Anal. Chem. 1988, 6 0 , 1498-1502
1498
of target preparation, other types of compounds, and other bombarding energies.
CONCLUSION The desorption yield for molecular ions from peptides decreases more rapidly as a function of molecular weight for 8-keV Cs+ ion bombardment than for 252Cffission fragment bombardment. Absolute yields were not determined, but normalized to leucine-enkephalin (556 u) the measured yield for ACTH (1-24) (2933 u) is about 10 times lower for kiloelectronvolt ion bombardment than for fission fragment bombardment; when normalized to leucine enkephalin, the yield for bovine insulin (5733 u) is at least 80 times lower. Also the signal-to-background ratio for the quasi-molecular ion peaks falls off more quickly with increasing mass for lower energy bombardment than for fission fragment bombardment. The results suggest a higher upper mass limit for high-energy (- 100 MeV) particle bombardment than for kiloelectronvolt particle bombardment. Note Added in Proof. Quasi-molecular ions with m / z 34 000 have recently been observed with fission fragment bombardment (15). Molecular ions in the same mass range have also been observed recently from photon bombardment (16). Registry No. ACTH (1-lo), 2791-05-1; ACTH (1-24), 16960-16-0;leucine-enkephalin, 58822-25-6;substance P, 3350763-0; a-endorphin, 61512-76-3; @-endorphin,60617-12-1; renin tetradecapeptide substrate, 64315-16-8; dynorphin, 74913-18-1; bovine insulin, 11070-73-8.
-
LITERATURE CITED (1) Macfarlane, R. D.; Torgerson, D. F. Science (Washington, D.C.) 1978. 191, 81. (2) Benninghoven, A.; Sichtermann, W. Anal. Chem. 1978, 50, 1180.
(3) Ens, W.; Standing, K. G.; Chait, B. T.; Field, F. H. Anal. Chem. 1981, 53, 1241. (4) McNeal, C. J.; Macfarlane, R . D.; Thurston, E. L. Anal. Chem. 1979, 5 , 2038. (5) Chait, B. T.; Standing, K. G. Int. J. Mass Spectrom. I o n Phys. 1981, 40, 185. (6) Chalt, B. T.; Aoosta, W. C.; Field, F. H. Int. J. Mass SDectrom. Ion Phys. 1981. 39, 339. (7) Beuhler, R. J.; Friedman, L. Nucl. Instrum. Methods 1980, 170, 309. (8) Chait, B. T.; Field, F. H. Int. J. Mass Spectrom. I o n Processes 1985, 65,169. (9) Ens, W.; Beavis, R.; Bolbach, G.; Main, D. E.;Schueler, 6.; Standing, K. G. I n Secondary I o n Mess Spectrometry SIMS V ; Benninghoven, A., Colton, R. J., Simons, D. S.,Werner, H. W., Eds.; Springer-Veriag: Berlin, 1986; p 185. (10) Hedin, A,; Hakansson, P.; Sundqvist, B. I n t . J. Mass Spectrom. Ion Processes 1987, 77, 123. (1 1) Ens, W.; Hakansson, P.; Sundqvist, B. U. R. Presented at the 6th International Conference on Secondary Ion Mass Spectrometry SIMS VI, proceedings in press. (12) Johnsson, G. P.; Hedin, A.; Mkansson, P.; Sundqvist, 6.; Save, G.; Nieisen, P.; Roepstorff, P.; Johansson, K. E.; Kamensky, I.; Lindberg, M. Anal. Chem. 1988, 58, 1084. (13) Kamensky, I.; Hakansson, P.; Sundqvist, 6.; McNeal, C. J.; Macfarlane, R. D., Nucl. Instrum. Methods 1982, 198, 65. (14) Lafortune, F.; Beavis, R.; Tang, X.; Standing, K. G.; Chait, B. T. Rapid Commun. Mess Spectrom. 1987, 1 114. (15) Craig, A. 0.; Engstrom, A.; Bennich, H.; Kamensky, I. Presented at the 35th ASMS Conference on Mass Spectrometry and Allied Topics, Denver, CO, May 24-29, 1987. (16) Tanaka, K.; Ido, Y.; Akita, S.;Yoshida, Y.; Yoshida, T. 2nd JapanChina Joint Symposium on Mass Spectrometry, Osaka, Japan, Sept. 1987. I
RECEIVED for review May 19, 1987. Resubmitted February 22,1988. Accepted March 11,1988. The Rockefeller portion of this work was supported in part by a grant from the U S . National Institutes of Health, Division of Research Resources. The Manitoba portion was supported by grants from the U.S. National Institutes of Health, Institute of General Medical Sciences (GM 30605-05), and from the Natural Sciences and Engineering Research Council of Canada.
