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The analysis of aflatoxins by high-performance liquid chromatography
ANALYTICAL BIOCHEMISTRY 93,409--418 (1979)
The Analysis of Aflatoxins by High-Performance
Liquid Chromatography P. J. COLLEY AND G . E . N E A L *
MRC Toxicology Unit, Medical Research Council Laboratories, Woodmansterne Road, Carshalton, Surrey, SM5 4EF, United Kingdom Received July 6, 1978 The potential of high-performance liquid chromatography as a technique for separating aflatoxins B1, B~, Gt, G2, B2a, Q1, MI, P~, aflatoxicol, and a degradation product of aflatoxin B~, 2,3-dihydrodiol, has been assessed. A microparticulate silica adsorption column used with a 1:1 chloroform-dichloromethane eluant provided good resolution of aflatoxins B1, B2, GA, and G2 but the addition of 1% propan-2-ol was necessary for the elution of aflatoxins M~ and Q1. By selecting appropriate solvent mixtures, good resolution of all of the aflatoxins studied was obtained using columns containing an octadecyl (C~8) reversed-phase bonded to a microparticulate support. Details are given for resolving: (1) aflatoxins B~, B2, G1, and G2 using a 5% tetrahydrofuran-15% dimethylformamide in water eluant and (2) aflatoxins B1, B2a, Q~, M~, PI, aflatoxicol, and a product of aflatoxin B~, 2,3-dihydrodiol treated with Tris-buffer, using either 15% dimethylformamide in water or 10% tetrahydrofuran in water as eluant.
Aflatoxins are fungal metabolites found as contaminants in a wide range of food and agricultural products. The most commonly occurring aflatoxin, aflatoxin B1, is a potent mutagen and hepatocarcinogen to a wide range of animal species. The mutagenic and possibly the hepatocarcinogenic activities of aflatoxin B1 appear to require its metabolic activation, primarily by liver microsomes, to a reactive, highly unstable product, currently thought to be a 2,3-epoxide, while other metabolic reactions occur which may detoxify the carcinogen [for review see (1)]. The origins and structures of the aflatoxins referred to in this communication are summarized in Fig. 1. The aflatoxins studied may be grouped into four classes on the basis of their polarity: (1) Aflatoxicol (Fig. la), produced from aflatoxin B~ by a cytoplasmic reductase (avian liver is a particularly rich source of this enzyme) (1), is highly nonpolar being soluble in both chloroform and diethyl ether; (2) * To whom reprint requests should be addressed.
aflatoxins B1, B2, G1, and G2 (Fig. lb), the main fungal metabolites, are also nonpolar being soluble in chloroform but insoluble in diethyl ether; (3) aflatoxins M1, Qx, B2a, and P1 (Fig. la) are all soluble in chloroform but, being monohydroxylated products of aflatoxin B1, show increased polarity over the aflatoxins in group 2. (It is noteworthy that this group also includes aflatoxin M~ which, besides being an important mammalian metabolite of aflatoxin Bx, also occurs as a fungal metabolite.); and (4) aflatoxin B1 2,3-dihydrodiol is polar, being water soluble. This presumed metabolite degrades spontaneously in the presence of the Trisbuffer used in the microsomal incubation medium to a product (Tris-diol) which is also water soluble and which is chromatographically identical to a major microsomal metabolite of aflatoxin B 1 (9). Conjugates of aflatoxin B1 metabolites with, for instance, glutathione, glucuronic acid, or sulfate, would also be polar, water-soluble compounds. Work in this laboratory is concerned with 409
0003-2697/79/040409-10502.00/0 Copyright© 1979by AcademicPress, Inc. All rightsof reproductionin any formreserved.
410
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ANALYSIS OF AFLATOXINS BY HPLC
studying aflatoxin B~ metabolism in relation to its toxicity and hepatocarcinogenicity to the rat. Most current methods for studying such metabolism, and indeed for assaying naturally occurring ariatoxins, are based on organic-solvent extraction followed by separation and quantification using tic ~ techniques (1). However such techniques are often only semiquantitative and invariably time consuming. More recently, methods involving silica-gel adsorption high-performance liquid chromatography (hplc) have been developed (2-7) which offer rapid and reliable means of separating and quantifying the microsomal metabolites of ariatoxin B1 as well as the aflatoxins arising from fungal contamination of foodstuffs. These methods do have limitations however: (i) polar ariatoxins which may be present in the sample are only slowly eluted, if at all, from the silica column and hence subsequent sample injections must be delayed or interference with subsequent analyses will result; (ii) highly polar constituents in the eluting solvent, including eluant modifiers such as alcohols and water, are strongly adsorbed onto the silica gel leading to a progressive reduction in column performance necessitating time-consuming column regeneration or repacking procedures; (iii) since ariatoxin B~, often the major peak in the chromatogram, has the shortest retention time, any broadening or tailing of this peak could interfere with the detection and quantitatioa of minor sample constituents having only a slightly longer retention time; (iv) perhaps of greatest importance in metabolic studies, aflatoxin B~ is metabolized to a range of products, as illustrated in Fig. 1, including those which are water soluble and not, therefore, readily amenable to separation by adsoption hplc. As some of these metabolites may be degradation products of the active metabolite(s) of the toxin, their importance may be considerable and it was considered 1 Abbreviations used: tic, thin-layer chromatography; hplc, high-performance liquid chromatography.
