Anal. Chem. 1994,66, 2656-2668
Differentiation of Isomeric Alditol Hexaacetates and Identification of Aldohexoses by Electron Impact Mass Spectrometry Melinda K. Hlggins, Robert S. Bly, and Stephen L. Morgan' Department of Chemistry and Biochemistry, The University of South Carolina, Columbia, South Carolina 29208 Alvin Fox Department of Microbiology & Immunology, School of Medicine, The University of South Carolina, Columbia, South Carolina 29208
Isomeric aldohexosesugars derivatized using the alditol acetate method produce diastereomeric products that can be differentiated by electron impact (E[)-mass spectrometry. The use of sodium borodeuteride for reduction in the derivatization scheme enables identification of aldohexoses because their hexitol hexaacetate products retain the chirality of the starting sugar. The observed differences in the EI-mass spectra were visualized using principal component analysis and cluster analysis. The statistical significance of these differences was evaluated using Hotelling's TL statistic for the comparison of multivariate cluster means. The electron impact mass spectra of nondeuterated and deuterated hexitol hexaacetates permit differentiation of the diastereomeric nondeuterated hexitol hexaacetates as well as identification of all of the aldohexoses. Carbohydrates have been widely employed as chemical markers for identification and trace detection of bacteria.1.2 A common approach is to analyze bacterial isolates by gas chromatography/mass spectrometry (GC/MS), which requires hydrolysis of cell samples to release the monomeric carbohydrates and derivatization of carbohydrates to a volatile form. The alditol acetate procedure3q4is a popular method of carbohydrate derivatization which we have modified to provide better separation of individual sugars, to enhance cleanup of the sample from interfering components in the background, and to allow analysis of both neutral and amino sugar^.^-^ Applications have included carbohydrate profiling of whole microbial cells as well as trace detection of carbohydrates in complex m a t r i c e ~ . ~ - l ~ * Author to whom correspondence should be addressed. (1) Morgan, S. L.; Fox, A.; Gilbart, J. J. Microbiol. Meth. 1989, 9, 57-69. (2) Fox, A.; Morgan. S.L. In AnalyticalMicrobiology Methods: Chromatography and Mass Spectrometry: Fox, A., Morgan, S. L., Larsson, L., Odham, G., Eds.; Plenum Press: New York, 1990; Chapter 1, pp 1-17. (3) Gunner, S. W.; Jones, J. K. N.; Perry, M. E. Chem. Ind. (London) 1961,
255-256.
(4) Sawardeker, J. S.;Sloneker, J. H.; Jeannes, A. Anal. Chem. 1965.37, 16021604. ( 5 ) Fox, A.; Morgan, S. L.; Gilbart, J. In Analysis ojCarbohydrates by GLCand
MS; Bicrmann, C. J., McGinnis, G. D., Eds.;CRC Press: Boca Raton, FL, 1989; pp 87-115. (6) Fox, A.; Gilbart, J.; Morgan, S. L. In Analytical Microbiology Methods: Chromatography and Mass Spectrometry: Fox, A., Morgan, S.L., Larsson, L., Odham, G., Eds.; Plenum Press: New York, 1990; Chapter 5, pp 71-87. (7) Whiton, R. S.; Morgan, S.L.; Gilbart, J.; Fox, A. J. Chromatogr. 1985,347, 109-1 20. (8) Fox, A.; Morgan, S.L.; Hudson, J. R.; Zhu, Z. T.;Lau, P. Y. J. Chromarogr. 1983, 256, 429-438.
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Ana&ticaiChemistry, Vol. 66, No. 17, September 1, 1994
The chemistry involved in the alditol acetate method is outlined in Figure 1 using as an example the derivatization of D-glucose. Following release of the monomeric sugar from a polymer matrix, the monomer undergoes hydrolysis in aqueous solution to produce an equilibrium mix of cyclic and acyclic monomers. The aldehyde is next reduced with sodium borodeuteride (NaBD4) or sodium borohydride (NaBH4). If NaBD4 is used, carbon 1 (C( l), Figure 1) becomes stereogenic due to the presence of deuterium and adds a fifth chiral center to the molecule, which results in two alditols: ( l R ) - l - d - ~ glucitol and (1s)- 1-d-D-glucitol (where the notation 1-d represents a deuterium on carbon 1). Reduction with NaBH4 produces only one product, D-glucitol. Acetylation of the hydroxyl groups produces a volatile stable alditol hexaacetate derivative (D-glucitol hexaacetate without deuterium and a mixture of (1R)- and (lS)-l-d-o-glucitol hexaacetates with deuterium). In contrast to certain other derivatization methods (such as trimethylsilyl derivatization), the alditol acetate method using NaBH4 produces a single derivative compound from each sugar that usually can be well resolved by GC, simplifying chromatographic interpretation and quantitation. When NaBD4 is used, only two products are formed and these are diastereomers that co-elute on most stationary phases. Figure 2 shows all possible compounds resulting from derivatization of the aldohexoses by sodium borohydride (producing nondeuterated hexitol hexaacetates) and sodium borodeuteride (producing deuterated hexitol hexaacetates). Nondeuterated alditol hexaacetate derivatives of the three common hexoses (mannose, galactose, and glucose) can be resolved by GC using cyanopropylsilicone stationary phases. If a mixture contains these three hexoses in combination with the other five less common hexoses (allose, talose, altrose, gulose, idose), structural similarity of these molecules makes separation and identification difficult. For example, after reduction of the aldehyde group by sodium borohydride and (9) Fox, A,; Lau, P. Y.; Brown, A,; Morgan, S. L.; Zhu,Z.-T.; Lema, M. J. Clin. Microbiol. 1984, 19, 326-332. (10) Walla, M. D.; Lau, P. Y.; Morgan, S. L.; Fox, A. J. Chromatogr. 1984,288, 399-413. (1 1) Gilbart, J.; Fox, A.; Whiton, R. S.;Morgan, S. L. J. Microbiol. Merh. 1986, 5, 271-282. (12) Gilbart, J.; Fox,A.; Morgan, S.L. Eur. J. Clin. Microbiol. 1987,6,715-723. (13) Fox, A.; Rogers, J. C.; Fox, K.F.; Schnitzer, G.;Morgan, S.L.; Brown, A.; Aono, A. J . Clin. Microbiol. 1990, 28, 546-552.
