ficity would not be expected to prevail for alkenyl ethers described above. Rather, hydrogen transfer reactions closely akin to aliphatic ethers (17) and thioethers (18) would be anticipated. To obtain the most information from an alkenyl ether mass spectrum, it should be interpreted in conjunction with the mass spectrum of the aldehyde precursor. In most cases only the mass of beta substituent(s) will be discernible, though at times a difference in leaving aptitude may indicate the structure of the fragment. However, even in the former situation, data provided by alkenyl ether spectra can reduce the number of structures that must be considered for a particular aldehyde. (17) C. Djerassi and C . Fenselau, J. Amer. Chem. SOC.,87, 5141 (1965). (18) S. D. Sample and C. Djerassi, ibid., 88, 1937 (1966).
ACKNOWLEDGMENT
The authors thank Dr. Fred Regnier, Department of Biochemistry, Purdue University, for his assistance in obtaining mass spectral data, and Dr. J. E. Sinsheimer, College of Pharmacy, University of Michigan, for measuring the NMR spectra of alkenylethers. RECEIVED for review November 7, 1969. Accepted October 16, 1970. This work represents a portion of the Dissertation of William G. Andrus, Jr., Purdue University, 1969. A brief account of these data was presented at the 158th National Meeting of the American Chemical Society, New York City, September 8, 1969. This work was supported, in part, by a National Institute of General Medical Sciences Fellowship No. 1-Fl, GM-34, 131-01, and by the American Foundation for Pharmaceutical Education.
Aflatoxin Detection by Thin=Layer Chromatography-Mass Spectrometry W. F. Haddon, Mabry Wiley, and A. C. Waiss, Jr. Western Regional Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Albany, Calq. 94710
NUMEROUS RECENT PUBLICATIONS describe the isolation and identification of aflatoxins at low levels (1-4). These papers reflect concern over the possible presence of these carcinogenic compounds in agricultural commodities. It is now common practice to use thin-layer chromatography (TLC) to identify aflatoxins on the basis of R , value and fluorescence at levels which can be as low as several parts per billion (1,3). A common complaint against all TLC methods used for aflatoxin detection is their vulnerability to interference from nonaflatoxin compounds (3). Nonfluorescing materials of coincident R , value can mask the presence of fluorescent spots from aflatoxins below threshold values which can be surprisingly high (5). Fluorescing artifacts can, on the other hand, falsely indicate the presence of aflatoxins. These problems have led to chemical confirmatory tests for aflatoxin BI, which is the most toxic known aflatoxin. A confirmatory method proposed originally by Andrellos and Reid (6) and based on the reaction of aflatoxin B1 with volatile acids has been dramatically successful in this regard, but often requires more than 1 pg of aflatoxin and cannot be used to confirm aflatoxins B2, GP,and MI. We have found that low and nigh resolution mass spectral techniques can provide unambiguous qualitative identification of aflatoxins isolated from individual TLC spots with sensitivity approaching that of the TLC-fluorescence method. The mass spectral method is not limited to particular aflatoxins and appears to have approximately equal sensitivity for BI, B2, GI, G2,and MI. The mass spectrometer is now used in (1) W. A. Pons, Jr., and L. A. Goldblatt, J . Amer. Oil Chem. SOC., 42,471 (1965). (2) M. Wiley, J. Ass. ODc. Anal. Chem.,49, 1223 (1966). (3) L. Stoloff, ibid., 50, 354 (1967). (4) I. F. H. Purchase and M. Steyn, ibid., p 363. (5) A. D. Campbell, ibid., p 343. (6) P. J. Andrellos and G. R. Reid, ibid., 47,801 (1964). 268
this laboratory to confirm the presence or absence of aflatoxins in a variety of agricultural commodities in amounts typically as low as 20 ppb. Positive identification can be obtained in many cases with 50 ng or less of aflatoxin. The use of mass spectrometry extends the lower limit of reliable detection two orders of magnitude or more below existing chemical confirmatory tests for these compounds. EXPERIMENTAL
TLC Analysis. TLC analysis reported here utilizes the procedure for aflatoxin isolation described by Pons and Goldblatt for cottonseed products (1). The samples used in this study were experimental aflatoxin-contaminated cottonseed meal. TLC plates are prepared from pure silica gel (G-HR, Brinkmann Instrument Company). The TLC plates are developed using 9 parts of CHCI, and 1 part acetone. The R , value for aflatoxin B1under these analytical conditions is 0.56. Mass Spectral Analysis. Mass spectra were obtained on a CEC Model 21-110 mass spectrometer with electron multiplier detection. Samples were introduced directly into the ion source using a heated direct introduction probe. The aflatoxins volatilize between 185 and 205 "C in the mass spectrometer. Aflatoxins in pure form used to establish mass spectral fragmentation patterns were obtained by column chromatography on silica gel. The purity of BI, B1, GI, and G 2was greater than 98 by mass spectral analysis. Aflatoxin MI contained about 10% aspertoxin. Because aspertoxin is more volatile than MI, it could be distilled from MI within the mass spectrometer, thus yielding a spectrum of pure MI. TLC-Mass Spectrometry. Aflatoxins cannot be run directly from TLC plates because such compounds adsorb strongly on silica gel and cannot be volatilized in the mass spectrometer without thermal decomposition. Developed chromatograms are first spotted with 1 p1 of distilled water to dislodge the aflatoxins from the most active sites of the silica gel. The gel is then loaded into a microcolumn formed by
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partially closing one end of a 1-inch length of 1.5-mm 0.d. melting Point tube. Ten to 15 ~1 of reagent grade acetone are used to elute the sample from this column into an open-ended melting point capillary which is subsequently placed in the direct introduction probe and inserted into the mass spectrometer following evaporation of the acetone. It is essential to exclude silica gel particles from this capillary. Exposure to ultraviolet light is held to less than 20 sec where possible to minimize Dossible Dhotochemical decomDosition. After insertion into the mass spectrometer, the *direct probe is programmed upward at 5-10 "C per min and spectra are recorded between 170 and 220 "C. The ion source temperature is 230 "C. RESULTS AND DISCUSSION
Figure 1 gives the partial mass spectra of aflatoxins B,, B2, GI, GB,and MI and a related compound aspertoxin (7). Peaks below mje 200 comprise less than 2 total ionization and are not shown. Intense molecular ions are obtained for (7) A. C . Waiss, Jr., M. Wiley, D. R. Black, and R. E. Lundin, Tetrahedron Lett., No. 28, 3207 (1968).
