758
Anal. Chem. 1983, 55, 758-761
Chemical and Toxicologic Characterization of Fossil Fuel Combustion Product Phenalen-I-one Julie A. Leary, Arthur L. Lafleur, Howard L. Llber, and Klaus Blernann* Department of Chemistry, Department of Nutrition and Food Science, and Energy Laboratory, Massachusetts Institute of Technology, 77 Massachusetts A venue, Cambridge, Massachusetts 02 130
I n the course of anaiysls of the combustlon products of fossil fuels, phenalen-I-one has been identlfled as one of the components. It was dlfferentiated from fluoren-%one and benzo[c]clnnollne, all of which exhlblt very slmllar mass spectra, by gas chromatographic mass spectrometry, hlgh-resolutlon mass spectrometry, and hlgh-performance liquid chromatography wlth hlgh-speed spectrophotometric detectlon. Ultravloiet spectra are reported along with retentlon data for both gas and liquid chromatography. Mutagenlc activlty was determined in Salmonella fyphlmurlum,using resistance to the purine analogue 8-araguanlne as a genetlc marker. Phenalen-I-one was found to be a potent mutagen, while benro[c]clnnoilne was SIXtlmes less active. Fiuoren-9-one was completely inactive as a bacterial mutagen.
The chemical analysis of the combustion products of fossil fuels has become of increasing importance because of the concern that some of the components of these complex mixtures may be toxic, mutagenic, or both. The identification of the constituents is usually accomplished by gas chromatography (GC) either alone or in conjunction with mass spectrometry (GC/MS). Although mass spectra in general contain a high degree of structural information, they often lack the capability to differentiate certain aromatic isomers or, in the case of low-resolution mass spectrometry, even isobars. Fortunately, their GC retention behavior is often sufficiently different to allow identification, if such data are available in the literature or can be determined with an authentic sample. If this is not possible other methods must be employed and we have found a combination of conventional capillary GC-MS, high resolution mass spectrometry (HRMS), and high-performance liquid chromatography coupled to a fast recording UV spectrophotometer (HPLC/UV) to be particularly useful for this purpose, eliminating the need for laborious isolation of the pure component. In this paper we report methodology for the identification of combustion products of fossil fuels employing a combination of techniques rather than GC/MS alone. This is particularly important for compounds which are mass spectrometrically difficult to differentiate. As an example, the unambiguous identification of phenalen-1-one is discussed in detail. This compound is present in the combustion products of No. 2 fuel oil (1) and was subsequently also identified in the same way in the effluent of a fluidized bed combustor burning bituminous coal (2). Phenalen-1-one turned out to be quite mutagenic and may have been previously misidentified as benzo[c]cinnoline ( 3 , 4 ) ,a compound which exhibits a much lower mutagenicity. EXPERIMENTAL SECTION Apparatus. The gas chromatographicmass spectrometer used was a Finnigan-MAT Model 212 double-focusing instrument interfaced to a Varian SS-200 data system running on a Digital Equipment Corp. PDP 11/34A computer. Coupled to the mass spectrometer through a capillary interface was a Varian Model 0003-2700/83/0355-0758$01.50/0
3700 gas chromatograph. It was equipped with a 15-m DB-5 fused silica capillary column with an internal diameter of 0.32 mm and a film thickness of 0.25 pm (J & W Scientific, Rancho Cordova, CA) and an on-column injector. The high-resolution mass spectrometer was a CEC 21-llOB instrument of the Mattauch-Herzogtype. Spectra were recorded on IONOMET evaporated silver bromide plates and measured with a computer-controlled microdensitometer. The liquid chromatographic system employed was a Varian Model 5060 ternary gradient pumping system with a Model UV-1 fixed wavelength (254 nm) detector (Varian/Instrument Group, Palo Alto, CA). The spectrophotometric system consisted of a Hewlett-Packard Model 8450A diode-array UV-Vis spectrophotometer with 32K word memory, Model 7225B graphics plotter, and adjustable flow cell holder (Hewlett-PackardCorp., Palo Alto, CA). The