OOOS-2700f 94/030&2650$04.50/0 0 1994 American Chemical Society
k a-D-Glucopyranose
D-Glucose
p-D-Glucopyranose
/ /aBD4 (NaBH,)
r
r HO-------D(H)
HAOH H+H
H
I4
W
-
OH
CH20H
(1R)-l-dD-gluCItol (IS)-l-4-D-glUCItol (D-glucitol)
AcO--H
AcOTH
H--0AC
H+OA~
H-
H ~H~OH
OAc
(CH,CO),O
OAc
I H T O A c
CH20Ac
CH20Ac
(1R)-I-dD-glucitol hexaacetate (1S)-I -6D-glucitol hexaacetate (D-glucitolhexaacetate)
Figure 1. Derlvatlzatlon scheme for the aldltol acetate methodIllustrated by applicationto Dglucose. The symbol D on C( 1) representsdeuterium, and the symbol OAc represents an acetate (OCOCY) group.
acetylation by acetic anhydride, D-glucose and D-gulose produce enantiomeric hexitol hexaacetates which co-elute and are indistinguishable by MS. In a similar fashion, enantiomeric and co-eluting hexitol hexaacetates are produced from L-glucose and L-gulose. The two nondeuterated hexitol hexaacetates possible from gulose and glucose are 4 and 5 (shown in Figure 2): one hexitol hexaacetate from either D-gulose or L-glucose (5) and the other possible enantiomer from either L-gulose or D-glucose (4). Similarly, D-altrose and D-talose produce the same hexitol hexaacetate (2) whose enantiomer (3) is produced from either L-altrose or L-talose. As a means of retaining asymmetry of the reduced sugar and simplifying mass spectral interpretation of the final alditol acetate product, reduction with sodium borodeuteride has been employed."'6 When carbon 1 (Figure 1) is labeled with deuterium, the asymmetry of carbons 1-5 is preserved throughout the derivatization. This permits differentiation of the products from D-glucose (17 and 18) from those of D-gulose (19 and 20) and the products of D-altrose (13 and 14) from those of D-talose (15 and 16). The same differentiation is possible for the products of L-glucose (33 and 34) from those of L-gulose (35 and 36) and the products of L-altrose (29 and 30) from those of L-talose (31 and 32). Close inspection in our laboratories of the E1 mass spectra of the deuterated products from D-mannose (23 and 24), D-altrose (13 and 14), and D-talose (15 and 16) revealed distinctive differences in relative abundances for certain ions of these three compounds. 1 3 9 1 7 Similar differences were also (14) Klok, J.; COX,H. C.; DcLceuw, J. W.; Schenck, P. A. J . Chromatogr. 1982, 253, 55-64. (15) Park, J. R.; Jankowski, K.; ApSimon, J. W. In Aduances in Heterocyclic Chemistry; Katrinsky, A. R., Ed.;Academic Press: Orlando, FL, 1987; Vol. 42, pp 335-349. (16) Rauvala, H.; Finne, J.; Krusius, T.; KHrkkiinen, J.; J h e f e l t , J. In Advances in Carbohydrate Chemistry and Biochemistry; Tipon, R. S., Horton, D., Us.; Academic Press: New York, 1981, Vol. 38; pp 399-400. (17) Rogers, J. C. Ph.D. Dissertation, The University of South Carolina, Columbia, SC. 1990.
noticed in the E1 mass spectra of the deuterated products from D-glucose (17 and 18) and D-gulose (19 and 20). Although variations in absolute ion intensity can be due to instrumental artifacts, the relative ion abundance differences have been reproduced and have been consistent for several years on several different MS instruments. The objective of the present work was the validation of these observed differences using a designed experiment coupled with appropriate multivariate statistical analyses. We have interpreted these differences in E1 mass spectra of hexitol hexaacetates using principal component analysis (PCA), cluster analysis, and hypothesis tests for the equality of the multivariate cluster means. No previous study has provided unambiguous identification of all hexitol hexaacetates in a single GC/MS run. The fundamental question addressed by this work was whether aldohexoses can be differentiated and identified on the basis of the E1 mass spectra of their hexitol hexaacetate products alone.
MATERIALS AND METHODS Chemicals. Each of the hexoses, allose, altrose, galactose, glucose, gulose, idose, mannose, and talose (eight D- and seven L-hexoses) was purchased from Sigma Chemicals (St. Louis, MO). L-Altrose was not commercially available. No distinction was made as to whether the sugars were a or 0 anomers since the configuration of C ( l ) is lost in the first step of derivatization. The following derivatization reagents and sample solventswere glass distilled: acetic anhydride (Alltech, Deerfield, IL), Ultrex grade acetic acid and ammonium hydroxide (J. T. Baker, Phillipsburgh, NJ), and chloroform and methanol (Burdick 8c Jackson Labs, Muskegon, MI). Reagent grade sodium borohydride and sodium borodeuteride were obtained from Sigma (St. Louis, MO). Chem Elut hydrophilic extraction columns were purchased from Analytichem International (Harbor City, CA). Analytical Chemistry, Vol. 66, No. 17, September 1, 1994
2657
Non-deuterated Hexitol Hexaacetates Allitol
Altritol
Talitol
CH20Ac
Glucitol
CH20Ac
Gulitol
CH20Ac AcOT%f
D series H Y A c
Galactitol CH20Ac
AcO%Ac
X O A c
H---OAc
rz: -0AC
Mannitol CH20Ac
Iditol CH20Ac
A C O q H Y O A c OAc AcO-H W O A c -0Ac
CH20Ac
CH,OAc
CH20Ac
CH,OAc
CH20Ac
CH,OAc
CH20Ac
CH20Ac
1
2
2
4
5
6
7
9
CH,OAc
CH20Ac
CH20Ac
CHzOAc
CHpAc
F O A c AcO-H W O A c I t - O A c *Ac Am-H AcOTH A c O I H
,
CH20Ac
I
AH AcO--+-H K-OAC AcO-H W O A c -0Ac W O A C -0Ac Ac0-f-H AcO-H AcO-H A T H
H-OAc AcO-H Hf-OAc A c O I H
I
CH,OAc
CH,OAc
CH20Ac
CHpAc
CHpAc
CH20Ac
3
5
4
6
8
10
Galactitol
Mannitol
Iditol
H
H
Deuterated Hexitol Hexaacetates Allitol
Altritol
Talitol
Glucitol
H
H
H
H D-Ac
Gulitol
D t O A c AcO-tH AcO
(IR)
AcO+H K-OAc W O A c
H AC H--+I)Ac I
CH,OAc