A Pyrolysis-Mass Spectrometry Investigation of Pectin Methylation Rupert E. Aries,*?'Colin S. Gutteridge, and William A. Laurie Cadbury-Schweppes plc Group Research, The Lord Zuckerman Research Centre, The University, Whiteknights, P.O. Box 234, Reading RG6 2LA, United Kingdom Jaap J. Boon and Gert B. Eijkel FOM-Institute for Atomic and Molecular Physics, Kruislaan 407, 1089 SJ Amsterdam, The Netherlands Pyrolysis-mass spectrometry (Py-MS), In conjuctlon wlth multivariate data h a d l n g procedures, was Investigated as a potentlal method for the rapld determlnatlon of the degree of methylatlon (DM) In pectln. Good dlscrlmlnatlon between pectins of various DM was achleved. Masses m / z 85 and 98 were ldentlfled as belng slgnlflcantly Important to the dlscrhlnatlon. Factor analysls and pyrdysle-gas chromatography-mass spectrometry (Py-GC-MS) were employed to study the origin of masses m / z 85 and 96. Based upon these flndlngs pyrolysls mechanlsms for galacturonic acid and methylated galacturonlc subunlts wlthln pectin are proposed.
Pectins are a major group of heterogeneous polysaccharides that are of considerable importance to the food industry as gelling and thickening agents ( I ) . Pectin, as illustrated in Present address: Perkin-Elmer, Ltd., Post Office Lane, Beaconsfield, Bucks HP9 lQA, United Kingdom. 0003-2700/88/0360-1498$01.50/0
Figure 1,consists predominately of a-1,4 linked D-gdaCtWOniC acid units with varying degrees of methylation. Neutral sugars such as galactose and arabinose are associated as side chains and rhamnose units are dispersed within the polygalacturonic acid backbone ( I , 2). The ability of pectin to gel depends largely on the degree of methylation (DM). Determination of DM is normally achieved by analysis (titration) of the carboxyl groups before and after hydrolysis ( I ) and/or gas chromatographic analysis of methanol released on hydrolysis (3, 4). The degree of methylation has also been estimated by the ratio of carboxymethyl to carboxyl resonances using 13C NMR (5). All these techniques require extraction/isolation of the pectin prior to analysis necessitating large quantities of sample and lengthy preparation/analysis times. Pyrolysis is an analytical technique well suited to the analysis of nonvolatile materials such as pectins. The technique requires minimal sample and little or no sample preparation and is readily combined with separation techniques 0 1988 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 60, NO. 15, AUGUST 1, 1988
\
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Flgure 1. Illustration of pectin structure, adapted from Nelson et al., ref 1.
