Environ. Sci. Technol. 1991, 2 5 , 342-346
sampling site and time. It is conceivable that in pristine atmospheres or in relatively unpolluted air masses the problems may not be as severe. Some test procedure should nevertheless be devised to safeguard the reliability of the measurement data.
(9) Calvert, J. G.; Lazrus, A. L.; Kok, G. L.; Heikes, B. G.; Walega, J. G.; Lind, J. A.; Cantrell, C. A. Nature 1985,317, 27. (10) Tanner, R. L.; Markovits, G. Y.; Ferreri, E. M.; Kelly, T. J. Anal. Chem. 1986, 58, 1857. (11) Jacob, P.; Tavares, T. M.; Klockow, D. Fresenius 2. Anal. Chem. 1986, 325, 359. (12) Slemr, R.; Harris, G. W.; Hastie, D. R.; Mackay, G. I.; Schiff, H. I. J . Geophys. Res. D 1986, 91, 5371. (13) Hartkamp, H.; Blachhausen, P. Atmos. Enuiron. 1987,21, 2207. (14) Kleindienst, T. E.; Shepson, P. B.; Hodges, D. N.; Nero, C. M.; Arnts, R. R.; Dasgupta, P. K.; Hwang, H.; Kok, G. L.; Lind, J. A.; Lazrus, A. L.; Mackay, G. I.; Mayne, L. K.; Schiff, H. I. Enuiron. Sci. Technol. 1988, 22, 55. (15) Heikes, B. G.; Walega, J. G.; Kok, G. L.; Lind, J. A.; Lazrus, A. L. Global Biogeochem. Cycles 1988,2, 57. (16) MacKay, G. I.; Mayne, L. K.; Schiff, H. I. Aerosol Sci. Technol. 1990, 12, 56. (17) Sakugawa, H.; Kaplan, I. R. Aerosol Sci. Technol. 1990,12, 77. (18) Tanner, R. L.; Shen, J. Aerosol Sci. Technol. 1990,12,86. (19) Dasgupta, P. K.; Dong, S.; Hwang, H. Aerosol Sci. Technol. 1990, 12, 98. (20) Lazrus, A. L.; Kok, G. L.; Lind, J. A.; Gitlin, S. N.; Heikes, B. G.; Shetter, R. E. Anal. Chem. 1986, 58, 594. (21) Dasgupta, P. K.; Hwang, H. Anal. Chem. 1985, 57, 1009. (22) Lind, J. A.; Kok, G. L. J . Geophys. Res. D 1986,91, 7889.
Acknowledgments We are indebted to D. F. Leahy for his valuable assistance and gratefully acknowledge the receipt of meterological data from M. J. Leach. Many helpful discussions, suggestions, and encouragements expressed by P. H. Daum, R. F. Fajer, L. Newman, and J. M. Roberts throughout the work are also deeply appreciated. Registry No. HzOz,7722-84-1; HF, 7664-39-3; Teflon, 900284-0.
Literature Cited (1) Crutzen, P. J.; Fishman, J. Geophys. Res. Lett. 1977,4,321. (2) Logan, J. A.; Prather, M. J.; Wofsy, S. C.; McElroy, M. B. J . Geophys. Res. C 1981,86, 7210. (3) Kleinman, L. I. J . Geophys. Res. D 1986, 91, 10889. (4) Hoffmann, M. R.; Edwards, J. 0. J . Phys. Chem. 1975, 79, 2096. (5) Penkett, S. A.; Jones, B. M.; Brice, K. A.; Eggleton, A. E. J. Atmos. Enuiron. 1979, 13, 123. (6) Moller, D. Atmos. Enuiron. 1980, 14, 1067. (7) Martin, L. R.; Damschen, D. E. Atmos. Enuiron. 1981,15, 1615. (8) Kunen, S. M.; Lazrus, A. L.; Kok, G. L.; Heikes, B. G. J . Ceophys. Res. C 1983, 88, 3671.
Received for review March 19, 1990. Revised manuscript received August 7, 1990. Accepted September 18, 2990. This research was performed under the auspices of the U.S. Department of Energy under Contract DE-ACO2-76CH00016.
