Quantitative Determination of 1-Naphthylamine and 2-Naphthylamine in Cigarette Smoke Yoshito Masuda and Dietrich Hoffmann Division of Environmental Cancerigenesis, Sloan-Kettering Institute for Cancer Research, New York, N . Y. IO021 A method is described for the analysis of l-naphthylamine (I) and 2-naphthylamine (11) in cigarette smoke. The basic nonvolatiles of the smoke of 300 cigarettes are reacted with pentafluoropropionic anhydride and the resulting neutral components are chromatographed on Florisil. The concentrates of N-pentafluoropropionamides of I and II are analyzed by gas chromatography with an electron capture detector, which has a sensitivity limit of less than 1 ng. 14C4abelledI and II served as internal standards. The mainstream smoke of an 85-mm U.S. nonfilter cigarette contained 2 7 ng of I and 22 ng of II. Although the identification of 2-naphthylamine in cigarette smoke represents the first isolation of a known bladder carcinogen from a non-occupational respiratory environment, no biological significance can as yet be attached to this finding.
THEISOLATION, identification and quantitative determination of primary amines of polynuclear aromatic hydrocarbons in non-occupational respiratory environments presents a difficult and arduous task. In fact, none of these components was so far detected in urban air ( I ) , and only recently Pailer, Hiibsch, and Kuhn ( 2 ) found 1-naphthylamine, an aminofluorene and an aminostilbene in cigarette smoke. Miller and Stedman (3) failed to detect 2-naphthylamine in cigarette smoke. The relatively high reactivity of primary amines, especially of naphthylamines ( 4 , 5 ) , as well as their presumably low concentrations in respiratory environments require special methods and sensitive detection techniques for their analysis. This paper reports the first method for the qualitative and quantitative determination of nanograms of 1-naphthylamine and 2-naphthylamine in cigarette smoke, EXPERIMENTAL
Apparatus. A Wilkens gas chromatograph Model 1525 with flame ionization detector was used to assess the purity of free primary aromatic amines. A Varian Aerograph Model 1200 with an electron capture detector (tritiated titanium as psource) was employed for the quantitative analysis, and a Perkin-Elmer gas chromatograph Model 800 with flame ionization detector for the isolation of unknowns. The 6-radiation of the 1Clabelled internal standards was counted with a Nuclear Chicago Scintillation System 720. The cigarettes were smoked with a Cigarette Components CSM-10 for the quantitative analysis and with a multiple-unit automatic machine for the isolation of naphthylamines (6). 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 70 eV.
(1) D. Hoffmann and E. L. Wynder in "Air Pollution, Vol. II", A. C. Stern Ed., Academic Press, New York, 1968, Chapter 20. (2) M. Pailer, W. J. Hiibsch and H. Kuhn, Fachliche Mitt. Oesterr. Tabakregie, 7, 109 (1967). (3) R. L. Miller and R. L. Stedman, Tobacco Sci., 11, 111 (1967). (4) S. F. D. Orr, P. Sims, and P. Manson, J . Chem. Soc., 1956,1337. ( 5 ) Q. Quick, R. F. Layton, H. R. Harless, and 0. R. Haynes, J . Gas Chromatogr., 6, 46 (1968). (6) E. L. Wynder and D. Hoffmann,"Tobacco and Tobacco Smoke, Studies in Experimental Carcinogenesis," Academic Press, New York, N.Y., 1967. 650
ANALYTICAL CHEMISTRY
The laboratories were illuminated with yellow light (Sylvania Electric Tubes F-40 G.O.) which excludes radiation below 450 mp. Reagents. All organic solvents were spectrograde, the other chemicals of analytical reagent grade. Pentafluoropropionic anhydride (ng = 1.273) was obtained from Peninsular Chemresearch, Inc. and used without further purification. Florisil (6C-100 mesh) was obtained from Fisher Scientific Co., basic alumina Woelm (activity 111) from Alupharm Chemicals, Gas Chrom Q (80-100 mesh), Chromosorb W (60-80 mesh), Silicon QF-1, DC-200 and Siponate DS-10 from Applied Science Laboratories. Internal Standards. 1-Naphthylamine-l-1C (sp. activity 2.0 mCi/mmol) and 2-naphthylamine-8-14C (sp. activity 3.58 mCi/m mol) were purchased from Mallinckrodt Nuclear, purified before use and their purity ascertained by GLPC. Toluene solutions with 0.4 % PPO (2.5-diphenyloxazole) and 0.005 POPOP (p-bis [2(5-phenyloxazole)] benzene) as scintillators gave counting efficiencies of 72.5% for the unquenched 14C-labelled naphthylamines. Pentafluoropropionamides of Naphthylamines. The electron capture system has been reported to be most sensitive for the detection of certain polyfluorinated organic compounds (7). The freshly purified amines were dissolved in dry ether and reacted with magnetic stirring with a 5 0 x excess of pentafluoropropionic anhydride. The solution was kept for 18 hr at room temp., extracted with 2N hydrochloric acid, sat. sodium bicarbonate solution and then dried (NazS04). The residue was recryst. from n-hexaneibenzene (4:l). The yields for the pentafluoropropionamides (PF) of 1-and 2-naphthylamines were better than 95%; the same high yields were observed in the presence of nicotine in an excess of 1000 parts. Anal. N-1-Naphthylpentafluoropropionamide mp 132 "C cor. Calcd for Cl3H8F5NO:C,53.98; H, 2.79; F, 32.84; N, 4.84. Found: C, 54.10; H, 3.04; F, 32.65; N, 4.72. N-2-Naphthylpentafluoropropionamidemp 150 "C cor. Calcd for CllH8FsNO:C,53.98. H, 2.79; F. 32.84; N, 4.84. Found C, 53.74; H, 3.00; F, 32.55; N, 4.66. Gas Chromatography. The primary amines were purified on basic alumina, recrystallized, and checked for purity by GLPC at 175 "C on 3mm by 2.5-m column filled with 10% Siponate DS-10 on Chromosorb W. The P F of 1-and 2naphthylamine can be separated at 145 "C on glass columns (3-mm by 1.7-m) filled with 7.5% QF-1 and 5% DC-200 on Gas Chrom Q. The retention times were 7.1 and 9.5 min, respectively. Figure 1 presents the electron capture sensitivity to nanograms of the PF of 1- and 2-naphthylamine. For the gas chromatographic separation of the N-1 and N-2 naphthylpentafluoropropionamides from a concentrate from cigarette smoke, a 2-m glass column of 6-mm diameter was used and the liquid phases and support were as described above; the temperature was kept at 160 "C; the column exit was connected with a glass splitter which led 10% of the effluent to a flame ionization detector and the remainder was collected. Procedures. A. ISOLATION OF THE PF OF 1-AND 2-NAPHTHYLAMINES. A total of 12,000 cigarettes were smoked with a multiple unit automatic machine at a rate of one puff per
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( 7 ) D. D. Clarke, S. Wilk, and S. E. Gitlow, J . Gas Chromatogr., 4,
310 (1966).
