Field detection of bacillus spore aerosols with stand-alone pyrolysis-gas chromatography and ion...

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FIELD ANALYTICAL CHEMISTRY AND TECHNOLOGY 3(4–5):315–326, 1999 CCC1086-900X/99/040315-12

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q 1999 John Wiley & Sons, Inc.

Field Detection of Bacillus Spore Aerosols withStand-Alone Pyrolysis–Gas Chromatography–IonMobility Spectrometry

A. Peter Snyder,1 Waleed M. Maswadeh,2 John A. Parsons, 2 Ashish Tripathi, 2 Henk L. C. Meuzelaar,3

Jacek P. Dworzanski, 3 and Man-Goo Kim3

1U.S. Army Edgewood Chemical Biological Center, Aberdeen Proving Ground, Maryland 210102GEO-CENTERS, INC., Gunpowder Branch, P. O. Box 68, Aberdeen Proving Ground, Maryland 210103Center for Micro Analysis and Reaction Chemistry, University of Utah, Salt Lake City, Utah 84112

Received 19 May 1999; revised 23 June 1999; accepted 14 July 1999

Abstract: A commercially available, hand-held chemicalvapor detector was modified to detect gram-positive Ba-cillus subtilis var. globigii spores (BG) in outdoor fieldscenarios. An airborne vapor monitor (AVM) ion mobilityspectrometry (IMS) vapor detector was interfaced to abiological sample processing and transfer introductionsystem. The biological sample processing was accom-plished by quartz tube pyrolysis (Py), and the resultantvapor was transferred by gas chromatography (GC) tothe IMS detector. The Py-GC/IMS system can be de-scribed as a hyphenated device where two analytical di-mensions, in series, allow the separation and isolation ofindividual components from the pyrolytic decompositionof biological analytes. Gram-positive spores such as BGcontain 5–15% by weight of dipicolinic acid (DPA), andpicolinic acid is a pyrolysis product of DPA. Picolinicacid has a high proton affinity, and it is detected in asensitive fashion by the atmospheric pressure-basedIMS device. Picolinic acid occupies a unique region in theGC/IMS data domain with respect to other bacterial py-rolysis products. A 1000 to 1, air-to-air aerosol concen-trator was interfaced to the Py-GC/IMS instrument, andthe system was placed in an open-air, western UnitedStates desert environment. The system was tested withBG spore aerosol releases, and the instrument was re-motely operated during a trial. A Met-One aerosol particlecounter was placed next to the Py-GC/IMS so as to obtaina real-time record of the ambient and bacterial aerosolchallenges. The presence/absence of an aerosol event,determined by an aerosol particle counter and a slit-sam-pler–agar-plate system, was compared to the presence/absence of a picolinic acid response in a GC/IMS datawindow at selected times in a trial with respect to a BG

Correspondence to: A. P. Snyder

challenge. In the 21 BG trials, the Py-GC/IMS instrumentexperienced two true negatives and no false positives,and developed a software failure in one trial. The remain-ing 18 trials were true positive determinations for thepresence of BG aerosol, and a limit of detection for thePy-GC/IMS instrument was estimated at approximately3300 BG spore-containing particles. QQ John Wiley &Sons, Inc. Field Analyt Chem Technol 3: 315–326, 1999Keywords: pyrolysis; gas chromatography; ion mobilityspectrometry; Bacillus subtilis spores (BG); field biode-tection; outdoor biodetection; biodetection biologicalaerosols; aerosol concentrator

Introduction

Recent and current events around the world have high-lighted the possibilities for deliberate outdoor disseminationof harmful biological substances,1–6 and at least 12 countriesare known to have some degree of biological warfare pro-gram capabilities.7,8 Alleged biological terrorism attacks inJapan9,10 and threats on U.S. domestic commercial estab-lishments have increased significantly in the past fiveyears.11–14 Reports of alleged localized aerosol releases andhoax domestic biological terrorism in the form of postal mailpackages, allegedly with spores of the pathogenic Bacillusanthracis and Yersinia pestis (bubonic plague) organisms,serve to exacerbate the problem.15–19

Desirable goals in effectively countering the biologicalwarfare and terrorism applications of harmful biologicalagents include their ready detection and possible identifica-tion in a relatively short period of time. The detection ofbiological aerosols, particularly that of bacterial cells andspores, is an important component of U.S. military biologi-

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316 FIELD ANALYTICAL CHEMISTRY AND TECHNOLOGY—1999

FIG. 1. Schematic of the complete XM-2 aerosol concentrator–Py-GC/IMS system.

cal programs.20–23 A portion of these programs consists ofanalytical instrumentation to effect trigger, detection, andidentification responses for the presence of bacterialaerosols.