Chromatographic Separation and Determination of Carcinogenic Benz[ clacridines in Creosote Oils Noboru Motohashl,
*st
Kunlhlro Kamata,' and Roger Meyer§
Meiji College of Pharmacy, Yato-cho, Tanashi-shi, Tokyo 188, Japan, Metropolitan Research Laboratory of Public Health, 24-1, Hyakunincho 3-chome, Shinjuku-ku, Tokyo 169, and Herbert Laboratories, 2525 Dupont Drive, Irvine, California 927 15
A separation and detection scheme has been developed for the unambiguous identification of benz[c]acridines and their methyl-substituted congeners (BAcs) found in creosote oils. After selective enrichment (including liquid/ liquid-acid/base partition, column chromatography on Sephadex LH 20 and ion-exchange chromatography), basic fractions of a creosote oil sample were analysed by highperformance liquid chromatography (HPLC) with fluorescence detection and subsequent capillary gas chromatography with a thermionic specific detector (TSD). A basic fraction was further separated by HPLC and followed by thin-layer chromatography (HPTLC). The subfractions were examined in detail by gas chromatography-mass spectrometry (GC-MS) and fluorescence spectrophotometry. This approach leads to the positive identification of many specific BAcs. The concentrations of identified BAcs (benz[c]acridine, 9-methylbenz[c]acridine, and 10-methylbenz[c]acridine)in creosote oil were at the levels of 7.7, 18.4, and 192.7 pg/g, respectively.
Introduction It is well-known that only a few of the theoretically possible isomers of methyl-substituted benz[c]acridines Meiji College of Pharmacy. Metropolitan Research Laboratory of Public Health. 5 Herbert Laboratories. t
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Environ. Sci. Technol., Vol. 25, No. 2, 1991
(BAcs) exhibit high carcinogenic activity, which has been shown to be related to the location of the methyl substitution (1-5). Recently, there has been renewed interest in the BAcs, with investigations of marine sediments (6) and lake sediments (7)revealing the wide-spread occurrence of BAcs in the biosphere. Environmental risks due to BAcs may result from tobacco smoke ( 8 , 9 ) ,automobile exhaust (10, l l ) , shale oil (12), coal tar (13, 14), coal liquefaction products (15, 161, and petroleum products (17, 18). Numerous studies have been made on the occurrence of BAcs in environmental samples and several separation and identification schemes of these BAcs have been reported. Separation of BAcs has been attempted by paper chromatography (19 ) , thin-layer chromatography (TLC) (20, 23), and conventional liquid chromatography (LC) using either adsorption or ion-exchange packings (8, 17, 18,24-26). Gas chromatography (GC) has also been applied with a flame ionization detector or with a nitrogenspecific detector (6-9, 11, 13-15, 18, 21, 26-29). Various modes of high-performance liquid chromatography (HPLC) have also been used for the separation of BAcs (8,21, 23, 30-32). One of the most challenging problems arising from the analysis of such substances is the identification of individual isomers in the BAc molecules. Creosote oil is a complex mixture of chemicals obtained from the distillation of coal tar. It contains many individual compounds with differing molecular weights and
0013-936X/91/0925-0342$02.50/0
0 1991 American Chemical Society
polarities. The major components contained in the creosote oils are arenes, phenols, and a large variety of carcinogenic and mutagenic azaarenes. The creosote oils are widely used as a wood preservative. Recently, Krone et al. (6) worked on the identification and quantitation of BAcs in creosote oil. Creosote oils were subjected to silica-alumina column chromatography to obtain fractions greatly enriched in BAcs. These were then characterized by GC with nitrogen-specific detection and gas chromatography-mass spectrometry (GC-MS). BAcs have been recognized as major nitrogen bases of creosote oil; however, no unambiguous identification of specific BAcs could be achieved. It has now become clear that we must know more about the composition of BAcs in creosote oil samples because of the mutagenic and carcinogenic properties associated with their continued use. Consequently, this paper describes a more extensive study of the isolation and identification of BAcs in the basic fraction of creosote oil samples. The BAcs were isolated by liquid/liquid extraction, column chromatography on Sephadex LH 20, ion-exchange chromatography and quantified by HPLC with fluorescence detection and capillary GC with a thermionic specific detector (TSD). The application of capillary GC can provide the superior resolution of BACs needed in the presence of more abundant compounds. After separation of the basic fraction of HPLC into 20 subfractions and purification of each subfraction of HPTLC in sequence, each fraction was independently characterized by GC-MS and fluorescence spectrophotometry.
