Field Ionization Mass pectaometry: l
Michael Anbar antI William H. Aberth Stanford Research Ins,tilute Menlo Park, Calif. 940 25
Although field ionization mass spectrometry is still in its infancy, its unique characteristics will m a k e it a powerful tool for solving critical problems in such diverse fields as medicine, criminalistics, and environmental research. Reliability, efficiency, and simplicity of operation will beperfected in the next few years to the point where t h e field ionization mass spectrometer will be standard equipment in m a n y analytical laboratories where mass spectrometry has never been used Field ionization was first applied to mass spectrometry about 20 years ago by R. Gomer and M. G. Inghram. Field ionization mass spectrometry has since developed and is now a t the stage of evolving from a laboratory curiosity into a practical analytical instrument. Once this technique hecomes routine, the analytical chemist will have an exciting new tool capable of resolving problems that were previously beyond reach. The unique features of field ionization mass spectrometry in providing nonfragmented and isotopically nonscrambled mass spectra will open up a t least two important areas of application-analysis of complex multicomponent mixtures without preseparation and isotope dilution analysis by use of multilabeled molecular tracers. In a previous article of this series entitled, “Ionization Sources in Mass Spectrometry,” Chait ( 1 ) reviewed the technique of field ionization, as well as other forms of ionization applied to mass spectrometry. The general description of the principle of operation and uses of field ionization in that paper can serve as a background for the present article. Field ioniza-
tion and many of its analytical applications were the subiect of a recent monograph (2).Her13, we shall describe in detail the niultinoint field ionization sources t h a nave aeen ueveloped a t SRI during the past few years. These sources, which are still heinrIimmoved. alreadv nromise sir. nificant advances in anaiytical mass spectrometry. In addition, we will discuss some new applications of mass spectrometry made possihle by field ionization.
The sample feed system is designed to handle efficientlv both hiehlv volatile and less volatile constit;ents. When used for the latter tvve of sam pies ,r igurt: A ) , m e sys~srriCunsisLb
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Multipoint Field Ionization
a small hollow rod-shaped sample holder threaded on one end and closed off a t the other. The holder containing the sample is inserted into the vacuum system and screwed into the hack end of the source by means of a long support rod that is passed throueh a vacuum lock into the vacuurn system. The support rod is then disconnected from the s a m d e holder
Present test; indicate that these points can have long lifetimes. One set of points has been subjected to over 1000 hr of diverse operation with little disceruihle loss of efficiency.
entering the ionization sour‘ce ( 5 ) . Typical source ion currents are beamp. We have tween 10-9 and also examined the effect of Idilution with air on the efficiency of idnization
Y
Fiaure 1. ScanninQ electron microaraDh of new multilloint source
Figure 2. Schematic cross section of multipoint source showing method of introducing solid sample and heating
Figure 3. Field ionization spectrum of I-tryptophan
(mol wt 204) at 200°C
technique of oreanic molecules. We diluted tolueneto 20 ppm in air without any significant loss in ionization efficiency, which was about in this particular case. This result shows that air does not interfere with the ionization process of trace constituents. In an analogous experiment, toluene was diluted in methanol down to 1part per thousand, again without change in its ionization efficiency. A similar result was obtained when methanol was diluted with toluene down to 1:1000. Again, the ionization efficiency of methanol remained constant and equal to that of pure methanol. Thus, the SRI source is able to ionize minor constituents of a mixture containing dissimilar chemicals without significant changes in the efficiency of ionization. This result suggests that most of the ionization takes place without chemisorption a t the tips. From the practical standpoint, this finding indicates that field ionization may be applied as a quantitative method of analysis of mixtures, after the ionization efficiencies have been calibrated. Such a calibration is not necessary when we are interested solely in pattern recognition and comparison of spectra. In another series of experiments (51, we compared the relative ionization efficiencies of organic compounds heavily substituted with deuterium atoms with their normal analogs. In the cases of ds-amphetamine, dlobarbital, and de-heroin, the ionization efficiency of the polydeuterated compounds was lower by not more than 10%.This small isotope effect (which can he readily corrected for by measuring standard mixtures), in addition to the high yield of the parent ion, makes field ionization an ideal method for multilaheled molec ular tracer applications. The nonfragmented nature of the 60 A
spectra obtained with our source is exemplified in Figures 3 4 . An ionization source that exclusively generates parent molecular ions produces interpretable mass spectra of mixtures because each constituent contributes to the ion beam only a t a single mass number. Figure 7 is an example of a single slow scan of a sample of fuel oil; each peak represents one compound or a number of compounds with the same mass number. Multiscan Field Ionization Mass Spectrometers Although a single slow scan provides us with useful qualitative information on the composition of the sample, it cannot he used as a quantitative measure. The evaporation of a sample through the ionization region takes a few minutes while the source is beine heated UD. Thus. a continuous change in the composition of the sample in the ion source takes place during this period, owing to differences in volatility of the different constituents of the mixture. A slow scan, say of 3 min, going from heavy to light masses is biased by the preferential evaporation of some of the more volatile components, especially in the low-molecular-weight range. A rapid scan, say of 1 sec, gives a fairly true picture of the composition of the sample's vapor, but only a t the given instance. A repeated scan a few minutes later could produce a significantly different spectrum. One way to obtain reproducible and quantitative mass spectra of slowly evaporating mixtures is to carry out many repeated fast scans over the whole spectral range of interest until the sample is completely consumed. The multiscan data are then integrated into a composite spectrum, which is a t first approximation independlent of the amount of the sample analyzed and of the rate of heating. The r~ssults are quantita-.
