Quantitative Determination of Nitrobenzenes in Cigarette Smoke’ Dietrich Hoffmann and Gunter Rathkamp Division of Environmental Toxicology, American Health Foundation, New York, N. Y. 10021
A method for the determination of nitrobenzenes in cigarette smoke is described. The nonvolatile particulate matter of the smoke collected in solvent is suspended in diluted acid and water steam distilled. The neutral portion of the distillate is chromatographed on alumina, and the concentrate of the nitrobenzenes is analyzed by gas chromatography. Nitrobenzene-U14C served as internal standard. The mainstream smoke of an 85-mm U.S. nonfilter cigarette contained 25.3 ng of nitrobenzene, 21.4 ng of 2-nitrotoluene, 10.4 ng of 3-nitrotoluene, 19.6 ng of a 2:l mixture of 4-nitrotoluene and 2-nitro-1,4-dimethylbenzene, 18.5 ng of 4nitro-1,3-dimethylbenzene, 5.3 ng of 4-nitrocumene, and 6.5 ng of 4-nitro-1,2-dimethylbenzene. Since already one nanogram of a nitrobenzene gives a significant electron capture response, a quantitative analysis required only 50 cigarettes. This communication reports the first identification of nitrobenzenes in tobacco smoke. Cigarette tobacco artificially enriched with KNOa produces increased yields of nitrobenzenes in the mainstream smoke. EXPERIMENTAL DATA suggest a correlation between the nitrate content of tobacco and the yield of pyrosynthesized polynuclear aromatic hydrocarbons (PAH) in the smoke ( I ) . For example, the mainstream smoke of cigarettes made of lownitrate, top leaves of a Burley variety contains more particulate matter (TPM) and PAH than the mainstream smoke of cigarettes made of high-nitrate, lower leaves of the same plant. The formation of benzo[a]pyrene is significantly more inhibited by nitrates than the formation of TPM. Since such selective reduction of PAH in the “tar” is associated with a significant reduction of its tumorigenicity in the mouse skin bioassay ( I ) , explanations are sought for possible mechanism(s). We hypothesized that in the hot zones of a burning tobacco product C,H-radicals are pyrolytically formed. These radicals react with each other and form PAH among other compounds. However, the last step, the actual pyrosynthesis, may be partially inhibited by an excess of thermically activated nitrogen oxides, which may react as scavengers. Our hypothesis is supported by the observation that with elevated nitrate content of the tobacco, the yield of primary and secondary nitroparaffins in the smoke increases whereas the yield of PAH decreases (2). Benzene and alkylated benzenes in the smoke are most likely formed by pyrolysis of derivatives of the aromatic hydrocarbons in tobacco or by pyrosynthesis from C,H-radicals. One would expect, therefore, that with ascending nitrate content of the tobacco, the yield in the smoke of other nitro compounds such as the nitrobenzenes should also be increased. Since nitrobenzenes had not been identified in tobacco smoke (3-3, we initiated the study presented here. 1
No. XI1 of “Chemical Studies on Tobacco Smoke.”
