88
ANALYTICAL CHEMISTRY, VOL. 50, NO. 1, JANUARY 1978
Isolation of Indoles and Carbazoles from Cigarette Smoke Condensate M. E. Snook," R. F. Arrendale, H. C. Higman, and 0. T. Chortyk Tobacco Laboratory, Agricultural Research Service, United States Department of Agriculture, P.O. Box 5677, Athens, Georgia 30604
smoke was trapped by dry-ice traps. The procedures for obtaining the organic solubles (Figure 1) and the chromatography on a silicic acid column have been described (15). Briefly, the CSC (from 90 cigarettes) was dissolved in a 600 mL (2:1:2)mixture of benzene (B), methanol (MI, and ethyl ether (E) and the solution was extracted with water (3 X 200 mL). The organic phase was concentrated and the residue adsorbed onto 20 g of silicic acid (100 mesh, Mallinckrodt, washed with methanol, and activated in a forced air oven at 150 "C for 18 h) and the silicic acid-sample mixture was placed on top of a 100-g silicic acid column packed in petroleum ether (PE) (15). The column was eluted with 500 mL of P E (Fraction A), 1 L of B:PE (1:3 v/v) (Fraction B), and 1 L of B:PE (1:l v / v ) (Fraction C). All solvents were Burdick and Jackson Laboratories, glass-distilled type. Large quantities of CSC were prepared similarly at the Roswell Park Memorial Institute, Buffalo, N.Y., and shipped to us packed in dry ice. The organic solubles were obtained from 97 g of this CSC as described above, except volumes of solvent were doubled. One half of the organic solubles was chromatographed on 1000 g of silicic acid and eluted with 7.5 L of P E (Fraction A), 7.5 L of B:PE (1:3 v/v) (Fraction B), and 6 L of B:PE (1:l v/v) (Fraction C). Fraction C weighed 0.7 g or 1.5% of the original CSC. This material was used for identification studies by GC-MS, GC-UV techniques. The gel chromatography system consisted of four 1.25 X 109 cm glass, Cheminert LC columns (Laboratory Data Control, Riviera Beach, Fla.) connected in series and packed with BioBeads SX-12 (a neutral, porous, styrene-divinylbenzene copolymer, M. exclusion 400, Bio-Rad Laboratories, Richmond, Calif.) in benzene to yield a wet gel bed of about 400 cm. The beads were purified by refluxing three times with fresh CH2Cl2, followed by refluxing twice with B. Fraction C was reduced in volume to about 5 mL on a rotary evaporator, and quantitatively transferred with B to a 1-mL volumetric flask. Fraction C was placed on the columns with a 0.9-mL loop injection valve. Benzene was pumped (Chromatronix CMP-3 pump) through the columns at 120 mL/h and 8-mL fractions were collected. The effluent was monitored at 280 nm with a Chromatronix Model 230 dual channel absorbance detector. 2,3,5-Trimethylindole was used to indicate the beginning of the elution of the indoles. Gel fractions were evaporated by a gentle stream of nitrogen and analyzed by GC. Fraction C from the large scale (97 g) fractionation of CSC was diluted to 3 mL with B and three 1-mL portions were chromatographed on the gel columns. Gel fractions with the same number were combined. The gel fractions were analyzed with a Hewlett-Packard 5750 gas chromatograph equipped with a 15 ft X ' / g inch stainless steel column packed with 3% Dexsil 300 GC on 100/120 mesh Chromosorb W-AW (temperature program: 90 "C for 5 min, 9C-325 "C at 2"/min, and constant at 325 O C until all compounds of interest eluted; 40 mL/min He; injector, 290 "C; detector, 350 "C). Compounds were identified by UV spectral analyses of preparative GC samples and by GC-mass spectral (GC-MS) analyses on a Varian 1400 GC instrument interfaced to a DuPont 21-492 mass spectrometer. 'Cindole (specific activity 50 mCi/mmol) was obtained from ICN Pharmaceuticals, Inc., Isotope & Nuclear Division, Irvine, Calif. A 5.00-pg sample of I4C-indole (activity: 4.74 X lo6 dpm) in B was added to CSC from 90 Kentucky 1R1 cigarettes and fractionated as described above. Recovery of radioactivity was determined by standard liquid scintillation counting techniques. The recovery of I4C-indole from the gel filtration step was determined by adding a sample of I4C-indole directly to Fraction C. Fraction C was placed in a 1.0-mL volumetric flask as described
A new method for the isolation of indoles and carbazoles from cigarette smoke condensate (CSC) is described. The four-step procedure involves water extraction, silicic acid chromatography, gel filtration chromatography, and gas chromatography (GC). Silicic acid chromatography effectively separated the indoles and carbazoles from polynuclear aromatic hydrocarbons (PAH), compounds that would interfere In the lndole/carbazole analysis. Gel filtration chromatography on Bio-Beads SX-12, in benzene, produced a relatively pure indole/carbazole isolate by an adsorption phenomena similar to that reported for PAH. The indole/carbazole isolate was sufficiently refined for identification of its components by GCUV and GC-mass spectrometry. Carbazole was quantitatively recovered by the method. The identification of all three possible isomers of benzocarbazole and their alkyl derivatives in cigarette smoke is reported for the first time. The method should be applicable to the analysis of indoles and carbazoles formed by other combustion processes in the environment.
