Table I. Chromatographic Recovery of 4-Aminobiphenyl
Table 11. Analysis of Various Commercially Available Lots of 2-Aminobiphenyl
Applied, wg
Rocovered, ug
0.020 0.100 0.200 1-00 10 .o 30.0
0.019 0.096 0.220
Lot
% 4-Aminobiphenyl
1 2
1.5
1.04 9.9 29.8
3 4
3 .O
50.0
45 .O
have shown that maximum fluorescence is obtained from the unionized state of each amine. Therefore, all determinations for 4-ABP are made using an excitation wavelength of 290 nm, an emission wavelength of 380 nm, and with absolute ethanol as the solvent. Employing the above conditions, we obtain a linear calibration curve over a concentration range of from 0.2 ppb to 0.5 ppm. Over the linear region, the calculated leastsquares slope of the log-log curve is 1.0. The detection limit, defined as the concentration at which the signal-tonoise is 2, is 0.2 ppb. The extent to which 4-ABP could be recovered from the plate was determined using the calibration curve. The results of this experiment are listed in Table I. From the data in Table I, it is obvious that the extraction procedure is essentially quantitative. Most of the imprecision in the data arises from the use of the micropipets which are reproducible to only about 5%. Obviously, the precision of the method can be increased with the use of more precise sampling devices. Having characterized the procedure, the technique was applied to the analysis of 4-ABP in four different lots of 2-ABP obtained from different commercial manufacturers. The results of this analysis are shown in Table 11. It is ap-
1.1 2.7
parent that all the lots tested contained an appreciable amount of 4-ABP. Since 2-ABP is used as an analytical reagent and since 4-ABP is a potent carcinogen, we recommend that the analyst use this procedure to determine the concentration of 4-ABP in the reagent. LITERATURE CITED (1) J. H. Stender, Fed. Reg/%,38, 20074 (1973). (2) J. H. Stender, Fed. Regis.,39, 3756 (1974). (3) W. F. Melick, H. M. Escue, J. J. Naryka, R. A. Mezera, and E. P. Wheeler, J. Urob, 74, 760 (1955). (4) T. S. Scoti and M. H. C. Williams, Brlt. J. ind. Med., 14, 150 (1957). (5) M. R. Melamed, L. Q. KOSS, A. Rlccl, and W. F. Whitmore, Cancer, 13, 67 (1960). (6) W. F. Melick and J. J. Naryka, Acta Unlo lnt. Contra Cancrum, 18, 277 (1960). (7) J. W. Gorrod and M. J. Carey, Blochem.J., 119, 52P (1970). (8) T. E. Tlmell, C. P. J. Glaudemans, and A. S. Currie, Anal. Chem., 28, 1916 (1956). (9) H. S.Gordon, W. Thornburg, and L. N. Werum, Anal. Chem., 28, 649 (1956). (IO) H. Nakai, H. Demura, and M. Koyama, J. Chromatogr.,68, 87 (1972). (11) S. Shlbata and S. Mlshlma, Bull. YamaguchlMed.Sch., 8, 13 (1962). (12) H. E. Stagg and R . H. Reed, Ana/yst(London),82, 503 (1957). (13) 0.Norman and 0. A. Vaughan, Ana/yst(London),91, 653 (1966). (14) D. A. Rellly, Ana/yst(London), 92, 642 (1967). (15) Y. Masuda and D. Hoffmann, J. Chromatogr.Scl., 7, 694 (1969). (16) J. W. Bridges, P. J. Creaven, and R. T. Williams, Biochem J., 98, 872 (1865).
RECEIVEDfor
review March 12, 1975. Accepted June 19,
1975.
