Vol. 37, No. 3
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1979, p. 358-361
0099-2240/79/03-0358/04$02.00/0
Differentiation of Aflatoxins from Territrems KUO HUANG LING,* CHUNG-KUANG YANG, AND HUI-CHUAN HUANG Institute of Biochemistry, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China
Received for publication 7 December 1979
Three methods were adopted for differentiation of aflatoxins B1 and B2 from territrems A and B. They were as follows. (i) Thin-layer chromatography coupled with chemical confirmation. A significant decrease in the Rf value of trifluoroacetic acid-treated aflatoxin B1 developed in chloroform-acetone (85:15, vol/vol) was satisfactory in differentiating this toxin from the other three. (ii) High-pressure liquid chromatography monitored synchronously at two wavelengths, 365 and 335 nm. The ratio derived from this double-wavelength detection could serve as an indicator of the presence of each toxin. (iii) Velasco's flurotoxin meter method, which is used for the determination of aflatoxins within the range of 0 to 50 ng/ ml, was not significantly affected by territrems even when they were present in quantities at the microgram-per-milliliter level. There are several fungal metabolites which behave like aflatoxins in some solvent systems of thin-layer chromatography (TLC). For example, territrem A (C28H3oO9) and territrem B (C29H3409), produced by Aspergillus terreus and previously designated as C1 and C2, respectively, reveal blue fluorescence under long-wave ultraviolet (UV) light and have Rf values similar to that of aflatoxin B1 on TLC plates when developed in certain solvent systems (4, 6). According to Tung et al. (10) and Chung et al. (1), Rhizopus, Penicillium, and Aspergillus were the major genera of fungi contaminating the stored unhulled rice in Taiwan during 1968; and among 206 isolates of aspergilli, 7 A. flavus isolates produced aflatoxin, whereas 11 A. terreus isolates were able to produce aflatoxin-like compounds. These compounds are designated as territrems in this report. A comparison of the properties of territrems (data established in this laboratory) and aflatoxins (2) suggested that the fluorescence emission of territrems at 420 nm is close to that at 425 nm for aflatoxins B1 and B2; the maximum wavelengths of UV absorbance of territrems at 331 to 338 nm are within the neighborhood of 362 to 363 nm for aflatoxins B1 and B2. It is noteworthy that blue fluorescence is used in TLC and in the Velasco method and UV absorbance is used in high-pressure liquid chromatography (HPLC) for aflatoxin determination. To avoid overestimation of aflatoxins when territrems are present, their differentiation is of practical importance. The present investigation is concerned with this question.
MATERLALS AND METHODS Aflatoxins. Aflatoxins B, and B2 were purchased from Markor Chemicals Ltd., Jerusalem, Israel. Individual stock solutions of 10 ,g/ml in benzene-acetonitrile (98:2, vol/vol) were prepared according to the procedure of the Association of Official Analytical Chemists (3). The concentration of the working standard solution for TLC was 1 Ag/ml, prepared by direct dilution of proper aliquots of its stock solution with the same solvent mixture. For preparation of the working standard solution for HPLC, proper aliquots of the stock solution were dried under a stream of nitrogen gas and redissolved in the elution solvent of HPLC to give a final concentration of 10 pg/nil. Territrems. Territrems A and B were isolated by the method of Ling (5), except that benzene-ethyl acetate (7:3, vol/vol) was used as the solvent system of preparative TLC, and the recrystallization of each toxin was carried out by precipitation of a chloroform solution of the toxin with n-hexane. The UV spectrum of each toxin in chloroform was determined with a Jasco Uvidec-1 spectrophotometer. Their molar absorptivities were calculated from the slopes of the regression lines calibrated at the maximum wavelengths of UV absorbance. The results