Environ. Sci. Technol. 1982, 16, 206-213 Turner, D. B. “Workbook of Atmospheric Dispersion Estimates”; Air Resources Field Research Office, Environmental Science Services Administration, Public Health Service: Cincinatti, OH, 1970; Publication 999-AP-26. Bullin, J. A,; Polasek, J. C.; Green, N. J. “Analytical and Experimental Assessment of Highway Impact on Air Quality”; Texas Transportation Institute, Texas A & M University: College Station, TX, 1979; Research Report 218-5. Cadle, S. H.; Chock, D. P.; Heuss, J. M.; Monson, P. R. “Results of the General Motors Sulfate Dispersion Experiments”; General Motors Corp.: Warren, MI, 1976; Research Publication GMR-2107.
(16) Noll, K. E.; Miller, T. T.; Claggett, M. Atmos. Environ. 1978, 12. 1323.
Received for review February 2,1981. Revised manuscript received October 26,1981. Accepted November 20,1981. This work was sponsored by the Texas State Department of Highways and Public Transportation in cooperation with the United States Department of Transportation, Federal Highway Administration. T h e contents of this paper reflect the views of the authors who are responsible for the facts and accuracy of the data presented herein. T h e contents do not necessarily reflect the official views or policies of the Federal Highway Administration, nor does this paper constitute a standard, specification, or regulation.
Identification of Nitroaromatics in Diesel Exhaust Particulate Using Gas Chromatography/Negative Ion Chemical Ionization Mass Spectrometry and Other Techniques David L. Newton,+Mitchell D. Erickson,” Kenneth B. Tomer,t Edo D. Peilizzarl, and Pamela Gentry5
Analytical Sciences Division, Chemistry and Life Sciences Group, Research Triangle Institute, Research Triangle Park, North Carolina 27709 Roy B. Zweidinger
Mobile Source Emissions Research Branch, Environmental Sciences Research Laboratory, US Environmental Protection Agency, Research Triangle Park, North Carolina 277 11
rn A series of nitroaromatic compounds were identified in diesel exhaust particulate extract. Isomers of nitroanthracene (and/or nitrophenanthrene) aqd nitropyrene (and/or nitrofluoranthene) were unequivocally identified. Alkyl homologues of nitroanthracene through CB-alkylnitroanthracene were tentatively identified, In addition, a ClsHllNOz isomer was tentatively identified. The nitro-substituted polynuclear aromatic hydrocarbons (PAHs) were found in two fractions of diesel exhaust particulate extract collected from a low-pressure liquid chromatography (LPLC) column. One of the two fractions containing nitroaromatic constitutents accounted for a large percentage of the mutagenicity of the crude particulate extract. Initial identifications were made by using highresolution gas chromatography/electron impact mass spectrometry/computer (GC/EIMS) and negative ion chemical ionization mass spectrometry/computer (GC/ NICIMS). These identifications were confirmed by direct probe high-resolutionmass spectrometry (HRMS) and gas chromatography/Fouier transform infrared spectrometry (GC/FT IR). The relative merit of each analytical technique for the determination of nitroaromatics is discussed with emphasis on the usefulness of GC/NICIMS as a means of analyzing for nitro-substituted PAHs.
Introduction A variety of studies of the physical and chemical characteristics of diesel exhaust particulate emissions have been published (1-8). The findings of mutagenicity of diesel exhaust particulate extracts in the Ames microbial assay (9)have generated concern about possible health effects t Present address: Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, NC 27514. * Present address: Analytical Chemistry Department, Midwest Research Institute, 425 Volker Blvd., Kansas City, MO 64113. t Present address: Midwest Center for Mass Spectrometry, Department of Chemistry, University of Nebraska, Lincoln, NE 68588. 5 Present address: Applied Biology, Inc., DeKalb Industrial Way, Decatur, GA 30033.
