XENOBIOTICA,
1990, VOL. 20,
12, 1353-1356
NO.
Identification of cis-3’-hydroxycotinine as a urinary nicotine metabolite P. VONCKEN, K. RUSTEMEIER and G. SCHEPERS INBIFO Institut fur biologische Forschung, Fuggerstrasse 3, D-5000 Koln 90, FRG Xenobiotica Downloaded from informahealthcare.com by University New South Wales on 08/26/15 For personal use only.
~
Received 2 Janua~y1990, accepted 30 June 1990 1. cis-3’-Hydroxycotinine was detected as an S(-)-nicotine metabolite in the urine of smokers as well as in the urine of rats and hamsters dosed with nicotine.
2. The excreted amount of cis-3’-hydroxycotinineis lower than that of the trmrs-isomer.
Introduction Hydroxycotinine, a nicotine metabolite, was isolated three decades ago from the urine of dogs (McKennis et al. 1959), rats (McKennis et al. 1962), and humans (Bowman and McKennis 1962) dosed with nicotine or cotinine. The exact structure and stereoconformation of metabolic hydroxycotinine was established by Dagne and Castagnoli (1972) to be tran~-3‘-hydroxycotinine. However, it was not until recently that trans-3‘-hydroxycotininewas recognized as a major nicotine metabolite in the urine of smokers (Neurath et 01. 1987, Jacob et al. 1988) as well as in the urine of guinea pigs, Syrian hamsters, and rabbits following i.p. nicotine administration (Nwosu and Crooks 1988). The presence of cis-3’-hydroxycotinine as a urinary nicotine metabolite has not been reported to date, although O’Doherty et al. (1988) analysed the urine of a smoker for the presence of the cis-isomer but did not detect it, probably due to the detection limit of their h.p.1.c. method. Using capillary g.1.c.-mass spectrometry and h.p.l.c., we were able to separate and identify cis-3‘-hydroxycotinine as an S( -)-nicotine metabolite in the urine of rats and hamsters dosed with nicotine, as well as in the urine of smokers.
Materials and methods cis- and truns-3’-Hydroxycotinine were obtained from the Institut fur Biopharmazeutische Mikroanalytik, G. Neurath (Hamburg, FRG). For g.1.c.-mass spectrometric analyses a gas chromatograph (Hewlett-Packard, Model 5890) coupled to a computer-controlled mass spectrometer (Hewlett-Packard, Model 5990B) was used. A DB-1701 or DB-210 fused silica capillary column (30m x 025 mm; J&W via Carlo Erba, FRG) was directly coupled to the ion source (ionization voltage 70eV). For the h.p.1.c. analysesbased on the method described by Barlow et al. (1987) a liquid chromatograph (Hewlett-Packard, Model 1090M) and a Nova-Pak C18 column (15 cm x 3.9mm; Millipore, FRG) were used. As the mobile phase, sodium pentane sulphonate in phosphoric acid and the solvents methanol, tetrahydrofuran, and acetonitrilewith a ternary gradient were used. U.V.detection was performed at 532 nm. Full details of the g.1.c. and h.p.1.c. method will be reported elsewhere. Following the i.v. administration of 0.8 mg S(-)-nicotine/kg body weight to male Sprague-Dawley rats and 2.5 mg S( -)-nicotine/kg body weight to male Syrian hamsters, urine samples were collected for 24 h. From smokers with a daily consumption of 05-2 cigarette packs, random samples of urine were taken. Extraction of the urine samples for g.1.c. analyses was performed as described for the simultaneous determination of trans-3’-hydroxycotininewith nicotine and cotinine (Voncken et ul. 1989). 0049-8254/90 $3.00 0 1990 Taylor & Francis Ltd
P. Voncken et al.
1354
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u 61 c Xenobiotica Downloaded from informahealthcare.com by University New South Wales on 08/26/15 For personal use only.
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Figure 1 . Capillary gas chromatogram of hamster urine ( 0 ) and mass spectra of ~&3’-hydroxycotinine(6). (a)Chromatogram of a hamster urine sample following intravenous administration of nicotine (mass selective detection, selected ion monitoring, m / z 192): peaks: 1 = trans-3’-hydroxycotinine; 2 = cis-3’-hydroxycotinine.(6) Mass spectra of cis-3’-hydroxycotinine:A = from urine sample of an
S( -)-nicotine dosed hamster; B =from reference substance.
IdentiJication of urinary cis-3'-hydroxycotinine
1355
Xenobiotica Downloaded from informahealthcare.com by University New South Wales on 08/26/15 For personal use only.
