(26) R. F. Lane and A. T. Hubbard, J . Phys. Chem., 79, 808 (1975). (27) N. Tanaka and R. Tamamushi, Electrochim. Acta. 9, 1963 (1964). (28) J. Jordan and R. A . Javick, Electrochim. Acta, 8, 23 (1962). (29) C. E. Shoemaker, Anal. Chern., 27, 552 (1955). (30) H. Gerischer, Z.Nektrochem., 54, 366 (1950). (31) E. W. Resnick, M.S. Thesis, Iowa State University, Ames. Iowa, 1970. (32) S.Bruckenstein and B. Miller. J. Electrochem. SOC.,117,1040 (1970). (33) F. Anson, Anal. Chem., 33, 934 (1961). (34) S.W. Feldberg, C. G. Enke, and C. E. Bricker, J . Nectrochem. SOC., 110,826 (1963). (35) S.Gilman, “Electroanalytical Chemistry”, A. J. Bard, Ed.. Vol. 2,Chap. 2, Marcel Dekker, New York, N.Y., 1967.
(36) R . N. Adams, “Electrochemistry at Solid Electrodes”, Marcel Dekker, New York, N.Y., 1969,Chap. 7 . (37) K. Schwabe, Nectrochlm. Acta, 8 , 223 (1962). (38) V. A. Zakharov and 0. A. Songina, Russ. J . Phys. Chem., 38, 1336 (1964). (39) R. F. Lane and A. T. Hubbard, Anal. Chern., 48, 1287 (1976).
RECEIVED for review April 8, 1977. Accepted August 8, 1977. The authors are grateful for the support of the National Science Foundation through grant GP-40646X.
Redox Potential and Quinone Content of Cigarette Smoke Irwin Schmeltz,
Jeff Tosk, Genie Jacobs, and Dietrich Hoffmann
Naylor Dana Institute for Disease Prevention, Amerjcan Health Foundation, Valhalla, New York 10595
The redox potential of cigarette smoke Is measured puff by puff electrometrically in a specially designed apparatus. The smoke Is shown to have reducing activity which increases wRh puff number. Smoke constituents, especlally qulnones, which contribute to or are affected by the reducing activity, are examlned. A method Is described for the Isolation, ldentlfication, and quantitatlon of qulnones In smoke, leaf, and pyrolyzates. I t uses solvent partitlan, column chromatography, GWMS technlques, and 9,10-anthraq~inone-9-’~C as Internal standard (80-90 % recovery). 2,3,S-Trlmethyl-1,4-naphthoquinone is the major quinone present In the smoke (220 pgkigarette). 9,lO-Anthraquinone (88 pg), 2-methyl-9,lOanthraquinone ( 190 pg), other naphthoqulnones, and smaller amounts of 1,4-benzoqulnones are also present. I n tobacco leaf, 2-methyl-9,lO-anthraquinone (36 ppm) Is the major quinone. Transfer from the leaf rather than pyrolytlc formatlon appears to be a principal route by which qulnones enter the smoke stream.
