Anal. Chem. 1990, 62,2566-2573
RECEIVEDfor review June 6,1990. Accepted August 6,1990. This work was supported in part by Grant CHE8901382 from the National Science Foundation and by the cooperative agreement between the University of Tennessee and the Oak
2565
Ridge National Laboratory and by the Office of Health and Environmental Research, US.Department of Energy, under Contract DE-AC05-84-OR21400with Martin Marietta Energy Systems, Inc.
Characterization of Underivatized Lipid Biomarkers from Microorganisms with Pyrolysis Short-Column Gas Chromatography/ Ion Trap Mass Spectrometry A. Peter Snyder* U S . A r m y Chemical Research, Development and Engineering Center, Aberdeen Proving Ground, Maryland 21010-5423 William H. McClennen, Jacek P.Dworzanski, a n d Henk L. C. Meuzelaar
Center for Micro Analysis and Reaction Chemistry, University of Utah, Salt Lake City, U t a h 84112
A microvolume Curie-point pyroiysls shortcolumn (5 m) gas chromatographylmass spectrometry (Py-GClMS) procedure was developed for the characterizationof various lipid moieties in microorganisms. High linear flow rates (approximately 175 cmls) characterized the GC conditions in order to effect an efficient chromatographic transfer and elution of the underlvatized diglycerides and monoglycerides, and small modifications were necessary to the ion trap MS system in order for it to accommodate the relatively high gas load. During a typical analysis run anhydrodiacyigiycerides eluted within a 5-6min t h e frame. Gram-positive bacilli and Gram-negative species were differentiated from each other by the pyrolysis patterns of thelr iipld components. I n spite of the complexity of the analyte, a straightforward vlsuai analysis was achleved with the aid of slmpie computerized data display procedures. These procedures Included examination of (1) total ion current (TIC) profiles of the lipid region of the reconstructed chromatogram, (2) the integrated mass spectrum of this region, (3) selected reconstructed ion chromatograms (RICs), (4) RIC intensity dlstrlbutions, and (5) corresponding mass spectra. An appealing aspect of the lipid data reduction procedure Is that most of it can be accomplished visually without requMng computerized pattern recognition technlques.
INTRODUCTION The detection and determination of microorganisms have been topics of major concern in many scientific areas. Medical/clinical monitoring of humans and animals and, in related fields, monitoring of drinking water, wastewater, and other environmental aspects are applications that fuel the broad array of bacterial testing and determination techniques. Furthermore, the need for a determination of bacterial presence is important for the energy-related fields such as oil, gas, and coal (1,2),the biodeterioration effects of many natural and man-made items (3),and the agriculture and foodstuff industries. A broad spectrum of microorganism characterization and determination methods has been developed that encompasses rapid (minutes to hours, ref 4-7, Staphaurex, Wellcome Diagnostics, Research Triangle Park, NC, and PathoDx, Diagnostics Products Corp., Los Angeles, CA) and slow (hours to 0003-2700/90/0362-2565$02.50/0
1day, (8,9)) single-step microbiological tests along with the classical albeit time-consuming test tube and membrane filtration bacterial growth/culture procedures (10). These techniques generally approach the organism with respect to its intact, viable form, and one or several portions of the bacteria are usually targeted, such as their intracellular and extracellular enzymes and macromolecular surface markers. These techniques are either specific or general in terms of the types of bacteria that can be ascertained. Usually, the more uncertain the identity of the microorganism, the larger the number of tests required in order to pinpoint the identity of the organism culture in question. These characteristics are not necessarily true for analytical techniques such as fluorescence (refs 11-13 and Vitek Systems, McDonnell Douglas, Hazelwood, MO), laser-induced fluorescence (14,151, microsensor analysis (16,17),electrochemistry (18),electrical impedance (19, 20), and Raman scattering (21). These methods can provide a convenient and relatively rapid vehicle for bacterial analyses while taking advantage of the microbiological characteristics of viable cells. The sensitivity to a detection event plays an important role here when compared to the positive/negative visual response that typically characterizes classical microbiological assays, and analytical techniques can be applied to many types of bacteria, while a microbiological assay is usually more specific in scope. There exists a class of analytical instrumentation technologies that involve some kind of processing of the organisms prior to analysis, and hence their viable or microbiologically active nature is usually compromised. With respect to the detection concepts of analytical instrumentation such as Fourier-transform infrared spectrometry, gas chromatography (GC), mass spectrometry (MS), time-of-flight MS, and nuclear magnetic resonance spectrometry, the sheer size and mass of a microorganism necessitates a sample processing step(s). In effect, an analysis consists of monitoring the "pieces and fragments" of the originally intact, viable bacterial cell. Shaw (22) has stated that of the myriad of components that can be found in microorganisms, the determination of their lipid composition is the closest to an ideal chemotaxonomic method. With regard to this concept, a typical analytical instrumentation-based investigation can be thought of as producing either an uninterpretive or microbiologically interpretive pattern or patterns of data, where the latter analysis is based on established biochemical compounds found in microor0 1990 American Chemical Society
2566
ANALYTICAL CHEMISTRY, VOL. 62, NO. 23, DECEMBER 1, 1990
ganisms. A number of analytical techniques belong to the latter category. In these pages, Fenselau et al. (23-25) have shown the merit of desorption techniques such as fast atom bombardment and laser desorption in the discrimination of bacteria as well as their mixtures. The intact phospholipid species that are ubiquitous to bacteria were desorbed from the cell surfaces and Gram-positive and -negative species could be distinguished by their respective phospholipid phosphate polar head groups. Direct mass measurements as well as the polar head group neutral losses from tandem mass spectrometry experiments provided the direct biochemical link in the interpretation of the desorption mass spectra. The fatty acid methyl ester (FAME) approach (refs 26, 27 and MIDI Microbial Identification System, Newark, DE) characterizes milligram amounts of bacteria through the saponification, derivatization, and extraction (SDE) of the lipid material to yield the methyl ester forms of the fatty acids. Injection onto a GC column serves as the analytical technique with either flame ionization or MS as the detector. The FAMES provide a diverse pattern and naturally provide a clear biochemical basis for either visual or