The analytical technique of ion mobility spectrometry(IMS) had its inception approximately three decades ago.24,25

An ion mobility spectrometer can be characterized as an at-mospheric pressure-based, time-of-flight system, and it isused as a detector and monitor for vapors.26–28 Atmosphericair is ionized by a 63Ni ring through protonation of a neutralwater molecule. The proton is transferred to a sample com-pound that has a higher proton affinity than water. A voltagegate pulses the resulting ions as packets into a drift tube, andthe ions are detected by a Faraday plate detector. The drifttime between the ion gate and Faraday detector characterizesthe ions. Different molecular masses and cross sections of

ions primarily determine the drift time and degree of sepa-ration of a mixture of ions. All these phenomena take placeat atmospheric pressure.

Prototype portable gas chromatography (GC)/IMS con-cept systems were shown to successfully separate headspacevapors from complex liquid mixtures of analytes.29–31 Thefirst Py-GC/MS analyses of biological materials, includingBacillus spores and nucleic acids, appear to have been re-ported by Meuzelaar et al. in 1991.32 This was followed bysystematic Py-GC/IMS studies of various biopolymers rel-evant to bioagent detection33 as well potential interferants,culminating in a recent Ph.D. thesis by Thornton.34

Biological compound information was generated by aquartz tube pyrolysis (Py)-GC/IMS breadboard system un-der controlled sample introduction of bacterial suspen-sions,35 and a preliminary presentation of a spore aerosol

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investigation in the field was reported recently with the Py-GC/IMS system.36 A compact portable briefcase Py-GC/ionmobility spectrometer was built. Aerosols were collectedand concentrated by a commercially available, air-to-airaerosol concentrator, and this was interfaced to the quartztube pyrolysis port of the Py-GC/IMS device. The bacteriawere collected on a frit inside the quartz tube and processedby pyrolysis, and a portion of the vapor was transferredthrough the GC separation stage. The ion mobility spectrom-eter analysis stage detected the separated components fromthe GC.

Aerosols of gram-positive spores of Bacillus subtilis var.globigii bacteria, which are a nonpathogenic surrogate of B.anthracis, were generated in an outdoor western UnitedStates desert test site, and the aerosol concentrator-Py-GC/IMS system was used to interrogate for the presence of theaerosols.

The tandem analytical technique of GC/IMS allowed forthe separation of bacterial pyrolysis products in the secondsdimension for GC and in the milliseconds time frame for theion mobility spectrometer. Picolinic acid, the compound in-dicative of the presence of gram-positive spore bacterial aer-osols, was effectively separated in a defined window in theGC/IMS data domain from other pyrolysis products. Currentresults provide evidence for a maturing, stand-alone, Py-GC/IMS biological point detector technology.

Experiment

Figure 1 presents a schematic of the complete aerosolconcentrator-Py-GC/IMS device, and Figure 2 shows a pho-tograph of the briefcase Py-GC/IMS unit. The major com-ponents of the system are as follows: 1, aerosol inlet; 2,pyrex filter aerosol collector in a quartz tube/pyrolysissource; 3, high-temperature three-way GC-injection valveand pump connection to draw aerosol particulate onto thequartz filter; 4, programmable GC column ring; 5, voltage-gated ion source and ion mobility spectrometer componentsof the airborne vapor monitor (AVM) (Graseby-Dynamics,Watford, Herts, U. K.); 6, coaxial cable to radio antenna; 7,wireless transceiver; 8, RS232 connection; 9, AC power in;10, Samsung Sens Pro 500 laptop computer with PCMCIAdata acquisition card from National Instruments; 11, coaxialcable for Ethernet/serial PC card communications; 12, dualdiaphragm vacuum pump; 13, electronic hardware andpower supplies; 14, 50–pin interface to PCMCIA data ac-quisition card; 15, molecular sieve packs; 16, 18 3 12.5 36-in. heavy-duty briefcase.