206
Environ. Sci. Technol., Vol. 16, No. 4, 1982
from exposure to particulate matter generated by diesel engines (1,2, 10-12). The importance of identifying the mutagenic components of the soluble organic compounds in diesel exhaust particulate has led to the separation of extracts into less complex fractions so that analytical techniques can be focused on the more mutagenic fractions. It has been observed that much of the mutagenic activity of diesel particulate extracts is caused by compounds in the moderately polar fractions and not in the polynuclear aromatic fractions (2, 7,8,10-12). Some of the mutagens in these moderately polar fractions have been found to be mutagenic without enzymatic activation (2, 7,8,10-12). Some nitroaromatic species have also been characterized as direct mutagens (2,8,11,13-15). Mutagenic nitrobenzo[a]pyrenes and nitroperylenes have been shown to form when benzo[a]pyrene and perylene are exposed to nitrogen dioxide and nitric acid (13). Nitropyrenes have been identified as direct mutagens in xerographic toners (14, 15). Recently, Pederson and Siak identified l-nitropyrene in a mutagenic fraction of diesel exhaust particulate extract using thin-layer chromatography (TLC) and ultraviolet spectrophotometry (UV) (8). Schuetzle and co-workers identified l-nitropyrene as a mutagenic component of a moderately polar fraction of diesel exhaust by combining techniques of high-performance liquid chromatography (HPLC), HRMS, and GC/EIMS (2). Previous studies in this laboratory indicated that alkyl-9-fluorenoneswere major components of a mutagenic fraction of diesel exhaust particulate extract although no fluorenone derivatives have been shown to exhibit mutagenicity. It was shown in the case of alkyl-9fluorenones that it is sometimes necessary to use multiple analytical techniques to confirm identities of unknowns in complex environmental mixtures. The use of more than one analytical method improves the confidence of identifications of unknowns. Multiple analytical methods have been used again for the analysis of diesel exhaust particulate extract. The
0013-936X/82/0916-0206$01.25/0
(a,
0 1982 American Chemical Society
Table I. Sample Fractionation and Bioassay Data for LPLC of Diesel Exhaust Particulate Extract of Lobar Size C Silica Column estimated revertants/ pg with TA98 straina
fraction
sub fraction
F1 F1A F 1B F1C F1D F1E F1G F2 F3
F4 F5 F6 F7 F8 F9 F10 F11 hexane insoluble
eluting solvent 10% CH,C1,/90% hexane 100% hexane 100%hexane 100%hexane 100% hexane 100% hexane 10%CH2C1,/9O% hexane 10% CH,Cl,/SO% hexane 50% CH,C1,/50% hexane 50% CH,Cl,/BO% hexane 100% CH,Cl, 100% CH,Cl, 10% MeOH/9O% CH,Cl, 10%MeOH/9O% CH,Cl, 50% MeOH/50% CH,Cl, 50% MeOH/50% CH,Cl, 100% MeOH 100% MeOH material insoluble in initial mobile phase of 10%CH,Cl, /90% hexane
whole extract
eluted material, wt %
without metabolic activation
with metabolic activation
50.6 47.1 1.3 1.0 0.8 0.2 0.2 3.3
0 0 0 0.1 +. 0.08 0.2 +. 0.03 0.3 i: 0.3 1.7 -t 0.4 190 f 1 4
0.1 r 0.5 0 0.4 +. 0.1 0.7 t 0.2 0.7 t 0.2 1.1+. 0.5 1.0 f 0.6 21 I2.8
1.6 1.6 0.4 5.9 0.8 9.9 0.5 1.3 0.3 23.7
14.6 +. 1.1 11 +. 1.3 4.9 +. 0.6 1.7 f 0.2 1.6 f 0.1 1.7 t 0.3 1.4 +. 0.2 1.0 t 0.1 1.9 i: 0.5 42 + 4.1
4.4 +. 1.4 3.7 ?r 0.6 3.9 * 0.3 1.2 f 0.2 1.9 f 0.2 1.0 +. 0.2 0.8 f 0.2 0.4 * 0.2 1.1f 0.9 1 6 t 1.6
8.0
i:
0.7
1.7 t 0.7
Estimated values reported i: standard error. Samples assayed in duplicate with at least five concentrations. Negative controls (Me,SO) had a mean of 28 * 4 and 1 4 i: 6 revertantslplate with and without activation, respectively. Positive controls using 2-anthramine had 335 +. 9 3 and 21 +. 4 revertants/pg with and without activation, respectively. When 2nitrofluorene was used, 1.18 i 1 9 revertants/pg were measured without activation. All controls were run in quadruplicate.
purpose of this study was to unequivocably identify nitroaromatics in diesel exhaust particulate extract. Samples were analyzed by using GC/NICIMS, GC/EIMS, HRMS, and GC/FT IR. While the latter three techniques have been used more frequently, the use of NICIMS has become increasingly popular for analyses requiring more selectivity. Bowie and co-workers first studied nitroaromatic compounds by using negative ion MS (16,17).Yinon et al. took advantage of increased sensitivity for nitroaromatics under negative ion MS conditions to monitor trace amounts of trinitrotoluene (18). On the basis of the sensitivity of NICIMS for the molecular ion of nitroaromatics and for the m/z 46 ion characteristic of the NO2 fragment, GC/NICIMS was used to monitor selected fractions of diesel exhaust particulate extract for the presence of nitroaromatics.