all of the compounds and comprise up to 47 % of the total ion current. Sequential losses of CHO and CO and loss of C2H30 account for most of the significant fragment peaks in all the compounds~ The relatively high percentage of total ion current carried by the molecular ion as indicated in Figure 1 is a Particularly favorable feature of the mass spectra for the detection of these ComPounds at low levels. Aflatoxins GI and MI, although coincident in molecular weight and elemental composition, differ dramatically in the abundance of the peaks for loss of CHO and CHO CO from the molecular ion, so that these compounds can be differentiated easily from their mass spectra alone. The ability of the mass spectrometer to detect compounds at low levels depends importantly both on the characteristics of the mass spectral fragmentation pattern and on the degree of adsorption on the walls of the mass spectrometer and associated inlet lines. For example, for simple organic compounds introduced into the mass spectrometer directly from a gas chromatograph using a Watson-Biemann separator, the limits of detection vary from 1 ng to 1 pg depending on the amount of oxygen and the polarity of the organic compound
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ANALYTICAL CHEMISTRY, VOL. 43, NO. 2, FEBRUARY 1971
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(8). This problem of adsorption limits severely the sensitivity for measurement of aflatoxin compounds in the mass spectrometer, but the high fraction of total ion current carried by the molecular ions partly compensates for this effect. Attempts to obtain spectra of aflatoxins directly from the silica gel support were not successful at sample loading above 10 pg compared to sensitivities below 50 ng when the compounds were run in the absence of silica gel from melting point capillaries. Careful elution from the silica gel permits detection at levels considerably below l pg. We are able to obtain usable spectra routinely on as little as 30 ng of aflatoxin after elution from the silica gel support. At sample levels above 0.25 pg, high resolution mass spectral techniques are used to increase the certainty of identification by exact mass measurement of the molecular ion. With 0.5 Mg, measurable ion current at greater than 25 to 1 signal-to-noise ratio is obtained for approximately 7 min for aflatoxin B1at resolution above 10,000. The sensitivity of the mass spectral method is best when the rate of heating the sample is high, because the signal-to-noise ratio of the electron multipler output increases with increasing rate of volatilization of sample into the ion source. This is true even for instruments having integrating photoplate detectors because of the presence of background peaks. Heating rates of 5 to 10 "C per min gave good results on 50-ng samples in this work. Figure 2 shows a series of mass spectra recorded at different RIvalues from the same thin-layer chromatogram containing aflatoxins extracted from 25 grams of the aflatoxin-contaminated cottonseed meal which contained 750 ppb of aflatoxin B1 and 20 ppb of aflatoxin Bz. The first spectrum, Figure 2a, obtained at R f 0.60 shows the presence of a small peak at mass 312 corresponding to aflatoxin BI. The second spectrum recorded at R 0.56 near the center of the B1 spot shows a 50-fold enhancement of intensity for the mass 312 peak. The spectrum of B1 shown in Figure 26 is attenuated by a factor of 10 over the previous spectrum. The next spectrum (Figure 2c) at R f 0.53 was obtained between the BI and BZ (8) W. D. MacLeod, Jr., and B. Nagy, ANAL.CHEM., 40,841 (1968).
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spots and shows both compounds at extremely low level. The final spectrum (Figure 2 4 at R f 0.50 shows an intense molecular ion peak at mass 314 corresponding to aflatoxin Bz and several interfering compounds of different mass. The amount of aflatoxin B2 on the spot for this spectrum was less than 50 ng. An additional useful feature of mass spectral analysis is that temperature-programming the direct-introduction probe of the mass spectrometer actually introduces a second dimension to the mass spectral analysis based on the differing volatility of the various components of a single TLC spot. Aflatoxins are known to decompose photochemically after exposure to UV light (9). We investigated this effect for aflatoxin B1and found that none of the photochemical decomposition products volatilize over the temperature range 180 to 220 "C in the mass spectrometer, so that no serious interference results from photochemical decomposition. It appears, however, that BI degrades rapidly during exposure to UV light (420 nm) under conditions typical for TLC analysis although the rate of degradation is such that 20-sec exposure produced no significant loss of sample at the 1-pg level. We are currently exploring the extension of these mass spectral techniques to quantitative analysis of aflatoxins, both as isolated spots and as mixtures, and the use of real-time mass spectral-computer techniques to increase the versatility of mass spectrometry applied to aflatoxin detection. ACKNOWLEDGMENT
The authors thank Dr. A. C. Key1 for encouragement and helpful discussion. RECEIVED for review September 1, 1970. Accepted October 27, 1970. Reference to a company or product name does not imply approval or recommendation of the product by the U. S. Department of Agriculture to the exclusion of others that may be suitable. (9) P. J. Andrellos, A. C . Beckwith, and R. M. Eppley, J . Ass. Ofic.Anal. Chem., 50, 346 (1967).
ANALYTICAL CHEMISTRY, VOL. 43, NO. 2, FEBRUARY 1971
