Quantitative Determination of Fluorenes in Cigarette Smoke and Their Formation by Pyrosynthesis Dietrich Hoffmann and Gunter Rathkamp Division of Enuironmental Carcinogenesis, American Health Foundation, N e w Y o r k , N . Y . 10021
A method for the determination of fluorenes in cigarette smoke is described. The nonvolatile particulate matter of cigarette smoke is distributed between three solvent pairs, chromatographed on alumina, and the fluorene concentrate is analyzed by gas chromatography. Fluorene-9-14C served as internal standard. The mainstream smoke of an 85-mm U S . blended nonfilter cigarette contained 417 ng of fluorene, 62.1 ng of 1-methylfluorene, 254 ng of 2- and 3-methylfluorene, 79.4 ng of 4-methylfluorene, traces of I)-methylfluorene, and several dimethylfluorenes. The latter are estimated to amount to more than 30 ng per cigarette. Pyrolysis experiments suggest that in cigarette smoke the fluorenes are partially formed via the Diels-Alder addition of indenes with volatile dienes. This hypothesis is supported by the correlation between the smoke concentration of fluorenes with that of isoprene. NEUTRAL SUBFRACTION BI constitutes about 0.6% of the dry particulate matter of cigarette smoke and shows the highest carcinogenic activity of all tobacco portions bioassayed (1). Fractionation of BI revealed that tobacco smoke contains relatively high concentrations of alkylated polynuclear aromatic hydrocarbons (PAH), especially in comparison to gasoline and urban pollutants (2). Methylfluorenes, methylfluoranthenes, methylchrysenes, and methylbenzo[a]pyrenes are among the alkylated PAH we have identified in BI ( I ) . Methylfluorenes were identified in cigarette smoke as early as 1957 (3-5). The identification of the fluorenes was based primarily on their ultraviolet absorption spectra. This method, however, does not distinguish satisfactorily between individual alkylated PAH. Recently, gas chromatography and mass spectrometry were employed for the analysis of alkylated fluorenes (6,7). Quantitative data on the concentration of these hydrocarbons in tobacco smoke have not yet been published. In this communication we report a quantitative method for the determination of alkylfluorenes in cigarette smoke. The analytical method consists of three solvent distribution steps, column chromatography, and, finally, gas chromatography. Model studies support the hypothesis that alkylfluorenes in cigarette smoke are at least partially pyrosynthesized by the Diels-Alder reaction of volatile dienes with indenes followed by aromatization. The terpenes of the processed tobacco most likely serve as precursors for the dienes as well as for the
(1) D. Hoffmann and E. L. Wynder, Cancer, 27, 848 (1971). (2) D. Hoffmann and E. L. Wynder, in “Air Pollution, Vol. 11,” A. C. Stern, Ed., Academic Press, New York, N.Y., 1968, Chap. 20. (3) S. Neukomm and J. Bonnet, Strahlentherapie, Sonderb., 31, 128 (1957). (4) E. L. Wynder and D. Hoffmann, “Tobacco and Tobacco
Smoke, Studies in Experimental Carcinogenesis,” Academic Press, New York, N.Y., 1967. (5) R. L. Stedman, Chem. Rec. 68, 153 (1968). (6) J. H. Bell, M. S. Ireland, F. J. Schultz and A. W. Spears, 24th Tobacco Chemists’ Res. Conf., 1970,21. (7) G . Rathkamp and D. Hoffmann, ibid., p 20.