Aerosol particulate with a diameter in the 2–10mm rangewas collected by a four-stage, 1000 liter/min XM-2 aerosolcollector (SCP Dynamics, Minneapolis, MN 55432). TheXM-2 has a 50% and 15% efficiency for aerosol collectionof particulate with 5- and 2-mm diameters, respectively (un-published data). The particulate is drawn out of the fourthstage at 250–300 milliliters or ml/min and onto the quartzfrit inside the quartz pyrolysis tube. The three-way valve(No. 3, Figure 2) is switched to a vacuum pump (No. 12,

Figure 2) in order to admit the particulate onto the quartzfrit. The particulate was pyrolyzed at 3507C, and the high-temperature three-way valve admitted a 1-s pulse of pyrol-ysis products into the GC column. Clean, dry air was usedas carrier gas, and molecular sieve packs (No. 15, Figure 2)were used to scrub the ambient recirculation air. The eluatewas ionized by the 63Ni ring at the entrance to the AVM ionmobility spectrometer. Analyte ionization is effected by pro-ton transfer from reagent protonated water molecule clusters.Table 1 provides analytical parameter operation values forthe Py-GC/IMS system. Temperature measurements of thepyrolyzer, three-way injection valve, and GC were moni-tored with the use of type-K thermocouples connected to in-house-constructed control electronic boards.

The laptop computer in the Py-GC/IMS system was re-motely controlled from approximately 150 ft with the use ofPC ANYWHERE software (Symantec Corp., Cupertino, CA95014) from a second Samsung Sens Pro 500 laptop com-puter by way of the Ethernet coaxial cable.

The events of a typical detection cycle started with theaerosol collection phase, and the duration was between 1and 2 min. After the aerosol particulate was collected on thequartz frit, the latter was heated to 1207C in 5 s, and a sub-

TABLE 1. Experimental operating conditions of the Py-GC/IMSsystem.

Overall properties:Weight (lb) 30Avg. power consumption (W) ,120 (Pyrolysis)

60 (Normal run, no Py)

PyrolyzerWire Nichrom (0.015 in. OD)Resistance (V): 3.3Tube diameter (mm): 6 3 4Length (mm): 65Pyrolysis time (s): 4–10Wire temp (7C): 700–900 (estimated)

FilterType Quartz micro fiberDiameter (mm): 4Particle size (mm) .1.0

GC column:Ultra-alloy stainless steel column (high temperature)Liquid Phase Methyl silicone (0.25mm)Length (m) 4.0

Inside diameter (mm) 0.5Temperature (7C): 50–150 @ 1207C/minCarrier gas Clean dry airFlow rate (ml/min): 20 @ GC column 607CSample injection pulse (s) 0.5–2.0 (select)Injection valve temp. (7C) 150

Ion mobility spectrometer:Ionization source 63Ni, 10 mCiGating pulse rate (Hz) 30 (internal)Cell temperature (7C) 35Cell pressure (torr): 520Drift gas Clean dry airMode Positive ion


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sequent 10-s drying of the aerosol particulate was adminis-tered on the quartz frit. The pyrolysis event then occurred(9 s), and consisted of 4 s of heating and 5 s for temperatureequilibration inside the pyrolyzer tube. At the time of theinitiation of the pyrolysis event, a 12-s switching of flowsoccurred. This included turning off the vacuum that admittedaerosol particulate on the frit, transferring of pyrolyzate tothe three-way injection valve, and the GC injection. The

temperature programming of the GC column began at theend of the pyrolysis event, and a portion of the pyrolyzatewas injected into the GC column for approximately a 1-sduration after initiation of the GC temperature programmingramp.

A vehicle-mounted Micronair agricultural sprayer assem-bly disseminated bioaerosols at approximately 800 m fromwhere the Py-GC/IMS biological detector was placed in the

FIG. 2. Picture of the Py-GC/IMS briefcase unit. Refer to the Experiment section for the legend.

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desert. A slurry of dry BG spores in water, stored in a res-ervoir tank, was drawn through a tube and forced into aspinning wire mesh. The spinning mesh partitioned the liq-uid slurry into approximately 50-mm droplets, and thesedroplets were disseminated into the air by a fan. The bac-terial particles evaporated water as they traversed the 800-m distance and resulted in particle diameters in the 0.7–10-mm range.

