Anal. Chem. 1907, 59, 197-200 (7) Heller, D. N.; Chen, T. S.; Hansen, G.; Fenseleau, C. I n Proceedlng of the 29th Conference on Mass Spectrometry and Allled Toplcs ; Mlnneapolls, MN, 1981; p 574. (8) Bowen, D. V.; Field, F. H. Anal. Chem. 1975, 47, 2289. (9) Dzidic, L.; DesMerlo, D. M.; Wilson, M. S.; Crain. P. F.; McCloskey, J. A. Anal. Chem. 1971, 43, 77. (10) Gross, M. L.; Chess, E. K.; Lyon, P. A.; Chow, F.; Evans, S.; Tudge, H. Int. J . Mass Spectrom. Ion Phys. 1982. 42, 243. (11) Taylor, L. C.; Evans, S.; Compson, K. R.; Ryan, P. I n Proceedlng of the 31th Conference on Mass Spectromeby and Allled Topics; Boston, MA, 1983; p 615. (12) Bone, W.; Marasco, J.; Doctor, B. P. I n Proceeding of the 30th Conference on Mass Spectrometry and Allled Toplcs; Honolulu, HI, 1982 p 632. (13) Biemann, K. I n Mass Spectrometry Organlc Chemical Appllcatlons; McGraw-Hill: New York, 1962, p 171.
197
(14) Klevan, M.; Munson, 0. Int. J . Mass Spectrom. Ion Phys. 1974, 13, 261. (15) Krause, J. R.; Potzinger, P. Int. J . Mass Spectrom. Ion phys. 1975, 18, 303. (18) Adlorne, T. J.; Harvey, D. J.; Bouros, P. J . Phys. Chem. 1972, 76, 3217. (17) Clemens, D.; Munson, B. Anal. Chem. 1985, 5 7 , 2022. (18) Orlov, V. Yu.; Narnetkin. N. S.; Gusel'Nikov, L. E.; Islamov, T. H. Org. Mass Spectrom. 1970, 4 , 195.
RECEIVED for review April 18,1976. Accepted August 6,1986. The authors gratefully acknowledge the National Science and Engineering Research Council of Canada for financial assistance (NSERC Grant A-1106).
Laser-Desorption Fourier Transform Mass Spectra of Polysaccharides Mildred L. Coates and Charles L. Wilkins* Department of Chemistry, University of California, Riverside, Riverside, California 92521
Mass spectra of nlne polysaccharides have been obtalned by use of laserdesorptlonFourier transform mass spectrometry. Extensive fragmentatbn of the saccharide chains, both wtthin the sugar rings and between them, Is observed. Although slmflarltles are seen in the spectra of m e compounds, each dlsplays a characteristic fragmentatlon pattern. The patterns are analogous to pyrolysls mass spectra of the same polysaccharides.
Polysaccharides, obtained from such biological sources as algae, yeasts, and bacteria, have long been used for a variety of commercial purposes (1, 2). Development of industrial applications has frequently preceded structural determination for newly isolated polysaccharides because of the complexities of the samples, generally obtained as mixtures, and of the determination procedures. Analysis has usually involved acid and/or enzymatic hydrolyses, followed by identification of the resulting monosaccharides. The use of mass spectrometry in this process has traditionally been confined to gas chromatography-mass spectrometric (GC-MS) determination of these monosaccharides following chemical derivatization (3,4). For some time, the only direct mass spectral alternative was pyrolysis mass spectrometry (5-9). However, recently developed desorption ionization methods have expanded the role of mass spectrometry in these applications (10-15). Laser-desorption Fourier transform mass spectrometry (LD-FTMS) of polymers, peptides, and other molecules of biological interest has been reported (16-21). Polysaccharides are good candidates for this technique due to their high molecular weights (2000 to >500 000) and low solubilities in neutral solvents. Earlier LD-FTMS studies of a series of malto-oligosaccharides revealed distinctive fragmentation patterns for those compounds (19). The present study was undertaken to examine the LD-FTMS behavior of polysaccharides for similarly distinct patterns which might have value for their structural characterization.
