Instrumentation
20
30
40
50
60
70
80
Relative Isotope Abundance (Atom %)
Figure 1. Percent accuracy and precision of isotope abundance measurements as functions of relative abundance on gases introduced from reservoir
Direct Analysis of Stable Isotopes with a Quadrupole Mass Spectrometer R. M. Caprioli Department of Chemistry Purdue University West Lafayette, Ind. 47907 W . F. Fies and M. S. Story Finnigan Instrument Corp. 595 N. Pastoria Avenue Sunnyvale, Calif. 94086
Isotope ratios can be determined with high precision by quadrupole mass spectrometry even under the dynamic conditions imposed by combined gas chromatography-mass spectrometry. Applications abound especially in chemistry, biology, and medicine
Interest in the use of stable isotopes has sharply increased in recent years in all fields of chemistry, biology, and medicine as a combined result of a decrease in the cost of stable isotopes, advances in the design and performance of magnetic resonance and mass spectrometric instrumentation, and the general concern over the hazards of radioactive isotopes. Of particular utility in isotope methodology is the emergence of mass spectrometric techniques designed to simultaneously determine the abundance of a particular isotope and also its location within the molecule. This direct analysis thus provides much structural information and has considerable advantage over other isotope techniques which require complex degradative procedures to locate the isotopic atom. Stable isotopes have been used in a wide variety of applications, most of which involve their use as tracers either to follow the fate of a particular atom in a reaction series or an entire molecule in a complex system. For example, tracing particular atoms of a molecule is vital to the elucidation of the origin of certain atoms of cellular metabolites in biosynthetic stud-
ies (1, 2) or in the study of reaction mechanisms where the fate of one or more atoms is diagnostic of a particular mechanism (3, 4). Specific isotopic labeling is also used when the fate of the entire molecule is of interest, such as in studies of the metabolism of drugs or other compounds in living cells (5) or in isotope dilution experiments in which quantitative analyses are sought. In view of the increasing use of stable isotopes, it is becoming of critical importance to develop mass spectrometric techniques which attain better accuracy in the measurement of isotope abundances in complex molecules. In addition, the use of the technique of mass fragmentography in which samples containing mixtures are analyzed by continuous ion monitoring also requires accurate ion intensity measurements if quantitative data are to be obtained. This is of significant value in the analysis of drugs, drug metabolites, and other compounds at subnanogram levels (6). Direct Analysis of Stable Isotopes
Direct analysis of stable isotopes involves measurement of ion intensi-
ANALYTICAL CHEMISTRY, VOL. 46, NO. 4, APRIL 1974 · 453 A
ties of the several isotopic species of a molecular or fragment ion formed in a mass spectrometer. In addition, if structural information is required, a study of fragmentation reactions through the use of normal and specifically labeled compounds may also be necessary. The alternative method for the mass spectrometric analysis of stable isotopes is indirect and involves the combustion of the sample to a gas, usually H2 for deuterium analysis, N2 for 1 5 N analysis, and C 0 2 for i 3 C and 1 8 0 analyses, followed by measurement of the isotope ratios of these gases (7). However, this method has the disadvantages of providing no structural information; requiring relatively large amounts of sample, usually at least 1 mg; and poor accuracy as a result of the simultaneous combustion of contaminants or incomplete combustion, even though the mass spectrometric ratio measurement may have a precision as high as ±0.001%. Although direct analysis of stable isotopes is the method of choice in many cases, it has not become a routine tool because of the relatively poor precision obtained in isotope ratio determinations by use of magnetic mass spectrometers. To be of general utility, methods for direct analysis must be able to achieve a precision of approximately ±0.1% or better with microgram or nanogram quantities of sample injected into the GC inlet of a mass spectrometer. Many biomedical experiments involve the isolation of small amounts of samples containing low isotope abundances. In many cases, even high initial isotope concentrations give low abundance products owing to the enormous dilution of the isotopic compound by the system. In addition, better precision in measurement would also permit the use of isotope methodology in low enrichment experiments where, previously, the high cost of high enrichments was prohibitive. To achieve high precision in the measurement of the intensities of two or more ions, it is desirable to measure these ions simultaneously in the manner of double collector techniques used with isotope ratio mass spectrometers. However, the fixed focus of magnetic instruments and use of a Faraday cup collector, which are essential to the high precision achieved with this method, do not lend themselves to ratio measurements on high mass and low abundance ions. The potential solution to these problems lies in the utilization or repetitive scanning techniques. Thus, Hites and Biemann (8) used continuous rapid scanning over a given mass range to measure ion intensities. Although this was an improvement over data ob-
tained from individual mass spectra, scanning a portion of the mass spectrum in this manner results in poor ion statistics since a great deal of time is spent between peaks where ions are not collected. In another approach, Sweeley et al. (9, 70), Klein et al. (11), and, more recently, Holmes et al. (12) and Watson et al. (13) used an accelerating voltage alternating (AVA) device to continuously switch the accelerating voltage in a cyclical manner so as to successively focus a series of ions at the collector. The precision of isotope ratio measurements achieved with this method was approximately ± 1 % . Limitations of the AVA technique are detuning of the ion source when the accelerating voltage is changed, relatively slow switching rates demanded by the high voltages involved, and the requirement that the mass difference in ions to be compared may not be greater than about 40%. Advantages of Quadrupoles Quadrupole mass analyzers present some attractive features in the pursuit of higher precision isotope ratio measurements. First, the electrostatic voltages used to produce mass dispersion can be switched rapidly and measured accurately. Thus, in a high switching rate mode, each ion of an isotope series would be collected for a time on the order of milliseconds with continuous cycling over the series of ions to be measured. Such a system would approach the ideal of simultaneous collection of ions used with the double collector method. Thus, with rapid switching, only those instabilities or pressure changes comparable to the switching rate will affect the abundance measurements. Since the dead time or settling time between masses is small ( ~ 1 msec), almost all the time the sample is in the instrument is spent collecting the ions of interest. Thus, compounds emerging from the GC having a duration of several seconds can also be analyzed. Second, ions in a particular series may be collected for different amounts of time depending on their relative abundances, maximizing ion statistics. Third, any ions in the spectrum, no matter their mass difference, may be measured in a particular analysis and need not lie within a given percentage of the mass range. During the past five years, the improvement of quadrupole mass spectrometer design and performance has virtually eliminated difficulties generally associated with these instruments, such as lack of high mass sensitivity and peak-tailing, and has provided capabilities which are comparable to magnetic deflection instruments in this regard. Quadrupole mass spec-
454 A · ANALYTICAL CHEMISTRY, VOL. 46, NO. 4, APRIL 1974
