Anal. Chem. 1996, 68, 3244-3249
Positional Isomer Differentiation of Monoalkylated Naphthalenes Using Principal Components Analysis and Mass Spectrometry Dorothy Swain,*,†,‡,§ Randall E. Winans,† and William J. Dunn, III‡
Division of Chemistry, Argonne National Laboratory, Argonne, Illinois 60439, and Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, Illinois 60612
Tandem mass spectrometry has been used to differentiate positional isomers of some monoalkylated naphthalenes. The basis of the distinction is the cluster of peaks from m/z 150 to 155. Principal components analysis can extract isomer-specific information that is not obvious from simple visual inspection of the spectra. The analysis led to the observation of similar trends in single-stage electron impact mass spectra. Aromatic positional isomer differentiation has long been recognized as a difficult challenge to mass spectrometrists. White and Bursey described the effort as “Sisyphean”.1 Some of the efforts in the area are described in the literature on the mass spectrometry of polychlorinated biphenyls,1,2 alkylbenzenes,3-8 and other substituted benzenes.9-12 There are a number of problems related to alkylated aromatics where isomer information can be important and is difficult to obtain.13 For example, the carcinogenicity of these compounds is strongly dependent on the structure of the ring system and the nature of the substitution pattern. Isomers found in complex mixtures derived from coal or petroleum will behave differently in chemical and thermal reactions. Even for a small aromatic compound, such as naphthalene, synthesizing different alkyl substituents of various carbon numbers and isomers would be very time-consuming and expensive. It would be useful to have tools for extracting isomer information from mass spectra and tandem mass spectra of model systems. In general, it is possible to separate aromatic positional isomers with an appropriate chromatography system due to their different †
shapes and physical properties, and infrared spectrometry is known for its ability to distinguish between positional isomers. We believe that the unique features of mass spectrometry (its sensitivity, its speed of data acquisition, and its high information content) make the exploration of positional isomer differentiation by mass spectrometry a worthwhile problem. For heteroaromatic systems (e.g., the n-pentadecylpyridines) positional isomer differentiation can be obtained by single-stage electron-impact mass spectrometry.14 The heteroatom in pyridine breaks the symmetry of the aromatic system and tends to localize the positive charge on the less electronegative atoms. The asymmetric charge distribution has the potential to yield different fragmentation patterns in different positional isomers. For aromatic molecules containing only carbon and hydrogen no such asymmetry exists, and the general problem of positional isomer differentiation of alkylated aromatic hydrocarbons (CxHy) has not been adequately solved and tested in the literature. Principal components analysis (PCA) is a multivariate technique15,16 which has been used since the 1970s in the analysis of mass spectra.3-6 This technique compresses large data matrices into smaller model matrices with dimensionality reduced according to the number of principal components in the model. If the dimensionality is reduced to three components or less, a set of mass spectra can be viewed as points in three-dimensional component space. The components represent the mutually orthogonal directions of maximum variation in the data and are composed of linear combinations of the variables (m/z values) that constitute the original mass spectra. The main advantage to this approach is that it allows rapid and systematic visualization of correlations between peaks, thus allowing the extraction of informative trends not apparent on simple visual inspection of one variable at a time. We report here a technique based on tandem mass spectrometry which results in positional isomer differentiation of 1- and 2-monoalkylated naphthalenes. The isomer-specific information is not apparent on cursory visual inspection of the data but it shows up clearly and reproducibly on transformation of the data by PCA. The technique relies on the fragmentation of the peak at m/z 155 (common to all alkylated naphthalenes higher than methyl) in the electron-impact mass spectrum of each compound.