D series
11 H
(IS)
(IR)
Ac0-D H-OAc H-OAc
L series
CH,OAc
CH20Ac
CH20Ac
13
15
17
19 ';I
21
H AcO-+D
H AcO-D
';I
H-OAc A~O+H It--OAc AcO-H H + ~ O A C -0Ac
Aco&A :~
K-OAc AcO$EA~ W O A c AcO H A ~ O - ~ H AH W O A c +OAc
q;;
I
CH20Ac
12
14
16
18
H
H
AcACO+H A c O Y
H D-Ac -0Ac H-t-OAc K----OAc A~O+H
H
AcOTD ACO-LD
CH20Ac
H D+OAc AcO-f-H W A AcO-H A~O-H
CH20Ac
W O A c AcO--"H A l T ACO-H H+-OAC K t O A c AcO-+H I+--OAC H-OAC CH20Ac
25
H
7
20
22
H
H
D ~ O A C D-OAC AcO-H AcO-H +OAc C AcO-H +OAc W O A c A~O-H A~O--CH
CH20Ac
26
H
H
D-OAC D-OAc W O A c H-OAc AcObr-H It-OAc A b H H--LOAC A~O--H A~O-H
CH,OAc
CH20Ac
27
29
31
33
35
37
39
H
ti
H
H
H
H
H
ET CH,OAc
28
Ac-D 4 Q A c AcAcOl-H CH20Ac
30
AcO+D Ac-D It-OAc AcO+H -0Ac W A W A C AcO--C-H AcO-H AcO-H CH20Ac
32
A c T D A c W D Ac-D &OAc AcO-H AcO-H +OAc I+--OAC C Ac0-H W O A c Ac(t-H AcOH A G F AcO---H
CH20Ac
34
CH20Ac
I
CH20Ac
24
CH20Ac
CH20Ac
%z:
AcO-D AcO-H A~O-H H+OAC W O A c Ac0-t-H It--OAc H-+-OAc
I
CH20Ac
P
CH20Ac
23
CH20Ac
A~O+ AcO-H
(1S)
CH20Ac
CH20Ac
CH20Ac
,
CH20Ac
CH20Ac
&OAc AcO-H A~O+H AcO-H AcO+
H ';I D-~OAC D+OAC W O A c W O A c A c O T Y AH A W H W O A c W O A c
CH20Ac
CHpAc
CH,OAc
36
38
40
CH20Ac
41 H Ac0-D H-OAc A~O-H
H-OAc Ac0-H CH20Ac
42
Figure 2. Flscher projections of the chemical structures for all of the possible hexkoi hexaacetate derhrathres of the aldohexoses. 1-10 are nondeuterated products. 11-42 are deuterated products.
Alditol Acetate Derivatization. The alditol acetate method was modified from that used previously.5.7-13 Samples of 10 pg (1 mLof each lOpg/mLaqueous solution) of each D-hexose were used to start the derivatization. To increase the final product for the L-hexoses, 50 pg (1 mL of each 50 pg/mL aqueous solution) was used. Changing the starting amount for the L-hexoses did not affect the amounts of any other 2058 Analytical Chemistry, Vol. 66, No. 17, September 1, 1994
reagents, since all were still in excess. The hexose remained as the limiting reagent. Four samples of each hexose were reduced, two with 50-pL sodium borohydride (NaBH4, 100 mg/mL) and two with 50-pL sodium borodeuteride (NaBD4, 100mg/mL). A 2.5-mL solution of acetic acid and methanol (1:200 v/v) was added and the sample dried under nitrogen with heat (100 "C)to remove the acylation-inhibiting borate.
This step was repeated four times and followed by a 3-h final drying under vacuum. A 300-pL aliquot of acetic anhydride was added to each sample and acylation allowed to occur overnight (for at least 15 h and no more than 19 h) in sealed vials at 100 OC. The vials were cooled in an ice bath. Excess acetic anhydride was degraded to acetic acid by vortexing the mixture with 0.75 mL of water for 1 h. A 1-mL amount of chloroform was added to each vial and vortexed for approximately 1 min. The aqueous phase was discarded, and 0.8 mL ofa cold (5-10 "C) concentratedsolution ofammonium hydroxide in water (80% v/v) was added to the remaining solution. This mixture was then poured into a Chem Elut column, and the hexitol hexaacetates were eluted with gn additional 3.5 mL of chloroform.8 The chloroform was evaporated under a flow of nitrogen. Prior to analysis by GC/MS, the samples were redissolved in 100p L of chloroform and transferred to autosampler vials equipped with 100-pL glass inserts. Gas Chromatography/Mass Spectrometry. A HP-5890 GC (Hewlett-Packard, Palo Alto, CA) with a HP-7673A autosampler interfaced to a HP-5970 mass-selective detector (MSD) was used to carry out the GC/EI-MS analyses. Hexitol hexaacetates were separated on a 30-m SP-2380 (Supelco, Bellefonte, PA) capillary column (bis(cyanopropy1)phenyl polysiloxanestationary phase, 0.32-mm inner diameter, film thickness of 0.20 mm). The injection port temperature was set at 250 OC, and the transfer line temperature to the MSD was set at 280 OC. The GC oven temperature was initially held at 100 OC for 1 min and then ramped at 20 OC/min to 230 OC and then ramped at 5 OC/min to 265 OC, which was held for 3.5 min, for a total run time of 18 min. Blank vials containing chloroform were placed at the beginning, end, and after every four samples in the autosampler tray. Three injections were done of each sample, and a single run was made of each blank. Each vial contained the product(s) from only one hexose. Mass spectral data were collected in the total ion monitoring (TIC) mode from 10 to 15 min over the range from 40 to 440 mass units. The instrument was operated daily under "Autotune" conditions at 70 eV. Data Pretreatment. For each of the eight D-hexoses, two samples were derivatized with NaBH4 and two were derivatized with NaBD4 for a total of 32 samples. The seven L-hexoses were derivatized likewise for a total of 28 additional samples. Each sample was injected three times providing three GC traces of one peak each and their corresponding EI-mass spectra, which produced 180 mass spectra. Mass spectra were tabulated and results stored in an ASCII file listing each ion detected and its corresponding abundance. Four data sets were produced as follows: Data set I , 42 mass spectra of the nondeuterated L-hexitol hexaacetates; data set 2,48 mass spectra of nondeuterated D-hexitol hexaacetates; data set 3,42 mass spectra of deuterated hexitol hexaacetate derivatives from the L-hexoses; data set 4,48 mass spectra of deuterated hexitol hexaacetate derivatives from the D-hexoses. The H P 59970 MS ChemStation records mass ions in tenths of mass units as the abundance of ions exceeds a threshold set by the user. The ion abundance of all ions for one mass fragment sometimes splits into several entries in the data table, e.g., ion m / z 51.1, abundance 4352, and ion m / z 51.4, abundance 564 783, for a mass fragment at m / z 5 1. Tocorrect