trometers, already in the forefront of GC/MS methodology, further hold the potential of becoming a primary instrument for the direct analysis of stable isotopes. In the work described here, isotope abundances were determined on a variety of samples with a quadrupole mass spectrometer. These include some of the inert gases and 1 8 0-labeled sugars. The inert gases are ideal samples for testing both precision and accuracy of isotope abundance measurements because their abundances are known exactly and because there is a single isotopic species per mass, unlike organic compounds where there may be mixtures of carbon, hydrogen, nitrogen, and oxygen isotopes at a particular mass. In addition, the range in the relative abundances is large, being nearly 1600:1 for m/e 40:m/e 38 for argon and 1:1 for certain isotopes of krypton and xenon. Thus, in the experiments which follow, Ar, Kr, and Xe were employed as standards. Their isotope abundances were measured by use of a data system to analyze samples introduced both from a reservoir inlet in which the pressure is stable and from the GC inlet in which the pressure is constantly varying. The measurements obtained from the data system were also compared with those obtained with the programmable multiple ion monitor (PROMIM) coupled to digital integrators. A second series of experiments was performed involving measurement of isotope ratios in fragment ions of natural abundance and 18 0-enriched pentaacetylglucose samples introduced into the mass spectrometer via the GC inlet. This experiment was also performed with the multiple ion monitor-integrator system, and the results were compared with those obtained from the data system. The precision and accuracy of the measurement techniques are given together with a discussion of critical parameters involved in such measurements . Experimental The inert gases (analyzed grade) were obtained from Mathieson Gas Products. (2- 18 0) and 6- 18 0) pentaacetylglucose were prepared and analyzed as previously described (12). A Finnigan Model 3100 quadrupole mass spectrometer equipped with a Model 9500 gas chromatograph and a Model 6000 data system was used for the determination of isotope abundances. For ion detection, a 14-stage beryllium-copper electron multiplier was employed. The mass spectrometer was equipped with a 1-liter reservoir inlet system for the introduction of gases. For GC studies, a 3-m x 2-mm
(i.d.) OV-1 column was used. A mass defect adjust device was installed in the mass calibrate circuit so that after calibration with perfluorotributylamine, the mass set point could then be moved the proper fraction of a mass unit, depending on the mass defect of the ion to be measured, to monitor the top of the mass peak. That this indeed occurred was veri fied by using the "diagnostic" mode of the data system which allows the operator to observe on the oscillo scope display the peaks of interest and the point on these peaks at which the instrument is calibrated. Ion intensities for the isotope abun dance determinations were obtained by using the mass fragmentography program of the data system in which, for a given mass, ions are collected through a series of sampling times, 1, 2, 4, 8, 16, 32, and 64 msec or until the analog-digital convertor satu rates. At this point, the computer will store the total signal intensity accumulated at the time prior to sat uration and then shift to the next ion in the isotope series and repeat those operations. Thus, the time spent col lecting ions of a given mass varied from 1 to 64 msec, depending upon the intensity of the ion current. After the last isotope in a series was mea sured, the system immediately recy cled to the first, providing continuous monitoring throughout the analysis. Isotope ratios were determined by using the area under the peaks pro duced by the mass chromatograms, i.e., the curve produced from a plot of ion intensity vs. time for a given iso tope. A total of four ions could be monitored at one time. For isotope series containing more than four ions, one ion was arbitrarily chosen as unity, and the others were calculated as a ratio of this. The abundance measurement of the ion normalized on was repeated in each set of deter minations until all isotopic species were measured. The oscilloscope dis play of the data system allows the mass chromatogram to be observed as the data are being acquired. When this is complete, the operator can then choose the two points on the mass chromatogram through which a base line should be drawn. The two points between which the peak area is to be calculated are then chosen, and finally the peak area is obtained in arbitrary units or as a voltage, with the portion of the background under the peak subtracted. Other ions in the isotope series are then similarly measured and their relative abun dances calculated. The precision of the measurements presented in the tables which follow is given in terms of the mean devia tion as a percent of the average value