such as gas chromatography and/or mass spectrometry. Despite these attractions, few studies of the pyrolysis of pectins have been reported in the literature. Zamorani et al. (6) indicate that DM and possibly other structural information may be obtained from pyrolysis gas chromatograms. This work has recently been revived by Barford e t al. (7) who developed a multiparameter model for determining DM. Little assignment of the chromatographic peaks employed in the determination is given in either study although both correlate DM with methanol evolution. Barford et al. (7) also negatively correlate DM with formaldehyde evolution. This minimal assignment is probably due to the lack of suitable standards and/or instrumentation necessary to identify the pyrolysis products. Direct pyrolysis-mass spectrometry (Py-MS) displays a number of advantages over pyrolysis-gas chromatography (Py-GC) including speed of analysis (5 min vs 1h), improved long-term stability (no column to degrade), and the ease of presenting the data for computer analysis (Aries et al., 1985). In addition, chemical interpretation of the differences between spectra may be achieved by application of factor analysis techniques. In this investigation, the pyrolysis of pectins with varying DM was studied by Curie-point pyrolysis-mass spectrometry and Curie-point pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS) in an attempt to provide a rapid method for determining DM and an understanding of the pyrolysis chemistry of pectin.
EXPERIMENTAL SECTION Pyrolysis-Mass Spectrometry. Pyrolysis-mass spectrometry (Py-MS) was performed on two instruments; a Pyromass 8-80 (VG Gas Analysis, Middlewich, U.K.) and a commercial prototype (Prutec PYMS System; Prutec, London, U.K.). Both instruments have been described in detail elsewhere by Shute et al. (8)and Aries et al. (9),respectively. The two systems differ primarily in that the Prutec PYMS system is based on a quadrupole mass analyzer (m/z 51-140) and employs a Curiepoint foil (temperature rise time, 0.4s to 510 "C) as the sample carrier while the Pyromass 8-80 has a magnetic mass analyzer (mlz 12-300) and uses Curie-point wires (temperature rise time, 0.2 s to 510 "C). The Prutec PYMS system also has the advantage of an automated sample inlet. Both systems were operated with an electron energy of 20 eV. A critical evaluation of the two instruments has been carried out by Aries et al. (9). Seven citrus pectins of varying DM (kindly donated by H.P. Bulmers, Ltd., Hereford, U.K.) were gelled in distilled water. Quantities (ca. 20 fig)were smeared onto both 510 "C Curie-point foils and wires and vacuum dried for 30 min. Four replicates of each pectin were analyzed by using a 2-s pyrolysis time.
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Data were transferred from floppy diskettes to an IBM main-frame computer, either as normalized (PYMS system) or raw (Pyromass 8-80) mass intensities. Data were analyzed by using a batch GENSTAT program which contains routines for normalization, principal components analysis (PCA), and canonical variates analysis (CVA) and a type of "factor analysis" for interpretation of the chemical differences between the spectra. The GENSTAT analysis is essentially that described by Windig et al. (lo),in which PCA is used primarily as a data reduction technique and CVA is applied to some or all of the principal components with nonzero variances in an attempt to discriminate between the groups of sample replicates. The "factor analysis" is a technique based on the work of Windig et al. (11,lZ)and has been described in detail elsewhere (13). Pyrolysis-Gas Chromatography-Mass Spectrometry. Pyrolysis-gas chromatography-mass spectrometry was carried out at the FOM Institute with a FOM 3LX Curie-point pyrolyzer mounted on a Packard 4388 gas chromatograph interfaced to a Jeol DX 300 mass spectrometer. The system has been described in detail elsewhere (14). Chromatography was carried out on a 50-m fused silica thick film 1pm CPSil5 column (0.32 mm id.) employing a temperature gradient (4 "C min-') between 30 and 300 "C. Pectin samples of high (ca. 81%) and low (ca. 18%)DM were smeared onto 510 "C Curie-point wires and dried as for Py-MS. Individual samples were analyzed by using a 4-spyrolysis time. Both electron impact (EI, 70 eV) and chemical ionization (CI, reagent gas, isobutane) mass spectrometry were carried out.
