoxin B 1 was evaporated to dryness and the residue dissolved in 100 ml of hexane :chloroform (95:5). After the sample had been added to the column, an additional 100 ml volume of hexane :chloroform was used to wash the flask and to wash the impurities through the column. To elute the aflatoxin B1, 200 ml of hexane :chloroform (90:10) were passed through the column. The eluate was evaporated to dryness, and the residue was dissolved in 2 ml of chloroform and transferred to a 12-ml centrifuge tube with a bulb type syringe. The flask was rinsed with three 2-ml portions of chIoroform and the rinsings were transferred to the centrifuge tube. The tube was placed in a heating block at 60 C and the chloroform removed with a jet of water-pumped nitrogen. A 1-ml volume of chloroform was used to wash down the sides of the tube and evaporated to dryness. The tube was cooled and the residue dissolved in 0.1 ml of chloroform. A volume sufficient to contain 1-7 ng of aflatoxin B 1 was spotted on a TLC plate with the apparatus shown in Figure :l. The aflatoxin B 1 standard was spotted on the plate in varying quantities and the spots were dried with cool air from a heat gun. The plate was developed with benzene saturated with formamide and viewed with an ultraviolet light provided with a 365-nm filter. The concentration was measured by the fluorescence of the spots relative to that of the standard spots. The aflatoxin IV[1 fraction from the Celite:
CaCI2 column was similarly evaporated to dryness, dissolved in chloroform, reduced to a 0.l-rot volume, and spotted on TLC plates along with the aflation M 1 standard in varying quantities. The plates were developed with benzene:chloroform (25:75). The concentration was estimated by visual comparison of the fluorescence of standard and test spots under ultraviolet light. Results and Discussion
Roberts and Allcroft (6) recently described a method for determining M1 in fluid milk at 0.3 ppb with aflatoxin B1 as the comparison standard. F o r more accurate comparisons of R~ values, it is desirable to use aflatoxin MI as a standard. Recoveries of afiatoxins B I and M1 added to milk at the 0.50 ppb level were satisfactory, subject to the errors of visual comparison; values are shown in Table 1. The equivalent of 13.4 ml of milk was spotted on the plate for the aflatoxin )/[1 recoveries, and 12.5 ml of milk for the B1 recoveries. The relatively little chromatographic background produced at these plate loadings permitted measurement of 1.25 ng of aflatoxin without difficulty. We consider that milks containing aflatoxins at 1.25 ng/12.5 ml (0.1 ppb) can be measured with confidence by this method. Although Roberts and Allcroft (6) have reported a change in the brightness of the fluorescence of the aflatoxin M 1 spots during or after short exposure to ultraviolet light, on these TLC plates a ]0-ng spot of aflatoxin M1 irradiated for five minutes showed only a minor residual spot after re-development; the major amount was advanced by the solvent. Afiatoxin B1 under similar circumstances is almost totally immobilized by irradiation. References
(1) Adye, J., and 12. I. Mateles. 1964. Incorporation of labelled compounds into ariatoxins. Biochim. Biophys. Acta, 86: 418. (2) Allcroft, R., and R. B. A. Carnaghan. 1963. Groundnut toxicity: An examination for toxin in human food products from animals fed toxic groundnut meal. Vet. Record. 75 : 259.
Fief. 1. Thin-layer chromatographic plate spotter. Screw-fed metering syringe on adjustable tilting stage.
(3) Allcroft, R., and B. A. Roberts. 1968. Toxic groundnut meal: The relationship between ariatoxin 131 intake by cows and excretion of aflatoxin Mx in milk. Vet. Record, 82: 116. (4) De Iongh, H., R. O. Vies, and 3". G. van Pelt. 1964. Milk of mammals fed an ariatoxin-containing diet. Nature, 202: 466. JOURNAL OF DAIRY SCIRI~CR ~OL. 54, 1~0. 1
24
JACOBSON ET AL.
(5) Holzapfel, C. W., P. S. Steyn, and I. F. H. Purchase. 1966. Isolation and structure of aflatoxins M 1 and M 2. Tetrahedron Letters No. 25, 2799. (6) Roberts, B. A., and R. Allcroft. 1968. A note on the semi-quantitative estimatioa of aflatoxin M 1 in liquid milk by thln-layer
JOURNAL OF DAIRY SCIENCE YOL. 54, ~S~O. 1
chromatography. Food Cosmet. Toxicol., 6: 339. (7) Wiseman, H. G., W. O. Jaeobson, and W. C. Harmeyer. 1967. Note on removal of pigments from chloroform extracts of aflatoxin cultures with copper carbonate. J . Ass. Offici. Anal. Chem., 50 : 982.