Field desorption mass spectrometry

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TIBS -June

1980

Emerging Techniques Field desorption mass spectrometry

syringe as shown in Fig. 2b. After the dissolved or suspended sample has been transferred to the microneedles outside the ion H.-R. Schulten and W. D. Lehmann source of the mass spectrometer, most of the solvent is evaporated at atmospheric The need for specialized ionization tech- rate evaporation process prior to the for- pressure. Then the emitter coated with the niques in mass spectrometry (MS) has mation of ions. Second, the ions are formed solid sample, is introduced into the mass arisen from the difficulty in obtaining clear, with only very little transfer of electronic spectrometer and adjusted for the FD unequivocal spectra for many biologically excitation energy, either by ionization measurements. important molecules. The conventional through the very strong electric field Relatively volatile compounds start analysis procedure - electron impact ion- leading to M ° ions - or, more frequently, desorbing from the emitter immediately ization - transfers a relatively large amount by field-promoted surface reactions which the high voltage is applied, but for of thermal and electronic excitation energy attach a cation such as H , N a etc. to the biochemical compounds the emitter wire to the molecules being analysed. Hence, molecule - leading to cationized molecules has to be heated to initiate an ionic desorpthe electron impact mass spectra of such such as (M + H ) , (M + N a ) . Once these tion of the sample. Usually, an electric curcompounds are characterized by many and positively charged ions are formed on the rent of up to 40 mA is passed through the varied fragment ions and few (or even no) surface of an FD emitter they are effec- emitter wire and thus the desorption promolecular ions. For the majority of analy- tively removed from the emitter surface cess can be controlled by varying the tical purposes, however, just the opposite which is at a positive potential of around emitter heating current. The FD ion beam characteristics are to be preferred, namely 10,000 V relative to a slotted counter elec- produced in the ion source is analysed in a the formation of molecular ion species to trode positioned 2-3 mm away from the conventional mass spectrometer which can the exclusion of fragment ions. A techni- surface. Fig. 1 gives a schematic view of the be a single- or double-focusing magnetic que capable of producing a much higher instrumental set-up of an FD ion source. system or a quadrupole mass filter. proportion of molecular ions would faciliThe FD emitters generally employed are Among the various techniques which tate the determination of molecular 10/zm diameter tungsten wires on which. have been developed to allow the producweights for many biochemical compounds. carbonaceous microneedles 30-70 p,m in tion of ions without separate evaporation Furthermore as each compound would length are grown by a separate activation of the sample molecules - such as electhen be represented by just one signal, its process. A typical, well-activated FD emit- trohydrodynamic ionization, secondary ion molecular ion peak, it would be possible to ter is shown in Fig. 2a. It should be men- mass spectrometry, plasma desorption, identify and quantify each component in a tioned that the production of efficient, laser ionization and direct chemical ionizamixture. The structural information which chemically- and mechanically-resistant tion - FD has to be regarded as the most cannot be given by such a 'one-peak spec- emitters is a prerequisite for FD-MS. Once widely developed and accepted ionization trum' would be provided, when necessary, this emitter is prepared, the sample is technique. Nevertheless, FD is not yet a by complementary chemical or physico- applied in solution either by simple dip- routine technique in most mass specchemical methods or by the use of an ion- ping, or better, by means of a microlitre trometric laboratories with about 150 ization technique which produces structurally significant fragment ions. I I Counter -~ I-,---10 IJm No ionization technique can yet fulfil electrode I these requirements completely. Field I desorption (FD), however, already allows I I us to obtain spectra with characteristics I near to the 'ideal' ones mentioned above. FD I FD is a very 'soft' ionization technique emitter t giving mass spectra that generally show Ion Mass ions containing the intact molecules as base i-beam spectrometer peaks even when thermally labile or highly polar compounds (e.g. amino acids, I oligopeptides, sugars, oligosaccharides I L 70etc.) are analysed [1-3]. Two unique prin? 150 iJm ciples of the FD process make this possible. I I First, the sample is not subjected to a sepaH.-R. Schulten is at the lnstitut far Physikalische Chemie, Universitiit Bonn, Wegelerstr. 12, D-5300 Bonn 1, F.R.G. and W. D. Lehmann is at the lnstitut fi~r Physiologische Chemie, UniversitiitsKrankenhaus-Eppendorf, Martinistr. 52, D-2000 Hamburg 20, F.R.G.

