Anal, Chem. 1994,66, 2820-2828
Pyrolysis-Gas Chromatography/Mass Spectrometry Analyses of Biological Particulates Collected during Recent Space Shuttle Missions Marilyn L. Matney’ and Thomas F. Limero KRUG Life Sciences Inc., 1290 Hercules, Houston, Texas 77058 John T. James Biomedical Operations and Research Branch, NASAIJohnson Space Center, Houston, Texas 77058
Biological particulates collected on air filters during shuttle missions (STS-40 and STS-42) were identifiedusing pyrolysisgas chromatography/mass spectrometry (Py-GCIMS). A method was developed for identifying the atmosphericparticles and their sources through the analysis of standard materials and the selectionof “marker”compounds specific to the particle type. Pyrolysis spectra of biological standardswere compared with those of airborne particles collected during two space shuttle missions; marker compounds present in the shuttle particle spectra were matched with those of the standards to identify the source of particles. Particles of 0.5-1-mm diameter and weighing as little as 40 pg could be identified using this technique. The Py-GUMS method identified rat food and soilless plant-growth media as two sources of particles collected from the shuttle atmosphere during flight. The relative absence of gravity in space allows particles of human food and skin flakes, paint chips, plastics, lint, dust, and hair to remain suspended in the spacecraft atmosphere until the air flow moves them to air filters in the shuttle’s middeck and spacelab. These free-floating particulates and those from payload experiments are a potential source of eye, skin, or respiratory tract irritation; moreover, particles released from animal payload experiments can pose infectious-disease hazards. Concern regarding potential particulate releases from animal and other payload experiments carried aboard the shuttle led to the collection of atmospheric particulates for ground-based analysis. During analysis, particles of interest in the filter debris can be separated using a microscope, but positive microscopic identification was often impossible, particularly for the foodlike particles having similar microscopic appearances. Therefore, a reliable chemical technique was needed to positively identify the origin of such particles, so that containment of potentially hazardous particles could be improved on future flights. Limited chemical analysis of flight particles by other investigators had been performed before this study. Particulates, collected by size on impactors during flight, were analyzed for elemental composition using scanning electron microscopy with energy dispersive spectroscopy (SEM/EDS) and X-ray fluorescence (XRF).’ The X R F analyses of particles less than 10 pm in diameter suggested that 95% were organic in nature, since the identified elements accounted for less than 5% of the total particulate mass. However, neither 2820
Analytical Chemistry, Vol. 66,No. 18, September 15, 1994
the identity nor the source of organic particles could be determined with these techniques; consequently, an alternative method was sought. Pyrolysis coupled with gas chromatography, mass spectrometry, or both has been applied successfully to the study of a variety of biological materials2 such as polysaccharide^,^ lignin^,^ amino acids,5 and soils.6 Particulates have also been studied with pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS). In oneair pollution study,’the insolubleportion of particulates collected from the atmosphere (total sample weight was 100-500 pg) was analyzed by Py-GCIMS. The numerous types of particulates present in the sample produced a complex chromatogram, making identification of the particle sources difficult. The particles collected during the present investigation were presumed to be larger than those of the air pollution study, since individual particles had typical diameters of 0.5-1.5 mm and weights of 35-200 pg. Because of the larger particle sizes, no more than four microscopically similar particles from the filter debris were analyzed a t a time, and it was often possible to analyze individual particles, simplifying identification of both the particle and its source. In this paper, the Py-GC/MS method used to analyze and identify unknown particles and their sources from air-filter debris collected during two space shuttle flights is described.
EXPERIMENTAL SECTION Instrumental Analysis. A Ruska Pyran system8-10 (prototype model) was used to analyze all standards and debris (1) Liu, B.Y. H.; Rubow, K. L.; McMurry, P. H.; Kotz, T. J.; Russo, D. Airborne
Particulate Matter and Spacecraft Internal Environments (SAE Technical Papers Series No. 91 1476). Presented at the 2lst International Conference on Environmental Systems, San Francisco, CA, July 15-18, 1991. (2) Irwin, W. J. Analyrical Pyrolysis: A Comprehensiue Guide; Marcel Dekker: New York, 1982. (3) Pouwels, A. D.; Eijkel, G. B.; Boon. J. J. Curie-Point Pyrolysis-Capillary Gas Chromatography-High-Resolution Mass Spectrometry of Microcrystalline Cellulose. J . Anal. Appl. Pyrolysis 1989, 1 4 , 237-80. (4) Faix, 0.;Meier, D.: Fortmann, I. Thermal Degradation Products of Wood. Holr Roh- Werkst 1990, 48, 281-5. (5) Boon, J . J.; De Leeuw, J . W. Amino Acid Sequence Information in Proteins and Complex Proteinaceous Material Revealed by Pyrolysis-Capillary Gas Chromatography-Low and High Resolution Mass Spectrometry.J . Anal. Appl. Pyrolysis 1987, 11, 313-27. (6) Hempfling, R.; Schulten, H.-R. Chemical Characterization of the Organic Matter in Forest Soils by Curie Point Pyrolysis-GC/MS and Pyrolysis-Field Ionization Mass Spectrometry. Org. Geochem. 1990, 15(2), 131-45. (7) Kunen, S. M.; Voorhees, K. J.; Hill, A. C.; Hileman, F. D.; Osborne, D. N. Chemical Analysis of the Insoluble Carbonaceous Components of Atmospheric Particulates with Pyrolysis/Gas Chromatography/Mass Spectrometry Techniques, 70thAnnual Meeting ofthe Air PollutionControl Association,Toronto, ON, June 2&24, 1977; paper 77-36.4.