ANALYTICAL CHEMIST RY, VOL. 46. NO. 1, JANUE\RY 1974
tive as long as the efficiency of ion. ization is independent of the rate of sample vapor throughput. This condition is fulfilled over a wide range of sample flow rates. We have used two types of mass analyzers to obtain rapid multiple mass scans. One mass analyzer used in our system is an advanced version of a Wien ion velocity filter ( 6 ) .By use of electrical guard shims and tapered pole caps, the electric and magnetic field can he shaped so that the focusing properties of the filter are removed (7, 8).This type of filter can produce higher mass dispersions for a given size magnet than any other available mass analyzer, while allowing essentially 100% transmission of the mass-analyzed ion beam. Using this analyzer, we can perform a mass scan by varying the electric field only. In the SRI machine, over 1000 mu can be scanned in less than 1sec. Repetitive mass scans are performed by supplying an amplified ramp voltage from a sweep generator (following conversion of the ramp shape to provide a linear time-mass relationship) to the electrostatic deflection plates of the ion velocity filter. The mass range covered is ohtained by adjusting the camp voltage amplitude and dc offset. A pulse synchronized to the ramp voltage triggers a multichannel analyzer (9),which operates in the multichannel scaling mode of data acquisition and obtains its pulsed signal input from a Bendix Model 4700 continuous dynode electron multiplier ion beam detector. This arrangement permits the storage of signals from a particular mass into a corresponding channel during repetitive mass scanning. The mass resolution of the system is determined by the ion beam optics, the stability of electronics, and the number of storage channels in the multichannel analyzer per mass unit. Present resolution is limited by electronic stability
and is in the range of 300-1000, depending on the mass number. Figure 8 exemplifies an integrated multiscan spectrum and can be compared with the spectrum of the same sample analyzed by a single scan in Figure 7 . Parallel to our work with the ion velocity filter system were efforts to interface our field ionization source with a quadrupole analyzer (IO).The quadrupole analyzer has several advantages over the Wien filter. I t is more compact, it has a built-in linear mass scale and rapid scanning mode, and its technology is far more advanced, owing to the greater development effort thus far expended. Its relatively lower throughput compared with the Wien filter is not critical in the multicomponent analysis of mixtures, where we are generally not sample limited. Significant improvements have been made recently in the resolution of quadrupole analyzers. The new ELF model of Extranuclear Corp. has achieved a resolution of 7000 a t mass 500 by use of a conventional electron bombardment source, compared with a resolution of 2500 a t mass 500 claimed by Colutron Corp. for its best ion velocity filter. Applying Extranuclear Model 270-9 with the ELF modification, we obtain multiscans similar in quality to those described above.