numerical interpretation schemes (28-30). Related to this technique are the recent methods of in situ derivatization (31)or on-line derivatization (32) of bacterial fatty acids with GC or GC/MS analyses. Essentially, an in situ SDE of the fatty acids takes place within seconds by using Curie-point pyrolysis (Py) with microgram amounts of bacteria. Trimethylanilinium hydroxide (31)and trimethylammonium hydroxide (32) have been used as the derivatization reagents. The characterization of bacteria with pyrolysis techniques has largely been approached with Py-GC (33-37), Py-MS (38, 391, and Py-GC/MS (40-42). Pyrolysis of large complex analytes usually produces data too complex to be effectively evaluated without some type of matrix-based numerical data reduction technique (38, 41, 43-45). Overwhelmingly, the pyrolysis literature (38,41-48) characterizes differentiation and discrimination processes of bacterial data sets either as dealing with uninterpretive patterns with respect to their microbiological/biochemical origin or as containing identifiable, yet nonspecific, low molecular weight compounds. Reproducible patterns of GC or mass spectral data are typically subjected to one or more forms of multivariate data analysis, and conclusions are derived therefrom. The differentiation of two groups of Streptococci have been achieved by PyGC/MS (49);however this was based on the presence/absence of a single compound in the pyrolysis mass chromatogram and general distinctions between streptococcal groups were facilitated by the aid of multivariate methods (50). With pyrolysis direct chemical ionization MS, Tas et al. (51-53) have used multivariate analysis techniques for bacterial differentiation purposes. Discriminant spectra clearly portrayed mass spectral features in the 500-650-amu range and were ascribed to the lipid component. The raw mass spectra, however, showed the lipid component as very low abundant signatures in the high-mass range while a "chemical noise" envelope of masses devoid of mass spectral structure was produced in the 250-400-amu range. Discrimination and differentiation of a number of different microorganisms were addressed in the present work by maximizing the quality, type, and analytical depth of useful information that Py-GC/MS can offer. The lipid material was targeted as the informative biomarker from whole, underivatized bacteria and an emphasis was placed on a data reduction process that did not require complex numerical analysis methods. Optimization of a Py-GC system was necessary whereby a microvolume Curie-point pyrolysis reactor (54-56) was interfaced to a short (4-5 m) gas chromatography column. Uncharacteristically high linear carrier gas velocities
(approximately 175 cm/s) were used to provide for an efficient chromatography of the lipid material, and the effluent was directed via a splitless interface into an ion trap mass spectrometer that had its Teflon spacer rings removed. These conditions were found to provide for a straightforward interpretation of the bacterial mass chromatograms. The chromatograms could be visually dissected to yield a wealth of biochemically interpretive layers of information that were decidedly useful for microbial differentiation purposes. EXPERIMENTAL SECTION A total of fourteen strains representing nine bacterial species were investigated. Bacillus anthracis (BA) (a virulent and lowvirulent strain, BO463 and B0464, respectively),Bacillus cereus (BC) (B0037), Bacillus thuringiensis (BT) (BO158 and B0150), Bacillus licheniformk (BL) (B0017 and B0089),Bacillus subtilis (BS) (B0014 and B0095), Staphylococcus aureus (SA), Escherichia coli (EC) (type 0127), and Francisella tularensis (FA) (strains A362/67 (MRE 618) and Schu 4 (MRE 609)) were supplied by Tony P. Phillips (Center for Defence Establishment, Porton Down, U.K.) and Leslie A. Shute (University of Bristol, Bristol, U.K.). Legionella pneumophila (LP) serogroup 1 was provided by Luc Berwald (Rijkinstitute, Bilthoven, The Netherlands). Bacteria were grown in Lab M nutrient broth for 3 days at 37 "C. The cells were harvested by centrifugation, washed with sterile water, and then resuspended in water. Cells were killed by adding an equal volume of 6% H202and incubating overnight. Samples were then centrifuged, washed, and freeze-dried. L. pneumophila (1mg/mL suspension) were heat-killed at 120 "C and then lyophilized. Suspensions of the organisms (1.7 mg/mL) were prepared by adding 0.15 mL of methanol to 0.5 mg of the lyophilized bacteria, sonicating to effect a uniform dispersion, and then adding 0.15 mL of deionized distilled water. A 3-pL sample, or approximately 5 pg of bacteria, was applied to the tip of a Curie-point wire, and the suspension was dried in a stream of warm (40 "C) air. Phospholipid standards for lipid analyses were DL-1,2-dipalmitoyl-rac-glycerol and ~-1,2-dipalmitoyl-a-glycerophosphoryl-N,N-dimethylethanolamine and were obtained from Sigma Chemical Co., St. Louis, MO. ~-a-1,2-Dimyristoyl-snglycero-3-phosphocholine(lecithin)was purchased from LaMotte Chemical Products Co., Baltimore, MD. A 2-pL aliquot of a 1.5 mg/mL suspension/solution of a phospholipid in methylene dichloride or a 5050 methano1:methylene dichloride solution were applied to the Curie-point wire and dried. Py-GC/MS experiments were performed on a system including a Hewlett-Packard 5890 gas chromatograph (Palo Alto, CA) and a Finnigan-MAT ion trap mass spectrometer (ITMS) (SanJose, CA). The pyrolyzer consisted of our own (BPC-1000) low dead volume Curie-point pyrolysis GC inlet (54-56) and a 1-MHz, 1.5-kW Curie-point power supply (Fischer 0310). The sample on the 610 "C Curie-point ferromagnetic wire was placed in the 280 O C pyrolysis head inlet and then heated to the Curie temperature in a 1-s pyrolysis. A 5 m X 0.32 mm i.d. capillary column coated with a 0.25-pm film of dimethylsilicone (J & W Scientific, SE-30) was operated with a He carrier gas at 175 cm/s linear velocity. The column was temperature-programmed from 100 to 300 "C at 40 "C/min and held isothermal for 5 min. The column was connected directly into the ITMS vacuum through a 1-m transfer line maintained at 300 "C with the vacuum manifold at 180 "C. The ion trap was run without the normal Teflon spacer rings to allow better conductance of the high (8 mL/min) carrier gas flow. The long-term reproducibility studies and the phospholipid standards analyses were performed on a Finnigan-MAT 700 ion trap detector system. Different pyrolysis heads from that of the bacterial experiments were used with 610 "C ferromagnetic Curie-point wires. A 5-m BP-1 column (Chrompack) with a 0.25-pm film thickness was used that had a helium linear velocity of approximately 175 cm/s. The GC inlet was at 8 psig, the transfer line was maintained at 280 "C, and the Model 8500 Perkin-Elmer GC system was operated from 100 to 300 "C at 30 OC/min. To accommodate the high gas flows, several holes were drilled into the spacers of the ion trap detector and the trap was held isothermal at 225 "C. For both systems, spectra were accumulated over the mass
ANALYTICAL CHEMISTRY, VOL.