EXPERIMENTAL SECTION The operating principles of FTMS have previously been described in detail (22). The LD-FTMS system used here consists 0003-2700/87/0359-0197$01.50/0
of a Nicolet FT-1000 Fourier transform mass spectrometer, equipped with a 3-T superconducting magnet and a 5.08-cm cubic cell, interfaced to a Tachisto 215-G pulsed C 0 2 laser, with the tunable grating replaced by a reflector passing all COz emission lines. The laser desorption interface has been described previously (16). The laser pulse width used is 80 ns. Samples are prepared by grinding them together with KBr dopant and a f f i i g them to the tip of a direct introduction probe by means of double-sided adhesive tape. (Chitin resists grinding, so pieces are placed manually onto the tape.) The sample layer is perforated by the laser beam, although the tape itself is not. Ejection sweeps are used to remove from the cell ions coming from the tape, tape adhesive, and dopant. The vacuum system is allowed to return to -2 x IO4 torr following sample introduction before analysis. A 15-8 delay after the laser pulse allows most of the desorbed neutrals to be pumped away, while ions are retained in the cell by a trapping potential of 0.7 V. Signal averaging is made possible by turning the probe handle after each laser shot. For each mass spectrum reported here, 36 transients, each containing 64K data points, were co-added and augmented by one zero-fill. After base-line correction and apodization using a sine-bell function, a fast Fourier transform is performed to obtain a magnitude mode spectrum. The polysaccharides examined in this study are locust bean gum, white dextrin, dextran, cellulose, starch, xanthan gum, agarose, agar, and chitin. Locust beam gum was obtained from Sigma Chemical Co., starch from Mallinckrodt, xanthan gum from Kelco, Division of Merck & Co., Inc., and white dextrin, dextran, cellulose, agarose, agar, and chitin from Polysciences. Samples were used as received, without further purification.
RESULTS AND DISCUSSION The mass spectra obtained from KBr-doped samples of polysaccharides exhibit extensive fragmentation. The K+attachment ions result from fragmentations within the sugar rings and between the rings at the glycosidic linkages. Although ring fragmentations have not been reported generally by workers using other desorption techniques, similar fragmentations have been found with laser-desorption time-offlight (TOF) mass spectrometery (23). Comparison of fragmentation patterns of different hexose polymers reveals similarities, despite structural differences. The ions observed occur in series, with the ions in each series separated by 162 m u , corresponding to the mass of a single 0 1986 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 1, JANUARY 1987
MASS I N A M U
0
0
Figwe 1. LD-FT mass spectra of KBrdoped samples of (a)dextran, (b) cellulose, and (c) starch. Letters correspond to series designations given
in Table I. hexose ring. The general composition of these series can thus be described as [(162), + X K]+ or [(Hex), + X + K]+. Values for n, which correspond to the number of sugar rings in the saccharide chain, are 2-6 for locust bean gum and xanthan gum, 2-8 for white dextrin, cellulose, and starch, and 3-9 for dextran. X is an integer, corresponding to the mass of a ring fragment left on the saccharide chain after cleavage. Although the exact nature of these fragments cannot be determined without labeling studies, one can speculate that they result from simple cleavages across the ring, without extensive rearrangement. The ion series and their relative contributions to the spectra of different polyhexoses are summarized in Table I. Laserdesorption mass spectra of dextran, cellulose, and starch (Figure 1)demonstrate that subtle structural differences influence mass spectral fragmentation. These compounds are polyglucoses, differing structurally only in the way the glucose rings are linked together, yet the spectral differences are quite apparent. The mechanism of LD is not fully understood but is believed to be a combination of processes, some thermal in nature. If thermal processes are important, then one would expect that ions derived from thermal cleavages would dominate LD mass spectra and that such spectra would be similar to pyrolysis mass spectra. In fact, the masses of X in the ion series observed in the mass spectra of the polyhexoses do correspond to those masses of pyrolysis fragment ions (5-9). This suggests that, for these compounds, laser desorption proceeds by thermolysis of polysaccharide chains by the laser, followed by attachment of K+ ions to the neutral species arising from that process. Chitin is a naturally acetylated polyglucosamine. Because its mass spectrum (Figure 2) was obtained in the absence of dopant, H+-,Na+-, and K+-attached ions are all observed, the latter two apparently due to adventitious salts. As with the polyhexoses, the observed ions occur in series, the general composition of which is [(203), + X + C]', where 203 is the mass of the acetylated glucosamine unit, X is an integer, and C is H, Na, or K. Values of n are 2-8 for chitin.