calculated from four to six indepen dent determinations of each isotopic species. The percent accuracy of the measurements is given as the per centage by which the average abun dance differs from its true value. In the experiments where the pro grammable multiple ion monitor (PROMIM) was used, each isotopic species was monitored on a separate channel of the unit, and the ion cur rent measured by individual digital integrators (Autolab Model 6300). The ion monitor was modified to sweep over the top of each peak cov ering a range of approximately 0.25 amu, rather than remain stationary. Sampling times of 1, 10, and 100 msec could be chosen for each sweep. As before, the instrument switched from peak to peak continuously throughout the analysis. Results Reservoir Samples. The relative abundances measured for the inert gases are given in Table I. The gases were admitted from a 1-liter reservoir at room temperature through a mo lecular leak to give an indicated ion source pressure of approximately 5 x 10 7 torr. Measurements were ob tained with the data system in the mass fragmentography mode, as de scribed earlier, with a total sampling time of 5 min. Since a maximum of four ions could be monitored simulta neously, for krypton and xenon, iso tope abundances were determined as ratios of m/e 82 and 129, respective
ly, and then relative abundances cal culated. The precision and accuracy attained as a function of isotope abundance are shown in Figure 1. GC Samples. To make a direct comparison of the precision and accu racy possible between the reservoir gas sample where the pressure is not changing and a more dynamic case of GC samples where sample pressures are constantly changing, xenon and krypton were introduced via the GC inlet at room temperature by using a gas-tight syringe. The results are given in Table II. As a typical exam ple, Figure 2 shows the mass chro matogram of xenon at m/e 124, the lightest and least abundant isotope, taken on continuous monitoring with the injection of four different sam ples. Each GC peak was of approxi mately 8-sec duration. The abun dances of each of the four peaks shown in Figure 2 were, from left to right, 0.098%, 0.093%, 0.095%, and 0.095%, with the true value being 0.096%. The greater precision of the last two reflects the effect of better ion statistics. Comparison of the data given in Table II for GC analyses to that in Table I for reservoir analyses shows comparable accuracy and precision. Thus, it can be concluded that with the rapid peak-switching system used here, changes in sample pressure en countered with GC samples are suffi ciently slow compared to the switch ing time so as to have no significant effect on abundance measurements. Isotope abundances of fragment
Table I. Isotope Abundance Measurements on Some Inert Gases from Reservoir Sample System
Sample
Argon
Krypton
Xenon
m/e
Theoretical rel abundance,
Measd rel abundance, 'Λ
Accuracy,
%
36 38 40
0.337 0.063 99.600
0.344 •;• 0.303 0.06/ - 0.0005 99.587 -. 0.003
+2.1 +fi.n
78 80 82 83 84 86
0.3=5 2.27 11.5b 11.55 56.90 17.37
0.39 2.31 11.51 11.63 56.69 17.47
+11.4 +1.8 -0.4 +0.7 -0.4 +0.6
124 J 26 128 1?!) 130 131 132 134 136
0.096 0.090 1.92 26.44 4.08 21.18 26.89 10.44 8.87
0.098 : 0.001 0.095 _-L- 0.001 1.98 -u 0.01 26.41 4.18 Λ- 0.02 21.3/ :- 0.U/ 27.05 1 0.04 10.35 J- 0.U2 8.53 =. 0.02
-
0.003 : 0.015
+ 0.015 •' 0.12 ·. 0.05
—0.01
+2.1 +5.5 +3.1 -•0.1 +2.4
+0.;) +0.6 -0.9 -4.0
Precision,
τΊΐ.Ο
4-0.7 •0.03 ::0.83 :0.64 -0.13 -L-0.22 -J 0.28 : 1.0 ±1.0 ±0.50
'' ±0.48 ^•0.33 h 0.14 :-0.20 ±-0.23
" " H a n d b o o k of Chemistry and Physic».," ••1st pri.. c.hpmic.il Hubber Cn., 19/1 4 Inn normalized o n .
456 A · ANALYTICAL CHEMISTRY, VOL. 46, NO. 4, APRIL 1974
ions of organic molecules were also measured. In the mass spectrum of pentaacetylglucose, the ion of m/e 242 is derived from the molecular ion by the successive loss of acetic anhy dride and formic acid {13). Thus, the