Determination of N’=Nitrosonornicotine in Tobacco by High Speed Liquid Chromatography Stephen S. Hecht, Raphael M. Ornaf, and Dietrich Hoffmann Division of Environmental Carcinogenesis, Naylor Dana Institute for Disease Prevention, American Health Foundation, Valhalla, N.Y. 10595
N’-Nitrosonornicotine (NNN) causes esophageal tumors in rats and lung adenomas in mice ( I , 2). I t has previously been identified in unburned commercial U.S.tobacco products, in amounts ranging from 0.3 to 90 ppm. These concentrations are exceptionally high for an environmental nitrosamine (3-5). The analytical method used so far, however, was somewhat lengthy and not amenable to the routine determination of many samples, such as will now be necessary for further studies concerning the relationship of NNN in tobacco to the observed biological activity of tobacco products. In particular, a rapid method is needed for the study of the formation of NNN, as well as of its reduction in tobacco products. We now report a new and rapid method for the routine determination of NNN, using high speed liquid chromatography. EXPERIMENTAL Apparatus. A Model ALC/GPC-202 high speed liquid chromatograph (Waters Associates, Milford, Mass.) equipped with a Model 6000 solvent delivery system, and a Model LC-25 UVivisi2046
ble detector was employed throughout this study. Mass spectra were run a t 70 eV on an Hitachi Perkin-Elmer RMU-6D instrument (Morgan Schaefer, Montreal, Canada), and on a HewlettPackard Model 5980A Mass Spectrometer. Liquid scintillation counting was done with a Nuclear Chicago Isocap 300 Scintillation System. Reagents. All solvents were spectroquality. NNN-2’-14C and unlabeled NNN were synthesized as described previously (6). Chromatographic cleanup was done by using activity 11-111 basic alumina (Woelm). Toluene solutions with 0.4% PPO (2,5-diphenyloxazole) and 0.005% POPOP (p-bia[2(5-phenyloxazolyl)] benzene) as scintillators gave efficiencies of 8 1 4 3 % for the unquenched 14C-labeled NNN. High Speed Liquid Chromatography. A 6-mm X 30-cm microporasil column (Waters Associates, Milford, Mass.) was used for HSLC determinations of NNN in tobacco. The eluting solvent was cyclohexane-chloroform-methanol 30:68.6:1.4, with a flow rate of 1.0 ml/min, a t a system pressure of 1000 psi. The sample to be injected was dissolved in cyclohexane-2-propanol 3:1, and introduced through a Model U6K septumless injector. The size of the . retention time for NNN was injections ranged from 5 to 8 ~ 1The 13 minutes. For separation of the syn and anti conformers of NNN, a 6-mm X 30-cm microbondapak/Cls column was used with
ANALYTICAL CHEMISTRY, VOL. 47, NO. 12, OCTOBER 1975
TOBACCO EXTRACT IN AOUEOUS ASCORBIC ACID
2) extract (CHCI ))
l l a d i ~ s 10 t DU 5.0 2 ) e i t r o c t (CHCl,)
1
CUROMLtTOGRAPHY
1
Figure 1. Scheme for analysis of N '-nitrosonornicotine in tobacco
' d l
5
10
15
TIME Lmnuterl
20
25
30
Figure 3. High speed liquid chromatogram of N '-nitrosonornicotine in scrap leaf chewing tobacco
fractions (-150 ml) eluted after ~ 2 0 0ml of cold eluant. The NNN fractions were combined and concentrated. An aliquot was counted to determine recovery, and another aliquot was then analyzed by HSLC.
0
5
IO
15
2b
25
30
TIME ( M I N U T E S )
Flgure 2. High speed liquid chromatogram of N '-nitrosonornicotine in fine cut chewing tobacco
50:50 acetonitrile-water as the mobile phase a t a flow rate of 1 ml/min. The retention times for the syn- and anti-NNN conformer were 4.6 and 4.3 min, respectively. Procedure. Tobacco products were obtained on the open market. Snuff and fine cut chewing tobacco were analyzed directly, without grinding. Other products were ground in a blender before extraction. An aliquot was taken for water determination ( 7 ) .For NNN analysis, samples of 5-10 grams were extracted overnight by stirring at room temperature with 50-100 ml 5mM ascorbic acid solution at pH 4.5 (citric acid-sodium phosphate buffer) (4, 8). I4C-NNN (4 X lo5 dpm, 2.5 Fg) was added as internal standard. The resulting suspension was filtered through a 5-micron nylon filter cup (Liquiflo, Inc., Plainview, N.Y.) and brought to pH 5 by addition of a few drops of 10N sodium hydroxide. The filtrate was extracted four times with an equal volume of CHC13. During this step, emulsions were sometimes encountered. These could usually be broken by passing the emulsion through the 5-micron nylon filter again, and allowing the filtrate to separate. The combined CHC13 layers were then concentrated to 70 ml by evaporation a t reduced pressure. The CHC13 solution was then extracted three times with 70 ml of 1N hydrochloric acid. The aqueous phase was again brought to pH 5 a t ice bath temperatures, and then extracted four times with 210 ml of CHC13. The organic phase was dried (NazS04) and concentrated, yielding about 0.2 gram of material, which was then applied to a 10- X 1.5-cm basic alumina column (30 grams alumina) with elution by 3:l benzene-ether. The radioactive
RESULTS AND DISCUSSION The partition scheme used in this method (Figure 1) was designed to take advantage of the difference in basicity between NNN, which is a pyridine derivative (pK = 6), and nicotine, which has both a pyridine and a pyrrolidine ring (pK1 = 6.16; pK2 = 10.96). At pH 5, nicotine is 100% monoprotonated, whereas NNN is less than 5% protonated (9). The partition coefficient between CHCl3 and H20 for nicotine a t this p H is 0.15:1.0, whereas for NNN it is 1.2:l.O. Thus, according to the partition scheme shown, the final CHCls solution (weakly basic fraction) contains 91.5% of the NNN and 18.3% of the nicotine. This partition scheme now allows the use of a short alumina column for final cleanup before high speed liquid chromatography. The response of the UV detector at 254 nm to NNN is linear with peak height throughout the range of interest (>0.5 pg). Typical chromatograms for NNN in fine cut and scrap leaf chewing tobacco are shown in Figures 2 and 3. For positive identification of NNN, the appropriate peak was collected and its mass spectrum determined, as shown previously ( 4 ) . In addition, this material was reexamined using reverse phase high speed liquid chromatography, and the syn and anti conformers of isolated NNN were separated, as shown in Figure 4. The syn and anti assignments were made by comparison to synthetic NNN, for which the ratio of conformers (65:35/anti-syn) could be determined by NMR ( 4 , IO). This aspect of the problem is now under further investigation. The results obtained for five determinations of NNN on snuff tobaccos are summarized in Table I, and the data obtained for other tobacco products are listed in Table 11. All values were determined by using the isotope dilution meth-