Argonne National Laboratory. University of Illinois. § Present address: Linfield College, Portland, OR 97210. (1) White, E. L.; Bursey, M. M. Biomed. Environ. Mass Spectrom. 1989, 28, 413-5. (2) Guevremont, R.; Yost, R. A.; Jamieson, W. D. Biomed. Environ. Mass Spectrom. 1987, 14, 435-41. (3) Rozett, R. W.; Petersen, E. M. Anal. Chem. 1975, 47, 1301-8. (4) Rozett, R. W.; Petersen, E. M. Anal. Chem. 1975, 47, 2377-84. (5) Rozett, R. W.; Petersen, E. M. Anal. Chem. 1976, 48, 817-25. (6) Rozett, R. W.; Petersen, E. M. Am. Lab. 1977, 2, 107-16. (7) Curtis, J. M.; Boyd, R. K.; Shushan, B.; Morgan, T. G.; Beynon, J. H. Org. Mass Spectrom. 1984, 19, 207-16. (8) Hawthorne, S. B.; Miller, D. J. Anal. Chem. 1985, 57, 694-8. (9) Riley, J. S.; Baer, T.; Marbury, G. D. J. Am. Soc. Mass Spectrom. 1991, 2, 69-75. (10) Donovan, T.; Brodbelt, J. Org. Mass Spectrom. 1992, 27, 9-16. (11) Yinon, J. Org. Mass Spectrom. 1992, 27, 689-94. (12) Yinon, J. Rapid Commun. Mass Spectrom. 1993, 7, 67-70. (13) Harvey, R. G. Polycyclic Aromatic Hydrocarbons: Chemistry and Carcinogenicity; Cambridge University Press: Cambridge, England, 1991.
(14) Hardy, D. R.; Mushrush, G. W.; Stalick, W. M.; Beal, E. J.; Hazlett, R. N. Rapid Comm. Mass Spectrom. 1988, 2, 16-8. (15) Wold, S.; Esbensen, K.; Geladi, P. Chemom. Intell. Lab. Syst. 1987, 2, 3752. (16) Meglen, R. R. J. Chemom. 1991, 5, 163-79.
3244 Analytical Chemistry, Vol. 68, No. 18, September 15, 1996
S0003-2700(96)00011-X CCC: $12.00
‡
© 1996 American Chemical Society
MATERIALS AND METHODS Mass spectra were collected using a Kratos MS-50 TC threesector (EBE) tandem mass spectrometer,17 modified with a fastscan magnet and a PAD collector. 1-Ethylnaphthalene, 2-ethylnaphthalene, 1-tert-butylnaphthalene, 2-tert-butylnaphthalene, and 2-n-butylnaphthalene were obtained commercially and used without further purification. Metastable spectra,18 based on first field-free region dissociation, were collected by utilizing a linked scan at constant B/E in MS1 and leaving E2 at a fixed voltage. Fragments of the parent ion at m/z 155, 156, or 157 were collected for the ethylnaphthalenes. For the butylnaphthalenes, parents at m/z 184 or 185 were used. Collisionally activated dissociation (CAD) spectra,18 based on third field-free region dissociation, were collected in the presence of helium gas under conditions of 50% transmission, by scanning the third sector (E2) after mass selection in MS1 (E1-B). Fragments of the parent ion at m/z 156 were collected for the ethylnaphthalenes. In all cases, unit mass resolution or better was obtained in both MS1 and MS2. The data analysis was restricted to the six peaks from m/z 150 to 155, and the data were normalized so that 100% corresponded to the base peak within that cluster. Data processing was performed on a SUN computer dedicated to the operation of the Kratos instrument. Additional processing for the CAD data was performed using programs written in Mathematica. The UNIPALS software19,20 was used for PCA. Mathematica and UNIPALS computations were carried out on a Gateway-486 personal computer. RESULTS AND DISCUSSION Representative electron-impact mass spectra (MS1) for ethylnaphthalene, n-butylnaphthalene, and tert-butylnaphthalene are shown in Figure 1. These three classes of alkylnaphthalenes are readily distinguished on the basis of their MS1 spectra. However, there were no differences between positional isomers observed at this stage of the analysis. Likewise, the full metastable (MS2) spectra were not useful for differentiating positional isomers. The problem lay largely in the lack of quantitative reproducibility of peak areas. The problem is represented in Figure 2 , which shows replicate spectra for 1-ethylnaphthalene and 2-ethylnaphthalene collected roughly three months apart. The variability in the identity of the base peak (at either m/z 156 or 141) is the dominant feature of the spectra, and it precludes other useful information from being extracted. Seeking a more reproducible feature of the spectra, the cluster of peaks from m/z 150 to 155 was extracted and normalized to the base peak within the cluster. The resulting spectra from representative acquisitions of 1- and 2-ethylnaphthalenes are shown in Figure 3. The base peak remains constant at m/z 152 for all such ethylnaphthalene spectra obtained. However, there were no consistent visual differences between positional isomers. (17) Gross, M. L.; Chess, E. K.; Lyon, P. A.; Crow, F. W.; Evans, S.; Tudge, H. Int. J. Mass Spectrom. Ion Phys. 1982, 42, 243-54. (18) Busch, K. L.; Glish, G. L.; McLuckey, S. A. Mass Spectrometry/Mass Spectrometry: Techniques and Applications of Tandem Mass Spectrometry; VCH Publishers: New York, 1988. (19) Glen, W. G.; Dunn, W. J., III; Scott, D. R. Tetrahedron Comput. Methods 1989, 2, 349-76. (20) Glen, W. G.; Sarker, M.; Dunn, W. J., III; Scott, D. R. Tetrahedron Comput. Methods 1989, 2, 377-96.