Bacterial aerosol particle counts were enumerated by thetest personnel, and this was accomplished by interfacing aslit sampler to petri dishes situated on automated revolvingdisks. This provided an aerosol record for the temporal pres-ence of bacterial particles. The slit sampler provided no dis-crimination as to the size of the particles that impacted onthe petri dishes. Thus, a wide range of particle sizes wasdirected onto the petri dishes, which included the 0.7–10-mm respirable-particle size range. The dishes containingtrypticase soy agar were incubated at 377C for 24 h, andcolonies were enumerated for BG. Each bacteria-containingparticle that impacted on the agar plate produced a spot orcolony-forming unit (CFU) upon growth of the bacteria dur-ing incubation. The particle can contain one or more indi-vidual bacterial cells; however, only one CFU will be pro-duced. Thus, a graph was constructed that described timeversus CFU per liter of air or analyte-containing particlesper liter of air (ACPLA), and the graph was not subjectedto a smoothing routine. The interpretation of an ACPLA isbased on the volume of a spherical particle of a given di-ameter and the maximum number of analyte organisms thatcan fit in the spherical volume.37 Usually, nonbiological or-ganic and inorganic salts and debris/growth media are alsocontained in a representative aerosol particle generated froma bacterial suspension.

Aerosol collectors such as a Met-One device measure to-tal particles per liter of air (PLA), whereas an agar dish onlymeasures particles containing viable bacteria. Thus, the Met-One aerosol information is usually equal to or higher in par-ticle counts than that of the petri dish method. A Met-Onedevice measures the presence of a particle when the particleintersects a helium–neon laser beam. The amount of scat-tered laser light determines the size of the particle. The Met-One was programmed to measure particles of 1–10 mm indiameter. The data output was smoothed by a three-pointsmoothing routine. This reduced the amount of data to one-third of that of the original data set. The reduced set wasthen subjected to a standard cubic spline smoothing routine.

The picolinic acid signal (S) was calculated by averagingthe maximum picolinic acid signal and four equidistant near-est-neighbor points. The same process was used for thenoise, except this was accomplished when no sample analytesignal was present in the GC/IMS data domain. Three timesthe standard deviation of the mean of the noise signal wasdefined as the noise (N) component. The signal-to-noise ra-tio (S/N) of the picolinic acid analyte signal resulted fromthese two individual values.

During the 4-week series of aerosol trials, the same GCcolumn was used, and the pyrolysis quartz filters were re-

placed every 8–10 h of operation. No significant GC columndegradation was noticed (i.e., no significant shift of analyteretention time), and the same air-scrubbing molecular sievepackage was used.

Results and Discussion

The XM-2 aerosol concentrator-Py-GC/IMS system wasplaced on a table in an outdoor desert site in the westernUnited States. The aerosol collector/concentrator was turnedon prior to a trial and then turned off after the end of thetrial window. A trial window typically encompassed a 20min–1.0 h time frame, and a bacterial aerosol was releasedfor a 3–14-min duration at a selected time within the trialwindow. This particular time was chosen by the test directorsand was not made available to the instrument operators.Thus, the aerosol concentrator/detection system was cycledin a continuous manner during a trial. A successful executionof a bacterial aerosol release from the point of disseminationto the Py-GC/IMS analytical detection device, whichspanned approximately 800 m, significantly relied on asteady current of wind.

For a given aerosol collection time, different amounts ofparticulate are introduced to the quartz tube pyrolysis sampleprocessing chamber, depending on the ambient aerosol par-ticulate challenge. For example, a total of 100,000 BG spore-containing particles are collected onto the pyrolysis quartztube filter from a 2-min air sampling of an ambient, real-time 100 PLA with a particle diameter of 5 mm. Thus, theanalytical system performs aerosol measurements on the col-lected, concentrated particles, rather than the ambient, real-time particulate burden. The series of trials consisted of 21BG aerosol releases, and a description and conclusion of twotrials, along with a bacterial standard analysis, follow.

BG Bacterial Standard

It is known that the Bacillus spore microorganism speciescontain dipicolinic acid (DPA) as the calcium dipicolinatechelate at levels of 5–15% by weight.38 Upon pyrolysis, thiscompound thermally fragments to picolinic acid and pyri-dine. Both compounds can be detected by IMS at very lowamounts because of their high proton affinity. Aromatic ni-trogen compounds are known to possess high proton affin-ities.39,40

In order to determine the signature of the BG material, apuff or release of approximately 24 mm of BG spores froma hand-held pressurized cylindrical container was directed atthe entrance of the aerosol collector/concentrator. Figure 3shows a contour plot of the GC/IMS data domain of thepyrolysis of BG, where the intensity parameter is the thirddimension (out of the page). The discontinuous peak at 4.25ms is the reactant ion peak (RIP), which consists of proton-ated water molecule clusters. Proton transfer from watermolecules to analyte species affects the sample ionization.The feature representing a coalescence of a number of rap-idly eluting compounds between drift times (tD) of 4.6–5.25


