Anal. Chem. 1985, 57, 1470-1472
1470
CORRESPONDENCE Fast Atom Bombardment Mass Spectrometry: A Screening Technique for Mixtures of Secondary Metabolites from Fungal Extracts of Fusarium Species Sir: Applications of fast atom bombardment mass spectrometry (FAB-MS) have increased rapidly since the inception of this technique in 1981 ( 1 , 2 ) and cover a variety of compounds ( 3 , 4 ) .These applications deal mainly with the analysis of pure samples in which FAB-MS is used for structural confirmation or to further characterize a tentative structure. We wish to report another application of FAB-MS that involves the qualitative analysis of mycotoxins present in crude fungal extracts by direct determination of the [M H]+ and other ions. Trichothecene mycotoxins (I) are secondary metabolites
+
1
produced by various Fusarium species. They inhibit protein synthesis and induce feed refusal in animals (5,6). Their rapid identification is thus of great interest in agriculture as well as other fields. For the analysis of trichothecene mycotoxins, most spectral techniques are not suitable for rapid screening purposes since they are moiety rather than compound specific (e.g., IR, UV) (7,8),or lack sensitivity (e.g., NMR) (9). Mass spectrometries, both chemical ionization (CI) and electron impact (EI) are definitve and sensitive techniques, but as with IR and UV require the resolution of mixtures by GC or HPLC prior to detection. However, the E1 mass spectra of the trichotheeenes are very complex with no single characteristic fragmentation pattern emerging that would allow the ready identification of the trichothecene ring system (6). In constrast, their FAB mass spectra are relatively simple and show little fragmentation apart from the loss of water or acetic acid from the protonated molecular ion [M + HI+ for species containing hydroxy- or acetoxy moieties, respectively. The simplicity of FAB mass spectra makes the technique useful for the determination of trichothecene mycotoxins in crude fungal extracts (10). Few MS/MS studies involving trichothecenes have been reported, possibly because of the complexity of the E1 spectra (10). Earlier MS/MS work was performed strictly to detect a given substance in mixtures (11, 12) or in other ionization modes (13, 14) where, of course, fragmentation is greatly reduced.
EXPERIMENTAL SECTION FAB mass spectra were recorded on a Finnigan MAT 312 double focusing reversed geometry instrument mounted with a saddlefield atom gun (Ion Tech., Ltd.) and coupled to a INCOS data system. Xenon (99.99570, Matheson) was used as the bombardment gas at 8 kV, and the resulting sputtered positive ions were extracted into the mass analyzer at an acceleration potential of 3 kV. The scan range was set between 110 and 1350 daltons at a rate of 10 s per scan and the electron multiplier was
set at 2.2 kV. The samples were introduced onto the target source as solutions or dispersions in glycerol (Fisher). Other support matrices (e.g., polyethylene glycol-200)were tested, but the spectra contained appreciably more solvent peaks and resulted in reduced signal-to-noise ratios. Although definitive detection limits are yet to be established, typical sample loads of mixtures included 500 ng of trichothecene standards in 1-2 pL of support matrix. The resulting spectra lasted for 5-10 min and had a signal-to-noise ratio better than 1O:l (for [M + HI+). Crude fungal extracts containing a variety of trichothecene mycotoxins were obtained by extraction of the beers from fermentation of Fusarium spp. using ow previously described scheme (15).
RESULTS AND DISCUSSION Figure 1 shows the FAB mass spectrum of calonectrin, a typical trichothecene mycotoxin. In contrast, the FAB mass spectrum of a crude extract described earlier (15) is shown in Figure 2. Only the region of 200-400 daltons is shown here for clarity reasons. All major peaks can be assessed readily by comparison with those of standards. The following compounds were detected: m / z 383, [DHDAD + HI+; 365, [DHDAD H - HzO]+; 351, [CAL + HI+; 339, [ ~ A c D O N+ HI+; 323, [DHDAD H - HOAc]+; 309, [? + HI+; 267, [SOL HI+; 251, [SON HI+; 237, [COL + HI+; 221, [CUL + H - H,O]+; 203, [CUL H - 2HzO]+;where DHDAD stands for 3,15-diacetoxy-7,8-dihydroxy-12,13-epox~richothec-9-ene (I, R, = R3 = OAc, Rz = R, = H, R4 = R5 = OH), CAL for calonectrin (I, R1 = R3 = OAc, Rz = R4 = R5 = R6 = H), 3AcDON for 3-acetyldeoxynivalenol (I, R1 = OAc, R2 = H, R3 = R4 = OH, R5 and Rs are a ketone), CUL for culmorin (11), SOL for sambucinol (111),SON for sambucoin (IV), and
+
+
+ + +
@
HO
I1
Ill
IV
COL for culmorone (11, with one of the OH oxidized to the ketone) (15). The peak at m / z 309 corresponds to a substance not yet characterized. The presence of these toxins in the crude fungal extract was confirmed by GC analysis of the mixture after derivatization with heptafluorobutyrylimidazole (16). GC retention times on a Varian Vista 6000 series gas chromatograph equipped with a "Ni electron-capture detector, 3% OV-3 glass column, temperature program 140-240 "C at 2 OC/min) were as follows: DHDAD, 24.4 min; 3-AcDON, 17.5 min; SOL, 10.3 min; COL, 5.7 min; CUL, 4.9 min. CAL and SON were not detected by the GC method under these con-
0003-2700/85/0357-1470$01.50/0 Published 1985 by the American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 57, NO. 7, JUNE 1985 [M + HIt
-
3: 2
lBQ.0
1471
65928.