spectrophotometer flow cell had a 1.0 mm diameter circular aperture and a volume of 8 KL(Hellma Cells Inc., Forest Hills, NY).The spectrophotometerflow cell was coupled in series with the fixed wavelength detector. The following HPLC columns were used: (1) For reversed-phase analysis of the chloroform fractions Apex octadecylsilyl-bonded silica columns were employed. The columns had an internal diameter of 4.6 mm and a length of 250 mm. The packing material consisted of spherical totally porous particles 5 pm in diameter. (2) Preparative separations were performed by using a 250-mm column with an internal diameter of 10.0 mm packed with 10-pm Spherisorb ODs. (Both obtained from Jones Chromatography, Columbus OH.) Chemicals. Mobile phases used in the column fractionation of the combustion effluent and in the HPLC analysis were HPLC grade solvents obtained from Burdick and Jackson Laboratories, Muskegon, MI. The neutral aluminum oxide used in the column chromatography (Brockman Activity I, 80-200 mesh) was purchased from Fisher Scientific. Reference samples of benzo[c]cinnoline [230-17-11and phenalen-1-one [548-39-01 (also called perinaphthenone), were obtained from Aldrich Chemical, Milwaukee, WI. Fluoren-9-one [486-25-91was obtained from Chem Service, Inc., West Chester, PA. All chemicals used in preparing the NADPH generating system were purchased from Sigma Chemical Co. The postmitochondrial supernatant (PMS) used in the assay was obtained from Litton Bionetics. (Numbers in brackets are Chemical Abstracts Service Registry Numbers.) Sample Collection and Preparation. The combustion products from No. 2 fuel oil fired in a hot air furnace operating at smoke number 5 (Bachqach scale) were obtained by sampling in an adsorbent collection train equipped with Teflon-coatedglass fiber filters and XAD-2 sorbent resin. The system used was based on a design previously reported (5). The total sampling time was approximately 50 h, after which the filters and XAD-2 resin were extracted with dichloromethane in a Soxhlet extractor. The extracts were evaporated to dryness and weighed. Portions of both the filter (52 mg) and XAD-2 (207 mg) extracts were separately fractionated by gravity column liquid chromatography on neutral alumina according to previously published methods (6, 7) although the procedure employed here varied slightly from those, During elution with chloroform, two fractions of approximately 40 mL each were collected instead of one large 80-mL fraction (X-3(1)and X-3(2) for the XAD-2 extract and F-30) and F-3(2) for the filter extract). The four chloroform fractions were then concentrated by evaporation under nitrogen and analyzed by the methods outlined below. Gas Chromatographic Mass Spectrometry. Approximately 0.5 to 1.0 pL of sample was injected into the GC/MS. The column was held isothermal at 45 OC for 45 s and then programmed at 0 1983 Amerlcan Chemical Soclety
ANALYTICAL CHEMISTRY, VOL. 55, NO. 4, APRIL 1983
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Table I. HRMS Data of Components with Molecular Weight 180 from Fractions 3(1) and 3 (2) fraction
determined mass
calcd mass
difference, mmu
3(1) 3(2)
180.0562 180.0552
180.05752 180.05752 180.06874
2.3 13.5
4 "C/min to 280 "C and held for 2 min. The mass spectrometer operating conditions were as follows: 70 eV electron energy, 4.5 A filament current, 3000 V accelerating voltage, and an ion source temperature of 200 O C . The resolution was 1:lOOOand the range from mlz 50 to 500 was scanned in 2 s with an interscan period of 0.8 8. High-Resolution Mass Spectrometry. The high-resolution instrument was set for 8000 V accelerating potential, 70 eV electron energy, 2.0 mA emission current, and 3.0 A filament current while the ion source temperature was maintained at 200 "C and the resolution 1:12 000. Mass spectra were recorded on vacuum-deposited silver bromide photoplates (IONOMET, Lincoln, MA) for a 1-min exposure and developed photoplates were measured on a computer-controlled microdensitometer. Liquid Chromatography. Analytical scale HPLC was performed with the Apex ODS column described above. The mobile phase consisted of acetonitrile and water at a flow rate of 1.2 mL/min. For the complete HPLC analysis of the chloroform fraction, the