Technical Notes

Correlation of Mass Spectrometry IdentifiedBacterial Biomarkers from a Fielded Pyrolysis-GasChromatography-Ion Mobility SpectrometryBiodetector with the Microbiological Gram StainClassification Scheme

A. Peter Snyder,*,† Jacek P. Dworzanski,‡ Ashish Tripathi,‡ Waleed M. Maswadeh,† andCharles H. Wick†

Research and Technology Directorate, RDECOM, Edgewood Chemical Biological Center, Aberdeen Proving Ground,Maryland 21010-5424, and Geo-Centers, Inc., Aberdeen Proving Ground, Maryland 21010-5424

A pyrolysis-gas chromatography-ion mobility spectrometry(Py-GC-IMS) briefcase system has been shown to detectand classify deliberately released bioaerosols in outdoorfield scenarios. The bioaerosols included Gram-positiveand Gram-negative bacteria, MS-2 coliphage virus, andovalbumin protein species. However, the origin andstructural identities of the pyrolysate peaks in the GC-IMS data space, their microbiological information content,and taxonomic importance with respect to biodetectionhave not been determined. The present work interrogatesthe identities of the peaks by inserting a time-of-flight massspectrometry system in parallel with the IMS detectorthrough a Tee connection in the GC module. Biologicalsubstances producing ion mobility peaks from the pyroly-sis of microorganisms were identified by their GC reten-tion time, matching of their electron ionization massspectra with authentic standards, and the National Insti-tutes for Standards and Technology mass spectral data-base. Strong signals from 2-pyridinecarboxamide wereidentified in Bacillus samples including Bacillus an-thracis, and its origin was traced to the cell wall pepti-doglycan macromolecule. 3-Hydroxymyristic acid is acomponent of lipopolysaccharides in the cell walls ofGram-negative organisms. The Gram-negative Escheri-chia coli organism showed significant amounts of 3-hydroxymyristic acid derivatives and degradation prod-ucts in Py-GC-MS analyses. Some of the fatty acidderivatives were observed in very low abundance in theion mobility spectra, and the higher boiling lipid specieswere absent. Evidence is presented that the Py-GC-ambient temperature and pressure-IMS system generatesand detects bacterial biochemical information that can

serve as components of a biological classification schemedirectly correlated to the Gram stain reaction in micro-organism taxonomy.

Outdoor analytical systems that detect biological and chemicalsubstances usually trace their roots to the configuration andperformance concepts of their laboratory-based brethren. Fieldedinstruments are usually compromised in the performance figuresof merit because of logistics and parameters such as operatorportability/transportability, small configuration and footprint, nobottled gas, extended times of unattended operation, low main-tenance, turnkey start-up, no chemical and biological substanceexpendables, and low power requirements.

Analytical ion mobility spectrometry (IMS) has been appliedin field and laboratory research and technology applications andis straightforward in concept and design.1,2 When operated in thelow-Torr regime under laboratory conditions interfaced in serieswith a time-of-flight mass spectrometry (TOF-MS) system, separa-tion of complex mixtures can be obtained. Examples can be foundin the literature from Clemmer et al.3-5 with bacterial proteomeextracts, Woods et al.6-9 with lipids and peptides, Hill et al.10-12

on amino acids, peptides, and protein conformation, and Bowers

* To whom correspondence should be addressed. Phone: 410-436-2416.Fax: 410-436-1912. E-mail: apsnyder@apgea.army.mil.

† Edgewood Chemical Biological Center.‡ Geo-Centers, Inc.

(1) Eiceman, G. A., Karpas, Z., Eds. Ion Mobility Spectrometry; CRC Press: BocaRaton, FL, 1994.

(2) Turner, R. B. Pure Appl. Chem. 2002, 74, 2317-2322.(3) Hoaglund-Hyzer, C. S.; Clemmer, D. E. Anal. Chem. 2001, 73, 177-184.(4) Barnes, C. A. S.; Hilderbrand, A. E.; Valentine, S. J.; Clemmer, D. E. Anal.

Chem. 2002, 74, 26-36.(5) Myung, S.; Lee, Y. J.; Moon, M. H.; Taraszka, J.; Sowell, R.; Koeniger, S.;

Hilderbrand, A. E.; Valentine, S. J.; Cherbas, L.; Cherbas, P.; Kaufmann, T.C.; Miller, D. F.; Mechref, Y.; Novotny, M.; Ewing, M. A.; Sporleder, C. R.;Clemmer, D. E. Anal. Chem. 2003, 75, 5137-5145.

(6) Gillig, K. J.; Ruotolo, B.; Stone, E. G.; Russell, D. R.; Fuhrer, K.; Gonin, M.;Schultz, A. J. Anal. Chem. 2000, 72, 3965-3971.

(7) Woods, A. S.; Koomen, J. M.; Ruotolo, B. T.; Gillig, K. J.; Fuhrer, K.; Gonin,M.; Egan, T. F.; Schultz, J. A. J. Am. Soc. Mass Spectrom. 2002, 13, 166-169.

(8) McLean, J. A.; Russell, D. H. J. Proteome Res. 2003, 2, 427-430.(9) Woods, A. S.; Ugarov, M.; Egan, T.; Koomen, J.; Gillig, K. J.; Fuhrer, K.;

Gonin, M.; Schultz, J. A. Anal. Chem. 2004, 76, 2187-2195.

Anal. Chem. 2004, 76, 6492-6499

6492 Analytical Chemistry, Vol. 76, No. 21, November 1, 2004 10.1021/ac040099i CCC: $27.50 © 2004 American Chemical SocietyPublished on Web 10/02/2004

et al.13-16 on carbon cluster compounds. At the opposite end ofthe spectrum, where an IMS detector is operated at close toatmospheric pressure, lower performance characteristics becomeevident,1 yet meaningful information can still be obtained.

Atmospheric pressure IMS is well suited in outdoors, fieldapplications.1,2,17-23 Field utility of IMS is attractive, because it hasa high degree of portability and low logistics and consumablerequirements.