the data tabulation, all ions whose abundances had been split were recombined into a new data table. For example, for the mass fragment at m / z 5 1, the abundance was recombined to be 569 135. Each ion's abundance was calculated to be a percentage relative to the ion with the highest abundance ( m l z 115 for all). Ions with relative percent abundance below a 5% threshold were discarded in the data pretreatment program. Other percentages were also evaluated (1% 10% 20%). Below 5%, too much background noise was included with each spectrum. Above 5%, too much of the unique mass spectral differences tended to be discarded. Using this threshold approach, the number of mass spectral features (ion masses) retained for data sets 1-4 were 32, 39, 57, and 66, respectively. The number of mass spectral features does not include ion m / z 115, which was deleted because it was used as the reference peak (constant 100% relative percent abundance in all spectra). The resulting data was arranged in a matrix, X, whose rows represent n different spectra and columns represent m selected ion masses:
x=
[
'11 Xil
xu
Xlj
Xn1
Xnj
"I
(1)
Xnm
where each element x i j is the relative percent abundance for ion mass j of spectrum i. For each data set, the mean and standard deviation of the relative percent abundance for each ion in the data set was calculated. Using this information for each data set, a column autoscaled data matrix Z was produced whose elements were calculated by
where Zj
=
1 " -(Exij) n is1
is the mean relative percent abundance for ion j , and
is the standard deviation of relative percent ion abundance for ionj . Data pretreatment (thresholding, autoscaling, etc.) was performed using programs written in Turbo Pascal (version 6.0, Borland International, Inc., Scotts Valley, CA). Plots of mass spectra showing average relative percent ion abundances and standard deviations were also produced by a Turbo Pascal program. Principal Component Analysis. Principal component analysis and its application in chemistry were reviewed by Geladi et a1.18and by Auf der Hyde.lg The sample covariance matrix, C, was calculated for each data set: (18) Wold, S.; Ebensen, K.; Geladi, P. Chemom. Intell. Lab. Sys. 1W7.2,37-52. (19) Auf der Heyde, T. P. E. J . Chem. Educ. 1990, 67, 461-469.
Analytical Chemistty, Vol. 66,No. 17, September 1, 1994
2659
1 c = -(Z’Z) n- 1
(3)
where n is the number of spectra and Z is the matrix of autoscaled values whose columns have been centered about the mean. The ordered eigenvalues of C (A,, AI, AS, ..., A,, where r is the matrix rank of C) give the relative proportion of variance explained by each corresponding principal component (PC). The eigenvectors of C (a1,a2, 83, ..., ak, ..., a,) contain the loadings a i k for the ith ion projected onto the kth principal component. The eigenanalysis involved in PCA can be efficiently computed using singular value decomposition.2°.21 If the eigenvectors are arranged as columns in a matrix A = [a,, 82, aj, ..., ak, ...,a,] for k = 1, 2,...,r, then the projections of the data points onto the principal components (also called the scores) can be calculated by S = ZA
(4)
These scores are linear combinations of the original autoscaled data values for each ion, weighted by the loadings or eigenvalues. For each data set, the scores for the data points on the first three PCs were plotted to permit relationships among the clusters of data points to be visualized in a space of lower dimensionality. In every case, three PCs explained the variability in the data sufficiently to provide adequate visual discrimination of the relevant differences in the mass spectra. Further testing to determine the number of significant PCs was not performed. In this work, PCA was done using SYSTAT (version 5.0, SYSTAT Inc., Evanston, IL). Cluster Analysis. Cluster analysis was performed using a similarity matrix based on Euclidean distances from the autoscaled data set. The multidimensional Euclidean distance, db2,between two pattern objects i a n d j (e.g., two mass spectra) is calculated by the formula
(5)
where x refers to the autoscaled intensity at each ion mass and m is the number of ion masses. Several clustering algorithms were evaluated including the single linkage, centroid, and the group average linkage clustering algorithm~.~~,~~ The various clustering algorithms differ in the manner in which distances between clusters are defined. The simplest method is the single-linkage algorithm (also known as nearest neighbor), which defines the distance between two clusters as the minimum distance between an observation in one cluster and an observation in another. Starting withoneobject defined as a cluster, at each succeeding stage of the algorithm, the nearest neighbor is joined to the existing cluster(s). The ~~
(20) Green, P. E. Analyzing Multiuariate Data; The Dryden Press: Hinsdale, IL, 1978; Chapter 4. (21) Davis, J. C. Statistics and Data Analysis in Geology, 2nd ed.;John Wiley and Sons: New York, 1986; Chapter 6. (22) Sokal, P. H. A.; Sneath, R. R. Numerical Taxonomy: The Principles and Practice of Numerical Classification; Freeman: San Francisco, CA, 1973. (23) Massart, D. L.; Kaufman, L. The Interpretation of Analytical Data by the Use of Cluster Analysis; John Wiley and Sons: New York, 1983.