Quinones in tobacco and/or tobacco smoke have received little attention. Stedman’s 1968 review on the chemical composition of tobacco and tobacco smoke (1)lists only two quinones, 9,lO-anthraquinone in tobacco (Z),and 2,3,6-trimethyl-1,4-naphthaquinonein smoke ( 3 ) . Enzell and coworkers reported the presence in sun-cured Greek tobacco of t h e same trimethylnaphthaquinone, in addition to 2,3-dimethyl-l,4-naphthoquinone( 4 ) . We noted that both the terpenoid fraction of tobacco, and a-tocopherol generate tetramethyl-1,4-benzoquinoneon pyrolysis (51,and subsequently this same quinone, as well as 2,5-dimethyl-1,4benzoquinone, was reported in cigarette smoke (6). With more than 2500 compounds known to be present in tobacco and tobacco smoke ( I , 6, 7), it is surprising that so few quinones have been reported in these materials. T h e answer may lie in the redox potential (or reducing activity) of the smoke. Although this activity has been noted before (8, 91, its relation to the possible presence or absence of quinones has not been discussed (10). In this paper we describe a method for directly measuring puff by puff the redox potential of the smoke aerosol. This measurement has heretofore been made only on aqueous solutions of the trapped portion of cigarette smoke (8, IO). Moreover, we examine the contribution of several smoke 1924
ANALYTICAL CHEMISTRY, VOL. 49, NO. 13, NOVEMBER 1977
constituents to this activity and, in turn, note how the reducing activity affects various quinones. Some of the latter that are not fully reduced by the smoke and occur there were isolated, identified, and quantitated by a method described herein that uses solvent partition, column chromatography, GC/MS, and a “C-internal standard. The method wm also applied to cured tobacco and pyrolyzates. Our interest in the quinones and in the reducing activity of tobacco smoke is more than academic in that the contribution of the quinones to the tumorigenic activity of the smoke has not been evaluated even though quinones are known to occur in the most active fraction (11). Moreover, the reducing activity of the smoke serves to demonstrate again how dynamic and subject to “change” the chemical composition of the smoke is. Furthermore, the possibility exists that the reducing and biological activities of the smoke are interrelated.
EXPERIMENTAL Apparatus. A 30-channel automatic smoker (Borgwaldt Co., Hamburg, Germany) was chosen to smoke cigarettes for the collection of cigarette “tar”. A single port Borgwaldt piston-type smoker was used for the generation of cigarette smoke aerosol, the redox potential of which was measured with a platinumcalomel electrode pair system in conjunction with an Orion Research Ionalyzer. The potential was determined in a glass apparatus through which the aerosol was directed during smoking (Figure 1). A Hewlett-Packard 7620 A research GC with FID was used for qualitative and quantitative analyses. GC/MS data were recorded on a Hewlett-Packard 5980A Mass Spectrometer interfaced with a Hewlett-Packard 5710A gas chromatograph and a Hewlett-Packard 5933A data system. A Nuclear Chicago Isocap 300 System was used for scintillation counting. Pyrolyses were conducted in a Lindberg Hevi-Duty furnace which housed a Vycor glass tube [(2 X 30 cm; 1511. Reagents. All organic solvents were spectrograde quality, and the other chemicals were of analytical reagent grade. Woelm neutral alumina (Waters Associates, Inc.) was adjusted to the required activity at least 24 h before column packing. Sephadex LH-20 (Pharmacia Co.) was swollen in appropriate solvents 24 h prior to use. Anthracene-9-14C(2.09 mCi/mM) was obtained from Cal Bionuclear Inc. Toluscint, containing 1.2 g/L POPOP (p-bis[2-(5-phenyloxazolyl)]benzene) and 100 g/L PPO (2,5-diphenyloxazole), was diluted with toluene 1:40 and used as the scintillation “cocktail” for measuring @-activity. Reference Compounds. The following reference compounds were obtained from commercial sources: 1,4-benzoquinone, methyl-1,4-benzoquinone, 2,5- and 2,6-dimethyl-l,4-benzoquinone, trimethyl- and tetramethyl-1,4-benzoquinone,1,4naphthoquinone, 2-methyl-l,4-naphthoquinone, 9,lO-anthraquinone, 2-methyl- and
v Salt Bridge ir(C1)
To Recorder
Voltmeter
ilornel
P t Electrode
Cigarette
f
s/.’
Flgure 1. Apparatus for measuring redox potential of cigarette smoke 250
{
was always encountered in the 90% benzene-hexane and benzene fractions. These were combined, reduced in volume, by careful distillation, to about 1 mL, and the concentrate was chromatographed on a Sephadex LH-20 column (2 X 30 cm) using 2propanol as eluting solvent. Fractions of 5 mL were collected. Those containing radioactivity were combined. For purposes of quantitation, the combined solutions were concentrated and rechromatographed on alumina 11-111 using the same elution schedule as for the silica gel column. Radioactivity was found in the benzene fraction. This was again reduced in volume and submitted to GC/MS. Recovery of label was 80-90%. This method was also used to determine quinones in tobacco leaf and in various pyrolyzates. Gas Chromatography. GC analysis (of quinone fractions) was performed on a 10 f t X 0.13 in. (0.d.) stainless steel column containing 10% UCW-98 on Chromosorb W.; column temperature was maintained at 100 “C for 10 min and then programmed to 250 O C at 2’/min. Injector and detector temperatures were set at 275 “C and the helium flow rate at 50 niL/min. Quantitation was achieved by triangulation of appropriate GC peaks. Pyrolysis. Pyrolyses were performed as described previcusly (15).