were 18,300 at 340 nm for territrem A and 17,700 at 334 nm for territrem B. These data served to prepare the stock solution of territrems A and B, 20 rig/ml in chloroform each. Proper aliquots of each stock solution were dried under a stream of nitrgoen gas and redissolved in benzene-acetonitrile (98:2, vol/vol) to give a final concentration of 10 ,Ag/ml for the TLC study, or, in the elution solvent of HPLC, a final concentration of 20 ,ug/ml for the HPLC study. Preparation of the mixture of toxins. Equal volumes of stock solutions of aflatoxins B, and B2 and territrems A and B were mixed and dried under a stream of nitrogen gas. The dried mixture was redis-
358
359
DIFFERENTIATION OF AFLATOXINS FROM TERRITREMS
VOL. 37, 1979
solved in benzene-acetonitrile (98:2, vol/vol) for the TLC study or in the elution solvent of HPLC for the HPLC study. The final concentrations of the toxins were 1 ,ug each of aflatoxins B1 and B2 and 2 yg each of territrems A and B per ml for TLC and 10 itg each of aflatoxins BI and B2 and 20 iLg each of territrems A and B per ml for HPLC. TLC. Each precoated plate (20 by 20 cm) of Silica Gel 60 (0.25-mm thicknesw; Merck, no. 5721) was activated for 2 h at 80°C before use. Each working standard solution and the mixture of toxins were separately applied in 5 id as a spot at an equal distance. For the formation of aflatoxin B2. from B1 (4), 1 ,ul of
trifluoroacetic acid (TFA) was added to each spot of the specimens. After 5 min of reaction, excess TFA was removed by warm air (35 to 40°C) from a hair dryer. The development in an unlined and unequilibrated tank was carried out ascendingly at a 15-cm distance at 23 ± 2°C, using the following solvent systems: toluene-ethyl acetate-85% formic acid (6:3:1, vol/vol/vol) and benzene-methanol-acetic acid (24:2: 1, vol/vol/vol) for the plate without TFA treatment (9), or chloroform-acetone (85:15, vol/vol) and ethyl acetate presaturated with water for the plate with TFA treatment (4). The developed plate was air dried and visualized under long-wave UV light. After estimation of R( values, the plate was sprayed with a freshly prepared mixture of 0.5 ml of p-anisaldehyde, 85 ml of methanol, 10 ml of glacial acetic acid, and 5 ml of concentrated sulfuric acid and then heated at 100°C (9), or the plate was sprayed with 25% sulfuric acid (4). HPLC. The mixture of water-saturated chloroform-cyclohexane-acetonitrile (25:7.5:1, vol/vol/vol) with 0, 0.2, 0.25, or 0.4% ethanol added was used as the elution solvent of HPLC (8). An instrument equipped with a Ai-Porasil porous silica gel column (10 Am, 4-mm ID by 30 cm; Waters Associates, Inc., Milford, Mass.) was used. The elution rate was 2.0 ml/ min. The toxin was detected separately by a Waters Associates model 440 absorbance detecter at 365 nm and also by a Schoeffel SF-770 Spectroflow Monitor at 335 nm. These two systems of detection were operated separately but synchronously at a sensitivity of 0.02 absorbance unit full scale. Chromatograms were recorded on an Omniscribe 10-inch (ca. 25-cm) recorder (Houston Instruments, Bellair, Tex.) with a
chart speed of 0.2 inch/min. Preliminary experiments carried out with a 5- or 10-pl working standard solution to mark the individual retention times and to calculate the ratio of peak height measured at 365 nm to that at 335 nm. Afterwards, 5 or 10 Ad of the toxin mixture was subjected to analysis. Velasco method. The procedure used was the modified Velasco method (7). A properly packed microcolumn was wetted from the bottom through various layers of adsorbents by being dipped in chloroform. One milliliter of aflatoxin B1 standard (20 ng/ml in chloroforrn-methanol [96:4, vol/vol]) was applied to the column. Once the solution was drained, an additional 1 ml of the chloroform-methanol mixture was added and allowed to drain. This wetted standard microcolumn and a blank microcolumn were used to calibrate the 20-ppb and zero readings of the Velasco flurotoxin meter (Neotec Instruments, Inc.) The other wetted