Experimental Section Reagents. Reference compounds were synthesized according to the previous paper (21) with the exception of papaverin, which was obtained from a commercial source (Sigma). Control of Blank. The wood creosote oil used was Japan Pharmacopeia grade (Shiseido Seiyaku Co., Tokyo, Japan). It was prepared with distillation to remove objectionable organics. Sample Preparation. A creosote oil sample (Yoshida Seiyu Co., Tokyo, Japan) (5 g) was added to a 250-mL separatory funnel containing 100 mL of hexane and partitioned twice with 50 mL of methanol-water (4:l). The aqueous layer was discarded. The hexane layer was extracted twice with 15 mL of 50% sulfuric acid solution. The acidic layers were combined, cooled in an ice bath, and neutralized to pH 12 (saturated sodium hydroxide solution). This solution was then back-extracted twice with 50-mL portions of chloroform. The combined chloroform portion was washed with 25 mL of 5% sulfuric acid solution and 100 mL of water, transferred to a round-bottom flask, and evaporated to 5 mL under reduced pressure in a rotary evaporator. After addition of 10 mL of 2-propanol, the solvent mixture was evaporated again to ca. 2 mL. This was transferred to the Sephadex LH 20 (10 g) column. The column was then eluted with 2-propanol, with the first 50 mL discarded and the second 50-mL 2-propanol fraction collected. Following evaporation, the BAcs that eluted from the Sephadex column were transferred in methanol to the cation-exchange column (SP-Sephadex C25, 4 8). After the flask was rinsed with 5 mL of methanol, the extract and rinse were run down the column until the solvents were adsorbed on the gel bed. The column was eluted with 200 mL of methanol, which was discarded. The BAcs were then recovered by elution with 50 mL of buffer solution (mixture of 30 mL of 5 N ammonium chloride, 10 mL of 5 N aqueous ammonia, 10 mL of water, and 50 mL of methanol). This fraction was diluted with
100 mL of water and extractzedtwice with 50-mL portions of chloroform. The chloroform layer was then washed with 100 mL of water and evaporated. Preseparation. The preseparation steps were performed by HPLC and HPTLC. The HPLC consisted of a Jasco pump, a Rheodyne injector, a Jasco column oven, and a Jasco UV spectrophotometer. The separation was achieved with a prepacked Ultron S C18 column (7 pm; 250 X 10 mm i.d.; Sinwakako) a t a column oven temperature of 35 "C. The elution solvent was 70% acetonitrile-water. The eluent was pumped at a flow rate of 3.0 mL/min. Twenty subfractions were collected, concentrated, and redissolved in 100 pL of chloroform. The subfractions were further purified by HPTLC (precoated RP-18) with the developer (80% acetonitrile-chloroform) (22). The zone with the BAc fraction was marked under UV light at 254 nm and scraped from the plate into a small centrifuge tube. The extraction of BAcs was carried out ultrasonically with 2.0 mL of methanol for 10 min. The supernatant was submitted for analysis of the BAcs by fluorescence spectroscopy after centrifuging for 10 min at 3000 rpm. GC. The sample was analyzed by GC using a Varian Model 3600 gas chromatograph equipped with TSD, a fused-silica capillary column (bound OV-1, ca. 25 m X 0.20 mm i.d.). Conditions were as follows: oven temperature 18C-260 "C at 3 "C/min after 5 min of initial hold; detector current 3.2 A, injector temperature 250 "C; split injection; and helium as the carrier gas. GC-MS. The identification of BAcs in the creosote oil were obtained by GC-MS employing a Hewlett-Packard Model 5890 gas chromatograph coupled to a HewlettPackard Model 5970 mass selective detector. The GC column used was the same as with the GC analysis above. Gas chromatographic conditions were as follows: oven temperature 180-260 "C at 2 "C/min after 5 min of initial hold; injector temperature 250 "C; splitless injection; and helium carrier gas. MS operating conditions were as follows: electron impact 70 eV; scan speed 200 amu/s; electron multiplier 2000 V; and scan range of 100-400 m u . HPLC. A TSK gel 120T reversed-phase column (5 pm; 250 X 4.6 mm id.; Toso) was used on the same pump and injector described above. The detector was a Hitachi fluorescence spectrophotometer. The mobile phase consisted of acetonitrile-water (70:30) at a flow rate of 1.0 mL/min. All separations were performed at 35 "C. For purpose of quantitation, the peak height a t fluorescence (excitation X 289 nm, emission X 400 nm) was measured and compared with that of calibration curves obtained from standard mixtures. The calibration curves of BAcs in the range of 0.01-0.1 ng were linear and relative standard deviations (RSD) of peak height for 0.1 ng of BAcs were confirmed within 0.5%.