411
to be valuable to develop a technique capable of separating all of the known microsomal metabolites of aflatoxin Ba on a single chromatogram. Many of the problems associated with silica adsorption hplc can be overcome by using reversed-phase hplc. To date this technique appears only to have been applied to the separation of naturally occurring ariatoxins (8). The reversed-phase hplc method described in this communication was primarily developed to study the in vitro microsomal metabolism of ariatoxin B1. However, it would also appear to have much to recommend it as a hplc method for assaying ariatoxins in the environment. MATERIALS AND METHODS Aflatoxins. Aflatoxin Bx was obtained from Makor Chemical Company, Jerusalem, Israel. The aflatoxin standards used were generous gifts from the following sources: aflatoxin Mi, Dr. P. L. Schuller, Rijks Instituut voor de Volksgezondheid, Bilthoven, The Netherlands; aflatoxins Q1, Dr. D. P. H. Hsieh, University of California; ariatoxins P1 and M1, Dr. G. N. Wogan, M.I.T.; aflatoxins B2, G1, and G2 were present in a fungal extract obtained from M.R.E., Potton, United Kingdom. After confirmation of their identities by reference to the authentic standards, ariatoxins Q1 and M~ produced in in vitro microsomal incubations (9) were purified on hplc and preparative tlc plates (precoated silicagel G plates, 2 mm thick, obtained from Anachem, Luton, United Kingdom) and subsequently used as standards. Aflatoxicol was prepared by metabolism of ariatoxin Ba by the 100,000g supernatant prepared from homogenates of chicken liver (10), and also by the reduction of aflatoxin B1 using sodium borohydride (11). Aflatoxin B2a was prepared by reriuxing aflatoxin Ba with 0.1 N citric acid for 4 h (11). Aflatoxin 2,3-dihydrodiol was prepared from aflatoxin Bi by reaction of aflatoxin B1 with m-chloro-
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COLLEY AND NEAL
perbenzoic acid in dichloromethane and subsequent hydrolysis with methanolic KOH (12). As reported (12) this compound spontaneously degraded in the presence of the Tris-HCl buffer, pH 7.4, which was employed as the microsomal incubation buffer, forming a fluorescent derivative which was also water soluble. The identities of all these products were confirmed by comparing their uv spectra and tic behavior with published data. Solvents. Chloroform, dichloromethane (AR grade) and methanol (hplc grade) were obtained from Fisons Ltd., Loughborough, United Kingdom. Dimethylformamide and tetrahydrofuran (AR grade) were obtained from B.D.H., Poole, United Kingdom. Solvents were filtered through a 0.22 ~m filter (Millipore Ltd., London). Water was purified by passage through a 4-bowl Milli-Q ultra-pure water system (Millipore Ltd., London) immediately prior to use. In order to maintain a constant pH, all of the eluants used in the reversed-phase studies contained 0.01% phosphoric acid.
High-performance liquid chromatography. The following equipment was obtained from Du Pont (U.K.) Ltd., England: a Model 830 constant-pressure liquid chromatograph, 838 programmable gradient and 837 variable
wavelength spectrophotometer. The column compartment was modified to allow temperature control of the column between 30°-69°C within __+0.5°C. Samples (5-30/xl in chloroform or methanol) were introduced via a model 7120 syringe loading sample injector (Rheodyne, California) fitted with a 50 /zl loop. Results were displayed on a Model 3380S recording integrator (HewlettPackard Ltd., England). The columns used are described in Table 1 and were obtained either from Du Pont (U.K.) Ltd., England or from Whatman Labsales Ltd., England. The generosity of these companies in loaning some of these columns is gratefully acknowledged. Peaks were identified on chromatograms by their retention time, co-chromatography with added standards and by collection for subsequent tic analysis. RESULTS AND DISCUSSION
The results obtained in this work are summarized in Table 2. A fungal extract in chloroform, containing aflatoxins B1, B~, G1, and G2 was resolved (Fig. 2) using a microparticulate silica-adsorption column (Zorbax SIL) and chloroform-dichloromethane (1:1) as eluant. Problems in the analysis of aflatoxins
TABLE 1 hplc COLUMNS INVESTIGATED Column Zorbax SIL a Zorbax ODS a
Dimension
Packing material
25 cm x 2.1 mm i.d. 25 cm x 2.1 mm i.d.
Silica a d s o r b e n t - p o r o u s spheres 5 - 7 / x m diameter Octadecyl silane hydrocarbon phase bonded to Zorbax SIL ° support Octadecyl silane hydrocarbon phase bonded to Zipax a 30/zm pellicular silica support. Aliphatic Ether polar phase bonded to Zipax a support. May be used in normal partition or reversed phase. Octadecyl silane hydrocarbon phase bonded to Partisil-10, ~ l 0 / x m "structured irregular" silica particles " C y a n o - t y p e " polar phase bonded to Partisil-10 b support. May be used in normal partition or reversed phase.
Permaphase ODS ~
l m x 2.1 mm i.d.
Permaphase E T H a
lm x 2.1mmi.d.
Partisil-10 ODS ~
25 c m x
Partisil- 10 PAC b
25 cm x 4.6 mm i.d.
a Du Pont. b Whatman.
4.6 mm i.d.
ANALYSIS
OF AFLATOXINS
TABLE CAPABILITIES OF VARIOUS
Column Silica adsorption
hplc C O L U M N S
Chloroform:dichloromethane. Room temperature Chloroform:diehloromethane: propan-2-ol. Room temperature Water: methanol or Water:acetonitrile gradient or Water:dimethylformamide
Reversed phase on microparticulate packing
Water:methanol gradient below 55°C Water:methanol gradient above 55°C Water:dimethyl formamide 69~C Water :tet rahydrofuran 69°C
2
A N D S O L V E N T SYSTEMS TO S E P A R A T E A F L A T O X I N S
Solvent and conditions
Intermediate polarity or pellicular reversed phase
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BY HPLC
Aflatoxins resolved B~, B2, G~, G2
Aflatoxins not resolved & other disadvantages Metabolites not eluted in reasonable time B2, G~, Gz water soluble metabolites not eluted
B~, Qa, M~
Aflatoxins not well retained on column
AItOH," B~, B~, G~, G2, Q~, Bza, (MI or Pt), Tris-diol AflOH, B1, B2, GI, Gz, QI, MI, (P1 or B2~), Tris-diol Bz, B2, G~, G2,or AflOH, B~, P~, M~, (Q~ or Bz~), Tris-diol B~, Bz, G~, G2 or AflOH, Ba, P~, (M~ or Q0, B2~, Tris-diol
P~ co-chromatographs with M~ P1 co-chromatographs with B2a B~a co-chromatographs with Q~ M1 poorly resolved from Q1
" AflOH, aflatoxieol.