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143

TIBS -June 1980

optimized with respect to the molecular ion abundance, a photographically recorded mass spectrum generally enhances the relative abundance of fragment and cluster ion species, and, as is demonstrated by the FD mass spectrum in Fig. 3, unambiguous information on the molecular weight is obtained from photographically recorded FD mass spectra. These can thus provide the desired final confirmation of a molecular structure.

Fig,

2. (a) Scanning electron micrograph o f an activated FD emitter. Tungsten wire aj%r activation with benzonitrile vapour at approximately 120t ~oC. The mwroneedles are about 301am long [3]. (b) Sample loading to the FD emitter by the syringe technique.

groups around the world using this technique. Details such as emitter preparation, ion source adjustment, sample application, desorption control, and highly sensitive detection of the weak and fluctuating ion currents add up to make FD a sop]~isticated technique. However, if performed with the required expertise FD-MS can provide clear-cut, reliable, and unique analytical data and here we want to highlight the potential of the technique wilh a few instructive examples.

Identification of drug metabolites The analytical power of FD for qualitative analyses in biochemistry can be demonstrated by its use in the identification of unknown compounds, e.g. drug metabolites. As a typical example we considered the elucidation of the structure of a urinary metabolite of I - (2,4,6 - trichlorophenyl) - 3,3 - dimethyltriazene (2,4,6 - C13 PDMT), a compound exhibiting a pronounced tumour-inhibiting activity against TLX5 murine lymphoma. The tumourinhibiting activity of triazenes depends on metabolic activation involving oxidation in vivo of the terminal dimethylamino group of the triazene side-chain. Since the symmetrically trichlorinated ring in 2,4,6C13PDMT is not hydroxylated in vivo, one can expect enhanced side-chain oxidation and/or conjugation. Following subcutaneous injection of 2,4,6-C13PDMT to rats, a single metabolite containing the intact triazene moiety was detected on thin-layer chromatograms. The metabolite was isolated by ion exchange chromatography and purified by repeated gel filtration. Fig. 3 shows the FD mass spectrum of the metabolite with its exact molecular weight of 443.005 which led to confirmation of the proposed structure as [1 - methyl - 3 - (2,4,6 - trichloro-

phenyl) - 2 - triazeno]methyl/3 - D - glucopyranoside uronic acid [4]. In the FD mass spectrum in Fig. 3 the protonated molecular ion is present with signals showing saturated blackening on the photoplate and the molecular weight identification is further confirmed by the occurrence of two characteristic ion groups at higher mass values which are identified as the (M + N a f and (M + K f ion groups. The abundant ion signals in the lower mass region are partly due to impurities present in the sample and partly to the integrating characteristic of the photoplate which accumulates the signals for all ions detected during the complete desorption process. Thus, compared with a mass spectrum recorded electrically under conditions

Quantification of drugs The highly sensitive and reliable quantification of endogenous and exogenous organic compounds in biological fluids or tissue is an area of biochemical analysis where mass spectrometry can provide highly specific and accurate data. Indeed, mass spectrometry combined with a chromatographic separation procedure is often the method of choice for the solution of these problems [5,6]. For quantitative mass spectrometric analyses, electrical registration of the ion currents is nearly always used in preference to the quantitative evaluation of photographically recorded ion signals, the latter being very tedious and the quantitative data obtained having less precision. The characteristics of FD make it promising for quantitative studies because as each component gives rise to only one major ion group there is less chance that signals from different components will be superimposed. Thus the technique shows considerable potential for the quantification of crude extracts.

