Anal. Chem. 1980, 52,49-52
(32) J. Franzen, K. H. Maurer, and K. D. Schuy, 2. Naturforsch A , 21, 37 (1966). (33) C. W. Hull, Proc. 70th Annu. Conf. Mass Spectrom. Allied Topics, New Orleans, La., 1962. (34) J. R. Woolston, RCA Rev., 26, 539 (1965). (35) R. L. Davison, D.F.S. Natusch, J. R. Wallace, and C. A. Evans, Jr., Environ. Sci. Techno/., 8, 1107 (1974). (36) H. H. Willard, L. L. Merrit, Jr., and J. A. Dean, "Instrumental Methods of Analysis", 5th ed.,D. Van Nostrand, New York, 1974, pp 671-702. (37) L. 9. Rodgers, Anal. Chem., 22, 1386 (1950). (38) H. J. Gluskoter and P. C. Lindahl, Science, 181, 264 (1973). (39) J. A. Campbell, J. C. Laul, K. K. Nielson, and R. D. Smith, Anal. Chem., 50, 1033 (1979). (40) D. W. Koppenaai, R. G. Lett, and F. R. Brown, Pittsburgh Energy Tech-
49
nology Center, US. Dept. of Energy, unpublished research, 1978.
RECEIVED for review May 29, 1979. Accepted October 9, 1979. One Of the authors (D.W*K.)gratefully in the form of a Laboratory Graduate Participantship by Oak Ridge Associated universities, presented in part at the 1979 Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, March 5-9, 1979, Cleveland, Ohio. Reference to trade names does not imply Government endorsement of commercial products.
Reduction of Keto Acids and Differentiation between Isomers by Chemical Ionization Mass Spectrometry David Issachar and Jehuda Yinon" Department of Isotope Research, The Weizmann Institute of Science, Rehovot, Israel
Mass spectrometric reduction has been found to occur in chemical Ionization (CI)of some keto compounds. Although the reagent gas is the major reducing agent, in a-ketoisovaleric and a-ketoisocaproic acid also the sample molecule itself participates actively in the reduction process. M f 3/M -k 1 ion ratios have been measured for all investigated compounds and serve as an indication of reduction rate. Large differences in reduction rates have been observed which can be used as a unique method for the differentiation of some Isomers.
In the analysis of carboxylic acids in body fluids by mass spectrometry, one of the major problems is the identification of the ion peaks in the mass spectrum, especially the differentiation between isomers. This problem becomes even more acute when using chemical ionization (CI) because the mass spectrum contains mainly the (M + 1) ion with the support of only a few fragments. During our work on the CI mass spectrometry of carboxylic acids in urine ( I ) , we observed M 3 ions in the mass spectra of some of the keto carboxylic acids. Mass spectrometric reduction, occurring in an electron impact (EI) ion source, forming M + 2 ions, has been observed years ago in benzoquinones (2, 3 ) and in naphthoquinones ( 2 ) . It was believed ( 2 , 3 ) that the origin of the reducing hydrogen was from the water which is always present in trace amounts in the inlet system and ion source of the mass spectrometer. The purpose of this paper is to report on the reduction of some keto carboxylic acids in chemical ionization and on the application of these results for the differentiation of isomers in the CI mass spectra of these acids.
