A Technique for Fast and Reproducible Fingerprinting of Bacteria by Pyrolysis Mass Spectrometry H. L. C. Meuzelaar and P. G. Kistemaker FOM-Instituut uoor Atoom- en Molecuulfysica, Amsterdam/ Wgm., The Netherlands
VERYLITTLE, IF ANY, work has been reported on the fingerprinting of complex biological material by pyrolysis mass spectrometry (Py-Ms) since Zemany ( I ) published Py-Ms fingerprints of albumin and pepsin in 1952. These proteins were pyrolyzed by filament heating in a closed borosilicate glass flask which was subsequently coupled to a mass spectrometer. No data on the reproducibility of the procedure were given. The apparent absence of activity in this field since then, must probably be explained by the advance of cheaper instrumental techniques for preparing pyrolysis fingerprints, notably pyrolysis gas-liquid chromatography (PyGLC). Although significant progress has been made in the development of Py-GLC techniques during the past ten years, the usefulness of these techniques for the fingerprinting of organic solids is still considerably limited by an obvious lack of standardization and an inherent lack of interlaboratory reproducibility ( 2 ) . Nevertheless, some of the more sophisticated pyrolysis techniques, such as the Curie-point pyrolysis technique developed by Simon and Giacobbo (3) and the filament pyrolysis technique developed by Levy ( 4 ) , are in principle suitable for standardization since nearly all parameters influencing the temperature/time profile can be adequately described. Our experience with Curie-point Py-GLC of complex biological material such as bacteria (5) although confirming the basic reproducibility of the method, indicates that at present the usefulness of Py-GLC techniques for these samples is probably more limited by a number of practical gas chromatographic problems than by small imperfections in pyrolysis techniques. Problems, such as insufficient column resolution, gradual deterioration of column performance, or sudden differences introduced by column replacement pose formidable obstacles to the computer processing of Py-GLC data and the compilation of reference libraries of standard fingerprints even if the use of such a library would be restricted to a single laboratory. Therefore, with the exception of a recent report by Menger, Epstein, Goldberg, and Reiner (6) describing an experiment on computer matching of fingerprints of Salmonella bacteria, most Py-GLC studies of complex biological samples have thus far relied on direct visual classification and identification of the obtained pyrograms. Because of the above-mentioned problems and in view of the great potentials of pyrolysis methods for the identification of complex biological material (7-II), we felt prompted (1) P. D. Zemany, ANAL.CHEM.,24, 1709 (1952). (2) N. B. Coupe, C. E. R. Jones, and S. G. Perry, J . Chromatogr., 47,291 (1970). (3) W. Simon and H. Giacobbo, Cliem.-l/zg.-Tech~z.,37, 709 (1965). (4) R. L. Levy, Thesis, Israel, Inst. Technol., Haifa, 1963. ( 5 ) H. L. C. Meuzelaar and R.,A. in 't Veld, J. Chromatogr. Sci., 10,213 (1972). (6) F. M. Menger, G. A. Epstein, D. A. Goldberg, and E. Reiner, ANAL.CHEM., 44,424 (1972). (7) R. L. Levy, Cliromntogr. Rea., 8 , 48 (1966). (8) A. Myers and L. Watson, Nature (Londo/z),223,965 (1969).