Pyrolysis (Py) converts a solid sample to vapors by rapidheating, and gas chromatography (GC) separates a vapor mixtureinto its individual constituents. Thus, large solid biologicalsubstances such as proteins and bacteria17-20,24 can be interrogatedby IMS through their thermal conversion into low mass species.

GC-electron ionization (EI)-MS can provide unambiguousidentification of a sample from the retention time of a substanceand its fragmentation pattern. GC-IMS, however, consists of twoanalytical separation stages, in series, where the information inboth dimensions is time. This situation does not allow for theidentification of a compound compared to that of GC-MS. Alaboratory Curie point wire, Py-GC-heated IMS-MS system wasused to investigate the presence of dipicolinic acid (DPA) inmicroorganisms.25 A Tee was placed in the GC to partition andtransfer the biological sample at the same retention time into theion trap MS and IMS detectors. The presence (Gram-positiveorganisms) and absence (Gram-negative organisms) of the pi-colinic acid thermal fragment of DPA in the GC-IMS and GC-MS data spaces were determined. DPA, as the calcium dipicolinatesalt, is found only in Gram-positive bacterial spores and constitutes5-15 wt %.26,27

The Py-GC-IMS laboratory concept25,28 was transformed intoa field-operational, briefcase bioaerosol detector, and an aerosolconcentrator was interfaced to the pyrolysis source of the system.The IMS detector component is a modified ambient pressure andtemperature chemical agent monitor (CAM) that is used by NorthAtlantic Treaty Organization (NATO) armed forces for thedetection and screening of chemical agent vapor. Outdoor bio-aerosols consisting of Gram-positive Bacillus atrophaeus (formerlyBacillus subtilis var. globigii) spores, Gram-negative Pantoeaagglomerans (formerly Erwinia herbicola) cells, ovalbumin protein(OV), and MS-2 coliphage virus were released during formal trialsat Western desert and prairie test sites in the United States andCanada.18-20 Differentiation of the four bioaerosols was possibleby visual18-20 and multivariate factor analysis determinations(unpublished data) of the dispersion of pyrolysate peaks in theGC-IMS data space. These series of tests documented the firstsuccessful detection and differentiation of outdoor-released bio-aerosols with an ambient temperature and pressure IMS detectorinterfaced to a biological sample Py-GC processing system.

Identification of the biological responses in the GC-IMS dataspace of the briefcase, fielded Py-GC-ambient temperature-IMSbiodetector is investigated herein by interfacing an EI-TOF-MSsystem in a parallel configuration similar to that of the Py-GC-IMS-MS laboratory system.25 The analytical mass spectrometerwas used to directly identify the compounds produced andtransferred by the Py-GC module in the fielded Py-GC-IMSdevice. Mass spectral information provided evidence that polarpyrolysate compounds with significant biological classificationinformation (biomarkers) were passing through the torturous Py-GC pathway. However, some of these compounds were spectrallysilent in the IMS detector. It is suggested that heating the IMScell would allow the observation of more and biologically relevant,GC-IMS data space peak information in the Py-GC-IMS system.

Evidence is presented that the outdoor fielded Py-GC-ambienttemperature and pressure IMS bioaerosol detector producesbiochemical information that can be correlated with the Gram stainreaction used for Gram-positive and Gram-negative microorganismtaxonomy.

EXPERIMENTAL SECTIONMaterials. All protein and biochemical standards were ob-

tained from Sigma-Aldrich (St. Louis, MO), except 2-pyridinecar-boxamide, which was purchased from Lancaster Synthesis(Windham, NH), and Staphylococcal enterotoxin B (SEB), whichwas purchased from List Biological Laboratories, Inc. Peptido-glycan from Staphylococcus aureus and lipopolysaccharide (LPS)from Escherichia coli K-235, the latter prepared by phenolextraction, were suspended in methanol (1 mg/mL) while modelamino acids, sugars, peptides, proteins, and nucleic acids wereused as water solutions (1 mg/mL).

Bacteria Preparation. Gram-positive vegetative cells of Bacil-lus anthracis and spores of Bacillus cereus ATCC 6464, Bacillusmegaterium, and B. atrophaeus were prepared as suspensions indeionized, distilled water. The B. anthracis strains included VNR1-∆1, ∆ Texas, and ∆ Ames. The Bacilli and suspensions of Gram-negative P. agglomerans ATCC 33248 and E. coli K-12 were heat

(10) Matz, L. M.; Asbury, G. R.; Hill, H. H., Jr. Rapid Commun. Mass Spectrom.2002, 16, 670-675.

(11) Matz, L. M.; Steiner, W. E.; Clowers, B. H.; Hill, H. H., Jr. Intl. J. MassSpectrom. 2002, 213, 191-202.

(12) Steiner, W. E.; Clowers, B. H.; English, W. A.; Hill, H. H., Jr. Rapid Commun.Mass Spectrom. 2004, 18, 1-7.

(13) Lee, S.; Gotts, N. G.; von Heldon, G.; Bowers, M. T. Science 1995, 267,999-1001.

(14) Bowers, M. T.; Kemper, P. R.; von Helden, G.; van Koppen, P. A. M. Science1993, 260, 1446-1451.

(15) Dugourd, Ph.; Hudgins, R. R.; Clemmer, D. E.; Jarrold, M. F. Rev. Sci.Instrum. 1997, 68, 1122-1129.

(16) Fye, J. L.; Jarrold, M. F. Int. J. Mass Spectrom. 1999, 185/186/187, 507-515.

(17) Dworzanski, J. P.; McClennen, W. H.; Cole, P. A.; Thornton, S. N.;Meuzelaar, H. L. C.; Arnold, N. S.; Snyder, A. P. Field Anal. Chem. Technol.1997, 1, 295-305.

(18) Snyder, A. P.; Maswadeh, M. M.; Parsons, J. A.; Tripathi, A.; Meuzelaar,H. L. C.; Dworzanski, J. P.; Kim, M.-G. Field Anal. Chem. Technol. 1996,3, 315-326.