Results and Discussion The accuracy of the procedure was determined by spiking a control blank with known concentrations of reference BAcs. The recovery of the reference BAcs is present in Table I. This assay for BAcs on GC has been developed by using the internal standard technique. Papaverin was added as an internal standard for quantification. The application of capillary GC can give superior resolution of BAcs in the presence of more abundant compounds. The TSD detection was found to be more sensitive than flame ionization detection by at least a factor of 10. The limit of detection for BAcs was 2 ng at a signal to noise ratio of 2. Recoveries of BAcs averaged 82.3% or better ( n = 3) with a SD within 4.8 for BAc levels of 0.5 pg/g. Environ. Sci. Technol., Vol. 25, No. 2, 1991 343
229
I
I
I
10
0
I
I
1
I
I
I
40
30
20
Retention time (min.) Figure 1. Capillary gas chromatogram of the basic constituents iso-
lated from creosote oil. Peak numbers refer to the compounds identified in Table 11.
Table I. Recovery of Benz[ c ]acridine Standards 30 40 50 Retention time (min.) Flgure 2. Single-ion (GC-MS) chromatograms (mlz 229 and 243) of the basic constituents isolated from creosote oil.
10
compound
replicates
added, wg/g
benz[c]acridine 7-methylbenz[c]acridine 11-methylbenz[c]acridine 7,10-dimethylbenz[c]acridine 7,9,10-trimethylbenz[c]acridine 7,9,11-trimethvlbenzlclacridine
3 3 3 3 3 3
0.5 0.5 0.5 0.5 0.5 0.5
recovery, 86.9 86.8 92.2 85.5 82.3 93.6
%
20
methylbenz[c]acridine (C18H,,N, mlz 243) are isomeric with methyldibenzoquinolines and methylbenzphenanthridine (6). HPLC and HPTLC techniques were very effective in removing interfering compounds for the determination of BAcs by GC. With this approach, after the preseparation procedure of 20 BAc subfractions by HPLC, each fraction was completely separated by HPTLC. Figure 3 shows a typical preseparation chromatogram. Fractions of highly enriched BAcs were obtained in this way and individual components identified in each subfraction by fluorescence spectroscopy. Some of the fluorescence spectra showed the presence of BAcs. The three spectra of the subfractions obtained are very similar to that of BAcs. Figure 4 shows one of them. The spectra of 9-methylbenz[c]acridine and 10methylbenz[c]acridine showed results similar to that of benz[c]acridines. The fluorescence spectrum by HPLC and HPTLC is more accurate. The detected and identified BAcs are listed in Table 11, where molecular weight (M+) data from GC-MS and fluorescence excitation and emission maximum absorption bands of the reference BAcs are also indicated. The ability of HPLC to separate BAcs is important because 9-methylbenz[c]acridine and 10-methylbenz[c]acridine have the same chromatographic behavior on GC.