Analysis of Multicomponent Mixtures Figure 8 demonstrates the potential of analysis of complex multicomponent mixtures by field ionization mass spectrometry. At present, we are using this methodology to identify metabolic aberrations in urine, to determine the purine and pyrimidine composition of nucleic acids, and to determine the composition of crude and fuel oils. Other examples of multicomponent systems to which the same technique could be applied include analysis of food flavors, paint, forensic samples, and air and water pollutants. There is hardly a field in analytical chemistry to which this technique could not be applied. It is as universal as gas-liquid chromatography, but it has several additional advantages: it is more sensitive, it provides higher resolution in a much
shorter time, and its information is much more meaningful because each isolated compound is identified by a most characteristic property-its molecular mass. The mass spectrometric multicomponent analysis has another unique feature. By subjecting the analyzed mixture to a certain derivatization procedure-e.g., acetylation-followed by mass spectrometric analysis, we can identify all the constituents which carry a given functional group (a hydroxy group in this example). Those constituents which carry the functional group disappear from the spectrum; instead, we find new peaks of their derivatives (in the case of acetylation, these will be heavier by 42 mu for each hydroxyl on a derivatized constituent). Olefins can thus be identified by bromination, carboxy groups by methylation, carbonyl
Figure 6. Field ionization spectrum of 1 :30 mixture of d5-phenobarbital and unlabeled phenobarbital
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Figure 4. Field ionization spectrum of methaqualone (mol wt 250) at room temperature
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Figure 5. Field ionization spectrum of 7-OH norchlorpromazind (mol wt 320) at 60°C
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220 230 240 250 260 270 280
Figure 7. Single scan fingerprint spectrum of No. 6 fuel oil, mid-cut sample ANALYTICAL CHEMISTRY, VOL. 46, NO. 1, JANUARY 1974
61 A
7 6 FUEL OIL I
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192 206 220 234 248 262 276 290 304 318
Figure 8. Integrated fingerprint of No. 6 fuel oil, vacuum distilled, mid-cut
groups by the formation of oximes. and so on. The characteristic A M for each derivatization reaction plus the knowledge of the molecular weight of the original underivatized compound make it easy to identify individual mono- and polyfunctional constituents in a mixture. We believe that clinical analysis will be the first discipline to adopt mass spectrometric analysis of complex multicomponent mixtures on a routine basis. The clinical laboratory is heavily exposed to this type of problem in the analysis of blood or urine. and the gains in time and added information should amply justify the capital investment in the new instrumentation. Multilabeled Molecular Tracers ( 1 7 )
As stated above, field ionization mass spectrometry allows us to measure the abundance of isotopically labeled compounds without the artifacts of fragmentation and isotopic scrambling. Figure 9 represents a mass scan of a 20:1 mixture of toluene and octadeuterated toluene. We see here mass 92 of toluene. mass 93 of 13C toluene (owing to naturally occurring 1 3 C ) , mass 100 of CD3C6D5, mass 101 of its 13C analog, and mass 99 of the heptadeuterated toluene present as an impurity. The abundance ratios of mass 92 and 100 can readily be measured by a double-collector abundance ratio mass spectrometer. We have constructed two such multicollector instruments-a magnetic sector triple-collector instrument with a dynamic range of 105, and a Wien filter double-collector instrument with a dynamic range of 106. In view of the severe limitations in using radioactive tracers in biological systems. it became desirable to reconsider the use of stable isotopic tracers. With standard electron impact mass spectrometry, the isotopic composition of a given organic compound could be determined only after its conversion into simple molecules. For instance, a D-. 13C-, or 15N-la62 A
beled histidine would have to be converted into D-labeled H2, 13C-labeled C o g , or 15N-labeledNz. If the organic sample could be effectively ionized without fragmentation or isotopic scrambling of the original organic compound, the parent ions could be used for isotopic mass analysis. The use of parent ions for isotopic mass analysis not only alleviates chemical preprocessing, it also provides us with a means to overcome the background limitations owing to the natural abundance of stable isotopes. This can be achieved by using multilabeled molecules as tracers. Let us take histamine (C5H9N3) as an example and synthesize an imidazole ring, which consists of three 13C carbons and two I5N nitrogens, and let us substitute deuterium atoms for the hydrogens a t positions 2 and 5 . The multilabeled histamine molecule will have a molecular mass that is i mu higher than the nonisotopic species (mass 111).The abundance of mass 112 will be about 6.7% of that of mass 111, owing to the natural abundance of 13C, 15X, and 2D. Mass 113 will be about 5.8% of that of mass 112