(19) Snyder, A. P.; Maswadeh, M. M.; Tripathi, A.; Dworzanski, J. P. Field Anal.Chem. Technol. 2000, 4, 111-126.

(20) Snyder, A. P.; Tripathi, A.; Maswadeh, M. M.; Ho, J.; Spence, M. Field Anal.Chem. Technol. 2001, 5, 190-204.

(21) Snyder, A. P.; Maswadeh, M. M.; Tripathi, A.; Eversole, J.; Ho, J.; Spence,M. Anal. Chim. Acta, 2004, 513, 365-377.

(22) Eiceman, G. A. Crit. Rev. Anal. Chem. 1991, 22, 471-490.(23) Baumbach, J. I.; Eiceman, G. A. Appl. Spectrosc. 1999, 53, 338A-355A.(24) Vinopal, R. T.; Jadamec, J. R.; deFur, P.; Demars, A. L.; Jakubielski, S.;

Green, C.; Anderson, C. P.; Dugas, J. E.; DeBono, R. F. Anal. Chim. Acta2002, 457, 83-95.

(25) Snyder, A. P.; Thornton, S. N.; Dworzanski, J. P.; Meuzelaar, H. L. C. FieldAnal. Chem. Technol. 1996, 1, 49-58.

(26) Breed, R. S., Murray, E. G. D., Smith, N. R., Eds. Bergey’s Manual ofDeterminative Bacteriology, 7th ed.; The Williams & Wilkins Co.: Baltimore,MD, 1957; pp 295-305.

(27) Gould, G. W., Hurst, A., Eds. The Bacterial Spore; Academic Press: NewYork, 1969.

(28) Snyder, A. P.; Harden, C. S.; Brittain, A. H.; Kim, M.-G.; Arnold, N. S.;Meuzelaar, H. L. C. Anal. Chem. 1993, 65, 299-306.

Analytical Chemistry, Vol. 76, No. 21, November 1, 2004 6493

killed in an autoclave. A 1-µL aliquot of 0.1-5 mg/mL suspensionswas used for all bacteria. B. atrophaeus was chosen because itproduces spore-containing DPA and is a surrogate for B. anthracisin outdoor aerosol biodetection scenarios. P. agglomerans waschosen because it does not contain DPA, and it is the only Gram-negative organism approved by the Environmental ProtectionAgency for outdoor, military bioaerosol dissemination studies.Furthermore, P. agglomerans is used as a surrogate for pathogenicGram-negative organisms.

Gram-positive bacterial growth and spore formation are de-scribed in the following procedures. A Bacillus culture wasstreaked for isolation onto an agar plate containing trypticase soyagar (TSA) and 5% sheep’s blood and incubated overnight at 37C. A single colony was streaked onto an agar plate and incubatedat 37 C for about 4-6 h or until there was visible growth on theplate. The bacterial growth was scraped from the agar plate andwas suspended in sterile broth or saline. A 500-µL aliquot of thebacterial suspension was pipetted onto the surface of nutrientsporulation media in an agar plate and spread evenly with a cellspreader. These plates were incubated at 37 C for 3 days untilspore production reached a maximum, and the spore generationwas monitored using phase contrast microscopy. Vegetative cellsappeared as dark, oblong cells in short or long chains except forB. atrophaeus. B. atrophaeus spores appeared as smaller, highlyrefractile bodies either within vegetative cells or free-floating.Growth was scraped from the agar plates and washed twice with200 mL of sterile water, resuspended in sterile water, heat-killed,and lyophilized. Vegetative cells were prepared by suspendingisolated colonies from TSA and sheep’s blood agar plates in sterilewater to a density corresponding to a 1 McFarland standard.29

This was diluted 1:10, and 2 mL of the 1:10 suspension was addedto 200 mL of nutrient broth. The nutrient broth was incubatedovernight at ambient temperature, centrifuged, and washed twicewith 200 mL of sterile water. The bacterial pellet was resuspendedin sterile water, heat-killed, and lyophilized. All cultures wereautoclaved for 20 min to kill both vegetative cells and spores. B.anthracis preparations were cultured and killed under BiosafetyLevel 3 (BL-3) facilities, and all other organisms were handledand processed under minimal safety level BL-1 conditions.

For the Gram-negative vegetative organisms, E. coli wasstreaked onto nutrient agar plates, and P. agglomerans was grownin Brucella broth. Both organisms were incubated for 24 h at 37C. Ten milliliters of nutrient broth was placed in a 15-mL conicaltube and seeded with the Gram-negative bacteria. The tube wasincubated at 37 °C for 24 h. A 250-mL aliquot of nutrient brothwas placed in a 500-mL Erlenmeyer flask and was seeded with a1:10 dilution of a 1 McFarland preparation from the bacterialgrowth in the 15-mL conical tube. The flask was placed in ashaking incubator at ambient temperature. Aliquots of bacteriain broth were centrifuged at 10 000 rpm for 25 min and washedtwice with 200 mL of sterile water. The pellet was resuspended,and aliquots were placed into the pyrolysis module.

Pyrolysis-Gas Chromatography-Ion Mobility Spectrometry-Mass Spectrometry. The pyrolysis sample processor consistedof a Pyrex tube (4-mm i.d., 8-mm o.d., and 5-cm length) with aquartz microfiber filter (type QM-A, thickness 0.45 mm, from

Whatman, Hillsboro, OR) supported by a Pyrex frit inside the tube(Figure 1). The Pyrex tube was wrapped with a resistive heaterfilament (Omega Engineering, Stamford, CT). The heating ap-paratus was used in two modes of operation: (a) a 10-s modewith the maximum temperature in the tube at 120 °C for dryingthe moisture and other low-boiling volatiles after a sample wasdeposited on the filter and (b) a subsequent pyrolysis mode witha maximum temperature of 400 °C in 7 s.18-20

Samples were deposited as water suspensions on the quartzfilter. The resulting pyrolysis products were transferred througha heated Silcosteel tube, and the injection valve transferred aportion of the sample into the GC column (stainless steel, 4 m ×0.50 mm i.d. Ultra Alloy) coated with a 0.10-µm layer of poly-(dimethylsiloxane) from Quadrex (New Haven, CT). The GC waskept in an isothermal mode at 40 °C prior to the pyrolysis event.Ten seconds after pyrolysis, the GC was ramped from 40 to 140°C at 120 °C/min. The temperatures of the pyrolyzer, three-wayinjection valve, and GC column were monitored by using type Kthermocouples connected to in-house-constructed control elec-tronic boards interfaced to a laptop computer.

