12 m l ( 5 grams) column of AGl-X8 resin. Elements such as the alkali metals, alkaline earths, rare earths, Ti(IV), Zr, Hf, Th, and AI, which in aqueous solution have no or only very slight tendencies to the formation of bromide complexes, have not been investigated, but it seems to be reasonable t o assume that they also can be separated quantitatively from C d by the above procedure. Au(III), Tl(III), Hg(II), Pd(II), Pt(IV), and Bi(II1) accompany Cd quantitatively and Pb(I1) does so partially. Pb(I1) can be eluted with 0.30M H N 0 3 containing 0.025M HBr, while Cd is retained by the column, but a larger column (23 ml) is required and only limited amounts of C d can
be separated (12). Ge(IV), Sn(IV), and Sb(II1) can be eluted with 0.1 M HBr according to literature information (6). With 0.20M HNOI containing 0.05M HBr, separation should be even better. However, as n o quantitative separations seem t o have been carried out with 0.1M HBr as eluent, the quantitative aspects of these separations still will have t o be investigated. RECEIVED for review May 14, 1968. Accepted July 15, 1968. (12) F. W. E. Strelow and F. von S. Toerien, ANAL.CHEM.,38, 545 (1966).
Luminescence Characteristics of Aflatoxins B1 and G, B. L. V a n Duuren, Tze-Lock Chan, and F. M. I r a n i Laboratory of Organic Chemistry and Carcinogenesis, Institute of Enuironmental Medicine, New York University Medical Center, New York, N . Y . 10016
A study on the fluorescence and phosphorescence characteristics of aflatoxins B, and GIwas carried out. A self-correcting luminescence spectrophotometer was used and it was found that characteristic fluorescence spectra of these toxins could be recorded for submicrogram quantities to pg/ml). The sensitivities for the detection of these substances by fluorescence and thin-layer chromatography were compared. The corrected fluorescence emission and excitation spectra of both aflatoxins were measured at room temperature and at 77 O K and in potassium bromide. In their fluorescence, they exhibit a blue shift when measured at 77 O K compared to their room temperature solution spectra. Phosphorescence spectra and decay curves were also recorded.
THEAFLATOXINS are metabolites produced by the mould Aspergillus frcrvus ( I ) . These compounds have been implicated as toxic contaminants in human and animal foodstuffs (2-4), in milk (3, and possibly in tobacco leaf and smoke (6). There is, therefore, a wide interest in sensitive analytical procedures for the qualitative detection and quantitative analysis of these materials in a variety of products. Several brief reports have described their fluorescence emission maxima ( 7 , 81, but a detailed study of their luminescence characteristics and limits of detection by spectroscopic methods has not been reported. Although the materials can be detected by thin-layer chromatography, it is desirable to have such identifications, which depend solely on RF value, confirmed beyond doubt by a spectroscopic method. Of these, luminescence is by far the most sensitive. The present report describes the following luminescence characteristics for aflatoxins B1 and GI: corrected fluorescence (1) K. Sargent, R. B. A. Carnaghan, and R. Allcroft, Chem. Ind. (London), 1963, 50. ( 2 ) K . Sargent and R. B. A. Carnaghan, Brit. Vet. J., 119, 178 (1963). (3) B. H. Armbrecht, F. A. Hodges, H.R. Smith, and A. A. Nelson, J . Assoc. Ofic.Agr. Chemists, 46, 805 (1963). (4) . . N. D. Davis, U. L. Diener, and D. W. Eldridge, - . Apul. . - Microbiol., 14, 378 (1966). (.5 )_H. de Ioneh. R. 0. Vles. and J. W. van Pelt. Nature. 202. 466 (1964). (6) T. C. Tso and T. Sorokin, Beitrzge zur Tubakforschung, 4, 18 (1967). ( 7 ) R. B. A. Carnaghan, R. D. Hartley, and J. O'Kelly, Nature, 200, 1101 (1963). (8) J. A. Robertson, W. S. Pons, and L. A. Goldblatt, J . Agr. Food Chem., 15, 799 (1967). I