dienophiles. Until now, the Diels-Alder synthesis has not been tested as a possible route for the pyrosynthesis of PAH in tobacco smoke. EXPERIMENTAL
Apparatus. Cigarettes were smoked with a 20-channel automatic smoker R M 20/68 for the fluorene analysis and with a twenty-port automated Phipps and Bird machine for the isoprene analysis (8, 9). A Perkin-Elmer gas chromatograph Model 800 with dual-flame ionization detector was used for the quantitative analysis of fluorenes. For the smoke analysis of isoprene, we used a Perkin-Elmer gas chromatograph Model 154 with flame ionization detector. Counting was done with a Nuclear-Chicago Scintillation System 720. Ultraviolet absorbance measurements were obtained with a Cary Model 11 and infrared spectra with a Perkin-Elmer Model 21. Mass spectra were determined with a Hitachi Perkin-Elmer RMU-6D instrument by the Morgan-Schaffer Corporation (Montreal, Canada) at 70 eV. Evaporations were carried out under reduced pressure with water bath temperatures below 45 “C. Melting points were corrected. A Perkin-Elmer Pyrolysis Unit, which was adjusted to the chosen temperatures by a Chromel-alumel thermocouple and differential voltmeter (J. Fluke Co.), was used for the pyrolysis experiments. Reagents. All organic solvents were spectrograde, the other chemicals of analytical reagent grade. Alumina Woelm neutral (activity 11, except as indicated) was purchased from Waters Associates, Sephadex LH-20 from Pharmacia Fine Chemicals, OV-17,0V-l, Carbowax 550, and Gas Chrom P from Applied Science Laboratories. Reference Compounds. Commercial fluorene, 1-, 2-, and 9-methylfluorene were purified by repeated chromatography on alumina, activity I. Their purity was ascertained by melting point, by ultraviolet absorption spectrometry, and by gas chromatography with the systems described below. 3-Methylfluorene. 4-Methyldiphenylmethane (0.8 gram) and 5% platinum on charcoal (1.0 gram) were heated in a sealed tube at 400 “C for 3 hours. After cooling, the solid material was repeatedly extracted with ether and filtered. Gas chromatography showed a 50 % yield of 3-methylfluorene. The crude product was chromatographed on alumina (activity I). 3-Methylfluorene (180 mg) was eluted from the column with n-hexanelbenzene (4 :1). After recrystallization from n-hexanelbenzene (10 :l), 3-methylfluorene (150 mg) was obtained as white crystals, mp 85.5-86.5 “ C [Lit. (10) 88 “C]. 4-Methylfluorene. Indene (1 1.6 grams), 1,3-pentadiene (9.0 grams), and hydroquinone (20 mg) were heated in a sealed tube at 180 “C for 24 hours (10). The reaction product was distilled under reduced pressure (20 mm Hg). After a forerun of indene (5.1 grams), we obtained a fraction with bp 151-1 55 “C, 1,4,4a,9a-tetrahydr0-4-methylfluorene(6.9 grams, 38%). The latter compound (6.5 grams) and 5 % PdjC (0.65 grams) were heated in a sealed tube for five hours at 270 “C. The benzene extract of the reaction mixture was filtered and chromatographed twice on alumina (activity I). (8) G. Lipp, Beitr. Tabakforsch., 5 , 39 (1969). (9) Y. Pillsbury, C . C. Bright, H. J. O’Connor, and F. W. Irish, J. Ass. Ofic.Agr. Chem. 52,458 (1969). (10) E. D. Bergmann, Bull. Res. Council Zsr., Sect. A, 5 , 147 (1956). ANALYTICAL CHEMISTRY, VOL. 44, NO. 6, MAY 1972