RESULTS A N D D I S C U S S I O N Reducing Activity. The apparatus developed to measure the redox potential of tobacco smoke consists of a n enclosed smoking chamber containing a platinum electrode which . 1 _ operates in conjunction with a reference calomel electrode O l 2 3 4 5 6 7 8 9 l C I5 PO 25 1c 35 I 2 3 1 5 6 7 8 3 3FDJFFS TlYE i MIN outside the chamber (Figure 1). Only the platinum electrode comes in contact with the smoke aerosol. Unlike electroFlgure 2. Redox potential of the smoke aerosol (from a commerical chemical methods used previously (a),this one measures puff 85 mm, U.S. cigarette, non-filter)as a function of puff number and time by puff the potential of the actual aerosol rather than that 2-ethyl-9,lO-anthraquinoneand 2,3-dimethyl-9,1O-anthraquinone, of the aqueous solution of the smoke. 2,3,6- and 2,6,7-trimethyl-1,4-naphthoquinone were synthesized Redox measurements were recorded during the smoking of from 2,3,6-trimethylnaphthalene,and 1-methyl-9,lO-anthraindividual cigarettes to produce a plot of potential vs. puff quinone from 1-methylanthracene (12). 9,10-Anthraq~inone-9-’~C number (Figure 2). Just prior to the first puff, the observed was synthesized from anthracene-9-14C(12). The counting efpotential (-240 mV) is that of the reference calomel electrode. ficiency of the synthesized material (unquenched) was about 749~. Then, with the first puff, the effect of the smoke aerosol, now Purity of the reference compounds was achieved, where necessary, in contact with the sensing electrode, is noted on the potential. by various chromatographic techniques (TLC, GC) and confirmed A minor rise in potential is seen for the early puff, which is by melting point and by mass spectrometry. possibly due to the initial influx of air into the smoke stream. Procedure, Redox Measurements. The puff by puff As the cigarette is puffed intermittently, the curve slopes measurement of the redox potential of whole smoke was made in an apparatus consisting of a smoke chamber housing a platinum down, indicative of an increase in reducing activity. The electrode (Figure 1). The platinum electrode was connected by increase in reducing activity continues in our setting for about way of a KCl salt bridge to an externally placed calomel electrode 7 min after the cessation of smoking. Then the curve begins Both electrodes were connected to a voltmeter (pH meter). A to rise (indicative of a loss of reducing activity) returning close single port smoking machine drew the cigarette smoke into the t o its initial value about 25 min later (Figure 2). T h e shape chamber so that the smoke stream impinged upon the platinum of the curve may be related to the levels of oxygen present salt bridge interface. The difference in potential between the in the smoke stream during smoking, higher in initial puffs, redox-sensitive interface and the calomel electrode was then but decreasing as the cigarette is smoked down (16-18). On recorded either manually or automatically (Figure 2). Test the other hand, there are likely reducing agents in the smoke chemicals were incorporated into cigarettes by the syringe technique (13) to determine their effect on the redox potential that also affect the reducing activity, in addition to the of the smoke. hydrogen radicals generated during pyrolysis. Moreover, the Quinone Analysis. Two hundred cigarettes were used per concentration of these agents in the smoke is expected to be analysis. These were humidified prior to smoking (24 h, 60% RH, a function of puff number, with higher concentrations present 22 f 2 “C). They were then smoked under standard smoking in later puffs (19). conditions, and the smoke “tar” was collected as previously In an effort to determine which components contribute to (1 wg = 2 X lo4 cpm) described (14). 