Gas Chromatograph-Mass Spectrometer-Accelerating Voltage Alternator System for the Measurement of Stable Isotope Ratios in Organic Molecules Peter D. Klein, Joseph R. Haumann, and William J. Eisler Division of Biological ana' Medical Research, Argonne National Laboratory, Argonne, Ill. 60439
A gas chromatograph-mass spectrometer with accelerating voltage alternation (GMA) for analysis of stable isotope ratios in organic molecules is described. The system provides independent focusing for each mass, magnet stabilization, and sweep options, and converts the output signal to digital form. The system shows excellent stability and can measure carbon isotopic ratios with an accuracy of 1 part in 104. I N 1966, SWEELEY AND COWORKERS described the measurement of two unresolved gas chromatographic components in a gas chromatograph-mass spectrometer equipped with an accelerating voltage alternator ( I ) . This GC-MS-AVA system (more conveniently abbreviated GMA) focuses alternately on two ion peaks by withdrawing and adding an increment of the accelerating voltage; at present it is commercially available only on the LKB 9000 instrument. Although Sweeley et al. used this system to measure the proportion of perdeuterio glucose in ordinary glucose, the main thrust of papers citing this reference has been in the direction of ion-specific detection (designated mass fragmentography) (2) that permits the estimation of extremely small quantities of compounds by reference to an internal standard. This standard may be an isotopically labeled derivative of the pure compound (3) or an isotopically labeled version of the compound itself (4). By this means of reverse isotope dilution, the compound serves as its own standard and one can eliminate problems, of losses during purification as well as of the efficiency of detection. An alternative (and seemingly neglected) use of the GMA system is to measure the stable isotope ratio of a metabolic compound after administration of the labeled precursor. measured the Sweeley and Vance, in unpublished work (3, appearance of dideuterioglucose in plasma after the administration of glucose to normal patients and those with Fabre's disease, and followed its incorporation into glycolipids, using their GMA system. Instrumental limitations prevented them from measuring less than 0.5 atom per cent of normal glucose as dideuterioglucose. In comparison with conventional 3H and 14C detection with liquid scintillation counting, such a dilution measurement is insensitive and could not be considered competitive in tracer studies. Nevertheless, the significant advantage of eliminating radiochemical hazard from metabolic studies in pregnant women, infants, and young children makes the development of such a GMA system imperative. (1) C. C. Sweeley, W. H. Elliott, I. Fries, and R. Ryhage, ANAL.
CHEM., 38, 1549 (1966). (2) C. G. Hamrnar, B. Holmstedt, and R. Ryhage, Anal. Biochern., 35, 532 (1968). (3) B. Samuelson, M. Hamberg, and C. C. Sweeley, ibid., 38, 301 (1970). (4) T. E. Gaffney, C. G. Hamrnar, H. Holmstedt, and R. E. Mc43, 307 (1971). Mahon, ANAL.CHEM., (5) C. C. Sweeley and J. Vance, Michigan State University, East Lansing, Mich., unpublished work, 1968. 490
ANALYTICAL CHEMISTRY, VOL. 44, NO. 3, MARCH 1972
ACCELERATING VOLTAGE M 1 COARSE FOCUS ELECTRON MULTIPLIER
I
1
Figure 1. Block diagram of mass spectrometer modifications We have recently described the design considerations for a GMA system to be used for stable isotope analysis (6) and report here on the construction, operation, and practical experience of such a system. EXPERIMENTAL
Instrumentation. The basic gas chromatograph-mass spectrometer used in this system is the Perkin-Elmer double focusing MS 270. It is a low resolution instrument with a Biemann molecular separator between the gas chromatograph and the ion source of the mass spectrometer. A block diagram of the modifications to the accelerating voltage and magnet supply is given in Figure 1. The original high voltage power supply was replaced by an ultra stable system with separate focusing controls for each mass. These separate controls permit the voltage to be switched between widely different masses (greater than 30%) without degradation of ion optics. The magnetic field is monitored by an external Hall effect probe (F. W. Bell, Inc., Columbus, Ohio) with temperature-compensated high linearity response characteristics, and is read either on a five-digit gaussmeter or on an associated mass marker. When the focus of the instrument has been set at , Hall effect probe is used in a the desired lower mass ( M I ) the second mode as the sensor for a supplementary magnet stabilization circuit. This fine-tuning servo circuit enables the magnetic field to be held constant to within 10 ppm. We have found it advisable to encase the magnet air gap in Styrofoam to reduce air currents in the vicinity of the probe. With the peak selection switch on the upper mass (Mz), the location of the second mass may directly set in units of A M / M . The focus of the second mass may be separately adjusted without changing the original settings for M I . In the intermediate AUTO position, the high voltage power supply alternates between M 2 and M I . The display, digitization, storage, and control features of the system are shown in Figure 2. The signal from the electron multiplier is presented to an attenuator with precision resistors rated at 10 ppm. The signal from either M I or M2 may be attenuated over a range from 0.9999 to 0.0001 prior to entering a variable bandwidth amplifier and 10 bit analog-to~
_
(6) P. D. Klein, J. R. Haumann, and W. J. Eisler, Jr., Clin. Chem., 17, 735 (1971).
