Signal Averaging Method for High Resolution Mass Spectral Measurement of Nitrogen-I 5 William F. Haddon, Harold C. Lukens, and Richard H. Elsken Western Regional Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Berkeley, Calif. 94710
By averaging repetitive electric sector scans on a doublefocusing mass spectrometer, accurate abundance ratios of stable isotopes can be obtained on microgram samples using the isotopic multiplets at a single nominal mass. The method is useful for 15N measurement for most nitrogen-containing organic compounds, and for 3C measurement in compounds which also contain nitrogen. Repetitive scanning increases the accuracy of measurement under conditions of changing sample concentration, thus permitting accurate isotope ratio determination on samples admitted by direct probe or chromatographic inlets. An application to the measurement of l5N*-enriched creatinine is given. A limit of detection of 0.002 atom per cent excess 15N2 at the 95% confidence level is demonstrated.
The use of mass spectral techniques in measurement of stable isotopes is well established, and has assumed greatly increased importance recently, particularly in biological and medical research (1 -3). For nitrogen isotope studies, no long-lived radioisotopes are available so that most studies involve incorporation of nonradioactive I5N.The conventional approach to 15Nmeasurement by mass spectrometry has been to convert the samples to Nz by Kjeldah1 digestion, followed by isotope determination in an isotope ratio mass spectrometer. Some of the limitations of the Kjeldahl method with regard to sample size, memory effects, erros introduced during combustion, and nonrandomization of the isotope label have been discussed previously (4-8). An alternate mass spectral approach to isotope measurement is to introduce the intact organic compound into the mass spectrometer and measure the isotope ratio of the molecular ion or other suitable peaks in the electron impact spectrum. Waller et al. (9) first described the use of this method to elucidate the biosynthetic pathways for ricinine and refinements of their method of direct ratio recording have been subsequently described (1 0-12). (1) (2) (3) (4)
(5) (6)
Jacob Sacks, "Isotopic Tracers in Biochemistry and Physiology." McGraw-Hill, New York, N . Y . , 1953. G . Waller, "Biochemical Applications of Mass Spectrometry,"WileyInterscience, New Y o r k , N.Y.. 1972. G . W . A. Milne. "Mass Spectrometry, Techniques and Applications," Wiley-Interscience, New Y o r k . N . Y . , 1972. D. Rittenberg, A . S. Keston: F. Rosebury, and Rudolf Schoenheimer. J. Bo/. Chem., 127, 291 (1939). J. W . Taylor and I-Jen Chen, Anal. Chem., 42, 224 (1970). D. Desaty, R. McGrath, and L. C. Vining, Anal. Biochem., 29, 22
(1969). (7) R. A. Saunders, J . Sci. instrum., Ser. 2, 1, 1053 (1968). (8) J. H . Beynon, "Mass Spectrometry and Its Applications to Organic Chemistry." Elsevier, Amsterdam, 1960. ( 9 ) G . R. Waller, R . Ryhage. and S. Meyerson, Anal. Biochem., 16, 277 (1966). ( 1 0 ) H . M . Fales, M . Greifner. D. Steinberg, and G. W . A . Milne, ASTM E-14 Conference on Mass Spectrometry, Denver, Colo., May 1967. (11) P. D. Klein, J . R . Haumann, and W. J. Eisler, Anal. Chem.. 44, 490 (1972). (12) N. M . Frew and T . L. isenhour, ibid., 44, 659 (1972).
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This paper describes a real-time computer method for measuring isotope ratios on a double-focusing spectrometer. The proposed method is extremely sensitive, both with regard to sample size and level of measurable isotope incorporation. The measurement technique used is to derive the isotope ratio from the abundance of the component peaks of a multiplet at a single mass containing the required isotopes. The isotopic multiplet is scanned repetitively by the computer and time-aveiaged to obtain enhanced signal-to-noise ratio and correct for changing sample concentration. This technique can greatly reduce interference from small impurities or from ion-molecule reaction products because of the high resolution employed. Time-averaging techniques have been reported previously for low resolution (23, 14) and high resolution (15) mass spectra for enhanced sensitivity. Recently described accelerating voltage alternation systems by Klein (11) and Frew (12) provide a time-average benefit for stable isotope measurement similar to that described here.