2800
Analytical Chemistry, Vol. 66, No. 17, September 1. 1994
centroid method defines the distance between two clusters as the Euclidean distance between their centroids or means and is more robust to outliers. At each stage, the new data point closest to a cluster mean is added to that cluster. Clustering algorithms may imposed incorrect structure on the data because of the way clusters and distances between clusters are defined. For example, single-linkage algorithms are susceptible to “chaining” nearest data points into clusters regardless of the existence of more global cluster structure. In the present application, the single linkage and centroid methods showed too many misclassifications to be useful. Results presented here were based on the group average linkage algorithm, which is an intermediate choice between the single linkage and complete linkage. The group average linkage algorithm averages all distances between pairs of objects in different clusters to decide how far apart they are.24 Cluster analysis output is often displayed as a dendrogram or tree structure that depicts, from left to right on a horizontal scale, the similarity (distance) between adjoining data points &e., mass spectra in our case) and cluster(s). The SYSTAT package was used for all cluster analysis computations and dendrograms. SignificanceTesting for the MultivariateMeans of Clusters. PCA and cluster analysis allow visual discrimination of the groups of samples for each hexitol hexaacetate. These methods of data display, however, do not provide statistical tests of hypotheses regarding the significance of the differencesin the cluster locations. The multivariate generalization of the univariate two-gruop Student’s t test is Hotelling’s Tz test.20J.25 Hotelling’s Tz statistic was used here to perform pairwise tests of equality for the centroids of all possible combinations of two groups of sample clusters. Hotelling’s 7’2 statistic is defined as follows:
where n, is the number of samples in group a, nb is the number of samples in group b, d is the difference vector of centroids, C,-1 is the inverse of the pooled within-groups samples covariance matrix, and p is the number of multivariate dimensions in which the clusters are characterized.20121 In this case, the projections of the data points on the first three principal components (i.e., the scores) were employed; thus, p was 3 for all tests of hypotheses. The test statistic used is na + n b - p - 1
p(na + nb - 2,
-id
(7)
which is distributed as an F@l,df2) distribution with the number of the first degrees of freedom (df I ) equal t o p and the second degrees of freedom (df 2 ) equal t (n, inb -p 1). The calculated value of the test statistic is compared to the critical value of the F distribution at the stated number of degrees of freedom and stated % confidence level 100( 1 CY).The null hypothesis of equivalence of group centroids is rejected with lOO(1 - CY)%confidence when the calculated (24) Sokal, R. R.; Michener, C. D. Uniu. Kansas Sci. Bull. 1958.38, 1409-1438.
(25) Hotelling, H . Ann. Math. Sfor. 1931, 2, 360-378.
Table 1. Selectd Crltlcal Valuer of x2 and F Dktrlbutlonsz7(n. = 8, % = 8) ( 1 - a): level of confidence
x2(6) F(3.8)
0.95
0.99
12.59 4.07
16.81 7.59
0.995
where ni is the number of samples in the ith group, g is the number of groups (g was 2 for the cluster pair tests done in this paper), C, is the pooled within-groups covariance matrix, Ci is the covariance matrix for the ith group, and 1x1denotes the determinant of matrix X.20*21B E 1 is approximately distributed as X2(dr) with degrees of freedom df = l/2(g l)p(p l), where p is the number of features (principal components) (p was 3 for all tests done in this paper), and
+
2p2
+ 3p- 1
6@ +
5
18.55 9.60
value of the test statistic is greater than the critical value of F. A program was written in Turbo Pascal to implement Hotelling’s P test. An assumption of Hotelling’s P test is that the two withingroup sample covariance matrices are equal, which is similar to the assumption of equal variance in the univariate Student’s t test. Bartlett has provided a test statistic for testing the equality of two or more covariance matrices prior to computing discriminant functions.20*21.26 This test statistic is
p = 1 -
1
X
1)
This approximation is good if p and g do not exceed about 5 and each covariance estimate is based on at least 20 observations.21 The calculated value of the test statistic is compared to the critical value of the x2 distribution at the stated number of degrees of freedom and stated ’31confidence level 100( 1 - a). The null hypothesis of equivalence of group covariances is rejected with 100( 1 - a)% confidence when the calculated value of the test statistic is greater than the critical value of x2. Table 1 lists selected critical values of x2 and F distributions for comparisons with the results for the pairwise significance testing of cluster separations to be shown below. A program written in Turbo Pascal implemented Bartlett’s x2 test. RESULTS AND DISCUSSION Stereochemistry of Hexitol Hexaacetates. Before discussing the differentiation of carbohydrate isomers by MS,it is instructive to review the possible isomeric forms of carbohydrates generated by the alditol acetate method. (26) Bartlett, M. S.J. R. Star. SOC.1947, 9, Setics B, 176-197. (27) Fisher, R. A.; Yatcs, F. Staristical Tables for Biological, Agricultural, and Medical Research, 4th 4.; Oliver and Boyd, Limited: Edinburgh, 1953.
Retention Time Figure 9. Capillary gas chromatogram of the eight nondeutefated Mexltol hexaacetates. Peak identlflcatlon: (1) allitol; (2) co-eluting mannitol, aitritol, and tallol; (3) galactitoi; (4) co-elutlng gluclol and gulitol; (5) Iditol hexaacetate.
Consider again the derivatization of D-glucose (Figure 1). When a sample is dissolved in water, the a and /Ianomers of D-glucopyranoseexist in equilibrium with each other and with acyclic D-glucose. The first step in the alditol acetate method is the reduction of the aldehyde at C(1). As mentioned previously, reduction by sodium borodeuteride instead of sodium borohydride places a deuterium on either side of carbon 1 and yields two products, (1R)- and (1s)-l-d-D-glucitol. Reduction by sodium borohydride produces only one product, D-glucitol. The second step is the acetylation of the hydroxyl groups. Use of NaBH4 for reduction in the alditol acetate method for all 16 aldohexoses yields a total of 10 hexitol hexaacetates (Figure 2,l-IO). Of these 10 compounds, only 6 are diastereomeric. Use of NaBD4 for reduction, however, yields a total of 32 C(1) monodeuterated hexitol hexaacetates (Figure 2, 11-42).28 These 32 compounds comprise 16 enantiomeric pairs of diastereomers. There are fewer products in the nondeuterated case for two reasons. First, only one alditol product is possible after reduction of the aldehyde with NaBH4. Second, the two ends of the molecule are identical and some of the resulting derivatives are identical even though they originated from different aldohexoses. Of the 10 possible nondeuterated hexitol hexaacetates shown in Figure 2, the following compounds are enantiomers of one another: 2 and 3,4 and 5 , 7 and 8, and 9 and 10. Thus there are six groups of diastereomers: 1,6, and four pairs of enantiomers, 2 and 3 , 4 and 5,7 and 8, and 9 and 10 (see Figure 2). As stated above, adding a deuterium to C(1) makes that carbon stereogenic and adds a fifth chiral center to the deuterated products. Because C(1) is stereogenic, (1R) and (1s)anomers are possible. From the 16 aldohexoses, 32 deuterated hexitol hexaacetates are possible: (1R) and (1s) anomers for the D series (11-26) and (1R) and ( 1 s ) anomers for the L series (27-42). Of these 32 compounds, only 16 are diastereomers: either the (1R)and (1s)from the D series, or the (1R) and (1s) from the L series. Each series of compounds constitutes enantiomers with respect to the other series. As seen in Figure 3, a mixture of the isomericnondeuterated hexitol hexaacetates cannot be completely resolved by GC using (cyanopropy1)silicone stationary phases.” Zand 7 (from (28) Solomons, T. W. G. Organic Chemistry, 5th ed.; John Wiley and Sons, Inc.:
New York, 1992; p 1023.