RESULTS AND DISCUSSION Pyrolysis mass spectra of the seven citrus pectins analyzed on the Prutec PYMS system (m/z 51-140) are given in Figure 2. Changes in the relative intensities of the mass groups centered a t mlz 85 and 96 with decreasing DM are evident. At high DM, m / z 85 dominates, while a t low DM mlz 96 is the more intense mass. Application of PCA to the data resulted in a linear relationship between DM and the first principal component as evidenced by a plot of the first two principal components (98% variance) given in Figure 3a. Examination of the principal component loadings (Figure 3b) reveals that this analysis is dominated by two pairs of ions, m / z 95 and 96 and mlz 85 and 86. Clearly these masses are important in the pyrolysis of pectin. Masses mlz 95 and 96 may be attributed to 2-furaldehyde (molecular weight 96), a major pyrolysis product of pectin previously identified by Ohnishi et al. (15). The origin of mass mlz 85 was unknown, although it had been observed in other Py-MS studies (16)and this prompted Py-GC-MS investigation. Figure 4 shows mass chromatograms (total ion current (TIC), m/z 96 and m / z 85) obtained for pectins of high (Figure
ANALYTICAL CHEMISTRY, VOL. 60, NO. 15, AUGUST 1, 1988
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The corresponding low DM mlz 85 chromatogram shows little of the high DM lactone but contains an additional, unidentified mlz 85 component. The low DM m / z 96 chromatogram contains one major peak which was confirmed by its full mass spectrum as 2-furaldehyde. The corresponding high DM mlz 96 chromatogram contains a considerably smaller 2-furaldehyde peak. A pyrolysis mechanism for the production of 2-furaldehyde from galacturonic acid subunits within the pectin is given in Figure 6a. Formation proceeds via decarboxylation of the free acid at C-6, cleavage of the glycosidic bond a t C-1 and two successive dehydrations. The precise sequence cannot be predicted, but the proposed route is consistent with the known dehydration chemistry of galacturonic acid (17). A possible pyrolysis mechanism for the generation of the hydroxymethyllactone from methylated galacturonic acid subunits within the pectin is given in Figure 6b. Production of the lactone proceeds via elimination of methanol and lactone formation, cleavage of the glycosidic bond at C-5, and elimination of carbon dioxide from C-1. This pyrolytic evolution of carbon dioxide from C-1 has been reported elsewhere (18, 19). The mechanism is similar to that proposed by Ohnishi et al. (20,21) for the formation of 3-hydroxy-2-penteno-1,5lactone from xylan. No logical pyrolysis mechanism for the formation of methoxy lactone from methylated galacturonic acid subunits within the pectin has been proposed. This suggests the origin of the mlz 85 is 4-(hydroxymethyl)-l,4butyrolactone (I),although synthesis is required to confirm the hypothesis. Analysis of the seven citrus pectins on the Pyromass 8-80 (mlz 12-300) and subsequent PCICVA revealed a linear relationship (F = 0.99) between DM and the first canonical variate scores (Table I). Construction of the factor spectrum (Figure 7) across CV1 allows chemical interpretation of the relationship. The high DM factor spectrum contains only four major contributing masses. Masses m / z 31 and 32 are attributed to methanol while m/z 85 is attributed to the lactone ion (111). Mass m / z 31 may also be due to loss of hydroxy-
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Flgure 4. Mass chromatograms(TIC, m l r 96, m l r 85) of pectin: (a) high methoxypectln (DM 81%); (b) low methoxypectin (DM 18%).
4a) and low (Figure 4b) DM, respectively. The high DM m / z 85 chromatogram contains one major, broad peak, which provided the full E1 mass spectrum given in Figure 5. This
ANALYTICAL CHEMISTRY, VOL. 60, NO. 15, AUGUST 1, 1988 a
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Table 1. Correlation between DM and the First Canonical Variate Scores for the Seven Citrus Pectins Analyzed on the Pyromass 8-80 pectin DM
CV1 score
pectin DM
CV1 score
18 33 36 49
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61 68 81
6.91 7.01 13.34
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methyl from the lactone (I). The origin of m / z 97 is not known. The low DM factor spectrum contains five major masses and is essentially the same as the mass spectrum of 2-furaldehyde. Masses m/z 96,95, and 39 are attributed to the 2-furaldehyde molecular ion, the deprotonated species, and cyclopropene respectively. Mass m / z 29 is due to loss of the aldehyde functions with subsequent loss of a proton resulting in carbon monoxide (mlz 28). The origin of m / z
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Flgure 8. Determination of the degree of methylation (DM), plot of nominal DM vs estimated DM (DM = ( m l z 85)l(m/z85 96)). Data are mean of four replicates. r = 0.955.