e
CrLOAc ~]
ion at m/e 242 contains the C-2, C-4, and C-6 oxygen atoms of the original glucose molecule. Samples of (2- 18 0) and (6- 18 0) pentaacetylglucose, as well as unenriched pentaacetylglu cose, were dissolved in methanol to a concentration of approximately 1 μg/μl and were injected into the GC inlet system at a column temperature of 220°C. The ion intensities at m/e 242 and 244 were measured, and the isotope abundances of m/e 244 are given in Table III. The mass chro matogram for unenriched pentaac etylglucose is given in Figure 3. The precision obtained with these samples was the same as that obtained from the analyses of the inert gases. A final set of experiments was per formed in which the multiple ion monitor-integrator system was used to measure isotope abundance of GC samples. Since each channel of the
CrLOAc ~*
)Ac OAc
t
CHO
AcOV
(+ \>Ac
/
OAc m/e 242
Table I I . Isotope Abundance Measurements on Some Inert Gases as GC Samples Theoretical measd abundance, Sample
Krypton
Xenon
M e a s d rel abundance,
%
m/e
78 80 82 83 84 86
0.35 2.27 11.56 11.55 56.90 17.37
0.34 2.24 11.45 11.59 56.89 17.48
124 126 128 129 130 131 132 134 136
0.096 0.090 1.92 26.44 4.08 21.18 26.89 10.44 8.87
0.095 ± 0.090 ± 1.98 ± 26.49 4.10 ± 21.05 ± 26.81 ± 10.49 ± 8.89 ±
± ± ± ± ±
Accuracy,
%
0.004 0.003 '· 0.034 0.09 0.02
-2.9 -1.3 -1.0 +0.3 -0.02 +0.6
0.001 0.001 0.016
-0.1 0.0 +3.0 +0.2 +0.5 -0.6 -0.3 +0.4 +0.2
0.025 0.04 0.03 0.08 0.06
Precision,
% ±1.2 ±0.14 b
±0.29 ±0.16 ±0.11 ±1.1 ±1.1 ±0.8 1,
±0.61 ±0.19 ±0.11 ±0.76 ±0.67
1U' Intnqiatur A I M Units
460 A ·
J 0.06 r-0.41 :.'.0.51
(2-^0) pentaacetyl glucose
&**-L' :rv:
Figure 4. Calibration curve for multi ple ion monitor-integrator system with m/e 242 of pentaacetylglucose introduced via GC inlet as a stan dard. Curve plots arbitrary integrator area units for each mass chromato gram vs. ratio of these area units for two channels used
-i.-0.36
ANALYTICAL CHEMISTRY, VOL. 46, NO. 4, APRIL
1974
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A comparison of T a b l e I (reservoir inlet) a n d T a b l e II (GC inlet) shows t h a t a l t h o u g h t h e precision of t h e m e a s u r e m e n t s is a b o u t t h e s a m e in b o t h cases, t h e accuracy of t h e low a b u n d a n c e m e a s u r e m e n t s is b e t t e r for s a m p l e s i n t r o d u c e d via t h e GC inlet. T h i s is n o t surprising since t h e d a t a system allows a b a s e line t o be d r a w n for t h e m a s s c h r o m a t o g r a m p e a k a n d b a c k g r o u n d s u b t r a c t e d from t h e area of t h i s p e a k t o a close a p p r o x i m a t i o n . T h i s could n o t be done for t h e reservoir s a m p l e s , a n d a n y re sidual b a c k g r o u n d w a s included in the measurements. Thus, these d a t a show t h a t r a p i d switching t e c h n i q u e s with a q u a d r u p o l e m a s s s p e c t r o m e t e r can be used to analyze GC p e a k s for isotope a b u n d a n c e w i t h o u t sacrificing accuracy. P e r h a p s a more rigorous t e s t of t h e t e c h n i q u e used here is t h e isotope a b u n d a n c e analysis of t h e f r a g m e n t ions of n o r m a l a n d 1 8 0 - l a b e l e d p e n taacetylglucoses i n t r o d u c e d via t h e GC inlet. T h e d a t a in T a b l e III again show excellent precision, in agree m e n t w i t h t h a t o b t a i n e d with t h e inert gases. T h i s , of course, provides t h e c a p a b i l i t y of s i m u l t a n e o u s l y ob t a i n i n g s t r u c t u r a l information a n d higher precision isotope r a t i o a n a l y ses, giving b o t h t h e position of t h e isotope within t h e molecule a n d its a b u n d a n c e a t t h a t position. The experiments with the multiple ion m o n i t o r - i n t e g r a t o r system show t h a t such a system m a y also b e e m ployed for high-precision isotope m e a s u r e m e n t s , as shown in T a b l e IV. T h i s system h a s t h e m a i n a d v a n t a g e of being less expensive t h a n t h e d a t a s y s t e m . O n t h e other h a n d , with t h e d a t a system it is easier to focus t h e masses t o be m o n i t o r e d . T h i s system