AnalvticaIChemistty, Vol. 66,No. 17, September 1, 1904
2661
H
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mannose, altrose, and talose) co-elute in peak 2. 4 and 5 (from glucose and gulose) also co-elute in peak 4. This lack of resolution, however, was not unexpected since only six nondeuterated D-hexitol hexaacetates are possible. The enantiomeric pair of 4 and 5, of course, cannot be separated by the achiral bis(cyanopropy1)phenylpolysiloxanestationary phase or be distinguished by their E1 mass spectra. Only peak 2 contains unresolved diastereomers. For six diastereomers, five peaks are obtained. For the 32 deuterated hexitol hexaacetates, 16 deuterated hexitol hexaacetates originate from the D-aldohexoses and 16 originate from the L-aldohexoses. Because the addition of deuterium does not change chromatographic retention time, the chromatogram appearance is identical for the deuterated and nondeuterated compounds. In this case, however, five peaks are obtained for 16 diastereomeric compounds. In the studies reported here, two samples of derivative product(s) from each single aldohexose were analyzed in triplicate to obtain mass spectra which were evaluated for differentiating characteristics. Hexitol Hexaacetate Mass Spectral Patterns. The Fischer projection of (lR)-l-d-~-mannitolhexaacetate is shown in Figure 4. The masses of ions formed by fragmentation from the C ( l ) end are shown to the right and from the C(6) end to the left of the structure. In general, for nondeuterated compounds, both C( 1) and C(6) are bonded only to hydrogen (H-) and acetate (-OAc). Ion masses for equivalent fragments containing either C ( l ) or C(6) are the same. Compounds with a deuterium on C(1) fragment to form ion mass pairs: fragments containing C(6) without deuterium and fragments containing C( I), which will be one mass unit higher due to the deuterium on C(1). These effects are illustrated in Figure 5 for nondeuterated D-mannitol hexaacetate (7) and deuterated D-mannitol hexaacetates (23 and 24). Comparisonsbetween the mass spectra of the nondeuterated and deuterated forms of D-mannitol hexaacetate establish a baseline for later comparisons. The relative percent abundances of ions m/z 217 and 259 in the mass spectrum in Figure SA of nondeuterated D-mannitol hexaacetate (7) are almost equal to each other. These two ion abundances are also slightly higher than the abundance of ion m / z 298, which is slightly higher than the abundance for ion m / z 361. This same pattern and the similar relative abundances are also 2602 Analytlcel Chemistry, Vol. 66, No. 17, September 1. 1994
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present in the mass spectrum of the deuterated D-mannitol hexaacetates (Figure 5B, 23 and 24). The mass spectrum of deuterated compounds differs from that of the nondeuterated compound in the presence of an additional paired ion mass peak at one mass unit higher for nearly every peak. Within experimental variability indicated by the boxes on each ion peak, the relative percent abundance in the mass spectrum of deuterated compounds for the first ion of each pair was the same as the abundance of the corresponding ion mass for the nondeuterated D-mannitol hexaacetate spectrum. The fundamental pattern present in the nondeuterated hexitol hexaacetate mass spectrum is reproducibly repeated in the mass spectrum of the deuterated compounds. This trend is true of all comparisons of these mass spectra. ELMS can not distinguish between the enantiomeric forms of hexitol hexaacetates. For example, Figure 6 shows mass spectra of the nondeuterated hexaacetate product from L-idose (49) and the nondeuterated hexaacetate product from D-idose (48). Within the range of variability for each ion abundance, no significant differences are seen. Similarly, as expected, all enantiomers yield identical mass spectra within experimental variability . In a similar vein, it is not surprising that nondeuterated and deuterated forms of the same molecule have the same basic underlying mass spectral pattern. What is novel is the ability to differentiate diastereomeric hexitol hexaacetates on the basis of their E1 mass spectra alone. The remainder of this discussion focuses on the differentiation of compounds within the two groups: (a) nondeuterated L-hexitol hexaacetates (data set 1) and (b) deuterated D-hexitol hexaacetates (data set 4). Differentiation within the group of deuterated
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L-hexitol hexaacetates and within the group of nondeuterated D-hexitol hexaacetates mirrors these results, and their presentation would be redundant. Differentiation within the Groupof Nondeuterated L-Hexitol Hexaacetates (Data Set 1). The seven E1 mass spectra for the (nondeuterated) L-hexitol hexaacetates (Figure 2,1,3-6, 8, and 10) are presented in Figure 7. Only seven spectra are shown because L-altrose was not commercially available. Although seven spectra are shown, 4 and 5 originating from L-gulose and L-glucose are enantiomers, hence their mass spectra (Figure 7G and F, respectively) must be identical within experimental variability (indicated by the plus/minus one standard deviation box drawn about each ion peak). For example, the relative percent abundances of ions at m / z 187, 217, 259, and 289 are seen to be virtually identical. At first glance, these spectra of diastereomers seem very similar. Similarities and differences seen in the Fischer projections (Figure 2) were also observed in the mass spectra. For example, the E1 mass spectra of 1 originating from L-allose (Figure 7A) and 10 originating from L-idose (Figure 7C) are similar, especially in the relative percent abundances of ions at m/a 145,217,259, and 289. However, the relative percent abundances of the ion at m / z 187 for 1 was lower than the abundances for the ions at m / z 217 and 145. For 10 the abundances of ions at m / z 145, 187, and 217 were all approximately the same. We have selected for discussion here just one of the possible pairwise comparisons. There are subtle but reproducible differences between the mass spectra of all six nondeuterated L-hexitol hexaacetate diastereomers shown in Figure 7. Principal component analysis was selected to visualize the differences in these E1 mass spectra more clearly. The score
plots, or projections of the mass spectra, onto the first three principal components, were effective for displaying the separation of each diastereomer group in the data set into distinct clusters. Pairwise comparisons of these clusters using Hotelling's T2 test were also done to provide statistical confidence that the observedclustering was not subjective.20*2' Cluster analyses were also performed to lend further evidence that the observed clustering was real and not a chance occurrence. The projection of the mass spectral data for each sample into the space of the first three principal components (the PCA scores plot) is shown in Figure 8 for the nondeuterated L-hexitol hexaacetates. Six separate clusters are visible, with the cluster of six replicate samples from L-glucose (5, labeled L) overlapping the cluster of six replicate samples from L-gulose (4, labeled U). This overlap is required since 4 and 5 are enantiomers and must have statistically identical mass spectra. There appears to be some overlap of the combined L/U cluster and the cluster of group T samples (for the six replicate samples from L-talose (3)) from the perspective shown. However, another perspective view of this plot eliminates this overlap while other clusters appear to overlap one another; the best perspective for display is a compromise. Table 2 presents results from the Hotelling's T2 tests for equivalence of the multivariate means of the six clusters. The first question is whether the clusters have equivalent variances. At the 95% level of confidence the value of x2 that must be exceeded to reject the null hypothesis of equivalent variances is 12.6 (Table 1). For none of the comparisons is this value exceeded; thus, the