Instrumentation Field Ionization Mass Spectrometry: A New Tool for the Analytical Chemist Michael Anbar and William H. Aberth Stanford Research Institute Menlo Park, Calif. 94025
Although field ionization mass spectrometry is still in its infancy, its unique char acteristics will make it a powerful tool for solving critical problems in such di verse fields as medicine, criminalistics, and environ mental research. Reliability, efficiency, and simplicity of operation will be perfected in the next few years to the point where the field ioniza tion mass spectrometer will be standard equipment in many 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 at the stage of evolving from a laboratory curiosity into a practical analytical instrument. Once this technique be comes routine, the analytical chemist will have an exciting new tool capable of resolving problems t h a t were pre viously beyond reach. T h e unique features of field ionization mass spec trometry in providing nonfragmented and isotopically nonscrambled mass spectra will open up at least two im portant 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 ap plied to mass spectrometry. T h e gen eral description of the principle of op eration and uses of field ionization in t h a t paper can serve as a background for t h e present article. Field ioniza-
tion and many of its analytical appli cations were the subject of a recent monograph (2). Here, we shall de scribe in detail the multipoint field ionization sources t h a t have been de veloped at SRI during the past few years. These sources, which are still being improved, already promise sig nificant advances in analytical mass spectrometry. In addition, we will discuss some new applications of mass spectrometry m a d e possible by field ionization. Multipoint Field Ionization Sources and Their Performance T h e multipoint field ionizing de vice recently developed at SRI (3, 4) consists of an array of about 1000 points spaced 25 μηι apart and depos ited by evaporation techniques over a 2-mm 2 area (1.6-mm diameter) on a gold-plated copper grid (Figure 1). E a c h point has a tip radius of about 10~ 5 cm and stands 40 μτη above the substrate surface. A 250-mesh gold grid electrically insulated from the points is placed about 100 μηι above the array. A potential difference of 2000-5000 V between the points and the grid is sufficient to initiate field ionization, and the ionization effi ciency increases rapidly with voltage. T h e entire structure is rigid and shock resistant and requires no spe cial handling procedures except the standard requirements of cleanliness. Present tests indicate t h a t these points can have long lifetimes. One set of points has been subjected to over 1000 hr of diverse operation with little discernible loss of efficiency.
T h e sample feed system is designed to handle efficiently both highly vola tile and less volatile constituents. When used for the latter type of sam ples (Figure 2), the system consists of a small hollow rod-shaped sample holder threaded on one end and closed off at the other. The holder containing t h e sample is inserted into the vacuum system and screwed into the back end of the source by means of a long support rod t h a t is passed through a vacuum lock into the vacu um system. The support rod is then disconnected from the sample holder and removed from the system. T h e sample holder can be heated from the outside to any programmed tempera ture. Temperatures as high as 500°C are reached in a few minutes. This system thus permits rapid sample change, efficient sample heating, and m i n i m u m memory in the ion source. The sample holder may be replaced with an electrically insulating ceram ic t u b e . This allows the introduction of volatile samples from the outside, via a molecular leak valve. Solid samples can be changed or the sys tem converted from the solid sam pling mode to the gas sampling mode in a few minutes while maintaining the source under a high vacuum. Our source ionized most organic compounds tested with an efficiency from 10 ~4 to 5 x 1 0 - 3 ; t h a t is, we ob tain 1-50 ions for every 10* molecules entering the ionization source (5). Typical source ion currents are be tween 10~ 9 and 10~ 8 a m p . We have also examined the effect of dilution with air on the efficiencv of ionization
Figure 1. Scanning electron micrograph of new multipoint source
Figure 2. Schematic cross section of multipoint source showing method of introducing solid sample and heating technique of organic molecules. We diluted toluene to 20 p p m in air without any significant loss in ionization efficiency, which was about 10 ~ 3 in this particular case. This result shows t h a t air does not interfere with the ionization process of trace constituents. In an analogous experiment, toluene was diluted in methanol down to 1 part 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 cons t a n t a n d equal to t h a t of pure methanol. T h u s , 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 t h a t most of the ionization takes place without chemisorption at the tips. From the practical standpoint, this finding indicates t h a t field ionization may be applied as a quantitative method of analysis of mixtures, after t h e ionization efficiencies have been calibrated. Such a calibration is not necessary when we are interested solely in pattern recognition a n d comparison of spectra, In another series of experiments (5), we compared t h e relative ionization efficiencies of organic compounds heavily substituted with deuterium atoms with their normal analogs. In the cases of dg-amphetamine, diobarbital, and de-heroin, t h e ionization efficiency of t h e polydeuterated compounds was lower by not more t h a n 10%. This small isotope effect (which can be readily corrected for by measuring standard mixtures), in addition to the high yield of the parent ion, makes field ionization an ideal method for multilabeled molecular tracer applications. T h e nonfragmented nature of the