o i l diff pump
011
diff
rotary pump
pump
Figure 1. Curie-point pyrolyzer/quadrupole mass spectrometer combination (1) liq. N2-cooled brass screen, (2) electron impact ionization cage, (3) quadrupole rods, (4) electron multiplier, (5) gold diaphragm with
central 0.4-mm 6 pinhole, (6) expansion chamber (gold coated), (7) heating element, (8) three-way metal ball valves, (9) sample probe, (10) W o n O-ring, (11)hf-coil, (12) sample region, (13) quartz reaction chamber, (14) ferromagnetic pyrolysis wire, (15) PTFE reaction tube holder
to consider a different analytical approach to the preparation of pyrolysis fingerprints. We report here some of the initial results obtained with direct coupling of a Curie-point pyrolysis system to a fast scanning quadrupole mass filter and recording the spectra by means of a multi-channel signal averager. EXPERIMENTAL
Apparatus. The pyrolysis mass spectrometry system shown in Figure 1 was designed and constructed in our laboratory. Two 70 l./sec capacity oil diffusion pumps, fitted with Freon-cooled baffles and backed up by rotary pumps, maintain the entire system at better than 10W Torr. The vacuum lock for the insertion of the sample probe consists of two three-way metal ball valves (Argus GmbH, Ettlingen/ Baden, Germany) and two Viton sliding seals. A quartz reaction tube is connected to the stainless steel probe rod by a special polytetrafluoroethylene (PTFE) holder. This PTFE mantle also centers and retains the ferromagnetic Curie-point pyrolysis wire which carries the sample. If the probe is fully inserted, the sample zone of the wire is located exactly at the center of the hf-coil which can be energized by a Fischer Labortechnik hf-generator with a measured power output of 1.5 kW at 1.1 MHz and a range of pyrolysis times selectable between 0.2 and 4 seconds. In this position, (9) V. I. Oyama and G. C. Carle, J . Gas Chromatogr., 5, 151 (1967). (10) E. Reiner, ibid., p 65. ( 1 1 ) P. G. Simmonds, Appl. Microbiol., 20, 567 (1970). ANALYTICAL CHEMISTRY, VOL. 45, NO. 3, MARCH 1973
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Quadrupole mass - spectrometer
DC. o f f s e t
1024 Figure 2. Mass spectra obtained under identical instrumental conditions as the spectra in Figure 4 a : before pyrolysis (residual gas background) b: second pyrolysis of 510 "Cwire for 1 second, demonstrating that the first pyrolysis was essentially complete
the pyrolysis reactor makes a gastight connection to the expansion chamber by means of a Viton O-ring. This expansion chamber is maintained at 150 "C to prevent condensation of pyrolysis products. Furthermore, the inside of the chamber is coated with three layers of gold, interspaced with layers of rhodium, to minimize degradation of pyrolysis product molecules. The latter enter the electron impact ionization region of the quadrupole head through a 0.4-mm pinhole in the wall of the expansion chamber. The quadrupole mass filter used in these initial experiments is a simple residual gas analyzer probe (Varian Model 974002) with a fixed electron energy of 100 eV. This quadrupole mass filter is capable of unit resolution up to mje 50 and is equipped with an electron multiplier. Pyrolysis product molecules passing through the ion source without being ionized are trapped by a large, liquid Nz cooled, brass screen or by the diffusion pump. The cold screen assists the diffusion pump in counteracting a general pressure built-up outside the beam of pyrolysis products, thus maintaining an optimal signal/background ratio. Moreover the residual gas pressure is reduced to less than 2 X lo-' Torr and the corresponding mass spectrum shows only a few minor peaks (Figure 2a) apart from the peaks at m/e 18, 28, and 44. The current from the electron multiplier is amplified by a Keithley preamplifier and fed into a 1024-channel signal averager (Varian C 203). The latter also generates the sweep ramp which directs the mass scan of the quadrupole, thus synchronizing the mass scan with the memory address cycle of the signal averager. This arrangement is schematically depicted in Figure 3. The duration of the scans can be selected between 25 msec and 100 sec but the actual scanning speed will, in practice, be limited by the bandwidth of the pre-amplifier at the gain chosen. In principle, any mass region within the mass range of the quadrupole filter can be selected by adjusting the amplitude of the sweep ramp and adding a dc offset voltage. However, actually the mass 588
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signal
start
I
1:"'"'l channel averager
t
1r-l pulse
H E pyrolyser
X - Y recorder