62, NO. 23, DECEMBER 1, 1990 2567
range 100-620 m / z at a rate of 1scan/s for approximately 8 min.
RESULTS AND DISCUSSION Pyrolysis is a sample processing technique that generally produces amounts of detectable products significantly greater than those which are informative for the sample under investigation. Essentially, a pyrogram can be thought of as consisting of chemical information and chemical noise. The term chemical noise in this case refers to the multitude of chemical compounds generated from the pyrolysis event that provide no real value in the data reduction process. This chemical noise actually hinders an analysis and is particularly acute for complex high molecular weight substances such as those in bacteria. The pyrolysis concept was reinvestigated not only in terms of the pyrolysis reactor but also in terms of the efficient transfer of pyrolyzate species to the detector proper with microorganisms as the analytes. The literature generally shows that lipid components are among the most diverse in terms of numbers of different compounds per organism, among the most specific for chemotaxonomic purposes (22,28,57,58),and among the larger nonpolymeric components of a bacterial cell. Therefore it was proposed that with an efficient pyrolysis technique coupled to a GC/MS system designed for an efficient chromatographic transfer and detection of the informative lipid species of a bacterial pyrolyzate, a desirable procedure could be established for the discrimination and differentiation of microorganisms. With this goal in mind, a microvolume Curie-point reactor was designed (54-56) such that the residence time of the pyrolysis products in the reaction zone was very short (0.1 s). The low effective dead volume of the pyrolysis reactor (0.02 mL), the high linear carrier gas velocity (approximately 10 cm/s) and the high temperature (approximately 300 "C) around the reactor head ensure a speedy delivery of the pyrolysis products to the GC column with a minimal amount of pyrolyzate condensation on the walls of the reactor and minimal bandbroadening of the GC peaks. A splitless capillary column interface to an ion trap mass spectrometer was used because with the original open split interface, many of the high-boiling compounds were found to be absent from the reconstructed chromatograms. As noted in the Experimental Section, holes were drilled and Teflon spacers were removed from the ion trap detector and ion trap mass spectrometer, respectively, in order to handle the increased helium flow rates. Phospholipid Standards. An important consideration in the analysis of lipid materials is the type of information that is generated. For underivatized high molecular weight lipids, thermal desorption pyrolysis products could range from intact, highly informative compounds (e.g., phospholipid moieties) to small (a*-*
Y
1 ' " ' ~ ' " ' I " '
% :
R%!
" '
'
"
"
,
'
'
'
r I
\
#I %6
Figure 4. Py-GC/MS lipid T I C of (A) E . anthracis 80464, thuringiensis BO 150, and (C) E . thuringiensis 80158.
(B) E .
presents the lipid TIC regions of the pyrolysis mass chromatograms of a low-virulent strain of B. anthracis and two strains of B. thuringiensis. The FAME procedure can differentiate between B. anthracis, B. cereus, and B. thuringiensis, also known as group E by Kaneda (53, and PyGC/MS can distinguish these bacteria by the lipid TIC portion of their pyrolysis mass chromatograms (Figures 3 and 4). At this relatively simple level of data analysis, one cannot distinguish between the BO463 (Figure 3) and BO464 (Figure 4) strains of B. anthracis while, remarkably, this can be accomplished with the BO150 and BO158 strains of B. thuringiensis. The FAME procedure, however, has a great deal of difficulty in distinguishing between the E . subtilis and B. licheniformis species, which are members of Kaneda's group A bacterial nomenclature (57). This is not the case with Py-GC/MS in that both organisms can be distinguished from each other with respect to their lipid TICs (Figure 5 ) , except that the lipid TIC patterns of the B. licheniformis BO089 (Figure 5B) and B. subtilis (Figure 5D) strains appear very similar. In general, all five bacillus species (Figures 3-5) can be easily distinguished by the lipid TIC portion of their pyrolysis TICs. The two B. anthracis and B. subtilis strains, however, cannot be distinguished from each other by a visual inspection of their respective lipid TICs (FiguresSA, 4A, and 5C,D). Figure 6 presents the lipid TIC portion of the pyrolysis
.a*
-,,
T ' ' " ' I ' '' '
388 5'81
S C ~ Nn TIME
6yg
Figure 0. Py-GC/MS lipid TIC of (A) S. aureus, (B) E . colitype 0127, (C) L . pneumphila (D)F . tularensls A326167, and (E) F . tularensis Schu 4. Note the different temperature ramp for the F. fularensls species. mass chromatogram of the Gram-positiveS. aureus and three Gram-negative organisms. These organisms along with the five bacilli can be readily differentiated at the species level by their lipid TICs. Lipid TIC Mass Spectra. Figure 7 provides strong evidence of the lipid nature of the high-boiling components in the bacterial pyrolysis mass chromatograms as well as a modicum of differentiation. Figure 7 presents the integrated, background-subtracted mass spectrum of the entire lipid TIC (lipid TIC mass spectrum) of each species. Note in particular the 14-amu methylene repeat unit throughout most of the mass spectra. The strains within each respective species provided the same lipid TIC mass spectrum except for B. licheniformis. The B. licheniformis BOO17 strain (data not shown) essentially had its m/z 522 and 550 relative intensities reversed from that of the BOO89 strain (Figure 7). The five bacilli provide virtually identical lipid TIC mass spectra and the E. coli and S. aureus organisms produce lipid TIC mass
ANALYTICAL CHEMISTRY, VOL. 62, NO. 23, DECEMBER 1, 1990
2589 n
494
I
564
Flgure 7. Py-GClMS lipid TIC mass spectra: BA, B. anthracis ; BC, B . cereus ; BT, 8.thuringknsis; BS, B. subtilis; BL, B. licheniformis ; SA, S . aweus; EC, E . coli; LP, L . pneumophih; FT, F.tuhrensis. All strains of each species produced the same mass spectrum except for 8.licheniformis . Shown is the mass spectrum for the BO089 strain, and the BO017 strain has a somewhat higher relathre intensity for m l z 522 with respect to m l z 550 (not shown).