by silica adsorption arise, however, due to difficulties in the elution of more polar aflatoxins. For instance, aflatoxin M1, which commonly occurs as a fungal metabolite, is only slowly eluted (retention time ca. 30 min) in this eluant system. Although this hplc chromatographic system appears to have limited practical application it has in fact been recommended for the analysis of naturally occurring aflatoxins (2). The addition of a polar modifier (1% propan-2-ol) to the above eluant system reduced both the retention time and resolution of aflatoxins B1, B~, G1, and G2. Nonpolar rat liver microsomal metabolites of aflatoxin B1 (aflatoxins M~ and Q0 were however eluted within 12 min although the aflatoxin M~ peak was rather broad (Fig. 3). Summarizing, although this column may be suitable for the analysis of the nonpolar aflatoxins, it was not considered suitable for the analysis of the wide polarity range of aflatoxin B1 metabolites which may be produced by mammalian systems (Fig. 1). Attempts were made to resolve the microsomal metabolites of aflatoxin B~ (Fig. 1) on columns having polarities intermediate between those of the silica, normal phase, and
the reversed-phase columns (Partisil-10 PAC and Permaphase-ETH, operated in a reversed-phase mode). Water-methanol was used as elnant and column temperatures of up to 40°C were employed. However, retention of aflatoxin B1 was achieved only at high water contents (greater than 60%) when excessive peak tailing was encountered, presumably due to insolubility of the sample in the aqueous solvent. Similar results were obtained using a pellicular ODS reversed-phase column (Permaphase ODS) with methanol (30-50%) or dimethylformamide (5-15%) in water as eluant and column temperatures up to 40°C. This result is in agreement with that of Seitz (7) who discounted the use of pellicular reversed-phase columns in aflatoxin separations. When a microparticulate reversed-phase column (Zorbax ODS or Partisil-10-ODS) was used, the microsomal metabolites of aflatoxin B1 together with the fungal aflatoxins B1, B2, G1, and G~ were separated when a water-methanol linear gradient (10% to 60% methanol over 40 min) was used as eluting solvent (Fig. 4a). In this system, the resolution of aflatoxins B2a, P1, and M1 was
414
COLLEY AND NEAL
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5
5
10
Retention time (min)
FIG. 2. Separation of aflatoxins B1, B~, G1, and (32 by silica-adsorption hplc. Isocratic elution of 1.6 /~g of a fungal aflatoxin mixture (% composition aflatoxin B~--55, G~--25, B2--13, Gz--7) from Zorbax SIL column using chloroform: dichloromethane, l:l, v/v, at 0.45 cm ~ min-~ and ambient temperature. Effluent monitored at 365 nm. Detector sensitivity indicated by vertical bar.
found to be extremely sensitive to small changes in column temperature, the effect being due to the temperature sensitivity of the retention time of aflatoxin PI: At temperatures below 55°C aflatoxin P~ co-chromatographed with aflatoxin M 1 while at temperatures above 55°C but at the same flow rate the retention time of aflatoxin P~ was reduced so that it then co-chromatographed with ariatoxin B2a. Although aflatoxins B2a, Px, and Ma are not well resolved by this method, this eluant system can be used if only one or two of these aflatoxins are present in the sample. Furthermore, this gradient system might allow metabolites even more polar than the Tris-diol e.g., conjugates (Fig. 1), to be resolved from the less polar metabolites. Rat liver microsomal sys-
tems in vitro do not form free aflatoxin B2a to a detectable level but do produce ariatoxins Q1, M1, and a small amount of P1 together with the more polar, water-soluble Tris-diol (Fig. 4b). This chromatographic system allows good resolution of these metabolites and has been used in previous studies (9). The main disadvantages of this system are its inability to resolve fully ariatoxins B2a, P1, and M1; the requirement for a gradient; and the fairly long analysis time involved. A rapid, isocratic separation of aflatoxins B1, P~, M~, Q~, ariatoxicol, and the Trisdiol was achieved when 15% dimethylformamide in water was used as eluant (Fig. 5a). This eluant system also gave reasonable resolution ofaflatoxins B~, B2, G1, G~, and more polar constituents in the fungal extract (Fig. 5b). Column temperature was again found to be important, good peak shape and resolution being achieved only at elevated column temperatures. A disadvantage of this sysQ! 81
M1
5
10
15
Retention time (min}
FIo. 3. Separation of aflatoxins B~, B~, G~, G2, Q~, and M~ by silica-adsorption hplc. Conditions as in Fig. 2 except that the eluant contained 1% propan-2-ol.
ANALYSIS OF AFLATOXINSBY HPLC
415
B1
Trisdi01
Gl
QI P1 o-,
o Afl~oxicol
30
20
10
Retention
timeImin) Q1
Trisdl0f
li
MI
10
20
30
Retention time (mini FIG.