Experimental Section Materials. All solvents were “distilled in glass”, purchased from Burdick and Jackson (Muskegon, MI). 9Nitroanthracene was purchased from Aldrich Chemical Co. (Milwaukee, WI). 1-Nitropyrene was provided by the US Environmental Protection Agency (US EPA) (Research Triangle Park, NC). Diesel particulate samples were collected and extracted by Roy B. Zweidinger, Sylvestre B. Tejada, and co-workers at the US EPA (Research Triangle Park, NC). Nitropyrene and dinitropyrene isomers were synthesized by Roy B. Zweidinger at the US EPA. Sample Collection and Fractionation. Diesel exhaust particulate samples were collected on T60 A20 Teflon-impregnated glass-fiber filters (Pallflex Corp., Putnam, CT) from a Nissan light-duty diesel using a chassis dynamometer and dilution tunnel. Details of sample collection qre provided elsewhere (3,12,19). The filters were extracted with methylene chloride in a Soxhlet extractor. The ex-
tract was concentrated to dryness by using nitrogen blowdown and then weighed. The extracted organics were dissolved in 10% methylene chloride/90% hexane and filtered to remove a “hexaneinsoluble” (HI) fraction. The material in solution was injected onto a Lobar size C silica gel column (E. Merck Laboratories, Inc., Darmstadt, Germany) connected to a Fluid Metering Inc. (Oyster Bay, NY) Model RP-54 lowpressure pump operated at a flow rate of 21 mL/min. Fractionation was achieved by using a step gradient beginning with a mobile phase of 10% methylene chloride/90% hexane and progressing to 100% methanol. The various mobile phases used in the step gradient appear in Table I, along with other pertinent data on the fractions collected. Eluants were monitored with a Gilson (Middleton, WI) Model 260 UV detector with a wavelength setting of 254 nm. A total of 11fractions (Fl-F11) were collected from the column, the first of which was refractionated on the same silica column into six subfractions (FlA-FlG). An initial mobile phase of 100% hexane and a final mobile phase of 10% methylene chloride/90% hexane were used for the refractionation of F1. The fractions collected from the LC separation were concentrated to dryness by using rotary evaporation and nitrogen blowdown. Masses were then obtained for the fractions. Samples were dissolved in either hexane or acetonitrile for analysis by GC/NICIMS. All samples were protected from photoxidation by wrapping containers with aluminum foil and working under yellow lights which do not emit radiation below 500 nm. Samples were stored in solution in a freezer until ready for analysis. Bioassay of Fractions. Dried aliquots of the chromatography fractions were packed in dry ice and sent to Stanford Research Institute, Menlo Park, CA, for bioassay in Salmonella typhimurium strain TA98. Using Me2S0 Environ. Sci. Technol., Vol. 16,No. 4, 1982 207
as the solvent, all assays were conducted in duplicate, with and without metabolic activation. Bioassay data were statistically analyzed by using the nonlinear model of Stead et al. (20, 211, which calculates the mutagenicity in revertants/pg with correction for toxicity. Gas Chromatography/Electron Impact Mass Spectrometry. Analyses using a gas chromatograph interfaced with a mass spectrometer were performed on an LKB (Bromma, Sweden) 2091 gas chromatograph/mass spectrometer equipped with a PDP-11 data system. The samples were chromatographed on a 25 m X 0.20 mm i.d. SE-54 fused silica WCOT capillary which was purchased from Applied Science, Inc. (State College, PA). The column temperature was initially 110 "C, temperature programmed at 4 "C/min to a final temperature of 260 "C. The injector and separator temperatures were both 250 "C. A scan interval of 2 s for scans from m/z 40 to m/z 492 was used in the mass-spectrometric analysis. The accelerating voltage was 3500 V, the ionizing energy 70 eV, and the trap current 50 PA. The ion source temperature was 210 "C. The data obtained were permanently stored on magnetic tape. Gas Chromatography/Negative Ion Chemical Ionization Mass Spectrometry. Negative ion chemical ionization mass-spectral analyses were performed on an LKB 2091 GC/MS equipped with the LKB negative ion modification. Methane was used as a moderating gas. Under these conditions, negative ion formation occurs by electron capture of thermal electrons. The moderating gas is used to help create a population of thermal electrons rather than an actual chemical ionization occurring. Gas-chromatographic conditions for GC/NICIMS were the same as for GC/EIMS, as were the scan interval and the source temperature. The box current was 250 