spectra which are similar to those of the bacilli. The Legionella and Francisella species, however, display quite different lipid TIC mass spectra. Even though the lipid TICs of the bacilli, E. coli, and S. aureus are significantly different from each other, a simple mathematical "average" of the lipid TICs shows how similar these organisms are with respect to their overall lipid content and electron ionization fragments thereof. An interesting occurrence in the lipid TIC mass spectra is that most of the bacteria display an m / z 299 signal, most likely an electron ionization fragment, as the base peak. The origin of this mass appears to be the 1-pentadecyl-3dehydroxyglycerol ion, [CI4H2&O2CH2CH(OH)CH2J'+. Reconstructed Ion Chromatograms (RICs). Visual inspection of the lipid TIC profiles and the lipid TIC mass spectra provides the first two steps in the analysis of the bacterial pyrolysis mass chromatograms. The third layer of information is that provided by the RICs. The ions in the middle mass range (mlz 250-350) produce very broad RICs which are on the order of that of the entire lipid TIC (60), and these ions are specifically found in the high-boiling lipid TIC region (60). This indicates that these middle mass ions represent fragments resulting from the electron ionization process as opposed to being formed in the pyrolysis event. The relatively higher mass ions, however, produce RICs of approximately 10-20 s in width (Figure 8). For the selected RICs shown in Figure 8, note that m / z 494, 508, and 578 produce different impressions for different bacteria. This suggests that, at least for these ions, isomers of a particular lipid compound can be found that are more or less characteristic for a given bacterial species. It is interesting to note, however, that with the exception of the B. thuringiensis strain B0150, both m / z 494 and 508 RIC patterns partition the five bacilli into their respective Kaneda A and E groups (57). Generally, as the ions increase in mass, the retention time also increases (Figure 8). However, certain m / z 494 and 508 isomers elute in the higher boiling 330-350 scan number range, which overlaps with that of the m / z 550 RIC. The RIC also provides two further inherent modes of data analysis, as outlined in detail in the following sections. RIC Intensity Distribution. The maximum intensities of the selected RICs (Figure 8) for each organism, regardless of retention time, were plotted according to their m / z and are displayed in Figure 9. For these relatively few ions, a strong impression is achieved in terms of the bacterial differentiation
Figure 8. Representative RICs from the Py-GClMS bacterial lipid TIC regions: (A) B. anthracis , B. cereus, and B. thuringiensis (BO158); (B) B . subtilis , B . licheniformis, B . thuringiensis (BO150), and E . coli (S. aureus only showed the more intense, higher boiling peak); (C) 8. anthracis, B. cereus, and B. thwtngiensis (B0158) (E. mionly shows the earlier eluting peak, and 8.thdngknsis (B0150) displays the earlier eluting and dashed-line peaks); (D) B. subtilis, 8.licheniformis, and S . aureus; (E) L . pneumophila; (F) ail strains (except that only S. aweus shows the dashed peak in addition to the earlier eluting peak); (G) all strains (except m l r 536 is absent in S. aureus and F . tulsrensis); (H) all strains; (I) 8.anthracfs, L . pneumophh, and S . aweus ; (J) L . pneumophila; (K) F . tularensis; (L) L . pneumophila
.
potential of the lipid components. As observed in the m / z 494 and 508 RICs (Figure 8), the bacilli tend to correlate among themselves according to the Kaneda A and E groups. It is observed that the RIC intensities of the B. anthracis, B. cereus, and B. thuringiensis BO158 strains produce smoother patterns than the disjointed intensity patterns found for the B. thuringiensis BO150 strain and the Kaneda group A B. subtilis and B. licheniformis species. Note that the two B. anthracis and B. subtilis strains produce similar intensity distributions within their respective species. Despite the very similar lipid TIC impressions of the B. licheniformis BO089 and B. subtilis BOO14 strains (Figure 5), the RIC intensity distributions (Figure 9) of these organisms show a reproducible, significantly higher mlz 5221550 intensity ratio for the former as compared to the latter strain. S. aureus and the three Gram-negative organisms have quite different RIC intensity patterns, and most of the species in general can be differentiated by this parameter. Figure 9 also displays a plot of the B. anthracis BO463 RIC intensity distribution along with a superimposed replicate analysis. This second analysis not only took place 6 months later, but a different microvolume pyrolysis reactor head, GC column, heating rate, and ion trap mass spectrometer unit were used. This finding would seem to attest to the inherent reproducibility and robustness of the technique even with different instrumental systems and experimental parameters. As reported by Tas et al., however, this was not the case with pyrolysis direct chemical ionization of bacteria (52). The relatively low-abundant, high-mass lipid signatures (500-650 m u ) were observed to vary considerably during the 2-4 month long reproducibility studies. The variations were ascribed to ion source contamination as opposed to biological or bio-
2570
ANALYTICAL CHEMISTRY, VOL. 62, NO. 23, DECEMBER 1, 1990
0 A
Seh" 4 A 362i67
404 S M 122 190 5W 564 570 M 2 E M
404 508 512 534 550 164 578
m/z
Figure 9. Plots of the R I C intensity distribution of selected masses for each organism. The bottom plot represents a long-term reproducibility study of B.snthracb 80463. See text for details. Intensities were normalized on m l z 522. Adapted with permission from ref 60.