An exemplary gas chromatogram of the basic components isolated from commercial creosote oil is presented in Figure 1. The peaks were identified by comparing their retention times with reference BAcs and GM-MS. Numbers in Figure 1 correspond to the compounds listed in Table 11. The two molecular weight groups of mlz 229 and 243 as BAcs were present in the chromatogram of the creosote oil. Figure 2 shows the typical single-ion chromatograms for the BAcs ( m l z 229 and 243) in the creosote oil. GC-MS and GC identification of BAcs is complicated by the presence of the same molecular ion and similar chromatographic retention times. The GC analysis showed an overlap of 9-methylbenz[clacridine and 10-methylbenz[c]acridine. These compounds could not be separated by use of other stationary phases (27). In some cases a number of isomeric-type compounds were possible for a given molecular weight; for example, benz[c]acridine (CI7Hl1N,mlz, 229) is isomeric with dibenzoquinolines and benzophenanthridines. Mono-
Table 11. Identified and Detected Benz[c]acridines i n Creosote Oil identification method no. 1 2 3 4
5 6 7 8 9 10
11
12
compound benz[c]acridine 7-methylbenz[c]acridine 8-methylbenz[c]acridine 9-methylbenz[c]acridine
10-methylbenz[c]acridine 11-methylbenz[c]acridine
5,7-dimethylbenz[c]acridine 7,9-dimethylbenz[c]acridine 7,10-dimethylbenz[c]acridine 7,1l-dimethylbenz[c]acridine 7,9,10-trimethylbenz[c]acridine 7,9,11-trimethylbenz[c]acridine
GC
+ -
-
+ + + -
-
GC/MS (M+) + (229) - (243) - (243)
+ (243) + (243) + (243) - (257) - (257) - (257) - (257)
Environ. Sci. Technol., Vol. 25, No. 2, 1991
+ -
-
+ +
-
(271)
-
- (271)
-
-
Maximum excitation wavelength and maximum emission wavelength in MeOH. 344
HPLC
fluorescence0 (Ex A, nm; Em A, nm) + (287; 399) - (288; 400) (288; 401) + (289; 396) + (287; 403) - (288; 395) - (292; 397) - (294; 397) - (291; 404) - (290; 396) - (293; 400) - (294; 393)
concn, p g / g 192.7 7.7 18.4
the creosote oils. These BAc concentrations in the creosote oils were 192.7, 7.7, and 18.4 pg/g, respectively. Dimethylbenz[c]acridine and trimethylbenz[c]acridinewere not found a t detection limits within 0.1 pg/g in creosote oil.
0
20
40
60
80
100
Retention time (min.) 44.5
1
Conclusions The separation scheme presented in this paper is useful in concentrating and isolating the BAcs in creosote oil. The developed cleanup method yields clean extracts for analysis and is compatible with both GC and HPLC analytical procedures. The GC and HPLC chromatograms of BAcs were more clear compared with any known cleanup methods and their recoveries were better (6-9, 13, 18, 25). A combination of GC, HPLC, HPTLC, GC-MS, and fluorescence spectrophotometric methods must be applied before accurate results can be obtained for the routine quantitative determination of BAcs in creosote oil. Three types of BAcs in creosote oil have been characterized, which contained benz[c]acridine, 9-methylbenz[c]acridine, and 10-methylbenz[c]acridine. Registry No. 1, 225-51-4; 2, 3340-94-1; 3, 13911-90-5; 4, 33942-93-7; 5,7230-71-9; 6,67028-20-0; 7,10567-95-0; 8,963-89-3; 9, 2381-40-0; 10, 32740-01-5; 11, 58430-01-6; 12, 51787-42-9.
Literature Cited (1) Laccasage, A.; Buu-Hoi, N. P.; Daudel, R.; Zajdela, F. Adv. Cancer Res. 1956, 4 , 315. (2) Lacassagne, A.; Buu-Hoi, N. P.; Zajdela, F.; Giaao, N. B.;
Jacquignon, P.; Dufour, M. C. R. Acad. Sci. Paris, Ser. D. 0
20
40
60
80
100
Retention time (min.) Figure 3. Liquid chromatograms of benz[c]acridines (top) and prefractionation of the creosote oil (bottom). Peak numbers refer to the compounds identified in Table 11.
1968,267, 981. (3) Liebermann, C.; Lazar, P.; Chouroulinkov, I.; Guerin, M. C. R. Seances SOC.Biol. Ses Fil. 1968, 162, 835. (4) Walker, B. A,; Rogan, E. G.; Cromwell, N. H. Anticancer Res. 1984, 4 , 399.