Indoles and carbazoles are present in cigarette smoke condensate (CSC) (1-3). Several members of this class of compounds possess biological activity ( 4 ) ,and consequently their identification in CSC is of importance. Until now, carbazoles and indoles have generally been isolated in the same CSC subfraction as t h e polynuclear aromatic hydrocarbons (PAH). Also, since indoles and carbazoles occur in amounts equal t o or greater than those of the PAH, it is important that they be cleanly separated from t h e P A H in order t o analyze for both classes of biologically active compounds. This is especially true for gas chromatographic (GC) analysis of the PAH, where t h e indoles and carbazoles are found t o co-elute with many PAH. Recent procedures for the isolation of P A H from CSC result in either t h e presence of both classes of compounds in the final isolate [e.g., the Rosen method (5, 611 or in the nonreproducible recovery of indoles and carbazoles (7). Hoffmann and co-workers have published procedures for the isolation and quantitation of indoles ( 4 9 ) and carbazoles ( I O , 1 1 ) from CSC by methanol/water extractions and Florisil chromatography. Although Hoffmann e t al. reported a n 85-9070 recovery of indoles from CSC, application of their procedure t o t h e similar smoke condensate from marijuana resulted in only a 30% recovery (12). W e now describe a four-step method for t h e isolation of indoles a n d carbazoles from CSC. T h e method effectively separated t h e indoles a n d carbazoles from t h e P A H and allowed both classes of compounds t o be analyzed. T h e method was quantitative for carbazole and yielded alkylated indoles a n d carbazoles which are lost by other methods (8, 10).
EXPERIMENTAL CSC was prepared from 90 experimental cigarettes (University of Kentucky, type 1R1). The cigarettes were conditioned at 6070 relative humidity for 48 h and smoked under standard conditions: 2-s puff, 1 puff/min, 35-mL draw/puff, 23-mm butt length (13, 14). A Borgwaldt 30-port smoking machine was used and the 0003-2700/78/0350-0088$01 OO/O
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1977 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 50, NO. 1, JANUARY csc i n B:M:E
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Organic Solubles ( 50%)
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Silicic Acid
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C
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Figure 1. Fractionation of cigarette smoke condensate (CSC) to obtain polynuclear aromatic hydrocarbons (PAH isolate) and indoles/carbazoles (sp3-N-PAH isolate) above and evaporated to about 0.4 mL under nitrogen. One-half mL of 14C-indole(activity: 900 000 dpm/mL) was added and the flask made to volume. The sample was chromatographed on the gels as above. The gel fractions corresponding to the elution of indole were collected and analyzed for radioactivity. Carba~ole-5,6,7,8,12,13-'~C was synthesized from ring-uniformly-labeled ''C-phenylhydrazine hydrochloride (ICN Pharmaceuticals, Inc., Isotope & Nuclear Division, Irvine, Calif.) and cyclohexanone by the method of Hoffmann et al. (IO)as modified by Haq et al. (12). The 14C-carbazolewas purified by silicic acid chromatography. The crude product was placed on a 100 g silicic acid column, slurry packed in PE. The column was eluted with 1 L of B:PE (1:3 v/v) and 1 L of B:PE (1:l v/v). A UV spectrum of the 50% B:PE eluate confirmed the presence of carbazole. The residue from the 50% B:PE fraction was dissolved in B and chromatographed on the gel columns as described above. (Previously, nonradioactive carbazole was shown to be eluted in gel fractions 44-50). Gel fractions 45-50 from the 14C-carbazole gel run were combined and analyzed by UV, which confirmed the purity of the 14C-carbazole. A sample of 14C-carbazole (30 mL of a B solution; activity: 604496 dpm) was added to the smoke condensate from 90 Kentucky 1R1 cigarettes and fractionated as described above. Recovery of radioactivity was determined by liquid scintillation counting techniques.