0003-2700/94/0366-2820$04.50/0 0 1994 American Chemical Society
COMI'UTCR/MSD
MASS
SI'RCTRAL LIBRARY
DETECTOR
GC COLUMN
3-
VENT
CARRIER GAS
Figure 1. Diagram of Ruska Pyran system. The sample loader (SIis lowered via computer control to accept cruclble samples. Once the sample has been loaded, the sample loader moves inside the pyrolysis cell (P) until the o-ring at the base of the sample loader is compressed against the bottom of the pyrolysis cell. Helium flow through the sample loader base and porous sample crucible carries the pyrolysis products into the splitter region where 10% of the products are split Into the -55 "C GC column.
particles by Py-GC/MS. The Pyran's quartz pyrolysis cell and splitter are directly in line with a capillary GC column coupled to a Hewlett-Packard 5971 mass selective detector (MSD), as shown in Figure 1. Pyran software was used to develop a method for precisely controlling and timing events such as sample loading and pyrolysis, split ratios and helium flow, and column cooling and heating. Sample crucibles were loaded automatically into the pyrolysis cell, where they were heated at 30 "C for 7 min. After this temperature-stabilization period, the pyrolysis cell was heated to 600 "C at the rate of 30 "C/min, resulting in a total pyrolysis time of -20 min. A helium flow rate of 10 mL/min was maintained through the pyrolysis cell to carry the evolved pyrolysates into a liquid CO2-cooled column held at -55 "C. The flow rate through this J&W DB-5 column (30 m, 0.32-mm i.d., 0.25-pm film thickness) was 1 mL/min; the remaining 9 mL/min flow was vented through a charcoal filter. The use of the 0.25-pm film column allowed higher molecular weight compounds to elute within a reasonable time. Because the Pyran system permits only split injections, a low split ratio (1O:l) was used to optimize the amount of pyrolysis products injected into the column, which was especially important for particles yielding low concentrations of products. After pyrolysis was complete, the column was heated by means of a temperature program from -55 to 200 "C at the rate of 10 "C/min, and then from 200 to 300 " C a t 5 "C/min. Eluted pyrolysates were subsequently detected by the mass spectrometer, which scanned at a rate of 1.3 scans/s over the
-
(8) Overton, E. B.; Martin, S.J. A Field Deployable Analytical Instrument for Analysis of Semivolatile Organic Compounds of Superfund Sites. Proc. 3rd Ann. Symp., U S . EPA Symp. on Solid Waste Testing and Quality Assurance, 1987; Vol. 11, July 13-17. (9) Zumberge, J. E.; Sutton, C.; Martin, S.J.; Worden, R. D. Determining Oil Generation Kinetic Parameters by Using a Fused-Quartz Pyrolysis System. Energy Fuels 1988, 2, 264-6. (IO) Overton, E. B.; Henry, C. B.; Sutton, C. Field Deployable Instrument for the Analysis of Semivolatile Organic Compounds. Field Screening Methods for Hazardous Waste Site Investigations, October 11-13, 1988, U S . EPA: Las Vegas, NV, 1989.