hypotheses of equal variance cannot be rejected. At the 95% level of confidence the value of F (at 3 and 8 degrees of freedom) that must be exceeded to reject the null hypothesis of equivalence of multivariate means is 4.07. At the 99.5%level of confidence this value is 9.60 (Table 1). All calculated F-statistics but one are much higher than thesevalues, indicating that these null hypotheses of equivalent means can be rejected. Naturally, the exception is the comparison of the mass spectra of enantiomers L-glucitol hexaacetate (5, labeled L) and L-gulitol hexaacetate (4, labeled U), marked by bold text in Table 2. The value of the test statistic for this comparison was only 2.03. This low value indicates the null hypothesis that the mean mass spectrum of the L-glucitol hexaacetate samples is equivalent to the mean mass spectrum of the L-gulitol hexaacetate samples cannot be rejected. It should be noted that the multiple pairwise tests made here are not completely independent of one another. The probability of incorrectly rejecting the null hypothesis (the error of the first kind, a)is increased by the number of comparisons made.29.30One further complication in this work is the rather low number of replicates used to characterize the covariance matrix of each cluster. The average linkage clustering dendrogram is displayed in Figure 9 for the nondeuterated L-hexitol hexaacetates. The clusters of the projections of the mass spectra of 1 originating from L-allose (labeled A), 10 originating from L-idose (I), 6 originating from L-galactose (G), and 3 originating from (29) Netcr, J.; Wasserman, W.; Kutner, M. H. Applied Linear Statistical Models, 3rd 4.; Richard D. Irwin, Inc.: Homewood, IL, 1990; p 579. (30) Bercnson, M. L.; Lcvin, D. M.; Goldstine, M. InrermedlateSratlsticulMet~~s and Applicarions; Prcntice-Hall, Inc.; Englewood Cliffs, NJ, 1983; p 86.
AMlytlcal Chemism, Vol. 66, No. 17, September 1, 1994
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239 302 365 mlz Figure 7. E1 mass spectra of nondeuterated Lhexltol hexaacetates (data set 1): (A) allltol; (B) galactltol; (C) idltol; (D)mannltol; (E) talltol; (F) glucltol; (0)guiltol. The top of each peak represents the average relative percent abundance for six samples; the top and bottom of the boxes drawn on each peak Indicate plus or minus one standard devlation. 50
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L-talose (T) are well separated from one another. 4 originating from L-gulose (U) and 5 from L-glucose (L) are indistinguishable from each other as was expected and could be distinguished from the other five clusters. 8 originating from L-mannose (M)was well separated from the other five groups except for one sample (M6)whose calculated distance put it on the outside of four other groups. Differentiation within the Group of Deuterated D-Hexitol Hexaacetates (DataSet 4). E1 mass spectra for the deuterated 2664
Ana&ticalChemistry, Vol. 66,No. 17, September 1, 1994
hexitol hexaacetate products from each of the eight Daldohexoses are presented in Figure 10. Once again, these spectra seem very similar but repeat the similarities and differences seen above in data set 1. For example, the E1 mass spectra of the mixture of 11and 12 from D-allose (Figure 1 OA) and the mixture of 25 and 26 from D-idose (Figure 1 OC) are similar in the relative percent abundances of ion pairs at m / z 11511 16,217/218,259/260, and 2891290. Therelative percent abundances of ions m / z 1871188 in Figure 10A are
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A
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0.00 0.00 6.50 413.92 3.58 638.18 2.35 524.17 3.74 120.39 6.77 452.25 5.46 244.70
0.00 0.00 9.74 272.62 6.08 386.19 6.49 187.57 6.38 203.27 10.69 229.40
0.00 0.00 5.10 2.03 4.67 305.56 6.69 63.02 4.04 181.93
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lower than the abundances for m/z 2171218 and m/z 145/ 146. In Figure lOC, the abundances of ions m/z 1451146, 1871188, and 217/218 were all approximately the same. For the remaining compounds, the mass spectra are clearly distinguishable from one another by similar subtle, but reproducible, differences. Of special interest is the distinction of the mass spectra of 13 and 14 originating from D-altrose (Figure 10E) from the massspectraof l5and 16from~-talose (Figure 10F). Without deuterium on C( l), these compounds are identical (i.e., are 2) and will have identical E1 mass spectra. The basic underlying mass spectral pattern is the same for these compounds. However, the ratio of relative percent abundances for ion pairs m/z 1871188 and 2891290 reverses significantly from Figure 10E to F. In the mass spectra shown in Figure 10E the relative percent abundance for ion m/z 188 is much greater than the abundance of ion m l z 187. The abundance for ion m l z 290 is also significantly greater than the abundance for ion mlz 289. In Figure 10F,the abundance for ion m/z 187 is greater than ion m/z 188 and the abundance for ion
m/z 289 is greater than ion m/z 290. Ion pairs m/z 2171218 and 259/260 alsoshow this relative percent abundance reversal but to a lesser degree. Also of interest is the distinction of the mass spectrum of 17 and 18 originating from D-glucose (Figure 10G) from that of 19and 2Ooriginating from D-gulose (Figure 10H). Without deuterium these compounds are enantiomers (i.e., 4 and 5) and have identical E1 mass spectra. The same ion pairs, m/z 187/188 and 2891290, exhibit ratio reversal of their relative percent abundances from Figure 10G to H. The score plot for the deuterated D-hexitol hexaacetate mass spectra projected on the first three principal components (PC’s) is shown in Figure 1 1 . Differentiation for all eight D-aldohexoses was readily seen in the eight clusters of their deuterated hexitol hexaacetate products. The apparent overlap between some of the clusters seen in this particular plot can be removed by rotating the plot, but other views obscure different clusters. Table 3 gives the results of each pairwise comparison made of these eight clusters. A few of the clusters exhibit slightly nonequal variances as indicated by the values of the Barlett’s test statistic. For example, clusters G and M appear to have different variances. In this case, however, the PCA scores plot shows clearly that these two clusters are well Anaiyticai Chemistry, Voi. 66, No. 17, September 1, 1994
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separated withthe M cluster grouped tighterthan theGcluster. All pairs of cluster means were different from one another at a high level of significance (i.e., all Hotelling's P test statistics led to rejection of the null hypothesis that the multivariate means were the same). The cluster analysis dendrogram for data set 4 shown in Figure 12 complements these conclusions. The near perfect cluster groupings confirm the correctness of the PCA results and the pairwise comparison tests. The single discrepancy was the gulitol sample (U6)that clustered on the 2666
AnalytlcalChemktry, Vol. 66, No. 17, September 1, 1994
outside of the gulitol (U), mannitol (M), and glucitol (L) groups. CONCLUSIONS Enantiomers have identical capillary GC retention times on an achiral stationary phase. They also have identical mass spectra. Thus D and L isomers of the sugars when derivatized to alditol acetates are indistinguishable chromatographically on a bis(cyanopropy1)phenyl polysiloxane (SP-2380)station-
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Figure 11. Projection of 48 mass spectra in the three-dimensional space of the first three principal components for deuterated hexltol hexaacetate products from the c-aidohexoses (date set 4). These three principal components accounted for 55.9% of the variance (32.8% by PC 1, 12.1% by PC 2, and 11.0% by PC 3). Symbols: (A) allose; (0)galactose; (I) idose; (M) mannose; (R) altrose; (T) talose; (L) glucose; (U) gulose. Circles do not represent statistical information and are provided only for visual identification of clusters.