899
Table I. Mass Spectra of Fluorenes Relative Ahndance of Characteristic Ions mle 194 180 179 166 165 164 152 139 115 89 82.5 76 63 51
Fluorene
100 86 3 0 8
5 3 23 1 8 3
1-Methyl- 2-Methyl- 3-Methyl- 4-Methyl- 9-Methyl100 25 15 98 10 10 4 3
28 18 21 9 6
98 28 13
100 4
91 29 16 100
10
4 3 30 16 27 10 5
Table 11. Fluorenes: Partition coefficients
5
10 5 4 33 18 28 12 7
91 25 15 100 4 11 5 4 29 19 25 10 6
66 16 16 100
6 7 5
3 16 12 12 6 4
1,9-Dimethyl46 14 100 0
4 1 8 2 2 13 3
9 4
2,3-Dimethyl 76 16 100 1 9 2 8 2 2 20 6 12 9
3
5
9,9-Dimethyl39 16 100 1 3
1 9 2 2 20 3 14 6 5
2-Ethyl84 17 100 14 81 22 10 5 4 21 6 13 6 3
Partition Coefficients and Retention Times Retention times
11.4 4.5 12.7 Fluorene 8.5 7.2 4.2 I-Methylfluorene 5.2 9.1 9.7 2-Methylfluorene 6.8 3-Methylfluorene 8.4 4.4 7.1 4-Methylfluorene 8.5 4.4 12.8 5.9 9.4 9-Methylfluorene 6.1 4.8 7.5 1,9-Dimethylfluorene 10.4 7.8 9.6 2,3-Dimethylfluorene 3.2 5.9 5.2 9,9-DimethyIfluorene 5.9 8.2 5.1 2-Ethylfluorene CH = Concentration in n-hexane. C.W= Concentration in methanol/water (4: 1). CD = Concentration in dimethylformamide/water (4 :1). Cw = Concentration in dimethylformamide/water (1 :4) (all solvents were pre-equilibrated), a = 3-mm x 6-m stainless steel column, 5z OV-17 on GCP (60/80); 190 "C. b = 3-mm x 4.5-m stainless steel column, 5 z OV-1 on GCP (60/80); 160 "C.
The yield of 4methylfluorene, pure by gas chromatography, was 7 0 z , mp 69-70 "C [Lit. (IO)68 "C]. 1,9-Dimethylfluorene. 1-Methylfluorene (0.85 gram) and potassium (0.2 gram) were boiled under reflux in dioxane (15 ml) for 90 minutes. After cooling, methyl iodide (0.5 ml) was added, and the reaction mixture was stirred a t room temperature for 1 hour. Water (5 ml) and ether (15 ml) were added. The ether solution was washed with water (5 X 10 ml), dried (CaCl,), and evaporated to give a n orange solid (0.66 gram), mp 61-63 "C. Crystallization from ethanol gave a first crop (95 mg), mp 69-70 "C [Lit. (11) 71-72 "C]. According to gas chromatography, this material contained 1-methylfluorene. Repeated chromatography on alumina (activity I) gave pure 1,9-dimethylfluorene (mp 71-71.5 "C). 2,3-Dirnethylfluorene. Indene (1 1.6 grams), 2,3-dimethylbutadiene (1 1.6 grams), and hydroquinone (0.1 gram) were heated in a sealed tube at 180 "C for 20 hours. The benzene extract was distilled under reduced pressure (12 mm Hg). The fraction of bp 168-172 "C consisted primarily of 1,4,4a,9a-tetrahydr0-2,3-dimethylfluorene (12.5 grams, 63 1,4,4a,9a-Tetrahydr0-2,3-dimethylfluorene (6.0 grams) and 5 PdjC (1 .O gram) were heated in a sealed tube a t 180 "C for two hours. The product was extracted with benzene and the resulting residue crystallized twice from n-hexanelbenzene (8 : 1). The silvery leaflets were purified by chromatography
z).