mass range 25-450 amu. After each sample analysis, a blank was run to clean the pyrolysiscell and establish that the column was free of high molecular weight compounds from the previous run. Pyrolysis spectra were obtained using HP's Chemstation software; the mass spectra of pyrolysis products were identified by spectral interpretation, comparison with standard compounds, library databases,11.12and literature s o ~ r c e s . ~ * ~ J ~ Biological Standards. Several types of biological materials were collected and analyzed in anticipation of their potential presence in flight samples. An experiment with rats was part of the first of two missions from which filter debris samples were analyzed; a plant-growth experiment using soilless media was part of the second. Rat food, feces, and bedding from the animal cages were collected for use as standards before the rats (Sprague-Dawley general purpose research model, supplied by Taconic, Germantown, NY) were stowed aboard the STS-40 Spacelab module (STS refers to space transportation system; -40 denotes the mission number). A soilless media standard was received from the same lot of soilless media used in an experiment aboard the STS-42 mission. The rat food was a modified Teklad Premier diet containing casein, wheat flour and gluten, corn starch, corn syrup, corn oil, sucrose, cellulose, vitamins, and minerals molded into a bar. It was assumed that the composition of the fecal pellets would be consistent from animal to animal since their genetic makeup and diet were similar. The bedding was coarse-ground corncob composed of lignin, cellulose and hemicellulose (or xylans).14 Soilless media consisted of 60% peat moss and 40% vermiculite. Because the shuttle particles underwent drying during and after the mission and were therefore received in a dehydrated state, the moist standards (rat food and feces, soilless media) were crumbled into glass petri dishes and allowed to dry before analysis. Before the drying process, food and fecal samples were obtained by removing sections from the interior of the bars and pellets, respectively, to avoid potential contamination from the polyethylene and paper containers. Bedding samples did not require drying and were obtained by shaving particles off larger pieces of bedding. Biological samples weighing 200 pg produced the optimal pyrolysis spectra; Le., the ion abundances were sufficient to allow qualitative identification of most compounds. In some cases, as many as five particles of the standard were placed in a single crucible to achieve this sample weight. Particles weighing > 150pg were analyzed individually. Because shuttle debris particles resembling soilless media weighed only 2090 pg, smaller standard samples (-40 pg) were analyzed to match the spectra for unknown materials more closely. In general, rat food and feces particles weighing 200 pg had diameters that ranged from 0.7 to 1.5 mm. Particle diameters were estimated under the microscope using a visual micrometer (note: these diameters have not been converted into aero(1 1) Wiley 130K MassSpectralDatabase, John Wiley & Sons,Inc., 1986. Licensed
to Hewlett-Packard Co. (12) NIST/EPA/MSDC Mass Spectral Database, v. 2.5, National Institute of Standards and Technology, 1990. (13) van Smeerdijk, D. G.; Boon, J. J. Characterisation of Subfossil Sphagnum Leaves, Rootlets of Ericaceae and Their Peat by Pyrolysis-High-Resolution Gas Chromatography-Mass Spectrometry. J. Anal. Appl. Pyrolysis 1987, l I , 377-402. (14) The Carbohydrates: Chemistry,Biochemistry, Physiology; Pigman, W . ,Ed.; Academic Press Inc.: New York, 1957; p 666.
AnalflicalChemistry, Vol. 66, No. 18, September 15, 1994
2021
1.8e+07 1.6e+07
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Flgure 2. (A) Pyrolysis spectrum of particulate rat food. Important chromatographic peaks are (identified by their mass spectra): (1) 2,3butanedbneand 2-methylfuran (overlapped), (2) benzene, (3) acetic acid, (4) 2,5dimethylfuran, (5) toluene, (7) 2-furancarboxaklehyde, (8) l-hydroxy2-propanone, (10) 2-furanmethanol, (11) 5-methyC2-furancarboxaMehyde,(12) unknown compound and 3-hydroxy-2-methyl-4Kpyren-4-one, (13) 1,4:3,6dianhydro-a-~-gglucopyranose, (16)hexadecanenitrile, (17) hexadecanoicacid, and ( 18) coeluting 9,1 P-octadecadienoic and g-octadecenoic acids. (B) Comparison of pyrolysis spectra of STS-40 shuttle particles (B) and rat food standard (A) shows that the rat food marker compounds are common to both spectra: (5) toluene, (10) 2-furanmethanol, (16) hexadecanenltrlle, and (18) 9,12-octadecadienoic and g-octadecenoic acids. Formic acid (6)and pyruvic acid methyl ester (9) were observed in the shuttle particles spectrum (B) at 36.38 and 37.41 min, respectively. Although the 5-methyl-2-furancarboxaldehyde is more abundant in the shuttle particles, it is within the range of concentrations listed in Table 1 for the rat food standards. (C) The pyrolysis spectrum of another STS-40 shuttle particle (C) is similar to the rat food standard, but contains addltional compounds (14) dodecanoic acid and (15) tetradecanoic acid. The compound asterlsked In A-C is a siloxane contaminant. The time axis (min) includes the pyrolysis run time; to obtain the actual retention time, a pyrolysis delay time of 25.8 min should be subtracted from the observed retention time.