EXPERIMENTAL Instrumentation. The high resolution isotope measurement method utilizes a CEC 21-llOA mass spectrometer capable of routine operation a t 1 part in 20,000 resolution, equipped with a standard CEC peak matching system. The electron multiplier output of the spectrometer is interfaced directly to an IBM-1800 process control computer which is shared with other laboratory instrumentation and with batch processing 116, I;). The spectrometer was modified for computer-controlled multiple scans by integrating a crystal clock with the peak matching function generator and the 14-bit 1800 analog-to-digital converter (ADC) as shown in Figure 1. The clock is an Anadex Model CF6117 (Anadex Instruments, Inc., 7833 Haskell Avenue, Van Nuys, Calif. 91406) located a t the spectrometer. The clock is externally programmable such that a string of pulses can be started and stopped by solid state or contact closure. The ramp functioh circuitry of the peak matcher was modified to produce a pulse a t the start of each electric sector scan. This pulse gates the crystal clock on and thus initializes a series of pulses which control the digitization of the signal by the ADC at a predetermined rate. To reset the crystal clock after collecting a fixed number of data points from a single scan, we use a digital output ECO (electronic contact operate) from the 1800 which gates the string of pulses off. In this fashion the computer is controlled by the peak matcher, as required for multiple-scan averaging. Computer Program. The data acquisition programming is under the M P X (multi-programming executive) operating system of the IBM-1800. To average multiple scans, the operator adjusts the mass range of the peak matcher appropriately, sets the scan repetition rate to 0.6 sec per scan and the crystal clock ( i e , data acquisition rate) to 2 KHz, and enters the number of scans desired in the operator console. Data acquisition is initiated by a control interrupt from the console. For each scan, 960 data points are taken under analog input data channel control, thus freeing the central processor of the 1800 for other tasks. End-of-table in-
(13) F. J. Biros, ibid.. 42, 537 (1970). (14) J . R . Plattner and S. P. Markey, J. Org. Mass Spectrom., 5, 463 (1971). (15) F. W . McLafferty, R . Venkataraghavan, J . E. Coutant, and B. G . Giessner. Anal. Chem., 43,969 (1971). (16) J. Scherer and S. K i n t , Appl. Opt., 9, 1615 (1970) (17) W. F. Haddon. D. R. Black, and R. H . Elsken, ASTM E-14 Conference on Mass Spectrometry, San Francisco, Calif..June 1970.
E L E C T R I C SECTOR
YD,
TIME-AVERAGED M U L T I P L E SCANS
,
r-----l
1
I
RESET E.C.O.
I
4
.. ..
R
g 4 "
t
I t
C R Y S T A L CLOCK
1
P
m
Creatinine, C,H,ON,
I
A DC
rn
103
m
MASS
Figure 3. 70 eV mass spectrum of creatinine. Ion source temperature: 200 "C. Direct probe temperature 170 "C
"PEAK MATCHER" RAMP FUNCTION
-A
GENERATOR
E L E C . SECTOR
Figure 1. Hardware mass spectrometer-computer interface for computer controlled electric sector scans M/E 114 M/AM
I
Figure 2.
10 , I
Time averaged scans of toluene-xylene doublet at
mass 93 terrupts are generated twice by the data channel, after collecting 480 and 960 words of data, and these interrupts initiate the scan averaging calculations. The second interrupt for a scan also resets the clock and checks for the end of the experiment. Buffering the calculations in this way allows the scan averaging to take place a t a lower (programmed) interrupt level than that of the end-oftable interrupt from the ADC. Double-word integer format is used for the accumulated sums. At the conclusion of the experiment, the data are divided by the number of scans, thus restoring single-word format, written to tape with experimental parameters, and plotted on the laboratory storage oscilloscope. The data acquisition program is written in IBM symbolic assembly language and requires 4,000 words of core, including data tables. The program and data tables remain core resident for the duration of the experiment in our system. Specific programming and hardware details are available from the authors.