+
EXPERIMENTAL General. All mass spectra were recorded with a DuPont 21-490 B mass spectrometer equipped with a dual EI/CI source. Reagent gas pressure in the source was 0.2-0.5 Torr. Electron emission current was 300 WAand electron energy, 300 eV. Multiplier gain was lo6 and electrometer amplifier input resistor, 10' n. The sample is in the free acid form after extraction with ether from the salt. About 2 ILLof the ether solution is introduced with 0003-2700/80/0352-0049$01.00/0
the solid probe into the ion source of the mass spectrometer, after the ether has been pumped away by the vacuum pump of the probe introduction system. Chemicals and Reagent Gases. Carboxylic acids and naphthoquinones were analytically pure and obtained from Sigma Chemical Company, St. Louis, Mo.; Fluka AG, Buchs, Switzerland; and BDH Chemicals Ltd., Poole, United Kingdom. Isobutane, methane, and ammonia were high purity grade and obtained from Liquid Carbonic, Chicago, Ill. Methane-d, (99% isotopic purity) and isobutane-dl0(98% isotopic purity) were obtained from Merck Sharp & Dohme, Pointe Claire-Dorval, Quebec:, Canada. R E S U L T S AND D I S C U S S I O N The appearance of reduced ions in the CI mass spectrometry of keto carboxylic acids raises the question whether part of the introduced sample molecules was already in reduced form before entering the ion source. From NMR and IR spectra of these compounds, it was found that they were not in reduced form before introduction into the mass spectrometer. According to the NMR analysis the maximum amounts of hydroxy acids that could be present in the samples is less than 0.1 mol 70. Table I shows the M + 3/M + 1 ratios of a series of isomeric keto acids and naphthoquinones. The experimental results as shown in this table indicate the possible use of C1 reduction to differentiate between isomers of keto compounds by observing the M + 3 / M + 1 ratios in the CI mass spectra. This reduction process could also be used to differentiate between some acids having approximately the same molecular weight without using high resolution mass spectrometry. A few examples are shown in Table 11. Reduction seems to occur not only in a-ketocarboxylic acids but also in compounds which have an a-carboxy double bond like fumaric and maleic acids. From the results shown in Tables I and 11, it can be seen that the described reduction behavior is characteristic of keto compounds and that the a-ketoisoacids are reduced to a larger extent than the other compounds. CI mass spectra were also recorded with CD4 and i-C4Dlo as reagents. The results substantiated the assumption that the (M 3)+ ions are (M + 3H)+ ions. The next question is what is the origin of the reducing hydrogen atoms?
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0 1979 American Chemical Society
50
ANALYTICAL CHEMISTRY, VOL. 52, NO. 1, JANUARY 1980
Table I. Reduction Ratios in CI-Isobutane of Some Isomeric Ketocarboxylic Acids and Other Keto Compounds
compound
MW
a-ketovaleric
reduction ratio,'
base peak
+
M+
structure 0 0 /I 11
source temp, 3/M t 1 "C
CIS I,,
I,, I,,
0.17 i 0.05
70
0.1
9
18.0i 4.0
70
0.1
9
no detectable M + 3 ion
70
0.1
9
100
0.1
9
0.05
100
0.1
9
0.15 i 0.05
70
0.1
9
a-ketoisocaproic
3.0
70
0.1
9
a-keto-p-methylvaleric
0.30
0.05
70
0.1
9
1,2-naphthoquinone
0.10 -r 0.01
70
0.1
9
1,4-naphthoquinone
0.02
70
0.1
9
116
M
CH3CH,CH,C-COH
1
00
116
a-ketoisovaleric
M+ 3
/I 11
CH'\
cH?/Cb3-c3r
0
7-ketovaleric
116
M-t 1
fumaric ( truns-l,2-ethylenedicarboxylic)
116
M+3
0 I1
!I
CH,CCH,CH,COH HOOC
HOOC \
maleic (cis-1,2-ethylenedicarboxylic)
116
M + 1
,c=/
a l80
130
contribution to the ( M
+
M
+
1
0.08
"
H
a-ketocaproic
1.5 t 1.0
\cool. coot-
d'
00 /I I/
CH,CH,CH,CH,C-COH
i
0.4
i
A
i
0.01
3)' ion has been subtracted.
Table 11. Reduction Ratios in CI--Isobutane of Some Carboxylic Acids
compound ketomalonic
MW
117.990
base peak
M+
1
reduction ratio,
HOC-C-COH 0
succinic
118.026
M
+
1
0
I1
HOCCH,CH,COH
LIS -
15, -
I,,
143
la
c
0.10 i 0.02 M t 3 ion in range of experimental error
70
0.1
9
70
0.1
9
no detectable M + 3ion
70
0.1
9
0.10
70
0.1
9
70
0.1
9
M + 3/M +
structure 0 00 I1 /I ' 1
source temp,
0
2-hydroxyisovaleric
118.063
M
+
II
1
'CHCH( oH)COH I
CH, a-ketoglutaric
146.021
Mt 1
0 I1
00 /I I1
HOCCH,CH,C-COH 0
0
11
I/
adipic 146.058 M+ 1 HOCCH,CH,CH,CH,COH ' l80contribution t o the (M+ 3)' ion has been subtracted.