+
300
400
500
600
700
MASS
IN
A M U
MASS
IN
A M U
800
900
1000
2 2
&
0
Flgure 2. LD-FT mass spectrum of an undoped sample of chitin. Letters correspond to series designations given in Table I, based upon 203 amu losses instead of 162 amu.
Fewer ion series are observed in the chitin spectrum than in the polyhexose spectra, specifically only those for which X = 0, 60, 74, and 185. The series for which X = 185 is equivalent to the polyhexose series for which X = 144 shifted in mass to reflect the acetylated amine substituent on carbon 2. The fact that the series for which X = 60 and 74 do not shift establishes that carbon 2 is not present in those fragments after cleavage across the sugar ring. Agarose is composed of chains of galactose rings alternating with 3,6-dehydrogalactose rings. Although agar consists of both agarose and agaropectin, the LD mass spectrum of agar (Figure 3) is similar to that of agarose. The major ions observed reflect the alternating sugar sequence and appear to arise from cleavages at the glycosidic linkages. Except for overall intensity, the LD-FT mass spectra of these samples do not change with laser power. Despite torr resulting from the laser pressure bursts of up to -5 X pulse, ion intensities are low. It is necessary to operate the laser at a high applied voltage and the maximum aperture and to signal average in order to achieve signal-to-noise levels sufficiently high to observe the lower intensity fragment ions.
ANALYTICAL CHEMISTRY, VOL. 59, NO. 1, JANUARY 1987
I
795
I
199
I
c Y
MASS I N A M U
n
Lr?
w
3 4
h
m
n
w
w
2
Lr?
m
Flguro 3. LD-FT mass spectrum of a KBrdoped sample of agar. Letters correspond to series designations given in Table I.
h
n
v
*
The heterogeneity of the sample surface and the laser power variation cause the amount of sample vaporized and ionized to vary from pulse to pulse. Thus, signal averaging also contributes to overall spectral reproducibility.
h
W
w
N cy
h
3. co h
m
w
b
CONCLUSION The LD-FT mass spectra of all the polysaccharides examined exhibit distinct fragmentation patterns. The variation between the fragmentation patterns of different polysaccharides suggests that these patterns could be useful in characterizing these compounds. The regularity of the ion series suggests they are governed by an orderly cleavage process. In order to establish detailed mechanisms necessary to extend these observations to determination of unknown polysaccharide structure, isotopic labeling studies and further analyses of model compounds must be completed. ACKNOWLEDGMENT M.L.C. thanks Annella J. Ford for helpful discussions. We are grateful to Kelco, Division of Merck & Co., Inc., for a
h
m
I
m
generous donation of xanthan gum. Registry NO. Locust bean gum, 9000-40-2; white dextrin, 9004-53-9; dextran, 9004-54-0; cellulose, 9004-34-6; starch, 900525-8;=than g ~ m11138-66-2; , 9012-36-6; agar, 9002-18-0; chitin, 1398-61-4.