35 1/BASE =e.=
58.0 277.1
I
[M t H - HOAc]'
215.1
t
369.2
237.2 249.1
291.1
c
[MtGly+Hlt
443.2
[M t 2Gly t HIt
Figure 1. FAB mass spectrum of calonectrin standard (I, R, = R, = OAc, R2 = R, = R, = R, = H). t HIt
r 10.0X
2
188.0
177152.
2674ASE =e.9178
50.0
[CUL+ H
[DHDAD t HIt
- H20]'
383.2
22t.2
I
237.2
1
251.2
I
ME
Flgure 2.
200
220
240
[3AcDON t HIt
309.2
280
300
323.2
320
33?m2
340
380
41
Typical FAB mass spectrum of a crude fungal extract of Fusarium spp. (see text for extraction procedure).
ditions demonstrating an advantage that FAB offers over conventional GC analytical procedure. The presence of SON and CAL in this specific crude fungal extract was proven by their subsequent characterization. Similar FAB mass spectral analyses of the fractions obtained from column chromatography of the crude fungal extracts during isolation permitted
the rapid monitoring of minor constituents that are masked by the large proportions of the major constituents. In summary, FAB mass spectra can be used to screen crude fungal extracts for trichothecene mycotoxins. I t allows for the direct molecular weight determination and subsequent identification of known trichothecenes and can indicate the
1472
Anal. Chem. 1905, 57, 1472-1474
presence of new species and provide limited structural information. Registry No. I (R, = R3 = OAc, R2 = Re = H, R4 = R5 = OH), 95673-99-7;I (R1= R3 = OAC,& = R4 = Rb = & = H), 38818-51-8; I [R, = OAC,Rz = H, R3 = R4 = OH, R5 = & = (*)I, 50722-38-8; 11, 18374-83-9; 111, 90044-33-0; IV, 90044-34-1; culmorone, 95648-62-7.
(13) Brumley, W. C.; Andrezejewski, D.; Trucksess, E. W.; Dreifuss, P. A,; Roach, J. A. G.; Eppley, R. M.; Thomas, F. s.; Thrope, C. W.; Sphon, J. A. Blomed. Mass SDectrom. 1982. 9 . 451-458. (14) Smith, R. D.; Usdeth, H. R. Anal. Chem. 1983, 55, 2266. (15) Greenhalgh, R.; Meier, R.-M.; Blackwell, B. A,; Miller, J. D.; Taylor, A,; ApSimon, J. W. J. Agrlc. Food Chem. 1984, 32, 1261-1264. (16) Scott, P. M.; Lau, P.-Y.; Kanhere, S. R. J. Assoc. Off. Anal. Chem. 1981, 64,1364.