Figure 3. BIock diagram of the experimental set-up
range of our quadrupole was restricted by the low resolution available. The function of the 1024 channel signal averager needs some discussion here since the combination of this instrument with a quadrupole mass filter, to our knowledge, has not received detailed attention before. After the initiation of the pyrolysis reaction, the beam of pyrolysis products emerging from the expansion chamber sustains a detectable ion current for about 30 seconds. During this period, the composition of the beam is changing profoundly, mainly as a consequence of the different diffusion rates of the components through the pinhole. The multichannel signal averager affords the summation of a relatively large number of spectra during these 30 seconds, thus integrating the total ion current at each m/e value. The final spectrum has not only an improved S/N ratio but is largely independent of temporary fluctuations in the composition of the product beam and reflects the original composition of the pyrolysate more closely than can be achieved by any single mass spectrum. This arrangement also makes it possible to study the effect of changing such parameters as pyrolysis time or sample amount on the composition of the pyrolysate, while automatically compensating accompanying non-essential changes in the temporary composition of the product beam. In our experience, signal averagers are excellently compatible with quadrupole mass filters in both technical and analytical respects and, as such, probably merit a more widespread use than they seem to have gained thus far. Procedure. The bacterial strains analyzed in this experiment were kindly provided by C. Walig and H. Dikken, Laboratorium voor Gezondheidsleer, Amsterdam. The Neisseriu strains were cultivated and harvested under identi-
NEISSER SICCA
a
1: 2-
j1, Ij I
I
~
0
30
LO
LO
Figure 4. Pyrolysis mass spectra of three bacterial strains ((a, b, and c). n and a' are duplicate analyses which demonstrate the high degree of reproducibility and are further evaluated in Table I. For conditions see text
c,al circumstances and their fingerprints (Figure 4, c1 and 6 ) may be directly compared. The Leptospira strain was cultured under different circumstances which may have influenced the fingerprint pattern (9) but was included in our study t o demonstrate the striking differences in the fingerprint patterns of taxonomically unrelated bacterial strains. All strains were washed several times in isotonic salt solutions, centrifuged, resuspended in distilled water, and freeze-dried. Fine suspensions of these freeze-dried samples were made in carbon disulfide (CS?) through mild sonification. By applying small drops of these suspensions t o the ferromagnetic Curie-point pyrolysis wires, which were slowly rotated t o ensure uniform distribution of the sample, and allowing the C S 2 t o evaporate, about 20 micrograms of the sample were tightly coated onto the wires. This suspension coating was developed by one of us (H.L.C.M.) and a detailed description of the method can be found elsewhere ( 5 ) . From the available range of ferromagnetic wires with different Curie-points, ( e . g . , 358, 480, 510, 610, 770, and 980 " C ) ,510 O C wires were chosen since at this temperature more characteristi': mass peaks were found with our samples than a't the higher temperatures. Moreover, at 510 "C, pyrolysis is still essentially complete within 1 sec, as demonstrated by the "repeated pyrolysis" spectrum in Figure 2 b, while a t lower temperatures the yield of pyrolysis products decreases a,nd much longer pyrolysis times would be necessary. All mass spectra were obtained by the accumulation of 60 scans of 0.5-sec duration in the signal averager memory. The scanning sequences were started by a trigger signal available from the hf-generator at the initiation of the pyrolysis procedure (see Figure 3). The electron multiplier gain was 5 x l o 3 and the preamplifiler setting 1 x 10P A. Minimum time needed for completing one analysis, including sample exchange, was less than 5 minutes. RESULTS AND DISCUSSION
The Py-Ms spectra shown in Figure 4 illustrate the reproducibility of two successive analyses of the Neisseria sicca sample. Comparison of the peak heights of these two spectra (see Table I) shows that the standard deviation of the peak ratios is less than 4z. The peaks at mle 18,28, and 44, which
Table I. Quantitative Evaluation of Duplicate Analyses of Neisseria sicca bacteria (a and a' in Figure 4) Ion current (arb. units) mle
a