+
71 is unknown. Chemical interpretation of the observed relationship between DM and the first canonical variate scores supports the proposed pyrolysis products for methylated galacturonic acid and galacturonic acid subunits within pectin. The absence of carbon dioxide (mlz 44) in the fador spectrum suggests that decarboxylation occurs in both pyrolysis mechanisms as proposed. The analysis also suggests that some electron impact fragmentation occurs despite employing low (ca. 16 eV) electron energies. Since the formation of 2-furaldehyde and 4-(hydroxymethyl)-l,Cbutyrolactoneare specific to the galacturonic acid and methylated galacturonic acid subunits within the pectin, respectively, a simple combination of masses m / z 85 and 96 may provide an estimate of DM. A plot of the ratio of the ion count for m / z 85 to the sum of the ion counts for mlz 85 and 96 (i.e. m / z 8 5 / x m / z 85 and 96) versus DM resulted
1502
ANALYTICAL CHEMISTRY, VOL. 60, NO. 15, AUGUST 1, 1988
in a linear relationship ( r = 0.955), as shown in Figure 8. However, the plot does not intersect the origin as would be expected if the samples were solely composed of galacturonic acid and methylated galacturonic acid subunits. A t high DM the estimated and “true” values agree well but as DM decreases the plot diverges and the error between the values increases. Since the divergence results in a positive error, this indicates an m/z 85 “impurity” in the pectin preparation. This impurity is probably the additional, unidentified m / z 85 component observed in the low DM mass chromatogram (Figure 4b). The PCA (Figure 3) suggests that the low DM pectin used for Py-GC-MS (DM ca. 18%) is atypical (sample revealed as outlier) supporting the hypothesis of an impurity in the sample. The principal component variate loadings (Figure 3b) also indicate that this impurity is associated with m/z 85 and 86. In hindsight a better choice of low DM pectin might have been pectin 2 (DM ca. 33%) since the estimated and “true” DM values agree well. In Py-MS any mass may have multiple origin, that is, be derived from a number of sources, which prevents the accurate determination of DM if impure samples are analyzed. In addition to positive “impurity” errors, negative errors may also result. 2-Furaldehyde is formed by a large number of carbohydrates on pyrolysis and hence m / z 96 may contain contributions from sources other than the galacturonic subunits within the sample. Indeed, a small negative error might have been expected since pectin contains other sugars in its polysaccharide backbone. Any fragmentation caused by the mass spectrometer would also affect the analysis. Nonspecific m898 detection is a limitation of Py-MS if single ion comparisons are used. However, if multivariate data handling techniques are employed, correlated masses are identified, leading to a better estimate of the sample DM. Comparison of the regression coefficients obtained by single ion (r = 0.955, Figure 8) and multivariate ( r = 0.990, Table I) techniques clearly shows the benefit of using a number of correlated parameters in the determination of DM. This work confirms the multiparameter approach employed by Barford et al. ( 7 ) for determining DM in pectin by using Py-GC. In this study, all the major parameters (masses) contributing to the analysis may be attributed to three compounds (methanol, 2-furaldehyde, and butyrolactone). Barford et al. ( 7 ) also used three parameters (peaks) in their DM determination but only conclusively identified methanol as one of the parameters. Their tentatively identified negative correlation of DM with formaldehyde evolution is difficult to explain on the basis of the proposed pyrolysis mechanisms of pectin unless formaldehyde is derived from secondary reactions of 2-furaldehyde under the conditions employed. Further work with a model system of characterized pectins is required to confirm the proposed pyrolysis mechanisms and overcome the problem of impurities. This model system might be prepared by either controlled enzymic treatment of a high DM pectin or stepwise methylation of polygalacturonic acid.