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ms and retention times (tR) of 1.9–4.9 s includes the pyridinecompound.35

Picolinic acid can be found as weak monomer and intensedimer peaks at the drift time (tD, ms), GC retention time (tR,s) values of 5.40, 16.8, and 6.63, 16.8, respectively, in Figure

3 (arrows). A confirmation of the identity of these peaks canbe found by comparing the reduced mobility (k0) terms ofthe drift-time values to established, documented results.35

The reduced mobility is calculated by the following equa-tion:

P 273 dobsk 50 760 T Etobs D

where k0 is reduced mobility in cm2/Vs; tD is the drift timein seconds; Pobs, is the pressure of the IMS cell in torr (520torr); Tobs, is the temperature of the IMS cell in degrees Kel-vin (308 K); d is the effective drift cell length in centimetersmeasured between the ion gate and ion collector grid (3.6cm); E5V/d, where V is the applied voltage (810 V) acrossthe effective drift cell length. Inserting the monomer anddimer drift times (in seconds) into the equation yields pi-colinic acid monomer and dimer reduced mobility values of1.80 and 1.46, respectively. The dimer value compares fa-vorably to that of the field-portable prototype Py-GC/IMSsystem from Table 2 in Reference 32 (average of 1.45). Thek0 value of the picolinic acid monomer (1.80) differs fromthe 1.94 average value in Reference 32. The 35-7C temper-ature in the present system is considerably lower than that(647C) used in Reference 32. This decrease in temperaturecan cause a relative increase in clustering of water moleculesabout the monomer ion, and a larger ion size leads to a higherdrift time (i.e., 5.4 ms) versus that of Reference 32 (i.e.,

FIG. 4. Met-One aerosol (PLA) and ACPLA distributions and temporal disposition of the Py-GC/IMS cycles for Trial 4.

FIG. 3. GC/IMS data domain of a standard BG spore aerosol challengeto the XM-2 concentrator Py-GC/IMS system.

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average of 4.92 ms). Thus, the decrease in k0 in the presentinvestigation is reasonable with respect to that at a higherIMS cell temperature.

The GC column was operated at a 607C temperature, andduring an analysis cycle, the column was linearly pro-grammed from 607C to 1307C at 2.07C/s. Scrubbed, ambientair was used as carrier gas for all operations. Note that thebacterial pyrolyzate contains relatively few compounds withintense signals. This can be caused by the generation of rel-atively few compounds with a high proton affinity and/orthe production of a majority of pyrolyzate compounds at lowconcentration. Another possibility is that most polar, high-proton affinity compounds condensed on the relatively lowtemperature surfaces between the pyrolysis source and the63Ni ionization source/ion-gate region of the IMS detector.

Preliminary outdoor tests have been reported with respectto Bacillus spore detection with the use of Py-GC/IMS in-strumentation.36 A characterization of select BG spore aero-sol trials follows.

Trial 4

Figure 4 presents a particle count record of Trial 4, andtwo significant aerosol events are labeled as Peaks 1 and 2.The Py-GC/IMS chromatograms in Figure 5 provide infor-mation as to the origin of these peaks, and the contour plotsprovide a successive series of snapshots as to the presence/absence of the BG spore species in discrete time windowsof the trial. Cycles 61 and 62 (data not shown) (Figure 5)show no biological activity whereas Cycle 63 (Figure 5)

FIG. 5. GC/IMS data domains for cycles 62–65 in Trial 4.


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yields an intense picolinic acid dimer signal at a tD , tR (ms,s) of 6.70, 16.5. Cycle 64 shows a lower intensity picolinicacid dimer signal, and both Cycles 63 and 64 correlate wellin the characterization of the aerosol particle event peak la-beled 2 in Figure 4. Cycle 65 shows the absence of biologicalaerosols. Thus, the disappearance of the picolinic acid signalfrom Cycle 64 to 65 shows the relatively rapid clear-downtime of the system. Each cycle in this trial appears as a boxbelow the graph, and the left-hand portion of the box withrespect to the tick mark represents the aerosol collectionphase of that particular cycle. The right-hand portion of thebox represents the pyrolysis processing and GC elutionphases. This trial presents a high concentration challenge ofBG spores at 87 PLA. This value is calculated as the differ-ence between the aerosol event maximum and average back-ground level. Note that the Met-One aerosol background isat a level of 100 PLA and is due to the 1–10-mm-diameterparticles.