dynamic diameters). A minimum of three pyrolysis spectra were collected for each standard to ensure that reproducible spectra were obtained. 2822
Analytical Chemistry, Vol. 66, No. 18, September 15, 1994
Shuttle Sample Preparation. Debris samples from the Spacelab modules were collected in a bag by vacuuming the air filters from the laboratory modules after landing. The
Figure 3. Pyrolysis spectrum of rat bedding. Major chromatographic peaks are (1) 2,3-butanedione and 2-methylfuran (overlapped), (2) acetic acid, (3) 3-pentanone, (4) formic acid, (5) l-hydroxy-2-propanone, (6) 2-furancarboxaidehyde, (7) 2-methoxyphenoi, (8) unknown compound overlapped wRh siloxane, (9) methylbenzaldehyde, (10) 4-ethyi-2-methoxypheno1, (1 1) 4-vinyi-2-methoxyphenol, and (12) 2,ddlmethoxyphenol.
filter debris was transferred to disposable petri dishes and weighed. The particles were separated manually from lint (the major component of the debris) and examined under a dissecting microscope (magnification power ranged from 1OX to 60X). Particles that resembled the reference standards were collected from the debris particles and placed in storage vials. Because of the large number of foodlike particles resembling rat food in the debris, debris particles were grouped together by color and texture, and then each group was placed in a storage vial. Typically, one to four foodlike particles from the same vial were pyrolyzed together in the crucible. Shuttle particles resembling soilless media were not combined to give a 200-pg sample weight because it was difficult to determine visuallywhether debris particles, such as plant stems, were constituents from peat moss or another plant source. Particles were placed in a fritted quartz crucible and weighed on a Cahn C-30 microbalance (0-200-mg scale) without further sample preparation before analysis.
RESULTS AND DISCUSSION Standards: Pyrolysis Spectra. Rut Food. The pyrolysis spectrum of rat food particles weighing a total of 217 pg is shown in Figure 2A. The major peaks in the spectrum (2,3butanedione, 2-methylfuran, toluene, 1-hydroxy-2-propanone, 2-furancarboxaldehyde, 2-furanmethanol) were produced from the pyrolyzed constituents of the rat food, Le., casein, cellulose, sucrose, corn syrup, corn starch, and wheat flour and gluten. Because the pyrolysis temperature is ramped relatively slowly, semivolatile compounds are released at temperatures generally below 300 OC, and pyrolysis generally occurs at higher temperatures. Consequently, the pyrolysis spectrum contains a mixture of thermally extracted and pyrolysis products. Hexadecanoic acid and the coeluting 9,12-octadecadienoic and 9-octadecenoic acids, found in wheat flour, wheat gluten, corn starch, and corn oil, were extracted thermally, because their boiling points are 390 and 360 OC, respectively. Rut Bedding. Bedding was used before flights but not during missions. However, the rats chewed on the bedding; therefore it was necessary to identify bedding marker compounds that would be present in the rat feces standard. The pyrolysis spectrum of 224 pg of bedding is shown in Figure
-
-
3. The major peaks in this spectrum are formic acid, l-hydroxy-2-propanone, and 2-furancarboxaldehyde (xylan and cellulose pyrolysis products) and 2-methoxyphenol and 4-vinyl-2-methoxyphenol (lignin pyrolysis products). Rut Feces. The rat feces (232 pg) pyrolysis spectrum shown in Figure 4A contains peaks common to both rat food and bedding. Since bedding was not present in the rat cages during the flight, feces produced during flight should contain little or no bedding-specific compounds. If the bedding peaks are eliminated from the standard feces spectrum, the pyrolysis spectra of the feces and food contain many of the same compounds, albeit with subtle differences. The amount of toluene (casein pyrolysis product) differed greatly between the feces and food spectra, with the toluene peak being nearly 10 times smaller in the feces. Hexadecanenitrile, another casein pyrolysis product observed in the rat food, was not detected in the feces spectrum. The overlapped peaks of 9,12-octadecadienoic and 9-octadecenoic acids were very weak in fecal samples (Figure 4C); in later samples, which were dried for longer periods (-8 weeks), they