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TIBS -June 1980

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also for the toxic heavy metals, thallium and cadmium. Although traces of metals can be analysed by a number of wellestablished techniques, in many cases the use of FD-MS is attractive because of the following characteristics: (a) First, only minute amounts of the sample (a few ~i) are needed and many samples such as physiological fluids or tissue homogenizates can be analysed w i t h o u t

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Two inherent properties of FD which could, in theory, hamper quantitative measurements - the low reproducibility of absolute FD ion current intensities and the sometimes pronounced fluctuations of FD ion currents - have been shown not to hinder quantitative FD work. The first difficulty was overcome by applying the wellestablished technique of internal standardization and the second difficulty could be eliminated through the use of integrating ion recording techniques, in particular by accumulation of electrically recorded ion signals via a multichannel analyser. In a series of recent publications on quantitative FD-MS (e.g. [7-11]) it has been demonstrated that FD provides quantitative data with a precision and accuracy that compares well with the data generated by more common ionization techniques such as electron impact or chemical ionization. The internal standards of choice for FD quantifications are compounds labelled with stable isotopes as these show a virtually identical desorption behaviour compared with their non-labelled analogues; however, desorption behaviour may be altered if the compounds are labelled with deuterium at very many sites [7]. As a practical example we consider here the determination of the cancer drug cyclophosphamide in body fluids by FD, for which the [2Hlo]-analogue is a suitable internal standard. In cancer chemotherapy this drug is generally applied in massive pulse doses. Recently, therapeutic effects

in multiple sclerosis patients have been observed after continued daily intake of considerably smaller doses of cyclophosphamide (four doses of 100 mg). Using FD-MS and the [2Hlo]-analogue as internal standard, the steady-state concentration of cyclophosphamide in urine, serum and cerebrospinal fluid of three multiple sclerosis patients could be determined at the end of a three-week treatment [8]. Fig. 4 shows the FD molecular ion pattern of cyclophosphamide and the internal standard [2Hlo]cyclophosphamide in an analysis of an extract from cerebrospinal fluid. This quantification of the drug in cerebrospinal fluid, which gave a concentration of 280 ng ml ', could be performed after careful extraction of lipids with n-hexane but without any further chromatographic purification. In summary, the investigation of the different body fluids gave the lowest values for cyclophosphamide in cerebrospinal fluid. However, this concentration was still c. 80% of the serum level.

(b) Second, in combination with stableisotope enriched internal standards, the technique exhibits an u n m a t c h e d reliability since matrix effects are effectively excluded. Thus, there is either no result (where no ions are detected) or an accurate result is obtained (if FD ions are detected). The intermediate situation, namely an incorrect result which looks accurate, appears much less probable than with other techniques using external standardization. (c) Third, the same mass spectrometer with combined EI/FD ion source can be utilized for an unequalled variety of analytical problems, e.g. identification of thermally labile drug metabolites, trace determinations of biocides, molecular weight determination of natural products, pyrolysis studies of polymers and microorganisms, etc. a n d metal assay w i t h o u t a n y m o d i f i c a t i o n . T h e FD technique offers the option to investigate organic components in the first step and, consecutively, inorganic traces in one and the same sample (see [5], p. 72). The utility of FD-MS for the quantification of metals in biological samples has been demonstrated, for instance for lithium [9] and thallium [10,11]. Thallium has two stable isotopes with abundances of 29.5% at mass 203 and 70.5% at mass 205. Stable-isotope enriched thallium with

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Identification and determination of metals by FD-MS Originally developed for 'soft' ionization of organic compounds of low volatility, FD unexpectedly proved to be a powerful Fig. 5. FD quantification o f thallium in uterus tissue ofla technique for the analysis of a number of pregnant mouse intoxicated with 8 mg kg ~ thallium. The monovalent and divalent metal cations measured isotope distribution is intermediate between the natural abundances and the abundances o f the pure including the alkali metals (Li, Na etc.), the 2°~Tl-enriched internal standard and thus allows the calalkaline earth series (Be, Mg etc.), a variety culation o f the thallium concentration in the original of the rare earth elements (Ce, Pr etc.) and sample [11 ].