J. R. Jocelyn Pare* Roy Greenhalgh Pierre Lafontaine
LITERATURE CITED Surman, D. J.; Vickerman, J. C. J. Chem. SOC.,Chem. Commun. 1981, 324-325. Barber, M.; Bordoli, R. S.; Sedgwick, R. D.; Tyler, A. N. J. Chem. SOC., Chem. Commun. 1981, 325-326. Rinehart, K. L., Jr. Science 1982, 218, 254-260. Barber, M.; Bordoll, R. S.; Elliot, G. J.; Sedgwick, R. D.; Tyler, A. N. Anal. Chem. 1982, 54, 645A-657A. Joffe, A. Z.I n "Microbial Toxins, Vol. VII"; Kadls, S., Ciegler, A,, Ajl, S. J., Eds.; Academic Press: New York, 1971: Chapter 5. Bamburg, J. R.; Strong, F. M. I n "Microbial Toxins, Vol. VII"; Kadis, S., Clegler, A., Ajl, S. J., Eds.; Academic Press: New York, 1971; Chanter 7. COG-R. J.; Cox, R. H. "Handbook of Toxic Fungal Metabolites"; Academic Press: New York. 1981. Mirocha, C. J.; Pathre, S:V.; Christensen, C. M. I n "Mycotoxic Fungi, Mycotoxicoses: An Encyclopedic Handbook"; Willie, T. D., Morehouse, L. G., Eds.; Marcel Dekker: New York, 1978: Vol. I, pp 365-420. Blackwell, B. A.; Greenhalgh, R.; Bain, A. D. J. Agrlc. Food Chem. 1984, 32, 1078-1083. Par& J. R. J. Ph.D. Thesis, Carleton University, Ottawa, ON, Canada, 1984. Busch, K. L.; Cooks, R. G. Anal. Chem. 1983, 55,38A. Plattner, R. D.; Bennett, G. A. J. Assoc. Off. Anal. Chem. 1983, 66, 1470.
Chemistry and Biology Research Institute Agriculture Canada Ottawa, Ontario, Canada K1A OC6
John W. ApSimon The Ottawa-Carleton Institute for Research and Graduate Studies in Chemistry Carleton University Ottawa, Ontario, Canada K1S 5B6
RECEIVED for review October 18, 1984. Resubmitted January 28,1985. Accepted February 19,1985. We thank the Natural Sciences and Engineering Research Council-Canada for the award of a Visiting Fellowship in Biotechnology to J.R.J.P. This paper is C.B.R.I. contribution No. 1495 and was taken in part from the Ph.D. Thesis of J. R. J. Par& Carleton University, July 1984.
Effect of Concentration Gradients on Spectra in Gas Chromatography/Fourier Transform Infrared Spectrometry Sir: One problem generally disregarded in GCIFT-IR is the question of whether changing analyte concentration during the collection of a single interferogram will detrimentally affect the spectral results. Lephardt and Vilcins addressed this problem theoretically and came to the conclusion that changing concentration has the same effect as an increased apodization (2). With the relatively broad peak widths of packed column GC, modern FT-IR scan rates arre sufficiently rapid to ensure negligible concentration gradients during single interferometric scans. However, the growing popularity of smaller, slower scanning FT-IR spectrometers and the development of capillary GC/FT-IR have made it possible for significant concentration changes to occur during the collectior of a single interferogram. With capillary peak elution times of 2-5 s and FT-IR spectrometer scan cycles of 2 s, only one to two interferograms will be collected over a single GC peak. Under these conditions, a noticeable concentration gradient will occur across each sample interferogram. The effects of this gradient upon spectral results are examined in this correspondence. EXPERIMENTAL SECTION Instrumentation. Reference and sample interferogramswere collected using an IBM Instruments IR-85 FT-IR spectrometer with a DTGS detector. The sample was a thin polystyrene film. The interferometric data were transferred to a Vax 11/780 minicomputer for spectral calculations. Procedure. A Gaussian chromatographic profile was used to model the changing analyte concentration. By variation of the width of this Gaussian function, it was possible to study the effects of different interferometric scan rates. Representation of the concentration gradient was established by assuming a valid Beer's
law relation. From this it follows that
where Ti and Ai represent the transmittance and absorbance of the sample at time i. The ratio of absorbances at a given frequency can be determined from the Gaussian profile &/Ap = G(6)
(2)
where G(6) is the Gaussian value at mirror displacement 6, A , is the maximum sample absorbance, and A6 is the sample absorbance at displacement 6. Substitution of eq 2 into eq 1results in T&u)= T , ( v ) ~ ( * )
(3)
Equation 3 simulates the change in transmittance as a function of mirror displacement 6 and the peak transmittance T,(v). It is the sample concentration gradient through the light pipe that causes transmittance to change with mirror displacement. To represent this change in the interferogram domain requires the calculation of a new transmittance spectrum for each mirror displacement. Sample interferograms containing concentration gradients can then be calculated Z(6) = ~ T , ( U ) ~ ' * )cos R(U (27rvS) )
(4)
where R(u)represents the reference power spectrum. In this work, these calculated sample interferograms were transformed and ratioed with the original reference power spectrum, R(u), to produce the sample gradient spectra which result from a changing concentration profile. To evaluate the spectral distortions caused by this concentration gradient, the sample gradient spectra were fit to a sample spectrum (containing no gradient). This was accomplished by utilizing the least-squares method described by
0 1985 American Chemical Society 0003-2700/85/0357-1472$01.50/0