_
SCOPE
PRECISION
-... . .-, - ..
$,u
VARIABLE BANDWIDTH AMPLIFIER
SCALER-MASS1
I
IO B I T ADC
I
L
J
1
SCALER-MASS2
1 Figure 2. Block diagram of data acquisition and control system
ELECTRON CHANNEL MEMORY ADDRESS CYCLE
i HIGH SPEED U V GALVANOMETER
CHANNEL ADVANCE +I
1
SAMPLES PER CHANNEL
DATA STORAGE MODE C H A N E L 0-199 CHANNEL 200-399 M7
T I ME
Figure 4. Galvanometer recording of peak-switching in static mode Upper: dwell time 2 sec per frame Lower: dwell time 200 msec per frame Note ringing and slewing effect in output amplifier voltage of changes in accelerating voltage
Figure 3. Multichannel analyzer display of digitized ion-specific gas chromatographic peaks Upper: individual mass records Lower : overlapped display showing coincidence of gas chromatographic peaks and absence of isotope fractionation
digital converter. This attenuation increases the sensitivity of the digitization process by ensuring that the signal voltages corresponding to the peaks of different intensities are comparable. The output of the A / D conversion is converted to counts by gating pulses from a I-MHz clock into a comparator until the value of the voltage is attained. A full scale conversion provides 1028 counts and requires 2 msec. The total counts resulting from the A / D conversion of each ion peak is determined by two quantities: the dwell time on each peak, which is variable in step fashion from 1 psec up to 100 seconds and is determined by the time base generator; and the number of conversion cycles per dwell time, which can be varied from 1 to 100 cycles. Thus, with a 200 msec dwell time, up to 100 conversions or 102,800 counts full scale can
be obtained from each sampling of an ion peak. A logic interlock assures that equal numbers of conversions are made for both peaks. The counts are stored in two forms: as integral counts for each mass in separate scales, or in a modified 400-channel analyzer (Packard Instrument Company) operated in the multiscaler mode. Those values stored in the analyzer are routed alternately to the first 200 channels ( M I ) and to the second 200 channels (Mz); e.g. the address sequence is 0,200,1,201,2,202, etc. This alternation is synchronized to the switching of the high voltage power supply and provides an ion-specific digital record of the gas chromatographic peak. Such a record is shown in Figure 3. The data stored in the analyzcr may also be used in studies of isotope fractionation during gas chromatography. In addition to the high speed ultraviolet galvanometer recorder which is part of the Perkin-Elmer DF/MS 270, we have provided an oscilloscope and have incorporated a sweep circuit into the high voltage supply. The sweep circuit contains controls for both the range and symmetry of the ramp voltage and when activated will cause a repetitive sweep through the region of mass at an interval determined by the time base generator. By narrowing the range, a single mass peak may be isolated and centered in the sweep. The sweep circuit used in conjunction with the multiple A / D conversion option confers a number of advantages on the system. The tuning procedure is easier since the peak may be directly viewed and isolated, and there is no requirement, as in static tuning, to find the exact peak maximum. ANALYTICAL CHEMISTRY, VOL. 44, NO, 3, MARCH 1972
491
0400
20
25
TIME, minutes
Figure 6. Instrument stability of magnetic field and isotope ratio Upper line: attentuated mje 2gjrnle 32 ratio Lower line, fluctuation in magnetic field IO TI ME
-
9-