columns were separately applied with aflatoxin BI, territrem A or B, or a mixture of these, in various concentrations. Each column was inserted into the sample holder of the calibrated Velasco flurotoxin meter, and its reading was recorded. The second reading was obtained by turning the column around 1800. The average of these two readings was taken as the final reading of the specimen. When territrem was applied to the column, the eluate was also determined by absorbance at 334, 360, and 365 nm with a Jasco Uvidec-1 spectrophotometer to calculate the average percentage of applied territrem that appeared in the eluate. were
RESULTS AND DISCUSSION Rf values of TLC, including those before and after TFA treatment, and colors of spraying tests are summarized in Table 1. The four toxins could be more clearly resolved in toluene-ethylacetate-85% formic acid than in the other solvent systems, among which benzene-methanol-acetic acid showed poor resolution and chloroform-acetone showed interference between aflatoxin BI and territrem A or between aflatoxin B2 and territrem B. However, these two classes of toxins revealed different colors upon spraying with p-anisaldehyde reagent but could not be
TABLE 1. Differentiation of aflatoxins B1 and B2 from territrems A and B by TLC coupled with chemical confirmation Colorb
R, in solvent system:'
Mycotoxin
Territrem A Territrem B
TEF
BMA
CA
T-CA
EW
T-EW
0.42 0.38 0.27 0.22
0.26 0.25
0.63 0.57 0.64 0.57
0.70
0.88 0.82 0.65 0.58
0.89 0.83 0.28 0.46
0.65
PAA gn gn
SA
yl-gn yl-gn gn to yl gn to yl
pk 0.14 0.22 Aflatoxin Bi pk 0.61 0.22 Aflatoxin B2 acid; CA, benzene-methanol-acetic Abbreviations: TEF, toluene-ethyl acetate-85% formic acid; BMA, chloroform-acetone; T-CA, spots pretreated with TFA and then developed in CA; EW, ethyl acetate saturated with water, T-EW, spots pretreated with TFA and then developed in EW. b Color (gn, green; pk, pink; yl, yellow) visualized under long-wave UV light after spraying with the reagent p-anisaldehyde (PAA) or 25% sulfuric acid (SA).
360
APPL. ENVIRON. MICROBIOL.
LING, YANG, AND HUANG
differentiated from each other by treatment with 25% sulfuric acid. The most satisfactory and rapid test in this study for differentiating aflatoxin B1 from the other toxins was TFA treatment followed by TLC developed in the chloroform-acetone solvent system. TFA treatment converts aflatoxin B1 into aflatoxin Ba., which is located on the plate at about one-fourth of the original Rf value of aflatoxin B1 (4). On the other hand, Rf values of the territrems and aflatoxin B2 were hardly changed after TFA treatment. The solvent water-presaturated ethyl acetate could substitute for chloroform-acetone and gave similar results. The preliminary HPLC tests indicated that each toxin had, in addition to its distinctive retention time, a fairly constant ratio of peak heights recorded at 365 nm to those recorded at 335 nm. The mean ratios were 1.1, 1.2, 0.5, and 0.4 for aflatoxins B1 and B2 and territrems A and B, respectively. When the toxin mixture was applied, it showed incomplete resolution. For instance (Fig. 1), territrem A (peaks b, b') appeared as shoulder peaks adhering to aflatoxin B2 (peaks c, c'). However, the ratio of peak heights at 365 nm to those at 335 nm of each toxin measured from the individual base line could be matched with its original value. These characteristic properties led us to suggest that both constants, i.e., the retention time and the ratio of peak heights at 365 nm to those at 335 nm, are necessary conditions for demonstrating the presence of aflatoxins in HPLC analysis. TABLE 2. Determination of aflatoxin B, and territrems by the Velasco flurotoxin meter (VFM)
Mycotoxin
ead-Concn eluted (%)b
ConcnVFM Cocn VF rea ing (ppb) applied
Aflatoxin B1
10 15 25 35 40
Territrem A
10 15 25 35 40
Territrem B
10 15 25 35 40
10 16 25 37 42 0 0 0 0 0.3
0.2 0 0.2 0.5 0 a Nanograms per milliliter for aflatoxin grams per milliliter for territrems. b Absorbance in the eluate/absorbance 100 = %.