following mobile phase program was employed 0-40 min, 50:50 acetonitrile/water; 40-60 min, 50% to 100% acetonitrile; 60-80 min, 100% acetonitrile. The absorbance signal represents the integrated absorbance between 252 and 256 nm. For the comparison of HPLC chromatograms of the standards with the chloroform fraction, the HPLC was operated isocratically at 1.0 mL/min with a mobile phase composition of 1:l acetonitrile/water. The detection parameters were the same as above. Bioassay. The mutagenicity of phenalen-1-one,fluoren-9-one, and benzo[c]cinnoline was determined in S. typhimun'um utilizing 8-azaguanine resistance as the genetic end point (8,9). Frozen aliquots of strain TM677 were grown in minimal media supplemented with 2% brain heart infusion and treated for 2 h at 37 OC in a liquid suspension with both compounds at concentrations of 0-200 pg/mL. This was done in both the presence and absence of a 5% (v/v) aroclor-induced rat liver postmitrochondrial supernatant (PMS). Cultures treated with PMS contained an NADPH generating system. Stock solutions were prepared in MezSO and a 10 fiL aliquot was added to 0.99 mL of bacteria with or without PMS and the NADPH generating system. Bacteria were plated in triplicate on minimal agar plates in the presence or absence of 50 p g / d of 8-azaguanine (8AG),incubated at 37 "C, and counted 2 days later. The number of mutant colonies observed divided by the plating efficiency of the culture times the dilution factor yielded an estimate of the mutant fraction (MF). The operational definition of a bacterial mutagen in this type of assay is a sample which induces a response greater than the 99% upper confidence limit for the background. The level of significance is the concentration at which the dose response curve crosses that limit. The batch of bacteria used in these experiments has an average background of (10.2 i 5.1) X loi, and therefore, the upper 99% confidence limit is 23.5 X 10".
RESULTS AND DISCUSSION In the course of our studies of the chemical identification of combustion products of fossil fuels, we are turning our attention more and more toward heteroatom-containing polyaromatic systems because of the likelihood that they could contribute considerably to the mutagenicity of these products. In the course of that work we have found in a number of sources the presence of two compounds of molecular weight 180 which are well separated by gas chromatography. One elutes before and the other after phenanthrene and both have practically identical mass spectra. From the mass chromatogram of m/z 180, one can conclude that the abundance ratio of these two components is 2:1. Computer comparison of each of the two with the NIH/ EPA collection of 38 000 mass spectra retrieved in both cases
1.3
elemen tal compn C,,H,O C,,H*O C,,H,N,
fluoren-9-one (I) and benzo[c]cinnoline (11) as the two closest matches. The gas chromatographic retention behavior of the
I
63 Ill
first peak agreed with that of I but the value for I1 was not known and therefore this parameter could not be used to confirm the identity of the second component. The presence of I1 could be compatible with the fact that the component in question appeared in the more polar fraction in which oxygen- and nitrogen-containing heterocyclic compounds elute. Furthermore, this compound has been previously reported to be present in the effluent from a domestic oil burner (3) as well as in the extractable portion of diesel particulate matter (4). In an effort to confirm the identity of the earlier eluting compound of molecular weight 180 as I and to definitely identify the later eluting component, a larger sample was obtained by collection of the effluent from a dosmestic oil burner. It was fractionated on alumina by batch elution with hexane, benzene, chloroform, and methanol, respectively. The chloroform fraction which elutes oxygen- and nitrogen-containing aromatic systems was collected in two portions when it was observed that a yellow band migrated down the column with the solvent. When the two filter fractions (3(1) and 3(2)) were subjected to GC/MS, it was found that the component eluting earlier from the gas chromatograph was in the first fraction while the fraction which included the yellow band contained the