is also m o r e flexible in t h a t it h a s t h e ability t o adjust for m a x i m u m integra tion t i m e w i t h o u t prior knowledge of t h e isotope a b u n d a n c e , t h e c a p a b i l i t y of directly d e t e r m i n i n g t h e ratio of areas by a n " a r e a r a t i o " function, a n d t h e capability of allowing t h e operator to visually choose t h e t w o points t h r o u g h which t h e b a s e line is d r a w n . T h e l a t t e r is of major i m p o r t a n c e a n d is illustrated by t h e c o m p a r i s o n of t h e isotope a b u n d a n c e s of t h e 1 8 0 enriched glucose s a m p l e s from t h e d a t a system a n d ion m o n i t o r s y s t e m . In b o t h cases, t h e a b u n d a n c e s ob t a i n e d with t h e ion m o n i t o r a r e lower b y 0.2 t o 0.3 a t o m % 1 8 0 t h a n those with t h e d a t a s y s t e m . T h i s w a s d u e t o t h e presence of an i m p u r i t y in t h e s a m p l e , giving a small p e a k at m/e 242 on t h e leading edge of t h e s a m p l e peak, a n d resulting in a base line change, b u t t h e r e was no correspond ing p e a k a t m/e 244. T h u s , t h e digi t a l integrators on t h e ion monitor a d d e d t h i s shoulder into t h e p e a k
462 A · ANALYTICAL CHEMISTRY, VOL. 46, NO. 4, APRIL 1974
a r e a a t m/e 242, giving a lower a b u n d a n c e for m/e 244. T h e d a t a system, on t h e o t h e r h a n d , allows visual inspec tion of t h e p e a k a n d provides t h e ability t o go b a c k after all t h e d a t a a r e t a k e n a n d choose t h e base line t o a closer a p p r o x i m a t i o n . T h u s , in t h e above case for t h e d a t a system, t h i s shoulder was n o t a d d e d t o t h e p e a k area for m/e 242. One further point dealing with iso tope a b u n d a n c e m e a s u r e m e n t s w i t h a q u a d r u p o l e m a s s s p e c t r o m e t e r is worth n o t i n g a n d concerns m a s s dis c r i m i n a t i o n . Of t h e several isotopic series s t u d i e d , t h e accuracy a t t a i n e d with t h e low m a s s species w a s t h e s a m e as t h a t for t h e high m a s s species, w i t h i n t h e a b u n d a n c e limits discussed above. T h u s , no m a s s dis c r i m i n a t i o n w a s observed, e.g., in T a bles I a n d II, t h e relative a c c u r a c y o b t a i n e d for t h e xenon isotopes as one proceeds from low t o high m a s s shows n o t r e n d a s a function of m a s s within these 12 m a s s u n i t s . In conclusion, t h e work p r e s e n t e d here d e m o n s t r a t e s t h e c a p a b i l i t y of a q u a d r u p o l e m a s s s p e c t r o m e t e r for use as a high-precision i n s t r u m e n t for t h e d e t e r m i n a t i o n of isotope ratios, even u n d e r t h e d y n a m i c conditions im posed b y c o m b i n e d gas c h r o m a t o g r a phy-mass spectrometry. Acknowledgment T h e a u t h o r s t h a n k A u t o l a b Inc., for t h e u s e of two of their Model 6300 digital integrators. References (1) G. Waller, R. Ryhage, and S. Meyerson, Anal. Biochem., 16, 277 (1966). (2) R. M. Caprioli and D. Rittenberg, Bio chemistry, 8,3375(1969). (3) M. Cohn, Biochim. Biophys. Acta, 37, 344(1960). (4) H. F. Fisher, E. E. Conn, B. Vennesland, and F. H. Westheimer, J. Biol. Chem., 202,687(1952). (5) H. Eriksson, J. A. Gustafsson, and J. Sjovall, Eur. J. Biochem., 9, 550 (1969). (6) C. G. Hammay, B. Holinstedt, and R. Ryhage, Anal. Biochem., 25, 532 (1968). (7) R. M. Caprioli, in "Biochemical Ap plications of Mass Spectrometry," G. Waller, Ed., ρ 735, Wiley-Interscience, New York, N.Y., 1972. (8) R. A. Hites and K. Biemann, Anal. Chem., 42,855(1970). (9) C. C. Sweeley, W. H. Elliott, I. Fries, and R. Ryhage, ibid., 38, 1549 (1966). (10) J. F. Holland, C. C. Sweeley, R. E. Thrush, R. E. Teets, and M. A. Bieber, ibid., 45,308(1973). (11) P. D. Klein, J. R. Haumann, and W. J. Eisler, ibid., 44, 490 (1972). (12) W. F. Holmes, W. H. Holland, B. L. Shore, D. M. Bier, and W. R. Sherman, ibid., 45,2063(1973). (13) J. T. Watson, D. R. Pelster, B. J. Sweetman, J . C. Frolich, and J. A. Oates, ibid., ρ 2071. (14) R. M. Caprioli and W. E. Seifert, Jr., Biochim. Biophys. Acta, 297, 213 (1973) R. M. C. thanks the American Cancer Society for partial support.
