Chem. Res. Toxicol. 1991,4, 513-516
513
The Nitrosation of Nicotine: A Kinetic Study William S. Caldwell,* Jackie M. Greene, David R. Plowchalk, and J. Donald deBethizy Research and Development, R. J. Reynolds Tobacco Company, Winston-Salem, N o r t h Carolina 27102 Received J a n u a r y 30, 1991
Introduction It has been recommended that cigarettes which yield low "tar" and medium levels of nicotine be developed to minimize "tar" exposure in smokers (1-3). However, this recommendation has been called into question partly on the basis of the hypothesis that nicotine can be nitrosated in vivo (4,5). The reaction of nicotine with nitrous acid in aqueous solution to produce three tobacco-specific nitrosamines (TSNA)' has been reported (6),but to date, no kinetic studies of this reaction have appeared. We now report such a study. The reaction of nicotjne with nitrous acid results in the formation of three products, 44Nmethylnitrosamino)-4-(3-pyridyl)-l-butanal(NNA), nitrosonornicotine (NNN), and 4-(N-methylnitrosamino)1-(3-pyridyl)-l-butanone(NNK) (Scheme I). For these three parallel reactions, the rate constants are kmA, k m , and kNNK. In their study of this reaction, Hecht et al. (6)found that when nicotine was reacted with nitrite at pH 3.4 and 20 O C , 80-90'70 of the nicotine remained unreacted after 17 h. Under these conditions, the yield of NNA was over 5-fold higher than the yield of NNN and NNK. When the reaction was allowed to proceed under more vigorous conditions, NNA was no longer detected, and they identified 14 other products resulting from secondary reactions of NNA and NNK and from fragmentation and oxidation of the pyrrolidine ring. In this study, we determined the order of the reactions in nicotine and nitrite and the rate constants for the three parallel reactions at pH 3.5 and 37 "C. Materials and Methods Materials. Caution: Nitrosamines are animal carcinogens and should be handled with care. 4-(N-Methylnitrosamino)4-(3-pyridyl)-l-butanal (NNA), 4-(N-methylnitrosamino)-1-(3pyridy1)-l-butanone (NNK), and N-nitrosonornicotine (NNN) were obtained from the NCI Chemical Carcinogen Repository, Midwest Research Institute (Kansas City, MO) and used as supplied. Sodium nitrite, dibasic sodium phosphate, citric acid, ammonium sulfamate, boric acid, sodium hydroxide, triethylamine, and acetic acid were obtained from Aldrich Chemical Co. (Milwaukee, WI) and were of the highest purity available. High-purity methanol, methylene chloride, and acetonitrile were obtained from Burdick and Jackson Labs (Muskegan, MI). All water used for this study was purified with a Milli-Q Water System (Millipore Corp., Bedford, MA) and had a resistance of a t least 18 MQ-cm. Nicotine was obtained from Eastman Kodak Co. (Kingsport, TN), distilled in vacuo from solid sodium hydroxide (bp 89.0-90.5 OC at 2-3 Torr), and stored in amber vials under dry nitrogen at -20 OC until used. The purity of the nicotine used for this study was >99.9% as determined by capillary gas chromatography. Nitrosation of Nicotine. Nicotine was reacted with sodium nitrite in a round-bottom flask equipped with a reflux condenser, Abbreviations: TSNA, tobacco-specific nitrosamines; NNA, 4-(Nmethylnitroeamino)-4-(3-pyridyl)-l-butanal; NNN, nitrosonornicotine; HPLC,highNNK, 4-(N-methylnitrosamino)-1-(3-pyridyl)-l-butone; performance liquid Chromatography;ODs, octadecylsilane; GC-MS, gas chromatography-mass spectrometry.