4. (a) Separation of aflatoxicol, aflatoxins Bx, B2, G1, (32, P1, M,, B~a, Q1, and aflatoxin Bx 2,3-
dihydrodiol treated with Tris-buffer by reversed-phase hplc. Elution from Zorbax-ODS column using a 10 to 60% linear gradient of methanol in water o v e r 40 min. Eluants contain 0.01% phosphoric acid. F l o w rate 0.45 cm3 min-~ at 55°C. (b) Separation of microsomal metabolites of aflatoxin Bx by r e v e r s e d - p h a s e hplc. Conditions as in Fig. 4a. Liver microsomes w e r e isolated from phenobarbitone pretreated rats and
incubated with 40/xg aflatoxin B, and NADPH in Tris buffer. See (9) for details.
tern, however, was that aflatoxin B2a cochromatographed with aflatoxin Q1- Good resolution of these metabolites was achieved using 10% tetrahydrofuran in water as eluant, but in this system aflatoxins Q1 and M1 were only poorly resolved (Fig. 6a). This solvent system also showed a different se-
lectivity from the dimethylformamide-water system when aflatoxins B1, B2, G1, and G2 were chromatographed (Fig. 6b). It was not possible to obtain good resolution of all of the aflatoxin metabolites studied using ternary mixtures of the solvents used in this study, but a very good and rapid
416
COLLEY AND NEAL B1
G1
Q~B2a
E
G2 m
E
Aflatoxicol
o
L_ r---"10
20
10
Retention time (min)
20
Retention time (rain)
FIG. 5. (a) Separation of aflatoxicol, aflatoxins B~, P~, M1, Bza, and aflatoxin B1, 2,3-dihydrodiol treated with Tris-buffer by reversed-phase hplc. Isocratic elution from Partisil-10-ODS column using 15% dimethylformamide and 0.01% phosphoric acid in water at 1.3 cm3 min-~ and 69~C. (b) Separation of altatoxins B1, Bz, G~, and G~ by reversed-phase hplc. Conditions as in Fig. 5a.
G1 Q1
B1
B:
G Afletoxicol
s
1'0
i5
2b
Retention time (min)
~ Retention time (min)
FIG. 6. (a) Separation of aflatoxicol, aflatoxins B1, PI, M1, Q~, B2a, and aflatoxin B1 2,3-dihydrodiol treated with Tris-buffer by reversed-phase hplc. Conditions as in Fig. 5a but using 10% tetrahydrofuran and 0.01% phosphoric acid in water as eluant. (b) Separation of atlatoxins B~, B2, G~, and (32 by reversed-phase hplc. Conditions as in Fig. 6a.
417
ANALYSIS OF AFLATOXINS BY HPLC
separation of aflatoxins B1, B2, G,, and G2 was achieved using a ternary eluant system containing 15% dimethylformamide and 5% tetrahydrofuran in water (Fig. 7). This system offers a potentially useful assay method for aflatoxins in the environment and overcomes the problems associated with established methods of silica-adsorption hplc already referred to. The uv detection limit of the methods described is about 5 ng of ariatoxin B1 and the spectrophotometer used gives a linear response to increasing amounts of aflatoxin B~ over at least 1000-fold range (Fig. 8). A further potential advantage of the reversed-phase methods described is that detection limits might be improved by the technique of trace enrichment analysis (10), a technique becoming increasingly important in reversed-phase hplc studies. In summary, although all of the aflatoxin metabolites studies could not be resolved in a single chromatographic step, a combination of the dimethylformamide-water and tetrahydrofuran-water systems using the re-
B1
62 e~
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i
i
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2
3
10gngB~injected FIG.8.Linearityofrelationshipofdetectorresponseto amountof aflatoxinB1.(a) Areaof detectorpeak.(b) Height of detector peak. The hplc conditions as in Fig. 3. Results of duplicate determinations at each level of aflatoxin B, are given.
versed-phase column does allow their resolution and quantitation. The water-methanol system described offers a flexible method which might allow the study of metabolites which are more polar even than the Tris-treated aflatoxin B1 2,3-dihydrodiol (e.g., conjugates), together with the nonpolar metabolites. Our current studies on the metabolism of aflatoxin B1 by rodent liver microsomes have shown the 15% dimethylformamide in water eluant-Partisil10-ODS column system to be a most useful routine procedure which we have now automated by the use of a Du Pont Model 834 automatic sample injector.
REFERENCES 5
10
15
Retent{0ntime(rain) FIG. 7. Separation of aflatoxins B1, B2, G1, and Gz by reversed-phase hplc. Isocratic elution from Partisil-10ODS column using 5% tetrahydrofuran:15% dimethylformamide:0.01% phosphoric acid in water at 0.7 cm 3 rain-' and 69"C.
1. Campbell, T. C,, and Hayes, J. R. (1976) Toxicol. Appl. Pharmacol. 35, 199-222. 2. Pons, W. A. (1976) J. Ass. Offic. Anal Chem. 59, 101-105. 3. Rao, G. H., and Anders, M. W. (1973)J. Chromatog. 84, 402-406. 4. Garner, R. C. (1975)J. Chromatog. 103, 186-188. 5. Hsieh, D. P. H., Fitzell, D. L., Miller, J. L., and
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COLLEY AND NEAL Seiber, J. N. (1976) J. Chromatog. 117, 474479.
6. Unger, P. D., Mehendale, H, M., and Hayes, A. W. (1977) Toxicol. Appl. Pharmacol. 41, 523-534. 7. Seitz, L. M. (1975)J. Chromatog. 104, 81-89. 8. Takahashi, D. M. (1977)J. Chromatog. 131, 147156.
9. Neal, G. E., and Colley, P. J. (1978)Biochem. J. 174, 839-851. 10. Patterson, D. S. P., and Roberts, B. A. (1971)Food Cosmet. Toxicol. 9, 829-837. 11. Garner, R. C., Miller, E. C., and Miller, J. A. (1972) Cancer Res. 32, 2058-2066. 12 Garner, R. C. (1973) FEBS Lett. 36, 261-264. 13. Schauwecker, P., Frei, R. W., and Erni, F. (1977) J. Chromatog. 136, 63-72.