The pyrolyzer was kept at atmospheric pressure with nitrogengas, and the pyrolysate was transferred into the GC column duringa pyrolysis event using a three-way valve. The exit of thetemperature programmable GC column was inserted into a Teeto provide a split of the carrier gas into an EI-TOF-MS system(Tempus, ThermoFinnigan, San Jose, CA) and to a CAM IMSdetector (General Dynamics, DeLand, FL) (Figure 1). Thesubambient pressure, generated by the IMS pump, at the GCcolumn outlet was utilized as a driving force for the carrier gasflow during the pyrolysate transfer and injection steps into andthrough the GC column. Further details of the Py-GC-IMSsystem configuration and parameters can be found elsewhere.20

Alternatively, pyrolysis was conducted under ambient atmosphericair conditions, and the pyrolysate was captured at the entranceof the GC column at 40 °C. The carrier gas was then changed tonitrogen. The pyrolysate portion that entered the IMS cell wasdiluted with an additional nitrogen gas supply to prevent saturationof the Faraday plate detector. The mass spectral and ion mobilitychromatograms produced equivalent GC-MS and GC-IMSinformation whether sample was pyrolyzed under air or nitrogencarrier gas conditions.

The flows of nitrogen gas to the MS and IMS detectors were3 and 15 mL/min, respectively. These flow rates were deter-mined by the transfer line i.d., temperature, and pressure differ-

(29) Lennette, E. H.; Balows, A.; Hausler, W. J., Jr., Eds. Manual of ClinicalMicrobiology; American Society for Microbiology: Washington, DC, 1980;Chapter 98.

Figure 1. Schematic of the Py-GC-IMS-MS system.

6494 Analytical Chemistry, Vol. 76, No. 21, November 1, 2004

ence between the Tee and each detector. The carrier gas flowrates and both transfer line i.d. and length dimensions wereadjusted so that the GC eluate entered both detectors at the sametime.

Data Analysis. Multivariate data analysis was applied to theGC-IMS data space information from biological substance solu-tions and suspensions. However, GC-IMS produces a three-dimensional (3-D) data set that includes ion drift time, GCretention time, and intensity. Multivariate data analysis can onlyinterrogate two-dimensional (2-D) information; therefore, the GC-IMS data space was converted from 3-D to 2-D data as follows.30,31

An automated peak search was performed on a raw GC-IMS dataspace where the search was conducted at longer drift times thanthe protonated, water reactant ion peak (RIP)1. A 7 × 14 cell gridwas placed over the GC-IMS data space domain where 14successive cells were positioned between 5.2- and 11-ms drift timesat 0.41-ms intervals on the abscissa. Seven cells were partitionedbetween 5 and 50 s after pyrolysis in the GC retention timeordinate at 6.4-s intervals, and this yielded 98 cells. All the peakintensities in each cell were summed to produce one intensityvalue per cell. Therefore, one GC-IMS data space from theanalysis of a sample is represented by a row of 7 × 14 ) 98summed intensity values. The 98 cells are transformed into vectorsor dimensions in multivariate data space that are mathematicallyorthogonal to each other. The summed intensity value for eachcell represents the magnitude for its respective vector or dimen-sion axis.

The first nine principal component (PC) factors described 95%of the data set variance and were used as the variables fordiscriminant function (DF) analysis of the biological substances.The PC factors were used as variables, because the very low cases(experiments) to dimension ratio yielded poor standard deviationstatistics.32 The number of cases (experimental replicates) was20 for each of 12 different biological substances or categories (videinfra). There were an additional 24 cases for the “blank” category,and this consisted of two experiments for each of the 12 samplecategories where the amount of sample deposited into thepyrolysis region was below the IMS detection threshold of <0.05µg/µL. The number of dimensions was 98. The case/dimensionratio ) 264/98 ) 2.7, and only three cases per dimension wouldyield poor standard deviation statistics. Instead of significantlyincreasing the number of experimental cases, a reduced numberof variables representing the first nine PC factors were chosen.

RESULTS AND DISCUSSIONThe utility of Py-GC-IMS to detect and provide bacterial

aerosol differentiation in outdoor environments has been pre-sented.17-20 However, there remain fundamental performanceconcerns such as the microbiological meaning of the various peaksin the GC-IMS data space. Additional concerns are the degreeof biochemical analyte throughput in the processing/transfermodules and the ability of the IMS detector to respond to the GCeluate components.

Gram-Positive Bacterial Spores. Dworzanski et al.33 de-scribed the performance of the Py-GC module, in the briefcasePy-GC-IMS biodetector, interfaced to a TOF-MS detector in theanalyses of biochemicals and microorganisms. Plausible pyrolysisdecomposition mechanisms for peptidoglycan macromoleculestandards and model biochemical compounds were postulated inorder to identify the pyrolysis products and deduce their bacterialorigin.

Figure 2 presents data showing the GC-MS total ion chro-matogram (TIC) and the equivalent peaks in the GC-IMS dataspace for a B. atrophaeus spore sample. Direct correlations areobserved between the dominant GC and IMS peaks and arehighlighted by arrows. All major and minor GC peaks havediscernible matching peaks in the IMS chromatograms. Therefore,a high degree of visual correlation exists between the twochromatograms. The TIC is labeled with numbers, and thecompound identities are listed in Table 1 along with theirstructures and known bacterial origin. Most of the compoundslisted in Table 1 were identified by the National Institutes ofStandards and Technology (NIST) EI mass spectral database,mass spectral comparison to commercially available authenticstandards, or both methods. For authentic standard identification,GC retention times were matched with the respective experimen-tally observed GC peaks. The mass spectra for compounds 16,17, 21, and 24 did not produce a satisfactory match with theNIST database, and standards were not commercially available.The identities of the compounds were tentatively identified basedon mass spectral interpretation.