z
11.3 17.2 16.4 16.2 18.2 11.5 17.4 27.8 10.8 22.6
6.8 11.6 11.3 11.5 12.0 7.6 12.4 20.6 7.1 14.2
on alumina (activity I). The yield was 6 0 x ; mp 124-124.5 "C [Lit. (12) 124-125 "C]. 9,9-Dimethylfluorene. Fluorene (16.6 grams) and potassium (7.8 grams) were boiled under reflux in dioxane (80 ml) for 5 hours under nitrogen, giving a deep red solution. Methyl iodide was added dropwise to the cooled solution, producing a white precipitate (KJ) in a vigorous, exothermic reaction. The reaction mixture was stirred under nitrogen a t room temperature overnight. Water (50 ml) and ether (100 ml) were added and the aqueous phase was separated. The ether solution was washed with water (5 X 50 ml) to remove the dioxane, dried (CaClJ, and evaporated to give a yellow oil (20.2 grams). Thin layer chromatography (silica gel, benzene elution) indicated the presence of three components including fluorene and 9-methylfluorene. The oil was chromatographed on alumina (300 grams; activity I). Elution with n-hexane gave 9,9-dimethylfluorene (7.7 grams) as the first compound eluted. Infrared spectra showed that subsequent fractions were contaminated by fluorene. Recrystallization from methanol gave pure 9,9-dimethylfluorene (4.60 grams), mp 92-93 "C [Lit. (13) 95-96 "C]. 2-EthJ lfluorene. According to Ray and Rieveschi (14, fluorene (40 grams) was dissolved in carbon disulfide (175 ml). (12) L. Chardonnens, B. Laroche, and G. Gamba, Helc. Chim
Acta,48,1801 (1965). (13) G. W. H. Scherf and R. K. Brown, Can. J. Chem., 38, 697
(11) B. L. McDowell, G. Smolinsky, and H. Rapoport, J. Amer. Chem. Soc., 84,3531 (1962). 900
ANALYTICAL CHEMISTRY, VOL. 44, NO, 6, MAY 1972
(1966). (14) F. E. Ray and G. Rieveschi, Jr., Org. Syn. 3, 23 (1955).
The solution was stirred while aluminum chloride (64 grams) was added in one portion. Acetic anhydride (25 grams) was added dropwise at such a rate as to maintain a gentle reflux. Vigorous stirring was maintained throughout. Effluent HCI was led through a calcium chloride drying tube to a funnel inverted over water for trapping. After addition of the acetic anhydride was complete, the reaction mixture was boiled under reflux for one hour and filtered. The solid was stirred with fresh CS2 (150 ml) and refiltered. The filter cake was washed with CS2 (three 50-ml portions) and petroleum ether (100 ml). This AlC18 complex was decomposed by adding it in small portions to a stirred solution of concentrated hydrochloric acid (15 ml) and water (400 ml). The precipitate was collected and dried overnight to give a quantitative yield of crude 2-acetylfluorene, mp 12C124 "C [Lit. (14) 128129 "C]. 2-Acetylfluorene (12 grams), powdered KOH (5 grams), 60% hydrazine hydrate (18 ml), and ethylene glycol (125 ml) were refluxed for 8 hours using a modified Dean-Stark trap (15). The water separator was removed when the boiling point of the mixture rose to 185 "C. The mixture was poured into water and the solid was collected to give crude 2-ethylfluorene (10.5 grams, 9373. Sublimation (80°/0.2 mm) gave purer material, mp 92.94 "C [Lit. (15) 99.5-100 "C]. Gas chromatography showed two minor impurities which mass spectral analysis suggested were a diethylfluorene and vinylfluorene. The 2-ethylfluorene was purified by chromatography on alumina (activity I) and had mp 99-100 "C. The major ions of the mass spectra of the fluorenes used as references are listed in Table I . Internal Standard. Fluorene-9-14C (12.11 mCi/mM ;TracerLab) was purified by column chromatography and its purity ascertained by repeated chromatography and GLPC. Toluene solutions with 0.4% PPO (2,5-diphenyloxazole) and 0.005 POPOP (p-bis[2(5-phenyloxazolyl)]-benzene) as scintillators
gave efficiencies of 71-72z for the unquenched l4C-Iabelled fluorene. Distribution. Preliminary studies have shown that the presence of paraffin hydrocarbons in a fluorene concentrate diminishes the ability of the adsorbent to separate alkylfluorenes. Therefore, three distributions between preequilibrated solvent pairs were carried out to separate fluorenes from the bulk of the tobacco smoke paraffins (Table 11). Distribution step 1 separates the hydrophilic constituents from the rest of the particulate matter, steps 2 and 3, the fluorenes from the paraffins. Gas Chromatography. The most satisfactory separation of methylfluorenes was obtained at 190 "C on a 3-mm by 6-m column packed with 5 % OV-17 on Gas Chrom P. The separation of 9-methylfluorene from fluorene, however, was best achieved at 160 "C on a 3-mm by 6-m column of 5 % OV-1 on Gas Chrom P. Both systems employed helium as carrier gas with a flow rate of 40 ml/min. The retention times are given in Table 11. About 0.5 pg of fluorene reaches the full scale of a 1-mV recorder. A 1 :4 splitter was installed for the isolation of the individual fluorenes from a concentrate from cigarette smoke, so that 80% of the effluent could be collected in a glass capillary. The effluent was rechromatographed before mass spectral analysis. Procedure. ISOLATION OF FLUORENES. The 8000 cigarettes (85-mm) were smoked in eight lots. The standard conditions and the apparatus for the collection of the nonvolatiles of the mainstream smoke were previously reported (16). The acetone suspension and rinsings of the "tar" were evaporated under reduced pressure. The residue (200 grams) was dissolved in 2 X 1.5 I. of methanol/water (4:l) and was extracted three times with 1.5 1. of n-hexane. The combined hexane layers were concentrated in cucuo to 3 1. The latter was extracted three times with equal volumes of dimethyl(16) D. Hoffmann, G. Rathkamp, and 41, 1256 (1969).