The Analysis of Aflatoxins by High-Performance
Liquid Chromatography P. J. COLLEY AND G . E . N E A L *
MRC Toxicology Unit, Medical Research Council Laboratories, Woodmansterne Road, Carshalton, Surrey, SM5 4EF, United Kingdom Received July 6, 1978 The potential of high-performance liquid chromatography as a technique for separating aflatoxins B1, B~, Gt, G2, B2a, Q1, MI, P~, aflatoxicol, and a degradation product of aflatoxin B~, 2,3-dihydrodiol, has been assessed. A microparticulate silica adsorption column used with a 1:1 chloroform-dichloromethane eluant provided good resolution of aflatoxins B1, B2, GA, and G2 but the addition of 1% propan-2-ol was necessary for the elution of aflatoxins M~ and Q1. By selecting appropriate solvent mixtures, good resolution of all of the aflatoxins studied was obtained using columns containing an octadecyl (C~8) reversed-phase bonded to a microparticulate support. Details are given for resolving: (1) aflatoxins B~, B2, G1, and G2 using a 5% tetrahydrofuran-15% dimethylformamide in water eluant and (2) aflatoxins B1, B2a, Q~, M~, PI, aflatoxicol, and a product of aflatoxin B~, 2,3-dihydrodiol treated with Tris-buffer, using either 15% dimethylformamide in water or 10% tetrahydrofuran in water as eluant.
Aflatoxins are fungal metabolites found as contaminants in a wide range of food and agricultural products. The most commonly occurring aflatoxin, aflatoxin B1, is a potent mutagen and hepatocarcinogen to a wide range of animal species. The mutagenic and possibly the hepatocarcinogenic activities of aflatoxin B1 appear to require its metabolic activation, primarily by liver microsomes, to a reactive, highly unstable product, currently thought to be a 2,3-epoxide, while other metabolic reactions occur which may detoxify the carcinogen [for review see (1)]. The origins and structures of the aflatoxins referred to in this communication are summarized in Fig. 1. The aflatoxins studied may be grouped into four classes on the basis of their polarity: (1) Aflatoxicol (Fig. la), produced from aflatoxin B~ by a cytoplasmic reductase (avian liver is a particularly rich source of this enzyme) (1), is highly nonpolar being soluble in both chloroform and diethyl ether; (2) * To whom reprint requests should be addressed.
aflatoxins B1, B2, G1, and G2 (Fig. lb), the main fungal metabolites, are also nonpolar being soluble in chloroform but insoluble in diethyl ether; (3) aflatoxins M1, Qx, B2a, and P1 (Fig. la) are all soluble in chloroform but, being monohydroxylated products of aflatoxin B1, show increased polarity over the aflatoxins in group 2. (It is noteworthy that this group also includes aflatoxin M~ which, besides being an important mammalian metabolite of aflatoxin Bx, also occurs as a fungal metabolite.); and (4) aflatoxin B1 2,3-dihydrodiol is polar, being water soluble. This presumed metabolite degrades spontaneously in the presence of the Trisbuffer used in the microsomal incubation medium to a product (Tris-diol) which is also water soluble and which is chromatographically identical to a major microsomal metabolite of aflatoxin B 1 (9). Conjugates of aflatoxin B1 metabolites with, for instance, glutathione, glucuronic acid, or sulfate, would also be polar, water-soluble compounds. Work in this laboratory is concerned with 409
0003-2697/79/040409-10502.00/0 Copyright© 1979by AcademicPress, Inc. All rightsof reproductionin any formreserved.
410
COLLEY AND NEAL
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ANALYSIS OF AFLATOXINS BY HPLC
studying aflatoxin B~ metabolism in relation to its toxicity and hepatocarcinogenicity to the rat. Most current methods for studying such metabolism, and indeed for assaying naturally occurring ariatoxins, are based on organic-solvent extraction followed by separation and quantification using tic ~ techniques (1). However such techniques are often only semiquantitative and invariably time consuming. More recently, methods involving silica-gel adsorption high-performance liquid chromatography (hplc) have been developed (2-7) which offer rapid and reliable means of separating and quantifying the microsomal metabolites of ariatoxin B1 as well as the aflatoxins arising from fungal contamination of foodstuffs. These methods do have limitations however: (i) polar ariatoxins which may be present in the sample are only slowly eluted, if at all, from the silica column and hence subsequent sample injections must be delayed or interference with subsequent analyses will result; (ii) highly polar constituents in the eluting solvent, including eluant modifiers such as alcohols and water, are strongly adsorbed onto the silica gel leading to a progressive reduction in column performance necessitating time-consuming column regeneration or repacking procedures; (iii) since ariatoxin B~, often the major peak in the chromatogram, has the shortest retention time, any broadening or tailing of this peak could interfere with the detection and quantitatioa of minor sample constituents having only a slightly longer retention time; (iv) perhaps of greatest importance in metabolic studies, aflatoxin B~ is metabolized to a range of products, as illustrated in Fig. 1, including those which are water soluble and not, therefore, readily amenable to separation by adsoption hplc. As some of these metabolites may be degradation products of the active metabolite(s) of the toxin, their importance may be considerable and it was considered 1 Abbreviations used: tic, thin-layer chromatography; hplc, high-performance liquid chromatography.