( 5 ) Papadsopoulo, D.; Levy, S.; Poirier, V.; Pene, C.; Markovits, P.; Hurbert-Habart, M. Eur. J . Cancer 1981, 17, 179. (6) Krone, C. A,; Burrows, D. G.; Robisch, P. A.; Friedman, A. J.; Mallns, D. C. Enuiron. Sci. Technol. 1986, 20, 1144. (7) Wakeham, S. G. Enuiron. Sci. Technol. 1979, 13, 1118. (8) Snook, M. E.; Fortson, P. J.; Cbortyk, 0. T. Beitr. Tabakforsch. Int. 1981, l l , 67. (9) Grimmer, G.; Naujack, K.-W.; Dettbarn, G. Toxicol. Lett. 1987, 35, 117. (10) Handa, T.; Yamauchi, T.; Sawai, K.; Yamamura, T.; Koseki, Y.; Ishii, T. Enuiron. Sci. Technol. 1984, 18, 895. (11) Williams, P. T.; Bartle, K. D.; Andrews, G. E. Fuel 1986, 65, 1150. (12) Pelroy, R. A.; Petersen, M. R. Enuiron. Health Perspect. 1979, 30, 191. (13) Burchill, P.; Herod, A. A.; Mahon, J. P.; Pritchard, E. J . Chromatogr. 1983, 265, 223. (14) Lauer, J. C.; Valles, D. H.; Cagniant, D. Fuel 1988,67,1446. (15) Later, D. W.; Pelroy, R. A,; Mahlum, D. D.; Wright, C. W.;
250
300
350
Wavelength ( n m )
350
400
450
Wavelength (nrn)
Figure 4. Fluorescence excitation (A) and emission (B) spectra of benz[c]acridine standard (solid line) and sample benz[c]acridine (dashed line) Isolated from HPTLC In subfraction d.
HPLC would measure both BAcs. Furthermore, HPLC equipped with a fluorescence detector is the detection method of choice owing to its greater sensitivity than GC. This combination of GC, HPLC, HPTLC, GC-MS, and fluorescence spectrophotometric methods has produced absolute confirmation of the presence of benz[c]acridine, 9-methylbenz[c]acridine,and 10-methylbenz[c]acridinein
Lee, M. L.; Weimer, W. C.; Wilson, B. W. Polynuclear Aromatic Hydrocarbons,International Symposium, 1982, 7th; Battelle Press: Columbus, OH, 1983; p 771. (16) Kershaw, J. R. Fuel 1983, 62, 1430. (17) Mckay, J. F.; Weber, J. H.; Latham, D. R. Anal. Chem. 1976, 48, 891. (18) Grimmer, G.; Naujack, K.-W. Fresenius 2. Anal. Chem. 1985, 321, 27. (19) Caroli, S.; Lederer, M. J . Chromatogr. 1968, 37, 333. (20) Sawicki, E.; Stanley, T. W.; Elbert, W. C. Occup. Health Rev. 1964, 16, 8. (21) Motohashi, N.; Kamata, K. Yukugaku Zasshi 1983, 103, 795. (22) Kamata, K.; Motohashi, N. J. Chromatogr. 1987,396,437. (23) Yamauchi, T.; Handa, T. Enuiron. Sci. Technol. 1987,21, 1177. (24) Sawicki, E.; Meeker, J. E.; Morgan, J. J. Chromatogr. 1965, 17, 252. Environ. Sci. Technol., Vol. 25, No. 2, 1991
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(25) Klimisch, H.-J.; Beiss, A. J . Chromatogr. 1976, 128, 117. (26) Grimmer, G.; Naujack, K.-W. J.-Assoc. Off.Anal. Chem. 1986, 69, 537.
(27) Kamata, K.; Motohashi, N. J . Chromatogr. 1985,319,331. (28) Lee, M. L.; Vassilarros, D. L.; White, C. M.; Novotny, M. Anal. Chem. 1979,51, 768. (29) Schmitter, J. M.; Ignatiadis, I.; Guiochon, G. J. Chromatogr. 1982, 248, 203.
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(30) Colin, H.; Schmitter, J.-H.; Guiochon, G. Anal. Chem. 1981, 53, 625. (31) Siouffi, A. M.; Righezza, M.; Guiochon, G. J. Chromatogr. 1986, 368, 189. (32) Kamata, K.; Motohashi, N. J . Chromatogr. 1990,498, 129.
Received f o r review December 22, 1989. Revised manuscript received June 19, 1990. Accepted August 2, 1990.