12 141 13 25 14 119 15 233 16 692 17 1175 22 63 25 18 26 98 27 300 29 339 30 62 31 60 32 102 33 43 34 115 37 16 38 22 39 67 40 34 41 100 42 91 43 225 45 46 46 10 47 22 48 29 Total 4246 Mean value of n'la
a'
160 28 130 262 794 1265 72 20 109 3 20 363 69 68 109 47 115 18 24 72 38 108 100 248 50 10 22 32 4653
a'/o
1.13 1.12 1.09 1.12 1.15 1.08 1.14 1.11 1.11 1.07 1.07 1.11 1.13 1.07 1.09 1.00 1.13 1.09 1.07 1.12 1.08 1.10 1.10 1.09
1 .oo
1 .oo 1.10 -
1.09
=I=0.04
have a substantial contribution from the residual gas background, have been omitted from the table. Although a certain degree of quantitative reproducibility has also been obtained in other Py-Ms experiments ( I , 12, 13), the high degree of quantitative reproducibility achieved by our method is perhaps unique. To our opinion, this improvement should be ascribed to the use of a better defined pyrolysis technique and the summation of a series of rapid mass scans by the signal averager instead of recording a single spectrum a t arbitrarily chosen time points. Furthermore, on visual inspection of the spectra in Figure 4, the differences between the three bacterial strains are obvious and the extent of these differences seems t o agree with the degree of taxonomic relationship as established by conventional methods. This is, however, by no means true for bacterial pyrolysis patterns in general (14). Although we are fully aware of the preliminary nature of our experiments and more work needs to be done to establish clearly the value and limitations of the method, it is tempting t o make a tentative comparison with Py-GLC, a t least insofar as both techniques can be regarded as fingerprinting methods. The prospects of chemical identification of PyG L C peaks by direct coupling to a mass spectrometer (PyGLC-MS) (11) will not be considered therefore. To start with the conditions of the pyrolysis reaction: the fact that this reaction proceeds at much lower pressures than in PyGLC may be expected, among other effects, to increase the maximum size of the fragments which are volatile enough to escape from the reaction zone and to decrease the chance of secondary reactions in the gas phase. However, since (12) H. D. R. Schhddemage, Thesis, Universitat Koln, 1967. (13) E. Bua and P. Manaresi, ANAL.CHEM., 31, 2022 (1959). (14) E. Reicer and W. H. Ewing, Nuliafure(Lo/?do/i),217, 191 (1968).
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the reaction mechanisms of solid phase pyrolysis are poorly understood, even for comparatively simple substances (15), the net effect of a low pressure environment on the course and reproducibility of the pyrolysis process is hard to predict. Nevertheless any effect that would be noticeable at all is probably beneficiary, a speculation borne out by the high degree of reproducibility encountered in this experiment. All pyrolysis products volatile enough to pass through the expansion chamber and subsequently ionized in the ion source, will contribute to the final mass spectrum at the appropriate mje values. With Py-GLC, this situation is different because of the chemical selection imposed by the column, which may exclude specific chemical classes-e.g., free acids or free amines-from contributing to the obtained fingerprint. I n a separate experiment, the existence of such classes of bacterial pyrolysis products was established by high resolution field ionization mass spectrometry in cooperation with a group a t the University of Bonn, Germany (16). The advantage of high analysis speed needs little comment. Thirty seconds after initiation of the pyrolysis reaction, the complete spectrum is recorded in digital form in the signal averager memory and if provisions are made for fast computer readout of this memory and the sample introduction system is modified so as to permit the introduction of several samples a t once, then the effective analysis time should be less than 1 minute. This is about 30-60 times as fast as can be accomplished by Py-GLC when taking into account that the use of high resolution columns and wide range temperature programming will generally be necessary for complex biological samples. Because of the stable and linear mass scale of the quadrupole spectrometer, which is not plagued by problems analogous to column deterioration by Py-GLC or different performance of replacement columns, the ease of data-processing is striking in comparison with Py-GLC. Moreover, the subject of computer coding, filing, retrieving, and matching of mass spectra has received widespread (15) J. Q. Walker and C. J. Wolf, J. Clirornafogr.Sci., 8, 513 (1970). (16) H. R. Schulten, H. D. Beckey, H. L. C. Meuzelaar, and A. J. H.