Figure 3. Field ionization spectrum of /-tryptophan (mol wt 204) at 200°C
spectra obtained with our source is exemplified in Figures 3-6. An ionization source t h a t exclusively generates parent molecular ions produces interprétable mass spectra of mixtures because each constituent contributes to the ion beam only at 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 be used as a quantitative measure. T h e evaporation of a sample through t h e ionization region takes a few minutes while the source is being heated u p . Thus, a continuous change in t h e composition of t h e 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 t h e more volatile components, especially in t h e low-molecular-weight range. A rapid scan, say of 1 sec, gives a fairly true picture of the composition of t h e sample's vapor, b u t only at 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 t h e whole spectral range of interest until t h e sample is completely consumed. T h e multiscan d a t a are then integrated into a composite spectrum, which is at first approximation independent of the amount of the sample analyzed and of the rate of heating. T h e results are quantita-
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tive as long as t h e efficiency of ionization 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 be shaped so t h a t the focusing properties of the filter are removed (7, 8). This type of filter can produce higher mass dispersions for a given size magnet t h a n 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 t h e electric field only. In the SRI machine, over 1000 m u can be scanned in less t h a n 1 sec. Repetitive mass scans are performed by supplying an amplified r a m p voltage from a sweep generator (following conversion of the r a m p shape to provide a linear t i m e - m a s s relationship) to the electrostatic deflection plates of the ion velocity filter. T h e mass range covered is obtained by adjusting t h e r a m p voltage amplitude and dc offset. A pulse synchronized to t h e r a m p voltage triggers a multichannel analyzer (9), which operates in t h e multichannel scaling mode of d a t a 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. T h e mass resolution of the system is determined by the ion beam optics, t h e stability of electronics, and the n u m b e r 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 (10). The quadrupole analyzer has several advantages over the Wien filter. It 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 at mass 500 by use of a conventional electron bombardment source, compared with a resolution of 2500 at 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 phénobarbital
Figure 4. Field ionization spectrum of methaqualone (mol wt 250) at room temperature
Figure 5. Field ionization spectrum of 7-OH norchlorpromazine (mol wt 320) at 60°C
Figure 7. Single scan fingerprint spectrum of No. 6 fuel oil, mid-cut sample ANALYTICAL CHEMISTRY, VOL. 46, NO. 1, JANUARY
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Figure 8. Integrated fingerprint of No. 6 fuel oil, vacuum distilled, mid-cut
groups by the formation of oximes, and so on. The characteristic ΔΜ 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 constitu ents in a mixture. We believe that clinical analysis will be the first discipline to adopt mass spectrometric analysis of com plex 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 jus tify the capital investment in the new instrumentation. Multilabeled Molecular Tracers (11)
As stated above, field ionization mass spectrometry allows us to mea sure the abundance of isotopically la beled compounds without the arti facts of fragmentation and isotopic scrambling. Figure 9 represents a mass scan of a 20:1 mixture of tolu ene and octadeuterated toluene. We see here mass 92 of toluene, mass 93 of 13 C toluene (owing to naturally oc curring 13 C), mass lOOof CD 3 C 6 D 5) mass 101 of its 13 C analog, and mass 99 of the heptadeuterated toluene present as an impurity. The abun dance ratios of mass 92 and 100 can readily be measured by a double-col lector abundance ratio mass spec trometer. We have constructed two such multicollector instruments—a magnetic sector triple-collector in strument with a dynamic range of 105, and a Wien filter double-collec tor 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 im pact mass spectrometry, the isotopic composition of a given organic com pound could be determined only after its conversion into simple molecules. For instance, a D-, 13 C-, or 15 N-la
beled histidine would have to be con verted into D-labeled H 2 , 1 3 C-labeled C0 2 , or 15 N-labeled N 2 . 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 pro vides us with a means to overcome the background limitations owing to the natural abundance of stable iso topes. This can be achieved by using multilabeled molecules as tracers. Let us take histamine (C5H9N3) as an example and synthesize an imid azole ring, which consists of three 13 C carbons and two 15 N nitrogens, and let us substitute deuterium atoms for the hydrogens at positions 2 and 5. The multilabeled histamine molecule will have a molecular mass that is 7 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 abun dance of 1 3 C, 1 5 N, and 2 D. Mass 113 will be about 5.8% of that of mass 112
Figure 9. Mass scan of toluene plus d 8 -toluene20:1