The latter is probably to be preferred since pectinesterases generally act in a blockwise manner which tends to produce a wide product distribution. Furthermore, they are not easily employed unless the reaction conditions (salt concentration, pH, etc.) are carefully controlled. Further work is also required to assess the potential of pyrolysis for other structural elucidation of pectins, such as the degree of polymerization within the polysaccharide backbone. In this case, enzymes may prove very useful in the preparation of different samples for analysis although removal of salts, etc. prior to pyrolysis may prove a problem unless chelating agents and/or ultrafiltration is employed.
ACKNOWLEDGMENT The authors thank H.P. Bulmers, Ltd., for provision of the pectin samples, h m a r i e Anderson for running the Pyromass 8-80, and David McHale, Roger Evans, and John Sheridan for helpful discussions. Registry No. High methoxypectin, 65546-99-8; low methoxypectin, 9049-34-7.
LITERATURE CITED Nelson, D. B.; Smtt, C. J. B.; Wiles, R. R. In FocdColio~s;Graham, H. D., Ed.; AVI Publishing: Westport, CT, 1977. Rouau, X.; Thibautt, J. F. Carbohydr. Polym. 1984, 4 , 111-125. Walter, R. J.; Sherman, R. M.; Lee, C. Y. J . Food Sci. 1983. 4 8 , 1006- 1007. McFeeters, R. F.; Armstrong, S. A. Anal. Biochem. 1984, 139, 212-217. Fishman, M. L.; Pfetter, P. E., Barford, R. A.; Doner, L. W. J . Agric. Food Chem. 1984, 32, 372-378. Zamorani, A.; Rcda, 0.;Lanzarini, G. Indusbje Agric. 1971, 9 , 35-41. Barford, R. A.; Magidman, P.; Phillips, J. G.; Fishman, M. L. Anal. Chem. 1966, 58, 2576-2570. Shute, L. A.; Gutteridge, C. S.;Norris, J. R.; Berkeley, R. C. W. J . Gen. Microbioi. 1984, 130, 343-355. Arbs. R. E.; Gutteridge, C. S.; Ottley, T. W. J . Anal. Appl. Pyrol. 1986, 9 . 01-90. Windig, W.; Haverkamp, J.; Klstemaker, P. G. Anal. Chem. 1983, 55, 387-391. Windig, W.; Kistemaker, P. G.; Haverkamp, J. J . Anal. Appl. Fyrol. 1982. 3 . 199-212. Windig, W.; de Hoog, G. S.; Haverkamp, J. J . Anal. Appl. Fyrol. 1982, 3 , 213-220. Aries, R. E.; Gutteridge, C. S.; Macrae, R. J . Chromatow. 1985, 319, 205-287. Boon, J. J.; Pouwels, A. D.; Eijkel, G. 8. Biochem. SOC. Trans. 1987, 15, 170-174. Ohnishi, A.; Tagaka, E.; Kato, K. Carbohydr. Res. 1978, 6 7 , 281-280. van der Vaik, F.; Boon, J. J.; Hertley, R. D. In Advances in Mass Spectrometfy; Todd, J. F. J., Ed.; Wiley: Chlchester, 1986; pp 655-656. Feather, M. S.;Harris, J. F. Adv. Carbohydr. Res. 1973, 2 8 , 16 1-224. Shafizadeh, F.; La, Y. 2. J . Org. Chem. 1972, 3 7 , 278-284. Shafizadeh, F.; Lia, Y. 2. Carbohydr. Res. 1975, 42, 39-53. Ohnishi, A.; Kato, K.; Tagaki, E. Carbohydr. Res. 1967, 50, 275-276. Ohnishi, A.; Kato, K.; Tagaki, E. Carbohydr.Res. 1977, 58, 387-395.
RECEIVED for review July 13, 1987. Resubmitted February 18,1988. Accepted March 4,1988. R.E.A. is in receipt of a research contract from the Ministry of Agriculture, Fisheries and Food (No. CSA 829).