(1) D. Hoffmann and E. L. Wynder, Nui. Curicerbzst., Monogr., 28, 151 (1968). (2) D. Hoffmann and G. Rathkamp, Beiir. Tubukforsch., 3, 124
For the identification and determination of nitrobenzenes, the nonvolatile particulate matter of cigarette smoke is watersteam distilled from a suspension in diluted acid. The neutral distillate is chromatographed and the resulting concentrate is separated into individual components by gas chromatography. Using a n electron capture detector, one obtains a significant response with as little as 1 nanogram of a nitrobenzene; and an even higher response with some 2alkylnitrobenzenes. Nitrobenzene-14C is employed as internal standard for the quantitative determination. EXPERIMENTAL Apparatus, For the isolation of nitrobenzenes, we employed a 30-channel automatic smoker with a vibrating liquid trap; for the quantitative analysis we smoked the cigarettes individually with the twenty-port automated Phipps and Bird machine (6, 7). A Perkin-Elmer gas chromatograph Model 800 with dual-flame ionization detector and a 4 :1 splitter was used for the isolation of unknowns, and a Varian Aerograph Model 1200 with an electron capture detector (tritiated titanium as @-source) for the quantitative analysis. The P-radiation of the l4C-labelled internal standard was counted with a Nuclear Chicago Scintillation System 720. The mass spectra were obtained with a Hitachi-Perkin-Elmer RMU-6D by the Morgan-Schaffer Corporation (Montreal, Canada). The energy of the bombarding electrons was kept at 50 eV. The laboratories were illuminated with yellow light (Sylvania Electric Tubes F-40 G.O.), which excludes radiation below 450 mp. Evaporations were completed at reduced pressure with water bath temperatures below 50 “C. Reagents. All organic solvents were spectrograde, the other chemicals of analytical reagent grade. Alumina Woelm neutral (activity IT, except as indicated) was obtained from Alupharm Chemicals, Gas Chrom P (80-100 mesh) and OV-225 from Applied Science Laboratories. Reference Compounds. The commercial nitrobenzenes were purified by repeated chromatography on alumina. 4Nitroethylbenzene, 2- and 4-nitrocumenes and 2-nitro-tertbutylbenzene required purification by gas chromatography with the system described below. The purity of the references was ascertained by refractometry and by gas chromatography using an electron capture detector as well as a flame ionization detector. 2-Nitro-tert-butylbenzene; mass spectrum (50 eV); m/e (re1 X intensity) 179(12.6), 165(7.7), 164(60.1), 162(5.4), 149(4.8), 147(7.4), 146(6.4), 134(25.2), 132(4.3), 119(28.4), 118(14.0), 117(19.2), 116(11,2), 115(36.8), 106(14.0), 105(12.7), 94(18.7), 92(20.0), 91(100), 90(6.3), 78(17.9), 77(46.3). Internal Standard. Nitr~benzene-U-~~C (3.2 mCi/mM; Amersham/Searle) was purified by column chromatography and its purity assured by GLPC with electron capture detector. Toluene solutions with 0.4% PPO (2.5-diphenylPOPOP (p-bis[2(5-phenyloxazolyl)]oxazole) and 0.005 benzene) as scintillators gave efficiencies of 71-72% for the unquenched 14C-labelled nitrobenzene. Gas Chromatography. The most satisfactory separation of the nitrobenzene was obtained at 200 “C on a 3-mm by
(1968). (3) G. Neurath, Arzneim. Forsch., 19, 1093 (1969). (4) R. L. Stedman, Chem. Rev., 68, 153 (1968). (5) E. L. Wynder and D. Hoffmann, “Tobacco and Tobacco
Smoke, Studies in Experimental Carcinogenesis,” Academic Press, New York, 1967.
(6) F. Seehofer, J. E. Miller, and E. Elmenhorst, Beiiv. Tubakforscli., 3, 75 (1965). (7) Y . Pillsbury, C. C. Bright, H. J. O’Connor, and F. W. Irish, J . Ass. Ofic.Cliemists, 52,458 (1969).