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excitation and emission spectra, corrected phosphorescence spectra, quantum efficiency of fluorescence, phosphorescence decay times, and limits of detectability. EXPERIMENTAL
Aflatoxins BI and GI. Chromatographically-pure substances used in this investigation were supplied by Professor G. N. Wogan of Massachusetts Institute of Technology (9). Each compound showed a single spot by thin-layer chromatography o n silica gel G (Merck) using chloroformmethyl alcohol (97:3) as solvent. The RF values were: 0.52 for aflatoxin B, and 0.46 for aflatoxin G1. Solvents. Luminescence spectra were recorded by using freshly prepared solutions in methyl alcohol and in frozen ethyl alcohol-methyl alcohol (4 :1). Fluorometric grade methyl alcohol (Hartman-Leddon Co., Philadelphia, Pa.) was used without further purification. Ethyl alcohol was repeatedly distilled until the fluorescence background was minimal. Potassium Bromide. Infrared-quality potassium bromide (Harshaw Co., Cleveland, Ohio) was used for the preparation of pellets. The previously described procedure ( I O ) was followed. Quinine Bisulfate. Commercial grade material (K & K Laboratories, Plainview, N. Y . )was purified by three crystallizations from water. Purity was established by the absorption spectrum and thin-layer chromatography. Thin-Layer Chromatography Adsorbent. Merck silica gel G was continuously extracted with ethyl acetate-methyl alcohol (1 :1) for two days and dried overnight at 110 "C prior to use. Absorption Instrumentation. Absorption spectra were obtained with a Cary Model-14 ultraviolet-visible spectrophotometer using 1-cm path-length cuvettes. Luminescence Instrumentation. A multipurpose self-correcting luminescence spectrophotometer (Farrand Optical CO., New York, N . Y . ) was used for all fluorescence and phosphorescence measurements in this work. The details of its design and performance are described elsewhere (11, 12). This instrument is equipped with a high-intensity 150-watt dc xenon arc (Hanovia Lamp Division, Newark, N. J.),
,
(9) T. Asoa, G. Buchi, M. M. Abdel-Kader, S . B. Chang, E. L. Wick, and G. N. Wogan, J . Amer. Chem. SOC.,87, 882 (1965). (10) B. L. Van Duuren and C. E. Bardi, ANAL.CHEM.,35, 2198 (1963). (11) S . Cravitt and B. L. Van Duuren, Chem. Instrum. 1, 71 (1968). (12) S. Cravitt and B. L. Van Duuren, Abstracts, Pittsburgh Conf. on Anal. Chem. and Applied Spectroscopy, 1968, p 90.
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Table I. Luminescence Characteristics of Aflatoxins Bl and GI Bi
Gi
Mediuma Excitation, mp Emission, mp Excitation, mp Emission, mp Measurement 363 450 363 426 Fluorescence, Methyl alcohol (1) 23 i 0 . 5 "C 365 408 365 423 Fluorescence, Ethyl alcohol-Methyl alcohol 77 "K ( 4 : ~ (2) 38OC 52OC 363b 49-56 Fluorescence. Potassium bromide (3) . . 23 f 0 . 5 'C 365 517, 489(sh), Phosphorescence, Ethyl alcohol-Methyl alcohol 365 498, 474 48 1 77 "K (4:l) (2) 71 < 1M sec, 7 2 = 1.2 sec. (exc. Phosphorescence, Ethyl alcohol-Methyl alcohol T I < 1M sec, 1 2 = 2.0 s a . (exc. decay time (41) (2) 365 mp; emiss. 498 mp) 365 mp; emiss. 516 mp) 6 Fluorescence Methyl alcohol (4) 4 = 0.078 (exc. 363 mp) 4 = 0.24 (exc. 363 rnp) 23 i 0 . 5 "C a Concentrations: B,: (1) 3.1 X 10-5M; (2) 2.5 X 10-5M; (3) 65 /.4g/200mg KBr; (4) 1.8 X G,: (1) 2.9 X IO-jM; (2) 2.4 X IO-jM; (3) 491 4 2 0 0 mg KBr; (4) 1.7 X b Primary filter 7-39; secondary filter 3-75. c Primary filter 7-51; secondary filter 3-75.