RESULTS AND DISCUSSION Advantages of Time Averaging. The need to time average multiple scans in this work is principally to reduce the effects of changing rate of volatilization and depletion of sample on the measured isotope ratio for compounds int,roduced via the direct introduction probe. For example, a 10% change in rate of volatilization over 30 sec leads to a 5 to 10% error in peak ratio for a single scan us. 0.1 to 0.2% for multiple scan averaging a t a 0.6-sec repetition rate. Much faster scanning rates appear feasible for isotope scanning of chromatographic peaks. Figure 2 illus-
25,000
1
Figure 4. Creatinine isotopic multiplet at mass 114. Left scan: Torr. Right scan: ion source ion source pressure 2 X Torr. Resolution: 25000, 64 time-averaged pressure 3 X scans
trates the enhancement in signal-to-noise obtained for multiple scans of the C513C2H-13C CeH7 doublet ( M I A M = 18,600) in a mixture of toluene and xylene at mass 93. There is some loss in resolution after 100 scans (60 sec) due to drift in the spectrometer. A 64-scan average (40 sec) is feasible a t 15-20,000 resolution when sample size permits and has given satisfactory results on several hundred samples with less than 10% loss of resolution. Method of Isotope Measurement. The high resolution isotope measurement is based on the separation of different isotopic components having the same nominal mass. For example, the C15NJ3CN mass difference (6.31 milli1 in an mass units) leads to a resolvable doublet at P organic compound with nitrogen. One component of this 13C-15N doublet can thus be considered an internal standard for measuring the abundance of the isotopic component present in excess. For example, Figure 3 shows the low resolution mass spectrum of unlabeled creatinine, C4H70N3, obtained from the direct introduction probe (170 "C) a t an ion source temperature of 200 "C. The scans shown in Figure 4 were obtained on the P 1 isotopic peak ( m / e 114) a t 1 part in 25,000 resolution. The principal peaks a t this mass are those due to the naturally occurring 13C and 15N isotopes, and these peaks are the basis for isotope ratio measurement by the high resolution technique. There are additional unresolved contributions
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Table I. Exact Masses and Abundance Valuesfor the P 1 Isotopic Multiplet (mass 114) of Creatinine Elemental composition
Cq H6DON3 C4H7170N3 C3’3CH70N3 3C2C2H60N3b C4H70N215N C4Hs1’ON3’ C3’3CH60N215Nb C ~ H ~ O N ~ ~ N ~ ~
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Mass
Abundance”
114.06519 114.06308 114.06226 114.05758 114.05595 114.05534 114.05172 114.04516
‘0.101 0.037 4.497 0.028 1.105 0.075 0.018 0.001
Relative to most abundant isotopes as 100.0. See Ref. 8 and 77. 2 isotopic contributions from M-1 peak.