Scheme I. Reaction of Nicotine and Nitrous Acid 0 N,N/C
I
Ha
NNA
NNN
N I COT I NE
R
NO
I
N NNK
a sideann stopcock, and a thermometer. The reaction was carried out under an atmosphere of dry, oxygen-free nitrogen. Stirring was accomplished with a pneumatically driven magnetic stirrer, and the reaction vessel was immersed in a thermostated water bath maintained at 37 "C. Solutions of the two reactants in degassed citrate-phosphate buffer (400 mM citrate, 200 mM phosphate) were prepared separately, and the pH was adjusted to 3.5 with saturated citric acid or 10% sodium hydroxide. The reactions were started by mixing the two solutions which had been prewarmed to 37 OC. Aliquots were withdrawn through the side-arm stopcock initially, and a t appropriate intervals throughout the course of the reaction. Aliquots used for the determination of nitrosamines were quenched by mixing with 10 parts of 500 mM ammonium sulfamate in 100 mM borate buffer, pH 9.0, and aliquots for nitrite determination were diluted with 10 parts of water and analyzed immediately. The concentration of nitrite in the aliquots was determined with a Model EA940 ion analyzer using a nitrite ion selective electrode (Orion Research, Inc., Boston, MA). The concentrations of nicotine, NNA, N", and NNK in the aliquots were determined with a System 42 analytical HPLC (Gilson Medical Electronics, Inc., Middleton, WI). Samples for HPLC analysis were contained in amber vials and placed in a thermostated sample holder maintained at 5 "C to eliminate the problem of sample degradation during analysis. The HPLC was equipped with a Spherisorb ODS 5-pm column (4.6 X 250 mm) (Regis Chemical Co., Morton Grove, IL). The solvents used for HPLC were as follows: solvent A, 25% acetonitrilelmethanol (1:l)-75% 29.4 mM acetate buffer (pH 4.5) adjusted to pH 7.4 with triethylamine; solvent B, 99% acetonitrile1 % triethylamine. Analytes were eluted with the following gradient profile: 100% solvent A for 5 min; 2-min linear gradient to 80% solvent A-20% solvent B; isocratic elution for 6 min; and return via linear gradient to initial conditions over a 2-min period. Detection was accomplished with a Model 116 W detector ( G h n Medical Electronics, Inc., Middleton, WI) a t 254 nm, and data collection and analysis were performed with a Peak Pro 1.1 chromatography system (Beckman, Waldwick, NJ) using external standardization. To confirm the identities of the nitrosamine peaks,the appropriate fractions were collected and extracted with methylene chloride. The extracts were concentrated and analyzed by GC-MS using a Model HP5890 gas chromatograph (Hewlett Packard Co., Avondale, PA) equipped with a fused silica DB-5 column (30 m X 0.32 mm i.d., film thickness 1.0 pm) (J&W
0893-228~/91/2704-0513$02.50/0 0 1991 American Chemical Society
514 Chem. Res. Toxicol., Vol. 4, No. 5, 1991 700
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1
0
Communications A
w
5
10
15
20
Retention Time (min) Figure 1. HPLC chromatograms of mixtures of NNA, NNN, NNK, and nicotine. (A) Mixed standard ([NNA] = 0.50 mM, [NNN] = 0.65 mM, [NNK] = 0.50 mM, [nicotine] = 10 mM). (B) Aliquot taken from a nicotine nitroeation reaction mixture (100 mM nicotine, 400 mM nitrite, pH 3.5 citrate-phosphate buffer) after 3 h at 37 O C .
Scientific,Fohm, CA) and coupled to a Model 5970 mass-selective detector (Hewlett Packard Co., Avondale, PA).
Results Typical chromatograms of a mixed standard of NNA, NNN, NNK, and nicotine and an aliquot from a nitrosation reaction mixture are shown in Figure 1. Adequate resolution of nicotine and the three TSNA products was achieved by using this chromatographic method. A shoulder is evident on the NNA and NNK peaks in chromatograms of both the standard and the nitrosation reaction mixture. The HPLC separation of E-(anti) and 2-(syn) isomers of NNN and NNK has been reported (7, 8),and in both cases the E / Z ratio was approximately 73/27. The ratio of the area of the large NNK peak to that of the shoulder is 74/26, so it is likely that the isomers have been partially separated by using this method. The sum of the areas of the major peak and its corresponding shoulder was used for quantification of NNA and NNK. The chromatogram of the nitrosation reaction mixture contains as yet unidentified peaks early in the chromatographic run. These peaks, which are clearly resolved from the TSNA peaks, probably represent some of the products of oxidation and fragmentation of the pyrrolidine ring and the secondary reaction products identified by Hecht et al. (6). To confirm the identities of the nitrosamine peaks in the HPLC, appropriate fractions were collected from the HPLC of nitrosation reaction mixtures, extracted with methylene chloride, and analyzed by GC-MS. The GCMS chromatograms of reaction mixtures contained peaks with the same retention times and mass spectra as authentic TSNA standards. While GC-MS revealed the presence of a number of impurities (most notably octyl phthalates) in samples isolated from reaction mixtures,
-z E
Y
Y
z
0.4
-L-J +100 mM Nitrite
02
200 mM Nitrite
0.0
0
1
2
3
4
5
8
Reaction Time (min x 10 .3) Figure 2. Time course of formation of NNA, NNN, and NNK in reaction mixtures consisting of 100 mM nicotine, pH 3.5 citrate-phosphate buffer, and 100 and 200 mM nitrite at 37 O C .