chemical variations of the Salmonella organisms. Extracted Ion Mass Spectra from t h e RICs. Each of the selected RICs for each organism was integrated and background-subtracted in order to display the relevant corresponding mass spectral information. Figure 10 presents the extracted ion mass spectra for the ions in Figure 9 for each of the bacterial species. Except for the B. anthracis and B. thuringiensis m / z 494 extracted mass spectra, all respective strains produced identical extracted ion mass spectra. The extracted mass spectra for m / z 494 and 508 were obtained from the most intense peak in their RICs (Figure 8). m / z 494. The bacilli can be divided into two groups where B. anthracis, B. cereus, and the BO158 strain of B. thuringiensis share a similar mass spectrum while the BO150 strain of B. thuringiensis and the other two bacilli share a common mass spectrum that is different from the former bacterial group. m / z 271 dominates the B. cereus group, and mlz 327 is found in the B. subtilis group. For eight replicate analyses of the B. anthracis strains and two for B. cereus, the latter organism provided a consistently higher m l z 2711299 intensity ratio (noted by the arrows in Figure 10). The last four m / z 494 extracted mass spectra (Figure 10) display the higher mass mlz 507,508, and 550 ions. This occurs because of the overlap with these four ions with the most intense feature (the higher boiling peak) in their RIC traces (Figure 8B). This overlap does not occur with that of B. anthracis and B. cereus because the peak that was analyzed was the more intense lower boiling peak (Figure 8A). Hence the ion of highest mass for these species in the m / z 494 extracted mass spectrum is m / z 494. Even though the lipid TIC is quite different between the bacilli, S. aureus, and E. coli (Figures 3-6), the m / z 494 extracted mass spectra of the two latter organisms are very
similar to those of the Kaneda group A bacilli. In addition, E . coli displays a prominent mlz 313, which is not found in the m / z 494 extracted mass spectra of the other organisms. m / z 508. Here, the Kaneda group A and E bacilli can be clearly differentiated from each other mainly by the differences in the intensities of m l z 271,285,327,493,494,and 550. Furthermore, the low-virulent and virulent forms of B. anthracis were reproducibly distinguished between each other by the m / z 2991313 intensity ratio (note arrows in Figure 10). The low-virulent form provides equivalent relative intensities of m / z 299 and 313, while mlz 299 is of a relatively greater abundance for the virulent organism. E. coli provides an m/z 508 extracted mass spectrum similar to that of the group E bacilli; however, instead of an mlz 313, E . coli displays an m / z 317 peak of low abundance. This could suggest the presence of a hydroxyacyl species (61). S. aureus displays mass spectral features similar to those of B. subtilis and B. licheniformis. The extracted mass spectrum of m l z 508 for the Legionella organism appears quite different from the other bacteria. m / z 522. The bacilli generally display very similar mlz 522 extracted mass spectra (Figure 10). However, the relative intensity of m / z 465 roughly divides the bacilli into their two Kaneda groupings. The B. anthracis strains show a minor, but reproducible, difference in that the low-virulent form provides an m / t 285 mass marker and the virulent organism lacks this ion (note arrows in Figure 10). The mass spectral slice of the E. coli and S. aureus lipid TICs (Figures 5 and 6) are very similar to those of the Kaneda group A bacilli. The Legionella organism displays relatively intense mlz 285 and 313 signlas that are of minor to negligible abundance in the other bacteria. m / z 536. It appears that the m / z 536 extracted mass spectra are very similar for the bacilli, except that the m / z 299 fragment ion is found only in the BO464 strain of B. anthracis. The two strains of B. anthracis can still be distinguished from each other in that the low-virulent strain provides an m l z 299 as the base peak while the virulent strain lacks this ion (Figure 10). The E. coli extracted mass spectrum of m / z 536 is similar in appearance to those of the bacilli, except that it lacks an m / z 493. The Legionella organism displays a mass spectrum similar to the low-virulent strain of B. anthracis, except that it exhibits an abundant m l z 479 peak that the other organisms lack. m / z 550. Here also the bacilli display similar mass spectra, except that they can be distinguished into their proper Kaneda groups by the abundance of mlz 299. Once again, E. coli displays a similar mass spectrum to those of the bacilli, specifically to the Kaneda group E organisms while s. aureus displays an extracted mass spectrum similar to those of the group A organisms. Legionella and the F. tularensis strain A 362167 both show quite different mlz 550 extracted mass spectra from the other organisms as well as between themselves. m / z 564. Only three species that were investigated displayed an m / z 564 ion, and their extracted mass spectra of this ion are shown in Figure 10. Each provides significant differences in terms of their electron ionization fragmentation patterns in that the base peak for each organism is a different mass. Hence a different isomer of m / z 564 is found in each microorganism. m / z 578. Only the Legionella and Francisella species produced this mass (Figure lo), and the fragmentation pattern is different for each. This indicates a different isomer of m / z 578 is found in each bacterium. It appears that the extracted ion mass spectra, or mass spectra that represent individual slices of the bacterial lipid TICs, offer a fair amount of information in terms of bacterial discrimination. In most of the extracted ion mass spectra,
m/z 522
m/z 564
BA, LV and V sm7
=y
LP b N
Flgure 10. PyQC/MS extracted ion mass spectra for the cited masses. m /I 494: the BO158 and BO 150 stralns of 8 . h&ngkn& had extracted mass spectra taken from their earlier and later eluting m l z 494 RIC peaks (Flgure E), respectively. The A362/67 straln of F . fukrensisis shown for m l z 550 and 578. Key: LV, low virulent; V, virulent. See text for details.