disappeared completely (Figure 4A,B). Pyruvic acid methyl ester, which was weakly present in only one of the three rat food standards, was present in the feces at a concentration nearly 3 times higher than that observed in the bedding. Soilless Media. Peat moss was the only constituent of the soilless media that generated thermal products, since vermiculite neither off-gassed nor thermally degraded at the temperatures used. According to a Py-GC/MS study by van Smeerdijk and Boon, macromolecules present in peat moss consist of polysaccharides, lignin, polyphenols (non-lignin), and suberin.13 The pyrolysis spectrum of 48 pg of soilless media is shown in Figure 5A. Prominent peaks are 1-hydroxy2-propanone and 2-furancarboxaldehyde (polysaccharide pyrolysis products) and phenol (polyphenol pyrolysis product). Thirty-two of the compounds detected in pyrolysis spectra of the rat and soilless media reference standards (and identified by their mass spectra) are shown in Table 1, with peak intensities normalized relative to the largest peak in each spectrum. More than 100 compounds were detected in the spectra of all standard materials, but only major components of the standards or those unique to a particular standard are reported in Table 1. Although additional pyrolysis spectra were collected for rat food and feces and soilless media, only AnalyticalChemistry, Vol. 66, No. 18, September 15, 1994
2823
6
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Flgure 4. (A) Pyrolysis spectrum of particulate rat feces. The chromatogram consists of peaks common to rat food and bedding: (1) 2,3butanedione and 2-methylfuran (overlapped), (2) benzene, (3) acetic acid, (4) 3-pentanone, (5) toluene, (6) l-hydroxy-2-propanone, (7) 2-furancarboxaldehyde, (8) pyruvic acid methyl ester, (9) 2-furanmethanol, (10) cyclohexanone, (11) 2-methoxyphenol, (12) unknown compound, (13) 4-vinyl-2-methoxyphenol. and (14) hexadecanoic acid. Comparison of particulate (A, B) and ground rat feces (C) shows that the pyrolysis spectra are similar. Retention times in (C) are -0.35 min longer than In (A) and (B) because of a carrier flow-rate change. Comparison of (A) and (B) (data collected under the same flow rate) shows that compounds (2) benzene, (3) acetic acid, (5) toluene, (9) 2-furanmethanol, and (14) hexadecanoic acid have reproducible retention times. Compounds (6) l-hydroxy-2-propanone, (7) 2-furancarboxaldehyde, and (8) pyruvic acid methyl ester have shifted retention times due to interaction with contaminant water.
three pyrolysis spectra of each standard were selected for analysis. Only compounds detected in all three pyrolysis spectra analyzed for each standard are listed in Table 1. Standards: Reproducibility. In order to use the pyrolysis spectra of standard materials as the basis for identifying unknown particles, it was necessary to demonstrate that the bulkstandards yielded particles of homogeneous composition. 2824
Analytical Chemistty, Vol. 66, No. 78, September 15, 1994
Variability in particle composition in the standards would limit the applicability of this method for identifying unknown particles. Thus, an important step in analyzing the standards was to determine whether consistent spectra could be obtained. For the rat food analyses, sample homogeneity appeared to be dependent on the size of the particle. It was observed that small rat food particles (C1OO-Fg weight, 300-500-pm
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Flgure 5. (A) Pyrolysis spectrum of soilless media. Significant peaks are (1) benzene, (3) l-hydroxy-2-propanone, (4) 3-furancarboxaklehyde, (5) 2-furancarboxaldehyde,(7) 2(5H)-furanone and cyclohexanone, (8) 5-methyl-2-furancarboxaldehyde,(9) phenol, and (1 1) levoglucosenone and 3-hydroxy-2-methyl-4Kpyran-4-one. Lignin pyrolysis products are (10) 2-methoxyphenol and (13) 4-vinyl-2-methoxyphenol. Comparison of PyGClMS spectra of STS-42 shuttle partlcle (e) and soilless media standard (A) shows that the major compounds of soilless media are present in both spectra: (1) benzene, (3) l-hydroxy-2-propanone, (5) 2-furancarboxaldehyde, and marker compound (9) phenol. The retention tlme of l-hydroxy-2-propanone is again shifted. The siloxane contaminant is marked by an asterisk.