ANALYTICAL CHEMISTRY, VOL. 42, NO. 13, NOVEMBER 1970
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Table I. Relative Retention Times and Relative Molar Retention Volumes of Several Nitrobenzenes“
ii 3
lo[8
Relative retention time
E.C. RANGE 10
Y
0
ATTENUATION 8
$2
01 0
I
I
I
40
60
I
I
I
I
I
80 100 120 140 160 Picomole Figure 1. Linearity curves for nitrobenzene (NB), 2-nitro-
20
toluene (2-NT), fnitrotoluene (SNT), and Cnitrotoluene (4NT) 12-
2-N-1,4 DMB 4-N-1,3 DMB
10-
Relative molar retention volume 1.00 1.03 0.83 0.72 0.87 0.70 0.93
Name Nitrobenzene 1.oo 2-Nitro-I ,3-dimethylbenzene 1.05 2-Nitrotoluene 1.10 2-Nitroethylbenzene 1.23 3-Nitrotoluene 1.29 2-Nitro-l,3,54rimethylbenzene 1.37 2-Nitro-l,4-dimethylbenzene 1.43 4-Nitrotoluene 1.43 0.88 2-Nitrocumene 1.49 0.73 4-Nitro-I ,3-dimethylbenzene 1.58 1.04 5-Nitro-l,3-dimethylbenzene 1.69 0.91 4-Nitroethylbenzene 1.85 0.94 LCNitrocumene 2.08 0.70 4-Nitro-l,2-dimethylbenzene 2.25 0.63 a Flame ionization detector The average, absolute retention time for nitrobenzene was 5.6 min after injection
8-
cu
5
+N-1,2 DMB 2-N-1,3 DMB
E.C.RANGE 10
h
ATTENUATION 8
10N
2-NEB
8-A-MCI~
E 0 0
6-
Y
4-
t! 0
a“ 2-4 0
0
4
7
1
20
1
40
1
1
1
1
1
1
1
60 80 100 120 140 160 Picomole
Figure 3. Linearity curves for 2-nitroethylbenzene (2-NEB), Cnitroethylbenzene (CNEB), and 2-nitrocumene (2-NCu) and 4-nitrocumene (4-NCu) 6.1-m column filled with 5% OV-225 o n Gas Chrom P. The relative retention times and relative retention volumes for nitrobenzenes are given in Table I, the linearity curves for the electron capture detector sensitivity to picomoles of some nitrobenzenes are shown in Figures 1 to 3. For the gas chromatographic isolation of individual nitrobenzenes from the concentrate a 4 : l splitter was installed. The column effluents of the gas chromatographic separation which had retention times comparable to those of the references and which 1644
induced in low concentrations significant electron capture response, were collected in glass capillaries and rechromatographed for mass spectral analysis. Procedures: Isolation of Nitrobenzenes. A total of 4100 cigarettes without filter tips (85-mm) were smoked in four portions. The standard conditions and the setup for the collection of the nonvolatiles of the mainstream smoke were previously reported (8). The acetone suspensions and rinsings with the collected “tar” were evaporated under reduced pressure and low water bath temperatures (40-45 “C) from a 2-1. flask with a 50-cm distillation column. To the residue (154 grams) were added 500 ml of 2 N HCI. This “tar” suspension was water-steam distilled until 1.5 1. of liquid were condensed (model studies with nitrobenzene-l4C and alkylated nitrobenzenes demonstrated that the first 0.5 1. of condensate represents the quantitative transfer of these compounds), The condensate was saturated with NaCl and four times extracted with 1 1. of ether. The ether layers were concentrated to 200 ml, twice extracted with 100 ml of 2 N NaOH, washed twice with 50 ml of saturated NaCl solution, dried (Na2S04),and evaporated. The neutral residue (4.15 grams) was dissolved in 30 ml of n-hexane-benzene (10 : 1) and chromatographed with n-hexane on 200 grams of alumina (column 2.5 x 75 cm). After 100 ml of forerun, the first 10 fractions, each 50 ml, were eluted with n-hexane-benzene (6 :l), and fractions 11-25 with n-hexane-benzene (4 :1). The 50-1111 fractions were concentrated to about 0.5 ml from which 1 p1 was injected into the gas chromatograph with the 5 OV-225 column and electron capture detector. Fractions 12-18 gave significant electron capture responses a t retention times for nitrobenzene, nitrotoluenes, and nitrodimethylbenzenes. The residue (49.4 mg) was rechromatographed with n-hexanebenzene (6:l) on 50 grams of alumina (column 10 mm by 1.2 rn); 2.5-ml fractions were collected each hour. The concentrate of fractions 52-57 (about 0.2 ml) gave an indication for the presence of nitrobenzene when tested by gas chromatography as outlined above. These fractions were combined and evaporated at 12 mm of Hg from a small flask, which was cooled from the outside by ice water, into a condensing flask immersed in dry ice-acetone, The residue (1.1 mg) was dissolved in 0.1 ml of hexane and separated by gas chromatography. We collected and rechromatographed for mass (8) D. Hoffmann, G. Rathkamp, and S. Nesnow, ANAL.CHEM., 41, 1256 (1969).