Results and discussion Using the capillary g.1.c. method we were able to separate the trans- and cisisomers of 3'-hydroxycotinine. The retention times of the trans- and cis-isomer on the DB-1701 column were 20.8 and 21.2 min. On the DB-210 column the retention times were 1 6 2 and 1 6 9 min, respectively. The cis-isomer was detected as an S( -)nicotine metabolite in the urine of smokers and in the urine of rats dosed with nicotine intravenously, orally, or by inhalation, and in hamsters dosed intravenously (see figure 1(a)). The cis-isomer was identified in the urine of rats and hamsters by g.1.c.-mass spectrometry. Its full scan mass spectrum was compared with that of the pure, chemically synthesized reference substance (spectra: see figure 1(b)). The properties of the reference substance ('H-n.m.r., m.p.) matched those described by Dagne and Castagnoli (1972). For confirmation the reference substance was added to urine samples which were analysed on the two capillary columns with different polarities. The chromatograms showed a single, pure cis-isomer peak on both columns, thereby confirming the presence of ci~-3'-hydroxycotininein the samples. Furthermore, the occurrence of both isomers in the urine of rats, hamsters, and smokers was confirmed by h.p.1.c. The retention time on the C18 column was 14.0min for the cis-isomer and 14.6min for the trans-isomer. Following the i.v. administration of S( -)-nicotine to rats (n= 5 ) and hamsters (n= 4), both metabolites were determined in the urine samples by g.1.c.-mass spectrometry. In rat urine 11*2-15-8% (mean 12.8%) of the excreted 3'-hydroxycotinine was present as the cis-isomer. In hamster urine the range was 9*7-13*0% (mean 11.7%). In the urine of smokers (n=4, random samples) only 0.18423% (mean 0.21%) of the excreted 3'-hydroxycotinine was the cis-isomer. T o date there are no indications that the excretion of cis-3'-hydroxycotinine is dose-dependent. Further investigations will be performed to check if additional cis-3'-hydroxycotinine is excreted as a conjugate, as was reported for the transisomer by Kazemi Vala et af. (1990).
References BARLOW, R. D., THOMPSON, P. A., and STONE, R. B., 1987,Simultaneous determination of nicotine, cotinine and five additional nicotine metabolites in the urine of smokers using pre-column derivatization and high-performance liquid chromatography. Journal of Chromatography, 419, 375-380. BOWMAN, E. R., and MCKENNIS, H., 1962,Studies on the metabolism of (-)-cotinhe in the human. Journal of Pharmacology and Experimental Therapeutics, 135, 306-31 1. DAGNE, E., and CASTAGNOLI, N., 1972,Structure of hydroxycotinine, a nicotine metabolite. Journal of Medicinal Chemistry, 15, 356360. JACOB, P.,BENOWITZ, N. L., and SHULGIN, A. T., 1988,Recent studies of nicotine metabolismin humans. Pharmacology, Biochemistry and Behavior, 30, 249-253. KAZEMI VALA,E., ENGLUND, G., and CURVALL, M., 1990,Excretion of nicotine and its major metabolites in urine (meeting abstract). Fourth European Congress of Biopharmaceutics and Pharmacokinetics, Geneva, April. MCKENNIS, H., TURNBULL, L. B., BOWMAN, E. R., and WADA,E., 1959,Demethylation of cotinine in vivo. Journal of the American Chemical Society, 81, 3951-3954. MCKENNIS,H., TURNBULL, L. B., SCHWARTZ, S. L., TAMAKI, E., and BOWMAN, E. R., 1962, Demethylation in the metabolism of (-)-nicotine. Journal of Biological Chemistry, 237, 541-546. NEURATH, G. B., DUNGER, M., ORTH,D., and &IN, G. F., 1987, Trans-3'-hydroxycotinine as a main metatolite in urine of smokers. International Archives of Occupational and Environmental Health, 59, 199-201. NWOSW, C. G.,and CROOKS, P. A., 1988, Species variation and stereoselectivity in the metabolism of nicotine enantiomers. Xenobiotica, 18, 1361-1372.
1356
Identification of urinary cis-3’-hydroxycotinine
O’DOHERTY, S. O., REVANS, A., SMITH, C. L., MCBRIDE, M., and COOKE, M., 1988, Determination of cis-
Xenobiotica Downloaded from informahealthcare.com by University New South Wales on 08/26/15 For personal use only.
and trans-3-hydroxycotinine by high performance liquid chromatography. Journal of High Resolution Chromatography and Chromatography Communications, 11, 723-725. VONCKEN,P., SCHEPERS, G., and SCHXFER, K.-H., 1989, Capillary gas chromatographicdetermination of tram-3’-hydroxycotininesimultaneously with nicotine and cotinine in urine and blood samples. Journal of Chromatography, 479, 41W18.
1990, VOL. 20,
12, 1353-1356
NO.