Figure 1. Single-stage electron impact mass spectra of ethylnaphthalene, n-butylnaphthalene and tert-butylnaphthalene.
Figure 4 shows a comparison of peak areas within the cluster for 1- and 2-ethylnaphthalene replicate spectra taken over a fourmonth period which illustrates both the consistency in the identity of the base peak and the difficulty in visually extracting positional isomer-specific data from the spectra. The results from PCA of these spectra are shown in Figure 5. A three-component model was derived from a 13 × 6 matrix in which the 6 columns represent m/z values 150-155 and the 13 rows represent the replicate spectra used for modeling purposes (six 1-ethylnaphthalene replicates and seven 2-ethylnaphthalene replicates, represented as filled and open squares, respectively in Figure 5). Figure 5 represents a plot of the scores from all three components, as seen from the perspective of a 100° rotation around component 2 (pc2). This perspective gave the best, and most consistent, visual isomer differentiation in component space, as seen in the clustering of all of the 1-isomer scores to the right of the plot and all of the 2-isomer scores to the left of the plot. We could draw a vertical decision line just to the right of the y-axis and use it to classify an ethylnaphthalene mass spectrum as arising from either the 1-isomer or the 2-isomer. Figure 5 also includes projected scores from spectra not included in the model. Filled and open diamonds represent test metastable spectra of 1- and 2-ethylnaphthalenes, respectively, whereas filled and open circles represent CAD spectra of 1- and 2-ethylnaphthalenes. The ability of the model to cluster the spectra according to positional isomer class across two different types of experiment (first FFR metastable and third FFR CAD) Analytical Chemistry, Vol. 68, No. 18, September 15, 1996
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Figure 2. Replicate metastable fragmentation spectra of the parent at m/z 157 for 1- and 2-ethylnaphthalenes.
Figure 4. Variation in areas for each of the 6 peaks from m/z 150 to 155 for 9 replicate 1-ethylnaphthalene and 11 replicate 2-ethylnaphthalene spectra recorded between September 1993 and January 1994. Dark circles represent the average intensity value for a given peak, and horizontal lines delineate the error bars as defined by the high and low observed intensity value for each peak.
Figure 3. Metastable fragmentation spectra of the parent at m/z 155 for 1- and 2-ethylnaphthalene, showing the cluster of renormalized peaks from m/z 150 to 155 only.
