2283
Anal. Chem. 1985, 57, 2283-2286
Acetone Chemical Ionization Mass Spectrometry of Monosaccharides Guanghui Wang* and Yixian Sha
Institute of Chemistry, Academia Sinica, Beijing, China Zhiling Xu and Jiongguang Pan
Institute of Chinese Materia Medica, Academy of Traditional Chinese Medicine, Beijing, China
It has been found that acetone chemical ioniratlon (CI)mass spectra of many stereoisomers of monosaccharides show apparent differences which do not appear In the corresponding isobutane or ammonla CI mass spectra. Factors affecting the CI mass spectra, such as source temperature, acetone pressure, surface condition of the sample tube, and amount of sample have been investigated to find the optimum experimental conditions for the ldentiflcatlon of these isomers.
The electron ionization (EI) mass spectra of monosaccharides and their volatile derivatives, such as methylates and acetates, are very insensitive to the stereochemistry of the molecules. Biemann (I)proposed a method to determine the structure of stereoisomeric pentoses and hexoses, in which the stereoisomers were condensed with acetone to form a cyclic 0-isopropylidene derivative prior to E1 mass spectrometry analysis. Later on, the E1 mass spectrometry of the cyclic alkaneboronate derivatives of sugars was studied for a similar purpose (2). By using negative ion fast atom bombardment mass spectrometry, Rose (3)made an interesting study of in situ reaction of sugar with boronic acid and obtained analytical information on stereochemistry. Each of these approaches is based on a certain appropriate bimolecular reaction which converts the stereoisomers to compounds with different actual bonding leading to significantly different E1 mass spectra. A well-known example is the reaction of D-galactose and Dglucose with acetone in which the former produces mainly pyranoid 1,2:3,4-di-O-isopropylidene while the latter producs 1,2:5,6-di-0-isopropylidene-~-glucofuranose. Although this approach is effective, it is not convenient. Chemical ionization (CI) mass spectrometry is another promising approach to get information on stereochemistry, where intramolecular reaction, such as hydrogen rearrangement, plays an important role. It has been demonstrated that many partially methylated glucitol, galactitol, and mannitol compounds can be differentiated by isobutane CI mass spectra of their acetates (4). However, by using methane, isobutane, or ammonia as the reagent gas, the CI mass spectra of the stereoisomeric monosaccharides were too similar for reliable differentiation of the isomers. A number of recent studies have already shown parallels between some well-known ionic reactions in solution and their gaseous counterparts (5-7). This encouraged us to study the gas-phase bimolecular reaction of protonated acetone with monosaccharides under CI mass spectrometric conditions with the hope that it would give more stereochemical information than the unimolecular reaction.
EXPERIMENTAL SECTION The mass spectrometer employed was a VG 7070H coupled to a VG 2035 data system. The accelerating voltage was maintained at 4 kV, the electron energy at 200 eV, and the electron
emission current at 500 FA. The exponential scan was 2 s/decade. Acetone was introduced to the source through the septum inlet. The monosaccharide sample (Packard-Becker, Ltd.) put in a quartz sample tube (shallow cup) was introduced to the ion source by direct insertion probe and heated by the ionization cage maintained at 150-170 "C (depending on the volatility of the sample). All of the mass spectral data of the monosaccharides presented were obtained from the average spectra of ten successive scans recorded during the time when the maximum total ion currents were observed.
RESULTS AND DISCUSSION Reactant Ion. The mass spectrum of acetone at ionization cage pressure of 0.5 mbar (estimated) and source temperature of 170 "C is shown in Figure 1. The conspicuous feature of the spectra is that the base peak does not correspond to the protonated acetone (mlz 59)but to its dimer (mlz 117)which amounts to about 60% of the total ion current and no trimer was observed. Figure 2 shows the mass chromatograph of the reactant ions as glucose was introduced into the source. It illustrates that mlz 117 ion is the major reactant ion, and another reactant ion of interest is the less abundant m / z 59 ion. Both of them may undergo condensation with monosaccharides to give stereochemical information. The abundant ratio of mlz 59 and mlz 117 ions changes with the acetone pressure and reaches a minimum value of about 0.15 around source housing pressure of 2.5 X mbar (Figure 3) which corresponds to ionization cage pressure of 0.5 mbar. Reaction of Protonated Acetone with Monosaccharides. With acetone as the ionizing gas, a variety of reactions between the reactant ions and the sample molecules as well as the consequent unimolecular reactions will occur. We consider that the following consecutive reactions will most likely give useful stereochemical information:
iH
M t CH3CCH3 ( o r d i m e r )
(M + 59)+
-
-H,O
( M t 59;
(M + 41)+
(1)
(2) where M is the molecule of the monosaccharide, ( M 59)+ is the protonated acetone-monosaccharide adduct ion, and (M+ 41)' is its dehydrated product. Reaction 2 is verified by linked scan at constant BIE. For different stereoisomers of the parent monosaccharides, the dehydration favorability of (M + 59)' may be different, and thus markedly different relative abundances of (M + 41)' ion vs. ( M + 59)+ ion can be obtained. For example, the value of the ( M + 41)+/(M + 59)' ratio is 1.50 for glucose and 0.51 for galactose. It should be noted that the (M + 41)' ion can also be formed by dehydration of monosaccharide prior to formation of the protnated acetone adduct ion. This reaction route does not give useful stereochemical information, since dehydration favorability of different stereoisomers of monosaccharides does
0003-2700/85/0357-2283$01.50/00 1985 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 57, NO. 12, OCTOBER 1985
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111
3001
100
50
150
mtz
Flgure 1. Mass spectra of acetone at ionizatlon cage pressure of 0.5 mbar and source temperature of 170 "C.