If we believe that the origin of the reducing hydrogen is from water traces like in E1 ( 2 , 3 ) , then CI with water as reagent should enhance the reduction process. Figures 1 and 2 show the CI-water and CI-isobutane mass spectra of ketomalonic acid and 1,2-naphthoquinone, respectively. Comparison of these mass spectra shows that in CI reduction, water is not the major contributor of the reducing hydrogen atoms. The M + 3 / M + 1 ratios in the CI-water mass spectra are even smaller than in the CI-isobutane mass spectra.
i
0.02
no detectable M + 3 ion
Measurements of the M + 3 / M + 1 ratios of the keto compounds as a function of reagent gas pressure showed an increase in the ratio intensity with increasing reagent gas pressure. Figure 3 shows the M + 3 / M + 1 ratio in CI-isobutane of a-ketoisovaleric acid as a function of the isobutane pressure which is represented by the ratio of the t-C4Hgtand C3Hit ion intensities ( 4 ) , Z57/143. This dependence indicates that the reagent gas participates actively in the CI reduction process. We have also observed that with isobutane as reagent the rate of reduction is greater
ANALYTICAL CHEMISTRY, VOL. 52,
NO. 1, JANUARY
LJM+’ I
+
I
Figure 1. CI mass spectra of ketomalonic acid with isobutane and water as reagents. In the CI-isobutane spectrum, 157/I.,3 = 9 and E I s / I 5 ,= 0.1; in the C1-water spectrum, CI,/I,9= 0.1
than with water or methane as reagents. Examples comparing the reduction ratios using isobutane and water as CI reagents are given in Figures 1 and 2. Although the source pressure is by one order of magnitude lower when using water as reagent than with isobutane or methane (5,6),the increase of water pressure does not increase the M + 3/M 1 ratio. Another important factor in CI reduction is the amount of sample introduced into the source. As the samples were introduced through the solid probe, it was very difficult to measure the actual pressure of the sample in the ion source.
+
C I YRTER
MtI
i
*O
1
l
50
j
Y + 3
I
1 oc
i
5C
,/
230
,,
.25C , .
. ,
,
3c:
WE COMPOJNO
I,2-NRPYTYOPUINONE
SOURCE TEMP. ‘00
i 20-
t
I,/”*‘ I
51
The sample pressure was represented in our measurements by the ratio of the total sample ion current over the reagent ion current. In isobutane the reagent ion is tK4Hg+,therefore in methane the reagent ions are CH5+ this ratio was CZs/Z57; and CzHSt,therefore the ratio was ZZ8/Zli Z29; and in water, where the reagent ion is H30t, the ratio was xZ8/Zlg. We observed a decrease in the M + 3/M + 1 reduction ratio with increasing sample pressure for most of the studied compounds. An example is given in Figure 4 which shows the CI reduction ratio M 3 / M + 1 of ketomalonic acid and of 1,2-naphthoquinoneas a function of relative total ion current. Exceptions to this behavior are two compounds: a-ketoisocaproic and a-ketoisovaleric acid which show an increase in the reduction ratio with increasing sample pressure. An example is given in Figure 5 which shows the M + 3/M + 1 reduction ratio of a-ketoisovaleric acid in CI-isobutane as a function of relative total ion current. It seems that in a-diketo systems having an isopropyl group, like a-ketoisovaleric and a-ketoisocaproic acid, the sample molecule itself is active in the CI ion-molecule reduction process. In the other investigated compounds the sample itself is passive in the reduction process while the reagent gas is the major reducing agent. Therefore when increasing the relative sample pressure in the ion source, the M + 3/M + 1 ratio will decrease because the amount of available reducing species is limited. There is insufficient data to suggest a founded mechanism for the presented reduction reactions. Therefore it is difficult to state what ionic species of the reagent participate in the reduction process and what is their relative concentration. The reduction enhancement of compounds having isopropyl groups can also explain the higher reduction capability of isobutane over methane and water. An example is given in Figure 6 which shows the CI mass spectra of cx-ketoisovaleric acid with isobutane and methane as reagents. The M + 3/M + 1 reduction ratio is larger with isobutane as reagent than with methane.
+
i
1980
,
E
52
ANALYTICAL CHEMISTRY, VOL. 52, NO. 1, JANUARY 1980 I5
l
l
l
l
a-Ketotsovaieric
l
l
l
l
acid
14
~
1
!
C1 ISOBU‘RNE
0c
t , . 6C.
: e
.
1
0
1
+
2
3
4
5
6
7
8
I57’14,
+
Figure 3. M 3/M 1 ratio in CI-isobutane of a-ketoisovaleric acid as function of reagent gas pressure. X I s = constant. Source temperature, 70 OC r
I
I
I
I
I
CI-I s o b uta ne 0 - Ketomalonic a c i d ~-1.2-Naphtoquinone
3.“101\
4.0
2
I
‘I I 1
----T