+,
id
+ + f
h h C
LITERATURE CITED
Y
*
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w
2
ei
(1) Sandford, P. A.; Balrd, J. I n The POlyseccherMes, Aspinall, G. O., Ed.; Academic Press: New York, 1963; Vol. 2. (2) Marchessauk, R. H. CHEMECH 1984, 74, 542-552. (3) DeJongh, D. C. I n The Carbohydrates Chemlstry and Biochemlstry; Plgman, W., Horton, D., Wander, J. D.,Eds.; Academic Press: New York. 1980; Vol. IB. 1 Relnhokl, V. N.; Carr, S. A. Mass Spectrom. Rev. 1983, 2 , 153-221. Schulten, H.-R.; Gzirtz, W. Anal. Chem. 1918, 5 0 , 428-433. Schulten, H.4. Int. J. Mass Spectrom. Ion Phys. 1979, 32, 97-283. Schulten, H A ; Bahr, U.; Gijrtz, W. J. Anal. Appl. Pyrowsis W81, 3 , 137- 150. Schulten, HA.; Bahr, U.; Gijrtz, W. J. Anal. Appl. Pyro/ysis 18811 1982. 3 . 229-241. van der Kaaden, A.; Boon, J. J.; Haverkamp, J. Biomed. Mass Spec@om. 1984, 7 1 , 466-492. Llnschekl, M.; D'Angona, J.; Burllngame, A. L.; Dell, A.; Ballou. C. E. R o c . Natl. Acad. Sci. U . S . A . 1981, 78, 1471-1475. Forsberg, L. S.; Dell, A.; Walton, D. J.; Ballou, C. E. J. Biol. Chem. 1982. 257, 3555-3563. Dell, A.; Ballou, C. E. Homed. Mass Spectrom. 1985, 70, 50-56. Kamerllng, J. P.; Heerma. W.: Vllegenthart, J. F. Q.: Green, B. N.; Lewis, I . A. S.; Strecker, G.; Spik, G. Biomed. Mass Spectrom. 1983, 10, 420-425. Dell, A.; Ballou, C. E. Carbhydr. Res. 1983, 720, 95-1 11. Bosso, C.; Defaye. J.; Hevraud, A.; Ulrlch, J. Carbohvdr. Res. 1984. 725,309-317. Wllklns, C. L.; Well, D. A.; Yang, C. L. C.; Ijames, C. F. Anal. Chem. 1985. 67. 520-524. Brown, R: S;. W i l , D. A.; Wllklns, C. L. Macromolecules 1988, 79, 1255-1260. Brown, R. S.; Wllklns. C. L. J. A m . Chem. SOC. 1988. 708, 2447-2448. Gates, M. L.; Wilklns, C. L. Blomed. Mass Spectrom. 1985, 72, 424-428.
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Anal. Chem. i987, 5 9 , 200-202
(20) Coates, M. L.; Wilkins, C. L. Biomed. Environ. Mass Spectrorn. 1986, 13, 199-204. (21) Lam, D. A., Jr.; Johiman, C. L.; Brown, R. S.; Weil. D. A.; Wilkins, C. L. Mass Spectrom. Rev. 1988, 5 . 107-166. (22) Wilkins, C. L.; Gross, M. L. Anal. Chem. 1981, 53, 1661A-1668A. (23) Honovlch. J.; Qureshl, N.; Takayama, K.; Cotter,R. J. Presented at the 34th Annual Conference on Mass Spectrometry and Allied Topics, Cin-
cinnati, OH,June, 1986.
RECEIVED for review July 15, 1986. Accepted September 10, 1986* Support under Institutes of Health Grant GM-30604 is gratefully acknowledged.