demonstrates the generality of the mass spectral features extracted by the model. The accurate projection of the CAD spectra onto the metastable model is particularly noteworthy and deserves some emphasis. The CAD clusters were clearly visually different from the metastable clusters, most notably in containing only four peaks rather than all six: m/z 150 was consistently missing from the CAD spectra, as was either 151 or 152 depending on the specific acquisition. Also, the m/z 155 peak was always the base peak in the CAD data whereas the m/z 152 peak was always the base peak in the metastable data. In spite of these differences, however, the CAD data do separate accurately on the basis of positional isomer in Figure 5. Principal components analysis was also performed on metastable daughter spectra of m/z 155 for some butylnaphthalenes: 3246 Analytical Chemistry, Vol. 68, No. 18, September 15, 1996
1-tert-butylnaphthalene, 2-tert-butylnaphthalene, and 2-n-butylnaphthalene. The m/z 155 peak appears consistently in butylnaphthalene mass spectra; it is 29 mass units lower than the molecular ion at m/z 184. The n-butyl isomer spectrum was clearly visually distinct from the tert-butyl isomers, but once again, positional isomer differences were not visually obvious. Here, as with the ethylnaphthalenes, a three-component model was derived based on a 5 × 6 matrix in which the six columns represent m/z values 150-155 and the five rows represent the replicate spectra used for modeling purposes (two replicates each of 1- and 2-tertbutylnaphthalenes and one 2-n-butylnaphthalene spectrum). Figure 6 represents a plot of the scores from all three components, as seen from the perspective of a 45° rotation around pc2. From this perspective, it can be seen that a combination of pc1 and pc3 separates n-butyl from tert-butyl isomers, whereas pc2 separates the positional isomers. Unfortunately, we did not have 1-n-butylnaphthalene available for analysis, but we can predict that those spectra would fall into the upper right-hand quadrant of Figure 6.
Figure 5. Scores from metastable and CAD spectra of 1- and 2-ethylnaphthalene projected onto a 3-pc model based on six 1-EN metastable replicates (filled squares) and seven 2-EN metastable replicates (open squares). Filled and open diamonds represent the projection of 1-EN and 2-EN metastable test spectra onto the model, and filled and open circles represent the projection of CAD spectra of 1-EN and 2-EN onto the model. From a starting point of pc2 vs pc1, the plot has been rotated 100 degrees around pc2 in order to show optimal separation based on all three components. The dotted line shows a decision line that could be used to distinguish a 1-EN from a 2-EN. The axes are scaled in arbitrary units.
Figure 6. Scores from 1-tert-butyl-, 2-tert-butyl-, and 2-n-butylnaphthalene metastable spectra projected onto a 3-pc model based on five of the spectra. The five spectra used to derive the model are represented by squares and circles, and the seven spectra projected onto the model are represented by diamonds and triangles. From a starting point of pc2 vs pc1, the plot has been rotated 45° around pc2 in order to show optimal separation based on all three components. The dotted lines show decision lines that could be used to distinguish a 1-BUN from a 2-BUN and a tert-BUN from a n-BUN. The axes are scaled in arbitrary units.
Now the question arises of whether the ethylnaphthalene and butylnaphthalene models exhibit reciprocity; i.e., can the butyl spectra be projected accurately onto the ethyl model, and vice versa? Figure 7 shows the 20 metastable ethylnaphthalene spectra (represented as triangles) of Figure 5 projected onto the metastable butylnaphthalene model of Figure 6 with the 1-isomers falling higher in pc2 than the 2-isomers for a given value in pc1. We could draw a diagonal decision line across the scores plot to get a good separation of the spectra by positional isomer. Figure 7 also shows projected scores from single-stage electron-impact mass spectra (MS1) of butylnaphthalenes obtained from the Wiley/NBS registry of mass spectral data. All of the replicate spectra in the database which had a recorded, nonzero intensity at each m/z value from 150 to 155 were analyzed and then treated as were the metastable and CAD data previously discussed. The data cluster accurately in terms of positional isomer information: higher in pc2 for 1-isomers than for 2-isomers.