diameter) tended to be mostly proteinaceous. If the particle was larger (-200 pg) or if the sample contained two or three particles (total weight 200 pg), reproducible rat food spectra were obtained. Therefore, the smallest rat food particles were not representative of the bulk standard, but particles weighing 200 pg did appear to be homogeneous. Reproducible spectra were obtained for 9 out of 10 rat food samples having -200pg weights. Chromatographic “patterns” matched with slight variations in peak intensities. Only one rat food standard was mostly proteinaceous; peak intensities in its spectrum were significantly different from the other rat food standards. As further evidence of reproducibility in particle analysis, the pyrolysis spectra of three rat feces samples are shown in Figure 4. Analysis of the fecal pellets was expected to be particularly challenging because the individual particulates were thought to be heterogeneous. The pyrolysis spectrum of fecal material that had been ground and mixed with a mortar and pestle (Figure 4C) was compared to those of unground feces that had been separated into particles (Figure 4A,B). Generally, the particulates seemed homogeneous, and unground particulate fecal material was analyzed for the remainder of the study. The minor variances in peak intensity among the spectra in Figure 4 are most likely a result of slight
-
variations in the composition and surface area of the fecal particles. Minor variations noted in compound retention times of the fecal standards were caused by slight changes in the carrier flow rate (Figure 4C) and an untraceable source of water contamination in the Pyran system,I5 which seemed to shift retention times for some compounds eluting between 35 and 37 min (Figure 4A,B). Although some variances in peak intensity and retention time were observed in the standards spectra, the compounds detected in the standards spectra were reproducible. For example, in the rat food reference standards, 72 of the 107 compounds detected were common to all three food standards. All of the remaining compounds (those not detected in all three standards) had peak intensities of very weak to weak; nearly 80% of them had very weak peak intensities. Variations in sample homogeneity could determine whether a very weak component would be present in the spectrum at a concentration high enough for detection. Not surprisingly, the percentage of common compounds among the standards decreased as more weak components were detected. However, major and minor pyrolysis products (along with a high number of weak (15) Note: water contamination was observed in blank runs as well.
AnalyticalChemistty, Vol. 66,No. 18, September 15, 1994
2825
Table 1. Thermally Extracted and Pyrolysls Products Found In Reference Standards’ normalized peak intensities compounds found 2,3-butanedione 2-methylfuran benzene acetic acid 2,s-dimethylfuran 3-pentanone 1-hydroxy-2-propanone toluene 2( 3H)-furanone formic acid 3(2H)-furanone 2-furancarboxaldehyde pyruvic acid methyl ester 1-(2-furanyl)ethanone 2( SH)-furanone
S-methyl-2-furancarboxaldehyde
rat food
m-s m-s w-m m w-m
rat bedding m w-m vw
m-s vw
m
rat feces w-m w-m vw w-m vw w-m
w-m
m-s
S
m-s
S
m-s*
vw
vw-w* w-m w-s (varies) w-m m w-m* vw
W
m-s* vw-w
m-s W
w-m w-m
m vw-m vw-w vw-w vw W
W
W
w-m vw-w
w-m vw
vw-w w-m vw-w
m*
W
3-hydroxy-2-methyl-4H-pyran-4-one MW unknown
m
vw w-m
vw-w w-m w-m w-s (varies) vw-m w-m vw-w
m m-s* w-m
1,4:3,6-dianhydro-a-~-glucopyranose
w-m
2,6-dimethoxyphenol hexadecanenitrile hexadecanoic acid 9,12-octadecadienoic acid 9-octadecenoic acid
W* w-m vw-w* vw*
w-m
w-s (varies) w-m W
W
vw-w W
m-s vw-m w-m w-m
vw-w
m-s* vw
methylbenzaldehyde 4-vinyl-2-methoxyphenol 4-ethyl-2-methoxyphenol
W
W
furanmethanol phenol cyclohexanone methylphenol 2-methoxyphenol
m
soilless media
m-s* w-m w-m vw-w W
w-m W
vw-w
a A range of peak intensities is reported for some compounds because of minor variations in peak intensities among t h e standards. T h e asterisk denotes marker compounds. Key: vw, very weak; w, weak; m, moderate; s, strong.
components) were detected consistently in the three standards analyzed for a material. This consistency in the pyrolysis spectra allowed the selection of “marker” compounds to represent the material of interest. Marker Compound Selection. The large number of pyrolysis products common to the rat and soilless media standards made it necessary to find some way to distinguish among the standards. Major pyrolysis products 2-furancarboxaldehyde and 1-hydroxy-2-propanone were common to all of the standards (Table 1); moreover, a number of minor products were common to all standards as well. Overall, the rat and soilless media standards shared many of the same pyrolysis products in spite of a dissimilar appearance under microscopic examination. In a previous Py-GC/MS study by Wuepper,16 materials were characterized by “diagnostic” peaks, which were often the largest in the spectrum. Many of the peaks in these pyrolysis spectra were unique to a material because the composition of the materials varied widely (Le., rubber, paint resin, epithelial tissue, textile fibers, polymers, and gum); consequently, materials were more easily distinguished from one another. However, in this work, the largest peaks in the spectra were generally common to all standards, as evidenced by the presence of major peaks 2-furancarboxaldehyde and 1-hydroxy-2-propanonein each standard spectrum. Therefore, compounds unique to the standard or having distinctive peak intensities were selected as markers to distinguish among the standards. (16) Wuepper, J L Pyrolysis Gas Chromatographic-Mass Spectrometric Identification of Intractable Materials Anal Chem 1979, 51, 997-1000