(15) J. D. Dickinson and C . Eaborn, J. Chem. SOC.,1959, 2337.
S. Nesnow, ANAL.CHEM.,
n
2+3 Methyl-F
I- Methyl-Fl, 4- Methyl-FI Phenanthrene
Figure 1. Gas chromatograms of concentrates of fluorenes from large scale separation of cigarette smoke condensate
U
0
I-Methyl-FI]
I--
B
u w
-2-Methyl-
FI
A.
I--
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35
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25
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ANALYTICAL CHEMISTRY, VOL. 44, NO. 6, MAY 1972
901
100
5
9 5
r,H3
A.REFERENCE
T
76
02.5 152
63 139
I I
s
152
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115
I
u
IIL
57
63
89
57
I39
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2010-
0 - l 1
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m/e formamide/water (4:1). The combined aqueous extracts were concentrated to 100 ml, and 500 ml water were added and extracted three times with n-hexane (600 ml). The residue of the hexane extracts (13.6 grams) was dissolved in 100 ml of n-hexanelbenzene (8 :1) and chromatographed on 1.3 kg of alumina (5 X 120-cm column). After a 4.4-1. forerun with n-hexane, fraction 1 was eluted by 1.6 1. of n-hexanelbenzene (lO:l), fraction 2 by 0.8 1. of n-hexanelbenzene (8:1), fraction 3 by 1.6 1. of n-hexanelbenzene (6:1), fraction 4 by 0.8 1. of n-hexanelbenzene ( 5 :l), fraction 5 by 0.88 1. of n-hexanel benzene (4 :1). Gas chromatography indicated that fraction 6 [n-hexane/benzene (3 :l)] contained the fluorenes. The residue of fraction 6 (183 mg) was dissolved in 2-propanol and chromatographed on 60 grams of Sephadex LH-20 (2 X 100-cm column) at a column temperature of 32 “ C and a flow rate of 0.8 ml per hour. Fractions 11-20, 2.0 ml each (total residue 61 mg), contained fluorene and fractions 21-25 (total residue 25 mg) contained alkylated fluorenes. These fractions were analyzed by gas chromatography. The materials 902
a
ANALYTICAL CHEMISTRY, VOL. 44, NO. 6, MAY 1972
which were characterized by well resolved maxima were collected and rechromatographed for mass spectral analysis and for ultraviolet and infrared absorption spectra. QUANTITATIVE ANALYSIS.Three hundred cigarettes were smoked with a 20-channel automatic smoker under standard conditions (16). The smoke suspension and rinsings were combined and 2.3 p g of fluorene-9-14Cwere added as internal standard. The “tar” was dissolved in 100 ml of methanol/ water (4 :1) and extracted three times with 100 ml of n-hexane. The combined extracts were concentrated in uucuo to about 50 ml. and, subsequently, extracted three times with equal volumes of dimethylformamide/water (4 :1). The combined lower layers were concentrated to 30 ml and, after adding water (120 ml), were extracted three times with n-hexane. The residue (average 0.5 gram) was dissolved in about 2 ml of n-hexanelbenzene (10 :1) and chromatographed with hexane/benzene (1O:l) on 350 grams of alumina. The eluate was collected in 50-ml fractions. On the average, fractions 23-29 (3-6 mg) contained the radioactivity and were com-
qOL'T
100
80
ma:::
I
h
A REFERENCE
UNKNOWN A
9
20
[L
10