P -t
+
to the P 1 peak from the deuterium containing molecular ions, and various “P + 2” isotopic combinations of the M - 1 ion ( m / e 112). The smaIl peak of composition C4He02N2 indicated in the Figure is an impurity, and the C4HsON3 peak is from an ion-molecule reaction discussed below. Table I lists the elemental compositions, exact masses and abundances for the expected components of the P + 1 isotope peak in creatinine. The values are calculated from isotope abundance data summarized in Kiser (28) using the exact formulas of Beynon (8). For the ions a t mass 114 which are “P 2” isotope peaks from M - 1 ions at mass 113, the abundances are computed from the ratio of the m / e 112 to m / e 113 peaks after correcting the peak height at m l e 113 for the I3C and 15Ncontributions of M - 1ions. The additional peak observed on the high mass side of the I5N isotope component (left scan of Figure 4) corresponds to C4H80N3, mass 114.06673. The peak is pressure dependent and thus arises from an ion-molecule reaction probably involving proton transfer to form a quasi molecular ion of creatinine. Figure 4 shows the enhanced abundance of the peak at higher source pressure observed by increasing the direct probe temperature. Decreasing the probe temperature on the same sample (right scan of Figure 4) restored the original spectrum where the ion molecule peak is of lower abundance. Isotope ratio measurements reported previously on the ricinine molecular ion show pressure dependence (9), probably from a comparable reaction, and such reactions are known to occur for a variety of nitrogen compounds even a t modest ion source pressure 18). An advantage of the high resolution method over low resolution peak ratio recording (9-12) is that the contribution of such peaks can be removed and precise control of sample volatilization is unnecessary. Precalibration with an unlabeled compound would remove this source of error only if the volatilization profile could be duplicated exactly for each sample. Isotope Ratio Calculation. Peaks obtained by multiply scanning the electric sector can be closely approximated by a Gaussian function, as with mass spectral data recorded on photoplates (19) An advantage of electron multiplier detection over photoplate recorded data is that multiplet peaks of the same mass have the same Gaussian sigma values and this greatly simplifies the recognition and fitting for partially resolved bands. Figure 5 shows three Gaussian curves fitted to the creatinine 13C, I5N peak envelope at m / e 114. The residual curve indicates that a 3-band system adequately describes the multiplet with less than 1% error. The other isotopic components at mle 114 (Table I) are not revealed on this
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(18) R. W. Kiser. “Introduction to Mass Spectrometry and I t s Applications,” Prenttce-Hall. Englewood Cliffs, N.J., 1965. (19) R . Venkataraghavan, F . W. McLafferty. and J. W. Amy. A n a / . Chem.. 39, 178 (1967).
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Figure 5.
Computer-fitted Gaussian curves for mass 114 of cre-
atinine. PeakA = C4HBON3;Peak 6 = C313CH70N3;Peak C = C4H70 N215N
curve because of their low abundance. Frequently the peaks obtained are not completely separated and in these cases greatly improved accuracy and precision for isotope measurement can be obtained from the use of iterative band separation procedures. We use two versions of band separation programs, which are derived from the infrared programs of R. N. Jones (20). The first computer program calculates the band parameters automatically by iterative least-squares procedures. For a 3-band, all-Gaussian system, about 40 min of computer time are required on the Il3M 1800. In a second much faster version, the operator controls the iterative fitting process by inputting changes in band parameters to obtain the best visual fit, as indicated by storage oscilloscope display in the format of Figure 5 . This latter method has been particularly useful for revealing unsuspected, partially resolved peaks such as that of the ion-molecule reaction shown in Figure 4 and is a valuable prelude to use of the more refined program. Accuracy a n d Precision of Isotope Measurement. Table II indicates the precision and accuracy achieved for the 13C-15N ratio of unlabeled creatinine using computerfitted Gaussian curves. Gaussian peak areas and peak heights were both used to compute the 13C-15N isotope ratio. The peak height ratio measurement yields an isotope ratio within one standard deviation of the value calculated from the known average abundance values (18). Peak area measurements give a value about 5 to 6% below the true value, but with somewhat greater precision. For a simpler case, that of pyrimidine, C ~ H ~ Nthe Z , same magnitude of difference between peak height and peak area measurement was observed for the 13C, I5N doublet at m / e 81. This error appears to be due to imperfect focusing of the mass spectrometer, and the peak area measurements appear somewhat sensitive to the spectrometer adjustments, particularly the alignment of the source and collector slits. By carefully adjusting these slits we could fit a Gaussian curve to mass 69, CF3+, in perfluorokerosene within 0.1%. The magnitude of the difference in computed isotope ratio between peak height and area values is constant under the same spectrometer conditions, so that the ability to calculate the degree of isotope incorporation is not adversely affected when an unlabeled (20) J. Pitha and R. N . Jones, NRC lNat. Res. Counc. Can.) Buii., 12, 13 (1968).