The GC retention clock times of picolinic acid for Cycles63 and 65 are 19:57:22 (7:57:22 p.m.) and 20:03:49 (8:03:49 p.m.), and the BG bacterial aerosol event may be char-acterized as occurring between these two times. Therefore,the experimentally reported time of the first observation ofthe presence of BG can be listed as the clock time tR of thepicolinic acid dimer in Cycle 63. The absence of the sporefrom the sampled air can be ascertained in the first blankcycle (Cycle 65) after the last cycle containing a picolinicacid signal (Cycle 64). Note the similarity in picolinic aciddimer drift time between Cycle 63 and that in Figure 3 ofthe BG standard. The drift time between the picolinic acidstandard (6.63 ms, Figure 3) and that in Cycle 63 (6.70 ms)in Figure 5 varies by only 0.07 ms.

Cycle 60 (data not shown) occurred in the time frame ofpeak 1 (Figure 4) and provided no evidence of biologicalpresence in the GC/IMS data domain. Therefore, the originof peak 1 may consist of nonbiological aerosols such as in-organic debris and dust generated from the wheels of themoving aerosol dissemination vehicle.

The first cycle containing a picolinic acid signal and thefirst blank cycle after the last picolinic-acid–containing cy-cle are contained in Cycles 63–65, respectively, and withrespect to clock time, these cycles comprise the 19:57:22–20:03:49 time region, respectively. These times do not nec-essarily reflect the true times of the leading edge and trailingedge of the aerosol cloud with respect to the Py-GC/IMSinstrument. The leading edge of the cloud could have oc-curred at a time between the beginning of the pyrolysis andprocessing phase in Cycle 62 and near the end of the aerosolcollection phase in Cycle 63. The leading edge of the bac-terial cloud could not have occurred during the aerosol col-lection phase in Cycle 62. Within the parameters of the Py-GC/IMS analytical information in Figure 5, an exact time ofarrival of the leading edge of the bacterial aerosol cloudcannot be obtained. The clock time of the trailing edge ofthe aerosol cloud can be analyzed in a similar manner. Ablank cycle must follow a cycle containing picolinic acidsignatures in order to provide for a proper trailing edge anal-

ysis, and in this case, Cycle 65 shows no picolinic acid sig-nals in Figure 5. The aerosol trailing edge could have oc-curred at a time after the beginning of the aerosol collectionphase in Cycle 64, and no later than at the end of the GCelution phase of Cycle 64. Thus, the exact time of the trailingedge of the aerosol which passed over the Py-GC/IMS sys-tem could not be obtained.

The close correlation of the leading edge of Peak 2 andthe beginning of Cycle 63 observed in Figure 4 is coinci-dental. Likewise, the close correlation of the trailing edge ofPeak 2 and the beginning of Cycle 64 is coincidental. Thebeginning of Cycle 65, which is the first cycle after Peak 2,where no biological marker is observed, and the trailing edgeof Peak 2 are separated by a gap in time which is that ofCycle 64. Correlations between an aerosol event and selecttimes in cycles are coincidental, because the timing of thePy-GC/IMS cycles is independent of the motion of the aero-sol cloud, including when the cloud arrives and departs theinstrument.

Figure 4 provides the clock time versus the PLA graphwith a superimposed graph of the ACPLA distribution fromthe agar plate growth. Note that the ACPLA response, whichshows a zero ACPLA baseline signal at the leading and trail-ing edges, occurs within the same general time frame as Peak2. The shaded regions in Figure 4 represent the completeaerosol collection phase for Cycle 63 and the first portion ofthe aerosol collection phase for Cycle 64. These shadedregions overlap in time with the ACPLA aerosol distributionin Figure 4. The juxtaposition of the XM-2 aerosol collectionphase in a cycle and the ACPLA aerosol distribution effectsthe picolinic acid indicator of biological presence in the re-spective GC/IMS data window. This observation reduces thedegree of importance that is placed on the presence/absenceof a biological aerosol event as measured by a particlecounter device. Thus, without an ACPLA-cultured biologi-cal aerosol determination, the presence/absence of a bacterialaerosol cloud is predicated on the presence/absence of abiomarker signal in the GC/IMS data domain and is an al-ternative to the presence/absence of an aerosol event in theMet-One aerosol particle count record.