these impurities were also present in samples isolated from HPLC chromatograms of blank injections prepared in the same fashion. None of these impurities were pyridinecontaining materials. To confirm that no materials formed from nonnitrosative reactions during nitrosation coeluted with the TSNA's in the HPLC, two control reactions were carried out. One reaction contained nicotine and no nitrite and the other nitrite with no nicotine. In both cases, HPLC analyses of the coptrol reaction mixtures over time revealed no UV-absorbing peaks that coeluted with authentic TSNA standards. The time course for formation of "A, NNN, and NNK during nicotine nitrosation revealed complex kinetics (Figure 2). As the reaction proceeded, the rate of product formation decreased, due to decomposition of nitrite and the aforementioned side reactions of nicotine, NNA, and NNK. Due to the complex chemistry occurring under the reaction conditions, it was necessary to use the initial rate method for studying the kinetics of nicotine nitrosation. For this method, the reaction was allowed to proceed to no more than 10% completion and plot of product concentration vs time was made. The slope of the line at zero time gave the initial rate. The initial rate of NNA formation was on the order of lo4 mol/(L.min) and the initial rates of NNN and NNK formation were on the order of lo-' mol/(L.min). The variations of the initial rates of product formation with reactant concentrations were used to determine the order
Chem. Res. Toxicol., Vol. 4, No. 5,1991 515
Communications
b -
2
x 150
.-C E
2
Table I. Rate Constants for Formation of NNA, NNN, NNK, and TSNA from Nicotine at pH 3.5 and 37 O c a product k, Xl@ k Ib NNA 50 f I 38 6 5.2 k 1.2 3.9 0.8 NNN NNK 6.8 k 1.2 5.1 f 0.9
180 L
120
-
0
'
+
-
NNA NNK NNN
**
-
E 9 0
Y
TSNA
k ' = k( 1 0
30
60
90
120
150
210
180
[Nicotine] (mM) Figure 3. Variation of initial rate of formation of NNA, NNN, and NNK with nicotine concentration at 37 O C and pH 3.5. [Nitrite] = 200 mM.
s)
+e lO-PKNc) 1+ (
270
HNO,
+ HX
R3N + ONX
0
+
.E 210
2 - 180 150 ; 120
F$NNO'
i_/
K, z== ONX + H20 K2
H'+X-
= F$NNO'+ k3
%
-'' k4
R~=CHR'
c
(2)
Scheme 11. Proposed Kinetic Mechanism for the Nitrosation of Nicotine
HX b -
41 f 6
62 i 8
'Values in L/(moLmin) f standard deviation (n = 8). *k'was calculated from the equation:
X'
+
R2N=CHR+ HNO PRODUCTS
$ 9 0
X- = NO; , H20 0
(6) that NNA is the dominant nitrosamine formed from nicotine a t pH 3.4 and 20 "C.