RESULTS AND DISCUSSION T h e present method for t h e isolation of indoles and carbazoles from CSC evolved from our recently developed method for t h e quantitation of PAH in CSC (Figure 1) (15). Smoke P A H were isolated from the organic-soluble portion of CSC by silicic acid and gel filtration chromatography. Radioactive tracer studies with ''C-BaP and I4C-anthracene showed t h a t t h e P A H were quantitatively removed from t h e silicic acid with a 25% B:PE solvent system (15). Gel filtration chromatography of Fraction B o n Bio-Beads SX-12 produced a P A H isolate (Figure 1) of unusual purity, suitable for identification and quantitation. Identification studies on the PAH isolate showed t h a t only minor amounts of oxygen analogues (15, 16) (benzo[b]furans, dibenzofurans, a n d naphthobenzofurans) of t h e P A H were present. Preliminary examination of Fraction C indicated the presence of nitrogen analogues of the PAH, specifically, indoles and carbazoles (for convenience designated sp3-N-PAH in Figure 1 and Table 11). I t was decided t o characterize these N-PAH by t h e same approach employed for t h e PAH. Fraction C was separated by gel filtration chromatography on Bio-Beads SX-12 in benzene on the same gel system used for t h e separation of t h e P A H fraction. T h e characteristics of the resulting gel chromatographic separation are shown in Figure 2. T h e elution (curve 1)of 2,3,5-trimethylindole, one of t h e earliest-eluting indoles, was used to mark the start of t h e indole-carbazole fraction. In a manner similar to t h a t found for the PAH (17),indoles and carbazoles were adsorbed
~~
Table I. Indole/Carbazole Identifications Peak NO.^ 1
2 3 4, 5 6 7,8 9-1 1
12 13, 1 4 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 a Peak ponent.
Compoundb Indole Skatole 3-Ethylindole Dimethylindole Propylindole Ethylmethylindole Trimethylindole Propylmethylindole Ethyldimethylindole Tetrame thylindole Tetramethylindole/propyldimethylindole
Propylethylindole Carbazole 1-Methylcarbazole 3-Methylcarbazole 2-Methylcarbazole 4-Methylcarbazole Ethylcarbazole Dimethylcarbazole Dimethylcarbazole/ 3-phenylindole Dimethylcarbazole Dimethylcarbazole Dimethylcarbazole/trimethylcarbazole
Trimeth ylcarbazole Trimethylcarbazole/methylphenylindole Trimethylcarbazole/tetramethylcarbazole
Benzo[ a ]carbazole Benzo[ blcarbazole Benzo[ c ]carbazole Methylbenzocarbazole Dimethylbenzocarbazole numbers refer t o Figure :3.
Major corn-
by the Bio-Bead gels and eluted after t h e majority of t h e weight of Fraction C. T h e elution of trimethylindole before indole illustrates the so-called "methyl effect", where increasing numbers of methyl or alkyl groups facilitate earlier elutions (18). This methyl effect on elution produced the two maxima in the 280-nm elution curve in gel fraction (GF) 43-50. T h e first maximum was due to multialkylated indoles and carbazoles; the second was due almost entirely to the elution of the more abundant parent compounds indole and carbazole a n d skatole. Subsequent identification studies on constituents of gel fractions 41-55 were conducted by GC-UV and GC-MS. T h e major components identified are given in Table I and t h e resulting chromatogram of combined G F 41-56 is shown in Figure 3. Identifications were achieved by a correlation of UV spectra of preparative GC peaks and GC-MS data.
90
ANALYTICAL CHEMISTRY, VOL. 50, NO. 1, JANUARY 1978
-____ 10
60
40
TlYl
(-8")
10
100
110
Figure 3. Gas chromatogram of t h e indoles and carbazoles in cigarette smoke condensate. (Peak identities are given in Table I )
Table 11. Recovery "C-Indole and Carbazole
Fraction
csc
a
Percent "C activity recovered Indole Carbazole 100 100
Organic solubles 97.9 Fraction A ... Fraction B 0.2 Fraction C 65.7 sp3-N-PAHIsolate ... 14C-indoleadded to Fraction C.
... I
100a 98.3a
.
.