The dotted-line graph in Figure 4 is a plot of the averagesignal intensity (S/N) of picolinic acid from its GC/IMS datawindow from Figure 5. Note that each point lies above theGC processing phase for each cycle. Because approximately60 BG ACPLA were contained in the real-time ambient 87PLA event, 69% of the ambient aerosol consisted of BGspores. Thus at least 30% of the signal in Peak 2 is actuallycomposed of nonbacterial, inorganic debris and dust. Ap-proximately 60,000 spore-containing particles were deliv-ered to the pyrolysis processor from the 1000 l/min XM-2concentrator during the 2-min aerosol collection time in Cy-cle 63, assuming particle diameters of 5 mm.

Trial 6

A visual analysis of Figure 6 shows a Met-One spectrumwhere no clear aerosol event is detected, and Cycles 85–92

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provide no biological signals in the GC/IMS data domain(not shown). Cycle 93 in Figure 7 also has no bacterial sporesignatures; however, Cycles 94 and 95 present picolinic acidmonomer and dimer signatures (arrows). Cycle 96 has nomonomer or dimer picolinic acid information, and a char-acterization of the presence of BG spores is contained in thetime frame of 22:35:41–22:42:35. The former represents theclock time tR of the picolinic acid biomarker analyte in Cycle94, because it is the first cycle containing picolinic acid. The22:42:35 value is the tR value in Cycle 96, where a picolinicacid signal would be expected to occur, because this is thefirst cycle after Cycle 94 that displays no picolinic acid an-alyte response. The delay time was 2 min and 43 s, andsubtraction of this value from both tR values yields BG aero-sol cloud arrival and departure times (as defined in the Trial4 analysis) of 22:32:58 and 22:39:52, respectively. The av-erage PLA is approximately 80 in the region of Cycles 94–96 (Figure 6). However, the noise component of the aerosoldistribution graph in the region of Cycles 94–96 is approx-imately 20 PLA. A number of factors can contribute to theparticle-counter sensitivity and hence random noise dis-placement from the overall background particulate level.These include the range of particle diameters admitted intothe particle counter and/or an increased particle signal av-eraging time. An example of the latter is that a longer timefor particle counting prior to signal output creates a smooth-ing effect. It is possible that the 80 particles per liter of aircould contain not only spore analyte, but also inorganic, non-

biological particulates. Figure 6 shows that a cultured AC-PLA aerosol distribution of BG occurred in the same timeframe as Cycles 94–95. The shaded regions constitute theoverlap in time between the aerosol-collection phase of theaerosol concentrator and the presence of an actual BG aero-sol cloud.

The average intensities of the picolinic acid dimer peaksin Figure 7, Cycles 94 and 95, show an overlap with theACPLA distribution in Figure 6. Indeed, the ACPLA valueof 5 is considerably lower than the total PLA value of 80,and approximately 6% of the aerosol particulate was BG.Only two-thirds of the 2-min aerosol concentration time forCycles 94 and 95 (shaded areas of 80 s in duration insteadof 120 s in each cycle) effectively overlapped the ACPLAdistribution; thus, approximately 3,300 spore-containingparticles were deposited in the quartz pyrolyzer for analysisin each cycle, assuming BG particle diameters of 5 mm.Leading- and trailing-edge analyses with respect to the Met-One particle count graph in Figure 6 are not practical, be-cause a clear aerosol event is not observed.

Summary of Trials

The suite of 18 outdoor trials spanned BG aerosol chal-lenges from 5–140 ACPLA. A summary of the trials can befound in Figure 8. The area of the ACPLA curves (boldfacepoints in Figures 4 and 6) are plotted versus the area of thedetection event in the S/N curves (dotted-line graphs in Fig-

FIG. 6. Met-One (PLA) and ACPLA distributions and temporal disposition of the Py-GC/IMS cycles 93–96 in Trial 6.


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ures 4 and 6). The S/N component derives from the additionof the intensities of the monomer and dimer peaks in theirrespective windows in the GC/IMS data domain.