Figure 9. Mass scan of toluene plus da-toluene 20:l
ANALYTICAL CHEMISTRY, VOL.. 4 6 , NO. 1, JANUARY 1974
or about 0.4% of that of mass 111. The contributions of the natural abundance to mass 118. the mass of the heptalabeled histamine, is merely that of mass 111. If the mass ratio l s / l Z could be measured down to one part in lo8, we could measure a millionfold dilution of labeled histamine with 1% precision without interference from the natural occurrence of stable isotopes, If we ionize histamine and mass analyze it with an overall efficiency of 10-5, which can be achieved by our systems, we can determine IOs heptalabeled molecules (=1.6 x 10-16 moles gram) with a 3% or about 1.9 x precision (which is sufficient for most practical purposes). However, the quantitative handling of 2 x gram of material is not feasible because of adsorption to surfaces. The addition of an overwhelming amount of the unlabeled compound as carrier overcomes this artifact. As we can readily measure the multilabeled compound in the presence of a millionfold excess of the unlabeled carrier, the quantitative assay of subfemtamole ( l O - I 5 ) quantities of organic molecules becomes feasible. The sensitivity of detection of the heptalabeled histamine exceeds that of histamine labeled in both the 2 and 5 positions with carrier-free tritium, by an order of magnitude. Moreover, the measurement can be carried out in less than 5 min with a negligible background (compared with over 2 hr counting time for the analogous tritiated sample). Mass analysis of the multilabeled tracer molecule not only provides us with a more sensitive and faster analytical methodology, it also alleviates all the major restrictions of the radiotracer methodology. First. the tracer molecule is positively identified by its molecular weight, which is by far more specific than beta or gamma radiation. The probability is extremely low that a compound will occur with both the same molecular mass as the multilabeled molecules and a similar TLC, I?,, or GLC retention time. Second, there are no artifacts resulting from radiolytic autodecomposition or from radiobiological damage to the tested cells. Third, unlike the case of radiotracers, it is possible to assess the extent of isotope effects on the process investigated. Spectra of multilabeled molecules generally contain satellite peaks owing to the limited purity of the heavy isotopes and the natural abundance of isotopes. Thus, if our heptalabeled histamine is synthesized from 95% pure 13C, 98% pure 15N,and 99% pure D, the mass r a t i o g T / l % will be 24/76 = 0.32, 116/118 = 0.10, lIs/lB = 0.032, and ls/lg= 0.026 (owing to 13C and I5N in the side
chain). These ratios can be determined in the synthesized heptalabeled tracer and reexamined following the biological or biochemical process under investigation. Any isotope effect that tends to discriminate against the heavier tracer molecules will distort the sequence of ratios and thus increase the relative abundance of the lower masses. From the change in these relative mass ratios, we can calculate the correction for any kinetic isotope effect on the biological or chemical process under investigation. In a demonstration experiment we were able to measure the incorporation of hexalabeled thymidine (natuYO)into ral abundance 8 X DNA of lung tissue of rats. Thirty minutes after an injection of the multilabeled thymidine, 0.019% of the DNA originated from the labeled tracer. Lung tissue challenged for 48 hr by breathing of 20-ppm NO2 had a much higher metabolism and incorporated six times as much of the molecular tracer. The present limit of detection of uptake of multilabeled thymidine by our method, measuring 50 ng of thymine, is 0.00‘2%. This limit, which is likely to decrease with further development of our instrumentation, is already lower than the limit of sensitivity attainable with “carrier free” tritiated thymidine. An obvious use ofmultilabeled molecular tracers is isotope dilution analysis, which has been extensively applied by using radioactive tracers. However, this methodology does not allow the addition of, say, a million times excess of a radioactive carrier, because specific activities can hardly be determined with a precision of Mass abundance better than k0.270. ratio measurement by use of multilabeled tracers thus increases the dynamic range of isotope dilution analysis by over 3 orders of magnitude. With the sole exception of immunoassay, the subpicogram sensitivity of molecular tracer isotope dilution analysis exceeds that of all conventional analytical methods. Dilution analysis has a broad range of applications. In pharmacology, for instance, the use of this method permits examination of biological availability, clearance rate, and degradation pathways of pure or formulated drugs a t subpharmacological doses. An important advantage of molecular tracer methodology is the infinite shelflife of the multilabeled molecules, which allows their synthesis in relatively large quantities. The cost of the tracer in dilution analysis, where microgram quantities are used, is trivial. Even in biological tests where the multilabeled molecules are used as tracers in milligram quantities, their cost will constitute less than $5.00 per diagnostic test. 64 A