replica gratings with 28,800 lines per inch, and an EM1 95584 photomultiplier detector. Excitation energy is monitored by a thermistor bolometer which is an absolute detector. Spectral correction is made by a ratio method and emission spectra can be recorded in either relative energy o r quantum units with a Moseley Model 7100B recorder. Luminescence measurements of solutions of the aflatoxins were made using a fused quartz cell (1 X 1 X 5 cm). I n order to minimize oxygen quenching, samples were flushed with nitrogen for 5 minutes immediately before measurement and a steady flow of nitrogen was maintained in the sample chamber during measurements. Low-temperature luminescence spectra were obtained at 77 OK in a frozen ethyl alcohol-methyl alcohol (4 :1) matrix. For these measurements, the sample cell was placed in a specially-constructed quartz Dewar vessel (11) into which liquid nitrogen was introduced. When making measurements at 77 OK, the sample area was continuously flushed with dry nitrogen to prevent fogging of the Dewar. Phosphorescence decay curves were viewed on a Tektronix Model 503 oscilloscope and photographed by a Polaroid camera using 3000-speed, Type 107 film. Pellet samples (1 mm thick, 13.0 mm in diameter) were placed in a nonfluorescent Teflon holder (11). The pellet was positioned at an angle of 45" with respect t o the exciting beam and emission was measured at right angle to excitation. Proper glass color filters (Corning Glass Works, Corning, N. Y . ) were selected t o eliminate extraneous peaks due to surface reflection and scattering phenomena. The resolution of bands was controlled by the size of the slits. The spectra were in most instances measured with 5-mp or 2.5-mp bandwidths. Detection of Aflatoxins on Silica Gel Plates. Standard solutions of the aflatoxins were prepared in fluorometric grade methyl alcohol. In determining the limits of detection for these substances o n a plate, aliquots containing 0.001-0.4 pg were spotted over areas -2-3 mm in diameter. Development was carried out with chloroform-methyl alcohol (97 :3). The fluorescence intensities of the developed spots were visually compared under a n ultraviolet lamp. Photolysis of Aflatoxin B1. Irradiation of aflatoxin B1 in ethyl alcohol solution was conducted in the sample chamber of the luminescence instrument. A 1.9 X 10-jM solution was photolyzed at 363 m p (5-mm slitwidth) at ambient temperature (23 =t 0.5 "C)for 5 hours. Fluorescence excitation and emission spectra were recorded at regular intervals during and after irradiation. F o r solid-state photolysis, spots containing 0.018 pg of aflatoxin B1 on a silica gel plate were exposed to ultraviolet light (320-400 mp) for periods varying from 5 minutes t o 1 hour. The plate was then developed and the chromatograms were compared with that of a fresh sample. O n a preparative
scale, 37 pg of aflatoxin B1 was deposited on a plate over an 8X 13-cm area. After irradiation for 3.5 hours, the adsorbent was removed and successively extracted with 30 ml of chloroform-methyl alcohol (1 :1) and 20 ml of methyl alcohol. The combined extracts were evaporated under reduced pressure and the residue was taken up in 3 ml of fluorometric grade methyl alcohol. The resulting solution was examined by thin-layer chromatography, ultraviolet-visible absorption spectrometry, and fluorescence spectrometry. RESULTS AND DISCUSSION
The luminescence characteristics of aflatoxins B1 and GI are summarized in Table I. Solution Spectra at Room Temperature. Because aflatoxins B1 and GI show striking similarities in their ultravioletvisible absorption spectra, fluorescence spectrometry appeared t o be the method of choice for the characterization of these substances, particularly because of the sensitivity of this method. The corrected fluorescence emission and excitation spectra of aflatoxin B1 in methyl alcohol are shown in Figure 1. For aflatoxins B1 and GI there is a close correspondence in both peak position and relative intensities between the ultraviolet-visible absorption and automatically corrected fluorescence excitation spectra. Because the aflatoxins are isolated by thin-layer chromatography from contaminated food and other products in amounts which are generally insufficient for absorption measurements, the usefulness of corrected excitation spectra for determining the ultraviolet characteristics of these compounds is obvious. The fluorescence intensities of the aflatoxins are insensitive to oxygen quenching; air-saturated and deoxygenated samples of the compounds show the same fluorescence intensities. Both compounds displayed the typical concentration-relative fluorescence intensity curves (13) at concentrations ranging from 1.8 x 1O-M to 6.2 X 10-jM for aflatoxin B1 and from 1.2 x lO-7M to 1.2 x 10d4M for aflatoxin Gl in methyl alcohol. The maximum relative fluorescence intensity regions were at 2.0 x 10-jM-3.0 x 10+M and 3.0 X 10-jM -4.5 x lO+M for B1 and GI, respectively. At concentrations beyond these upper limits, fluorescence intensities decreased with increasing concentration. Neither substance showed shifts in excitation or emission maxima at high con(13) s. Udenfriend, "Fluorescence Assay in Biology and Medicine," Academic Press, New York, 1962, p 15. VOL. 40, NO. 13, NOVEMBER 1968