411
to be valuable to develop a technique capable of separating all of the known microsomal metabolites of aflatoxin Ba on a single chromatogram. Many of the problems associated with silica adsorption hplc can be overcome by using reversed-phase hplc. To date this technique appears only to have been applied to the separation of naturally occurring ariatoxins (8). The reversed-phase hplc method described in this communication was primarily developed to study the in vitro microsomal metabolism of ariatoxin B1. However, it would also appear to have much to recommend it as a hplc method for assaying ariatoxins in the environment. MATERIALS AND METHODS Aflatoxins. Aflatoxin Bx was obtained from Makor Chemical Company, Jerusalem, Israel. The aflatoxin standards used were generous gifts from the following sources: aflatoxin Mi, Dr. P. L. Schuller, Rijks Instituut voor de Volksgezondheid, Bilthoven, The Netherlands; aflatoxins Q1, Dr. D. P. H. Hsieh, University of California; ariatoxins P1 and M1, Dr. G. N. Wogan, M.I.T.; aflatoxins B2, G1, and G2 were present in a fungal extract obtained from M.R.E., Potton, United Kingdom. After confirmation of their identities by reference to the authentic standards, ariatoxins Q1 and M~ produced in in vitro microsomal incubations (9) were purified on hplc and preparative tlc plates (precoated silicagel G plates, 2 mm thick, obtained from Anachem, Luton, United Kingdom) and subsequently used as standards. Aflatoxicol was prepared by metabolism of ariatoxin Ba by the 100,000g supernatant prepared from homogenates of chicken liver (10), and also by the reduction of aflatoxin B1 using sodium borohydride (11). Aflatoxin B2a was prepared by reriuxing aflatoxin Ba with 0.1 N citric acid for 4 h (11). Aflatoxin 2,3-dihydrodiol was prepared from aflatoxin Bi by reaction of aflatoxin B1 with m-chloro-
412
COLLEY AND NEAL
perbenzoic acid in dichloromethane and subsequent hydrolysis with methanolic KOH (12). As reported (12) this compound spontaneously degraded in the presence of the Tris-HCl buffer, pH 7.4, which was employed as the microsomal incubation buffer, forming a fluorescent derivative which was also water soluble. The identities of all these products were confirmed by comparing their uv spectra and tic behavior with published data. Solvents. Chloroform, dichloromethane (AR grade) and methanol (hplc grade) were obtained from Fisons Ltd., Loughborough, United Kingdom. Dimethylformamide and tetrahydrofuran (AR grade) were obtained from B.D.H., Poole, United Kingdom. Solvents were filtered through a 0.22 ~m filter (Millipore Ltd., London). Water was purified by passage through a 4-bowl Milli-Q ultra-pure water system (Millipore Ltd., London) immediately prior to use. In order to maintain a constant pH, all of the eluants used in the reversed-phase studies contained 0.01% phosphoric acid.
High-performance liquid chromatography. The following equipment was obtained from Du Pont (U.K.) Ltd., England: a Model 830 constant-pressure liquid chromatograph, 838 programmable gradient and 837 variable
wavelength spectrophotometer. The column compartment was modified to allow temperature control of the column between 30°-69°C within __+0.5°C. Samples (5-30/xl in chloroform or methanol) were introduced via a model 7120 syringe loading sample injector (Rheodyne, California) fitted with a 50 /zl loop. Results were displayed on a Model 3380S recording integrator (HewlettPackard Ltd., England). The columns used are described in Table 1 and were obtained either from Du Pont (U.K.) Ltd., England or from Whatman Labsales Ltd., England. The generosity of these companies in loaning some of these columns is gratefully acknowledged. Peaks were identified on chromatograms by their retention time, co-chromatography with added standards and by collection for subsequent tic analysis. RESULTS AND DISCUSSION
The results obtained in this work are summarized in Table 2. A fungal extract in chloroform, containing aflatoxins B1, B~, G1, and G2 was resolved (Fig. 2) using a microparticulate silica-adsorption column (Zorbax SIL) and chloroform-dichloromethane (1:1) as eluant. Problems in the analysis of aflatoxins
TABLE 1 hplc COLUMNS INVESTIGATED Column Zorbax SIL a Zorbax ODS a
Dimension
Packing material
25 cm x 2.1 mm i.d. 25 cm x 2.1 mm i.d.
Silica a d s o r b e n t - p o r o u s spheres 5 - 7 / x m diameter Octadecyl silane hydrocarbon phase bonded to Zorbax SIL ° support Octadecyl silane hydrocarbon phase bonded to Zipax a 30/zm pellicular silica support. Aliphatic Ether polar phase bonded to Zipax a support. May be used in normal partition or reversed phase. Octadecyl silane hydrocarbon phase bonded to Partisil-10, ~ l 0 / x m "structured irregular" silica particles " C y a n o - t y p e " polar phase bonded to Partisil-10 b support. May be used in normal partition or reversed phase.
Permaphase ODS ~
l m x 2.1 mm i.d.
Permaphase E T H a
lm x 2.1mmi.d.
Partisil-10 ODS ~
25 c m x
Partisil- 10 PAC b
25 cm x 4.6 mm i.d.
a Du Pont. b Whatman.
4.6 mm i.d.
ANALYSIS
OF AFLATOXINS
TABLE CAPABILITIES OF VARIOUS
Column Silica adsorption
hplc C O L U M N S
Chloroform:dichloromethane. Room temperature Chloroform:diehloromethane: propan-2-ol. Room temperature Water: methanol or Water:acetonitrile gradient or Water:dimethylformamide
Reversed phase on microparticulate packing
Water:methanol gradient below 55°C Water:methanol gradient above 55°C Water:dimethyl formamide 69~C Water :tet rahydrofuran 69°C
2
A N D S O L V E N T SYSTEMS TO S E P A R A T E A F L A T O X I N S
Solvent and conditions
Intermediate polarity or pellicular reversed phase
413
BY HPLC
Aflatoxins resolved B~, B2, G~, G2
Aflatoxins not resolved & other disadvantages Metabolites not eluted in reasonable time B2, G~, Gz water soluble metabolites not eluted
B~, Qa, M~
Aflatoxins not well retained on column
AItOH," B~, B~, G~, G2, Q~, Bza, (MI or Pt), Tris-diol AflOH, B1, B2, GI, Gz, QI, MI, (P1 or B2~), Tris-diol Bz, B2, G~, G2,or AflOH, B~, P~, M~, (Q~ or Bz~), Tris-diol B~, Bz, G~, G2 or AflOH, Ba, P~, (M~ or Q0, B2~, Tris-diol
P~ co-chromatographs with M~ P1 co-chromatographs with B2a B~a co-chromatographs with Q~ M1 poorly resolved from Q1
" AflOH, aflatoxieol.