pA, and the ionizing energy was 50 eV. High-Resolution Electron Impact Mass Spectrometry. To determine the exact masses of the molecular ions of selected components, we analyzed fraction 2 (F2) by direct probe high-resolution mass spectrometry (HRMS). The direct probe HRMS data were obtained on an A.E.I. (Manchester, England) MS-902 double focusing mass spectrometer operated at a resolution of 15000. Peaks of interest were matched to those of a standard to yield exact masses to f10 ppm accuracy. Possible molecular formulas were calculated on the basis of these exact masses. The direct probe sample was heated to 100 OC. The standard, heptacosafluorotributylamine (Heptacosa) was introduced through the gas inlet system at room temperature. Gas Chromatography/Fourier Transform Infrared Spectroscopy. Samples were analyzed by GC/FT IR using a Varian (Palo Alto, CA) 3700GC with direct injection and thermal conductivity detection interfaced with a Nicolet (Madison, WI) 7199 FT IR spectrometer. The samples were chromatographed on a 16 m X 0.54 mm i.d. WCOT capillary deactivated with BaC03 and coated with Silar 1OC (22,23),except for the 3-nitropyrene standard, which was injected onto a 25 m X 0.32 mm i.d. OV-101 fused silica WCOT capillary purchased from HewlettPackard (Avondale, PA). The initial column temperature was 170 "C, held for 1min, then temperature programmed at 4 "C/min to a final temperature of 270 "C. The column effluent was transferred to the FT IR light pipe through a gold-coated nickel capillary at 250 "C. Other GC/FT IR analytical conditions used are presented elsewhere (7). Interpretation of Mass-Spectral Data. GC/EIMS data were manually interpreted by comparison of unknown spectra with standard reference spectra (24, 25). When 208
Environ. Sci. Technol., Vol. 16, No. 4, 1982
A
7 6 9 75
45
24
I
.
-_
!
125
150
175
M -.
203
Column Temperature 'C
250 260
225
I
5do
1co3
1;03
7 m .
2
h
,
3003
3ico
Time lsec I
Figure 1. Reconstructed ion chromatograms: (A) total ion current of GClEIMS analysis of F2; (8)single ion current ( m l z 46) of GC/NICIMS analysis of F2; (C) total Ion current of GClNICIMS analysis of F2. (Relative intensitles of A-C are not comparable.)
standard spectra were not available, "tentative" identifications were made on the basis of parent ions and fragmentation patterns. When identifications of sample components were made by additional methods such as GC/NICIMS, GC/FT IR, and HRMS, as in the case of nitroanthracene and nitropyrene, identifications were considered to be more certain.
Results and Discussion The LPLC fractionation of the diesel exhaust particulate extract initially produced 11 eluted fractions plus one fraction, labeled hexane insolubles (HIS), consisting of material that would not dissolve in the volume of initial mobile phase injected onto the column. Of the total sample injected onto the column, -98% of the material was recovered. Pertinent data on the various fractions appear in Table I. Bioassay of the diesel exhaust fractions indicated that the majority of the mutagenic activity was associated with the moderately polar fractions F2-F4. The hexane insolubles (HIS)also exhibited a high degree of mutagenicity. The moderately polar fractions F2-F4 were initially investigated by GC/NICIMS. The reconstructed ion chromatograms of F2 are shown in Figure 1. The data from F3 and F4 did not indicate the presence of nitroaromatic species and were of substantially less interest. Several components of F2 were observed with the correct molecular weights for nitroaromatic compounds (see, e.g., Figures 2 4 ) . The NICI mass spectra of authentic samples of 9-nitroanthracene, nitropyrene, and dinitropyrene (isomers unknown) were also obtained. Comparison of the NICI mass spectra and retention times for authentic 9nitroanthracene and nitropyrene with unknowns in Figures 2 and 3 shows excellent agreement, strongly suggesting the presence of nitroanthracene and nitropyrene in F2. Fraction F2 was also analyzed by GC/EIMS. For comparative purposes, the reconstructed ion chromatogram
C
B 80 I
I
I
I
I
i
I
/
I
200
100
1
rnlz rnlz mh Flgure 2. (A) E1 mass spectrum of eluant no. 53, identified as nitroanthracene or nitrophenanthrene isomer (mol wt 223). (6) NICI mass spectrum of eluant no. 53. (C) NICI mass spectrum of 9-nitroanthracene standard.
I-_- _40
100
200 rniz
300
400
33
100
200
300
'
0~ 1
I
I 200
100 rniz
rniz
Flgure 3. (A) E1 mass spectrum of eluant no. 80, identified as nitropyrene or nitrofluoranthene isomer (mol wt 247). (6) NICI mass spectrum of eluant no. 80. (C) NICI mass spectrum of nitropyrene standard.