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TIBS -June 1980

approximately the reverse abundances is commercially available and was successfully used for FD quantifications of thallium. Very recently the teratogenic toxicity of elevated thallium concentrations in pregnant mice was studied. The developing foetuses were screened for skeletal malformations and the mice were then killed and the thallium concentrations were determined in the foetuses and in uterus tissue. Fig. 5 shows the original •trace of an FD-MS thallium quantification in uterus tissue of a pregnant mouse which had been fed with a single dose of 8 mg kg ~thallium which caused pronounced skeletal malformations in the foetus [11]. On the basis of the results in trace (ppm) and ultratrace (ppb, ppt) analysi:s of metals obtained by FD-MS so far, it can be expected that the technique will find a wide and successful application in the biLosciences. For instance, it may be used for trace element analysis, toxicological stud]es, or the analysis of metal-containing organic substances. In addition, FD-MS offers the unique possibility of analysing organic and inorganic constituents in one process. Organic samples desorb from t]he emitter surface between ambient temperature and about 300°C whereas most metal cations require higher temperatures for desorption and for this purpose temperatures up to about 2200°C can be generated without damaging the emitter wire. Use of internal standards labelled with stable isotopes makes possible simultaneous ,quantification of organic and inorganic constituents in one sample by FD-MS.

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Interlaboratory reproducibility In the first years of FD-MS poor reproducibility of the analytical res'alts was a severe problem; decisive factors such as the quality of the emitters, the sample loading procedure, and the desorption rate could not be controlled adequately. Since then, substantial progress has been achieved through the use of standard hightemperature activated emitters, the syringe loading technique, and the introduction of emission-controlled desorption. Fig. 6 shows an encouraging example of the reproducibility of FD-MS obtainable today. The two FD-MS spectra of the same sample were recorded in two different laboratories using two different types of mass spectrometers [12]. With respect to the essential results, such as cationized molecular ion, the appearance of multiply charged ions and structurally significant fragment ions, the two mass spectra are in good agreement and

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give the same analytical information. The most pronounced differences are found in the relative abundances of the fragment ions; their greater abundance in spectrum b is probably due to a higher emitter temperature during desorption. In spite of these improvements, significant differences in the quality of FD mass spectra of the same

compound still may occur when samples produced by different separation and purification steps are analysed, because the surface processes responsible for the production and desorption of the ions are strongly dependent on inorganic and organic impurities in the sample solutions. Field desorption, without any doubt, has

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TIBS - J u n e 1980

extended the range of applicability of mass spectrometry in the biochemical sciences. However, further basic investigations, and instrumental and methodological improvements are still required. Current work aims to produce automated and simplified emitter activation procedures, more efficient emitter heating techniques using laser heating, higher sensitivity in ion detection, and straightforward work-up procedures for biological samples which are specially adapted to the requirements of FD, particularly in combination with high performance liquid chromatography [8,13,14].

References 1 Beckey, H. D. (1977) Principles of Field Ioniz-

ation and Field Desorption Mass Spectrometry, Pergamon, Oxford 2 Sehulten, H.-R. (1977 ) in Methods of Biochemical Analysis (Glick, D. ed.), Vol. 24, p. 313, WileyInterscience, New York 3 Schulten, H.-R. (1979)1nt. J. Mass Spectrom. Ion Phys. 32, 97-283 and references cited therein 4 Kolar, G. F. and Carubelli, R. (1979) CancerLett. 7,209 5 De Leenheer, A. P., Roncucci, R.R. and van Peteghem, C. (eds) (1978) in Quantitative Mass Spectrometry in Life Sciences Vol. H, Elsevier Scientific, Amsterdam 6 Millard, B. J. (1978) Quantitative Mass Spectrometry, Heyden, London