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Marker compounds are identified with an asterisk in Table 1. The markers for rat food, toluene and 2-furanmethanol, were chosen for their distinctive peak intensities; these compounds were also observed in the other standards but at greatly reduced peak intensities. Hexadecanenitrile, 9,12octadecadienoic acid, and 9-octadecenoic acid were present only in the rat food particles. Rat bedding markers were 2-methoxyphenol, 4-vinyl-2-methoxypheno1, and formic acid. After the bedding markers were removed from the feces pyrolysis spectrum, the feces spectrum contained many of the same compounds as the rat food. However, fecal spectra can be distinguished from food spectra by the presence of a very small toluene peak (the normalized toluene peak is 10 times less than in the rat food) and a moderately strong pyruvic acid methyl ester peak. Because chromatographic peaks are normalized to the strongest peak(s) in the spectrum, pyruvic acid methyl ester levels seem to be comparable in the rat bedding and feces. When the ratio of peak areas to sample weight is taken, pyruvic acid methyl ester is 3 times stronger in the feces. Hexadecanenitrile, 9,12-octadecadienoic acid, and 9-octadecenoic acid were not present in the rat feces. Soilless medium was characterized by a moderately strong phenol peak. Although phenol was also detected in the rat standards, its concentration was higher in the soilless media relative to the other soilless media pyrolysis products. Identifying Unknown Particles from Space Shuttle Atmospheres. Pyrolysis chromatograms of the shuttle particles first were screened by comparing them with chromatograms generated for the reference standards. If the chromatographic “pattern” of a shuttle particle seemed to match a standard,
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-
Table 2. Comparlson of Rat Food and STS-40 Shuttle Particles Thermal Extractlon and Pyrolyslr Productsa
compounds found butanone 2-buten-2-one 2,3-butanedione 2-methyl fur an 3-methyl furan
1,3,5-hexatriene or 1,3-cyclohexadiene benzene C6-dialkene acetic acid 3-pentanone 2,3-pentanedione 2- heptene 2,3-dimethylfuran 3-penten-2-one MW82 pyrazine toluene 1H-pyrrole 2( 3H)-furanone cyclopentanone (E)-4-octene octane 1-(3-furanyl)ethanone 3-furancarboxaldehyde formic acid 1-hydroxy-2-propanone 2-furancarboxaldehyde MW82 pyridine ethylbenzene 1,2-dimethyIbenzene MW98 pyruvic acid methyl ester 3 (2H)-furanone styrene 2-cyclohexen- 1-one 1,3-dimethylbenzene alkane with unknown M W C2-substituted cyclopentene 2-furfuryl formate 2-furanmethanol
I*atfood
STS-40 shuttle particles
w-s vw-w m-s m-s vw vw w-m vw w-m vw-w vw-w vw-w w-m vw vw vw m-s w-m
vw vw m m vw vw vw vw w-m vw vw
W
W
vw vw m W
W
vw vw vw vw vw
vw-w b
m
S
S
m-s vw vw-w
m vw vw vw vw vw
vw vw-w W
W
vw-w vw-w b b
W W
W
vw
vw-w vw-w vw vw-w vw-w m-s
W
vw vw vw m
compounds found 1-(2-furanyl)ethanone dihydro-2(3H)-furanone 2( 5H)-furanone 5-methyl-2-furancarboxaldehyde cyclohexanone C2-substituted cyclopentene benzofuran M W 128 MW 110 C3-substituted benzene 2-hydroxy-3-methyl-2-cyclopenten1-one phenol C4-substituted benzene C5-substituted benzene M W 124 methylphenol MW 128 M W 132 M W unknown 3-hydroxy-2-methyl-4H-pyran-4-one pentylbenzene MW 148 M W 162 M W 162 M W unknown 1,4:3,6-dianhydro-a-~-glucopyranose hexylbenzene eth y1ph e no1 C6-substituted benzene lH-indole 3-methyl-1H-indole M W unknown M W unknown M W unknown hexadecanenitrile hexadecanoic acid M W unknown M W unknown 9,12-octadecadienoic acid 9-octadecenoic acid
rat food
STS-40 shuttle particles
W
w-m
b w-m w-m w-m vw-w vw vw vw vw vw vw vw vw
W W
vw-w b vw m m vw-w vw vw-w vw-w vw w-m vw b vw vw b
m w-m vw vw vw vw W
vw vw vw vw W
vw vw w-m m vw vw W W
vw w-m vw vw vw
W
vw vw vw vw vw
w-m vw vw vw-w vw
vw vw vw vw
W
vw
W
Summary: 8 1 compounds total; 66 compounds common to both; >80% compounds common to both. These compounds were present in one or two of the three rat food standards. (1