is in
*Z k.
r
o
o
notetlo tb
(min)
FIG. 1. HPLC chromatogram of a mixture of aflatoxins B, and B2 and territrems A and B on a ,uPorasil column; elution solvent, water-saturated chloroform-cyclohexane-acetonitrile (25:7.5:1, vol/vol/vol) with 0.4% ethanol; flow rate, 2.0 ml/min. The upward peaks were measured at 365 nm and located in the coordinate system shown in the lower right corner, whereas the downward peaks were measured at 335 nm and located in the coordinate system shown in the upper left corner. The peaks are alphabetically labeled and recognized at the individual base lines: a, a' (aflatoxin Bi); b, b' (territrem A); c, c' (aflatoxin B2); d, d' (territrem B).
The Velasco flurotoxin meter did not respond to the application of territrems even at a 1,000fold concentration of aflatoxins (Table 2). It also indicated that more than 80% of the territrems applied was recovered in the eluate under the experimental conditions used. Experiments on the mixture of toxins showed similar results (data not presented). It was conclusive that territrems cannot interfere in the determination of aflatoxin B1 by the Velasco method.
84.4 90.0 91.3 92.9 94.9 91.3 88.0 96.1 97.8 92.8
B1; niicro-
applied
a-. the (WAR)
x
ACKNOWLEDGEMENT This work was supported by grant NSC-66B-0412-02 (31) to K.H.L. from the National Science Council, Taipei, Taiwan, Republic of China.
DIFFERENTIATION OF AFLATOXINS FROM TERRITREMS
VOL. 37, 1979
LITERATURE CITED 1. Chung, C. H., K. H. Ling, S. S. Tung, and T. C. Tung. 1971. Study on fungi of the stored unhulled rice of Taiwan. (2) Aflatoxin B, like compounds from the culture of Aspergillus genus. J. Formosan Med. Assoc. 70: 258-266. 2. Detroy, R. W., E. B. Lillehoj, and A. Ciegler. 1971. Aflatoxin and related compounds, p. 3-178. In A. Ciegler, S. Kadis, and S. J. Ajl (ed.), Microbial toxins, vol. 6. Academic Press Inc., New York. 3. Horwitz, W. (ed.). 1975. Determination of aflatoxin concentration. Official methods of analysis, 12th ed., p. 3, sect. 26.009. Association of Official Analytical Chemists,
Washington, D.C. 4. Horwitz, W. (ed.). 1975. Identification of aflatoxin by derivative formation on TLC plate. Official methods of analysis, 12th ed., p. 14, sect. 26.A17. Association of Official Analytical Chemists, Washington, D.C. 5. Ling, K. H. 1976. Study on mycotoxins contaminated in food in Taiwan. (2) Tremor inducing compounds from Aspergillus terreus, p. 121-129. In Proc. Natl. Sci.