by silica adsorption arise, however, due to difficulties in the elution of more polar aflatoxins. For instance, aflatoxin M1, which commonly occurs as a fungal metabolite, is only slowly eluted (retention time ca. 30 min) in this eluant system. Although this hplc chromatographic system appears to have limited practical application it has in fact been recommended for the analysis of naturally occurring aflatoxins (2). The addition of a polar modifier (1% propan-2-ol) to the above eluant system reduced both the retention time and resolution of aflatoxins B1, B~, G1, and G2. Nonpolar rat liver microsomal metabolites of aflatoxin B1 (aflatoxins M~ and Q0 were however eluted within 12 min although the aflatoxin M~ peak was rather broad (Fig. 3). Summarizing, although this column may be suitable for the analysis of the nonpolar aflatoxins, it was not considered suitable for the analysis of the wide polarity range of aflatoxin B1 metabolites which may be produced by mammalian systems (Fig. 1). Attempts were made to resolve the microsomal metabolites of aflatoxin B~ (Fig. 1) on columns having polarities intermediate between those of the silica, normal phase, and
the reversed-phase columns (Partisil-10 PAC and Permaphase-ETH, operated in a reversed-phase mode). Water-methanol was used as elnant and column temperatures of up to 40°C were employed. However, retention of aflatoxin B1 was achieved only at high water contents (greater than 60%) when excessive peak tailing was encountered, presumably due to insolubility of the sample in the aqueous solvent. Similar results were obtained using a pellicular ODS reversed-phase column (Permaphase ODS) with methanol (30-50%) or dimethylformamide (5-15%) in water as eluant and column temperatures up to 40°C. This result is in agreement with that of Seitz (7) who discounted the use of pellicular reversed-phase columns in aflatoxin separations. When a microparticulate reversed-phase column (Zorbax ODS or Partisil-10-ODS) was used, the microsomal metabolites of aflatoxin B1 together with the fungal aflatoxins B1, B2, G1, and G~ were separated when a water-methanol linear gradient (10% to 60% methanol over 40 min) was used as eluting solvent (Fig. 4a). In this system, the resolution of aflatoxins B2a, P1, and M1 was
414
COLLEY AND NEAL
B1 61
m o
I
-5
5
5
10
Retention time (min)
FIG. 2. Separation of aflatoxins B1, B~, G1, and (32 by silica-adsorption hplc. Isocratic elution of 1.6 /~g of a fungal aflatoxin mixture (% composition aflatoxin B~--55, G~--25, B2--13, Gz--7) from Zorbax SIL column using chloroform: dichloromethane, l:l, v/v, at 0.45 cm ~ min-~ and ambient temperature. Effluent monitored at 365 nm. Detector sensitivity indicated by vertical bar.
found to be extremely sensitive to small changes in column temperature, the effect being due to the temperature sensitivity of the retention time of aflatoxin PI: At temperatures below 55°C aflatoxin P~ co-chromatographed with aflatoxin M 1 while at temperatures above 55°C but at the same flow rate the retention time of aflatoxin P~ was reduced so that it then co-chromatographed with ariatoxin B2a. Although aflatoxins B2a, Px, and Ma are not well resolved by this method, this eluant system can be used if only one or two of these aflatoxins are present in the sample. Furthermore, this gradient system might allow metabolites even more polar than the Tris-diol e.g., conjugates (Fig. 1), to be resolved from the less polar metabolites. Rat liver microsomal sys-
tems in vitro do not form free aflatoxin B2a to a detectable level but do produce ariatoxins Q1, M1, and a small amount of P1 together with the more polar, water-soluble Tris-diol (Fig. 4b). This chromatographic system allows good resolution of these metabolites and has been used in previous studies (9). The main disadvantages of this system are its inability to resolve fully ariatoxins B2a, P1, and M1; the requirement for a gradient; and the fairly long analysis time involved. A rapid, isocratic separation of aflatoxins B1, P~, M~, Q~, ariatoxicol, and the Trisdiol was achieved when 15% dimethylformamide in water was used as eluant (Fig. 5a). This eluant system also gave reasonable resolution ofaflatoxins B~, B2, G1, G~, and more polar constituents in the fungal extract (Fig. 5b). Column temperature was again found to be important, good peak shape and resolution being achieved only at elevated column temperatures. A disadvantage of this sysQ! 81
M1
5
10
15
Retention time (min}
FIo. 3. Separation of aflatoxins B~, B~, G~, G2, Q~, and M~ by silica-adsorption hplc. Conditions as in Fig. 2 except that the eluant contained 1% propan-2-ol.
ANALYSIS OF AFLATOXINSBY HPLC
415
B1
Trisdi01
Gl
QI P1 o-,
o Afl~oxicol
30
20
10
Retention
timeImin) Q1
Trisdl0f
li
MI
10
20
30
Retention time (mini FIG.