Figure 5. Galvanometer recording of peak-switching in sweep mode
0-
Dwell time in all frames is 200 msec. Sweep range: A , 100%; B, 50%; C,40%; D,30%; E,20% of total range
7-
c W z
E
6-
0 3
Moreover, slight changes in tuning position are without effect on the quantitation of the peak because base-line values lost on one side of the sweep are gained on the other side. Finally, as shown in Figures 4 and 5, the dwell time on each peak required for stable values in the static mode (Figure 4) can be greatly reduced in the sweep mode (Figure 5 ) because the sweep begins and ends at base-line values and the slewing effects of rapid switching are absent. Because of these advantages, the sweep option was routinely employed for all ratio measurements. Instrument Operation. The gas chromatograph-mass spectrometer is set up in the conventional manner to carry out gas chromatographic analyses. After zeroing the amplifiers for the electron multipler and A/D converter, the following procedure readies the instrument for isotope ratio analyses: Set approximate mass number for gaussmeter. With sweep at widest range, inject reference compound, set peak selection at MI. When spectrum appears in sweep, reduce sweep range while adiusting magnet fine control to keep peak centered in sweep. Lock magnet stabilizer in position. With peak selection switch in M Z position, inject labeled reference compound. When peak appears in sweep, set AMJM to proper value, observe positioning of M Z in sweep. Return peak selection switch to AUTO position. The measurement of the isotope ratio in a sample requires the knowledge of four quantities. These are: The ratio M2/Mlfor the pure unlabeled compound. The ratio M2/Ml for the pure labeled compound. The counts at M iand M Zper unit time in the absence of sample (background). The counts at M l and M Zand the total time of measurement for the sample. Users of liquid scintillation counters will recognize the direct parallel to the use of channel ratios for determining the proportion of 3H to I4C in a sample by beta spectrometry. In the simplest situation, where the labeled material does not contribute to the counts at MI, the isotope ratio is simply: 492
ANALYTICAL CHEMISTRY, VOL. 44,
NO. 3, MARCH 1972
2
0
5-
-J
2 P
4-
3-
2I-
TIME, minutes
Figure 7. Total ion record of bile acid chromatogram, showing periods of isotope ratio measurement of 1-minute duration
Atom Per Cent Excess
=
(")M I
asmplo
-(%) /(")MI MI normal
normal
where all readings are corrected for any background at MI and M2. In a series of analyses, the first two quantities need be determined only at the beginning and end of the measurements. Although the background values are stable in the absence of excessive contamination from column bleed or sample overload, the most precise measurement is obtained when they are determined during each analysis. Gas Chromatographic Studies. Tetradeuterio-(2,2,4,4, d4-)chenodeoxycholic acid was prepared by enolic exchange of the 3-keto derivative on alumina deactivated with 3 z by weight of 99.6x D20by the method of Hofmann, Szczepanik, and Klein (7). Both protio- and deuterio- chenodeoxycholic acids were chromatographed as the acetates (8) of their methyl esters on a 1-mm x 6-ft column packed with 3 AN600 on Gas Chrom Q, 80-100 mesh. The helium carrier gas flow was 15 ml/min at 60 psi, the oven temperature was (7) A. F. Hofmann, P. A, Szczepanik, and P. D. Klein, J . Lipid Res., 9, 707 (1968). (8) J. Roovers, E. Evrard, and H. Vanderhaeghe, Clin. Chim. Acta, 19, 449 (1968).