compound with the longer retention time. Figure 1represents the total ion chromatogram and the mass chromatogram of m / z 180 for fraction 3(1) (top) and 3(2) (bottom). The mass spectra of these two components are practically identical (Figure 2). The presence of a compound containing two nitrogen atoms (such as 11) would be of particular interest because it might imply interaction of molecular nitrogen with a polycyclic aromatic hydrocarbon during combustion. In order to positively confirm or rule out the presence of a nitrogen-containing molecule, high-resolution spectra of fractions 3(1) and 3(2) were obtained. Exact mass measurement (Table I) revealed that both components were of the composition CI3H8O. The CI2H8N2combination was clearly eliminated because the mass calculated for this compound was well outside the limits of error (f0.003 amu) for the experimentally determined value. Since the compound in fraction 3(2) was determined not to be 11, it remained to be determined which isomer of I this compound represented. Clearly, this question could not be answered by either low- or high-resolution mass spectrometry but required a technique than can differentiate isomeric
760
ANALYTICAL CHEMISTRY, VOL. 55, NO. 4, APRIL 1983 I
,-J
0
3
3
TIHE ( . d
Flgure 3. HPLC trace of fraction 3(2) superimposed on that of three
standards: phenalen-1-one, benzene, and floren-9-one.
Flgure 1. Total Ion plots and mass chromatogram of m l r 180 for fraction 3(1) (top) and fraction 3(2) (bottom). 1130
(A) *u
40
T
l
'4
60
Flgure 2. Mass spectra of components with molecular weight 180 (A) from fraction 3(1)and (8)from fraction 3(2).
aromatic systems. For this reason fraction 3(2) was separated by HPLC using a continuously recording UV detector which allowed the acquisition of the complete UV spectrum of this component during the separation. The resulting spectrum was then compared with the UV spectra of compounds of the elemental composition CI3H80, and it was found that it
W
W
I
P
I
P
%
I
&
I
UAYELENGlh ln3
Flgure 4. UV spectra of authentic phenaien-bone superimposed on that of the major component in fraction 3(2).
matched quite closely that of phenalen-1-one (111). An authentic sample of 111, which is commercially available, was acquired, and a mixture of 11,111, and benzene was separated under identical conditions by HPLC and the UV spectra recorded. The resulting chromatogram (Figure 3) is shown superimposed on that of fraction 3(2) demonstrating the same retention behavior of authentic 111. As expected, the UV spectrum recorded for authentic 111 showed a high degree of correspondence with that of the unknown as shown in Figure 4. The identity of the two isomers was also checked by comparing the gas chromatographic retention indexes (10) with those of the compounds present in fractions 3(1) and 3(2). Authentic standards of I, 11, and 111 were obtained and their gas chromatographic retention indexes calculated. Five measurements were made and mean values of 294.20,320.52, and 323.50 obtained with standard deviations of 0.02, 0.07, and 0.10 for I, 111, and 11, respectively. The values for the compounds eluting in fraction 3(1) and 3(2) were 294.41 and 320.53 and therefore matched that of fluoren-9-one and phenalen-1-one. It turns out that the retention indexes of I1 and I11 are quite close and I11 could well be misidentified as I1 unless one coinjecta the standard and sample on a capillary column. Because of this coincidence and the fact that the mass spectrum of I11 is not contained in the NIH/EPA collection of mass spectra, it is not unlikely that the previous identification of I1 ( 3 , 4 )may well have been in error. Since both I1 and I11 are commercially available, coinjection is the simplest
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 4, APRIL 1983
the same 2:l abundance ratio in the corresponding fractions of the XAD-2 extract. In the experiments described in this paper, both compounds appear in both extracts. One has to keep in mind, however, that the distribution of the combustion products between filter and XAD-2 trap depends to a great extent on the temperature at which the fiter is held. Methods are currently being developed to quantitate both compounds at several different temperatures and smoke number.