4. (a) Separation of aflatoxicol, aflatoxins Bx, B2, G1, (32, P1, M,, B~a, Q1, and aflatoxin Bx 2,3-
dihydrodiol treated with Tris-buffer by reversed-phase hplc. Elution from Zorbax-ODS column using a 10 to 60% linear gradient of methanol in water o v e r 40 min. Eluants contain 0.01% phosphoric acid. F l o w rate 0.45 cm3 min-~ at 55°C. (b) Separation of microsomal metabolites of aflatoxin Bx by r e v e r s e d - p h a s e hplc. Conditions as in Fig. 4a. Liver microsomes w e r e isolated from phenobarbitone pretreated rats and
incubated with 40/xg aflatoxin B, and NADPH in Tris buffer. See (9) for details.
tern, however, was that aflatoxin B2a cochromatographed with aflatoxin Q1- Good resolution of these metabolites was achieved using 10% tetrahydrofuran in water as eluant, but in this system aflatoxins Q1 and M1 were only poorly resolved (Fig. 6a). This solvent system also showed a different se-
lectivity from the dimethylformamide-water system when aflatoxins B1, B2, G1, and G2 were chromatographed (Fig. 6b). It was not possible to obtain good resolution of all of the aflatoxin metabolites studied using ternary mixtures of the solvents used in this study, but a very good and rapid
416
COLLEY AND NEAL B1
G1
Q~B2a
E
G2 m
E
Aflatoxicol
o
L_ r---"10
20
10
Retention time (min)
20
Retention time (rain)
FIG. 5. (a) Separation of aflatoxicol, aflatoxins B~, P~, M1, Bza, and aflatoxin B1, 2,3-dihydrodiol treated with Tris-buffer by reversed-phase hplc. Isocratic elution from Partisil-10-ODS column using 15% dimethylformamide and 0.01% phosphoric acid in water at 1.3 cm3 min-~ and 69~C. (b) Separation of altatoxins B1, Bz, G~, and G~ by reversed-phase hplc. Conditions as in Fig. 5a.
G1 Q1
B1
B:
G Afletoxicol
s
1'0
i5
2b
Retention time (min)
~ Retention time (min)
FIG. 6. (a) Separation of aflatoxicol, aflatoxins B1, PI, M1, Q~, B2a, and aflatoxin B1 2,3-dihydrodiol treated with Tris-buffer by reversed-phase hplc. Conditions as in Fig. 5a but using 10% tetrahydrofuran and 0.01% phosphoric acid in water as eluant. (b) Separation of atlatoxins B~, B2, G~, and (32 by reversed-phase hplc. Conditions as in Fig. 6a.
417
ANALYSIS OF AFLATOXINS BY HPLC
separation of aflatoxins B1, B2, G,, and G2 was achieved using a ternary eluant system containing 15% dimethylformamide and 5% tetrahydrofuran in water (Fig. 7). This system offers a potentially useful assay method for aflatoxins in the environment and overcomes the problems associated with established methods of silica-adsorption hplc already referred to. The uv detection limit of the methods described is about 5 ng of ariatoxin B1 and the spectrophotometer used gives a linear response to increasing amounts of aflatoxin B~ over at least 1000-fold range (Fig. 8). A further potential advantage of the reversed-phase methods described is that detection limits might be improved by the technique of trace enrichment analysis (10), a technique becoming increasingly important in reversed-phase hplc studies. In summary, although all of the aflatoxin metabolites studies could not be resolved in a single chromatographic step, a combination of the dimethylformamide-water and tetrahydrofuran-water systems using the re-
B1
62 e~
~°° a2~_ o×~_~~.o3l,o / ~ 31 [
I
I
]
t 4
z= zl
i
i
I
I
2
3
10gngB~injected FIG.8.Linearityofrelationshipofdetectorresponseto amountof aflatoxinB1.(a) Areaof detectorpeak.(b) Height of detector peak. The hplc conditions as in Fig. 3. Results of duplicate determinations at each level of aflatoxin B, are given.
versed-phase column does allow their resolution and quantitation. The water-methanol system described offers a flexible method which might allow the study of metabolites which are more polar even than the Tris-treated aflatoxin B1 2,3-dihydrodiol (e.g., conjugates), together with the nonpolar metabolites. Our current studies on the metabolism of aflatoxin B1 by rodent liver microsomes have shown the 15% dimethylformamide in water eluant-Partisil10-ODS column system to be a most useful routine procedure which we have now automated by the use of a Du Pont Model 834 automatic sample injector.
REFERENCES 5
10
15
Retent{0ntime(rain) FIG. 7. Separation of aflatoxins B1, B2, G1, and Gz by reversed-phase hplc. Isocratic elution from Partisil-10ODS column using 5% tetrahydrofuran:15% dimethylformamide:0.01% phosphoric acid in water at 0.7 cm 3 rain-' and 69"C.
1. Campbell, T. C,, and Hayes, J. R. (1976) Toxicol. Appl. Pharmacol. 35, 199-222. 2. Pons, W. A. (1976) J. Ass. Offic. Anal Chem. 59, 101-105. 3. Rao, G. H., and Anders, M. W. (1973)J. Chromatog. 84, 402-406. 4. Garner, R. C. (1975)J. Chromatog. 103, 186-188. 5. Hsieh, D. P. H., Fitzell, D. L., Miller, J. L., and
418
COLLEY AND NEAL Seiber, J. N. (1976) J. Chromatog. 117, 474479.
6. Unger, P. D., Mehendale, H, M., and Hayes, A. W. (1977) Toxicol. Appl. Pharmacol. 41, 523-534. 7. Seitz, L. M. (1975)J. Chromatog. 104, 81-89. 8. Takahashi, D. M. (1977)J. Chromatog. 131, 147156.
9. Neal, G. E., and Colley, P. J. (1978)Biochem. J. 174, 839-851. 10. Patterson, D. S. P., and Roberts, B. A. (1971)Food Cosmet. Toxicol. 9, 829-837. 11. Garner, R. C., Miller, E. C., and Miller, J. A. (1972) Cancer Res. 32, 2058-2066. 12 Garner, R. C. (1973) FEBS Lett. 36, 261-264. 13. Schauwecker, P., Frei, R. W., and Erni, F. (1977) J. Chromatog. 136, 63-72.