the two Kaneda groups of bacilli could be separated from each other, except that B. thuringiensis appears to fall into the group A bacterial category when the extracted mass spectra of mlz 494 and 536 were examined. The ambivalent nature of B. thuringiensis within this parameter and the RICs (Figure 8) and RIC intensity distribution (Figure 9) data reduction
methods provide an interesting comparison to the FAMEsderived Kaneda grouping of the bacilli (57). Another unexpected but interesting occurrence is the similarity of the E. coli and S. aureus extracted ion mass spectra to that of either the Kaneda group A or E bacilli (Table I). This occurs despite the dramatic visual differences of their lipid TICS (Figures
2572
ANALYTICAL CHEMISTRY, VOL. 62, NO.
23, DECEMBER 1, 1990
Table I. Similarity of t h e Extracted Ion M a s s Spectra of E . coli and S. aureus with Respect t o t h e K a n e d a G r o u p Bacilli
ion 494 508 522 536 550
E. coli
S. aureus
E A
A A A
E
A
t r v1
E 3-6). This phenomenon can be explained by the relative differences in abundances of the parent ions and their electron ionization fragments as they occur across the lipid TICs and is generally reflected in the RIC intensity distribution (Figure 9) of the organisms. B. thuringiensis, B. cereus, and B. anthracis strains could be distinguished by an extensive series of biochemical tests (621,and the last two organisms could be separated by canonical variate analysis of their Py-MS data (63). However, these same tests could not adequately separate low-virulent from virulent B. anthracis strains. Multivariate analysis techniques were decidedly helpful in the discrimination of FAME GC profiles of B. subtilis, B. licheniformis, B. cereus, and B. thuringiensis (64). Combinations of parameters in the present investigation could provide a visual discrimination between the microbiologically similar B. thuringiensis, B. cereus, and B. anthracis species (57). These visual analyses were found to extend into the other bacilli, S. aureus, and the selected Gram-negative species. Furthermore, for the B. anthracis species, a low-virulent and virulent strain could be differentiated by the fragmentation pattern of three separate extracted ion mass spectra, i.e. from m / z 508, 522, and 536. The subtle mass spectral differences associated with the B. anthracis and B. cereus species prompted an investigation of the relative abundance of m / t 271,285, and 299 throughout their lipid TICs (Figure 11). The RIC profiles were reproducible when compared in the short- and long-term studies. ml.7 211 clearly differentiated the B. anthracis and B. cereus species in that the latter organism portrayed a prominent peak a t 300-310 scan numbers while this was a minor feature for B. anthracis. All three strains could be distinguished by the m / z 285 RIC (Figure 11). The first two peaks 290-300 and 300-310 scan numbers) are opposite in relative abundance between the B. anthracis strains, while a distinct peak centered about scan number 320 is observed only in the B. cereas organism. Replicate RIC analyses are shown for the three organism strains for m / z 299 (Figure 11). The organisms can be distinguished from each other by the relative intensity distribution of the three peaks between 300-310,320-330 and 330-340 scan numbers. However, it should be noted that more strains of each organism must be investigated before any definitive conclusions can be reached concerning their PyGC/MS differentiation. CONCLUSIONS Curie-point pyrolysis was reinvestigated for its potential with regard to effective and meaningful differentiation of bacterial species with respect to the classical Py-GC, Py-MS, and Py-GC/MS procedures. A microvolume Curie-point pyrolysis reactor coupled to a short-column GC/MS system, along with high linear gas velocities, provided a framework for the visualization of an important class of biochemical information from whole, underivatized microorganisms. This information could be identified unambiguously as the lipid component. Furthermore, the chromatographic and mass spectral information were shown to outline various subcomponents of the lipids such as the dehydrated mono- and diacylglycerides. T o this end, data reduction techniques in-
n/z
...A'.'
285
'I,
.,,..,__.
.I
1 " " " " ' I "
i
!+
aie
5:BI
S C ~ Na
TIME
4ie
6:41
Figure 11. Reconstructed ion current chromatograms for m / z 2 7 1, 285,and 299 for B.anthracis and B . cereus. Short-term (one day) replicate analyses are presented for the three strains for m l z 299.
cluding the examination of the lipid TIC visual impression, lipid TIC mass spectrum, RIC, RIC intensity distribution, and extracted ion mass spectra were used to produce different perspectives of the extensive population of data in a bacterial lipid TIC. Taken together, a straightforward blueprint of a microorganism could be achieved with respect to its lipid biochemical component. These bacterial lipid blueprints appear to offer a major reservoir for microorganism discrimination and differentiation purposes despite the inherent complexity of the extremely high molecular weight bacterial analyte. The appealing aspect of the lipid data reduction procedure presented herein is that most of it can be accomplished visually without requiring computerized pattern recognition methods. It appears that the bacterial lipid information observed by the microvolume Curie-point pyrolysis short-column GC/MS approach complements analytical procedures such as the FAME and desorption MS techniques. However, a limitation of the ion trap approach is that only masses 5650 m u can be observed. Intact phospholipids have molecular weights of >650 amu and therefore the potential exists that these compounds might perhaps be observed by using a microvolume pyrolysis short-column GC system with a mass spectrometer that can detect these relatively higher masses. ACKNOWLEDGMENT We thank Linda G. Jarvis for the preparation and editing of the manuscript. LITERATURE CITED ( 1 ) Fukushima, K. J . Anal. Appi. Pyrolysis 1983, 5 , 245-256. (2) Philp, R. P.; Gilbert, T. 0. J . Anal. Appl. Pyrolysis 1987, 7 , 93-108.