Table 3. Palrwlre Comparison Teats for the Deuterated Hexitol Hexaactate Products of the D-Hexoses (Data Set 4)’ A R G L U I M T 0.00 0.00 R 15.64 159.72 G 17.54 357.57 L 4.02 48.20 U 16.18 112.11 I 11.56 23.08 M 12.79 207.38 T 8.13 202.84 A
0.00 0.00 0.00 11.68 144.37 0.00 11.64 0.00 8.88 168.87 149.85 0.00 6.94 8.17 0.00 2.86 75.12 73.23 65.05 0.00 14.94 4.42 8.59 0.00 6.51 217.67 225.58 45.65 232.79 0.00 23.09 12.47 13.87 11.54 0.00 8.25 242.70 84.64 49.23 31.62 231.81 0.00 10.81 5.72 4.96 7.38 8.50 0.00 5.27 406.52 210.67 80.68 254.89 72.26 261.10 0.00
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The topvalue listed is Bartlett’s test statisticBK-Lfor theequivalence of variances calculated from eqs 8 and 9. The bottom value listed is the Hotelling’s test statistic test for differences between two multivariate group means given by eq 7. Hexose products group identification: A, allose; R, altrose; G, galactose;L,glucose;U,gulose;I, idose; M, mannose; T, talose. Six replicate mass spectra were taken in each group.
L4 LS L6 U6 Figure 12. Cluster analysis dendrogram for mass spectra of the deuterated hexito1hexaacetateproducts from the c-aidohexoses (data set 4) based on Euclideandistances and the average linkage algorithm. Symbols: (A) allose; (0)galactose; (I) idose; (M) mannose; (R) altrose: (1)taiose; (L) glucose; (U) gulose. Numbers indicate the six different samples In each cluster.
ary phase and appear identical by E1 mass spectrometry. These results are of course entirely expected. From the 16 aldohexoses, 10 nondeuterated compounds are generated by the alditol acetate method, of which only 6 are diastereomers of one another. The nondeuterated hexitol hexaacetates from L-glucose and L-gulose are enantiomers with respect to one another; they have the same retention time and identical mass spectra. The same is true for D-glucose and D-gulose. L-Altrose and L-talose produce the same hexitol hexaacetate; thus, their derivatization products also cannot be distinguished from one another by their E1 mass spectra (likewise for D-altrose and D-talose). These a priori expectations were fulfilled by the results of the studies reported here. Unfortunately, on the SP-2380 capillary column, nondeuterated altritol and talitol hexaacetates co-elute with mannitol hexaacetate. Five chromatographic peaks are observed for the six diastereomeric nondeuterated compounds. Remarkably, the E1 mass spectra also support these conclusions in
that, within experimental variability, six different mass spectra are found. Principal component analysis, cluster analysis, and hypothesis tests for the equivalence of the multivariate cluster means support the mass spectral differences between these six groups of diastereomers. With the deuterated hexitol hexaacetates, similar structural considerations apply. However, the addition of a deuterium label maintains the chiral nature of the anomeric carbon from the parent sugar. As a result, 32 compounds are possible by the alditol acetate method (of which 16 are diastereomers) from the 16 aldohexoses, 8 from the D series, and 8 from the L series. Once again the results verify these conclusions: eight different mass spectra can be distinguished within each series. Results from principal component analysis, cluster analysis, and hypothesis tests substantiate the ability of EI-MS to differentiate these aldohexoses by the mass spectra of their hexitol hexaacetate products. It is possible to identify unambiguously all eight aldohexoses (within the D or L series) Analytical Chemlstry, Vol. 66, No. 17, September 1, 1994
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using the distinctive E1 mass spectra of their hexitol hexaacetate products. Further, these differences in the mass spectra of the hexitol hexaacetates also suggest the possibility of numerically resolving overlapped peaks for mixtures of these compounds. Visual discrimination of the mass spectra is easily possible without the assistance of statistical analysis. In this work we have employed a suite of three different and complementary multivariate statistical techniques. PCA and cluster analysis were employed to reduce the dimensionality of the data and to allow easier visual and numerical discrimination of the samples. Multivariate tests of the equality of the cluster means were performed on the PCA scores to confirm the statistical significance of differences observed. Our results substantiate
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Analytical Chemistry, Vol. 66, No. 17, September 1, 1994
the conclusion that EI-MS can be used successfully for the discrimination of the diastereomeric nondeuterated hexitol hexaacetates as well as for the identification of the aldohexoses by their deuterated hexitol hexaacetate products.
ACKNOWLEDGMENT This work was supported in part by Army Research Office through Grant DAAL-90406. The assistance of Dr. James C. Rogers (R. J. Reynolds, Inc., Winston-Salem, NC) on GC separation and mass spectral interpretation of carbohydrates is also acknowledged. Received for review January 3, 1994. Accepted March 22, 1994. e Abstract
published in Advance ACS Absrracrs, May 1, 1994.