9,10-Anthraq~inone-9-’~C the reducing activity of the smoke, we spiked cigarettes with was added to the “tar” in the collection traps as an internal ammonia, 1,4-benzoquinone, and hydroquinone (Figure 3). standard. The ‘‘tar”was dissolved in 100 mL 4 1 methanol/water, Ammonia and hydroquinone had little effect on the reducing and the latter extracted four times with cyclohexane (4 X 50 mL). activity, whereas 1,4-benzoquinone had the effect of stabilizing The dried cyclohexane solu.cion was carefully concentrated by fractional distillation, and the resulting 50 mL concentrate was the redox potential, and minimizing the reducing activity. The extracted with dimethylsulfoxide (4 X 50 mL). The dimethylreducing activity of the gas phase was practically nil. (Gas sulfoxide (DMSO) solution was adjusted by the addition of water phase is arbitrarily defined as that portion of the whole smoke to 95% DMSO, and was then washed with cyclohexane (4 X 200 which is not retained by the inert Cambridge glass-fiber filter.) mL). The cyclohexane washings were dried and reduced in volume This indicates that the reducing activity resides primarily in to 5 mL. The cyclohexane extract was chromatographedon a silica the particulate phase of the smoke. Although nicotine has gel (J. T. Baker Chemical Co.) column (100 g, 2 X 30 cm) using been shown to be a reducing agent in certain instances (ZO), the following elution schedule: n-hexane (100 mL), 10% and we observed in model studies that it reacted with 1,4benzene-hexane (100 mL), 25% benzene-hexane (100 mL), 50% benzoquinone to yield hydroquinone and other unidentified benzene-hexane (100 mL), 75% benzene-hexane (100 mL), 90% benzene-hexane (100 mL), benzene (200 mL). The radioactivity species, its contribution to the reducing activity of the smoke :
:
YL
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ANALYTICAL CHEMISTRY, VOL. 49, NO. 13, NOVEMBER 1977
1925
___ --
-
REDOX POTENTIALS _ _ _ ~ ~ 240 GAS PHASE
220
Table I. Effect of Cigarette Smoke “Tar” on Various Quinones and Other Compounds Present in Reduced “smoke” 1,4-Benzoquinone + + Methyl-l,4-benzoquinone + + 2,5-Dimethyl-l,4-benzoquinone +a 2,6-Dimethyl-l,4-benzoquinone Trimethyl-l,4-benzoquinone Tetramethyl-l,4-benzoquinone 1,4-Naphthoquinone Z-Methyl-l,4-naphthoquinone Trimethyl-1,4-naphthoquinonec 9,lO-Anthraquinone 2-Methyl-9,lo-anthraquinone 2,3-Dimethyl-9,lo-anthraquinone Furfural Nitrate
CONTROL COMMERCIAL 85rnrr, NON-FILTER U S CIGARETTEWHOLE SMOKE
-
+ +
+
-
+b
+ + + +b +
-
+ +
-m a
2,3-Dimethyl-1,4-benzoquinone has also been char-
Specific isomer present in Mixture of 2,3,6- and 2,6,7trimethyl-l,4-naphthoquinone.Only the 2,3,6-trimethyl isomer has been identified in the smoke.
acterized in the smoke. smoke not determined.
\
Table 11. Major Quinones in Fresh Cigarette SmokeQ (nglcgt) Peak NO.^ (4) 2,3,6-Trimethyl-1,4-napth~quinone~ 220 i. 17e ( 6 ) 2-Methyl-9,10-anthraquinoneC 190 t 36e ( 5 ) 9,lO-Anthraquinone 88i. 13e ( 3 ) 2,3-Dimethyl-l,4-naphthoquinoned 3 4 i Z e
160 140
a
,
1
1
1
300 7
(1) Tetramethyl-1,4-benzoquinone
U.S. commercial, non-filter cigarette
35f
2 3 4 5 6 7 8 9 PUFF N U M B E R Figure 3. Redox potential of the gas phase of cigarette smoke, and of smoke modified by the presence of added hydroquinone, NH, or 1,4-benzoquinone
mm). * Identified by retention time and mass spectrum, both of which are identical to those of synthetic 2,3,6-trimethyl1,4-naphthoquinone, one of two isomers obtained by the oxidation of 2,3,6+rimethylnaphthalene ( 1 2 ) . Smaller amounts of other trimethylnaphthoquinones, with different retentior. times, are also present in smoke. Identification confirmed by retention time and mass spectrum which are identical to those of authentic Z-methylanthraquinone. The other possible isomer, l-methyl-9,lOanthraquinone, synthesized from 1-methylanthracene ( 1 2 ) , has a shorter retention time. Characterized on basis of mass spectrum; other dimethylnaphthoquinones with different retention times are also present. e Average of 3 values, obtained by the isotope dilution technique. (Internal standard: 9,1O-anthraq~inone-9-‘~C). f Average of 2 values: 3 0 , 40. Calculated as tetramethyl-1,4benzoquinone. g See Figure 6.