~
~~
Table I I . 13C/15NRatio in Unlabeled Creatinine Method Measd 13C/151*1”
Height Area
3.833 3.524
Sb
Sm
True 13C/15NC
0.074 0.033
0.022 (0.58%) 0.010 (0.29%)
3.848 3.753
Table I V . Atom Per Cent Excess I 5 N 2in Labeled Creatinine Isolated from Human Urine
Average of 12 determinations of 64 averaged scans at 25,000 resolution. Standard deviation based on 5-kg sample consumption. Calculated from data of Table I , assuming all peaks to have the same Gaussian half width at 25,000 resolution.
Sample
Atom % excess of 15N2
Sma
2-24 2-27 2-32 2-36 2-42 2-47 2-63
0 638 0 559 0 524 0 517 0 479 0 442
0 0037, 0 58% 0.0019, 0.34% 0.0031, 0 59% 0 0044, 0 85% 0 0034, 0 71% 0 0029, 0.65% 0 0025,O 70%
0 357
Relative standard error based on 6-10 separate 64-scan averages on the same sample Total sample consumption of less than 25 fig a
Table 1 1 1 . Exact Masses and Abundance Values for the P -t 2 Isotopic Multiplet (mass 115) of Creatinine Elemental composition C4 H 7 0 N 1 5 N 2
C313CH70N215N C4H 7 1 8 0 N 3 Czl 3C2H 7 0 N 3 a
Mass
115.05298 115.05955 115.06316 115.06562
Abundancea
0.0041 0.0491 0.2043 0.0756
Relative to most abundant isotopes as 100.0. See Ref. 8 and 17.
standard is used for comparison, as is usually the case. Note that for the high resolution multiplet method, the usual correctiQns for isotopic fractionation of the sample through the molecular leak or by volatilization and for inherent mass discrimination of the spectrometer (2) need not be applied to the measured isotope ratio because the peaks which are ratioed have essentially the same mass. There are, however, two potential errors of the method which were not evident in the creatinine case: discrimination of the electron multiplier to the different isotopic components of a multiplet. and isotope effects on fragmentation. The latter problem will affect the results if there is an isotope effect for a particular fragmentation pathway of the molecular ion. For example, in creatinine, if the probability of He loss to produce the M - 1 peak is different for 13C or I5N isotopic molecular ions, this would lead to different populations of unfragmented isotopic molecular ions and result in an error in the computed isotope ratio. Conversely, the method could be used to calculate these isotope effects in special cases. In any case, the multiplier discrimination and isotope effect errors cancel when the unlabeled compound is used as a standard, and were not observed at all in the work reported here. Sensitivity. The creatinine P + 1 peak of Figure 4 was generated from 64 time-averaged scans at 0.6 sec per scan and represents a sample consumption of about 5 micrograms. The standard deviation (1 a) for a measurement based on this amount of sample is 1.1% (see Table 11). Thus for I3C, a 0.07 atom 70 excess can be measured at the 95% confidence level on 5 micrograms of sample. When sample size permits, improved precision is possible. However, it appears better on our spectrometer to make repeated averages of 64 scans rather than increase the number of scans because arcs or slow drift in the spectrometer can degrade the peak shape as shown in Figure 2 . The real-time display of the results of time averaging on the laboratorv storage oscilloscope is an important factor for obtaining valid isotope data, particularly when the experiment approaches the resolution limit of the mass spectrometer. IWz-Labeled Creatinine. The specific objective of this work was to measure the degree of 15N2 incorporation in creatinine isolated from the urine of human subjects who had been labeled with creatine-l5Nz of high isotopic purity. The results of this study will be described separately (21) The high resolution method provided excellent precision for these measurements on about 300 separate samples, allowed small samples to be used, and avoided ex-
r -Figure 6. 15000, 64
~
CREATININE M/E 115
Creatinine isotopic multiplet at mass 115. Resolution: time-averaged scans
tensive routine Kjeldahl digestion, as required by conventional isotope methods (4). Creatinine was isolated from the urine as the Zn complex, which was subsequently hydrolyzed to give pure creatinine (21). The m l e 115 “P 2” peak in creatinine was used for measuring the degree of 15N2incorporation. The 13Cz, l8O and I5NI3C components of the resulting isotopic multiplet are used as internal standards. The abundances and masses of the contributing peaks at mass 115 in unlabeled creatinine are given in Table 111. The low natural abundance for 15N2 leads to a very high precision for 15Nz measurement in the labeled samples. For routine measurement of 15N2 isotope a resolution of 15,000 was used, and a typical resulting peak profile shown in Figure 6. A simplified computer program was used to calculate the amount of 15N2present. In this program the computer located the valley between the 15N2 component and the other components used as internal standards, and integrated each peak from a threshold of 5% of the peak maxima to the minimum of the valley. A correction for overlap was applied assuming Gaussian peak envelopes and based on the sigma value calculated for the 15N2 component. Two successive 9-point digital smooths using the Savitzky-Golay coefficients 122) were employed to improve the quality of the raw data and aid the location of the minimum of the peak envelope. Table IV lists a number of results for measurements a t different levels of I5Nz incorporation in the creatinine samples.