361
Council, Republic of China, no. 9, part 2. 6. Ling, K. H., and M. T. Huang. 1975. Study on mycotoxins contaminated in food in Taiwan. (1) Study on pseudo-aflatoxin B, from Aspergillus terreus, p. 65-74. In Proc. Natl. Sci. Council, Republic of China, no. 8, part 2. 7. Neotec Instruments, Inc. 1974. Velasco flurotoxin meter, instruction manual. Neotec Instruments, Inc., Silver Spring, Md. 8. Pons, W. A., Jr. 1976. Resolution of aflatoxins B,, B2, G, and G2 by high-pressure liquid chromatography. J. As8oc. Off. Anal. Chem. 59:101-105. 9. Scott, P. M., J. W. Lawrence, and W. V. Walbeek. 1970. Detection of mycotoxins by thin-layer chromatography: application to screening of fungal extracts. Appl. Microbiol. 20:839-842. 10. Tung, S. S., K. H. Ling, S. E. Tsai, C. H. Chung, J. J. Wang, and T. C. Tung. 1971. Study on fungi of the stored unhulled rice. (1) Mycological survey of the stored unhulled rice. J. Formosan Med. Assoc. 70:251257.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1979, p. 358-361
0099-2240/79/03-0358/04$02.00/0
Differentiation of Aflatoxins from Territrems KUO HUANG LING,* CHUNG-KUANG YANG, AND HUI-CHUAN HUANG Institute of Biochemistry, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China
Received for publication 7 December 1979
Three methods were adopted for differentiation of aflatoxins B1 and B2 from territrems A and B. They were as follows. (i) Thin-layer chromatography coupled with chemical confirmation. A significant decrease in the Rf value of trifluoroacetic acid-treated aflatoxin B1 developed in chloroform-acetone (85:15, vol/vol) was satisfactory in differentiating this toxin from the other three. (ii) High-pressure liquid chromatography monitored synchronously at two wavelengths, 365 and 335 nm. The ratio derived from this double-wavelength detection could serve as an indicator of the presence of each toxin. (iii) Velasco's flurotoxin meter method, which is used for the determination of aflatoxins within the range of 0 to 50 ng/ ml, was not significantly affected by territrems even when they were present in quantities at the microgram-per-milliliter level. There are several fungal metabolites which behave like aflatoxins in some solvent systems of thin-layer chromatography (TLC). For example, territrem A (C28H3oO9) and territrem B (C29H3409), produced by Aspergillus terreus and previously designated as C1 and C2, respectively, reveal blue fluorescence under long-wave ultraviolet (UV) light and have Rf values similar to that of aflatoxin B1 on TLC plates when developed in certain solvent systems (4, 6). According to Tung et al. (10) and Chung et al. (1), Rhizopus, Penicillium, and Aspergillus were the major genera of fungi contaminating the stored unhulled rice in Taiwan during 1968; and among 206 isolates of aspergilli, 7 A. flavus isolates produced aflatoxin, whereas 11 A. terreus isolates were able to produce aflatoxin-like compounds. These compounds are designated as territrems in this report. A comparison of the properties of territrems (data established in this laboratory) and aflatoxins (2) suggested that the fluorescence emission of territrems at 420 nm is close to that at 425 nm for aflatoxins B1 and B2; the maximum wavelengths of UV absorbance of territrems at 331 to 338 nm are within the neighborhood of 362 to 363 nm for aflatoxins B1 and B2. It is noteworthy that blue fluorescence is used in TLC and in the Velasco method and UV absorbance is used in high-pressure liquid chromatography (HPLC) for aflatoxin determination. To avoid overestimation of aflatoxins when territrems are present, their differentiation is of practical importance. The present investigation is concerned with this question.