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Figure 4. Mass spectra of 2,3-dimethylfluorene and isolated dimethy lfluorenes bined with the next eight fractions. Aliquots of these combined fractions were taken for gas chromatographic analysis and liquid scintillation counting. Pyrolysis Experiments. For these tests, the standard Y-tube of the pyrolysis unit was modified by adding a sidearm at a 45" angle from the main tube. This sidearm is 1.5 cm long and is closed with a silicon septum through which the reaction solution can be introduced into the hot zone by a microsyringe with a 5-cm needle. For the actual pyrolysis experiments, we injected 5-pl test solutions. After 1 minute, the unit was cooled to room temperature and the tube was washed with benzene and acetone. To the combined washings of three equal pyrolysis experiments, 1.0 pg of fluorene-I4Cwas added as internal standard. The residue was dissolved in n-hexane and chromatographed on 2 grams of alumina (0.6 X 15-cm column). The radioactive eluate and the subsequent 30 ml were combined, evaporated, dissolved in 0.5 ml of n-hexane and analyzed by gas chromatography and liquid scintillation counting. Isoprene Analysis. A single cigarette (*20 mg within the average weight of 200 cigarettes) was smoked under standard conditions into a specially designed trap (4). After sampling, during which the trap is immersed in a dry ice-acetone bath, the glass vessel was connected with the gas chromatograph by using it as a loop for the sampling valve. A stream splitter with a ratio of 1 : 24 is built into the gas chromatograph. Preheated helium was used as carrier gas with a flow rate of 40 ml/minute. The trap was brought to room temperature and then kept in a water bath of 30 OC. After five minutes, 4 z of the trapped gas phase was introduced onto the column.
I
850
I
800
I
753
I
700
FREQUENCY (cm9 Figure 5. Infrared spectra (700-850 cm-' region) of 2-methylfluorene, 3-methylfluorene, and mixtures of 2- and 3-methylfluorenes isolated from cigarette smoke The column conditions were as follows: 3-mm X 6.6-m column of 1 5 x Carbowax 550 on Gas Chrom P, column temperature 65 "C isothermal. The retention time for isoprene is 4.5 i 0.2 min. (It is possible that the isoprene values are somewhat low, since we cannot exclude some losses in the trap by interactions of isoprene with other smoke constituents. However, our values compare favorably with results from other groups). RESULTS AND DISCUSSION
Gas chromatograms of the fluorene and post-fluorene fractions from a large scale separation of the particulate matter of cigarette smoke are shown in Figure 1. The maxima which were identified by retention times, ultraviolet, and mass spectra are so labeled. Figures 2, 3, and 4 compare mass spectra of authentic and isolated fluorenes. As can be deduced from Table I, mass spectra are helpful for the identification of fluorenes. Fluorene and 9-methylfluorene were separated and isolated with a second gas chromatography system (Table 11). ANALYTICAL CHEMISTRY, VOL. 44, NO. 6, MAY 1972
903
Table 111. Fluorenes in Cigarette Smoke. ng/cigarette Analysis number
Fluoreneb 390 380 420 450 445 417 31.2
1-Methylfluorene 63.9 54.6 57.7 68.2 66.3 62.1 5.78
I I1 I11 IV V Average Standard deviation Deviation 7.5z 9.3z coefficient a Calculated with the isotope dilution using fl~orene-9-'~C as internal standard. 9-Methylfluorene