(3) Barry, S.;Houghton, D. R. BlOdeterhKeHon 6;CAB International, Cambrian News Ltd.: Great Britain, 1966. (4) Karachewski,N. 0.; Busch, E. L.; Wells, C. L. J . Clin. Microbioi. 1985, 27, 122-126. (5) Dennehy, P. H.; Gauntlett, D. R.; Tente, W. E. J . Clin. Microbiol. 1988, 26, 1630-1634. (6) Sepulveda, J. L.; Stager, C. E.; Davis, J. R. J . Ciin. Microbioi. 1990, 26, 177-181. (7) Ezzeli, J. W., Jr.; Abshire, T. G.; Little, S.F.; Lidgerding, 8. C.; Brown, C . J . Clin. Microbiol. 1990, 28, 223-231. (8) Stratman, S.Am. Lab. 1988, 20, 60-64. (9) Covert, T. C.; Shadix, L. C.; Rice, E. W.; bines, J. R.; Freyberg, R. W. Appi. Environ. Microbioi. 1989, 55, 2443-2447. 10) Standstti Methods tw the Examinatbn of Water and Wastewater; American Publlc Health Association: Washington, DC, 1985; Part 900. 11) Shelly, D. C.; Quarles, J. M.; Warner, I . M. Anal. Len. 1981, 74(813), 1 111-1 124. 12) Snvder. A. P.; Wang, T. T.; Greenberg, D. E. Appl. Environ. Microbioi. 1988. 57, 969-977. 13) Berg, J. D.; Fiksdal. L. Appi. Environ. Microbioi. 1988, 5 4 , 2 118-21 22.
Anal. Chem. 1090, 62, 2573-2580
(32) (33) (34) (35) (36) (37) (38) (39) (40) (41) (42) (43)
Coburn, J. T.; Lytle, F. E.; Huber, D. M. Anal. Chem. 1085, 57, 1669- 1673. Hughes, K. D.; Lytle, F. E.; Huber, D. M. Anal. Chem. WSg, 67, 1656-1660. Hafeman, D. G.; Parce, J. W.: McConnell, H. M. Science 1988, 240, 1182-1185. Libby, J. M.: Wada, H. G. J. Clin. Microbiol. 1989, 27, 1456-1459. MaOyu, Y.; Zhang, Y. Appl. Envbn. Miembbl. 1989, 55, 2082-2085. Cody, P.: Dufour, S. W.; Shaw, J.; Kraeger, S. J. J. Clln. Microbiol. 1978. 7 . 265-272. Silverman, M. P.; Munoz, E. F. Appl. Environ. Microbiol. 1979, 37, 521-526. Britton, K. A.; Dalterio, R. A.; Nelson, W. H.; Britt, D.; Sperry, J. F. Appl. Spectrosc. 1988, 42, 782-788. Shaw, N. Adv. Appl. Mlcrobiol. 1974, 77, 63-108. Heller, D. N.; Cotter, R. J.; Fenselau, C.; Uy, 0. M. Anal. Chem. 1987, 59, 2806-2809. Platt. J. A.; Uy, 0. M.; Heller, D. N.; Cotter, R. J.; Fenselau, C. Anal. Chem. 1988, 60, 1415-1419. Heller, D. N.; Murphy, C. M.; Cotter, R. J.; Fenselau, C.; Uy, 0. M. Anal. Chem. 1988, 60, 2787-2791. Moss, C. W.: Dees. S. B.; Guerrant, G. 0. J. Clin. Microbiol. 1980, 12, 127-130. Miller, L.; Berger, T. Bacteria identification by gas chromatography of whole cell fatty acids. Hewlett-Packard Appllcatlon Note 228-41; Hewlett Packard: Palo Alto, CA, 1985. Austin, D. J.; Blake, P. S.; Fletcher, D. A,; Garrett, C. M. E. Chemotherap. Int. Lab. Systems 1888, 5 , 53-63. Eerola, E.; Lehtonen, 0.-P. J. Clin. Mlcrobiol. 1988, 26, 1745-1753. Mukwaya, G. M.; Welch. D. F. J. Clin. Microblol. 1989, 2 7 , 2640-2646. Holzer, G.; Bourne, T. F.; Bertsch, W. J. Chromatogr. 1989, 468, 181-190. Dworzanski, J.; Meuzelaar, H. L. C. Proceeding from the 36th American Soclety for Mass Spectrometry Conference on Mass Spectrometry end Allied Topics, San Francisco. CA, June 1988; pp 401-402. Relner, E. Nature 1965, 200, 1272-1274. Reiner. E.; Hicks, J. J.; Beam, R. E.; David, H. L. Am. Rev. Respir. Disease 1971, 104, 656-660. Reiner, E.; Hicks, J. J.; Ball, M. M.; Martin, W. J. Anal. Chem. 1972, 44, 1058-106t. Gutteridge, C. S.; Norris, J. R. J. Appl. Bacteriol. 1979, 47, 5-43. Papa, G.; Balbl, P.; Audisio, G. J. Anal. Appl. Pyroiysis 1987, 1 7 , 539-548. Meuzelaar, H. L. C.; Windig, W.; Harper, A. M.; Huff, S. M.; McClennen, W. H.; Richards, J. M. Science 1984, 226, 268-274. Voorhees, K. J.; Durfee, S. L.; Holtzciaw, J. R.; Enke, C. G.; Bauer, M. R. J . Anal. Appl. Pyrolysis 1988, 14, 7-15. Abbey, L. E.; Hlghsmlth, A. K.; Moran, T. F.; Reiner, E. J. J. Clin. Microblol. 1981, 73, 313-319. Engman, H.; Mayfield, H. T.; Mar, T.; Bertsch, W. J. Anal. Appl. pvrolysis 1984, 6 , 137-156. Medley, E. E.; Simmonds, P. G.; Manatt, S. L. Biomed. Mass Spectrom. 1975, 2 , 261-265. Irwin, W. J. Analytical Pyrolysis; Chromatographic Science: New York, 1982; Vol. 22, Chapter 2.