I
I
I
170
150
1 oc
Source temperature
+
190
(QC)
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Flgure 4. Relation between (M 41)+/(M 59)' ratlo and the source temperature for glucose and galactose: 0, galactose; V, glucose.
tween (M + 41)+/(M + 59)+ ratio and source temperature for D-glucose and D-galactose is shown in Figure 4 (acetone pressure 0.5 mbar). The two curves are almost parallel to each other, each with a minimum value of (M + 41)+/(M 59)+ ratio at about the same source temperature. Thus, in spite of the apparent change of (M + 41)+/(M 59)+ ratio with the source temperature, the difference between values of (M + 41)+/(M + 59)' ratios for glucose and galactose at the same source temperature is significant enough for reliable differentiation of the stereoisomers. Lower source temperature is unfavorable to dehydration of (M + 59)+ ion. Thus, if water elimination from (M 59)+ ion were the only pathway to produce the (M + 41)' ion, the (M + 41)+/(M + 59)+ ratio would have decreased with the decrease of source temperature. However, Figure 4 shows that as the source temperature goes down below 160 "C the curves concave upward. This may be attributed to the fact that
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5c
+
I . . . P
Scan number
40
Flgure 2. Mass chromatograph of the reactant ions as glucose was introduced into the source: A, m l z 117; B, m l z 101; C, m l z 73; D, m l z 59; E, m / z 43; F, total ion current 2125
I
(M + 41)+/(M + 59)+ = (M + 59 - H,O)+/ (M + 59)' + (M - H2O + 59)+/(M + 59)+ where (M + 59 - H20)+is the dehydrated product of (M +
+
59)' ion and (M - H 2 0 59)' is the dehydrated hexoseprotonated acetone adduct ion. As mentioned above (M + 59 - H20)+/(M 59)" ratio should decrease with the decrease of source temperature. However, as the source temperature goes down below 160 "C, the rate of vaporization may decrease more rapidly than the rate of dehydration for the hexoses; that is, the abundance of (M 59)+ ion decreases more rapidly than that of (M - H 2 0 + 59)+ ion. Conesequently (M - HzO + 59)+/(M 59)+ ratio may increase with the decrease of source temperature, and when the amount of increase exceeds that of the decrease of (M 59 - HzO)+/(M+ 59)+, (M + 41)+/(M + 59)+ ratio may also increase to give the upward curve. If the source temperature is kept within the range of 160-170 "C, the value of (M 41)+/(M 59)' ratio changes only slightly, and good reproducibility can be obtained. (2) Acetone Pressure. The CI mass spectra are often affected markedly by the pressure of the reagent gas. In order to assure the reproducibility of the mass spectra, it is necessary to investigate the influence of acetone pressure on (M + 41)+/(M + 59)+ ratio in order to find the optimum pressure for stereoisomer identification. Due to the high accelerating voltage (4kV) of the magnetic mass spectrometer, it is difficult to measure accurately the ionization cage pressure when the mass spectrometer is running. Therefore both the source housing pressure and (59)+/(117)+ ratio were used to indicate the relative cage pressure. Figure 5 illustrates (M 41)+/(M + 59)' as the function of the acetone pressure for D-glucose and D-galactose (source
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I
'"1
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1
2
3
4
5
6
7
Source housing pressure (XI0-5mbar)
Flgure 3. Abundance ratlo of ml z 59 and m lz 1 17 Ions as a function of the source housing pressure. (Source housing pressure of 2.5 X lo-' mbar corresponds to ionization cage pressure of -0.5 mbar.)