MATERLALS AND METHODS Aflatoxins. Aflatoxins B, and B2 were purchased from Markor Chemicals Ltd., Jerusalem, Israel. Individual stock solutions of 10 ,g/ml in benzene-acetonitrile (98:2, vol/vol) were prepared according to the procedure of the Association of Official Analytical Chemists (3). The concentration of the working standard solution for TLC was 1 Ag/ml, prepared by direct dilution of proper aliquots of its stock solution with the same solvent mixture. For preparation of the working standard solution for HPLC, proper aliquots of the stock solution were dried under a stream of nitrogen gas and redissolved in the elution solvent of HPLC to give a final concentration of 10 pg/nil. Territrems. Territrems A and B were isolated by the method of Ling (5), except that benzene-ethyl acetate (7:3, vol/vol) was used as the solvent system of preparative TLC, and the recrystallization of each toxin was carried out by precipitation of a chloroform solution of the toxin with n-hexane. The UV spectrum of each toxin in chloroform was determined with a Jasco Uvidec-1 spectrophotometer. Their molar absorptivities were calculated from the slopes of the regression lines calibrated at the maximum wavelengths of UV absorbance. The results were 18,300 at 340 nm for territrem A and 17,700 at 334 nm for territrem B. These data served to prepare the stock solution of territrems A and B, 20 rig/ml in chloroform each. Proper aliquots of each stock solution were dried under a stream of nitrgoen gas and redissolved in benzene-acetonitrile (98:2, vol/vol) to give a final concentration of 10 ,Ag/ml for the TLC study, or, in the elution solvent of HPLC, a final concentration of 20 ,ug/ml for the HPLC study. Preparation of the mixture of toxins. Equal volumes of stock solutions of aflatoxins B, and B2 and territrems A and B were mixed and dried under a stream of nitrogen gas. The dried mixture was redis-
358
359
DIFFERENTIATION OF AFLATOXINS FROM TERRITREMS
VOL. 37, 1979
solved in benzene-acetonitrile (98:2, vol/vol) for the TLC study or in the elution solvent of HPLC for the HPLC study. The final concentrations of the toxins were 1 ,ug each of aflatoxins B1 and B2 and 2 yg each of territrems A and B per ml for TLC and 10 itg each of aflatoxins BI and B2 and 20 iLg each of territrems A and B per ml for HPLC. TLC. Each precoated plate (20 by 20 cm) of Silica Gel 60 (0.25-mm thicknesw; Merck, no. 5721) was activated for 2 h at 80°C before use. Each working standard solution and the mixture of toxins were separately applied in 5 id as a spot at an equal distance. For the formation of aflatoxin B2. from B1 (4), 1 ,ul of
trifluoroacetic acid (TFA) was added to each spot of the specimens. After 5 min of reaction, excess TFA was removed by warm air (35 to 40°C) from a hair dryer. The development in an unlined and unequilibrated tank was carried out ascendingly at a 15-cm distance at 23 ± 2°C, using the following solvent systems: toluene-ethyl acetate-85% formic acid (6:3:1, vol/vol/vol) and benzene-methanol-acetic acid (24:2: 1, vol/vol/vol) for the plate without TFA treatment (9), or chloroform-acetone (85:15, vol/vol) and ethyl acetate presaturated with water for the plate with TFA treatment (4). The developed plate was air dried and visualized under long-wave UV light. After estimation of R( values, the plate was sprayed with a freshly prepared mixture of 0.5 ml of p-anisaldehyde, 85 ml of methanol, 10 ml of glacial acetic acid, and 5 ml of concentrated sulfuric acid and then heated at 100°C (9), or the plate was sprayed with 25% sulfuric acid (4). HPLC. The mixture of water-saturated chloroform-cyclohexane-acetonitrile (25:7.5:1, vol/vol/vol) with 0, 0.2, 0.25, or 0.4% ethanol added was used as the elution solvent of HPLC (8). An instrument equipped with a Ai-Porasil porous silica gel column (10 Am, 4-mm ID by 30 cm; Waters Associates, Inc., Milford, Mass.) was used. The elution rate was 2.0 ml/ min. The toxin was detected separately by a Waters Associates model 440 absorbance detecter at 365 nm and also by a Schoeffel SF-770 Spectroflow Monitor at 335 nm. These two systems of detection were operated separately but synchronously at a sensitivity of 0.02 absorbance unit full scale. Chromatograms were recorded on an Omniscribe 10-inch (ca. 25-cm) recorder (Houston Instruments, Bellair, Tex.) with a