0 99.0 98.5
Specific notice is directed t o the successful isolation of multialkylated indoles and carbazoles by the present method. Previous isolation methods for indoles (8) and carbazoles ( I O ) apparently failed to recover these compounds, possibly because of unfavorable partitioning coefficients. Also, benzocarbazoles and their methyl and dimethyl derivatives were found in the gel fractions. All three possible isomers of benzocarbazole (a, b, a n d c isomers) were detected. T o our knowledge, the benzocarbazoles have not been reported in smoke previous t o this report ( 3 ) . T h e quantitative aspects of t h e isolation method were determined with 14C-indole and 14C-carbazole (Table 11). 14C-carbazole was synthesized from 14C-labeled phenylhydrazine hydrochloride and cyclohexanone ( I 0,12). Samples of ''C-carbazole were added t o t h e CSC of 90 research cigarettes and t h e CSC was submitted to the described isolation scheme. Percent recovery of radioactivity was determined by standard liquid scintillation counting methods and the results are given in Table 11. Carbazole was quantitatively recovered through the entire isolation procedure. However, a significant amount of indole was lost by silicic acid chromatography. Apparently, indole underwent oxidation on silicic acid because the remaining radioactivity could be recovered by elution of t h e column with more polar solvents, such as ether or methanol. T o determine the extent of losses in the silicic acid step, a mixture of equal amounts of unlabeled indole, skatole, and carbazole (each purified by recrystallization from ethanol) was chromatographed on silicic acid. T h e amounts of recovered indole a n d skatole were compared to t h e recovered amount of carbazole. Assuming carbazole to be quantitatively recovered, the recovery of indole was 65% (in agreement with t h e radioactive work), while t h a t of skatole was 79%. T h e higher skatole recovery was expected as it is more stable than indole. Haq a n d co-workers also found higher skatole recoveries in their isolations (12). Thus, although some indole was lost in the silicic acid chromatography, recovery was high enough for identification studies.
A further problem with the recovery of indole was its high volatility. When Fraction C was reduced in volume on a rotary evaporator, and then concentrated to 1 m L with a gentle stream of nitrogen, about 1470 of l4C-indole was lost. However, when the solvent was carefully removed from Fraction C with a small, vacuum-jacketed Vigreux column, loss of indole was negligible. Radioactive indole was used to determine t h e recovery of indoles from the gel filtration step. An aliquot of Fraction C was combined with a 14C-indole solution, separated on the Bio-Beads, and t h e resulting gel fractions 41-60 were combined and t h e recovery of 14C-indole determined. T h e d a t a showed that more than 98% of the indole was recovered from the gels. Recovery experiments are summarized in Table 11. In summary, a four-step method has been developed for the isolation of indoles and carbazoles from cigarette smoke condensate. The method effectively separated the indoles and carbazoles from the P A H of smoke and allowed analyses of both classes of compounds from the same sample of CSC. Gel filtration chromatography purified t h e indole/carbazole fraction, producing an isolate suitable for identification and quantitation. Carbazole was quantitatively recovered by the method. The method should be applicable to the analysis of indoles and carbazoles resulting from other combustion processes of nitrogen-containing material in our environment.
LITERATURE CITED ( 1 ) G. 6.Neurath, Beitr. Tabakforsch.,5, 115 (1969). (2) R. L. Stedman, Chem. Rev., 68, 153 (1968). (3) I. Schmeltz and D Hoffmann, Chem. Rev., 7 7 , 295 (1977). (4) J. C. Arcos and M. R. Araus. "Chemical Induction of Cancer", Academic Press, New York, N.Y.,-1974. (5) A. A. Rosen and F. M. Middleton, Anal. Chern., 2 7 , 790 (1955). (6) R. C. Lao, R. S. Thomas, H. Oja, and L Dubois, Anal. Chem., 45, 908
119731. (7) M. L. Lee, M. Novotny, and K. D. Bartle, Anal. Chem., 48, 405 (1976). (8) D. Hoffmann and J. Rubin, Beitr. Tabakforsch., 3, 409 (1966). (9) D. Hoffmann and G. Rathkamp, Anal. Chem., 42, 366 (1970). 10) D. Hoffmann and G. Rathkamp, and H.Woziwodzki. Be&. Tabakforsch., 4, 253 (1968). 11) D. Hoffmann, G. Rathkamp, and S. Nesnow, Anal. Chern., 41, 1256 (1969). 12) M. Haq,S. Rose, L. W i r i c h , and A. Patel, Anal. Chem., 46, 1781 (1974). 13) H. C. Pillsbury, C. C. Bright, K. J. O'Corron, and F. W. Irlsh, J . Assoc. Off. Anal. Chem., 5 2 , 458 (1969). 14) K. Rothweil, Ed., "Standard Methods foc the Analysis of Tobacco Smoke", Research Paper 11, Tobacco Research Council, London, 1972. 15) R. F. Severson, M. E. Snook, R. F. Arrendale, and 0. T. Chortyk, Anal. Chem., 48, 1866 (1976). (16) M. E. Snook, R. F. Severson, H. C. Higman, R. F . Arrendale. and 0. T. Chortyk, Eeitr. Tabakforsch., 8, 250 (1976). (17) M. E. Snook, J . Chrornatogr. Sci., submitted for publication. (18) M. E. Snook, Anal. Chim. Acta.. 81, 423 (1976).
RECEIVED for review August 25, 1977. Accepted October 11, 1977. This paper was presented in part a t the 30th Tobacco Chemists' Research Conference, Nashville, Tenn., October 1976.