Peaks 11 and 19 are particularly noteworthy, because thesepyrolysis products predominately originate from Gram-positivebacteria. Peak 11 is picolinic acid,25 and it originates from thecalcium dipicolinate compound that is found only in Gram-positivespores.26,27 The vegetative Gram-positive organism does notcontain calcium dipicolinate; therefore, the observation of picolinicacid signifies the presence of a Gram-positive spore. Peak 19 isrelatively novel,33 because it has not been previously observed inthe bacterial pyrolysis literature. This compound is 2-pyridine-carboxamide, and it was shown to originate from the lysine peptide

(30) Kroonenberg, P. M., Ed. Three-Mode Principal Component Analysis: Theoryand Applications; DWO Press: Leiden, The Netherlands, 1983.

(31) Marengo, E.; Leardi, R.; Robotti, E.; Righetti, P. G.; Antonucci, F.; Cecconi,D. J. Proteome Res. 2003, 2, 351-360.

(32) Hair, J. F., Jr.; Anderson, R. E.; Tatham, R. L.; Black, W. C. MultivariateData Analysis; Prentice-Hall: Upper Saddle River, NJ, 1998.

(33) Dworzanski, J. P.; Tripathi, A.; Snyder, A. P.; Maswadeh, M. M.; Wick, C.H., submitted to J. Anal. Appl. Pyrolysis.

Figure 2. Py-GC-IMS and Py-GC-MS chromatograms of Gram-positive B. atrophaeus spores. The major peaks are numbered in theGC chromatogram, and the vertical arrows indicate the analogousion mobility peaks. Select compound structures are shown. Identitiesof the numbered peaks are listed in Table 1.

Analytical Chemistry, Vol. 76, No. 21, November 1, 2004 6495

cross-links in peptidoglycan.33 The relative amount of the com-pound is high enough to form an intense dimer ion1 at GC:IMS

coordinates of 53 s:8.4 ms. A weak monomer ion is observed atthe same GC retention time and at a faster drift time of 6.3 ms. Itis not clear why a significant amount of the substance is formedas well as its general absence in the bacterial pyrolysis literature.However, the pyrolysis tube air flow operational conditions aregenerally different from that in the standard biological pyrolysisand Py-GC literature. A drying step is used, and a fast 20 mL/min sweep and flushing of the tube during pyrolysis was utilizedin order to facilitate the rapid removal of pyrolysis products fromthe heating zone. This prevents secondary pyrolysis mechanismsfrom taking place51 that cause degradation of the primary productsto lower molecular weight fragments. Peak 1 is pyridine and isobserved in the thermal processing of organisms in general. Itoriginates from the protein, nucleic acid, and peptidoglycancomponents of microorganisms.25,34-37 However, it is a significantproduct from Gram-positive spores, because it is formed predomi-nately from the DPA chelate in calcium dipicolinate. Note thatsecondary pyrolysis mechanisms on 2-pyridinecarboxamide (19)can also contribute to the generation of pyridine (1).

Compounds 6, 9, 12, and 13 are indicative of proteinaceouspyrolysis products, and they are listed in Table 1. The appearanceof protein peaks is expected, because proteins constitute 50-70%of the dry weight of bacteria. However, their presence is mostlygeneric with respect to bacterial biomarker utility. No direct usefulbacterial distinction or discrimination information was obtainedwith these substances.

The pyrolysis compounds eluted in under 0.75 min, and thiscan be characterized as a relatively rapid analysis of a complexpyrolysate. Except for 19, the pyrolysate products generally havehigh mobilities (low ion drift times) compared to the RIP.

Gram-Positive Vegetative Cells. The appearance of the TICand GC-IMS data space of B. anthracis ∆ Texas in Figure 3 is

(34) Fox, A.; Morgan, S. L. In The Chemotaxonomic Characterization ofMicroorganisms by Capillary Gas Chromatography and Gas Chromatography-Mass Spectrometry; Nelson, W. H., Ed.; VCH Publishers: Deerfield Beach,FL, 1985; Chapter 5, pp 135-164.

(35) Stankiewicz, B. A.; van Bergen, P. F.; Duncan, I. J.; Carter, J. F.; Briggs, D.E. G.; Evershed, R. P. Rapid Commun. Mass Spectrom. 1996, 10, 1747-1757.

(36) Beverly, M. B.; Voorhees, K. J.; Hadfield, T. L. Rapid Commun. MassSpectrom. 1999, 13, 2320-2326.

(37) Beverly, M. B.; Basile, F.; Voorhees, K. J.; Hadfield, T. L. Rapid Commun.Mass Spectrom. 1996, 10, 455-458.

(38) Eudy, L. W.; Walla, M. D.; Morgan, S. L., Fox, A. Analyst 1985, 110, 381-385.

(39) Medley, E. E.; Simmonds, P. G.; Manatt, S. L. Biomed. Mass Spectrom.1975, 2, 261-265.

(40) Grassie, N.; Murray, E. J.; Holmes, P. A. Polym. Degrad. Stab. 1984, 6,127-134.

(41) Chiavari, G.; Galletti, G. J. Anal. Appl. Pyrolysis 1992, 24, 123-137.(42) Smith, G. G.; Reddy, G. S.; Boon, J. J. J. Chem. Soc., Perkin Trans. 2 1988,

203-211.(43) Schrodter, R.; Baltes, W. J. Anal. Appl. Pyrolysis 1991, 19, 131-137.(44) van der Kaaden, A.; Haverkamp, J.; Boon, J. J.; de Leeuw, J. W. J. Anal.

Appl. Pyrolysis 1983, 5, 199-220.(45) Tsuge, S.; Matsubara, H. J. Anal. Appl. Pyrolysis 1985, 8, 49-64.(46) Boon, J. J.; de Leeuw, J. W. J. Anal. Appl. Pyrolysis 1987, 11, 313-327.(47) Voorhees, K. J.; Zhang, W.; Hendricker, A. D.; Murugaverl, B. J. Anal. Appl.