ANALYTICAL CHEMISTRY, VOL. 47, NO. 12, OCTOBER 1975
2047
Table I. N'-Nitrosonornicotine in Snuff Tobacco Sample
1 2 3 4
NNN,
u&/ga
11.7 12.8 11.9 11.4 12.9 12.1 0.7 6 .O%
5 Average St dev Re1 std dev a Calculated with the isotope dilution method.
Table 11. N'-Nitrosonornicotinc in Commercial U.S. Tobacco Products
3
5
IO
15
20
Time ( M i n u t e r )
speed liquid chromatogram of N '-nitrosonornicotine isolated from tobacco and reinjected for separation of anti and syn conformers. The anti conformer elutes first
Figure 4. High
od, and recoveries ranged from 60-80%. Using the high speed liquid chromatographic system with UV detection at 254 nm, one can routinely determine amounts as low as 10 ng of pure NNN. For quantitation of NNN within our standard deviation for samples of typical U S . commercial tobacco products, the detection limit is 500 ng of NNN. This limit allows facile measurement of NNN a t the levels now observed. The method described here requires approximately one day per sample, but with multiple samples the analyses can be performed at a considerably quicker rate. Each high speed liquid chromatographic run requires approximately 20 minutes. The major time-consuming step is the partition, in which emulsions are usually encountered. The chromatographic clean-up on alumina generally requires two hours. The method previously used required 2 to 3 days per sample and involved a relatively lengthy alumina chromatography, followed by preparative TLC, and finally by gas chromatography. Based on this study, it appears that high speed liquid chromatography could be used for routine analysis of other relatively nonvolatile nitrosamines, found not only in tobacco products, but also in other environmental materials. Since many nonvolatile nitrosamines, such as "-nitroso-
Product
NNN, t a l &
Cigarette (filter) Cigar Little cigar Scrap leaf chewing tobacco Plug chewing tobacco Fine cut chewing tobacco Snuff
9 .o
5.5 11.2 8.2 4.3
90.6 12.1
nornicotine, are carcinogenic, rapid and quantitative methods are needed for studies concerning their determination and reduction in the environment. LITERATURE CITED (1)E. Boyland, F. J. C. Roe, and J. W. Gorrod. Nature (London). 202, 1126 (1964). (2)D. Hoffmann, R. Raineri, S. S.Hecht, R. R. Maronpot, and E. L. Wynder. J. Natl. Cancer Inst., in press.
(3) D. Hoffmann, S.S. Hecht, R. M. Ornaf. and E. L. Wynder, Science, 188, 265 (1974). (4)S. S. Hecht, R. M. Ornaf, and D. Hoffmann, J. Natl Cancer Inst., 54, 1237 (1975). (5)N. P. Sen, in "Toxic Constituentsin Animal Foodstuffs", I. E. Liener, Ed., Academic Press, New York, N.Y.. 1974,p 131. (6)M. W. Hu, W. E. Bondinell, and D. Hoffmann, J. LabelledCompd., 10,79 (19741. (7)M. Von Bethmann, G. Lipp. and H. Van Nooy, Beitr. Tabakforsch., 1, 19 (1961). (8)S. S. Mirvish. L. Wallcave. M. Eagen. and P. Shubik, Science, 177,65 (1972). (9)K. D. Brunnemann and D. Hoffmann, food Cosmet. Toxicol., 12, 115 ( 1974). (IO)G.J. Karabatsos and R. A. Tailer, J. Am. Chem. Soc., 88, 4673 (1964). ~I
RECEIVEDfor review June 12, 1975. Accepted July 17, 1975. This study was supported by National Cancer Institute Grant SHP-74-106. No. XLI of "Chemical Studies on Tobacco Smoke".
Preparative Trinitrofluorenone Charge-Transfer Chromatography of Petroleum Aromatics D. M. Jewel1 Gulf Research & Development Company, Pittsburgh, Pa. 15230
Although polynitroaromatic molecules have been used frequently as coatings for analytical TLC or HPLC separations ( I ) of aromatic molecules, little effort to develop and apply this area of chemistry preparatively has been made. 2048
Mair ( 2 ) successfully utilized 2,4,7-trinitrofluorenone (TNF) for separating naphthalenes from biphenyls, and Eisenbraun ( 3 ) has found synthetic applications of picrate derivatives. T o better understand the complex structures
ANALYTICAL CHEMISTRY, VOL. 47, NO. 12, OCTOBER 1975