The point plot of all the trials in Figure 8 defines tworegions. The first region (shaded circles) can be character-ized as a relatively linear increase in S/N of picolinic acidas the amount of the ambient aerosol challenge increasesgiven a constant aerosol collection time of 2 min. Thesetrials had BG aerosol challenges of # 70 ACPLA. Threetrials show that the S/N of picolinic acid from its GC/IMSdata window provides a saturation value, and the aerosolchallenge for Trials 22, 14, and 16 were 80, 100, and 140ACPLA, respectively. The water RIP for these three trials

were depleted during the analysis (data not shown) such that,despite the GC module partitioning of the total pyrolyzate,relatively large amounts of sample analyte entered into theion mobility spectrometer. RIP deletion and nonlinear inten-sity effects are undesirable yet common phenomena inIMS.28 Thus the product of relative intensity and duration ofthe gram-positive Bacillus spore aerosol event versus theaerosol challenge burden yields an approximate linear cor-relation until an excess of aerosol particulate is attained. Thisis a common phenomenon with chemical sample vapors,30

and to the best of the author’s knowledge, this is the firsttime that the IMS signal saturation phenomenon has beendocumented for pyrolyzates of biological aerosols.

FIG. 7. GC/IMS data domains for cycles 93–96 in Trial 6.

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Aerosol Detection Limits

It was discovered that in a comparison between the pres-ence/absence of picolinic acid in its GC/IMS data windowand the presence/absence of a BG ACPLA aerosol event, arelative picolinic acid intensity value of 15% of the reactantion peak (RIP) intensity provided a satisfactory correlation.This means that the presence of a signal in the GC/IMS datawindow for picolinic acid, when the intensity gain was setat 15% of that of the RIP, indicated the presence of a BGcloud, while no signal inferred the absence of a BG aerosolcloud. From these observations, a limit of detection has beenestimated at approximately 3300 BG spore-containing par-ticles for the current version of the Py-GC/IMS system forparticle diameters of 5 mm. This estimate was made in con-junction with three other ambient real-time 5 BG ACPLA(low concentration) aerosol trials (data not shown). The GC/IMS monomer and dimer picolinic acid features providedlow intensity, yet relatively consistent signatures from low-level biological aerosol challenges. The 3300-particle esti-mate is with respect to an aerosol that also contained non-bacterial debris and inorganic dusts. As was presented, a

mixture of particles containing bacterial spore and nonsporematter typically characterized the type of aerosol analyzedin the field trials. Trial 1 and 7 provided true negatives, be-cause no picolinic acid peaks were observed in all of thecycles in each trial (data not shown). In addition, aerosolevents were not observed in the PLA or ACPLA particlecount recordings (data not shown).

Conclusions

Py-GC/IMS has been portrayed as an analytical technol-ogy that can provide a measure of ambient bioaerosol inter-rogation. The Py-GC/IMS instrument was shown to be acredible complement to an aerosol particle counter. Aerosolparticle events in an aerosol record can originate from dif-ferent types of materials such as biological and nonbiolog-ical particles. An aerosol event can also consist of a mixtureof these different kinds of materials, and different classes ofbiomaterials also can be present. The current Py-GC/IMSsystem takes advantage of the sensitive nature of IMS bydetecting a substituted amine functional group compound in

FIG. 8. Each trial is represented by the total picolinic acid signal versus the total biological aerosol burden. Shaded circles represent trials having #70ACPLA, and the numbers represent Trials 22, 14, and 16, having 80, 100, and 140 ACPLA, respectively.


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gram-positive bacterial spores such as the protonated formof picolinic acid. This compound was shown to be a reliableindicator of spore presence in an aerosol cloud containingas low as 5 BG ACPLA. This ambient real-time aerosolchallenge yielded an effective 3,300 spore-containing par-ticles at the analytical system’s pyrolysis processor with thegiven aerosol concentrator parameters.

The system was shown to be reliable and rugged (withthe exception of the pyrolysis filter) over the testing periodunder outdoor temperatures routinely in the 0–57C range.The memory effect in the Py-GC/IMS system was relativelylow, and the GC column performed in a satisfactory mannerdespite the constant 60–1307C temperature cycling underscrubbed air carrier gas conditions for a period of 4 weeks.The trials spanned biological challenges from between 5 and140 BG ACPLA. A summary of the 4-week, 21 BG fieldtrials produced two true negatives, no false positives, andone software failure, and the remaining 18 trials were truepositive determinations.

Acknowledgments

We wish to thank Dr. Douglas Andersen for helpful dis-cussions and information concerning the biological aerosoloutdoor trials, and Alice I. Vickers for the preparation andediting of the manuscript.

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