the mass spectra of shuttle particle compounds were then analyzed to match them with spectra of marker compounds. A tentative match was assigned if a set of marker compounds was found in a shuttle particle chromatogram. Next, the remaining compounds in the shuttle particle chromatogram were identified, and a match was confirmed if a high percentage of compounds was common to both the standard and the shuttle particle. A match was considered to be “definite” if the marker compounds, major and minor pyrolysis products, and many of the weak compounds were present in the standard and shuttle particle chromatograms. Using this method, rat food and soilless media particles were identified in the shuttle filter debris. Fifty-eight foodlike particles from the STS-40 filter debris were analyzed in 44 Py-GC/MS runs (in some cases, two or more particles were analyzed at a time); it was estimated that -13 of the 5 8 particles were rat food. Nine particles collected from the STS-42 debris were analyzed in seven Py-GC/MS runs. One soilless media particle was identified. A rat food and soilless media match with STS-40 and STS-42 particles is described below. Rat Food March. The pyrolysis spectrum of particles collected on STS-40 that were identified as rat food is shown
in Figure 2B. The 245-pg sample consisted of three particles from the same vial that looked alike. The marker compounds for rat food shown in the Figure 2A spectrum match those in the unknown sample. However, the shuttle sample seems to have had more cellulose, as indicated by higher peaks of formic acid, l-hydroxy-2-propanone, hydroxypropanone, 2-furancarboxaldehyde, and pyruvic acid methyl ester. Formic acid was detected in two out of the three rat food standards used in this study; pyruvic acid methyl ester was detected in one of the three rat food standards. Moreover, another available rat food standard not used in this study contained these same compounds at levels comparable to those observed in the shuttle sample. Therefore, even though two minor components of the shuttle sample, formic acid and pyruvic acid methyl ester, were not consistently observed in the rat food pyrolysis spectra, additional evidence suggested that the shuttle particles were rat food. Although another shuttle particle had microscopic similarities to rat food, its spectrum in Figure 2C shows that it contained additional compounds, dodecanoic and tetradecanoic acids. These fatty acids, which were moderately strong in intensity, were not detected in any of the rat food standards; therefore, it was concluded that the shuttle particle in Figure 2C did not originate from the rat food. Analytical Chemistry, Vol. 66,No. 18, September 15, 1994
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The results of a mass spectral analysis of all components found in the chromatogram of the shuttle sample (Figure 2B) are listed in Table 2. Rat food components reported in Table 2 were detected consistently in all three standards analyzed for this study. Of the detected pyrolysis products, >80% of the compounds were common to both the rat food standard and the rat food shuttle particles. The 20%of the compounds that did not match were typically very weak in intensity (except for formic acid and pyruvic acid methyl ester, as discussed earlier). Soilless Media Match. A particle from STS-42 (Figure 5B) weighing 46 pg was identified as soilless media when its Py-GC/MS spectrum was compared with that of the reference standard (Figure 5A). In addition to phenol, other pyrolysis products were detected in the shuttle particle spectrum to indicate a match with soilless media. Further mass spectral analysis of the shuttle particle pyrolysis products showed that nearly 70% of the total detected compounds were observed in both the standard and the shuttle particle spectra. A 70% match percentage was also observed among the three soilless media standards. Possible explanations for this lower match percentage could be that soilless medium has a higher degree of heterogeneity or that contaminants were introduced when the soilless medium was mixed with a wetting agent before flight.
CONCLUSION The Py-GC/MS method can be used to identify the source of biological particles for which relatively homogeneous
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Analytical Chemistry, Vol. 66, No. 18, September 15, 1994
standards exist. Particles weighing 40 pg or more can produce identifiable spectra. Marker compounds unique to each standard were selected to aid the identification process because of the large number of common compounds among the standards. Marker compounds proved to be a good indicator of particle identity, since the results of mass spectral analysis of the remaining pyrolysis products confirmed the initial match. By identifying the type of atmospheric particulates in spacecraft environments, the source of potentially hazardous particles can be determined, and subsequently, the effectiveness of animal and experimental enclosure facilities can be evaluated.
ACKNOWLEDGMENT We are grateful for financial support from the Office of Space Science and Applications. We also thank Dr. Louis Ostrach (Lockheed Engineering & Sciences Co., Ames Research Center) for helpful information regarding rat food and feces composition, Ms. Bonnie P. Dalton (Ames Research Center) for supplying the rat food, feces, and bedding standards, and Mr. John L. Frazier (Marshall Space Flight Center) for providing the soilless media standard. Received for review December 16, 1993. 1994.*
Accepted May 20,
Abstract published in Aduance ACS Absrmcrs, July 1, 1994