Identification of cis-3’-hydroxycotinine as a urinary nicotine metabolite P. VONCKEN, K. RUSTEMEIER and G. SCHEPERS INBIFO Institut fur biologische Forschung, Fuggerstrasse 3, D-5000 Koln 90, FRG Xenobiotica Downloaded from informahealthcare.com by University New South Wales on 08/26/15 For personal use only.
~
Received 2 Janua~y1990, accepted 30 June 1990 1. cis-3’-Hydroxycotinine was detected as an S(-)-nicotine metabolite in the urine of smokers as well as in the urine of rats and hamsters dosed with nicotine.
2. The excreted amount of cis-3’-hydroxycotinineis lower than that of the trmrs-isomer.
Introduction Hydroxycotinine, a nicotine metabolite, was isolated three decades ago from the urine of dogs (McKennis et al. 1959), rats (McKennis et al. 1962), and humans (Bowman and McKennis 1962) dosed with nicotine or cotinine. The exact structure and stereoconformation of metabolic hydroxycotinine was established by Dagne and Castagnoli (1972) to be tran~-3‘-hydroxycotinine. However, it was not until recently that trans-3‘-hydroxycotininewas recognized as a major nicotine metabolite in the urine of smokers (Neurath et 01. 1987, Jacob et al. 1988) as well as in the urine of guinea pigs, Syrian hamsters, and rabbits following i.p. nicotine administration (Nwosu and Crooks 1988). The presence of cis-3’-hydroxycotinine as a urinary nicotine metabolite has not been reported to date, although O’Doherty et al. (1988) analysed the urine of a smoker for the presence of the cis-isomer but did not detect it, probably due to the detection limit of their h.p.1.c. method. Using capillary g.1.c.-mass spectrometry and h.p.l.c., we were able to separate and identify cis-3‘-hydroxycotinine as an S( -)-nicotine metabolite in the urine of rats and hamsters dosed with nicotine, as well as in the urine of smokers.
Materials and methods cis- and truns-3’-Hydroxycotinine were obtained from the Institut fur Biopharmazeutische Mikroanalytik, G. Neurath (Hamburg, FRG). For g.1.c.-mass spectrometric analyses a gas chromatograph (Hewlett-Packard, Model 5890) coupled to a computer-controlled mass spectrometer (Hewlett-Packard, Model 5990B) was used. A DB-1701 or DB-210 fused silica capillary column (30m x 025 mm; J&W via Carlo Erba, FRG) was directly coupled to the ion source (ionization voltage 70eV). For the h.p.1.c. analysesbased on the method described by Barlow et al. (1987) a liquid chromatograph (Hewlett-Packard, Model 1090M) and a Nova-Pak C18 column (15 cm x 3.9mm; Millipore, FRG) were used. As the mobile phase, sodium pentane sulphonate in phosphoric acid and the solvents methanol, tetrahydrofuran, and acetonitrilewith a ternary gradient were used. U.V.detection was performed at 532 nm. Full details of the g.1.c. and h.p.1.c. method will be reported elsewhere. Following the i.v. administration of 0.8 mg S(-)-nicotine/kg body weight to male Sprague-Dawley rats and 2.5 mg S( -)-nicotine/kg body weight to male Syrian hamsters, urine samples were collected for 24 h. From smokers with a daily consumption of 05-2 cigarette packs, random samples of urine were taken. Extraction of the urine samples for g.1.c. analyses was performed as described for the simultaneous determination of trans-3’-hydroxycotininewith nicotine and cotinine (Voncken et ul. 1989). 0049-8254/90 $3.00 0 1990 Taylor & Francis Ltd
P. Voncken et al.
1354
1
u 61 c Xenobiotica Downloaded from informahealthcare.com by University New South Wales on 08/26/15 For personal use only.
0
a iA
u Q!
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f 56
IJ
c II
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135
/
192
73
C
/
3
Y
a I .
1DD 12D Ma 9 s F C h P r
148
16W
1BB
38D
e
B
Figure 1 . Capillary gas chromatogram of hamster urine ( 0 ) and mass spectra of ~&3’-hydroxycotinine(6). (a)Chromatogram of a hamster urine sample following intravenous administration of nicotine (mass selective detection, selected ion monitoring, m / z 192): peaks: 1 = trans-3’-hydroxycotinine; 2 = cis-3’-hydroxycotinine.(6) Mass spectra of cis-3’-hydroxycotinine:A = from urine sample of an
S( -)-nicotine dosed hamster; B =from reference substance.
IdentiJication of urinary cis-3'-hydroxycotinine
1355
Xenobiotica Downloaded from informahealthcare.com by University New South Wales on 08/26/15 For personal use only.