ANALYTICAL CHEMISTRY, VOL. 42, NO. 13, NOVEMBER 1970
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A. REFERENCE 134
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1 “I-
B. ELECTRONCAPTURE DETECTOR
0
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O i 18
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9
12
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3
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Figure 4. Gas chromatographs for concentrates of nitrobenzenesfrom cigarette smoke
100
do
60
do
A. REFERENCE
yo2
I tkCH3
65
A. REFERENCE 120
800t-
I20
m/e Figure 6. Mass spectra of Cnitro-l,3-dimethylbenzene
Minutes
loo?
140
c.
500, 1
.-
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-I 0
I 70
m/e
Figure 7. Mass spectra of Cnitrocumene
160
I40
I20
IO0
m/e
BO
60
40
Figure 5. Mass spectra of 2-nitrotoluene spectral analysis those column effluents that gave signals with retention times comparable to those of nitrobenzenes and that also induced high responses in an electron capture detector. Quantitative Analysis. Fifty cigarettes (85-mm) without filter tips were smoked individually under standard conditions
with the Phipps and Bird machine. The mainstream smoke was directed through a Cambridge filter and two gas wash bottles filled with 0.2N NaOH. After smoking of each 4 cigarettes, the Cambridge filter was replaced. The loaded filters and the trapping solutions from the gas wash bottles were transferred into a 50-ml flask and 1 pg of nitrobenzeneU - l T was added as internal standard. The suspension was acidified with 5N HzS04and was water steam distilled. After 500 ml of condensate were collected, NaCl was added to saturation. The resulting oily suspension was four times extracted with 300 ml of ether. The combined ether layers were extracted with 50 ml of 2N NaOH and washed once with 50 ml of water, saturated with NaC1. The ether layer was dried (Na2S04), 3 ml of n-hexane were added, and the ether was evaporated. The remaining dry n-hexane concen-
ANALYTICAL CHEMISTRY, VOL. 42, NO. 13, NOVEMBER 1970
1645
- I -
'
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1'
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CH3
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0
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140
120
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100
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Figure 8. Mass spectra of 4-nitro-1,2-dimethylbenzene trate of the neutral, organic portion of the water steam distillate was chromatographed on 60 grams of alumina. Fractions 1-10 (30 ml each) were eluted with n-hexane-benzene (6 :l), fractions 10-15 with n-hexane-benzene (4 :1). In general, the P-activity was found in fractions 7-11. These were combined, evaporated by dry-freezing (residue 0.4-0.6 mg) and aliquots were taken for gas chromatographic analysis with electron capture detector and liquid scintillation counting. Experimental Cigarettes. In order to verify our working hypothesis that the alkali nitrate content in tobacco is a determining factor for the yield of pyrosynthesized PAH, we enriched artificially the alkali nitrate content of the tobacco of a cigarette made entirely from a Bright tobacco variety low in nitrate. For this purpose we placed each time 45 kg of the Bright tobacco, cut at 0.8 mm, in a rotating cylinder and sprayed slowly onto it 50% KN03 solutions (45 "C) which contained 1.1,2.3, 3.2, and 3.7 kg of the nitrate. The moisture content of the sprayed tobacco was adjusted to about 11 by blowing an air stream with the relative humidity of 45% through the tobacco in the rotating cylinder. These nitrate enriched tobaccos were then machine-made into 85-mm cigarettes without filter tip using the same cigarette paper as for the control cigarettes. From 200 cigarettes (about 11.O % moisture content) of equal nitrate content we selected by weight 40 cigarettes, removed the paper, and took three samples for the nitrate determination according to Broaddus et al. (9). For each analysis, we smoked 50 cigarettes individually, as described above. RESULTS AND DISCUSSION
In the mainstream smoke of a U.S. blended cigarette, we detected eight nitrobenzenes. These compounds were identified by retention times and retention volumes in a gas chroma(9) G. M. Broaddus, J. E. York, Jr., and J. M. Moseley, Tobacco Sci., 9, 149, (1965). 1646
1
160
I
140
I
120
I
100
m/e
l!L 60
40
Figure 9. Mass spectra of Cnitrotoluene, 2-nitro-1,Cdimethylbenzene, and a mixture of 4nitrotoluene nd 2-nitro-1,4dimethyblenzene isolated from cigarette smoke
tography system with a flame ionization detector as well as an electron capture detector and by mass spectra (Figures 4-8). During the final gas chromatography, we separated all the individual nitrobenzenes except 4-nitrotoluene and 2-nitro-l,4dimethylbenzene. However, the mass spectral analysis of the effluent with the retention time corresponding to these two compounds proved that both nitrobenzenes were present in cigarette smoke. As derivatives of 2-nitrotoluene and 4-nitrotoluene these compounds gave well distinguishable mass spectral fragmentation patterns (Figure 9). In the case of 2nitroalkylbenzenes, which have in a-position an alkyl group with a primary or secondary carbon, the mass spectra show a base peak due to the loss of a hydroxyl radical. The M-OH ion dissociates further by ejection of carbon monoxide ( 1 0 , I I ) . These fragments are not observed with 3- and 4-nitroalkylbenzenes and occur only to a minor degree with 2-nitroalkyl(10) H. Budzikiewicz, C. Djerassi, and D. H. Williams, "Mass
Spectrometry of Organic Compounds," Holden-Day, Inc., San Francisco, Calif., 1967. (11) J. H. Beyon, R. A. Saunders, and A. E. Williams, Z/zd Chim. Belge, 29 (4), 311 (1964).
ANALYTICAL CHEMISTRY, VOL. 42, NO. 13, NOVEMBER 1970
Number of analysis 1
2 3 4 5
Nitrobenzene 25.6 24.8 26.7 25.0 24.2 25.3
Table 11. Nitrobenzenes in Cigarette Smoke" (ng/cig) 4-Nitrotoluene 2-nitro-1,44-Nitro-1,34-Nitro- 1,23-Nitrotoluene dimethylbenzeneb dimethylbenzene 4-Nitrocumene dimethylbenzene 2-Nitrotoluene 11.7 20.8 17.9 5.7 6.3 21.7 9.8 18.4 18.4 5.1 6.9 22.0 10.4 19.8 19.4 5.0 7.1 19.8 10.2 18.6 19.0 4.9 6.4 21.0 9.9 20.4 17.9 5.6 6.0 22.4 10.4 19.6 18.5 5.3 6.5 21.4
+
Average Standard 1.02 0.77 1.o 0.675 0.95 deviation Deviation coefficient 3.7% 4.8% 7.4% 5.1% 3.7% a Calculated with the isotope dilution method using as internal standard nitrobenzene-U- 14C. b Calculated as composite of 36 % of 4-nitrotoluene and 64% of 2-nitro-1,4-dimethylbenzene.
0.392
0.450
7.4%
7.0%
Table 111. Nitrobenzene in the Mainstream Smoke of Cigarettes with KNOs-Addition (ng/W CNitrotoluene 2-nitro4-Nitro-1,3CigaNitro3-Nitro3-Nitro- 1,4-dimethyl- dimethyl4-Nitrorette Weight, mg KNO,, % No. puffs benzene toluene toluene benzene benzene cumene