chart speed of 0.2 inch/min. Preliminary experiments carried out with a 5- or 10-pl working standard solution to mark the individual retention times and to calculate the ratio of peak height measured at 365 nm to that at 335 nm. Afterwards, 5 or 10 Ad of the toxin mixture was subjected to analysis. Velasco method. The procedure used was the modified Velasco method (7). A properly packed microcolumn was wetted from the bottom through various layers of adsorbents by being dipped in chloroform. One milliliter of aflatoxin B1 standard (20 ng/ml in chloroforrn-methanol [96:4, vol/vol]) was applied to the column. Once the solution was drained, an additional 1 ml of the chloroform-methanol mixture was added and allowed to drain. This wetted standard microcolumn and a blank microcolumn were used to calibrate the 20-ppb and zero readings of the Velasco flurotoxin meter (Neotec Instruments, Inc.) The other wetted columns were separately applied with aflatoxin BI, territrem A or B, or a mixture of these, in various concentrations. Each column was inserted into the sample holder of the calibrated Velasco flurotoxin meter, and its reading was recorded. The second reading was obtained by turning the column around 1800. The average of these two readings was taken as the final reading of the specimen. When territrem was applied to the column, the eluate was also determined by absorbance at 334, 360, and 365 nm with a Jasco Uvidec-1 spectrophotometer to calculate the average percentage of applied territrem that appeared in the eluate. were
RESULTS AND DISCUSSION Rf values of TLC, including those before and after TFA treatment, and colors of spraying tests are summarized in Table 1. The four toxins could be more clearly resolved in toluene-ethylacetate-85% formic acid than in the other solvent systems, among which benzene-methanol-acetic acid showed poor resolution and chloroform-acetone showed interference between aflatoxin BI and territrem A or between aflatoxin B2 and territrem B. However, these two classes of toxins revealed different colors upon spraying with p-anisaldehyde reagent but could not be
TABLE 1. Differentiation of aflatoxins B1 and B2 from territrems A and B by TLC coupled with chemical confirmation Colorb
R, in solvent system:'
Mycotoxin
Territrem A Territrem B
TEF
BMA
CA
T-CA
EW
T-EW
0.42 0.38 0.27 0.22
0.26 0.25
0.63 0.57 0.64 0.57
0.70
0.88 0.82 0.65 0.58
0.89 0.83 0.28 0.46
0.65
PAA gn gn
SA
yl-gn yl-gn gn to yl gn to yl
pk 0.14 0.22 Aflatoxin Bi pk 0.61 0.22 Aflatoxin B2 acid; CA, benzene-methanol-acetic Abbreviations: TEF, toluene-ethyl acetate-85% formic acid; BMA, chloroform-acetone; T-CA, spots pretreated with TFA and then developed in CA; EW, ethyl acetate saturated with water, T-EW, spots pretreated with TFA and then developed in EW. b Color (gn, green; pk, pink; yl, yellow) visualized under long-wave UV light after spraying with the reagent p-anisaldehyde (PAA) or 25% sulfuric acid (SA).
360
APPL. ENVIRON. MICROBIOL.
LING, YANG, AND HUANG
differentiated from each other by treatment with 25% sulfuric acid. The most satisfactory and rapid test in this study for differentiating aflatoxin B1 from the other toxins was TFA treatment followed by TLC developed in the chloroform-acetone solvent system. TFA treatment converts aflatoxin B1 into aflatoxin Ba., which is located on the plate at about one-fourth of the original Rf value of aflatoxin B1 (4). On the other hand, Rf values of the territrems and aflatoxin B2 were hardly changed after TFA treatment. The solvent water-presaturated ethyl acetate could substitute for chloroform-acetone and gave similar results. The preliminary HPLC tests indicated that each toxin had, in addition to its distinctive retention time, a fairly constant ratio of peak heights recorded at 365 nm to those recorded at 335 nm. The mean ratios were 1.1, 1.2, 0.5, and 0.4 for aflatoxins B1 and B2 and territrems A and B, respectively. When the toxin mixture was applied, it showed incomplete resolution. For instance (Fig. 1), territrem A (peaks b, b') appeared as shoulder peaks adhering to aflatoxin B2 (peaks c, c'). However, the ratio of peak heights at 365 nm to those at 335 nm of each toxin measured from the individual base line could be matched with its original value. These characteristic properties led us to suggest that both constants, i.e., the retention time and the ratio of peak heights at 365 nm to those at 335 nm, are necessary conditions for demonstrating the presence of aflatoxins in HPLC analysis. TABLE 2. Determination of aflatoxin B, and territrems by the Velasco flurotoxin meter (VFM)
Mycotoxin
ead-Concn eluted (%)b
ConcnVFM Cocn VF rea ing (ppb) applied
Aflatoxin B1
10 15 25 35 40
Territrem A
10 15 25 35 40
Territrem B
10 15 25 35 40
10 16 25 37 42 0 0 0 0 0.3
0.2 0 0.2 0.5 0 a Nanograms per milliliter for aflatoxin grams per milliliter for territrems. b Absorbance in the eluate/absorbance 100 = %.