may not be major. In cellulose cigarettes (85 mm) spiked with nicotine, only a slight increase in reducing activity (decrease of potential) was noted (Figure 4). Pyrrolidines react with 1,4-benzoquinone in a manner similar to that of nicotine, and since many of these are present in tobacco and tobacco smoke, they may also contribute to the reducing activity of the smoke. Because of its over-all redox potential, smoke “tar” was observed to convert 1,4-benzoquinone to hydroquinone quantitatively (Table I), and this may explain why 1,4benzoquinone has been so elusive a component of cigarette smoke to detect. Other compounds are also reduced by the smoke. Nitrate (in leaf) is known to be converted to ammonia during smoking (22). In the present study, we also observed t h e reduction of methyl-1,4-benzoquinone,1,4-naphthoquinone, and furfural by smoke tar. Quinones. For the isolation of a quinone fraction from cigarette smoke, we developed the scheme outlined in Figure 5 . Initially, 14C-labeled 1,4-benzoquinone was used as an internal standard; however, following either silica gel or Sephadex chromatography, the label appeared in two distinct
fractions. This was due to a partial reduction of the benzoquinone to hydroquinone. Nonetheless, GC/MS analysis of the two fractions resulted in the identification of a number of quinones in aged cigarette “tar”: 2 3 - and 2,5-dimethyll&benzoquinone, trimethyl-1,4-benzoquinone,tetramethyl-1,4-benzoquinone, 1,4-naphthoquinone, and methyl-, dimethyl-, and trimethyl-1,4-naphthoquinones(Table I). Because of the instability of 1,4-benzoquinone in cigarette smoke environments, another internal standard was chosen, which was synthesized from namely 9,10-anthraq~inone-9-~~C anthracene-9-14C(12). The analytical scheme was otherwise identical to that shown in Figure 5. In addition to the quinones identified earlier, GC/MS of this “quinone” fraction of smoke “tar” revealed the presence of anthraquinone and methylanthraquinones; trace amounts of compounds other than quinones were also identified and these included aromatic ketones, aromatic nitriles, indoles, fluorenones, dibenzopyrone, solanone(s), acridine, and others. The method was used for the determination of quinone concentration levels in freshly generated cigarette smoke.
180
160 140
1 4
’r
I
I
1926
ANALYTICAL CHEMISTRY, VOL. 49, NO. 13, NOVEMBER 1977
(85
245 244 24 3 24 2 dmV=
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3 237 236 235
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4
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Figure 4. Redox potential of smoke from ce//u/osecigarette with (A)and without ( 0 )10 mg added nicotine Table 111. Productsa from Pyrolysis of Vitamin K , ( m d g pyrolyzed) 2-Methy1-9,lO - a n t h r a q ~ i n o n e ~ , ~ 12.1 2-Methyl -1,4-naphthoquinonec 9.5 2,3-Dimethyl-l ,4-naphthoquinonec 9.G 2,3,6-Trimethyl-l,4-naphthoquinonec 1.7 9,lO-Anthraquinonec 1.2 See a Isolated amounts; identifications by GC/MS. footnote c , Table 11. Calculated as 9,lO-anthraquinone.