CORRESPONDENCE Physiologically Relevant Pseudophase High-Performance Liquid-Liquid Chromatography Sir: It is well-established that the lipophilicity of a chemical agent is one of the important determinants of its toxicology, since it determines its partition into physiological fluids or cells and interaction with biologically relevant molecules. Many researchers have proposed approaches to quantify the lipophilicity of chemical agents with the view to measure their bioavailabilities and bioaccumulations, and hence predict aspects of their toxicologies. Lipophilicity has been traditionally quantified on the basis of the logarithm of the partition coefficient of the agent between immiscible lipophilic and hydrophilic solvents ( 1 , 2). It is now generally accepted that the logarithm of the partition coefficient between l-octanol and water is a reasonable model for the prediction of agent transport from a hydrophilic phase into a biophase (3, 4). The selection of 1-odanol as a model for a lipophilic phase has often been questioned but, since there is a large data base for these partition coefficients, there has been little incentive for research into alternative solvent systems. The major criticism of the use of 1-octanol is that it does not include the quasi-fluidic character found in lipoprotein structures in biological systems. Hansch and co-workers (5) proposed a method, based on Martin's concept of additivity of linear free energy (6), for calculating partition coefficients using quantitative structure activity relationships (QSAR). Various substituent constants (n),determined experimentally, were used to predict the logarithm of the partition coefficient (for 1-octanol/water) on the basis of molecular structure. However, the QSAR approach cannot discriminate between isomers and congeners of complex molecules, since it assumes that the values of functional groups or atoms are constant. In fact, values of n in molecules that contain multiple functional groups can vary considerably; therefore, the partition coefficient must be determined experimentally. Reverse-phase (CIS)high-performance liquid chromatography (HPLC) is an alternate approach to the traditional static method for determination of partition coefficients (7-11) and has the advantage of being experimentally easier and quicker. The HPLC data can be linearly related to the static data by an experimental constant determined with both static and dynamic approaches using a compound with properties similar to those of the molecules under study. Currently, there is discussion on whether to determine the partition coefficients or capacity factors from HPLC data (10-12), since both approaches can be related to the partition coefficients for 1octanol/water obtained by the static method. One of the limitations of using reverse-phase HPLC to measure partition coefficients is that i t is not a good model for biological systems. Pseudophase liquid chromatography 0003-2700/87/0359-0200$0 1.50/0
was an attempt to remedy this deficiency by using an aqueous sodium dodecyl sulfate (SDS) micellar mobile phase (13). While this approach represents an exciting extension to the field of liquid chromatography, the ionic SDS micelles do not have the zwitterionic character found in physiological systems. This paper reports on preliminary studies that show that it is possible to use optically opaque micelles that have zwitterionic character as mobile phases for high-performance liquid chromatography using UV detection. In this communication the term micelle is not used in the strict physicochemical sense since the model chylomicrons may be microemulsions.
EXPERIMENTAL SECTION Materials. The following materials (Sigma Chemical Co. and Aldrich Chemical Co.) were used as received corn oil, albumin (dog), cholesterol, L-a-phosphatidylcholine,sodium chloride, benzaldehyde, acetophenone, nitrobenzene, 1-propanol, nickel sulfate, and p-aminobenzoic acid. Equipment. A liquid chromatograph with a variable-wavelength UV detector (Varian 2000 Series), an additional HPLC pump (Waters 6000),a liquid sampling valve (Rheodyne 7125), and a short CI8 column (2 cm, 40-pm Pelliguard packing, Supelco) were used in this study. Procedure. The concentrated micellar mobile phase was prepared as follows: Albumin (0.2 g), cholesterol (0.6 g), ~ - a phosphatidylcholine (1.8 g), and corn oil (17.4 g) were added to a conical tube and homogenized with a Polytron (PT 10/35 Brinkmann Instruments). The composition of this mixture is based on the published composition of the chylomicron fraction of exogenous lipoproteins contained in human plasma (14-1 7). The resulting solution was then rehomogenized with an aqueous solution of sodium chloride (40 mL, 0.9%). This opaque white micellar concentrate was filtered through a membrane filter (1 pm) to remove any metallic particles from the homogenizer and stored at 0 "C until used. This concentrate was found to be stable for at least 1week. The micellar mobile phases were prepared by vortexing known dilutions of the concentrate with aqueous solutions of sodium chloride (0.970, w/v). Opaque mobile phases cannot be used directly with UV detectors and therefore the mobile phase was clarified postcolumn by the addition of 1-propanolvia a second solvent delivery system in a 1.02.8 ratio (v/v) (micellar mobile phase, 1-propanol). The column effluent and the 1-propanol were combined in a low volume tee (18), passed into a mixer (The Lee Co. Visco Jet Micro-Mixer, 250-pL internal volume) with one inlet blocked, followed by an empty column (internalvolume 100 pL), and fmally into the UV detector via a low dispersion connecting tube (19). RESULTS AND DISCUSSION The use of opaque micelles that model the exogenous blood plasma lipoprotein fraction, chylomicron, as mobile phases for HPLC represents a unique approach for the investigation 0 1986 American Chemical Society