100200300400500600
[Nitrite] (mM) Figure 4. Variation of initial rate of formation of NNA, NNN, and NNK with nitrite concentration at 37 "C and pH 3.5. [Nicotine] = 100 mM.
of the reactions in nicotine and nitrite. The nitrosation of nicotine was first order in both nicotine (Figure 3) and nitrite (Figure 4) for the three parallel reactions. These reactions follow the general rate law: rate = k[nicotine][NaN02] (1) This differs from the nitrosation of secondary amines, which is first order in amine and second order in nitrite, but is consistent with the findings of Gowenlock et al. (9), who found that the nitrosation of a number of trialkylamines is first order in both reactants. An intrinsic rate constant, k', which reflects the rate of the reaction in terms of un-ionized amine and nitrous acid, can be calculated from the rate constant k by
(
k ' = k 1+- 1 0 - p ~ 10-PKNr
)(
l+-
j
Dlscusslon The kinetic mechanism for the nitrosation of tertiary amines developed by Smith and Loeppky (12) and Gowenlock et al. (9)can be applied to the nitrosation of nicotine (Scheme 11). This mechanism includes the equilibrium formation of an activated nitrosating species, here desig nated ONX, which may be NzO3 or H2N02+. Nicotine (R3N)undergoes a reversible nitrosation to form &NNO+, and in the rate-determining step, this intermediate undergoes a slow elimination of HNO to produce an iminium ion. The iminium ion then rapidly reacts to form products. This mechanism differs from that for nitrosation of secondary amines in that here the rate-limiting step is not the initial nitrosation (k3), but rather the loss of HNO (k4). From this mechanism an equation for the rate of product formation can be derived:
(
rate = K1K3k4 _)IH*I[R3NJ[HNO2]
(3)
(2)
where pK, is the pK of nitrous acid and pKNic is the pK of nicotine. We used eq 2 to calculate k'for the formation of each product from o w experimentally determined values of k and pKs of 3.37 and 8.02 for nitrous acid (10) and nicotine (11),respectively, which were obtained from the literature. The values of k and k'for the formation of NNA, NNN, NNK, and total tobacco-specific nitrosamines by the reaction of nicotine with nitrite at pH 3.5 and 37 "C are presented in Table I. The rate constants for formation k"N, and k"K. of TSNA are merely the sum of k"A, The rate constant for formation of NNA is an order of magnitude greater than that for formation of NNN and NNK, in agreement with the observation of Hecht et al.
This mechanism is consistent with our results in that it predicts an order of 1for both nicotine and nitrite. It also predicts a pH dependence for k', which is currently being tested. Recently, Hecht et al. (13) reported that no hemoglobin adducts of NNN and NNK could be detected in rata treated with nicotine, implying that no in vivo nitrosation of nicotine occurred in that animal model in the absence of added nitrite. Nornicotine, which is nitrosated much more rapidly than nicotine (141, has been reported to be a metabolite of nicotine in a number of species (15-17); however, the relative importance of nornicotine, formed metabolically from nicotine, as a precursor to NNN remains to be determined. Given the very slow rate of nicotine nitrosation as determined in this study and the
516 Chem. Res. Toxicol., Vol. 4, No. 5, 1991
observation that nicotine is not nitrosated in simulated or fresh gastric juice and saliva even at a nicotine concentration of 0.5 mg/mL (18, 19), it is unlikely that nicotine itself contributes to exposure to nitroso compounds due to chemically mediated intragastric nitrosation.
Acknowledgment. We thank Gary Byrd for performing the mass spectral analyses.
References (1) Russell, M. A. H. (1980)The case for medium-nicotine, low-tar, low carbon monoxide cigarettes. In Banbury Report 3. A Safe Cigarette? (Gori, G. B., and Bock, F. G., Eds.) pp 297-310,Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. (2) Froggatt, P. (1988)The product modification program. In Fourth Report of the Independent Scientific Committee on Smoking and Health, pp 1-15,Her Majesty's Stationary Office, London. (3) Editorial (1991)Nicotine use after the year 2000. Lancet 337, 1191-1192. (4) Hoffmann, D., and Hecht, S. S. (1985)Nicotine-derived Nnitrosamines and tobacco-related cancer: Current status and future directions. Cancer Res. 45, 935-944. (5) Hecht, S.S.,and Hoffmann, D. (1988)Tobacco-specific nitrosamines, an important group of carcinogens in tobacco and tobacco smoke. Carcinogenesis 9, 875-884. (6) Hecht, S.S.,Chen, C. B., Ornaf, R. M., Jacobs, E., Adams, J. D., and Hoffmann, D.(1978)Reaction of nicotine and sodium nitrite: Formation of nitrosamines and fragmentation of the pyrrolidine ring. J. Org. Chem. 43, 72-76. (7) Hecht, S. S.,Chen, C. B., Dong, M., Ornaf, R. M., Hoffmann, D., and Tso, T. C. (1977)Chemical studies on tobacco smoke LI: Studies on non-volatile nitrosamines in tobacco. Beitr. Tabakforsch. 9, 1-6. (8) Hecht, S.S.,Adams, J. D.,and Hoffmann, D. (1983)HPLC-TEA of tobacco-specific nitrosamines. In Environmental Carcinogens Selected Methods of Analysis, N-Nitroso Compounds (Egan, H., Preussmann, R., O'Neill, I. K., Eisenbrand, G., Spiegelhalder, B., and Bartsch, H., Eds.) pp 429-436,International Agency for Research on Cancer, Lyon, France. (9) Gowenlock, B. G., Hutchison, R. J., Little, J., and Pfab, J. (1979) Nitrosative dealkylation of some symmetrical tertiary amines. J.