Pyrolysis 1994, 30, 1-16.(48) Hendricker, A. D.; Voorhees, K. J. J. Anal. Appl. Pyrolysis, 1998, 48, 17-

33.(49) Hendricker, A. D.; Voorhees, K. J. J. Anal. Appl. Pyrolysis 1996, 36, 51-

70.(50) Wilkinson, S. G. In Microbial Lipids; Ratledge, C., Wilkinson, S. G., Eds.;

Academic Press: London, 1988; Chapter 7.(51) Tripathi, A.; Maswadeh, M. M.; Snyder, A. P. Rapid Commun. Mass

Spectrom. 2001, 15, 1672-1680.

Table 1. GC-MS Identified Pyrolysis Products

a N, NIST EI-mass spectrum identification. S, standard commerciallyobtained compound used for identification.

6496 Analytical Chemistry, Vol. 76, No. 21, November 1, 2004

similar to that of B. atrophaeus in Figure 2. An intense, fast GCpeak is initially observed, and it primarily consists of pyridine (1)and 2-butenoic acid or crotonic acid (4). Pyridine is found in theTICs of both Bacilli (Figures 2 and 3), but crotonic acid is foundonly in the B. anthracis chromatogram. Pyridine can originate fromthe peptidoglycan cell wall layer in B. anthracis. 3-Hydroxy-butyric acid is the monomeric subunit of the poly(hydroxy-butyric acid) (PHBA) polymer, and the latter is an energy storagecompound in certain bacteria. Crotonic acid is the monomericsubunit pyrolysis product of PHBA. The microbiological literatureshows that B. atrophaeus synthesizes very low amounts of PHBA,52

but B. anthracis produces appreciable quantities of the com-pound.36,53

Under pyrolysis conditions, linear organic polymers usuallythermally fragment into the monomeric repeat unit, compoundscontaining multiples of the monomer, and fragments of themonomer. The dimer pyrolysis product of PHBA contains twomolecules of 3-hydroxybutyric acid minus a water molecule.54 Themass spectrum of the dimer (18 in Table 1) was observed inFigure 3 and was not observed for B. atrophaeus in Figure 2.Confirmation of the presence of the dimer in B. anthracis wasobtained from the same GC retention time and matching massspectrum to that of the PHBA model compound (mass spectraldata not shown).

In Figure 2, picolinic acid (11) is observed from the B.atrophaeus spores, and it is absent in the Gram-positive vegetativecells of B. anthracis in Figure 3. The Py-MS,36,37,51 Py-GC-MS,17,18,20,25 and microbiological literature26,27 provide extensiveevidence that Gram-positive spores and vegetative cells do anddo not generate picolinic acid, respectively.

Compounds 21 and 24 were observed in the mass spectralchromatogram in Figure 3, but equivalent signals were not evidentin the GC-IMS data space (dotted lines). These compounds weredetermined to be diketopiperazine (DKP) species by mass spectral

interpretation. Pyrolysis has been documented to convert proteinsand peptides into cyclic diamino acid products48,49,42,55 such as 21and 24 (Table 1). The amino end of an amino acid residue andthe carboxyl moiety of the adjacent residue cyclize by a dehydra-tion reaction. The N atom of each residue is resident in the ringin a para position, and the R groups are in the para positions onthe ring. Compounds 21 and 24 were tentatively identified asthe His-Val and Pro-Pro DKP species, respectively.

In addition to the DKP species, 16 was observed in the masschromatogram but not in the ion mobility chromatogram. Com-pound 16 was tentatively identified as isotridecanenitrile by massspectral interpretation. Fatty acid species in Gram-positive organ-isms are predominately branched56 while those in Gram-negativeare linear in nature.50 This fatty acid derivative most likelyoriginated from intracellular lipoprotein species resident in theB. anthracis vegetative cells.

Compounds 16, 21, and 24 traversed the torturous Py-GCpathway and were registered in the mass spectrometer. However,the IMS cell was not able to respond to the compounds. Ionizationis effected by transferal of a proton in the proton-water hydratespecies, formed in the 63-Ni source, to the neutral analyte only ifthe latter has a proton affinity higher than water. All threecompounds display high proton affinity nitrogen moieties, whichare amenable to proton ionization and therefore capable of beingdetected by the IMS detector. The literature by Clemmer et al.5

supports this where small and large peptide species are observedin electrospray IMS-TOF-MS proteomics studies. Woods et al.9

described the characterization of a number of biochemicalcompounds including peptides and lipids such as sphingomyelinand cerebroside sulfate in their matrix-assisted laser desorption/ionization (MALDI)-IMS-TOF-MS investigations.

The absence of certain pyrolysate species in the ion mobilitychromatogram is also a phenomenon with Gram-negative bacteriaas outlined in the next section, and possible reasons are delin-eated.

Gram-Negative E. coli. Figure 4 presents the Py-GC-IMS-MS mass spectra for E. coli. The protein pyrolysis speciesdistribution appears very similar to that of the Gram-positive

(52) Reusch, R. A.; Sadoff, H. L. J. Bacteriol. 1983, 156, 778-788.(53) Watt, B. E.; Morgan, S. L.; Fox, A. J. Anal. Appl. Pyrolysis 1991, 19, 237-

249.(54) Morikawa, H.; Marchessault, R. H. Can. J. Chem. 1981, 59, 2306-2313.

(55) Noguerola, A. S.; Murugaverl, B.; Voorhees, K. J. J. Am. Soc. Mass Spectrom.1992, 3, 750-756.

(56) O’Leary, W. M.; Wilkinson, S. G. In Microbial Lipids; Ratledge, C., Wilkinson,S. G., Eds.; Academic Press: London, 1988; Chapter 5.