Of particular note is the fact that we were able to obtain a 1-nbutylnaphthalene electron-impact spectrum from the database and that its projected spectrum fell onto the model close to what was predicted: with relatively high values in both pc1 and the combination of pc2 and pc3 although it did not land in the first quadrant. Figure 8 shows the 12 butylnaphthalene metastable spectra (represented as circles and diamonds) of Figure 6 projected onto the ethylnaphthalene model of Figure 5. Projections of MS1 data from the Wiley/NBS registry of mass spectral data are also displayed for the ethylnaphthalenes. With two exceptions (a 2-nbutylnaphthalene metastable spectrum and the 1-tert-butylnaphthalene MS1 spectrum), the 1-isomers fall to the right of the y-axis and the 2-isomers fall to the left. Figures 7 and 8 demonstrate that there is considerable generality in the isomer-specific differences found in the principal components models of the m/z 150-155 cluster of ethyl- and Analytical Chemistry, Vol. 68, No. 18, September 15, 1996
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Figure 7. Scores from ethylnaphthalene metastable spectra, as well as from Wiley/NBS database electron-impact spectra for butylnaphthalenes, projected onto the 3-pc metastable butylnaphthalenes model described for Figure 6. Squares and crosses represent the five spectra used to derive the model. Triangles represent the 20 ethylnaphthalene metastable spectra of Figure 5 projected onto that model. Diamonds and circles represent butylnaphthalene Wiley/NBS MS1 spectra projected onto the model. The plot has the same 45° rotation around pc2 described for Figure 6. The dotted line shows a decision line that could be used to distinguish a 1-isomer from a 2-isomer. The axes are scaled in arbitrary units.
Figure 8. Scores from butylnaphthalene metastable spectra, as well as from ethylnaphthalenes spectra in the Wiley/NBS database, projected onto the ethylnaphthalene metastable model described for Figure 5. Squares represent the 13 spectra used to derive the model. Triangles represent the 12 butylnaphthalene metastable spectra of Figure 6 projected onto that model. Circles and diamonds represent Wiley/NBS MS1 spectra of ethylnaphthalenes projected onto the model. The plot has the same 100° rotation around pc2 described for Figure 5. The dotted line shows a decision line that could be used to distinguish a 1-isomer from a 2-isomer. The axes are scaled in arbitrary units.
butylnaphthalene metastable spectra. The same features that are extracted from ethyl spectra can be found in butyl spectra, and vice versa. Moreover, the differences found in the metastable data can also be found in MS1 data, in spite of the fact that there are also some rather consistent spectral differences between data from the two types of scan (the m/z 155 peak is consistently larger in the MS1 spectra than in the metastable spectra, for example). Our attempts to extract isomer-specific information from visual inspection of the intensities, without subjecting them to PCA, were unsuccessful; also, when PCA was applied to either type of spectrum as a whole, rather than just the m/z 150-155 cluster, no positional isomer differentiation was obtained. CONCLUSIONS We have found that the m/z 150-155 cluster in several types of mass spectral scan of monoalkylated naphthalenes is uniquely informative as far as positional isomer differentiation is concerned. 3248 Analytical Chemistry, Vol. 68, No. 18, September 15, 1996
This should be a general phenomenon for monoalkylated naphthalenes since all should form the m/z 150-155 cluster of peaks in both MS1 and MS2 scans. We have studied ethyl- and butylnaphthalenes and have found consistency in our results between the two models. PCA was able to extract isomer-specific information from this cluster of peaks that was not at all obvious on visual inspection of the data, and long-term reproducibility has been established. FURTHER WORK We are interested in expanding on the work reported here in the following areas: (1) We would like to compare the results we obtained from the Wiley database to include mass spectral data from other compendia. (2) We would like to look at different (larger) aromatic ring systems and see whether similar results could be obtained.
(3) Eventually, we would like to explore the area of multicomponent mixtures to see whether mixtures of different isomers can be resolved using these techniques. ACKNOWLEDGMENT This work partially fulfilled the requirements of D.S. for the Ph.D. degree in Medicinal Chemistry at the University of Illinois at Chicago. This project was carried out at Argonne National Laboratory while D.S. was a Laboratory-Graduate Participant. The program is administered by the Argonne Division of Educational
Programs with funding from the U.S. Department of Energy. This work was performed under the auspices of the Office of Basic Energy Sciences, Division of Chemical Sciences, U.S. Department of Energy, under Contract W-31-109-ENG-38. Received for review January 4, 1996. Accepted June 7, 1996.X AC9600114 X
Abstract published in Advance ACS Abstracts, August 1, 1996.
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