105,
1
Figure 8. Digital representation of gas chromatographic peak obtained from mle 374 (tetradeuterio chenodeoxycholic acid) and from m/e 370 (protio form) Dotted line: isotope ratio from integrals of individual ion peaks. Attentuation of rnie 370 peak, 0.10, nominal concentration of tetradeuterioiprotiomixture, 10 %
PERCENT DEUTERIUM LABELED B I L E A C I D
Figure 9. Dilution curve for mixtures of do and ddabeled chenodeoxycholic acid
270 OC, manifold (molecular separator) 300 "C and ion chamber 150 OC. The ion source was ouerated with an accelerating voltage of 2000 volts, an election energy of 70 electron volts, a filament current of 3.5 A, and an emission current of 100 PA. RESULTS AND DISCUSSION The stability of this system is illustrated in the following experiment: The instrument was tuned on mje 32 and mje 28, the latter being attenuated to 2 0 z of its full intensity. The ratio of ion intensities was measured at 1-min intervals and plotted in parallel with the output signal of the Hall effect probe used to monitor the stabilized magnetic field. The Hall effect probe signal was offset with a bucking voltage to magnify the actual excursions of the field strength in the milligauss range. These data are shown in Figure 6 and il-
Table I. Analysis of mje 44, rnle 45, 102 CtS 102 CtS 53795 61780 48930 56351 46950 53959 46095 53101 46955 53104 47539 54629
Abundance Ratio Attenuation, Abundance, % mle 44, Z 1,1484 1.Ooo 1.1516 1.Ooo 1,1492 1.Ooo 1.1519 1.Ooo 1,1470 1.ooO 1,1491 1.ooO Av 1,1495 =t 0 .GO29
13C1602/12C1602
lustrate the absence of drift in either tuning or magnetic field under these conditions. In a second demonstration of the sensitivity of the isotope ratio measurements, a gas sampling loop with a capacity of 250 p1 was used to introduce a sample of COSinto the carrier gas stream. In place of the usual gas chromatographic column, a dilution chamber of 15-ml capacity was used to broaden the gas pulse and provide a counting time of several minutes at mje 45 and rnje 44. The data shown in Table I for the natural abundance of ' T O 2 illustrate that six measurements show an extreme range from 1.1470 to 1.1519 % with a standard error of 0.0029 %. This corresponds to an absolute error of 3 X in the isotope ratio measurement. The gas chromatographic record obtained from the total ion monitor shown in Figure 7 is for a 5-pg sample of a mixture of normal and tetradeuterio chenodeoxycholic acids as the methyl ester acetates. Shortly after the solvent peak emerged, the accelerating voltage alternation was turned on, causing the wider excursions of the recorder pen. At I-min intervals, the scalers were read and reset to zero; during this time the alternation was turned off, as can be seen in the total ion record. Figure 8 shows in bar graph manner the total counts and net counts above a background determined prior to the run. The dotted line indicates the isotope ratio obtained from the integrals of both peaks and the individual points represent the isotope ratio in successive time intervals. Note that each peak had a total area of approximately 3.5 X lo6 counts, permitting good counting statistics even with a moderate-sized sample. A dilution series was carried out with normal and tetradeuterio chenodeoxycholic acid and the results are shown in Figure 9. The log-log scale was chosen for this representation to permit the linearity and reproducibility below 1 % to be seen with the same clarity as that for higher concentrations. The limiting concentration of tetradeuterio chenodeoxycholic acid that can be measured with significance is between 0.1 and 0.2%. This instrumentation has been used in a comparison of deuterium and tritium labeled bile acids used for the study of bile acid kinetics in man (9); a fuller report of its use in clinical applications will appear elsewhere. ACKNOWLEDGMENT We thank Charles M. Stevens, of the Chemistry Division for his numerous helpful discussions and suggestions of design alternatives.
RECEIVED for review August 23, 1971. Accepted October 26, 1971. Work supported by the U S . Atomic Energy Commission. (9) R. G. Danzinger, A. F. Hofmann, L. J. Schoenfield, 0. W. Berngruber, P. A. Szczepanik, and P. D. Klein, Gastroenterology, 60, 192 (1971). ANALYTICAL CHEMISTRY, VOL. 44, NO. 3, MARCH 1972
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