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Figure 1. Linearity curve for N-l-naphthylpentafluoropropionamide (a)and N-2-naphthylpentafluoropropionamide(p) minute of 2-sec duration and an average puff volume of 35 ml; butt length was 23 mm. The mainstream smoke was drawn through a series of 4 wash bottles (500 ml each) filled with 7 HCl in methanol/water (1 :1) and immersed in ice water. The traps were washed with methanol and the combined suspensions filtered and concentrated; the filtrate (1 1) was diluted with an equal volume of water, three times extracted with ether, 1 1 each, was made basic with 3N sodium hydroxide, and seven times extracted with ether (1 1 each). The ethereal extract (-30 grams) was dried (Na2S04) and concentrated to 250 ml. To this solution was added 100 grams of pentafluoropropionic anhydride under ice cooling and magnetic stirring; the solution was then kept at room temperature for 20 hr. The neutral portion was separated and the basic portion dissolved in 200 ml of ether and treated again with pentafluoropropionic anhydride (20 grams). The combined neutral portions were dried (Na2S04)and their residues (-8 grams) dissolved in n-hexanelbenzene (4:l; 50 ml) for chromatography on Florisil (300 grams) with n-hexane, n-hexanelbenzene (8:1, 4:1, 2:1, 1 :1) and benzene as eluting solvents. 250 fractions (25 ml each) were obtained of which all those eluted with n-hexane, benzene 1:l were combined. Their concentrate was rechromatographed on 30 Cigarette Smoke
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Figure 3. Mass spectra of N-l-naphthylpentafluoropropionamide grams of Florisil. Every fifth fraction of 100 fractions (20 ml each) of the n-hexane-benzene (2:1, 1 :1) and benzene eluates was analyzed for PF by a gas chromatograph with an electron capture detector. The fractions with maxima corresponding to retention times of the PF of 1-and 2-naphthylamine were combined for the isolation of the derivatives. B. QUANTITATIVE ANALYSIS. A total of 300 cigarettes were smoked individually under standard conditions with a Model CSM-10 (6). The mainstream smoke was directed through a series of 3 gas wash bottles filled with 150 ml of 3.5 HCI in methanol. The smoke suspensions in the gas wash bottles were combined with the rinsing solvent (-500 ml) and 1.5 pg of 1-naphthylamine lJ4C (or 1.5 pg 2-naphthylamine-8-14C) in 1 ml of toluene was added and filtered. The filtrate and washings were concentrated to 50 ml, diluted with 100 ml of H 2 0 and extracted three times with 100 ml ether each. The acidic layer was made basic with 3N sodium hydroxide and seven times extracted with 100 ml ether. The combined extracts were dried (Na2S04),evaporated to about 30 ml and reacted with 1.5 grams of pentafluoropropionic anhydride. After standing for 20 hr, the solution was extracted with 2N hydrochloric acid and 5 % sodium bicarbonate solution. The ether containing the neutral portion was dried, evaporated, dissolved in 5 ml of n-hexanelbenzene (4:l) and chromatographed on Florisil (30 grams). The fractions were eluted with n-hexane, n-hexanelbenzene (8:1, 4:1, 2:1, 1:l) and benzene. The eluates with n-hexanelbenzene 2:1,1:1 contained in separate fractions the PF of I-and 2-naphthylamine. The fractions with P-radioactivity were combined, concentrated to 1-2 ml, and five 1-pl samples were injected for GLPC analysis. Three aliquots of 100-200 pl were taken for liquid scintillation counting. Figure 2 summarizes the analytical separation procedure.
z
RESULTS AND DISCUSSION
Figures 3 and 4 compare mass spectra of synthetic and isolated N-1 , and N-2-naphthylpentafluoropropionamides. These data and the retention times and retention volumes from two gas chromatography systems served for the identification of 1-naphthylamine and 2-naphthylamine. For the VOL. 41, NO. 4, APRIL 1969
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quantitative analysis, three times 300 cigarettes were smoked for each of the amines. From the final gas chromatograms (Figure 5) it was calculated that the smoke from an 85-mm, U.S. blended cigarette without filter tip contained 27 ng of 1-naphthylamine and 22 ng of 2-naphthylamine (Table I). As experienced before for trace components in cigarette smoke (