not differ significantly. However from the relative abundance of (M + NH4)+and (M + NH4- H20)+ ions in the ammonia CI mass spectra of the monosaccharides, it seems unlikely that under proper experimental conditions the dehydration of monosaccharides in the ion source would occur to a significant extent. Factors Influencing the Abundance Ratio of (M + 41)+ and (M 59)+. (1) Source Temperature. The relation be-
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ANALYTICAL CHEMISTRY, VOL. 57, NO. 12, OCTOBER 1985
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.IO0 A/
-e a
U
C
50
2 0
c
'
2
1
4
3
5
Source housing pressure ( x l 0.5 mbor)
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20
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Figure 5. Relation between (M 41)+/(M 59)' ratio and the source housing pressure for glucose and galactose: 0,galactose; 0, glucose; V, (59)+/(117)+ ratio of acetone.
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Table I. Value of (M 41)'/(M 59)' Ratio Obtained from Different Amounts of Glucose and Galactose sample
amt of sample, rg
(M + 41)'/(M ratio
D-glucose D-glucose D-glUCOSe
10 30 60
152/100 133/100 148/100
D-galaCtOSe D-galaCtOSe D-galaCtOSe
10 30 60
72/100 71/100 75/100
+ 59)'
+
sample
+ 59)'
+
and (M
+ 41)'
re1 abundance (M + 59)' (M + 41)+
25 30 Scan number
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35
Flgure 6. Variation of (M 41)+/(M 59)' ratio during the whole process of the vaporization of 30 pg of galactose: 0, (M 41)+/(M 59)+ ratio; V, total ion current.
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smooth, it is recommended that it be cleaned with water and organic solvents each time after analysis. Burning off the sample residue by heating the sample tube in the flame is not recommended because it will lead to a rough surface. Use of a quartz rod instead of the sample tube with the sample deposited on its surface will also give satisfactory results. (4)Amount of the Sample. Table I lists the values of the (M 41)+/(M 59)+ ratio for different amounts of D-glucose and D-galaCtOSe (source temperature 170 "c,source housing pressure 2 X mbar and (59)+/(117)+ = 4/10). The data show that the amount of the sample does not significantly influence the value of the (M + 41)+/(M + 59)' ratio. The variation of the (M 41)+/(M + 59)' ratio during the process of the vaporization of 30 pg of D-galactose is shown in Figure 6. The use of the average spectrum of ten successive scans recorded during the time when the maximum total ion current were observed improves the reproducibility of the spectra. R e l a t i v e A b u n d a n c e o f (M + 41)+ and (M + 59)+ I o n s f o r Some Monosaccharides. The acetone CI mass spectra of D-glucose and D-galaCtOSe (Figure 7) and L-rhamnose and L-fucose (Figure 8) are presented for intuitive comparison. Table I1 compares relative abundances of (M + 59)+ and (M + 41)+ ions for some common monosaccharides. With glucose and galactose as the samples, good reproducibility of the mass spectral data was obtained at the source temperature of 170 "C and acetone pressure of -0.5 mbar. The average value and standard deviation from five measurements of the (M + 41)+/(M + 59)+ ratio obtained on different dates is 1.51 f 0.15 for glucose and 0.63 f 0.14 for galactose. The fact that the acetone but not the isobutane or ammonia CI mass spectra of some stereoisomers of monosaccharides shows apparent difference implies that the difference might
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temperature 170 "C). The two curves are parallel to each other and also to the curve of (59)+/(117)+w. acetone pressure with a minimum value at almost the same acetone pressure. This means that the more abundant the m / z 117 ion is, the higher the relative abundance of (M + 59)' vs. (M + 41)' ions will be. The phenomenon implies that (M + 59)' ion formed through the reaction of m / z 117 ion with the sample molecule possesses lower internal energy. The acetone pressure for the minimum value of (59)+/(117)+ ratio is used for the identification of the stereoisomeric aldohexoses, since under this condition change of (M + 41)+/(M + 59)+ ratio with the pressure is reduced to a minimum and good reproducibility of (M + 41)+/(M 59)+ ratio can be obtained. (3) Surface Condition of the Sample Tube. It has been found that a sample tube with rough surface will cause tremendous increase and poor reproducibility of the value of (M + 41)+/(M + 59)' ratio. This is not surprising since a rough surface is unfavorable to the vaporation of the monosaccharide molecule. In order to keep the surface of the sample tube Table 11. Relative Abundance of (M