55c 500 0
2
* 450
0
c0 400 a LL
2 350 2 3
ACKNOWLEDGMENT
1 300
c-
2-
OPEN SYMBOLS : NO PMS CLOSED SYMBOLS’WITH 5 % P M S
250
v)
c W W
761
200
6 z
0 ,0
PHENALENONE
A , A
FLUORENONE BENZO i c i CINNOLINE
0
2 150
,
W LI
For the fuel oil combustion sample, the authors are indebted to William A. Peters who directs the domestic oil burner project with the assitance of Edward Kruzel. We also thank Joan Jackman, Rita Klibanov, and Greg Vasquez for carrying out the mutagenicity tests. Registry No. Fluoren-9-one, 486-25-9; benzo[c]cinnoline, 230-17-1; phenalen-1-one, 548-39-0.
IO0
LITERATURE CITED 50 0 0
IO 20
50
100
CONCENTRATION, pg/rnL
Flgure 5. Mutagenicity data for phenalen-bone, fluoren9-one, and benzo[c]cinnoline wlth and wlthout postmltochondrial supernatant activation.
check to determine which one of the two, or both, are present. The identification of I11 as a combustion product of fossil fuels became even more significant when a sample of I11 was tested for mutagenicity. From Figure 5 it can be seen that I is completely inactive as a bacterial mutagen. I1 is weakly mutagenic in the presence of PMS and inactive in the absence of metabolic activation a t the concentrations tested. I11 is mutagenic both with and without metabolic activation and is clearly more active after metabolism with PMS, since the dose response curve crossed the 99% upper confidence level for the background at 10 bg/mL with PMS and 50 hg/mL without PMS. Although only the chloroform fractions of the filter extract are discussed here, compounds I and I11 were also found in
(1) Leary. J. A,; La Fleur, A. L.; Peters, W. A.; Kruzel, E. L.; Longwell, J. P.; Biemann, K. “Polycycllc Aromatic Hydrocarbons”, 7th Internatlonai Symposlum; Batteile Press: Columbus, OH, In press. (2) Chiu, K. S.; Walsh, P. M.; Bier, J.; Blemann, K. “Polycycilc Aromatic Hydrocarbons”, 7th Internatlonal Symposium; Battelle Press: Columbus, OH, In press. (3) Suprenant, N. F.; Hall, R. R.; McGregor, K. T.; Werner, A. S. “Emissions Assessment of Conventional Stationary Combustion Systems; Volume I . Gas- and Oil-fired Resldential Heating Sources”; U.S. Environmental Protectlon Agency, Report No. EPA-600/7-79-029b, May 1979. (4) Yergey, J. A.; Rlsby, T. H.; Lestz, S. S. Anal. Chem. 1982, 5 4 , 354-357. (5) Strup, P. E.; Giammar, R. D.; Stanford, T. B.; Jones, P. W. Carcinogenesis-A Comprehensive Survey”; Freudenthal, R.I., Jones, P.W., Eds.; Raven Press: New York, 1976 Vol 1, pp 241-251. (6) Schiller, J. E.; Mathiason, D. R. Anal. Chem. 1977, 49, 1225-1228. (7) Later, D. W.; Lee, M. L.; Bartle, K. D.; Kong, R. C.; Vassilaros, D. L. Anal. Chem. 1981, 5 3 , 1612-1620. (8) Skopek, T. R.; Liber, H. L.; Kroiewski, J. J.; Thllly, W. G. Proc. Nafl. Acad. Sci. U . S . A . 1978. 7 5 , 410-414. (9) Skopek, T. R.; Liber, H. L.; Kaden, D. A.; Thilly, W. G. Roc. Natl. Amd. SCI. U . S . A . 1978, 75, 4465-4469. ( I O ) Lee, M. L.; Vassilaros, D. L.; Whlte, C. M.; Novotny, M. Anal. Chem. 1979, 5 1 , 766-773.
RECEIVED for review December 6, 1982. Accepted January 14, 1983.