100
200
300
mlz
33
200
100
300
mlz
Flgure 4. (A) E1 mass spectrum of eluant no. 61, identified as methylnitroanthracene or C15H1,N02isomer (mol wt 237) with background interference from eluant no. 60, tentatlvely identified as a methylanthraldehyde Isomer (mol wt 220). (6) NICI mass spectrum of eluant no. 61.
appears with that obtained by GC/NICIMS in Figure 1. The positive ion spectra of the tentatively identified nitroaromatics appear along with their respective negative ion spectra in Figures 2-6. Comparison of the positive ion spectrum of the unknown nitroanthracene with a standard reference spectrum (24) of 9-nitroanthracene gave excellent
agreement. The positive ion mass spectrum of the eluant tentatively identified as nitrochrysene or a CIBHllNOZ isomer was similar to, though not identical with, a standard reference spectrum (25) for nitrochrysene. No other positive ion reference spectra of nitroaromatics tentatively identified in the samples were available, but molecular Environ. Sci. Technol., Vol. 16, No. 4, 1982
209
_____ Table 11. Compounds Identified in F2 of Diesel Exhaust Particulate Extract by GC/EIMS and GC/NICIMS chromatographic peak no.
210
elution temp, C
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
131 139 147 149 150 151 152 155 158 160 161 162 163 163 164 165 166 169 169 170 170 173 174 175 177 179 180 181 184 184 187 188 190 190 191 192 193 196 197 200 201 20 3 203 205
45 46
208 210
47 48 49 50 51 52
210 210 211 213 213 216
53
217
54 55 56
219 220 221
57 58
221 223
59 60
223 226
61 62 63 64
227 227 228 229
compd siloxane (bkg) unknown unknown unknown naphthaldehyde isomer (tent) naphthaldehyde isomer (tent) unknown unknown 2,2,4-trimethylpenta-l , 3 -diol diisobutyrate (bkg ) unknown methylnaphthaldehyde isomer (tent) methylnaphthaldehyde isomer (tent) methylnaphthaldehyde isomer (tent) methylnaphthaldehyde isomer (tent) methylnaphthaldehyde isomer (tent) unknown unknown unknown anthracene or phenanthrene (trace) unknown phenylbenzaldehyde isomer (tent) C,-alkylnaphthaldehyde isomer (tent) C,-alkylnaphthaldehyde isomer (tent) 9-fluorenone or C,,H,O isomer C,-alkylnaphthaldehyde isomer (tent) unknown unknown unknown methyl-9-fluorenone isomer or C,,H,,O isomer unknown C,-alkylnaphthaldehyde isomer (tent) + unknown methyl-9-fluorenone isomer or C,,H,,O isomer unknown methyl-9-fluorenone isomer or C,,H,,O isomer unknown Cz-alky1-9-fluorenone isomer or C,,H,,O isomer C2-alkyl-9-fluorenoneisomer or C,,H,,O isomer + unknown C2-alkyl-9-fluorenoneisomer or C,,H,,O isomer C2-alkyl-9-fluorenone isomer or C,,H,,O isomer 9,lO-anthraquinone t unknown (trace) unknown Cz-alky1-9-fluorenone isomer or C,,H,,O isomer C3-alkyl-9-fluorenoneisomer or C,,H,,O isomer C3-alkyl-9-fluorenone isomer or C,,H,,O isomer + unknown 4H-cyclopenta [ de f ] phenanthren-4-one (tent) C3-alkyl-9-fluorenone isomer or C,,H,,O isomer + C4-alkyl-9-fluorenone isomer or C,,H,,O isomer t unknown (trace) unknown unknown C,-alkyl-g-fluorenone isomer or C,,H,,O isomer (tent) + unknown pyrene-d,, (int std) C,-alkyl-g-fluorenone isomer or C,,H,,O isomer (tent) t unknown anthraldeh yde isomer + methyl-4H-cyclopen ta [def ]phenanthren4-one isomer (tent) nitroanthracene isomer or nitrophenanthrene isomer + anthraldehyde isomer methyl-4H-cyclopenta [ d e f ] phenanthren-4-one isomer (tent) unknown C4-alkyl-9-fluorenone isomer or C,,H,,O isomer (tent) t C5-alkyl-9-fluorenone isomer or C,,H,,O isomer (tent) C,-alkyl-4H-cyclopenta[ d e f ] phenanthren-4-one isomer (tent) C4-alkyl-9-fluorenoneisomer or C,,H,,O isomer (tent) + C5-alkyl-9-fluorenone isomer or C,,H,,O isomer (tent) + unknown (trace) C,-alkylnaphthaldehyde isomer (tent) methylanthraldehyde isomer (tent) + C,-alkylnaphthaldehyde isomer (tent) methylnitroanthracene isomer or C,,H,,NO, isomer (tent) methylnitroanthracene isomer or C,,H,,NO, isomer (tent) methylnitroanthracene isomer or C,,H,,NO, isomer (tent) C2-alkyl-4~-cyclopenta [ d e f ] phenanthren-4-one isomer (tent) t methvlnitroanthracene isomer or C,.H,, . . ." ..NO,- isomer (tent)
Envlron. Scl. Technol., Vol. 16, No. 4, 1982
EIMS'
+ + + + + + t + + + + + + + + + t + + + + + + + + + + t + + t + t + + + + + + + + + t
-
+ + -
+ + + + + + t
+
+ + t
-
+ + + t
-
t
-
NICIMS'
Table I1 (Continued) chromatographic peak no,
a
+
elution temp, "C
EIMSa NICIMSa
compd
65 66 67 68 69 70
230 231 234 237 237 240
unknown C2-alkyl-4H-cyclopenta[def] phenanthren-4-one isomer (tent) unknown benzanthrone isomer benzanthrone isomer (tent) benzanthrone isomer + C,-alkylnitroanthracene isomer (tent) + unknown
71 72
242 246
benzofluorenone isomer (tent) C,,H,,O ketone isomer (tent) + unknown (trace)
73 74 75 76 77 78 79 80 81 82 83 84 85 86
246 247 248 248 252 255 257 260 260 260 260 260 260 260
87 88 89 90 91 92 93 94 95 96 97 98 99 100 101
260 260 260 260 260 260 260 260 260 260 260 260 260 260 260
unknown benzofluorenone isomer (tent) + C,,H,,O isomer (tent) bis(ethylhexy1) phthalate isomer (tent) (bkg?) C,,H,,O isomer (tent) unknown C,,H,,O, dione isomer (tent) C,,H,,O, dione isomer (tent) nitropyrene isomer or nitrofluoranthene isomer C,,H,,O isomer (tent) GH-benzo[cd] pyrenone isomer o r C,,H,,O isomer (tent) 6H-benzo [ c d ] pyrenone isomer or C,,H,,O isomer (tent) GH-benzo[cd] pyrenone isomer or C,,H,,O isomer (tent) methylnitropyrene isomer or C,,H,,NO, isomer (tent) nitrochrysene isomer or C,,H,,NO, isomer (tent) + unknown, m / z 299 unknown, mlz 256 unknown, mlz 280 C,,H,,O ketone isomer (tent) unknown, mlz 280 unknown, mlz 294 (trace) unknown unknown, mlz 282 unknown, mlz 308 unknown, mlz 306 unknown, m / z 306 unknown, mlz 280 unknown, mlz 280 unknown, mlz 278 unknown, mlz 278 unknown
-+
+ + + + '+ + + + t + + + + t + + + + + + + + + + + + + + + + + + +
-
4-
= identified; - = not identified.
80
80
E60
.-c Em
.-c
-Bal
-BalC
C
m -g40
-8 -m 40
20
20
E
CE
0 40
100
200 rnh
300
0 33
100
200
300
mh
Figure 5. (A) E1 mass spectrum of eluant no. 85, tentatively identified as methylnitropyrene or CI7HllNO2 isomer (mol wt 261) with background Interference from eluant no. 84, tentatively identified as 6H-benzo[cd]pyrenone Isomer or CI8Hl00 isomer (mol wt 254). (B) NICI mass spectrum of eluant no. 85 with background interference from eluant no. 84.
Envlron. Sci. Technol., Vol. 16, No. 4, 1982
211
A
' L 80
100
200
300
mlz
mlz
Flgure 6. (A) E1 mass spectrum of eluant no. 86, tentatively identified as nttrochrysene or C,8HllN02 isomer (mol wt 273). (B)NICI mass spectrum of eluant no. 86, tentatively identified as CI8Hl1NO2 isomer (mol wt 273) along with unknown eluant at mlz 299 not apparent in E1 mass spectrum.