is in
*Z k.
r
o
o
notetlo tb
(min)
FIG. 1. HPLC chromatogram of a mixture of aflatoxins B, and B2 and territrems A and B on a ,uPorasil column; elution solvent, water-saturated chloroform-cyclohexane-acetonitrile (25:7.5:1, vol/vol/vol) with 0.4% ethanol; flow rate, 2.0 ml/min. The upward peaks were measured at 365 nm and located in the coordinate system shown in the lower right corner, whereas the downward peaks were measured at 335 nm and located in the coordinate system shown in the upper left corner. The peaks are alphabetically labeled and recognized at the individual base lines: a, a' (aflatoxin Bi); b, b' (territrem A); c, c' (aflatoxin B2); d, d' (territrem B).
The Velasco flurotoxin meter did not respond to the application of territrems even at a 1,000fold concentration of aflatoxins (Table 2). It also indicated that more than 80% of the territrems applied was recovered in the eluate under the experimental conditions used. Experiments on the mixture of toxins showed similar results (data not presented). It was conclusive that territrems cannot interfere in the determination of aflatoxin B1 by the Velasco method.
84.4 90.0 91.3 92.9 94.9 91.3 88.0 96.1 97.8 92.8
B1; niicro-
applied
a-. the (WAR)
x
ACKNOWLEDGEMENT This work was supported by grant NSC-66B-0412-02 (31) to K.H.L. from the National Science Council, Taipei, Taiwan, Republic of China.
DIFFERENTIATION OF AFLATOXINS FROM TERRITREMS
VOL. 37, 1979
LITERATURE CITED 1. Chung, C. H., K. H. Ling, S. S. Tung, and T. C. Tung. 1971. Study on fungi of the stored unhulled rice of Taiwan. (2) Aflatoxin B, like compounds from the culture of Aspergillus genus. J. Formosan Med. Assoc. 70: 258-266. 2. Detroy, R. W., E. B. Lillehoj, and A. Ciegler. 1971. Aflatoxin and related compounds, p. 3-178. In A. Ciegler, S. Kadis, and S. J. Ajl (ed.), Microbial toxins, vol. 6. Academic Press Inc., New York. 3. Horwitz, W. (ed.). 1975. Determination of aflatoxin concentration. Official methods of analysis, 12th ed., p. 3, sect. 26.009. Association of Official Analytical Chemists,
Washington, D.C. 4. Horwitz, W. (ed.). 1975. Identification of aflatoxin by derivative formation on TLC plate. Official methods of analysis, 12th ed., p. 14, sect. 26.A17. Association of Official Analytical Chemists, Washington, D.C. 5. Ling, K. H. 1976. Study on mycotoxins contaminated in food in Taiwan. (2) Tremor inducing compounds from Aspergillus terreus, p. 121-129. In Proc. Natl. Sci.
361
Council, Republic of China, no. 9, part 2. 6. Ling, K. H., and M. T. Huang. 1975. Study on mycotoxins contaminated in food in Taiwan. (1) Study on pseudo-aflatoxin B, from Aspergillus terreus, p. 65-74. In Proc. Natl. Sci. Council, Republic of China, no. 8, part 2. 7. Neotec Instruments, Inc. 1974. Velasco flurotoxin meter, instruction manual. Neotec Instruments, Inc., Silver Spring, Md. 8. Pons, W. A., Jr. 1976. Resolution of aflatoxins B,, B2, G, and G2 by high-pressure liquid chromatography. J. As8oc. Off. Anal. Chem. 59:101-105. 9. Scott, P. M., J. W. Lawrence, and W. V. Walbeek. 1970. Detection of mycotoxins by thin-layer chromatography: application to screening of fungal extracts. Appl. Microbiol. 20:839-842. 10. Tung, S. S., K. H. Ling, S. E. Tsai, C. H. Chung, J. J. Wang, and T. C. Tung. 1971. Study on fungi of the stored unhulled rice. (1) Mycological survey of the stored unhulled rice. J. Formosan Med. Assoc. 70:251257.