It is expected that the multilabeled molecular tracer methodology will be useful in many different applications: clinical diagnosis and feedback control of therapy; pharmacological research and evaluation of new drugs; the tracing of nutrients and metabolites in physiological and nutritional research; the determination of compounds of interest in small samples of tissues in toxicology and forensic medicine; the determination of subpicogram quantities of insecticides, herbicides, and industrial pollutants in environmental research; and the determination of residual hormones or insecticides in processed food. This nonexhaustive list demonstrates the universal usefulness of this methodology. As in the case of multicomponent analysis, we believe that the clinical laboratory will be the first to pick up this instrumentation. I t is likely that isotope dilution analysis by use of multilabeled dilutants will be used on a large scale to determine a large variety of metabolites in blood and urine. These could include estrogens (pregnancy testing), 17-ketosteroids (pituitary and adrenal cortex disorders), methylated purines and polyamines (cancer indicators), vitamins (deficiencies), thyroxine and triiodothyronine (thyroid disorders), free amino acids (liver disorders), galactose (passive liver function in infants), betahydroxybutyric acid (diabetes), indican (bacterial processes), and phenyl pyruvic acid (phenyl ketonuria). These are just a few examples of chemical tests on urine and blood that are carried out on materials present a t the ,ug/ml level and that are identified by classical analytical techniques. The availability of a universal analytical method that can determine any metabolite including simple polypeptides, oligosaccharides, or porphyrins down to the pg/ml level (i.e., one million times lower levels than attainable by the classical technique) could lead to the development of hundreds of new diagnostic tests. The molecular tracer methodology will probably become extremely useful in the feedback control of therapy. The simplest case is the optimization of therapeutic doses of drugs. By use of this methodology, it is easy to determine the level of a given drug in the plasma and to keep it constant by appropriate dosage. This would be done first as part of pharmacological research to establish the optimal doses of new drugs. For instance, the bioavailability of a given constituent in a “patented” drug formulation could thus be readily established. For therapies that have a high risk of overdose, e.g., radiation therapy, cancer chemotherapy, or hormone substi-
ANALYTICAL CHEMISTRY, VOL. 46, NO. 1, JANUARY 1974
tution therapy, the level of a given drug could preferably be determined frequently on an individual basis. Likewise, if the concentration of a given metabolite in blood or urine can be used as an index of health, this factor could be routinely monitored by use of dilution analysis. The cost of an abundance ratio field ionization mass spectrometer is not expected to exceed the cost of an advanced model of a UV or IR spectrometer. It could, therefore, be readily adopted by clinical and other analytical laboratories. Conclusion We strongly believe that these new modes of application of multilabeled molecular tracers made possible by recent developments in mass spectrometry will have a far greater impact on the biomedical fields in the coming 20 years than the use of radioactive tracers has had in the last 60 years. Although field ionization mass spectrometry is still in its infancy, its unique characteristics will make it a powerful tool for solving critical problems in such diverse fields as medicine, criminalistics, and environmental research. If the rate of progress during the past few years continues, we can expect many new and exciting developments in the near future. In addition, the reliability, efficiency, and simplicity of operation will be perfected in the next few years to the point where the field ionization mass spectrometer will be standard equipment in many analytical laboratories where mass spectrometry has never been used. References (1) E;”. Chait,Anal. Chem., 44 (3), 77A (19 ( 2 ) . (2) H. D. Beckey, “Field Ionization Mass Spectrometry,” Pergamon Press, Elmsford, N.Y., 1971. (3) C . A. Spindt, J. A p p l . Phys., 39, 3504 (1968). (4) W . Aberth, C . A. Spindt, and R. R. Sperry, 21st Annual Conference on Mass Spectrometry and Allied Topics, San Francisco, Calif., May 1973. (5) G . A. St. John and M . Anbar, 21st Annual Conference on Mass Spectrometry and Allied Topics, San Francisco, Calif., May 1973. (6) Colutron Model 300-6, Colutron Corp., Boulder, Colo. 80302. ( 7 ) L. Wahlin, Nucl. Instrum. Meth., 27, 55 (1964). (8) R. L. Seliger, 11th Symposium on Electron, Ion, and Laser Beam Technology, p 183, Boulder, Colo., San Francisco Press, San Francisco, Calif., 1971. (9) Nuclear Data Model ND 2400, Nuclear Data, Inc., San Leandro, Calif. 94577. (10) H. L. Brown, W. Aberth, and M . Anbar, 21st Annual Conference on Mass Spectrometry and Allied Topics, San Francisco, Calif., May 1973. (11) M . Anbar, J. H. McReynolds, W . H. Aberth, and G. A. St. John, Conference on Stable Isotopes in Chemistry, Biology, and Medicine, Argonne National Lab, Ill., May 1973.