7 Lehmann, W. D. and Schulten, H.-R. (1978) Biomed. Mass Spectrom. 5,208 8 Bahr, U., Schulten, H.-R., Hommes, O. R. and Aerts, F. (1980) Clin. Chim.Acta (in press) 9 Lehmann,W. D., Bahr,U., Schulten,H.-R.(1978) Biomed. MassSpectrom. 5,536 10 Schulten,H.-R., Lehmann,W. D. and Ziskoven, R. (1978) Z. Naturforsch. 33c,484 11 Achenbach,C., Ziskoven,R., Koehler,F., Bahr, U. and Schulten,H.-R. (1979)Angew. Chem. 91, 944; (1979)Angew. Chem.Int. Ed. Engl. 18,882 12 Komori, T., Kawamura, M., Miyahara, K., Kawasaki,T., Tanaka, O., Yahara, S. and Schulten, H.-R. (1979)Z. Naturforsch. 34c, 1094 13 Schulten,H.-R. and Kuemmler,D. (1980)Anal Chim. Acta 113,253 14 Schulten,H.-R. and Stoeber, I. (1978) Fresenius Z. Anal. Chem. 293,370

Open Question Revolutionary concepts in evolutionary cell biology W. Ford Doolittle Developments in micropaleontology, R N A and protein sequencing, and the analysis o f genome organization suggest a view o f early cellular evolution radically different from that accepted as recently as ten years ago.

It is difficult to remember exactly how one thought about cellular evolution ten or fifteen years ago and certainly risky to assume that everyone thought about it in that same way. However, it is probably fair to assume that most biochemists and molecular biologists then accepted the following three notions. (i) The appearance of the first living cell was the cumulative result of very many random events of extremely low probability, and thus the period of biological (cellular) evolution was preceded by a very much longer period of prebiotic evolution involving chance associations of non-biologically synthesized macromolecules accumulating in the Oparin [1] ocean. (ii) The first living cells 'were what we would now call prokaryotes, and these and their modem prokaryotic descendants, for all their biochemical diversity, represent a single monophyletic assemblage of entities which are essentially similar at the most fundamental levels of cellular organization, genetic organization and expression. (iii) Even though the 'line W. Ford Doolittle is at the Department of Biochemistry, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7.

of demarcation between eukaryotic and prokaryotic cellular organisms is the largest and most profound single evolutionary discontinuity in the contemporary biological world' [2], eukaryotes arose rather recently from among the prokaryotes (Fig. 1). The remarkable differences in genetic organizationl mechanisms of gene expression and evolutionary versatility which tempt us to consider eukaryotes more advanced than prokaryotes were to be seen as in part the cause and in part the consequence of the transition between prokaryotic and eukaryotic levels of cellular organization. Each of these three earlier views can now be seriously challenged. Although none of the points I make below is uniquely mine, none can be taken as proven, and some remain highly controversial, they deserve to be considered together because together they represent a radical revision of the way in which we think about cellular evolution. The points are these. (i) Living cells arose very early in the history of the earth. (ii) Living cells diverged very early into three (organizationally prokaryotic) lineages, the 'archaebacterial' lineage, the

'eubacterial' lineage (as defined by Woese and Fox [3]), and that lineage which gave rise to the nucleus of eukaryotic cells (termed the 'urkaryotic' lineage by Woese and Fox, and here called the nuclearcytoplasmic lineage). (iii) Eukaryotic cells themselves resulted from the repeated fusion of representatives of two of the three lineages. Living cells arose very early in the history of the earth The fossil record for multiceUular eukaryotes is some 700 million years old and, until the late 1950s or early 1960s, was the only fossil record we had [4]. Since then, microfossils which are cellular by several criteria have been discovered in deposits of increasing and almost unbelievable antiquity. Organic microstructures from the Swaziland system of S. Africa which, at 3.5 billion years, is more than three-fourths as old as the earth itself, have now been shown by Knoll and Barghoom [5] to be almost unquestionably biogenic, on five grounds: (i) chemical composition, (ii) unimodal size distribution, (iii) morphology, (iv) sedimentary context, and (v) the preservation of cells in the process of division. The oldest (3.8 billion years) sedimentary rocks are those of the Isua formation of Greenland. These too may show evidence of biological activity [6]. The earth itself is only some 700 million years older than these rocks [7]. Even if we assume that conditions conducive to the accumulation of non-biologically synthesized precursors of biological systems existed from the very beginning, we must conclude that life arose precipitously; a long period of biological (cellular) evolu© Elsevier/North-HollandBiomedicalPress 1980