2
+
+
Ions for Some Common Monosaccharides
source temp, O C
acetone pressure source housing pressure x mbar (59)'/(117)'
D-galactose D-glucose D-mannose
100 100 100
51 150 132
170 170 170
2.4 2.4 2.4
2/10 2/10 2/10
D-fructose L-sorbose
100 100
480 1500
170 170
2.4 2.4
2/10 2/10
L-arabinose D-xy1ose D-ribose D-lyxose
100 100 100 100
110 90 50 38
160 160 160 160
1.3 1.3 1.3 1.3
4/10 4/10 4/10 4/10
L-rhamnose L-fucose
100 100
53 178
150 150
2.4 2.4
2/10 2/10
ratio
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ANALYTICAL CHEMISTRY, VOL. 57, NO. 12, OCTOBER 1985
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D-GLUCOSE 221 239
the range of 1.51 f 0.15. Aqueous solution and methanol solution of @-D-glucosegive values of (M + 41)+/(M+ 59)+ very close to those by solid fl-D-glucose (within *lo% deviation) and samples of D-glucose and D-galactose collected from HPLC column (using acetonitrile-water as mobile phase) also show the values of (M 41)+/(M 59)+ ratio within the ranges of 1.51 f 0.15 and 0.63 f 0.14 respectively, which are very consistent with those from pure compounds. In the acetone CI mass spectra of the stereoisomers of monosaccharides, there are actually some peaks other than (M + 41)' and (M 59)+which also show difference in relative abundance in the spectra. For example, D-glucose shows a peak at mlz 201 which is much weaker in D-galactose spectrum, while the later exhibits a much more prominent peak a t m/z 181 (Figure 7). Similarly, the peak at m / z 165 from rhamnose is a very weak one in the spectrum of fucose which instead shows a prominent peak at mlz 169 (Figure 8). In addition the protonated monosaccharide dimer may also produce a dehydration ion series with different relative abundances for different isomers. Figure 8 shows the relative abundances of mlz 329 (protonated dimer), m/z 311 (m/z329 - H,O), mlz 293 (m/z 329 - 2H20),m/z 275 (m/z 329 -3H20), and m/z 257 ( m / z 329 - 4H20) for rhaminose which are significantly different from those of fucose. The above results may also serve as information for the differentiation of stereoisomers of monosaccharides. Registry No. D-Galactose, 59-23-4; D-glucose, 50-99-7; Dmannose, 3458-28-4; D-fructose, 57-48-7; L-sorbose, 87-79-6; Larabinose, 5328-37-0;D-xylose, 58-86-6;D-ribose, 50-69-1;D-lyxose, 1114-34-7;L-rhammose, 3615-41-6;L-fucose, 2438-80-4;acetone, 67-64-1.
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D-GALACTOSE
239
E 0
I
J
+
221
50,
150
250
350
mIz
Flgure 7. Acetone C I mass spectra of Pglucose and Pgalactose: m l z 239 is (M 59)+; m l z 221 is (M 41)+.
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+
RHAMNOSE 223 . I
.-cP
150
$100
293
250
I
z
+
350 FUCOSE
LITERATURE CITED 150
250 ml z
350
Flgure 8. Acetone C I mass spectra of L-rhamnose and L-fucose: rnlz 223 is (M 59)'; rnlz 205 is (M 41)'.
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+
be brought about by the condensation of two hydroxyl groups (adjacent or accessible in some other way) on the sugar molecules with the protonated acetone. No significant difference has been found among the acetone CI mass spectra of a-D-glucose, P-D-glucose, and L-glucose (samples from Packard-Becker; Ltd). All values of (M + 41)+/(M 59)+ratio obtained from these spectra are within
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(1) DeJongh, D. C.; Blemann, K. J . Am. Chem. SOC. 1984, 86, 67 (2) Radford, T.; DeJongh, D. C. "Biochemical Applicatlans of Mass Spectrometry"; Waller, G. R., Dermer, 0. C., Eds.; Wlley-Interscience: New York, 1980; 1st supplementary volume, pp 259-282. (3) . . Rose, M. E.: Lonastaff. C.; Dean, P. D. G. Biomed. Mass SDecfrom. 1983. 10, 512 (4) McNell, M.; Albersheim, P. Carbohydr. Res. 1977, 56, 239 (5) Wesdemiotis, C.;McLafferty, F. W. Org. Mass Specfrom. 1981, 16,
-
RR 1
(6) ;ish, G. L.; Cooks, R. G. J . Am. Chem. SOC. 1978, 100,6720 (7) Audier, H. E.; Flammay, R.; Maquestiau, A.; Milliet, A. N o w . J . Chim. 1980, 4 , 531
RECEIVED for review October 26,1984. Resubmitted May 28, 1985. Accepted May 28, 1985.