Figure 3. Py-GC-IMS and Py-GC-MS chromatograms of theGram-positive ∆ Texas vegetative strain of B. anthracis. The majorpeaks are numbered in the GC chromatogram, and the vertical arrowsindicate the analogous ion mobility peaks. A solid arrow indicates acompound observed in both chromatograms, and compounds presentin the GC chromatogram and absent in the IMS chromatogram areindicated by dashed lines. Select compound structures are shown.Identities of numbered peaks are listed in Table 1.

Figure 4. Py-GC-IMS and Py-GC-MS chromatograms of Gram-negative E. coli K-12. Refer to Figure 3 caption for details.

Analytical Chemistry, Vol. 76, No. 21, November 1, 2004 6497

organisms (Figures 2 and 3). Except for 12, all protein pyrolysatespecies have the core benzene ring with an attached side chain(Table 1).

There are a few volatile pyrolysis species that can originatefrom the carbohydrate-containing biomolecules in the E. coli cells,and these include 2, 3, and 8. Pyrolysis products 4 and 18,formed from PHBA in vegetative B. anthracis (Figure 3), werenot observed in the E. coli mass chromatogram (Figure 4). It isknown that E. coli does not manufacture and store PHBA57,58 undernormal growth conditions, and this supports its absence in Figure4. A relatively small amount of 2-pyridinecarboxamide (19) isobserved and appears on the fast side of peak 20 as a low-intensityshoulder. The amount of peptidoglycan in Gram-negative cells is∼10% of that in Gram-positive cells; therefore, 19 is expected tobe of very low abundance.30 Pyridine (1) is present and canoriginate from the low amount of 19 observed in the chromato-gram. The relative amount of pyridine is low compared to B.atrophaeus spores (Figure 2) and B. anthracis (Figure 3). SinceE. coli does not contain calcium dipicolinate, picolinic acid (11)is absent in the chromatogram.

The mass chromatogram in Figure 4 is particularly rich in lipidsubstances. These include free fatty acids and fatty acid deriva-tives, and the mass spectral confirmation details are presentedelsewhere.33 The outer surface layer of Gram-negative organismssuch as E. coli is covered with an LPS complex macromolecule.This layer produces the endotoxin material that circulates in thebloodstream when a human is infected by a Gram-negativemicroorganism.

The two earliest eluting fatty acid derivatives (17, 20) havevery low intensities in the ion mobility chromatogram (verticalarrows), and the analogous peaks in the mass chromatogram showsignificant abundances. These compounds are 1-tridecene (C13,17) and dodecanal (C12, 20), and 17 was determined by massspectral interpretation. C12 and C13 indicate that the moleculeshave a backbone containing 12 and 13 carbon atoms, respectively(Table 1). The thermal decomposition mechanisms that lead tothe appearance of these hydroxy fatty acid derivatives are outlinedin a companion paper,33 and they originate from the 3-hydroxy-myristic acid portion of the LPS macromolecule. Four more lipidspecies are evident in the mass chromatogram, and they are2-tridecanone (C13, 22), n-dodecanoic acid (C12) or lauric acid(23), 2-tetradecenenitrile (C14, 25), and tetradecanoic acid (C14)or myristic acid (26). It can be noted that the yields of thesefour high-boiling compounds are much lower than the two lowerboiling fatty acid derivatives. Their thermal decomposition mech-anisms and generation are also outlined elsewhere,33 and theserelatively high boiling lipid substances are not observed in theion mobility chromatogram from the fielded Py-GC-IMS system.

Compounds 5, 7, 10, 12, 14, and 15 indicate proteinaceoussubstances (Figure 4); however, 17, 20, 22, 23, 25, and 26indicate Gram-negative, LPS derived lipid substances. This lattergroup of compounds is an important indicator for the presenceof Gram-negative organisms; however, they either are relativelylow in intensity or are not observed by the IMS detector.

Biomarker Taxonomic Utility. Thermal processing of bacte-rial substances produces biochemical information that can providemore than a biological or nonbiological determination with respectto differentiation and biodetection goals and objectives. The fieldedPy-GC-IMS system has shown that it indeed can produce asignificant number of direct biochemical products from thethermal processing of whole, intact microorganisms. More specif-ically, a number of biochemical polar species, originating in cellwalls that react either in a positive or negative fashion with theGram stain, are created in the pyrolysis region, are swept rapidlyenough from the heated pyrolysis region and into the GC column,and elute from the column. The Py-GC is a torturous path forpolar biomolecules and has many sites for compound degradationand adsorption. Secondary thermal degradation reactions mayoccur in the pyrolysis source, and adsorption may occur in thepyrolyzer, Py-GC interface, and GC column. The operatingconditions of the pyrolysis module, however, were optimized tolimit the loss of biomaterial.51 The mass chromatograms, alongwith authentic standards and NIST mass spectral databasematching, provided confirmatory support for the biodetectionpotential of the system.

The Py-GC-ambient temperature and pressure-IMS system hasbeen shown to discriminate between deliberately released Gram-positive B. atrophaeus spore, Gram-negative P. agglomerans, andOV protein18-20 aerosols between 200 and 800 m from the stand-alone operational system in outdoor scenarios. The present workprovides mass spectral evidence of distinct biochemical com-pounds from microorganisms that contain Gram-positive andGram-negative taxonomic information, which are produced by thepyrolysis module. This lends credibility to the outdoor perfor-mance of the Py-GC-IMS biodetector.

Improvement of Py-GC-IMS Performance. The Py-GCmodule was operated at the same conditions for the IMS and MSanalyses, yet differences were observed between the two chro-matograms primarily in the relatively higher temperature regionof the GC column. A fundamental concern of the Py-GC-IMSsystem is that the temperature of the IMS detector is relativelylow and is somewhat controlled by the ambient environment aboutthe briefcase housing.18-20 When operated in an outdoor fieldapplication, the IMS cell is subject to the ambient environmentaltemperature and that of the heat generated by the internalcomponents including the Py-GC module, electronics, and pump-ing system. This results in an uneven, nonuniform temperaturefor the IMS analyzer. Good laboratory practices generally requirethat an analytical detector should be just as hot if not hotter intemperature than the sample analyte introduction, processing, andtransfer modules. Therefore, a constant uniform temperatureabout the IMS cell is warranted. Informal, unpublished workshowed that a CAM IMS detector irreversibly fails at temperaturesgreater than 150 °C, because the internal components and solderconnections are not high-temperature compatible. A heated IMScell in the briefcase biodetector could allow the observation ofcompounds such as 16 and 21-26 as well as a greaterabundance of crucial Gram-positive and Gram-negative bacterialindicator compounds such as 4, 11, 17, 19, and 20.