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WAVELENGTH, rnv
Figure 1. A . Corrected fluorescence excitation spectrum of aflatoxin B1emission at 425 mp
centrations (3.5 X lOU3M) and, hence, they d o not form fluorescent aggregates or undergo excited state dimerization in methyl alcohol (14). The fluorescence quantum efficiencies of the aflatoxins in methyl alcohol were determined by the method of Parker and Rees (15) using quinine bisulfate in 0.1N sulfuric acid as standard. For the calculation of quantum efficiencies, the value of 0.55 (16) was used for the quantum efficiency of the reference compound although it has recently been suggested that this value may be too high (17). As suggested by Parker and Rees ( 1 3 , the concentrations of the samples were kept within the range corresponding to 0.04 absorbance unit per cm at the excitation wavelengths used in order to avoid selfabsorption; the corrected emission spectra were recorded directly in quantum units. The fluorescence values found were: 0.078 for B1 and 0.24 for GI. Thus, the quantum yields are in the order of GI > BI, with the ratio of 3 :1. Previous findings on the basis of relative fluorescence intensity as compared with quinine sulfate indicate the same order, but the ratios of G1:B1expressed in terms of KQ, were 5.0:l (7) and 1.4:l (8). (These values were obtained by comparing the fluorescence intensities of the aflatoxins with that of a quinine sulfate standard.) The quantum efficiency values found in this study seem surprisingly low inasmuch as both substances show intense visible fluorescence on thin-layer plates. These observations suggest that there is a significant difference between solid state and solution fluorescence and this aspect merits further study. Limits of Detectability. The sensitivity for the detection of the aflatoxins by fluorescence and thin-layer chromatography are compared in Table IT. The minimum quantities
+
(14) E. L. Wehry and L. B. Rogers, “Fluorescence and Phosphorescence Analysis,” D. M. Hercules, Ed., Interscience, New York, 1965, p 96. (15) C . A. Parker and W. T. Rees, Analyst, 85, 587 (1960). (16) W. H. Melhuish, J . Opt. SOC.Am., 54, 183 (1964). (17) R. Rusakowicz and A. C . Testa, J . Pliys. Cliem., 72, 793 (1968). 2026
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B. Corrected fluorescence emission spectrum excitation at 363 mp Both curves were obtained for a 3 X 10-SMsolution in methyl alcohol at 23 i 0.5 “C. For the excitation spectrum 2.5 mp excitation and 5.0 mp emission bandwidths were used. For the emission spectrum the slits were reversed. The second line on both curves is an instrumental tracing of the correction factor
for detection by fluorescence are 0.07 pg for B1and 0.002 pg for G1using a 1- X 1-cm cell holding 3 ml of solution; by thin-layer chromatography, the corresponding quantities are 0.2 pg and 0.1 pg per spot. I n addition to its high sensitivity, fluorescence spectrometry is preferable to thin-layer chromatography for the identification of these substances because the characteristic fluorescence spectra are more definitive. Pons et af. (18) have reported the detection of 2 X low4 pg of B1 or G1 with a densitometer, but these identifications again are based solely on the RE.on a thin-layer chromatogram and cannot be considered definitive. In measuring the fluorescence spectra of B1 and GI in methyl alcohol at low concentrations, it was observed that the Raman bands for the solvent become significant. The positions of the Raman bands vary from the excitation wavelength by a constant frequency difference (19), and, for methyl alcohol, they fall within the emission regions of both the aflatoxins when the samples are excited at 363 mp. Consequently, it was necessary to use shorter wavelength excitation bands (264 mp) with wide slits in order to obtain true emission spectra. Fluorescence Spectra in Potassium Bromide. The spectra of powdered aflatoxins B1 and G1 measured in potassium bromide pellets indicate a shift to longer wavelength as compared to those in solution (Table I), This red shift is -70 mp for both compounds. Similar results were noted in previous work with aromatic hydrocarbons (10). Luminescence Spectra of Frozen Solution. Aflatoxins BI and GI in ethyl alcohol-methyl alcohol ( 4 : l ) at room tem(18) W. A. Pons, J. A. Robertson, and L. A. Goldblatt, J . Am. Oil Cliemists’ Soc., 43, 665 (1966). (19) C . A. Parker, Analyst, 84, 446 (1959).