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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 10 ~ 10 that of mass 111. If the mass ratio 118/1 l i could be measured down to one part in 108, we could measure a millionfold dilution of la beled 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 ~ s , which can be achieved by our sys tems, we can determine 108 heptala beled molecules ( = 1.6 x 10~ 16 moles or about 1.9 x 10~ 14 gram) with a 3% precision (which is sufficient for most practical purposes). However, the quantitative handling of 2 X 10~ 14 gram of material is not feasible be cause 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 mil lionfold excess of the unlabeled car rier, the quantitative assay of subfemtamole (10 - 1 5 ) quantities of or ganic 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 tri tium, 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 ana lytical methodology, it also alleviates all the major restrictions of the ra diotracer methodology. First, the tracer molecule is positively identi fied 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, Rf, or GLC reten tion time. Second, there are no arti facts resulting from radiolytic autodecomposition or from radiobiological damage to the tested cells. Third, un like the case of radiotracers, it is pos sible 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 abun dance of isotopes. Thus, if our hepta labeled histamine is synthesized from 95% pure 13 C, 98% pure ^N,_and 99% pure D, the mass ratio Π.7/118 will be_24/76 = 0.32, 116/118 = 0.10, 115/118 = 0.032, and 119/118 = 0.026 (owing to 13 C and 1 5 N in the side
chain). These ratios can be deter mined in the synthesized heptalabeled tracer and reexamined fol lowing the biological or biochemical process under investigation. Any iso tope 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 kinet ic isotope effect on the biological or chemical process under investigation. In a demonstration experiment we were able to measure the incorpora tion of hexalabeled thymidine (natu ral abundance 8 x 10" 1 3 %) into 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 incorpo rated six times as much of the molec ular tracer. The present limit of de tection of uptake of multilabeled thy midine by our method, measuring 50 ng of thymine, is 0.002%. This limit, which is likely to decrease with fur ther development of our instrumenta tion, is already lower than the limit of sensitivity attainable with "carrier free" tritiated thymidine. An obvious use of multilabeled mo lecular 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 better than ±0.2%. Mass abundance ratio measurement by use of multila beled tracers thus increases the dy namic range of isotope dilution analy sis by over 3 orders of magnitude. With the sole exception of immunoas say, the subpicogram sensitivity of molecular tracer isotope dilution analysis exceeds that of all conven tional analytical methods. Dilution analysis has a broad range of applications. In pharmacology, for instance, the use of this method per mits examination of biological avail ability, clearance rate, and degrada tion pathways of pure or formulated drugs at subpharmacological doses. An important advantage of molecu lar tracer methodology is the infinite shelflife of the multilabeled mole cules, 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 con trol of therapy; pharmacological re search and evaluation of new drugs; the tracing of nutrients and metabo lites in physiological and nutritional research; the determination of com pounds of interest in small samples of tissues in toxicology and forensic medicine; the determination of subpi cogram 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 methodol ogy. As in the case of multicomponent analysis, we believe that the clinical laboratory will be the first to pick up this instrumentation. It is likely that isotope dilution analysis by use of multilabeled dilutants will be used on a large scale to determine a large va riety of metabolites in blood and urine. These could include estrogens (pregnancy testing), 17-ketosteroids (pituitary and adrenal cortex disor ders), methylated purines and polyamines (cancer indicators), vita mins (deficiencies), thyroxine and tri iodothyronine (thyroid disorders), free amino acids (liver disorders), ga lactose (passive liver function in in fants), betahydroxybutyric acid (dia betes), indican (bacterial processes), and phenyl pyruvic acid (phenyl ketonuria). These are just a few ex amples of chemical tests on urine and blood that are carried out on materi als present at the Mg/ml level and that are identified by classical ana lytical techniques. The availability of a universal analytical method that can determine any metabolite includ ing simple polypeptides, oligosacchar ides, 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 develop ment of hundreds of new diagnostic tests. The molecular tracer methodology will probably become extremely use ful 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 de termine 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, can cer 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 moni tored 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 spec trometer. It could, therefore, be read ily adopted by clinical and other ana lytical laboratories. Conclusion We strongly believe that these new modes of application of multilabeled molecular tracers made possible by recent developments in mass spec trometry will have a far greater im pact on the biomedical fields in the coming 20 years than the use of ra dioactive 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 prob lems in such diverse fields as medi cine, criminalistics, and environmen tal 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 equip ment in many analytical laboratories where mass spectrometry has never been used. References (1) E. M. Chait, Anal. Chem., 44 (3), 77A (1972). (2) H. D. Beckey, "Fieid Ionization Mass Spectrometry," Pergamon Press, Elmsford, N.Y., 1971. (3) C. A. Spindt, J. Appl. Phvs., 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 Spectrome try and Allied Topics, San Francisco, Calif., May 1973. (6) Colutron Model 300-6, Colutron Corp., Boulder, Colo. 80302. (7) L. Wahlin, Nucl. lustrum. Meth., 27, 55(1964). (8) R. L. Seliger, 11th Symposium on Electron, Ion, and Laser B^am Technol ogy, ρ 183, Boulder, Colo., San Francisco Press, San Francisco, Calif., 1971. (9) Nuclear Data Model ND 2400, Nucle ar 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, Biolo gy, and Medicine, Argonne National Lab, 111., May 1973.