weights and fragmentation patterns of remaining unknowns often permitted tentative identifications. A comparison of negative ion spectra and positive ion spectra in Figures 2-6 and a comparison of the two reconstructed ion chromatograms illustrate several advantages of NICI mass spectrometry over E1 mass spectrometry for an analysis of this type. In the NICI mode, spectra obtained for nitroaromatics have only two major peaks consisting of the molecular ion and the m / z 46 ion (NO2 fragment). In addition, the selectivity of the NICI method eliminates the hydrocarbon background from the spectrum.The NICI spectra of the unknown and the standard, while not identical because of background, are comparable and much easier to interpret than the E1 spectra. The E1 mass spectra of diesel exhaust components nearly always have interfering peaks in the lower mass regions. The compounds identified in F2 by GC/EIMS and GC/NICIMS are listed in Table 11. The compounds identified included 16 aldehydes, 38 ketones, 3 diketones, 1 polynuclear aromatic, and 9 nitroaromatics. Fraction F1G was also analyzed by GC/NICIMS and GC/EIMS. A homologous series of alkylnitroanthracenes (or alkylnitrophenanthrenes) were the major component of this fraction. The bioassay data indicated that F1G did not contribute significantly to the overall mutagenicity of the sample. HRMS analysis of F2 by direct probe techniques indicated the presence of two compounds with the same molecular weights as nitroanthracene and nitropyrene. A measured mass of 247.0637 was obtained as compared to a calculated mass of 247.0633 for CI6HgNO2(nitropyrene). A measured mass of 223.0630 was obtained as compared to a calculated mass of 223.0632 for C14H9N02(nitroanthracene). Fraction F2 was analyzed by GC/FT IR also. For comparison with unknown eluants, standard GC/FT IR spectra of 9-nitroanthracene and 1-nitropyrene were also obtained. The standard spectra and the unknown spectra are compared in Figures 7 and 8. As can be seen in the superimposed FT IR spectra, major absorbance peaks occur a t identical wavenumbers (f4 cm-l) for standards and unknowns. Of the complementary analytical techniques employed, GC/NICIMS was found to be the most useful for detecting 212
Environ. Scl. Technol., Vol. 16, No. 4, 1982
I
P
m 0 N
Figure 7. Comparison of GC/FTIR spectra of (A) compound in F2 identifled as nitroanthracene or nitrophenanthrene isomer and (6)9nitroanthracene standard. I
A
r 0
l O h b d
32b0
'28bO
2kbO ZObO WAVENUMBERS
l6bO
I2bO
BbO
Figure 8. Comparison of GC/FTIR spectra of (A) compound in F2 identifled as nitropyrene or nitrofluoranthene isomer and (6)3-nitropyrene standard.
nitroaromatic compounds in a complex sample. The superiority of the NICI technique over the other methods lies in its selectivity. Characteristic spectra can be obtained for nitroaromatic compounds by using GC/NICIMS, while interfering hydrocarbons and other coeluanta which do not undergo electron capture are eliminated from the mass spectrum. GC/EIMS is useful primarily because of elucidation of structures through fragmentation patterns and because of the many standard reference spectra available for comparison with unknowns (24,25). The problem with using GC/EIMS for identification of nitroaromatics in complex samples is that coeluting components may interfere with the identification of nitro compounds present in trace quantities. Comparison of nitroaromatics identified in F2 by GC/NICIMS and GC/EIMS in Table I1 points out that some nitro components were completely masked by coeluants when using GC/EIMS. When dealing with complex mixtures, direct probe HRMS is useful only as a tool for confirming previous identifications obtained by other analytical techniques. Many possible formulas of molecules and fragments can often be calculated for a single high-resolution peak, any of which is plausible in a given sample. GC/FT IR was found to be especially useful for identifying functional groups present in eluting components. GC/FT IR analysis alone was less practical for these analyses, mainly because of its lower sensitivity. Column overloading was often necessary to provide a sufficient quantity of compounds for detection. There are several general problems encountered in the analysis of diesel exhaust particulate fractions by the methods used in this study. First, the analytical techniques employed provided little capability for differentiating among structural isomers. Since structural isomerism can have a major effect on mutagenicity, methods need to be developed which are capable of distinguishing between isomers of trace organics in complex mixtures. Conclusions