&sy? T / M - W
5%
tip
,F
'
DMSO- H
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,
H
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C H = CYCLOHEXANE
25%
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,
90%
I . SEPHADEX (i-:rn*j
M - W z M E T H A N O L - WATER
H = HEXANE
BzBENZENE
2 A1203 (E) GC/MS
Figure 5. Scheme for isolation of quinone fraction from cigarette smoke (or tobacco)
Here, following chromatography on Sephadex, .the quinone fraction was further enriched on an alumina column giving the gas chromatogram shown in Figure 6. Quinone levels were determined (Table 11) showing that 2,3,6-trimethyl-l:4naphthoquinone is the major quinone present (220 ng/cigarette) in cigarette smoke. Overall quinone levels were relatively low, in the ppb range relative to the weight of tobacco consumed. We expected to find higher levels of tetramethyl- 1,4-benzoquinone based on pyrolysis studies with a-tocopherol in which tetramethyl-l,4-benzoquinone(3.5 mg/g pyrolyzed) is the major product in addition to smaller amounts
of tetramethyl-, trimethyl-, and dimethyl-1,4-naphthoquinones, and C5 and C3-1,4-benzoquinones. The situation in a burning cigarette is somewhat different from that in the pyrolysis furnace, and in the former only minor oxidative degradation of a-tocopherol to quinones occurs and a suhstantial portion of the tocopherol transfers intact into the smoke stream (22). Similarly, although in this study trimethyland dirnethylnaphthoquinones were shown to arise from the pyrolysis of vitamin I(,(Table III), it is conceivable that these compounds, as well as the anthraquinones, are direct transfer products from the leaf ( 4 ) . Interestingly, the major product, 2-methyl-9,10-anthraquinone, from the pyrolysis of vitamin K1 is likely the result of a cyclization mechanism involving the side chain of the vitamin. Our studies with 9,1O-anthraquinone-9-'4L"have shown that by trapping and separation this quinone is not reduced (or otherwise changed), and, therefore, we can assume that ail those quinones with similar or lower redox potentials, as compared to anthraquinone, are likewise not altered in the analytical procedure. When we applied the "quinone" method to processed leaf, we identified relatively large quantities of quinones there (Figure 7, Table IV), the major one being 2-methyl-9,10anthraquinone (36 bg/g tobacco). This is not unusual since 2-methyl-9,lO-anthraquinone is known to occur widely in the plant kingdom (23). In addition to the analytical aspects of redox potential and ANALYTICAL CHEMISTRY, VOL. 49, NO. 13, NOVEMBER 1977
1927
TOOACCO SMOKE ‘QUINONE” FRACTION 4
I . TffRAMETHYLBENZOOUINNE
2. SOLANOM
3. DIYTHYLNAPHTHOOUINONE 4. TRIYETHYLNAPHTHOOUINONE
5 . ANTHRAQUINONE 6. YETHYLANTHRAOUINONE
2
1 2 4
20
6810
40
30
50
60 TIME (min.1-
Figure 6. Gas chromatogram of “quinone” fraction of cigarette smoke
TOB*CCO LEAF oOUINONE‘ FRACTION 1.
YETHY LNAPWTWINONE
2 . DIWETHYLNAPHTHOOUINONE
3 . TRIMETHYLNAPHTHOOUlNOllE 4. YOL.WT.I94(METHYLFWOREI(O(IE-?)
5. ANTHRAQUlNONE 6. 2-METHYLANTHRAOUINON
2
7. DI YETHY LANTH RAWINOM€
0 2 4 6 8 0
20
30
$0
50
h
60
TIME(min)-
Figure 7. Gas chromatogram of “quinone” fraction of processed tobacco Table
IV.
Q u i n o n e s I s o l a t e d from Processed L e a @ ( p p m P
Peak
N0.d (6) (5 ) (2)
(7) (1) (3)
2-Methyl-9,10-anthraq~inone~.~
36.0
9,lO-Anthraquinonec 2,3-Dimethyl-1 ,4-naphthoquinonec C , -Anthraquinonec C,-NaphthoquinoneC 2,3,6-Trimethyl-l ,4-naphthoquinonec
3.0 5.2 2.2