Boerboom, ANAL.CHEM., 45, 191 (1973).
attention in literature during the past few years (17) and PyMS may certainly benefit from the activities in this field. If Py-MS thus appears to be a promising method for fingerprinting of complex biological samples, it seems nevertheless regrettable that virtually all information o n the chemical composition of the sample, in principle obtainable from the pyrolysis process (It?), is lost in the electron impact ionization and fragmentation processes occurring in the ion source of the quadrupole. The use of ionization techniques causing negligible fragmentation such as field ionization (19), chemical ionization .(20), or low-voltage electron-impact ionization (21) seems to be the obvious experimental approach to this problem. Apart from the earlier mentioned experiments with high resolution field ionization mass spectrometry ( I @ , we are at present engaged in low-voltage electron-impact ionization studies, which also encompass higher mass ranges than the study reported here (22). ACKNOWLEDGMENT The authors thank J. Kistemaker and A. J. H. Boerboom for their invaluable support and advice. They also gratefully acknowledge the expert assistance of M. Hoogervorst, W. J. Barsingerhorn, and R. Heubers, with the design and construction of the pyrolysis mass spectrometry system.
RECEIVED for review July 17, 1972. Accepted October 16, 1972. This research is part of a project sponsored by the Organization for Fundamental Research on Matter (FOM) and the Dutch Ministry of Health. (1 7) H. S. Hertz, R. A. Hites, and K. Biemann, ANAL.C H E M . ,681 ~~,
(1971). (18) W. Simon, P. Kriemler, J. A. Vollmin, and H. Steiner, J. Gas Chromatogr., 5 5 3 (1967). (19) H. D. Beckey, “Field Ionization Mass Spectrometry,” Pergamon Press, Oxford, and Akademie Verlag, Berlin, 1971. 43 (13), 28A (1971). (20) M. S. B. Munson, ANAL.CHEM., (21) F. H. Field and H. S. Hastings, ibid.,28, 1248 (1956). (22) H. L. C. Meuzelaar, M. A. Posthumus, P. G. Kistemaker, and J. Kistemaker, ANAL.CHEM.,in press.
Construction of Optimum Variables for Spectral Interpretation (Pattern Recognition) C. F. Bender and B. R. Kowalski’ Lawrence Licemore Laboratory, Licermore, Calif. 94550 PATTERN RECOGNITION techniques have recently been used to extract chemical information from spectroscopic data ( I ) . Basically the problem is the following: Using spectra where the sought-for property is known, construct a rule for “recognizing’’ the property (or classification). The adopted rule can then be applied for predictive purposes o n unknown spectra. Although pattern recognition techniques have proved to be quite effective for this type of problem, a few difficulties Present address, Department of Chemistry, Colorado State University, Fort Collins, Colo.
still remain. The most pressing problem is related to the fact that for most classification problems, standard representation of the spectral information does not lead to linearly separable subspaces. This can cause erroneous predictions from linear learning machines (2). In some cases, clusters d o not even exist and hence a nearest neighbor technique (3) will also yield misleading classifications. This problem can be minimized by improving the classification rule or changing the representation of the information. (2) N. J. Nilsson, “Learning Machines,” McGraw-Hill, New York,
(1) T. L. Isenhour and P. C. Jurs, ANAL.CHEM.,43 (lo), 20A (1971); also see references within.
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N.Y., 1965. (3) B. R. Kowalski and C. F. Bender, ANAL.CHEM., 44,1405 (1972).