Nitropyrene and nitroanthracene isomers were identified in diesel exhaust particulate extracts with a high degree of certainty, although isomers of nitrofluoranthene and nitrophenanthrene are also possibilities. While there is little doubt as to the presence of nitroaromatics in diesel exhaust particulate extracts, the possibility that they are artifacts of the process of collection on filters has not been eliminated. Further investigations in this area need to be conducted. GC/NICIMS was found to be a useful technique for monitoring complex mixtures for the presence of nitroaromatics. Multiple complementary analytical techniques were found to be useful in verifying initial identifications, especially in cases where standards were not available for comparison to unknowns. Acknowledgments
We thank Sylvestre Tejada and Ronald Bradow of EPA for helpful comments. Fred Williams of RTI is thanked for HRMS analysis. SRI International is thanked for performing bioassay on the diesel exhaust particulate samples. Literature Cited (1) Lipkea, W. H.; Johnson, J. H.; Vuk, C. T. “The Physical and Chemical Character of Diesel Particulate Emissions-Measurement Techniques and Fundamental Considerations”, SAE Special Publication 430; Society of
Automotive Engineers Inc.: Warrendale, PA, 1979. (2) Schuetzle, D.; Lee, J. S.-C.; Prater, T. J.; Tejada, S. B. Znt. J . Environ. Anal. Chem. 1981,9, 93-144. (3) Hare, C. T.; Springer, K. J.; Bradow, R. T. “Fuel and Additive Effects on Diesel Particulate Development and Demonstration of Methodology”, SAE Technical Paper Series No. 760130; Society of Automotive Engineers, Inc.: Warrendale, PA, 1976. (4) Hare, C. T.; Bradow, R. T. “Characterization of Heavy-Duty Diesel Gaseous and Particulate Emissions and Effects of Fuel Composition”, SAE Technical Paper Series No. 790490, Society of Automotive Engineers, Inc.: Warrendale, PA, 1979. (5) Funkenbusch, E. F.; Leddy, D. G.; Johnson, J. H. “The Characterization of the Soluble Organic Fraction of Diesel Particulate Matter”, SAE Technical Paper Series No. 790418; Society of Automotive Engineers, Inc.: Warrendale, PA, 1979. (6) Karasek, F. W.; Smythe, R. J.; Laub, R. J. J. Chromatogr. 1974,101, 125-36. (7) Erickson, M. D.; Newton, D. L.; Pellizzari, E. D.; Tomer, K. B.; Dropkin, D. J. Chromatogr. Sei. 1979,17,449-54. (8) Pederson, L. J.; Siak, J.-S. “Characterization of Direct Acting Mutagens in Diesel Exhaust Particulates by Thin Layer Chromatography”, presented at the Division of Environmental Chemistry of the American Chemical Society, Las Vegas, NV, Aug 1980. (9) Ames, B. N.; McCann, J.; Yamasaki, E. Mutat. Res. 1975, 31, 347-64. (10) Barth, D. S.; Blacker, S. M. J. Air Pollut. Control Assoc. 1978, 28, 769. (11) Wei, E. T.; Wang, Y. Y.; Rappaport, S. M. J . Air Pollut. Control Assoc. 1980, 30, 267-71. (12) Huisingh, J.; Bradow, R.; Jungers, R.; Claxton, L.; Zweidinger, R. B.; Tejada, S.; Bumgarner, J.; Duffield, F.; Waters, M.; Simmon, V. F.; et. al. In “Application of Short-Term Bioassays in the Fractionation and Analysis of Complex Environmental Mixtures”; Waters, M. D., Nesnow, S., Huisingh, J. L., Sandhu, S. S., Claxton, L., Eds.; Plenum Press: New York, 1978; pp 381-418. (13) Pitts, J. N.; Van Cauwenberghe, K. A.; Grosjean, D.; Schmid, J. P.; Fitz, D. R.; Belser, W. L.; Knudson, G. B.; Hynds, P. M. Science 1978,202, 515-9. (14) Lofroth, G.: Hefner. E.: Alfheim. I.: Merller. M. Science 1980. 209, 1037-9. Rosenkranz, H. S.; McCoy, E. C.; Sanders, D. R.; Butler, M.; Kiriazides, D. K.; Mermelstein, R. Science 1980,209, 1039-43. Bowie, J. H. Org. Mass Spectrom. 1971, 5 , 945. Blumenthal, T.; Bowie, J. H. Aust. J. Chem. 1971,24,1853. Yinon, Y.; Boettger, H. G.; Weber, W. P. Anal. Chem. 1972, 44, 2235-7. Hare, C. T. “Characterization of Diesel Gaseous and Particulate Emissions”, Final Report on EPA Contract No. 68-02-1777, Sept 1977. Stead, A.; Hasselblad, V.; Creason, J.; Claxton, L. Mutat. Res. 1981, 85, 13-27. Myers, L.; Sexton, N.; Southerland, L.; Wolff, T. Environ. Mutagenesis, in press. Grob, K.; Grob, G.; Grob, K., Jr. Chromatographia 1977, IO, 181-7. Bouche, J.; Verzele, M. J . Gas Chromatogr. 1968,6, 501. Stenhagen, E.; Abrahamsson, S.; McLafferty, F. W. “Registry of Mass Spectral Data”; Wiley: New York, 1974; VO~.1-4. ”Eight Peak Index of Mass Spectra”, compiled by Mass Spectrometry Data Centre in Collaboration with IC1 Ltd., 2nd ed.; AWRE: Aldermaston, Reading RG 7/4PR, United Kingdom, 1970; Vol. 1-3.
Received for review April 4, 1981. Revised manuscript received September 23,1981. Accepted November 19, 1981. The work upon which this publication is based was performed pursuant to Contract No. 68-02-2767 with the US Environmental Protection Agency. Environ. Sci. Technol., Vol. 16, No. 4, 1982
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