The IMS detector temperature may not be the only parameterthat causes minimal to negligible appearance of certain polar,biochemical compounds from the pyrolysis of microorganisms.

(57) Reusch, R.; Hiske, T.; Sadoff, H.; Harris, R.; Beveridge, T. Can. J. Microbiol.1987, 33, 435-444.

(58) Reddy, C. S. K.; Ghai, R.; Rashmi, V. C. K. Bioresource Technol. 2003, 87,137-146.

6498 Analytical Chemistry, Vol. 76, No. 21, November 1, 2004

Improvement in the signal of certain compounds may also beinfluenced by the pressure, sweep gas flow rate, internal gas flowpatterns, and internal geometry in the IMS cell to remove excess,contaminating neutral species. For example, relatively high(atmospheric) pressure and low, ambient cell temperature canlead to condensation conditions. However, relatively lower pres-sures and higher temperatures can significantly reduce the samplecondensation phenomenon on the inner surfaces of the IMS cell.

Multivariate Data Analysis. Figure 5 shows two DF plotsthat provide a significant degree of differentiation for the GC-IMS data spaces of bacterial and protein species. Even thoughprotein species are not part of the Gram classification scheme,inclusion of this class of biological molecule in the multivariatedata analyses provides a relatively more comprehensive biologicaldiscrimination capability for the Py-GC-ambient temperature andpressure-IMS system. The percent variance or information contentof the data set expressed by the first three DF vectors is DF1,36%; DF2, 20%; and DF3, 17.9%. Both DF plots in Figure 5 showthat all three Bacillus spore species can be distinguished betweenthemselves and the vegetative B. anthracis strains group. Therelatively low molecular weight Y and L proteins cluster as a group,while the higher molecular weight SEB and OV species are clearlyseparated in multivariate DF space. The Gram-negative speciescluster as a group, while background samples are clearly sepa-rated. DF3 has the ability to distinguish the OV protein from theB. megaterium spore.

It appears that the differentiation and distinction of the bacterialgroups in Figure 5 have a high correlation with the Gram taxon-omic information from the bioanalytical results presented inFigures 2-4. The Gram-negative species cluster as a group, andthe Gram-positive vegetative and spore species are clearly dif-ferentiated from each other. This is a significant finding, becauseit is possible that the biochemical species identified in Figures2-4 can provide discrimination attributes with respect to micro-biological Gram stain taxonomy. This is shown on a broader scale

in Figure 5 compared to Figures 2-4, and the addition of proteinanalytes strengthens the argument for biological classificationcapabilities of the Py-GC-IMS system. Figure 5 provides a degreeof credibility to the documented performance of the Py-GC-IMSbiodetector at the outdoor bioaerosol field trials for the determi-nation of bacterial and protein the presence and discrimination.

CONCLUSIONThe Py-GC module in the fielded Py-GC-IMS system gener-

ated biomolecules from bacterial species, and a portion of thepyrolysate was transferred to an MS detector. Mass spectralanalyses confirmed that these biomolecules contained micro-biological discrimination information. This information appearsto augment and confirm the outdoor bioaerosol performancecapabilities of the briefcase Py-GC-IMS, because taxonomicallyuseful pyrolysate species were generated and detected by thebiodetector. The 2-pyridinecarboxamide and 3-hydroxymyristicacid derivative biomolecules can be utilized as biomarker indica-tors for certain classes of bacteria and contain microbiologicaltaxonomic information. These biomarkers individually and col-lectively correlate to a significant degree with the microbiologicalGram stain classification scheme. 2-Pyridinecarboxamide, pro-duced by the fielded Py-GC-IMS, can be directly correlated toGram-positive classification information, and the 3-hydroxymyristicacid derivative compounds can be directly correlated to Gram-negative bacteria. An added benefit is that these specific bio-markers are invariant to microbiological growth and cultureparameters such as growth media conditions, age of the cells,growth temperatures, and harvest conditions. Observation of thesebiomarkers can confirm the presence of either Gram-positive orGram-negative microorganisms. Picolinic acid and PHBA pyroly-sate species are not directly related to the Gram stain phenom-enon. However, they correlate with a Gram-positive or Gram-negative organism and are added benefits to the informationcontent in a Py-GC-IMS spectrum.

Specific instrumental parameter modifications were sug-gested to improve the detection of taxonomically useful informa-tion such as maintaining an elevated temperature in the analyti-cally challenged ambient temperature and pressure IMS cell.Certain relatively high boiling lipid and DKP polar compoundsthat are observed in the mass chromatogram are of very lowintensity or absent in the corresponding ion mobility chromato-gram. The mass spectral information generated and transferredfrom the Py-GC module, the DF analyses of the GC-IMS dataspaces of a suite of biological samples, and the documentedperformance of the fielded Py-GC-IMS system together providemicrobiological, scientific, technical, and operational support forthe utility of the system in biological classification and discrimina-tion scenarios.

Received for review May 20, 2004. Accepted August 5,2004.

AC040099I

Figure 5. DF plots of the analysis of liquid suspensions of Gram-positive, Gram-negative, and protein biological species. Twentyreplicates were conducted for each biological substance: O, oval-bumin (OV); S, Staphylococcal enterotoxin B (SEB); L, lysozyme(Pro); Y, myoglobin (Pro); B, B. atrophaeus; C, B. cereus; M, B.megaterium (mega); E, E. coli (G-); P, P. agglomerans (G-); T, B.anthracis ∆ Texas, A, B. anthracis ∆ Ames, and V, B. anthracis ∆VNR1 are collectively labeled anthrax.

Analytical Chemistry, Vol. 76, No. 21, November 1, 2004 6499