Results and discussion Using the capillary g.1.c. method we were able to separate the trans- and cisisomers of 3'-hydroxycotinine. The retention times of the trans- and cis-isomer on the DB-1701 column were 20.8 and 21.2 min. On the DB-210 column the retention times were 1 6 2 and 1 6 9 min, respectively. The cis-isomer was detected as an S( -)nicotine metabolite in the urine of smokers and in the urine of rats dosed with nicotine intravenously, orally, or by inhalation, and in hamsters dosed intravenously (see figure 1(a)). The cis-isomer was identified in the urine of rats and hamsters by g.1.c.-mass spectrometry. Its full scan mass spectrum was compared with that of the pure, chemically synthesized reference substance (spectra: see figure 1(b)). The properties of the reference substance ('H-n.m.r., m.p.) matched those described by Dagne and Castagnoli (1972). For confirmation the reference substance was added to urine samples which were analysed on the two capillary columns with different polarities. The chromatograms showed a single, pure cis-isomer peak on both columns, thereby confirming the presence of ci~-3'-hydroxycotininein the samples. Furthermore, the occurrence of both isomers in the urine of rats, hamsters, and smokers was confirmed by h.p.1.c. The retention time on the C18 column was 14.0min for the cis-isomer and 14.6min for the trans-isomer. Following the i.v. administration of S( -)-nicotine to rats (n= 5 ) and hamsters (n= 4), both metabolites were determined in the urine samples by g.1.c.-mass spectrometry. In rat urine 11*2-15-8% (mean 12.8%) of the excreted 3'-hydroxycotinine was present as the cis-isomer. In hamster urine the range was 9*7-13*0% (mean 11.7%). In the urine of smokers (n=4, random samples) only 0.18423% (mean 0.21%) of the excreted 3'-hydroxycotinine was the cis-isomer. T o date there are no indications that the excretion of cis-3'-hydroxycotinine is dose-dependent. Further investigations will be performed to check if additional cis-3'-hydroxycotinine is excreted as a conjugate, as was reported for the transisomer by Kazemi Vala et af. (1990).
References BARLOW, R. D., THOMPSON, P. A., and STONE, R. B., 1987,Simultaneous determination of nicotine, cotinine and five additional nicotine metabolites in the urine of smokers using pre-column derivatization and high-performance liquid chromatography. Journal of Chromatography, 419, 375-380. BOWMAN, E. R., and MCKENNIS, H., 1962,Studies on the metabolism of (-)-cotinhe in the human. Journal of Pharmacology and Experimental Therapeutics, 135, 306-31 1. DAGNE, E., and CASTAGNOLI, N., 1972,Structure of hydroxycotinine, a nicotine metabolite. Journal of Medicinal Chemistry, 15, 356360. JACOB, P.,BENOWITZ, N. L., and SHULGIN, A. T., 1988,Recent studies of nicotine metabolismin humans. Pharmacology, Biochemistry and Behavior, 30, 249-253. KAZEMI VALA,E., ENGLUND, G., and CURVALL, M., 1990,Excretion of nicotine and its major metabolites in urine (meeting abstract). Fourth European Congress of Biopharmaceutics and Pharmacokinetics, Geneva, April. MCKENNIS, H., TURNBULL, L. B., BOWMAN, E. R., and WADA,E., 1959,Demethylation of cotinine in vivo. Journal of the American Chemical Society, 81, 3951-3954. MCKENNIS,H., TURNBULL, L. B., SCHWARTZ, S. L., TAMAKI, E., and BOWMAN, E. R., 1962, Demethylation in the metabolism of (-)-nicotine. Journal of Biological Chemistry, 237, 541-546. NEURATH, G. B., DUNGER, M., ORTH,D., and &IN, G. F., 1987, Trans-3'-hydroxycotinine as a main metatolite in urine of smokers. International Archives of Occupational and Environmental Health, 59, 199-201. NWOSW, C. G.,and CROOKS, P. A., 1988, Species variation and stereoselectivity in the metabolism of nicotine enantiomers. Xenobiotica, 18, 1361-1372.
1356
Identification of urinary cis-3’-hydroxycotinine
O’DOHERTY, S. O., REVANS, A., SMITH, C. L., MCBRIDE, M., and COOKE, M., 1988, Determination of cis-
Xenobiotica Downloaded from informahealthcare.com by University New South Wales on 08/26/15 For personal use only.
and trans-3-hydroxycotinine by high performance liquid chromatography. Journal of High Resolution Chromatography and Chromatography Communications, 11, 723-725. VONCKEN,P., SCHEPERS, G., and SCHXFER, K.-H., 1989, Capillary gas chromatographicdetermination of tram-3’-hydroxycotininesimultaneously with nicotine and cotinine in urine and blood samples. Journal of Chromatography, 479, 41W18.