2573
(44) Voorhees, K. J. Analytical Pyrolysis; Butterworths: London, 1984. (45) Liebman, S. A.; Levy, E. J. q.rdysis and Gas Chrometogaphy in polymer Science; Chromatographic Science: New York, 1985; Vol. 29, Chapter 2. (46) Meuzelaar, H. L. C.; Haverkamp, J.; Hileman, F. D. +o/ysis Mass Spectrometry of Recent and Fossil Biomaterials ; Compendium and Atlas; Elsevier: Amsterdam, 1982. (47) Boon, J. J.; Brandtde Boer, B.; Eljkel, G. B.; Vlegels, E.; Sijtsma, L.; Wouters, J. T. M. I n h4ass Spectrometry in Biotechnological Process Analysis and Control; Heinzle, E., Reuss, M., Eds.; Plenum: New York, 1987; pp 187-208. (48) Simmonds, P. G. Appl. Microbiol. 1970, 20, 567-572. (49) Smith, C. S.;Morgan, S. L.; Parks, C. D.; Fox, A,; Pritchard, D. G. Anal. Chem. 1987, 59, 1410-1413. (50) Smfih, C. S.; Morgan, S. L.; Fox, A. J. Anal. Appl. molysis, In press. (51) Tas, A. C.; van der Greef, J.; de Waart, J.; Bouwman, J.; ten Noever de Brauw, M. C. J. Anal. Appl. Pyrolysis 1985, 7 , 249-255. (52) Tas, A. C.; de Waart, J.; Bouwman, J.; ten Noever de Brauw, M. C.; van der Greef, J. J. Anal. Appl. Pyrolysis 1987, 7 7 , 329-340. (53) Tas, A. C.; Wieten, G.; de Waart, J.; Berwald, L.; van der Greef, J. J. Microbiol. Methods 1988, 8 , 333-345. (54) Meuzelaar, H. L. C.; McClennen, W. H.; Yun, Y. Proceeding from the 35th American Society for Mass Spectrometry Conference on Mass Spectrometry and Allled Topics, Denver, CO, May 1987; pp 1142-1143. (55) McClennen, W. H.; Richards, J. M.; Meuzelaar, H. L. C. Proceeding from the 36th American Society for Mass Spectrometry Conference on Mass Spectrometry and Allied Topics; San Francisco, CA, June 1988; pp 403-404. (56) Richards, J. M.: McClennen, W. H.; Bunger, J. A.; Meuzelaar, H. L. C. Proceeding from the 36th American Society for Mass Spectrometry Conference on Mass Spectrometty and Allied Topics, San Francisco, CA, June 1988; pp 547-548. (57) Kaneda, T. Bacteriol. Rev. 1977, 4 7 , 391-418. (58) Ratledge, C.; Wilkinson, S. 0. MicrobialLipMs; Academic Press: London, 1988; Vol. 1. (59) Batrakov, S. G.; Sadovskaya, V. L.; Rozynov, B. V.; Bergelson, L. D. Chem. Phys. LipMs 1983, 33, 331-353. (60) Snyder, A. P.; McClennen, W. H.; Meuzelaar, H. L. C. I n Analyfical Microbiology Methods : Oas Chromatographyand Mass Spectrometry; Fox, A., Morgan, S. L., Larsson, L., Odham, G., Eds.; Plenum: New York, 1990; Chapter 13, pp 201-217. (61) Ikawa, M.; Koepfli, J. B.; Mudd, S. G.; Niemann, C. J. Am. Chem. SOC. 1953, 75, 1035-1038. (62) Logan, N. A.; Carman, J. A.; Melling, J.; Berkeley, R. C. W. J. Med. Microbiol. 1085, 20, 75-85. (63) O’Donnell, A. G.; Norris, J. R. I n The Aerobic Endospore-forming Becterie; Berkeley, R. C. W., Goodfellow, M., Eds.; Academic Press: London, 1981; pp 141-179. (64) O’Donnell, A. G. I n Computer-Assisted Bacterial Systematics; Goodfellow, M., Jones, D., Priest, F. G., Eds.; Academic Press: London, 1985; pp 403-414.
RECEIVED for review March 26, 1990. Accepted August 22, 1990.
Particle Beam Interface for Liquid ChromatographylMass Spectrometry Woodfin V. Ligon, Jr.* and Steven B.Dorn General Electric Company, Corporate Research and Development, Schenectady, New York 12301 A modlfled partlcle-beam-type llquld chromatographylmass spectrometry Interface has been developed that Is designed to be fully compatible with the vacuum requirements of a hlgh-resolutlon double focusing ma08 spectrometer. Important features Include a three-stage momentum separator, a nebullzer that Is both ultrasonlc and pneumatic, and an Inchamber desolvation heater. The momentum separator provldes Ion source pressures of (2-3) X lo-’ Torr for most solvents. The nebullzer Is capable of handllng high llquld chromatography flow rates while belng much less sensltlve to dlsperslon gas flow rate than Is a purely pneumatic nebulizer. The In-chamber desolvation heater Is feedback controlled by downstream gas temperature In order to automatically compensate for changes In solvent encountered durlng gradlent elutlons.
INTRODUCTION Browner and co-workers (1-3)have described an interface for liquid chromatography/masa spectrometry (LC/MS) based on aerosol formation. In their interface, a nebulizer is used to convert the LC effluent into an aerosol. After addition of a dispersion gas, the aerosol is allowed to drift through a “desolvation chamber” in which the aerosol particles tend to lose volatile solvents by evaporation. A t the downstream end of this chamber, dry particles of solute remaining from the desolvation process are formed into a high-velocity beam by a capillary nozzle. The central portion of this beam of dry particles is directed into a mass spectrometer by using two skimmers with strong pumping between the nozzle and the first skimmer and between the fiit and second skimmers. The combined effect of the pumping and skimmers results in
0003-2700/90/0362-2573$02.50/00 1990 American Chemical Society