Table 11. Limits of Detection of Aflatoxins B1 and G1 B1 GI X 10-gM (5.5 X 1.7 7.2 x 10-*M(2.2 X 10-2pg/ml) By fluorometry (in methyl alcohol): pg/ml) with excitation at 264 mp and with excitation at 264 mp and emission at 450 mp emission at 426 mp. By thin-layer chromatography visible Blue fluorescence: 0.2 pg/spot Green fluorescence: 0.1 pg/spot fluorescence Noncharacteristic pale-white Noncharacteristic pale-white fluorescence: 0.01 pglspot fluorescence: 0.01 pg/spot
perature displayed fluorescence emission maxima a t 426 m p and 450 mp, respectively, and showed identical excitation spectra with peaks at 363 and 264 mp. At 77 OK, both excitation peaks were shifted 2 mp to longer wavelength while the fluorescence emission peaks were at 408 mp for BI and 423 mp for G1(Table I). In addition, a three-fold increase in relative fluorescence intensity and sharpening of fluorescence bands were observed for both compounds at 77 OK compared to the room temperature curves. The observed “blue shift” in going from room temperature to 77 OK is similar t o the results reported for hydroxynaphthalenes (20). This effect was attributed to emission from a n excited solute molecule without solvent reorientation (20). Unlike the low-temperature fluorescence spectra, the phosphorescence spectra of the aflatoxins exhibited resolution into several peaks (Table I). Phosphorescence decay measurements, observed on the oscilloscope, revealed two decay times for both aflatoxins. The mean lifetimes for the fast decays were less than 1 msec. These values could not be determined more accurately because of instrumental limitations ( I / ) . The slow decay times showed mean lifetimes of 1.2 and 2.0 sec for B1 and G1, respectively. These results were reproducible regardless of the wavelength of phosphorescence emission at which the decay curves were traced. As a representative case, the slow decay curve of B1is given in Figure 2. Other examples of two luminescence decay times have been noted in 1-indanone (11, 2 / ) and in guanine at pH 1 (22). It is not clear whether the observed multiple exponential decay is a n intrinsic property of the aflatoxins or owes its existence t o other phenomena. The usefulness of low-temperature luminescence measurements for characterization of this series of compounds is exemplified in the case of the so-called “milktoxin” (aflatoxin M), a hydroxy derivative of aflatoxin B1 ( 5 ) . Aflatoxins M and B1 show very similar absorption, excitation, and emission spectra at room temperature in methyl alcohol; however, there are distinct differences in their lowtemperature fluorescence and phosphorescence spectra and decay times (23). Photo-Decomposition of Aflatoxin B1. Andrellos ef al. (24) have reported on the photochemical degradation of aflatoxin B1 on silica gel plates and in methyl alcohol solution. I n the course of the present investigation, similar observations were made. When a spot of 0.018 pg of aflatoxin B1 on a silica gel plate was exposed to ultraviolet light for periods of 0.5 hour or
(20) D. M. Hercules and 1,. B. Rodgers, J . Phys. Chem., 64, 397 (1960). (21) N. C . Yang, and S. Murov, J. Chem. Phys., 45,4358 (1966). (22) R. 0. Rahn and R. G. Shulman, ibid.,45, 2930 (1966). (23) B. L. Van Duuren and T. L. Chan, New York University Medical Center, unpublished data, 1968. (24) P. J. Andrellos, A. C . Beckwith, and R. M. Eppley, J . Assoc. Ofic.Agr. Chemists, 50 (2), 347 (1967).
Figure 2. Phosphorescence decay curve of aflatoxin B1a t 77 O K Excitation and emission wavelengths at 365 and 498 rnk, respectively; 5.0 r n M excitation and emission bandwidths. Solvent system and concentration are given in Table I. Each box on the abscissa represents 1 second longer and the resulting plate developed with chloroformmethyl alcohol (97 :3), a new blue fluorescent spot, with a RF of 0.15, appeared. This result agrees with the earlier findings (24). However, irradiation carried out on a preparative scale resulted in the formation of a complex mixture which was not separable by thin-layer chromatography and gave inconsistent absorption and fluorescence excitation and emission spectra. Irradiation of a 1.9 x 10-jM solution of Bl in ethyl alcohol gave a n unidentified substance which showed a single spot ( R p = 0.60) in chloroform-methyl alcohol (97:3). I n ethyl alcohol, the photoproduct and starting material showed identical fluorescence emission curves with maxima at 429 mp. A slight change appeared in the absorption spectrum of the photoproduct; a new weak shoulder appeared at 400 mp in addition to the peaks characteristic of B1at 363, 264, and 223 m p (9). Furthermore, in the corrected fluorescence excitation spectrum of the photoproduct, the 400 rnp peak was anomalously more intense than the 363 mp peak. Using the combination of thin-layer chromatography and luminescence spectrometry, studies are currently underway o n the possible presence of aflatoxins in tobacco leaf extracts. ACKNOWLEDGMENT The authors are indebted t o G. N. Wogan (M.I.T., Cambridge, Mass.) for the samples of aflatoxins and to s. Cravitt (Farrand Optical Co., New York, N.Y.) for his interest and helpful discussions.
RECEIVED for review J L ~5,Y1968. Accepted August 19, 1968. This investigation was supported by Contract No. PH4364-938 from the National Cancer Institute, National Institutes of Health, and ES-00260 from the National Institutes of Health. VOL. 40, NO. 13, NOVEMBER 1968
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