Communications Chem. Soc., Perkin Trans. 2, 1979, 1110-1114. (10) Weast, R. C., Astle, M. J., and Beyer, W. H., Eds. (1984)Dissociation constants of inorganic acids in aqueous solutions. In CRC Handbook of Chemistry and Physics, . 65th ed.,. D - D167, CRC Press, Boca Raton. (11) Weast, R. C., Astle, M. J., and Beyer, W. H., Eds. (1984)Dissociation constants of organic bases in aaueous solution. In CRC Handbook of Chemistriand Physics, (5th ed., pp D163-D165, CRC Press, Boca Raton. (12)Smith, P. A.S., and Loeppky, R. N. (1967)Nitrosative cleavage 89, 1147-1157. of tertiary amines. J. Am. Chem. SOC. (13) Hecht, S. S.,Kagan, M., Kagan, S. S., and Carmella, S. G. (1989)Quantitation of 4-hydroxy-l-(3-pyridyl)-l-butanone in human hemoglobin as a dosimeter for exposure to carcinogenic tobacco specific nitrosamines. Poster No. P-34,Tenth Intemational Meeting on N-Nitroso Compounds, Mycotoxins and Tobacco Smoke: Relevance to Human Cancer. IARC, Lyon, France, Sept 25-28. (14) Mirvish, S. S.,Sams, J., and Hecht, S. S. (1977)Kinetics of nornicotine and anabasine nitrosation in relation to "-nitrosonornicotine occurrence in tobacco and to tobacco-induced cancer. J . Natl. Cancer Znst. 59, 1211-1213. (15) Kyerematen, G. A,, Taylor, L. H., deBethizy, J. D., and Vesell, E. S. (1988)Pharmacokinetics of nicotine and 12 metabolites in the rat. Application of a new radiometric high performance liquid chromatography assay. Drug Metab. Dispos. 16, 125-129. (16) Nwosu, C. G., Godin, C. S., Houdi, A. A,, Damani, L. A., and Crooks, P. A. (1988)Enantioselective metabolism during continuous administration of s-(-)- and r-(+)-nicotine isomers to guinea-pigs. J . Pharm. Pharmacol. 40, 862-869. (17) Kyerematen, G. A.,Morgan, M. L., Chattopadhyay, B., deBethizy, J. D.,and Vesell, E. s. (1990)Disposition of nicotine and eight metabolites in smokers and nonsmokers: Identification in smokers of two metabolites that are longer lived than cotinine. Clin. Pharmacol. Ther. 48,641-651. (18) Tricker, A.R., Haubner, R., Spiegelhalder, B., and Preusamann, R. (1988)The occurrence of tobacco-specific nitrosamines in oral tobacco producta and their potential formation under simulated gastric conditions. Food Chem. Toricol. 26, 861-865. (19) Tricker, A. R., and Preussmann, R. (1991)Exposure to nicotine-derived N-nitrosamines from smokeleas tobacco and evidence against their endogenous formation. In Effect of Nicotine on Biological Systems (Adlkofer, F., and Thurau, K., Eds.) pp 109-113,Birkhauser Verlag, Basel.