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(21) M. Crim and D. Calloway, Department of Nutritional Sciences, University of Cailfornia, Berkeley. Calif., unpublished results.1972. (22) A. Savttzkyand M. J. E. Golay. Anal. Chem., 36, 1627 (1964)
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creatinine. However, at 15,000 resolution used for these scans, it was not possible to obtain consistently accurate 1eO-13C2 ratios because of insufficient separation of these peaks, and poor statistical definition of the curve.
t t
J
Figure 7.
Computer-fitted Gaussian curves for mass 115 of cre-
atinine Peak 1 =
15N2;Peak
2 = l°C15N; Peak 3
= 1 8 0 ; Peak 4 = 13C2
Sensitivity for 15N2. The standard errors of Table IV indicate a sensitivity of about 0.002 atom per cent excess for diJ5N labeled creatinine. To verify this extremely low limit of detection an unlabeled creatinine sample was used to calculate the natural abundance of di-15N creatinine, using the IS0,13C2 and 13C15N components of the P + 2 multiplet as internal standards. Six separate meabsurements, using peak areas, yielded an abundance value of 0.0089 & 0.0009 (standard deviation of the mean, 1 u ) , compared to a calculated value of 0.0041, as shown in Table 111. Thus the limit of detection for measurement of di-15N in creatinine is 0.0018 at the 95 per cent confidence level. I 8 0 Measurement. In principle it is possible to measure the I80content by comparing the peak heights of the 2 isotope peak. In Figure 7, 180-13C2 doublet of the P and Gaussian curves corresponding to 15N2, 13C15N, I80 have been fit to the isotopic multiplet a t m / e 115 of
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CONCLUSIONS The time averaging experiments described here are a sensitive. new method for measuring the abundance of stable isotopes in organic compounds. The method can be applied to stable 15N measurement when naturally-occurring 13C is also present for use as the internal standard. For such compounds containing nitrogen, the 13C stable isotope level can also be measured by considering naturally-occurring 15N as the internal standard. Routine measurements with excellent accuracy and precision are possible on microgram samples of a wide variety of compounds. The particular advantage of the time averaging is to correct for changing sample concentration, which occurs with samples admitted via the direct insertion probe or gas chromatographic inlets. Two advantages result from the use of isotopic multiplets for such measurements; the “unknown” and internal standard peaks, e.g. 13C and 15N, are of roughly comparable magnitude so that the measurement statistics for both peaks are similar, and interference from small sample impurities or ion-molecule reaction products can be eliminated since these will generally have different mass values. The method is not applicable to compounds containing no heteroatoms, and requires very high spectrometer resolution for I80 measurement. ACKNOWLEDGMENT The authors thank R. E. Lundin and J. Bartulovich for helpful discussions and programming assistance, and James H. Scherer for assistance with the band separation programs. Received for review June 19, 1972. Accepted December 7, 1972. Reference to a company or product name does not imply approval or recommendation of the product by the U.S.Department of Agriculture to the exclusion of others that may be suitable.
